Page 1


Volume 7 2003 Published by Gene Therapy Press


!!!!!!!!!!!!!!!!!!!!!!!! ! Editor

Teni Boulikas Ph. D., CEO Regulon Inc. 715 North Shoreline Blvd. Mountain View, California, 94043 USA Tel: 650-968-1129 Fax: 650-567-9082 E-mail:

Teni Boulikas Ph. D., CEO, Regulon AE. Gregoriou Afxentiou 7 Alimos, Athens, 17455 Greece Tel: +30-210-9853849 Fax: +30-210-9858453 E-mail:

!!!!!!!!!!!!!!!!!!!!!!!! ! Assistant to the Editor Maria Vougiouka B.Sc., Gregoriou Afxentiou 7 Alimos, Athens, 17455 Greece Tel: +30-210-9858454 Fax: +30-210-9858453 E-mail:

!!!!!!!!!!!!!!!!!!!!!!!! ! Associate Editors

Aguilar-Cordova, Estuardo, Ph.D., AdvantaGene, Inc., USA Berezney, Ronald, Ph.D., State University of New York at Buffalo, USA Crooke, Stanley, M.D., Ph.D., ISIS Pharmaceuticals, Inc, USA Crouzet, Joël, Ph.D. Neurotech S.A, France Gronemeyer, Hinrich, Ph.D. I.N.S.E.R.M., IGBMC, France Rossi, John, Ph.D., Beckman Research Institute of the City of Hope, USA Shen, James, Ph.D., Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China & University of California at Davis, USA. Webb, David, Ph.D., Celgene Corporation, USA Wolff, Jon, Ph.D., University of Wisconsin, USA

!!!!!!!!!!!!!!!!!!!!!!!! ! Editorial Board Akporiaye, Emmanuel, Ph.D., Arizona Cancer Center, USA Anson, Donald S., Ph.D., Women's and Children's Hospital, Australia Ariga, Hiroyoshi, Ph.D., Hokkaido University, Japan Baldwin, H. Scott, M.D Vanderbilt University Medical Center, USA Barranger, John, MD, Ph.D., University of Pittsburgh, USA Black, Keith L. M.D., Maxine Dunitz Neurosurgical

Institute, Cedars-Sinai Medical Center, USA Bode, Jürgen, Gesellschaft für Biotechnologische Forschung m.b.H., Germany Bohn, Martha C., Ph.D., The Feinberg School of Medicine, Northwestern University, USA Bresnick, Emery, Ph.D., University of Wisconsin Medical School, USA Caiafa, Paola, Ph.D., Università di Roma “La Sapienza”, Italy Chao, Lee, Ph.D., Medical University of South Carolina, USA

Cheng, Seng H. Ph.D., Genzyme Corporation, USA Clements, Barklie, Ph.D., University of Glasgow, USA Cole, David J. M.D., Medical University of South Carolina, USA Chishti, Athar H., Ph.D., University of Illinois College of Medicine, USA Davie, James R, Ph.D., Manitoba Institute of Cell Biology;USA DePamphilis, Melvin L, Ph.D., National Institute of Child Health and Human, National Institutes of Health, USA Donoghue, Daniel J., Ph.D., Center for Molecular Genetics, University of California, San Diego, USA Eckstein, Jens W., Ph.D., Akikoa Pharmaceuticals Inc, USA Fisher, Paul A. Ph.D., State University of New York, USA Galanis, Evanthia, M.D., Mayo Clinic, USA Gardner, Thomas A, M.D., Indiana University Cancer Center, USA Georgiev, Georgii, Ph.D., Russian Academy of Sciences, USA Getzenberg, Robert, Ph.D., Institute Shadyside Medical Center, USA Ghosh, Sankar Ph.D., Yale University School of Medicine, USA Gojobori, Takashi, Ph.D., Center for Information Biology, National Institute of Genetics, Japan Harris David T., Ph.D., Cord Blood Bank, University of Arizona, USA Heldin, Paraskevi Ph.D., Uppsala Universitet, Sweden Hesdorffer, Charles S., M.D., Columbia University, USA Hoekstra, Merl F, Ph.D., Epoch Biosciences, Inc., USA Hung, Mien-Chie, Ph.D., The University of Texas, USA Johnston, Brian, Ph.D., Somagenics, Inc, USA Jolly, Douglas J, Ph.D., Advantagene, Inc.,USA Joshi, Sadhna, Ph.D., D.Sc., University of Toronto Canada Kaltschmidt, Christian, Ph.D., Universität Witten/Herdecke, Germany Kiyama, Ryoiti, Ph.D., National Institute of Bioscience and Human-Technology, Japan Krawetz, Stephen A., Ph.D., Wayne State University School of Medicine, USA Kruse, Carol A., Ph.D., La Jolla Institute for Molecular Medicine, USA Kuo, Tien, Ph.D., The University of Texas M. D. Anderson Cancer USA Kurachi Kotoku, Ph.D., University of Michigan Medical School, USA Kuroki, Masahide, M.D., Ph.D., Fukuoka University School of Medicine, Japan Lai, Mei T. Ph.D., Lilly Research Laboratories USA

Latchman, David S., PhD, Dsc, MRCPath University of London, UK Lavin, Martin F, Ph.D., The Queensland Cancer Fund Research Unit, The Queensland Institute of Medical Research, Australia Lebkowski, Jane S., Ph.D., GERON Corporation, USA Li, Jian Jian, Ph.D., City of Hope National Medical Center, USA Li, Liangping Ph.D., Max-Delbrück-Center for Molecular Medicine, Germany Lu, Yi, Ph.D., University of Tennessee Health Science Center, USA Lundstrom Kenneth, Ph.D. , Bioxtal/Regulon, Inc. Switzerland Malone, Robert W., M.D., Aeras Global TB Vaccine Foundation, USA Mazarakis, Nicholas D. Ph.D., Oxford BioMedica, UK Mirkin, Sergei, M. Ph.D., University of Illinois at Chicago, USA Moroianu, Junona, Ph.D., Boston College, USA Müller, Rolf, Ph.D., Institut für Molekularbiologie und Tumorforschung, Phillips-Universität Marburg, USA Noteborn, Mathieu, Ph.D., Leiden University, The Netherlands Papamatheakis, Joseph (Sifis), Ph.D., Institute of Molecular Biology and Biotechnology Foundation for Research and Technology Hellas, USA Platsoucas, Chris, D., Ph.D., Temple University School of Medicine, USA Rockson, Stanley G., M.D., Stanford University School of Medicine, USA Poeschla, Eric, M.D., Mayo Clinic, USA Pomerantz, Roger, J., M.D., Tibotec, Inc., USA Raizada, Mohan K., Ph.D., University of Florida, USA Razin, Sergey, Ph.D., Institute of Gene Biology Russian Academy of Sciences, USA Robbins, Paul, D, Ph.D., University of Pittsburgh, USA Rosenblatt, Joseph, D., M.D, University of Miami School of Medicine, USA Rosner, Marsha, R., Ph.D., Ben May Institute for Cancer Research, University of Chicago, USA Royer, Hans-Dieter, M.D., (CAESAR), Germany Rubin, Joseph, M.D., Mayo Medical School Mayo Clinic, USA Saenko Evgueni L., Ph.D., University of Maryland School of Medicine Center for Vascular and Inflammatory Diseases, USA Salmons, Brian, Ph.D., (FSG-Biotechnologie GmbH), Austria Santoro, M. Gabriella, Ph.D., University of Rome Tor Vergata, USA Sharrocks, Andrew, D., Ph.D., University of Manchester, USA

Shi, Yang, Ph.D., Harvard Medical School, USA Smythe Roy W., M.D., Texas A&M University Health Sciences Center, USA Srivastava, Arun Ph.D., University of Florida College of Medicine, USA Steiner, Mitchell, M.D., University of Tennessee, USA Tainsky, Michael A., Ph.D., Karmanos Cancer Institute, Wayne State University, USA Sung, Young-Chul, Ph.D., Pohang University of Science & Technology, Korea Taira, Kazunari, Ph.D., The University of Tokyo, Japan Terzic, Andre, M.D., Ph.D., Mayo Clinic College of Medicine, USA Thierry, Alain, Ph.D., National Cancer Institute, National Institutes of Health, France

Trifonov, Edward, N. Ph.D., University of Haifa, Israel Van de Ven, Wim, Ph.D., University of Leuven, Belgium Van Dyke, Michael, W., Ph.D., The University of Texas M. D. Anderson Cancer Center, USA White, Robert, J., University of Glasgow, UK White-Scharf, Mary, Ph.D., Biotransplant, Inc., USA Wiginton, Dan, A., Ph.D., Children's Hospital Research Foundation, CHRF , USA Yung, Alfred, M.D., University of Texas, USA Zannis-Hadjopoulos, Maria Ph.D., McGill Cancer Centre, Canada Zorbas, Haralabos, Ph.D., BioM AG Team, Germany

!!!!!!!!!!!!!!!!!!!!!!!! ! Associate Board Members

Aoki, Kazunori, M.D., Ph.D., National Cancer Center Research Institute, Japan Cao, Xinmin, Ph.D., Institute of Molecular and Cell Biology, Singapore Falasca, Marco, M.D., University College London, UK Gao, Shou-Jiang, Ph.D., The University of Texas Health Science Center at San Antonio, USA Gibson, Spencer Bruce, Ph.D., University of Manitoba, USA Gra•a, Xavier, Ph.D., Temple University School of Medicine, USA

For submission of manuscripts and inquiries: Editorial Office Teni Boulikas, Ph.D./ Maria Vougiouka, B.Sc. Gregoriou Afxentiou 7 Alimos, Athens 17455 Greece Tel: +30-210-985-8454 Fax: +30-210-985-8453 and electronically to

Gu, Baohua, Ph.D., The Jefferson Center, USA Hiroki, Maruyama, M.D., Ph.D., Niigata University Graduate School of Medical and Dental Sciences, Japan MacDougald, Ormond A, Ph.D., University of Michigan Medical School, USA Rigoutsos, Isidore, Ph.D., Thomas J. Watson Research Center, USA

Instructions to authors: Gene Therapy and Molecular Biology (GTMB) FREE ACCESS Scope Gene Therapy and Molecular Biology, bridging various fields is one of the most rapid with free access at The scope of Gene Therapy and Molecular Biology is to promote interaction between researchers in the fields of Gene Therapy and Molecular Biology providing rapid publication of review articles and research papers. Articles (both invited and submitted) review or report novel findings of importance to a general audience in gene therapy, molecular medicine, gene discovery, and molecular biology with emphasis to molecular mechanisms. The journal will accept papers on all aspects of gene therapy, including gene delivery systems, gene therapy of cancer and other diseases (e.g. CFTR, hemophilia, AIDS, restenosis) at the clinical, preclinical or cell culture stage, gene discovery, cancer immunotherapy, DNA vaccines, use of DNA regulatory elements in gene transfer, cell therapy and transplantation, arraying technologies & DNA chips, peptide libraries and drug discovery related to gene therapy, cell targeting, gene targeting, therapy with oligonucleotides (antisense, ribozymes, triplex). The authors are encouraged to elaborate on the molecular mechanisms that govern a gene therapy approach. Gene Therapy and Molecular Biology will also publish articles on, transcription factors, DNA replication, recombination, repair, chromatin, nuclear matrix, DNA regulatory regions, locus control regions, protein phosphorylation, signal transduction, development, and on molecular mechanism of human disease. To make the publication attractive authors are encouraged to include color figures.

Type of articles Both review articles and original research articles will be considered. In addition, short 1-2 page news & views will also be considered for publication. Original research articles should contain a generous introduction in addition to experimental data. The articles contain information important to a general audience as the volume is also addressed to researches outside the field. There is no limit on the length of the articles provided that the subject is interesting to a general audience and covers exhaustively a field. The typical length of each manuscript is a approximately 4-20 printed page including Figures and Tables. This is 12-60 manuscript pages. Charges, Complimentary reprints & Subscriptions There are no charges for color figures or page numbers. Corresponding authors get a one-year free subscription (hard copy) plus 25 reprints free of charge. The free subscription can be renewed for additional years by having one paper per year accepted for publication. The free electronic access to articles published in " Gene Therapy and Molecular Biology " to a big general audience, the attractive journal title, the speed of the reviewing process, the no-charges for page numbers or color figure reproduction, the 25 complimentary reprints, the rapid electronic publication, the embracing of many fields in gene therapy (from molecular mechanisms to clinical trials), the high quality

in depth reviews and first rate research articles and most important, the eminent members of the Editorial Board being assembled are prognostic factors of a big success for GTMB.

Sections of the manuscript Each manuscript should have a Title, Authors, Affiliation, Corresponding Author (with Tel, Fax, and Email), Summary, key words , running title and Introduction; review articles are subdivided into headings I, II, III, etc. (starting with I. Introduction) subdivided into A, B, C, and further subdivided using 1, 2, 3, etc. You can further subdivide into 1, 2, 3, etc. Research articles are divided into Summary; I. Introduction; II. Materials and Methods III. Results; IV. Discussion; Acknowledgments; and References. Please include in your text citations the name of authors and year in parenthesis; for three or more authors use: (name of first author et al, with year); for two authors please use both names. Please delete hidden text for references. In the reference list, please, type references with year and Journal in boldface and provide full title of the article such as: Buschle M, Schmidt W, Berger M, Schaffner G, Kurzbauer R, Killisch I, Tiedemann J-K, Trska B, Kirlappos H, Mechtler K, Schilcher F, Gabler C, and Birnstiel ML (1998) Chemically defined, cell-free cancer vaccines: use of tumor antigen-derived peptides or polyepitope proteins for vaccination. Gene Ther Mol Biol 1, 309-321. To avoid delays it is essential to submit an electronic and a hard copy version of your manuscript via email and mail in a floppy, CD-ROM or ZIP, containing the manuscript that will be used to typeset the paper. Please include in the digital media: Tables, if any, (preferably as a Microsoft Word text) and Figure legends. Please use Microsoft Word, font “Times” (Mac users) or “Times New Roman” (PC users) and insert Greek or other characters using the “Insert/Symbol” function in the Microsoft Word rather than simple conversion to font “Symbol”. Please boldface Figure 1, 2, 3 etc. as well as Table 1, 2, etc. throughout the text. Please provide the highest quality of prints of your Figures; whenever possible, please provide in addition an electronic version of your figures. Article contributors are kindly requested to provide a color (or black/white) photo of themselves (preferably 4x5 cm or any size) or a group photo of the authors, as we shall include these in the publication Submission and reviewing Peer reviewing is by members of the Editorial Board and external referees. Please suggest 2-3 reviewers providing their electronic addresses, mailing addresses and telephone/fax numbers. Authors are sent page proofs. Gene Therapy and Molecular Biology is published in on high quality paper, hardbound, and with excellent reproduction of color figures. Reviewing is completed within 5-15 days from receiving the manuscript. Articles accepted without revisions (i.e., review articles) will be published online ( in approximately 1 month following submission.

Please submit an electronic version of full text and figures preferably in jpeg format. The electronic version of the figures will be used for the rapid reviewing process. High quality prints or photograph of the figures and the original with one copy should be sent via express mail to the Editorial Office. Editorial Office Teni Boulikas, Ph.D./ Maria Vougiouka, B.Sc. Gregoriou Afxentiou 7 Alimos, Athens 17455 Greece Tel: +30-210-985-8454 Fax: +30-210-985-8453 and electronically to The free electronic access to articles published in "GTMB" to a big general audience, the attractive journal title, the speed of the reviewing process, the no-charges for page numbers or color figure reproduction, the 25 complimentary reprints, the rapid electronic publication, the embracing of many fields in cancer, the anticipated high quality in depth reviews and first rate research articles and most important, the eminent members of the Editorial Board being assembled are prognostic factors of a big success for the newly established journal.

Table of contents

Gene Therapy and Molecular Biology Vol 7, December 2003 Pages

Type of Article

Article title

Authors (corresponding author is in boldface)


Review Article

Dynamic histone acetylation and its involvement in transcription

Virginia A. Spencer and James R. Davie


Review Article

Tumor therapy using radiolabelled antisense oligomers- aspects for antiangiogenetic strategy and positron emission tomography

Kalevi JA Kairemo, Mark Lubberink, Mikko Tenhunen, Antti P Jekunen


Review Article

Strategy of sensitizing tumor cells with adenovirus-p53 transfection

Jekunen Antti, Miettinen Susanna, Mäenpää Johanna, Kairemo Kalevi


Review Article

Antigenicity and immunogenicity of HIV envelope gene expressed in baculovirus expression system

Alka Arora, Pradeep Seth


Review Article

Characterization of genes transcribed in an Ixodes scapularis cell line that were identified by expression library immunization and analysis of sequence tags

Consuelo Almazan, Katherine M. Kocan, Douglas K. Bergman, Jose C. GarciaGarcia, Edmour F. Blouin and José de la Fuente


Research Article

Delayed intratracheal injection of manganese superoxide dismutase (MnSOD)-plasmid/liposomes provides suboptimal protection against irradiationinduced pulmonary injury compared to treatment before irradiation

Michael W. Epperly, Hongliang Guo, Michael Bernarding, Joan Gretton, Mia Jefferson, Joel S. Greenberger


Mini Review

Regulation of vascular endothelial growth factor by hypoxia

Ilana Goldberg-Cohen, Nina S Levy, Andrew P Levy


Review Article

Gene therapy antiproliferative strategies against cardiovascular disease.

Marisol Gasc!n-Ir"n, Silvia M. SanzGonz#lez and Vicente Andrés


Review Article

Regulation of the Sp/KLF-family of transcription factors: focus on posttranscriptional modification and proteinprotein interaction in the context of chromatin

Toru Suzuki, Masami Horikoshi, and Ryozo Nagai


Research Article

Detection of MET oncogene amplification in hepatocellular carcinomas by comparative genomic hybridization on microarrays

W.L. Robert Li, Nagy A. Habib, Steen L. Jensen, Paul Bao, Diping Che, Uwe R. Müller


Research Article

HMG-CoA-reductase inhibitiondependent and independent effects of statins on leukocyte adhesion

Triantafyllos Chavakis, Thomas Schmidt-Wรถll, Peter. P. Nawroth, Klaus T. Preissner, Sandip M. Kanse


Review Article

Current progress in adenovirus mediated gene therapy for patients with prostate carcinoma

Ahter D. Sanlioglu,, Turker Koksal, Mehmet Baykara, Guven Luleci, Bahri Karacay and Salih Sanlioglu


Review Article

Gene therapy for vascular diseases

Sarah J. George, Filomena de Nigris, Andrew H. Baker, Claudio Napoli


Review Article

Angiogenic gene therapy for improving islet graft vascularization.

Nan Zhang, Karen Anthony, Katsunori Shinozaki, Jennifer Altomonte, Zachary Bloomgarden and Hengjiang Dong


Research Article

G-CSF Receptor-mediated STAT3 activation and granulocyte differentiation in 32D cells.

Ruifang Xu, Akihiro Kume, Yutaka Hanazono, Kant M. Matsuda, Yasuji Ueda, Mamoru Hasegawa, Fumimaro Takaku, and Keiya Ozawa


Research Article

Calcium induces apoptosis and necrosis in hematopoetic malignant cells: Evidence for caspase-8 dependent and FADDautonomous pathway

Christof J. Burek Malgorzata Burek, Johannes Roth, and Marek Los


Review Article

The current status and future direction of fetal gene therapy

Anna L David, Michael Themis, Simon N Waddington, Lisa Gregory, Suzanne MK Buckley, Megha Nivsarkar, Terry Cook, Donald Peebles, Charles H Rodeck, Charles Coutelle


Research Article

The role of EBV and genomic sequences in gene expression from extrachromosomal gene therapy vectors in mouse liver

Stephanie M. Stoll, Leonard Meuse, Mark A. Kay, and Michele P. Calos


Review Article

Site-specific kidney-targeted plasmid DNA transfer using nonviral techniques

Hiroki Maruyama, Noboru Higuchi, Shigemi Kameda, Gen Nakamura, Junichi Miyazaki, and Fumitake Gejyo


Research Article

Hepatocyte-targeted delivery of Sleeping Beauty mediates efficient gene transfer in vivo

Betsy T. Kren, Siddhartha S. Ghosh, Cheryle L. Linehan, Namita RoyChowdhury, Perry B. Hackett, Jayanta Roy-Chowdhury, and Clifford J. Steer


Research Article

PRL-3 as a target for cancer therapy

Koh Vicki, Fu Jianlin, Guo Ke, Lip Kuo Ming, Li Jie and Zeng Qi


Review Article

Protective effect of heat shock proteins: potential for gene therapy

David S. Latchman


Review Article

Lung cancer gene therapy


Review Article

Advances in cationic lipid-mediated gene delivery

Kexia Cai, Mai Har Sham, Paul Tam, Wah Kit Lam and Ruian Xu Benjamin Martin, Abderrahim Aissaoui, Matthieu Sainlos, Noufissa Oudrhiri, Michelle Hauchecorne, Jean-Pierre


Research Article

Unusual chemical hypersensitivity of the d(GA)n• d(TC)n repeat in vivo dependent on functional lactose repressor

Vigneron, Jean-Marie Lehn and Pierre Lehn Gerald L. Buldak and Sergei M. Mirkin

Gene Therapy and Molecular Biology Vol 7, page 1 Gene Ther Mol Biol, Vol 7, 1-13, 2002

Dynamic histone acetylation and its involvement in transcription Review Article

Virginia A. Spencer and James R. Davie! Manitoba Institute of Cell Biology, 675 McDermot Avenue, Winnipeg, Manitoba, R3E 0V9 CANADA

__________________________________________________________________________________ Correspondence: Dr. J.R. Davie; Manitoba Institute of Cell Biology; 675 McDermot Avenue Winnipeg, MB, R3E 0V9; Tel: (204) 7872391; Fax: (204) 787-2190; E-mail: Key words: histone acetylation, histone acetyltransferases and deacetylases, transcription Received: 10 January 2002; accepted: 22 January, 2002; electronically published: July 2003

Summary Histones are subject to a variety of posttranslational modifications, the most studied being acetylation of the N terminal lysine residues. Acetylation is a dynamic event mediated by the actions of histone acetyltransferases and deacetylases. The exact function of this event in transcription has remained an enigma for several reasons. The enzymes that catalyze this dynamic event act on histones, but are also capable of affecting the properties of nonhistone proteins including transcription factors. Also, some histone acetyltransferases can acetylate the histones along a specific gene, while simultaneously acetylating the histones over an entire region of the genome. More confusing are the observations that the acetylation pattern of histones along one transcriptionally active gene may differ significantly from that along another. Perhaps some of these discrepancies can be explained by the dynamic interplay between histone acetyltransferases and deacetylases, and the proximity of a gene to these enzymes. Thus, to fully appreciate the role of acetylation in transcription, we must further understand the dynamic nature of this event. with proteins or DNA (Hansen et al, 1998). The DNA extending from one nucleosome to another varies in length and is referred to as linker DNA (Spencer and Davie, 1999). A fifth type of histone called linker histone H1 contains a central globular domain surrounded by N and C-terminal tails. Histone H1 binds to the regions of linker DNA that enter and exit the nucleosome, as well as to nucleosomal DNA near the dyad axis of symmetry (Spencer and Davie, 1999).

I. The organization of nuclear DNA The DNA within a cell is packaged into chromatin. The basic structural repeating unit of chromatin is the nucleosome which is composed of an octamer of two histone H2A-H2B dimers bound to a histone H3 and H4 tetramer (Spencer and Davie, 1999). During nucleosome assembly, histones H3 and H4 first associate with DNA, followed by histones H2A and H2B (Kimura and Cook, 2001). The association of H3 and H4 with nucleosomal DNA appears to be more stable than that for H2A and H2B. The end result is the wrapping of one hundred and forty-six base pairs of DNA around each histone octamer. The four core histones have a basic N terminal tail, a central globular domain organized into a histone fold and a C terminal tail (Figure 1). The central histone fold domain is involved in histone-histone and histone-DNA interactions, and, therefore is important in histone octamer and nucleosome formation (Spencer and Davie, 1999). The histone N terminal tails protrude from the core particle in all directions and vary in length from 16 to 44 amino acids (Davie and Spencer, 2001). Evidence showing that H3 and H4 display "-helical structures in their N terminal domains when bound to nucleosomal DNA has lead to the belief that these domains fold upon contact

II. Chromatin structure & organization At physiological ionic strength, chromatin assumes the form of a 30 nm fiber and higher order structures (Davie, 1995). This fiber is a dynamic structure that is continually condensing and unfolding. For example, a chromatin fiber composed of nucleosomes spaced at physiological intervals is in equilibrium between an unfolded, moderately folded, highly folded and oligomerized conformation (Annunziato and Hansen, 2000). The proteolytic removal of the N terminal domains does not significantly change nucleosome structural integrity, and, instead, prevents the formation of the 30 nm fiber (Davie and Spencer, 2001). Thus, the stability of this


Spencer and Davie: Dynamic histone acetylation and its involvement in transcription 30 nm fiber is maintained by the N terminal tails (Davie and Spencer, 2001). The chromatin fiber becomes moderately folded by the H3 and H4 N terminal tails at physiological ionic strength. However, the N terminal tails of the four core histones are required for the chromatin fiber to undergo extensive folding (Tse and Hansen, 1997; Logie et al, 1999). At low ionic strength, the chromatin fiber assumes a three-dimensional irregular shape that is stabilized by the globular domain of H1 and either the H1 tails or the H3 N terminal tail (Zlatanova et al, 1998; Leuba et al, 1998a). The N terminal tails from histones H2A, H2B and H4 do not have the same effect as H3 on the chromatin fiber. However, the N terminal tail of H3 is 44 amino acids long, whereas histones H4, H2B and H2A have N terminal tails that are only 26, 32, and 16 amino acids long, respectively. As a result, the N terminal tail of histone H3 can extend over a significantly larger portion of linker DNA compared to the other core histones (Leuba et al, 1998b). The H3 N terminus is also positioned close to the point where linker DNA enters and exits the nucleosome, and, therefore, it can undergo extensive interactions with the linker DNA (Zlatanova et al, 1998). The chromatin fibers within a cell interdigitate with neighboring fibers into a higher order fibrous mass that impedes the access of transcription factors to their target sequences, thereby preventing transcription initiation (Schwarz et al, 1996). At physiological ionic strength, the interaction of these neighboring fibers with one another is partly dependent on either the H2A and H2B or the H3 and H4 core histone N terminal tails (Davie and Spencer, 2001). These fibrous masses are then further organized into compact chromosome territories within interphase nuclei (Verschure et al, 1999).

In addition to binding linker DNA, the histone N terminal tails are capable of interacting with other histones and non-histone chromosomal proteins. The N terminus of H4 binds to the H2A-H2B dimer of neighboring nucleosomes, and, as such, is thought to assist in chromatin folding (Luger et al, 1997). In yeast, the transcriptional repressors Sir3, Sir4, and Ssn6/Tup1 interact with the H3 and H4 N terminal domains, causing the associated chromatin to become transcriptionally repressed (Grunstein, 1998). Likewise, the Drosophila Groucho and its mammalian homologues bind to the N terminal domain of H3 and repress transcription (Palaparti et al, 1997; Fisher and Caudy, 1998). These domains also interact with non-histone proteins such as HMG-14 and HMG-17 that promote the unfolding of higher order chromatin structures (Bustin, 1999).

III. Acetylation of the histone N terminal tails The N terminal tails can undergo a series of posttranslational modifications at specific amino acids including acetylation, phosphorylation, ubiquitination and methylation (Spencer and Davie, 1999) (Figure 1). The most extensively studied of these modifications is dynamic acetylation, a reversible process catalyzed by acetyltransferases and deacetylases which mediate the transfer of acetyl groups on to and off of the #-amino group of N terminal lysine residues, respectively (Kuo and Allis, 1998).

Figure 1. General structure of the core histones and their sites of post-translational modifications. The central globular domain of each histone is depicted as a circle with the N and C terminal tails extending towards the left and right sides, respectively. Me, Ac, P, and Ub represent methylation, acetylation, phosphorylation, and ubiquitination, respectively. HAT A (histone acetyltransferase) and HDAC (histone deacetylase) represent the enzymes that catalyze the reversible acetylation of lysine residues along the histone N terminal tails. H3 kinase and PP1 (protein phosphatase 1) represent the enzymes responsible for the reversible phosphorylation of H3 serine residue.


Gene Therapy and Molecular Biology Vol 7, page 3 HDACs 1,2,3 and 8. These class I members are nuclear transcriptional co-repressors with homology to the yeast Rpd3 deacetylase. The class II histone deacetylases are larger proteins of approximately 1000 amino acids with structural homology to yeast Hda1 and include HDACs 4,5,6,7,9 and 10 (Davie and Moniwa, 2000; Bertos et al, 2001; Guardiola and Yao, 2002). Class III histone deacetylases are encoded by genes similar to the yeast silent information regulator (Sir 2) gene (Afshar and Murnane, 1999; Frye, 1999). These deacetylases are dependent on NAD+ and ADP-ribosylase activity (Frye, 2000; Imai et al, 2000; Landry et al, 2000). Class I deacetylases are ubiquitously expressed, while class II deacetylases are tissue-, cell-and differentiation-specific (Davie and Moniwa, 2000). Both classes of deacetylases can deacetylate the four core histones, however, each deacetylase has a site preference (Davie and Spencer, 2001). Similar to histone acetyltransferases, the yeast Rpd3 and Hda1 deacetylases exist in distinct multi-protein complexes, suggesting that class I and II deacetylases have distinct biological functions. Furthermore, the components of these complexes influence the substrate specificity of these enzymes (Davie and Moniwa, 2000). For example, the free form of avian HDAC1 preferentially deacetylates free but not nucleosomal H3. When assembled into a multi-protein complex, this deacetylase preferentially deacetylates free H2B and histones assembled into a nucleosome (Sun et al, 1999). Class I deacetylases reside in the nucleus (Davie and Moniwa, 2000). However, the sub-cellular distribution of class II deacetylases is not as straight forward. HDACs 4 and 5 shuttle between the cytoplasm and the nucleus (Bertos et al, 2001). HDAC7 is predominantly nuclear but binds to the membrane-associated endothelin receptor A and most likely functions in the cytoplasm (Lee et al, 2001). HDAC6 is strictly cytoplasmic, and HDAC9 appears to be both nuclear and cytoplasmic (Zhou et al, 2001). HDACs 4,5, and 7 are transcriptional co-repressors that interact with MEF2 transcription factors, as well as the co-repressors N-CoR, BCoR, and CtBP (Bertos et al, 2001; Guardiola and Yao, 2002). Similarly, HDAC9 interacts with MEF-2 and represses MEF-2-mediated transcription (Zhou et al, 2001). HDAC10 resides in the nucleus and the cytoplasm (Guardiola and Yao, 2002). In the nucleus, this deacetylase functions as a transcriptional repressor when tethered to a promoter (Guardiola and Yao, 2002). Interestingly, HDAC6 can interact with ubiquitin. As well, the mammalian homologue of UFD3, a yeast protein involved in protein ubiquitination, is part of the cytoplasmic mammalian HDAC6 complex (SeigneurinBerny et al, 2001).

This modification typically occurs on up to five lysine residues along the H3 and H4 N terminal tails, four residues along H2B, and one residue along H2A (Davie and Spencer, 1999). Whether a histone is hypo- or hyperacetylated depends on the net activities of neighboring histone acetyltransferases and deacetylases.

IV. Histone acetyltransferases The following is only a brief summary of the histone acetyltransferases identified to date. For a more detailed description of histone acetyltransferases and their substrates, please refer to the following reviews (Sterner and Berger, 2000; Davie and Spencer, 2001; Marmorstein and Roth, 2001; Bertos et al, 2001). Numerous transcription co-activators including yGcn5, P/CAF, CBP/p300, Esa1, NuA4, and ACTR/SRC-1 have been identified as having intrinsic histone acetyltransferase activity (Sterner and Berger, 2000; Davie and Spencer, 2001; Klochendler-Yeivin and Yaniv, 2001; Marmorstein and Roth, 2001). In addition, the DNA-binding transactivator ATF-2, the general transcription factors TAFII250 and Nut1, and the elongation factor Elp3 are histone acetyltransferases (Marmorstein and Roth, 2001). Histone acetyltransferases generally exist in large complexes (Spencer and Davie, 1999). Each histone acetyltransferase has a different target substrate, and the specificity for this substrate depends on the proteins associated with the histone acetyltransferase (Grant et al, 1999). For example, the free full-length form of yeast Gcn5 preferentially acetylates H3 in vitro and H3 and H4 in vivo (Zhang et al, 1998; Sterner and Berger, 2000; Davie and Spencer, 2001). However, the acetylating efficiency of yeast Gcn5 for nucleosomal histones increases when assembled into high molecular weight, multi-protein complexes referred to as SAGA (Spt-AdaGcn5-acetyltransferase) and Ada (Grant et al, 1999). In addition, the pattern of histone acetylation for Gcn5 assembled into the SAGA complex is distinct from that exhibited by Gcn5 when assembled into Ada (Grant et al, 1999). Similarly, the histone substrate specificity of individual human PCAF and yeast Esa1 acetyltransferases becomes altered when these enzymes are assembled into multi-protein complexes (Davie and Spencer, 2001). The phosphorylation of CBP by ERK1 enhances the activity of this acetyltransferase, suggesting that the function of histone acetyltransferases may be regulated by phosphorylation events (Liu et al, 1999).

V. Histone deacetylases As many as 10 histone deacetylases have been identified to date (Bertos et al, 2001). Refer to the following reviews (Sterner and Berger, 2000; Bertos et al, 2001; Davie and Spencer, 2001; Marmorstein and Roth, 2001) for a more detailed description of histone deacetylases. These deacetylases are divided into 3 classes defined by their size and sequence homologies to yeast deacetylases. The class I histone deacetylases are approximately 400-500 amino acids in length and include

VI. The acetylation




Studies of histone acetylation dynamics indicate that both acetylation and deacetylation occur at more than one rate (Covault and Chalkley, 1980; Zhang and Nelson, 1988a). In human fibroblasts and mature avian


Spencer and Davie: Dynamic histone acetylation and its involvement in transcription erythrocytes, there are two populations of acetylated histones. The first population, which accounts for approximately 15% of acetylated core histones in hepatoma tissue culture cells, is rapidly hyperacetylated (t1/2= 7 to 15 min for monoacetylated H4) and rapidly deacetylated (t1/2= 3 to 7 min). The second population, which accounts for up to 50% of acetylated histones, is slowly acetylated (t1/2= 140-300 min for monoacetylated H4) and then slowly deacetylated (t1/2= 30 min) (Covault and Chalkley, 1980; Zhang and Nelson, 1988a). Similarly, MCF-7 human breast cancer cells also display two populations of acetylated H3, H4 and H2B histones: a rapidly acetylated one comprising 10% of the total nuclear acetylated histones and a slowly acetylated one that includes approximately 50% of acetylated histones (Sun et al, 2001). In immature chicken erythrocytes, approximately 2% of the genome is dynamically acetylated, while the rest is either frozen in a state of mono- or di-acetylation or unacetylated (Zhang and Nelson, 1988a). The acetylated histones in immature avian erythrocytes are divided into two populations. In contrast to mature avian erythrocytes, both populations within the immature erythrocytes display the same rate of histone acetylation (t1/2=12 min for monoacetylated H4). However, in the case of H4, one population is hyperacetylated to tri- or tetra-acetylated isoforms and then rapidly deacetylated (t1/2= 5 min) (referred to as class I). The other population, however, is only mono- or di-acetylated, and subsequently deacetylated at a slower rate (t1/2=90 min) (referred to as class II)(Zhang and Nelson, 1988a; Zhang and Nelson, 1988b). Histones H3 and H2B are also class I acetylated since butyrate-treated immature chicken erythrocytes display a drastic and rapid decline in tri- and tetraacetylated H3 and H2B within 10 minutes of incubation in

the absence of butyrate (Spencer and Davie, 2001) (Figure 2).

VII. The effect of histone acetylation on chromatin structure Histone acetylation affects chromatin structure in several ways. One theory suggests that histone acetylation alters nucleosome structure and weakens the interaction of histone N terminal tails with DNA (Turner, 1991; Norton et al, 1989). Histone acetylation also maintains the open conformation of the transcriptionally active nucleosome (Walia et al, 1998). Thus, histone acetylation may neutralize the positive charges on the N terminal lysine residues, and loosen the contacts between histones and DNA. However, Gcn5 similarly affects transcription and cell growth whether H3 contains a lysine, arginine, or glutamine at position 14 of its N terminal tail. Similarly, replacement of lysine 8/16 residues with arginine or glutamine does not alter the affect of Gcn5 on transcription or cell growth (Zhang et al, 1998). This suggests that histone acetylation may influence transcription by mechanisms other than the neutralization of N terminal lysine residues. Histone acetylation is also thought to disrupt the higher order folding of chromatin fibers (Garcia-Ramirez et al, 1995; Moore and Ausio, 1997; Hansen, 1997). At physiological salt concentrations, acetylated chromatin fibers are salt-soluble, while unacetylated fibers are insoluble (Ridsdale et al, 1990). However, these fibers are incapable of interacting with other fibers by the process of oligomerization, and, therefore, are unable to form higher order structures (Annunziato and Hansen, 2000).

Figure 2. Immunoblot analyses of H2B deacetylation. Avian immature erythrocytes were incubated with sodium butyrate for 1 h, and then incubated in the absence of butyrate for 0, 5, 10, 15 or 30 min. The total nuclear histones from erythrocytes at each time point were extracted. Twenty Âľg of acid-extracted histones were electrophoresed on an Acid-Urea-Triton 15% polyacrylamide gel. The resolved proteins were then transferred to nitrocellulose and immunostained with an antibody to hyperacetylated H2B (Serotec, UK). 0, 1, 2, 3, and 4 designate un-, mono-, di-, tri-, and tetra-acetylated histone isoforms, respectively .


Gene Therapy and Molecular Biology Vol 7, page 5

VIII. The effect of histone acetylation on ATP-dependent chromatin remodeling

The acetylation of only 12 out of 28 lysine residues per histone octamer promotes transcription approximately 15 fold in vitro, and affects chromatin similar to the proteolytic removal of the core histone N terminal tails (Tse et al, 1998; Annunziato and Hansen, 2000). As a result, acetylation of the histone N terminal tails is thought to facilitate transcription by disrupting the folding of the chromatin fiber, as well as inter-fiber interactions. Such an event would allow transcription factors access to their target DNA binding sites. In support of this, the treatment of estrogen-responsive cells with estrogen induces H3 and H4 acetylation along the TATA sequence of the PS2 promoter, subsequently, exposing the TATA binding site and allowing the TATA binding protein to bind to this site (Sewack et al, 2001). In addition, chromatin immunoprecipitation studies show an enrichment of hyperacetylated H3 and H4 along the promoter regions of several genes including the vitamin A and vitamin D genes when transcriptionally activated (Chen et al, 1999; Kadosh and Struhl, 1998; Parekh and Maniatis, 1999; Krebs et al, 1999). As well, the binding of estrogen to its receptor leads to the recruitment of p300/CBP to the promoter of estrogen-responsive genes (Chen et al, 1999). In addition to disrupting chromatin fiber-fiber interactions, histone acetylation disrupts the interactions between the histone N terminal tails and non-nucleosomal proteins or DNA. For example, H3 and H4 hyperacetylation abolish Ssn6-Tup1-mediated transcriptional repression (Watson et al, 2000). The histone N terminal domains display "-helical structures when assembled into the nucleosome (Annunziato and Hansen, 2000). This "-helical character increases upon acetylation (Wang et al, 2000). Histone acetyltransferases may positively influence transcription by altering the structure of the N terminal tails and perturbing the interactions of these tails with proteins that repress transcription. However, histone acetylation may also be associated with transcriptional repression since the heterochromatin of several organisms contains H4 acetylated at lysine 12 (Turner, 2000; Turner et al, 1992). As well, loss of the yeast RPD3 histone deacetylase causes an increase in the silencing of telomeric DNA (De Rubertis et al, 1996). It has also been suggested that histone acetylation plays a role in marking the state of genetic activity or inactivity from one cell generation to the next, thereby epigenetically determining the long-term transcriptional competence of a gene (Turner, 1998). However, recent evidence shows that catalytically active histone acetyltransferases and histone deacetylases are unable to acetylate or deacetylate chromatin in situ during mitosis (Kruhlak et al, 2001). Moreover, these enzymes become spatially reorganized and displaced from condensing chromosomes. Instead, it appears that the spatial organization of these enzymes relative to euchromatin and heterochromatin plays an important role in determining the post-mitotic activation of a gene (Kruhlak et al, 2001).

Besides playing a role in transcription factor binding, histone acetylation may also be fundamental for ATPdependent chromatin remodeling. These type of complexes use ATP hydrolysis as a source of energy to alter nucleosome and chromatin structure and enhance transcription factor binding to nucleosomal DNA-binding sites (Davie and Moniwa, 2000). For a more detailed description of ATP-dependent chromatin remodeling factors refer to the following reviews (Kingston and Narlikar, 1999; Davie and Moniwa, 2000). While these complexes can alter the chromatin structure of transactivator binding sites, they are unable to activate transcription alone (Gregory et al, 1999). The recruitment of the SWI/SNF chromatin remodeling complex to nuclear receptor and BRCA1-regulated genes is thought to increase nucleosome fluidity, and facilitate the subsequent binding of transcription factors to affected regions (Singh et al, 2000). In the case of the yeast HO gene, the binding of the chromatin remodeling factor, SWI/SNF leads to the recruitment of the SAGA histone acetyltransferase complex (Krebs et al, 1999). These two complexes facilitate the binding of a second activator, SBF, which most likely recruits TBP and other components of the preinitiation complex. ATP-dependent chromatin remodeling are also involved in transcription repression (Davie and Moniwa, 2000). Because of this, ATP-dependent chromatin remodeling complexes may increase the rate at which a chromatin region fluctuates between an active and repressed structure (Kingston and Narlikar, 1999). If factors are present that stabilize chromatin structure and promote transcriptional repression, then the remodeling complex will drive the chromatin into a repressed state by allowing the transcriptional repressors to associate with the chromatin. However, if transcriptional activators bind to the remodeled chromatin instead, then the remodeling complexes will drive the chromatin structure to a transcriptionally active state. The subsequent binding of histone acetyltransferases and activating complexes to this chromatin structure will then “fix� it in an active state (Kingston and Narlikar, 1999). In support of this, the elimination of SAGA acetyltransferase activity prevents proper chromatin remodeling at the PHO8 promoter in vivo (Gregory et al, 1999). However, ATP-dependent chromatin remodeling complexes do not always bind chromatin before histone acetyltransferases. In the case of the interferon $ promoter, the enhanceosome assembles at a nucleosome-free enhancer region of this gene and initially recruits Gcn5 to acetylate the nucleosome positioned over the TATA box and transcription start site (Agalioti et al, 2000). This leads to the recruitment of the CBP-PolII holoenzyme complex, and CBP subsequently recruits SWI/SNF. Therefore, in some cases, the SWI/SNF complex prefers acetylated chromatin as a substrate (Agalioti et al, 2000). The BRG1 sub-unit of the SWI/SNF complex contains a bromodomain, and this type of domain can interact with acetylated histones (Winston and Allis, 1999; Cairns et al, 5

Spencer and Davie: Dynamic histone acetylation and its involvement in transcription 1999). The presence of acetylated histones along a promoter may increase the affinity of the SWI/SNF complex to this gene region. In support of this, SWI/SNF was recruited to a promoter by a transactivator, however, its retention was enhanced when the histones along this region were acetylated (Hassan et al, 2001). Incubation of these nucleosomal arrays with SAGA and NuA4 increased this retention (Hassan et al, 2001). Furthermore, histone acetyltransferases have been shown to increase the rate of gene induction by accelerating ATP-dependent chromatin remodeling (Barbaric et al, 2001). The order of recruitment for chromatin-remodeling activities and the function of these complexes in gene activation or repression is most likely gene-specific, and dependent on the combination of transcription factors bound to the promoter.

1 by p300 reduces its ability to bind DNA, as well as its nuclease activity, while acetylation of importin-alpha by CBP promotes its interaction with importin-beta in vitro (Hasan et al, 2001; Bannister et al, 2000). Furthermore, the acetylation of ACTR by another acetyltransferase suggests that acetylation may be a cascading event involved in signal transduction (Kouzarides, 2000; Marmorstein and Roth, 2001).

X. Global versus targeted histone acetylation Numerous studies have displayed an enrichment of acetylated H3 and H4 along the promoter regions of transcriptionally active genes. For example, activation of the human interferon gene induces H3 and H4 hyperacetylation over 2-3 nucleosomes within the promoter region (Parekh and Maniatis, 1999). Likewise, the yeast Gcn5 histone acetyltransferase complex acetylates histones only in the HO gene promoter (Krebs et al, 1999). Hormone-mediated transcriptional activation also involves the H3 and H4 hyperacetylation over the promoter regions of hormone-responsive genes (Chen et al, 1999; Sewack G.F. et al, 2001). A similar scenario occurs for histone deacetylation where the yeast Sin3-Rpd3 histone deacetylase complex deacetylates histones over a 1-2 nucleosome range within the promoter of a repressed gene (Kadosh and Struhl, 1998). In a recent study, the CpG island of the transcriptionally active chicken carbonic anhydrase gene was associated with higher levels of acetylated histones compared to the near-by promoter region (Myers et al, 2001). The acetylation of H3 and H4 along this gene was greatest at the CpG island and showed a drastic drop at approximately 1.5 kilobases into the transcribed region. Similarly, the chicken thymidine kinase gene displayed elevated levels of hyperacetylated histones along its CpG island (Crane-Robinson et al, 1999). High levels of hyperacetylated histones were also mapped to the chicken GAPDH promoter, which is located within a CpG island (Myers et al, 2001). The regions downstream of this promoter that do not contain CpG islands displayed a sharp drop in the levels of hyperacetylated H3 and H4. As well, chromatin fragments containing CpG islands are enriched in highly acetylated H3 and H4 isoforms (Tazi and Bird, 1990). These findings suggest that histone hyperacetylation is a feature of CpG islands. In a recent study, acetylated histones were mapped to CpG islands located both within the promoter and regions downstream from the transcription start site of a reporter gene (Cervoni and Szyf, 2001). The significance of histone acetylation along CpG islands is not known. However, when associated with acetylated histones, a methylated DNA sequence will become demethylated (Cervoni and Szyf, 2001). Because the interaction of demethylase with DNA is thought to be the limiting step in DNA demethylation, the acetylation of histones associated with CpG islands may increase the accessibility of demethylase to its target DNA sequence (Cervoni and Szyf, 2001). However, histone hyperacetylation does not always appear to be promoter- or CpG island-targeted. H4

IX. The effect of acetylation on nonhistone proteins Histone acetyltransferases can also acetylate transcription factors (p53, ACTR, EKLF, estrogen receptor, MyoD, GATA-1, E2F1), non-histone chromosomal proteins (HMG), components of the transcription machinery (TFIIE, TFIIF), the nuclear import protein importin, tubulin, and flap endonuclease-1 (Fen-1), an enzyme involved in DNA metabolism (Bannister et al, 2000; Chen et al, 1999; Imhof et al, 1997; Munshi et al, 1998; Hasan et al, 2001; Wang et al, 2001; Polesskaya et al, 2000; Herrera et al, 1999; Zhang and Bieker, 1998; Hung et al, 1999; L'Hernault and Rosenbaum, 1985; Martinez-Balbas et al, 2000). The acetylation of p53 and MyoD increases their binding affinity for DNA (Gu and Roeder, 1997; Polesskaya et al, 2000). As well, acetylation of E2F1 extends the half-life of this protein (MartinezBalbas et al, 2000). Thus, along with modifying chromatin structure, acetyltransferases may function in transcription by altering the DNA-binding properties of transcription factors or enhancing the stability of transcription factors. The acetylation of HMGI(Y) plays an important role in viral-induced interferon $ gene activation as well as the inactivation of this event (Parekh and Maniatis, 1999). Upon infection, the enhanceosome assembles at the interferon gene promoter with the help of HMGI(Y). At the same time, CBP and P/CAF are recruited to the interferon $ gene promoter where they acetylate H3 and H4 and, in combination with the enhanceosome, activate transcription of the interferon $ gene. Following induction, CBP acetylates HMGI(Y) which decreases its DNA binding affinity and causes the disruption of the enhanceosome complex. In addition, p300 binds to estrogen receptor " in the absence of estrogen and acetylates lysine residues within the hinge/ligand binding domain of this receptor. This event suppresses the sensitivity of the receptor to ligand (Wang et al, 2001). The evidence from these studies suggests that the theory of acetylation stimulating transcriptional activity is not always true. Acetyltransferases may also function in other biological processes. The acetylation of flap endonuclease-


Gene Therapy and Molecular Biology Vol 7, page 7 acetylated at lysine 16 (H4Ac16) is distributed along the entire length of X-linked genes targeted by the malespecific lethal dosage compensation. The promoter regions of these genes are associated with lower levels of H4Ac16 compared to the middle and 3’ regions (Smith et al, 2001). Similarly, pol I- and pol II-transcribed genes contain elevated levels of H4Ac16, while the levels of H4Ac12 are significantly elevated in yeast and Drosophila heterochromatin (Johnson et al, 1998; Braunstein et al, 1996). As well, the chicken $A-globin gene does not contain a CpG island, but displays high levels of widespread H3 and H4 acetylation (Myers et al, 2001). Acetylated lysine residues are also located throughout the c-myc gene, as well as the entire adult chicken $-globin domain (Hebbes et al, 1994; Madisen et al, 1998; Myers et al, 2001). While a particular histone acetyltransferase can be recruited to and acetylate the histones along a specific gene, recent evidence suggests that some histone acetyltransferases can also globally affect the acetylation of many genes in a non-targeted manner. Depletion of Esa1, an acetyltransferase specifically recruited to the ribosomal protein and heat shock promoters, causes a dramatic decrease in H4 acetylation over many regions of the genome without affecting the transcription of many genes (Reid et al, 2000). Similarly, the acetylation of the yeast PHO5 promoter by Esa1 and Gcn5, and the subsequent deacetylation of this region by HDA1 and Rpd3 also results in the widespread histone acetylation/deacetylation of three separate chromosomal regions making to 22 kb of DNA (Vogelauer et al, 2000). Thus, the promoter-targeted acetylation activity of some histone acetyltransferases and deacetylases may occur in a background of non-targeted histone acetylation that is mediated by these same enzymes and not required for transcription. However, this global acetylation can, in some cases, be targeted to particular regions of the genome. The expression of the C/EBP" transcription factor in GHFT1-5 pituitary cells causes an increase in the levels of acetylated H3 at pericentromeric chromatin domains (Zhang et al, 2001). CBP may be the histone acetyltransferase associated with C/EBP", since this enzyme concentrates at pericentromeric chromatin during C/EBP" expression (Schaufele et al, 2001). The global activity of these enzymes may maintain the balance of acetylated and deacetylated histones throughout the genome or regions of the genome and prevent the histones along a gene from becoming transiently or permanently fully acetylated. The hyperacetylation of histones on regions downstream from the promoter suggests that histone acetylation may function in transcriptional elongation. For example, Elp3, a 60-kilodalton subunit of the elongator/RNAPII holoenzyme has histone acetyltransferase activity and is able to acetylate all four core histones in vitro (Wittschieben et al, 1999). This histone acetyltransferase activity is essential for the elongator function of Elp3 in vivo (Wittschieben et al, 2000). Furthermore, the removal of Gcn5 and Elp3 acetyltransferase activity from yeast cells causes

widespread transcription defects (Wittschieben et al, 2000). Gcn5 functions in the transcription of only a subset of genes. Therefore, Elp3 histone acetyltransferase activity must be important for the transcription of a significant number of genes. Other evidence suggesting a role for histone acetylation in transcriptional elongation comes from observations that transcription by T7 RNA polymerase through a nucleosome occurs at a similar rate on nucleosomal templates containing either tailless or hyperacetylated histones (Protacio et al, 2000). As well, H3 and H4 hyperacetylation is necessary to maintain the transcriptionally active nucleosome in an open conformation for transcriptional elongation (Walia et al, 1998). As a result, a cell may contain two types of histone acetyltransferases with respect to the transcriptional process: those involved in initiation, and those involved in elongation. Histone acetyltransferases required for the initiation process would either enhance transcription factor binding to promoter/enhancer target regions by one or several of the mechanisms previously described, while acetyltransferases required for elongation would increase the accessibility of elongation factors to the DNA within coding regions. In support of this theory, the p300 histone acetyltransferase interacts specifically with initiationcompetent form of RNA polymerase II, while PCAF interacts with the elongation-competent form (Cho et al, 1998). Furthermore, p300 associates with the promoter region of an estrogen-responsive gene only during immediate exposure to estrogen when transcription is initiated rather than during subsequent re-initiation stages of transcription (Shang et al, 2000). Salt-soluble chromatin fragments enriched in active genes are associated with several unidentified histone acetyltransferases (Hebbes and Allen, 2000). Whether these acetyltransferases function in initiation and/or elongation remains to be determined. Different histone acetyltransferases have different histone substrates along certain regions of specific target genes. The histone deacetylase Rpd3 preferentially acetylates lysine 5 of H4 at only a select number of genes (Rundlett et al, 1998). As well, the yeast histone acetyltransferase, Esa1, interacts only with the promoter regions of ribosomal protein genes (Reid et al, 2000). Histone deacetylases along with nuclear receptor corepressors can exist in discrete nuclear bodies (Downes et al, 2000). Similarly, nuclear matrix-associated promyelocytic leukemia bodies contain PML proteins that bind and concentrate CBP into discrete domains (Boisvert et al, 2001). The differential levels of hyperacetylated histones observed on different regions of active genes may be explained by the proximity of histone acetyltransferases and deacetylases to specific regions of these genes. Regions situated close to regions of high acetyltransferase activity are more frequently acetylated than deacetylated, while regions close to deacetylases are deacetylated more often than acetylated. As well, cellular context may influence the acetylation status of histones along specific gene regions. Histone acetyltransferases and deacetylases exist in large


Spencer and Davie: Dynamic histone acetylation and its involvement in transcription multi-protein complexes, and the types of proteins associated with these enzymes can determine their substrate specificity (Grant et al, 1999). For example, in one cell type a specific histone acetyltransferase may exist in a complex capable of acetylating H4, while, in another cell type this same enzyme may be associated with different proteins and have a substrate specificity for H3. In some cases, the ability of histone acetyltransferases and deacetylases to occupy a particular gene region may be transient (Shang et al, 2000). Within 15-20 minutes following estradiol exposure, the histone acetyltransferases AIB1 and p300 within MCF-7 human breast cancer cells associate with the estrogen-responsive cathepsin D promoter. RNA polymerase associates shortly following this event. This association most likely initiates transcription since significant levels of transcription are observed 45 min after estrogen stimulation. The association of these factors then starts to decline 60 min from the initial time of estrogen treatment. A few minutes before these acetyltransferases are removed, the levels of CBP and PCAF histone acetyltransferases associated with the cathepsin D promoter starts to rise and peak between 60 and 75 minutes. However, the levels of cathepsin D transcription are significantly reduced after 75 minutes. The levels of CBP and PCAF and the rate of transcription then drop sharply at 90 minutes. Approximately 100 minutes after estrogen stimulation, the AIB1, CBP and PCAF acetyltransferases all assemble on the promoter in the same order as before, and the rate of transcription simultaneously increases. Similar results were also observed for the PS2 estrogen-responsive promoter in MCF-7 cells, and the cathepsin D promoter in ECC-1 endometrial cells, showing that estrogen-induced transcription involves the cyclical assembly of histone acetyltransferases along the promoters of estrogenresponsive genes. Even though the association of histone acetyltransferases with estrogen-responsive promoters is cyclical after estrogen stimulation, the levels of acetylated histones along the promoter region never drop to the levels observed in estrogen-deplete conditions when the acetyltransferases are displaced. Once transcription has been initiated, histone acetylation may maintain the open structure of an entire gene, and increase the accessibility of the promoter and downstream regions to the RNA polymerase complex for subsequent rounds of initiation and elongation. Such an event may increase the rate of transcription (Orphanides and Reinberg, 2000). Determining the structure of chromatin after initiation, but before and after elongation will help elucidate the function of acetylation in elongation.

(Hendzel et al, 1991). The majority of histone acetyltransferase and deacetylase activity, class I acetylated histones, and transcriptionally active $-globin and histone H5 genes are located in the insoluble nuclear material which contains the nuclear matrix (Hendzel et al, 1991). As well, the nuclear matrix is the site of transcription (Davie, 1995). We recently showed that intronic regions of the transcriptionally active $-globin gene, and transcriptionally competent, DNAse I-sensitive but inactive #-globin genes are associated with class I acetylated histones (Spencer and Davie, 2001). This association was shown for chromatin fragments in both salt-soluble and nuclear matrix-containing nuclear fractions. Of the two sequences, the $-globin intron appeared to have a higher concentration of class I acetylated histones, while the #-globin intron was associated with a mosaic of class I and class II acetylated histones. These findings suggest that the N terminal tails of the core histones situated on transcriptionally active genes contact nuclear-matrix associated histone acetyltransferases and deacetylases in a rapid and transient manner, while the frequency of contact between these enzymes and the histones along transcriptionally competent genes is less. In support of this, the entire chicken $-Aglobin gene, which has a high rate of transcription, was associated with higher levels of H3 and H4 acetylation when compared to genes transcribed at slower rates (GAPDH, carbonic anhydrase (Myers et al, 2001). As well, multiple histone acetyltransferases are associated with chromatin fragments enriched in transcriptionally active genes (Hebbes and Allen, 2000). Thus, dynamic histone acetylation may function to selectively retain transcriptionally active genes at sites of transcription within the nuclear matrix (Spencer and Davie, 2001). In fact, evidence from a recent study on estrogenresponsive human breast cancer cells suggests that exposure to estrogen changes the dynamics of histone acetylation by altering the balance of histone acetyltransferases and deacetylases along different regions of estrogen-responsive genes (Sun et al, 2001). In human breast cancer cells, exposure to estradiol causes the recruitment of acetyltransferases and the subsequent hyperacetylation of histones at the promoter region of estrogen-responsive genes (Chen et al, 1999). In addition, exposure of hormone-responsive human breast cancer cells to estrogen reduces the rate of histone deacetylation without affecting the rate of histone acetylation, or the sub-nuclear location, level or activity of class I and II histone deacetylases (Sun et al, 2001). Instead, exposure to estrogen alters the distribution of the estrogen receptor and histone acetyltransferases (SRC-1 and SRC-3) by causing both types of factors to become tightly associated with the nuclear matrix (Stenoien et al, 2001; Sun et al, 2001). Thus, the binding of estrogen to the estrogen receptor may cause the estrogen receptor to recruit histone acetyltransferases from other nuclear regions to the promoter region of estrogen-responsive genes (Figure 3). At present, a large emphasis is placed on the role of

XII. Transcription and the dynamics of histone acetylation The exact function of dynamic histone acetylation in transcription is unknown. Nuclear fractionation studies indicate that the nuclear distribution of class I, but not class II, acetylated histones closely follows that of the transcriptionally active $-globin and histone H5 genes


Gene Therapy and Molecular Biology Vol 7, page 9 histone acetyltransferases in transcriptional initiation and elongation. However, as previously mentioned, histone acetylation is a dynamic event resulting from the combined activities of histone acetyltransferases and deacetylases. Thus, more attention must be given to understanding how acetyltransferases and deacetylases function together at specific sites along transcriptionally active genes to fully appreciate the role of dynamic histone acetylation in transcription.

Moreover, Gcn5 preferentially associates with a Ser10 phosphorylated form of H3 over a non-phosphorylated form (Cheung et al, 2000). Recently, the phosphorylation of H3 Ser10 by the Snf1 kinase was shown to lead to Gcn5-mediated acetylation at the INO1 promoter (Lo et al, 2001). Thus, the recruitment of a kinase complex to specific promoters may cause Ser10 phosphorylation and either increase the affinity of histone acetyltransferase complexes for nucleosomes or increase acetyltransferase catalytic activity (Lo et al, 2000). However, the affect of one post-translational modification on another may not always be positive. Heterochromatic silencing requires the methylation of Lys9 on H3 by the lysine methyltransferase Su(var)39 (Rea et al, 2000). The methylation of Lys9 inhibits phosphorylation of H3 at Ser10 possibly by hindering the access of kinases to this serine residue (Rea et al, 2000). Thus, methylation of Lys9 may impair transcription by inhibiting phosphorylation events required for transcriptional stimulation (Berger, 2001). This finding, however, needs to be further investigated since immunoprecipitation studies have identified an association between CBP and a histone methyltransferase that specifically targets lysines 4 and 9 of H3 without significantly affecting the ability of CBP to efficiently acetylate other H3 lysine residues (Vandel and Trouche, 2001).

XII. The histone code The histone N terminal tails undergo several posttranslational modifications mediated by a variety of enzymes. Research in the field of gene expression has focussed primarily on determining the function of each modification in transcription. However, a new concept has emerged referred to as the “histone code� (Strahl and Allis, 2000; Jenuwein and Allis, 2001). This term proposes that the different post-translational modifications occurring on one or more histone tails act either together or in sequence to form recognition sites for specific proteins involved in distinct cellular functions. Furthermore, these modifications may positively or negatively influence the affect of one another on specific cellular functions. Evidence from several recent studies suggests that histone phosphorylation and acetylation may function together to promote gene expression. For example, the stimulation of mammalian cells by epidermal growth factor causes the sequential phosphorylation of Ser10, and acetylation of Lys14 on H3 (Cheung et al, 2000).

Figure 3. Proposed model for the effect of estradiol on the distribution of histone acetyltransferases and histone deacetylases in human breast cancer cells. In the absence of estradiol (left), histone acetyltransferases (HAT) such as CBP, SRC-1, SRC-3, and PCAF occupy the same chromatin regions as histone deacetylases (HDAC) such as HDAC1, and HDAC2. Upon addition of estradiol (right), the estrogen receptor (ER) is recruited to nuclear matrix sites and associates with the estrogen response element of estrogen responsive genes. When bound to estradiol, the ER recruits histone acetyltransferases from other nuclear regions, thereby altering the balance of histone acetyltransferases and deacetylases along specific chromatin regions.


Spencer and Davie: Dynamic histone acetylation and its involvement in transcription distinct forms of the RSC nucleosome-remodeling complex, containing essential AT hook, BAH, and bromodomains. Mol Cell 4, 715-723. Cervoni N and Szyf M (2001) Demethylase activity is directed by histone acetylation. J Biol Chem 276, 40778-40787. Chen H, Lin RJ, Xie W, Wilpitz D, and Evans RM (1999) Regulation of hormone-induced histone hyperacetylation and gene activation via acetylation of an acetylase. Cell 98, 675686. Cheung P, Tanner KG, Cheung WL, Sassone-Corsi P, Denu JM, and Allis CD (2000) Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation. Mol Cell 5, 905-915. Cho H, Orphanides G, Sun X, Yang XJ, Ogryzko V, Lees E, Nakatani Y, and Reinberg D (1998). A human RNA polymerase II complex containing factors that modify chromatin structure. Mol Cell Biol 18, 5355-5363. Covault J and Chalkley R (1980) The identification of distinct populations of acetylated histone. J Biol Chem 255, 91109116. Crane-Robinson C, Myers FA, Hebbes TR, Clayton AL, and Thorne AW (1999). Chromatin immunoprecipitation assays in acetylation mapping of higher eukaryotes. Methods Enzymol 304, 533-547. Davie JR (1995) The nuclear matrix and the regulation of chromatin organization and function. Int Rev Cytol 162A, 191-250. Davie JR and Moniwa M (2000) Control of chromatin remodeling. Crit Rev Eukaryot Gene Expr 10, 303-325. Davie JR and Spencer VA (1999) Control of histone modifications. J Cell Biochem Suppl 32-33, 141-148. Davie JR and Spencer VA (2001) Signal transduction pathways and the modification of chromatin structure. Prog Nucleic Acid Res Mol Biol 65, 299-340. De Rubertis F, Kadosh D, Henchoz S, Pauli D, Reuter G, Struhl K, and Spierer P (1996) The histone deacetylase RPD3 counteracts genomic silencing in Drosophila and yeast. Nature 384, 589-591. Downes M, Ordentlich P, Kao HY, Alvarez JG, and Evans RM (2000) Identification of a nuclear domain with deacetylase activity. Proc Natl Acad Sci USA 97, 10330-10335. Fisher AL and Caudy M (1998) Groucho proteins: transcriptional corepressors for specific subsets of DNA-binding transcription factors in vertebrates and invertebrates. Genes Dev 12, 1931-1940. Frye RA (1999) Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein ADPribosyltransferase activity. Biochem Biophys Res Commun 260, 273-279. Frye RA (2000) Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins. Biochem Biophys Res Commun 273, 793-798. Garcia-Ramirez M, Rocchini C, and Ausio J (1995) Modulation of chromatin folding by histone acetylation. J Biol Chem 270, 17923-17928. Grant PA, Eberharter A, John S, Cook RG, Turner BM, and Workman JL (1999) Expanded lysine acetylation specificity of Gcn5 in native complexes. J Biol Chem 274, 5895-5900. Gregory PD, Schmid A, Zavari M, Munsterkotter M, and Horz W (1999) Chromatin remodeling at the PHO8 promoter requires SWI-SNF and SAGA at a step subsequent to activator binding. EMBO J 18, 6407-6414.

A recent study mapping the distribution of di-methylated lysine 9 on H3 across the chicken $-globin domain during erythropoiesis showed that regions enriched in methylated lysine 9 were depleted of di-acetylated H3 (K9 and K14). However, H3 acetylation correlated with lysine 4 methylation, suggesting that transcriptional activation is associated with H3 methylated at K4, as well as with acetylated H3 and H4 isoforms (Litt et al, 2001). Likewise, in Tetrahymena, methylated Lys4 of H3 is found only in transcriptionally active macronuclei (Strahl et al, 1999).

Acknowledgments Research supported by grants from the Canadian Institutes of Health Research (CIHR) (MT-9186,RO15183), CancerCare Manitoba, and the U.S. Army Medical and Materiel Command Breast Cancer Research Program (#DAM17-00-1-0319), and the National Cancer Institute of Canada with funds from the Canadian Cancer Society. A CIHR Senior Scientist Award to J.R.D. and a U.S. Army Medical and Materiel Command Fellowship to V.A.S. are gratefully acknowledged.

References Afshar G, and Murnane JP (1999) Characterization of a human gene with sequence homology to Saccharomyces cerevisiae SIR2. Gene 234, 161-168. Agalioti T, Lomvardas S, Parekh B, Yie J, Maniatis T, and Thanos D (2000) Ordered recruitment of chromatin modifying and general transcription factors to the IFN-$ promoter. Cell 103, 667-678. Annunziato AT, and Hansen J C (2000) Role of histone acetylation in the assembly and modulation of chromatin structures. Gene Expr 9, 37-61. Bannister AJ, Miska EA, Gorlich D, and Kouzarides T (2000) Acetylation of importin-alpha nuclear import factors by CBP/p300. Curr Biol 10, 467-470. Barbaric S, Walker J, Schmid A, Svejstrup JQ, and Horz W (2001) Increasing the rate of chromatin remodeling and gene activation--a novel role for the histone acetyltransferase Gcn5. EMBO J 20, 4944-4951. Berger SL (2001) An embarrassment of niches: the many covalent modifications of histones in transcriptional regulation. Oncogene 20, 3007-3013. Bertos NR, Wang AH, and Yang XJ (2001) Class II histone deacetylases: structure, function, and regulation. Biochem Cell Biol 79, 243-252. Boisvert FM, Kruhlak MJ, Box AK, Hendzel MJ, and BazettJones DP (2001) The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body. J Cell Biol 152, 1099-1106. Braunstein M, Sobel RE, Allis CD, Turner BM, and Broach JR (1996) Efficient transcriptional silencing in Saccharomyces cerevisiae requires a heterochromatin histone acetylation pattern. Mol Cell Biol 16, 4349-4356. Bustin M ( 1999) Regulation of DNA-dependent activities by the functional motifs of the high-mobility-group chromosomal proteins. Mol Cell Biol 19, 5237-5246. Cairns BR, Schlichter A, Erdjument-Bromage H, Tempst P, Kornberg RD, and Winston F (1999) Two functionally


Gene Therapy and Molecular Biology Vol 7, page 11 Grunstein M (1998) Yeast heterochromatin: regulation of its assembly and inheritance by histones. Cell 93, 325-328. Gu W and Roeder RG (1997) Activation of p53 sequencespecific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595-606. Guardiola AR and Yao TP (2002) Molecular cloning and characterization of a novel histone deacetylase HDAC10. J Biol Chem 277, 3350-3356. Hansen JC (1997) The core histone amino-termini: combinatorial interaction domains that link chromatin structure with function. Chemtracts Biochem Mol Biol 10, 56-69. Hansen JC, Tse C, and Wolffe AP (1998) Structure and function of the core histone N-termini: more than meets the eye. Biochemistry 37, 17637-17641. Hasan S, Stucki M, Hassa PO, Imhof R, Gehrig P, Hunziker P, Hubscher U, and Hottiger MO (2001) Regulation of human flap endonuclease-1 activity by acetylation through the transcriptional coactivator p300. Mol Cell 7, 1221-1231. Hassan AH, Neely KE, and Workman JL (2001) Histone acetyltransferase complexes stabilize swi/snf binding to promoter nucleosomes. Cell 104, 817-827. Hebbes TR and Allen SC (2000) Multiple histone acetyltransferases are associated with a chicken erythrocyte chromatin fraction enriched in active genes. J Biol Chem 275, 31347-31352. Hebbes TR, Clayton AL, Thorne AW, and Crane-Robinson C (1994) Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken $-globin chromosomal domain. EMBO J 13, 1823-1830. Hendzel MJ, Delcuve GP, and Davie JR (1991) Histone deacetylase is a component of the internal nuclear matrix. J Biol Chem 266, 21936-21942. Herrera JE, Sakaguchi K, Bergel M, Trieschmann L, Nakatani Y, and Bustin M (1999) Specific acetylation of chromosomal protein HMG-17 by PCAF alters its interaction with nucleosomes. Mol Cell Biol 19, 3466-3473. Hung HL, Lau J, Kim AY, Weiss MJ, and Blobel GA (1999) CREB-Binding protein acetylates hematopoietic transcription factor GATA-1 at functionally important sites. Mol Cell Biol 19, 3496-3505. Imai S, Armstrong CM, Kaeberlein M, and Guarente L (2000) Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403, 795-800. Imhof A, Yang XJ, Ogryzko VV, Nakatani Y, Wolffe AP, and Ge H (1997) Acetylation of general transcription factors by histone acetyltransferases. Curr Biol 7, 689-692. Jenuwein T and Allis CD (2001) Translating the histone code. Science 293, 1074-1080. Johnson CA, O'Neill LP, Mitchell A, and Turner BM (1998) Distinctive patterns of histone H4 acetylation are associated with defined sequence elements within both heterochromatic and euchromatic regions of the human genome. Nucleic Acids Res 26, 994-1001. Kadosh D and Struhl K (1998) Targeted recruitment of the Sin3Rpd3 histone deacetylase complex generates a highly localized domain of repressed chromatin in vivo. Mol Cell Biol 18, 5121-5127. Kimura H and Cook PR (2001) Kinetics of core histones in living human cells: little exchange of H3 and H4 and some rapid exchange of H2B. J Cell Biol 153, 1341-1353. Kingston RE and Narlikar GJ ( 1999) ATP-dependent remodeling and acetylation as regulators of chromatin fluidity. Genes Dev 13, 2339-2352.

Klochendler-Yeivin A and Yaniv M (2001). Chromatin modifiers and tumor suppression. Biochim Biophys Acta 1551, M110. Kouzarides T (2000) Acetylation: a regulatory modification to rival phosphorylation? EMBO J 19, 1176-1179. Krebs JE, Kuo MH, Allis CD, and Peterson CL (1999) Cell cycle-regulated histone acetylation required for expression of the yeast HO gene. Genes Dev 13, 1412-1421. Kruhlak MJ, Hendzel MJ, Fischle,W, Bertos NR, Hameed S, Yang XJ, Verdin E, and Bazett-Jones DP (2001) Regulation of global acetylation in mitosis through loss of histone acetyltransferases and deacetylases from chromatin. J Biol Chem 276, 38307-38319. Kuo MH and Allis CD (1998) Roles of histone acetyltransferases and deacetylases in gene regulation. Bioessays 20, 615-626. L'Hernault SW and Rosenbaum JL (1985) Chlamydomonas alpha-tubulin is posttranslationally modified by acetylation on the epsilon-amino group of a lysine. Biochemistry 24, 473-478. Litt MD, Simpson M, Gaszner M, Allis CD, and Felsenfeld G (2001) Correlation between histone lysine methylation and developmental changes at the chicken $-globin locus. Science 293, 2453-2455. Landry J, Slama JT, and Sternglanz R (2000) Role of NAD(+) in the deacetylase activity of the SIR2-like proteins. Biochem Biophys Res Commun 278, 685-690. Lee,HJ, Chun M, and Kandror KV (2001) Tip60 and HDAC7 interact with the endothelin receptor a and may be involved in downstream signaling. J Biol Chem 276, 16597-16600. Leuba SH, Bustamante C, van Holde K, and Zlatanova J (1998a) Linker histone tails and N-tails of histone H3 are redundant: scanning force microscopy studies of reconstituted fibers. Biophys J 74, 2830-2839. Leuba SH, Bustamante C, Zlatanova J, and van Holde K (1998b) Contributions of linker histones and histone H3 to chromatin structure: scanning force microscopy studies on trypsinized fibers. Biophys J 74, 2823-2829. Liu YZ, Thomas NS, and Latchman DS (1999) CBP associates with the p42/p44 MAPK enzymes and is phosphorylated following NGF treatment. Neuroreport 10, 1239-1243. Lo WS, Duggan L, Tolga NC, Emre, Belotserkovskya R, Lane WS, Shiekhattar R, and Berger SL. (2001) Snf1--a histone kinase that works in concert with the histone acetyltransferase Gcn5 to regulate transcription. Science 293, 1142-1146. Lo WS, Trievel RC, Rojas JR, Duggan L, Hsu JY, Allis CD, Marmorstein R, and Berger SL (2000) Phosphorylation of serine 10 in histone H3 is functionally linked in vitro and in vivo to Gcn5-mediated acetylation at lysine 14. Mol Cell 5, 917-926. Logie C, Tse C, Hansen JC, and Peterson CL (1999) The core histone N-terminal domains are required for multiple rounds of catalytic chromatin remodeling by the SWI/SNF and RSC complexes. Biochemistry 38, 2514-2522. Luger K, Mader AW, Richmond RK, Sargent DF, and Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251-260. Madisen L, Krumm A, Hebbes TR, and Groudine M (1998) The immunoglobulin heavy chain locus control region increases histone acetylation along linked c-myc genes. Mol Cell Biol 18, 6281-6292.


Spencer and Davie: Dynamic histone acetylation and its involvement in transcription Marmorstein R and Roth SY (2001) Histone acetyltransferases: function, structure, and catalysis. Curr Opin Genet Dev 11, 155-161. Martinez-Balbas MA, Bauer UM, Nielsen SJ, Brehm A, and Kouzarides T (2000) Regulation of E2F1 activity by acetylation. EMBO J 19, 662-671. Moore SC and Ausio J (1997) Major role of the histones H3-H4 in the folding of the chromatin fiber. Biochem Biophys Res Commun 230, 136-139. Munshi N, Merika M, Yie J, Senger K, Chen G, and Thanos D (1998) Acetylation of HMG I(Y) by CBP turns off IFN-$ expression by disrupting the enhanceosome. Mol Cell 2, 457-467. Myers FA, Evans DR, Clayton AL, Thorne AW, and CraneRobinson C (2001) Targeted and extended acetylation of histones H4 and H3 at active and inactive genes in chicken embryo erythrocytes. J Biol Chem 276, 20197-20205. Norton VG, Imai BS, Yau P, and Bradbury EM (1989) Histone acetylation reduces nucleosome core particle linking number change. Cell 57, 449-457. Orphanides G and Reinberg D (2000) RNA polymerase II elongation through chromatin. Nature 407, 471-475. Palaparti A, Baratz A, and Stifani S (1997) The Groucho/transducin-like enhancer of split transcriptional repressors interact with the genetically defined aminoterminal silencing domain of histone H3. J Biol Chem 272, 26604-26610. Parekh BS and Maniatis T (1999) Virus infection leads to localized hyperacetylation of histones H3 and H4 at the IFN$ promoter. Mol Cell 3, 125-129. Polesskaya A, Duquet A, Naguibneva I, Weise C, Vervisch A, Bengal E, Hucho F, Robin P, and Harel-Bellan A (2000) CREB-binding protein/p300 activates MyoD by acetylation. J Biol Chem 275, 34359-34364. Protacio RU, Li G, Lowary PT, and Widom J (2000) Effects of histone tail domains on the rate of transcriptional elongation through a nucleosome. Mol Cell Biol 20, 8866-8878. Rea S, Eisenhaber F, O'Carroll D, Strahl BD, Sun ZW, Schmid M, Opravil S, Mechtler K, Ponting CP, Allis CD, and Jenuwein T (2000) Regulation of chromatin structure by sitespecific histone H3 methyltransferases. Nature 406, 593599. Reid JL., Iyer VR, Brown PO, and Struhl K (2000) Coordinate regulation of yeast ribosomal protein genes is associated with targeted recruitment of Esa1 histone acetylase. Mol Cell 6, 1297-1307. Ridsdale JA, Hendzel MJ, Delcuve GP, and Davie JR (1990) Histone acetylation alters the capacity of the H1 histones to condense transcriptionally active/competent chromatin. J Biol Chem 265, 5150-5156. Rundlett SE, Carmen AA, Suka N, Turner BM, and Grunstein M (1998) Transcriptional repression by UME6 involves deacetylation of lysine 5 of histone H4 by RPD3. Nature 392, 831-835. Schaufele F, Enwright JF, Wang X, Teoh C, Srihari R, Erickson R, MacDougald OA, and Day RN (2001) CCAAT/Enhancer Binding Protein alpha Assembles Essential Cooperating Factors in Common Subnuclear Domains. Mol Endocrinol 15, 1665-1676. Schwarz PM, Felthauser A, Fletcher TM, and Hansen JC (1996) Reversible oligonucleosome self-association: dependence on divalent cations and core histone tail domains. Biochemistry 35, 4009-4015.

Seigneurin-Berny D, Verdel A, Curtet S, Lemercier C, Garin J, Rousseaux S, and Khochbin S (2001) Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways. Mol Cell Biol 21, 8035-8044. Sewack GF, Ellis TW, and Hansen U (2001) Binding of TATA binding protein to a naturally positioned nucleosome is facilitated by histone acetylation. Mol Cell Biol 21, 14041415. Shang Y, Hu X, DiRenzo J, Lazar MA, and Brown M ( 2000) Cofactor dynamics and sufficiency in estrogen receptorregulated transcription. Cell 103, 843-852. Singh H, Sekinger EA, and Gross DS (2000) Chromatin and cancer: causes and consequences. J Cell Biochem Suppl 35, 61-68. Smith ER, Allis CD, and Lucchesi JC (2001) Linking global histone acetylation to the transcription enhancement of Xchromosomal genes in Drosophila males. J Biol Chem 276, 31483-31486. Spencer VA and Davie JR ( 1999) Role of covalent modifications of histones in regulating gene expression. Gene 240, 1-12. Spencer VA and Davie JR (2001) Dynamically acetylated histone association with transcriptionally active and competent genes in the avian adult $-globin gene domain. J Biol Chem 276, 34810-34815. Stenoien DL, Patel K, Mancini MG, Dutertre M, Smith CL, O'Malley BW, and Mancini MA (2001) FRAP reveals that mobility of oestrogen receptor-" is ligand- and proteasomedependent. Nat Cell Biol 3, 15-23. Sterner DE and Berger SL (2000) Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev 64, 435-459. Strahl BD and Allis CD (2000) The language of covalent histone modifications. Nature 403, 41-45. Strahl BD, Ohba R, Cook RG, and Allis CD (1999) Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena. Proc Natl Acad Sci USA 96, 14967-14972. Sun JM, Chen HY, and Davie JR (2001) Effect of Estradiol on histone acetylation dynamics in human breast cancer cells. J Biol Chem 276, 49435-49442. Sun JM, Chen HY, Moniwa M, Samuel S, and Davie JR (1999) Purification and characterization of chicken erythrocyte histone deacetylase 1. Biochemistry 38, 5939-5947. Tazi J and Bird A (1990) Alternative chromatin structure at CpG islands. Cell 60, 909-920. Tse C and Hansen JC (1997) Hybrid trypsinized nucleosomal arrays: identification of multiple functional roles of the H2A/H2B and H3/H4 N-termini in chromatin fiber compaction. Biochemistry 36, 11381-11388. Tse C, Sera T, Wolffe AP, and Hansen JC (1998) Disruption of higher-order folding by core histone acetylation dramatically enhances transcription of nucleosomal arrays by RNA polymerase III. Mol Cell Biol 18, 4629-4638. Turner BM (1991) Histone acetylation and control of gene expression. J Cell Sci 99 ( Pt 1), 13-20. Turner BM (1998) Histone acetylation as an epigenetic determinant of long-term transcriptional competence. Cell Mol Life Sci 54, 21-31. Turner BM (2000) Histone acetylation and an epigenetic code. Bioessays 22, 836-845.


Gene Therapy and Molecular Biology Vol 7, page 13 Turner BM, Birley AJ, and Lavender J (1992) Histone H4 isoforms acetylated at specific lysine residues define individual chromosomes and chromatin domains in Drosophila polytene nuclei. Cell 69, 375-384. Vandel L and Trouche D (2001) Physical association between the histone acetyl transferase CBP and a histone methyl transferase. EMBO Rep 2, 21-26. Verschure PJ, van Der Kraan, I, Manders EM, and van Driel R (1999) Spatial relationship between transcription sites and chromosome territories. J Cell Biol 147, 13-24. Vogelauer M, Wu J, Suka N, and Grunstein M (2000) Global histone acetylation and deacetylation in yeast. Nature 408, 495-498. Walia H, Chen HY, Sun JM, Holth LT, and Davie JR (1998) Histone acetylation is required to maintain the unfolded nucleosome structure associated with transcribing DNA. J Biol Chem 273, 14516-14522. Wang C, Fu M, Angeletti RH, Siconolfi-Baez L, Reutens AT, Albanese C, Lisanti MP, Katzenellenbogen BS, Kato S, Hopp T, Fuqua SA, Lopez GN, Kushner PJ, and Pestell RG (2001). Direct acetylation of the estrogen receptor " hinge region by p300 regulates transactivation and hormone sensitivity. J Biol Chem 276, 18375-18383. Wang X, Moore SC, Laszckzak M, and Ausio J (2000) Acetylation increases the "-helical content of the histone tails of the nucleosome. J Biol Chem 275, 35013-35020. Watson AD, Edmondson DG, Bone JR, Mukai Y, Yu Y, Du W, Stillman DJ, and Roth SY (2000) Ssn6-Tup1 interacts with class I histone deacetylases required for repression. Genes Dev 14, 2737-2744. Winston F and Allis CD (1999) The bromodomain: a chromatintargeting module? Nat Struct Biol 6, 601-604. Wittschieben BO, Fellows J, Du W, Stillman DJ, and Svejstrup JQ (2000) Overlapping roles for the histone acetyltransferase activities of SAGA and elongator in vivo. EMBO J 19, 3060-3068. Wittschieben BO, Otero G, de Bizemont T, Fellows J, Erdjument-Bromage H, Ohba R, Li Y, Allis CD, Tempst P, and Svejstrup JQ (1999) A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme. Mol Cell 4, 123-128. Zhang DE and Nelson DA (1988b) Histone acetylation in chicken erythrocytes. Rates of acetylation and evidence that histones in both active and potentially active chromatin are rapidly modified. Biochem J 250, 233-240.

Zhang DE and Nelson DA (1988a) Histone acetylation in chicken erythrocytes. Rates of deacetylation in immature and mature red blood cells. Biochem J 250, 241-245. Zhang W and Bieker JJ (1998) Acetylation and modulation of erythroid Kruppel-like factor (EKLF) activity by interaction with histone acetyltransferases. Proc Natl Acad Sci USA 95, 9855-9860. Zhang W, Bone JR, Edmondson DG, Turner BM, and Roth SY (1998) Essential and redundant functions of histone acetylation revealed by mutation of target lysines and loss of the Gcn5p acetyltransferase. EMBO J 17, 3155-3167. Zhang WH, Srihari R, Day RN, and Schaufele F (2001) CCAAT/enhancer-binding protein alpha alters histone H3 acetylation at large subnuclear domains. J Biol Chem 276, 40373-40376. Zhou X, Marks PA, Rifkind RA, and Richon VM (2001) Cloning and characterization of a histone deacetylase, HDAC9. Proc Natl Acad Sci USA 98, 10572-10577. Zlatanova J, Leuba SH, and van Holde K (1998) Chromatin fiber structure: morphology, molecular determinants, structural transitions. Biophys J 74, 2554-2566.

Virginia A. Spencer and James R. Davie


Spencer and Davie: Dynamic histone acetylation and its involvement in transcription


Gene Therapy and Molecular Biology Vol 7, page 15 Gene Ther Mol Biol Vol 7, 15-23, 2002

Tumor therapy using radiolabelled antisense oligomers- aspects for antiangiogenetic strategy and positron emission tomography Review Article 1*



Kalevi JA Kairemo , Mark Lubberink , Mikko Tenhunen , Antti P Jekunen


Department of Nuclear Medicine1 and Hospital Physics2 Uppsala University, Uppsala Sweden Department of Oncology3 and Department of Clinical Pharmacology4, Helsinki University Central Hospital, Helsinki, Finland

__________________________________________________________________________________ *Correspondence: Kalevi J A Kairemo, MD, PhD, MSc (Eng); Professor, Department of Nuclear Medicine, Uppsala University Hospital, Sweden; Tel. +46-18-611 1006; Fax. +46-18-611 4124; e-mail: Key words: antisense therapy, oligonucleotides, phosphorus radioisotopes, sulphur radioisotopes, AIDS, cancer, dosimetry, positron emission tomography Received: 17 January 2002; accepted: 29 January, 2002; electronically published: July 2003

Summary Angiogenesis provides a putative target for radiochemotherapy as endothelial cells on vascular wall are sensitive for radiation and by destructing of one endothelial cell may lead to death hundred of tumor cells. Endothelial cells in the angiogenic vessels within solid tumors express several proteins that are absent or faintly expressing in established blood vessels, including !v integrins (Hammes, 1996) and receptors for certain angiogenic growth factors (Hanahan, 1997) (Risau, 1997). Recently, vascular endothelial cell growth factor (VEGF)-induced invasiveness has been inhibited specifically by ETS-1 antisense oligonucleotide. ETS-1 gene expression can be induced, while there are several other systems with constant expression. In this paper, we extent use of oligos from conventional biokinetic studies to therapeutic use by comparing radioactive oligos to peptide counterparts. Radiolabelled oligos have a potential of having both direct antisense inhibition and radiation effects. Previously we have shown theoretically that oligonucleotide therapy may be effective with internally labelled (P-32, P-33 and S-35) oligodeoxynucleotide phosphorothioates. This has also been demonstrated in vitro using P-33 (Kairemo et al, 1999). We investigate also the possibility of using 15-mer oligodeoxynucleotide phosphorothioates (oligos) or oligomers in which the phosphate-ribose backbone has been replaced with polyamide backbone (peptide nucleic acids). The absorbed organ doses of these radiolabelled compounds were estimated from biodistribution data. Subcellular biodistribution was used in evaluation of the best targeting inside the cell with one oligomer. Our results indicate that oligos can give significantly up to 130-fold higher absorbed organ doses in oligos than in peptides. Mainly this is due to slower biokinetics of oligos (35-fold slower half-lives). For imaging, positron emitters such as F-18 and Br-76, offer an advantage for radiopharmacokinetic studies (Wu at al., 2000). We have therefore calculated the subcellular dosimetry for these isotopes in different cell dimensions (nuclear diameter 6-16Âľm, cellular diameter 12-20Âľm). angiogenetic factors in our model; tie tyrosine kinase receptor and ets, representing a factor participating and inducing angiogenesis On the basis of amino acid sequence and structural similarities, receptor tyrosine kinases can be divided into several families (Ullrich and Schlessinger, 1990). Tie is the protein product of a recently described receptor tyrosine kinase cDNA, which together with tek defines a new subfamily. The tie gene is mandatory for the normal growth and differentiation of endothelial cells during fetal

I. Introduction Angiogenesis is a cascade of processes involving both soluble angiogenic factors and insoluble extracellular matrix factors (Jekunen and Kairemo, 1997). Soluble multiple molecules, that induce angiogenesis, are released by both tumor cells and host cells, including endothelial cells, epithelial cells, mesothelial cells, and leukocytes. These processes provide several targets for development of angiogenesis inhibitors. We have used two


Kairemo et al: Oligonucleotide radiotherapy development (Korhonen, 1992). It is abundantly expressed in vascular endothelia during development, and in some megakaryoblastic and erythroleukemia cell lines; as well as tieRNA accumulates in the epithelium of local vessels during ovulation and wound healing (Korhonen, 1992). Tie receptor has an important role in the angiogenesis associated with melanoma metastasis (Kaipainen, 1994). Radioantibodies against tie receptor have been used in targeting studies in vivo with success (Kairemo et al, 1996). As the location of tie receptor is at the outer cell membrane, receptor is easily reachable and effects of radiation and receptor blocking should occur immediately, which may be beneficiary for the radioantibody treatment. For further development of these receptors the crucial point is to find inducers for normally low levels. Ligands for endothelial cell receptors tyrosine kinases, Tie-1 and Tie-2 are not known. Ligands with agonistic and antagonistic activities for Tie-2 have now been identified: angiopoetin 1 is an activating ligand for Tie 2 and regulates blood vessel maturation (Suri, 1996), while angiopoetin 2 serves as antagonist (Maisonpierre, 1997). The ETS family proteins are transcription factors that bind to the regulatory control region of certain genes via ETS binding motif, which has been found in numerous genes including proteases and receptor tyrosine kinases (Wasylyk, 1993). ETS regulates the expression of proteases and migration of endothelial cells, and in fact, the induction of ETS-1 mRNA is a mutual phenomenon in endothelial cells stimulated with angiogenic growth factors (Iwasaka, 1996). It has also been shown that ETS 1 antisense oligo markedly reduced the DNA- ETS complex diminishing the responsiveness to the stimulus of

angiogenic factor (Iwasaka, 1996). Induction of expression of ETS gene is faster and more prominent than protein expression providing better although transient target for therapy. The specificity resides in the sequence of oligo, which interacts with its complementary mRNA, but only minimally with noncomplementary structures. The antisense oligo, through the formation of a mRNA-DNA duplex, specifically prevents the translation of that mRNA into protein (Figure 1). For oligos to be effective antisense agents, they first must enter the cells and achieve appropriate concentration in the correct intracellular compartment. Cellular nucleases are highly potent in digesting phosphodiester oligos. Thus several nuclease resistant oligos have been developed. Phosphorothioate oligo has a non-bridging oxygen atom replacing a sulphur atom. Peptide nucleic acid (PNA) is an oligomer in which the charged phosphate-ribose backbone has been eliminated and replaced with an uncharged backbone (Egholm 1992) and PNAs have been reported to resist nuclease and protease degradation (Egholm 1993). Oligos bind to serum albumin and other proteins with low affinity and distribute to all peripheral tissues with the kidneys and liver accumulating most of the drug. They are cleared by slow metabolism with an elimination half-life up to 50 hrs. The biokinetics of GEM 91 phosphorothioate oligodeoxynucleotide has been evaluated in six AIDS patients, where the plasma mean residence time varied from 24.7 to 49.6 hrs, the mean being 41.7 Âą 3.6 hrs (Zhang, 1995a).

Figure 1. Schematic presentation of radionanotargeting


Gene Therapy and Molecular Biology Vol 7, page 17 Case 1: rapid kinetics compared with physical decay:

Phosphorothioate oligodeoxynucleotides have several advantages: they are relatively resistant to destruction by nucleases; they have good aqueous solubility; they hybridize efficiently with target RNA with relatively high specificity; they are relatively efficiently taken up by cells; and they are widely used in automated oligonucleotide synthesizers (Zhang, 1995b). Phosphorothioate oligodeoxynucleotides labelled internally either with sulphur or phosphorus do not require any extra coupling techniques as in the case with transition metals. The therapeutic possibilities of radiolabelled antisense oligodeoxynucleotides or peptides are still unknown, and one of the basic questions in radiotherapy is the optimal source of radiation. Here we have estimated dosimetric properties of different radiolabels on oligonucleotides and peptides at cellular level, that could be predicted from existing data. The aim of this study was to calculate internal radiation dose from the known data and assess the suitability of different isotopes for the labels. Macroscopic doses were calculated for oligonucleotides labelled with 76 Br, 111In, 90Y and 211At, as examples of positron emitters, Auger-electron emitters, high-energy beta radiation emitters, and alpha emitting nuclides. We have previously shown by using calculations from the biodistribution data of oligonucleotide phosphorothioates in a xenograft model that oligonucleotide radiotherapy can optimally be given with P-32 and P-33 (Kairemo et al, 1996). Calculations can suggest recommendable source of radiation, and thus allow a proper selection of the optimal label. By selecting a radiation source the penetrability of radiation can be controlled and severe side effects may be avoided efficiently.

T f >> Tb1 , Tb 2

TT T + Tb2 D1 Ã1 A01 = = " f b1 " f = D2 Ã2 A02 Tf + Tb1 Tf Tb2 A T = 01 " b1 A02 Tb2

Case 2: rapid kinetics compared with very slow kinetics:

Tb 2 >> T f >> Tb1

Tf Tb1 Tf + Tb2 D1 Ã1 A01 = = " " = D2 Ã2 A02 Tf + Tb1 Tf Tb2 A T = 01 " b1 A02 Tf


The absorbed dose of P-32, P-33 and S-35 labelled oligonucleotides were estimated using published biodistribution data with several oligonucleotides and mouse models. (Crooke et al, 1996) have investigated pharmacokinetics of a 20-mer oligodeoxynucleotide phosphorothioate (ISIS 3082) and its 2_-propoxy phosphorothioate (ISIS 9045) in mice. This oligodeoxynucleotide inhibits the expression of mouse intercellular adhesion molecule (Crooke et al, 1996). (Dewanjee et al, 1994a) have published the data in mouse for 15-mer oligonucleotide sequence coupled with diethylenetriamine pentaacetate (DTPA)-isothiocyanate. (Mardirossian et al, 1997) have published the pharmacokinetic and stability data for radiolabeled aminederivatized 15-base DNA oligomer in mice. The pharmacokinetics of the compounds were expected not to change depending on P-32, P-33 or S-35 labelling. Here we also studied positron emitters F-18 and Br-76, betaemitter Y-90, Auger-emitter In-111 and alpha-emitter At211. The whole organ uptakes as percent of the injected activity were used.

II. Dosimetric calculations The accumulated dose from radionuclides used internal labelling of oligos, phosphorus-32 (P-32), phosphorus-33 (P-33) and sulphur-35 (S-35) was estimated using the MIRD (Medical Internal Radiation Dose) formalism, the basic equations of which are

D= Ã"S



III. Dosimetric data

and x

à = # A(t)dt = A0 0

Teff ln2

Table I summarizes actual delivered doses in liver, kidney and tumor. Data was collected from different published reports on pharmacokinetic data with different S-35 labelled oligonucleotides and mouse models. The liver doses in mouse models varied from 0.003 to 30 Gy/MBq. The kidney doses in the same animal models varied from from 0.01 to 35 Gy/MBq. The values in these modelswere all within tolerance limits of radiotoxicity except those for ISIS 9045. In the mammary tumor model the observed kidney dose of 9.1 Gy/MBq for P-32 (not shown) is close to the maximum tolerated dose, whereas for S-35 the absorbed radiation dose in kidneys was acceptable 1.3 Gy/MBq. The tumor dose was 1.0 Gy per administered MBq. Table I shows that oligos deliver up to 130-fold higher organ doses (including tumors) than peptide nucleic


where D, A, S and Teff refer to absorbed doses, activities, geometric factors and effective half-lives. The effective half-life can be calculated using monoexponential kinetics by

Teff =

Tb T f Tb + T f


where Tb is the biological half-life of the oligomer and T f physical half-life of the specific radionuclide. Two different situations were investigated to calculate the relative dose. 17

Kairemo et al: Oligonucleotide radiotherapy acids of the same size. The PNAs have rapid biokinetics; the half-lives are approximately 35-fold faster than those of oligo phosphorothiates. The lipophilic oligo phosphorothiate 9045 with is 2´-propoxy modification gives very high organ doses. All other 15-21-mer oligos give identical liver doses. The smallest kidney dose was calculated for the 15-mer oligo, and both ISIS 3082 and 2105 had 3.2-fold higher kidney dose. Despite the heterogeneity of the origin of the input data and used approximations of the time-activity distribution, consistent results were obtained. Subcellular dosimetry was applied in situations as described in Figure 2. The following results were obtained as shown in Figure 3. It demonstrates subcellular dosimetric data in different cell dimensions (nuclear diameter 3-8 µm,cellular diameter 610 µm) for positron emitters F-18 and Br-76 in four different oligodeoxynucleotide target systems. If high nuclear DNA target is used,large variation especially in Br-76 dose can be observed. This means that the cell nuclear dose is very much dependent on cell dimensions. If highly inductable RNA target is used, variation is much smaller as as in less extreme subcellular concentrations of oligodeoxynucleotide. Kinetics of oligonucleotides are highly dependent on the chemistry of the sugar-phosphate backbone of the molecules, and of the length of the molecules. Here, the 20h SUVs and cellular distribution reported by (Wu et al, 2000) for antisense 76Br-phosphorothioate oligonucleotides of length 20 mer was used, combined with octreotide kinetics. For tumour, a SUV of 17.5 was used, as likely for octreoscan, since no oligonucleotide data was found. Cellular uptake values in tumour are assumptions. The only data on oligonucleotide kinetics found was made (Tavitian et al, 1998), describing only the first 90 min after administration of three different oligonucleotides in baboons as measured by PET with 18F.

Macroscopic doses were calculated for oligonucleotides labelled with 76Br, 111In, 90Y and 211At, as examples of positron emitters, Auger-electron emitters, high-energy beta radiation emitters, and alpha emitting nuclides (Table III). Absorbed doses were calculated using the Mirdose 3.1 program by Stabin (Stabin, 1996), except for 211At where gamma radiation was ignored and local absorbtion of all alpha and beta radiation energy was assumed. Kidney, liver, spleen and remainder of the body were used as source organs. Using cellular S-value data (Bolch, 1999), nucleus to nucleus absorbed doses were calculated for the subcellular distributions (Table II, IV), and compared to macroscopic doses. The mean number of decays in each cell was calculated assuming a uniform distribution of the activity within each organ, and assuming spherical cells with a diameter of 14 µm and a nucleus diameter of 10 µm.

IV. Discussion Here, we have emphasized the possible role of radiolabelled antisense oligos in the anti-angiogenetic therapy. It is known that new tumor vessels due to angiogenesis differ from capillaries in normal tissues due to properties of regulation of blood flow and also interstitial fluid pressure in tumors is elevated. Molecules, related to angiogenesis in tumors may retain longer in tumors and thus give for a longer effect for therapeutic agents. The ETS1 gene has a direct role in angiogenesis: the antisense oligonucleotides directed against the ETS1 gene thus altered a cellular property of endothelial cells that is correlated with the ability of the cells to migrate through basement membranes (Chen 1997). While ETS1 regulates the expression of various proteins by endothelial cells related their growth, it is also regulating various proteins affecting coagulation and other factors which perform important endothelial functions.

Table I. The calculated organ doses for different oligomers in mouse models Oligomer

Initial activity (% of Biologic half- Liver dose (S-35) injected dose) life, Tb (hours) Gy/ MBq

Kidney dose (S35) Gy/ MBq


Peptide nucleic acid, 15-mer

0.19% (liver) 1.45 % (kidney)

5.1% (liver) 4.8 (kidney)

0.79% 0.01 Gy/ MBq

Mardirossian et al, 1997

c-myc, antisense, 15-mer

6.95 % (liver) 5.15 % (kidney)

178.2 (liver) 100 % 170.7 (kidney) 0.4 Gy/ MBq

100 % 1.3 Gy/ MBq

Dewanjee et al, 1994

ISIS 3082 20-mer

18 % (liver) 25 % (kidney)

62 (liver) 112 (kidney)

90% 0.4 Gy/ MBq

320 % 4.0 Gy/ MBq

Crooke et al, 1996

ISIS 9045, 20-mer

45 % (liver) 12 % (kidney)

$ (liver) $ (kidney)

7620 % (S-35) 30 Gy/ MBq

2710 % (S-35) 35 Gy/ MBq

Crooke et al, 1996

ISIS 2105, 21-mer

18 % (liver) 25 % (kidney)

62 (liver) 112 (kidney)

90% 0.4 Gy/ MBq

320 % 4.0 Gy/ MBq

Crooke et al, 1996

c-myc, antisense, 15-mer

11.0 % (tumor)

194 (tumor)

100 % (tumor) 1.0 Gy/ MBq


0.078 % 0.003 Gy/ MBq

Dewanjee et al, 1994

Gene Therapy and Molecular Biology Vol 7, page 19 Figure 2: Schematic model for cellular calculations in real and extreme situations. Subcellular dosimetry was applied in these situations

Dose calculations

Figure 3. It demonstrates subcellular dosimetric data in different cell dimensions (nuclear diameter 3-8 Âľm, cellular diameter 6-10 Âľm) for positron emitters F-18 and Br-76 in four different oligodeoxynucleotide target systems.


Kairemo et al: Oligonucleotide radiotherapy Table II. Shows subcellular distributions calculated by the nucleus to nucleus absorbed doses

The following SUVs at 20h after injection were given by Wu et al, 1999: Kidney Liver Spleen

6 mer 53.1 0.5 0.5

12 mer 13.3 0.5 0.5

20 mer 17.8 8.6 3.4

30 mer 1.9 12.3 5.1

The following subcellular distribution was assumed for 20 mer, approximately as in Wu et al, 1999: Nucleus 30% 30% 80%, 50%

Kidney Liver Tumour

Rest 70% 70% 20%, 50%

Table III shows the calculated absorbed doses for a number of organs and tumours. Macroscopic absorbed doses (mGy/MBq) Organ Liver Spleen Kidney Whole body (mGy/MBq)


In 0.63 0.43 0.95 0.13


Y 4.41 3.59 10.1 0.57


Br 1.29 0.96 2.35 0.23


Tumour, 100g Tumour, 0.01g Organ Liver

1.03 0.54 111 In 0.63

12.0 3.29 90 Y 4.41

2.65 0.59 76 Br 1.29

10.9 10.9 211 At 4.90

At 4.90 1.91 8.74 0.54

Table IV shows the cellular doses Average nucleus self-dose (mGy/MBq), and percentage of average nucleus absorbed dose 111 90 76 211 Organ In Y Br At Liver 0.03 (4.0%) 0.004 (0.09%) 0.006 (0.5%) 0.21 (4.4%) Kidney Tumour, 100g Tumour, 20g

0.05 (4.8%) 0.15 (14.5%), 0.09 0.15 (27.8%), 0.09

0.007 (0.04%) 0.011 (0.5%) 0.023 (0.19%), 0.015 0.036 (1.4%), 0.023 0.023 (0.71%), 0.015 0.036 (6.1%), 0.023

0.38 (4.4%) 1.26 (11.6%), 0.79 1.26 (11.6%), 0.79

transplanted into nude mice: growth of the antisenseVEGF cell lines was inhibited compared to control cells, despite the fact that they have a faster division time in vitro. These tumors had fewer blood vessels and a higher degree of necrosis explaining the reduced tumor size (Saleh 1996). Also, human melanoma cells transfected with sense vascular permeability factor (VPF)/VEGF expressed and secreted large amounts of mouse VPF/VEGF and formed well-vascularized tumors with hyperpermeable blood vessels and minimal necrosis in nude/SCID mice (Claffey, 1996).

Furthermore, ETS1 has expression in B and T lymphocytes and thymus. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that promotes angiogenesis in solid tumors. The VEGFinduced invasiveness was inhibited by ETS1 antisense oligonucleotides but not by a sense control (Chen 1997). Antisense-VEGF has been successfully used to control tumor growth and it may provide another basis for the development of antiangiogenic gene therapy (Saleh 1996). Rat glioma cells were transfected with a eukaryotic expression vector bearing an antisense-VEGF cDNA and 20

Gene Therapy and Molecular Biology Vol 7, page 21 VPF/VEGF promoted melanoma growth by stimulating angiogenesis and constitutive VPF/VEGF expression dramatically promoted tumor colonization in the lung up to 50-fold of that of controls (Claffey, 1996). Minimal sequence information required for high-affinity binding to VEGF is contained in 29-36-nucleotide motifs for the development of potent and specific VEGF antagonists (Jellinek, 1994). Transforming growth factor alpha (TGF-alpha) has been shown to induce VEGF/VPF in normal human epidermal keratinocytes in vitro (Smyth, 1997). By using a 19-mer antisense phosphorothioate oligodeoxynucleotide complementary to bases 6-24 relative to the translational start site of the VEGF/VPF mRNA, modulation of VEGF/VPF induction by TGFalpha was examined in vitro. The anti-sense oligo was capable of inhibiting VEGF/VPF RNA and protein to near-basal levels providing an antiangiogenetic strategy (Smyth, 1997). Previously, it was shown that phosphorothioate antisense oligonucleotides directed against basic fibroblast growth factor (bFGF) mRNA inhibited both the growth of Kaposi's sarcoma (KS) cells derived from different patients and the angiogenic activity associated with these cells, including the induction of KS-like lesions in nude mice (Ensoli, 1994). These effects were due to the block of the production of bFGF which is required by AIDS-KS cells to enter the cell cycle and which, after release, mediates angiogenesis (Ensoli, 1994). We describe oligos to be superior to peptide oligos in vehicle characteristics of radiation. While phosphorothioate oligos have rapid disappearance from plasma within an hour, and a biexponential elimination, their half lives apparently longer than in the peptide oligos. Although a phosphorothioate oligo leaves plasma rapidly, it requires days to leave the whole body. There is also significant extravascular accumulation of greater than 50 % of the injected dose over a period of 3 to 12 hr. Furthermore, uptake into tissues is not saturated, as some uptake is happening even at 28 days during continuos infusion (Iversen et al, 1994). The oligos are extensively eliminated in the urine over first 3 days after bolus injection. Distribution to, and tissue accumulation and distribution is tissue-specific (Iversen et al, 1992, 1994). It can be addressed that the behavior of the radiation at small distancies is crucial. This would be crucial in oligoradiotherapy with highest possible uptake in the target cell and minimal radiation toxicity to surrounding normal cells. Here, oligos are transferring radioactive source inside the cell and finally to close contact with target RNA macromolecule. We have shown earlier that for subcellular targeting internal labels give the lowest variation in estimated absorbed nuclear doses in our cell model with given dimensions (nuclear diameter 6-16 Âľm, cellular diameter 12-20 Âľm) (Kairemo et al, 1996). From the published data (Crooke et al, 1995) for ISIS 2105,21-mer oligonucleotide the following subcellular distribution was obtained: the nuclearuptake 0.2 %, cytoplasmic uptake 1.3 %, and cell surface uptake 0.3 % of injected dose. In this anti-human papilloma virus (HPV) model these uptakes as % cell

volume are 11 % for nucleus, 72% for cytoplasm and 17 % for cell surface. We calculated concentration distributions including the uniform distribution and published biodistribution. We normalized the results relative to the uniform distribution and the effect of the activity outside the cell was not taken into account, which assumption lead to the maximal possible inhomogeneity in absorbed dose distribution within a single cell. We have also calculated in vivo subcellular tissue distribution for oligodeoxynucleotide phosphorothioates with some Auger emitting radionuclides. Auger emittersare low-range electrons with high biological efficiency with a tendency of becoming more and more frequently used, at least theoretically. The doses vary considerably depending on cellular dimensions when using Auger-emitting isotopes; however, in small cells they may give a high dose. In tumors cell dimensions may vary and therefore these Auger-emitting isotopes should be applied only when nuclear target circumstances are well characterized. High energy %-emitter P-32 gives the nuclear dose closest to uniform distribution in cell sizes, but this is due to high energy. We have previously shown (Kairemo) that when using P-32 labelled oligos other than target cells will be destroyed because of long range. This is not the case when using %-emitters, P-33 and S-35, which are optimal when targets are smaller than 300 Âľm in diameter. P-33 was not studied here separately because its characteristics are very close to those of S-35. Now we also demonstrate that calculations related to positron emitters F-18 and Br-76, beta-emitter Y-90, Auger-emitter In-111 and alpha-emitter At-211 add substantial information to radionanotargeting dosimetry. Calculations using Br-76 demonstrate up to 5-fold differences in cell nuclear dose only in different cellular dimensions. This indicates the importanc of careful selection of a proper radionuclide. It is possible to use a mixture of radioisotopes to ensure a complete coverage of targets in more than one locations, e.g. targeting nuclear related and cellular RNA at the same time. In addition, modern imaging technique allows visual control over kinetic events. Dual labelling may provide therapeutic benefits when treating smaller and larger targets simultaneously. Further in vivo development, especially with various labels for oligos is highly indicated.

References Agrawal S, Temsamani J, Galbraith W, Tang J. (1995) Pharmacokinetics of antisense oligonucleotides. Clin Pharmacokinet 28, 7-16. Agrawal S, Temsamani J, Tang JY. (1991) Pharmacokinetics, biodistribution and stability of oligodeoxynucleotide phosphorothioates in mice. Proc Natl Acad Sci USA 88, 7595-7599. Bolch WE, Bouchet LG, Robertson JS, Wessels BW, Siegel JA, Howell RW, Erdi AK, Aydogan B, Costes S, Watson EE, Brill AB, Charkes ND, Fisher DR, Hays MT, Thomas SR. (1999) MIRD pamphlet No. 17, the dosimetry of nonuniform activity distributions--radionuclide S values at the voxel


Kairemo et al: Oligonucleotide radiotherapy level. Medical Internal Radiation Dose Committee. J Nucl Med 40, 11S-36S. Chen Z, Fisher RJ, Riggs CW, Rhim JS, Lautenberger JA. (1997) Inhibition of vascular endothelial growth factor-induced endothelial cell migration by ETS1 antisense oligonucleotides. Cancer Res 57, 2013-9 Claffey KP, Brown LF, del Aguila LF, Tognazzi K, Yeo KT, Manseau EJ, Dvorak HF. (1996) Expression of vascular permeability factor/vascular endothelial growth factor by melanoma cells increases tumor growth, angiogenesis, and experimental metastasis. Cancer Res 56, 172-81 Crooke RM, Graham MJ, Cooke ME, Crooke ST. (1995) In vitro pharmacokinetics of phosphorothioate antisense oligonucleotides. J Pharmacol Exp Ther 275, 462-473. Crooke ST, Graham MJ, Zuckerman JE, Brooks D, Conklin BS, Cummins LL, Greig MJ, Guinosso CJ, Kornburst D, Manorahan M, Sasmor HM, Schleich T, Tivel KL, Griffey RH. (1996) Pharmacokinetic properties of several novel oligonucleotide analogs in mice. J Pharmacol Exp Ther 277, 923-937 Dewanjee M.K, Ghafouripour A.K, Kapadvanjwala M, Dewanjee S, Serafini AN, Lopez DM, and Sfakianakis GN. (1994a) Noninvasive imaging of c-myc oncogene messenger RNA with indium-111-antisense probes in a mammary tumor-bearing mouse model. J. Nucl. Med. 35, 1054-1063. Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, Driver DA, Berg RH, Kim SK, Norden B, Nielsen PE. (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365, 566-568. Egholm M, Burchardt O, Nielsen PE, Berg RH. (1992) Peptide nucleic acids (PNA), oligonucleotide analogs with an achiral peptide backbone. J Am Chem Soc 114, 1895-1897. Ensoli B, Markham P, Kao V, Barillari G, Fiorelli V, Gendelman R, Raffeld M, Zon G, Gallo RC. (1994) Block of AIDSKaposi's sarcoma (KS) cell growth, angiogenesis, and lesion formation in nude mice by antisense oligonucleotide targeting basic fibroblast growth factor. A novel strategy for the therapy of KS. J Clin Invest 94, 1736-46 Geselowitz DA, Neckers LM. (1992) Analysis of oligonucleotide binding, internalization and intracellular trafficking utilizing a novel radiolabeled crosslinker. Antisense Res Dev 2, 1725. Hammes HP, Brownlee M, Jonczyk A, Sutter A, Preissner KT. (1996) Subcutaneous injection of a cyclic peptide antagonist of vitronectin receptor-type integrins inhibits retinal neovascularization. Nat Med. 2, 529-33. Hanahan D. (1997) Signaling vascular morphogenesis and maintenance. Science 277, 48-50. Iversen PL, Mata J, Tracewell WG, and Zon G. (1994) Pharmacokinetics of an antisense phosphorothioate oligodeoxynucleotide against rev from human immunodeficiency virus type 1 in the adult male rat following single injections and continuos infusion. Antisense Res. Dev 4, 43-52. Iversen PL, Shu S, Meter A, and Zon G. (1992) Cellular uptake and subcellular distribution of phosphorothioate oligonucleotides into cultured cells. Antisense Res. Dev 2, 211-222. Iwasaka C, Tanaka K, Abe M, Sato Y. (1996) Ets-1 regulates angiogenesis by inducing the expression of urokinase-type plasminogen activator and matrix metalloproteinase-1 and migration of vascular endothelial cells. J Cell Physiol 169, 522-531

Jekunen AP,Kairemo KJA. (1997) Inhibition of malignant angiogenesis. Cancer Treat Rev 23, 263-86. Jellinek D, Green LS, Bell C, Janjic N. (1994) Inhibition of receptor binding by high-affinity RNA ligands to vascular endothelial growth factor. Biochemistry 33, 10450-6 Kaipainen A, Vlaykova T, Hatva E, Bรถhling T, Jekunen A, Pyrhรถnen S, Alitalo K. (1994) Enhanced expression of the tie receptor tyrosine kinase messenger RNA in the vascular endothelium of metastatic melanomas. Cancer Res 54, 6571-6577 Kairemo KJA, Jekunen A, Karnani P. (1996) Modulation of antibody kinetics by the cell membrane active agent Tween 80 in vivo.Anticancer Res 16, 3542-3550 Kairemo KJA, Jekunen AP, Tenhunen M. (1999) Essentials of radionanotargeting using oligodeoxynucleotides. Gene Ther Mol Biol4, 171-176 Kairemo KJA, Tenhunen M, Jekunen AP. (1996) Oligoradionuclidetherapy using radiolabelled antisense oligodeoxynucleotide phosphorothioates. Anti-Cancer Drug Design 11, 439-449 Kairemo KJA,Tenhunen M, Jekunen AP. (1996) Dosimetry of radionuclide therapy using radiophosphonated antisense oligodeoxynucleotide phosphorothioates based on animal pharmacokinetic and tissue distribution data. Antisense Nucl Acid Drug Dev 6, 215-220. Kairemo KJA, Thorstensen K,Mack M, Tenhunen M, Jekunen AP. (1999) Ets-1 mRNA as target for antisense radiooligonucleotide therapy in melanoma cells. Gene Ther Mol Biol 4, 177-182 Korhonen J, Partanen J, Armstrong E, Vaahtokari A, Elenius K, Jalkanen M, Alitalo K. (1992) Enhanced expression of the tie receptor tyrosine kinase in cells during neovascularization. Blood 20, 2548-2555 Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Compton D, McClain J, Aldrich TH, Papadopoulos N, Daly TJ, Davis S, Sato TN, Yancopoulos GD. (1997) Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277, 55-60. Mardirossian G, Lei K, Rusckowski M, Chang F, Qu T, Egholm M, Hnatowich DJ. (1997) In vivo hybridization of technetium-99m-labeled peptide nucleic acid (PNA). J Nucl Med 38, 907-913 Masood R, Cai J, Zheng T, Smith DL, Naidu Y, Gill PS. (1997) Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma. Proc Natl Acad Sci U S A. 94, 979-84 Risau W. (1997) Mechanisms of angiogenesis Nature 386, 6714. Saleh M, Stacker SA, Wilks AF. (1996) Inhibition of growth of C6 glioma cells in vivo by expression of antisense vascular endothelial growth factor sequence. Cancer Res 56, 393-401 Sands H, Gorey-Feret LJ, Cocuzza AJ, Hobbs FW, Chidester D, Trainor GL. (1994) Biodistribution and metabolism of internally 3H-labeled oligonucleotides. I. Comparison of a phosphodiester and a phosphorothioate. Mol Pharmacol 45, 932-943. Shoji Y, Akhtar S, Periasamy A. (1991) Mechanism of cellular uptake of modified oligonucleotides methylphosphonate linkage. Nucl Acid Res 19, 5543-5550. Smyth AP, Rook SL, Detmar M, Robinson GS. (1997) Antisense oligonucleotides inhibit vascular endothelial growth factor/vascular permeability factor expression in normal


Gene Therapy and Molecular Biology Vol 7, page 23 human epidermal keratinocytes. J Invest Dermatol 108, 523-6 Stabin MG. (1996) MIRDOSE, personal computer software for internal dose assessment in nuclear medicine. J Nucl Med 37,538-46. Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, Yancopoulos GD. (1996) Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87, 1171-80. T Wu J, Zhou L, Tonissen K, Tee R, Artzt K. (1999) The quaking I-5 protein (QKI-5) has a novel nuclear localization signal and shuttles between the nucleus and the cytoplasm. J Biol Chem 274,29202-10. Tavitian B, Terrazzino S, Kuhnast B, Marzabal S, Stettler O, Dolle F, Deverre JR, Jobert A, Hinnen F, Bendriem B, Crouzel C, Di Giamberardino L. (1998) In vivo imaging of oligonucleotides with positron emission tomography. Nat Med 4,467-71. Ullrich A, Schlessinger J. (1990) Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203-212

Wasylyk B, Hahn SL, Giovane A. (1993) The ets family of transcription factors. Eur J Biochem 211, 7-18. Wu F, Yngve U, Hedberg E, Honda M, Lu L, ErikssonB, Watanabe Y, Bergstrรถm M, L_ngstrรถm B. (2000) Distribution of 76Br-labelled antisense oligonucleotides of different lengthdetermined ex vivo in rats. Eur J Pharm Sci 10, 179-186 Zhang R, Diasio RB, Lu Z, Liu T, Jiang Z, Galbraith WM, and Agrawal S. (1995) Pharmacokinetics and tissue distribution in rats of an oligodeoxynucleotide phosphorothioate (GEM 91) developed as a therapeutic agent for human immunodeficiency virus type-1. Biochem Pharmacol 49, 929-939. Zhang R, Yan J, Shahinian H, Amin G, Lu Z, Liu T, Saag MS, Jiang Z, Temsamani J, Martin RR, et al (1995) Pharmacokinetics of an anti-human immunodeficiency virus antisense oligodeoxynucleotide phosphorothioate (GEM 91) in HIV-infected subjects. Clin Pharmacol Ther 58, 44-53.


Kairemo et al: Oligonucleotide radiotherapy


Gene Therapy and Molecular Biology Vol 7, page 25 Gene Ther Mol Biol Vol 7, 25-35, 2003

Strategy of sensitizing tumor cells with adenovirusp53 transfection Review Article

Jekunen Antti1*, Miettinen Susanna2, M채enp채채 Johanna3, Kairemo Kalevi4 1

Department of Clinical Pharmacology, Helsinki University, and Department of Oncology, Turku University, and Aventis Pharma Finland, Finland. 2Department of Anatomy, Tampere University, Finland. 3Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Tampere University Hospital and Tampere University, Finland. 4 Department of Nuclear Medicine, Uppsala University Hospital, Sweden

__________________________________________________________________________________ *Correspondence: Antti Jekunen, MD, PhD, PL96, 00241 Helsinki, Finland; Tel. +358400 755208; Fax. +3589 47638140; Received: 29 January 2002; accepted: 06 March 2002; electronically published: July 2003

Summary Loss or malfunction of the p53-mediated apoptotic pathway has been proposed as one mechanism by which tumors become resistant to chemotherapy. While it may be the most frequently mutated gene in human tumor samples, the function of p53 is critical for maintaining the integrity of the cellular genome in its responses to treatment with cytotoxic agents. Intact p53 protein in nuclei of normal cells acts as a transcriptional activator for a group of genes involved in cell cycle arrest, DNA repair and apoptosis. The transfection of adenovirus p53 (adeno-p53) alone has been shown in ovarian cancer cell culture models to inhibit cell growth and to promote apoptosis regardless of the endogenous p53 status of the cells. Both mutant p53 in the tumor cells and the loss of p53 function were associated with resistance to chemotherapeutic agents. There are various reports of at least additive interactions between adeno-p53 and several chemotherapeutic agents in a number of cancers, e.g. bladder cancer, NSCLC, prostate cancer, breast cancer, and ovarian cancer both in vitro and in vivo. The mechanisms of these interactions are unknown, but they may depend on the chemotherapeutic agents used, the targets and critical tissues, and the intracellular signal transduction pathways affected.Results obtained with a speculative treatment regimen consisting of oligonucleotide therapy and p53 transfection suggest that p53 expression in tumor cells may improve their sensitivity to routine chemotherapy, e.g. docetaxel and irinotecan, which are efficacious drugs possessing different modes of action: prevention of depolymerization of tubulin and specific DNA topoisomerase I inhibition, respectively. It is known, however, that even these new agents cannot achieve responses in all tumors, and that in some tumors the efficacy, once established, diminishes along with the treatment. In these cases of resistant tumors or recurrences and relapses, combined treatment with adeno-p53 and chemotherapeutic agents may be an attractive strategy for inhibiting the progression of local cancers. In fact, the ground is ready for a rapid practical development of adeno-p53, which itself causes only minimal side-effects after administration, e.g. injection site rashes and fever, and an immunostimulation that seems to be quite mild and transient in nature. Future cancer therapy strategies may consist of effective chemotherapy coupled to molecular medicine specifically targeting tumor cells. So far, we do not have proper means in molecular medicine for achieving high enough tumor access with any of the current systemic virus vectors having the proper level of selectivity between tumor and normal cells. We have already some clinical experience, however, with intratumoral approaches that ensure the highest possible concentrations inside NSCLC, ovarian cancer and head and neck cancer tumors. It seems that there is clear evidence of good tolerability at non-maximal doses, but unfortunately, only modest activity when the construct is used alone. We review here the published data on the use of adenovirus p53 for sensitizing tumors to chemotherapeutic agents and outline perspectives for the future. Its basic function is to control the entry of the cell into the S phase of the cell cycle. p53 extends the time available for DNA repair before S phase entry (Fan et al, 1995). The wild- type gene product regulates cell growth and division negatively. Although not essential for progression of the

I. Introduction A. Function of p53 The p53 protein, a nuclear phosphoprotein, is indispensable for genomic integrity and cell cycle control. 25

Jekunen et al: Strategy of sensitizing tumor cells with adenovirus-p53 transfection cell cycle, it is critical as a checkpoint that blocks uncontrolled cell division (Levine, 1992). In the nuclei of normal cells, the intact p53 protein acts as a transcriptional activator for a group of genes involved in cell cycle arrest (p21cip1/waf1), DNA repair (GADD45), and apoptosis (Bax) (O'Connor et al, 1997; Sugrue et al, 1997; Yin et al, 1997; Carrier et al, 1999). In addition to this, p53 is a potent inducer of programmed cell death (apoptosis) within a cell in which the DNA has been damaged. Normally, the p53 gene is inactive. When, after DNA damage, the normal p53 is activated, the levels of p21, p27, and GADD 45 may become very high (Sherr, 1994). DNA damage in cells induces expression of p53 and interruption of the cell cycle in both G1 and G2 (Chu and DeVita, 2001). If DNA repair is successful, the cell continues its cycle. If repair does not succeed, the cell undergoes apoptosis.

to chemotherapeutic agents (Lowe et al, 1994; Righetti et al, 1996; Blandino et al, 1999). A recent study of ovarian cancer shows that women with tumors having the p53 null mutation have a survival disadvantage over those with p53 missense mutations (Shahin et al, 2000).

II. Evidence of the role of p53 in chemosensitizing A. p53 and chemotherapeutic agents Dysregulation of the p53 pathway may lead to drug resistance due to overproduction of the gene products responsible for entry into the S phase and rapid cell growth (Figure 1). Activation of these genes could theoretically increase the resistance of cells to the following chemotherapeutic agents: methotrexate, 2-chlorodeoxyadenosine, hydroxyurea, fludarabine, cytosine arabinoside, and 5fluorouracil. Under some experimental circumstances, cell death in response to exposure to DNA-damaging agents may require an intact p53-dependent apoptotic mechanism. Some of the genes that are transcriptionally activated by p53 belong to a class of proteins known to inhibit cyclin-dependent kinases (cdk). p21 forms a complex with proliferating cell nuclear antigen or inhibits cdk’s, e.g. cdk4 (Polyak et al, 1997). Activated p53 can cause a G1 cell cycle arrest by increasing the transcription of the cdk inhibitor p21 (Figure 2), which block cdk4 activity, preventing reitinoblastoma gene product (RB) phosphorylation (Sherr, 1994) and release of E2F blocking the transcription of a number of genes, and inhibiting entry into S phase (Kirsch, 1998). The E2F family of transcription factors bind to the regulatory regions of a number of genes that participate in the synthesis of DNA (Figure 2).

B. Mutation of p53 Mutations in the p53 gene are among the most common genetic alterations observed in human tumor samples (Oren, 1992). The specific cytotoxic treatment, the conditions of treatment, the p53 status, and other elements of cell-cycle regulation may all contribute to the outcome of exposure of a cell to DNA-damaging agents (Chu and DeVita, 2001). p53 can activate an apoptotic response to DNA damage, especially in hematopoietic and lymphoid cells, which often overrides the G1 checkpoint response (Fan et al, 1995). In cell types programmed for apoptosis, loss of p53 function decreases their sensitivity to a wide variety of DNA-damaging agents, while in cell apoptosis, it has been more difficult to establish a clear relationship between p53 gene status and chemosensitivity types of some solid tumors not inherently programmed for (Fan et al, 1995). If the DNA is damaged, the cell with intact p53 function will undergo p53-dependent apoptosis (Chu and DeVita, 2001). In tumor cells with mutated p53, the loss of p53 function, is thought to result in resistance

Figure 1. Effect of chemotherapy via p53 pathway. After chemotherapy has induced DNA damage, p53 protein is activated and transcription of many genes is increased, resulting in cell cycle arrest and apoptosis. For apoptotically sensitive cells, genotoxic damage can signal an immediate apoptotic response, while for apoptotically insensitive cells, the primary apoptotic decision point is disabled. Cells that avoid apoptotic or necrotic death after DNA repair can survive and grow. (Kirsch 1998; Brown and Wouters 1999)


Gene Therapy and Molecular Biology Vol 7, page 27 vivo and may strongly suggest the presence of synergy in vivo. Nielsen et al, used three-dimensional statistical modeling to evaluate the presence of synergistic, additive, or antagonistic efficacy between adenovirus-mediated p53 gene transfer and paclitaxel in a panel of human tumor cell lines, including those for ovarian, head and neck, prostate, and breast cancer (Nielsen et al, 1998). Cells were either pretreated with paclitaxel 24 h or not, before proliferation was measured 3 days later. Paclitaxel had synergistic or additive efficacy with p53 transfer, independently of whether the cells expressed mutant p53 protein or no p53 protein at all. Cell cycle analysis demonstrated that, prior to apoptotic cell death, p52 transfection arrested cells in the G0/G1 stage, whereas paclitaxel arrested cells in the G2-M stage. When combined, the relative concentrations of the two agents determined the dominant cellular response. The observed synergy remained unexplained; however, some speculations were offered. P53 has been shown to down regulate the expression of the antiapoptotic bcl-2 gene and up regulate the expression of the proapoptotic bax gene in other tumor cells (Selter and Montenarh 1994). Thus, p53 and paclitaxel may potentiate each other in stimulating the apoptotic pathway in neoplastic cells (Nielsen et al, 1998). It may also be that paclitaxel increased the number of cells transfected by the adenovirus. Particularly, the concentrations of paclitaxel responsible for increased adenovirus transduction are lower than the concentrations required for microtubule condensation. Moreover, the rate of change in the number of cells transduced by adenovirus appears to be independent of paclitaxel-induced cell death. The authors also determined the efficacy of the combination therapy in vivo. In some instances, it seems that loss of p53 may increase resistance to one agent, while simultaneously increasing sensitivity to another. Bunz et al, (1999) have reported that deletion of p53 in colorectal cancer cell lines maintained the cells that were resistant to 5-fluorouracil, but increased the sensitivity to doxorubicin and radiation in vitro. If the compound exerts it effects by apoptosis, as does 5-fluorouracil, loss of the apoptotic pathway may lead to resistance.

These genes include ribonucleotide reductase, dihydrofolate reductase, DNA-dependent RNA polymerase, thymidylate synthase, c-myc, c-fos, and cmyb. Activation of these gene products facilitates the entry of the cell into the S phase. There is much evidence in support of the idea that a mutation in p53 may lead to resistance to cytotoxic agents. In premenopausal women with node negative breast cancer, it has been shown by immunohistochemistry that p53(+) tumors are less sensitive to treatment with a regimen including 5-fluorouracil, doxorubicin, and cyclophosphamide than p53 (-) tumors. (Clahsen et al, 1998). Under in vitro conditions Koechli et al, have shown that mutant p53 can increase chemoresistance to 5fluorouracil, cyclophosphamide, and methotrexate (Koechli, 1994). Cisplatin resistance seems to be connected with p53 mutations, and in advanced ovarian cancer, the p53 mutational status is a predictor of the responsiveness to platinum-based chemotherapy (Calvert, 1999). However, there are also reports that apparently disagree with the chemoresistance effect of p53 (Fan et al, 1995; Stal 1995; Hawkins et al, 1996). Human fibroblasts lacking functional p53 were more sensitive to cisplatin, carboplatin, paclitaxel, nitrogen mustard or melphalan than cells with functional p53 (Hawkins et al, 1996). Similar results, loss of p53 function and the sensitizing effect of cisplatin, have been demonstrated in MCF-7 breast cancer cells and RKO methotrexate, and 5-fluorouracil have been reported in colon cancer cell lines with or without disruption of p53 function by a dominant negative p53 transgene (Fan et al, 1995). Increased rates of response to cyclophosphamide, patients with breast cancer who were determined to be immunohistochemically p53(+) (Stal,1995).

B. In vitro interactions Synergy between two chemical agents in vitro is an empirical phenomenon, in which the observed effect of the combination is greater than would be predicted from the effect of each agent working alone. While synergy is not directly measurable in clinical practice, it may predict a favorable outcome when two treatments are combined in

Figure 2. Two examples of cell cycle arrest via p53 activation. P53 mediated cell-cycle arrest is demonstrated with two examples: A) inhibition of cdk4 and cdk2 resulting G1-S and G2-M arrest, respectively. B) p53 activation increases the transcription of the cyclindependent kinase (cdk) inhibitor p21. Increase levels of p21 protein prevent cdk’s from phosphorylating their substrates, such as the retinoblastoma protein (RB) and thus block cell-cycle progression from G1 into S phase. (Kirsch 1998; Brown and Wouters 1999)


Jekunen et al: Strategy of sensitizing tumor cells with adenovirus-p53 transfection enhanced in cells that expressed wild-type p53 and were able to trigger their own cell death program. In cell culture models, adenovirus-mediated p53 gene transfer alone inhibits cell growth and promotes apoptosis, regardless of the endogenous p53 status of the ovarian cancer cells (Santoso et al, 1995). In tumor cells, mutated p53 and also loss of p53 function were associated with resistance to chemotherapeutic agents. There are several reports of at least an additive interaction between adenop53 and cisplatin in bladder cancer (Miyake et al, 2000), between adeno-p53 and cisplatin, SN-38 (a metabolite of irinotecan), 5-fluorouracil, taxanes, bleomycin, and cyclophosphamide in NSCLC (Fujiwara et al, 1994) (Horio et al, 2000), and between adeno-p53 and paclitaxel in ovarian cancer (Nielsen et al, 1998). In the ovarian cancer model, enhanced efficacy has been reported in a three-drug combination of adeno-p53, cisplatin, and paclitaxel (Gurnani et al, 1999). There is some evidence that chemosensitivity can be increased by replacement of the p53 gene. Roth (Roth, 1996) reported that recombinant-adenovirus-mediated transfer of the wild-type p53 gene into several human cells with homozygous deletions of p53 markedly increased cellular chemosensitivity to the major chemotherapeutic drugs. An additive antiproliferative effect was reported in p53null H358 lung cancer cells when cultured with cisplatin for 24 h before transduction with adeno-p53 (Fujiwara et al, 1994). Enhanced apoptosis, detected by DNA fragmentation, was reported for the combination compared with each agent alone. A viability assay demonstrated that a replicationdefective adenovirus encoding the wild-type p53 gene (INGN 201, Introgen Therapeutics, Inc.) suppresses growth and enhances sensitivity to DNA-damaging chemotherapeutic drugs (5-fluorouracil, doxorubicin, cisplatin) in p53-mutant-expressing cell lines (Gjerset and Mercola, 2000). These cells lines represent DLD-1 colon cancer, T47D breast cancer, PC-3 prostate cancer, and T98G glioblastoma. Transfection efficiencies were 6070%. It seems that restoration of the wild-type p53 to mutant p53-expressing or p53null cells results in marked enhancement of sensitivity to several DNA damaging agents. This enhancement of sensitivity was not observed in two wild-type p53-expressing cell lines, MCF7 and LS174T, suggesting that, in this model, wild-type p53 gene transfer is effective as therapy sensitization only in tumors that have lost wild-type p53 function.

Recently, a report using isobologram modelling have showed that the combination of adeno-p53 + radiation produced significantly synergistic effects in NSCL cell lines, whereas the combination of docetaxel + adeno-p53 and docetaxel + radiation produced mixed effects ranging between additive and synergistic (Nguyen al., 1996). The three-agent combination also produced significantly synergistic effects. Brown and Wouters have criticized the sensitizing results obtained in cell cultures. They have pointed out the need for further evidence in relating p53 to the sensitivity of anticancer agents (Brown and Wouters, 1999). Because apoptosis, particularly p53窶電ependent apoptosis, can occur rapidly after drug exposure, short-term growth rate assays tend to underestimate overall death of cells with mutant p53 or of cells not undergoing apoptosis. This may result in a situation where short-term assays may incorrectly assess overall cell death in tumor cells with different probabilities of undergoing early apoptosis. Thus, results may have a bias toward increased cell death in wild-type p53 cells and decreased cell kill in mutant p53 cells. Results of experiments with normal cells transformed with dominant oncogenes have often been extrapolated to tumor cells, instead of initially using cancer cell models. Transformed normal cells are usually apoptotically more sensitive than cancer cells. Therefore, in sensitizing experiments, both long term clonogenic assays and tumor cell models with solid tumors should be used rather than growth rate assays and transformed normal cells. However, the more widely accepted conclusion drawn from studies conducted in cancer cell lines and tumors of different origin is still that restoration of normal p53 function in tumors restores the apoptotic pathway and leads to an increased response to chemotherapy (Peller, 1998; Ferreira, 1999; Chang, 2000).

C. Transfection of cell cultures with the adenovirus p53 gene construct Adenovirus vectors have many advantages over other viral and non-viral vectors. Their transfection efficacy is high, in both dividing and resting cells, and they show high expression levels (Hwu, 2001). As adenoviral DNA is not incorporated into the cell genome, expression of the transgene is transient, but adenoviral vectors can be produced at high titers. Introduction of wild-type p53 into tumors with non functional p53 offers a novel strategy for treating cancer, by inducing apoptotic death in neoplastic cells. Genomic instability accompanied by loss of p53mediated apoptosis can also lead to therapy resistance. The support for this rationale is that loss of p53 could desensitize cells to the damaging effects of drugs. Normal transgenic hematopoetic cells (Lotem and Sachs, 1993), E1A-expressing transgenic fibroblasts (Lowe et al, 1993), and transformed transgenic fibroblasts (Lowe et al, 1994) were all more resistant to apoptosis following treatment with any of a wide variety of anticancer agents, than were comparable cells from the parental strain of mice, which expressed wild-type p53. Apoptosis seemed to be

1. Glioma and pancreatic cancer Somatic gene therapy based on the reintroduction of p53 limits the proliferation of human malignant glioma cells, but is unlikely to induce clinically relevant sensitization to chemotherapy in these tumors. Wild-type p53 failed to sensitize glioma cells to cytotoxic drugs including BCNU, cytarabine, doxorubicin, teniposide, and vincristine. The combined effects of the wild-type p53 gene transfer and drug treatment were less than additive rather than synergistic, suggesting that the intracellular cascades activated by p53 and chemotherapy were redundant. Unexpectedly, forced expression of mutant28

Gene Therapy and Molecular Biology Vol 7, page 29 p53 reduced 3H-thymidine incorporation by about 90% at 48 hr, cell viability at 6 days was reduced by only about 50% relative to controls. Although apoptosis is detectable in the adeno-p53-treated cultures, these results suggest that a large fraction of adeno-p53-treated cells merely undergo reversible cell cycle arrest. Combined treatment with adeno-p53 and doxorubicin results in a greater than additive loss of viability in vitro and increased apoptosis. These data indicate an additive to synergistic effect of adeno-p53 and doxorubicin for the treatment of primary and metastatic breast cancer. However, in breast cancer cell lines results without any clear cut link between transfection of p53 and a sensitizing effect have been reported. Two human breast cancer cell lines, MDA-MB-231 and MDA-MB-435, both with p53 mutations, were transduced with adenoviral vectors containing wild-type p53 and the effects on growth were determined by clonogenic assays (Parker et al, 2000). Combining VP-16 and paclitaxel with Ad5CMV-p53 did not consistently or significantly decrease clonogenic survival.

p53-modulated drug sensitivity enhanced the toxicity of some drugs but attenuated the effects of others (Trepel et al, 1998). Likewise, in p53-null pancreatic carcinoma cells, wild-type p53 gene transduction had no effect on in vitro chemosensitivity to cisplatin, etoposide, 5fluorouracil and paclitaxel (Kimura et al, 1997). Moreover, in anaplastic thyroid cancer cells, adeno-p53 increased the sensitivity to doxorubicin with a 10-fold decrease in IC50 values.

2. Hepatocellular cancer One of the goals of gene therapy for treating cancer is selective expression of cytotoxic gene products in tumor cells. When replication-defective retroviruses were constructed containing p53 cDNA that was transcriptionally regulated by the human hepatocellularcarcinoma-associated alpha-fetoprotein gene transcriptional control elements, the expression of exogenous wild-type p53 from this retroviral vector was limited to the cells producing alpha-fetoprotein. Introduction of wild-type p53 into alpha-fetoprotein positive human hepatocellular carcinoma cells by retroviral infection markedly inhibited their clonal growth in a monolayer and increased the sensitivity of these cells to the chemotherapeutic drug cisplatin (Xu et al, 1996).

5. Bladder cancer Combined treatment with Ad5CMV-p53 and cisplatin could be an attractive strategy for inhibiting progression of bladder cancer. In human bladder cancer KoTCC-1 cells, transfer of an adenovirus-mediated p53 gene enhances cisplatin cytotoxicity in vitro, and Ad5CMV-p53 and cisplatin synergistically inhibit growth and metastasis in vivo. Ad5CMV-p53 substantially enhances cisplatin chemosensitivity in a dose-dependent manner, reducing the median IC50 by more than 50%. Furthermore, orthotopic injection of adeno-p53 combined with cisplatin therapy synergistically inhibits growth of subcutaneous KoTCC-1 tumors and the incidence of metastasis (Miyake et al, 2000). In contrast, p21cip1/waf1 gene therapy had no effect on in vitro or in vivo chemosensitivity to cisplatin (Miyake et al, 1998).

3. Ovarian cancer In cell culture models adenovirus-mediated p53 gene therapy is one way to inhibit cell growth and promotes apoptosis, regardless of the endogenous p53 status of the ovarian cancer cells (Santoso et al, 1995) (Wolf et al, 1999). Adeno-p53 gene transfer, combined with cisplatin, doxorubicin, 5-fluorouracil, methotrexate, or etoposide, inhibited cell proliferation more effectively than chemotherapy alone in head and neck, ovarian, prostate and breast tumor cell lines. Of particular significance, in an ovarian cancer model enhanced efficacy was noted when using the three-drug combination of adeno-p53, cisplatin, and paclitaxel (Gurnani et al, 1999). In human head and neck, ovarian, prostate, and breast cancer cells, low concentrations of paclitaxel also increase the number of cells transduced by recombinant adeno-p53 in a dosedependent manner (Nielsen et al, 1998). The concentration of paclitaxel responsible for increased adenovirus transduction is lower than that required for microtubule condensation.

6. Lung cancer Recombinant adenovirus-mediated transfer of the wild-type p53 gene into monolayer cultures or multicellular tumor spheroids of the human NSCLC cell line H358, in which there is homozygous deletion of p53, markedly increased the cellular sensitivity of these cells to cisplatin (Fujiwara et al, 1994). In a study made by Osaki et al,(Osaki et al, 2000), an alteration in drug chemosensitivity caused by the adenovirus-mediated transfer of the wild-type p53 gene in human lung cancer cells was tested on a human pulmonary squamous cell carcinoma cell line, NCI-H157, and a human pulmonary large-cell carcinoma cell line, NCI-H1299. Based on isobologram data, a supra-additive effect was observed for 5-fluorouracil and SN-38 on NCI-H157 cells. An additive effect was also observed for cisplatin, paclitaxel, bleomycin, and cyclophosphamide on NCI-H157 cells. Cisplatin, paclitaxel, 5-fluorouracil, and SN-38 had an additive effect on NCI-H1299 cells. No drug showed any subadditive or protective effects. These findings suggest

4. Breast cancer Transduction of cells using replication-deficient adenovirus vectors can induce endogenous p53 expression in cells containing the wild-type p53 gene and this response is different from the p53 induction observed after DNA damage (McPake et al, 1999). Lebedeva et al, have examined the effects of a replication-defective adenovirus encoding p53 (INGN 201, Ad5CMV-p53), alone or in combination with the breast cancer therapeutic doxorubicin, in suppressing growth and inducing apoptosis in breast cancer cells in vitro (Lebedeva et al, 2001). They found that whereas in vitro treatment of cells with adeno29

Jekunen et al: Strategy of sensitizing tumor cells with adenovirus-p53 transfection that CPT-11 and 5-fluorouracil may be useful as anticancer agents for use in a combination therapy regimen, using wild-type p53 gene transfer. These results indicate that CPT-11, as well as cisplatin, is a candidate for the combination of chemotherapy and gene therapy for NSCLC. Adeno-p53 and DNA-damaging agents, cisplatin, etoposide and CPT-11 showed synergistic effects in NSCLC, but, in contrast had additive effects with antitubulin agents such as paclitaxel and docetaxel (Horio, Hasegawa et al, 2000). Perdomo et al, (Perdomo et al, 1998) have demonstrated that human NSCLC cells having a mutant form of p53 grow faster in vivo than wild-type p53 cell lines and the treatment with cisplatin or radiation does not reduce the size of mutant p53 tumors, although wild-type p53 tumors regress markedly. Apoptosis occurred in mutant p53 cell types only at high cisplatin doses and not at the magnitude detected in wild-type tumors.

III. In vivo evidence chemosensitization by adenovirus p53

later by doxorubicin or mitomycin-C, but not by vincristine (Blagosklonny and El-Deiry 1996). In the p53 null SK-OV-2 xenograft model of ovarian cancer, a dosing schedule of the p53 therapy that, by itself, had a relatively minimal effect on the tumor burden (16%) caused a much greater decrease in tumor burden (55%) when combined with paclitaxel (Nielsen et al, 1998). Further, in nude mice implanted intraperitoneally with 2774 human ovarian cancer cells (mutated p53), the response to adeno-p53 gene therapy showed significant survival duration, with a survival time greater than that of untreated animals. However, no statistically significant survival advantage was observed between adeno-p53- and adenovirus-!gal-treated mice (von Gruenigen et al, 1998). In another ovarian cancer study using nude mice, the adeno-p53 treatment effectively suppressed the growth of peritoneal tumors and prolonged the survival of the treated group, especially when the tumor burden was small (Kim et al, 1999). Greater combined efficacy was observed in the p53null DU-145 prostate, p53Mut MDA-MB-468 breast, and p53met MDA-MB-231 breast cancer xenograft models in vivo. The authors concluded that their data, taken together, offer the possibility of enhanced antitumor activity with lower than normal doses of paclitaxel and adenovirus p53, when the two drugs are administered in combination (Nielsen et al, 1998). They noted that this could potentially decrease the chemotherapy-induced side effects, increasing the quality of life of the patients and, perhaps, reducing the overall expense of a complete course of cancer treatment.


These observations have been extended to in vivo models. Tumors have been treated in vivo with replication-defective p53 adenovirus and chemotherapy. Nguyen et al, have reported convincing in vivo studies, in which p53null H1299 lung tumor xenografts were given i.p. cisplatin before, concurrently with, or after intratumoral adenovirus p53 (Nguyen et al, 1996). The most effective dosing regimen was cisplatin given two days before p53 therapy. Cisplatin and CPT-11 had a significant antitumoral effect on lung cancer H157 cell xenografts of nude mice in vivo. Human head and neck cancer and colon cancer (Gjerset et al, 1997) and prostate cancer (Gjerset and Mercola 2000) in nude mice models in vivo have been found to exhibit a similar sensitization effect with adenovirus plus cisplatin as in studies in vitro. Gjerset et al, demonstrated increased sensitivity to cisplatin cytotoxicity in p53mut T98G glioblastoma and p53 mut H23 small cell lung carcinoma cells transduced with p53 expression vectors one or two days before exposure to cisplatin (Gjerset et al, 1995). These results are consistent with other in vivo studies in animal models showing a combined benefit of p53 and chemotherapy (Badie et al, 1998), (Fujiwara et al, 1994), (Miyake et al, 1998), (Nielsen et al, 1998), (Nguyen et al, 1996). Gjerset and Mercola are convinced that these results support the clinical application of adenovirus p53 combination approaches to tumors expressing mutant p53 (Gjerset and Mercola 2000). Chemosensitization by p53 has also been studied using ex vivo modified cells in an orthotopic model of glioblastoma in Fisher rats (Dorigo et al, 1998). The combination of p53 with 5-fluorouracil and topotecan has been studied in p53mut SW480 colorectal tumor cells transfected with an inducible p53 construct (Yang et al, 1996). Dose-dependent enhancement of cytotoxicity was observed with these drugs by the concurrent expression of wild-type p53. Increased cytotoxicity has been reported in p53mut SkBr3 mammary tumor cells when transduction with p53 was followed 8 hr

IV. Clinical results of adenovirus p53 transfection with chemotherapy The first evidence of the efficacy of p53 gene therapy for cancer was given by a pilot study in which retroviral p53 expression vectors were directly injected into small endobronchial lesions of NSCLC patients (Roth et al, 1996). Tumor regression was noted in three patients out of nine, and tumor growth stabilized in three other patients. The safety and feasibility of the intratumoral injection of adenoviral wild-type p53 expression vectors have been established in NSCLC patients, with clear evidence for transgenic expression, and possibly induction of apoptosis (Swisher et al, 1999; see Table 1). The antitumor activity in this trial was consistent with the activity of retroviral p53 injection in NSCLC patients. Twenty-four patients received intratumor injections of adenovirus p53 and two patients achieved a partial response, while 17 patients achieved stable disease as the best clinical response. A nonrandomized, phase I, dose-escalating study by Clayman et al expanded these findings into head and neck squamous cell carcinoma (Clayman et al, 1998). Patients with incurable recurrent local or regionally metastatic HNSCC received multiple intratumoral injections of adeno-p53, either with or without tumor resection. P53 expression was detected in tumor biopsies despite antibody responses after injections. prevent the appearance of adeno-p53 in blood and urine. were seen in the study As expected, almost Neither dose-limiting effects nor serious


Gene Therapy and Molecular Biology Vol 7, page 31 adverse events all the patients developed anti-adenovirus antibodies in the course of treatment, but this immune response did not treatment. The most common treatmentrelated adverse event was pain at injection site. Other reported adverse events were transient fever, headache, pain, and edema. No evidence of systemic hypersensitivity or allergic reactions was seen, despite the fact that patients received many repeated courses of treatment. In some patients, adenovirus p53 administration led to objective antitumor activity. Two out of 17 patients showed objective tumor regressions greater than 50% and six patients showed stable disease for up to 3.5 months. In addition, one patient showed a complete pathologic response. The median survival for responding patients was 13.6 months, and the overall median survival was 267 days, which is about 60% longer than that reported in chemotherapy trials with a similar patient profile (Schornagel et al, 1995). Of course, it is impossible, for a phase-one study with limited numbers of patients to state anything more than that these results are promising and that further studies are needed, and are underway, to determine the actual role of adenovirus-mediated p53 intratumoral injections as a treatment option for HNSCC. The next step in the development of p53 treament is to include combination therapy with cytotoxic agents. There is also a negative trial published by Schuller and coworkers (2001). Twenty-five patients with nonresectable NSCLC were enrolled in an open-label, multicenter, phase II study of three cycles of chemotherapeutics with intratumoral injection of recombinant adenovirus p53. The main idea of this small study was to compare the isolated responses of a tumor lesion treated by transfer of the adenoviral wild-type p53 gene with a comparable lesion not receiving any injections in patients undergoing first-line chemotherapy for NSCLC. In the 13 patients receiving carboplatin and paclitaxel, there was no obvious difference between the mean response of gene-therapy-treated and the reference lesions. In contrast, the mean regression of the reference lesions in patients treated with cisplatin and vinorelbine was 15%, whereas it amounted to 55% in lesions that were additionally injected with the gene construct. There was no difference between the responses of lesions treated with p53 gene therapy in addition to chemotherapy (52%) and those of lesions treated with chemotherapy alone (48%). The authors concluded that, in these patients

the therapy appears to provide no additional benefit. However, there were several possible shortcomings in the clinical set-up: no injections to the reference lesions, highly restrictive inclusion criteria may result in selection bias, a higher response rate (50%) than is normally achieved in this disease, a chance of having a biologically inactive virus construct, and insufficient spreading of the replication-defective adenoviral vectors within the tumors after only one central intralesional injection. Recently, attemps have been made to overcome the problem of ineffective vector spreading by administration of replication-competent adenoviruses (Heise, Sampson et al, 1997) and encouraging clinical results have been reported (Khuri et al, 2000). There were concerns about the safety, which, however, turned out to be exaggerated. Khuri et al, (2000) demonstrated an acceptable safety pattern with no sign of any dissemination to the environment. A Phase II trial of a combination of intratumoral ONYX-015 injection with cisplatin and 5fluorouracil was carried out with patients having recurrent squamous cell cancer of the head and neck. Only pain at the injection site (45%), mucous membrane disorder (21%), syncope (5%), kidney failure (5%), and anorexia (3%) could not be ruled out as attributable to Onyx-015. In addition, the injected tumors achieved objective responses at a substantially higher rate (9 of the 11) than the non-injected tumors (3 of the 11) within the same patients. In six patients, the injected tumor responded and the non injected tumor did not respond. The time to tumor progression was also longer for the injected tumors than for the non-injected tumors. There was no correlation between the response and the baseline tumor size, baseline neutralizing antibody titer, p53 gene status, or prior treatment. It was also clear that the efficacy of the intratumoral injection was not prevented by neutralizing antibodies. There has been discussion about whether or not enough evidence about viral replication of ONYX-015 in patients, as along experience based on 190 patients treated by a replication-defective adenovirus demonstrating similar biodistribution (Clayman et al, 1998; ConstenlaFigueiras et al, 1999). It may simple be that Taqman realtime polymerase chain reaction technology is not sufficient to prove that viral reproduction is taking place (Yver et al, 2001).

Table 1. Sensitising effect of adenovirus-p53 on chemotherapeutic agents, major clinical treatment results Disease




Treatment responses

Reference (first author year)





2 PR, 17 SD_

(Swisher et al, 1999)

Head & neck




1CR, 2 PR, 6 SD_

(Clayman et al, 1998)

NSCLC Heach & neck (_) on patients


Cisplatin + vinorelbin Cisplatin +5-FU

25 11

13 PR* 9 PR*

(Schuler et al, 2001) (Khuri et al, 2000)

(*) on measurable lesions


Jekunen et al: Strategy of sensitizing tumor cells with adenovirus-p53 transfection strategy for inhibiting progression of local cancers. It is clear that even a modest change in drug sensitivity may bring some refractory tumors within a range that is treatable with conventional chemotherapy. Future therapy might couple standard cytotoxic agents with new biologic agents that attack specific molecular targets to reregulate the cell-cycle checkpoint. Human data supporting the effect of sensitizing chemotherapy with adenovirus p53 is still maturing, although we have not found a way to use systemic administration. We know that is s safe to perform intratumoral gene therapy with adenovirus either with a replication non-competent or replication competent vector. As yet, there is no clinical evidence to support a definite conclusion that adenovirus p53 provides a clinically meaningful improvement on conventional chemotherapy. However, it is clear that in some trial set ups it has been possible to demonstrate encouraging results and the possibility of a clinical sensitizing effect of p53 gene therapy on the chemotherapy used when specifically indicated. Intratumoral expression of transgenes and tumor-selective tissue destruction have been documented in phase I and phase II clinical trials of adenovirus p53 mediated gene therapy. However, durable responses and the clinical benefit seen have been limited, with of 10-15% response rates. The rationale of combining p53 gene therapy with a chemotherapeutic agent in the clinical setting has been noted to be as follows: combinations of agents with different toxicologic profiles can result in increased efficacy without increased overall toxicity, they may thwart the development of resistance to the single agents, they may offer a solution to the problem of heterogeneous tumor cell populations with different drug sensitivity profiles and they allow the physician to take advantage of possible synergies between drugs, resulting in increased anticancer efficacy in patients (Nielsen, Lipari et al, 1998). Several phase III clinical trials with adenovirus p53 therapy in head and neck cancer, NSCLC, and ovarian cancer, will be completed in the near future, and the role of gene therapy may become routine a part of treatment regimens.

V. Conclusion Several subsequent studies have confirmed that various malignant cell lines and tumors expressing mutant or deleted p53 are chemoresistant to a wide range of anticancer agents. However, other studies disagree suggesting that cells with impaired p53 function can become sensitized to various anticancer agents. Thus, the relationship between p53 status and chemosensitivity is complex and presumably depends on a number of factors, including the specific cytotoxic stimuli, tissue-specific differences, and the specific cellular context that incorporates the overall genetic machinery and the various intracellular signaling pathways (Chu and DeVita 2001). The relationship between p53 and chemotherapy depends on the chemotherapeutic agents used, the target and the critical tissues, and the intracellular signal transduction pathways affected. The theoretical basis of the sensitizing effect of chemotherapeutic agents in combination with adenovirus p53 has been presented and so have a number of supportive data. As adenovirus p53 has its own activity, there seems to be a possibility that the cytotoxicity may be enhanced at least in some cell lines by transfer of the gene into the tumor cells. This concept has reached the level of proof in some, although not all, experimental conditions. This leaves a room for doubt, as all spontaneous solid tumors are heterogeneous and there may always remain cell clones that fail to obey the sensitizing principle. It is clear that more evidence is needed to support this principle, especially clonogenic assays and classical interaction studies. Although the in vivo experiments are convincing and strongly positive, it may not be altogether correct to extrapolate these results into clinical practice. There is a relative lack of pharmacokinetic studies and pharmacokinetic interaction studies in adenovirus p53 gene therapy. Several strategies may be used to develop p53-based anticancer therapies, with the goal of resensitizing tumor cells to conventional chemotherapy (Chang 2000). These include reintroduction of the gene encoding wild-type p53 and methods for restoring normal p53 function to mutant p53. In addition, methods are being developed that target the p53-mdm-2 interaction of using lack of wild-type p53 in tumors to protect normal tissue from the adverse effects of chemotherapy. Replacement of the wild-type p53 by intratumoral transfection has already reached the phase III stage of clinical trials. Transfection of p53 can be combined with radioimmunotherapy as part of a tumor manipulation scheme (Kairemo, Jekunen et al, 1999). Increasing suppressor gene p53 expression in tumor cells improves the sensitivity of the tumor cells to routine chemotherapy. In a variety of tumor types, docetaxel and irinotecan are efficacious drugs with a new mode of action: prevention of depolymerization of tubulin and inhibition of specific DNA topoisomerase I, respectively. But we cannot obtain responses from all tumors, and in some tumors the efficacy, although established, diminished with time. In these cases of resistant tumors or recurrences and relapses, combined treatment with adenop53 and chemotherapeutic agents may be an attractive

Acknowledgments We would like to thank Aventis Pharma Finland for supporting this work.

References Badie B, Kramar MH, Lau R, Boothman DA, Economou JS, Black KL. (1998). “Adenovirusmediated p53 gene delivery potentiates the radiation-induced growth inhibition of experimental brain tumors.” J Neurooncol 37: 217-222. Blagosklonny, M. V. and W. S. El-Deiry (1996). “In vitro evaluation of a p53-expressing adenovirus as an anti-cancer drug.” Int J Cancer 67: 386-392. Blandino G, Levine AJ, Oren M. (1999). “Mutant p53 gain of function: differential effects of different p53 mutants on resistance of cultured cells to chemotherapy.” Oncogene 18(2): 477-85.


Gene Therapy and Molecular Biology Vol 7, page 33 Brown, M. J. and B. G. Wouters (1999). “Apoptosis, p53, and tumor cell sensitivity to anticancer agents.” Cancer Res 59: 1391-1399. Bunz, F. ( 1999). “Disruption of p53 in human cancer cells alters the responses of chemotherapeutic agents.” J Clin Invest 104: 263-269. Calvert, A. H. (1999). “Carboplatin and paclitaxel, alone and in combination: dose escalation, measurement of renal function, and role of the p53 tumor supressor gene.” Semin. Oncol. 29: 90-94. Carrier, F., Georgel, P.T., Pourquier, P., Blake, M., Kontny, H.U., Antinore, M.J., Gariboldi, M., Myers, T. G, Weinstein, J.N., Pommier, Y,and Fornace, A.J., Jr. (1999). “Gadd45, a p53-responsive stress protein, modifies DNA accessibility on damaged chromatin.” Mol Cell Biol 19(3): 1673-85. Chang, E. H. (2000). “Tp53 gene therapy: akey to modulating resistance to anticancer therapies?” Molecular Medicine Today 6: 358-364. Chu, E. and V. T. J. DeVita (2001). Apoptosis, cell-cycle control, and resistance to chemotherapy. Cancer. Principles & Practice of Oncolgy. V. T. J. DeVita, S. Hellman and S. A. Rosenberg. Philadelphia, Lippincott Williams & Wilkins. 6: 289-306. Clahsen PC, van de Velde CJ, Duval C, Pallud C, Mandard AM, Delobelle-Deroide A, van den Broek L, Sahmoud TM, van de Vijver MJ. (1998). “p53 protein accumulation and response to adjuvant chemotherapy in premenopausal women with node-negative early breast cancer.” J Clin Oncol 16(2): 470-3945. Clayman GL, el-Naggar AK, Lippman SM, Henderson YC, Frederick M, Merritt JA, Zumstein LA, Timmons TM, Liu TJ, Ginsberg L, Roth JA, Hong WK, Bruso P, Goepfert H. (1998). “Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcionoma.” JCO 16: 2221-2232. Constenla-Figueiras M, Betticher DC, DelCampo JM, Hitt R, Rochlitz C, Dhondt V, Gautier E, Saulnier P, Yver A, Badri N. (1999). “A phase II trial with Ad5CMV-p53 as a single agent in recurrent/refractory SCCHN looking at vector biodistribution and horizontal trnasmission under normal life conditions.” Proc Am Soc Clin Oncol 18: 444a. Dorigo O, Turla ST, Lebedeva S, Gjerset RA. (1998). “Sensitization of rat gliblastoma multiforme to cisplatin in vivo following restoration of wild-type p53 function.” J Neurosurg 88: 535-540. Fan S, Smith ML, Rivet DJ 2nd, Duba D, Zhan Q, Kohn KW, Fornace AJ Jr, O'Connor PM. (1995). “Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline.” Cancer Res 55(8): 1649-907. Ferreira, C. G. (1999). “p53 and chemosensitivity.” Ann Oncol 9: 1011-1021. Fujiwara T, Grimm EA, Mukhopadhyay T, Zhang WW, OwenSchaub LB, Roth JA. (1994). “Induction of chemosensitivity in human lung cancer cells in vivo by adenovirus-mediated transfer of the wild-type p53 gene.” Cancer Res 54(9): 2287-91. Gjerset, R. A., O. Dorigo, et al, (1997). “Tumor regression in vivo following p53 combination therapy.” Cancer Gene Therapy 4: 0-2. Gjerset, R. A. and D. Mercola (2000). “Sensitizing of tumors to chemotherapy through gene therapy.” Cancer Gene Therapy 465: 273-291. Gjerset RA, Turla ST, Sobol RE, Scalise JJ, Mercola D, Collins H, Hopkins PJ. (1995). “Use of wild-type p53 to achieve

complete treatment sensitization of tumor cells expressing endogenous mutant p53.” Mol Carcinog 14: 275-285. Gurnani M, Lipari P, Dell J, Shi B, Nielsen LL. (1999). “Adenovirus-mediated p53 gene therapy has greater efficacy when combined with chemotherapy against human head and neck, ovarian, prostate, and breast cancer.” Cancer Chemother Pharmacol 44(2): 143-51. Gurnani M, Lipari P, Dell J, Shi B, Nielsen LL. (1998). “Adenovirus-mediated p53 gene therapy and paclitaxel have synergistic efficacy in models of human head and neck, ovarian, prostate, and breast cancer.” Clin Cancer Res 4: 835-846. Hawkins DS, Demers GW, Galloway DA. (1996). “Inactivation of p53 enhances sensitivity to multiple chemotherapeutic agents.” Cancer Res 56: 892-898. Heise C, Sampson-Johannes A, Williams A, McCormick F, Von Hoff DD, Kirn DH. (1997). “ONYX-015, an E1B geneattenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficaacy that can be augmented by standard chemotherapuetic agents.” Nature Medicine 3: 639-645. Horio Y, Hasegawa Y, Sekido Y, Takahashi M, Roth JA, Shimokata K. (2000). “Synergistic effects of adenovirus expressing wild-type p53 on chemosensitivity of non-small cell lung cancer cells.” Cancer Gene Ther 7(4): 537-44. Hwu, P. (2001). Gene therapy. Cancer. Principles & Practice of Oncology. V. T. J. DeVita, S. Hellman and S. A. Rosenberg. Philadelphia, Lippincott Williams & Wilkins. 6: 3161-3180. Kairemo KJ, Jekunen AP, Tenhunen M. (1999). Dosimetry and optimization of in vivo targeting with radiolabeled antisense oligonucleotides: oligonucleotide radiotherapy. Methods of Enzymology. New York, Academic Press. 314: 506-525. Khuri FR, Nemunaitis J, Ganly I, Arseneau J, Tannock IF, Romel L, Gore M, Ironside J, MacDougall RH, Heise C, Randlev B, Gillenwater AM, Bruso P, Kaye SB, Hong WK, Kirn DH. (2000). “A controlled trial of intraumoral ONYX015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer.” Nature Medicine 6: 879-885. Kim J, Hwang ES, Kim JS, You EH, Lee SH, Lee JH. (1999). “Intraperitoneal gene therapy with adenoviral-mediated p53 tumor suppressor gene for ovarian cancer model in nude mouse.” Cancer Gene Ther 6(2): 172-8. Kimura M, Tagawa M, Takenaga K, Yamaguchi T, Saisho H, Nakagawara A, Sakiyama S. (1997). “Inability to induce the alteration of tumorigenicity and chemosensitivity of p53-null human pancreatic carcinoma cells after the transduction of wild-type p53 gene.” Anticancer Res 17(2A): 879-83. Kirsch, D., Kastan, M.B. (1998). “Tumor-suppressor p53: implications for tumor development and prognosis.” JCO 16: 3158-3168. Koechli, O. (1994). “Mutant p53 protein associated with chemosensitivity in breast cancer specimens.” Lancet 344: 1647-1648. Lebedeva S, Bagdasarova S, Tyler T, Mu X, Wilson DR, Gjerset RA. (2001). “Tumor suppression and therapy sensitization of localized and metastatic breast cancer by adenovirus p53.” Hum Gene Ther 12(7): 763-772. Levine, A. J. (1992). “The p53 tumour suppressor gene and product.” Cancer Surveys 12: 59-79. Lotem, J. and L. Sachs (1993). “Hematopoietic cells from mice deficient in wild-type p53 are more resistant to induction of apoptosis by some agents.” Blood 82: 1092-1096.


Jekunen et al: Strategy of sensitizing tumor cells with adenovirus-p53 transfection Lowe SW, Bodis S, McClatchey A, Remington L, Ruley HE, Fisher DE, Housman DE, Jacks T. (1994). “p53 status and the efficacy of cancer therapy in vivo.” Science 266(5186): 807-10. Lowe SW, Ruley HE, Jacks T, and Housman DE. (1993). “p53mediated apoptosis modulates the cytotoxicity of anticancer agents.” Cell 74: 957-967. McPake CR, Shetty S, Kitchingman GR, Harris LC.. (1999). “Wild-Type p53 Induction Mediated by Replication-deficient Adenoviral Vectors.” Cancer Res 59(17): 4247-907. Miyake H, Hara I, Gohji K, Yamanaka K, Arakawa S, Kamidono S. (1998). “Enhancement of chemosensitivity in human bladder cancer cells by adenoviral-mediated p53 gene transfer.” Anticancer Res 18(4C): 3087-92. Miyake H, Hara I, Hara S, Arakawa S, Kamidono S. (2000). “Synergistic chemosensitization and inhibition of tumor growth and metastasis by adenovirus-mediated P53 gene transfer in human bladder cancer model.” Urology 56(2): 332-6. Nguyen DM, Spitz FR, Yen N, Cristiano RJ, Roth JA (1996). “Gene therapy for lung cancer: enhancement of tumor suppression by a combination of sequential systemic cisplatin and adenovirus-mediated p53 transfer.” J Thorac Cardiovasc Surg 112: 1372-1376. O'Connor PM, Jackman J, Bae I, Myers TG, Fan S, Mutoh M, Scudiero DA, Monks A, Sausville EA, Weinstein JN, Friend S, Fornace AJ Jr, Kohn KW. (1997). “Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents.” Cancer Res 57(19): 4285-300. Oren, M. (1992). “p53: the ultimate tumor suppressor gene?” Faseb J 6(13): 3169-76. Osaki S, Nakanishi Y, Takayama K, Pei XH, Ueno H, Hara N. (2000). “Alteration of drug chemosensitivity caused by the adenovirus-mediated transfer of the wild-type p53 gene in human lung cancer cells.” Cancer Gene Ther 7(2): 300-7. Parker LP, Wolf JK, Price JE. (2000). “Adenoviral-mediated gene therapy with Ad5CMVp53 and Ad5CMVp21 in combination with standard therapies in human breast cancer cell lines.” Ann Clin Lab Sci 30(4): 395-405. Peller, S. (1998). “Clinical implications of p53: effect on prognosis, tumor progression and chemotherapy response.” Semin. Cancer Biol. 8: 379-387. Perdomo JA, Naomoto Y, Haisa M, Fujiwara T, Hamada M, Yasuoka Y, Tanaka N. (1998). “In vivo influence of p53 status on proliferation and chemoradiosensitivity in nonsmall-cell lung cancer.” J Cancer Res Clin Oncol 124(1): 10-8. Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B. (1997). “A model for p53-induced apoptosis.” Nature 389: 300. Righetti SC, Della Torre G, Pilotti S, Menard S, Ottone F, Colnaghi MI, Pierotti MA, Lavarino C, Cornarotti M, Oriana S, Bohm S, Bresciani GL, Spatti G, Zunino F. (1996). “A comparative study of p53 gene mutations, protein accumulation, and response to cisplatin-based chemotherapy in advanced ovarian carcinoma.” Cancer Res 56(4): 689-93. Roth, J. A. (1996). “Modification of tumor-suppressor-gene expression and induction of apoptosis in non-small-cell lung cancer (NSCLC) with an adenovirus vector expressing wildtype p53 and cisplatin.” Hum Gene Ther 7: 1013-1030. Roth JA, Nguyen D, Lawrence DD, Kemp BL, Carrasco CH, Ferson DZ, Hong WK, Komaki R, Lee JJ, Nesbitt JC, Pisters KM, Putnam JB, Schea R, Shin DM, Walsh GL, Dolormente

MM, Han CI, Martin FD, Yen N, Xu K, Stephens LC, McDonnell TJ, Mukhopadhyay T, Cai D. (1996). “Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer.” Nature Medicine 2(9): 985991. Santoso JT, Tang DC, Lane SB, Hung J, Reed DJ, Muller CY, Carbone DP, Lucci JA 3rd, Miller DS, Mathis JM. (1995). “Adenovirus-based p53 gene therapy in ovarian cancer.” Gynecol Oncol 59(2): 171-8. Schornagel JH, Verweij J, de Mulder PH, Cognetti F, Vermorken JB, Cappelaere P, Armand JP, Wildiers J, de Graeff A, Clavel M, et al, (1995). “Randomized phase III trial of adatrexate versus methotrexate in patients with metastatic and/or recurrent squamous cell carcinoma of the head and neck: A European Organization for Research and Treatment of Cancer Head and Neck Cancer Cooperative Group study.” JCO 13: 1649-1655. Schuler M, Herrmann R, De Greve JL, Stewart AK, Gatzemeier U, Stewart DJ et al (2001). “Adenovirus-Mediated WildType p53 Gene Transfer in Patients Receiving Chemotherapy for Advanced Non-Small-Cell Lung Cancer: Results of a Multicenter Phase II Study.” J Clin Oncol 19(6): 1750-1758. Selter, H. and M. Montenarh (1994). “The emerging picture of p53.” Int J Biochem 26: 154-154. Shahin MS, Hughes JH, Sood AK, Buller RE. (2000). “The prognostic significance of p53 tumor suppressor gene alterations in ovarian carcinoma.” Cancer 89(9): 2006-17. Sherr, C. J. (1994). “G1 phase progression: cycling on cue.” Cell 79: 55-62. Stal, O. (1995). “p53 expression and the result of adjuvant therapy of breast cancer.” Acta Oncologica 34: 767-770. Sugrue MM, Shin DY, Lee SW, Aaronson SA. (1997). “Wildtype p53 triggers a rapid senescence program in human tumor cells lacking functional p53.” Proc Natl Acad Sci U S A 94(18): 9648-53. Swisher SG, Roth JA, Nemunaitis J, Lawrence DD, Kemp BL, Carrasco CH, Connors DG, El-Naggar AK, Fossella F, Glisson BS, Hong WK, Khuri FR, Kurie JM, Lee JJ, Lee JS, Mack M, Merritt JA, Nguyen DM, Nesbitt JC, Perez-Soler R, Pisters KM, Putnam JB Jr, Richli WR, Savin M, Waugh MK, et al, (1999). “Adenovirus mediated p53 gene tranfer in advanced non-small-cell lung cancer.” J Natl Cancer Inst 91: 763-771. Trepel M, Groscurth P, Malipiero U, Gulbins E, Dichgans J, Weller M. (1998). “Chemosensitivity of human malignant glioma: modulation by p53 gene transfer.” J Neurooncol 39(1): 19-32. Wolf JK, Mills GB, Bazzet L, Bast RC Jr, Roth JA, Gershenson DM. (1999). “Adenovirus-mediated p53 growth inhibition of ovarian cancer cells is independent of endogenous p53 status.” Gynecol Oncol 75(2): 261-6. von Gruenigen VE, Santoso JT, Coleman RL, Muller CY, Miller DS, Mathis JM. (1998). “In vivo studies of adenovirus-based p53 gene therapy for ovarian cancer.” Gynecol Oncol 69(3): 197-204. Xu GW, Sun ZT, Forrester K, Wang XW, Coursen J, Harris CC. (1996). “Tissue-specific growth suppression and chemosensitivity promotion in human hepatocellular carcinoma cells by retroviral-mediated transfer of the wildtype p53 gene.” Hepatology 24(5): 1264-8. Yang B, Eshleman JR, Berger NA, Markowitz SD. (1996). “Wild-type p53 protein potentiates cytotoxicity of


Gene Therapy and Molecular Biology Vol 7, page 35 therapeutic agents in human colon cancer cells.” Clin Cancer Res 1996: 1649-1657. Knudson CM, Tung KSK, Tourtellotte WG, Brown, GAJ, Korsmeyer SJ. (1997). “Bax suppresses tumorigenesis and stimulates apoptosis in vivo.” Nature 385(6617): 637-40.

Yver, A., J. J. Nemunaitis, et al, (2001). “Does detection of circulating ONYX-015 genome by polymerase chain reaction indicate vector replication.” JCO 19(12): 3155-3157.


Jekunen et al: Strategy of sensitizing tumor cells with adenovirus-p53 transfection


Gene Therapy and Molecular Biology Vol 7, page 37 Gene Ther Mol Biol Vol 7, 37-42, 2003

Antigenicity and immunogenicity of HIV envelope gene expressed in baculovirus expression system Research Article

Alka Arora1, Pradeep Seth2* 1 2

Post Doctoral Fellow, Department of Medical Genetics and Microbiology, University of Toronto, Canada. Professor and Head Department of Microbiology, All India Institute of Medical Sciences, India.

__________________________________________________________________________________ *Correspondence: Dr. Pradeep Seth, Professor and Head, Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India -110029.Tel: 91-11-652 6814; Fax: 91-11-686 2663; E mail: Received: 28 December 2002; Accepted: 5 February 2003; electronically published: July 2003

Summary Human immunodeficiency virus type I (HIV-1) envelope gene was expressed in Spodoptera frugiperda (Sf21) cells. DNA constructs encoding env-tat-rev genes were cloned into the baculovirus expression vector pBacPAK9. Recombinant baculovirus was prepared by cotransfection with linearized wild type virus DNA. Western blotting of cell extracts containing recombinant HIV-1 proteins demonstrated expression of HIV-1 gp160 and its complete cleavage products gp120 and gp41. A time course experiment suggested that the maximum expression was observed at 48-hrs post infection. In order to measure the biological activity recombinant HIV envelope proteins were used for lymphocyte proliferation assay. The results demonstrated that recombinant gp160 and its cleavage products were antigenically and functionally authentic. tend to decrease with progression of clinical symptoms (Lange et al, 1986; Goudsmit et al, 1987). Recombinant antigen based EIAs have been shown to be more sensitive, especially in detecting early seroconverters and specific than peptide or virus lysate based EIAs (Johnson 1992; Galli et al, 1996). The main objective of this study was to obtain large quantities of purified recombinant protein, suitable to be used as an immunogen and for development of HIV-1 detection kit. We used Baculovirus expression vector system for expressing HIV-1 Gp160 as this system results in efficient processing of the protein, posttranslational modifications and is known to give high yields of expressed protein.

I. Introduction HIV genome, like other retroviruses encode for Gag, Pol and Env. In addition, it also encodes for 6 regulatory and accessory proteins Tat, Rev, Nef, Vif, Vpr and Vpu. The major structural protein encoded by env gene of HIV1 consists of a protein of 850-880 amino acids. Extensive glycosylation of this precursor protein results in the production of Gp160 monomers, which then assemble into oligomers for transport from ER to the plasma membrane (Earl et al, 1991). During transport from Golgi, intracellular cleavage of Gp160 yields an outer envelope glycoprotein Gp120 and trans-membrane glycoprotein Gp41 (Kozarsky et al, 1989). Specifically, the HIV viral envelope protein Gp120 is important for virus-receptor interaction and virus entry (Kowalski et al, 1987, Hill et al, 1997). Gp41 is known to play a central role in the envelope glycoprotein oligomerization and fusion function (Poumbourios et al, 1997). HIV infection results in the production of HIV specific antibodies, therefore detection of these antibodies by ELISA and Western blot assay remains the basis of blood donor and patient screening. Serum specimen from HIV infected people regardless of their clinical stage react efficiently with precursor glycoprotein Gp160 or its cleavage product Gp120 and Gp41 (Lange et al, 1986; Goudsmit et al, 1987). Antibodies to gag protein p24 are the earliest protein detectable by Western blot after infection, however, these

II. Materials and Methods A. Plasmids, cells, reagents and peptides pCR-Script SK (+) cloning vector was purchased from Stratagene, LaJolla, CA, USA. pBRU plasmid containing complete genome of BRU strain of HIV-1 cloned in pUC18 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, MD, USA. BacPAK, Baculovirus expression system was purchased from Clontech (BD Biosciences Clontech, Palo Alto, CA). Plasmids were grown in DH5! strains of Escherichia coli (Life Technologies, Gaithesburg, MD, USA), and purified using Wizard miniprep columns (Promega Corp, Madison, WI). TNMFH media for insect cell culture was obtained from HyClone (Genetix, New Delhi, India). TNM-FH medium contains Grace's


Arora and Seth: Antigenicity and immunogenicity of HIV envelope gene medium, lactalbumin hydrolysate and yeast extract. Sf 21 cells were cultured at 27oC in TNM-FH medium supplemented with 10% FBS (TNM-FH/FBS). Vaccinia expressed recombinant gp120 and gp160 (vPE8 and vPE16) were obtained through NIH AIDS Research and Reference Reagent Program.

20µl of 10M-ammonium acetate. Samples were then spotted on to the nitrocellulose membrane by loading on the wells of the dot blot manifold apparatus (Bio Rad Laboratory, Richmond, CA). Vacuum suction was applied to drain off the entire solution. Membrane was dried at room temperature for 5-10 min and then baked for 2 hrs at 80oC. Hybridization was performed using !32P-dCTP labeled envelope probe prepared by random primer labeling using Klenow fragment of DNA polymerase 1 (Amersham Biosciences, Piscataway, NJ). The membrane was then washed and exposed to a Kodak-X film overnight at -70oC.

B. DNA constructs 3kb env-tat-rev gene segment (nt 5352- nt 8354) of HIV-1 subtype B strain, BRU, was PCR amplified using primers API (5352-5390) TTATTCTAGAGAGAAGAGCAAGAAATGGA TCCAGTAGAT and APII (8316-8354) TTTTTGAGCTCTTGCCACCCATTTTAAAGTAAAGACCTT and cloned into pCR-Script (SK+) cloning vector to produce pSBRU-TRE as described earlier (Arora and Seth, 2001). The 3kb HIV-1 env-tat-rev gene segment was released by restriction digestion of pSBRU-TRE with Xba I, Not I and Bgl I. The env, tat and rev gene fragment was then purified from low melting point agarose gel and subcloned into baculovirus transfer vector, pBacPAK9 predigested with Xba I and Not I to generate pBacBRU-TRE. Recombinant clone was screened by colony hybridization followed by restriction enzyme analysis. pCIBRUTRE, mammalian expression vector expressing 3kb HIV-1 envtat-rev gene under the control of Immediate-Early Promoter/Enhancer of CMV, used in this study for immunizing Balb/c mice has been described earlier (Arora and Seth, 2001).

F. In vitro expression A time course experiment was performed to examine the expression of HIV-1 env gene in Sf 21 cells infected with the recombinant virus. Cells were harvested at various time intervals post infection. SDS PAGE, immunofluorescence and Western Blot analysis of cell lysate were conducted to study expression of proteins. SDS-PAGE was performed according to Laemmli. For Western Blot analysis proteins were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane using Trans-blot SD semi-dry electrophoretic transfer Cell (Bio Rad Laboratories) The membrane was treated with non-fat powdered milk in TTBS (Tween 20- Tris buffer Saline) for 1 hr at room temp. and reacted with HIV-1 positive human polyclonal serum (at a dilution of 1:200) in TBS for 1h at room temperature. After washing thrice with TTBS, the membrane was incubated at room temperature for 1 hr. with anti-human IgG conjugated with alkaline phosphatase (1:10,000). Membrane was then washed thrice with TTBS and incubated in the substrate solution (Sigma fast BCIP/NBT tablet dissolved in 10ml of deionized water, Sigma Chemicals Co., St. Louis). For Immunofluorescence, P4 (recombinant baculovirus) infected cells, uninfected cells (control) and AcNPv (wild type virus) infected cells were harvested at different time points and washed thrice with PBS. 1x104 cells were spotted onto the wells of a teflon-coated slide and fixed with acetone: methanol (1:1) at -20oC for 30 min. For staining, cells were allowed to react with HIV-1 positive human polyclonal serum (1:50) for 1h at 37oC. Cells were then washed with PBS and incubated with FITC conjugated anti-human IgG (Sigma) and incubated for 1hr at 37oC. Thereafter, the cells were washed and mounted with glycerol buffer and visualized under fluorescent microscope.

C. Generating a recombinant virus Recombinant virus was prepared as per manufacturer's instructions. Briefly, 35mm tissue culture dishes were seeded with 1x106 Spodoptera frugiperda cells (Sf21) (Vaughn et al, 1977) in 1.5 ml of complete TNM-FH/FBS medium and incubated overnight at 27oC in a humid chamber. 500ng of plasmid pBacBRU-TRE DNA, along with Bsu 361 digested BacPAK6 viral DNA was mixed with 5µg of lipofectin and incubated at room temperature for 15 min. Culture medium in the tissue culture dishes containing Sf21 cells was replaced with 1.5 ml of serum free TNM-FH. Lipofectin-DNA complex was then gently added to Sf21 cells. Plates were incubated at 27oC for 5 hrs. Thereafter, serum free TNM-FH medium was replaced with TNM-FH/FBS medium and the plates were returned for incubation at 27oC for 4 days.

G. T cell proliferation assay

D. Isolation of recombinant virus


H thymidine uptake assay was used to measure the proliferation of splenocytes after antigenic stimulation. Balb/c mice were immunized intramuscularly with pCIBRU-TRE or pCI (control vector) DNA as described earlier (Arora and Seth, 2001). Six groups of Balb/c mice were taken (each group comprising 5 mice) (Table 1). In-group D3 three doses of 100 µg DNA each were given at bi-weekly intervals. In D0P2 group animals were immunized with 2 doses of P4 with no DNA priming. In-group D3P2 animals were immunized with 3 doses of pCIBRU-TRE DNA followed by 2 doses of P4. Group D3V2 consisted of mice immunized with 3 doses of pCIBRU-TRE followed by 2 doses of recombinant vaccinia virus expressed gp120 and gp160 (vPE8 and vPE16). D0V2 group consisted of mice immunized with 2 doses of vPE8 and vPE16 with no priming with DNA construct and control group. Stimulating antigens included vaccinia expressed recombinant gp160/gp120 (vPE16/ vPE8) and baculovirus expressed gp160 (P4). Splenocytes from various groups of mice were harvested and resuspended at a concentration of 2x106 cells/ml in RPMI 1640 medium supplemented with 10% FCS. Cells were stimulated in

Plaque assay was performed using co-transfection supernatant to generate a pure clone of recombinant virus. 1x106 Sf21 cells were seeded in 35mm tissue culture dishes and incubated overnight at 27oC. These cells were then infected with 100µl of neat or 10-1 dilution of co-transfection supernatant. One hour later, the virus inoculum was removed and infected cells were overlaid with 1.5ml of agarose (1.5% in TNM-FH/FBS). After agarose was set 1.5 ml of TNM-FH/FBS medium was added to each dish and incubated for 4 days at 27oC. Plaques were stained with .03% of neutral red solution. 4 plaques were picked up and transferred into an eppendorf tube containing 500µl of TNM-FH/FBS and stored at 4oC overnight.

E. Virus propagation and evaluation The plaque picks were used as a source of virus to infect cells in a 96 well plate. Infections were performed in duplicate. Cells were harvested 4 days following infection and cell lysate was used to perform dot blot analysis to detect the recombinant virus. Each sample was suspended in 200µl of 0.5N NaOH and


Gene Therapy and Molecular Biology Vol 7, page 39 used as control to study the non due to wild type vaccinia/vero baculovirus/Sf21 cell protein in index was calculated by

triplicate. Five Âľg/ml of vPE16/vPE8 infected vero cell lysates/ P4 infected Sf21 cell lysates was used in cell proliferation assay. Lysates of wild type vaccinia virus (WR) infected Vero cells/ wild type baculovirus (AcNPv) infected Sf21 cell lysate was

SI =

specific 3H-thymidine uptake cell proteins or wild type the cell lysates. Stimulation the following formula.

Mean cpm of 3H thymidine incorporated in the presence of stimulating antigen (vPE 16, vPES or P4) Mean cpm of 3H thymidine incorporated in wild type virus (VacWR or AcNPv) control

allows its insertion into the genome of the wild type virus. The BacPAK6 DNA is missing an essential portion of the baculovirus genome, ORF1629, that is essential for viral replication (Possee et al, 1991) When the DNA recombines with the vector (the transfer vector carries the missing ORF1629 sequence), the essential element is restored and the target gene is transferred to the baculovirus genome. Recombinant viruses were collected and selected by plaque purification. Recombinant phenotype of the plaques is verified by Dot-Blot analysis. Two of the plaques were found to be positive by Dot-Blot analysis and were termed as P4 and P5 (Figure 3). Plaque P4 gave the stronger signal and was therefore amplified and used for further infections.

II. Results A. Generating a Recombinant Baculovirus: Complete HIV-1 envelope glycoprotein along with the regulatory protein Tat and Rev were PCR amplified from subtype B, BRU strain of HIV-1 and cloned into pBacPAK9, baculovirus transfer vector, downstream to the baculovirus polyhedrin gene promoter (Figure 1). Recombinant baculovirus transfer vector was screened by colony hybridization followed by restriction enzyme analysis and was termed as pBacBRU-TRE (Figure 2). Following co-transfection, recombinant baculovirus was formed by the homologous recombination between pBacBRU-TRE and Bsu361 digested BacPAK6 viral DNA in the region flanking the chimeric gene, which

Figure 2 a) Autoradiograph showing recombinant colonies as detected by colony hybridization, b) Restriction enzyme analysis of the recombinant plasmid pBacBRU-TRE with different enzymes. Lanes M: Lambda DNA digested with Hind III enzyme. Positions of the molecular weight markers are indicated, 1: uncut; 2: pBacBRU-TRE digested with Bam H1; 3: pBacBRU-TRE digested with Hind III; 4: pBacBRU-TRE digested with Pvu II

Figure 1. a) pBacPAK9 baculovirus transfer vector, b) Recombinant plasmid pBacBRU-TRE. HIV-1 env, tat and rev gene released on digestion of pSBRU-TRE was gel purified and subcloned into baculovirus transfer vector pBacPAK9 predigested with restriction enzymes Xba 1 and Not 1.


Arora and Seth: Antigenicity and immunogenicity of HIV envelope gene

Figure 3. Autoradiograph showing dot blot analysis of cell lysates from plaque picks infected Sf21 cells. 2 plaques labeled as P4 and P5 were found to be positive. Cells infected with wild type baculovirus AcNPv served as the negative control. pBRU plasmid DNA served as the positive control.

Figure 4. The photograph showing Immunofluorescence microscopy of the recombinant baculovirus infected Sf21 cells at 48h-post infection. HIV-1 positive human polyclonal serum served as the source of primary antibody.

B. Expression of HIV-1 Envelope glycoprotein by Recombinant Baculovirus Expression of gp160 in Sf21 cells was examined by indirect immunofluorescence and western blot analysis of infected cells using HIV-1 positive human polyclonal sera. A 3+ fluorescence was observed at 48-hrs post infection on a scale of 0 to 4+ that is from no fluorescence to intense fluorescence (Figure 4). These results were supported by western blot analysis of the infected cells at 48hrs-post infection. Gp160 and its cleavage products, Gp120 and Gp41, could be detected after immunostaining. Since the total carbohydrate load added to the insect cell expressed glycoprotein is marginally less than that added during secretion from a mammalian cell, the baculovirus expressed glycoprotein are correspondingly smaller (105 kDa) than their mammalian counterparts (120 kDa) No corresponding protein bands were detected on from wild type baculovirus (AcNPv) infected cells and uninfected cells (Figure 5).

Figure 5. Western blot analysis of recombinant baculovirus expressed gp160. Lanes M: protein high range molecular weight marker; 1: uninfected cell lysate; 2: cell lysate from AcNPv infected cells; 3 & 4: cell lysate from recombinant baculovirus infected cells.

C. Lymphocyte Proliferation Assay In vitro T cell proliferative activity of splenocytes from animals immunized with DNA vaccine pCIBRU-TRE alone (group D3), boosted with P4 or vPE8/vPE16 (groups D3P2, D3V2) or P4 and vPE8/vPE16 alone (groups D0P2, D0V2) was studied. (Table 1). Splenocytes from all the animal groups showed positive proliferative response on in vitro stimulation (Figure 6). Splenocytes from group D0P2 mice demonstrated proliferation in response to P4 cell lysate (SI-8.16), as well as to vPE8 and vPE16 antigens (SI of 4 and 5.6). Splenocytes from DNA vaccine immunized mice group D3 and D3P2 proliferated with SI of 8.8 on stimulation with vPE8 and with SI of 3.8 and 4.4 respectively on stimulation with P4. Splenocytes from mice immunized with 2 doses of vaccinia expressed recombinant Gp120/Gp160 with no DNA priming (Group D0V2) showed better proliferation with vPE8, as compared with vPE16 and P4. However, splenocytes from mice immunized with 3 doses of DNA followed by 2 doses of vaccinia expressed recombinant Gp120/Gp160 (Group D3V2) gave almost equal proliferation with P4, vPE8 and vPE16 respectively (Figure 6).

Figure 6. In vitro T cell proliferative response to P4, vPE8 & vPE16 (recombinant baculovirus expressed gp160) of splenocytes from Balb/c mice immunized with pCIBRU-TRE (3 doses at biweekly intervals) and boosted with 2 doses of either recombinant baculovirus (P4) or recombinant vaccinia virus (vPE8 & vPE16). These groups of mice were marked as D3P2 or D3V2 respectively. Animals from groups D0P2 and D0V2 were injected only with recombinant baculovirus or recombinant vaccinia virus (no DNA priming).


Gene Therapy and Molecular Biology Vol 7, page 41 Table 1. Different groups of mice primed with pCIBRUTRE DNA and boosted with baculovirus expressed (P4) or vaccinia expressed (vPE8 and vPE16) recombinant gp160. Group



3 doses

D0P2 D3P2

vPE8 and vPE16

Acknowledgments 2 doses

3 doses

D0V2 D3V2


baculovirus expressed envelope protein was also demonstrated by lymphocyte proliferation assays. Largescale protein purification is being pursued for further studies.

The Department of Biotechnology, Ministry of Science and Technology, Government of India has provided financial support for this research. Ms Alka Arora received Research Fellowship from CSIR during this study.

2 doses 2 doses

3 doses

2 doses

IV. Discussion


The main objective of this study was to prepare large amounts of HIV-1 envelope protein, which may be used as a source of antigen for studying immune response against HIV-1. HIV-1 gp160 with its signal sequence along with the regulatory genes tat and rev was used to produce recombinant baculovirus (Malim et al, 1989; Ruben et al, 1989 Rosen and Pavlakis; 1990, Roy et al, 1990). This system has several advantages over other systems including high level of protein production and posttranslational modification, which cannot be achieved in bacterial system (Luckow and Summers 1988, 1989). We observed poor expression of envelope proteins following infection of Sf21 cells as no protein was observed after SDS-PAGE of the P4 infected Sf21 cell lysate followed by coommassie blue staining. Several other studies have indicated that env protein is refractory to efficient recombinant expression (Lasky et al, 1986 Hu et al, 1987; Hu et al, 1987). Replacement of the signal sequence of the HIV-1 envelope protein with those of herpes simplex virus glycoprotein or human tPA results in efficient expression (Lasky et al, 1986; Berman et al, 1988). These studies therefore suggest that the signal sequence of HIV-1 envelope gene, which consists of 5 positively charged amino acids, may be responsible for the poor expression. Li et al, (1994), showed that substitution of the gp120 natural signal sequences with the signal sequences from honeybee mellitin or murine interleukin 3 promotes a high level of expression of a glycosylated form of gp120 and efficient secretion. These heterologous signal sequences contain one (mellitin) or no (IL-3) positively charged amino acids. These workers also demonstrated that on stepwise substitution of positively charged amino acids with neutral amino acids resulted in enhanced expression of HIV-1 gp120. Similarly, Golden et al, 1998, compared three different signal sequences [human tissue plasminogen activator (tPA), human placental alkaline phosphatase (pap), or baculovirus envelope glycoprotein (gp67)] and found that the tPA leader yielded the highest level of secreted protein, followed by the gp67 and pap sequences. In this study, however, HIV-1 gp160 and its complete cleavage products were observed on Western Blot analysis using HIV-1 positive human polyclonal sera. Suggesting thereby that the envelope protein retained its antigenicity and may be used as a source of antigen for Western Blot analysis. Immunogenicity as well as antigenicity of this

Arora A and Seth P (2001). Immunization with HIV-1 Subtype B gp160-DNA Induces Specific as well as cross-reactive Immune Responses in Mice. Indian J Med Res 114, 1-9. Berman PW, Nunes WM and Haffar OK (1988) Expression of membrane-associated and secreted variants of gp160 of human immunodeficiency virus type 1 in vitro and in continuous cell lines. J Virol 62, 3135-42. Earl PL, Moss B and Doms RW (1991) Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type 1 envelope protein. J Virol 65, 2047-55. Galli RA, Castriciano S, Fearon M, Major C, Choi KW, Mahony J and Chernesky M (1996) Performance Characteristics of Recombinant Enzyme Immunoassay To Detect Antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-2 and To Measure Early Antibody Responses in Seroconverting Patients. J Clin Microbiol 34, 999–1002. Golden A, Austen DA, van Schravendijk MR, Sullivan BJ, Kawasaki ES, Osburne MS (1998) Effect of promoters and signal sequences on the production of secreted HIV-1 gp120 protein in the baculovirus system. Protein Expr Purif 14, 812. Goudsmit J, Lange JMA, Paul DA, Dawson GJ (1987) Antigenemia and antibody titers to core and envelope antigens in AIDS, AIDS-related complex, and subclinical human immunodeficiency virus infection. J Infect Dis 155, 558-60. Hill CM, Deng H, Unutmaz D, Kewalramani VN, Bastiani L, Gorny MK, Zolla-Pazner S, Littman DR (1997) Envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus can use human CCR5 as a co-receptor for viral entry and make direct CD4dependent interactions with this chemokine receptor. J Virol 71, 6296-304. Hu SI, Kosowski SG and Schaaf KF (1987) Expression of envelope glycoproteins of human immunodeficiency virus by an insect virus vector. J Virol 61, 3617-20. Johnson JE (1992) Detection of human immunodeficiency virus type 1 antibody by using commercially available whole-cell viral lysate, synthetic peptide, and recombinant protein enzyme immunoassay systems. J Clin Microbiol 30, 216–218. Kozarsky K, Penman M, Basiripour L, Haseltine W, Sodroski J and Krieger M (1989) Glycosylation and processing of the human immunodeficiency virus type 1 envelope protein. J Acquir Immune Defic Syndr 2, 163-9. Kowalski M, Potz J, Basiripour L, Dorfman T, Goh WC, Terwilliger E, Dayton A, Rosen C, Haseltine W, Sodroski J


Arora and Seth: Antigenicity and immunogenicity of HIV envelope gene (1987) Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science 237, 1351-5. Lange JM, Paul DA, Huisman HG, de Wolf F, van den Berg H, Coutinho RA, Danner SA, van der Noordaa J, Goudsmit J (1986) Persistent HIV antigenemia and decline of HIV core antibodies associated with transition to AIDS. Brit Med J 293, 1459-62. Lasky LA, Groopman JE, Fennie CW, Benz PM, Capon DJ, Dowbenko DJ, Nakamura GR, Nunes WM, Renz ME, Berman PW (1986) Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein. Science 233, 209-12. Li Y, Luo L, Thomas DY, Kang CY (1994) Control of expression, glycosylation, and secretion of HIV-1 gp120 by homologous and heterologous signal sequences Virology 204, 266-78. Luckow VA and Summers MD (1988) Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors. Virology 167, 56-71 Luckow VA and Summers MD (1989) High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors. Virology 170, 31-9. Malim MH, Hauber J, Le SY, Maizel JV and Cullen BR (1989) The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338, 254-257. Possee RD, Sun TP, Howard SC, Ayres MD, Hill-Perkins M, Gearing KL (1991) Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9.4 kbp EcoRI-I and -R (polyhedrin gene) region. Virology. 185, 229-41. Poumbourios P, Wilson KA, Center RJ, El Ahmar W and Kemp BE (1997) Human immunodeficiency virus type 1 envelope glycoprotein oligomerization requires the gp41 amphipathic

alpha-helical/leucine zipper-like sequence. J Virol 71, 20419. Rosen CA and Pavlakis GN (1990) Tat and Rev: positive regulators of HIV gene expression. AIDS 4, A51 Roy S, Delling U, Chen CH, Rosen CA, Sonenberg N (1990) A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation. Genes Dev 4, 1365-1373. Ruben S, Perkins A, Purcell R, Joung K, Sia R, Burghoff R, Haseltine WA, Rosen CA (1989) Structural and functional characterization of human immunodeficiency virus tat protein. J Virol 63, 1-8. Vaughn JL, Goodwin RH, Tompkins GJ, McCawley P (1977) The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae).In Vitro. 13, 213-7.

Pradeep Seth


Gene Therapy and Molecular Biology Vol 7, page 43 Gene Ther Mol Biol Vol 7, 43-59, 2003

Characterization of genes transcribed in an Ixodes scapularis cell line that were identified by expression library immunization and analysis of expressed sequence tags Research Article

Consuelo Almazán, Katherine M. Kocan, Douglas K. Bergman, Jose C. GarciaGarcia, Edmour F. Blouin and José de la Fuente* Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.

__________________________________________________________________________________ *Correspondence: José de la Fuente, Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078; Phone: (405) 744-0372; Fax: (405) 744-5275; e-mail: Key words: tick, vaccine, tick cell culture, cDNA library immunization, EST, expression library immunization Received: 23 May 2003; Accepted: 06 June 2003; electronically published: June 2003

Summary Expression library immunization (ELI) combined with analysis of expressed sequence tags (ESTs) were used to identify genes transcribed in a cell line (IDE8) that was originally derived from embryos of Ixodes scapularis. A cDNA expression library was constructed from the IDE8 cells and cDNA clones were screened by ELI. Mice injected with cDNA clones were then infested with I. scapularis larvae. cDNA clones affecting larval feeding or development were subjected to single pass 5’ sequence analysis and the non-redundant sequences were putatively identified by sequence identity using the protein Basic Local Alignment Search Tool (BLAST) algorithm. Sequences of the clones were grouped according to the predicted function of the encoded proteins. 351 cDNAs that affected larval feeding and/or development were identified, of which 316 cDNA clones contained non-redundant sequences and 101 produced a significant identity to sequences reported previously. Gene ontologies could be assigned to 87 clones. Vaccination of mice with plasmid DNA followed by tick infestation resulted in identification of cDNA clones that inhibited tick infestation or promoted tick feeding. cDNAs that inhibited tick infestation were identical to nucleotidase, heat shock proteins, beta-adaptin, chloride channel, ribosomal proteins, and proteins with unknown function. cDNA clones that promoted tick feeding were identical to beta-amyloid precursor, block of proliferation, mannose-binding lectin, RNA polymerase III, ATPases and a protein of unknown function. Herein, we describe the sequence analysis of I. scapularis ESTs selected by ELI that affected larval tick feeding and/or development. These proteins may be useful for incorporation into vaccine preparations designed to interrupt the life cycle of I. scapularis and/or interfere with transmission of pathogens. garinii, Rickettsia helvetica, R. japonica and R. australis, Babesia divergens, as well as tick-borne encephalitis (TBE) and Omsk Hemorrhagic fever viruses (EstradaPeña and Jongejan, 1999; Parola and Raoult, 2001). Throughout eastern and southeastern United States and Canada, I. scapularis (the black legged tick) is the main vector of B. burgdorferi sensu stricto and A. phagocytophilum (Estrada-Peña and Jongejan, 1999; Parola and Raoult, 2001). Control of tick infestations is difficult, particularly for multi-host ticks such as Ixodes spp. Presently, tick

I. Introduction Ticks are ectoparasites of wild and domestic animals and humans, and are considered to be the most important vector of pathogens in North America (Parola and Raoult, 2001). Ixodes spp. (Acari: Ixodidae) are distributed worldwide and are vectors of human pathogens, including Borrelia burgdorferi (Lyme disease), Anaplasma phagocytophilum (human granulocytic ehrlichiosis), Coxiella burnetti (Q fever), Francisella tularensis (tularemia), B. afzelii, B. lusitaniae, B. valaisiana and B.


Almazán et al: Expressed sequence tags in Ixodes scapularis control is effected by integrated pest management in which different control methods are adapted in a geographic area against one tick species with due consideration to their environmental effects. Recently, development of vaccines against one-host Boophilus spp. has provided new possibilities for identification of protective antigens for use in vaccines for control of tick infestations (Willadsen, 1997; Willadsen and Jongejan, 1999; de la Fuente et al, 1999, 2000a; de Vos et al, 2001). Control of ticks by vaccination would avoid environmental contamination and selection of drug resistant ticks that can result from repeated acaricide application (de la Fuente et al, 1998; Garcia-Garcia et al, 1999). Anti-tick vaccines also allow for inclusion of multiple antigens in order to target a broad range of tick species, as well as pathogenblocking antigens. Development of high throughput DNA sequencing technologies and bioinformatic tools facilitate assignment of provisional function to expressed sequence tags (ESTs; Boguski et al, 1993). This approach has resulted in valuable information for the study of biological systems and for the identification of potential vaccine candidates (Lizotte-Waniewski et al, 2000; Knox et al, 2001; Tarleton and Kissinger, 2001; Touloukian et al, 2001; Kessler et al, 2002). In ticks, construction of EST databases has been reported for B. microplus (Crampton et al, 1998), Amblyomma americanum (Hill and Gutierrez, 2000) and A. variegatum (Nene et al, 2002). The application of EST technology has been used for characterization of gene expression in salivary glands of I. scapularis (Valenzuela et al, 2002), I. ricinus (Valenzuela, 2002), A. americanum and Dermacentor andersoni (Bior et al, 2002), for identification of genes differentially expressed in D. variabilis ovaries in response to rickettsial infection (Mulenga et al, 2003) and in I. ricinus salivary glands in response to blood feeding (Leboulle et al, 2002). A new technique, expression library immunization (ELI), in combination with sequence analysis of ESTs, provides an alternative approach for identification of potential vaccine antigens that is based on rapid screening of the expressed genes without prior knowledge of the antigens encoded by the cDNAs. ELI was first reported for Mycoplasma pulmonis (Barry et al, 1995) and since then has been used for unicellular and multicellular pathogens and viruses (Manoutcharian et al, 1998; Alberti et al, 1998; Brayton et al, 1998; Melby et al, 2000; Smooker et al, 2000; Moore et al, 2001; Singh et al, 2002; Leclercq et al, 2003). Recently, we reported the first application of ELI to arthropods, specifically to I. scapularis (Almazán et al, 2003) in a mouse model system. A combination of cDNA ELI and EST analysis resulted in the selection of 351 cDNA clones affecting tick larval development (Almazán et al, 2003). After grouping the clones according to the putative function of predicted proteins, some cDNA pools resulted in the inhibition of tick infestation and others promoted tick feeding after ELI (Almazán et al, 2003). Herein we describe the sequence analysis and characterization of I. scapularis ESTs that were identified

by Almazán et al. (2003) using cDNA ELI and a mouse model for tick infestation.

II. Materials and methods A. Construction of the I. scapularis expression cDNA library The cDNA library was constructed from I. scapularis cultured embryonic IDE8 cells (Munderloh et al, 1994) as reported previously (Almazán et al, 2003). The expression library was constructed in the vector pEXP1 containing the strong human cytomegalovirus major immediate early promoter/enhancer (CMVIE) (Clontech, Palo Alto, CA). The cDNA library contained 4.4 x 106 independent clones and a titer of approximately 1010 cfu/ml with more than 93% of the clones with cDNA inserts. The average cDNA size was 1.7 kb (0.5-4.0 kb).

B. DNA vaccination and tick infestation Vaccinations with plasmid DNA and tick infestations were done as reported previously for the screening of the expression cDNA library by ELI using the mouse model of I. scapualris infestations (Almazán et al, 2003). Briefly, plasmid DNA was purified (Wizard SV 96 plasmid DNA purification system, Promega, Madison, WI) and used to inject CD-1 female mice, 56 weeks of age at the time of first vaccination. Mice were cared for in accordance with standards specified in the Guide for Care and Use of Laboratory Animals. Mice were injected using a 1 ml tuberculin syringe and a 27!G needle at days 0 and 14. Three to 6 mice per group were each immunized IM in the thigh with 1 µg total DNA/dose in 50 µl PBS. Control mice were injected with 1 µg vector DNA alone. Two weeks after the last immunization, mice were infested with 100 I. scapularis larvae per mouse. For tick infestations, mice were retrained in a small wire cage in a cardboard carton. One hundred larvae were counted and applied to the mice with a brush. Ticks were reared at the Oklahoma State University Tick Rearing Facility by feeding larvae on mice, nymphs on rabbits and adults on sheep. For these experiments, larvae were obtained from the eggs oviposited by sister females. Twelve hours after tick infestation, larvae in the bottom of the cage that did not attach were counted in order to calculate the number of attached larvae per mouse. Mice were then transferred to individual cages in which they were placed on an elevated 1/4” mesh wire platform over water (1/2” deep). Replete larvae dropping from each mouse were collected daily from the water and counted during 7 days. Time for larval development was evaluated from the day of tick infestation to the day in which the maximum number of replete larvae was collected. The inhibition of tick infestation (I) for each test group was calculated with respect to vector-immunized controls as [1-(RLn/RLc x RLic/RLin)] x 100, where RLn is the average number of replete larvae recovered per mouse for each test group, RLc is the average number of replete larvae recovered per mouse for control group, RLic is the average number of larvae attached per mouse for control group, and RLin is the average number of larvae attached per mouse for each test group. Engorged larvae were held in a 95% humidity chamber and allowed to molt. Molting of engorged larvae was evaluated 34 days after the last larval collection by visual examination of ticks under a dissecting light microscope. The inhibition of molting (M) for each test group was calculated with respect to controls as [1-(MLn/MLc x RLc/RLn)] x 100, where MLn is the average number of nymphs for each test group, MLc is the average number of nymphs for the control group, RLc is the average number of larvae recovered for the control group, and RLn is the average number of larvae recovered for each test group.


Gene Therapy and Molecular Biology Vol 7, page 45 constructed based on a sequence distance method utilizing the Neighbor Joining algorithm of Saitou and Nei (1987). BLAST (Altschul et al, 1990) was used to search the NCBI databases to identify previously reported sequences with identity to those that we sequenced. Gene ontology assignments were made according to Ashburner et al. (2000) for non-redundant EST sequence data with the help of GoFish v.1.0 (Berriz et al, 2003).

C. Plasmid DNA preparation and sequencing Bacterial colonies were inoculated in Luria-Bertani with 50 µg/ml ampicillin, grown for 16 hr in a 96-well plate and plasmid DNA purified (Wizard SV 96 plasmid DNA purification system, Promega, Madison, WI) and partially sequenced with a 5’ vectorspecific primer (5’-CGACTCACTATAGGGAG-3’) at the Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University, using ABI Prism dye terminator cycle sequencing protocols developed by Applied Biosystems (Perkin-Elmer Corp., Foster City, CA). In most cases a sequence larger than 700 nucleotides was obtained.

III. Results The screening of the I. scapularis expression cDNA library by ELI and EST analysis resulted in 351 cDNAs affecting larval development in the mouse model of tick infestation (Almazán et al, 2003). Of them, 316 cDNA clones contained non-redundant sequences and 101 (32%) produced a significant identity to previously reported sequences by BLAST analysis of NCBI nucleotide and protein databases (Table 1). Gene ontologies could be assigned to 87 clones (27.5% of non-redundant sequences and 86.1% of clones with identity to sequences reported previously) (Table 2).

D. Data analysis Nucleotide sequences were analyzed using the program AlignX (Vector NTI Suite V 5.5, InforMax, North Bethesda, MD). Multiple sequence alignment was performed using an engine based on the Clustal W algorithm (Thompson et al, 1994). Nucleotides were coded as unordered, discrete characters with five possible character-states; A, C, G, T, or N (missing) and gaps were coded as missing data. Phylogenetic trees were

Table 1. cDNA clones with identity to previously reported sequences. EST clone

Predicted protein

GenBank accession number


V-ATPase E subunit



Na+/K+ ATPase, alpha subunit



NADH dehydrogenase



NADH dehydrogenase subunit 5 (nad5)



Aldehyde dehydrogenase



Translation initiation factor 5A (eIF5A)



Translation initiation factor 5C (eIF-5C)



Initiate factor 5 (if5)



Virilizer (vir)






Elongation factor 2



Elongation factor 1alpha







Nucleotide binding protein 1 (Nubp1)



Identity to D. melanogaster GH03607 full length cDNA coding for a putative membrane protein



Ribosomal protein S4 (RpS4)


Ribosomal protein S11 (RpS11)



Laminin receptor 1 (ribosomal protein SA)



Ribosomal protein L3 (RpL3)



Ribosomal protein L7A (RpL7A)



Putative membrane protein



Ribosomal protein S8 (RpS8)



Sterol carrier protein



Ribosomal protein L27A (RpL27A)



Cyclin C (CycC)



Alpha tubulin



QM homolog (DQM) ribosomal protein



Beta tubulin



Proteasome/Signalosome subunit



Notchless (Nle)



Proteasome subunit



Export factor binding protein 2 (Refbp2)



Proteasome subunit



G protein-coupled receptor



Ribophorin I



Ubiquitin-conjugating enzyme


Succinate dehydrogenase B (SdhB)


1B12 1D10




V-ATPase D subunit Contains microsatellite sequence


Beta-amyloid precursor protein (APP)





V-ATPase C subunit


Fructose-1,6-bisphosphatase (fbp gene)


DNA repair protein Rad1 (Rad1)



Identity to S. pombe dim1+, helicase protein 1


1B2 EST clone

Predicted protein

GenBank accession number


Almazรกn et al: Expressed sequence tags in Ixodes scapularis 2E8




Disulfide isomerase



Identity to AvGI TC255 (A. variegatum) & hypothetical protein FLJ12475 (H. sapiens)



Fumarate hydratase



Rab3D (member of the Ras superfamily of small GTPases)


Transmembrane G-proteinresponsive adenylyl cyclase



Chloride channel



Lysyl-tRNA synthetase



Solute carrier protein



Sodium- and chloride-dependent taurine transporter









RNA polymerase III






















Microtubule-associated protein, RP/EB family



Myosin II regulatory light chain



Unknown Zinc finger like protein



Mannose binding lectin (rhea)



Clathrin heavy chain (Chc)



Identity to M. musculus adult male testis cDNA



Identity to D. melanogaster Pelement somatic inhibitor (Psi)









NAD-dependent malate dehydrogenase



Cytochrome c oxidase I (COI)



Cytochrome c oxidase II (COII)




Cytochrome c oxidase III (COIII)


Identity to D. melanogaster BM40 extracellular basement membrane protein



Cytochrome b (cytb)



16S ribosomal RNA



Identity to D. melanogaster regulator of gene transcription (Chi)





Identity to D. melanogaster homeoprotein phtf


Unknown Identity to I. scapularis clone AC22 microsatellite sequence (AF331735)




Amino acid transporter system A (ATA2)


Unknown Contains microsatellite sequence







Unknown Contains a microsatellite sequence


4B2 4C9

Identity to D. melanogaster transducin (G protein)-like enhancer of split 3, homolog of E(spl)



Unknown Contains microsatellite sequence





Intracellular receptor of activated protein kinase C1 (Rack1)


Unknown Contains microsatellite sequence


Identity to D. melanogaster CG10395 cDNA



Identity to D. melanogaster LD23959 cDNA



Identity to D. melanogaster CG13597 cDNA



Identity to H. sapiens hypothetical protein FLJ10342



Pre-mRNA splicing factor



Receptor signaling protein serine/threonine kinase






Block of proliferation 1 (Bop1)



Identity to H. sapiens hypothetical protein MGC2404



LRP/alpha-2-macroglobulin receptor


NR, Not reported to the EST database for being identical to mitochondrial sequences The majority of clones with gene ontology assigned corresponded to non-nuclear gene products involved in cell growth and maintenance, including genes with ligand binding, carrier or enzymatic activities (Table 2). Seventeen clones contained sequences corresponding to tick mitochondrion and were not submitted to the EST database. Other clones such as 2A9 and 1D6, although probably coding for mitochondrial proteins, were analyzed and submitted to the EST database. Interestingly, 11 clones encoded gene products localized in the cell nucleus (Table 2). The average G + C content of the EST dataset (47,503 bases excluding the poly-A tails with 171 (0.4%) undetermined nucleotide positions) was 54%, but some sequences, such as clone 2A9 which probably codes for a mitochondrial protein, had only a 25% G + C content. 46

Gene Therapy and Molecular Biology Vol 7, page 47 Some short ESTs in clones 1D1 and 2D5 contained a long stretch of T. Vaccination of mice with plasmid DNA followed by tick infestation resulted in some cDNA clones that had an inhibitory effect on tick infestations, while others appeared to promote tick feeding (Table 3 ). The cDNAs inhibiting tick infestation were identical to nucleotidase, heat shock proteins, beta-adaptin, chloride channel, ribosomal proteins and proteins with unknown function. cDNA clones identical to beta-amyloid precursor, block of proliferation, mannose-binding lectin, RNA polymerase III, ATPases and a protein of unknown function enhanced tick feeding. Further characterization of cDNAs that affected larval development (Table 3) was conducted for all clones except for 4D8, 4F8, 4D6 and 4E6, which produced high inhibition of tick infestation and are currently being studied separately as recombinant proteins expressed in Escherichia coli. The pool of heat shock proteins hsp70 and hsp60 cDNAs conferred partial protection against tick

infestations and did not affect molting (Table 3). The cDNA sequences for hsp70 and hsp60 in clones 1C10 and 3F6, respectively, were partial and contained the region coding for the C-terminal of the protein, and were highly identical to other hsp70 sequences (data not shown). The sequence of hsp70 contained a 3’ untranslated region (UTR) of 299 bp before the poly-A tail. The clone 3E1 contained a cDNA identical to the beta-adaptin that produced a 27% inhibition of tick infestation and a 5% inhibition of molting to the nymphal stage after vaccination and tick challenge (Table 3). The complete sequence was determined for the clone 3E1 (Figure 1A), and contained an insert of 1,942 bp encoding for a predicted protein of 191 amino acids. The sequence of this protein was shorter than that for other beta-adaptins (Figure 1B), suggesting that it could encode for a betaadaptin appendage or it may be a partial cDNA sequence because of a long 3’ UTR of 1,334 bp located before the poly-A tail.

Table 2. I. scapularis gene ontology assignments. Category

Number of clones

% of 87 clones with gene ontology assignments

% of 101 clones with identity to reported sequences





Cellular component Mitochondria




Cell membrane




















Biological process Cell growth or maintenance




Physiological process




Developmental process




Cell communication








Molecular function Ligand binding or carrier
















Structural molecule








Gene ontology assignments were made according to Ashburner et al. (2000) for non-redundant EST sequence data with the help of GoFish v.1.0 (Berriz et al, 2003). The number of clone sequences falling into each category are listed and then calculated as a percent of clones for which gene ontology was assigned and the total number of clones for which identity was found to previously published sequences.


Almazán et al: Expressed sequence tags in Ixodes scapularis Table 3. Summary of results of DNA vaccination and challenge with I. scapularis larvae in the mouse model of tick infestations. EST cDNA clone

Predicted protein

Inhibition of tick infestation I (%)

Inhibition of molting M (%)


Identity to H. sapiens hypothetical protein FLJ10342 with unknown function

40 a




50 a

17 a

1C10 b


17 a






Identity to D. melanogaster CG10395 cDNA with unknown function




Identity to D. melanogaster CG13597 cDNA with unknown function








Chloride channel



17 clones b

Ribosomal proteins

15 a



Beta-amyloid precursor protein (APP)


c c


Block of proliferation Bop1



Mannose binding lectin

-48 a, c



RNA polymerase III


2F9 b

Identity to A. variegatum AvGI TC255 & Homo sapiens hypothetical protein FLJ12475 with unknown functions

1A9, 1B2, 4A4 b


a, c

-57 a, c




Data reported by Almazán et al. (2003). For all other experiments, mice were immunized with cDNA-containing expression plasmid DNA as described above. I and M were calculated as described in Materials and Methods section. ND, not determined. b Pooled together for vaccination experiments by ELI (Almazán et al, 2003) (1C10 and 3F6, cDNA pool “Heat shock”; 3C12 and 2F9, cDNA pool “Secreted protein”; ribosomal clones, cDNA pool “Ribosomal”; 1A9, 1B2 and 4A4, cDNA pool “ATPase”). c Resulted in enhanced tick feeding after mouse vaccination and tick challenge.

identical to fly and mosquito sequences (Figure 3). Vaccination with this cDNA resulted in 8% enhancement of larval feeding (Table 3). Vaccination with cDNA clone 4F1 resulted in enhanced larval feeding (Table 3). The complete sequence of clone 4F1 cDNA was determined and contained an insert of 2,475 bp with 30 bp and 66 bp of 5’ and 3’ UTR, respectively and a poly-A tail of 114 bases. An open reading frame of 2,265 bp encoded for a protein of 754 amino acids that was identical to mouse block of proliferation (Bop 1) (Figure 4). Similar proteins have been identified in other organisms including Drosophila melanogaster, Anopheles gambiae and humans (Figure 4), suggesting that this protein has been highly conserved during evolution. The clone 3E10 had a pronounced stimulatory effect on larval feeding (Table 3). This clone was completely sequenced and contained an insert of 1,848 bp with 50 bp and 279 bp of 5’ and 3’ UTR, respectively and a short poly-A tail of 24 bases. An open reading frame of 1,494 bp encoded for a protein of 497 amino acids that was identical to mannose-binding lectins found in many eukaryotes (Figure 5). A similar sequence was described in A. variegatum ESTs, which clustered together with the I. scapularis sequence (Figure 5).

The cDNA in clone 4G11 was identical to a chloride channel but it contained only a partial sequence (Figure 2A). This sequence protected against tick infestations and inhibited larval molting (Table 3). Chloride channels have been found in living organisms from bacteria to mammals, with some amino acid positions being conserved in all sequences (Figure 2A). As expected, phylogenetic analysis of chloride channel sequences demonstrated that the I. scapularis sequence comprised a sister group to other insect sequences that have been reported (Figure 2B). Vaccination with ribosomal sequences had some inhibitory effect on tick infestations but did not affect molting (Table 3). The pool of ribosomal cDNAs included EST sequences coding for cellular and mitochondrial ribosomal proteins and translation factors (Table 4), and these genes are highly conserved across species. However, proteins encoded by I. scapularis ESTs were 43% to 95% identical to arachnida or insect sequences and 36% to 85% identical to mouse sequences (Table 4). The cDNA in clone 2C12 that was found to be identical to the betaamyloid precursor protein (APP) contained a fragment encoding for the C-terminal of the protein (Figure 3), suggesting that it contains a partial cDNA with a long (1,400 bp) 3’ UTR. Nonetheless, the C-terminal sequence of the I. scapularis APP contained regions of amino acids 48

Gene Therapy and Molecular Biology Vol 7, page 49 A cgATGCAGGCGATGACGGGCTTTGCGGTGCAGTTCAACAAAAACAGTTTCGGGCTGACTCCAGCTCAGCCGCTGCAGTTGCAGATTCCCCT GCAGCCCAACTTCCCAGCTGATGCGAGCTTGCAGCTGGGAACCAACGGTCCCGTGCAGAAGATGGACCCCCTCACCAACCTTCAGGTGGCC ATCAAGAACAATGTGGACGTGTTCTACTTCAGCTGCCTGGTGCCCATGCACGTGCTGAGCACGGAGGACGGCCTGATGGACAAGCGGGTGT TCCTGGCCACCTGGAAAGACATCCCCGCCCAAAACGAGGTCCAGTACACCCTCGACAACGTCAACCTCACTGCAGACCAAGTTTCCCAGAA GCTGCAGAACAACAACATTTTCACGATAGCCAAGAGGAACGTGGACGGCCAGGACATGCTGTACCAGTCCCTGAAGCTCACCAACGGCATT TGGGTGTTGGCGGAGCTCAAGATACAGCCCGGCAATCCAAGGATCACGTTGTCTTTGAAGACAAGAGCACCTGAAGTGGCAGCAGGTGTAC AACAAACTTACGAACTCATTCTACACAGCTGAggctgctgtgaatgaaactcttctcccacccccttcttttgatggcagtcaatgtctcg tttcattttcttgttttcttttgcggcgtgctacggaacaaggtcctacattcccaagttatatggtgttgtcgcgtagggggcagagtgc cgctgagcccgcgacagccttgtttctgaggagagccgaacgcaccacttcgaaaaagaaaaagtgaaaacggaaaaatgaaaaattttcc agttgcttcaaattaacattcctcgtagtcagtctgtggccgttgagtttggtgtaaagaagaaaaaggtgtctcttttagtgaaaatggt tgctttttattggtatcccctatcacaccgagcacgaacataagaaatcctgacaaggattctcctttagttgtattatggtggctggagc acacgaggcacctgttgccaattcgacccagcaaatgcccaattctcaagatttgagttcattgaggttgttttgctcctccccccccacc ccccaactttgtcgttggattgtctaacagtgtaaatgggcgacgactcgttattctttttttcttcattctttctttttgttgtcacgcg ccccgggggacgcgacacaacttatgtgcataattgattttcacaggctgcgacgcagtctgtaaaagaaggggaagtgaaactctgctcc gccgctgctagtgtcatcacgggacgaccatcgcgttttctctgactatttaaacaaaactgcatagcttagggggcagtctgtgcaaagt ggaacaaccaaactgagccctgccctttcggtgtgtgtacaagcatctctgtgtaacatgaactactttacatgaactacattgcatgaac gggagaagtttagttgtttttttgttttttttttcaggtgactatgtcaacagattagaaccattttttggaacggctggaaagataaccg ctcattttgtttctactaaaagactacgaaaagtgttgactttttgcatcggtttggcaacgtttgtttggcatgcatgtagttgagcgta atggtatcacccctcgtaaacaataacagtgcaatggagcagtactgtagtgtccattaaagagcgagagtttggttaaaggttgttaatt gaggtccgtgttatcctttgagtaggagagcggcactttttgcaaatagcgctgctgggggcgtcatatctgccctccaaaacatgcacat tttaagtgtgaattgttgcggcggcttgtacaagtatgtgtgttatgtgtagaaaaagaactcttaattaaaatatttgtggccaaaacgt caaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

B M. musculus D. melanogaster H. sapiens I. scapularis Consensus

(747) (731) (68) (1) (748)


M. musculus D. melanogaster H. sapiens I. scapularis

(797) (780) (118) (51)


Consensus (798) MEPLNNLQVAVKNNIDVFYFSCLIPLNVLFVEDGKMDKRVFLATWKDIPN M. musculus D. melanogaster H. sapiens I. scapularis Consensus

(847) (830) (168) (101) (848)


M. musculus D. melanogaster H. sapiens I. scapularis Consensus

(897) (880) (218) (151) (898)


Figure 1. Analysis of clone 3E1 identical to beta-adaptin. (A) Nucleotide sequence of complete cDNA. Non-coding sequence is shown in lower case letters and coding sequence is shown in capital letters with translation initiation and termination codons in bold letters. (B) Alignment of M. musculus (GenBank accession number XP_109938), D. melanogaster (CAA53509) and Homo sapiens (AAA35583) protein sequences and the translation product of clone 3E1 identified as I. scapularis beta-adaptin appendage (AY296113). Protein sequences are shown in the single letter amino acid code. Identical amino acids are shown in red and amino acids conserved in 3 of 4 sequences are shown in blue.

A E. coli O. mossambicus X. laevis I. scapularis C. elegans D. melanogaster L. major A. gambiae

(4) (98) (146) (1) (141) (223) (114) (272)



Almazรกn et al: Expressed sequence tags in Ixodes scapularis M. musculus S. tuberosum S. cerevisiae Consensus

(155) (108) (102) (272)


E. coli O. mossambicus X. laevis I. scapularis C. elegans D. melanogaster L. major A. gambiae M. musculus S. tuberosum S. cerevisiae Consensus

(53) (130) (178) (32) (172) (254) (138) (303) (187) (123) (133) (322)


E. coli O. mossambicus X. laevis I. scapularis C. elegans D. melanogaster L. major A. gambiae M. musculus S. tuberosum S. cerevisiae Consensus

(103) (178) (226) (82) (221) (302) (185) (351) (235) (151) (182) (372)


E. coli O. mossambicus X. laevis I. scapularis C. elegans D. melanogaster L. major A. gambiae M. musculus S. tuberosum S. cerevisiae Consensus

(150) (227) (275) (131) (270) (351) (234) (400) (284) (200) (232) (422)


E. coli O. mossambicus X. laevis I. scapularis C. elegans D. melanogaster L. major A. gambiae M. musculus S. tuberosum S. cerevisiae Consensus

(188) (267) (315) (171) (310) (391) (273) (440) (324) (250) (272) (472)


E. coli O. mossambicus X. laevis I. scapularis C. elegans D. melanogaster L. major A. gambiae M. musculus S. tuberosum S. cerevisiae Consensus

(236) (315) (363) (219) (358) (439) (321) (488) (372) (298) (322) (522)



Gene Therapy and Molecular Biology Vol 7, page 51 B

Figure 2. Analysis of clone 4G11 identical to chloride channel. (A) Alignment of M. musculus (XP_134186), D. melanogaster (AAM76180), Solanum tuberosum (T07608), Oreochromis mossambicus (AAD56388), A. gambiae (EAA11899), C. elegans (NP_495940), Leishmania major (strain Friedlin) (T02805), Saccharomyces cerevisiae (P37020), Escherichia coli K12 (AAC73266), and Xenopus laevis (CAA71071) protein sequences and the translation product of clone 4G11 identified as a fragment of I. scapularis chloride channel (AY296114). Protein sequences are shown in the single letter amino acid code. Identical amino acids are shown in red and amino acids conserved in 6-10 of 11 sequences are shown in blue. (B) Phylogenetic tree constructed from analysis of chloride channel protein sequences based on a sequence distance method utilizing the Neighbor Joining algorithm of Saitou and Nei (1987).

D. melanogaster I. scapularis A. gambiae Consensus


Figure 3. Analysis of clone 2C12 identical to beta-amyloid precursor protein. Alignment of D. melanogaster (AF181628) and A. gambiae (EAA07868) protein sequences and the translation product of clone 2C12 identified as I. scapularis beta-amyloid peptide (Ă&#x;AP) (AY296115). Protein sequences are shown in the single letter amino acid code. Identical amino acids are shown in red and amino acids conserved in 2 of 3 sequences are shown in blue.

Table 4. Characterization of I. scapularis ESTs encoding for ribosomal proteins EST clone

Predicted protein

Identical amino acids


GenBank accession number

4F7 1A2

Elongation factor 1-alpha

95% 85%

Neacarus texanus

AAK12660 NP_031932


Elongation factor-2

88% 80%

Mastigoproctus giganteus Mus musculus

AAK12348 BAC26203



65% 59%

Drosophila melanogaster Mus musculus

AAM68297 XP_203336

1F6 2C3


79% 75%

Spodoptera frugiperda Mus musculus

AAL26580 AAH09100



92% 80%

Dermacentor variabilis Mus musculus

AAO92287 XP_133477


Laminin receptor 1 (RpSA)

66% 73%

Anopheles gambiae Mus musculus

EAA00413 NP_035159



70% 68%

Spodoptera frugiperda Mus musculus

AAL62468 AAH09655



55% 60%

Drosophila melanogaster Mus musculus

NP_511063 A30241


Ribophorin I

57% 50%

Drosophila melanogaster Mus musculus

AAN71150 BAC26679



70% 71%

Spodoptera frugiperda Mus musculus

AAL62472 XP_134904




Spodoptera frugiperda


Mus musculus


Almazรกn et al: Expressed sequence tags in Ixodes scapularis 36%

Mus musculus



Proteasome subunit

60% 55%

Drosophila melanogaster Mus musculus

NP_524115 NP_035315


Proteasome/Signalosome subunit

43% 56%

Anopheles gambiae Mus musculus

EAA11895 AAC33900


Proteasome subunit

84% 85%

Anopheles gambiae Mus musculus

EAA10351 NP_036096

The sequences of I. scapularis ESTs identical to ribosomal proteins pooled for DNA vaccination as described in Almazรกn et al. (2003), were compared to all non-redundant sequences in GenBank DNA and protein databases (1,419,727 sequences total; Apr-09-2003) using BLASTX 2.2.6 (Altschul et al, 1997). The percent of identical amino acids to arachnida or insect and mouse sequences are shown together with their corresponding GenBank accession number. The GenBank accession numbers for I. scapualris sequences are shown on Table 1. 1 M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus

(1) (1) (1) (1) (1) (1)

M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus

(27) (51) (23) (30) (29) (51)

M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus

(72) (98) (73) (77) (79) (101)

M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus

(108) (148) (109) (127) (129) (151)

M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus

(157) (197) (158) (176) (177) (201)

M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus

(207) (247) (208) (226) (227) (251)

M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus

(257) (297) (258) (276) (277) (301)

M. musculus D. melanogaster H. sapiens

(300) (346) (301)



Gene Therapy and Molecular Biology Vol 7, page 53 A. gambiae I. scapularis Consensus M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus


Figure 4. Analysis of clone 4F1 identical to block of proliferation (Bop1). (A) Alignment of M. musculus (AAH12693), D. melanogaster (NP_611270), A. gambiae (EAA04116), and H. sapiens (AAH07274) protein sequences and the translation product of clone 4F1 identified as I. scapularis Bop (AY296116). Protein sequences are shown in the single letter amino acid code. Identical amino acids are shown in red and amino acids conserved in 3-4 of 5 sequences are shown in blue.


Almazán et al: Expressed sequence tags in Ixodes scapularis The clone 3C12, together with clone 2F9, produced the greatest enhancement of tick feeding after vaccination and tick challenge (Table 3). The clone 3C12 was completely sequenced and contained an insert of 447 bp with 5 bp and 86 bp of 5’ and 3’ UTR, respectively and a short poly-A tail of 29 bases. An open reading frame of 327 bp encoded for a protein of 108 amino acids that was identical to RNA polymerase III, and had a high degree of identity with human and insect sequences (Figure 6A). The EST in clone 2F9 was identical to human and A. variegatum sequences coding for proteins of unknown function (Figure 6B). Vaccination with the pool of ESTs identical to ATPases resulted in a 57% increase in larval feeding (Table 3). This pool originally contained 6 sequences (Almazán et al, 2003) but only 3 were non-redundant (clones 1A9, 1B2 and 4A4). All sequences were identical to vacuolar proton pump ATPases (EC The sequence of 1A9 was identical to D. melanogaster (TC112371) V-ATPase subunit D, 1B2 was identical to A. americanum (AAU03374) V-ATPase subunit C and 4A4 was identical to D. melanogaster (TC112172) V-ATPase subunit E. Six clones of the I. scapularis ESTs contained short tandem repeat (STR) microsatellite sequences. STRs were found in 5 clones (1F4, 2C7, 3B6, 4G12 and 4H2) containing sequences of unknown function and in one clone (1A9) that was identical to the D. melanogaster VATPase subunit D (Table 1). Microsatellite sequences contained perfect and imperfect STRs (Table 5). Clones 1A9, 4G12 and 3B6 contained 9, 6 and 12 TA repeats, respectively. Clone 1F4 contained an imperfect repeat of 15 GC/T and the clone 2C7 contained 9 GT repeats. The clone 4G12 contained a second STR of 10 CA/GA/CT repeats.

Willadsen, 1997; Willadsen and Jongejan, 1999; de la Fuente et al, 1999, 2000a). However, a limiting step for development of effective anti-tick vaccines is the identification of tick protective antigens. In the past, tick protective antigens were identified by (a) evaluating proteins after host immunization and tick challenge that were derived from progressive fractionation of crude tick extracts, (b) immunomapping of tick antigens which elicit an antibody response in the infested host, and (c) testing tick proteins in vaccination experiments that were considered to be important for the parasite function and/or survival. However, construction of cDNA libraries and EST databases from different tick tissues, developmental stages and from genes expressed in response to various stimuli (i.e., tick feeding or infection of cDNAs encoding for tick immunosuppressants, anticoagulants and other proteins with low antigenicity that may enhance tick feeding. Alternatively, they may encode for proteins homologous to host proteins associated with anti-tick or growth suppression activity which neutralization results in a tick pro-feeding effect. The former could be the case for ATPases. These proteins are highly conserved across species and, therefore, could elicit a poor immune response. However, ATPases are expressed in tick embryos and salivary glands of unfed adults and adult females at all stages of feeding and some evidences suggest that these proteins may participate in salivary fluid secretion in A. americanum (McSwain et al, 1997). Therefore, although the mechanism is not known, DNA vaccination with ATPase-coding cDNAs could produce enhanced larval feeding. Although we presently do not have evidence to support the latter hypothesis, proteins of unknown function, such as the one encoded by clone 2F9 that is identical to host proteins of unidentified function, and Bop 1, a nonribosomal protein that is highly conserved from yeast to human with a growth suppressor function that plays a key role in the formation of mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit (Pestov et al, 1998; Strezoska et al, 2000), are examples that may enhance tick feeding.

IV. Discussion The feasibility of controlling tick infestations through immunization of hosts with tick antigens has been demonstrated previously for Boophilus spp. (reviewed by

Figure 5. Analysis of clone 3E10 identical to mannose-binding lectin. Phylogenetic tree constructed from analysis of C. elegans (NP_492548), A. gambiae (EAA11908), D. melanogaster (NP_524776), M. musculus (XP_128952), R. norvegicus (NP_446338), Cercopithecus aethiops (Q9TU32), H. sapiens (NP_005561), Polyandrocarpa misakiensis (BAB20045), X. laevis (AAC59755), Dictyostelium discoideum (AAL92589), A. variegatum (BM290898) and I. scapularis (AY296117) protein sequences based on a sequence distance method utilizing the Neighbor Joining algorithm of Saitou and Nei (1987).


Gene Therapy and Molecular Biology Vol 7, page 55 A 1 D. melanogaster H. sapiens A. gambiae I. scapularis Consensus

(1) (1) (1) (1) (1)

D. melanogaster H. sapiens A. gambiae I. scapularis Consensus

(51) (51) (51) (51) (51)

D. melanogaster H. sapiens A. gambiae I. scapularis Consensus

(101) (101) (101) (101) (101)


B I. scapularis A. variegatum H. sapiens Consensus

(78) (1) (115) (115)


I. scapularis A. variegatum H. sapiens Consensus

(128) (51) (165) (165)


Figure 6. Analysis of clones 3C12 and 2F9 identical to RNA polymerase III and a hypothetical protein of unknown function, respectively. (A) Alignment of D. melanogaster (AAF57437), A. gambiae (TC6088), and H. sapiens (AAK61210) RNA polymerase III protein sequences and the translation product of clone 3C12 identified as I. scapularis RNA polymerase III (AY296118). (B) Alignment of A. variegatum (TC255), H. sapiens (FLJ12475) and I. scapularis clone 2F9 (AY296119) partial protein sequences. Protein sequences are shown in the single letter amino acid code. Identical amino acids are shown in red and amino acids conserved in 2-3 of 4 (A) and 2 of 3 (B) sequences are shown in blue.

Table 5. Microsatellite STR sequences in I. scapularis ESTs. cDNA clone

Microsatellite sequence













(ß-AP), a "40 amino acids peptide derived from the APP protein found as the major component of dense plaques in brains of Alzheimer disease patients (reviewed by Cummings, 2003). Vaccination with ß-AP prevented the formation of ß-AP plaques in transgenic mice, opening a new possible approach for treatment of Alzheimer disease (McGeer and McGeer, 2003). However, we do not understand the apparent enhanced feeding effect of the tick ß-AP in cDNA-vaccinated mice. The lectin in clone 3E10 was identical to mannose-binding endoplasmic reticulum-Golgi intermediate compartment protein (Arar et al, 1995; Lahtinen et al, 1996). However, the carbohydrate-binding domain is shared by other lectins found in different cell compartments. The clone 3C12 encoded for an RNA polymerase III. Enhanced tick

Nonetheless, cDNAs associated with enhanced tick feeding could be made as recombinant proteins to modify their immunogenicity and then be evaluated as candidate protective antigens. Additionally, these antigens may also be good candidates for blocking the transmission of tickborne pathogens (Wikel et al, 1997; Labuda et al, 2002). The enhanced feeding effect of cDNA clones with identity to App (2C12), mannose-binding lectin (3E10) and RNA polymerase III (3C12) is difficult to explain. The beta-amyloid protein precursor is involved in different physiological processes, including development of the embryonic nervous system in D. melanogaster (Rosen et al, 1989) and pharyngeal pumping in Caenorhabditis elegans (Zambreano et al, 2002). The sequence contained in clone 2C12 corresponded to the beta-amyloid peptide 55

Almazán et al: Expressed sequence tags in Ixodes scapularis feeding was produced in mice vaccinated with a DNA pool containing this clone and clone 2F9 of unknown function. It is therefore possible that the enhanced feeding effect on tick larvae was due to clone 2F9 with little or no contribution of clone 3C12. Microsatellites are a class of genetic markers that are composed of STR sequences flanked by unique DNA sequences (Hearne et al, 1992). STRs are highly polymorphic and widely distributed through the genome. The analysis of tick STRs has been used for identification of strains of B. microplus (de la Fuente et al, 2000b) and for the development of a preliminary genetic linkage map of I. scapularis (Ullman et al, 2003). The STR sequences described in this study could be used for completion of the genetic map of I. scapularis as the first step toward the sequencing of this tick genome. Most sequences in the I. scapularis EST data set were relatively G + C rich, with an average G + C content of 54%, similar to the 52% reported by Nene et al. (2002) for A. variegatum. The few sequences with a high A + T content probably corresponded to mitochondrial genes, with pathogens) provides new exciting possibilities for screening and identifying antigens protective against tick infestations. This approach may also allow for identification of antigens that interfere with pathogen development and transmission. Recently, Almazán et al. (2003) used cDNA ELI combined with EST analysis as a rapid method for the identification of protective antigens against I. scapularis infestations, demonstrating the role of sequence information in conjunction with new technologies such as bioinformatics and ELI for a systematic and comprehensive approach to vaccine discovery. One of the advantages of ELI for identification of protective antigens is that a priori criteria are not introduced to direct the selection of candidate genes. This approach, as shown in this study, resulted in potential vaccine antigens otherwise not predicted, such as clone 4F8 that was found to be identical to a nucleotidase. However, nucleotidases are essential for cell growth and the inhibition of its enzymatic activity would be cytotoxic (Spiegelberg et al, 1999), providing a possible explanation for their protective properties against tick infestations. The I. scapularis sequence in clone 4F8 was different from the 5’-nucleotidase that was identified and characterized previously by Liyou et al. (1999, 2000) in B. microplus. However, the protective capacity of this protein has not been evaluated. As discussed previously by Almazán et al, (2003), a possible explanation for the inhibitory effect on larval tick development of other vaccine candidates that were identified in this study is based on the role that they play in cell growth and maintenance, which is evident for clones identical to beta-adaptin (3E1) and chloride channel (4G11). Beta adaptins are adaptor components required in the assembly of clathrin-coated plasma membrane pits that function in cell vesicular transport mechanisms including endocytosis (Camidge and Pearse, 1994; Boehm and Bonifacino, 2002), a process actively involved in blood digestion by ticks and other hematophagous arthropods

(Akov, 1982). Chloride channels are also involved in vital cell functions including the catalysis of counter ion currents that accompany primary proton fluxes in endosomal and lysosomal acidification (Koprowski and Kubalski, 2001; Iyer et al, 2002). Therefore, interference with the process of endocytosis may impair acquisition and digestion of the tick bloodmeal and result in inhibition of tick infestations. Another I. scapularis EST (clone 3E12) encoded for a protein identical to D. melanogaster clathrin heavy chain, a protein involved in synaptic vesicle endocytosis (Chang et al, 2002). This cDNA is also a candidate protective antigen because it interfers with endocytosis in feeding larvae. The protection capacity of ribosomal and heat shock protein preparations has been documented previously in other organisms (Elad and Segal, 1995; Silva, 1999; Melby et al, 2000; Cassataro et al, 2002). Recently, Hsp70 was demonstrated to be induced in I. ricinus salivary glands during blood feeding (Leboulle et al, 2002), documenting the role of heat shock proteins in physiological responses in ticks. Even in the case where substantial homology exists between tick proteins and host (mouse) proteins, analysis of ribosomal proteins suggests that differences in the amino acid sequence could direct the host immune response against distinctive, non-self epitopes, which could be sufficient to induce a protective response. The results of vaccination and tick infestation demonstrated that some cDNAs enhance tick feeding. This effect could be due to the expression corroborating the hypothesis that there is a marked difference in codon usage between mitochondrial and nuclear protein coding genes in the Ixodidae (Nene et al, 2002). Most of the ESTs in our database, although initially identified by ELI of cDNA pools that produced inhibition of tick infestation, were not characterized further and remain potential candidate antigens for vaccine development against I. scapularis infestations. Particularly interesting were cDNAs that may be involved in developmental processes. Clone 4B2, identical to D. melanogaster sequence NP_523710, encoded for calmodulin, a Ca++-binding protein of 149 amino acids that is involved in fly development. This protein was found to be expressed in several larval and adult tissues, including the larval midgut (Takamatsu et al, 2002). Clone 1C8 had a low degree of identity to D. melanogaster virilizer, a gene involved in Sex-lethal (Sxl) splicing and essential for fly male and female viability and embryonic development (Niessen et al, 2001). Clone 2A11 also had a low degree of identity to D. melanogaster developmental regulator, Notchless, a key player in the signaling by Notch family receptors that are involved in many cell-fate decisions during development (Royet et al, 1998). Similarly, clone 4A10 had partial identity to the putative homeodomain transcriptional factor, phtf, a member of a gene family that plays an important role during development and is conserved between fly, mouse and human (Manuel et al, 2000). Other clones with special interest as vaccine candidates may include those identical to membrane proteins (1D8, 1D11, 3G11) and those putatively involved


Gene Therapy and Molecular Biology Vol 7, page 57 identical to MR60, an intracellular mannose-specific lectin of myelomonocytic cells. J Biol Chem 270, 3551-3553. Ashburner M, Ball CA, Blake JA, et al (2000) Gene ontology: tool for the unification of biology. Nature Genet 25, 25-29. Akov S. Blood digestion in ticks. In: Obenchain FD, Galun R, editors (1982) Physiology of ticks. Current themes in tropical science (vol. 1). Oxford, Pergamon, pp. 197-211. Barry MA, Lai WC, Johnston SA (1995) Protection against mycoplasma infection using expression-library immunization. Nature 377, 632-635. Berriz GF, White JV, King OD, Roth FP (2003) GoFish finds genes with combinations of Gene Ontology attributes. Bioinformatics 19, 788-789. Bior AD, Essenberg RC, Sauer JR (2002) Comparison of differentially expressed genes in the salivary glands of male ticks, Amblyomma americanum and Dermacentor andersoni. Insect Biochem Mol Biol 32, 645-655. Boguski MS, Lowe TM, Tolstoshev CM (1993) dbEST-database for "expressed sequence tags". Nat Genet 4, 332-333. Boehm M, Bonifacino JS (2002) Genetic analyses of adaptin function from yeast to mammals. Gene 286, 175-186. Brayton KA, Vogel SW, Allsopp BA (1998) Expression library immunization to identify protective antigens from Cowdria ruminantium. Ann N Y Acad Sci 849, 369-371. Camidge DR, Pearse BM (1994) Cloning of Drosophila betaadaptin and its localization on expression in mammalian cells. J Cell Sci 107, 709-718. Cassataro J, Velikovsky CA, Giambartolomei GH, Estein S, Bruno L, Cloeckaert A, Bowden RA, Spitz M, Fossati CA (2002) Immunogenicity of the Brucella melitensis recombinant ribosome recycling factor-homologous protein and its cDNA. Vaccine 20, 1660-1669. Chang HC, Newmyer SL, Hull MJ, Ebersold M, Schmid SL, Mellman I (2002) Hsc70 is required for endocytosis and clathrin function in Drosophila. J Cell Biol 159, 477-487. Crampton AL, Miller C, Baxter GD, Barker SC (1998) Expressed sequenced tags and new genes from the cattle tick, Boophilus microplus. Exp Appl Acarol 22, 177-186. Cummings JL (2003) Alzheimer's disease: from molecular biology to neuropsychiatry. Semin Clin Neuropsychiatry 8, 31-36. de la Fuente J, Rodriguez M, Redondo M, Montero C, GarciaGarcia JC, Mendez L, Serrano E, Valdes M, Enriquez A, Canales M, Ramos E, Boue O, Machado H, Lleonart R, de Armas CA, Rey S, Rodriguez JL, Artiles M, Garcia L (1998) Field studies and cost-effectiveness analysis of vaccination with Gavac against the cattle tick Boophilus microplus. Vaccine 16, 366-373. de la Fuente J, Rodriguez M, Montero C, Redondo M, GarciaGarcia JC, Mendez L, Serrano E, Valdes M, Enriquez A, Canales M, Ramos E, Boue O, Machado H, Lleonart R (1999) Vaccination against ticks (Boophilus spp.): the experience with the Bm86-based vaccine Gavac. Genet Anal 15, 143-148. de la Fuente J, Rodriguez M, Garcia-Garcia JC (2000a) Immunological control of ticks through vaccination with Boophilus microplus gut antigens. Ann N Y Acad Sci 916, 617-621. de la Fuente J, Garc_a-Garc_a JC, Gonz_lez DM, Izquierdo G, Ochagavia ME (2000b) Molecular analysis of Boophilus spp. (Acari: Ixodidae) tick strains. Vet Parasitol 92, 209-222. de Vos S, Zeinstra L, Taoufik O, Willadsen P, Jongejan F (2001) Evidence for the utility of the Bm86 antigen from Boophilus

in G-protein-coupled signaling (2B7, 2F12, 4C9). In fact, the clone 3G11 was identical to D. melanogaster BM-40, a protein of the group of extracellular basement membrane proteins which includes the protective antigen p29 from Haemaphysalis longicornis (Mulenga et al, 1999). In summary, we have characterized I. scapularis EST sequences that were selected by cDNA ELI in the mouse/tick challenge model because they affected tick development. Characterization of these ESTs provides a basis for future research on ticks and is a source of candidate antigens for use in vaccine development designed to control tick infestations and/or reduce transmission of pathogens. The combination of ELI with EST appears to be a productive systematic and comprehensive approach to vaccine discovery.

Acknowledgments This research was supported by the project No. 1669 of the Oklahoma Agricultural Experiment Station, the Endowed Chair for Food Animal Research (K. M. Kocan, College of Veterinary Medicine, Oklahoma State University), NIH Centers for Biomedical Research Excellence through a subcontract to J. de la Fuente from the Oklahoma Medical Research Foundation, and the Oklahoma Center for the Advancement of Science and Technology, Applied Research Grant, AR00(1)-001 and AR02(1)-037. Consuelo Almazán is supported by a grantin-aid from the CONACYT, Mexico and an assistantship from the College of Veterinary Medicine, Oklahoma State University. J. C. Garcia-Garcia is supported by a Howard Hughes Medical Institute Predoctoral Fellowship in Biological Sciences. Jerry Bowman is acknowledged for providing tick larvae. Janet J. Rogers and Sue Ann Hudiburg (Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University) are acknowledged for DNA sequencing and oligonucleotide synthesis, respectively. We thank Joy Yoshioka for editorial assistance.

References Alberti E, Acosta A, Sarmiento ME, Hidalgo C, Vidal T, Fachado A, Fonte L, Izquierdo L, Infante JF, Finlay CM, Sierra G (1998) Specific cellular and humoral immune response in Balb/c mice immunised with an expression genomic library of Trypanosoma cruzi. Vaccine 16, 608-612. Almazán C, Kocan KM, Bergman DK, Garcia-Garcia JC, Blouin EF, de la Fuente J (2003) Identification of protective antigens for the control of Ixodes scapularis infestations using cDNA expression library immunization. Vaccine 21, 1492-1501. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215, 403-410. Altschul, SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nuc Acids Res 25, 3389-3402. Arar C, Carpentier V, Le Caer JP, Monsigny M, Legrand A, Roche AC (1995) ERGIC-53, a membrane protein of the endoplasmic reticulum-Golgi intermediate compartment, is


Almazán et al: Expressed sequence tags in Ixodes scapularis microplus in vaccination against other tick species. Exp Appl Acarol 25, 245-261. Elad D, Segal E (1995) Immunogenicity in calves of a crude ribosomal fraction of Trichophyton verrucosum: a field trial. Vaccine 13: 83-87. Estrada-Peña A, Jongejan F (1999) Ticks feeding on humans: a review of records on human-biting Ixodoidea with special reference to pathogen transmission. Exp Appl Acarol 23, 685-715. Garcia-Garcia JC, Gonzalez IL, Gonzalez DM, Valdes M, Mendez L, Lamberti J, D'Agostino B, Citroni D, Fragoso H, Ortiz M, Rodriguez M, de la Fuente J (1999) Sequence variations in the Boophilus microplus Bm86 locus and implications for immunoprotection in cattle vaccinated with this antigen. Exp Appl Acarol 23, 883-395. Hearne CM, Ghosh S Todd J (1992) Microsatellites for linkage analysis of genetic traits. Trends Genet 8, 288-294. Hill CA, Gutierrez JA (2000) Analysis of the expressed genome of the lone star tick, Amblyomma americanum (Acari: Ixodidae) using an expressed sequence tag approach. Microb Comp Genomics 5, 89-101. Iyer R, Iverson TM, Accardi A, Miller C (2002) A biological role for prokaryotic ClC chloride channels. Nature 419, 715-718. Kessler MM, Willins DA, Zeng Q, Del Mastro RG, Cook R, Doucette-Stamm L, Lee H, Caron A, McClanahan TK, Wang L, Greene J, Hare RS, Cottarel G, Shimer GH Jr (2002) The use of direct cDNA selection to rapidly and effectively identify genes in the fungus Aspergillus fumigatus. Fungal Genet Biol 36, 59-70. Knox DP, Redmond DL, Skuce PJ, Newlands GF (2001) The contribution of molecular biology to the development of vaccines against nematode and trematode parasites of domestic ruminants. Vet Parasitol 101, 311-335. Koprowski P, Kubalski A (2001) Bacterial ion channels and their eukaryotic homologues. Bioessays 23, 1148-1158. Labuda M, Trimnell AR, Lickova M, Kazimirova M, Slovak M, Nuttall PA (2002) Recombinant tick salivary antigens (64TRP) as a TRANSBLOK vaccine against tick-borne encephalitis virus. Abstracts 4th International Conference on Ticks and Tick-Borne Pathogens, Banff, Canada, p. 51. Lahtinen U, Hellman U, Wernstedt C, Saraste J, Pettersson RF (1996) Molecular cloning and expression of a 58-kDa cisGolgi and intermediate compartment protein. J Biol Chem 271, 4031-4037. Leboulle G, Rochez C, Louahed J, Ruti B, Brossard M, Bollen A, Godfroid E (2002) Isolation of Ixodes ricinus salivary gland mRNA encoding factors induced during blood feeding. Am J Trop Med Hyg 66, 225-233. Leclercq S, Harms JS, Oliveira SC (2003) Enhanced efficacy of DNA vaccines against an intracellular bacterial pathogen by genetic adjuvants. Curr Pharm Biotechnol 4, 99-107. Liyou N, Hamilton S, Elvin C, Willadsen P (1999) Cloning and expression of ecto 5’-nucleotidase from the cattle tick Boophilus microplus. Insect Mol Biol 8, 257-266. Liyou N, Hamilton S, Mckenna R, Elvin C, Willadsen P (2000) Localization and functional studies on the 5’-nucleotidase of the cattle tick Boophilus microplus. Exp Appl Acarol 24, 235-246. Lizotte-Waniewski M, Tawe W, Guiliano DB, Lu W, Liu J, Williams SA, Lustigman S (2000) Identification of potential vaccine and drug target candidates by expressed sequence tag analysis and immunoscreening of Onchocerca volvulus larval cDNA libraries. Infect Immun 68, 3491-3501.

Manoutcharian K, Terrazas LI, Gevorkian G, Govezensky T (1998) Protection against murine cysticercosis using cDNA expression library immunization. Immunol Lett 62, 131136. Manuel A, Beaupain D, Romeo PH, Raich N (2000) Molecular characterization of a novel gene family (PHTF) conserved from Drosophila to mammals. Genomics 64, 216-220. McGeer PL, McGeer E (2003) Is there a future for vaccination as a treatment for Alzheimer's disease? Neurobiol Aging 24, 391-395. McSwain JL, Luo C, deSilva GA, Palmer MJ, Tucker JS, Sauer JR, Essenberg RC (1997) Cloning and sequence of a gene for a homologue of the C subunit of the V-ATPase from the salivary gland of the tick Amblyomma americanum (L). Insect Mol. Biol 6, 67-76. Melby PC, Ogden GB, Flores HA, Zhao W, Geldmacher C, Biediger NM, Ahuja SK, Uranga J, Melendez M (2000) Identification of vaccine candidates for experimental visceral leishmaniasis by immunization with sequential fractions of a cDNA expression library. Infect Immun 68, 5595-5602. Moore RJ, Lenghaus C, Sheedy SA, Doran TJ (2001) Improved vectors for expression library immunization-application to Mycoplasma hyopneumoniae infection in pigs. Vaccine 20, 115-120. Mulenga A, Sugimoto C, Sako Y, Ohashi K, Musoke A, Shubash M, Onuma M (1999) Molecular characterization of a Haemaphysalis longicornis tick salivary gland-associated 29kilodalton protein and its effect as a vaccine against tick infestation in rabbits. Infect Immun 67, 1652-1658. Mulenga A, Macaluso KR, Simser JA, Azad AF (2003) Dynamics of Rickettsia-tick interactions: identification and characterization of differentially expressed mRNAs in uninfected and infected Dermacentor variabilis. Insect Mol Biol 12, 185-193. Munderloh UG, Wang YLM, Chen C, Kurtti TJ (1994) Establishment, maintenance and description of cell lines from the tick Ixodes scapularis. J Parasitol 80, 533-543. Nene V, Lee D, Quackenbush J, Skilton R, Mwaura S, Gardner MJ, Bishop R (2002) AvGI, an index of genes transcribed in the salivary glands of the ixodid tick Amblyomma variegatum. Int J Parasitol 32, 1447-1456. Niessen M, Schneiter R, Nothiger R (2001) Molecular identification of virilizer, a gene required for the expression of the sex-determining gene Sex-lethal in Drosophila melanogaster. Genetics 157, 679-688. Parola P, Raoult D (2001) Tick-borne bacterial diseases emerging in Europe. Clin Microbiol Infect 7, 80-83. Pestov DG, Grzeszkiewicz TM, Lau LF (1998) Isolation of growth suppressors from a cDNA expression library. Oncogene 17, 3187-3197. Rosen DR, Martin-Morris L, Luo LQ, White K.A (1989) Drosophila gene encoding a protein resembling the human beta-amyloid protein precursor. Proc Natl Acad Sci U S A 86, 2478-2482. Royet J, Bouwmeester T, Cohen SM (1998) Notchless encodes a novel WD40-repeat-containing protein that modulates Notch signaling activity. EMBO J 17, 7351-7360. Saitou N, Nei M (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425. Silva CL (1999) The potential use of heat-shock proteins to vaccinate against mycobacterial infections. Microbes Infect 1, 429-435.


Gene Therapy and Molecular Biology Vol 7, page 59 Singh RA, Wu L, Barry MA (2002) Generation of genome-wide CD8 T cell responses in HLA-A*0201 transgenic mice by an HIV-1 ubiquitin expression library immunization vaccine. J Immunol 168, 379-391. Smooker PM, Setiady YY, Rainczuk A, Spithill TW (2000) Expression library immunization protects mice against a challenge with virulent rodent malaria. Vaccine 18, 25332540. Spiegelberg BD, Xiong JP, Smith JJ, Gu RF, York JD (1999) Cloning and characterization of a mammalian lithiumsensitive bisphosphate 3'-nucleotidase inhibited by inositol 1,4-bisphosphate. J Biol Chem 274, 13619-13628. Strezoska Z, Pestov DG, Lau LF (2000) Bop1 is a mouse WD40 repeat nucleolar protein involved in 28S and 5. 8S RRNA processing and 60S ribosome biogenesis. Mol Cell Biol 20, 5516-5528. Takamatsu Y, Nakagoshi H, Rachidi M, Lopes C, Nishida Y, Ohsako S (2002) Characterization of the dCaMKII-GAL4 driver line whose expression is controlled by the Drosophila Ca(2+)/calmodulin-dependent protein kinase II promoter. Cell Tissue Res 310, 237-252. Tarleton RL, Kissinger J (2001) Parasite genomics: current status and future prospects. Curr Opin Immunol 13, 395-402. Thompson JD, Higgins DG, Gibson TJ (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nuc Acids Res 22, 4673-4680. Touloukian CE, Leitner WW, Robbins PF, Rosenberg SA, Restifo NP (2001) Mining the melanosome for tumor vaccine targets: P.polypeptide is a novel tumor-associated antigen. Cancer Res 61, 8100-8104. Ullmann AJ, Piesman J, Dolan MC, Iv WC ( 2003) A preliminary linkage map of the hard tick, Ixodes scapularis. Insect Mol Biol 12, 201-210. Valenzuela JG (2002) Exploring the messages of the salivary glands of Ixodes ricinus. Am J Trop Med Hyg 66, 223-224. Valenzuela JG, Francischetti IM, Pham VM, Garfield MK, Mather TN, Ribeiro JM (2002) Exploring the sialome of the tick Ixodes scapularis. J Exp Biol 205, 2843-2864.

Willadsen P (1997) Novel vaccines for ectoparasites. Vet Parasitol 71, 209-222. Willadsen P, Jongejan F (1999) Immunology of the tick-host interaction and the control of ticks and tick-borne diseases. Parasitol Today 15, 258-262. Wikel SK, Ramachandra RN, Bergman DK, Burkot TR, Piesman J (1997) Infestation with pathogen-free nymphs of the tick Ixodes scapularis induces host resistance to transmission of Borrelia burgdorferi by ticks. Infect Immun 65, 335-338. Zambrano N, Bimonte M, Arbucci S, Gianni D, Russo T, Bazzicalupo P (2002) feh-1 and apl-1, the Caenorhabditis elegans orthologues of mammalian Fe65 and beta-amyloid precursor protein genes, are involved in the same pathway that controls nematode pharyngeal pumping. J Cell Sci 115, 1411-1422

Back row from left to right: Jose C. Garcia-Garcia, Katherine M. Kocan, Jose de la Fuente; Front row: Consuelo Almazรกn and Edmour F. Blouin


Almazรกn et al: Expressed sequence tags in Ixodes scapularis


Gene Therapy and Molecular Biology Vol 7, page 61 Gene Ther Mol Biol Vol 7, 61-68, 2003

Delayed intratracheal injection of manganese superoxide dismutase (MnSOD)-plasmid/liposomes provides suboptimal protection against irradiationinduced pulmonary injury compared to treatment before irradiation Research Article

Michael W. Epperly, Hongliang Guo, Michael Bernarding, Joan Gretton, Mia Jefferson, Joel S. Greenberger* Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213

__________________________________________________________________________________ *Correspondence: Joel S. Greenberger, M.D., Professor and Chairman, Department of Radiation Oncology, University of Pittsburgh Cancer Institute, B346-PUH 200 Lothrop Street, Pittsburgh, PA 15213; Telephone: 412-647-3607; Fax: 412-647-6029; Email: Key words: MnSOD, reactive oxygen species, pulmonary fibrosis Abbreviations: OCT Optimum Cutting Temperature, ROS reactive oxygen species Received: 10 May 2003; Accepted: 10 June 2003; electronically published: June 2003

Summary Ionizing irradiation results in cellular production of reactive oxygen species (ROS), which cause DNA strand breaks, lipid peroxidation or other cellular damage leading to cell death. Antioxidant enzymes neutralize these ROS and provide cellular protection against sources of oxidative stress including ionizing irradiation. Intratracheal injection of the transgene for antioxidant protein MnSOD in plasmid/liposome (PL) complex 24 hours before irradiation has been shown to protect the murine lung from irradiation-induced organizing alveolitis/fibrosis. To determine whether intratracheal injection of MnSOD-PL at later times of macrophage infiltration and inflammation following irradiation had a detectable protective effect against irradiation fibrosis, control noninjected or MnSOD-PL complex injected C57BL/6J mice were irradiated to 20 Gy. Subgroups received a delayed injection of MnSOD-PL at day 1, 80, 90 or 100 after irradiation and all were followed for the development of organizing alveolitis/fibrosis. While mice injected with MnSOD-PL prior to irradiation demonstrated the best level of protection, we observed that mice injected with MnSOD-PL at 80 or 100 days after irradiation also showed significant protection of the lung compared to irradiated, control mice. Thus, delayed administration of MnSOD-PL has detectable radioprotective effects on C57BL/6J mouse lung but pre-irradiation injection remains the optimal treatment paradigm. irradiation induction of inflammatory cytokines including tumor necrosis factor-alpha (TNF-!), interleukin (IL)-1, and transforming growth factor-beta (TGF-") (Epperly et al, 1999c). Approximately 80 days after total lung irradiation C57BL/6J mice show increased TNF-! mRNA and this level decreases to background levels by 120 days following irradiation (Epperly et al, 1999c). As TNF-! mRNA expression decreases, that for TGF-" increases at 100 days after irradiation and continues to elevate during the development of the pathologic changes of organizing alveolitis/fibrosis (Epperly et al, 1999c). At the initiation

I. Introduction MnSOD is a mitochondrial localized enzyme which reduces superoxides produced during respiration (Quinlan et al, 1994; Fridovich, 1995). Therapeutic increase in expression of MnSOD by transgene administration protects tissues and organs from irradiation damage including lung (Epperly et al, 1998; 1999b; 2000a) esophagus, (Stickle et al, 1999; Epperly et al, 2001a; Epperly et al, 2000b) oral cavity (Guo et al, 2003) and bladder (Kanai et al, 2002). Increased expression of MnSOD at the time of irradiation also decreases the 61

Epperly et al: Late injection of MnSOD-PL protects against pulmonary fibrosis of organizing alveolitis/fibrosis, an increase in TGF-"1 is also detected (Epperly et al, 1999c). This late increase in TGF-"1 persists to day 120, the time at which TGF-"2 expression also increases (Epperly et al, 1999c). Levels of TGF-"2 remain elevated throughout the development of organizing alveolitis/fibrosis (Epperly et al, 1999c). We have previously demonstrated that intratracheal injections of MnSOD-PL complex or adenovirus containing the human MnSOD transgene 24 hours before irradiation protects the murine lung from irradiationinduced damage (Epperly et al, 1998; 1999b; 2000a, 2001b). Protection of the murine lung was measured as: (a) increased survival (Epperly et al, 1998, 1999b), (b) decreased pathologically quantifiable percent of lung showing organizing alveolitis/fibrosis, (Epperly et al, 1998; 1999b; 2000a, 2001b) and (c) decreased production of inflammatory cytokine mRNA for IL-1, TNF-!, and TGF-" (Epperly et al, 1998, Epperly et al, 2001b). The optimal schedule for administration of MnSOD-PL is not known. Injection prior to irradiation might be effective by preventing ROS mediated DNA damage or protecting against mitochondrial mediated apoptosis (Epperly et al, 1999a, 2002). However, injection following irradiation or at delayed time points when increases in TNF-! and TGF" mRNA are detected may reduce cytokine mediated production of ROS and also protect against tissue injury. To determine the optimal time of MnSOD-PL administration in the C57BL/6J mouse model, mice were injected with MnSOD-PL at 1, 80, 90, or 100 days after 20 Gy whole lung irradiation and data were compared to that with mice treated before irradiation. The mice were followed for development of organizing alveolitis/fibrosis and the percent of lung displaying organizing alveolitis/fibrosis was determined. Since MnSOD is a mitochondrial enzyme that dismutates superoxides only (Quinlan et al, 1994; Fridovich, 1995) the detection of increased survival in delayed injection groups of mice might indicate the presence of delayed increases in superoxide production, and thus be interpreted to play a role in the development of pulmonary fibrosis. In the present studies, we sought to determine whether delayed elevation of MnSOD by transgene therapy protects lungs from irradiation-induced pulmonary fibrosis.

B. Determination of organizing alveolitis/fibrosis When 80% of the control, irradiated mice had been sacrificed due to moribund condition as indicator of pulmonary organizing alveolitis/fibrosis, a subgroup of mice from each group was also sacrificed. The lungs were expanded with Optimum Cutting Temperature (OCT), removed, frozen in OCT, sectioned, and hematoxylin and eosin (H&E)-stained (Epperly et al, 1998; Epperly et al, 1999b). The sections were examined microscopically and the percent of organizing alveolitis/fibrosis was determined using an Optimus Image Analysis System (Epperly et al, 1998; Epperly et al, 1999b). In this system, the area of organizing alveolitis/fibrosis was compared to the area of the entire lobe, and the percent of lung developing organizing alveolitis/fibrosis calculated.

C. Statistics The irradiation survival curves of the different subgroups were compared with control irradiated mice using a Log Rank Test (Epperly et al, 1998; 1999b). The percent organizing alveolitis/fibrosis for the different subgroups of mice were compared using a Student’s t-Test (Epperly et al, 1998; Epperly et al, 1999b).

D. Animal protocols Protocols for animal usage were approved by the Institutional Animal care and Use Committee of the University of Pittsburgh. Veterinary support was provided by the Division of Laboratory Animal Research of the University of Pittsburgh.

III. Results A. Delayed injection of MnSOD-PL after lung irradiation improves survival To determine whether intratracheal injection of MnSOD-PL at delayed intervals following irradiation protected the murine lung from irradiation-induced damage, C57BL/6J mice were injected intracheally with 500 µg of plasmid DNA containing the MnSOD transgene at 1, 80, 90 or 100 days following 20 Gy irradiation to the pulmonary cavity. The mice were then followed for the development of organizing alveolitis/fibrosis and were sacrificed when moribund. Mice injected with MnSOD-PL at 80 or 100 days after irradiation showed a significant increase in survival compared to 20 Gy irradiated noninjected control mice while mice injected with MnSODPL at day 1 or 90 after irradiation showed a detectable but not significant increase in survival (Figure 1).

II. Materials and methods A. Injection of MnSOD-PL C57BL/6J were anesthetized using Nembutal and injected intratracheally with MnSOD-PL complexes (500 µg plasmid DNA in a volume of 50 µl plus 28 µl of lipofectant) (Epperly et al, 1998; 1999b) Twenty-four hours later the MnSOD-PLinjected mice plus control non-injected mice were irradiated to 20 Gy to the pulmonary cavity. The mice were shielded so that only the pulmonary cavity was irradiated. A subgroup of the control, irradiated mice was injected with MnSOD-PL 24 hours after irradiation. Other subgroups of each control irradiated or MnSOD-PL pre-irradiation injected mice were injected intratracheally a second time at day 80, 90 or 100 following irradiation. All mice were followed for development of organizing alveolitis/fibrosis, at which time the mice were sacrificed.

B. Pre-irradiation injection of MnSODPL affords optimal protection and is not further enhanced by a second delayed treatment Groups of mice were next injected with MnSOD-PL 24 hours before 20 Gy irradiation to the pulmonary cavity and then evaluated to determine whether a second injection of MnSOD-PL at 80, 90 or 100 days after irradiation resulted in an additional increase in survival compared to single pre-irradiation therapy. In this study,


Gene Therapy and Molecular Biology Vol 7, page 63 subgroups of mice received a second injection of MnSODPL at 80, 90 or 100 days later. The mice were then followed for the development of organizing alveolitis/fibrosis at which time they were sacrificed. As shown in Figure 2, there was no significant improvement in survival following a second injection of MnSOD-PL compared to the improvement seen with one preirradiation injection. A second injection at day 90 resulted in a significantly decreased survival compared to preinjection only. A comparison of these injection groups is shown in Figure 3. All subgroups of mice injected with MnSOD-PL 24 hours before irradiation had increased survival compared to mice that received no injection and only 20 Gy irradiation. Furthermore, mice injected with MnSODPL 24 hours prior to irradiation showed the best survival compared to all other groups of mice including those that received a second delayed injection.

The percent of lung displaying organizing alveolitis/fibrosis was calculated using an Optimus Image Analysis system as described in the Methods.

C. Decreased lung irradiation damage histopathologically correlates to MnSOD-PL mediated increased survival

Figure 2: Improved survival of mice injected with MnSOD-PL 24 hours before pulmonary irradiation is not further enhanced by a second delayed injection. C57BL/6J mice were injected with MnSOD-PL 24 hours before 20 Gy irradiation to the pulmonary cavity. Subgroups were injected with a second dose of MnSODPL at 80, 90 or 100 days after the initial irradiation. There was no significant improvement in the overall survival by a second injection 80, 90 or 100 days after irradiation (p=0.547, 0.039, and 0.309 respectively) compared to pre-irradiation administration above. A second injection at day 90 resulted in significantly decreased survival compared to pre-injection only. Groups contained #10 mice/group.

To determine whether the differences in survival of mice between groups correlated with histopathologic changes in the lung, specifically the development of organizing alveolitis/fibrosis, representatives of each subgroup of mice were euthanized at the time point when 80% of the 20 Gy irradiated, control mice were sacrificed due to moribund condition from developing organizing alveolitis/fibrosis. The lungs were expanded in OCT, removed, frozen in OCT, sectioned, and H&E-stained.

Figure 3: Pre-irradiation injection of MnSOD-PL provides optimal protection from lung irradiation damage. C57BL/6J mice were injected with MnSOD-PL and irradiated 24 hours later to 20 Gy to the lung, as were non-injected control mice. Subgroups of mice were subsequently injected with MnSOD-PL at day 1 (control, irradiated mice only), 80, 90 or 100 after irradiation. The mice were then followed for development of organizing alveolitis/fibrosis, and were sacrificed when moribund. All mice injected with MnSOD-PL 24 hours before irradiation had a significantly increased life span compared to control, irradiated mice (p $ 0.0066). Groups contained #10 mice/group.

Figure 1: Improved survival of pulmonary irradiated C57BL/6J mice injected with MnSOD-PL at day 1, 80, 90 or 100 following irradiation. C57BL/6J mice were irradiated to 20 Gy to the pulmonary cavity. Subgroups were subsequently injected with MnSOD-PL on day 1, 80, 90, or 100 following irradiation. The mice were followed for the development of organizing alveolitis/fibrosis, at which time they were sacrificed. These results demonstrated that injection of MnSOD-PL at day 80 or 100 following irradiation (or times when TNF-! and TGF-" production are increased) increases survival compared to irradiated, control mice (p = 0.0015 or 0.0005, respectively). Groups contained #10 mice/group.


Epperly et al: Late injection of MnSOD-PL protects against pulmonary fibrosis Mice injected with MnSOD-PL 24 hours before irradiation had a decreased percent of the lung displaying organizing alveolitis/fibrosis compared to the control, irradiated mice (Figures 4 and 5). Mice injected with MnSOD-PL on days 1, 80 or 100 following irradiation also had a decreased percent of the lung displaying organizing alveolitis/fibrosis compared to the control, irradiated mice. In mice injected with MnSOD-PL only on day 90 following irradiation there was no significant change detected in the percent of lung displaying organizing alveolitis/fibrosis. These results correlate to the survival curves in which mice injected on day 90 following irradiation showed no improvement in survival compared to 20 Gy control, irradiated mice. These data indicate that there is some protection against irradiation-induced organizing alveolitis/fibrosis afforded by delayed MnSOD-PL administration at the time of late cytokine elevation at 80-100 days after a single 20 Gy fraction to both lungs in the mouse model; however, MnSOD-PL administration prior to irradiation provides the optimal protection.

of neutralization of ROS might apply. Irradiation of bone marrow stromal cells in vitro results in continued production of nitric oxide for at least 24 hours following irradiation. The nitric oxide production from the irradiated cells was detected in attached, non-irradiated hematopoietic progenitor cells (Greenberger et al, 1996; Gorbunov et al, 2000). For the irradiation lung damage experiments in the present report it is known that pulmonary activated macrophages release nitric oxides and other ROS (Vujaskovic et al, 2002). Recent data suggest that macrophages are detectably present at delayed times at 100-120 days; however, we found detectable protection by MnSOD-PL injection at 80 days (Epperly et al, 2003). There was no histopathologically detectable irradiation damage to the lung at 80 to 100 days after irradiation, and only at 120-150 days were macrophages and fibrosis detected (Epperly et al, 1999c, 2003). There was no detectable migration of macrophages into areas of irradiation damage until after 100 days following irradiation (Epperly et al, 2003).

IV. Discussion MnSOD is one of three cellular superoxide dismutase enzymes responsible for reduction of superoxides produced in eukaryotes (Quinlan et al, 1994; Fridovich, 1995). Overexpression of MnSOD has been shown to protect cells and tissues from irradiation-induced damage, TNF-!, IL-1, serum factor withdrawal, and some chemotherapeutic drugs (Wong et al, 1989; Hirose et al, 1993; Urano et al, 1995; Li and Oberley, 1997; Epperly et al, 1999a)The enzyme MnSOD dismutates superoxides to hydrogen peroxide, which is then further reduced to oxygen and water by catalase, glutathione and glutathione peroxidase (Quinlan et al, 1994; Fridovich, 1995). It has been hypothesized that MnSOD protects irradiated cells by reducing superoxides that are produced during irradiation (Quinlan et al, 1994; Fridovich, 1995). Since irradiationinduced ROS are produced for less than a second, a protective effect of increased MnSOD expression has been logically thought to be required at the time of irradiation. Late injection of the MnSOD antioxidant transgene was carried out in the present studies to test the hypothesis that it might also be protective against ROS produced during the time of inflammatory cell mediated late effects of irradiation in the lung (Gossart et al, 1996; Bowler and Crapo, 2002). There is evidence showing that superoxides and ROS continue to be produced at delayed times following irradiation (Greenberger et al, 1996; Gorbunov et al, 2000) The hematopoietic line of 32D cl 3 cells and subclones 1F2 and 2C6 overexpressing MnSOD in vitro had similar levels of DNA strand breaks following irradiation; however, unlike the parent 32D cl 3 cells, 1F2 and 2C6 cells showed stabilized mitochondria and decreased mitochondrial membrane depolarization 3 to 6 hours after irradiation (Epperly et al, 2002). Protection of subclonal lines of cells with increased MnSOD expression at 3 to 6 hours following irradiation might indicate that superoxides were produced at this later time (Epperly et al, 2002). Alternatively, other actions of MnSOD independent

Figure 4: Pre-irradiation injection of MnSOD-PL provides optimal decrease in irradiation-induced pulmonary organizing alveolitis/fibrosis in 20 Gy irradiated mice. C57BL/6J mice were injected intratracheally with MnSOD-PL and irradiated 24 hours later along with non-injected control mice to 20 Gy. Subgroups of each large group were then injected with MnSOD-PL on day 1 (control, non-injected irradiated group only), 80, 90, or 100 after irradiation. The mice were then followed for the development of organizing alveolitis/fibrosis. When 80% of the control, irradiated mice had developed organizing alveolitis/fibrosis they were sacrificed, as were the surviving mice in all other subgroups. The lungs were expanded in OCT, removed, frozen in OCT, and sectioned. The sections were stained with H&E and examined for the percent of the lung developing organizing alveolitis/fibrosis using an Optimus Image Analysis System. All mice that received MnSOD-PL injection 24 hours before irradiation had reduced percent of the lung displaying organizing alveolitis/fibrosis compared to control, irradiated mice (p $ 0.0027). Control, irradiated mice injected with MnSOD-PL on day 1, 80 or 100 following irradiation also displayed reduced levels of organizing alveolitis/fibrosis compared to non-injected irradiated mice (p $ 0.0176). All groups of mice were #5 mice/group. Solid bars indicate MnSOD-PL pre-irradiation plus the other second time point. Open bars indicate delayed injection time point only.


Gene Therapy and Molecular Biology Vol 7, page 65

Figure 5: Delayed injection of MnSOD-PL provides detectable protection from irradiation-induced organizing alveolitis/fibrosis. C57BL/6J mice were injected with MnSOD-PL 24 hours before 20 Gy irradiation to the pulmonary cavity. Subgroups of the MnSODPL-injected mice were given a second injection of MnSOD-PL at day 80, 90 or 100. Subgroups of non-injected but 20 Gy irradiated control mice were injected with MnSOD-PL only at day 1, 80, 90 or 100 following irradiation. Once 80% of the non-injected control, irradiated mice had been sacrificed due to moribund condition resulting from organizing alveolitis/fibrosis, representative mice in each other group were sacrificed. The lungs were expanded in OCT, excised, frozen in OCT, sectioned, and H&E-stained. Representative photographs of the lungs at the time of sacrifice are shown for: non-irradiated mice (A); 20 Gy non-injected control, irradiated mice, (B); 20 Gy irradiated mice injected with MnSOD-PL at 80 days, ( C); MnSOD-PL-injected mice 24 hours before 20 Gy, (D); or mice injected with MnSOD-PL both 24 hours before irradiation and again at day 80 (E).


Epperly et al: Late injection of MnSOD-PL protects against pulmonary fibrosis The mechanism of action of TGF-" in cells of the lung may involve the mitochondria since TGF-"1 can lead to downregulation of Bcl-2 and Bcl-xl, which normally prevent apoptosis (Lafon et al, 1996; Herrera et al, 2001a). Overexpression of Bcl-2 suppresses the effects of TGF-" (Huang and Chou, 1998). Following exposure to TGF-" there is also a loss of mitochondrial membrane potential, release of cytochrome-C, and activation of caspase-3 (Herrera et al, 2001a). TGF-"1 activates caspase- 3, 8 and 9, which precede the loss of mitochondrial membrane potential (Herrera et al, 2001b). Activation of caspase-8 results in cleavage of Bid and Bcl-xl, which may lead to an amplification loop resulting in the mitochondrial mediated apoptosis (Zha et al, 2000). Irradiation of murine bone marrow stromal cell line D2XRII in vitro induces release of TGF-" into the culture medium (Greenberger et al, 1996). Co-cultivation of 32D cl 3 cells or subclones 1F2 or 2C6 overexpressing MnSOD with irradiated bone marrow stromal cells resulted in higher levels of intracellular ROS in the non-irradiated 32D cl 3, 1F2 or 2C6 cells compared to cells co-cultivated with nonirradiated stromal cell lines (Greenberger et al, 1996). The MnSOD overexpressing subclonal line 1F2 or 2C6 formed more cobblestone islands on the irradiated stromal cells than 32D cl 3 cells (Greenberger et al, 1996). Increased MnSOD activity in 1F2 or 2C6 cells may have resulted in a decrease in ROS, thus allowing for greater attachment of the MnSOD overexpressing cell lines to the irradiated stromal cells. Therefore, injections of MnSOD-PL into the lung at day 100 when TGF-" levels are beginning to increase may inhibit ROS production, and/or stabilize the bronchoalveolar cell or endothelial cell mitochondria, preventing some (but not all) of the late effects of irradiation damage to the lung. We are currently exploring this possible mechanism. The present report indicates that a single administration of MnSOD-PL 24 hours prior to 20 Gy lung irradiation is significantly more effective than administration at any of four post-irradiation time points ranging from 1-100 days after irradiation. We did not evaluate time points between 1 and 80 days as there was no histopathologic or other evidence to suggest that initiation steps in the late organizing alveolitis/fibrosis response began prior to 80 days. Our results may help explain the dynamics of late irradiation pulmonary injury. One interpretation of the results is that it represents evidence of a pleiotropic effect of ionizing irradiation on several cellular and physiologic targets within the lung. Initiation events at the time of irradiation may lead to a multiplicity of effectuating events beginning at around day 100 and leading to rapid organizing alveolitis/fibrosis. Prevention of some of the initiating events by MnSOD-PL administration prior to irradiation may have a significantly greater effect at reducing the overall outcome compared to modulation of some of the late effectuating events by MnSOD-PL administration at that time. For example, neutralization of free radical moieties induced by irradiation at day 0 by overexpression of MnSOD at that time may be a significant early event which impacts on multiple downstream/delayed effectuating targets (macrophage migration, fibroblast migration into the lung,

Therefore, MnSOD-PL action on ROS produced by inflammatory cells such as macrophages at 80 days does not appear to explain the present data implying superoxides might have been produced by macrophages and neutralized by injections of MnSOD-PL at 80 or 100 days after irradiation (Epperly et al, 2003). We previously demonstrated that at 80 days after irradiation of the mouse lung there is an increase in TNF-! mRNA expression which decreases to background level by day 120 (Epperly et al, 1999b). This increase is accompanied by increased expression of mRNA for TGF" at day 100. Initially, there is an increase in TGF-"1 isoform until day 120 at which time TGF-"1 expression decreases, and TGF-"2 expression increases and stays elevated until development of organizing alveolitis/fibrosis (Epperly et al, 1999b). The detectable protection by MnSOD-PL injection at day 80 might have been attributed to an effect on the TNF-! elevation at that time point. ROS production might increase TNF-! expression at day 80 leading to further increased ROS production (Haddad, 2002). Treatment of alveolar epithelial cells with a ROS generating system results in increased TNF-! expression and a depletion of glutathione (Haddad, 2002). TNF-! treatment inhibits myogenesis by causing a decrease in glutathione levels and elevation of ROS (Langen et al, 2002). Pre-treatment with the anti-oxidant N-acetyl-1cysteine (NAC) restored the formation of multi-nucleated myotubes, indicating that myogenesis inhibition was attributable to ROS expression (Langen et al, 2002). Pretreatment of HELA cells with gammaglutamylcysteinylglycine inhibits TRAIL-induced apoptosis (Lee et al, 2002). TNF-! expression may increase the production of ROS and result in a further increase in TNF-! expression. ROS response to and induction of TNF-! expression may be a cyclic mechanism in the lung at day 80, and MnSOD-PL treatment at this time point may have interrupted the cycle. Further studies will be required to explain the protection by injections of MnSOD-PL at 80 days after irradiation. Pulmonary increases in TGF-"1 and TGF-"2 mRNA at 100 to 120 days after irradiation have been detected (Epperly et al, 1999b). It has been demonstrated that ROS can also increase TGF-" expression (Bellocq et al, 1999) The treatment of human alveolar lung cell line A549 with xanthine and xanthine oxidase or nitric oxide generator Snitroso-N-acetyl-penicillamine (SNAP) leads to release of TGF-"1 (Bellocq et al, 1999). The xanthine-xanthine oxidase induced release of TGF-"1 can be inhibited by the addition of catalase but not superoxide dismutase, implicating the involvement of hydrogen peroxide (Bellocq et al, 1999) TGF-"1 has been demonstrated to induce production of extracellular hydrogen peroxide in human fibroblasts that mediate oxidative dityrosinedependent cross-linking of ECM (Larios et al, 2001). TGF-" and hydrogen peroxide have been observed to induce connective tissue factor (CTGF) that then induces collagen type 1 and fibronectin, a deposition leading to fibrosis (Park et al, 2001).


Gene Therapy and Molecular Biology Vol 7, page 67 (1999b) Intratracheal injection of adenovirus containing the human MnSOD transgene protects athymic nude mice from irradiation-induced organizing alveolitis. Int J Radiat Oncol Phys 43, 169-181. Epperly MW, Travis EL, Sikora C, Greenberger JS (1999c) Magnesium superoxide dismutase (MnSOD) plasmid/liposome pulmonary radioprotective gene therapy, Modulation of irradiation-induced mRNA for IL-1, TNF-!, and TGF-" correlates with delay of organizing alveolitis/fibrosis. Biol Blood Bone Marrow Transplant 5, 204-214. Epperly MW, Epstein CJ, Travis EL, Greenberger JS (2000a) Decreased pulmonary radiation resistance of manganese superoxide dismutase (MnSOD)-deficient mice is corrected by human manganese superoxide dismutaseplasmid/liposome (SOD2-PL) intratracheal gene therapy. Radiat Res 154, 365-374. Epperly MW, Sikora C, Defilippi S, Bray J, Koe G, Liggitt D, Luketich JD, Greenberger JS (2000b) Plasmid/liposome transfer of the human manganese superoxide dismutase (MnSOD) transgene prevents ionizing irradiation-induced apoptosis in human esophagus organ explant culture. Radiat Oncol Invest 90, 128-137. Epperly MW, Gretton JA, DeFilippi SJ, Greenberger JS, Sikora CA, Liggitt D, Koe G (2001a) Modulation of radiationinduced cytokine elevation associated with esophagitis and esophageal stricture by manganese superoxide dismutaseplasmid/liposome (SOD-PL) gene therapy. Radiat Res 155, 2-14. Epperly MW, Travis EL, Whitsett JA, Raineri I, Epstein CJ, Greenberger JS (2001b) Overexpression of manganese superoxide dismutase (MnSOD) in whole lung or alveolar type II (AT-II) cells of MnSOD transgenic mice does not provide intrinsic lung irradiation protection. Radiat Oncol Invest 96, 11-21. Epperly MW, Guo HL, Gretton JE, Greenberger JS. (2003) Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis. AJRCMB, 29, in press. Epperly MW, Sikora CA, DeFilippi SJ, Gretton JA, Zhan Q, Kufe DW, Greenberger JS (2002) MnSOD inhibits irradiation-induced apoptosis by stabilization of the mitochondrial membrane against the effects of SAP kinases p38 and Jnk1 translocation. Radiat Res 157, 568-577. Fridovich I (1995) Superoxide radical and superoxide dismutases. Annu Rev Biochem 64, 97-112. Gorbunov NV, Pogue-Geile KL, Epperly MW, Bigbee WL, Draviam R, Day BW, Wald N, Watkins SC, Greenberger JS (2000) Activation of the nitric oxide synthase 2 pathway in the response of bone marrow stromal cells to high doses of ionizing radiation. Radiat Res 154, 73-86. Gossart S, Cambon C, Orfila C, Seguelas MH, Lepert JC, Rami J, Carre P, Pipy B (1996) Reactive oxygen intermediates a regulators of TNF-alpha production in rat lung inflammation induced by silica. J Immun 156, 1540-1548. Greenberger JS, Epperly MW, Zeevi A, Brunson KW, Goltry KL, Pogue-Geile KL, Bray J, Berry L (1996) Stromal cell involvement in leukemogenesis and carcinogenesis. In Vivo 10, 1-18. Guo HL, et al. (2003) Prevention of irradiation-induced oral cavity mucositis by plasmid/liposome delivery of the human manganese superoxide dismutase (MnSOD) transgene. Radiat Res 159, 361-370. Haddad JJ (2002) Redox regulation of pro-inflammatory cytokines and IkappaB-alpha/NF-kappaB nuclear

endothelial upregulation of adhesion molecules, and other components of the fibrosis response not yet elucidated). In contrast, modulation of some of the effectuating events by MnSOD-PL administration at the late time points may have a significantly decreased effect in preventing late lesion simply due to the multiplicity of events already in progress, and that many of these may be unrelated to the free radical neutralization capacity of MnSOD at that late time point. The same mechanism explaining a greater effect of treatment prior to irradiation might also hold true for anti-apoptotic effects of MnSOD overexpression in the mitochondria. The present data also indicate that significant protective effects afforded by MnSOD-PL administration prior to irradiation were not significantly further improved by a second delayed administration. This result may simply be attributable to the dominant mechanism of prevention of initiating events compared to effectuating events. The present results add support to utilization of fractionated inhalation of freeze-dried MnSOD-PL during courses of fractionated radiotherapy which would be appropriate to the clinical translational model of normal lung irradiation protection in lung cancer patients receiving chemoradiotherapy over a 60-day time course. Fractionation experiments currently in progress incorporate twice weekly inhalation of freeze-dried MnSOD-PL by mice receiving 24 fractions of irradiation during 35 days. The present observation of a lack of toxicity of a second delayed administration of MnSOD-PL in the present data supports the concept that multi-fraction administration of this gene therapy technique should not exacerbate and may decrease pulmonary irradiation damage.

Acknowledgments This research has been supported by the National Institutes of Health, Grant #R01-HL-60132

References Bellocq A, Azoulay E, Marullo S, Flahault A, Fouqueray B, Philippe C, Cadranel J, Baud L (1999) Reactive oxygen and nitrogen intermediates increase transforming growth factor beta1 release from human epithelial alveolar cells through two different mechanisms. AJRCMB 21, 128-136. Bowler RP, Crapo JD (2002) Oxidative stress in airways, is there a role for extracellular superoxide dismutase? AJRCCM 166, 38-43. Epperly MW, Bray JA, Kraeger S, Swacka R, Engelhardt JF, Travis E, and Greenberger (1998) Prevention of late effects of irradiation lung damage by manganese superoxide dismutase gene therapy. Gene Ther 5, 196-208. Epperly MW, Bray JA, Esocobar P, Bigbee WL, Watkins S, Greenberger JS (1999a) Overexpression of the human MnSOD transgene in subclones of murine hematopoietic progenitor cell line 32D cl 3 decreases irradiation-induced apoptosis but does not alter G2/M or G1/S phase cell cycle arrest. Radiat Oncol Invest 7, 331-342. Epperly, MW, Bray, JA, Krager, S, Berry, LM, Gooding, W, Engelhardt, JF, Zwacka, R, Travis,EL, and Greenberger, JS


Epperly et al: Late injection of MnSOD-PL protects against pulmonary fibrosis translocation and activation. Biochem Biophys Res Commun 296, 847-856. Herrera B, Alvarez AM, Sanchez A, Fernandez M, Roncero C, Benito M, Fabregat I (2001) Reactive oxygen species (ROS) mediates the mitochondrial-dependent apoptosis induced by transforming growth factor (beta) in fetal hepatocytes. FASEB J 15, 741-751. Herrera B, Fernandez M, Alvarez AM, Roncero C, Benito M, Gil J, Fabregat I (2001) Activation of caspases occurs downstream from radical oxygen species production, Bcl-xl down-regulation and early cytochrome C release in apoptosis induced by transforming growth factor beta in rat fetal hepatocytes. Hepatology 34, 548-556. Hirose K, Longo DL, Oppenheim JJ, Matsushima K (1993) Overexpression of mitochondrial manganese superoxide dismutase promotes the survival of tumor cells exposed to IL-1, TNF, selected anticancer drugs and ionizing irradiation. FASEB J 7, 361-368. Huang YL, Chou CK (1998) Bcl-2 blocks apoptotic signal of transforming growth factor-beta in human hepatoma cells. J Biomed Sci 5, 185-191. Kanai AJ, Zeidel ML, Lavelle JP, Greenberger JS, Birder LA, de Groat WC, Apodaca GL, Meyers SA, Ramage R, Epperly MW (2002) Manganese superoxide dismutase gene therapy protects against irradiation-induced cystitis. Am J Physiol Renal Physiol. 283, 1304-1312. Lafon C, Mathieu C, Guerrin M, Pierre O, Vidal S, Valette A (1996) Transforming growth factor beta 1-induced apoptosis in human ovarian carcinoma cells, protection by the antioxidant N-acetylcysteine and Bcl-2. Cell Growth Diff 7, 1095-1104. Langen RC, Schols AM, Kelders MC, Van Der Velden JL, Wouters EF, Janssen-Heininger YM (2002) Tumor necrosis factor-alpha inhibits myogenesis through redox-dependent and independent pathways. Am J Physiol Cell Physiol 283, 714-721. Larios JM, Budhiraja R, Fanburg BL, Thannickal VJ (2001) Oxidative protein cross-linking reactions involving L-

tyrosine in transforming growth factor-beta1-stimulated fibroblasts. J Biol Chem 276, 17437-17441. Lee MW, Park SC, Kim JH, Kim IK, Han KS, Kim KY, Lee WB, Jung YK, Kim SS (2002) The involvement of oxidative stress in tumor necrosis factor (TNF)-related apoptosisinducing ligand (TRAIL)-induced apoptosis in HeLa cells. Cancer Letters 182, 75-82. Li JJ, Oberley LW (1997) Overexpression of manganesecontaining superoxide dismutase confers resistance to the cytotoxicity of TNF- ! and/or hyperthermia. Cancer Res 57, 1991-1998. Park SK, Kim J, Seomun Y, Choi J, Kim DH, Han IO, Lee EH, Chung SK, Joo CK (2001) Hydrogen peroxide is a novel inducer of connective tissue growth factor. Biochem Biophys Res Commun 284, 966-971. Quinlan T, Spivack S, Mossman BT (1994) Regulation of antioxidant enzymes in lung after oxidant injury. Environ Health Perspect 102, 79-87. Stickle RL, Epperly MW, Klein E, Bray JA, Greenberger JS (1999) Prevention of irradiation-induced esophagitis by intraesophageal plasmid/liposome delivery of the human manganese superoxide dismutase (MnSOD) transgene. Radiat Oncol Invest 7, 204-217. Urano M, Kuroda M, Reynolds R, Oberley TD, St Clair DK (1995) Expression of manganese superoxide dismutase reduces tumor control radiation dose, gene radiotherapy. Cancer Res 55, 2490-2493. Vujaskovic Z, Feng QF, Rabbani ZN, Anscher MS, Samulski TV, Brizel DM (2002) Radioprotection of lungs by amifostine is associated with reduction in profibrogenic cytokine activity. Radiat Res 157, 656-660. Wong GHW, Elwell JH, Oberley LW, Goeddel DV (1989) Manganese superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor. Cell 58, 923-931. Zha J, Weiler S, Oh KJ, Wei MC, Korsmeyer SJ (2000) Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis. Science 290, 1761-1765.


Gene Therapy and Molecular Biology Vol 7, page 69 Gene Ther Mol Biol Vol 7, 69-73, 2003

Regulation of vascular endothelial growth factor by hypoxia Mini Review

Ilana Goldberg-Cohen*, Nina S Levy, Andrew P Levy Technion Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

__________________________________________________________________________________ *Correspondence: Ilana Goldberg-Cohen, Technion Faculty of Medicine, Haifa, Israel; Tel 011-972-4-8295202; Fax 011-972-48514103; email: Key words: VEGF (vascular endothelial growth factor), hypoxia, HuR Received: 04 June 2003; Accepted: 27 June 2003; electronically published: July 2003

Summary The past few decades have singled out the growth of new blood vessels, termed angiogenesis, as a key process in the course of normal development as well as in pathological disease processes. VEGF, an endothelial cell specific mitogen, is now accepted as a key mediator of angiogenic events and as such may be a powerful tool in manipulating the growth of new blood vessels. VEGF expression is regulated to a great extent by hypoxia. The lack of oxygen to supply a tissue triggers several molecular mechanisms that increase VEGF mRNA transcription, stability and translation, and thus upregulate the expression of VEGF protein. This review focuses on the increase in VEGF mRNA stability through its recognition by the RNA binding protein HuR. Binding of HuR to its cognate site on the 3´UTR of VEGF mRNA results in a several fold increase in VEGF mRNA stability, possibly due to the masking of a nearby binding site for ribonucleases. Mastering the regulatory mechanisms of VEGF expression is of great importance for the future manipulation of VEGF and angiogenesis in the disease setting. different signal transduction cascades when activated and thus mediate separate responses to VEGF (Waltenberger et al, 1994; Yoshida et al, 1996). A third receptor family unrelated to the receptor families described above, the neuropillin receptor family, binds mainly to VEGF165 and its members are thought to act as coreceptors (Soker et al, 1996).

I. Introduction The ability to grow new blood vessels to supply the needs of a growing tissue is critical in both physiological processes such as embryogenesis and in pathological processes that include tumor growth and metastasis. Vascular Endothelial Growth Factor (VEGF), an endothelial cell specific mitogen, (Ferrara and Henzel, 1989; Plouet et al, 1989) is a critical mediator in the establishment of new blood vessels in both vasculogenesis, the de novo foundation of vascular systems (Risau, 1997), and angiogenesis, the development of new blood vessels from a pre existing network (Risau, 1997). The VEGF gene, found on chromosome 6p21 (Vincenti et al, 1996), consists of eight exons separated by seven introns and is alternatively spliced to form five different VEGF isoforms, the most prominent being VEGF165, that differ in length and ability to bind heparin (Houck et al, 1991). Two tyrosine kinase family receptors flt-1 (VEFGR1) and flk-1 (VEGFR2) were identified as VEGF receptors (de Vries et al, 1992; Terman et al, 1992). They have a similar structure of seven immunoglobulin-like loops in their extracellular domain, a transmembrane region and a tyrosine kinase consensus sequence (Shibuya et al, 1990; Terman et al, 1991). The two receptors induce

II. Regulation expression




In light of its potency and importance in vasculature development, VEGF itself is carefully regulated to provide for the appropriate amount of VEGF at the appropriate time. Growth factors, cytokines and other extracellular molecules such as PDGF, TNF! and others influence angiogenesis by governing VEGF expression (Deroanne et al, 1997; Finkenzeller et al, 1997; Frank et al, 1995; Pertovaara et al, 1994; Ryuto et al, 1996). Oncogenes and tumor suppressor genes also play a role in VEGF modulation as in the case of the von Hipple Lindau tumor suppressor gene whose absence or inactivation dramatically increases VEGF expression (Iliopoulos et al, 1996; Maher and Kaelin, 1997; Mukhopadhyay et al, 1997).


Goldberg-Cohen et al: Regulation of vascular endothelial growth factor by hypoxia One of the key factors, which controls VEGF expression, is oxygen tension. A growing mass such as an embryo or a tumor is in need of oxygen when it can no longer rely on diffusion to sustain itself. The lack of oxygen, termed hypoxia, induces a cascade of events, which increase VEGF expression and ultimately the growth of new blood vessels.

abolished the destabilizing properties of the entire AU rich element (Akashi et al, 1994; Chen et al, 1994). The degradation of mRNAs containing AU rich elements in their 3´ UTR is facilitated by the binding of trans-acting factors which may promote exonuclease as well as site-specific endonucleolytic events. Tristetraproline (TTP) and AUF1 are two such trans-acting RNA binding proteins that bind AU rich elements and destabilize the mRNAs carrying these sequences (Brewer, 1991; Carballo et al, 1998; Lai and Blackshear, 2001). While AU rich elements allow for the rapid degradation of mRNAs they also appear to be able to bind trans-acting factors that act to increase mRNA stability under certain circumstances as discussed below for VEGF mRNA. Like GM-CSF, the 3´UTR of VEGF mRNA consists of multiple AU rich elements that render it vulnerable to rapid degradation. However, under hypoxic conditions, RNA binding proteins recognize and bind to their cognate AU rich sites on the 3´UTR of VEGF mRNA, increasing its stability and thus its expression several fold.

III. Hypoxic regulation of VEGF Hypoxia increases VEGF expression by several mechanisms which act at the level of mRNA transcription, stabilization and translation.

A. Upregulation of VEGF mRNA transcription VEGF transcription, as well as that of several other hypoxia inducible genes such as the glycolytic enzymes and erythropoietin, is increased with hypoxia. Most of these genes have Hypoxia Response Elements (HREs) that bind a heterodimeric helix-loop-helix transcription factor called Hypoxia Inducible Factor 1 (HIF-1) (Wang and Semenza, 1995; Semenza et al, 1996). HIF-1 binds to its recognition site on VEGF 5´ promoter and together with other trans acting factors mediates the increase in VEGF transcription with hypoxia. Several other transcription factors such as AP-1 and CREB also appear to influence the hypoxic induction of VEGF transcription most likely via direct interaction with HIF-1 (Abate et al, 1990).

IV. HuR A prominent member of the ARE binding protein family that acts to increase mRNA stability with hypoxia is HuR. This RNA binding protein belongs to the Embryonic Letal Abnormal Visual (ELAV) protein family first described in Drosophila (Robinow et al, 1988). The founding member, ELAV, is expressed immediately following neuroblast differentiation into neurons and is involved in the subsequent neuronal differentiation and maintenance (Robinow and White, 1991; Campos et al, 1985). Further studies identified four human homologues that were characterized as tumor antigens (Szabo et al, 1991). Three of the human ELAV-like proteins are expressed solely in terminal differentiation of neurons and neuroendocrine tumors (King et al, 1994; Barami et al, 1995; Jain et al, 1997) while the fourth, termed HuR, is found in proliferating cells and in tumors throughout the body (Ma et al, 1996). Classification as tumor antigens gave rise to extensive research into the essence of their RNA binding properties and resulted in the identification of three highly conserved RNA recognition motifs. Two of the RNA recognition motifs are in tandem separated from the third by a basic segment (Kenan et al, 1991). Subsequent studies confirmed that the ELAV-like proteins are prone to bind AU rich elements present in the 3´UTRs of mRNAs as well as to their polyA tails, which may contribute to their ability to protect mRNAs from ribonuclease degradation (Ma et al, 1997). As discussed above, HuR, the only ELAV family member not restricted to the nervous system but rather expressed throughout the body, is involved in increasing VEGF mRNA stability with hypoxia by binding to an AU rich recognition site on the VEGF mRNA 3´UTR. A study investigating the binding of HuR to c-fos mRNA identified a high affinity site containing three AU rich motifs AUUUA, AUUUUA, and AUUUUUA, all of which are critical for maximal binding (Ma et al, 1996). The requirement for a nonspecific number of U residues in

B. Hypoxic regulation of VEGF mRNA translation VEGF mRNA has an unusually long 5´ untranslated region (5´ UTR) containing stable secondary structures and a short in-frame initiation and termination codons. This significantly inhibits initiation of protein synthesis by the classical model of the cap-dependant ribosome scanning. VEGF mRNA can also be translated in a capindependent manner through an Internal Ribosome Entry Site (IRES). Under hypoxic conditions, and other conditions of stress, cap dependant translation is reduced. The presence of an IRES site allows the translation of VEGF and other IRES containing mRNAs to continue (Akiri et al, 1998; Stein et al, 1998).

C. Hypoxic stabilization of VEGF mRNA The half life of VEGF mRNA, like that of several other cytokine and oncogene mRNAs, is very short. Increased stability of a mRNA renders it more accessible to the translational machinery and thus increases the amount of its gene product. Shaw and Kamen (1986) reported a considerable decrease in the stability of "globin mRNA when an AU-rich element (ARE) from the 3´UTR of GM-CSF was introduced 3´ to the "-globin gene (Shaw and Kamen, 1986). Further studies indicated that the pentameric sequence AUUUA is necessary but not sufficient to induce degradation of mRNAs and mutations that specifically interrupted this pentameric sequence


Gene Therapy and Molecular Biology Vol 7, page 71 the target sequence points to an inclination towards binding a particular structure rather than a primary sequence (Kim et al, 1974).

it binds and stabilizes VEGF.

VI. Perspectives The manipulation of angiogenic events as a therapeutic tool marks the dawn of a new era in treating numerous afflictions in medicine today. Whether angiogenesis is stimulated to feed the ischemic heart or anti angiogenic agents are applied to prevent tumor growth and metastases, understanding and controlling angiogeneic factors will be critical to the outcome. As a major participant in the angiogenic process, VEGF has been the target of a worldwide research effort in the past two decades. Cancer research has focused on the importance of VEGF in tumor growth and metastases and the findings support this notion. VEGF is now recognized as a key regulator in tumor induced angiogenesis and several anti VEGF treatments that utilize antibodies against VEGF and VEGF receptors have successfully blocked tumor growth in mouse models (Kim et al, 1993; Ferrara, 1999; Gerber et al, 2000; Lee et al, 2000). Cardiovascular research has also benefited from the advances in VEGF research. Gene therapy techniques that augment VEGF expression in the diseased heart or ischemic leg are currently under way. The success of these VEGF treatments will depend to a large extent on our understanding the complex regulation of VEGF. In addition, deciphering the molecular mechanisms that govern the expression of VEGF and other angiogenic factors will give rise to new possibilities not only in the ever growing field of vascular biology but also in the vast area of embryonic development and tissue transplantation.

V. HuR binding site on VEGF mRNA 3´UTR The 3´UTR of VEGF mRNA contains long stretches of AU residues, which confer rapid mRNA degradation through the binding of ribonucleases to the AU rich elements. However, under hypoxic conditions, these AU rich elements allow the binding of RNA binding proteins such as HuR, which block binding of ribonucleases and thus increase the stability of the VEGF mRNA and VEGF expression under hypoxia (Stein et al, 1995;; Damert et al, 1997; Claffey et al, 1998). Attempts to characterize the minimal binding site of HuR on the 3´UTR of VEGF mRNA that is still able to confer increased stability with hypoxia were carried out in our lab and resulted in the identification of a 40 base pair element at position 1285 of the 3´UTR of VEGF mRNA (nucleotides 1285-1325 of the VEGF 3´UTR, GenBank accession number U22372)(Goldberg-Cohen et al, 2002). Transient cotransfection of a vector carrying the 40 base pair element positioned 3´ to the luciferase reporter gene and a plasmid overexpressing HuR showed an increase in reporter activity that correlated with an increase in cotransfected HuR. Furthermore, when incubated overnight under hypoxic conditions, cells transfected with the reporter vector containing the 40 base pair element had greater reporter activity than cells transfected with reporter vector alone. These observations were confirmed in an in vitro model where the stability of an RNA containing the 40 base pair element was shown to be increased in an RNA degradation assay in the presence of HuR. RNase T1 and lead protection assays mapped the HuR binding site to nucleotides 23-39 of the 40 base pair element. Deletion of the HuR specific binding site dramatically reduced reporter activity in the transient transfection assay (Goldberg-Cohen et al, 2002). In view of the ability of HuR to bind and stabilize VEGF mRNA with hypoxia, a model was constructed. In this model, under normoxic conditions VEGF mRNA is extremely unstable by virtue of its recognition by ribonucleases that bind the VEGF mRNA 3´UTR and cause its rapid degradation. This labile character of VEGF mRNA can be overcome under hypoxic conditions through the binding of HuR to its recognition site on the 3´UTR of VEGF mRNA rendering it less vulnerable to ribonuclease digestion. The hypoxic regulation of HuR is not completely understood. Under normoxic conditions the bulk of HuR is sequestered in the nucleus. Under hypoxia, cytoplasmic HuR levels increase with no apparent increase in total HuR levels (Levy et al, 1998). This would suggest nucleocytoplasmic transport of HuR and indeed it was reported that HuR possess a shuttling signal termed HuR Nucleocytoplasmic Shuttling sequence (HNS) that may be significant to the process (Fan and Steitz, 1998). It remains to be investigated whether HuR binds VEGF mRNA in the nucleus and is transported to the cytoplasm as a complex or whether HuR is first transported to the cytoplasm where

References Abate C, Luk D, Gagne E, Roeder RG, Curran T (1990) Fos and jun cooperate in transcriptional regulation via heterologous activation domains. Mol Cell Biol 10, 5532-5535. Akashi M, Shaw G, Hachiya M, Elstner E, Suzuki G, Koeffler P (1994) Number and location of AUUUA motifs: role in regulating transiently expressed RNAs. Blood 83, 31823187. Akiri G, Nahari D, Finkelstein Y, Le SY, Elroy-Stein O, Levi BZ (1998) Regulation of vascular endothelial growth factor (VEGF) expression is mediated by internal initiation of translation and alternative initiation of transcription. Oncogene 17, 227-236. Barami K, Iversen K, Furneaux H, Goldman SA (1995) Hu protein as an early marker of neuronal phenotypic differentiation by subependymal zone cells of the adult songbird forebrain. J Neurobiol 28, 82-101. Brewer G (1991) An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro. Mol Cell Biol 11, 2460-2466. Campos AR, Grossman D, White K (1985) Mutant alleles at the locus elav in Drosophila melanogaster lead to nervous system defects. A developmental-genetic analysis. J Neurogenet 2, 197-218. Carballo E, Lai WS, Blackshear PJ (1998) Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin. Science 281, 1001-1005.


Goldberg-Cohen et al: Regulation of vascular endothelial growth factor by hypoxia Chen CY, Chen TM, Shyu AB (1994) Interplay of two functionally and structurally distinct domains of the c-fos AU-rich element specifies its mRNA-destabilizing function. Mol Cell Biol 14, 416-426. Claffey KP, Shih SC, Mullen A, Dziennis S, Cusick JL, Abrams KR, et al (1998) Identification of a human VPF/VEGF 3' untranslated region mediating hypoxia-induced mRNA stability. Mol Biol Cell 9, 469-481. Damert A, Machein M, Breier G, Fujita MQ, Hanahan D, Risau W, et al (1997) Up-regulation of vascular endothelial growth factor expression in a rat glioma is conferred by two distinct hypoxia-driven mechanisms. Cancer Res 57, 3860-3864. De Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams LT (1992) The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 255, 989-991. Deroanne CF, Hajitou A, Calberg-Bacq CM, Nusgens BV, Lapiere CM (1997) Angiogenesis by fibroblast growth factor 4 is mediated through an autocrine up-regulation of vascular endothelial growth factor expression. Cancer Res 57, 55905597. Fan XC, Steitz JA (1998) HNS, a nuclear-cytoplasmic shuttling sequence in HuR. Proc Natl Acad Sci U.S.A 95, 1529315298. Ferrara N (1999) Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int 56, 794-814. Ferrara N, Henzel WJ (1989) Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 161, 851858. Finkenzeller G, Sparacio A, Technau A, Marme D, Siemeister G (1997) Sp1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for platelet-derived growth factor-induced gene expression. Oncogene 15, 669-676. Frank S, Hubner G, Breier G, Longaker MT, Greenhalgh DG, Werner S (1995) Regulation of vascular endothelial growth factor expression in cultured keratinocytes. Implications for normal and impaired wound healing. J Biol Chem 270, 12607-12613. Gerber HP, Kowalski J, Sherman D, Eberhard DA, Ferrara N. (2000) Complete inhibition of rhabdomyosarcoma xenograft growth and neovascularization requires blockade of both tumor and host vascular endothelial growth factor. Cancer Res. 60, 6253-6258. Goldberg-Cohen I, Furneauxb H, Levy AP (2002) A 40-bp RNA element that mediates stabilization of vascular endothelial growth factor mRNA by HuR. J Biol Chem 277, 1363513640. Houck KA, Ferrara N, Winer J, Cachianes G, LI B, Leung DW (1991) The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol 5, 1806-1814. Iliopoulos O, Levy AP, Jiang C, Kaelin WGJ, Goldberg MA (1996) Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc Natl Acad Sci U.S.A 93, 10595-10599. Jain RG, Andrews LG, Mcgowan KM, Pekala PH, Keene JD (1997) Ectopic expression of Hel-N1, an RNA-binding protein, increases glucose transporter (GLUT1) expression in 3T3-L1 adipocytes. Mol Cell Biol 17, 954-962. Kenan DJ, Query CC, Keene JD (1991) RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci 16, 214-220.

Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, et al (1993) Inhibition of vascular endothelial growth factorinduced angiogenesis suppresses tumour growth in vivo. Nature 362, 841-844. Kim SH, Suddath FL, Quigley GJ, Mcpherson A, Sussman JL, Wang AH, et al (1974) Three-dimensional tertiary structure of yeast phenylalanine transfer RNA. Science 185, 435-440. King PH, Levine TD, Fremeau RTJ, Keene JD (1994) Mammalian homologs of Drosophila ELAV localized to a neuronal subset can bind in vitro to the 3' UTR of mRNA encoding the Id transcriptional repressor. J Neurosci 14, 1943-1952. Lai WS, Blackshear PJ. (2001) Interactions of CCCH zinc finger proteins with mRNA: tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur in the absence of a poly(A) tail. J Biol Chem 276, 23144-23154. Lee CG, Heijn M, Di Tomaso E, Griffon-Etienne G, Ancukiewicz M, Koike C, et al (2000) Anti-Vascular endothelial growth factor treatment augments tumor radiation response under normoxic or hypoxic conditions. Cancer Res 60, 5565-5570. Levy NS, Chung S, Furneaux H, Levy AP (1998) Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR. J Biol Chem 273, 64176423. Ma WJ, Cheng S, Campbell C, Wright A, Furneaux H (1996) Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein. J Biol Chem 271, 8144-8151. Ma WJ, Chung S, Furneaux H (1997) The Elav-like proteins bind to AU-rich elements and to the poly(A) tail of mRNA. Nucleic Acids Res 25, 3564-3569. Maher ER, Kaelin WGJ (1997) von Hippel-Lindau disease. Medicine 76, 381-391. Mukhopadhyay D, Knebelmann B, Cohen HT, Ananth S, Sukhatme VP (1997) The von Hippel-Lindau tumor suppressor gene product interacts with Sp1 to repress vascular endothelial growth factor promoter activity. Mol Cell Biol 17, 5629-5639. Pertovaara L, Kaipainen A, Mustonen T, Orpana A, Ferrara N, Saksela O, et al (1994) Vascular endothelial growth factor is induced in response to transforming growth factor-beta in fibroblastic and epithelial cells. J Biol Chem 269, 62716274. Plouet J, Schilling J, Gospodarowicz D (1989) Isolation and characterization of a newly identified endothelial cell mitogen produced by AtT-20 cells. EMBO J 8, 3801-3806. Risau W (1997) Mechanisms of angiogenesis. Nature 386, 671674. Robinow S, Campos AR, YAO KM, White K (1988) The elav gene product of Drosophila, required in neurons, has three RNP consensus motifs. Science 242, 1570-1572. Robinow S, White K (1991) Characterization and spatial distribution of the ELAV protein during Drosophila melanogaster development. J Neurobiol 22, 443-461. Ryuto M, Ono M, Izumi H, Yoshida S, Weich HA, Kohno K, et al (1996) Induction of vascular endothelial growth factor by tumor necrosis factor alpha in human glioma cells. Possible roles of SP-1. J Biol Chem 271, 28220-28228. Semenza GL, Jiang BH, Leung SW, Passantino R, Concordet JP, Maire P, et al (1996) Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxiainducible factor 1. J Biol Chem 271, 32529-32537.


Gene Therapy and Molecular Biology Vol 7, page 73 Shaw G, Kamen R (1986) A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46, 659-667. Shibuya M, Yamaguchi S, Yamane A, Ikeda T, Tojo A, Matsushime H, et al (1990) Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family. Oncogene 5, 519524. Soker S, Fidder H, Neufeld G, Klagsbrun M (1996) Characterization of novel vascular endothelial growth factor (VEGF) receptors on tumor cells that bind VEGF165 via its exon 7-encoded domain. J Biol Chem 271, 5761-5767. Stein I, Itin A, EinaT P, Skaliter R, Grossman Z, Keshet E (1998) Translation of vascular endothelial growth factor mRNA by internal ribosome entry: implications for translation under hypoxia. Mol Cell Biol 18, 3112-3119. Stein I, Neeman M, Shweiki D, Itin A, Keshet E (1995) Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes. Mol Cell Biol 15, 5363-5368. Szabo A, Dalmau J, Manley G, Rosenfeld M, Wong E, Henson J, et al (1991) HuD, a paraneoplastic encephalomyelitis antigen, contains RNA-binding domains and is homologous to Elav and Sex-lethal. Cell 67, 325-333.

Terman BI, Carrion ME, Kovacs E, Rasmussen BA, Eddy RL, Shows TB (1991) Identification of a new endothelial cell growth factor receptor tyrosine kinase. Oncogene 6, 16771683. Terman BI, Dougher-Vermazen M, Carrion ME, Dimitrov D, Armellino DC, Gospodarowicz D, et al (1992) Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem Biophys Res Commun 187, 1579-1586. Vincenti V, Cassano C, Rocchi M, Persico G (1996) Assignment of the vascular endothelial growth factor gene to human chromosome 6p21.3. Circulation 93, 1493-1495. Waltenberger J, Claesson-Welsh L, Siegbahn A, Shibuya M, Heldin CH (1994) Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem 269, 26988-26995. Wang GL, Semenza GL (1995) Purification and characterization of hypoxia-inducible factor 1. J Biol Chem 270, 1230-1237. Yoshida A, Anand-Apte B, Zetter BR (1996) Differential endothelial migration and proliferation to basic fibroblast growth factor and vascular endothelial growth factor. Growth Factors 13, 57-64.


Goldberg-Cohen et al: Regulation of vascular endothelial growth factor by hypoxia


Gene Therapy and Molecular Biology Vol 7, page 75 Gene Ther Mol Biol Vol 7, 75-89, 2003

Gene therapy antiproliferative strategies against cardiovascular disease Review Article

Marisol Gascón-Irún, Silvia M. Sanz-González and Vicente Andrés* Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy, Instituto de Biomedicina de Valencia, Spanish Council for Scientific Research (CSIC), Valencia, Spain

__________________________________________________________________________________ *Correspondence: Vicente Andrés, Ph.D; Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy, Instituto de Biomedicina de Valencia, Spanish Council for Scientific Research (CSIC), C/ Jaime Roig, 11 46010 Valencia (SPAIN); Tel.: +34-963391752 (office), +34-963391751 (lab), Fax: +34-963690800; e-mail: Key words: atherosclerosis, restenosis, bypass graft failure, cell cycle, gene therapy List of abbreviations: apoE, apolipoprotein E; AP-1, activator protein-1; BrdU, 5-bromodeoxyuridine; CDK, cyclin-dependent kinase; CKI, CDK inhibitory protein; EC, endothelial cell; ERK, extracellular signal-regulated kinase; IVUS, intravascular ultrasound; JNK, cjun NH2-terminal protein kinase; MAPK, mitogen-activated protein kinase; ODN, oligodeoxynucleotide; PCNA, proliferating cell nuclear antigen; PDGF, platelet-derived growth factor; pRb, retinoblastoma protein; PTCA, percutaneous transluminal angioplasty; SAPK, stress-activated protein kinase; TGF-!, transforming growth factor-!; VSMC, vascular smooth muscle cell. Received: 17 June 2003; Accepted: 27 June 2003; electronically published: July 2003

Summary Excessive cellular proliferation is thought to contribute to the pathogenesis of several forms of cardiovascular disease (e. g., atherosclerosis, restenosis after angioplasty, and vessel bypass graft failure). Therefore, candidate targets for the treatment of these disorders include cell cycle regulatory factors, such as cyclin-dependent kinases (CDKs), cyclins, CDK inhibitory proteins (CKIs), tumor suppressors, growth factors and their receptors, and transcription factors. Importantly, animal models of atherosclerosis have demonstrated an inverse correlation between neointimal cell proliferation and atheroma size, suggesting that excessive cell growth prevails at the onset of atherogenesis. Cell growth may also predominate at the onset of human atherosclerosis. Thus, given that affected humans often exhibit advanced atherosclerotic plaques when first diagnosed, the potential benefit of antiproliferative strategies for the treatment of atherosclerosis in clinic is doubtful. The antiproliferative approaches used so far in the setting of vascular obstructive disease have focused on restenosis and graft atherosclerosis, during which neointimal hyperplasia is spatially localized and develops over a short period of time (typically 2-12 months). Vascular interventions, both endovascular and open surgical, allow minimally invasive, easily monitored gene delivery. Thus, gene therapy strategies are emerging as an attractive approach for the treatment of vascular proliferative disease. In this review, we will discuss the use of gene therapy strategies against cellular proliferation in animal models and clinical trials of cardiovascular disease. inflammatory response also plays a critical role during restenosis after angioplasty and graft atherosclerosis. Thus, understanding the molecular mechanisms that control hyperplastic growth of vascular cells should help develop novel therapeutic strategies for the treatment of vascular obstructive disease. Although arterial cell proliferation occurs in animal models during all phases of atherogenesis (Ross, 1999; Díez-Juan and Andrés, 2001; Cortés et al, 2002), studies with hyperlipidemic rabbits have shown an inverse correlation between atheroma size and cellular proliferation within the atheromatous plaque (Spraragen et al, 1962; McMillan and Stary, 1968; Rosenfeld and Ross, 1990). Experimental angioplasty is also characterized by

I. Introduction Large-scale clinical trials conducted over the last decades have allowed the identification of independent risk factors that increase the prevalence and severity of atherosclerosis (e. g., hypercholesterolemia, hypertension, smoking). Cardiovascular risk factors initiate and perpetuate an inflammatory response within the injured arterial wall that promotes the development of atherosclerotic plaques (Ross, 1999; Lusis, 2000; Dzau et al, 2002; Steinberg, 2002) (Figure 1). Chemokines and cytokines secreted by leukocytes that accumulate within the injured arterial wall promote their own proliferation, as well as the growth and migration of the underlying vascular smooth muscle cells (VSMCs) (Figure 2). This 75

Gascón-Irún et al: Gene therapy antiproliferative strategies against cardiovascular disease abundant proliferation of VSMCs, followed by the reestablishment of the quiescent phenotype, typically within 2-4 weeks (Bauters and Isner, 1997; Libby and

Tanaka, 1997; Andrés, 1998). These animal studies suggest that vascular cell proliferation prevails at the onset of atherogenesis and restenosis.

Figure 1. Neointimal lesion development in response to cardiovascular risk factors and mechanical injury. Exposure of the arterial wall to cardiovascular risk factors and mechanical injury leads to endothelial damage. Recruitment of circulating leukocytes is promoted by the expression of adhesion molecules by the injured endothelial cells. Neointimal leukocites release a plethora of cytokines and chemokines that initiate and perpetuate an inflammatory response, which activates signal transduction pathways and transcription factors that promote the hyperplastic growth of the lesion. Accumulation of noncellular material also contributes to atheroma development.

Figure 2. Early atherogenesis is associated with abundant cell proliferation within the arterial wall. Immunohistochemical analysis of aortic arch cross-section of male New Zealand rabbits fed control chow or a cholesterol-rich diet for 2 months. Animals were injected with 5-bromodeoxyuridine (BrdU) prior to sacrifice. Specimens were incubated with anti-BrdU and anti-RAM11 antibodies to monitor cell proliferation and to identify macrophages, respectively (Cortés et al, 2002). Arrowheads indicate the internal elastic lamina. Note lack of atherosclerosis and undetectable immunoreactivity for BrdU and RAM11 within the aortic arch of control rabbits. In contrast, prominent fatty streaks enriched in lipid-laden macrophages are seen in cholesterol-fed animals. Some macrophages are also detected within the media. Abundant BrdU immunoreactivity demonstrates a high proliferative activity, particularly within the atherosclerotic lesion. All photomicrographs are at the same magnification.


Gene Therapy and Molecular Biology Vol 7, page 77 intima (46% versus 9.7% "-actin immunoreactive VSMCs, 14.3% ECs, 13.1% T lymphocytes), whereas VSMC proliferation prevailed in the media (44.4% versus 20% ECs, 13.0% monocyte/macrophages, and 14.3% T lymphocytes). It is also noteworthy that cell proliferation in human peripheral and coronary ateries is greater in restenotic versus primary lesions (O'Brien et al, 1993; 2000; Pickering et al, 1993). Furthermore, cultured VSMCs from human advanced primary stenosing disclosed lower proliferative capacity than cells from fresh restenosing lesions (Dartsch et al, 1990). Thus, similar to the situation in animal models, proliferation during human atherosclerosis and restenosis might peak at the onset of these pathologies and then progressively decline. Cell cycle progression is controlled by several cyclindependent kinases (CDKs) that associate with regulatory cyclins (Morgan, 1995) (Figure 3). Active CDK/cyclin holoenzymes hyperphosphorylate the retinoblastoma protein (pRb) and the related pocket proteins p107 and p130 from mid G1 to mitosis. Phosphorylation of pRb and related pocket proteins contributes to the transactivation of genes with functional E2F-binding sites, including several growth and cell-cycle regulators (i.e., c-myc, pRb, cdc2, cyclin E, cyclin A), and genes encoding proteins that are required for nucleotide and DNA biosynthesis (i. e., DNA polymerase ", histone H2A, proliferating cell nuclear antigen, thymidine kinase) (Dyson, 1998; Lavia and Jansen-Durr, 1999; Stevaux and Dyson, 2002). Interaction of CDK/cyclins with CDK inhibitory proteins (CKIs) attenuates CDK activity and promotes growth arrest (Philipp-Staheli et al, 2001). CKIs of the Cip/Kip family (p21Cip1, p27Kip1 and p57Kip2) bind to and inhibit a wide spectrum of CDK/cyclin holoenzymes, while members of the Ink4 family (p16Ink4a, p15Ink4b, p18Ink4c, p19Ink4d) are specific for cyclin D-associated CDKs.

Expression of proliferation markers in human primary atheromatous plaques and restenotic lesions has been well documented (Essed et al, 1983; Gordon et al, 1990; Burrig, 1991; Nobuyoshi et al, 1991; Katsuda et al, 1993; Kearney et al, 1997; O'Brien et al, 1993, 2000; Rekhter and Gordon, 1995; Wei et al, 1997; Orekhov et al, 1998; Tanner et al, 1998; Veinot et al, 1998). However, controversy exists regarding the magnitude of the proliferative response, ranging from a very low index of cell proliferation (Gordon et al, 1990; Katsuda et al, 1993; O'Brien et al, 1993; 2000; Rekhter and Gordon, 1995; Veinot et al, 1998) to abundance of dividing cells (Essed et al, 1983; Nobuyoshi et al, 1991; Pickering et al, 1993; Kearney et al, 1997). Aside from methodological issues (e. g., differences in the fixatives used for tissue preservation, antigen accessibility, diversity of proliferation markers analyzed in these studies), some of the reported variance with regard to the issue of cell proliferation might relate to differences in the arteries being analyzed (i. e., peripheral, coronary and carotid arteries) and variance in the stage of atherogenesis at the time of tissue harvesting (Isner, 1994). The cell types that undergo cell proliferation within human atherosclerotic tissue include VSMCs, leukocytes and endothelial cells (ECs) (Gordon et al, 1990; Burrig, 1991; Katsuda et al, 1993; O'Brien et al, 1993; Rekhter and Gordon, 1995; Orekhov et al, 1998; Veinot et al, 1998). Histological examination in 20 patients undergoing antemortem coronary angioplasty revealed that the extent of intimal proliferation was significantly greater in lesions with evidence of medial or adventitial tears than in lesions with no or only intimal tears (Nobuyoshi et al, 1991). Human carotid artery primary atherosclerotic tissue retrieved by endarterectomy surgery displayed greater proliferative activity in the intimal lesion versus the underlying media (Rekhter and Gordon, 1995). Moreover, monocyte/macrophage proliferation predominated in the

Figure 3. Control of mammalian cell cycle by CDK/cyclin holoenzyme and growth suppresssors of the CKI family. Sequential activation of specific CDK/cyclin complexes leads to progression through the different phases of the cell cycle. Inhibitory proteins of the CKI family (Cip/Kip and Ink4) inhibit CDK/cyclin activity.


Gascón-Irún et al: Gene therapy antiproliferative strategies against cardiovascular disease al, 1997), demonstrating the assembly of functional CDK/cyclin holoenzymes in the injured arterial wall. Expression of CDK2 and cyclin E was also detected in human VSMCs within atherosclerotic and restenotic tissue (Kearney et al, 1997; Wei et al, 1997; Ihling et al, 1999), suggesting that induction of positive cell-cycle control genes is a hallmark of vascular proliferative disease in human patients. In the following sections, we will discuss the use of gene therapy strategies targeting cellular proliferation in preclinical (Table 1) and clinical studies (Table 2) related to cardiovascular disease.

Mitogenic and antimitogenic stimuli affect the rates of synthesis and degradation of CKIs, as well as their redistribution among different CDK/cyclin pairs (PhilippStaheli et al, 2001). For example, p27Kip1 promotes the assembly of CDK4/cyclin D complexes by binding to them, thus facilitating CDK2/cyclin E activation through G1/S phase. VSMC proliferation in the balloon-injured rat carotid artery is associated with a temporally and spatially coordinated expression of CDKs and cyclins (Wei et al, 1997; Braun-Dullaeus et al, 2001). Importantly, augmented expression of these factors is associated with an increase in their kinase activity (Abe et al, 1994; Wei et

Table 1: Attenuation of neointimal thickening by antiproliferative gene therapy approaches in animal models of vascular proliferative disease. Strategy Antisense (ODN)

Target gene


Animal model


Balloon angioplasty (rat)

Abe et al, 1994; Morishita et al, 1994a


Balloon angioplasty (rat)

Abe et al, 1994; Morishita et al, 1994b

Cyclin B1

Balloon angioplasty (rat)

Morishita et al, 1994b


Graft arteriosclerosis (rabbit, rat)

Mann et al, 1995; Miniati et al, 2000


Balloon angioplasty (rat)

Morishita et al, 1993


Graft arteriosclerosis (mouse)

Suzuki et al, 1997

c-myb *

Balloon angioplasty (pig, rat)

Simons et al, 1992; Gunn et al, 1997

c-myc *

Balloon angioplasty (rat, pig, rabbit)

Bennett et al, 1994a; Shi et al, 1994b; Kipshidze et al, 2001, 2002

c-myc *

Graft arteriosclerosis (pig)

Mannion et al, 1998

PDGF! receptor

Balloon angioplasty (rat)

Antisense (retrovirus)

Cyclin G1

Balloon angioplasty (rat)

Cohen-Sacks et al, 2002 Zhu et al, 1997



Stent (pig)

Frimerman et al, 1999


Balloon angioplasty (rat)

Yamamoto et al, 2000


Balloon angioplasty (rat)

Kotani et al, 2003


Balloon angioplasty (rat)


Balloon angioplasty (rat, pig)

Gu et al, 2001 Morishita et al, 1995; Ahn et al, 2002a; Nakamura et al, 2002


Graft arteriosclerosis (rabbit, mouse, monkey)

Mann et al, 1997; Kawauchi et al, 2000; Ehsan et al, 2001


Balloon angioplasty (rat, rabbit, minipig)


Balloon angioplasty (rat, mouse, pig)

Ahn et al, 2002b; Buchwald et al, 2002; Kume et al, 2002 Chang et al, 1995a; Yang et al, 1996; Ueno et al, 1997a; Condorelli et al, 2001;


Graft arteriosclerosis (rabbit)

Bai et al, 1998


Balloon angioplasty (rat, pig)

Chen et al, 1997; Tanner et al, 2000


Balloon angioplasty (rat, pig)

Chang et al, 1995b; Smith et al, 1997b


Balloon angioplasty (rat)

Claudio et al, 1999


Balloon angioplasty (rabbit, rat)

Yonemitsu et al, 1998; Scheinman et al, 1999; Matsushita et al, 2000


Balloon angioplasty (rat, rabbit)

Maillard et al, 1997; Smith et al, 1997a; Perlman et al, 1999


Balloon angioplasty (rat)

Mano et al, 1999 Indolfi et al, 1995; Ueno et al, 1997b

‘Decoy’ ODN

Oveexpression of growth suppressors

Overexpression of RAS Balloon angioplasty (rat) dominant-negative ERK Balloon angioplasty (rat) mutants JNK Balloon angioplasty (rat) * These inhibitory effects might be caused by a nonantisense mechanism (Burgess 1995; Villa et al, 1995; Wang et al, 1996).


Izumi et al, 2001 Izumi et al, 2001 et al, 1995; Chavany et al, 1995; Guvakova et al,

Gene Therapy and Molecular Biology Vol 7, page 79 overexpression of negative regulators of cell growth (e. g., CKIs, p53, pRb, GAX, and GATA-6), and 3) overexpression of transdominant negative mutants of positive cell cycle regulators (e. g., Ras, mitogen-activated protein kinases).

II. Preclinical studies Antiproliferative gene therapy strategies designed for the treatment of experimental cardiovascular disease include the following: 1) inactivation of positive cell cycle regulators (e. g., CDK/cyclins, protooncogenes, E2F, growth factors) by antisense approaches, ribozymes, and transcription factor ‘decoy’ strategies (Figure 4), 2)

Table 2: Gene therapy clinical trials for vascular proliferative disease based on cytostatic strategies. Trial







Randomized, double-blinded, single center

E2F decoy ODN ex vivo transfection of vein graft

Autologous vein graft failure after peripheral artery bypass

70-74% decreases in the level of positive cell cycle regulators expressed by VSMCs in the vein, and reduction in primary graft failure

Mann et al, 1999


Randomized multicenter, double-blinded, placebo-controlled

E2F decoy ODN ex vivo transfection of vein graft

Autologous vein graft failure after coronary artery bypass

Larger patency and inhibition neointimal thickening

Dzau et al, 2002


Randomized, placebo-controlled


c-myc antisense In-stent No reduction in Kutryk et ODN delivery coronary angiographic al, 2002 after stent restenosis restenosis rate implantation Project of ex-vivo vein graft engineering via transfection Investigation by the thoraxcenter of antisense DNA using local delivery and IVUS after coronary stenting

Figure 4. Targeted gene inactivation by means of gene therapy strategies. Decoy approach by delivering a double-stranded ODN corresponding to the optimum DNA recognition sequence of the transcription factor of interest (TF) leads to attenuation of its interaction with the authentic cis-elements in cellular target genes, thus resulting in reduced gene transcription. Ribozymes inactivate the gene of interest by degrading their transcript. Antisense ODNs hybridize in a complementary fashion and stoicheometrically with the target mRNA, thus causing blockade of translation or synthesis of a truncated (inactive) protein.


Gascón-Irún et al: Gene therapy antiproliferative strategies against cardiovascular disease Guvakova et al, 1995; Villa et al, 1995; Wang et al, 1996). It has been recently shown that nanospheres containing antisense ODN against PDGF! receptor inhibit neointimal thickening in the rat carotid model of balloon angioplasty (Cohen-Sacks et al, 2002).

A. Antisense approach The gene of interest is inactivated by using a synthetic antisense oligodeoxynucleotide (ODN) that hybridizes in a complementary fashion and stoicheometrically with the target mRNA.

B. Ribozymes

1. CDKs and cyclins

Ribozymes represent a unique class of RNA molecules that catalytically cleave the specific target RNA, thus resulting in targeted gene inactivation. Su et al. (2000) designed a DNA-RNA chimeric hammerhead ribozyme targeted to human transforming growth factor!1 (TGF-!1) that significantly inhibited angiotensin IIstimulated TGF-!1 mRNA and protein expression in human VSMCs, and efficiently inhibited the growth of these cells. Likewise, cleavage of the platelet-derived growth factor (PDGF) A-chain mRNA by hammerhead ribozyme attenuated human and rat VSMC growth in vitro (Hu et al, 2001a,b) and inhibited neointima formation in the rat carotid artery model of balloon injury (Kotani et al, 2003). Studies using experimental models of angioplasty provided the first evidence that ribozymes might represent useful tools in cardiovascular therapy. Frimerman et al. (1999) reported the efficacy of chimeric hammerhead ribozyme to PCNA in reducing stent-induced stenosis in a porcine coronary model, and ribozyme strategy against TGF-!1 inhibited neointimal formation after balloon injury in the rat carotid artery model (Yamamoto et al, 2000). 12-Lipoxygenase products of arachidonate metabolism have growth and chemotactic effects in vascular smooth muscle cells, and ribozyme against this enzyme prevents intimal hyperplasia in balloon-injured rat carotid arteries (Gu et al, 2001).

The efficacy of antisense ODN strategies targeting CDKs and cyclins to reduce neointimal lesion formation has been demonstrated in several animal models of balloon angioplasty. These studies include antisense oligodeoxynucleotides against CDK2 (Abe et al, 1994; Morishita et al, 1994a), CDC2 (Morishita et al, 1993; 1994b; Abe et al, 1994) and cyclin B1 (Morishita et al, 1994b). Interestingly, cotransfection of antisense ODN against CDC2 kinase and cyclin B1 resulted in further inhibition of neointima formation, as compared to blockade of either gene target alone (Morishita et al, 1994b). Of note, Morishita et al. (1993) reported sustained inhibition of neointima formation in the rat carotid balloon-injury model after a single intraluminal molecular delivery of combined CDC2 and proliferating cell nuclear antigen (PCNA) antisense ODNs, whereas this approach had no effect in the coronary arteries of pigs after balloon angioplasty (Robinson et al, 1997). Downregulation of cyclin G1 expression by retrovirus-mediated antisense gene transfer inhibited VSMC proliferation and neointima formation after balloon angioplasty (Zhu et al, 1997). Attenuated graft atherosclerosis has been also observed upon inactivation of CDC2/PCNA (Mann et al, 1995; Miniati et al, 2000) and CDK2 (Suzuki et al, 1997) with antisense ODN.

2. Mitogen-responsive nuclear factors that promote cell growth

C. Transcription factor ‘decoy’ strategies

Several “immediate-early” genes (e. g., c-fos, c-jun, c-myc, c-myb, egr-1) are induced in serum-stimulated VSMCs, and their overexpression can promote VSMC proliferation in vitro (Castellot et al, 1985; Kindy and Sonenshein, 1986; Reilly et al, 1989; Brown et al, 1992; Campan et al, 1992; Rothman et al, 1994; Bennett et al, 1994b; Gorski and Walsh, 1995). VSMCs cultured from atheromatous plaques present higher levels of c-myc mRNA than in VSMCs from normal arteries (Parkes et al, 1991), and arterial injury induced the expression of several “immediate-early” gene (Lambert et al, 2001; Miano et al, 1990; 1993; Sylvester et al, 1998). Antisense ODNs against c-myc and c-myb reportedly inhibited in a sequence-specific manner both VSMC proliferation in vitro (Pukac et al, 1990; Brown et al, 1992; Ebbecke et al, 1992; Simons and Rosenberg, 1992; Biro et al, 1993; Shi et al, 1993; Bennett et al, 1994a; Shi et al, 1994a; Gunn et al, 1997), and neointima formation after angioplasty (Simons et al, 1992; Bennett et al, 1994a; Shi et al, 1994b; Gunn et al, 1997; Kipshidze et al, 2001, 2002) and vein grafting (Mannion et al, 1998) in vivo. However, these inhibitory effects may be mediated by a nonantisense mechanism (Burgess et al, 1995; Chavany et al, 1995;

This approach consists of delivering a doublestranded ODN corresponding to the optimum DNA target sequence of the transcription factor of interest, thus leading to the sequestration of the specific trans-acting factor and attenuation of its interaction with the authentic cis-elements in cellular target genes.

1. E2F E2F participates in the transcriptional activation of genes encoding proteins that are required for nucleotide and DNA biosynthesis (e. g., DNA polymerase ", histone H2A, pcna, thymidine kinase) (Dyson, 1998; Lavia and Jansen-Durr, 1999) and in several growth and cell-cycle regulators (e. g., c-myc, pRb, cdc2, cyclin E, cyclin A). Experimental neointimal thickening in ballooninjured arteries (Morishita et al, 1995; Nakamura et al, 2002), vein grafts (Mann et al, 1997; Ehsan et al, 2001), and cardiac allografts (Kawauchi et al, 2000) is prevented by the use of a synthetic ‘decoy’ ODN containing an E2F consensus binding site that inactivates the transcription factor E2F. Ahn et al. (2002a) developed a novel E2F 80

Gene Therapy and Molecular Biology Vol 7, page 81 ‘decoy’ ODN with a circular dumbbell structure (CD-E2F) and compared its properties with those of conventional phosphorothioated E2F ‘decoy’ ODN (PS-E2F). CD-E2F displayed more stability and stronger antiproliferative activity than PS-E2F when assayed in cultured VSMCs, and was more effective in inhibiting neointimal formation in vivo.

response of intimal and medial VSMCs towards basic fibroblast growth factor (bFGF or FGF2) (Olson et al, 2000). Intrinsic differences in the regulation of p27Kip1 might also play an important role in creating variance in the proliferative and migratory capacity of VSMCs isolated from different vascular beds, which might in turn contribute to establishing regional variability in atherogenicity (Castro et al, 2003). Tanner et al (1998) have reported more frequent expression of p27Kip1 and p21Cip1 within regions of human coronary atheromas not undergoing proliferation. Concordant expression of TGF-! receptors I and II in virtually all cells positive for p27Kip1 within human atherosclerotic plaques indicates that TGF-!1 present in these lesions may contribute to p27Kip1 upregulation (Ihling et al, 1999). Moreover, coexpression of p53 and p21Cip1 in human carotid atheromatous plaque cells that revealed lack of proliferation markers suggests that induction of p21Cip1 may occur via transcriptional activation by p53 (Ihling et al, 1997). Ectopic expression of p21Cip1 and p27Kip1, but not Ink4a p16 , significantly reduced neointimal thickening in several animal models of angioplasty (Chang et al, 1995a; Yang et al, 1996; Chen et al, 1997; Ueno et al, 1997a; Tanner et al, 2000; Condorelli et al, 2001). Overexpression of p21 Cip1 also attenuated neointimal lesion formation in a rabbit model of vein grafting (Bai et al, 1998).

2. Activator protein-1 (AP-1) Cell proliferation in the rat carotid artery model of angioplasty correlated with elevated expression and high DNA-binding activity of transcription factors of the AP-1 family (Miano et al, 1990; Miano et al, 1993; Hu et al, 1997; Sylvester et al, 1998; Andrés et al, 2001). Under conditions of PDGF stimulation, AP-1 ‘decoy’ ODN delivery into cultured human VSMCs significantly reduced cell number and TGF-!1 production (Kume et al, 2002), and attenuated neointimal thickening when applied at the site of balloon angioplasty in rabbit carotid artery (Kume et al, 2002) and minipig coronary arteries (Buchwald et al, 2002). Circular dumbbell AP-1 ‘decoy’ ODN was more effective in inhibiting the proliferation of VSMCs in vitro and neointimal hyperplasia in vivo compared to conventional phosphorothioated AP-1 decoy ODN, (Ahn et al, 2002b).

D. Overexpression of growth suppressors 1. CKIs

2. p53

The efficacy of CKIs in inhibiting CDK activity and cell cycle progression has been widely documented in a variety of normal and tumour cells in vitro. The first evidence that p21Cip1 and p27 Kip1 may function as negative regulators of neointimal hyperplasia was suggested in animal studies showing the upregulation of these CKIs at late time points following balloon angioplasty, coinciding with the restoration of the quiescent phenotype after the initial proliferative wave (Chen et al, 1997; Tanner et al, 1998). The protective role of p27Kip1 against neointimal thickening has been rigorously demonstrated in hypercholesterolemic apolipoprotein E (apoE)-deficient mice, in which genetic inactivation of p27Kip1 accelerated atherogenesis in a dose-dependent manner (Díez-Juan and Andrés, 2001). However, neointimal hyperplasia after mechanical damage of the arterial wall was similar in wild-type and p27Kip1-null mice (Roque et al, 2001b). Redundant roles between p21Cip1 and p27Kip1, or compensatory increase in p21Cip1 expression (or other CKIs) might account for the lack of phenotype of p27Kip1null mice in the setting of mechanical arterial injury. Several studies have suggested a role of CKIs in establishing regional phenotypic variance in VSMCs from different vascular beds. Using human VSMCs isolated from internal mammary artery and saphenous vein, Yang et al. (1998) suggested that sustained p27Kip1 expression in spite of growth stimuli may contribute to the resistance to growth of VSMCs from internal mammary artery and to the longer patency of arterial versus venous grafts (Yang et al, 1998). Likewise, different expression of p15Ink4b and p27Kip1 has been correlated with distinct proliferative

p53 is a transcription factor that functions as a tumor suppressor displaying both antiproliferative and proapoptotic actions. These effects result from complex regulatory networks, including transcriptional activation of antiproliferative and proapoptotic genes (e. g., p21Cip1 and Bax, respectively), transcriptional repression of proproliferative and antiapoptotic genes (e. g., IGF-II and bcl-2, respectively), and direct protein-protein interactions (e. g., with helicases and caspases). Increased VSMC proliferation has been shown as a result of antisense p53 ODN transfection (Aoki et al, 1999; Matsushita et al, 2000), and p53 gene transfer has the opposite effect (Yonemitsu et al, 1998). Mayr et al (2002) showed a higher rate of proliferation and migration of VSMCs isolated from p53-deficient mice than its wild-type counterparts. Consistent with these findings, early migration and proliferation of VSMCs happened in explanted porcine tunica media tissue after mitogeninduced downregulation of p53 (Rodriguez-Campos et al, 2001). p53 deficiency has been demonstrated to have a proatherogenic effect in studies of genetic inactivation in hypercholesterolemic apoE and apoE*3-Leiden mice, although the relative contribution of increased cellular proliferation and decreased apoptosis in these animal models remains obscure (Guevara et al, 1999; van Vlijmen et al, 2001). Mice deficient for p53 also disclosed accelerated vein graft atherosclerosis (Mayr et al, 2002). Regarding human atherosclerosis, p53 is overexpressed but not mutated in human atherosclerotic tissue (Iacopetta


Gasc贸n-Ir煤n et al: Gene therapy antiproliferative strategies against cardiovascular disease et al, 1995), and lack of proliferation markers in vascular cells coexpressing p53 and p21Cip1 within advanced human atherosclerotic lesions suggests that transcriptional activation of the p21Cip1 gene by p53 may be a protective mechanism against excessive vascular cell growth (Ihling et al, 1997). p53 appears to play an important role in the pathogenesis of restenosis, as suggested by both animal and human studies. Transfection of antisense p53 ODN into rat intact carotid artery decreased p53 protein expression and resulted in a significant increase in neointimal lesion growth at 2 and 4 weeks after balloonangioplasty (Matsushita et al, 2000). Evidence suggests that human cytomegalovirus (HCMV) infection contributes to the development of atherosclerosis and restenosis, and part of this effect may be due to increased VSMC proliferation and migration by inactivation of p53 (Speir et al, 1994; Zhou et al, 1996; 1999; Tanaka et al, 1999). It is also noteworthy that human VSMCs from restenosis or in-stent stenosis sites demonstrate normal or enhanced responses to p53 when compared to VSMCs from normal vessels (Scott et al, 2002). Moreover, p53 gene transfer effectively inhibited neointimal hyperplasia after experimental angioplasty (Yonemitsu et al, 1998; Scheinman et al, 1999; Matsushita et al, 2000), and in human saphenous vein (George et al, 2001).

expression and G1 cell cycle arrest (Perlman et al, 1998). Importantly, p21Cip1-null mouse embryonic fibroblasts were refractory to the GATA-6-induced growth inhibition (Perlman et al, 1998). The level of GATA-6 mRNA, protein, and DNA-binding activity is transiently downregulated at early time points after balloon angioplasty in the rat carotid artery, and reversal of GATA-6 downregulation by adenovirus-mediated GATA6 gene transfer to the vessel wall inhibited intimal hyperplasia in this animal model (Mano et al, 1999).

5. GAX Gax is a homeobox gene highly expressed in cultures of quiescent VSMCS, which is rapidly downregulated in vitro upon growth factor stimulation of VSMCs, and after balloon angioplasty in vivo (Gorski et al, 1993; Weir et al, 1995). Overexpression of GAX inhibited VSMC proliferation in vitro and attenuated neointimal thickening in balloon-injured rat carotid arteries in a p21Cip1dependent manner (Smith et al, 1997a; Perlman et al, 1999). Percutaneous delivery of the Gax gene also inhibited vessel stenosis in a rabbit model of balloon angioplasty (Maillard et al, 1997).

E. Overexpression of transdominant negative mutants of positive cell cycle regulators.

3. pRb

1. Ras

The complex interplay between pRb and transcription factors of the E2F family plays a critical role in the control of cell growth (Stevaux and Dyson, 2002). E2F-dependent transactivation of genes required for cell cycle progression is prevented in quiescent cells due to the accumulation of hypophosphorylated pRb. Hyperphorylation of pRb by mitogenic stimuli leads to E2F activation and cell growth. Transfer of antisense pRb ODN into human VSMCs resulted in the induction of the proapoptotic factors bax and p53, and this was associated with increased number of apoptotic cells and a higher rate of DNA synthesis (Aoki et al, 1999). Inhibition of VSMC proliferation in vitro and attenuation of neointima formation after balloon angioplasty can be achieved by adenovirus-mediated transfer of several forms of pRb, including full-length constitutively active (nonphosphorylatable) and phosphorylation-competent pRb, and truncated versions of pRb (Chang et al, 1995b; Smith et al, 1997b). Similarly, adenoviral transfer of the pRb related protein RB2/p130 inhibited VSMC proliferation in vitro and prevented neointimal hyperplasia after experimental angioplasty (Claudio et al, 1999).

Ras-dependent signaling plays an important role in mitogen-stimulated cell growth (Pronk and Bos, 1994). Ras is implicated in the activation of the G1 CDK/cyclin/E2F pathway (Winston et al, 1996;Aktas et al, 1997; Kerkhoff and Rapp, 1997; Leone et al, 1997; Lloyd et al, 1997; Peeper et al, 1997; Zou et al, 1997) and is critical for the normal induction of cyclin A promoter activity and DNA synthesis in mitogen-stimulated VSMCs (Sylvester et al, 1998). Consistent with these findings, local delivery of transdominant negative mutants of Ras attenuated neointimal thickening after experimental balloon angioplasty (Indolfi et al, 1995; Ueno et al, 1997b).

2. Mitogen-activated (MAPKs)



The MAPK pathway is critical in the transducction of proliferative signals in many mammalian tissues, including the cardiovascular system (Zou et al, 1998; Bogoyevitch, 2000). Several families of MAPKs have been described, including the stress-activated protein kinases/c-jun NH2terminal protein kinases (SAPKs/JNKs), extracellular signal-regulated kinases (ERKs), and p38. JNKs and ERKs disclosed persistent hyperexpression and activation in atherosclerotic lesions of cholesterol-fed rabbits, suggesting that these factors play critical roles in initiating and perpetuating cell proliferation during the development of atherosclerosis (Hu et al, 2000; Metzler et al, 2000). Likewise, angioplasty in porcine and rat arteries led to the

4. GATA-6 The GATA transcription factors play a critical role in the establishment of hematopoietic cell lineages and during the development of the cardiovascular system (Simon, 1995). GATA-6 is rapidly downregulated upon mitogen stimulation of quiescent VSMCs (Suzuki et al, 1996), and overexpression of GATA-6 induced p21Cip1 82

Gene Therapy and Molecular Biology Vol 7, page 83 rapid activation of ERKs and JNKs (Lai et al, 1996; Lille et al, 1997; Pyles et al, 1997; Koyama et al, 1998). Consistent with this notion, gene transfer of dominantnegative mutants of ERK or JNK prevented neointimal formation in balloon-injured rat artery (Izumi et al, 2001).

Kutryk et al. (2002) recently reported the results of the Investigation by the Thoraxcenter of Antisense DNA using Local delivery and IVUS after Coronary Stenting (ITALICS) trial. This randomized, placebo controlled study was designed to determine the efficacy of antisense ODN against c-myc in inhibiting in-stent restenosis. Eighty-five patients were randomly assigned to receive either c-myc antisense ODN or saline vehicle by intracoronary local delivery after coronary stent implantation. Follow-up included the percent neointimal volume obstruction measured by IVUS, clinical outcome and quantitative coronary angiography. There was no reduction in either the neointimal volume obstruction or the angiographic restenosis rate after treatment with 10 mg of phosphorothioate-modified ODN directed against cmyc as demonstrated by the analysis of 77 patients.

III. Clinical studies The antiproliferative approaches used so far for the treatment of cardiovascular disease have focused on restenosis and graft atherosclerosis, during which neointimal hyperplasia is rapid and localized. These disorders remain the major limitation of revascularization by percutaneous transluminal angioplasty (PTCA) and artery bypass surgery.

A. E2F ‘decoy’

IV. Conclusions

Encouraging results of the E2F ‘decoy’ strategy in animal models of balloon angioplasty and graft atherosclerosis (see above) led to the initiation of the first Project of Ex-vivo Vein graft Engineering via Transfection (PREVENT I) (Mann et al, 1999). In this single-centre, randomized, controlled gene therapy trial, 41 patients undergoing bypass for the treatment of peripheral arterial occlusions were randomly assigned untreated (n=16), E2F‘decoy’-ODN-treated (n=17), or scrambled-ODN-treated (n=8) human infrainguinal vein grafts. Ex vivo delivery of ODNs was achieved intraoperatively via pressuremediated transfection. This procedure was associated with a 70-74% decrease in the level of PCNA and c-myc mRNA expressed by the VSMCs in the vein, and a statistically significant reduction in primary graft failure compared to control groups. Following to this pilot trial, a randomized, double-blinded, placebo controlled Phase IIb trial (PREVENT II) was carried out in patients undergoing coronary artery bypass surgery. The results of quantitative coronary angiography and intravascular ultrasound (IVUS) showed larger patency and inhibition of neointimal thickening in treated patients at 12 months after intervention (Dzau et al, 2002).

Excessive cell proliferation within the arterial wall is thought to contribute to neointimal thickening during the pathogenesis of atherosclerosis, in-stent restenosis, and vessel bypass graft failure. Animal models of atherosclerosis have demonstrated an inverse correlation between neointimal cell proliferation and atheroma size, suggesting that excessive cell growth prevails at the onset of atherogenesis. Cell proliferation may also predominate at the early stages of human atheroma development. Thus, given that patients frequently exhibit advanced atherosclerotic plaques when first diagnosed, the potential benefit of antiproliferative strategies for the treatment of human atherosclerosis is uncertain. The antiproliferative approaches used so far in the setting of vascular obstructive disease have focused on restenosis and graft atherosclerosis, during which neointimal hyperplasia is spatially localized and develops over a short period of time (typically 2-12 months). Gene therapy is emerging as an attractive strategy in the treatment of vascular proliferative disease due to minimally invasive and easily monitored gene delivery in vascular interventions. Antiproliferative gene therapy strategies that have proven efficient in inhibiting neointimal thickening in animal models of vascular obstructive disease include the use of antisenseand ribozyme-mediated inactivation of positive cell cycle regulators, overexpression of negative regulators of cell growth, and ‘decoy’ strategies to inactivate transcription factors that promote cell cycle progression. Although some of these strategies have shown encouraging results in humans, further studies are required to override the current practical barriers and limitations placed on most clinical trials before gene therapy strategies exhibit wide application in clinic. These should include the clarification of safety issues, development of better gene delivery vectors, and improvement of transgene expression. Aside from these technical improvements, significant effort in basic research is warranted to identify more effective and safer treatment genes.

B. c-myc antisense ODN Pharmacokinetics and clinical safety of ascending doses of c-myc antisense ODN (LR-3280) administered after PTCA was assessed by Roque et al. (2001a). Seventy eight patients were randomized to receive either standard care (n = 26) or standard care and escalating doses (1 to 24 mg) of LR-3280 (n = 52), administered into target vessel through a guiding catheter. The peak plasma concentrations of LR-3280 occurred at 1 minute and decreasing rapidly after approximately 1 hour, with little LR-3280 detected in the urine between 0-6 hours and 1224 hours. The intracoronary administration of LR-3280 was well tolerated at doses up to 24 mg and produced no adverse effects in dilated coronary arteries, thus providing the basis for the evaluation of local delivery of c-myc antisense ODN for the prevention of human vasculoproliferative disease.


Gascón-Irún et al: Gene therapy antiproliferative strategies against cardiovascular disease Bogoyevitch, MA (2000). Signalling via stress-activated mitogen-activated protein kinases in the cardiovascular system. Cardiovasc Res 45, 826-842. Braun-Dullaeus, RC, Mann, MJ, Seay, U, Zhang, L, von Der Leyen, HE, Morris, RE, and Dzau, VJ (2001). Cell cycle protein expression in vascular smooth muscle cells in vitro and in vivo is regulated through phosphatidylinositol 3kinase and mammalian target of rapamycin. Arterioscler Thromb Vasc Biol 21, 1152-1158. Brown, KE, Kindy, MS, and Sonenshein, GE (1992). Expression of the c-myb proto-oncogene in bovine vascular smooth muscle cells. J Biol Chem 267, 4625-4630. Buchwald, AB, Wagner, AH, Webel, C, and Hecker, M (2002). Decoy oligodeoxynucleotide against activator protein-1 reduces neointimal proliferation after coronary angioplasty in hypercholesterolemic minipigs. J Am Coll Cardiol 39, 732738. Burgess, TL, Fisher, EF, Ross, SL, Bready, JV, Qian, YX, Bayewitch, LA, Cohen, AM, Herrera, CJ, Hu, SS, Kramer, TB, and et al. (1995). The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism. Proc Natl Acad Sci U S A 92, 4051-4055. Burrig, KF (1991). The endothelium of advanced arteriosclerotic plaques in humans. Arterioscler Thromb 11, 1678-1689. Campan, M, Desgranges, C, Gadeau, A-P, Millet, D, and Belloc, F (1992). Cell cycle dependent gene expression in quiescent, stimulated, and asynchronously cycling arterial smooth muscle cells in culture. J Cell Physiol 150, 493-500. Castellot, JJ, Cochran, DL, and Karnovsky, MJ (1985). Effect of heparin on vascular smooth muscle cells. I. Cell metabolism. J Cell Physiol 124, 21-28. Castro, C, Díez-Juan, A, Cortés, MJ, and Andrés, V (2003). Distinct regulation of mitogen-activated protein kinases and p27Kip1 in smooth muscle cells from different vascular beds. A potential role in establishing regional phenotypic variance. J Biol Chem 278, 4482-4490. Chang, MW, Barr, E, Lu, MM, Barton, K, and Leiden, JM (1995a). Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest 96, 2260-2268. Chang, MW, Barr, E, Seltzer, J, Jiang, Y, Nabel, GJ, Nabel, EG, Parmacek, MS, and Leiden, JM (1995b). Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science 267, 518-522. Chavany, C, Connell, Y, and Neckers, L (1995). Contribution of sequence and phosphorothioate content to inhibition of cell growth and adhesion caused by c-myc antisense oligomers. Mol Pharmacol 48, 738-746. Chen, D, Krasinski, K, Chen, D, Sylvester, A, Chen, J, Nisen, PD, and Andrés, V (1997). Downregulation of cyclindependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27Kip1, an inhibitor of neointima formation in the rat carotid artery. J Clin Invest 99, 2334-2341. Claudio, PP, Fratta, L, Farina, F, Howard, CM, Stassi, G, Numata, S, Pacilio, C, Davis, A, Lavitrano, M, Volpe, M, Wilson, JM, Trimarco, B, Giordano, A, and Condorelli, G (1999). Adenoviral RB2/p130 gene transfer inhibits smooth muscle cell proliferation and prevents restenosis after angioplasty. Circ Res 85, 1032-1039. Cohen-Sacks, H, Najajreh, Y, Tchaikovski, V, Gao, G, Elazer, V, Dahan, R, Gati, I, Kanaan, M, Waltenberger, J, and Golomb, G (2002). Novel PDGFbetaR antisense encapsulated in

Acknowledgments Work in the laboratory of V. Andrés is partially supported by the Ministerio de Ciencia y Tecnología of Spain (MCyT) and Fondo Europeo de Desarrollo Regional (grants SAF2001-2358 and SAF2002-1143), and from Instituto de Salud Carlos III (ISCIII) (Red de Centros C03/01). S. M. Sanz and M. Gascón are predoctoral fellows of the ISCIII and MCyT, respectively.

References Abe, J, Zhou, W, Taguchi, J, Takuwa, N, Miki, K, Okazaki, H, Kurokawa, K, Kumada, M, and Takuwa, Y (1994). Suppression of neointimal smooth muscle cell accumulation in vivo by antisense cdc2 and cdk2 oligonucleotides in rat carotid artery. Biochem Biophys Res Commun 198, 16-24. Ahn, JD, Morishita, R, Kaneda, Y, Kim, HS, Chang, YC, Lee, KU, Park, JY, Lee, HW, Kim, YH, and Lee, IK (2002a). Novel E2F decoy oligodeoxynucleotides inhibit in vitro vascular smooth muscle cell proliferation and in vivo neointimal hyperplasia. Gene Ther 9, 1682-1692. Ahn, JD, Morishita, R, Kaneda, Y, Lee, SJ, Kwon, KY, Choi, SY, Lee, KU, Park, JY, Moon, IJ, Park, JG, Yoshizumi, M, Ouchi, Y, and Lee, IK (2002b). Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo. Circ Res 90, 1325-1332. Aktas, H, Cai, H, and Cooper, GM (1997). Ras links growth factor signaling to the cell cyle machinery via regulation of cyclin D1 and the cdk inhibitor p27KIP1. Mol Cell Biol 17, 3850-3857. Andrés, V (1998). Control of vascular smooth muscle cell growth and its implication in atherosclerosis and restenosis. Int J Molec Med 2, 81-89. Andrés, V, Ure_a, J, Poch, E, Chen, D, and Goukassian, D (2001). The role of Sp1 in the induction of p27 gene expression in vascular smooth muscle cells in vitro and after balloon angioplasty. Arterioscl Thromb Vasc Biol 21, 342347. Aoki, M, Morishita, R, Matsushita, H, Hayashi, S, Nakagami, H, Yamamoto, K, Moriguchi, A, Kaneda, Y, Higaki, J, and Ogihara, T (1999). Inhibition of the p53 tumor suppressor gene results in growth of human aortic vascular smooth muscle cells. Potential role of p53 in regulation of vascular smooth muscle cell growth. Hypertension 34, 192-200. Bai, H, Morishita, R, Kida, I, Yamakawa, T, Zhang, W, Aoki, M, Matsushita, H, Noda, A, Nagai, R, Kaneda, I, Higaki, J, Ogihara, T, Sawa, Y, and Matsuda, H (1998). Inhibition of intimal hyperplasia after vein grafting by in vivo transfer of human senescent cell-derived inhibitor-1 gene. Gene Ther 5, 761-769. Bauters, C, and Isner, JM (1997). The biology of restenosis. Prog Cardiovasc Dis 40, 107-116. Bennett, MR, Anglin, S, McEwan, JR, Jagoe, R, Newby, AC, and Evan, GI (1994a). Inhibition of vascular smooth muscle cell proliferation in vitro and in vivo by c-myc antisense oligodeoxynucleotides. J Clin Invest 93, 820-828. Bennett, MR, Evan, GI, and Newby, AC (1994b). Deregulated expression of the c-myc oncogene abolishes inhibition of proliferation of rat vascular smooth muscle cells by serum reduction, interferon-g, heparin, and cyclic nucleotide analogues and induces apoptosis. Circ Res 74, 525-536. Biro, S, Fu, Y-M, Yu, Z-X, and Epstein, SE (1993). Inhibitory effects of antisense oligodeoxynucleotides targeting c-myc mRNA on smooth muscle cell proliferation and migration. Proc Natl Acad Sci U S A 90, 654-658.


Gene Therapy and Molecular Biology Vol 7, page 85 polymeric nanospheres for the treatment of restenosis. Gene Ther 9, 1607-1616. Condorelli, G, Aycock, JK, Frati, G, and Napoli, C (2001). Mutated p21/WAF/CIP transgene overexpression reduces smooth muscle cell proliferation, macrophage deposition, oxidation-sensitive mechanisms, and restenosis in hypercholesterolemic apolipoprotein E knockout mice. FASEB J 15, 2162-2170. Cortés, MJ, Díez-Juan, A, Pérez, P, Pérez-Roger, I, ArroyoPellicer, R, and Andrés, V (2002). Increased early atherogenesis in young versus old hypercholesterolemic rabbits by a mechanism independent of arterial cell proliferation. FEBS Letters 522, 99-103. Dartsch, PC, Voisard, R, Bauriedel, G, Hofling, B, and Betz, E (1990). Growth characteristics and cytoskeletal organization of cultured smooth muscle cells from human primary stenosing and restenosing lesions. Arteriosclerosis 10, 6275. Díez-Juan, A, and Andrés, V (2001). The growth suppressor p27Kip1 protects against diet-induced atherosclerosis. FASEB J 15, 1989-1995. Dyson, N (1998). The regulation of E2F by pRB-family proteins. Genes Dev 12, 2245-2262. Dzau, VJ, Braun-Dullaeus, RC, and Sedding, DG (2002). Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies. Nat Med 8, 1249-1256. Ebbecke, M, Unterberg, C, Buchwald, A, Stohr, S, and Wiegand, V (1992). Antiproliferative effects of a c-myc antisense oligonucleotide on human arterial smooth muscle cells. Basic Res Cardiol 87, 585-591. Ehsan, A, Mann, MJ, Dell'Acqua, G, and Dzau, VJ (2001). Long-term stabilization of vein graft wall architecture and prolonged resistance to experimental atherosclerosis after E2F decoy oligonucleotide gene therapy. J Thorac Cardiovasc Surg 121, 714-722. Essed, CE, Van den Brand, M, and Becker, AE (1983). Transluminal coronary angioplasty and early restenosis. Fibrocellular occlusion after wall laceration. Br Heart J 49, 393-396. Frimerman, A, Welch, PJ, Jin, X, Eigler, N, Yei, S, Forrester, J, Honda, H, Makkar, R, Barber, J, and Litvack, F (1999). Chimeric DNA-RNA hammerhead ribozyme to proliferating cell nuclear antigen reduces stent-induced stenosis in a porcine coronary model. Circulation 99, 697-703. George, SJ, Angelini, GD, Capogrossi, MC, and Baker, AH (2001). Wild-type p53 gene transfer inhibits neointima formation in human saphenous vein by modulation of smooth muscle cell migration and induction of apoptosis. Gene Ther 8, 668-676. Gordon, D, Reidy, MA, Benditt, EP, and Schwartz, SM (1990). Cell proliferation in human coronary arteries. Proc Natl Acad Sci U S A 87, 4600-4604. Gorski, DH, LePage, DF, Patel, CV, Copeland, NG, Jenkins, NA, and Walsh, K (1993). Molecular cloning of a diverged homeobox gene that is rapidly down-regulated during the G0/G1 transition in vascular smooth muscle cells. Mol Cell Biol 13, 3722-3733. Gorski, DH, and Walsh, K (1995). Mitogen-responsive nuclear factors that mediate growth control signals in vascular myocytes. Cardiovasc Res 30, 585-592. Gu, JL, Pei, H, Thomas, L, Nadler, JL, Rossi, JJ, Lanting, L, and Natarajan, R (2001). Ribozyme-mediated inhibition of rat leukocyte-type 12-lipoxygenase prevents intimal hyperplasia in balloon-injured rat carotid arteries. Circulation 103, 1446-1452. Guevara, NV, Kim, HS, Antonova, EI, and Chan, L (1999). The absence of p53 accelerates atherosclerosis by increasing cell proliferation in vivo. Nat Med 5, 335-339.

Gunn, J, Holt, CM, Francis, SE, Shepherd, L, Grohmann, M, Newman, CM, Crossman, DC, and Cumberland, DC (1997). The effect of oligonucleotides to c-myb on vascular smooth muscle cell proliferation and neointima formation after porcine coronary angioplasty. Circ Res 80, 520-531. Guvakova, MA, Yakubov, LA, Vlodavsky, I, Tonkinson, JL, and Stein, CA (1995). Phosphorothioate oligodeoxynucleotides bind to basic fibroblast growth factor, inhibit its binding to cell surface receptors, and remove it from low affinity binding sites on extracellular matrix. J Biol Chem 270, 2620-2627. Hu, WY, Fukuda, N, Kishioka, H, Nakayama, M, Satoh, C, and Kanmatsuse, K (2001a). Hammerhead ribozyme targeting human platelet-derived growth factor A- chain mRNA inhibited the proliferation of human vascular smooth muscle cells. Atherosclerosis 158, 321-329. Hu, WY, Fukuda, N, Nakayama, M, Kishioka, H, and Kanmatsuse, K (2001b). Inhibition of vascular smooth muscle cell proliferation by DNA-RNA chimeric hammerhead ribozyme targeting to rat platelet-derived growth factor A-chain mRNA. J Hypertens 19, 203-212. Hu, Y, Cheng, L, Hochleitner, BW, and Xu, Q (1997). Activation of mitogen-activated protein kinases (ERK/JNK) and AP-1 transcription factor in rat carotid arteries after balloon injury. Arterioscler Thromb Vasc Biol 17, 2808-2816. Hu, Y, Dietrich, H, Metzler, B, Wick, G, and Xu, Q (2000). Hyperexpression and activation of extracellular signalregulated kinases (ERK1/2) in atherosclerotic lesions of cholesterol-fed rabbits. Arterioscler Thromb Vasc Biol 20, 18-26. Iacopetta, B, Wysocki, S, Norman, P, and House, A (1995). The p53 tumor suppressor gene is overexpressed but not mutated in human atherosclerotic tissue. Int J Oncol 7, 399-402. Ihling, C, Menzel, G, Wellens, E, Monting, JS, Schaefer, HE, and Zeiher, AM (1997). Topographical association between the cyclin-dependent kinases inhibitor p21, p53 accumulation, and cellular proliferation in human atherosclerotic tissue. Arterioscler Thromb Vasc Biol 17, 2218-2224. Ihling, C, Technau, K, Gross, V, Schulte-Monting, J, Zeiher, AM, and Schaefer, HE (1999). Concordant upregulation of type II-TGF-beta-receptor, the cyclin- dependent kinases inhibitor p27Kip1 and cyclin E in human atherosclerotic tissue: implications for lesion cellularity. Atherosclerosis 144, 714. Indolfi, C, Avvedimento, EV, Rapacciuolo, A, Di Lorenzo, E, Esposito, G, Stabile, E, Feliciello, A, Mele, E, Giuliano, P, Condorelli, G, and Chiariello, M (1995). Inhibition of cellular ras prevents smooth muscle cell proliferation after vascular injury in vivo. Nature Med 1, 541-545. Isner, JM (1994). Vascular remodeling. Honey, I think I shrunk the artery. Circulation 89, 2937-2941. Izumi, Y, Kim, S, Namba, M, Yasumoto, H, Miyazaki, H, Hoshiga, M, Kaneda, Y, Morishita, R, Zhan, Y, and Iwao, H (2001). Gene transfer of dominant-negative mutants of extracellular signal- regulated kinase and c-Jun NH2terminal kinase prevents neointimal formation in ballooninjured rat artery. Circ Res 88, 1120-1126. Katsuda, S, Coltrera, MD, Ross, R, and Gown, AM (1993). Human atherosclerosis. IV. Immunocytochemical analysis of cell activation and proliferation in lesions of young adults. Am J Pathol 142, 1787-1793. Kawauchi, M, Suzuki, J, Morishita, R, Wada, Y, Izawa, A, Tomita, N, Amano, J, Kaneda, Y, Ogihara, T, Takamoto, S, and Isobe, M (2000). Gene therapy for attenuating cardiac allograft arteriopathy using ex vivo E2F decoy transfection by HVJ-AVE-liposome method in mice and nonhuman primates. Circ Res 87, 1063-1068.


Gascón-Irún et al: Gene therapy antiproliferative strategies against cardiovascular disease Kearney, M, Pieczek, A, Haley, L, Losordo, DW, Andrés, V, Schainfield, R, Rosenfield, R, and Isner, JM (1997). Histopathology of in-stent restenosis in patients with peripheral artery disease. Circulation 95, 1998-2002. Kerkhoff, E, and Rapp, UR (1997). Induction of cell proliferation in quiescent NIH 3T3 cells by oncogenic c-Raf-1. Mol Cell Biol 17, 2576-2586. Kindy, MS, and Sonenshein, GE (1986). Regulation of oncogene expression in cultured aortic smooth muscle cells: posttranscriptional control of c-myc mRNA. J Biol Chem 261, 12865-12868. Kipshidze, N, Keane, E, Stein, D, Chawla, P, Skrinska, V, Shankar, LR, Khanna, A, Komorowski, R, Haudenschild, C, Iversen, P, Leon, MB, Keelan, MH, and Moses, J (2001). Local delivery of c-myc neutrally charged antisense oligonucleotides with transport catheter inhibits myointimal hyperplasia and positively affects vascular remodeling in the rabbit balloon injury model. Catheter Cardiovasc Interv 54, 247-256. Kipshidze, NN, Kim, HS, Iversen, P, Yazdi, HA, Bhargava, B, New, G, Mehran, R, Tio, F, Haudenschild, C, Dangas, G, Stone, GW, Iyer, S, Roubin, GS, Leon, MB, and Moses, JW (2002). Intramural coronary delivery of advanced antisense oligonucleotides reduces neointimal formation in the porcine stent restenosis model. J Am Coll Cardiol 39, 1686-1691. Kotani, M, Fukuda, N, Ando, H, Hu, WY, Kunimoto, S, Saito, S, and Kanmatsuse, K (2003). Chimeric DNA-RNA hammerhead ribozyme targeting PDGF A-chain mRNA specifically inhibits neointima formation in rat carotid artery after balloon injury. Cardiovasc Res 57, 265-276. Koyama, H, Olson, NE, Dastvan, FF, and Reidy, MA (1998). Cell replication in the arterial wall: activation of signaling pathway following in vivo injury. Circ Res 82, 713-721. Kume, M, Komori, K, Matsumoto, T, Onohara, T, Takeuchi, K, Yonemitsu, Y, and Sugimachi, K (2002). Administration of a decoy against the activator protein-1 binding site suppresses neointimal thickening in rabbit balloon-injured arteries. Circulation 105, 1226-1232. Kutryk, MJ, Foley, DP, van den Brand, M, Hamburger, JN, van der Giessen, WJ, deFeyter, PJ, Bruining, N, Sabate, M, and Serruys, PW (2002). Local intracoronary administration of antisense oligonucleotide against c-myc for the prevention of in-stent restenosis: results of the randomized investigation by the Thoraxcenter of antisense DNA using local delivery and IVUS after coronary stenting (ITALICS) trial. J Am Coll Cardiol 39, 281-287. Lai, K, Lee, W-S, Jain, MK, Lee, M-E, and Haber, E (1996). Mitogen-activated protein kinase phosphatase-1 in rat arterial smooth muscle cell proliferation. J Clin Invest 98, 15601567. Lambert, DL, Malik, N, Shepherd, L, Gunn, J, Francis, SE, King, A, Crossman, DC, Cumberland, DC, and Holt, CM (2001). Localization of c-Myb and induction of apoptosis by antisense oligonucleotide c-Myb after angioplasty of porcine coronary arteries. Arterioscler Thromb Vasc Biol 21, 17271732. Lavia, P, and Jansen-Durr, P (1999). E2F target genes and cellcycle checkpoint control. Bioessays 21, 221-230. Leone, G, DeGregori, J, Sears, R, Jakoi, L, and Nevins, JR (1997). Myc and Ras collaborate in inducing accumulation of active cyclin E/cdk2 and E2F. Nature 387, 422-426. Libby, P, and Tanaka, H (1997). The molecular basis of restenosis. Prog Cardiovasc Dis 40, 97-106. Lille, S, Daum, G, Clowes, MM, and Clowes, AW (1997). The regulation of p42/p44 mitogen-activated protein kinases in the injured rat carotid artery. J Surg Res 70, 178-186.

Lloyd, AC, Obermüller, F, Staddon, S, Barth, CF, McMahon, M, and Land, H (1997). Cooperating oncogenes converge to regulate cyclin/cdk complexes. Genes Dev 11, 663-677. Lusis, AJ (2000). Atherosclerosis. Nature 407, 233-241. Maillard, L, Van Belle, E, Smith, RC, Le Roux, A, Denèfle, P, Steg, G, Barry, JJ, Branellec, D, Isner, JM, and Walsh, K (1997). Percutaneous delivery of the gax gene inhibits vessel stenosis in a rabbit model of balloon angioplasty. Cardiovasc Res 35, 536-546. Mann, M, Gibbons, GH, Kernoff, RS, Diet, FP, Tsao, PS, Cooke, JP, Kaneda, Y, and Dzau, VJ (1995). Genetic engineering of vein grafts resistant to atherosclerosis. Proc Natl Acad Sci USA 92, 4502-4506. Mann, MJ, Gibbons, GH, Tsao, PS, von der Leyen, HE, Cooke, JP, Buitrago, R, Kernoff, R, and Dzau, VJ (1997). Cell cycle inhibition preserves endothelial function in genetically engineered rabbit vein grafts. J Clin Invest 99, 1295-1301. Mann, MJ, Whittemore, AD, Donaldson, MC, Belkin, M, Conte, MS, Polak, JF, Orav, EJ, Ehsan, A, Dell'Acqua, G, and Dzau, VJ (1999). Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial. Lancet 354, 1493-1498. Mannion, JD, Ormont, ML, Magno, MG, O'Brien, JE, Shi, Y, and Zalewski, A (1998). Sustained reduction of neointima with c-myc antisense oligonucleotides in saphenous vein grafts. Ann Thorac Surg 66, 1948-1952. Mano, T, Luo, ZY, Malendowicz, SL, Evans, T, and Walsh, K (1999). Reversal of GATA-6 downregulation promotes smooth muscle differentiation and inhibits intimal hyperplasia in balloon-injured rat carotid artery. Circ Res 84, 647-654. Matsushita, H, Morishita, R, Aoki, M, Tomita, N, Taniyama, Y, Nakagami, H, Shimozato, T, Higaki, J, Kaneda, Y, and Ogihara, T (2000). Transfection of antisense p53 tumor suppressor gene oligodeoxynucleotides into rat carotid artery results in abnormal growth of vascular smooth muscle cells. Circulation 101, 1447-1452. Mayr, U, Mayr, M, Li, C, Wernig, F, Dietrich, H, Hu, Y, and Xu, Q (2002). Loss of p53 accelerates neointimal lesions of vein bypass grafts in mice. Circ Res 90, 197-204. McMillan, GC, and Stary, HC (1968). Preliminary experience with mitotic activity of cellular elements in the atherosclerotic plaques of cholesterol-fed rabbits studied by labeling with tritiated thymidine. Ann N Y Acad Sci 149, 699-709. Metzler, B, Hu, Y, Dietrich, H, and Xu, Q (2000). Increased expression and activation of stress-activated protein kinases/c-Jun NH(2)-terminal protein kinases in atherosclerotic lesions coincide with p53. Am J Pathol 156, 1875-1886. Miano, JM, Tota, RR, Vlasic, N, Danishefsky, KJ, and Stemerman, MB (1990). Early proto-oncogene expression in rat aortic smooth muscle cells following endothelial removal. Am J Pathol 137, 761-765. Miano, JM, Vlasic, N, Tota, RR, and Stemerman, MB (1993). Localization of Fos and Jun proteins in rat aortic smooth muscle cells after vascular injury. Am J Pathol 142, 715724. Miniati, DN, Hoyt, EG, Feeley, BT, Poston, RS, and Robbins, RC (2000). Ex vivo antisense oligonucleotides to proliferating cell nuclear antigen and Cdc2 kinase inhibit graft coronary artery disease. Circulation 102, III237-242. Morgan, DO (1995). Principles of CDK regulation. Nature 374, 131-134. Morishita, R, Gibbons, GH, Ellison, KE, Nakajima, M, von der Leyen, H, Zhang, L, Kaneda, Y, Ogihara, T, and Dzau, VJ (1994a). Intimal hyperplasia after vascular injury is inhibited


Gene Therapy and Molecular Biology Vol 7, page 87 by antisense cdk2 kinase oligonucleotides. J Clin Invest 93, 1458-1464. Morishita, R, Gibbons, GH, Ellison, KE, Nakajima, M, Zhang, L, Kaneda, Y, Ogihara, T, and Dzau, VJ (1993). Single intraluminal delivery of antisense cdc2 kinase and proliferating-cell nuclear antigen oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci USA 90, 8474-8478. Morishita, R, Gibbons, GH, Horiuchi, M, Ellison, KE, Nakajima, M, Zhang, L, Kaneda, Y, Ogihara, T, and Dzau, VJ (1995). A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle cell proliferation in vivo. Proc Natl Acad Sci USA 92, 5855-5859. Morishita, R, Gibbons, GH, Kaneda, Y, Ogihara, T, and Dzau, VJ (1994b). Pharmacokinetics of antisense oligodeoxyribonucleotides (cyclin B1 and CDC 2 kinase) in the vessel wall in vivo: enhanced therapeutic utility for restenosis by HVJ-liposome delivery. Gene 149, 13-19. Nakamura, T, Morishita, R, Asai, T, Tsuboniwa, N, Aoki, M, Sakonjo, H, Yamasaki, K, Hashiya, N, Kaneda, Y, and Ogihara, T (2002). Molecular strategy using cis-element 'decoy' of E2F binding site inhibits neointimal formation in porcine balloon-injured coronary artery model. Gene Ther 9, 488-494. Nobuyoshi, M, Kimura, T, Ohishi, H, Horiuchi, H, Nosaka, H, Hamasaki, N, Yokoi, H, and Kim, K (1991). Restenosis after percutaneous transluminal coronary angioplasty: pathologic observations in 20 patients. J Am Coll Cardiol 17, 433-439. O'Brien, ER, Alpers, CE, Stewart, DK, Ferguson, M, Tran, N, Gordon, D, Benditt, EP, Hinohara, T, Simpson, JB, and Schwartz, SM (1993). Proliferation in primary and restenotic coronary atherectomy tissue. Implications for antiproliferative therapy. Circ Res 73, 223-231. O'Brien, ER, Urieli-Shoval, S, Garvin, MR, Stewart, DK, Hinohara, T, Simpson, JB, Benditt, EP, and Schwartz, SM (2000). Replication in restenotic atherectomy tissue. Atherosclerosis 152, 117-126. Olson, NE, Kozlowski, J, and Reidy, MA (2000). Proliferation of intimal smooth muscle cells. Attenuation of basic fibroblast growth factor 2-stimulated proliferation is associated with increased expression of cell cycle inhibitors. J Biol Chem 275, 11270-11277. Orekhov, AN, Andreeva, ER, Mikhailova, IA, and Gordon, D (1998). Cell proliferation in normal and atherosclerotic human aorta: proliferative splash in lipid-rich lesions. Atherosclerosis 139, 41-48. Parkes, JL, Cardell, RR, Hubbard, FC, Hubbard, D, Meltzer, A, and Penn, A (1991). Cultured human atherosclerotic plaque smooth muscle cells retain transforming potential and display enhanced expression of the myc protooncogene. Am J Pathol 138, 765-775. Peeper, DS, Upton, TM, Ladha, MH, Neuman, E, Zalvide, J, Bernards, R, DeCaprio, JA, and Ewen, ME (1997). Ras signalling linked to the cell-cycle machinery by the retinoblastoma protein. Nature 386, 177-181. Perlman, H, Luo, Z, Krasinski, K, Le Roux, A, Mahfoudi, A, Smith, RC, Branellec, D, and Walsh, K (1999). Adenovirusmediated delivery of the Gax transcription factor to rat carotid arteries inhibits smooth muscle proliferation and induces apoptosis. Gene Ther 6, 758-763. Perlman, H, Suzuki, E, Simonson, M, Smith, RC, and Walsh, K (1998). GATA-6 induces p21Cip1 expression and G1 cell cycle arrest. J Biol Chem 273, 13713-13718. Philipp-Staheli, J, Payne, SR, and Kemp, CJ (2001). p27Kip1: regulation and function of a haploinsufficient tumor suppressor and its misregulation in cancer. Exp Cell Res 264, 148-168.

Pickering, JG, Weir, L, Jekanowski, J, Kearney, MA, and Isner, JM (1993). Proliferative activity in peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous revascularization. J Clin Invest 91, 1469-1480. Pronk, GJ, and Bos, JL (1994). The role of p21ras in receptor tyrosine kinase signalling. Biochim Biophys Acta 1198, 131-147. Pukac, LA, Castellot, JJ, Jr., Wright, TC, Jr., Caleb, BL, and Karnovsky, MJ (1990). Heparin inhibits c-fos and c-myc mRNA expression in vascular smooth muscle cells. Cell Regul 1, 435-443. Pyles, JM, March, KL, Mehdi, K, Wilenski, RL, and Adam, LP (1997). Activation of MAP Kinase in vivo follows balloon overstretch injury of porcine coronary and carotid arteries. Circ Res 81, 904-910. Reilly, CF, Kindy, MS, Brown, KE, Rosenberg, RD, and Sonenshein, GE (1989). Heparin prevents vascular smooth muscle cell progression through the G1 phase of the cell cycle. J Biol Chem 264, 6990-6995. Rekhter, MD, and Gordon, D (1995). Active proliferation of different cell types, including lymphocytes, in human atherosclerotic plaques. Am J Pathol 147, 668-677. Robinson, KA, Chronos, NA, Schieffer, E, Palmer, SJ, Cipolla, GD, Milner, PG, and King, SB, 3rd (1997). Endoluminal local delivery of PCNA/cdc2 antisense oligonucleotides by porous balloon catheter does not affect neointima formation or vessel size in the pig coronary artery model of postangioplasty restenosis. Cathet Cardiovasc Diagn 41, 348-353. Rodriguez-Campos, A, Ruiz-Enriquez, P, Faraudo, S, and Badimon, L (2001). Mitogen-induced p53 downregulation precedes vascular smooth muscle cell migration from healthy tunica media and proliferation. Arterioscler Thromb Vasc Biol 21, 214-219. Roque, F, Mon, G, Belardi, J, Rodriguez, A, Grinfeld, L, Long, R, Grossman, S, Malcolm, A, Zon, G, Ormont, ML, Fischman, DL, Shi, Y, and Zalewski, A (2001a). Safety of intracoronary administration of c-myc antisense oligomers after percutaneous transluminal coronary angioplasty (PTCA). Antisense Nucleic Acid Drug Dev 11, 99-106. Roque, M, Reis, ED, Cordon-Cardo, C, Taubman, MB, Fallon, JT, Fuster, V, and Badimon, JJ (2001b). Effect of p27 deficiency and rapamycin on intimal hyperplasia: in vivo and in vitro studies using a p27 knockout mouse model. Lab Invest 81, 895-903. Rosenfeld, ME, and Ross, R (1990). Macrophage and smooth muscle cell proliferation in atherosclerotic lesions of WHHL and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis 10, 680-687. Ross, R (1999). Atherosclerosis: an inflammatory disease. N Engl J Med 340, 115-126. Rothman, A, Woler, B, Button, D, and Taylor, P (1994). Immediate-early gene expression in respose to hypertrophic and proliferative stimuli in pulmonary arterial smooth muscle cells. J Biol Chem 269, 6399-6404. Scheinman, M, Ascher, E, Levi, GS, Hingorani, A, Shirazian, D, and Seth, P (1999). p53 gene transfer to the injured rat carotid artery decreases neointimal formation. J Vasc Surg 29, 360-369. Scott, S, O'Sullivan, M, Hafizi, S, Shapiro, LM, and Bennett, MR (2002). Human vascular smooth muscle cells from restenosis or in-stent stenosis sites demonstrate enhanced responses to p53. Implications for brachytherapy and drug treatment for restenosis. Circ Res 90, 398-404. Shi, Y, Dodge, GR, Hall, DJ, Desrochers, PE, Fard, A, Shaheen, F, Talbot, C, Yurgenev, L, and Zalewski, A (1994a). Inhibition of type 1 collagen synthesis in vascular smooth


Gascón-Irún et al: Gene therapy antiproliferative strategies against cardiovascular disease muscle cells by c-myc antisense oligomers. Circulation 90, I-147. Shi, Y, Fard, A, Galeo, A, Hutchinson, HG, Vermani, P, Dodge, GR, Hall, DJ, Shaheen, F, and Zalewski, A (1994b). Transcatheter delivery of c-myc antisense oligomers reduces neointimal formation in a porcine model of coronary artery balloon injury. Circulation 90, 944-951. Shi, Y, Hutchinson, HG, Hall, DJ, and Zalewski, A (1993). Downregulation of c-myc expression by antisense oligonucleotides inhibits proliferation of human smooth muscle cells. Circulation 88, 1190-1195. Simon, MC (1995). Gotta have GATA. Nat Genetics 11, 9-11. Simons, M, Edelman, ER, DeKeyser, J-L, Langer, R, and Rosenberg, RD (1992). Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature 359, 67-70. Simons, M, and Rosenberg, RD (1992). Antisense nonmuscle myosin heavy chain and c-myb oligonucleotides supress smooth muscle cell proliferation in vitro. Circ Res 70, 835843. Smith, RC, Branellec, D, Gorski, DH, Guo, K, Perlman, H, Dedieu, J-F, Pastore, C, Mahfoudi, A, Denèfle, P, Isner, JM, and Walsh, K (1997a). p21CIP1-mediated inhibition of cell proliferation by overexpression of the Gax homeodomain gene. Genes Dev 11, 1674-1689. Smith, RC, Wills, KN, Antelman, D, Perlman, H, Truong, LN, Krasinski, K, and Walsh, K (1997b). Adenoviral constructs encoding phosphorylation-competent full-length and truncated forms of the human retinoblastoma protein inhibit myocyte proliferation and neointima formation. Circulation 96, 1899-1905. Speir, E, Modali, R, and Huang, E-S (1994). Potential role of human cytomegalovirus and p53 interaction in coronary restenosis. Science 265, 391-394. Spraragen, SC, Bond, VP, and Dahl, LK (1962). Role of hyperplasia in vascular lesions of cholesterol-fed rabbits studied with thymidine-3H autoradiography. Circ Res 11, 329-336. Steinberg, D (2002). Atherogenesis in perspective: hypercholesterolemia and inflammation as partners in crime. Nat Med 8, 1211-1217. Stevaux, O, and Dyson, NJ (2002). A revised picture of the E2F transcriptional network and RB function. Curr Opin Cell Biol 14, 684-691. Su, JZ, Fukuda, N, Hu, WY, and Kanmatsuse, K (2000). Ribozyme to human TGF-beta1 mRNA inhibits the proliferation of human vascular smooth muscle cells. Biochem Biophys Res Commun 278, 401-407. Suzuki, E, Evans, T, Lowry, J, Truong, L, Bell, DW, Testa, JR, and Walsh, K (1996). The human GATA-6 gene: structure, chromosomal location and regulation of expression by tissuespecific and mitogen-responsive signals. Genomics 38, 283290. Suzuki, J-I, Isobe, M, Morishita, R, Aoki, M, Horie, S, Okubo, Y, Kaneda, Y, Sawa, Y, Matsuda, H, Ogihara, T, and Sekiguchi, M (1997). Prevention of graft coronary arteriosclerosis by antisense cdk2 kinase oligonucleotide. Nat Med 3, 900-903. Sylvester, AM, Chen, D, Krasinski, K, and Andrés, V (1998). Role of c-fos and E2F in the induction of cyclin A transcription and vascular smooth muscle cell proliferation. J Clin Invest 101, 940-948. Tanaka, K, Zou, JP, Takeda, K, Ferrans, VJ, Sandford, GR, Johnson, TM, Finkel, T, and Epstein, SE (1999). Effects of human cytomegalovirus immediate-early proteins on p53mediated apoptosis in coronary artery smooth muscle cells. Circulation 99, 1656-1659.

Tanner, FC, Boehm, M, Akyürek, LM, San, H, Yang, Z-Y, Tashiro, J, Nabel, GJ, and Nabel, EG (2000). Differential effects of the cyclin-dependent kinase inhibitors p27Kip1, p21Cip1, and p16Ink4 on vascular smooth muscle cell proliferation. Circulation 101, 2022-2025. Tanner, FC, Yang, Z-Y, Duckers, E, Gordon, D, Nabel, GJ, and Nabel, EG (1998). Expression of cyclin-dependent kinase inhibitors in vascular disease. Circ Res 82, 396-403. Ueno, H, Masuda, S, SNishio, S, Li, JJ, Yamamoto, H, and Takeshita, A (1997a). Adenovirus-mediated transfer of cyclin-dependent kinase inhibitor p21 suppresses neointimal formation in the balloon-injured rat carotid arteries in vivo. Ann N Y Acad Sci 811, 401-411. Ueno, H, Yamamoto, H, Ito, S-i, Li, J-J, and Takeshita, A (1997b). Adenovirus-mediated transfer of a dominantnegative H-ras suppresses neointimal formation in ballooninjured arteries in vivo. Arterioscler Thromb Vasc Biol 17, 898-904. van Vlijmen, BJ, Gerritsen, G, Franken, AL, Boesten, LS, Kockx, MM, Gijbels, MJ, Vierboom, MP, van Eck, M, van De Water, B, van Berkel, TJ, and Havekes, LM (2001). Macrophage p53 deficiency leads to enhanced atherosclerosis in APOE*3- Leiden transgenic mice. Circ Res 88, 780-786. Veinot, JP, Ma, X, Jelley, J, and O'Brien, ER (1998). Preliminary clinical experience with the pullback atherectomy catheter and the study of proliferation in coronary plaques. Can J Cardiol 14, 1457-1463. Villa, AE, Guzman, LA, Poptic, EJ, Labhasetwar, V, D'Souza, S, Farrell, CL, Plow, EF, Levy, RJ, DiCorleto, PE, and Topol, EJ (1995). Effects of antisense c-myb oligonucleotides on vascular smooth muscle cell proliferation and response to vessel wall injury. Circ Res 76, 505-513. Wang, W, Chen, HJ, Schwartz, A, Cannon, PJ, Stein, CA, and Rabbani, LE (1996). Sequence-independent inhibition of in vitro vascular smooth muscle cell proliferation, migration, and in vivo neointimal formation by phosphorothioate oligodeoxynucleotides. J Clin Invest 98, 443-450. Wei, GL, Krasinski, K, Kearney, M, Isner, JM, Walsh, K, and Andrés, V (1997). Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty. Circ Res 80, 418-426. Weir, L, Chen, D, Pastore, C, Isner, JM, and Walsh, K (1995). Expression of GAX, a growth-arrest homeobox gene, is rapidly down-regulated in the rat carotid artery during the proliferative response to balloon injury. J Biol Chem 270, 5457-5461. Winston, JT, Coats, SR, Wang, Y-Z, and Pledger, WJ (1996). Regulation of the cell cycle machinery by oncogenic ras. Oncogene 12, 127-134. Yamamoto, K, Morishita, R, Tomita, N, Shimozato, T, Nakagami, H, Kikuchi, A, Aoki, M, Higaki, J, Kaneda, Y, and Ogihara, T (2000). Ribozyme oligonucleotides against transforming growth factor-beta inhibited neointimal formation after vascular injury in rat model: potential application of ribozyme strategy to treat cardiovascular disease. Circulation 102, 1308-1314. Yang, Z, Oemar, BS, Carrel, T, Kipfer, B, Julmy, F, and Lüscher, TF (1998). Different proliferative properties of smooth muscle cells of human arterial and venous bypass vessels: role of PDGF receptors, mitogen-activated protein kinase, and cyclin-dependent kinase inhibitors. Circulation 97, 181187. Yang, Z-Y, Simari, RD, Perkins, ND, San, H, Gordon, D, Nabel, GJ, and Nabel, EG (1996). Role of p21 cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterial injury. Proc Natl Acad Sci USA 93, 7905-7910.


Gene Therapy and Molecular Biology Vol 7, page 89 Yonemitsu, Y, Kaneda, Y, Tanaka, S, Nakashima, Y, Komori, K, Sugimachi, K, and Sueishi, K (1998). Transfer of wild-type p53 gene effectively inhibits vascular smooth muscle cell proliferation in vitro and in vivo. Circ Res 82, 147-156. Zhou, YF, Leon, MB, Waclawiw, MA, Popma, JJ, Yu, ZX, Finkel, T, and Epstein, SE (1996). Association between prior cytomegalovirus infection and the risk of restenosis after coronary atherectomy. N Engl J Med 335, 624-630. Zhou, YF, Yu, ZX, Wanishsawad, C, Shou, M, and Epstein, SE (1999). The immediate early gene products of human cytomegalovirus increase vascular smooth muscle cell migration, proliferation, and expression of PDGF betareceptor. Biochem Biophys Res Commun 256, 608-613.

Zhu, NL, Wu, L, Liu, PX, Gordon, EM, Anderson, WF, Starnes, VA, and Hall, FL (1997). Downregulation of cyclin G1 expression by retrovirus-mediated antisense gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation. Circulation 96, 628-635. Zou, X, Rudchenko, S, Wong, K-k, and Calame, K (1997). Induction of c-myc transcription by the v-Abl tyrosine kinase requires Ras, Raf1, and cyclin-dependent kinases. Genes Dev 11, 654-662. Zou, Y, Hu, Y, Metzler, B, and Xu, Q (1998). Signal transduction in arteriosclerosis: mechanical stress-activated MAP kinases in vascular smooth muscle cells (review). Int J Mol Med 1, 827-834.


Gasc贸n-Ir煤n et al: Gene therapy antiproliferative strategies against cardiovascular disease


Gene Therapy and Molecular Biology Vol 7, page 91 Gene Ther Mol Biol Vol 7, 91-97, 2003

Regulation of the Sp/KLF-family of transcription factors: focus on post-transcriptional modification and protein-protein interaction in the context of chromatin Review Article

Toru Suzuki1,2*, Masami Horikoshi3,4 and Ryozo Nagai1 1

Department of Cardiovascular Medicine, 2 Department of Clinical Bioinformatics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan, 3 Laboratory of Developmental Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan, 4 Horikoshi Gene Selector Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Corporation, 5-9-6 Tokodai, Tsukuba, Ibaraki 300-2635 Japan

__________________________________________________________________________________ *Correspondence:Toru Suzuki, MD, PhD, Department of Cardiovascular Medicine, Department of Clinical Bioinformatics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; Tel: 81-3-3815-5411; Fax: 81-3-58008824; e-mail: Key words: transcription factors, gene regulation, chromatin, Sp1, acetyltransferase, nucleosome remodeling Received: 25 June 2003; Accepted: 10 July 2003; electronically published: July 2003

Summary The Sp1- and Kr端ppel-like zinc finger transcription factor family is a rapidly expanding and highlighted group of factors given important biological roles. Understanding specific regulation is important to dissect individual functions. In this collective review, the regulation of this family of transcription factors with a particular focus on post-transcriptional modification and protein-protein interaction in the context of chromatin will be discussed. Studies by ourselves and others show that the zinc finger DNA-binding domain region of these factors mediates important regulatory interactions and modifications which may explain at least in part their specific regulation. Their possible implications in gene therapy are discussed. Dang et al, 2000; Bieker, 2001; Black et al, 2001; Bouwman and Philipsen, 2002; Kaczynski et al, 2003). DNA-binding activators/repressors bind in a sequence-specific manner to their cognate binding sites in enhancers/silencers and core promoter regions and activate/repress transcription of genes through combinatorial effects with the general transcription machinery (Horikoshi et al. 1988a, b; Zawel and Reinberg 1995). The DNA-binding transcription factor has been classically shown to possess modular functional regions consisting of an activation/regulatory domain which regulates transcription through interactions with basal transcription machinery and the DNA-binding domain (DBD) which specifies the target promoter gene (Ptashne and Gann, 1990; Zawel and Reinberg, 1995). The DNA-binding transcription factor is regulated at multiple steps. Presence as dictated by spatial expression (e.g. ubiquitous versus restricted expression) in addition to temporal regulation (e.g. constitutive versus inducible expression) plays a primary regulatory role. Sequence-

I. Introduction The zinc finger motif (paired cysteine and histidine type) was discovered approximately two decades ago (Diakun et al, 1986). Since then, we have learnt that this is one of the major motifs for proteins in the cell ranging from enzymes to transcription factors. Recent analysis of the human genome showed that transcription factors with this zinc finger motif have evolved in cascading magnitude as shown by their increased genomic complexity in eukaryotes (Tupler et al, 2001). At present, the paired-cysteine and histidine-type (C2H2-type) zinc finger transcription factors are thought to be one of the most important type of regulatory transcription factor in the eukaryotic cell. Among these factors, the Sp/KLF (for Sp1- and Kr端ppel-like factor) family of transcription factors has received recent attention due to important roles in development, differentiation, and oncogenic processes (Philipsen and Suske, 1999; Turner and Crossley, 1999;


Suzuki et al: Regulation of the Sp/KLF-family of transcription factors specific DNA-binding is further critically important for dictating gene-specific actions. DNA-binding transcription factors with common DNA-binding domains often bind similar DNA sequences (e.g. basic helix-loop-helix proteins bind E-boxes, homeoproteins bind A/T-rich sites) but additional regulatory steps must be present as the complexity of these factors in undertaking specific functions cannot be readily explained by their expression patterns and sequence-specific DNA binding properties alone. Regulation through differential protein-protein interactions and/or chemical modifications (e.g. phosphorylation, acetylation) further contribute to their differential functions. In the present review, the regulation of the Sp/KLF-family of transcription factors with a particular focus on post-transcriptional modification and protein-protein interactions in the context of chromatin will be discussed.

the underlying mechanisms governing their specific functions and regulation are poorly understood.

III. Differential regulation of Sp/KLF factors The mechanisms underlying specificity of this family of factors have been the topic of great interest among concerned researchers to understand the basis for their individual functions. As the paired cysteine-histidine type zinc finger is a DNA-binding motif, initial studies began by investigations of DNA-binding characteristics. One of the hallmark features of the Sp/KLF factors is that they bind to similar GC-rich sites and/or CACC-boxes. Well studied crystal structure analyses of DNA-binding zinc finger transcription factors have allowed the prediction of the cognate DNA-binding sequence from the primary amino acid structure (Klevit, 1991; Suzuki et al, 1994). Amino acids which contact DNA reside in the !-helical region of the zinc finger. As these critical amino acids are highly conserved in Sp/KLF zinc finger transcription factors, it is tempting to assume that they likely share similar DNA binding properties. Closer examination of this zinc finger region, however, shows discrete yet distinct differences. For instance, the third amino acid critical for DNA binding of the third zinc finger, and in the amino acids N-terminal adjacent to the first amino acid critical for DNA binding and the third amino acid critical for DNA binding in each of the zinc fingers differ (Suzuki et al, 1998). The relevance of these differences in the context of DNAbinding specificity or affinity remains to be clarified. The optimal cognate binding sequence of selected factors have been shown experimentally which showed that Sp1 binds the sequence 5'-GGGGCGGGGT-3' (Thiesen et al, 1990) and KLF4/GKLF binds the sequence 5'G/AG/AGGC/TGC/T-3' (Shields and Yang, 1998) which is a derivative of the CACC-box and BTE-element (which is a GC-rich site which binds BTEB1). Collectively, it is generally thought that this family of factors bind similar GC-rich sequences in a sequence-specific manner with a binding selectivity which does not allow individual factors to be clearly discriminated based on their DNA-binding characteristics alone. It is important to note here, however, that DNAbinding characteristics likely differ in the context of chromatin DNA as separate from the naked DNA-state often used for biochemical experiments. One important example using transgenic mice showed that EKLF/KLF1 preferentially binds the beta-globin locus site in vivo which had been shown to bind both EKLF and Sp1 in biochemical studies (Gillemans et al, 1998). We too had been interested in understanding whether there is specific binding of factors to GC-rich sites in vivo which are not reflected in biochemical studies in vitro. For this, we used a yeast one-hybrid assay using the GC-rich sites of the HIV-1 core promoter which have been shown to bind Sp1 to investigate what factors actually bind this site. The binding site probe used for the assay was integrated into the yeast genome to better reflect cellular

II. Basic classification of Sp/KLF factors The Sp/KLF family of zinc-finger transcription factors are comprised of over 20 mammalian family members which have in common three contiguous C2H2type zinc fingers at the carboxyl-terminus which comprises the DNA-binding domain (Philipsen and Suske, 1999; Turner and Crossley, 1999; Dang et al, 2000; Bieker, 2001; Black et al, 2001; Bouwman and Philipsen, 2002; Kaczynski et al, 2003). Sp/KLF family members can be classified into Sp- and KLF-subsets based on their similarities. The Sp-subtype is based on the founding ubiquitous factor Sp1 (Dynan and Tjian, 1983), and the KLF-subtype is based on the Drosophila Kr端ppel gene (Preiss et al, 1985). The first systematic classification used to distinguish mammalian Kr端ppel-like factors was demonstrated in a distinction with the GLI subgroup, which defined the consensus amino acid finger sequence for the Kr端ppel subgroup to be [Y/F]XCX2CX3FX5LX2HXRXHTGEKP (Ruppert et al, 1988). The Sp subgroup is based on similarity to the founding factor Sp1. Among the KLFs are erythroid differentiation factor EKLF/KLF1 (Miller and Bieker, 1993) and the tumor suppressor gene KLF6/GBF/Zf9/COPEB which we and others identified as a cellular factor possibly involved in HIV-1 transcription (Koritschoner et al, 1997; Suzuki et al, 1998; Narla et al, 2001). We have recently shown by gene knockout studies that the protooncogene KLF5/BTEB2/IKLF (Sogawa et al, 1993; Shi et al, 1999) is important for cardiovascular remodeling in response to stress (Shindo et al, 2002). At present, the annotation of this family of factors uses a numbering system in order of identification in accordance with an international collaboration to unify the nomenclature. Factors of the Sp-subset have six to eight members, whereas the KLF-subset have approximately 15 members, and are still increasing in numbers. Contrary to initial expectations that this family of factors would likely have redundant functions, they in fact have important individual biological functions as shown by gene knockout studies (e.g. EKLF/KLF1, LKLF/KLF2, KLF5). However,


Gene Therapy and Molecular Biology Vol 7, page 93 conditions. Although a mammalian environment was not used and as there was limitation by overexpression of factors, we believed that the yeast environment would be better reflective of the eukaryotic intracellular environment as compared to the traditional southwestern filter hybridization or affinity chromatography techniques. Our studies interestingly resulted in the isolation of KLF6/GBF, a novel KLF factor which shows similar GCrich binding properties as Sp1 (Suzuki et al, 1998). This was the only Sp/KLF factor identified in our screen thus suggesting the possibility that distinct factors may bind GC-rich sites in the cellular environment. Therefore, at present, while biochemical studies do show that Sp/KLF factors bind similar GC-rich sites, the actual intracellular environment especially in the context of chromatin may allow for preferential binding of different factors. This issue on effect of intracellular context remains to be further explored.

profound effect on post-translational modifications in addition to protein-protein interactions.

A. Regulation by chemical modification Focusing on the regulatory role of acetylation on Sp/KLF transcription factors, we have shown differential regulation through interaction and acetylation on the DNA-binding domain by the coactivator/acetylase p300 (Suzuki et al, 2000). Acetylation is an important nuclear regulatory signal which regulates transcriptional processes, importantly with biological implications which include regulation of development, differentiation and oncogenesis (Brownell and Allis, 1996; Cheung et al, 2000; Nakatani 2001; Freiman and Tjian, 2003) which closely resembles the roles of Sp/KLF family members. We thought that the Sp/KLF-factors might be differently regulated by acetylation and showed that the coactivator/acetylase p300 but not the MYST-type acetylase Tip60 specifically interacts and acetylates Sp1 but not KLF6 through the zinc finger DNA-binding domain, and further that DNA binding inhibits this interaction and acetylation (Suzuki et al, 2000). Interaction of p300 acetyltransferase region and the Sp1 zinc finger DNA-binding domain stimulates the DNA-binding activity of the latter, while acetylation per se has only marginal effects. While much is known of acetylation in general, its regulation and implications are still poorly understood. A similar mechanism has been shown for KLF13/FKLF2. KLF13 is acetylated both by PCAF and CBP, as well as interact through the zinc finger DNAbinding domain of KLF13. The acetyltransferase regions of PCAF and CBP stimulate KLF13 binding to its cognate DNA-binding site. These findings suggest and further support that acetyltransferase interaction with the zinc finger DNA-binding domain of at least KLFs affects DNA-binding activity (Song et al, 2002). Acetylation of KLF13 by CBP has been further shown to inhibit KLF13 DNA-binding activity, and that PCAF

IV. Regulation through chemical modifications and/or differential proteinprotein interactions Regulation through differential protein-protein interactions and/or chemical modifications (e.g. acetylation) are further likely to contribute to the differential functions of Sp/KLF factors. We have focused our attention on the role of the DNA-binding domain (DBD) because it is most reasonable, if not optimal, for regulating DNA-associated events such as promoter access and topological changes given its ability and activity to bind DNA (Figure 1). Amino acid differences are evident in the zinc finger DNA-binding domain of Sp/KLF factors, although there is extensive conservation overall. Aside from the likelihood of affecting DNA-binding properties, these differences in primary structure and quite possibly in the overall conformation of the folded protein may have a

Figure 1. Regulation of DNA-binding transcription factors in general. Note that there are modular activation and DNA-binding domains. Regulation through interaction and modification of DNA-binding domains is poorly understood. We have focused our studies on the role of the zinc finger DNA-binding domain for Sp/KLF factors. The active role of the DNA-binding domain is suggested in DNA-binding processes not only for naked DNA but also in the context of nucleosomal DNA.


Suzuki et al: Regulation of the Sp/KLF-family of transcription factors There are other modifications such as phosphorylation, methylation, glycosylation, ubiquitination, and SUMOylation (SUMO; small ubiquitin-related modifier) among others. From the perspective of the DNA-binding domain, cell-cycle dependent phosphorylation by a putative kinase has been reported for Sp1 (Black et al, 1999). Casein kinase II also phosphorylates the second zinc finger of Sp1 resulting in a reduction in DNA-binding activity (Armstrong et al, 1997). PKC-zeta also binds and phosphorylates the zinc finger region of Sp1 which is suggested to result in transcriptional activation (Pal et al, 1998). Sp1 is also glycosylated (Jackson and Tjian, 1988). Much of our knowledge on the regulatory mechanisms of the Sp/KLF factors at present are centered on Sp1 as it was one of the first eukaryotic DNA-binding regulatory transcription factors ever identified and serves as an excellent molecular model to dissect and understand mechanisms of transcriptional activation. A recent report has further shown that Sp3 is SUMOylated at the same residue that is acetylated (Sapetschnig et al, 2002). While we still have much to learn on post-transcriptional modifications, cross-talk and co-regulation of signaling pathways not only for lysine modifications but also for coupling of pathways such as a phosphorylation-acetylation cascade will likely show the complex nature of regulation by chemical modifications.

blocks CBP acetylation and its disruption of DNA binding (Song et al, 2003). Our findings on Sp1 and further those on KLF13 provide an attractive model of promoter access by cooperative action of DNA-binding activator with coactivator/acetyltransferase. Important here is that there is a concerted interaction between these two factors which facilitates promoter access (Figure 2). The regulatory and activation domains likely play an additional role. This is in contrast to the extant model of recruitment of coactivator/acetyltransferase to the DNA-binding activator involving specific binding by the latter to its cognate binding site with subsequent recruitment of the former to the promoter (Ogryzko et al, 1996). Our interpretation and model explains one of the limitations of this prior model on how the DNA-binding activator accesses its cognate site or how interaction with coactivator/acetyltransferases affects this reaction which were issues which remained unclear. Other Sp/KLF factors are also acetylated in the zinc finger DNA-binding domain. EKLF/KLF1 is acetylated by p300 and its homologue CBP at two lysine residues, one residing in the DNA-binding zinc finger domain and the other in the transactivation domain. The mutation of the zinc finger acetylated residue does not affect DNAbinding activity and the individual role of its acetylation is unclear, but mutation of the transactivation domain lysine residue results in decreased transactivation and acetylation collectively increased affinity for the SWI/SNF chromatin remodeling factors (Zhang and Bieker, 1998; Zhang et al, 2001). Sp3 is acetylated in its inhibitory domain lying between the glutamine-rich activation domain and zinc finger DNA-binding domain. Acetylation of this lysine residue regulates transcriptional activity (Braun et al, 2001).

B. Regulation by protein-protein interaction The zinc finger DBD motif, while binding DNA, is also an interface for protein-protein interaction such as homo- and hetero-dimerization in addition to proteinprotein interactions with heterologous proteins (MacKay and Crossley 1998)

Figure 2. Model of promoter access as mediated by interaction betweeen the zinc finger DNA-binding domain (DBD) of the Sp/KLF transcription factor and catalytic region of acetyltransferase (HAT) (e.g. p300 for Sp1 and PCAF for KLF13). Interaction between the activation domain (AD) of the DNA-binding factor and regulatory domain (RD) of the acetyltransferase is unknown but is likely to play an additional role to retain the DNA-binding factor and HAT on the promoter.


Gene Therapy and Molecular Biology Vol 7, page 95 Bieker, 2001). From within the HDAC-associated corepressor complex, sin3A also binds EKLF through the zinc finger DNA-binding domain. Further, the zinc finger DNA-binding domains of Sp1 and that of EKLF interact with the ATP-dependent nucleosome remodeling enzyme Swi/Snf (Kadam et al, 2000). Two SWI/SNF subunits (BRG1 and BAF155) are required for targeted chromatin remodeling and transcriptional activation by EKLF in vitro. Remodeling is achieved with only the BRG1-BAF155 minimal complex and the EKLF zinc finger DBD, whereas transcription additionally requires an activation domain. We have recently shown that the zinc finger DNAbinding domain of Sp1 mediates interaction with the histone chaperone TAF-I (template activating factor)(Suzuki et al, 2003). Interaction is specific, as different subsets of DNA-binding factors do not bind TAF-I and as other ATP-independent nucleosome remodeling enzymes do not bind Sp1. TAF-I negatively regulates Sp1 activity by inhibiting DNA binding, and likely as a consequence of this, regulates Sp1-mediated promoter activation. Based on these findings, the Sp1 DBD interacts with all three major chromatin-related factors consisting of chemical modification enzymes (e.g. acetyltransferase p300), ATP-dependent nucleosome assembly factor (e.g. SWI/SNF) and histone chaperone (e.g. TAF-I)(Figure 3). This finding is of particular interest because it implicates the DBD to play a likely role in mediating transcriptional regulatory processes in eukaryotes at the chromatin level. Although interaction with individual chromatin remodeling factors has been documented for numerous proteins, as interaction with all three chromatin remodeling factors has only been reported previously for histones, the DNA-transcription factor, and importantly its DNA-binding domain, may, therefore, represent a vital target for chromatin-related transcriptional processes

which results in specific regulation. In general, while much research on transcription factors has focused on the role of the activation domain to mediate regulation (e.g. activation, repression, ligand-dependent modulation, etc.) (Horikoshi et al, 1988a,b; Roeder, 1996; Lemon and Tjian, 2000), functions of the DBD other than its DNA-binding activity have received little attention (Wagner and Green, 1994). Here the discussion will focus on the fact that numerous chromatin remodeling factors and other factors which act on transcription at the level of higher-order DNA interact and regulate through the zinc finger DBD (Figure 1). As mentioned in the above section on acetylation, Sp1 and KLF13 catalytically interact with acetyltransferase (e.g. p300 with Sp1, and PCAF and CBP with KLF13). Importantly, they also stably interact through the zinc finger DBD which results in stimulation of DNA-binding activity of the DNA-binding transcription factor. These findings allow for the model of promoter access as shown in Figure 1. While we assume a priori that DNA-binding factors recruit acetyltransferase and other chromatin remodeling factors to DNA after they are pre-bound to DNA, these results suggest that they in fact show interaction in solution and that DNA binding is inhibitory to interaction. This suggests that interaction promotes access of the DNA-binding factor to DNA but is released once bound to DNA. Deacetylases also bind Sp/KLF factors through the zinc finger DNA-binding domain. Both Sp1 and EKLF/KLF1 have been shown to associate with HDAC1. Both Sp1 and EKLF bind HDAC1 through the zinc finger DNA-binding domain. Interaction of Sp1 and HDAC1 is thought to be repressive on Sp1 transcription because coexpression of E2F1, which interferes with HDAC1 binding to Sp1, abolishes Sp1-mediated transcriptional repression (Doetzlhofer et al, 1999). EKLF also binds HDAC1 through its zinc finger DNA-binding domain which results in transcriptional regulation (Chen and

Figure 3. Model (deducted from Sp1 interactions) explaining how the DNA-binding domain of the transcription factor (DBP) interacts with all three classes of chromatin remodeling enzymes which has only been known for histones. Interactions include the chemical modification enzyme acetyltransferase (HAT)(Suzuki et al, 2000), the ATP-independent nucleosome remodeling enzyme histone chaperone (HC)(Suzuki et al, 2003), and the ATP-dependent nucleosome remodeling enzyme (ATPase)(Kadam et al, 2000).


Suzuki et al: Regulation of the Sp/KLF-family of transcription factors Black AR, Jensen D, Lin SY and Azizkhan JC (1999) Growth/cell cycle regulation of Sp1 phosphorylation. J Biol Chem 274, 1207-1215. Black, AR, Black, JD and Azizkhan-Clifford, J (2001) Sp1 and krüppel-like factor family of transcription factors in cell growth regulation and cancer. J Cell Physiol 188, 143-160. Braun H, Koop R, Ertmer A, Nacht S and Suske G (2001) Transcription factor Sp3 is regulated by acetylation. Nucleic Acids Res 29, 4994-5000. Brownell JE, Allis CD (1996) Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation. Curr Opin Genet Dev 6, 176-184. Bouwman P and Philipsen S (2002) Regulation of the activity of Sp1-related transcription factors. Mol Cell Endocrinol 195, 27-38. Chen X and Bieker JJ (2001) Unanticipated repression function linked to erythroid Krüppel-like factor. Mol Cell Biol 21, 3118-3125. Cheung WL, Briggs SD and Allis CD (2000) Acetylation and chromosomal functions. Curr Opin Cell Biol 12, 326-333. Dang DT, Pevsner J and Yang VW (2000) The biology of the mammalian Krüppel-like family of transcription factors. Int J Biochem Cell Biol 32, 1103-1121. Diakun GP, Fairall L and Klug A (1986) EXAFS study of the zinc-binding sites in the protein transcription factor IIIA. Nature 324, 698-699. Doetzlhofer A, Rotheneder H, Lagger G, Koranda M, Kurtev V, Brosch G, Wintersberger E and Seiser C (1999) Histone deacetylase 1 can repress transcription by binding to Sp1. Mol Cell Biol 19, 5504-5511. Dynan, WS and Tjian R (1983) The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell 35, 79-87. Freiman, RN and Tjian R (2003) Regulating the regulators: lysine modifications make their mark. Cell 112, 11-17. Gillemans N, Tewari R, Lindeboom F, Rottier R, de Wit T, Wijgerde M, Grosveld F and Philipsen S (1998) Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the beta-globin locus control region in vivo. Genes Dev 12, 2863-2873. Horikoshi M, Hai T, Lin YS, Green MR and Roeder RG (1988a) Transcription factor ATF interacts with the TATA factor to facilitate establishment of a preinitiation complex. Cell 54, 1033-1042. Horikoshi M, Carey MF, Kakidani H and Roeder RG (1988b) Mechanism of action of a yeast activator: direct effect of GAL4 derivatives on mammalian TFIID-promoter interactions. Cell 54, 665-669. Jackson SP, Tjian R (1988) O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation. Cell 55, 125-33. Kaczynski J, Cook T and Urrutia R (2003) Sp1- and Krüppel-like transcription factors. Genome Biol 4, 206. Kadam S, McAlpine GS, Phelan ML, Kingston RE, Jones KA and Emerson BM (2000) Functional selectivity of recombinant mammalian SWI/SNF subunits. Genes Dev 14, 2441-2451. Klevit RE (1991) Recognition of DNA by Cys2, His2 zinc fingers. Science 253, 1367. Koritschoner NP, Bocco JL, Panzetta-Dutari GM, Dumur CI, Flury A and Patrito LC (1997) A novel human zinc finger protein that interacts with the core promoter element of a TATA box-less gene. J Biol Chem 272, 9573-9580.

through cooperative interaction with chromatinremodeling factors. The zinc finger transcription factors are the most widely evolved family of transcription factors in eukaryotes. Given that this biological diversification was coupled with the evolution of nuclear structure in eukaryotes, it is conceivable that regulation of chromatin is a necessary process to further allow for efficient use and access of factors to the tightly packaged DNA genetic information. Important mechanisms of transcriptional regulation in the context of chromatin have been shown as discussed in this review. The mechanism that the DBD mediates important regulation of the DNA-binding transcription factors through interaction and modification with chromatin factors can certainly be generalized to DNA-binding transcription factors other than the described zinc finger factors. Selectivity may be found between interaction of subsets for chromatin factors and DBD motifs. Furthermore, although only three types of chromatin factors were described including modification enzymes (e.g. acetyltransferase), ATP-independent (e.g. histone chaperones) and ATP-dependent (Swi/snf) factors, other chromatin factors are likely also to participate in regulatory interactions. Understanding the hierarchy and network of regulation among DNA-binding transcription factors and chromatin factors will likely play an important role in understanding the complexity of eukaryotic transcriptional regulation. As the Sp/KLF factors are a key family important in mammalian biological processes ranging from development, differentiation, to oncogenic processes, further studies aimed at understanding the temporospatial regulation of chromatin centered on Sp/KLF factors will surely advance our understanding of eukaryotic transcriptional mechanisms of chromatin activation in a biological context. Future gene therapy approaches could use strategies of expressing such activator, modifier or factor genes individually or in complexed form to facilitate regulation of therapeutically important genes at the physiologically relevant chromatin DNA level.

Acknowledgements This study was supported by grants from the New Energy and Industrial Technology Development Organization, Ministry of Health, Labour and Welfare, Ministry of Education, Culture, Sports, Science and Technology, Japan Science and Technology Corporation, Sankyo Life Science Foundation, Takeda Medical Research Foundation, and the Applied Enzyme Association.

References Armstrong SA, Barry DA, Leggett RW and Mueller CR (1997) Casein kinase II-mediated phosphorylation of the C terminus of Sp1 decreases its DNA binding activity. J Biol Chem 272, 13489-3495. Bieker, JJ (2001) Krüppel-like factors: three fingers in many pies. J Biol Chem 276, 34355-34358.


Gene Therapy and Molecular Biology Vol 7, page 97 Lemon B and Tjian R (2000) Orchestrated response: a symphony of transcription factors for gene control. Genes Dev 14, 2551-2569. Mackay JP and Crossley M (1998) Zinc fingers are sticking together. Trends Biochem Sci 23, 1-4. Miller IJ and Bieker JJ (1993) A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Krüppel family of nuclear proteins. Mol Cell Biol 13, 2776-2786. Narla G, Heath KE, Reeves HL, Li D, Giono LE, Kimmelman AC, Glucksman MJ, Narla J, Eng FJ, Chan AM, Ferrari AC, Martignetti JA and Friedman S (2001) KLF6, a candidate tumor suppressor gene mutated in prostate cancer. Science 294, 2563-2566. Nakatani Y (2001) Histone acetylases--versatile players. Genes Cells 6, 79-86. Ogryzko VV, Schiltz RL, Russanova V, Howard BH and Nakatani Y (1996) The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87, 953-959. Pal S, Claffey KP, Cohen HT and Mukhopadhyay D (1998) Activation of Sp1-mediated vascular permeability factor/vascular endothelial growth factor transcription requires specific interaction with protein kinase C zeta. J Biol Chem 273, 26277-26280. Philipsen S and Suske G (1999) A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res 27, 2991-3000. Preiss A, Rosenberg UB, Kienlin A, Seifert E and Jackle H (1985) Molecular genetics of Krüppel, a gene required for segmentation of the Drosophila embryo. Nature 313, 27-32 Ptashne M and Gann AA (1990) Activators and targets. Nature 346, 329-331. Roeder RG (1996) The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem Sci 9, 327-335. Ruppert JM, Kinzler KW, Wong AJ, Bigner SH, Kao FT, Law ML, Seuanez HN, O'Brien SJ and Vogelstein B (1998) The GLI-Krüppel family of human genes. Mol Cell Biol 8, 31043113. Sapetschnig A, Rischitor G, Braun H, Doll A, Schergaut M, Melchior F and Suske G. (2002) Transcription factor Sp3 is silenced through SUMO modification by PIAS1. EMBO J 21, 5206-15. Shi H, Zhang Z, Wang X, Liu S, and Teng CT (1999) Isolation and characterization of a gene encoding human Krüppel-like factor 5 (IKLF): binding to the CAAT/GT box of the mouse lactoferrin gene promoter. Nucleic Acids Res 27, 4807-4815. Shindo T, Manabe I, Fukushima Y, Tobe K, Aizawa K, Miyamoto S, Kawai-Kowase K, Moriyama N, Imai Y, Kawakami H, Nishimatsu H, Ishikawa T, Suzuki T, Morita H, Maemura K, Sata M, Hirata Y, Komukai M, Kagechika H, Kadowaki T, Kurabayashi M, and Nagai R (2002) Krüppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling. Nat Med 8, 856-863. Shields JM and Yang VW (1998) Identification of the DNA sequence that interacts with the gut-enriched Krüppel-like factor. Nucleic Acids Res 26, 796-802. Sogawa K, Kikuchi Y, Imataka H and Fujii-Kuriyama Y (1993) Comparison of DNA-binding properties between BTEB and Sp1. J Biochem 114, 605-609.

Song CZ, Keller K, Murata K, Asano H and Stamatoyannopoulos G (2002) Functional interaction between coactivators CBP/p300, PCAF, and transcription factor FKLF2. J Biol Chem 277, 7029-7036. Song CZ, Keller K, Chen Y and Stamatoyannopoulos G (2003) Functional Interplay between CBP and PCAF in Acetylation and Regulation of Transcription Factor KLF13 Activity. J Mol Biol 329, 207-215. Suzuki M, Gerstein M and Yagi N (1994) Steriochemical basis of DNA recognition by Zn fingers. Nucleic Acids Res 22, 3397-3405 Suzuki T, Yamamoto T, Kurabayashi M, Nagai R, Yazaki Y and Horikoshi M (1998) Isolation and initial characterization of GBF, a novel DNA-binding zinc finger protein that binds to the GC-rich binding sites of the HIV-1 promoter. J Biochem 124, 389-395. Suzuki T, Kimura A, Nagai R and Horikoshi M (2000) Regulation of interaction between the acetyltransferase region of p300 and the DNA-binding domain of Sp1 on and through DNA binding. Genes Cells 5, 29-41. Suzuki T, Muto S, Miyamoto S, Aizawa K, Horikoshi M and Nagai R (2003) Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I. J Biol Chem 278, 2875828764 Thiesen HJ and Bach C (1990) Target Detection Assay (TDA): a versatile procedure to determine DNA binding sites as demonstrated on SP1 protein. Nucleic Acids Res 18, 32033209. Tupler R, Perini G and Green MR (2001) Expressing the human genome. Nature 409, 832-833. Turner J and Crossley M (1999) Mammalian Krüppel-like transcription factors: more than just a pretty finger. Trends Biochem Sci 24, 236-40. Wagner S and Green MR (1994) DNA-binding domains: targets for viral and cellular regulators. Curr Opin Cell Biol 6, 410414. Zawel L and Reinberg D (1995) Common themes in function of eukaryotic transcription complexes. Annu Rev Biochem 64, 533-561. Zhang W and Bieker JJ (1998) Acetylation and modulation of erythroid Krüppel-like factor (EKLF) activity by interaction with histone acetyltransferases. Proc Natl Acad Sci USA 95, 9855-9860. Zhang W, Kadam S, Emerson BM and Bieker JJ (2001) Sitespecific acetylation by p300 or CREB binding protein regulates erythroid Krüppel-like factor transcriptional activity via its interaction with the SWI-SNF complex. Mol Cell Biol 21, 2413-2422

Dr. Toru Suzuki


Suzuki et al: Regulation of the Sp/KLF-family of transcription factors


Gene Therapy and Molecular Biology Vol 7, page 99 Gene Ther Mol Biol Vol 7, 99-102, 2003

Detection of MET oncogene amplification in hepatocellular carcinomas by comparative genomic hybridization on microarrays Research Article

W.L. Robert Li!1, Nagy A. Habib¨*, Steen L. Jensen¨*, Paul Bao!2, Diping Che!3, Uwe R. Müller!2 !

Vysis Inc., Downers Grove, Illinois, USA, ¨Liver Surgery Section, Imperial College School of Medicine, Hammersmith Hospital Campus, London, UK. 1 Pharmacia Corporation, 700 Chesterfield Parkway North, Chesterfield, MO 63198, 2Corning Incorporated, SP-FR-01, Corning, NY 14831, 3 Illumina, Inc., 9390 Towne Center Drive, Suite 200, San Diego, CA 92121, USA

__________________________________________________________________________________ *Correspondence: Nagy A. Habib, ChM FRCS, Head of Liver Surgery Section, Imperial College London, Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, UK; tel: +44-20-8383-8574, fax: +44-20-8383-3212, e-mail: Key words: MET oncogene, amplification, hepatocellular carcinoma, microarrays, comparative genomic hybridization Abbreviations: HCC, hepatocellular carcinoma; CLM, colorectal liver metastases; FISH, fluorescent in situ hybridization; P1, phage P1; PAC, P1-derived artificial chromosome; BAC, bacterial artificial chromosome; CCD, charge coupled device. Received: 26 June 2003; Accepted: 10 July 2003; electronically published: July 2003

Summary The oncogene MET localized on human chromosome 7q21-31 encodes a transmembrane protein with tyrosine kinase activity and is believed to be implicated in progression of colorectal cancer. The aims of the study were to determine whether overexpression and amplification of the MET oncogene confers a selective growth advantage to hepatocellular carcinomas. Comparative genomic hybridization on microarrays was used in the analysis of DNA from 32 liver tumors (6 hepatocellular carcinoma; 16 colorectal liver metastases; 3 cholangiocarcinomas; 2 adenomas; 2 fibrolamellar; 3 unclassified) to screen for sequence copy number changes. The results revealed a MET gene amplification in hepatocellular carcinoma, cholangiocarcinoma, and colorectal liver metastases tumors. Moreover, one of the patients with hepatocellular carcinoma showed MET amplifications in both tumor and nontumor samples, with the tumor having approximately 12.8 copies of the MET target locus per cell. These findings suggest that amplifications in the MET gene may play an important role in hepatocarcinogenesis. amplifications have been reported in human gastric carcinomas (Soman et al, 1990; Ponzetto et al, 1991) and gliomas (Fischer et al 1995). Furthermore, MET gene amplification and the resulting over-expression are believed to be involved in progression of colorectal cancer (Di Renzo et al, 1995). Human hepatocellular carcinoma (HCC) is one of the most common and devastating cancers with a poor prognosis. It has been widely considered that hepatitis B virus (HBV) and environmental agents such as aflatoxin B1 are major risk factors. However, the molecular mechanism of hepatocarcinogenesis is poorly understood. Loss of heterozygosity (LOH) has been reported for several genomic loci, such as the region surrounding RB1 on 13q (Nishida et al, 1992; Zhang et al, 1994), or sequences on 11p (Rogler et al, 1985), and 6q (De Souza

I. Introduction The oncogene MET, localized on human chromosome 7q21-31 by in situ hybridization (Dean et al, 1985), encodes a transmembrane protein with tyrosine kinase activity (Dean et al, 1985; Park et al 1996). It was shown that this protein is the receptor of hepatocyte growth factor (HGF)/ Scatter factor (Giordano et al, 1989; Bottaro et al, 1991), and the signals of HGF are transduced through the receptor tyrosine kinase encoded by the MET proto-oncogene. The MET gene can be activated by the formation of a chimeric gene through fusing the translocated promoter region (TPR) on chromosome 1 to the N-terminally truncated MET kinase domain (Park et al, 1996). Gene amplification and mutation may be another path to MET proto-oncogene activation, since MET gene


Li et al: MET amplification in liver tumors was normalized by dividing with the average ratio of all "normal" targets, resulting in an estimate for the copy number change of that specific sequence compared to the rest of the genome.

et al, 1995). Mutation of the p53 gene was detected in approximately 36% of advanced HCC (Murakami et al, 1991) and was also implicated in tumor progression (Teramoto et al, 1994). Overexpression was reported for several oncogenes such as N-ras, c-myc and fos (Arbuthnot et al, 1991). However, oncogene amplification appears to be rarely the underlying mechanism of cancer development in these cases. Amplifications associated with HCC have been found on 11q13 (Nishida et al, 1994), involving both INT2 and cyclin D1. Nishida and colleagues (1994) showed that the cyclin D1 gene was amplified 3 to 16 fold in about 11% of HCC samples analyzed, with a concomitant 6 to 10 fold overexpression. Based on this finding they suggested that amplification and overexpression of the cyclin D1 gene might be responsible for rapid growth of a subset of HCC. The rapid emergence of microarray technology has allowed new approaches to tumor analysis. The most common application of this technology has focused on the use of cDNA arrays for the large-scale analysis of gene expression to monitor tumor progression (Sgroi et al, 1999) or for cancer typing (Anbazhagan et al, 1999). Oligonucleotide arrays have enabled rapid re-sequencing for genotyping or point mutation analysis, such as p53 mutation detection (Hacia, 1999). Applying Comparative Genomic Hybridization (CGH) to microarrays of large genomic clones has also been successful, allowing the detection of gross chromosomal abnormalities that result in copy number changes for a given sequence, such as gene amplifications or deletions (e.g. LOH), (SolinasToldo et al, 1997; Pinkel et al, 1998; Muller, 2001). Such sub-chromosomal aneuploidies are known to be fundamental causes of cancer and many other human diseases, often leading to the over- or under-expression of genes.

III. Results As shown in Figure 1, the DNA extracted from the tumor tissue of HCC patient #21 was found to have an average normalized ratio of 4.2 ± 1 by Genosensor analysis for the MET target locus (average of 5 experiments), and 6.4 ± 0.8 by Southern analysis (3 experiments; see below). Since the reference sample used here was from a normal human male and has 2 copies of the MET sequence, this ratio suggests that there are on average between 8.4 to 12.8 copies per cell (4.2 or 6.4 x 2) of the MET gene in the HCC tumor sample. This amplification is considered a significant finding, as it is the first time to be reported in this type of cancer. Since microarray or Southern analysis yields an estimate for the copy number of a sequence averaged over all cells from which the DNA was extracted, the MET amplification level was confirmed by fluorescent in situ hybridization (FISH). The tumor tissue from the same HCC patient was formalin-fixed, paraffin-embedded, and sectioned. FISH was performed with SpectrumGreen labeled DNA from a BAC clone containing the MET gene. SpectrumOrange labeled CEP 7 DNA (containing chromosome 7-specific centromere DNA sequences; Vysis) was co-hybridized as a control. The signal for both, the MET gene and chromosome 7 were counted under a fluorescent microscope after counterstaining with DAPI. As expected, the majority (60%) of the cells contained 2 copies of chromosome 7 per nucleus, while approximately 40 % of cells have an average of 25 copies of MET (Figure 2). Since the remaining 60% of cells have only 2 copies of the MET gene, the DNA extracted from this tumor section should have 11 copies of the MET gene, which is in good agreement with the microarray and Southern data. For further confirmation and comparisons additional Southern blot analyses were carried out with EcoR1digested DNA from 32 tumor samples including 6 HCC, 16 colorectal liver metastasis (CLM), 3 cholangiocarcinomas, 2 adenomas, 2 fibrolamellar (HCC variant), and 3 unclassified liver tumors. Normal human genomic DNA (control) and DNA from the non-tumor liver tissue of HCC patient #21 were also included in the Southern blot analyses. A 360bp DNA fragment (1) was amplified by polymerase chain reaction (PCR) in the presence of the following pair of primers, primer H1: 5'TCTTGATTACCTGCATTTGC-3' and primer H2: 5'TGGGGCAAGAAGGCCTCTCT-3' from a BAC clone containing the entire MET gene. The 360bp MET probe was labeled by PCR in the presence of 32P-labelled dCTP and hybridized to the Southern blot. A probe generated from a genomic clone on 11q13 was re-hybridized to the same Southern blot for normalization, after the MET probe was stripped from the blot.

II. Materials and methods We have developed a CGH-based microarray system (Genosensor System) and a microarray to specifically detect abnormalities of 52 genomic loci that have been associated with formation of various human solid tumors (Müller et al, 2002). The arrays consist of 3 repeats each of 52 P1, PAC or BAC clone DNAs that are arrayed on a chromium-coated glass surface. For hybridization to this array, genomic DNA samples were extracted from human liver tumors or from histopathologically non-tumor liver sections from the same patient. After purification (Gentra Kits, Gentra Systems, Inc., Minneapolis, MN), the genomic DNA samples were then labeled by nick translation (Nicktranslation Kit, Vysis, Inc., Downers Grove, IL) in the presence of Spectrum-Green dUTP (green fluorophore). Genomic DNA from a normal human male donor was chemically labeled with a red fluorophore (Vysis, Inc., Downers Grove, IL), and served as a reference. The test probe (green) and reference probe (red) were then mixed with unlabeled human cot-1 DNA and co-hybridized to the microarrays. After removal of un-hybridized probes, the array was imaged by a multi-color CCD based image analysis system, and fluorescence intensities were determined for each target spot. Under the assumption that the hybridization kinetics for a given sequence are equal for the test and reference DNA, the signal intensity is proportional to the copy number of that sequence in the hybridization mixture. The test/reference intensity ratio for each target genomic locus (average of 3 spots)


Gene Therapy and Molecular Biology Vol 7, page 101

Figure 1: Genosensor and Southern analysis of HCC samples. Genomic DNA (8 Âľg for DNA from tumor tissue and 8 Âľg for DNA from normal tissue) was digested with Eco R1, run on agarose gels and blotted. Southern hybridization was performed with a P32 labeled 360 bp MET probe as described in the text. A composite image (red, green and blue) of a Gneosensor oncogene array after hybridization with a mixture of sample 21T DNA (green) and normal refernce DNA (red) is shown after counterstaining with DAPI.

of the MET oncogene in hepatocellular carcinomas strongly suggests a role here as well. This finding in combination with multiple other reports of cancer associated gene amplifications underscores the need for a rapid, quantitative detection method for such genetic changes. The microarray based method described here is consistent (within a factor of 2) with other established methods (FISH, Southern blotting), and therefore suitable for the screening of gene amplifications. Since this method is non-radioactive, simpler, faster, and more economical than either FISH or Southern, especially when the mutated genetic locus is not known, it lends itself to applications in clinical diagnostics.

The level of MET gene amplification was determined using a PhosphoImager (Molecular Dynamics). Some of the results are shown in Figure 1. Among the 6 HCC samples analysed, 2 MET gene amplifications were observed (6.4 and 2.5 fold after normalization). MET gene amplifications were also observed in the cholangiocarcinoma and CLM samples. Two of the three cholangiocarcinoma patients had MET gene amplifications in their tumour specimens at a level of 6.5 fold and 1.6 fold, respectively. Of the 16 patients with CLM, three had MET gene amplifications of 2.3, 2.1 and 1.8 fold, respectively. Of specific interest is the finding that both, the tumor as well as non-tumor tissues from the same HCC patient (No. 21) showed a similar level of MET amplification (6.4 fold and 6.1 fold, respectively), suggesting that MET amplification may precede malignant histopathological changes. This patient developed HCC in the background of a cirrhotic liver complicating hepatitis C infection. Liver cirrhosis provides a pre-malignant field change for HCC development.

IV. Discussion Hepatocyte growth factor (HGF) plays an important role in the growth, progression and angiogenesis of various tumors and is known to specifically promote hepatocyte proliferation and liver regeneration. In addition, it may also be involved in tumor invasion and progression (Tamatani et al, 1999). Overexpression and amplification of the HGF receptor (MET gene) have been implicated in progression of colorectal cancer (Di Renzo et al, 1995), by a mechanism where the elevated level of the MET gene product confers a selective growth advantage to tumor cells (Di Renzo et al, 1991). In the context of this information, our finding of amplifications

Figure 2: FISH on interphase nuclei of patient #21. FISH was performed on formalin-fixed, de-parafinized tumor tissue sections. A BAC clone containing the MET gene was labeled with SpectrumGreen by nick translation and used as a probe. A SpectrumOrange labeled chromosome 7-specific centromere probe (CEP7; Vysis Inc.) was co-hybridized as reference.


Li et al: MET amplification in liver tumors human hepatocellular carcinoma. Cancer Res 52, 55205525. Nishida N, Fukuda Y, Kokuryu H, Sadamoto T, Isowa G, Honda K, Yamaoka Y, Ikenaga M, Imura H, Ishizaki K. (1992) Accumulation of allelic loss of arms of chromosomes 13q, 16q and 17p in the advanced stages of human hepatocellular carcinoma. Int J Cancer 51, 862-868. Nishida N, Fukuda Y, Komeda T, Kita R, Sando T, Furukawa M, Amenomori M, Shibagaki I, Nakao K, Ikenaga M. (1994) Amplification and overexpression of the cyclin D1 gene in agrressive human hepatocellular carcinoma. Cancer Res 54, 3107-3110. Park M, Dean M, Cooper CS, Schmidt M, O’Brien SJ, Blair DG,, Vande Woude GF. (1986) Mechanism of met oncogene activation. Cell 45, 895-904. Pinkel D, Segraves R, Sudar D, Clark S, Poole I, Kowbel D, Collins C, Kuo W-L, Chen C, Zhai Y, Dairkee S, Ljung BM, Gray JW, Albertson DG. (1998) High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nat Genet 20, 207-211 Ponzetto C, Giordano S, Peverali F, Della Valle G, Abate ML, Vaula G, Comoglio PM. (1991) c-met is amplified but not mutated in a cell line with an activated met tyrosine kinase. Oncogene 6, 553-559. Rogler CE, Sherman M, Su CY, Shafritz DA, Summers J, Shows TB, Henderson A, Kew M. (1985) Deletion in chromosome 11p associated with a hepatitis B integration site in hepatocellular carcinoma. Science 230, 319-322. Sgroi D, Teng S, Robinson G, LeVanglie R, Hudson JR, Jr, Elkahloun AG. (1999) In vivo gene expression profile analysis of human breast cancer progression. Cancer Res 59, 5656-5661. Solinas-Toldo S, Lampel S, Stilgenbauer S, Nickolenko J, Benner A, Dohner H, Crmer T, Lichter P. (1997) Matrixbased comparative genomic hybridization: biochips to screen for genomic imbalances. Genes Chromosom Cancer 20, 399-407. Soman NR, Wogan GN, Rhim JS. (1990) TPR-MET oncogenic rearrangement: detection by polymerase chain reaction amplification of the transcript and expression in human tumor cell lines. Proc Natl Acad Sci USA 87, 739-742. Tamatani T., Hattori K., Iyer A., Tamatani K, Oyasu R. (1999) Hepatocyte growth factor is an invasion/migration factor of rat urothelial carcinoma cells in vitro. Carcinogenesis 20, 957-962. Teramoto T, Satonaka K, Kitazawa S, Fujimori T, Hayashi K, Maeda S. (1994) p53 gene abnormalities are closely related to hepatovirus infections and occur at a late stage of hepatocarcinogenesis. Cancer Res 54, 231-235. Zhang X, Xu H-J, Murakami Y, Sachse R, Yashima K, Hirohasha S, Hu S-X, Benedict WF, Sekiya T. (1994) Deletions of chromosome 13q, mutations in Retinoblastoma 1, and retinoblastoma protein state in human hepatocellular carcinoma. Cancer Res 54, 4177-4182.

Acknowledgments We thank Ragai Mitry, Teresa Ruffalo and Anna Lublinsky for their excellent technical support. We would also like to thank The Pedersen Family Charitable Foundation for their financial support with this research.

References Anbazhagan R., Tihan T, Bornman DM, Johnston JC, Saltz JH, Weigering A, Piantadosi S, Gabrielson E. (1999) Classification of small cell lung cancer and pulmonary carcinoid by gene expression profiles. Cancer Res 59, 51195122. Arbuthnot P, Kew M, Fitschen W. (1991) c-fos and c-myc oncoprotein expression in human hepatocellular carcinoma. Anticancer Res 11, 921-924. Bottaro DP, Rubin JS, Faletto DL, Chan AM-L, Kmiecik TE, Vande Woude GF, Arronson SA. (1991) Identification of the hepatocyte growth factor receptor as the met proto-oncogene product. Science 251, 802-804. De Souza AT, Hankins GR, Washington MK, Fine RL, Orton TC, Jirtle RL. (1995) Frequent loss of heterozygosity on 6q at the mannose 6-phosphate/insulin-like growth factor II receptor locus in human hepatocellular tumors. Oncogene 10, 1725-1729. Dean M, Park M, Le Beau MM, Robins TS, Diaz MO, Rowley JD, Blair DG, Vande Woude GF. (1985) The human met oncogene is related to the tyrosine kinase oncogenes. Nature 318, 385-388. Di Renzo MF, Narsimhan RP, Olivero M, Bretti S, Giordano S, Medico E, Gaglia P, Zara P, Comoglio PM. (1991) Expression of the met/HGF receptor in normal and neoplastic human tissues. Oncogene 6, 1997-2003. Di Renzo MF, Olivero M, Giacomini A, Porte H, Chastre E, Mirossay L, Nordlinger B, Bretti S, Bottardi S, Giodarno S. (1995) Overexpression and amplification of the met/HGF receptor gene during the progression of colorectal cancer. Clin Cancer Res 1, 147-154. Fischer U, Muller H-W, Sattler H-P, Feiden K, Zang KD, Meese E. (1995) Amplification of the met gene in glioma. Genes Chromosom Cancer 12, 63-65. Giordano S, Ponzetto C, Di Renzo MF, Cooper CS, Comoglio PM. ( 1989) Tyrosine kinase receptor indistinguishable from the c-met protein. Nature 339, 155-156. Hacia JG. (1999) Resequencing and mutational analysis using oligonucleotide microarrays. Nat Genet 21 (1 suppl), 42-47. MĂźller U, Bao P, Che D, et al. (2002) DNA-Chips in the analysis of genetic aberrations. Springer Laboratory Manual FISH Technology ed. Rautenstrasse BW, Liehr T, Springer Verlag, Heidelberg, Germany. Murakami Y, Hayashi K, Hirohashi S, et al. (1991) Aberrations of the tumor suppressor p53 and retinoblastoma genes in


Gene Therapy and Molecular Biology Vol 7, page 103 Gene Ther Mol Biol Vol 7, 103-111, 2003

HMG-CoA-reductase inhibition-dependent and independent effects of statins on leukocyte adhesion Research Article

Triantafyllos Chavakis1,2*, Thomas Schmidt-WĂśll2, Peter. P. Nawroth1, Klaus T. Preissner2, Sandip M. Kanse2 1

Department of Internal Medicine I, University Heidelberg and 2Institute for Biochemistry, Justus-Liebig-Universität, Giessen, Germany

__________________________________________________________________________________ *Correspondence: Dr. T. Chavakis, Department of Internal Medicine I, University Heidelberg, Bergheimer Strasse 58, D-69115 Heidelberg, Germany; tel.: ++49 6221 56 4776; fax: ++49 6221 56 ; email: Key words: leukocyte, adhesion, !2-integrins, urokinase-receptor, statins, lovastatin, HMG-CoA reductase Abbreviations: BSA, bovine serum albumin, FBG, fibrinogen, HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme-A, ICAM-1, intercellular cell adhesion molecule-1, PBS, phosphate buffered saline, uPA, urokinase-type plasminogen activator , uPAR, urokinasetype plasminogen activator receptor, VN, vitronectin Received: 1 July 2003; Accepted: 10 July 2003; electronically published: July 2003

Summary Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase, a key enzyme for cholesterol biosynthesis and isoprenoid intermediates. Increasing evidence suggests that statins might affect inflammatory processes including leukocyte recruitment, yet, the underlying mechanisms are not defined. In this study two different pathways for inhibition of leukocyte adhesion by statins are described. (i) Coincubation with lovastatin inhibited adhesion of LFA-1 (CD11a/CD18, "L!2)-transfected K562 cells to ICAM-1 and of p150.95 (CD11c/CD18, "X!2)-transfected K562 cells to both ICAM-1 and fibrinogen (FBG), whereas adhesion of Mac-1 (CD11b/CD18, "M!2)-transfected K562 cells was not affected. Moreover, only LFA-1-mediated adhesion to ICAM1 but not Mac-1-mediated adhesion to FBG or urokinase-receptor (uPAR)-mediated adhesion to vitronectin (VN) of myelo-monocytic U937 cells was blocked by coincubation with lovastatin. The antiadhesive effect of lovastatin was independent of HMG-CoA-reductase inhibition, as it was not reversible in the presence of mevalonate, farnesylpyrophosphate or geranyl-pyrophosphate. In purified systems, lovastatin only blocked the ICAM-1/LFA-1 interaction but not the ICAM-1/Mac-1, FBG/Mac-1 or the VN/uPAR interactions. (ii) In contrast, preincubation of U937 cells for up to 18 h with lovastatin completely abrogated LFA-1-, Mac-1- and uPAR-dependent cell adhesion to the respective ligands. This anti-adhesive function of lovastatin was dependent on HMG-CoA reductase inhibition, since mevalonate or the isoprenoid intermediates restored adhesion, while no downregulation of integrinor uPAR-expression was observed. Thus, two distinct pathways, involving a direct interaction with LFA-1 and p150.95 and an indirect inhibition of cell adhesion through disruption of cholesterol and/or isoprenoid metabolite biosynthesis are induced by statins. These functions can explain at least in part the inhibition of leukocyte adhesion and the associated antiinflammatory role of statins such as VLA-4 ("4!1), that can bind to fibronectin, whereas adhesion to FBG is mediated by the !2 integrins Mac-1 (CD11b/CD18, "M!2, CR3) and p150.95 (CD11c/CD18). Mac-1 together with LFA-1 (CD11a/CD18, "L!2) also provide firm adhesion to and transmigration through the endothelium by recognition of their counter-receptor ICAM-1 on endothelial cells; evidence exists that p150.95 binds ICAM-1 as well (Springer, 1994; Carlos and Harlan, 1994; Stewart et al, 1995; Blackford et al, 1996; Gahmberg, 1997). The functional properties of integrins in general can be modulated by lateral (cis) interaction with integrin

I. Introduction When leukocytes emigrate from the blood-stream into sites of inflammation or injury, they undergo a complex sequence of adhesion and locomotion steps requiring the expression and upregulation of various adhesion receptors on the surface of leukocytes and vascular cells. During their transmigration phase leukocytes adhere to provisional matrix substrates such as fibrinogen (FBG), fibronectin or vitronectin (VN) at sites of increased vascular permeability or damage. The prominent adhesion receptors on leukocytes are integrins, 103

Chavakis et al: Leukocyte adhesion and statins associated protein (CD47), members of the tetraspanin family, syndecans, caveolin-1 or urokinase type plasminogen activator receptor (uPAR) (CD87), leading to the formation of transient multireceptor complexes that facilitate the dynamic recruitment of signaling molecules to sites of cellular contacts or focal adhesions (Ossowski and Aguirre-Ghiso, 2000; Preissner et al, 2000). Besides its ability to regulate integrin-dependent adhesion phenomena, uPAR can also directly mediate leukocyte adhesion to matrix-associated VN (Wei et al, 1994; Sitrin et al, 1996; May et al, 1998). Recently, attention has been drawn to the role of microdomain structures of the plasma membrane, denoted lipid rafts, in cell adhesion. Lipid rafts are enriched in glycosphingolipids, cholesterol, transmembrane proteins and signaling molecules. GPI-anchored proteins may become sequestered into the microdomains as well, which have a lower fluidity than the surrounding membrane allowing the formation of multireceptor adhesion complexes. On epithelial cells, caveolin is a unique raft component, that has the intrinsic propensity to oligomerize and, thereby, contribute to formation of membrane invaginations termed caveolae (Horejsi et al, 1999; Kurzchalia and Parton, 1999; Smart et al, 1999; Simons and Toomre, 2000). Although leukocytes lack caveolin expression, they still contain lipid rafts that may facilitate the formation of adhesion complexes. The possibility that lipid rafts might regulate leukocyte adhesion by modulating integrin avidity has already been suggested (Krauss and Altevogt, 1999). Statins inhibit the key enzyme of cholesterol biosynthesis 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG-CoA reductase). In addition to lowering plasma cholesterol, increasing evidence suggests that statins play a pleiotropic role in the vascular system by effects on nitric oxide synthesis, smooth muscle cell proliferation, fibrinolysis or the immune system (Soma et al, 1993; Aikawa et al, 1998; Essig et al, 1998; Guisarro et al, 1998; Laufs and Liao, 1998; Laufs et al, 1998, 1999; Kwak et al, 2000; Diomede et al, 2001; Kwak and Mach, 2001). In particular, statins could inhibit leukocyte recruitment by regulating the expression of monocyte chemoattractant protein-1 (Romano et al, 2000) and of adhesion receptors (Weber et al, 1997; Ganne et al, 2000; Yoschida et al, 2001) or they might modulate integrin affinity by preventing geranyl-geranylation of RhoA (Liu et al, 1999). Cholesterol depletion by statins might also disrupt lipid rafts and, thereby, affect cell adhesion (Kraus and Altevogt, 1999; Simons and Toomre, 2000). Finally, a recent report suggested that different statins selectively bind to LFA-1, thereby blocking LFA-1 mediated leukocyte adhesion (Kallen et al, 1999; Weitz-Schmidt et al, 2001). These observations prompted us to investigate in more detail the role of lovastatin in !2-integrin- and uPAR-mediated leukocyte interactions. Two distinct mechanisms, a HMG-CoA reductase-dependent and an –independent, for inhibition of leukocyte adhesion are described, which further help to understand the antiinflammatory role of statins.

II. Materials and methods A. Reagents Two-chain high molecular weight urokinase type plasminogen activator (uPA) was from American Diagnostica (Bergstrasse, Germany). VN was purified from human plasma and converted to the multimeric form as previously described (Chavakis et al, 1998). FBG and fibronectin were purchased from Sigma (Munich, Germany). Vitamin D3 was from Biomol (Hamburg, Germany), transforming growth factor-! was from R & D Systems (Boston, MA), and interleukin-3 was from PBH (Hannover, Germany). Phorbol 12-myristate 13-acetate (PMA) was from Gibco (Paisley, Scotland,UK). The blocking monoclonal antibody against human CD18, 60.3, was kindly provided by Dr. J. Harlan (University of Washington, Seattle, WA), the blocking monoclonal antibody against human CD11a, L15, was a generous gift from Dr. C. Figdor (University of Nijmegen, The Netherlands) and anti-uPAR monoclonal antibodies R3 and R4 (Chavakis et al, 1999) were given by Dr. G. Hoyer-Hansen (The Finsen Laboratory, Copenhagen, Denmark). Monoclonal antibodies K20 against !1-integrins (CD29), 6.5B5 against ICAM-1, 2LPM19c against CD11b, KB90 against CD11c, MHM24 against CD11a and polyclonal rabbit-anti-FBG were from Dako (Hamburg, Germany). Isolated Mac-1, LFA-1 and ICAM-1 were kindly obtained from Dr. S. Bodary (Genentech, San Francisco, CA). Recombinant soluble uPAR was kindly provided by Dr. D. Cines (University of Pennsylvania, Philadelphia, PA). Lovastatin, mevalonate, farnesyl-pyrophosphate and geranyl-pyrophosphate were from Sigma (Munich, Germany). Peroxidase-conjugated secondary anti-mouse and anti-rabbit immunoglobulins were from DAKO (Hamburg, Germany).

B. Cell culture Myelomonocytic cells (U937) obtained from American Type Culture Collection (ATCC) (Rockville, MD) were cultured in RPMI-1640 medium containing 10% (vol/vol) fetal calf serum. K562 cells transfected with Mac-1 were kindly provided by Dr. M. Robinson (Celltech Ltd, Slough, England) and K562 cells transfected with LFA-1 or p150.95 were a generous gift from Dr. Y. van Kooyk (University of Nijmegen, The Netherlands) and were cultivated in a mixture of 75% RPMI containing 10% fetal calf serum and 25% ISCOVE´s medium containing 5% fetal calf serum. Expression of the respective !2integrins was tested by FACS analysis (see below). All culture media were from Gibco (Eggenstein, Germany), and the cell culture plastic was from Nunc (Rocksilde, Denmark).

C. Cell adhesion assays Cell adhesion to VN, ICAM-1 and FBG coated plates (and to BSA-coated wells as control) was tested according to previously described protocols (Chavakis et al, 1999, 2000, 2001, 2002). Briefly, multiwell plates were coated with 5 µg/ml ICAM1, FBG or 2 µg/ml VN (dissolved in bicarbonate buffer, pH 9.6), respectively, and blocked with 3% (wt/vol) BSA. U937 cells, which had been differentiated for 24 h with vitamin D3 (100 nM) and transforming growth factor-! (2 ng/ml), or K562 cells were washed in serum-free RPMI and plated onto the precoated wells for 60-90 min at 37°C in the absence or presence of competitors in serum-free RPMI as indicated in the figure legends. Where indicated, U937 cells were preincubated for various time periods without or together with lovastatin in the absence or presence of mevalonate, farnesyl-pyrophosphate or geranyl-pyrophosphate. Following the incubation period for the adhesion assay, the wells were washed and the number of adherent cells was quantified by


Gene Therapy and Molecular Biology Vol 7, page 105 crystal violet staining at 590 nm.

inhibitory effect of lovastatin on ICAM-1 adhesion was unchanged in the presence of the isoprenoid metabolites mevalonate, farnesyl-pyrophosphate, or geranylpyrophospha te (Figur e 1B). None of these three metabolites alone could affect U937 cell adhesion to ICAM-1 (not shown). U937 cells engage both Mac-1 and LFA-1 for ICAM-1-dependent adhesion; however, the lack of inhibitory activity of lovastatin on Mac-1-related adhesion to FBG indicated that lovastatin interacts only with LFA-1 directly.

D. Analysis of uPAR and integrin expression by flow cytometry After incubation for 18 h in the absence or presence of lovastatin differentiated U937 cells were washed twice with HEPES-buffered saline and were incubated with saturating concentrations of primary antibody (10 µg/ml) for 60 min at 4°C. Cells were washed again, resuspended in HEPES buffer and phycoerythrin-conjugated F(ab ,)2 fragment of goat anti-rabbit (or mouse) IgG (Dianova, Hamburg, Germany) was added in saturating concentrations for 60 min at 4°C. After washing and resuspension, mean fluorescence of 10,000 cells was measured in a flow cytometer (Beckton Dickinson, Heidelberg, Germany). Nonspecific fluorescence was determined using control speciesand isotype-matched primary antibody.

E. ELISA for ligand-receptor interactions Maxisorp plates (high binding capacity; Nunc) were coated with Mac-1 or LFA-1 (5 µg/ml) dissolved in 20 mM HEPES, 150 mM NaCl, 1 mM Mn2+, pH 7.2 and then blocked with 3% (wt/vol) bovine serum albumin (BSA) in the same buffer. Binding of FBG (10 µg/ml) or ICAM-1 (10 µg/ml) to the immobilized integrin was performed in a final volume of 50 µl of the same buffer as above together with 0.05% (wt/vol) Tween-20 and 0.1 % (wt/vol) BSA in the absence or presence of different competitors as indicated in the figure legends. After incubation for 2 h at 22°C and a washing step, bound ligands were detected by the addition of polyclonal rabbit anti-FBG or monoclonal mouse anti-ICAM-1 followed by the addition of 1:1000 diluted peroxidase-conjugated antibody against rabbit or mouse immunoglobulins, respectively. The conversion of the substrate 2,2-azino-di(3-ethly)benzthiazoline sulphate (Boehringer, Mannheim, Germany) was monitored at 405 nm in a Thermomax microtitre plate reader (Molecular Devices, Menlo Park, CA). Nonspecific binding to BSA-coated wells was used as blank and was subtracted to calculate the specific binding. The same protocol was used when binding of multimeric VN (2 µg/ml) to immobilized uPAR (5 µg/ml, dissolved in bicarbonate buffer, pH 9.6) was tested, except that the binding buffer was TBS containing 0.05 % (wt/vol) Tween-20 0.1 % (wt/vol) BSA. Bound VN was detected with the anti-VN monoclonal antibody VN7 and additional steps of quantitation were the same as mentioned above.

Figure 1. U937 cell adhesion to ICAM-1, FBG and VN. (A) PMA (50 ng/ml)-stimulated U937 cell adhesion to immobilized ICAM-1 (5 µg/ml) and FBG (5 µg/ml) or uPA (50 nM)stimulated U937 cell adhesion to immobilized VN (2 µg/ml) was studied in the absence (open bars) or the presence of lovastatin (100 µM, filled bars) or the following blocking antibodies (hatched bars): anti-CD18 (15 µg/ml) for ICAM-1- and FBGmediated adhesion, anti-uPAR (10 µg/ml) for VN-dependent adhesion. (B) PMA (50 ng/ml)-stimu late d U937 cell adhesion to immobilized ICAM-1 (5 µg/ml) was studied in the absence (-) or presence of a blocking anti-CD18 antibody (15 µg/ml), a blocking anti-LFA-1 (CD11a) antibody (15 µg/ml), lovastatin alone (100 µM), or in combination with mevalonate (100 µM, MEV), farnesyl-pyrophosphate (100 µM, FP), or geranylpyrophosphate (100 µM, GP). Cell adhesion is expressed as percent of control, which is represented by the adhesion in the presence of PMA (or uPA, where adhesion to VN is shown) and in the absence of any competitor. Data are mean ± SEM (n=3) of a typical experiment; similar results were obtained in at least three separate experiments.

III. Results A. HMG-CoA reductase independent regulation of leukocyte adhesion by lovastatin As previously established, the adhesion of myelomonocytic U937 cells [differentiated with TGF! (2 ng/ml) and vitamin D3 (100 nM) for 24 h] to immobilized FBG is predominantly mediated by Mac-1, whereas both Mac-1 and LFA-1 mediate adhesion to immobilized ICAM-1. U937 cell adhesion to FBG and ICAM-1 is enhanced by Mn2+ or phorbol ester (PMA). Moreover, U937 cell adhesion to VN is uPAR-dependent; uPA can stimulate adhesion, as it increases the affinity of the uPAR/VNinteraction (Chavakis et al, 2000, 2001 Preissner et al, 2000). In the presence of lovastatin, adhesion of U937 cells to ICAM-1was markedly reduced, whereas adhesion to FBG or VN was not affected at all (Figure 1A). The 105

Chavakis et al: Leukocyte adhesion and statins In order to test this hypothesis in detail, the inhibitory capacity of lovastatin was tested in two further systems: (i) In a purified system, lovastatin inhibited only binding of ICAM-1 to LFA-1, whereas the binding of ICAM-1 to immobilized Mac-1, the binding of FBG to Mac-1 or the binding of VN to immobilized uPAR were not affected at all (Figure 2). ( ii) The effect of lovastatin on adhesion of differently transfected erythroleukemic K562 cells was studied: While non-transfected K562 cells did not adhere to FBG or ICAM-1, respectively, cells became adherent to both substrates upon transfection with Mac-1 or p150.95, whereas LFA-1 transfected cells only adhered to ICAM-1 (not shown). As expected, adhesion of Mac-1 transfected cells to ICAM-1 and FBG was not changed in the presence of lovastatin, whereas adhesion of LFA-1 transfected cells was completely inhibited by lovastatin with an IC50 of approximately 20 µM. Interestingly, adhesion of p150.95 transfected cells to both FBG and ICAM-1 was partially blocked by lovastatin with an IC50 of about 70 µM (Figure 3A and Figure 3B). The antiadhesive effect of lovastatin on adhesion of both LFA1- and p150.95- transfected cells was not abolished in the presence of mevalonate, farnesyl-pyrophosphate or geranyl-pyrophosphate (Figure 3C and Figure 3D). Taken together, these data indicate that lovastatin selectively interacts with LFA-1 and with a lower potency with p150.95 but not with Mac-1. Lovastatin thereby can block LFA-1-mediated cell adhesion to ICAM-1 and to a lower extent p150.95-mediated adhesion to FBG and ICAM-1 in a manner independent of inhibition of HMGCoA reductase.

Figure 3: Influence of lovastatin coincubation on the adhesion of K562 cells. PMA (50 ng/ml)-stimulated adhesion of Mac-1transfected K562 cells (filled squares), p150.95-transfected K562 cells (open circles) and LFA-1-transfected K562 cells (filled triangles) to immobilized ICAM-1 (5 µg/ml) (A) and PMA (50 ng/ml)-stimulated adhesion of Mac-1-transfected K562 cells (filled squares) and p150.95-transfected K562 cells (open circles) to immobilized FBG (5 µg/ml) (B) was studied in the presence of increasing concentrations of lovastatin. PMA (50 ng/ml)stimulated adhesion of Mac-1-transfected K562 cells, p150.95transfected K562 cells and LFA-1-transfected K562 cells to immobilized ICAM-1 (5 µg/ml) (C) and PMA (50 ng/ml)stimulated adhesion of Mac-1-transfected K562 cells and p150.95-transfected K562 cells to immobilized FBG (5 µg/ml) (D) was studied in the absence (open bars) or presence of lovastatin alone (100 µM, filled bars), or in combination with mevalonate (100 µM, hatched bars), farnesyl-pyrophosphate (100 µM, dotted bars), or geranyl-pyrophosphate (100 µM, vertical lines). Cell adhesion is shown as percent of control, which is represented by the adhesion of cells in the absence of any competitor. Data are mean ± SEM (n=3) of a typical experiment; similar results were obtained in at least three separate experiments.

Figure 2: Influence of lovastatin on different ligand receptor interactions. The binding of ICAM-1 (10 µg/ml) to immobilized Mac-1 (open squares) or to immobilized LFA-1 (filled triangles), the binding of FBG (10 µg/ml) to immobilized Mac-1 (filled squares) or the binding of VN to immobilized uPAR (open circles) is analyzed in the absence or presence of increasing concentrations of lovastatin. Specific binding is expressed as percent of control, which is represented by the binding of the ligand to the respective immobilized receptor in the absence of lovastatin. Data are mean ± SEM (n=3) of a typical experiment; similar results were obtained in at least three separate experiments.


Gene Therapy and Molecular Biology Vol 7, page 107 direct blocking effect on LFA-1 and a HMG-CoA reductase-dependent effect. The HMG-CoA reductase-dependent antiadhes ive effect of lova statin preincuba tion might result from a downregulation of the expression of respective adhesion receptors, namely !2-integrins or uPAR. However, lovastatin preincubation for 18 h did not affect the expression level of uPAR, !2-integrins (no change in CD11a, CD11b and CD18 expression) or !1 integrins (CD29) (Table 1 ). The CD11c chain was not detected on U937 cells, explaining the lack of inhibition of U937 cell adhesion to FBG by coincubation with lovastatin (Figure 1). In conclusion, these findings indicate that lovastatin preincubation can regulate both !2-integrin and uPARmediated leukocyte adhesion in a cholesterol biosynthesisdependent manner without changing the expression level of !2-integrins or uPAR.

B. HMG-CoA reductase-dependent regulation of leukocyte adhesion by lovastatin In contrast to the described direct antiadhesive effect of lovastatin on cells during coincubation, a completely different pattern of inhibition was observed when lovastatin was preincubated with leukocytes for up to 18 h followed by removal of excess reagent prior to the cell adhesion experiment. In particular, lovastatin preincubated for 18 h with U937 cells dose-dependently inhibited their adhesion to ICAM-1, FBG or VN. The inhibitory capacity was almost identical in all three systems (IC50 of about 12 µM) (Figure 4). Furthermore, the following differences were observed between U937 cell adhesion to ICAM-1 and adhesion to FBG and VN: In the time course, after 5 h of incubation with lovastatin about 30 % inhibition of U937 cell adhesion to FBG and VN was observed and inhibition was almost complete after 12 h. At all time points the effect of lovastatin was restored by mevalonate. Farnesyl-pyrophosphate or geranyl-pyrophosphate as well could completely reverse the antiadhesive effect of lovastatin on cell adhesion to FBG and VN (Figure 5). In contrast, already after 2 h of lovastatin preincubation adhesion to ICAM-1 was inhibited by 50% but could not be restored by mevalonate. Again, after 12 h lovastatin preincubation U937 cell adhesion to ICAM-1 was completely abolished However, this effect was only partially (50% of initial adhesion) reversed in the presence of mevalonate reaching a cell adhesion level that was comparable to cell adhesion after 2 h lovastatin preincubation (Figure 5). Thus, the action of lovastatin preincubation on U937 cell adhesion to ICAM-1 consists of two components, a HMG-CoA reductase-independent

Figure 5: Influence of preincubation of lovastatin and isoprenoid metabolites on U937 cell adhesion. Following preincubation for various time periods as indicated, PMA (50 ng/ml)-stimulated U937 cell adhesion to (A) immobilized ICAM-1 (5 µg/ml), to (B) immobilized FBG (5 µg/ml) or (C) uPA (50 nM)-stimulated U937 cell adhesion to immobilized VN (2 µg/ml) was studied in the absence (vertical lines) or presence of lovastatin (20 µM) alone (open bars) or in combination with mevalonate (100 µM, filled bars). In the 18 h preincubation setting lovastatin was also reacted together with farnesyl-pyrophosphate (100 µM, hatched bars) or geranyl-pyrophosphate (100 µM, dotted bars). Cell adhesion is expressed as percent of control, which is represented by the adhesion in the presence of PMA (or uPA, where adhesion to VN is shown) and in the absence of any competitor. Data are mean ± SEM (n=3) of a typical experiment; similar results were obtained in three separate experiments.

Figure 4: Influence of lovastatin preincubation on U937 cell adhesion. Following preincubation for 18 h in the absence or presence of increasing concentrations of lovastatin, adhesion of PMA (50 ng/ml)-stimulated U937 cells to immobilized ICAM-1 (5 µg/ml) (filled triangles), to immobilized FBG (5 µg/ml) (open squares) or uPA (50 nM)-stimulated U937 cell adhesion to immobilized VN (2 µg/ml) (open circles) was studied. Cell adhesion is expressed as percent of control, which is represented by the adhesion in the presence of PMA (or uPA, where adhesion to VN is shown) and in the absence of lovastatin. Data are mean ± SEM (n=3) of a typical experiment; similar results were obtained in three separate experiments.


Chavakis et al: Leukocyte adhesion and statins Table 1: Influence of lovastatin on integrin and uPAR expression. Receptors Control CD11a 100+8.2 CD11b 100+6.4 CD18 100+12.9 CD29 100+8.4 uPAR 100+8.9

Lovastatin 92.6+3.1 97.3+5.3 110.7+9.1 106.7+4.5 97.9+1.7

The expression of CD11a, CD11b, CD18, CD29 and uPAR on U937 cells that were preincubated for 18 h in the absence or presence of lovastatin (40 µM) as measured by FACS-analysis is shown. The expression of the various integrins or uPAR is presented as percent of control, which relates to the expression of the respective adhesion molecule in the absence of lovastatin. Data are mean ± SEM (n=3) of a typical experiment; similar results were obtained in three separate experiments.

affinity to p150.95, but not to Mac-1, thereby directly affecting leukocyte adhesion. When lovastatin was preincubated with monocytes for up to 18 h, a different inhibition profile was observed: Lovastatin completely blocked all three adhesive events, namely LFA-1/Mac-1-dependent adhesion to ICAM-1, Mac-1-dependent adhesion to FBG and uPAR-dependent adhesion to VN. Inhibition of ICAM-1-related adhesion could be partially attributed to the direct LFA-1 binding property of lovastatin, as (i) a significant inhibition by 50% occured already after 2 h, and was not reversed by mevalonate and (ii) complete inhibition was observed after longer preincubation times (12-18 h) and could be partially reversed by mevalonate up to the adhesion level obtained after 2 h preincubation with lovastatin. In contrast, both Mac-1- and uPAR-dependent cell adhesion were partially inhibited after 6 h preincubation with lovastatin and were completely blocked after 12-18 h. This effect of lovastatin was dependent on HMG-CoA reductase inhibition, as it was completely reversible in the presence of mevalonate. Interestingly, the IC50 of the HMG-CoA reductase-dependent effect of lovastatin was approximately 1 µM, which is about 20 times (LFA-1) or 70 times (p150.95) lower than the IC50 of the HMG-CoA reductase-independent direct abrogation of both integrinmediated adhesion reactions. Thus, the antiinflammatory action of statins implied in clinical studies are very likely attributable to the HMG-CoA reductase-dependent pathway, as the higher concentrations of statins required for the direct inhibition of the LFA-1/ICAM-1-, the p150.95/FBG- and the p150.95/ICAM-1-interactions may not be reached with the standard doses (nanomolar range) of approved statin drugs (Frenette, 2001). Indeed, a recent report demonstrated that mevalonate-derived isoprenoid metabolites mediate the antiinflammatory activity of statins in the in vivo air-pouch model of local inflammation (Diomede et al, 2001). Finally, the antiinflammatory capacity of statins may vary dependent on their individual structure (Weitz-Schmidt et al, 2001). While direct binding to LFA-1 and p150.95 sufficiently explains the HMG-CoA reductaseindependent antiadhesive effect of lovastatin, different mechanisms might be involved in the HMG-CoA reductase-dependent anti-adhesive property of lovastatin: (i) Lowering the plasma membrane cholesterol content can affect cell adhesion by disrupting lipid raft integrity (Krauss and Altevogt, 1999; Simons and Toomre, 2000). Recently, the assembly of adhesion complexes containing

IV. Discussion Leukocyte activation and adhesion to the endothelium and the subsequent transendothelial migration are pivotal steps in the recruitment of cells to the inflammatory /injured tissue. This highly coordinated multistep process requires tight regulation of adhesive events (Carlos and Harlan, 1994; Springer, 1994) including the induction of genes coding for participating adhesion receptors including integrins, their change in avidity as well as the modification of ligand-binding properties (Porter and Hogg, 1998; Woods and Couchman, 2000). Conversely, in pathological situations associated with organ transplantation, atherosclerosis and ischemia/reperfusion injury, arthritis and psoriasis the antagonism of these adhesive leukocytic interactions may become a promising therapeutic appproach (Nahakura et al, 1996; Issekutz, 1998; Kruegeret al, 2000; Martin et al, 2000; Poston et al, 2000). In this respect, recent evidence points to an immunomodulatory role of statins (Katznelson and Kobashigawa, 1995; Maron et al, 2000; Kwak and Mach, 2001) which are commonly used to reduce plasma cholesterol levels in order to decrease the risk of cardiovascular disease (Corsini et al, 1995). In this study we define the direct and indirect role of statins in leukocyte adhesion and the possible underlying mechanisms. Two distinct pathways, a HMG-CoA reductase-dependent and an –independent were distinguished and appear to be relevant for the antiadhesive effects of statins. In particular, coincubation of monocytes with lovastatin resulted in a dramatic reduction of LFA-1dependent cell adhesion to ICAM-1, but not of Mac-1dependent adhesion to FBG or uPAR-dependent adhesion to VN. This direct antiadhesive effect of lovastatin was unrelated to HMG-CoA reductase inhibition, as it was not reversed by mevalonate or other isoprenoid metabolites. Rather, it was attributed to the direct inhibition of the LFA-1/ICAM-1 interaction by lovastatin as corroborated in a purified system. Whereas Mac-1 binding to its ligands ICAM-1 and FBG as well as uPAR interaction with VN were not directly affected by lovastatin, binding of another !2-integrin, p150.95, to FBG and ICAM-1 was partially blocked directly by lovastatin. Our data are in accordance with and extend a recent report showing that statins inhibit LFA-1 by binding to an allosteric L-site located within the I-domain of the " chain (Weitz-Schmidt et al, 2001). Thus, lovastatin binds to LFA-1 as well as with lower


Gene Therapy and Molecular Biology Vol 7, page 109 adhesion receptors as well as signaling molecules such as focal adhesion kinase or src kinases has been proposed to be confined to glycosphingolipid- and cholesterol-rich, detergent insoluble microdomains of the cell membrane. The antiadhesive effect of lovastatin preincubation presented here could very well be due to raft disruption by cholesterol depletion, as other approaches to disrupt these membrane microdomains result in a very similar downregulation of !2-integrin and uPAR mediated leukocyte adhesion (Chavakis et al., unpublished observations). Moreover, as lipid rafts have been implicated in T-cell receptor-, EGF-receptor-, insulin receptor-, H-Ras-, eNOS- and integrin-dependent signalling phenomena (Simons and Toomre, 2000), the potential modulatory role of HMG-CoA-reductase inhibitors on raft integrity and associated vital cellular functions renders these drugs very attractive for several therapeutic interventions in vascular medicine. (ii) Although conflicting results have been reported as to the influence of statins on the cell type specific integrin and uPAR expression (Weber et al, 1997; Liu et al, 1999; Wojeiak-Stothard, 1999; Yoschida et al, 2001), our data are in accordance with these reports showing no change in integrin expression in e.g. myelo-monocytic U937 cells by lovastatin (Weber et al, 1995; Liu et al, 1999). (iii) It has been demonstrated that protein geranyl-geranylation is required for !1-integrin-dependent adhesion of leukocytes. It is thus conceivable that statin treatment may affect integrin-dependent leukocyte adhesion via inhibition of the geranyl-geranylation of RhoA, which is thought to be one of the most important effectors involved in regulation of the cytoskeleton network, including the clustering of adhesion molecules during monocyte adherence (Liu et al, 1999; Wojciak-Stothard et al, 1999; Kwak and Mach, 2001; Yoshida et al, 2001). The possibility that statin treatment could thereby directly inhibit RhoA activation and disrupt actin polymerization leading to failure of integrin clustering is a likely interpretation of the presented data, since isoprenoid metabolites could reverse the antiadhesive effect of lovastatin pretreatment. Together, our findings help to decipher the mechanisms underlying the postulated antiinflammatory effects of statins, which, besides atherothrombosis, may prove to be beneficial in arthritis, organ transplantation or psoriasis.

References Aikawa M, Rabkin E, Okada Y, Voglic SJ, Clinton SK, Brinckerhoff CE, Sukhova GK, Libby P (1998) Lipid lowering by diet reduces matrix metalloproteinase activity and increases collagen content of rabbit atheroma: a potential mechanism of lesion stabilization. Circulation 97, 2433–2444 Blackford J, Reid HW, Pappin DJ, Bowers FS, Wilkinson JM (1996) A monoclonal antibody, 3/22 to rabbit CD11c which induces homotypic T cell aggregation: evidence that ICAM-1 is a ligand for CD11c/CD18. Eur J Immunol 26, 525-531 Carlos TM, Harlan JM (1994) Leukocyte-endothelial adhesion molecules. Blood 84, 2068-2101 Chavakis T, Kanse SM, Yutzy B, Lijnen HR, Preissner KT (1998) Vitronectin concentrates proteolytic activity on the cell surface and extracellular matrix by trapping soluble urokinase receptor-urokinase complexes. Blood 91, 23052312 Chavakis T, May AE, Preissner KT, Kanse SM (1999) Molecular mechanisms of zinc-dependent leukocyte adhesion involving the urokinase receptor and !2-integrins. Blood 93, 29762983 Chavakis T, Kanse SM, Lupu F, Hammes HP, Muller-Esterl W, Pixley RA, Colman RW, Preissner KT (2000) Different mechanisms define the antiadhesive function of high molecular weight kininogen in integrin- and urokinase receptor-dependent interactions. Blood 96, 514-522 Chavakis T, Kanse SM, Pixley RA, May AE, Isordia-Salas I, Colman RW, Preissner KT (2001) Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen. FASEB J 15 2365-2376 Chavakis T, Hussain M, Kanse SM, Peters G, Bretzel RG, Flock JI, Herrmann M, Preissner KT (2002) Staphylococcus aureus extracellular adherence protein (Eap) serves as antiinflammatory factor by inhibiting the recruitment of host leukocytes. Nature Medicine 8, 687-693 Corsini A, Maggi FM, Catapano AL (1995) Pharmacology of competitive inhibitors of HMG-CoA reductase. Pharmacol Res 31, 9–27 Diomede L, Albani D, Sottocorno M, Polentarutti N, Donati MB, Bianchi M, Fruscella P, Salmona M (2001) The in vivo antiinflammatory effect of statins is mediated by nonsterol mevalonate products. Arterioscler Thromb Vasc Biol 21, 1327–1332 Essig M, Nguyen G, Prie D, Escoubet B, Sraer JD, Friedlander G (1998) 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors increase fibrinolytic activity in rat aortic endothelial cells: role of geranylgeranylation and Rho proteins. Circ Res 83, 683–690 Frenette PS (2001) Locking a leukocyte integrin with statins. N Engl J Med 345, 1419-1421 Gahmberg CG (1997) Leukocyte adhesion: CD11/CD18 integrins and intercellular adhesion molecules. Curr Opin Cell Biol 9, 643-650 Ganne F, Vasse M, Beaudeux JL, Peynet J, Francois A, Mishal Z, Chartier A, Tobelem G, Vannier JP, Soria J, Soria C (2000) Cerivastatin, an inhibitor of HMG-CoA reductase, inhibits urokinase/urokinase-receptor expression and MMP-9 secretion by peripheral blood monocytes-a possible protective mechanism against atherothrombosis. Thromb Haemost 84, 680-688 Guijarro C, Blanco-Colio LM, Ortego M, Alonso C, Ortiz A, Plaza JJ, Diaz C, Hernandez G, Edigo J (1998) 3-Hydroxy-3-

Acknowledgments This work was supported in part by a grant from the Novartis Foundation for Therapeutical Research to TC and KTP (Nürnberg, Germany), by a grant from the Deutsche Forschungsgemeinschaft to TC (CH279/1-1) and by a grant from Vascular Genomics-Kerckhoff Klinik GmbH to KTP (Bad Nauheim, Germany). We acknowledge the generous gift of reagents from Drs. D.B. Cines (Philadelphia, PA), G. Hoyer-Hansen and N. Behrendt (Copenhagen, Denmark), S. Bodary (San Francisco, CA) and J. Harlan (Seattle, WA) and Ms M. Economopoulou for help during manuscript preparation.


Chavakis et al: Leukocyte adhesion and statins methylglutaryl coenzyme A reductase and isoprenylation inhibitors induce apoptosis of vascular smooth muscle cells in culture. Circ Res 83, 490 –500 Horejsi V, Drbal K, Cebecauer M, Cerny J, Brdicka T, Angelisova P, Stockinger H (1999) GPI-microdomains: a role in signalling via immunoreceptors. Immunol Today 20, 356-361 Issekutz AC (1998) Adhesion molecules mediating neutrophil migration to arthritis in vivo and across endothelium and connective tissue barriers in vitro. Inflamm Res 47, S123–S132 Kallen J, Welzenbach K, Ramage P, Geyl D, Kriwacki R, Legge G, Cottens S, Weitz-Schmidt G, Hommel U (1999) Structural basis for LFA-1 inhibition upon lovastatin binding to the CD11a I-domain. J Mol Biol 292, 1-9 Katznelson S, Kobashigawa JA (1995) Dual roles of HMG-CoA reductase inhibitors in solid organ transplantation: Lipid lowering and immunosuppression. Kidney Int 48, S112–S115 Krauss K, Altevogt P (1999) Integrin leukocyte functionassociated antigen-1-mediated cell binding can be activated by clustering of membrane rafts. J Biol Chem 274, 3692136927 Krueger J, Gottlieb A, Miller B, Dedrick R, Garovoy M, Walicke P (2000) Anti-CD11a treatment for psoriasis concurrently increases circulating T-cells and decreases plaque T-cells, consistent with inhibition of cutaneous T-cell trafficking. J Invest Dermatol 115, 333 Kurzchalia TV, Parton RG (1999) Membrane microdomains and caveolae. Curr Opin Cell Biol 11, 424-431 Kwak BR, Mach F (2001) Statins inhibit leukocyte recruitment. New evidence for their anti-inflammatory properties. Arterioscler Thromb Vasc Biol 21, 1256-1258 Kwak B, Mulhaupt F, Myit S, Mach F (2000) Statins as a newly recognized type of immunomodulator. Nat Med 6, 13991403 Laufs U, La Fata V, Plutzky J, Liao JK (1998) Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 97, 1129–1135 Laufs U, Liao JK (1998) Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem 273, 24266–24271 Laufs U, Marra D, Node K, Liao JK (1999) 3-Hydroxy-3methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing rho GTPaseinduced down-regulation of p27(Kip1). J Biol Chem 274, 21926 –21931 Liu L, Moesner P, Kovach NL, Bailey R, Hamilton AD, Sebti SM, Harlan JM (1999) Integrin-dependent leukocyte adhesion involves geranylgeranylated protein(s). J Biol Chem 274, 33334–33340 Maron DJ, Fazio S, Linton MF (2000) Current perspectives on statins. Circulation 101, 207–213 Martin X, Da Silva M, Virieux SR, Hadj Aissa A, Buffet R, Tiollier J, Dubernard JM (2000) Protective effect of an antiLFA 1 monoclonal antibody (odulimomab) on renal damage due to ischemia and kidney autotransplantation. Transplant Proc 32, 481 May AE, Kanse SM, Lund LR, Gisler RH, Imhof BA, Preissner KT (1998) Urokinase receptor (CD87) regulates leukocyte recruitment via !2-integrins in vivo. J Exp Med 188, 10291037

Nakakura EK, Shorthouse RA, Zheng B, McCabe SM, Jardieu PM, Morris RE (1996) Long-term survival of solid organ allografts by brief anti-lymphocyte function-associated antigen-1 monoclonal antibody monotherapy. Transplantation 62, 547–552 Ossowski L, Aguirre-Ghiso JA (2000) Urokinase-receptor and integrin partnership: coordination of signaling for cell adhesion, migration and growth. Curr Opin Cell Biol 12, 613-620 Porter JC, Hogg N (1998) Integrins take partners: cross-talk between integrins and other membrane receptors. Trends Cell Biol 8, 390-396 Poston RS, Robbins RC, Chan B, Simms P, Presta L, Jardieu P, Morris RE (2000) Effects of humanized monoclonal antibody to rhesus CD11a in rhesus monkey cardiac allograft recipients. Transplantation 69, 2005–2013 Preissner KT, Kanse SM, May AE (2000) Urokinase receptor: a molecular organizer in cellular communication. Curr Opin Cell Biol 12, 621-628 Romano M, Diomede L, Sironi M, Massimiliano L, Sottocorno M, Polentarutti N, Guglielmotti A, Albani D, Bruno A, Fruscella P, Salmona M, Vecchi A, Pinza M, Mantovani A (2000) Inhibition of monocyte chemotactic protein-1 synthesis by statins. Lab Invest 80, 1095–1100 Simons K, Toomre D (2000) Lipid rafts and signal transduction. Nat Rev Mol Cell Bio 1, 31-39 Sitrin RG, Todd RF, Petty HR, Brock TG, Shollenberger SB, Albrecht E, Gyetko MR (1996) The urokinase receptor (CD87) facilitates CD11b/CD18-mediated adhesion of human monocytes. J Clin Invest 97, 1942-1951 Smart EJ, Graf GA, McNiven MA, Sessa WC, Engelman JA, Scherer PE, Okamoto T, Lisanti MP (1999) Caveolins, liquid-ordered domains, and signal transduction. Mol Cell Biol 19, 7289-7304 Soma MR, Donetti E, Parolini C, Mazzini G, Ferrari C, Fumagalli R, Paoletti R (1993) HMG CoA reductase inhibitors: in vivo effects on carotid intimal thickening in normocholesterolemic rabbits. Arterioscler Thromb 13, 571–578 Springer TA (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 76, 301-314 Stewart M, Thiel M, Hogg N (1995) Leukocyte integrins. Curr Opin Cell Biol 7, 690-696 Weber C, Erl W, Weber KS, Weber PC (1997) HMG-CoA reductase inhibitors decrease CD11b expression and CD11bdependent adhesion of monocytes to endothelium and reduce increased adhesiveness of monocytes isolated from patients with hypercholesterolemia. J Am Coll Cardiol 30, 1212–1217 Weber C, Erl W, Weber PC (1995) Lovastatin induces differentiation of Mono Mac 6 cells. Cell Biochem Funct 13, 273-277 Wei Y, Waltz DA, Rao N, Drummond RJ, Rosenberg S, Chapman HA (1994) Identification of the urokinase receptor as an adhesion receptor for vitronectin. J Biol Chem 269, 32380-32388 Weitz-Schmidt G, Welzenbach K, Brinkmann V, Kamata T, Kallen J, Bruns C, Cottens S, Takada Y, Hommel U (2001) Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat Med 7, 687692


Gene Therapy and Molecular Biology Vol 7, page 111 Wojciak-Stothard B, Williams L, Ridley AJ (1999) Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering. J Cell Biol 145, 1293–1307 Woods A, Couchman JR (2000) Integrin modulation by lateral association. J Biol Chem 275, 24233-24236

Yoshida M, Sawada T, Ishii H, Gerszten RE, Rosenzweig A, Gimbrone MA Jr, Yasukochi Y, Numano F (2001) HMGCoA reductase inhibitor modulates monocyte endothelial interaction under physiological flow condition in vitro: involvement of Rho GTPase-dependent mechanism. Arterioscler Thromb Vasc Biol 21, 1165–1171


Chavakis et al: Leukocyte adhesion and statins


Gene Therapy and Molecular Biology Vol 7, page 113 Gene Ther Mol Biol Vol 7, 113-133, 2003

Current progress in adenovirus mediated gene therapy for patients with prostate carcinoma Review Article

Ahter D. Sanlioglu 1,3, Turker Koksal 2,3, Mehmet Baykara 2,3, Guven Luleci 1,3, Bahri Karacay4 and Salih Sanlioglu1,3,* 1

Departments of Medical Biology and Genetics, 2Department of Urology and 3The Human Gene Therapy Unit of Akdeniz University, Faculty of Medicine, Antalya, Turkey, 07070; 4Department of Pediatrics, University of Iowa, College of Medicine, Iowa City, IA, 52240, USA

__________________________________________________________________________________ *Correspondence: Salih Sanlioglu V.M.D., Ph.D., Director of The Human Gene Therapy Unit of Akdeniz University, Faculty of Medicine, B- Block, 1st floor, Campus, Antalya, 07070 Turkey; Phone: (90) 242-227-4343/ext: 44359, Fax: (90) 242-227-4482; e-mail: Key words: Prostate cancer, adenovirus, gene therapy, immunomodulation, apoptosis, inducible promoters Received: 1 July 2003; Accepted: 11 July 2003; electronically published: July 2003

Summary Prostate cancer is the most frequently diagnosed male cancer in the world. Like all cancers, prostate cancer is a disease of uncontrolled cell growth. In some cases tumors are slow growing and remain local, but in others they may spread rapidly to the lymph nodes, other organs and especially bone. Although surgery and radiation can cure early stages of organ confined prostate carcinoma (stages I and II), there is no curative therapy at this time for locally advanced or metastatic disease (stages III and IV). The likelihood of postsurgical local recurrence increases with capsular penetration as detected in 30 % of the patients at the time of radical prostatectomy. Moreover, 10-15 % of patients have metastatic cancer at the time of diagnosis. Considering the fact that 60 % local recurrence is observed in patients receiving radiation therapy with or without adjuvant hormonal ablation therapy, it is generally believed that androgen ablation therapy simply delays the progression of prostate carcinoma to a more advanced stage. In addition, the overall ten-year survival rate of patients with locally recurrent prostate cancer is only around 35 %; thus; the ultimate progression into androgen independent prostate carcinoma appears to be inevitable. Gene therapy arose as a novel treatment modality with the potential to decrease the morbidity associated with conventional therapies. Therefore, gene therapy is expected to lower the incidence of tumor recurrence and finally improve the outcome of patients with recurrent and androgen independent prostate carcinoma. Viral vectors are most commonly used for the purpose of gene therapy. Currently, there are a total of 40 clinical trials being conducted using viral vectors for the treatment of prostate carcinoma. 22 out of 40 clinical protocols (55 %) approved for the treatment of prostate cancer utilize adenovirus vectors. Most of these adenovirus mediated therapeutic approaches employ either selectively replicating adenoviruses or suicide gene therapy approaches. In this review, we mainly concentrated on the progress in adenovirus mediated gene therapy approaches for prostate cancer. Analysis of the death ligand mediated gene therapy approach was also discussed in detail, while our novel findings were incorporated as an example for up-to-date approaches used for adenovirus mediated gene therapy against prostate carcinoma. male cancer in the United States (Powell et al, 2002). Despite the fact that there has been a considerable effort for screening and early detection of prostate cancer in recent years, the lifetime risk of being diagnosed with prostate cancer is still reported to be 1 in 5 (Grumet and Bruner, 2000). Several hundred clinical studies using experimental or approved chemotherapeutics failed to improve survival rates of patients with prostate cancer (Devi, 2002). Because prostate cancer is a heterogeneous

I. Introduction Prostate cancer is the second leading cause of death in men from cancer following lung carcinoma with an annual mortality rate of 38,000 (Yeung and Chung, 2002). There are 200,000 newly diagnosed cases of prostate carcinoma every year in the United States alone (Boring et al, 1994; Greenlee et al, 2001). As a result, prostate carcinoma is claimed to be the most frequently diagnosed


Sanlioglu et al: Adenovirus mediated gene therapy for prostate carcinoma disease, treating patients with prostate cancer still remains a formidable task. In addition, the molecular mechanism responsible for the onset of the disease is poorly understood. However, earlier detection of prostate cancer has been associated with an improved outcome (Perrotti et al, 1998). Thus, the detection of prostate cancer at an earlier stage remains to be the most realistic chance for therapy. For this purpose, different molecular screening methods (Ross et al, 2002a, 2002b) have been employed, but the most effective method is yet to be established. The most commonly used screening assays are based on the detection of up-regulated prostate specific markers such as prostate specific antigen (PSA). Currently, prostate specific antigen, (Farkas et al, 1998) when it is used in conjunction with other markers such as Gleason Scoring (Koksal et al, 2000) and TNM grading (Schroder et al, 1992), is considered to be a valuable tool to evaluate the histological grade of prostate carcinomas (Xess et al, 2001). As a result, patients were provided with various treatment options based on the results obtained with these parameters. These treatment options included but were not limited to operation, (Klotz, 2000b) radiotherapy, (Do et al, 2002) chemotherapy (Wang and Waxman, 2001) and hormone therapy (Klotz, 2000a; Smith et al, 2002). Regrettably, these conventional treatment modalities could not decrease the casualties from prostate cancer (Hsieh and Chung, 2001). Hence, there is a great need for development of novel treatment modalities to fight against prostate cancer. These remorseful facts ignited the initiation of gene therapy trials for prostate carcinoma (Sanda, 1997). So far, various viral vectors including lentivirus (Yu et al, 2001a), herpes simplex virus (Jorgensen et al, 2001), adeno-associated virus (Vieweg et al, 1995) and adenovirus (Loimas et al, 2001) were tested as carriers for therapeutic genes against prostate cancer. Other types of viruses such as Semliki Forest virus and Sindbis virus were also tested for gene delivery to prostate cancer cells (Loimas et al, 2001), but these viruses were unable to transduce prostate cells efficiently. Due to its antigenic properties and tissue transduction characteristics, adenovirus arose as a favored transporter vector. The exploitation of the tissue specific promoter in gene therapy especially eased adenovirus use in clinical trials (Lu and Steiner, 2000). In this review, we mainly highlighted the progress in adenovirus mediated prostate cancer gene therapy within the last three years with a particular emphasis in death ligand mediated gene therapy approach.

antiviral immunity barrier to increase the efficacy of adenovirus mediated gene delivery. One of these methods involves the testing of a collagen-based matrix (Gelfoam) (Siemens et al, 2001). Coinjection of Gelfoam with adenovirus vectors carrying prostate-specific antigen (Ad5-PSA) into mice naive to PSA but immune to adenovirus, relinquished the inhibitory effects of adenoviral immunity on CTL activation. Viral vectors are also being tested to deliver tumor specific peptides into dendritic cells (DCs) to evoke an immune response. The degree of immune response generated relies on the functionality of DCs following viral transduction. To prove this, adenovirus and retrovirus vectors were compared on the basis of their influence on the functionality of DCs (Lundqvist et al, 2002a). Adenovirustransduced monocyte-derived DCs (MO-DCs) stimulated allogenic lymphocytes and produced high levels of TNF and IL12. In addition, the expression of NF-!B and antiapoptotic molecules such as Bcl-X(L) and Bcl-2 (Lundqvist et al, 2002b) were also increased in adenovirus-transduced MO-DCs. Consequently, these cells became more resistant to spontaneous as well as Fasmediated cell death. In contrast, retroviruses failed even to transduce MO-DCs. Although CD34(+) cell-derived DCs were transducable with retroviruses to a lesser extent, they were less potent in their ability to stimulate allogenic lymphocytes in comparison to nontransduced DCs. These results suggest that adenovirus transduction of DCs increased the survival and the potency of DC mediated activation of the immune system. This might be important for prolonging the antigen presentation to generate a greater degree of immune response. Cytokine stimulated tumor infiltrating macrophages also play a major role in the generation of the cellular immune response against the tumor. The role of tumorinfiltrating macrophages in IFN-"-induced host defense against prostate cancer was revealed using xenograft mice models injected with adenovirus carrying IFN-" gene (Zhang et al, 2002a). Injection of an adenoviral vector encoding murine IFN-" (AdIFN-") directly into the tumor suppressed the growth of PC-3MM2 tumors as well as prevented metastasis and prolonged the survival of tumorbearing mice. Based on immunohistochemical staining, AdIFN-" infection resulted in the reduction of microvessel density of the tumor and increased apoptotic cell death (Cao et al, 2001). On the contrary, macrophage-selective anti-Mac-1 and anti-Mac-2 antibodies significantly reduced the antitumor effect of AdIFN-" induced therapy. Therefore, it was concluded that tumor-infiltrating macrophages must be involved in IFN-" induced suppression of tumor growth and metastasis.

II. Immunomodulation Tumors exhibit some degree of immunogenicity and the human immune system responds to these tumor specific antigens by mounting humoral and cellular responses, which are essential for the eradication of tumors. Adenovirus is commonly used for the delivery of genes encoding tumor-associated antigens in order to augment tumor-specific immune responses. However, antiviral immunity against adenovirus is a big concern, challenging its application in gene therapy. Various methods were employed in order to get around the

III. Suicide Gene Therapy Suicide strategy is a combined treatment modality involving chemotherapy and the gene transfer technology. The underlying principle is to limit the cytotoxicity of a drug to the local area of the tumor. To achieve this, the cDNA of a prodrug-converting enzyme is delivered into the tumor using viral vectors followed by regional or systemic application of the corresponding prodrug. As 114

Gene Therapy and Molecular Biology Vol 7, page 115 soon as the prodrug reaches the tumor, it is taken up and converted to a cytotoxic drug by tumor cells expressing the prodrug-converting enzyme. For example, 5Fluorouracil (5-FU) is widely used as a chemotherapeutic agent for the treatment of various malignancies. Although clinical trials have been conducted, so far 5-FU manifested a poor therapeutic index, which drastically limited its clinical use for cancer therapy. It is still not known whether the lack of success was due to problems associated with drug delivery or inherent insensitivity of cancer cells to this metabolite. However, adenovirus (Ad) vector-mediated cytosine deaminase (CD)/5fluorocytosine (5-FC) gene therapy had the potential to overcome pharmacokinetic issues associated with systemic 5-FU administration. Escherichia coli cytosine deaminase converts the prodrug 5-FC to the cytotoxic product 5-FU. Adenovirus encoding cytosine deaminase (AdCD) gene was injected into the prostate cancer cells transplanted orthotopically on mice followed by the systemic use of 5FC in order to investigate the antitumor and antimetastatic effects of this approach (Zhang et al, 2002c). An effective inhibition on tumor growth and metastasis was observed through in situ injection of AdCD followed by systemic use of 5-FC in the xenograft mouse model of prostate cancer. The use of E. coli uracil phosphoribosyltransferase (UPRT), a pyrimidine salvage enzyme, which modifies 5-FU into 5-fluorouridine monophosphate, improved the activity of AdCD through enhancing the anti-tumoral effect of 5-FU. In order to assess the efficacy of the combined suicide gene therapy approach, two separate adenovirus constructs expressing either the E. coli CD or E. coli UPRT genes were infected into androgen refractory prostate cancer cell line DU145 bearing mice. This combined gene therapy approach drastically regressed the growth of tumors in these animals better than what was achieved with AdCD alone (Miyagi et al, 2003). The most commonly used prodrug-converting enzyme for clinical approaches is the herpes simplex virus thymidine kinase gene (HSV-tk). The enzyme thymidine kinase phosphorylates the prodrug ganciclovir (GCV) to ganciclovir monophosphate, which is then further phosphorylated by cellular enzymes to ganciclovir triphosphate, a toxic metabolite and inhibitor of DNA polymerase. The efficacy of this approach was evaluated in an extended phase I/II study involving 36 prostate cancer patients with local recurrence after radiotherapy. These patients received single or repeated cycles of replication-deficient adenoviral mediated HSV-tk plus GCV in situ gene therapy (Miles et al, 2001). The study concluded that the repeated cycles of in situ HSV-tk plus GCV gene therapy can safely be administered to patients with prostate cancer who failed radiotherapy and have a localized recurrence. The therapeutic parameters such as PSA doubling time (PSADT), the mean PSA reduction (PSAR), and return to initial PSA (TR-PSA) values were all increased as a response to the treatment, indicating a therapeutic effect. A combined gene therapy approach using a recombinant adenovirus containing a fusion gene of CD and HSV-tk controlled by a cytomegalovirus (CMV) enhancer-promoter was designed to explore new

frontiers in prostate cancer gene therapy (Lee et al, 2002b). Both of the prostate carcinoma cell lines tested (DU-145 or PC-3 cells) were effectively transduced and killed by this replication-incompetent adenovirus encoding CD-TK fusion protein in the presence of prodrugs. The effect of radiation and heat treatment was also tested using this vector system. Interestingly, heat treatment not only increased the expression of CD-TK but sensitized prostate cancer cells to radiation as well. These results suggested that combining heat treatment with radiation therapy improved the efficacy of the adenovirus mediated suicide gene therapy approach for prostate carcinoma. The CDTK fusion fragment was also cloned into a lytic, replication-competent adenovirus (Ad5-CD/TKrep) and administered into patients with prostate carcinoma in a Phase I trial. This was the first gene therapy study in which a replication-competent virus was used to deliver a therapeutic gene to humans (Freytag et al, 2002a). This study demonstrated that intraprostatic administration of the replication-competent Ad5-CD/TKrep virus followed by 2 weeks of 5-fluorocytosine and ganciclovir prodrug therapy led to the destruction of tumor cells in patients without safety concerns. In addition, the efficacy and the toxicity of replication-competent adenovirus-mediated double suicide gene therapy (AdCD-TK) combined with an external beam radiation therapy (EBRT) approach was tested as a trimodal treatment modality in a preclinical study (Freytag et al, 2002b). Animals bearing prostate tumors were first injected with the lytic, replicationcompetent Ad5-CD/TKrep virus, then received 1 week of 5-fluorocytosine + ganciclovir (GCV) prodrug therapy supplemented with EBRT. The results from this study suggested that replication-competent adenovirus-mediated double suicide gene therapy combined with EBRT is very effective in eliminating tumors and reducing metastasis in an orthotropic mouse model of prostate carcinoma. The efficacy of another gene-directed enzyme prodrug therapy based on the Escherichia coli enzyme purine nucleoside phosphorylase (PNP) was tested in androgenindependent prostate cancer cells. PNP modifies the prodrug fludarabine to 2-fluoroadenine (Voeks et al, 2002). In this study, a recombinant ovine adenovirus vector (OAdV220) with a different receptor choice than that of human adenovirus type 5 carrying the PNP gene under the control of RSV promoter was used for functional studies. OAdV220 manifested a higher transgene expression compared to human Ad5 vector in infected murine RM1 prostate cancer cells during in vitro studies. Furthermore, the OAdV220 construct dramatically inhibited subcutaneous tumor growth when fludarabine phosphate was administered systemically in immunocompetent mice. Similar results were obtained using human PC3 xenografts in mice. PNP is also known to convert the prodrug 6MPDR to a toxic purine (6MP) causing cell death. In order to assess the efficacy of this approach for prostate cancer, replication-deficient human type-5 adenovirus (Ad5) carrying the PNP gene (Ad5SVPb-PNP) was directly injected into PC3 tumors (Martiniello-Wilks et al, 2002). The specificity and the level of transgene expression from this recombinant adenoviral vector were controlled by the promoter from 115

Sanlioglu et al: Adenovirus mediated gene therapy for prostate carcinoma the androgen-dependent, prostate-specific rat probasin (Pb) gene hooked up to the SV40 enhancer (SVPb). Unexpectedly, the SVPb element confirmed substantial prostate specificity even in the absence of androgens. Intratumoral delivery of Ad5-SVPb-PNP followed by 6MPDR administration significantly suppressed the growth of human prostate tumors in nude mice. These results suggested that Ad5-SVPb-PNP has therapeutic potential even in the absence of androgens for the treatment of prostate carcinoma. Another non-toxic prodrug, CB1954, which is converted to a toxic metabolite by the Escherichia coli nitroreductase gene (NTR), was tested as a suicide gene therapy approach for prostate cancer. Adenovirus vector expressing NTR (CTL102) was injected into subcutaneous prostate cancer xenografts followed by systemic CB1954 administration (Djeha et al, 2001). A clear anti-tumor effect of the approach was observed. In addition to all the methods mentioned above, a novel approach inspired from radioiodine therapy for thyroid cancer was developed using sodium iodide symporter (NIS). NIS is normally exclusively expressed in thyroid glands. Adenovirus carrying the NIS gene (AdCMVNIS) was constructed and tested for the treatment of prostate cancer following 131I administration (Spitzweg et al, 2001). Injection of AdCMVNIS construct to prostate cancer xenografts manifested highly active radioiodine uptake resulting in a drastic reduction in the tumor size following 131I administration in nude mice. This new approach represented an effective and potentially curative modality leading to the accumulation of therapeutically effective radioiodine in prostate. Diphtheria toxin (DT) is known to be a potent inhibitor of protein synthesis. The fact that a single molecule of DT can result in cell death complicated the utilization of DT as a suicide gene for cancer therapy. Thus, the feasibility of using DT gene therapy would greatly be influenced by tissue specific gene expression. Adenovirus vector carrying the catalytic domain (A chain) of DT under the control of the prostate-specific antigen (PSA) promoter (Ad5PSE-DT-A) induced apoptosis in PSA-positive prostate cancer cells in the presence of exogenous androgen (R1881) (Li et al, 2002a). In addition, Ad5PSE-DT-A injection regressed the growth of a PSApositive LNCaP xenograft in nu/nu mice. Non-PSAsecreting DU-145 cells did not manifest the same effect due to the lack of activation of PSA promoter in these cells. Therefore, the Ad5PSE-DT-A viral gene therapy approach might be a viable alternative in the treatment of PSA-secreting androgen-dependent prostate carcinoma.

by HSV-tk gene expression and ganciclovir (GCV) treatment (Hall et al, 2002). This dual treatment generated radical local and systemic growth suppression in a metastatic model of mouse prostate cancer (RM-1). The unification of AdHSV-tk/GCV + Ad.mIL-12 gene therapy approaches resulted in the induction of apoptosis due to increased expression of Fas and FasL and improved antimetastatic activity secondary to a strong NK effect. Intratumoral injection of AdHSV-tk vector followed by systemic ganciclovir or local radiation therapy or the combination of gene and radiation therapy was administered to subcutaneously transplanted mouse prostate tumors (Chhikara et al, 2001). The combined treatment reduced tumor growth by 61% compared to 38% obtained by single therapy modalities. Combined therapy also increased the mean survival time. In order to analyze systemic anti-tumor activity, lung metastases were generated by tail vein injection of RM-1 prostate cancer cells. While radiotherapy alone had no effect on the metastatic growth, the number of lung nodules was reduced by 37% following treatment with AdHSV-tk. The combinational therapy led to an additional 50% reduction in lung colonization. This was the first study demonstrating a significant systemic effect of AdHSV-tk administration combined with radiation. A Phase I/II study of radiotherapy and in situ gene therapy (adenovirus/herpes simplex virus thymidine kinase gene/valacyclovir) in combination with or without hormonal therapy in the treatment of prostate cancer was conducted recently (Teh et al, 2001). Based on the preliminary results, no serious side effect of the combined therapy was observed. This was reported as the first trial of its kind in the field of prostate cancer, and is expected to enlarge the curative index of radiotherapy by merging in situ gene therapy.

V. Molecular signaling pathways modulating the efficacy of adenovirus mediated therapeutic gene delivery Expression of certain hormone and growth factor receptors as well as cytokines and related downstream molecules can affect the efficacy of adenovirus-mediated gene therapy for prostate cancer. For example, gonadotrophin-releasing hormone (GnRH) restrains cell growth of reproductive tissue via gonadotrophin-releasing hormone receptors (GnRH-Rs) expressed in most cancers of reproductive tissues like that of prostate. Unfortunately, endogenous GnRH-R expression was not detected in PC3 cells, indicating that the cells are insensitive to GnRH. Exogenous expression of high affinity GnRH-R using adenovirus vectors (AdGnRH-R) facilitated antiproliferative effects of GnRH agonists in prostate cancer cells (Franklin et al, 2003). In addition, most of the prostate cancer cell lines overexpress fibroblast growth factors (FGFs). FGF signaling controls cell proliferation and inhibits cell death. A recombinant adenovirus expressing a dominant-negative FGF receptor (AdDNFGFR-1) was created in order to determine the biological significance of altered FGF signaling in human

IV. Joint approaches involving immunomodulation-hormonal or radiation therapy in combination with suicide gene approach AdHSV-tk suicide gene therapy was coupled to adenovirus-mediated IL-12 delivery as a combined gene therapy approach in order to enhance NK activity induced


Gene Therapy and Molecular Biology Vol 7, page 117 prostate cancer (Ozen et al, 2001). AdDNFGFR-1 infection of LNCaP and DU145 prostate cancer cells induced extensive cell death within 48 hours. Some of the prostate cancer cell lines are androgen dependent (LNCaP) whereas some are androgen independent (DU145 or PC3). Androgen ablation therapy, surgery, and radiation therapy are relatively effective in treating androgen dependent prostate carcinoma. However these treatments were ineffective for androgen-insensitive prostate carcinoma. Upregulation of IL6 cytokine induced by the constitutive NF-!B and Jun D activation is one of the distinctive parameters of androgen independent cell lines (Giri et al, 2001). IL6 is known to function as a proliferation and differentiation factor for prostate carcinoma. The infection with adenovirus vectors encoding either the dominant negative form of I!B# gene or Jun D reduced IL6 gene expression, leading to growth suppression of prostate cancer cells (Zerbini et al, 2003). Some but not all prostate cancer cells respond to vitamin D treatment. 1#, 25Dihydroxyvitamin D(3) (1#, 25-(OH)(2)D(3)) is known to have significant antiproliferative effects on certain prostatic carcinoma (PC) cell lines. 1#, 25-(OH)(2)D(3) inhibited cell growth and upregulated p21 expression in PC cell lines such as ALVA-31 and LNCaP (Moffatt et al, 2001). Stable transfection with a p21 antisense construct abolished the growth inhibition of ALVA-31 cells without altering vitamin D receptor expression. On the contrary, adenovirus-mediated expression of a sense p21 cDNA significantly reduced the proliferation of 1#, 25(OH)(2)D(3) unresponsive TSU-Pr1 and JCA-1 prostate cancer cell lines. Therefore, Adp21 gene therapy may be useful even for prostate cancer patients not responding to vitamin D treatment. Molecular signaling pathways are also altered in cancer cells. For instance, highly metastatic tumor cell lines display increased activity for focal adhesion kinase (FAK). The role of FAK in regulating migration of prostate carcinoma cell lines with increasing metastatic potential was studied in detail (Slack et al, 2001). Highly tumorigenic PC3 and DU145 cells displayed intrinsic migratory capacity correlating with an increased FAK expression and activity. On the contrary, poorly tumorigenic LNCaP cells required a stimulus to migrate. Inhibiting the FAK/Src signal transduction pathway by overexpressing FRNK (Focal adhesion kinase-Related Non-Kinase), an inhibitor of FAK activation, significantly inhibited migration of prostate carcinoma cells. Modulation of phosphatidylinositol 3'-kinase (PI3'-kinase), leading to Akt activation, frequently occurs in prostate cancer and disrupts apoptotic signaling induced by various cytokines such as tumor necrosis factor TNF and TNFrelated apoptosis-inducing ligand (TRAIL). Two prostate cancer cell lines with constitutively activated PI3'-kinase cascades (LNCaP and PC-3) were examined in order to study the role of PI3' phosphorylation in cellular response to TNF or TRAIL alone. Both TNF and TRAIL failed to activate apoptosis in either LNCaP or PC-3 cells. Interestingly, downregulation of PI3'-kinase/Akt signaling significantly enhanced the apoptotic activity of both TNF and TRAIL in LNCaP cells but not in PC-3 cells. Infection with adenovirus delivered PTEN/MMAC1 (phosphatase

and tensin homologue/mutated in multiple advanced cancers) reduced Akt activation, activated apoptosis and sensitized cells to TNF but not to TRAIL in LNCaP cell line (Beresford et al, 2001). Therefore, it was concluded that although PI3'-kinase signaling inhibits both TNF and TRAIL mediated apoptosis, this may only represent one of the several apoptotic resistance mechanisms in signaling pathways. Selenium compounds are known to be potential chemotherapeutic agents for prostate cancer. NF-!B has been categorized as the key antiapoptotic signaling molecule often activated in transformed cells. Testing of selenium compounds on DU145 and JCA1 prostate carcinoma cells revealed that these compounds induced apoptosis through the inhibition of NF-!B pathways in these cell lines (Gasparian et al, 2002b). Increased IKK activity was blamed for constitutive NF-!B activation responsible for survival of androgen independent prostate carcinoma cell lines (Gasparian et al, 2002a). 60-80 % of prostate cancers acquire the PTEN mutation during tumorigenesis. This results in the constitutive activation of the PI3'-kinase pathway and prostatic cell proliferation. The loss of PTEN activity is also correlated with the loss of activity of the FOXO family of forkhead transcription factors such as FKHRL1 and FKHR. Interestingly, these transcription factors are shown to control the expression of apoptosis inducing ligand TRAIL. Not surprisingly, the expression of TRAIL was also reduced in PTEN-lacking prostate cancer cells, leading to decreased apoptosis. Restoration of TRAIL expression using adenovirus-mediated overexpression of these transcription factors in LAPC4 prostate cancer cell line induced apoptosis (Modur et al, 2002).

VI. Apoptosis Modulators A. The exploitation of death ligands to induce apoptosis in cancer cells Apoptosis, known as programmed cell death (Reed, 2000) is defined as cell’s preferred form of death under hectic conditions (Sears and Nevins, 2002). In reality, it is also a key mechanism for homeostasis throughout embryonic and adult life. Genetic aberrations disrupting programmed cell death underpin tumorigenesis and drug resistance. Therefore, the specific activation of apoptosis within tumor cells could be a highly effective therapeutic intervention for prostate cancer. Currently, chemotherapy (Stein et al, 2002) and radiotherapy (Wang et al, 2002) are among the most commonly used treatment modalities against prostate cancer. The tumor suppressor gene, p53, is required in order for both of these treatment methods to work as anti-tumor agents (Levine, 1997). However, more than half of the human tumors acquire p53 mutations during tumorigenesis (Horowitz, 1999; Zeimet et al, 2000). As a result, tumors lacking p53 display resistance to both chemotherapy and radiotherapy (Obata et al, 2000). Intriguingly, death ligands induce apoptosis independent of p53 status of the cells (Ehlert and Kubbutat, 2001; Norris et al, 2001). Thus, these methods constitute somewhat of a complementary treatment modality to currently employed conventional treatments. 117

Sanlioglu et al: Adenovirus mediated gene therapy for prostate carcinoma At present, death ligands are being evaluated as potential cancer therapeutic agents (Herr and Debatin, 2001). Previously, several studies using external Fas agonists, anti-Fas antibodies and membrane-bound FasL failed to induce Fas L mediated apoptosis in prostate cancer cells. Although the down regulation of c-FLIP expression through the use of anti-sense oligonucleotides sensitized DU145 cells to an anti-Fas monoclonal antibody (Hyer et al, 2002), efficient cell killing was not observed by this approach. However, intracellular expression of FasL using adenoviruses efficiently killed 70-90% of various human prostate cancer cell lines tested (Hyer et al, 2000). Furthermore, part of this cell killing was attributed to the bystander effect mediated by FasL carried within the apoptotic bodies and cellular debris (Hyer et al, 2003). Despite the fact that human prostate cancer cells express apoptotic FasL, some of the cell lines, such as LNCaP, are resistant to Fas L mediated cell death. Even so, prior exposure to IFN$ sensitized orthotropic prostate primary tumors to recombinant adenovirus mediated FasL delivery (Selleck et al, 2003). Despite the fact that tumor necrosis factor (TNF) (Terlikowski, 2001) and FasL (Nagata, 1997) have been studied extensively and were shown to effectively induce apoptosis in cancer cells, their systemic use in cancer gene therapy is not recommended due to the systemic toxicity. With the discovery of a novel death ligand, TRAIL/Apo2L, (Wiley et al, 1995; Pitti et al, 1996) a new era emerged for the deployment of death ligands for cancer gene therapy (Nagane et al, 2001). The fact that TRAIL does not cause any harm to normal cells but can selectively induce apoptosis in cancer cells brought up the possibility of TRAIL testing for systemic use (Griffith and Lynch, 1998). Five different receptors were identified to interact with TRAIL; TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and osteoprotegrin (Abe et al, 2000; Sheikh and Fornace, 2000). TRAIL-R1 and TRAIL-R2 function as authentic death receptors inducing apoptosis while TRAIL-R3 and TRAIL-R4 are unable to induce such signaling but can serve as decoy receptors (Meng et al, 2000). However even today, no single mechanism has been found to account for TRAIL resistance observed in normal cells. The soluble form of TRAIL has successfully been tested and no toxicity due to systemic use was observed in animal models. However, large quantities of TRAIL were needed in order to suppress the tumor growth. A replication-deficient adenovirus encoding human TRAIL (TNFSF10; Ad5-TRAIL) was generated as an alternative to recombinant, soluble TRAIL protein (Griffith and Broghammer, 2001). Ad5-TRAIL infection into TRAIL-sensitive prostate tumor cells induced apoptosis through the activation of Caspase 8 pathways. Normal prostate epithelial cells were not harmed by Ad5TRAIL infection. Moreover, in vivo Ad5-TRAIL administration suppressed the outgrowth of human prostate tumor xenografts in SCID mice. Eight prostate cancer cell lines (CWR22Rv1, Du145, DuPro, JCA-1, LNCaP, PC-3, PPC-1, and TsuPr1) and primary cultures of normal prostate epithelial cells (PrEC) were tested for sensitivity to soluble TRAIL induced cell death in another study (Voelkel-Johnson et al, 2002). 100 ng/mL of soluble

TRAIL administration did not induce apoptosis in Du145, DuPro, LNCaP, TsuPr1, and PrEC. Interestingly, treatment with the chemotherapeutic agent doxorubicin sensitized almost all prostate cancer cells to TRAILinduced cell death. On the other hand, an adenoviral vector expressing full-length TRAIL (AdTRAIL-IRES-GFP) killed prostate cancer cell lines and, unexpectedly, PrEC as well, independent of doxorubicin cotreatment. This study suggested that the AdTRAIL-IRES-GFP gene therapy approach, complemented with tissue-specific promoters, would be useful for the treatment of prostate carcinoma. However, the mechanism of TRAIL resistance in normal cells is not understood and some prostate cancer cells appeared to be TRAIL-resistant (Nesterov et al, 2001). In one study, ALVA-31, PC-3, and DU 145 cell lines were highly sensitive to apoptosis induced by TRAIL, while TSU-Pr1 and JCA-1 cell lines were moderately sensitive, and the LNCaP cell line was resistant (Nesterov et al, 2001). Due to the lack of active lipid phosphatase PTEN, LNCaP cells demonstrated a constitutive Akt activity. Akt is a negative regulator of the phosphatidylinositol (PI)3-kinase/Akt pathway. PI3-kinase inhibitors sensitized LNCaP prostate cancer cells to TRAIL. In addition, adenovirus expressing a constitutively active Akt reversed the ability of wortmannin to potentiate TRAIL-induced BID cleavage. This suggested that constitutive Akt activity inhibits TRAIL-mediated apoptosis (Nesterov et al, 2001).

B. NF-!B inhibiting approaches used to breakdown TRAIL resistance in prostate cancer cells The mechanism of TRAIL induced apoptosis and resistance is outlined in Figure 1. So far, at least two different hypotheses that may partly explain TRAIL resistance are asserted. The first hypothesis advocates that normal cells carry decoy receptors (TRAIL-R3, TRAILR4), which compete with apoptosis inducing TRAIL receptors (TRAIL-R1, TRAIL-R2) for binding to TRAIL (Pan et al, 1997; Sheridan et al, 1997). In this hypothesis, it is believed that decoy receptors either function to dilute out TRAIL ligands (like TRAIL-R3) or supply antiapoptotic signals (like TRAIL-R4) to cells. As reported previously, TRAIL-R4 binding activates the anti-apoptotic NF-!B signaling pathway, leading to the blockade of TRAIL induced apoptosis (Degli-Esposti et al, 1997). In addition, the expression of decoy receptors is downregulated in cancer cells through promoter hypermethylation leading to differential sensitivity to TRAIL (van Noesel et al, 2002). However, the link between TRAIL resistance and the expression of decoy receptors has not been clearly established in human cells (Griffith and Lynch, 1998). Interestingly, activation of death receptors such as TRAIL-R1 and TRAIL-R2 also stimulated the NF-!B pathway (Chaudhary et al, 1997; Schneider et al, 1997). Under these circumstances, the reason(s) for cells undergoing apoptosis despite the induction of anti-apoptotic pathways through the same death receptors is not fully understood.


Gene Therapy and Molecular Biology Vol 7, page 119

Figure 1: A gene therapy strategy to block anti-apoptotic NF-!B signaling pathway to induce TRAIL sensitivity in prostate cancer cells. Activation of TRAIL receptor 1 (R1) or 2 (R2) by trimeric TRAIL ligands leads to the recruitment of Fas associated death domain protein (FADD) to the membrane. Then, FADD recruits procaspase 8 to form death inducing signaling complex (DISC). DISC induced signaling activates caspase pathway inducing cells into apoptosis. TRAIL receptor 3 (R1) and 4 (R4) serve as decoy receptors. R4 activates NF- !B signaling pathways as well. In addition, NF-!B pathway is also activated by R1 and R2 via TNFR-associated death domain protein (TRADD) and receptor interacting protein (RIP). Consequently, NF-!B activation augments expressions of various antiapoptotic genes such as cIAP, BclxL and cFlip in addition to R3. c-Flip, a procaspase 8 homologue, competes with procaspase 8 for binding to FADD. Thereby it inhibits apoptotic signaling. The expression of adenovirus delivered IKK"KA mutant prevented the activation of anti-apoptotic NF-!B signaling. This method sensitized prostate cancer cells to TRAIL.

The second hypothesis claims the presence of apoptosis inhibitory substances in these cells. Such a molecule, cFLIP (FLICE Inhibitory Protein), a caspase 8 homologue, has been shown to obstruct death ligand induced apoptosis (Irmler et al, 1997; Griffith et al, 1998). Intriguingly, NF!B activating agents up-regulated cFLIP synthesis (Kreuz et al, 2001). Furthermore, the NF-!B pathway has been proven to increase TRAIL-R3 synthesis, a decoy receptor for TRAIL, (Bernard et al, 2001) and the expression of apoptosis inhibitor Bcl-xL (Hatano and Brenner, 2001; Ravi et al, 2001) resulting in the obstruction of TRAIL mediated apoptosis. Apoptosis inhibitors such as cIAP are also activated by NF-!B pathways (Mitsiades et al, 2002). Based on these results, we can clearly state that the active NF-!B signaling pathway may provide cells with TRAIL resistance by at least four different ways (Figure 1). Additionally, it has been reported that a novel tumor suppressor gene, PTEN/MMAC1 (Steck et al, 1997; Simpson and Parsons, 2001) negatively regulated TNF induced NF-!B activity (Ozes et al, 1999; Mayo et al, 2002) through the IKK complex (Gustin et al, 2001). The observation in which IKK activity is required for PI3KAkt induced NF-!B activation (Burow et al, 2000; Demarchi et al, 2001) confirmed this report (Madrid et al, 2001; Sizemore et al, 2002). Due to a negative correlation between the expression of PTEN and the progression of prostate cancer, advanced prostate cancer cells might have intrinsically higher NF-!B activity due to the progressive

loss of PTEN. Absence of PTEN function may result in increased Akt activity induced by PI3K. Since NF-!B is a downstream target for Akt, (Kane et al, 1999; Romashkova and Makarov, 1999; Andjelic et al, 2000; Jones et al, 2000) TRAIL resistance would ultimately be ensured in cells by way of the NF-!B pathway. In agreement with this hypothesis, PTEN sensitized prostate cancer cells to TRAIL induced apoptosis (Yuan and Whang, 2002). Thus, these possible scenarios make NF!B inhibiting vectors such as Ad.IKK"KA (Sanlioglu et al, 2001a) or Ad.I!B#SR (Batra et al, 1999; Sanlioglu and Engelhardt, 1999) ideal candidates for overcoming the TRAIL resistance in PTEN mutant prostate cancer cells. In a similar manner, TNF induced apoptosis can also be prevented by NF-!B activation as reported (Beg and Baltimore, 1996; Van Antwerp et al, 1996). Previously, NF-!B inhibiting approaches such as adenovirus mediated transfer of IKK" (Ad.IKK"KA) (Sanlioglu et al, 2001a, 2001b) or I!B# (Ad.I!B#SR) (Batra et al, 1999; Sanlioglu and Engelhardt, 1999) dominant negative mutants were successfully deployed in order to sensitize lung cancer cells to TNF. Since some tumor cells have intrinsically high NF-!B activity, which might be responsible for TRAIL resistance, NF-!B blocking agents can potentially be useful to overcome TRAIL resistance. For example, a constitutive NF-!B activation was observed in renal carcinoma (Oya et al, 2001). Not surprisingly, melanoma cells having a constitutive NF-!B 119

Sanlioglu et al: Adenovirus mediated gene therapy for prostate carcinoma activity exhibit TRAIL resistance (Franco et al, 2001). Resistant melanoma cells were sensitized to TRAIL either with proteasome inhibitors or transfections with plasmids encoding degradation resistant I!B# protein (Franco et al, 2001). In accordance with these studies, we have tested if adenovirus mediated NF-!B inhibiting approach would sensitize prostate cancer cells to TRAIL. Consequently, adenovirus mediated delivery of IKK"KA mutant (Ad.IKK"KA) sensitized PTEN mutant prostate cancer cells (PC3) to TRAIL as shown in Figure 2. At first, PC3 cells appeared to be relatively resistant to pro-apoptotic effects of TRAIL when cells were infected with adenovirus vector encoding hTRAIL (Ad.hTRAIL) even at an MOI of 1000 DNA particles/cell (Figure 2 Panel A). Infection with Ad.IKK"KA vector alone did not yield any cell death either (Figure 2, Panel B). However, when the dose of Ad.hTRAIL vector was kept constant at an MOI of 1000 DNA particles/cell, increasing the amount of Ad.IKK"KA construct sensitized PC3 cells to TRAIL mediated apoptosis (Figure 2, Panel C).

cells. Cell death was mediated by replication-deficient adenoviral vector expressing conditional caspase-1 (AdG/iCasp1) or caspase-3 (Ad-G/iCasp3) and the caspase activation was achieved by nontoxic, lipid-permeable, chemical inducers of dimerization (CID) (Shariat et al, 2001). Aggregation and activation of these recombinant caspases occurred, leading to rapid apoptosis only after vector transduction followed by CID administration in both human (LNCaP and PC-3) and murine (TRAMP-C2 and TRAMP-C2G) prostate cancer cell lines. Subcutaneous TRAMP-C2 tumors displayed focal but extensive apoptosis following direct injection of AdG/iCasp1 in vivo. In order to express caspase 9 exclusively in prostate, a recombinant adenovirus carrying iCaspase-9 was constructed with two copies of the androgen response region (ARR) placed upstream of the probasin promoter elements (ADV.ARR(2)PB-iCasp9) (Xie et al, 2001b). AP20187 is a chemical dimeric ligand, which causes dimerization and thereby activation of iCaspase-9 leading to rapid apoptosis in both dividing and nondividing cells. Testing of ADV.ARR(2)PB-iCasp9 construct in LNCaP tumor xenografts demonstrated that this construct induces apoptosis in prostate cancer cells only in the presence of AP20187. The proapoptotic members of Bcl- 2 protein family including Bax, Bak, Bad, and Bik also mediate apoptosis. Apoptosis-inducing proteins were cloned into adenovirus constructs and shown to induce apoptosis in prostate cancer cell lines previously.

C. Intracellular proapoptotic regulators Although caspases are the effector mediators of apoptosis, the expression of proapoptotic molecules such as procaspase 3 or 7 using adenovirus constructs did not induce apoptosis in prostate cancer cells due to the inability of these caspases to undergo autocatalytic activation (Li et al, 2001). A novel suicide gene therapy approach was developed using chemically inducible effector caspases to trigger apoptosis in prostate cancer

Figure 2. Adenovirus mediated IKK"KA expression sensitized PC3 cells to TRAIL mediated apoptosis. PC3 cells were infected with increasing MOIs of either Ad5hTRAIL (Panel A) or Ad.IKK"KA (Panel B). In panel C, the dose of Ad.IKK"KA vector was increased gradually (stated just above each panel) while the amount of Ad5hTRAIL was kept constant (as indicated under the panel). Cell death was detected using molecular probe’s Live and Death Cellular viability and toxicity kit 48 hours following infection. Numbers indicate viral doses as MOI values of DNA particles/cell.


Gene Therapy and Molecular Biology Vol 7, page 121 However, overexpression of proapoptotic genes without the use of tissue specific promoters could result in unwanted apoptosis even in normal cells. In order to provide tissue specificity, an adenoviral construct was generated containing Bax cDNA under control of the probasin promoter that included two androgen response elements (Av-ARR2PB-Bax). Av-ARR2PB-Bax construct drove Bax overexpression in an androgen-dependent way in androgen receptor (AR)-positive cell lines of prostatic origin but not in others. The androgen dihydrotestosterone activated apoptosis in LNCaP cells infected with AvARR2PB-Bax but not in those infected with control vectors. These results demonstrated that Av-ARR2PB-Bax induced apoptosis was androgen dependent and limited to AR positive cells of prostatic epithelium. On the other hand, using a binary co-transfection strategy involving Ad/GT Bax and Ad/PGK-GV16; overexpression of proapoptotic Bax protein induced apoptosis both in androgen-insensitive (DU145 and PC3), and androgensensitive (LNCaP) cell lines (Honda et al, 2002). The same binary approach was tested to assess the consequences of Bcl-2 overexpression in the progression of prostate carcinoma leading to apoptosis-resistant and androgenindependent phenotype in DU145, PC3 and LNCaP cell lines which represent models of advanced prostate carcinoma. Bax expression generated by the adenoviral co-transfection system induced apoptosis even in these Bcl-2 overexpressing cell lines. These results suggest that the Ad/GT Bax and Ad/PGK-GV16 combined expression system might represent a powerful gene therapy strategy for the treatment of androgen-independent and apoptosisresistant prostate carcinoma. Moreover, monogene and polygene approaches were compared in an experimental prostate cancer model using apoptotic genes bad and bax driven by a prostate specific promoter (ARR(2)PB) in an adenovirus construct (Zhang et al, 2002b). The ARR(2)PB is a dihydrotestosterone (DHT)-inducible third-generation probasin-derived promoter. In this study, animals bearing tumors of prostatic origin responded better to combined bad and bax therapy than either of the vectors alone. Therefore, it was concluded that polygene therapy involving more than one apoptotic molecule is more effective in xenograft models of androgen-dependent or independent prostate cancer than monogene therapy alone. It is also known that overexpression of anti-apoptotic genes such as Bcl-2 in prostate carcinoma provides resistance to radiation therapy and androgen ablation. A second-generation adenoviral vector (ARR2PB.Bax.GFP) was constructed with the modified prostate-specific probasin promoter (ARR2PB) directing the expression of a HA-tagged Bax gene in order to restore the balance of Bcl-2 family members to induce apoptosis in prostate cancer cells (Lowe et al, 2001). ARR2PB.Bax.GFP vector induced significant levels of apoptosis in LNCaP cells 48 hours following infection even in the presence of high levels of Bcl-2 protein. No toxicity in liver, lung, kidney, and spleen was detected by systemic administration of ARR2PB.Bax.GFP in nude mice. Therefore, a secondgeneration adenovirus-mediated, prostate-specific Bax gene therapy appeared to be a very safe and efficient approach for the treatment of prostate cancer. Another

member of the proapoptotic Bcl-2 family, namely "Bik", was cloned into adenovirus vectors to explore its therapeutic potential. AdBik infection also induced apoptosis and suppressed the growth of PC-3 xenografts established in nude mice (Tong et al, 2001). Several other genes were also tested for their ability to induce apoptosis in prostate tumor cell lines as well as in xenograft models. The antiapoptotic protein CLN3 negatively regulates endogenous ceramide production, an inducer of apoptotic cell death. CLN3 protein is overexpressed in most of the cancer cell lines tested including those of prostate (Du145, PC-3, and LNCaP). An adenovirus-expressing antisense CLN3 (Ad-ASCLN3) blocked CLN3 protein expression in prostate cancer cell lines as demonstrated by Western Blotting (Rylova et al, 2002). Ad-AS-CLN3 infection resulted in the inhibition of cell growth and reduction in cell viability of cancer cells through elevation of endogenous ceramide production. This study revealed CLN3 as a novel target to induce apoptosis in prostate cancer cells. A recombinant adenovirus containing pHyde cDNA gene (AdpHyde), a novel gene cloned from Dunning rat prostate cancer cells, was constructed in order to study its function (Zhang et al, 2001). Surprisingly, the AdpHyde construct inhibited the growth of human prostate cancer cells and induced apoptosis involving the caspase-3 pathway in human prostate cancer tumor xenografts in nude mice. Ionic movement also influences apoptosis. For instance, K+ efflux is an early event in apoptosis, which is regulated by K+ channel-associated protein (KChAP). A recombinant adenovirus encoding KChAP (Ad/KChAP) was constructed in order to determine if KChAP expression could induce apoptosis in prostate cancer cells (Wible et al, 2002). The LNCaP cell line displayed a reduction in cell size upon infection with Ad/KChAP. The Ad/KChAP construct also induced apoptosis in DU145 cells in a p53 independent manner. In addition, infection with Ad/KChAP prevented growth of DU145 and LNCaP tumor xenografts in nude mice.

VII. Tumor suppressor genes Aberrations in the expression of tumor suppressor genes have been one of the key factors affecting the outcome of cancer therapy. Several studies examined the possible use of tumor suppressor genes as therapeutic agents for prostate cancer. Doxorubicin (Dx) is a commonly used chemotherapeutic agent in recurrent prostate cancer and is a strong inducer of p53 expression leading to p21(CIP1/WAF1) transactivation. As suggested by previous reports, p21 plays a role in the modulation of chemotherapy-induced apoptosis, prostate cancer progression and androgen regulation. Two androgenregulated human prostate cancer cell lines (MDA PCa 2b and LNCaP) were exposed to Dx and growth factor withdrawal in order to investigate if p21 plays a role in the survival of prostate cancer cells under stress (Martinez et al, 2002). Infection with adenovirus vectors encoding the antisense strand of p21 reduced p21 levels, sensitized prostate cancer cells to Dx and facilitated apoptosis in response to growth factor withdrawal. These results suggest that modulation of p21 pro-survival gene 121

Sanlioglu et al: Adenovirus mediated gene therapy for prostate carcinoma expression via adenovirus constructs sensitizes prostate cancer cells to chemotherapeutics and androgen withdrawal. Another tumor suppressor protein, p27, also known as cyclin-dependent kinase inhibitor (CDKI), is normally expressed in human prostate. However, the majority of human prostate cancers have reduced levels of p27. The down regulation of this putative tumor suppressor gene through proteolysis is mediated by SCFSKP2 ubiquitin ligase complex. Adenovirus-mediated overexpression of SKP2 induced ectopic down-regulation of p27 in LNCaP prostate carcinoma cells (Lu et al, 2002). This observation confirmed that SKP2 activity was the major determinant of p27 levels in human prostate cancer cells. Based on in vitro studies, it is believed that the overexpression of SKP2 might be one of the mechanisms allowing prostate cancer cells to escape growth control mediated by p27. Therefore, knocking out SKP2 function would be a logical novel approach to fight prostate cancer. In another study, an adenovirus construct carrying p27 coding sequences Adp27(Kip1) was generated to assess whether the overexpression of p27 has any affect on the prostatic tumor growth in vivo (Katner et al, 2002). Injection of Adp27(Kip1) vector reduced the growth of LNCaP tumor xenografts in mice. This study supported the idea that Adp27(Kip1) can serve as a potential therapeutic vector for the treatment of prostate carcinoma. p14(ARF), encoded by the human INK4a gene locus, is another tumor suppressor protein which is frequently inactivated in human cancer. p14(ARF) has recently been implicated in p53-independent cell cycle regulation and apoptosis. A replication-deficient adenoviral construct carrying p14(ARF) coding sequence (Ad-p14(ARF)) was generated in order to explore the pro-apoptotic function of p14(ARF) in relationship to p53 function (Hemmati et al, 2002). Ad-p14(ARF) construct induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells and HCT116 cells lacking functional Bax expression. This study demonstrated that overexpression of p14 through adenovirus vectors is sufficient to induce apoptosis in p53and bax-deficient prostate cancer cells. Prostate carcinoma with p53 mutant phenotype represents a clear obstacle for irradiation therapy. Ionizing radiation (IR) and adenoviral p53 gene therapy (Ad5CMV-p53) were utilized individually as well as in combination in order to assess the effectiveness of combined therapy for prostate cancer (Sasaki et al, 2001). In this study, IR alone did not induce significant levels of apoptotic cell death in DU145 and PC-3 cells. However, after combined therapy, the proportion of apoptotic cells was greatly amplified in both of the cell lines tested. Therefore, it was concluded that the observed synergistic effect might be useful for the treatment of radio-resistant prostate carcinoma. The loss of MMAC/PTEN tumor suppressor gene expression is frequently detected in human tumors. Survival signaling through the phosphatidylinositol-3 kinase/Akt pathway is constitutively activated in cells lacking functional PTEN expression. Therefore, the functional effect of MMAC/PTEN expression was examined in LNCaP cells, which are devoid of a functional PTEN product (Davies et al, 1999). Infection with an adenovirus construct driving the expression of

MMAC/PTEN resulted in a specific inhibition of Akt/PKB activation. This is consistent with the phosphatidylinositol phosphatase activity of MMAC/PTEN. Compared to adenovirus delivered p53 expression, MMAC/PTEN expression induced apoptosis in LNCaP cells to a lesser extent. Interestingly, the growth suppression properties of MMAC/PTEN were significantly greater than those accomplished with p53. Moreover, Bcl-2 overexpression in LNCaP cells blocked both the adenovirus mediated MMAC/PTEN- and p53-induced apoptosis, but it did not affect the growth-suppressive properties of MMAC/ PTEN. This is consistent with the fact that MMAC/PTEN may play multiple roles in the cell. Prostate cells were infected with adenovirus vector carrying PTEN coding sequence in order to determine if supplying PTEN function would sensitize these cells to various apoptotic stimuli (Yuan and Whang, 2002). As predicted, adenovirus-mediated PTEN delivery sensitized LNCaP prostate cancer cells to apoptosis through the inhibition of constitutive Akt activation. Since PTEN G129E mutant lacking lipid phosphatase activity was unable to sensitize cells to apoptosis, it was concluded that the lipid phosphatase activity of PTEN was required for apoptosis. The therapeutic effect of adenoviral delivery of MMAC/PTEN was tested on both the in vitro and in vivo growth of PC3 human prostate cancer cells (Davies et al, 2002). The in vitro growth of PC3 cells was repressed by adenovirus expression of MMAC/PTEN via blocking of cell cycle progression. Although this approach did not inhibit the tumor progression of orthotopically implanted PC3 cells, a significant reduction was observed in the tumor size in vivo, in addition to complete inhibition of metastases. Therefore, it was suggested that MMAC/PTEN might play a role mostly in the regulation of the metastatic potential of prostate cancer. A considerable fraction of prostate tumors display an alteration of Mxi1 expression, an antagonist to c-Myc. This was confirmed by transgenic approaches in which prostatic hyperplasia was observed in mice deficient for Mxi1. Mxi1-expressing adenovirus (AdMxi1) was generated to study the ability of Mxi1 to act as a growth suppressor in prostate tumor cells (Taj et al, 2001). Overexpression of Mxi1 using adenovirus vectors in the DU145 prostate carcinoma cell line resulted in growth arrest and decreased colony formation on soft agar. All these studies emphasize that the modulation of tumor suppressor gene function might be necessary for an optimum therapeutic response to fight against prostate cancer.

VIII. Cell adhesion molecules and antiangiogenic approaches Cell adhesion molecules play major roles especially in metastasis of cancer cells. Therefore, aberrant expression patterns of cell adhesion molecules are frequently associated with poor prognosis. For instance, the expression of a well-known cell adhesion molecule, CCAM1, is downregulated during the early stages of prostate carcinoma in an animal model (TRAMP) (Pu et al, 1999). C-CAM1 was cloned into an adenovirus 122

Gene Therapy and Molecular Biology Vol 7, page 123 construct and its efficacy was tested both in vitro and in vivo using PC3 xenograft murine model (Lin et al, 1999). AdC-CAM1 construct manifested a strong antitumoral activity on PC3 tumor cells grown in nude mice. Therefore, selective use of cell adhesion molecules might be beneficial for the treatment of prostate carcinoma. Moreover, combining C-CAM1-based therapy with TNP470, a potent angiogenesis inhibitor, induced greater growth suppression on DU145 tumor xenografts than by either Ad-C-CAM1 or TNP-470 application alone (Pu et al, 2002). Vascularization of a solid tumor is required for cancer growth. Recently, preventing vascularization through inhibition of angiogenesis was a popular target for cancer gene therapy. For example, a 16-kDa prolactin protein (PRL) has previously been shown to possess an antiangiogenic activity (Galfione et al, 2003). Not surprisingly, adenovirus delivery of PRL protein manifested a significant antitumoral activity in vivo (Kim et al, 2003). In addition, vascular endothelial growth factor (VEGF) receptor signaling is another relevant pathway, which modulates the vascularization of newly growing tumors. Interfering with such a signaling pathway might be valuable in controlling the tumor growth. In fact, when fused to an Fc domain and cloned into the recombinant adenovirus construct, the ligand-binding ectodomain of VEGF receptor 2 (Flk1) manifested a considerable reduction in tumor growth induced by a drastic decline in the microvessel density in SCID mice carrying human LNCaP xenografts (Becker et al, 2002). Growth factors are needed for survival of cancer cells and molecular chaperones are required for functional production of these molecules. A new member of the heat shock protein family functioning as a molecular chaperone in the endoplasmic reticulum was recently discovered and named as 150-kDa oxygen-regulated protein (ORP150). Since prostate cancer cells exhibited an upregulation of ORP150 protein and VEGF, adenovirus delivery of an antisense ORP150 cDNA approach was used to reduce angiogenicity and tumorigenicity through inhibition of VEGF secretion. This approach indeed suppressed the growth of DU145 prostate carcinoma cell line in a xenograft model (Miyagi et al, 2002).

mechanisms, the potential radiosensitizing effects of CV706 on prostate cancer cells were evaluated (Chen et al, 2001). The CV706 construct demonstrated a synergistic antitumoral effect both on irradiated human prostate cancer cells and tumor xenografts. Moreover, in order to investigate the safety and the functionality of intraprostatic delivery of CV706 for the treatment of patients with locally recurrent prostate cancer following radiation therapy, a Phase I dose-escalation study was conducted (DeWeese et al, 2001). Results from this study suggested that even at high doses, intraprostatic delivery of the CV706 was relatively safe for patients and CV706 construct demonstrated high therapeutic activity as reflected by the reduction in serum PSA. This was the first clinical trial of a prostate-specific, replication-restricted adenovirus for the treatment of prostate cancer. Another prostate-specific replication-competent adenovirus carrying not one, but two, cell type specific promoters (CV787) was constructed. This construct contained E1B gene driven by the human prostate-specific enhancer/promoter and the adenovirus type 5 (Ad5) the E1A gene under the control of prostate-specific rat probasin promoter. The Ad5 E3 region was also conserved in the vector to improve the efficacy. A single tail vein injection of CV787 eliminated LNCaP xenografts within 4 weeks in nude mice (Yu et al, 1999). When the prostate cancer-specific adenovirus CV787 was combined with chemotherapeutic agents like taxanes (paclitaxel and docetaxel), a synergistic antitumoral effect was observed in mice carrying human prostate cancer xenografts (Yu et al, 2001b). Heat-inducible gene expression is another approach used in the context of suicide gene therapy. A recombinant adenovirus containing the CD-TK fusion gene controlled by the human inducible heat shock protein 70 promoter (Ad.HS-CDTK) was generated for this purpose. Heat application at 41oC for 1 hour induced therapeutic gene expression from this vector. Despite the fact that the Ad.HS-CDTK construct induced CD-TK expression in human prostate cancer cells, a therapeutic benefit was not observed due to lower transduction efficiency of tumors in vivo. Instead, a replication-competent, E1B-attenuated adenoviral vector containing the hsp70 promoter-driven CD-TK gene (Ad.E1A+HS-CDTK) was generated to increase CD-TK gene expression to achieve a therapeutic effect (Lee et al, 2001). Contrary to replication incompetent Ad.HS-CDTK, replication competent Ad.E1A+HS-CDTK construct yielded severe cytotoxicity and greater levels of therapeutic index in the presence of prodrugs. This approach revealed the beneficial effects of using replication competent virus complemented with a heat inducible suicide gene therapy approach for prostate carcinoma.

IX. Replication competent adenovirus vectors Replication competent adenoviral vectors provide powerful means to kill cancer cells through cell lysis. Since they only replicate in tumor cells, the therapeutic range is limited to cancer cells. Two replication-competent adenoviruses, CV706 and CV787, were generated in order to selectively destroy PSA producing prostate cancer cells. It has been demonstrated earlier that prostate-specific antigen (PSA)-selective replication-competent adenovirus variant CV706 specifically eliminated tumors in human prostate cancer xenografts in preclinical models (Rodriguez et al, 1997). Since adenovirus E1A is known to be a potent inducer of chemosensitivity and radiosensitivity through p53-dependent and independent

X. Adenovirus vectors with cell type specific and inducible promoters Even though adenovirus-mediated HSVTK suicide gene therapy approach manifested a satisfactory toxicity profile in Phase I clinical trials, the toxicity studies using adenovirus vectors were very restricted in numbers. 123

Sanlioglu et al: Adenovirus mediated gene therapy for prostate carcinoma However, it was known that the promoter of choice might influence the level of toxicity. In order to study the promoter effect on adenovirus mediated toxicity the mouse caveolin 1 promoter was cloned into the adenovirus HSVtk vector (Adcav-1tk) because this promoter was highly active in metastatic and androgen-resistant prostate cancer cells (Pramudji et al, 2001). The efficacy of this vector for suicide gene therapy was compared to those of AdHSV-tk vectors carrying either cytomegalovirus (AdCMV-tk) or rous sarcoma virus (AdRSV-tk) promoters in mice transplanted with mouse prostate cancer cells. Following GCV administration, all the HSV-tk expressing vectors regressed the tumor growth in situ. Interestingly, the efficacy of Adcav-1tk vector was much greater in terms of inducing necrosis and microvessel density. In order to evaluate the toxicity profile of adenovirus vectors carrying CMV, RSV or mouse caveolin promoter-driven HSV-tk transgenes, these vectors were also injected systemically into mice (Ebara et al, 2002). Adenovirus vectors with CMV and RSV promoters, but not caveolin promoter, exhibited significant levels of liver damage. These results suggested that the promoter selection greatly influences the toxicity profile of adenovirus-mediated suicide gene therapy approach. In order to increase the number of promoters available for prostate specific gene expression, transgenic mice were generated expressing a reporter gene (SV40 Tag) directed by prostate secretory protein of 94 amino acids (PSP94) (Gabril et al, 2002). PSP94 gene promoter/enhancer region directed SV40 Tag expression exclusively in prostate leading to prostatic intraepithelial neoplasia and eventually to high-grade prostate carcinoma. These studies suggested that this PSP94 gene promoter/enhancer strategy could be employed for the treatment of prostate carcinoma. One conventional way to limit the toxicity of virus mediated suicide gene therapy is to use cell type specific promoters as suggested above. Although adenovirus vectors with the native PSA enhancer and promoter (PSAP) provided prostate-specific expression, lower transcriptional activity observed in prostate challenged its use in prostate-targeted gene therapy. To improve the activity and specificity of the prostate-specific PSA enhancer for gene therapy, various studies were carried out by exploring the properties of the natural PSA control regions. Chimeric PSA enhancer constructs were generated with tandem copies of the proximal ARE elements and then inserted into adenovirus constructs (AdPSE-BC-luc) (Wu et al, 2001). This construct was highly inducible with androgens as shown by systemic administration into SCID mice carrying LAPC-9 human prostate cancer xenografts while retaining prostate specific gene expression. Furthermore, the CreLoxP system was also utilized to enhance the activity of PSAP. CD suicide gene therapy approach using adenoviral vectors with CRELoxP augmented PSAP activity effectively inhibited subcutaneous LNCaP tumor growth in nude mice (Yoshimura et al, 2002). In addition, hormone refractory prostate cancer cells retain the expression of prostatespecific membrane antigen (PSMA) and prostate-specific antigen (PSA). An adenovirus construct with an artificial chimeric enhancer (PSES) composed of two modified

regulatory elements of PSA and PSMA genes (Ad-PSESluc) was generated and tested for its promoter activity for the treatment of prostate cancer (Lee et al, 2002a). Systemic injection of Ad-PSES-luc construct into mice produced very low levels of reporter gene expression in major organs. However, when injected directly into prostate, only the prostate but not other tissues produced high levels of reporter gene expression. These results encouraged the use of PSES for the treatment of androgenindependent prostate carcinoma. Even though prostatespecific antigen (PSA/hK3) provided prostate specific gene expression, its expression displayed an inverse correlation with prostate cancer grade and stage, giving reason to doubt its effectiveness for advanced stage of prostate carcinoma. A new approach was developed in order to generate gene therapy vectors targeting higher grades especially of prostate carcinoma. The human glandular kallikrein 2 (hK2) is upregulated in an advanced form of prostate cancer with a higher grade. Therefore the hK2 promoter was cloned into adenovirus construct in combination with EGFP reporter gene (ADV.hK2-E3/PEGFP) in order to obtain preferential expression of EGFP in prostate cancer (Xie et al, 2001a). Indeed ADV.hK2E3/P-EGFP injection led to a robust but tumor-restricted EGFP expression in subcutaneously generated LNCaP tumors. These results showed that adenovirus constructs with the hk2 multienhancer/promoter driven therapeutic genes might be a powerful tool for gene therapy of advanced prostate cancer. Previous studies have shown that the bone matrix protein osteocalcin is predominantly expressed in prostate cancer epithelial cells, fibromuscular stromal cells and osteoblasts. A conditional replication competent adenovirus vector carrying the osteocalcin promoter driven early E1A gene (AdOCE1A) was generated to cotarget both prostate cancer cells and their surrounding stromal cells (Matsubara et al, 2001). Both PSA-producing (LNCaP) and non-producing (DU145 and PC3) human prostate cancer cell lines as well as human stromal cells and osteoblasts were effectively killed by this recombinant virus in vitro. In addition a single systemic intravenous injection of the AdOCE1A construct significantly destroyed prostate tumor cells transplanted in SCID mice. This co-targeting strategy appeared to have a broader effect compared to other recombinant constructs tested on the preclinical models of human prostate cancer. These promising results initiated first gene therapy trial (phase I) in which adenoviruses carrying the osteocalcin promoter driven HSV-tk gene (AdOCHSVTK) were directly injected into prostate cancer lymph node and bone metastasis (Kubo et al, 2003). The results of this trial suggested that adenoviruses did not display any adverse effects and the treatment was well tolerated in all patients. In addition, 63 % of the patients had local cell death in treated lesions. Further studies are suggested in order to assess the efficacy of this approach for androgenindependent prostate carcinoma. A new treatment modality to enhance adenoviral replication by vitamin D3 in androgen-independent human prostate cancer cells and tumors was tested using a novel replication-competent adenoviral vector, Ad-hOC-E1, carrying the human 124

Gene Therapy and Molecular Biology Vol 7, page 125 osteocalcin (hOC) promoter to drive both the early viral E1A and E1B genes (Hsieh et al, 2002). While the replication properties of Ad-hOC-E1 vector were restricted to OC-expressing cells, vitamin D3 exposure further enhanced viral replication by 10 fold. The growth of both androgen-dependent and androgen-independent prostate cancer cells was suppressed by Ad-hOC-E1 infection, irrespective of the cells’ androgen responsiveness and PSA status. This is in contrast to AdsPSA-E1 vector, which only replicated in PSA-expressing cells with androgen receptor (AR). Ad-hOC-E1 injection inhibited the growth of DU145 (an AR and PSA-negative cell line) tumor xenografts in mice. Consequently, vitamin D3-enhanced Ad-hOC-E1 viral replication represented an alternative for the treatment of localized or osseous metastatic prostate cancer. Prostate specific antigen promoter (PSAP) and rat probasin (rPB) promoter are currently employed to drive the therapeutic transgene expression in prostate cancer cells. However, since these promoters require the binding of androgen to androgen receptor for activation, they were only functional in androgen-dependent prostate carcinoma cells. Because androgen refractory prostate carcinoma cells lose the expression of androgen receptor along the way, constructs with PSAP or rPB promoters are not useful for treating patients with androgen-independent prostate carcinoma. In order to circurment this problem, prostate specific promoters were modified so that they were activated in response to the retinoids-retinoid receptor complex in place of the androgen-AR complex. As a result, retinoid treated androgen-independent prostate cancer cells were sensitized to HSVTK-ganciclovir gene therapy using promoters responding to retinoids (Furuhata et al, 2003). Apart from promoters providing tissue specific gene expression, expression inducible promoters were cloned into adenovirus constructs to control the onset and the duration of gene expression. Tetracycline-inducible adenovirus vectors expressing the cytokine interleukin-12 were successfully tested in an immunotherapy model for prostate cancer (Nakagawa et al, 2001). Thus, recombinant adenovirus vectors with tetracycline-inducible gene expression opened up new avenues while improving the safety of viral vector administration for cancer gene therapy. Limitation of cytotoxic gene expression only to tumor cells is very much desired in adenovirus-mediated gene therapy approach for cancer. Unfortunately, the expression levels of many tumor and tissue-specific promoters are much lower than the constitutively active promoters. A complex adenoviral vector was generated by fusing the tetracycline transactivator gene to a prostatespecific ARR2PB promoter while placing a mouse FASLGFP fusion gene under the control of the tetracycline responsive promoter. This allowed the joining of cell-type specificity with high-level regulation of transgene expression (Rubinchik et al, 2001). The doxycycline regulated, ARR2PB driven FASL-GFP vector generated higher levels of prostate-specific FASL-GFP expression than FASL-GFP expression directed with ARR2PB alone, leading to apoptosis in LNCaP cells. Systemic delivery of both the prostate-specific and the prostate-specific/tetregulated vectors was well tolerated in animals at doses

that were lethal for adenovirus vectors with CMV-driven FASL-GFP expression. This approach improved the safety and efficacy of adenovirus-mediated cytotoxic gene delivery for the treatment of prostate carcinoma. The prostate-specific adenovirus gene expression technology can also be used for the identification of metastatic lesions of prostate cancer through the use of non-invasive imaging. A prostate-specific adenovirus vector expressing a luciferase reporter gene (AdPSE-BCluc) and a charge-coupled device-imaging system were employed for this purpose (Adams et al, 2002). A robust expression from AdPSE-BC-luc construct was found in the prostate, especially in the androgen-independent tumors. Furthermore, metastatic lesions in the lung and spine with prostatic origin were identified successfully through repetitive imaging over a three-week period after AdPSE-BC-luc injection into tumor-bearing mice. These results demonstrate that adenovirus gene delivery specific to the prostate can be coupled to a non-invasive imaging modality for therapeutic and diagnostic strategies for prostate cancer.

XII. Adenovirus vectors for vaccination and adjuvant gene therapy CAR receptors and MHC class I heavy chains are important mediators of adenovirus entry into tumor cells. Contrary to the cell lines derived from other malignancies, down regulation of CAR or MHC class I expression is relatively rare in both human and murine prostate carcinoma cells. This brought the possibility of developing vaccine strategies for prostate cancer based on the modification of prostate cancer cells using recombinant adenovirus vectors (Pandha et al, 2003). The expression of prostate-specific antigen (PSA) is highly restricted to prostatic epithelial cells. In fact, 95 % of patients with prostate carcinoma express PSA, making this antigen a good candidate for targeted immunotherapy. A recombinant PSA adenovirus type 5 (Ad5-PSA) was generated in order to activate PSA-specific T-cell response with the potential of eliminating prostate cancer cells (Elzey et al, 2001). Ad5-PSA immunized mice displayed a PSA-specific cellular immunity involving CD8+ T lymphocytes. This approach deterred subcutaneous tumor formation with RM11 prostate cancer cells expressing PSA (RM11psa). However, this did not affect the growth of existing RM11psa tumors. On the contrary, Ad5-PSA administration followed by intratumoral injection of recombinant canarypox viruses (ALVAC) encoding interleukin-12 (IL-12), IL-2, and tumor necrosis factor-# effectively eliminated established RM11psa tumors. Surgery is one of the conventional treatment modalities used against solid tumors. Due to the fact that minor residual tumors following surgical operation may result in local recurrence, surgery is neither efficient nor plausible for the treatment of metastatic disease. Although AdHSV-tk gene therapy followed by ganciclovir administration has been evaluated extensively as a potential treatment modality for numerous tumors, it has not yet been proven to achieve a complete cure on its own.


Sanlioglu et al: Adenovirus mediated gene therapy for prostate carcinoma Prostate-derived tumor models were used to evaluate the effects of AdHSV-tk gene therapy as an adjuvant to surgery (Sukin et al, 2001). Lung nodules of prostate cancer cells were generated by intravenous injection of tumor cells in order to evaluate systemic effects. Following resection of subcutaneous tumors, AdHSV-tk was delivered to the resection site. Toxicity, local tumor recurrence, survival, and lung nodule formation were evaluated in animals; increased survival and decreased recurrence accompanied by no systemic toxicity were observed. Adjuvant AdHSV-tk gene therapy resulted in a significant reduction in lung nodules as well. This study suggested that AdHSV-tk gene therapy might be beneficial as an adjuvant for patients undergoing surgical treatment of cancer.

vector (Roy et al, 2002). Infection of p53 negative human prostate cancer cells (LNCaP) by this approach generated very efficient gene delivery of p53, inducing apoptosis not only in the infected cells but also in the surrounding uninfected cells.

C. Enhancement of transgene expression through transcriptional regulation Although the use of prostate specific promoters is necessary to limit the transgene toxicity, the low level of transgene expression directed by these promoters represents a barrier to gene therapy. The observation, which led to the idea that chemotherapeutics enhanced the transgene expression from viral promoters, represented a new approach to overcome this barrier. Two recombinant adenovirus constructs were used to deliver p21WAF1/CIP1 and p53 protein c-DNA under the control of cytomegalovirus promoter to the metastatic androgen independent prostate cancer cells treated with chemotherapeutic agents docetaxel or paclitaxel (Li et al, 2002b). Both chemotherapeutics appeared to enhance adenovirus mediated transgene expression in androgen independent prostate cancer cell lines. This increase in transgene expression was attributed to the enhancement of CMV promoter activity rather than the increased viral uptake. Therefore, the observed synergy of gene therapy with these chemotherapeutics may become useful when the transgene expression is a limiting factor for the treatment of the metastatic androgen independent prostate cancer. The possible use of other chemotherapeutic agents and their effect on prostate specific promoters should also be explored.

XIII. Current progress to overcome rate-limiting steps in adenovirus-mediated gene therapy for prostate carcinoma The success of adenovirus mediated gene therapy for prostate carcinoma is effected by several factors including the level of expression of the receptor which facilitates the entry of the viral vectors into the cells, penetration of transgenes to surrounding tissues, and finally the expression of the delivered gene. Enhancing these factors has been the focus of many laboratories working on adenovirus-mediated gene therapy for prostate carcinoma. Although a limited number of studies have been completed regarding these issues, effectiveness of prostate cancer gene therapy will certainly benefit from the progress in this field.

A. Receptor abundance

XIV. Summary of clinical trials

The presence of the coxsackie adenovirus cell surface receptor, CAR, is required for an effective adenovirus infection of target cells. CAR expression patterns of normal prostate and prostate carcinoma were compared using immunohistochemical approaches in order to assess the feasibility of adenovirus mediated gene therapy for prostate cancer (Rauen et al, 2002). While a robust membrane staining for CAR was detected in the metastatic prostate specimens with higher Gleason scores, just lumenal and lateral cell membrane staining were detected in the benign prostate epithelia. Therefore, adenovirus mediated gene delivery should be more effective for aggressive prostate tumors than it is for benign cases.

There are 636 clinical protocols involving 3496 patients employed in gene therapy worldwide as reported to the Journal of Gene Medicine website by the year 2002. 403 clinical studies (63.4 %) with regard to gene therapy for cancer were tested on 2392 (68.5 %) patients. Adenovirus was the vector of choice in 171 of these protocols (27 %), and 644 patients (18.4 %) received the adenovirus vector for gene therapy. 22 out of 171 clinical protocols were engaged in adenovirus mediated gene therapies targeting the prostate only as summarized in Table 1. 13 of these were reported to be in Phase I, 3 trials in Phase II and the rest (5) were in Phase I/II. There is no Phase III clinical study reported using adenovirus vectors targeting prostate yet. Some of the adenovirus mediated gene therapy approaches were complemented either with radiotherapy or radical prostatectomy. The percentage of the choice of gene therapy modalities targeting prostate is provided in Figure 3. The use of selectively replicating adenovirus constructs leads other approaches followed by suicide gene therapy. This is partly because not long ago astonishing results were obtained with selectively replicating adenovirus constructs in the preclinical animal models. It is also interesting to note that two of these clinical trials utilize suicide gene therapy in combination with the selectively replicating adenovirus approach

B. Penetration of hybrid therapeutic transgenes to the surrounding tissue Despite the fact that adenovirus could transduce cells very efficiently in vitro, adenovirus mediated gene delivery is restricted by the inefficient transduction of surrounding cells for a given tumor. In order to overcome this obstacle, an important intercellular transport protein named VP22, was first fused to the therapeutic transgene of interest (p53 gene) and then cloned into adenovirus


Gene Therapy and Molecular Biology Vol 7, page 127 (Figure 3). No clinical studies have been carried out using the death ligand-mediated gene therapy approach and adenovirus vectors up to date. However we should not be surprised if such trials are being initiated and we encounter some of these in the near future. Although preliminary results are very encouraging from these clinical investigations, clear conclusions can be drawn only upon completion of these studies. Considering all these preclinical and clinical studies, we concluded that great progress in adenovirus mediated

gene therapy for prostate carcinoma has been made within the last 3 years. While the molecular mechanisms responsible for prostate carcinoma are not fully understood, the effectiveness of gene therapy is still quite amazing. As more data become available on the understanding of prostate carcinoma, we anticipate that more effective treatment modalities will be developed using adenovirus to target prostate cancer.

Table 1. A summary of ongoing clinical trials of adenovirus mediated gene therapy targeting prostate as of 2002. The data was collected from the Journal of Gene Medicine web site ( and published with the permission from %John Wiley and Sons 2002. Country Canada Canada USA USA

Investigator A. K. Stewart J. Dancey Peter T. Scardino Simon J. Hall

Mode of Therapy Immunotherapy (IL-2) Immunotherapy (IL-2) Suicide gene therapy (HSV-tk) + radiotherapy Neo-adjuvant suicide gene therapy (HSV-tk) + radical prostatectomy

Phase I I I I


Arie Belldegrun Christopher J. Logothetis

Tumor suppressor gene therapy (p53) Tumor suppressor gene therapy (p53)



Dov Kadmon

Neo-adjuvant suicide gene therapy (HSV-tk) + radical prostatectomy



Jonathan W. Simons

Selectively replicating adenovirus (CN706)



Thomas A. Gardner

Suicide gene therapy (HSV-tk)



Jae Ho Kim

Suicide gene therapy (CD/Tk) with selectively replicating adenovirus + radiotherapy



E. Brian Butler Jeffrey R. Gingrich Martha K. Terris George Wilding Alan Pollack Thomas A. Gardner

Suicide Gene Therapy (HSV-tk) + radiotherapy Neo-adjuvant CDK inhibitor (p16) + radical prostatectomy Selectively replicating adenovirus (CV787) + Radiotherapy Selectively replicating adenovirus (CV787) Tumor suppressor gene therapy (p53) + radiotherapy Selectively replicating adenovirus with osteocalcin promoter (Ad-OCE1A)



David M. Lubaroff Brian J. Miles Theodore L. DeWeese

Immunotherapy (PSA) Immunotherapy (IL-12) + radiotherapy Selectively replicating adenovirus (CV706)



Eric J. Small Svend O. Freytag



John M. Corman

Selectively replicating adenovirus (CV787) + chemotherapy Neo-adjuvant suicide gene therapy (CD/Tk) with selectively replicating adenovirus + Radiotherapy Selectively replicating adenovirus (CG7060) + radiotherapy



Sanlioglu et al: Adenovirus mediated gene therapy for prostate carcinoma

Figure 3. Adenovirus mediated clinical gene therapy modalities for prostate. The types of clinical gene therapy modalities for prostate are represented as percentages in a pie graph in order to better appreciate the contribution of each treatment modality. regulation of NF-!B signaling events in suppression of TNFinduced apoptosis. Biochem Biophys Res Commun 271, 342-345. Cao G, Su J, Lu W, Zhang F, Zhao G, Marteralli D, and Dong Z (2001) Adenovirus-mediated interferon-" gene therapy suppresses growth and metastasis of human prostate cancer in nude mice. Cancer Gene Ther 8, 497-505. Chaudhary PM, Eby M, Jasmin A, Bookwalter A, Murray J, and Hood L (1997) Death receptor 5, a new member of the TNFR family, and DR4 induce FADD- dependent apoptosis and activate the NF-!B pathway. Immunity 7, 821-830. Chen Y, DeWeese T, Dilley J, Zhang Y, Li Y, Ramesh N, Lee J, Pennathur-Das R, Radzyminski J, Wypych J, Brignetti D, Scott S, Stephens J, Karpf DB, Henderson DR, and Yu DC (2001) CV706, a prostate cancer-specific adenovirus variant, in combination with radiotherapy produces synergistic antitumor efficacy without increasing toxicity. Cancer Res 61, 5453-5460 Chhikara M, Huang H, Vlachaki MT, Zhu X, Teh B, Chiu KJ, Woo S, Berner B, Smith EO, Oberg KC, Aguilar LK, Thompson TC, Butler EB, and Aguilar-Cordova E (2001) Enhanced therapeutic effect of HSV-tk+GCV gene therapy and ionizing radiation for prostate cancer. Mol Ther 3, 536542. Davies MA, Kim SJ, Parikh NU, Dong Z, Bucana CD, and Gallick GE (2002) Adenoviral-mediated expression of MMAC/PTEN inhibits proliferation and metastasis of human prostate cancer cells. Clin Cancer Res 8, 1904-1914. Davies MA, Koul D, Dhesi H, Berman R, McDonnell TJ, McConkey D, Yung WK, and Steck PA (1999) Regulation of Akt/PKB activity, cellular growth, and apoptosis in prostate carcinoma cells by MMAC/PTEN. Cancer Res 59, 25512556. Degli-Esposti MA, Dougall WC, Smolak PJ, Waugh JY, Smith CA, and Goodwin RG (1997) The novel receptor TRAIL-R4 induces NF-!B and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain. Immunity 7, 813-820. Demarchi F, Verardo R, Varnum B, Brancolini C, and Schneider C (2001) Gas6 anti-apoptotic signaling requires NF-! B activation. J Biol Chem 276, 31738-31744. Devi GR (2002) Prostate cancer: status of current treatments and emerging antisense- based therapies. Curr Opin Mol Ther 4, 138-148. DeWeese TL, van der Poel H, Li S, Mikhak B, Drew R, Goemann M, Hamper U, DeJong R, Detorie N, Rodriguez R, Haulk T, DeMarzo AM, Piantadosi S, Yu DC, Chen Y,

Acknowledgments This work is supported by Akdeniz University Scientific Research Project Administration Division Grants (#2002.01.0122.06, #2002.01.0122.07 and #2002.01.0200.005 to Dr. Salih Sanlioglu).

References Abe K, Kurakin A, Mohseni-Maybodi M, Kay B, and KhosraviFar R (2000) The complexity of TNF-related apoptosisinducing ligand. Ann N Y Acad Sci 926, 52-63. Adams JY, Johnson M, Sato M, Berger F, Gambhir SS, Carey M, Iruela-Arispe ML, and Wu L (2002) Visualization of advanced human prostate cancer lesions in living mice by a targeted gene transfer vector and optical imaging. Nat Med 8, 891-897. Andjelic S, Hsia C, Suzuki H, Kadowaki T, Koyasu S, and Liou HC (2000) Phosphatidylinositol 3-kinase and NF-! B/Rel are at the divergence of CD40-mediated proliferation and survival pathways. J Immunol 165, 3860-3867. Batra RK, Guttridge DC, Brenner DA, Dubinett SM, Baldwin AS, and Boucher RC (1999) I!B# gene transfer is cytotoxic to squamous-cell lung cancer cells and sensitizes them to tumor necrosis factor-#-mediated cell death. Am J Respir Cell Mol Biol 21, 238-245 Becker CM, Farnebo FA, Iordanescu I, Behonick DJ, Shih MC, Dunning P, Christofferson R, Mulligan RC, Taylor GA, Kuo CJ, and Zetter BR (2002) Gene therapy of prostate cancer with the soluble vascular endothelial growth factor receptor flk1. Cancer Biol Ther 1, 548-553. Beg AA, and Baltimore D (1996) An essential role for NF-!B in preventing TNF-#-induced cell death. Science 274, 782-784. Beresford SA, Davies MA, Gallick GE, and Donato NJ (2001) Differential effects of phosphatidylinositol-3/Akt-kinase inhibition on apoptotic sensitization to cytokines in LNCaP and PCc-3 prostate cancer cells. J Interferon Cytokine Res 21, 313-322. Bernard D, Quatannens B, Vandenbunder B, and Abbadie C (2001) Rel/NF-!B transcription factors protect against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by up-regulating the TRAIL decoy receptor DcR1. J Biol Chem 276, 27322-27328. Boring CC, Squires TS, Tong T, and Montgomery S (1994) Cancer statistics, 1994. CA Cancer J Clin 44, 7-26. Burow ME, Weldon CB, Melnik LI, Duong BN, Collins-Burow BM, Beckman BS, and McLachlan JA (2000) PI3-K/AKT


Gene Therapy and Molecular Biology Vol 7, page 129 Henderson DR, Carducci MA, Nelson WG, and Simons JW (2001) A phase I trial of CV706, a replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy. Cancer Res 61, 7464-7472 Djeha AH, Thomson TA, Leung H, Searle PF, Young LS, Kerr DJ, Harris PA, Mountain A, and Wrighton CJ (2001) Combined adenovirus-mediated nitroreductase gene delivery and CB1954 treatment: a well-tolerated therapy for established solid tumors. Mol Ther 3, 233-240 Do LV, Do TM, Smith R, and Parker RG (2002) Postoperative Radiotherapy for Carcinoma of the Prostate: Impact on Both Local Control and Distant Disease-Free Survival. Am J Clin Oncol 25, 1-8. Ebara S, Shimura S, Nasu Y, Kaku H, Kumon H, Yang G, Wang J, Timme TL, Aguilar-Cordova E, and Thompson TC (2002) Gene therapy for prostate cancer: toxicological profile of four HSV-tk transducing adenoviral vectors regulated by different promoters. Prostate Cancer Prostatic Dis 5, 316325 Ehlert JE, and Kubbutat MH (2001) Apoptosis and its relevance in cancer therapy. Onkologie 24, 433-440. Elzey BD, Siemens DR, Ratliff TL, and Lubaroff DM (2001) Immunization with type 5 adenovirus recombinant for a tumor antigen in combination with recombinant canarypox virus (ALVAC) cytokine gene delivery induces destruction of established prostate tumors. Int J Cancer 94, 842-849 Farkas A, Schneider D, Perrotti M, Cummings KB, and Ward WS (1998) National trends in the epidemiology of prostate cancer, 1973 to 1994: evidence for the effectiveness of prostate-specific antigen screening. Urology 52, 444-448; discussion 448-449. Franco AV, Zhang XD, Van Berkel E, Sanders JE, Zhang XY, Thomas WD, Nguyen T, and Hersey P (2001) The role of NF-!B in TNF-related apoptosis-inducing ligand (TRAIL)induced apoptosis of melanoma cells. J Immunol 166, 53375345. Franklin J, Hislop J, Flynn A, and McArdle CA (2003) Signalling and anti-proliferative effects mediated by gonadotrophin- releasing hormone receptors after expression in prostate cancer cells using recombinant adenovirus. J Endocrinol 176, 275-284. Freytag SO, Khil M, Stricker H, Peabody J, Menon M, DePeralta-Venturina M, Nafziger D, Pegg J, Paielli D, Brown S, Barton K, Lu M, Aguilar-Cordova E, and Kim JH (2002a) Phase I study of replication-competent adenovirusmediated double suicide gene therapy for the treatment of locally recurrent prostate cancer. Cancer Res 62, 4968-4976 Freytag SO, Paielli D, Wing M, Rogulski K, Brown S, Kolozsvary A, Seely J, Barton K, Dragovic A, and Kim JH (2002b) Efficacy and toxicity of replication-competent adenovirus-mediated double suicide gene therapy in combination with radiation therapy in an orthotopic mouse prostate cancer model. Int J Radiat Oncol Biol Phys 54, 873-885 Furuhata S, Ide H, Miura Y, Yoshida T, and Aoki K (2003) Development of a prostate-specific promoter for gene therapy against androgen-independent prostate cancer. Mol Ther 7, 366-374. Gabril MY, Onita T, Ji PG, Sakai H, Chan FL, Koropatnick J, Chin JL, Moussa M, and Xuan JW (2002) Prostate targeting: PSP94 gene promoter/enhancer region directed prostate tissue-specific expression in a transgenic mouse prostate cancer model. Gene Ther 9, 1589-1599 Galfione M, Luo W, Kim J, Hawke D, Kobayashi R, Clapp C, Yu-Lee LY, and Lin SH (2003) Expression and purification of the angiogenesis inhibitor 16-kDa prolactin fragment from insect cells. Protein Expr Purif 28, 252-258.

Gasparian AV, Yao YJ, Kowalczyk D, Lyakh LA, Karseladze A, Slaga TJ, and Budunova IV (2002a) The role of IKK in constitutive activation of NF-!B transcription factor in prostate carcinoma cells. J Cell Sci 115, 141-151. Gasparian AV, Yao YJ, Lu J, Yemelyanov AY, Lyakh LA, Slaga TJ, and Budunova IV (2002b) Selenium compounds inhibit I !B kinase (IKK) and nuclear factor- !B (NF-!B) in prostate cancer cells. Mol Cancer Ther 1, 1079-1087. Giri D, Ozen M, and Ittmann M (2001) Interleukin-6 is an autocrine growth factor in human prostate cancer. Am J Pathol 159, 2159-2165. Greenlee RT, Hill-Harmon MB, Murray T, and Thun M (2001) Cancer statistics, 2001. CA Cancer J Clin 51, 15-36. Griffith TS, and Broghammer EL (2001) Suppression of tumor growth following intralesional therapy with TRAIL recombinant adenovirus. Mol Ther 4, 257-266. Griffith TS, Chin WA, Jackson GC, Lynch DH, and Kubin MZ (1998) Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells. J Immunol 161, 2833-2840. Griffith TS, and Lynch DH (1998) TRAIL: a molecule with multiple receptors and control mechanisms. Curr Opin Immunol 10, 559-563. Grumet SC, and Bruner DW (2000) The identification and screening of men at high risk for developing prostate cancer. Urol Nurs 20, 15-18, 23-14, 46. Gustin JA, Maehama T, Dixon JE, and Donner DB (2001) The PTEN tumor suppressor protein inhibits tumor necrosis factor- induced nuclear factor !B activity. J Biol Chem 276, 27740-27744. Hall SJ, Canfield SE, Yan Y, Hassen W, Selleck WA, and Chen SH (2002) A novel bystander effect involving tumor cellderived Fas and FasL interactions following Ad.HSV-tk and Ad.mIL-12 gene therapies in experimental prostate cancer. Gene Ther 9, 511-517. Hatano E, and Brenner DA (2001) Akt protects mouse hepatocytes from TNF-#- and Fas-mediated apoptosis through NK-!B activation. Am J Physiol Gastrointest Liver Physiol 281, G1357-1368. Hemmati PG, Gillissen B, von Haefen C, Wendt J, Starck L, Guner D, Dorken B, and Daniel PT (2002) Adenovirusmediated overexpression of p14(ARF) induces p53 and Baxindependent apoptosis. Oncogene 21, 3149-3161. Herr I, and Debatin KM (2001) Cellular stress response and apoptosis in cancer therapy. Blood 98, 2603-2614. Honda T, Kagawa S, Spurgers KB, Gjertsen BT, Roth JA, Fang B, Lowe SL, Norris JS, Meyn RE, and McDonnell TJ (2002) A recombinant adenovirus expressing wild-type Bax induces apoptosis in prostate cancer cells independently of their Bcl2 status and androgen sensitivity. Cancer Biol Ther 1, 163167 Horowitz J (1999) Adenovirus-mediated p53 gene therapy: overview of preclinical studies and potential clinical applications. Curr Opin Mol Ther 1, 500-509. Hsieh CL, and Chung LW (2001) New prospectives of prostate cancer gene therapy: molecular targets and animal models. Crit Rev Eukaryot Gene Expr 11, 77-120. Hsieh CL, Yang L, Miao L, Yeung F, Kao C, Yang H, Zhau HE, and Chung LW (2002) A novel targeting modality to enhance adenoviral replication by vitamin D(3) in androgenindependent human prostate cancer cells and tumors. Cancer Res 62, 3084-3092. Hyer ML, Sudarshan S, Kim Y, Reed JC, Dang JY, Schwartz DA, and Norris JS (2002) Downregulation of c-FLIP Sensitizes DU145 Prostate Cancer Cells to Fas- Mediated Apoptosis. Cancer Biol Ther 1, 401-406. Hyer ML, Sudarshan S, Schwartz DA, Hannun Y, Dong JY, and Norris JS (2003) Quantification and characterization of the bystander effect in prostate cancer cells following


Sanlioglu et al: Adenovirus mediated gene therapy for prostate carcinoma adenovirus-mediated FasL expression. Cancer Gene Ther 10, 330-339. Hyer ML, Voelkel-Johnson C, Rubinchik S, Dong J, and Norris JS (2000) Intracellular Fas ligand expression causes Fasmediated apoptosis in human prostate cancer cells resistant to monoclonal antibody-induced apoptosis. Mol Ther 2, 348358. Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner V, Bodmer JL, Schroter M, Burns K, Mattmann C, Rimoldi D, French LE, and Tschopp J (1997) Inhibition of death receptor signals by cellular FLIP. Nature 388, 190-195. Jones RG, Parsons M, Bonnard M, Chan VS, Yeh WC, Woodgett JR, and Ohashi PS (2000) Protein kinase B regulates T lymphocyte survival, nuclear factor !B activation, and Bcl-X(L) levels in vivo. J Exp Med 191, 1721-1734. Jorgensen TJ, Katz S, Wittmack EK, Varghese S, Todo T, Rabkin SD, and Martuza RL (2001) Ionizing radiation does not alter the antitumor activity of herpes simplex virus vector G207 in subcutaneous tumor models of human and murine prostate cancer. Neoplasia 3, 451-456 Kane LP, Shapiro VS, Stokoe D, and Weiss A (1999) Induction of NF-!B by the Akt/PKB kinase. Curr Biol 9, 601-604. Katner AL, Hoang QB, Gootam P, Jaruga E, Ma Q, Gnarra J, and Rayford W (2002) Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27(Kip1). Prostate 53, 77-87. Kim J, Luo W, Chen DT, Earley K, Tunstead J, Yu-Lee LY, and Lin SH (2003) Antitumor activity of the 16-kDa prolactin fragment in prostate cancer. Cancer Res 63, 386-393. Klotz L (2000a) Hormone therapy for patients with prostate carcinoma. Cancer 88, 3009-3014. Klotz L (2000b) Intraoperative cavernous nerve stimulation during nerve sparing radical prostatectomy: how and when? Curr Opin Urol 10, 239-243. Koksal IT, Ozcan F, Kadioglu TC, Esen T, KiliCaslan I, and Tunc M (2000) Discrepancy between Gleason scores of biopsy and radical prostatectomy specimens. Eur Urol 37, 670-674. Kreuz S, Siegmund D, Scheurich P, and Wajant H (2001) NF-!B inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling. Mol Cell Biol 21, 39643973. Kubo H, Gardner TA, Wada Y, Koeneman KS, Gotoh A, Yang L, Kao C, Lim SD, Amin MB, Yang H, Black ME, Matsubara S, Nakagawa M, Gillenwater JY, Zhau HE, and Chung LW (2003) Phase I dose escalation clinical trial of adenovirus vector carrying osteocalcin promoter-driven herpes simplex virus thymidine kinase in localized and metastatic hormone-refractory prostate cancer. Hum Gene Ther 14, 227-241 Lee SJ, Kim HS, Yu R, Lee K, Gardner TA, Jung C, Jeng MH, Yeung F, Cheng L, and Kao C (2002a) Novel prostatespecific promoter derived from PSA and PSMA enhancers. Mol Ther 6, 415-421. Lee YJ, Galoforo SS, Battle P, Lee H, Corry PM, and Jessup JM (2001) Replicating adenoviral vector-mediated transfer of a heat-inducible double suicide gene for gene therapy. Cancer Gene Ther 8, 397-404. Lee YJ, Lee H, and Borrelli MJ (2002b) Gene transfer into human prostate adenocarcinoma cells with an adenoviral vector: Hyperthermia enhances a double suicide gene expression, cytotoxicity and radiotoxicity. Cancer Gene Ther 9, 267-274. Levine AJ (1997) p53, the cellular gatekeeper for growth and division. Cell 88, 323-331.

Li X, Marani M, Yu J, Nan B, Roth JA, Kagawa S, Fang B, Denner L, and Marcelli M (2001) Adenovirus-mediated Bax overexpression for the induction of therapeutic apoptosis in prostate cancer. Cancer Res 61, 186-191. Li Y, McCadden J, Ferrer F, Kruszewski M, Carducci M, Simons J, and Rodriguez R (2002a) Prostate-specific expression of the diphtheria toxin A chain (DT-A): studies of inducibility and specificity of expression of prostate- specific antigen promoter-driven DT-A adenoviral-mediated gene transfer. Cancer Res 62, 2576-2582. Li Y, Okegawa T, Lombardi DP, Frenkel EP, and Hsieh JT (2002b) Enhanced transgene expression in androgen independent prostate cancer gene therapy by taxane chemotherapeutic agents. J Urol 167, 339-346. Lin SH, Pu YS, Luo W, Wang Y, and Logothetis CJ (1999) Schedule-dependence of C-CAM1 adenovirus gene therapy in a prostate cancer model. Anticancer Res 19, 337-340. Loimas S, Toppinen MR, Visakorpi T, Janne J, and Wahlfors J (2001) Human prostate carcinoma cells as targets for herpes simplex virus thymidine kinase-mediated suicide gene therapy. Cancer Gene Ther 8, 137-144. Lowe SL, Rubinchik S, Honda T, McDonnell TJ, Dong JY, and Norris JS (2001) Prostate-specific expression of Bax delivered by an adenoviral vector induces apoptosis in LNCaP prostate cancer cells. Gene Ther 8, 1363-1371. Lu L, Schulz H, and Wolf DA (2002) The F-box protein SKP2 mediates androgen control of p27 stability in LNCaP human prostate cancer cells. BMC Cell Biol 3, 22. Lu Y, and Steiner MS (2000) Transcriptionally regulated adenoviruses for prostate-specific gene therapy. World J Urol 18, 93-101. Lundqvist A, Choudhury A, Nagata T, Andersson T, Quinn G, Fong T, Maitland N, Pettersson S, Paulie S, and Pisa P (2002a) Recombinant adenovirus vector activates and protects human monocyte- derived dendritic cells from apoptosis. Hum Gene Ther 13, 1541-1 Lundqvist A, Nagata T, Kiessling R, and Pisa P (2002b) Mature dendritic cells are protected from Fas/CD95-mediated apoptosis by upregulation of Bcl-X(L). Cancer Immunol Immunother 51, 139-144. Madrid LV, Mayo MW, Reuther JY, and Baldwin AS, Jr. (2001) Akt stimulates the transactivation potential of the RelA/p65 Subunit of NF-!B through utilization of the I!B kinase and activation of the mitogen-activated protein kinase p38. J Biol Chem 276, 18934-18940 Martinez LA, Yang J, Vazquez ES, Rodriguez-Vargas Mdel C, Olive M, Hsieh JT, Logothetis CJ, and Navone NM (2002) p21 modulates threshold of apoptosis induced by DNAdamage and growth factor withdrawal in prostate cancer cells. Carcinogenesis 23, 1289-129 Martiniello-Wilks R, Tsatralis T, Russell P, Brookes DE, Zandvliet D, Lockett LJ, Both GW, Molloy PL, and Russell PJ (2002) Transcription-targeted gene therapy for androgenindependent prostate cancer. Cancer Gene Ther 9, 443-452. Matsubara S, Wada Y, Gardner TA, Egawa M, Park MS, Hsieh CL, Zhau HE, Kao C, Kamidono S, Gillenwater JY, and Chung LW (2001) A conditional replication-competent adenoviral vector, Ad-OC-E1a, to cotarget prostate cancer and bone stroma in an experimental model of androgenindependent prostate cancer bone metastasis. Cancer Res 61, 6012-6019 Mayo MW, Madrid LV, Westerheide SD, Jones DR, Yuan XJ, Baldwin AS, Jr., and Whang YE (2002) PTEN blocks tumor necrosis factor-induced NF-!B-dependent transcription by inhibiting the transactivation potential of the p65 subunit. J Biol Chem 277, 1111 Meng RD, McDonald ER, 3rd, Sheikh MS, Fornace AJ, Jr., and El-Deiry WS (2000) The TRAIL decoy receptor TRUNDD


Gene Therapy and Molecular Biology Vol 7, page 131 (DcR2, TRAIL-R4) is induced by adenovirus-p53 overexpression and can delay TRAIL-, p53-, and KILLER/DR5-dependent colon cancer apoptosis. Mol Ther 1, 130-144. Miles BJ, Shalev M, Aguilar-Cordova E, Timme TL, Lee HM, Yang G, Adler HL, Kernen K, Pramudji CK, Satoh T, Gdor Y, Ren C, Ayala G, Wheeler TM, Butler EB, Kadmon D, and Thompson TC (2001) Prostate-specific antigen response and systemic T cell activation after in situ gene therapy in prostate cancer patients failing radiotherapy. Hum Gene Ther 12, 1955-1967 Mitsiades N, Mitsiades CS, Poulaki V, Chauhan D, Richardson PG, Hideshima T, Munshi N, Treon SP, and Anderson KC (2002) Biologic sequelae of nuclear factor-!B blockade in multiple myeloma: therapeutic applications. Blood 99, 40794086. Miyagi T, Hori O, Koshida K, Egawa M, Kato H, Kitagawa Y, Ozawa K, Ogawa S, and Namiki M (2002) Antitumor effect of reduction of 150-kDa oxygen-regulated protein expression on human prostate cancer cells. Int J Urol 9, 577-585. Miyagi T, Koshida K, Hori O, Konaka H, Katoh H, Kitagawa Y, Mizokami A, Egawa M, Ogawa S, Hamada H, and Namiki M (2003) Gene therapy for prostate cancer using the cytosine deaminase/uracil phosphoribosyltransferase suicide system. J Gene Med 5, 30-37. Modur V, Nagarajan R, Evers BM, and Milbrandt J (2002) FOXO proteins regulate tumor necrosis factor-related apoptosis inducing ligand expression. Implications for PTEN mutation in prostate cancer. J Biol Chem 277, 47928-47937. Moffatt KA, Johannes WU, Hedlund TE, and Miller GJ (2001) Growth inhibitory effects of 1#, 25-dihydroxyvitamin D(3) are mediated by increased levels of p21 in the prostatic carcinoma cell line ALVA-31. Cancer Res 61, 7122-7129. Nagane M, Huang HJ, and Cavenee WK (2001) The potential of TRAIL for cancer chemotherapy. Apoptosis 6, 191-197. Nagata S (1997) Apoptosis by death factor. Cell 88, 355-365. Nakagawa S, Massie B, and Hawley RG (2001) Tetracyclineregulatable adenovirus vectors: pharmacologic properties and clinical potential. Eur J Pharm Sci 13, 53-60. Nesterov A, Lu X, Johnson M, Miller GJ, Ivashchenko Y, and Kraft AS (2001) Elevated AKT activity protects the prostate cancer cell line LNCaP from TRAIL-induced apoptosis. J Biol Chem 276, 10767-10774. Norris JS, Hyer ML, Voelkel-Johnson C, Lowe SL, Rubinchik S, and Dong JY (2001) The use of Fas Ligand, TRAIL and Bax in gene therapy of prostate cancer. Curr Gene Ther 1, 123136. Obata A, Eura M, Sasaki J, Saya H, Chikamatsu K, Tada M, Iggo RD, and Yumoto E (2000) Clinical significance of p53 functional loss in squamous cell carcinoma of the oropharynx. Int J Cancer 89, 187-193. Oya M, Ohtsubo M, Takayanagi A, Tachibana M, Shimizu N, and Murai M (2001) Constitutive activation of nuclear factor-!B prevents TRAIL-induced apoptosis in renal cancer cells. Oncogene 20, 3888-3896. Ozen M, Giri D, Ropiquet F, Mansukhani A, and Ittmann M (2001) Role of fibroblast growth factor receptor signaling in prostate cancer cell survival. J Natl Cancer Inst 93, 17831790. Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, and Donner DB (1999) NF-!B activation by tumour necrosis factor requires the Akt serine- threonine kinase. Nature 401, 82-85. Pan G, Ni J, Wei YF, Yu G, Gentz R, and Dixit VM (1997) An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 277, 815-818. Pandha HS, Stockwin LH, Eaton J, Clarke IA, Dalgleish AG, Todryk SM, and Blair GE (2003) Coxsackie B and

adenovirus receptor, integrin and major histocompatibility complex class I expression in human prostate cancer cell lines: implications for gene therapy strategies. Prostate Cancer Prostatic Dis 6, 6-11 Perrotti M, Rabbani F, Farkas A, Ward WS, and Cummings KB (1998) Trends in poorly differentiated prostate cancer 1973 to 1994: observations from the Surveillance, Epidemiology and End Results database. J Urol 160, 811-815. Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, and Ashkenazi A (1996) Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 271, 12687-12690. Powell IJ, Dey J, Dudley A, Pontes JE, Cher ML, Sakr W, Grignon DJ, and Wood DP (2002) Disease-free survival difference between African Americans and whites after radical prostatectomy for local prostate cancer: a multivariable analysis. Urology 59, 907Pramudji C, Shimura S, Ebara S, Yang G, Wang J, Ren C, Yuan Y, Tahir SA, Timme TL, and Thompson TC (2001) In situ prostate cancer gene therapy using a novel adenoviral vector regulated by the caveolin-1 promoter. Clin Cancer Res 7, 4272-4279. Pu YS, Do KA, Luo W, Logothetis CJ, and Lin SH (2002) Enhanced suppression of prostate tumor growth by combining C-CAM1 gene therapy and angiogenesis inhibitor TNP-470. Anticancer Drugs 13, 743-749. Pu YS, Luo W, Lu HH, Greenberg NM, Lin SH, and Gingrich JR (1999) Differential expression of C-CAM cell adhesion molecule in prostate carcinogenesis in a transgenic mouse model. J Urol 162, 892-896. Rauen KA, Sudilovsky D, Le JL, Chew KL, Hann B, Weinberg V, Schmitt LD, and McCormick F (2002) Expression of the coxsackie adenovirus receptor in normal prostate and in primary and metastatic prostate carcinoma: potential relevance to gene therapy. Cancer Res 62, 3812-3818. Ravi R, Bedi GC, Engstrom LW, Zeng Q, Mookerjee B, Gelinas C, Fuchs EJ, and Bedi A (2001) Regulation of death receptor expression and TRAIL/Apo2L-induced apoptosis by NF-!B. Nat Cell Biol 3, 409-416. Reed JC (2000) Mechanisms of apoptosis. Am J Pathol 157, 1415-1430. Rodriguez R, Schuur ER, Lim HY, Henderson GA, Simons JW, and Henderson DR (1997) Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells. Cancer Res 57, 2559-25 Romashkova JA, and Makarov SS (1999) NF-!B is a target of AKT in anti-apoptotic PDGF signalling. Nature 401, 86-90. Ross JS, Sheehan CE, Dolen EM, and Kallakury BV (2002a) Morphologic and molecular prognostic markers in prostate cancer. Adv Anat Pathol 9, 115-128. Ross JS, Sheehan CE, Fisher HA, Kauffman RA, Dolen EM, and Kallakury BV (2002b) Prognostic markers in prostate cancer. Expert Rev Mol Diagn 2, 129-142. Roy I, Holle L, Song W, Holle E, Wagner T, and Yu X (2002) Efficient translocation and apoptosis induction by adenovirus encoded VP22-p53 fusion protein in human tumor cells in vitro. Anticancer Res 22, 3185-3189. Rubinchik S, Wang D, Yu H, Fan F, Luo M, Norris JS, and Dong JY (2001) A complex adenovirus vector that delivers FASLGFP with combined prostate-specific and tetracyclineregulated expression. Mol Ther 4, 416-426. Rylova SN, Amalfitano A, Persaud-Sawin DA, Guo WX, Chang J, Jansen PJ, Proia AD, and Boustany RM (2002) The CLN3 gene is a novel molecular target for cancer drug discovery. Cancer Res 62, 801-808.


Sanlioglu et al: Adenovirus mediated gene therapy for prostate carcinoma Sanda MG (1997) Biological principles and clinical development of prostate cancer gene therapy. Semin Urol Oncol 15, 4355. Sanlioglu S, and Engelhardt JF (1999) Cellular redox state alters recombinant adeno-associated virus transduction through tyrosine phosphatase pathways. Gene Ther 6, 1427-1437. Sanlioglu S, Luleci G, and Thomas KW (2001a) Simultaneous inhibition of Rac1 and IKK pathways sensitizes lung cancer cells to TNF#-mediated apoptosis. Cancer Gene Ther 8, 897-905. Sanlioglu S, Williams CM, Samavati L, Butler NS, Wang G, McCray PB, Jr., Ritchie TC, Hunninghake GW, Zandi E, and Engelhardt JF (2001b) Lipopolysaccharide induces Rac1dependent reactive oxygen species formation and coordinates tumor necrosis factor-# secretion through IKK regulation of NF-!B. J Biol Chem 276, 30188-30198. Sasaki R, Shirakawa T, Zhang ZJ, Tamekane A, Matsumoto A, Sugimura K, Matsuo M, Kamidono S, and Gotoh A (2001) Additional gene therapy with Ad5CMV-p53 enhanced the efficacy of radiotherapy in human prostate cancer cells. Int J Radiat Oncol Biol Phys 51, 1336-1345. Schneider P, Thome M, Burns K, Bodmer JL, Hofmann K, Kataoka T, Holler N, and Tschopp J ( 1997) TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF-!B. Immunity 7, 831-836. Schroder FH, Hermanek P, Denis L, Fair WR, Gospodarowicz MK, and Pavone-Macaluso M (1992) The TNM classification of prostate cancer. Prostate Suppl 4, 129-138. Sears RC, and Nevins JR (2002) Signaling networks that link cell proliferation and cell fate. J Biol Chem 22, 22. Selleck WA, Canfield SE, Hassen WA, Meseck M, Kuzmin AI, Eisensmith RC, Chen SH, and Hall SJ (2003) IFN-gamma sensitization of prostate cancer cells to fas-mediated death: a gene therapy approach. Mol Ther 7, 185-192. Shariat SF, Desai S, Song W, Khan T, Zhao J, Nguyen C, Foster BA, Greenberg N, Spencer DM, and Slawin KM (2001) Adenovirus-mediated transfer of inducible caspases: a novel "death switch" gene therapeutic approach to prostate cancer. Cancer Res 61, 2562-2 Sheikh MS, and Fornace AJ, Jr. (2000) Death and decoy receptors and p53-mediated apoptosis. Leukemia 14, 15091513. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard AD, Godowski P, and Ashkenazi A (1997) Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 277, 8 Siemens DR, Elzey BD, Lubaroff DM, Bohlken C, Jensen RJ, Swanson AK, and Ratliff TL (2001) Cutting edge: restoration of the ability to generate CTL in mice immune to adenovirus by delivery of virus in a collagen-based matrix. J Immunol 166, 731-735. Simpson L, and Parsons R (2001) PTEN: life as a tumor suppressor. Exp Cell Res 264, 29-41. Sizemore N, Lerner N, Dombrowski N, Sakurai H, and Stark GR (2002) Distinct roles of the I!B kinase # and " subunits in liberating nuclear factor !B (NF-!B) from !B and in phosphorylating the p65 subunit of NF-!B. and in phosphorylating the p65 subunit of NF-!B. J Biol Chem 277, 3863-3869 Slack JK, Adams RB, Rovin JD, Bissonette EA, Stoker CE, and Parsons JT (2001) Alterations in the focal adhesion kinase/Src signal transduction pathway correlate with increased migratory capacity of prostate carcinoma cells. Oncogene 20, 1152-1163. Smith MR, Finkelstein JS, McGovern FJ, Zietman AL, Fallon MA, Schoenfeld DA, and Kantoff PW (2002) Changes in

Body Composition during Androgen Deprivation Therapy for Prostate Cancer. J Clin Endocrinol Metab 87, 599-603. Spitzweg C, Dietz AB, O'Connor MK, Bergert ER, Tindall DJ, Young CY, and Morris JC (2001) In vivo sodium iodide symporter gene therapy of prostate cancer. Gene Ther 8, 1524-1531. Steck PA, Pershouse MA, Jasser SA, Yung WK, Lin H, Ligon AH, Langford LA, Baumgard ML, Hattier T, Davis T, Frye C, Hu R, Swedlund B, Teng DH, and Tavtigian SV (1997) Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat Genet 15, 356-362. Stein R, Chen S, Reed L, Richel H, and Goldenberg DM (2002) Combining radioimmunotherapy and chemotherapy for treatment of medullary thyroid carcinoma. Cancer 94, 51-61. Sukin SW, Chhikara M, Zhu X, Ayala G, Aguilar LK, O'Brian Smith E, Miles BJ, Thompson TC, Kadmon D, and AguilarCordova E (2001) In vivo surgical resection plus adjuvant gene therapy in the treatment of mammary and prostate cancer. Mol Ther 3, 500-506. Taj MM, Tawil RJ, Engstrom LD, Zeng Z, Hwang C, Sanda MG, and Wechsler DS (2001) Mxi1, a Myc antagonist, suppresses proliferation of DU145 human prostate cells. Prostate 47, 194-204. Teh BS, Aguilar-Cordova E, Kernen K, Chou CC, Shalev M, Vlachaki MT, Miles B, Kadmon D, Mai WY, Caillouet J, Davis M, Ayala G, Wheeler T, Brady J, Carpenter LS, Lu HH, Chiu JK, Woo SY, Thompson T, and Butler EB (2001) Phase I/II trial evaluating combined radiotherapy and in situ gene therapy with or without hormonal therapy in the treatment of prostate cancer--a preliminary report. Int J Radiat Oncol Biol Phys 51, 605-613. Terlikowski SJ (2001) Tumour necrosis factor and cancer treatment: a historical review and perspectives. Rocz Akad Med Bialymst 46, 5-18. Tong Y, Yang Q, Vater C, Venkatesh LK, Custeau D, Chittenden T, Chinnadurai G, and Gourdeau H (2001) The pro-apoptotic protein, Bik, exhibits potent antitumor activity that is dependent on its BH3 domain. Mol Cancer Ther 1, 95-102. Van Antwerp DJ, Martin SJ, Kafri T, Green DR, and Verma IM (1996) Suppression of TNF-#-induced apoptosis by NF-!B. Science 274, 787-789. Van Noesel MM, van Bezouw S, Salomons GS, Voute PA, Pieters R, Baylin SB, Herman JG, and Versteeg R (2002) Tumor-specific down-regulation of the tumor necrosis factorrelated apoptosis-inducing ligand decoy receptors DcR1 and DcR2 is associated with dense promoter hypermethylation. Cancer Res 62, 2157-2161. Vieweg J, Boczkowski D, Roberson KM, Edwards DW, Philip M, Philip R, Rudoll T, Smith C, Robertson C, and Gilboa E (1995) Efficient gene transfer with adeno-associated virusbased plasmids complexed to cationic liposomes for gene therapy of human prostate cancer. Cancer Res 55, 23662372. Voeks D, Martiniello-Wilks R, Madden V, Smith K, Bennetts E, Both GW, and Russell PJ (2002) Gene therapy for prostate cancer delivered by ovine adenovirus and mediated by purine nucleoside phosphorylase and fludarabine in mouse models. Gene Ther 9, 759-7 Voelkel-Johnson C, King DL, and Norris JS (2002) Resistance of prostate cancer cells to soluble TNF-related apoptosisinducing ligand (TRAIL/Apo2L) can be overcome by doxorubicin or adenoviral delivery of full-length TRAIL. Cancer Gene Ther 9, 164-172. Wang J, and Waxman J (2001) Chemotherapy for prostate cancer. Clin Oncol 13, 453-454. Wang L, Yorke E, Desobry G, and Chui CS (2002) Dosimetric advantage of using 6 MV over 15 MV photons in conformal


Gene Therapy and Molecular Biology Vol 7, page 133 therapy of lung cancer: Monte Carlo studies in patient geometries. J Appl Clin Med Phys 3, 51-59. Wible BA, Wang L, Kuryshev YA, Basu A, Haldar S, and Brown AM (2002) Increased K+ efflux and apoptosis induced by the potassium channel modulatory protein KChAP/PIAS3" in prostate cancer cells. J Biol Chem 277, 17852-17862. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, Sutherland GR, Smith TD, Rauch C, Smith CA, and et al. (1995) Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3, 673-682. Wu L, Matherly J, Smallwood A, Adams JY, Billick E, Belldegrun A, and Carey M (2001) Chimeric PSA enhancers exhibit augmented activity in prostate cancer gene therapy vectors. Gene Ther 8, 1416-1426. Xess A, Singh M, Raghwendra KH, Sharma HP, and Shahi SK (2001) Prostate specific antigen as tumor marker: relationship with histologic grading. Indian J Pathol Microbiol 44, 261-264. Xie X, Zhao X, Liu Y, Young CY, Tindall DJ, Slawin KM, and Spencer DM (2001a) Robust prostate-specific expression for targeted gene therapy based on the human kallikrein 2 promoter. Hum Gene Ther 12, 549-561. Xie X, Zhao X, Liu Y, Zhang J, Matusik RJ, Slawin KM, and Spencer DM (2001b) Adenovirus-mediated tissue-targeted expression of a caspase-9-based artificial death switch for the treatment of prostate cancer. Cancer Res 61, 6795-6804. Yeung F, and Chung LW (2002) Molecular basis of co-targeting prostate tumor and stroma. J Cell Biochem Suppl Suppl, 65-72. Yoshimura I, Ikegami S, Suzuki S, Tadakuma T, and Hayakawa M (2002) Adenovirus mediated prostate specific enzyme prodrug gene therapy using prostate specific antigen promoter enhanced by the Cre-loxP system. J Urol 168, 2659-2664. Yu D, Chen D, Chiu C, Razmazma B, Chow YH, and Pang S (2001a) Prostate-specific targeting using PSA promoterbased lentiviral vectors. Cancer Gene Ther 8, 628-635. Yu DC, Chen Y, Dilley J, Li Y, Embry M, Zhang H, Nguyen N, Amin P, Oh J, and Henderson DR (2001b) Antitumor synergy of CV787, a prostate cancer-specific adenovirus, and paclitaxel and docetaxel. Cancer Res 61, 517-525. Yu DC, Chen Y, Seng M, Dilley J, and Henderson DR (1999) The addition of adenovirus type 5 region E3 enables calydon virus 787 to eliminate distant prostate tumor xenografts. Cancer Res 59, 4200-4203. Yuan XJ, and Whang YE (2002) PTEN sensitizes prostate cancer cells to death receptor-mediated and drug-induced apoptosis through a FADD-dependent pathway. Oncogene 21, 319327. Zeimet AG, Riha K, Berger J, Widschwendter M, Hermann M, Daxenbichler G, and Marth C (2000) New insights into p53 regulation and gene therapy for cancer. Biochem Pharmacol 60, 1153-1163. Zerbini LF, Wang Y, Cho JY, and Libermann TA (2003) Constitutive Activation of Nuclear Factor !B p50/p65 and Fra-1 and JunD Is Essential for Deregulated Interleukin 6 Expression in Prostate Cancer. Cancer Res 63, 2206-2215. Zhang F, Lu W, and Dong Z (2002a) Tumor-infiltrating macrophages are involved in suppressing growth and metastasis of human prostate cancer cells by INF-" gene therapy in nude mice. Clin Cancer Res 8, 2942-2951. Zhang X, Steiner MS, Rinaldy A, and Lu Y (2001) Apoptosis induction in prostate cancer cells by a novel gene product, pHyde, involves caspase-3. Oncogene 20, 5982-5990. Zhang Y, Yu J, Unni E, Shao TC, Nan B, Snabboon T, Kasper S, Andriani F, Denner L, and Marcelli M (2002b) Monogene

and Polygene Therapy for the Treatment of Experimental Prostate Cancers by Use of Apoptotic Genes bax and bad Driven by the Prostate- Specific Promoter ARR(2)PB. Hum Gene Ther 13, 2051-2064. Zhang Z, Yin L, Zhang Y, and Zhao F (2002c) In situ transduction of cytosine deaminase gene followed by systemic use of 5-fluorocytosine inhibits tumor growth and metastasis in orthotopic prostate cancer mouse models. Chin Med J (Engl) 115, 227-231.

Dr. Salih Sanlioglu


Sanlioglu et al: Adenovirus mediated gene therapy for prostate carcinoma


Gene Therapy and Molecular Biology Vol 7, page 135 Gene Ther Mol Biol Vol 7, 135-151, 2003

Gene therapy for vascular diseases Review Article

Sarah J. George1, Filomena de Nigris2, Andrew H. Baker3, Claudio Napoli4,5 1

Bristol Heart Institute, University of Bristol, Bristol, BS2 8H, UNITED KINGDOM; 2Department of Pharmacological Sciences, University of Salerno, 84084 Italy; 3Division of Cardiovascular and Medical Sciences, University of Glasgow, Western Infirmary, Glasgow G11 6NT, UNITED KINGDOM; 4Departments of Medicine and Clinical Pathology, University of Naples, Naples 80131, Italy; 5Department of Medicine-0682, University of California San Diego, CA92093, USA SJ George and F de Nigris contributed equally to this review.

__________________________________________________________________________________ *Correspondence: Claudio Napoli, MD, PhD, FACA, PO BOX 80131, Naples, Italy, e-mail: Key words: Atherosclerosis, gene therapy, adenoviruses, vascular diseases. Received: 2 July 2003; Accepted: 18 July 2003; electronically published: July 2003

Summary Currently, successful pharmacological treatments are unavailable for many vascular diseases. Many patients undergo surgical interventions and then present with recurrence of symptoms. Recently, gene therapy using both non-viral and viral delivery has emerged as a novel tool to treat patients with vascular diseases. Here we discuss the requirement to develop suitable gene delivery vectors for vascular diseases. Our expanding knowledge of the pathogenesis of vascular diseases has allowed the identification of several gene therapy strategies and many candidate genes. Gene therapy using both gene knockout and gene overexpression has been considered. In preclinical studies, antisense and decoy oligonucleotides have been successfully employed to knockout the expression of stimulatory genes such as cell cycle promoters and growth factors. Furthermore, overexpression of inhibitory genes such as cell cycle inhibitors and nitric oxide and overexpression of genes to promote therapeutic angiogenesis have been shown potential in animal models. The progress of pre-clinical studies to treat vein graft failure, restenosis, myocardial and peripheral ischemia and hypertension and the development of clinical trials will be discussed. Despite the quite promising findings with clinical trials, particularly with therapeutic angiogenesis, improved gene transfer vectors and methods for safe long-term gene transfer are still required to bring gene therapy to clinical practice. adenoviral vectors to a patient on a gene therapy clinical trial for ornithine transcarbamylase (OTC) deficiency as well as the evolution of leukaemia in severe combined immunodeficiency (SCID) patients involving retroviral vectors (Cavazzana-C et al, 2000; Somia et al, 2000; Fox 2003) have highlighted safety issues relating to gene delivery vectors. In vascular diseases, successful gene therapy will require the following: Identification of the optimal transgene cassette. Expression systems vary considerably for different gene therapy applications. Traditionally strong viral promoters have been used to provide maximal levels of expression in a multitude of recipient cell types. However, it is becoming increasing important to supply expression selectively to individual cell types or in a regulated manner through inducible promoters (such as tetracyclin system (Gossen et al, 1992; Vigna et al, 2002) thus circumventing potentially deleterious effects of transgene expression in non-target cell types. Additionally, viral promoters, particularly the cytomegalovirus immediate

I. Introduction Gene therapeutics have been proposed as a potential novel therapy for a host of diverse disease that encompass acquired conditions such as cancer, cardiovascular disease and arthritis as well as monogenic diseases through gene replacement strategies. In theory the concept has seemed relatively simply; in practice, however, gene therapy is extremely complex, both technically and clinically. It requires a multifaceted approach involving identification of suitable therapeutic gene(s), identification of a suitable gene delivery vehicle together with the availability of satisfactory pre-clinical models in which to evaluate the potential benefit of the gene therapeutic approach, particularly against alternative pharmacological therapies, if available. The issue of long-term safety of gene therapy approaches is still unclear. To date, major progress at the clinical level has been made in defined areas, particular cancer, cystic fibrosis, haemophilia and some vascular diseases. These advances have not been without major drawbacks. Tragic events involving high dose delivery of 135

George et al: Gene therapy for vascular diseases early promoter (CMV IEP) is prone to host-mediated silencing in vivo (De Geest et al, 2000) leading to a shut down in transgene expression, an effect not observed with cell-specific promoters. Further optimisation of expression cassettes can be made through incorporation of introns and enhancers to elevate promoter activity as well as posttranscriptional modifications including the Woodchuck post-transcriptional regulatory element (WPRE) which is thought to act through promoting mRNA stability (Loeb et al, 1999; Zufferey et al, 1999). Optimisation and evaluation of the gene delivery vehicle. At present the repertoire of gene delivery vectors available for human gene therapy is limited. Traditionally, non-viral vectors such as naked DNA and liposome DNA complexes provide low efficiency gene transfer and are restricted to the delivery of highly potent biological agents, such as angiogenic gene therapy (see below). Improvements in the efficiency of non-viral vectors, such as inclusion of targeting peptides into DNA liposome complexes (Hart et al, 1997; Parkes et al, 2002) have been realised but are still someway from the efficiency of viral vectors. Certain viruses, by virtue of evolution, infect human cells with high efficiency resulting in high potency gene transfer and overexpression of candidate therapeutic genes. For gene delivery to vascular tissues the current armoury of viral vectors includes adenoviruses (Ad), adeno-associated viruses (AAV), lentiviruses and Semliki forest viruses. Efficient modalities for gene delivery to the target site. Certain vascular diseases, such as vein grafting are optimal for gene therapyapy since the target tissue (i.e. the vein to be grafted) is harvested and is available ex vivo for gene delivery prior to grafting within a clinically relevant time window (approximately 30 minutes). This enables delivery of genes in a safe and efficient manner (Baker et al, 1997; Tamirisa et al, 2002). Due to the short time frame, however, efficient vectors are required. Adenoviral vectors have proven particularly suited for this application (Channon et al, 1997; George et al, 2000; Tamirisa et al, 2002). Conversely, gene delivery to blood vessels in vivo requires the use of devices to allow localised in vivo gene delivery. Specific catheter systems have been developed and utilised with high efficiency for post-angioplasty and in-stent restenosis in a variety of animal species and blood vessels (French et al, 1994; Klugherz et al, 2000, 2002). Additionally, local delivery technology has been applied for gene therapyapy aimed at the myocardium. Different applications, such as atherosclerosis or hypertension require alternate delivery systems and often rely on intravenous vehicle administration. Together, a combined approach to optimise the gene expression system, the delivery vehicle and the route of delivery are required for successful gene therapy. A number of key areas within vascular diseases have successfully exploited this and advanced to clinical trials while other areas have been severely limited due to deficiencies in one or more of the above requirements. Here, we discuss a number of these applications. There is no doubt that gene therapy may offer advantages above traditional pharmacological therapies in certain respects. Delivery of gene can be achieved locally

in the vasculature thereby increasing the selectivity and, potentially, the safety. This would be particularly important when the therapy may have an adverse effect if contact to non-target tissue in vivo occurred. Since many of the strategies that have been designed to be effective in vascular disease may be deleterious if exposed to nontarget tissue, this advantage becomes very important. For example, in development of gene therapy for vein graft failure (see later) pro-apoptotic genes are highly effective but clearly their expression in other tissues such as the liver, may be detrimental. Likewise, in restenosis postangioplasty (cytotoxic or cytostatic strategies) and angiogenesis gene therapy can be delivered locally and is a pre-requisite for clinical translation. A second (and equally important) advantage of gene therapy might be the requirement for only a single administration compared to the requirement for multiple administrations of conventional drugs, often daily for the lifetime of the patient. Again, this depends largely on the application and is to date unproven. Evidence suggests that beneficial effects of gene therapy for hypertension, vein grafting and restenosis can be elicited in the long term from single administrations (see later). This provides ample preclinical evidence to support these concepts. In the following review, we discuss gene therapy for some vascular diseases and its progression in different experimental and clinical applications.

II. Local gene delivery to the vessel wall It has been known for over a decade that gene delivery to the vessel wall can result in alterations in cell behaviour (Nabel et al, 1993 a, b, c) thereby initiating a plethora of studies that have evaluated and optimised gene delivery to the vessel wall. Although the first studies revealed that non-viral gene delivery could lead to phenotypic modulation of cell behaviour, it soon became clear that adenoviral vectors provided the most efficient means to achieve high-level gene delivery to the vessel wall in vivo (Lemarchand et al, 1993; French et al, 1994). Pioneering studies by Lemerchand and colleagues (1993) and French et al, (1994) showed that local exposure of high titre adenoviral vectors to normal and diseased blood vessels in vivo led to high-level transduction, in sheep and rabbit models, respectively. Catheter systems were rapidly developed and optimised for gene delivery postangioplasty resulting in transgene expression throughout the vessel wall in a geographical localisation defined by the mode of vector delivery by the catheter utilised. This initiated a host of studies and led to the use of adenoviral vectors as the most commonly used modality through which to deliver genes to the vessel wall in vivo. However, this is not without limitations since adenoviralmediated gene delivery was found to evoke an inflammatory response in the vessel wall leading to toxicity and endothelial cell activation (Newman et al, 1995). Furthermore, the use of these first-generation Ad vectors only resulted in transient gene expression lasting 7-14 days. Unlike other tissues, second generation vectors (that contained modifications of the Ad genome to reduce 136

Gene Therapy and Molecular Biology Vol 7, page 137 expression of Ad-related genes) did not lead to sustained transgene expression in the vessel wall in vivo (Engelhardt et al, 1994; Wen et al, 2000). Other vector systems have recently been tested including improved non-viral systems such as peptide-targeted DNA/liposome complexes (Hart et al, 1997; Parkes et al, 2002), HVJ-modified liposomes (Morishita et al, 1995; Von Der Leyen et al, 1995; Dzau et al, 1996) and ultrasound-enhanced systems (Lawrie et al, 1999; Taniyama et al, 2002). Likewise, other viral vectors (including adeno-associated viruses (Maeda et al, 1997; Richter et al, 2000), Semliki-forest viruses (Lundstrom et al, 2001) and lentiviruses (Dishart et al, 2003) have been utilised. Modified viral systems in particular provide opportunities to modify the longevity of transgene expression as well as the principle cell type transduced. As an example, adeno-associated viruses (AAV) transduce smooth muscle cells in the vessel wall, even in the presence of an intact endothelial layer (Richter et al, 2000). This is in direct comparison to Ad-mediate delivery since endothelial transduction is high when an intact endothelium is present and represents a barrier to transduction (Lemarchand et al, 1993). This finding may in part be due to different physical sizes of Ad and AAV and due to different vector tropisms of each, which is dictated by host expression of viral receptors and coreceptors (Wickham et al, 1993; Bergelson et al, 1997; Tomko et al, 1997; Summerford et al, 1998; Qing et al, 1999; Summerford et al, 1999; Dishart et al, 2003). Hence, these systems have provided researchers with a diverse

range of vectors through which to evaluate the phenotypic effects of overexpression of candidate therapeutic genes in the vessel wall in vivo.

III. Gene therapy and vein graft failure The failure of vein bypass grafts in the coronary or lower extremity circulation is a common clinical occurrence that incurs significant morbidity and mortality. Despite the very common use of saphenous vein grafts to treat coronary and lower extremity occlusions the failure rate is extremely high, approximately 50% and 70% of vein grafts fail within 5-10 years after surgery, respectively (Angelini 1992; Conte et al, 2001). To date, pharmacological approaches to prolong vein graft patency have produced very limited results. Consequently, genetic approaches to modulate bypass grafts are actively being studied both in vitro and in vivo and are progressing to clinical trials. Vein grafts are uniquely amenable to intraoperative genetic modification because of the ability to manipulate the tissue ex vivo with controlled conditions. We will describe how both gene overexpression and gene blockade strategies have been tested, and how the latter is now in clinical trials (see also Figure 1 for schematic summary of gene therapy strategies).

Restenosis Angioplasty Stent Placement Intimal proliferation Intimal proliferation Constrictive remodelling

Early Failure Thrombosis

Late Failure Intimal proliferation Constrictive remodelling

Gene Therapy Strategies

Gene Therapy Strategies

Gene Therapy Strategies

!Anti-VSMC proliferation: !-Cytostatic: cell cycle

inhibitors, antisense cell cycle genes !& growth factors !-Cytotoxic: tk, p53, !Anti-thrombotic: uPA, tPA, NO !Re-endothelialization:


migration & matrix remodelling: TIMPs

Vein Graft Failure

Gene Therapy Strategies !Anti-VSMC


cell cycle inhibitors, antisense cell cycle genes !& growth factors !Cytotoxic: tk, p53, !Anti-thrombotic: uPA, tPA, NO

!Anti-VSMC proliferation: !-Cytostatic: cell cycle




inhibitors, antisense cell cycle genes, transcription factors (E2F)* & growth factors


none tested !Re-endothelialization: Ctype natriuretic peptide



migration & matrix remodelling: TIMPs

Figure 1: Gene therapy strategies for the treatment of restenosis and vein graft failure. Many preclinical studies have been utilised to determine the potential of these various strategies * indicates those that have progressed to clinical trials.


George et al: Gene therapy for vascular diseases Recently, transfection of cis-element double-stranded oligonucleotides (decoy ODNs) has been reported as a new powerful tool in a new class of anti-gene strategies for gene therapyapy. Transfection of double-stranded ODN corresponding to the cis sequence will result in attenuation of the authentic cis-trans interaction, leading to removal of trans-factors from the endogenous cis-elements with subsequent modulation of gene expression. A decoy to E2F, which induces the coordinated expression of a number of critical cell cycle genes, including PCNA, cyclin-dependent kinase-1, cell division cycle-2 kinase, cmyc, c-myb, was used successfully. This E2F decoy ODN not only almost completely inhibited intimal thickening after balloon injury of the rat carotid at two weeks after injury (Morishita et al, 1995), but sustained inhibition was observed after eight weeks. This inhibition of intimal thickening was also observed using a porcine coronary artery model (Nakamura et al, 2002). Furthermore, a single intraoperative pressure-mediated delivery of E2F decoy effectively provided vein grafts with long-term (up to 6 months) resistance to intimal thickening and atherosclerosis (Ehsan et al, 2001). Interestingly, it has been demonstrated that although E2F decoy ODN treatment of vascular grafts inhibits VSMC proliferation and activation, it spares the endothelium, thereby allowing normal endothelial healing (Ehsan et al, 2002). A clinical trial (PREVENT) using intraoperative delivery of E2F decoy ODN to infrainguinal arterial bypass grafts demonstrated fewer graft occlusions, revisions, or critical stenoses in the E2F-treated group (Mann et al, 1999). Recently, a corporate-sponsored (Corgentech, Inc, Palo Alto, Calif) phase II trial of E2F decoy treatment of coronary vein grafts was completed (SoRelle 2001). This study, which involved 200 patients revealed a 30% reduction in critical stenosis and has formed the basis for design of a phase III trial in coronary bypass grafting. Furthermore, on the basis of this combination of preclinical and phase I/II clinical data, a phase III trial of E2F decoy ODN for the prevention of lower extremity vein graft failure involving 1400 patients was initiated in December 2001.

A. Biological processes involved in restenosis and molecular targets in vein graft failure A complex series of biological events is initiated in the vein immediately after implantation into the arterial circulation. Within the first few days after implantation many vein grafts fail due to thrombosis, stimulated by endothelial injury (Bryan et al, 1994). Furthermore, in the first 24 hours vein grafts undergo a period of ischemia followed by reperfusion, which leads to the generation of superoxide and other reactive oxygen species that triggers cytoxicity of endothelial and smooth muscle cells (Shi et al, 2001; West et al, 2001). The grafted vein is then targeted by an acute inflammatory response involving neutrophil and mononuclear cell recruitment and oxidative stress persists (West et al, 2001). In the first week after implantation matrix remodelling and migration of smooth muscle cells into the intima takes place; once in the intima the smooth muscle cells proliferate contributing further to the intimal thickening (Newby et al, 1996). Each of these processes offers a set of potential molecular targets for gene therapyapy.

B. Anti-thrombotic and accelerated reendothelialization strategies Anti-thrombotic strategies have been investigated as a relevant target for gene transfer to reduce thrombosis in various models of arterial injury and thrombosis formation. Thrombosis is dramatically reduced using natural anti-thrombotic, anti-aggregatory, and fibrinolytic pathways such as overexpression of thrombomodulin (Waugh et al, 1999), tissue factor pathway inhibitor (Nishida et al, 1999; Zoldhelyi et al, 2000), CD39 (Gangadharan et al, 2001) and tissue plasminogen activator (Waugh et al, 1999). Despite their proven success, the potential of these anti-thrombotic strategies has not been widely tested in vein graft models perhaps due to the availability of pharmacological treatments. However, acceleration of re-endothelialization by gene transfer of C-type natriuretic peptide in rabbit jugular vein grafts reduced both thrombosis and intimal thickening (Ohno et al, 2002). This illustrates that promoting reendothelialization and reducing thrombosis is a promising strategy to circumvent vein graft failure.

D. Pro-apoptotic strategy In addition to the above-mentioned cytostatic approaches, cytotoxic strategies have also been considered. Delivery of TIMP-3, which in addition to inhibiting MMP activity and VSMC migration promotes VSMC apoptosis significantly reduced intimal thickening in a porcine vein graft model (George et al, 2000). Adenoviral delivery of wild type p53 which promotes VSMC apoptosis has also been studied in human saphenous vein in vitro studies (George et al, 2001). Induction of VSMC apoptosis by overexpression of p53, without a detectable reduction in VSMC proliferation, led to a significant reduction, >70%, in intimal thickening (George et al, 2001). Studies using a porcine arteriovenous bypass model are currently been underway to determine if this cytostatic strategy reduces intimal thickening in vivo. Despite initial concerns, this proapoptotic strategy with TIMP-3 and p53 did not lead to a

C. Anti-proliferative strategy In an attempt to inhibit VSMC proliferation in vein grafts both overexpression of cell cycle inhibitory proteins and inhibition of cell cycle promontory genes using antisense has been investigated in arterial injury and vein graft models. In fact it is thought that strategies targeting multiple cell cycle genes offer greater potential than single targets. Rabbit vein grafts treated simultaneously with antisense oligonucleotides to proliferating cell nuclear antigen (PCNA) and cell division cycle-2 kinase showed reduced intimal thickening and diet induced atherosclerosis (Mann et al, 1995).


Gene Therapy and Molecular Biology Vol 7, page 139 loss of VSMC density or thinning of the graft wall that may lead to aneurysm (George et al, 2000, 2001).

mechanisms including heat shock protein-70 (Jayakumar et al, 2000), and scavenging enzymes such as catalase (Danel et al, 1998), superoxide dismutase (Li et al, 2001), and heme oxygenase-1 (Yang et al, 1999) have proven efficacy in models of arterial and lung injury and cardiac reperfusion but to date have not been used in vein grafts. Similarly, gene transfer of TIMPs has not been used in cerebral ischemia (Napoli, 2002). Although pre-treating the vein with anti-oxidant gene therapy is an attractive strategy it may be difficult in practice because of the immediate onset of reperfusion after implantation and the time delay before adequate transgene expression. However, antioxidant gene therapy might be advantageous for later stages of graft healing, as oxidative stress is a consequence of inflammation (West et al, 2001). Possible anti-inflammatory strategies include overexpression of nitric oxide synthase (NOS), soluble adhesion molecules and CC-chemokine blockade. By far the most progress has been made with NOS overexpression, probably since it also inhibits thrombosis formation and VSMC proliferation (Cable et al, 1997). Ex vivo gene transfer of endothelial (e)NOS to canine ipsilateral femoral vein grafts (Matsumoto et al, 1998) and inducible (i)NOS to porcine jugular (Kibbe et al, 2001) and intraoperative gene transfer of neuronal (n)NOS to jugular vein grafts in rabbits (West 2001) significantly reduced (30% to 50%) intimal thickening. However, only in the latter study was a reduction in inflammation observed. A current clinical trial (Cardion, Inc, Cambridge, Mass) is examining the effects of liposome-mediated iNOS gene transfer to coronary arteries after angioplasty for the prevention of restensosis but no such trials are currently examining the potential for prevention of vein graft failure. Despite demonstration of the ability to overexpress a soluble form of the vascular adhesion molecule in vein grafts and highlighting the potential for reducing vein graft failure (Chen et al, 1994), its efficacy has not been demonstrated. Furthermore, the ability of overexpression of 35K, a CCchemokine inactivator, to inhibit inflammation has only been demonstrated in the peritoneum of mice (Bursill et al, 2003).

E. Anti-migration/matrix remodelling Cell migration is critical to intimal thickening and requires remodelling of the matrix by proteolytic enzymes such as matrix-degrading metalloproteinases (MMPs) and plasmin. The tissue inhibitors of matrix-degrading metalloproteinases (TIMPs) regulate the proteolytic activity of MMPs whilst the balance of plasminogen activators and plasminogen activator inhibitor-1 (PAI-1) regulate plasmin. Increased MMP activity has been demonstrated both in vitro (George et al, 1997) and in vivo (Southgate et al, 1999) models of vein graft failure. Local overexpression of TIMPs (1, 2 and 3) reduced intimal thickening in a human in vitro model of vein graft failure (George et al, 1998a,b, 2000). Furthermore, ex-vivo delivery of TIMP-3 gene reduced MMP activity and intimal thickening in a porcine vein graft model (George et al, 2000), (Figure 2). Using the recently established mouse model of vein grafting the potential of gene therapy of TIMPs was further illustrated (Hu et al, 2001). Inhibition of plasminogen activators also inhibits intimal thickening in a human in vitro model of vein graft failure (Quax et al, 1997). Intimal thickening after balloon injury of the rat carotid was reduced by 35% at 4 weeks after adenoviral delivery of a hybrid protein which consists of the amino-terminal fragment of urokinase plasminogen activator linked to bovine pancreas trypsin inhibitor, a potent inhibitor of plasmin (Lamfers et al, 2001). Gene transfer of TIMPs has not been used yet in adversing cerebral ischemia (Napoli, 2002).

F. Anti-ischemia/reperfusion, oxidative stress, inflammation Molecular therapies targeted at scavenging the excess of reactive oxygen species generated locally or protecting resident cells from their downstream effects may be useful in the prevention of vein graft failure. Gene therapy using naturally occurring cytoprotective and anti-oxidant

Figure 2: Adenoviral-mediated gene transfer of TIMP-3 reduced intimal thickening in vein grafts. Transverse sections stained for !smooth muscle cell actin illustrate that intimal thickening was dramatically reduced in porcine arterio-venous vein grafts at one month by Ad-mediated over-expression of TIMP-3 compared to controls (AdlacZ). White dotted line indicates the intimal/medial boundary.


George et al: Gene therapy for vascular diseases Simari et al, 1996). Similarly, expression of cytosine deaminase in the presence of 5-fluorocytosine caused a 45% reduction of stenosis (Harrell et al, 1997). Endogenous inducers of cell death have also been utilized. Delivery of the tumour suppressor p53 to injured rat carotid arteries reduced intimal thickening (Yonemitsu et al, 1998), as did gene transfer of FasL (Luo et al, 1999). Some caution has been applied to the use of cytotoxic gene therapy for restenosis, since VSMC viability is essential for the integrity of the lesion, particularly the fibrous cap, and thereby the stability of atherosclerotic plaques. In addition, promotion of apoptosis in injured vessels may increase intimal thickening, since overexpression of fortilin, a recently characterised, negative regulator of apoptosis reduced intimal thickening in injured rat arteries (Tulis et al, 2003). It has been well documented that cytostatic genetic strategies using antisense oligonucleotides (ODN), decoy ODN and gene transfer of cell cycle inhibitory genes (Li et al, 1999) limit VSMC proliferation and inhibit intimal thickening following experimental injury. Despite encouraging results using antisense ODN to immediate early genes such as c-myb (Simons et al, 1992) and c-myc (Shi et al, 1994) and promoters of cell cycle such as cyclin B and CDK-2 (Morishita et al, 1994), where intimal thickening was inhibited between 40 and 84% to in rat and in some cases also porcine injured arteries some years ago, this strategy appears to have made little progress recently. This is despite the observation that co-transfection of combinations of these antisense resulted in further inhibition (Morishita et al, 1994). Transfer of retinoblastoma protein (Rb) to restrict the cell cycle, into rat and porcine injured arteries prevented intimal thickening (Chang et al, 1995). Similarly, overexpression of the CDK inhibitors p21 and p27 resulted in reduction of intimal thickening both in rat and porcine injured arteries (Chang et al, 1995; Yang et al, 1996; Chen et al, 1997). Furthermore, overexpression of a mutated form of p21 was able to reduce restenosis in hypercholesterolemic mice by enhancing vascular apoptosis and reducing VSMC proliferation (Condorelli et al, 2001). A further strategy that has been examined is the inhibition of signalling molecules. H-ras, a key protein in signal transduction, mediates mitogenic signals, therefore blocking this early signal transduction. Application of an adenoviral dominant negative H-ras and G"#-binding peptide affected downstream signalling events and reduced intimal thickening by 70-80% (Ueno et al, 1997; Iaccarino et al, 1999). Targeting of transcription factors by gene therapy is also a strategy of interest. Inhibition of NF$B and E2F, cytoplasmic transcription factor using antisense ODNs in balloon-injured rat carotid arteries reduced intimal thickening by approximately 70% (Autieri et al, 1995; Morishita et al, 1995). Overexpression of the growth arrest homeobox gene (GAX) reduced intimal thickening by 5070% in rat and rabbit injury models (Maillard et al, 1997; Smith et al, 1997). Although the use of transcription factors as targets for gene therapyapy in restenosis appeared promising, it should be noted that these transcription factors are also involved in several mechanisms regulating vascular wall homeostasis.

IV. Gene therapy and restenosis Treatment of symptomatic coronary artery atherosclerotic plaques by angioplasty leads to vascular responses including intimal thickening and constrictive remodelling causing restenosis in approximately 30% of initially successfully treated patients. Although stents prevent constrictive vascular remodelling, they induce vascular injury eventually leading to intimal thickening and thereby restenosis. Gene therapy has been perceived as attractive to treat restenosis as it can be delivered locally and appears to be able to treat excessive vascular cell proliferation. To date, a number of small (rat, mice) or large size animal modes (rabbit, pig) have been used to evaluate the potential of many gene therapy approaches for restenosis. The gene therapy strategies for treatment of restenosis are summarized below and also in Figure 1. However, despite the successful use of gene therapy to treat animal restenosis by various approaches, application of gene therapy to prevent restenosis in man has only been carried out using a re-endothelialization strategy with VEGF. Before further clinical trials are initiated a better understanding of vascular biology, gene expression, vector design, and catheter-tissue interactions is required. It must also be mentioned that the efficacy of sirolimus (rapamycin) for the treatment of in-stent restenosis (Serruys et al, 2002; Sousa et al, 2003) has reduced the impetus for designing gene therapy for in-stent restenosis.

A. Biological processes involved in restenosis and molecular targets in restenosis The two major components that lead to restenosis are intimal thickening and negative (constrictive) remodelling. Intimal thickening following experimental injury involves a combination of many processes, including VSMC and adventitial cell migration, proliferation, and matrix deposition. Negative remodelling, which only occurs after angioplasty and not after stent placement may also arise from many processes, including VSMC apoptosis, medial and adventitial fibrosis and matrix remodelling. However, restenosis, both in the absence and in the presence of stents, is primarily due to VSMC accumulation. Since mural thrombi may aggravate restenosis by contributing directly to cell proliferation, anti-thrombotic strategies have received attention. Finally, strategies that accelerate re-endothelialization of the injury artery have been investigated.

B. Inhibition of VSMC proliferation Cytotoxic strategies have been tested based on the expression of enzymes capable of converting nucleoside analogues into toxic metabolites that impair DNA replication and consequently cause death of transduced cells entering S phase. Adenoviral delivery of thymidine kinase (tk), a gene from herpes simplex virus (HSV), followed by ganciclovir treatment led to death of tkexpressing cells and reduced intimal thickening after injury of rat and rabbit arteries (Guzman et al, 1994; 140

Gene Therapy and Molecular Biology Vol 7, page 141 Control of VSMC proliferation has also been attempted by inhibition of growth factor expression and overexpression of inhibitory growth factors and cytokines. Delivery of basic fibroblast growth factor (bFGF) (Hanna et al, 1997) as well as platelet-derived growth factor-" (PDGF-") (Deguchi et al, 1999) antisense ODN and TGF" ribozyme ODN (Yamamoto et al, 2000) inhibited intimal thickening by 60-90% in injured rat carotid arteries. Similarly, adenoviral delivery of the extracellular region of the PDGF-" receptor and of endovascular PDGF-" receptor antisense ODN reduced intimal thickening in injured rat arteries (Sirois et al, 1997; Noiseux et al, 2000). Activin, a TGF-"-like factor that induces a contractile phenotype in VSMCs, reduced intimal thickening by more that 70% in injured mouse femoral arteries (Engelse et al, 2002). The inhibitory cytokine interferon-g delivery by Ad-mediated gene therapy reduced intimal thickening in a porcine model of arterial injury (Stephan et al, 1997).

porcine injured arteries (Shears et al, 1998), illustrating that the degree of response differs greatly between different animal models. Furthermore, administration of the iNOS Ad could not mediate regression of established intimal thickening.

D. Re-endothelialization As regeneration of the endothelium is associated with reduction in thrombotic and proliferative processes in the vessel wall it has been seen as a potential strategy of gene therapy for restenosis. Local intravascular and extravascular expression of vascular endothelial growth factor (VEGF), a potent endothelium specific angiogenic factor, using plasmid DNA accelerated reendothelialization and decreased intimal thickening after arterial injury in rabbit models (Asahara et al, 1996; Laitinen et al, 2000), and reduced in-stent restenosis by 50% (Van Belle et al, 1997). The feasibility of this approach was tested in a small clinical trial, in which VEGF plasmid/liposome gene transfer after angioplasty was seen to be safe and well tolerated (Laitinen et al, 2000). A recently published larger clinical trial was designed to test the feasibility, tolerability and efficacy of VEGF gene therapy to prevent restenosis after stenting (Hedman et al, 2003). The overall restenosis rate in this study was surprisingly low (6%), virtually precluding the detection of a difference among treatments. Nevertheless, the results establish feasibility and provide safety data on the used of naked DNA and Ad to express VEGF. This strategy is perceived attractive as it is trying to mimic nature’s inhibitory strategy to limit intimal thickening, but we await clinical evidence of its success. The use of VEGF is also attractive as it should be endothelial cell specific; however, there are safety concerns in respect to tumour growth as VEGF is involved in induction and progression (Huang et al, 2003).

C. Cell migration and matrix remodelling Constrictive (negative) remodelling plays a very important in human restenosis particularly in the absence of a stent (Mintz et al, 1996), therefore gene therapy strategies aimed at reducing intimal thickening alone are unlikely to be successful in humans following angioplasty. Post injury intimal thickening is also reliant on VSMC migration, which requires remodelling of the extracellular matrix that surrounds the VSMC. Adenoviral gene transfer of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 reduced intimal thickening (Cheng et al, 1998; Furman et al, 2002). A combination of anti-proliferative and anti-migratory approaches may therefore be useful.

D. Anti-thrombotic strategy A number of studies have focused on seeding stents with genetically modified endothelial cells with increased fibrinolytic of anticoagulant activity (Dichek et al, 1989, 1996; Dunn et al, 1996). Although seeding stented vessels with endothelial cells overexpressing tPA and uPA produced anti-thrombotic activity (Dichek et al, 1996), overexpression of tPA was associated with increased detachment of seeded cells (Dunn et al, 1996). Another strategy to prevent thrombosis as well as intimal thickening is to inhibit platelet activation or aggregation or to increase nitric oxide (NO). NO is vasoprotective by inhibiting platelet and leukocyte adhesion, inhibiting VSMC proliferation and migration and promoting endothelial cell survival and proliferation (Li et al, 1999); therefore, nitric oxide synthase (NOS) that increases NO production was proposed as a suitable candidate to treat restenosis. Delivery of endothelial (e)NOS by non-viral methods (von der Leyen et al, 1995) and adenoviruses (Chen et al, 1998; Janssen et al, 1998; Varenne et al, 1998) reduced intimal thickening by 3770% in rat and pig injured arteries. Interestingly, adenoviral delivery of inducible (i)NOS by adenoviruses to rat injured arteries almost completely (95%) inhibited intimal thickening, whilst reduced it by only 50% in

V. Gene therapy for hypertension Gene therapy for essential hypertension represents is an enormous challenge due to the complex polygenic trait that underlies human essential hypertension. Gene therapy is however attractive since it offers the opportunity to treat the disease with a single administration rather than daily drug regimens. Essential hypertension is associated with endothelial dysfunction and contributes significantly to cardiovascular risk. Gene therapy would, therefore, target specific systems with the explicit aim of lowering blood pressure and reducing end organ damage. Unlike other disease targets discussed above, gene therapy for hypertension requires the use of strategies to provide longterm effects on blood pressure. These have included antisense/ribozyme strategies to block systems that regulate blood pressure as well as vasodilator strategies using overexpression of pro-vasodilator genes. Preclinical studies on gene therapy for hypertension have taken two main approaches (Phillips, 2002). First, extensive studies on gene transfer to increase vasodilator proteins (kallikrein, atrial natriuretic peptide, adrenomedullin, and endothelin nitric oxide synthase) 141

George et al: Gene therapy for vascular diseases have been carried out in different rat models (Lin et al, 1995; Chao et al, 1996, 1997; Lin et al, 1997; Chao et al, 1998 a, b; Yayama et al, 1998; Alexander et al, 1999; Dobrzynski et al, 1999; Lin et al, 1999; Alexander et al, 2000; Dobrzynski et al, 2000; Wolf et al, 2000; Zhang et al, 2000; Wang et al, 2001; Emanueli et al, 2002). Using these approaches, blood pressure can be lowered for 3-12 weeks with the expression of these genes. Second, an antisense approach, which began by targeting angiotensinogen and the angiotensin type 1 (AT1) receptor, has now been tested independently by several different groups in multiple models of hypertension (Katovich et al, 1999; Tang et al, 1999; Wang et al, 2000; Kimura et al, 2001). Other genes targeted include the "1adrenoreceptor, TRH, angiotensin gene activating elements, carboxypeptidase Y, c-fos, and CYP4A1 (Gardon et al, 2000; Phillips, 2001; Tomita et al, 2002). There have been two methods of delivery antisense, short ODNs, and full-length DNA in viral vectors. All the studies show a decrease in blood pressure lasting several days to weeks or months. ODNs are safe and particular non-toxic. The decreased hypertension after systemic adeno-associated virus delivery antisense to AT1 receptors in adult rodents for up to 6 months, may constitute a good incentive for testing the antisense ODNs first and later the AAV (Kimura et al, 2001; Phillips 2001). Hypertension is also the presenting feature of some of these disorders, such as congenital adrenal diseases, and adrenal and pituitary tumors. Preclinical data indicate that gene transfer to both the adrenal gland and the pituitary is not only feasible but also quite efficient (Alesci et al, 2002).

highlighted the benefit of viral delivery of antisense (Wang C et al, 1995; Martens et al, 1998; Reaves et al, 1999; Tang et al, 1999; Wang H et al, 1999).

B. Vasodilator overexpression There are a number of candidate genes for overexpression that may provide therapeutic benefit of different aspects of hypertension. These include kallikrein, adrenomedullin, nitric oxide synthase and superoxide dismutase. Kallikrein cleaves kininogen producing kinin peptide, which in turn stimulates the release of the vasodilators prostacyclin, endothelium-derived hyperpolarising factor and nitric oxide. Based on this principle, infusion of naked DNA expressing kallikrein reduced blood pressure for 6 weeks (Wang et al, 1995). Comparative studies showed that naked DNA plasmids and adenoviral vectors both proved effective (Chao et al, 1997). Kallikrein delivery using viruses has also been established as an anti-hypertensive strategy in different models demonstrating the potential benefit of this strategy and the potency of the transgene (Dobrzynski et al, 1999; Wolf et al, 2000). Adrenomedullin also causes vasodilation. Adenoviral-mediated overexpression of adrenomedullin in hypertensive rats led to a blood pressure drop of 41 mm Hg 9 days after tail vein injection (Dobrzynski et al, 2000). This lasted nearly 20 days. Again, proof of this strategy was realised when other studies gained similar findings in different labs and models of hypertension (Zhang et al, 2000; Wang et al, 2001). Targeting endothelial dysfunction is highly attractive for gene therapyapy. Endothelial dysfunction is characterised by reduced nitric oxide (NO)-mediated vasodilation and a reduction in available NO. The loss of NO leads to deleterious effects on platelet aggregation and adhesion, smooth muscle proliferation, inflammation and increased oxidative stress in the vessel wall. Improving the bioavailability of NO, therefore, is a highly logical strategy to improve a number of key processes that are integral to vessel wall homeostasis in order to reduce blood pressure. This can be achieved by increasing NO production itself through nitric oxide synthase (NOS) gene delivery or by preventing NO degradation by superoxide dismutase (SOD) gene transfer. A number of studies have addressed these issues. An early study established such a concept by systemic delivery of naked DNA encoding the endothelial form of NOS (eNOS) with a significant reduction in blood pressure that lasted for at least 12 weeks (Lin et al, 1997). Again, such effects with naked DNA are astonishing since little uptake was achieved in vivo and the majority was sequestered to the liver. It is important to note that targeting gene delivery to the endothelium is extremely difficult using currently available vector systems when the delivery mode is intravenously. The liver sequesters the vast majority of all commonly used vector systems with relatively little uptake by the endothelium itself. This has restricted studies to local applications of gene delivery to selected blood vessels in vivo. Adenoviral delivery of eNOS or SOD3,

A. Inhibition of vasoconstrictor genes This has been achieved using antisense oligonucleotides to block the renin-angiotensin system. For example, Wielbo et al (1996) used DNA/liposomes complexes containing angiotensinogen antisense and lowered mean arterial pressure, angiotensinogen and angiotensin II levels in adult spontaneously hypertensive rats following systemic administration. These highly effective results are somewhat surprising when it is realised that the in vivo uptake of DNA/liposome complexes into the vasculature and organs is very poor when delivery intravenously. Not surprisingly viral vector systems have also been engineered to deliver antisense. Using a retroviral system to deliver antisense against the angiotensin type-1 receptor to young (5 day old) hypertensive and normotensive animals, blood pressure was significantly lowered selectively in the hypertensive animals (Lu et al, 1996). Interestingly, the effect of the antisense was sustained for 90 days while losartan had the expected transient effect of less than 24 hours. This does highlight the clinical relevance of such technology to provide sustained benefit compared to traditional pharmacological regimens. However, in the light of recent clinical experience using retroviral vectors with development of leukaemia on phase I trial (Cavazzana et al, 2000), the use of retroviral vectors is unlikely to be developed in this disease. Other studies have also 142

Gene Therapy and Molecular Biology Vol 7, page 143 but not SOD-1 or –2 are able to improve endothelial function in carotid arteries in the spontaneously hypertensive stroke-prone (SHRSP) rats (Alexander et al, 1999, 2000; Fennell et al, 2002).

are not suitable candidates for surgical endovascular approaches may be amenable to gene therapy for therapeutic angiogenesis. Diabetes impairs endogenous neovascularization of ischaemic tissues due to a reduced expression of VEGF (Rivard et al, 1999) and HGF (Taniyama et al, 2001). Consequently Ad-mediated overexpression of VEGF and plasmid HGF restored neovascularization in mouse and rat models of diabetes, respectively (Rivard et al, 1999; Taniyama et al, 2001). Enhanced angiogenesis by such strategies also improves neuropathy both when growth factors including VEGF, are given alone (Rissanen et al, 2001) or in conjunction with the prostacyclin synthase gene (Koike et al, 2003). Furthermore, a small clinical trial which included 6 diabetic patients with critical leg ischaemia, observed neurologic improvement and therapeutic angiogenesis after plasmid injections of VEGF165 in the muscles of the ischaemic limb (Simovic et al, 2001). Inhibition of angiogenesis may also have therapeutic potential for the treatment of retinopathy, since lentiviral delivery of angiostatin inhibited neovascularization in a murine proliferative retinopathy model (Igarashi et al, 2003). Although, this strategy has made great progress in the last decade there are still some unresolved issues. For example is administration of a single angiogenic molecule sufficient? Will administration of VEGF lead to toxic effects such as oedema? Will an angiogenic factor be suitable for myocardial and peripheral angiogenesis? Since the same adenoviral VEGF121 gave positive effects in the myocardium (Stewart et al, 2002) but failed in peripheral vascular disease (Rajagopalan et al, 2003), will VEGF be proven clinically benefial? Some caution has been cast on the potential of VEGF gene therapy by the observation that VEGF enhances atherosclerotic plaque progression in both mice and rabbits (Celletti et al, 2001). Are other VEGF homologues safer options? Increased lymphogenesis and reduced oedema is observed with VEGFC and VEGFD (Yla-Herttuala et al, 2003).

VI. Therapeutic angiogenesis Therapeutic angiogenesis represents a novel strategy for the treatment of vascular insufficiency. It is based on supplementation with angiogenic growth factors to enhance native angiogenesis in critical myocardial or peripheral ischaemia. Angiogenic growth factors have been delivered both as protein and by way of gene transfer and have demonstrated positive results (Yla-Herttuala et al, 2003). The recent insights in the molecular basis of angiogenesis have resulted in great interest in the gene therapy field. However, because of the rapid evolution and enthusiasm in the field, angiogenic molecules have been tested without a complete understanding of their mechanism of action. Among the angiogenic growth factors used in pre-clinical studies, VEGF165 and VEGF121, FGF1, FGF2 and hepatocyte growth factor (HGF) have all shown significant improvement of native angiogenic response to ischemia, resulting in accelerated rate of perfusion, (see reviews by Hammond et al, 2001) (Emanueli et al, 2001; Manninen et al, 2002). Besides growth factors a number of other substances have been investigated, such as human tissue kallikrein (Emanueli et al, 2001), angiopoietin (Shyu et al, 1998), leptin (Bouloumie et al, 1998) and thrombopoietin (Brizi et al, 1999). Although difficulties have been encountered in the field of gene therapy, great progress has been made in the field of pro-angiogenic gene therapy. It has been suggested that this is because the long-term gene expression is not required for therapeutic vascular growth and the current gene therapy vectors induce at least some physiological improvement (Yla-Herttuala et al, 2003). Over 23 clinical trials have been initiated; approximately half are for peripheral disease and the other half for coronary heart disease. The first set of clinical trials involved pioneering attempts to overexpress VEGF165 with naked DNA (Isner et al, 1996; Baumgartner et al, 1998; Losordo et al, 1998) and adenoviruses (Rosengart et al, 1999). The second phase of trials were small, uncontrolled trials using naked DNA and adenoviruses to overexpress VEGF165 and VEGF121; many of these had positive results (Symes et al, 1999; Laitinen et al, 2000; Rajagopalan et al, 2001). Only recently, the third set of clinical trials has begun to test the potential of this gene therapy fully. These randomised, controlled and blinded trials have involved larger numbers of patients and defined primary and secondary endpoints (Grines et al, 2002; Makinen et al, 2002; Stewart et al, 2002; Hedman et al, 2003; Rajagopalan et al, 2003). Several of these have been judged positive according to primary and secondary endpoints but it has been suggested that this may not be transferable to a clear-cut clinical benefit (Yla-Herttuala et al, 2003). Critically ischaemic lower limbs from diabetes that

VII. Future directions Recent advances through preclinical studies have raised the profile of gene therapy in some vascular diseases, particularly with respect to angiogenic gene therapy in the myocardium and peripheral vasculature as well as in vein graft disease. These studies, presently in phase II, highlight the potential of the technology for relieving symptoms of human vascular diseases. Despite the lack of dramatic cures, a decade of clinical trials has provided important news about the strengths and weaknesses of current vectors. Both adenoviruses and liposomal vectors have been shown to be able to transduce transgenes in patients with a variety of disorders. From this work, it is now extremely clear that the expression is temporary and is associated with an inflammatory response. However, there are some important points to consider. First, with respect to myocardial and peripheral vascular gene transfer clinical trials, these have been performed with single proangiogenic genes with gene delivery using sub-optimal vector systems (e.g. naked DNA/adenoviral vectors). With 143

George et al: Gene therapy for vascular diseases respect to the former, angiogenic gene therapy may be significantly more therapeutic with respect to collateral vessel formation with a combination of therapeutic genes rather than single gene therapy strategies. With recent advances in adenoviral vector technology [e.g. using "gutted" adenoviral vectors (Kochanek et al, 1996; Parks et al, 1996)] the cloning capacity required for such studies is now available. Equally, the gutted adenoviral vector systems are less immunogenic in vivo and would allow longer term overexpression of transgenes that in turn may promote sustained angiogenic effects. It is known that vascular cell uptake by these vectors (all based on serotype 5 adenoviruses) is extremely poor in comparison to other cells, such as hepatocytes in the liver (Nicklin et al, 2001). Indeed, pre-clinical experiments have shown that local delivery of adenoviruses serotype 5 vectors to the vasculature leads to virion dissemination, not only to the liver but also to testes and other organs posing additional safety concerns (Hiltunen et al, 2000; Baker, 2002). Given the limited ability of liposomes and adenoviruses to enable long-term gene expression, and given the poor in vivo performance of retroviruses, the AAV vectors are being developed. This virus is smaller than the adenovirus and has a relatively low-capacity size. However, it allows for long-term gene expression (ie, months to years) with only minimal induction of inflammation or antiviral immune responses. A better understanding of the life cycle of this virus, along with improved production techniques, has allowed investigators to conduct clinical trials with AAV in diseases such as hemophilia and cystic fibrosis (see uk/wileychi/genmed/clinical/). Preclinical data in mice injected intramuscularly with an AAV-human alpha-1antitrypsin (1AT) vector are encouraging (Xiao et al, 1998; Phillips et al, 2002). To date, the major problem in gene therapy remains the relative inefficiency of current vectors. Currently, this inefficiency, coupled with a relatively poor specificity of most vectors, requires the delivery of large doses of vector. This is both expensive and more likely to lead to side effects. Pathophysiological questions still remain about which and how many cells need to be transduced to obtain a clinical response. One new and very exciting area of gene therapy that has not yet reached clinical trials is the "gene correction" (Gamper et al, 2000; Metz et al, 2002). It is possible to design oligonucleotides that bind to areas of single-nucleotide changes that are associated with abnormal functions and to catalyze corrections of the nucleotide errors. This concept clearly has been demonstrated to work in cell cultures and in animal models, although the efficiency is still quite low. With the development of better oligonucleotides and improved delivery methods, this approach will likely be tested first in diseases such as hemophilia and 1AT. When it is considered that angiogenic gene therapy should be highly localised due to potential side effects [including potentiation of atherosclerosis (Celletti et al, 2001) and development of cancer (Lee et al, 2000)] other vector systems should now be considered. The choice of potential new vectors is broad and must be considered

with caution and evaluated based on current knowledge of existing systems (de Nigris et al, 2003). Additional evidence now suggests that the vast majority of AAV genomes remain in a non-integrative capacity within infected cells (Nakai et al, 2001; Schnepp et al, 2003) further supporting the safety of this vector system. Of equal potential are adenoviral vectors originating from different serotypes. Previous pre-clinical data support of the notion that novel vector systems can be isolated for the capacity to efficiently infect an individual tissue type (Havenga et al, 2001, 2002). For example, adenoviruses based on serotype 16 have a high propensity to transduce both endothelial cells and smooth muscle cells than serotype 5 vectors (Havenga et al, 2001). Again, like AAV-2, this may provide a system through which to optimise gene delivery for defined gene therapeutic applications. The use of cell selective promoters (tissuespecific expression) to drive transgene expression will add a further level of selectivity to such systems. The combined use of vectors and immuno-suppressors may be also reasonable. Gene therapy remains the key link between advances in genetics and genomics and the translation of this knowledge into useful outcomes for patients. Although progress has been slower than hoped for, clear advances are being made; gene therapy will probably find a number of key therapeutic niches. Together, these modifications will enhance the utility and safety of gene therapy as transition from pre-clinical to clinical gene therapy proceeds for the vascular system and its diseases.

References Alesci S Chrousos GP, Pacak K (2002) Genomic medicine: exploring the basis of a new approach to endocrine hypertension. Annals of the New York Academy of Sciences 970, 177-92 Alexander M.Y, Brosnan M, Hamilton C ( 2000) Gene transfer of endothelial nitric oxide synthase but not Cu/Zn superoxide dismutase restores nitric oxide availability in the SHRSP. Cardiovasc Res 47, 609-617. Alexander M.Y, Brosnan M, Hamilton CA, Downie P, Devlin A, Dowell F, Martin W, Prentice H, O'Brien T, Dominiczak, A.F (1999) Gene transfer of endothelial nitric oxide synthase improves nitric oxide-dependent endothelial function in a hypertensive rat model. Cardiovasc Res 43, 798-807. Angelini G (1992) Saphenous vein graft failure: etiologic considerations and strategies for prevention. Current Opinion in Cardiology 7, 939-944. Asahara T, Chen D, Tsurumi Y, Kearney, M, Rossow, S, Passeri J, Symes J.F, Isner J (1996) Accelerated restitution of endothelial integrity and endothelium-dependent function after phVDGF165 transfer. Circulation 94, 3291-3302. Autieri M.V, Yue T, Ferstein G.Z, Ohlstein E (1995) Antisense oligonucleotides to the p65 subunit of NF-kB inhibit human vascular smooth muscle cell adherence and proliferation and prevent neointima formation in rat carotid arteries. Biochem Biophys Res Commun 213, 827-836. Baker A, Mehta D, George S, Angelini G (1997) Prevention of vein graft failure: potential applications for gene therapyapy. Cardiovasc Res 35, 442-450.


Gene Therapy and Molecular Biology Vol 7, page 145 Baker A (2002) Development and use of gene transfer for treatment of cardiovascular disease. J Card Surg. 17, 543548. Baumgartner I, Pieczek, A, Manor O, Blair R, Kearney, M, Walsh, K, Isner J (1998) Constitutive expression of phVEGF (165) after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 97, 1114-1123. Bergelson J, Cunningham, JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong J, Horwitz, M, Crowell R, Finberg R (1997) Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science 275, 1320-1323. Bouloumie A, Drexler H, Lafontan M, Busse R (1998) Leptin the product of Ob gene promotes angiogenesis. Circ Res 83, 1059-1066. Brizi M, Battaglia E, Montrucchio G, Dentelli P, Del Sorbo L, Garbarino G, Pegoraro L, Camussi G (1999) Thrombopoietin stimulates endothelial cell motility and neoangiogenesis by a platelet-activating factor-dependent mechanism. Circ Res 84, 785-796. Bryan A, Angelini G (1994) The biology of saphenous vein graft occlusion: etiology and strategies for prevention. Current Opinion in Cardiology 9, 641-649. Bursill C, Cai S, Channon K, Greaves D (2003) Adenoviralmediated delivery of a viral chemokine binding protein blocks CC-chemokine activity in vitro and in vivo. Immunobiology 207, 187-196. Cable D, O'Brien T, Schaff H, Pompili V (1997) Recombinant veins: Gene Ther to augment nitric oxide production in bypass conduits. Circulation 96 (suppl 9), II173-II178. Cavazzana-C M, Hacein-Bey, S, Saint-Basile C, Gross F, Yvon E, Nusbaum, P, Selz, F, Hue C, Certain S, Casanova J.-L, Bousso P, Deist F, Fischer A (2000) Gene Therapy of human severe combined immunodeficiency (scid)-1x Disease. Science 288, 669 - 672. Celletti F, Waugh, J, Amabile P, Brendolan A, Hilfiker P, Dake M (2001) Vascular endothelial growth factor enhances atherosclerotic plaque progression. Nat Med 7, 425-429. Chang M, Barr E, Lu M, Barton K, Leiden J (1995) Adenovirusmediated over-expression of the cyclin cyclin-dependent kinase inhibitor p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest 96, 2260-2268. Chang M, Barr E, Seltzer J, Jiang Y, Nabel G, Nabel E, Parmacek, M, Leiden J (1995) Cytostatic gene-therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene- product. Science 267 (5197), 518-522. Channon K, Fulton G, Gray, J, Annex, B, Shetty, GA, Blazing MA, Peters K, Hagen P.O, George S.E (1997) Efficient adenoviral gene transfer to early venous bypass grafts: comparison with native vessels. Cardiovasc Res 35, 505513. Chao J, Chao L (1997) Experimental kallikrein Gene Therapy in hypertension cardiovascular and renal diseases. Pharmacological Research 35, 517-522. Chao J, Jin L, Chen L, Chen V Chao L (1996) Systemic and portal vein delivery of human kallikrein gene reduces blood pressure in hypertensive rats. Human Gene Ther 7, 901911. Chao J, Yang Z.R, Jin L, Lin K.F, Chao L (1997) Kallikrein Gene Therapy in newborn and adult hypertensive rats. Canadian Journal of Physiology and Pharmacology 75, 750-756.

Chao J, Zhang J, Lin K.F, Chao L (1998a) Human kallikrein gene delivery attenuates hypertension cardiac hypertrophy, and renal injury in Dahl salt-sensitive rats. Human Gene Ther 9, 21-31. Chao J, Zhang J, Lin K.F, Chao L (1998b) Adenovirus-mediated kallikrein gene delivery reverses salt-induced renal injury in Dahl salt-sensitive rats. Kidney International 54, 12501260. Chen D, Krasinski K, Sylvester A, Chen J, Nisen P, Andres V (1997) Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27 (KIP1), an inhibitor of neointima formation in the rat carotid artery. J Clin Invest 99 (10), 2334-2341. Chen L, Daum, G, Forough, R, Clowes M, Walter U, Clowes A (1998) Overexpression of human endothelial nitric oxide synthase in rat vascular smooth muscle cells and in ballooninjured carotid artery. Circ Res 82, 862-870. Chen S, Wilson J, Muller D (1994) Adenovirus-meidated gene transfer of soluble vascular cell adhesion molecule to porcine interposition vein grafts. Circulation 89, 1922-1928. Cheng L, Mantile G, Pauly, R, Nater C, Felici A, Monticone R, Bilato C, Gluzband YA, Crow, M Stetler-Stevenson W, Capogrossi M.C (1998) Adenovirus-mediated gene transfer of the human tissue inhibitor of metalloproteinase-2 blocks vascular smooth muscle cell invasiveness in vitro and modulates neointimal development in vivo. Circulation 98, 2195-2201. Condorelli GL, Aycock J, Frati G, Napoli C (2001) Mutated p21/WAF/CIP transgene overexpression reduces smooth muscle cell proliferation, macrophage deposition, oxidationsensitive mechanisms and restenosis in hypercholesterolemic apolipoprotein-E knockout mice. FASEB Journal 15, 21622170. Conte M, Belkin M, GR U, Mannick, J, Whittemore A, Donaldson M (2001) Impact of increasing comorbidity on infrainguinal reconstructions: a 20-year perspective. Ann Surg 233, 445-452. Danel C, Erzurum, S, Prayssac P, Eissa N, Crystal R, Herve P, Baudet B, Mazmanian M, Lemarchand P (1998) Gene therapy for oxidant injury-related diseases: adenovirusmediated transfer of superoxide dismutase and catalase cDNAs protects against hyperoxia but not against ischemiareperfusion lung injury. Hum Gene Ther 9 (10), 1487-1496. De Geest B, Van Linthout S, Lox, M, Collen D, Holvoet P (2000) Sustained expression of human apolipoprotein A-I after adenoviral gene transfer in C57BL/6 mice: role of apolipoprotein A-I promoter apolipoprotein A-I introns and human apolipoprotein E enhancer. Hum Gene Ther 11, 10112. Deguchi J, Namba T, Hamada H, Nakaoka T, Abe J, Sato O, Miyata T, Makuuchi M, Kurokawa K, Takuwa Y (1999) Targeting endogenous platelet-derived growth factor B-chain by adenovirus-mediated gene transfer potently inhibits in vivo smooth muscle proliferation after arterial injury. Gene Ther 6, 956-965. de Nigris F, Sica V, Herrmann J, Condorelli G, Chade A, Tajana G, Lerman A, Lerman LO, Napoli C (2003) c-Myc oncoprotein: cell cycle-related events and new therapeutic challenges in cancer and cardiovascular diseases. Cell Cycle 2, 325-328. Dichek DA, Neville R.F, Zwiebel JA, Freeman S, Leon M.B, Anderson W.F (1989) Seeding of intravascular stents with genetically engineered endothelial cells. Circulation 80, 1347-1353.


George et al: Gene therapy for vascular diseases Dichek DA, Anderson J, Kelly, A.B, Hanson S, Harker L (1996) Enhanced in vivo antithrombotic effects of endothelial cells expressing recombinant plasminogen activators transduced with retroviral vectors. Circulation 93, 301-309. Dishart K, Denby, L, George S, Nicklin S, Yendluri S, Tuerk, M, Kelley, M, Donahue B, Newby, A, Harding T, Baker A (2003) Third-generation lentivirus vectors efficiently transduce and phenotypically modify vascular cells: implications for gene therapyapy. J Mol Cell Cardiol 35, 739-748. Dobrzynski E, Yoshida H, Chao J (1999) Adenovirus-mediated kallikrein gene delivery attenuates hypertension and protects against renal injury in deoxycorticosterone-salt rats. Immunopharmacology 44, 57-65. Dobrzynski E, Wang C, Chao J (2000) Adrenomedullin gene delivery attenuates hypertension cardiac remodeling and renal injury in deoxycorticosterone acetate-salt hypertensive rats. Hypertension 36, 995-1001. Dunn P, Newman K.D, Jones M, Yamada I, Shayani V, Virmani R, Dicheck, D (1996) Seeding of vascular grafts with genetically modified endothelial cells. Secretion of recombinant TPA results in decrease seeded cell retention in vitro and in vivo. Circulation 93, 1439-1446. Dzau V, Mann M, Morishita R, Kaneda Y (1996) Fusigenic viral liposome for gene therapy in cardiovascular diseases. Proc Natl Acad Sci USA 93, 11421-11425. Ehsan A, Mann M, Dell'Acqua G, Dzau V (2001) Long-term stabilization of vein graft wall architecture and prolonged resistance to experimental atherosclerosis after E2F decoy oligoneucleotide Gene Ther. J Thorac Cardiovasc Surg 121, 714-722. Ehsan A, Mann M, Dell'Acqua G, Tamura K, Braun-Dullaeus R, Dzau V (2002) Endothelial healing in vein grafts. Proliferative burst unimpaired by genetic therapy of neointimal disease. Circulation 105 (1686-1692). Emanueli C, Madeddu P (2001) Angiogenesis Gene Ther to rescue ischaemic tissues: achievements and furture directions. Br J Pharm 133, 951-958. Emanueli C, Salis M, Pinna A, Stacca T, Milia A, Spano A, Chao J, Chao L, Sciola L, Madeddu P (2002) Prevention of diabetes-induced microangiopathy by human tissue kallikrein gene transfer. Circulation 106, 993 - 999. Engelhardt J.F, Litzky, L, Wilson J (1994) Prolonged transgene expression in cotton rat lung with recombinant adenoviruses defective in E2a. Human Gene Ther 5, 1217-1229. Engelse MA, Lardenoye JP, Neele J, Grimbergen J, de Vries M.R, Lamfers M, Pannekoek, H, Quax, P, deVries C M (2002) Adenoviral activin A expression prevents intimal hyperplasia in human and murine blood vessels by maintaining the contractile smooth muscle cell phenotype. Circ Res 90, 1128-1134. Fennell J.P, Brosnan M, Frater A, Hamilton CA, Alexander M.Y, Nicklin SA, Heistad D.D, BAker A, Dominiczak, A.F (2002) Adenovirus-mediated overexpression of extracellular superoxide dismutase improves endothelial dysfunction in a rat model of hypertension. Gene Ther 9, 110-117. Fox, J (2003) US authorities uphold suspension of SCID Gene Ther. Nat Biotechnol 21, 217-217. French, B, Mazur W, Ali N, Geske R, finnigan J, Rodgers G, Roberts R, Raizner A (1994a) Percutaneous transluminal in vivo gene transfer by recombinant adenovirus in normal porcine coronary arteries atherosclerotic arteries and two models of coronary restenosis. Circulation 90, 2402-2413.

French, BA, Mazur W, Geske R, Bolli R (1994b) Direct in vivo gene transfer into procine myocardium using replicationdeficient adenoviral vectors. Circulation 90, 2414-2424. Furman C, Luo Z, Walsh, K, Duverger N (2002) Sytemic tissue inhibitor of metalloproteinase-1 gene delivery reduces neointimal hyperplasia in balloon-injured rat carotid artery. FEBS Letters 531, 122-126. Gangadharan S, Imai M, Rhynhart K, Sevigny, J, Robson S, Conte M (2001) Targeting platelet aggregation: CD39 gene transfer augments nucleoside triphosphate diphosphohydrolase activity in injured rabbit arteries. Surgery 130, 296-303. George S, Capogrossi M Angelini G, Baker A (2001) Wild type p53 gene transfer inhibits neointima formation in human saphenous vein by modulation of smooth muscle cell migration and induction of apoptosis. Gene Ther 8, 668-676. George S, Johnson J, Angelini G.D, Newby, A, Baker A (1998) Adenovirus-mediated gene transfer of the human TIMP-1 gene inhibits SMC migration and neointima formation in human saphenous vein. Human Gene Ther 9, 867-877. George S, Lloyd C Angelini G.D, Newby, A, Baker A (2000) Inhibition of late vein graft neointima formation in human and porcine models by adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-3. Circulation 101, 296-304. George S, Zaltsman A.B, Newby, A.C (1997) Surgical preparative injury and neointima formation increase MMP-9 expression and MMP-2 activation in human saphenous vein. Cardiovasc Res 33, 447-459. George S, Baker A, Angelini G, Newby, A.C (1998) Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins. Gene Ther 5, 1552-1560. Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89, 5547-5551. Grines C, Watkins M, Helmer G, Penny, W, Brinker J, JD M, West A, Rade J, Marrott P, Hammond H, Engler R (2002) Angiogenic Gene Therapy (AGENT) trial in patients with stable angina pectoris. Circulation 105, 1291-1297. Guzman R, Hirschowitz, EA, Brody, S, Crystal R, Epstein S.E, Finkel T (1994) In-vivo suppression of injury-induced vascular smooth-muscle cell accumulation using adenovirusmediated transfer of the herpes-simplex virus thymidine kinase gene. Proc Natl Acad Sci USA 91 (22), 1073210736. Hammond H.K, McKirman M (2001) Angiogenic Gene Therapy for heart disease: a review of animal studies and clinical trials. Cardiovasc Res 49, 561-567. Hanna A.K, Fox, J Neschis D, Safford S.D, Swain J, Golden M (1997) Antisense basic fibroblast growth factor gene transfer reduces neointimal thickening after arterial injury. J Vasc Surg 25, 320-325. Harrell R, Rajanayagam, S, Doanes A, Guzman R, Hirschowitz, EA, Crystal R, Epstein S.E, Finkel T (1997) Inhibition of vascular smooth muscle cell proliferation and neointimal accumulation by adenovirus-mediated gene transfer of cytosine deaminase. Circulation 96, 621-627. Hart S, Collins L, Gustafsson K, Fabre J (1997) Integrinmediated transfection with peptides containing arginineglycine-aspartic acid domains. Gene Ther 4, 1225-1230. Havenga M E, Lemckert A C, Grimbergen J, Vogels R, Huisman L, Valerio D, Bout A, Quax, P.H (2001) Improved


Gene Therapy and Molecular Biology Vol 7, page 147 adenovirus vectors for infection of cardiovascular tissues. J. Virol. 75, 3335-3342. Havenga M, Lemckert A, Ophorst O, Meijer M, Germeraad W, Grimbergen J, Doel M, Vogels R, Duetekom, J, Janson A, Bruijn J, Uytdehaag F, Quax, P, Logtenberg T, Mehtali M, Bout A ( 2002) Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease. Virology 76, 4612 - 4620. Hedman M, Hartikainen J, Syvanne M, Stjernvall J, Hedman A, Kivela A, Vanninen E, Mussalo H, Kauppila E, S S, Narvanen O, Rantala A, Peuhkurinen K, Nieminen M, Laakso M, Yla-Herttuala S (2003) Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemai. Phase II results of the Kupoio Angiogenesis Trial (KAT) Circulation 107, 26772683. Hiltunen M.O, Turunen M.P, Turunen A.-M, Rissanen T Laitinen M, Kosma V.-M, Yla-Herttuala S (2000) Biodistribution of adenoviral vector to nontarget tissues after local in vivo gene transfer to arterial wall using intravascular and periadventitial gene delivery methods. FASEB Journal 14, 2230-2236. Hu Y, Baker A, Zou Y, Newby, A, Xu Q (2001) Local gene transfer of tissue inhibitor of metalloproteinase-2 influences vein graft remodeling in a mouse model. Arterioscler Thromb Vasc Biol 21, 1275-1280. Huang J, Frischer J, Serur A, Kadenhe A, Yokoi A, McCrudden K, New, T, O'Toole K, Zabski S, Rudge J, Holash, J, Yancopoulos G, Yamashiro D, Kandel J (2003) Regression of established tumors and metastases by potent vascular endothelial growth factor blockade. Proc Natl Acad Sci USA 100, 7785-7790. Iaccarino G, Smithwick, L, Lefkowitz, R, Koch, W (1999) Targetting Gbeta gamma signaling in arterial vascular smooth muscle proliferation: a novel strategy to limit restenosis. Proc Natl Acad Sci USA 96, 3945-3950. Igarashi T, Miyake K, Kato K, Watanabe A, Ishizaki M, Ohara K, Shimada T (2003) Lentivirus-mediated expression of angiostatin efficiently inhibits neovascularization in a murine proliferative retinopathy model. Gene Ther 10, 219-226. Isner J, Pieczek, A, Schainfeld R, Blair R, Haley, L, Asahara T, Rosenfield K, Razvi S, Walsh, E, Symes J.F (1996) Clinical evidence of angiogenesis after arterial gene transfer of phVEGF (165) in patient with ischaemic limb. Lancet 348 , 370-374. Janssen S, Flaherty, D, Nong Z, Varenne O, Van Pelt N, Haustermans C, Zoldhelyi P, Gerard R, Collen D (1998) Human endothelial nitric oxide synthase gene transfer inhibits vascular smooth muscle cell prolifeation and neointima formation after balloon injury in rats. Circulation 97, 1274-1281. Jayakumar J, Suzuki K, Khan M, Smolenski R, Farrell A, Latif N, Raisky, O, Abunasra H, Sammut I, Murtuza B, Amrani M, Yacoub M (2000) Gene therapy for myocardial protection: transfection of donor hearts with heat shock protein 70 gene protects cardiac function against ischemiareperfusion injury. Circulation 102 (19 suppl 3), III302III306. Katovich, M, Gelband C, Reaves P (1999) Reversal of hypertension by angiotensin II type 1 receptor antisense gene therapy in the adult SHR. Am J Physiol 277, H1260-H1264. Kibbe M.R, Tzeng E, Gleixner S, Watkins S, Kovesdi L, Lizonova A, Makaroun M, Billiar T, Rhee R (2001)

Adenovirus-meidated gene transfer of human inducible nitric oxide synthase in porcine vein grafts inhibits intimal hyperplasia. J Vasc Surg 34, 156-165. Kimura B, Mohuczy, D, Tang X, Phillips I (2001) Attenuation of hypertension and heart hypertrophy by adeno-associated virus delivering angiotensinogen antisense. Hypertension 37, 376 - 380. Klugherz, B, Song C, Defelice S, Cui X, Lu Z, Connolly, J, Hinson J, Wilensky, R, Levy, R (2002) Gene delivery to pig coronary arteries from stents carrying antibody-tethered adenovirus. Human Gene Ther 13, 443 - 454. Klugherz, B.D, Jones P, Cui X, Chen W, Meneveau N.F, DeFelice S, Connolly J, Wilensky, R, Levy, R (2000) Gene delivery from a DNA controlled-release stent in porcine coronary arteries. Nat Biotechnol 18 (11), 1181-4. Kochanek, S, Clemens P.R, Mitani K, Chen H.-H, Chan S, Caskey, C. (1996) A new adenoviral vector: replacement of all viral coding sequences with 28kb of DNA independently expressing both full-length dystrophin and B-galactosidase. Proc Natl Acad Sci USA 93, 5731-5736. Koike H, Morishita R, Iguchi S, Aoki M, Matsumoto K, Nakamura T, Yokoyama C, Tanabe T, Ogihara T, Kaneda Y (2003) Enhanced angiogenesis and improvement of neuropathy by cotransfection of human hepatocyte growth factor and prostacyclin synthase gene. FASEB Journal 17, 779-781. Laitinen M, Hartikainen J, Hiltunen M.O, Eranen J, Kiviniemi M, Narvanen O, Makinen K, Manninen H, Syvanne M, Martin J.F, Laakso M, Yla-Herttuala S (2000) Cathetermediated vascular endothelial growth factor gene transfer to human coronary arteries after angioplasty. Human Gene Ther 11, 263-270. Lamfers M, Lardenoye J, de Vries M.R, Aalders M, Engelse MA, Grimbergen J, van Hinsbergh, V, Quax, P (2001) In vivo suppression of restenosis in balloon-injured rat carotid artery by adenovirus-mediated gene transfer of the cell surface-directed plasmin inhibitor ATF.BPTI. Gene Ther 8, 534-541. Lawrie A, Brisken A, Francis S, Tayler D, Chamberlain J, Crossman D, Cumberland D, Newman C (1999) Ultrasound Enhances Reporter Gene Expression After Transfection of Vascular Cells In Vitro. Circulation 99, 2617-2620. Lee R, Springer M, Blanco-Bose W.E, Shaw, R, Ursell P Blau H (2000) VEGF Gene Delivery to Myocardium: Deleterious Effects of Unregulated Expression. Circulation 102, 898901. Lemarchand P, Jones M, Yamada I, Crystal R (1993) In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors. Circ Res 72, 1132-1138. Li JM, Brooks G ( 1999) Cell cycle regulatory molecules (cyclins cyclin-dependent kinases and cyclin dependent kinase inhibitors) and the cardiovascular system. Potential targets for therapy? Eur Heart J 20, 406-420. Li Q, Bolli R, Qiu Y, Tang X, Guo Y, French, B (2001) Gene therapy with extracellular superoxide sismutase protects conscious rabbits against myocardial infarction. Circulation 103, 1893-1898. Lin K.F, Chao J, Chao L (1995) Human atrial natriuretic peptide gene delivery reduces blood pressure in hypertensive rats. Hypertension 26, 847-853. Lin K.F, Chao L, Chao J (1997) Prolonged reduction of high blood pressure with human nitric oxide synthase gene delivery. Hypertension 30, 307-313.


George et al: Gene therapy for vascular diseases Lin K.F, Chao J, Chao L (1999) Atrial natriuretic peptide gene delivery reduces stroke-induced mortality rate in Dahl saltsensitive rats. Hypertension 33, 219-224. Loeb J.E, Cordier W, Harris M.E, Weitzman M, Hope T (1999) Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy. Hum Gene Ther 10, 2295-305. Losordo D, Vale P.R, Symes J.F, Dunnington C, Esakof D.D, Maysky, M, Ashare A.B, Lathi K, Isner J (1998) Gene therapy for myocardial angiogenesis - Initial clinical results with direct myocardial injection of phVEGF (165) as sole therapy for myocardial ischemia. Circulation 98, 28002804. Lu L, Bonham, CA, Chambers F, Watkins S Hoffman RA, Simmonds R, Thomson A (1996) Induction of nitric oxide synthase in mouse dendritic cells by IFN-gamma endotoxin and interaction with allogeneic T Cells. Immunology 157, 3577-3586. Lundstrom, K, Schweitzer C, Rotmann D, Hermann D, Schneider E, Ehrengruber M (2001) Semliki forest virus vectors: efficient vehicles for in vitro and in vivo gene delivery. FEBS Letters 504, 99 - 103. Luo Z, Sato M, Nguyen T, Kaplan J, Akita G, Walsh, K (1999) Adenovirus-mediated delivery of fas ligand inhibits intimal hyperplasia after balloon injury in immunologically primed animals. Circulation 99, 1776-1779. Maeda Y, Ikeda U, Ogasawara Y, Urabe M, Takizawa T, Saito T, Colosi T, Kurtzman G, Shimada K, Ozawa K ( 1997) Gene transfer into vascular cells using adeno-associated virus (AAV) vectors. Cardiovasc Res 35, 514-521. Maillard L, Van Belle E, Smith, R, Le Roux, A, Denefle P, Steg G, Barry, J, Branellec D, Isner J, Walsh, K (1997) Percutaneous delivery of the gax gene inhibits vessel stenosis in a rabbit model of balloon angioplasty. Cardiovasc Res 35, 536-546. Makinen K, Manninen H, Hedman M, Matsi P, Mussalo H, Alhava E, Yla-Herttuala S (2002) Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized placebocontrolled doubl-blinded phase II study. Mol Ther 6, 127133. Mann M, Gibbons G, Kernoff R, Diet F, Tsao P, Cooke J, Kaneda Y, Dzau V ( 1995) Genetic engineering of vein grafts resistant to atherosclerosis. Proc Natl Acad Sci USA 92, 4502-4506. Mann M, Whittemore A.D, Donaldson M Belkin M, Conte M, Polak, J.F, Orav, E, Ehsan A, Dell'Acqua G, Dzau V (1999) Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre randomised controlled trial. Lancet 354, 1493-1498. Manninen H, Makinen K (2002) Gene therapy techniques for peripheral arterial disease. Cardiovasc Intervent Radiol 25, 98-108. Martens J.R, Reaves P.Y, Lu D (1998) Prevention of renovascular and cardiac pathophysiological changes in hypertension by angiotensin II type 1 receptor antisense gee therapy. Proc Natl Acad Sci USA 95, 2664-2669. Matsumoto T, Komori K, Yonemitsu Y, Morishita R, Sueishi K, Kaneda Y, Sugimachi K (1998) Hemagglutinating virus of Japan-liposome-mdiated gene transfer of endothelial cell nitric oxide synthase inhibits intimal hyperplasia of canine grafts under conditions of poor runoff. J Vasc Surg 27, 135144.

Mintz, G, Popma J, Pichard A, Kent K, Satler L, Wong C.Y, Hong M, Kovach, J, Leon M (1996) Arterial remodeling after coronary angioplasty: a serial intravascular ultrasound study. Circulation 94, 35-43. Morishita R, Gibbons G, Kaneda Y, Ogihara T, Dzau V (1994) Pharmacokinetics of antisense oliogdeoxyribonucleotides (cyclin B1 and CDC 2 kinase) in the vessel wall in vivo: enhanced therapeutic utility for restenosis by HVJ-liposome delivery. Gene 149, 13-19. Morishita R, Gibbons G, Horiuchi M, Ellison K, Nakama M, Zhang L, Kaneda Y, Ogihara T, Dzau V (1995) A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo. Proc Natl Acad Sci USA 92, 5855-5859. Nabel E, Shum, L, Pompili V, Yang ZY, San H, Shu H.B, Liptay, S, Gold L, Gordon D, Derynck, R (1993) Direct transfer of transforming growth factor b1 gene into arteries stimulates fibrocellular hyperplasia. Proc Natl Acad Sci USA 90, 10759-10763. Nabel E, Yang Z, Liptay, S, San H, Gordon D, Haudenschild C Nabel G (1993a) Recombinant platelet-derived growth factor B expression in porcine arteries induces intimal hyperplasia in vivo. J Clin Invest 91, 1822-1829. Nabel E, Yang Z, Plautz, G, Forough, R, Zhan X, Haudenschild C Maciag T, Nabel G (1993b) Recombinant fibroblast growth factor-1 promotes intimal hyperplasia and angiogenesis in arteries in vivo. Nature 362, 844-846. Nakai H, Yant S, Storm, T, Fuess S, Meuse L, Kay, M (2001) Extrachromosomal recombinant adeno-associated virus vector genomes are primarily resposible for stable liver transduction in vivo. Virology 75, 6969 - 6976. Nakamura T, Morishita R, Asai T, Tsuboniwa N, Aoki M, Sakonjo H, Yamasaki K, Hashiya N, Kaneda Y, Ogihara T (2002) Molecular strategy using cis-element 'decoy' of E2F binding site inhibits neointimal formation in porcine ballooninjured coronary artery model. Gene Ther 9, 488-494. Napoli C (2002) MMP-inhibition and the development of cerebrovascular atherosclerosis: the road ahead. Stroke 33, 2864-2865. Newby A, George S (1996) Proliferation migration matrix turnover and death of smooth muscle cells in native coronary and vein graft atherosclerosis. Curr Opin Cardiol 11, 574582. Newman K.D, Dunn P.F, Owens J, Schulick A, Virmani R, Sukhova G, Libby, P, Dichek, D (1995) Adenovirusmediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation inflammation and neointimal hyperplasia. J Clin Invest 96, 2955-2965. Nicklin SA, Von Seggern D, Work, L, Pek, D.C.K, Dominiczak, A.F, Nemerow, G, Baker A (2001) Ablating adenovirus type 5 fiber-CAR binding and HI loop insertion of the SIGYPLP peptide generate an endothelial cell-selective adenovirus. Mol Ther 4, 534-542. Nishida T, Ueno H, Atsuchi N, Kawano R, Asada Y, Nakahara Y, Kamikubo Y, Takeshita A, Yasui H (1999) Adenovirusmediated local expression of human tissue factor pathway inhibitor eliminates shear stress-induced redurrent thrombosis in the injured carotid artery of the rabbit. Circ Res 84, 1446-1452. Noiseux, n, boucher C, Cartier R, Sirois M (2000) Bolus endovascular PDGFR-beta antisense treatment suppressed intimal hyperplasia in a rat carotid injury model. Circulation 102 (11), 1330-1336.


Gene Therapy and Molecular Biology Vol 7, page 149 Ohno N, Itoh, H, Ikeda T, Ueyama K, Yamahara K, Doi K, Yamashita J, Inoue M, Masatsugu K, Sawada N, Fukunaga Y, Sakaguchi S, Sone M, Yurugi T, Kook, H, Komeda M, Nakano K (2002) Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirusmediated gene transfer of C-type natriuretic peptide. Circulation 105, 1623-1626. Parkes R, Meng Q, Siapati K, McEwan J, Hart S (2002) High efficiency transfection of porcine vascular cells in vitro with a synthetic vector system. J. Gene Medicine 4, 292-299. Parks R, Chen L, Anton M, Sankar U, Rudnicki M, Graham, F (1996) A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci USA 93 (24), 13565-13570. Phillips M.I (2001) Gene therapy for Hypertension: The Preclinical Data. Hypertension 38, 543-548. Qing K, Mah, C, Hansen J, Zhou S, Dwarki V, Srivastava A (1999) Human fibroblast growth factor receptor 1 is a coreceptor for infection by adeno-associated virus 2. Nat Med 5, 71-7. Quax, PA, Lamfers M, Grimbergen J, Verheijen J, van Hinsbergh, V (1997) Inhibition of neointima formation in cultured human saphenous vein segments by an advenovirus expressing an urokinase receptor binding plasmin inhibitor. Circulation 96 (Suppl), I-669. Rajagopalan S, Shah, M, Luciano A, Crystal R, Nabel E (2001) Adenovirus-mediated gene transfer of VEGF121 improves lower extremity endothelial function and flow reserve. Circulation 104, 753-755. Rajagopalan S, Mohler E, Lederman R, Saucedo J, Mendelsohn F, Olin J, Bleba J, Goldman C, Trachtenberg J, Pressler M, Rasmussen H, Annex, B, Hirsch, A, trial R W.V.E.G.F (2003) Regional angiogenesis with vascular endothelial growth factor (VEGF) in peripheral arterial disease: Design of the RAVE trial. Am Heart J 145, 1114-8. Reaves P.Y, Gelband C, Wang H, Yang H, Lu D, Berecek, K, Katovitch, M, Raizada M.K (1999) Permanent cardiovascular protection from hypertension by the AT1 receptor antisense gene therapy in hypertensive rat offspring. Circ Res 85, e45-e50. Richter M, Iwata A, Nyhuis J, Nitta Y, Miller A, Halbert L, Allen M (2000) Adeno-associated virus vector transduction of vascular smooth muscle cells in vivo. Physiol. Genomics 2, 117-127. Rissanen T, Vajanto I, Yla-Herttuala S (2001) Gene therapy for therapeutic angiogenesis in critically ischaemic lower limb on the way to the clinic. Eur J Clin Invest 31, 651-666. Rivard A, Silver M, Chen D, Kearney, M, Magner M, Annex, B, Peters K, Isner J (1999) Rescue of diabetes-related impairment of angiogensis by intramuscular gene therapy with adeno-VEGF. Am J Pathol 154, 355-363. Rosengart T.K, Lee L.Y, Patel S.R, Sanborn TA, Parikh, M, Bergman G, Hachamovitch, R, Szulc M, Kligfield P.D, Okin P, Hahn R Devereux, R.B, Post M.R, Hackett N.R, Foster T, Grasso T, Lesser M, Isom, O, Crystal R (1999) Angiogenesis Gene Therapy - Phase I assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant severe coronary artery disease. Circulation 100, 468-474. Schnepp, B, Clark, K, Klemanski D, Pacak, C, Johnson P (2003) Genetic fate of recombinant adeno-associated virus vector genomes in muscle. J Virol 77, 3495-3504.

Serruys P, Degertekin M, Tanabe K, Abizaid A, Sousa J.E, Colombo A, Guagliumi G, Wijns W, Lindeboom, W, Ligthart J, de Feyter P, Morice M, Group, R (2002) Intravascular ultrasound findings in the multicenter randomized double-blind RAVEL (RAndomized study with the sirolimus-eluting VElocity balloo-expandable stent in the treatment of patients with de novo native coronary artery Lesions) trial. Circulation 106, 798-803. Shears L, Kibbe M, Murdock, A, Billiar T, Lizonova A, Kovesdi I, Watkins S, Tzeng E (1998) Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo. J Am Coll Surg 187, 295-306. Shi Y, Fard A, Galeo A, Hutchinson H, Vermani P, Dodge G, Hall D, Shaheen F, Zalewski A (1994) Transcatheter delivery of c-myc antisense oligomers reduces neointimal formation in a porcine model of coronary artery balloon injury. Circulation 90, 944-951. Shi Y, Patel S, Davenpeck, K, Niculescu R, Rodriguez, E, Magno M, Ormont M, Mannion J, Zalewski A (2001) Oxidative stress and lipid retention in vascular grafts: comparison between venous and arterial conduits. Circulation 15 (103), 19. Shyu K, Manor O, Magner M, Yancopoulos G, Isner J (1998) Direct intramuscular injection of plasmid DNA encoding angiopoietin-1 but not angiopoietin-2 augments revascularization in the rabbit ischemic hindlimb. Circulation 98, 2081-2087. Simari R.D, San H, Rekhter M, Ohno T, Gordon D, Nabel G, Nabel E (1996) Regulation of cellular proliferation and intimal formation following balloon injury in atherosclerotic rabbit arteries. J Clin Invest 98, 225-235. Simons M, Edelman E.R, DeKeyser J.-L, Langer R, Rosenberg R (1992) Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature 359, 67-70. Simovic D, Isner J, Ropper A, Pieczek, A, Weinberg D (2001) Improvement in chronic ischemic neuropathy after intramuscular phVEGF165 gene transfer in patients with critical limb ischemia. Arch Neurol 58, 761-768. Sirois M, Simons M, Edelman E (1997) Antisense oligonucleotide inhibition of PDGFR-beta receptor subunit expression directs suppression of intimal thickening. Circulation 95, 669-676. Smith, R, Branellec D, Gorski D, Guo K, Perlman H, Dedieu J, Pastore C, Mahfoudi A, Denefle P, Isner J, Walsh, K (1997) p21CIP1-mediated inhibition of cell proliferation by overexpression of gax homeodomain gene. Genes Dev 11, 1674-1689. Somia N, Verma I (2000) Gene therapy: trials and tribulations. Nat Rev Genet 1, 1-9. SoRelle R (2001) Late-breaking clinical trials at the American Heart Association's Scientific Session 2001. Circulation 104, c9046. Sousa J.E, Costa MA, Sousa A, Abizaid A, Seixas A, Abizaid A, Feres F, Mattos L, Falotico R, Jaeger J, Popma J, Serruys P (2003) Two-year angiographic and intravascular ultrasound follow-up after implantation of sirolimus-eluting stents in human coronary arteries. Circulation 107, 381-383. Southgate K, Mehta D, Izzat M.B, Newby, A, Angelini G (1999) Increased secretion of basement membrane-degrading metalloproteinases in pig saphenous vein into carotid artery interposition grafts. Arterioscler Thromb Vasc Biol 19, 1640-1649.


George et al: Gene therapy for vascular diseases Stephan D, San H, Yang Z, Gordon D, Goelz, S, Nabel G, Nabel E (1997) Inhibiton of vascular smooth muscle cell proliferation and intimal hyperplasia by gene transfer of betainterferon. Mol Med 3, 593-599. Stewart D, investigators o.b.o.ts (2002) A phase 2, randomized multicenter 26-week study to assess the efficacy and safety of BIOBYPASS (AdGVVEGF121.10) delivered through minimally invasive surgery versus maximum medical treatment in patients with severe angina advanced coronary artery disease and no options for revascularization. Circulation 106, 23-26. Summerford C, Samulski R (1998) Membrane-associated heparan sulfate proteoglycan is a receptor for adenoassociated virus yype 2 virions. J. Virol. 72, 1438-1445. Summerford C, Bartlett J, Samulski R (1999) aVb5 integrin: a co-receptor for adeno-associated virus type 2 infection. Nat Med 5, 78-82. Symes J.F, Losordo D, Vale P.R, Lathi K, Esakof D.D, Mayskiy M, Isner J (1999) Gene therapy with vascular endothelial growth factor for inoperable coronary artery disease. Annals of Thoracic Surgery 68, 830-836. Tamirisa K.P, Mukherjee D (2002) Gene therapy in cardiovascular diseases. Curr Gene Ther. 2:427-435 Tang X, Mohuczy, D, Zhang Y.C (1999) Intravenous angiotensinogen antisense in AAV-based vector decreases hypertension. Am J Physiol 277, H2392-H2399. Taniyama Y, Morishita R, Hiraoka K, Aoki M, Nakagami H, Yamasaki K, Matsumoto K, Nakamura T, Kaneda Y, Ogihara T (2001) Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat diabetic hind limb ischemia model: molecular mechanisms of delayed angiogenesis in diabetes. Circulation 104, 2344-2350. Taniyama Y, Tachinaba K, Hiroaka K, Namba T, Yamasaki K, Hashiya N, Aoki M, Ogihara T, Yasufumi K, Morishita R (2002) Local delivery of plasmid DNA into rat carotid artery using ultrasound. Circulation 105, 1233-1239. Tomko R.P, Xu R, Philipson L (1997) HCAR and MCAR: The human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc Natl Acad Sci USA 94, 3352-3356. Tulis DA, Mynjoyan Z, Schiesser R, Shelat H, Evans A, Zoldhelyi P, Fujise K (2003) Adenoviral gene transfer of fortilin attenuates neointima formation through suppression of vascular smooth muscle cell proliferation and migration. Circulation 107, 98-105. Ueno H, Yamamoto H, Ito S.I, Li J, Takeshita A (1997) Adenovirus-mediated transfer of a dominant-negative H-ras suppresses neointimal formation in balloon-injured arteries in vivo. Arterioscler Thromb Vasc Biol. 17, 898-904. Van Belle E, Tio F, Chen D, Maillard L, Chen D, Kearney, M, Isner J (1997) Passivation of metallic stents after arterial gene transfer of phVEGF165 inhibits thrombus formation and intimal thickening. J Am Coll Cardiol 29, 1371-1379. Varenne O, Pislaru S, Gillijns H, Van Pelt N, Gerard R.D, Zoldhelyi P, Van de Werf F, Collen D, Janssens S.P (1998) Local adenovirus-mediated transfer of human endothelial nitric oxide synthase reduces luminal narrowing after coronary angioplasty in pigs. Circulation 98 (9), 919-926. Vigna E, Cavalieri S, Ailles L, Guena M, Loew, R, Buyjard H, Naldini L (2002) Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors. Mol Ther 5, 252 -257. von der Leyen H.E, Gibbons G, Morishita R, Lewis N, Zhang L, Nakajima M, Kaneda Y, Cooke J, Dzau V (1995) Gene

therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene. Proc Natl Acad Sci USA 92, 1137-1141. Wang C, Chao L, Chao J (1995) Direct gene delivery of human tissue kallikrein reduces blood pressure in spontaneously hypertensive rats. J Clin Invest 95, 1710-1716. Wang C, Dobrzynski E, Chao J (2001) Adrenomedullin gene delivery attenuates renal damage and cardiac hypertrophy in Goldblatt hypertensive rats. Am J Physiol Renal Physiol 280, F964-F971. Wang H, Katovich, M, Gelband C, Reaves P.Y, Phillips M.I, Raizada M.K (1999) Sustanined inhibition of angiotensin Iconverting enzyme (ACE) expression and long-term antihypertensive action by virally mediated delivery of ACE antisense cDNA. Circ Res 85, 614-622. Wang H, Reaves P.Y, Gardon M (2000) Angiotensin Iconverting enzyme antisense gene therapy causes permanent antihypertensive effects in the SHR. Hypertension 35, 202208. Waugh, J, Kattash, M, Li J, Yuksel E, Kuo M.D, Lussier M, Weinfeld A.B, Saxena R, Rabinovsky, E.D, Thung S, Woo S.L Shenaq, S (1999a) Gene therapy to promote thromboresistance: local overexpression of tissue plasminogen activator to prevent arterial thrombosis in an in vivo rabbit model. Proc Natl Acad Sci USA 96, 1065-1070. Waugh, J, Yuksel E, Li J, Kuo M.D, Kattash, M, Saxena R, Geske R, Thung S.N, Shenaq, S, Woo S (1999b) Local overexpression of thrombomodulin for in vivo prevention of arterial thrombosis in a rabbit model. Circ Res 84, 84-92. Wen S, Schneider D, Driscoll R, Vassalli G, Sassani A, Dichek, D (2000) Second-generation adenoviral vectors do not prevent rapid loss of transgene expression amd vector DNA from the arterial wall. Arterioscler Thromb Vasc Biol. 20, 1452 - 1458. West N, Guzik, T, Black, E, Channon K (2001) Enhanced superoxide production in experimental venous bypass graft intimal hyperplasia. Role of NAD (P)H oxidase. Arterioscler Thromb Vasc Biol 21 (189-194). Wickham, T, Mathias P, Cheresh, D, Nemerow, G (1993) Integrins avb3 and avb5 promote adenovirus internalization but not virus attachment. Cell 73, 309-319. Wielbo D, Simon A, Phillips M.I (1996) Inhibition of hypertension by peripheral administration of antisense oligodeoxynucleotides. Hypertension 28, 147-151. Wolf W Yoshida H, Agata J (2000) Human tissue kallikrein gene delivery attenuates hypertension renal injury, and cardiac remodeling in chronic renal failure. Kidney Int 58, 730-739. Yamamoto K, Morishita R, Tomita N, Shimozato T, Nakagami H, Kikuchi A, Aoki M, Higaki J, Kaneda Y, Ogihara T (2000) Ribozyme oligonucleotides against transforming growth factor-b inhibited neointimal formation after vascular injury in rat model. Circulation 102, 1308-1314. Yang L, Quan S, Abraham, N (1999) Retrovirus-mediated HO gene transfer into endothelial cells protects against osidantinduced injury. Am J Physiol 277 (1 pt 1), L127-L133. Yang Z, Simari R, ND P, H, S, Gordon D, GJ, N, Nabel E ( 1996) Role of the p21 cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterila injury. Proc Natl Acad Sci USA 93 (15), 7905-7910. Yayama K, Wang C, Chao L (1998) Kallikrein gene delivery attenuates hypertension and cardiac hypertrophy and enhances renal function in Goldblatt hypertensive rats. Hypertension 31, 1104-1110.


Gene Therapy and Molecular Biology Vol 7, page 151 Yla-Herttuala S, Alitalo K (2003) Gene transfer as a tool to induce therapeutic vascular growth. Nat Med 9, 694-701. Yonemitsu Y, Kaneda Y, Tanaka S, Nakashima Y, Komori K, Sugimachi K, Sueishi K (1998) Transfer of wild-type p53 gene effectively inhibits vascular smooth muscle cell proliferation in vitro and in vivo. Circ Res 82, 147-156. Zhang J, Yoshida H, Chao L (2000) Human adrenomedullin gene delivery protects against cardiac hypertrophy, fibrosis and renal damage in hypertensive dahl salt-sensitive rats. Hum Gene Ther 11, 1817-1827.

Zoldhelyi P, McNatt J, Shelat H, Yamamoto Y, Chen Z, Willerson J. (2000) Thromboresistance of balloon-injured porcine carotid arteries after local gene transfer of human tissue factor pathway inhibitor. Circulation 101, 289-295. Zufferey, R, Donello J.E, Trono D, Hope T (1999) Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol. 73, 2886-2892.


George et al: Gene therapy for vascular diseases


Gene Therapy and Molecular Biology Vol 7, page 153 Gene Ther Mol Biol Vol 7, 153-165, 2003.

Angiogenic gene therapy for improving islet graft vascularization Review Article

Nan Zhang1, Karen Anthony1, Katsunori Shinozaki1, Jennifer Altomonte1, Zachary Bloomgarden2 and Hengjiang Dong1,3* 1

Carl Icahn Institute for Gene Therapy and Molecular Medicine, 2Department of Medicine, 3Division of Experimental Diabetes and Aging, Department of Geriatrics, Mount Sinai School of Medicine, New York, NY 10029.

__________________________________________________________________________________ *Correspondence: Hengjiang Dong, Ph.D., Mount Sinai School of Medicine, Box 1496, One Gustave L. Levy Place, New York, NY 10029; tel: 212-241-3662; fax: 212-241-0738; email: Key words: Type 1 diabetes, islet transplantation, islet revascularization, VEGF, gene transfer. Received: 3 July 2003; Accepted: 19 August, 2003; electronically published: August 2003

Summary Clinical islet transplantation is considered a curative treatment for type 1 diabetes, but long-term survival and function of implanted islets is greatly compromised by a number of adverse events. In addition to immune rejection and recurrent autoimmunity, the survival and function of islets is determined by the rate and degree of islet revascularization, an essential process termed angiogenesis that is required for the development of new vessels within islet grafts to derive blood from the host vasculature. Rapid and adequate revascularization is crucial for islet survival and function. Delay in islet revascularization can deprive islets of oxygen and nutrients, resulting in islet cell death and early graft failure. There is evidence that despite the infusion of sufficiently large amounts of islets (~11,000 islets/kg body weight) per diabetic recipient, less than 30% of islet mass becomes stably engrafted post transplantation. In this article, we will review the molecular basis of islet revascularization and highlight the importance of developing novel therapeutic strategies to stimulate angiogenesis within islet grafts and enhance islet graft vascularization post transplantation. Such strategies, when applied in conjunction with islet transplantation, are expected to improve the viability of transplanted islets and provide long-term survival of functional islet mass post transplantation, thereby increasing the overall success rate of islet transplantation. incidence of about 15 per 100,000 children in the US alone (Karvonen et al, 2000). This poses a tremendous burden on patients and healthcare economies.

I. Introduction A. Type 1 diabetes Type 1 diabetes is a metabolic disorder that is caused by insulin deficiency due to autoimmune destruction of ! cells, leading to chronic elevation of blood sugar levels. Because of its onset in children and young adolescents, type 1 diabetes was previously referred to as juvenile diabetes or insulin-dependent diabetes. Prior to the discovery and isolation of insulin for therapeutic use, patients with type 1 diabetes survived only for a period of months, with death caused primarily by the accumulation of ketones in the body, leading to diabetic ketoacidosis. Over the past century, the prevalence of type 1 diabetes has increased in a variety of populations with an incidence rate ranging from 1-3 per 100,000 children per year in the US at the beginning of the 20th century to 4-7 per 100,000 in Scandinavian countries between 1930-1950, and to approximately 20 per 100,000 in Scandinavia over the past two decades (Bloomgarden, 1998; Gale, 2002). Currently, there are about 1.7 million patients with an overall annual

B. Insulin therapy and limitations Type 1 diabetes is commonly treated with twicedaily injection of a mixture of delayed and short-acting insulin. Delayed-acting insulin is provided to maintain a relatively constant background level of plasma insulin for the basal requirement, on which short-acting insulin is imposed to meet the postprandial demand of insulin after meals. Nevertheless, such conventional insulin therapy typically leads to inadequate blood sugar control as most treated patients experience to a lesser or greater extent elevated blood sugar levels between meals and during the night, the cumulative effect of which can result in the development of diabetic complications at a late stage. There is clinical evidence that more than half of diabetic patients have eyes affected by diabetic retinopathy (Bloomgarden, 1998), with additional effects on the 153

Zhang et al: Angiogenic Gene Therapy for Improving Islet Graft Vascularization kidneys by diabetic nephropathy (Chaturvedi et al, 2000) and on nerves by diabetic neuropathy, together with about 4- and 10-fold lifetime increase in rates of cardiovascular mortality among men and women, respectively (Laing et al, 2003). To improve glycemic control, a number of insulin analogs, such as short-acting insulin lispro and aspart (Plank et al, 2002), as well as delayed-acting insulin glargine (Murphy et al, 2003) and detimir (Vague et al, 2003) have been developed. Nevertheless, implementation of treatment regimens with insulin analogs in different formulations to strive for normoglycemic control without risk of hypoglycemia can be very challenging and requires extraordinary efforts from both health care providers and diabetic patients (Bloomgarden et al, 2002).

sources by generating insulin-producing cells through genetic engineering of embryonic stem cells (Lumelsky et al, 2001; Soria et al, 2001). In addition, limited progress has been made to induce graft tolerance using immune modulation or allorecognition (Cote et al, 2001). An indepth discussion of these two outstanding issues in relation to the optimal clinical outcome of islet transplantation, which is beyond the scope of this article, has been reviewed elsewhere (Waldmann, 2002; Lechler et al, 2003; Lechner and Habener, 2003). Here we would like to highlight a third limiting factor, namely islet revascularization, which appears to play an important role in determining the long-term survival and optimal performance of functional islet mass post transplantation.

1. Islet revascularization post transplantation

C. Islet transplantation Of alternative insulin replacement therapies developed, islet transplantation offers the prospect of providing a curative treatment for type 1 diabetes without the need for exogenous insulin. The protocol of islet transplantation developed by Shapiro and colleagues at the University of Alberta at Edmonton, Canada, known as the Edmonton protocol, is relatively simple and minimally invasive, which is carried out under local anesthetics without surgery. Using fluoroscopic guidance, isolated human islets are implanted intraportally to a diabetic recipient, such that islets are engrafted in the liver and function to provide near physiological insulin release from an ectopic site. The success of this protocol has largely been attributed to technical advances in isolating highquality human islets in relatively large quantities and the application of more potent and less toxic non-steroidal immunosuppressants (Shapiro et al, 2000). Using the Edmonton protocol, long-term excellent glycemic control has been achieved with sustained freedom from insulin injection in type 1 diabetic patients (Shapiro et al, 2000). Currently, this protocol is being rigorously tested in clinical trials at multiple clinical centers to evaluate the safety and efficacy of islet transplantation and assess the benefit and risk ratio associated with long-term use of immunosuppressive drugs (Boker et al, 2001). Although promising for providing a curative option for type 1 diabetes, the Edmonton protocol is limited by two major factors: the lack of a sufficiently large source of islets due to the scarcity of cadaveric pancreas donors, and the presence of persistent immune rejection as well as the potential for recurrence of autoimmunity. Recent followup studies indicate that even with the rigorous application of steroid-free immunosuppressive regimens, there is still a slow and progressive loss of insulin production from transplanted islets in diabetic recipients over time, as evidenced by reports that 30-40% of islet recipients may experience recurrence of autoimmune diabetes with reacquisition of insulin dependence one to two years post transplantation (Shapiro et al, 2000; Boker et al, 2001; Ryan et al, 2001, 2002). To overcome these limitations, attempts have been made to develop alternative islet

a. Re-establishment of islet microvasculature. Native islets in the pancreas have a rich glomerularlike vascular system that consists of fine capillaries supplied by one to five feeding arterioles and drained by coalescing into an efferent plexus exiting the islet via one to five venules (Menger et al, 2001; Mattson et al, 2002). Such a rich microvasculature in pancreatic islets serves to provide efficient delivery of oxygen and nutrients to islet cells, and at the same time ensure rapid dispersal of pancreatic hormones to the circulation. However, isolated islets are avascular in both structural and functional entities, such that after transplantation, the survival and function of islets must rely on the re-establishment of new vessels in the grafts to derive blood flow from the host vessel system (Boker et al, 2001; Vasir et al, 2001). There is evidence that freely transplanted islets are associated with significantly reduced islet revascularization in comparison to native islets in the pancreas and this problem occurs irrespective of whether islets are transplanted intraportally in the liver, retrogradely into the spleen, or under the kidney capsule (Figure 1) (Mattson et al, 2002). What are the likely consequences of delayed or insufficient islet revascularization post islet transplantation? To answer this question, let us take a quantitative view of the relative partitioning of blood flow to islets vs. exocrine tissue in the pancreas. Using a modified microsphere technique, it has been shown that islets take up more than 10% of the total pancreatic blood flow despite their collectively comprising only about 1% of the tissue mass of the pancreas (Jansson and Carlsson, 2002). Thus, it is critically important to maintain adequate microvascular perfusion to islet cells for oxygen and nutrient supplies. While islets are transplanted either as single entities or as aggregated islet clusters under the kidney capsule or intraportally in the liver, adequate microvascular perfusion to islet cells does not resume immediately after islet transplantation.


Gene Therapy and Molecular Biology Vol 7, page 155

Figure 1. Intra-islet microvasculature. A. Microvasculature in the mouse pancreas, as visualized by immunostaining for the endothelium marker CD-31, also known as the platelet endothelial cell adhesion molecule-1 (PECAM-1). B. Microvasculature in engrafted islets under the renal capsule of a diabetic mouse following 16 days of islet transplantation. Islet grafts are indicated by arrows. Bar, 50 Âľm.

Instead, it can take up to three to five days for the formation of intra-graft microvessels to occur post islet transplantation and the re-establishment of intra-graft blood perfusion can take even longer time (>14 days) (Vasir et al, 2001, Jansson and Carlsson, 2002). This delay in the re-establishment of a functional microvasculature in newly grafted islets can starve islet cells of oxygen and nutrients. Indeed, several studies have shown that newly transplanted islets are hypoxic, causing islet cells to undergo apoptosis and/or necrosis, which attributes to the loss of functional !-cell mass post transplantation (Vasir et al, 2001; Jansson and Carlsson, 2002). Consistent with this interpretation, it has been shown that despite the administration of a large number of islets (11,000 islets/kg body weight) per diabetic recipient, only about 30% of transplanted islets become stably engrafted, corresponding to a total loss of about 70% of the functional islet mass in the early post transplantation phase (Boker et al, 2001). In addition, recent clinical data indicate that even when fasting blood glucose levels are restored to the physiological range post islet transplantation, the optimal performance of engrafted islets in terms of glucose-inducible insulin secretion is abnormal. In response to intravenous glucose infusion, the amplitude of the first phase insulin secretion is only about 20% of normal, which coincides with relatively slow glucose disposal rates following an oral glucose load in post-transplant subjects (Ryan et al, 2002). Although there is no direct proof suggesting that this observed suboptimal performance of transplanted islets in glycemic control is associated with insufficient vascularization, there is general agreement that impaired islet revascularization does adversely affect the optimal function of islets post transplantation. Recent preclinical studies have shown that even after transplanted islets are stably engrafted, the extent of vascularization, defined as microvascular density in transplanted islets is significantly lower than that in native islets in the pancreas (Jansson and Carlsson, 2002). In addition, engrafted islets in all three of the different transplantation organs (kidney cortex, liver and spleen) also exhibit markedly low oxygen tension, in comparison

to native islets in the pancreas, which is associated with a concomitant reduction in intra-graft blood perfusion (Carlsson et al, 2000, 2001). Currently, the extent to which this observed low oxygen tension and reduced blood perfusion in islet grafts, as a result of insufficient islet revascularization, adversely affect the long-term survival and optimal performance of functional islet mass and contribute to early graft failure is not known. An additional factor that might contribute to the metabolic abnormality in glucose tolerance in diabetic recipients is islet graft reinnervation post transplantation. However, little is currently known about its molecular basis in relation to islet revascularization and the optimal performance of islet function in glycemic control post transplantation. b. Mechanism of islet graft vascularization To date, the molecular mechanism of islet revascularization post islet transplantation remains poorly understood. In general, tissue graft vascularization depends on a coordinated process of angiogenesis and vasculogenesis, which are functionally governed by two key protein factors, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1). These two angiogenic/vasculogenic factors play separate but complementary roles in the de novo formation of blood vessels during embryonic development (vasculogenesis) as well as in the formation of new blood vessels from preexisting ones (angiogenesis) (Yancopoulos et al, 2000). VEGF acts in the early phase to stimulate the formation of primitive vascular networks by vasculogenesis and angiogenic sprouting, whereas Ang-1 functions subsequently for remodeling and maturation of the primary vascular system by integrating the endothelial cells of vessels with surrounding matrix and supporting cells (smooth muscle cells and pericytes) (Thurston et al, 1999). Thus, in terms of their specific roles in angiogenesis/vasculogenesis, VEGF seems to be a critical "driver" for initiating vascular formation, whereas Ang-1 works as a "stabilizer" to ensure subsequent maturation


Zhang et al: Angiogenic Gene Therapy for Improving Islet Graft Vascularization and stability of the newly formed blood vessels. These two factors act synergistically to ensure new blood vessel formation, growth and maturation. VEGF has four different isoforms in humans, consisting of 121, 165, 189 and 206 amino acid residues, all of which are generated by alternative splicing of a single gene. Rodents have only three isoforms, namely VEGF120, VEGF164 and VEGF188, each polypeptide one amino acid shorter than their corresponding human homologues (Kim et al, 2000; Vasir et al, 2000, 2001). The most abundant and widely distributed form is VEGF165 in humans (or VEGF164 in rodents). In concert with their respective functions in angiogenesis / vasculogenesis, the receptors for both VEGF (VEGFR1/Flt1 and VEGFR-2/Flk-1/KDR) and Ang-1 (Tie2) are selectively expressed in the vascular endothelium (Ferrara and Davis-Smyth, 1997; Otani et al, 1999; Kim et al, 2000). In addition, both VEGF and Ang-1 are expressed in the pancreas, suggesting their functional importance in pancreatic tissue angiogenesis / vasculogenesis (Vasir et al, 2001). However, due to limited data in the literature, little is known about the functional interplay between VEGF and Ang-1 in islet revascularization post transplantation.

types (Chegini, 1997; Asplin et al, 2001; Li et al, 2003). Although FGF and TGF have been implicated to play important roles in angiogenesis (Vasir et al, 2000; Kawakami et al, 2001), their functional contributions to islet revascularization remain unknown. HGF/SF is a mitogen that acts to stimulate cell division and proliferation of a variety of cell types, including smooth muscle cells and pericytes that are functionally involved in blood vessel formation (Bussolino et al, 1992; Ahmet et al, 2003; Ding et al, 2003; Sengupta et al, 2003). In addition, it has recently been shown that elevated HGF production in islet grafts significantly improves the outcome of marginal islet transplantation due to its proliferative effect on islet cells (Garcia-Ocana et al, 2003). c-Met is a tyrosine kinase receptor of HGF/SF, which is expressed in endothelial cells. In concert with the action of HGF/SF, the islet-specific expression of c-Met functions to mediate the mitogenic effect of HGF/SF on islet cell growth and proliferation (Weidner et al, 1993; Rosen et al, 1997). Vasir and colleagues (2000) showed that the expression of HGF/SF together with its receptor in newly transplanted islets is profoundly delayed in diabetic animals (Laing et al, 2003), which correlates with reduced islet graft vascularization. Nevertheless, its specific role in islet revascularization has not been defined. The urokinase plasminogen activator system, consisting of uPA and uPAR, plays a pivotal role in angiogenic sprouting. uPA binds to its cell surface receptor uPAR and converts plasminogen to plasmin, a serine protease with a broad specificity that functions to catalyze the degradation of extracellular matrix/basement membrane, an essential process that is required for clearing a path to facilitate endothelial cell migration and tissue remodeling in an angiogenic cascade (Saksela and Rifkin, 1988; Bacharach et al, 1992; Pepper et al, 1993). Consistent with their roles in angiogenesis, both uPA and uPAR expression are stimulated by VEGF and HGF/SF (Pepper et al, 1992; Mandriota et al, 1995). Like other angiogenic molecules, the expression of uPA and uPAR in newly engrafted islets is significantly delayed (Vasir et al, 2000). It has been suggested that impaired uPA and uPAR expression in newly transplanted islets also contributes to insufficient islet revascularization under diabetic conditions.

c. Genes involved in islet revascularization Of the genes whose functions are involved in angiogenesis, VEGF seems to play a crucial role in islet revascularization. Recent studies by Vasir and colleagues (2000, 2001) indicate that VEGF expression in islet cells is transiently induced, followed by significant decline twothree days post transplantation. This impaired expression of VEGF is further pronounced in the presence of prevailing hyperglycemia, which coincides with delayed expression profiles of VEGF receptor molecules, Flk1/KDR and Flt-1, in islet grafts post transplantation in diabetic animals (Hellerstrom et al, 1898; Korsgren and Jansson, 1989; Mattson et al, 2002). These results reflect to some extent an impaired angiogenesis of islet grafts in the diabetic milieu, which is contributable to the lack of adequate islet revascularization under hyperglycemic conditions. In addition to VEGF, there are a number of other angiogenic molecules whose expression in islet cells also seems to affect islet revascularization, including fibroblast growth factor (FGF), hepatic growth factor (or scatter factor) (HGF/SF) and its receptor c-Met, transforming growth factor-" (TGF-") and -! (TGF-!), and urokinase plasminogen activator (uPA) and its receptor uPAR. Like VEGF, FGF appears to be a positive regulator of angiogenesis, as it has been shown to induce endothelial cell proliferation, migration and angiogenesis (Bikfalvi et al, 1997; Vasir et al, 2000, 2001, Kawakami et al, 2001). Regarding the function of TGF in angiogenesis, TGF-" has been shown to stimulate the growth of microvascular endothelial cells (Tokuda et al, 2003). In addition, TGF-" is also a potent inducer of VEGF (Gille et al, 1997; Li et al, 2003). On the other hand, TGF-! is found to stimulate wound healing and regulate differentiation of certain cell

4. Factors affecting islet revascularization As discussed above, islet revascularization is an important determinant for the clinical outcome of islet transplantation. Unfortunately, transplanted islets are invariably associated with markedly reduced revascularization no matter whether islets are transplanted in the renal, splenic or hepatic subcapsular space (Jansson and Carlsson, 2002). What are the factors that adversely affect islet revascularization?. One potential factor that affects islet revascularization is the presence of prevailing hyperglycemia in diabetic recipients. Data in support of this view have been obtained by Vasir et al. (2000, 2001), who showed that the expression of several key angiogenic


Gene Therapy and Molecular Biology Vol 7, page 157 proteins and their respective receptor molecules in newly engrafted islets is significantly delayed in diabetic recipient mice, compared to that in nondiabetic recipient mice. These results suggest that islets transplanted under the renal capsule in a diabetic environment fare less well in terms of graft vascularization than those transplanted in a normoglycemic subject. In contrast, a different view of the possible impact of prevailing hyperglycemia on islet revascularization is provided by Menger et al, (1992), who showed that the relative microvascular blood perfusion is equivalent in islets engrafted in the striated skin muscle in hyperglycemic and normoglycemic Syrian golden hamsters. Unfortunately, there is no quantitative data regarding the functional vascular density in islet grafts in relation to the presence or absence of persistent hyperglycemia provided in these studies. Thus, whether and to what extent prevailing hyperglycemia affects islet revascularization still remain an issue of debate. A second factor that may potentially influence islet revascularization is the use of immunosuppressive agents associated with islet transplantation. One outstanding concern is that immunosuppressive agents are commonly associated with anti-proliferative activity and their clinical application in conjunction with islet transplantation may adversely affect islet revascularization. The immunosuppressants, sirolimus and tacrolimus, are shown to inhibit angiogenesis in a dose-dependent manner in both in vitro and in vivo angiogenesis assays (Eckhard et al, 2003). In the same sensitive assays, cyclosporine and prednisolone are also found to retain anti-angiogenic activities in counteracting the proliferative effect of FGF in angiogenesis (Eckhard et al, 2003), although it has been previously reported that the application of cyclosporin-A does not seem to alter microvascular perfusion to islet grafts (Mendola et al, 1997; Vajkoczy et al, 1999). These results raise a great deal of concern that clinical application of immunosuppressive drugs, which is intended to prevent islet graft loss, may actually compromise the viability of newly transplanted islets by hampering the process of islet revascularization. A third limiting factor for islet revascularization is the presence of contaminating exocrine cells in isolated islets, including macrophage, dendritic cells (DC) and endothelial cells. It has been suggested that exocrine cells perturb angiogenesis and islet revascularization (Heuser et al, 2000; Jansson and Carlsson, 2002). Consistent with this idea is the observation that culturing of islets prior to transplantation tends to improve the outcome of islet transplantation, as culturing helps eliminate contaminating cells, in particular, the antigen presenting cells (APC) in islet preparation (Gaber et al, 2001; Kuttler et al, 2002). However, culturing of freshly isolated islets also results in the loss of endothelium in islets. Interestingly, recent studies show that intra-islet endothelial cells serve as integrated components in angiogenesis and function together with recipient endothelium to facilitate the overall islet graft vascularization (Brissova et al, 2003; Linn et al, 2003). These results suggest that transplantation of freshly isolated islets may be favorable for islet viability because of the functional contribution of intra-islet endothelial

cells to islet revascularization post transplantation (Jansson and Carlsson, 2002). Finally, a less well-characterized factor that might affect islet revascularization is islet cryopreservation. This process is necessary as it can afford a great deal of flexibility and additional advantages to clinical islet transplantation. Cryopreservation allows pooling of marginal islets and subsequent distribution of islets to different islet transplantation centers/hospitals. It also allows sufficient time for pre-transplantation quality control testing of isolated islets to ensure islet cell viability and microbiological sterility prior to transplantation. In addition, cryopreservation also allows for genetic modification of islets by introducing angiogenic, cytoprotective or immunomodulatory genes via gene transfer to islets prior to islet transplantation to improve the clinical outcome of islet transplantation in the future. However, recovery of functional islets after cryopreservation has been technically challenging, as freezing and thawing can significantly reduce the viability of islet cells (Kuo et al, 2002). Up to 50% of functional islet loss has been reported after cryopreservation (Lakey et al, 2001). Furthermore, the extent to which cryopreservation affects islet revascularization remains to be determined.

B. Enhancing islet revascularization 1. Angiogenic gene transfer to enhance islet revascularization As discussed above, rapid and sufficient islet revascularization is crucial for long-term survival and function of islet grafts post transplantation. Delayed and inadequate revascularization of newly transplanted islets can deprive islet cells of oxygen and nutrients, resulting in islet cell death and premature graft failure. Given the fact that successful islet transplantation depends on the infusion of sufficiently large amounts of islets, which usually requires at least two cadaveric pancreata per recipient, increased islet revascularization is expected to reduce the number of islets and improve the pancreas donor to recipient ratio required for transplantation. In addition, rapid and adequate islet revascularization will protect islet grafts from hypoxia-induced inflammation and necrosis, thereby improving long-term graft survival and providing better preservation of functional islet mass. However, only limited efforts have been made in the past in this aspect of islet transplantation. VEGF is known to play a pivotal role in angiogenesis / vasculogenesis. To investigate its angiogenic effect on islet revascularization, Sigrist and colleagues (2002) have applied collagen-immobilized VEGF protein in encapsulated islets, followed by transplantation into the peritoneal cavity of streptozotocininduced diabetic mice. Blood glucose and plasma insulin levels were determined and animals were sacrificed two weeks post transplantation. It was found that islets transplanted in the presence of collagen-immobilized VEGF protein show significantly increased angiogenesis and microvasculature in islet grafts, which associated with increased insulin production and improved glycemic


Zhang et al: Angiogenic Gene Therapy for Improving Islet Graft Vascularization control, in comparison to control islets that are transplanted in the absence of VEGF protein. These results suggest that local VEGF delivery to islet grafts improves the outcome of islet transplantation by enhancing islet revascularization (Sigrist et al, 2002). To improve islet graft vascularization, we have delivered the human vascular endothelial growth factor (hVEGF) cDNA by adenoviral-gene transfer to mouse islets, followed by transplantation under the renal capsule in streptozotocin-induced diabetic mice (Zhang et al, 2003). We showed that all the renal capsules containing the hVEGF vector-transduced islets (250 islets) displayed significantly higher functional islet mass, as measured by insulin immunostaining, and greater vascular density, as determined by immunostaining of CD31, the platelet endothelial cell adhesion molecule-1 (PECAM-1) (Watanabe et al, 2000). As a result, diabetic mice receiving the hVEGF vector-treated islets exhibited normoglycemia with improved glucose tolerance. In contrast, diabetic mice receiving an equivalent islet mass that were pre-transduced with a control vector maintained moderate hyperglycemia with impaired glucose tolerance. These results provide the proof-of-principle that angiogenic gene transfer to islets prior to islet transplantation allows local production of VEGF in islet grafts, which in turn stimulates graft angiogenesis and augments islet revascularization (Zhang et al, 2003). While therapeutic angiogenesis, so called biobypass, has been considered an alternative modality for treating coronary and peripheral artery diseases, based on the efficacy and safety of plasmid- or adenoviral vectormediated VEGF delivery in angiogenesis in a number of preclinical studies and clinical trials (Isner, 2002; Koransky et al, 2002; Mercadier and Logeart, 2002; Rasmussen et al, 2002; Sylven, 2002, Khan et al, 2003; Kusumanto et al, 2003), our view is that a similar

angiogenic strategy should be explored to accelerate islet graft angiogenesis, allowing rapid and adequate islet revascularization post transplantation. Such an approach, when used in conjunction with islet transplantation, has the potential for improving the success rate and clinical outcome of islet transplantation with long-term glycemic control at a reduced cost of islets.

2. Ex vivo gene delivery to islets The rationale for enhancing islet graft vascularization by angiogenic gene transfer is as follows: islets are transduced in culture with a vector expressing angiogenic molecules, such as VEGF, followed by transplantation into a diabetic subject, as illustrated schematically in Figure 2. Using an adenoviral-mediated gene delivery system, we have validated this concept by showing that VEGF production in newly transplanted islets significantly improves islet revascularization and functional islet mass (Zhang et al, 2003). It is noteworthy that adenoviral vectors are associated with immunogenecity. In addition, islets are terminally differentiated post-mitotic cells, which poses a great challenge for ex vivo gene delivery to islets by vectors whose transduction depends on cell division (Ito and Kedes, 1997; Robbins and Ghivizzani, 1998). However, recent advances in both viral and nonviral vector development have made it feasible to transfer genes to intact islets ex vivo at reasonable efficiencies without adversely affecting the architecture and function of islets. Below is a focused review of a number of vector systems that are currently in use for ex vivo gene transfer to isolated islets.

Figure 2. Schematic representation of angiogenic gene transfer in conjunction with islet transplantation. Islets are isolated and incubated in culture media in the presence of a gene vector that expresses angiogenic molecules. After transduction, islets are transplanted intraportally into the liver of a diabetic subject.


Gene Therapy and Molecular Biology Vol 7, page 159 a. Adenovirus-mediated gene transfer to islets Adenovirus is the most commonly used vector system in preclinical studies due to its relatively high transduction efficiency for both dividing and nondividing cell types. Adenovirus is capable of accommodating large DNA inserts and can be produced in a large quantity and at a relatively high titer. Although adenoviral vectors have been shown to efficiently transduce islets without altering glucose-inducible insulin secretion from ! cells (Newgard, 1994; Csete et al, 1995; O'Brien et al, 1999), recent studies indicate that adenoviral-mediated transduction of islets induces the production of a number of chemokines and their respective receptors, resulting in subsequent recruitment of inflammatory cells to islet grafts. This may potentially impair islet engraftment (Zhang et al, 2003).

transduction (Girod et al, 1999; Wu et al, 2000). Using a rAAV-5 serotype vector, Flotte et al, (2001) showed that efficient transduction of isolated murine islets could be achieved with a 100-fold lower multiplicity of infection (MOI) than rAAV-2. More recently, rAAV-2 has been pseudotyped with capsids of any one of the eight known serotypes of AAV (Gao et al, 2002; Rabinowitz et al, 2002). In these recombinant rAAV vectors, the gene of interest is inserted between the AAV-2 ITRs and packaged into the serotype-specific capsids varying from AAV-1 to AAV-8. In this way, rAAV-2 pseudotyped with AAV-1 and AAV-5 or AAV-8 capsids is shown to transduce skeletal muscle and liver at a significantly higher efficiency than the native rAAV-2 (Gao et al, 2002; Mingozzi et al, 2002; Walsh et al, 2003). Using a rAAV vector encoding the green fluorescent protein (GFP), we showed that rAAV-1 and rAAV-2 are able to effectively transduce murine and human islets in culture, respectively (Figure 3).

b. rAAV-mediated gene delivery to islets Recombinant adeno-associated virus (rAAV) has become the vector of choice for gene transfer to a variety of cell types because of its ability to mediate long-term transgene expression in the absence of cytotoxicity (Flotte et al, 2001; Kapturczak et al, 2002; Mah et al, 2002; Vizzardelli et al, 2002). The most commonly used rAAV is derived from AAV-2, an AAV serotype that belongs to a group of non-pathogenic human parvoviruses. AAV-2 contains a 4.7-kb single-stranded genome encoding viral replication (rep) and capsid (cap) genes flanked by inverted terminal repeat sequences (ITRs) (Srivastava et al, 1994). Productive replication of AAV-2 depends on adenoviral or herpes viral helper functions, in the absence of which, AAV2 establishes a "rep-dependent" latent infection by integrating its genome site-specifically into the AAVS1 site in human chromosome 19 (Kotin et al, 1992; Rabinowitz and Samulski, 1998). In rAAV-2 vectors, the entire viral coding sequences are replaced with the therapeutic gene of interest (insertion size <4.7 kb) between the two ITRs. High titer infectious viral particles are produced using an "adenovirus helper-free" system by co-transfecting a permissive cell line with the rAAV-2 shuttle plasmid and plasmids that provide the necessary helper functions as well as the Rep and Cap proteins (Kay et al, 2001). Because of the lack of immunogenecity coupled with its non-pathogenic property, rAAV-2 has not been associated with toxicity and immune response in preclinical studies and clinical trials (Kay et al, 2001). Although rAAV-2 is able to transduce both dividing and non-dividing cells, its transduction efficiency varies significantly among different cell types (Kay et al, 2001; Qing et al, 2003). While both muscle and brain cells are efficiently transduced, only about 5% of hepatocytes can be transduced. In addition, several cell types, including murine fibroblasts and human leukemia cells, are refractory to rAAV-2 transduction (Hansen et al, 2001). This observed variability in rAAV-2 mediated transduction of different cell types is associated with the heterogeneity of cell surface receptors that are required for viral entry (Srivastava et al, 2002). To improve viral infectivity and expand AAV tropism to non-permissive cells, chimeric AAVs carrying different cell-specific ligands in their capsid proteins have been shown to transduce cells that were previously refractory to rAAV-2

c. Lentivirus-mediated gene transfer to islets Lentiviruses are related to retroviruses, but unlike retroviruses, lentiviral vectors retain the ability to efficiently transduce non-dividing cells, although cell cycle activation has been shown to improve significantly the efficiency of lentiviral-mediated transduction (Vigna and Naldini, 2000; Chang and Gay, 2001). Using a reporter gene expression system encoding either green fluorescent protein or !-galactosidase, lentivirus-mediated gene transfer is shown to result in sustained transgene expression in a variety of quiescent cell types including pancreatic endocrine cells (Ju et al, 1998; Giannoukakis et al, 1999; Leibowitz et al, 1999; Curran et al, 2000, 2002). Recently, a lentiviral-mediated gene transfer system, derived originally from feline immunodeficiency virus (FIV) (Wang et al, 1999), has been developed. The tropism of FIV is feline-specific with suggestive evidence of safety in humans, as veterinarians bitten and scratched by FIV-infected cats do not display signs of seroconversion or disease (Djalilian et al, 2002). FIV can mediate stable transgene expression because its chromosomal DNA is integrated into the host genome. In the literature, FIV-mediated transgene expression persisting for up to 6 months in vivo has been reported (Wang et al, 1999; Hughes et al, 2002). To test the ability of FIV to transduce islet cells, we have used the FIV-LacZ vector to transduce freshly isolated murine islets, demonstrating that FIV is effective in transducing islets in culture (Figure 4). Furthermore, FIV-mediated transduction of islets does not perturb islet function, as the characteristic feature of glucose-inducible insulin secretion from ! cells remains unchanged before and after FIV transduction (Zhang et al, 2002). Our results are consistent with Curran et al. who recently showed that FIV vectors efficiently transduce human and murine islets in vitro (Curran et al, 2002).


Zhang et al: Angiogenic Gene Therapy for Improving Islet Graft Vascularization

Figure 3. Ex vivo transduction of murine and human islets by rAAV. Prior to exposure to rAAV, islets were incubated with a helper adenovirus (Adv-5) at an MOI of 5 pfu/cell for 2 h in CMRL-1066 medium (Sigma-Aldrich, St. Louis, MO) in a 37 _C incubator with 5% CO 2. Subsequently, islets were transduced with the rAAV-GFP vector expressing the green fluorescent protein at an MOI of 1,000 pfu/cell and visualized in a fluorescent microscope. One islet contains about 1,000 cells on average. Shown are murine islets that were mock-treated (A) and rAAV1-GFP transduced (B), as well as human islets that were mock-treated (C) and rAAV2-GFP transduced (D). . .

Figure 4. Lentiviral-mediated transduction of islets. Freshly isolated mouse islets were mock-transduced (A) and transduced with the FIV-LacZ vector at an MOI of 100 transducing units/cell (B) and stained for !-gal after 24 h of incubation in the CMRL-1066 medium. In addition, after transduction with the FIV-LacZ vector, islets were paraffin-embedded and thin-sections of embedded islets were immuno-stained for insulin (C, brown) and stained with X-gal for !-gal (D, blue). Bar, 25 Âľm.

d. Nonviral vector-mediated gene transfer to islets In addition to viral-mediated gene delivery systems, nonviral systems such as liposome-mediated transfection have been used to deliver genes to a variety of cells both in vitro and in vivo (Ledley et al, 1995). Cationic liposomes are artificial membrane vesicles that can complex with DNA. The resulting liposome-DNA complex is thought to fuse with the negatively charged plasma membrane (Felgner and Ringold, 1991) or become endocytosed (Zhou and Huang, 1994), resulting in gene delivery to the nucleus. It has been shown that islet cells in a monolayer derived from dispersed islets or intact islets can be effectively transduced using the monoliposomal reagent

Lipofectin or the polycationic liposome Lipofectamine or adenovirus-polylysine (AdpL) DNA complexes (Welsh et al, 1990; Welsh and Andersson, 1994; Saldeen et al, 1996; Benhamou et al, 1997). Recently, Mahato and colleagues (Mahato et al, 2003) reported that human islets transduced with the hVEGF gene by nonviral-mediated gene transfer resulted in sustained hVEGF production for up to 10 days post transduction. Although nonpathogenic, nonviralmediated gene transfer is in general associated with a relatively low efficiency and short duration of transgene expression (Lakey et al, 2001). It has been suggested that after liposome-mediated endocytosis, a vast majority of lipid-DNA particles are retained in the perinuclear area and subsequently degraded (Zabner et al, 1995). Thus, the 160

Gene Therapy and Molecular Biology Vol 7, page 161 failure of DNA to leave the endosomal compartment represents a major hurdle to liposome-mediated gene transfer. Nonviral-mediated gene transfer systems are of a preferred choice when persistent transgene expression is not desirable. Recently, a novel system, known as protein transduction, is being developed. Unlike gene transfer systems, this protein transduction system allows selective delivery of proteins into cells, when linked to a specific protein transduction domain (PTD). PTD is a small peptide domain that can freely cross the cytoplasmic membrane through a receptor-mediated process, which is independent of ATP (Hawiger et al, 1999; Schwarze et al, 2000). In particular, a PTD designated PTD-5, which is originally selected from an M13 phage peptide display library, has been reported to successfully transduce both human and mouse islets without significant effects on islet function (Mi et al, 2000; Rehman et al, 2003). Likewise, Embury et al, (2001) also showed that a small peptide of 11 amino acid residues that constitute the PTD of the HIV/TAT protein, when fused to !-galactosidase, is able to transduce rat islets ex vivo with the fusion protein in a dose-dependent manner at a relatively high efficiency. However, such a protein transduction system is normally associated with a transient effect, depending on the relative stability of the fusion protein. In addition, for therapeutic protein delivery, caution should be taken to ascertain that the fusion of a PTD does not adversely affect the proper folding and compromise the function of the therapeutic protein. .

expected to ensure adequate microvascular perfusion to islet cells and protect implanted islet cells from hypoxiainduced inflammation and necrosis, which will ultimately improve the outcome of islet transplantation by reducing the donor/recipient ratio thus increasing the success rate of islet transplantation.

Acknowledgement We would like to thank Marcia Meseck for critical reading of this manuscript. This project is supported partly by the Juvenile Diabetes Research Center at Mount Sinai School of Medicine.

References Ahmet I, Sawa Y, Yamaguchi T, Matsuda H (2003) Gene transfer of hepatocyte growth factor improves angiogenesis and function of chronic ischemic myocardium in canine heart. Ann Thorac Surg 75, 1283-1287. Asplin IR, Wu SM, Mathew S, Bhattacharjee G, Pizzo SV (2001) Differential regulation of the fibroblast growth factor (FGF) family by alpha(2)-macroglobulin: evidence for selective modulation of FGF-2-induced angiogenesis. Blood 97, 34503457. Bacharach E, Itin A, Keshet E (1992) In vitro patterns of expression of urokinase and its inhibitor PA-1 suggest a concerted role in regulating physiological angiogenesis. Proc Natl Acad Sci U S A. 89, 10686-10690. Beger C, Cirulli V, Vajkoczy P, Halban PA, Menger MD (1998) Vascularization of purified pancreatic islet-like cell aggregates (pseudoislets) after syngerneic transplantation. Diabetes 47, 559-565. Benhamou PY, Moriscot C, Prevost P, Rolland E, Hallmi S, Chroboczek J (1997) Standardization of procedure for efficient ex vivo gene transfer into porcine pancreatic islets with cationic liposomes. Transplantation 63, 1798-1803, . Bikfalvi A, Klein S, Pintucci G, Rifkin DB (1997) Biological roles of fibroblast growth factor-2. Endocrine Reviews 18, 26-45. Bloomgarden ZT (1998) The Epidemiology of Complications. Diabetes Care 25, 924-932. Bloomgarden ZT (2002) Treatment Issues in Type 1 Diabetes. Diabetes Care 25, 230-238. Boker A, Rothenberg L, Hernandez C, Kenyon NS, Ricordi C, Alejandro R (2001) Human islet transplantation: update. World J Surg 25, 481-486. Brissova M, Fowler M, Shiota M, Radhika A, Shostak A, Wiebe P, Gannon M, Powers A (2003) Intra-islet endothelial cells contributor to revascularization of transplanted pancreatic islets. Diabetes 52 (suppl. 1), A13. Bussolino F, Di Renzo MF, Ziche M, Bocchietto E, Olivero M, Naldini L, Gaudino G, Tamagnone L, Coffer A, Comoglio PM ( 1992) Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol 119, 629-64. Carlsson P-O, Palm F, Andersson A, Liss P (2000) Chronically decreased oxygen tension in rat pancreatic islets transplanted under the kidney capsule. Transplantation 69, 761-766. Carlsson P-O, Palm F, Andersson A, Liss P (2001) Markedly decreased oxygen tension in transplanted rat pancreatic islets irrespective of implantation sites. Diabetes 50, 489-495.

III. Conclusion Rapid re-establishment of an appropriate microvascular system in newly transplanted islets is crucial for survival and function of islet grafts. Unfortunately, islets implanted at ectopic sites, such as under the renal capsule or in the liver and spleen, are invariably associated with markedly reduced vascularization, in comparison with native islets in the pancreas (Beger et al, 1998; Mattson et al, 2002). This impairment in islet revascularization accounts at least in part for the demand of sufficiently large quantities of islet mass for restoration of normoglycemia in type 1 diabetic subjects. In addition, delayed and inadequate islet graft vascularization can deprive islets of oxygen and nutrients, causing islet cells to undergo cellular apoptosis and subsequent cell death, particularly in the core of large islets or in the center of aggregated islet clusters post transplantation. Moreover, a lack of sufficient islet revascularization may also compromise the optimal performance of transplanted islets. Indeed, there are clinical data indicating that even after postabsorptive blood glucose homeostasis is restored to normal post islet transplantation, implanted islets do not seem to function at optimal levels, as reflected in their significantly impaired glucose tolerance in diabetic recipients in response to intravenous glucose challenge (Ryan et al, 2001, 2002). Thus, it is of great significance to define the molecular mechanism of islet revascularization and develop therapeutic angiogenesis approaches to enhance the process of islet revascularization. Such approaches are 161

Zhang et al: Angiogenic Gene Therapy for Improving Islet Graft Vascularization Chang LJ, Gay EE (2001) The molecular genetics of lentiviral vectors-current and future perspectives. Curr Gene Ther 1, 237-251. Chaturvedi N, Bandinelli S, Mangili R, Penno G, Rottiers RE, Fuller JH (2000) Microalbuminuria in type 1 diabetes: rates, risk factors and glycemic threshold. Kidney Int 60, 219â&#x20AC;&#x201C;227. Chegini N (1997) The role of growth factors in peritoneal healing: transforming growth factor beta (TGF-beta). Eur J Surg Suppl 577, 17-23 . Cote I, Rogers NJ, Lechler RI (2001) Allorecognition. Transfus Clin Biol. 8, 318-323. Csete ME, Benhamou PY, Drazan KE, Wu L, McIntee DF, Afra R, Mullen Y, Busuttil RW, Shaked A (1995) Efficient gene transfer to pancreatic islets mediated by adenoviral vectors. Transplantation 59, 263-269. Curran MA, Kaiser SM, Achacoso PL, Nolan GP (2000) Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors. Mol Ther 1, 31-38. Curran MA, Ochoa MS, Molano RD, Pileggi A, Inverardi L, Kenyon NS, Nolan GP, Ricordi C, Fenjves ES (2002) Efficient transduction of pancreatic islets by feline immunodeficiency virus 1 vectors. Transplantation 74, 299306. Ding S, Merkulova-Rainon T, Han ZC, Tobelem G (2003) HGF receptor up-regulation contributes to the angiogenic phenotype of human endothelial cells and promotes angiogenesis in vitro. Blood 101, 4816-4822. Djalilian HR, Tsuboi Y, Ozeki M, Tekin M, Djalilian AR, Obritch W, Lin J (2002) Feline immunodeficiency virusmediated gene therapy of middle ear mucosa cells. Auris Nasus Larynx 29, 183-186. Eckhard M, Erb D, Bretzel R, Brendel M, Linn T (2003) Inhibition of angiogenesis by immunosuppressive agents used in clinical transplantation. Diabetes 52 (supppl. 1), A14. Embury J, Klein D, Pileggi A, Ribeiro M, Jayaraman S, Molano RD, Fraker C, Kenyon N, Ricordi C, Inverardi L, Pastori RL (2001) Proteins linked to a protein transduction domain efficiently transduce pancreatic islets. Diabetes 50, 17061713. Felgner PL, Ringold GM (1991) Cationic liposome-mediated transfection. Nature 337, 387-388. Ferrara N, Davis-Smyth T (1997) The biology of vascular endothelial growth factor. Endocrine Rev 18, 4-25, . Flotte T, Agarwal A, Wang J, Song S, Fenjves ES, Inverardi L, Chesnut K, Afione S, Loiler S, Wasserfall C, Kapturczak M, Ellis T, Nick H, Atkinson M (2001) Efficient ex vivo transduction of pancreatic islet cells with recombinant adenoassociated virus vectors. Diabetes 50, 515-520. Gaber AO, Fraga DW, Callicutt CS, Gerling IC, Sabek OM, Kotb MY (2001) Improved in vivo pancreatic islet function after prolonged in vitro islet culture. Transplantation 72, 1730-1736. Gale EAM (2002) The Rise of Childhood Type 1 Diabetes in the 20th Century. Diabetes 51, 3353â&#x20AC;&#x201C;3361. Gao GP, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM (2002) Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A. 99, 11854-11859. Garcia-Ocana A, Takane KK, Reddy VT, Lopez-Talavera JC, Vasavada RC, Stewart AF (2003) Adenovirus-mediated hepatocyte growth factor expression in mouse islets

improves pancreatic islet transplant performance and reduces beta cell death. J Biol Chem 278, 343-351. Giannoukakis N, Mi Z, Gambotto A, Eramo A, Ricordi C, Trucco M, Robbins PD (1999) Infection of intact human islets by a lentiviral vector. Gene Ther 6, 1545-1551. Gille J, Swerlick RA, Caughman SW (1997) Transforming growth factor-alpha-induced transcriptional activation of the vascular permeability factor (VPF/VEGF) gene requires AP2-dependent DNA binding and transactivation. EMBO J 16, 750-759 . Girod A, Ried M, Wobus C, Lahm H, Leike K, Kleinschmidt J, Deleage G, Hallek M (1999) Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2. Nat Med 5, 1052-1056. Hansen J, Qing K, Srivastava A. (2001) Adeno-associated virus type 2-mediated gene transfer: altered endocytic processing enhances transduction efficiency in murine fibroblasts. J Virol 75, 4080-4090. Hawiger J (1999) Noninvasive intracellular delivery of functional peptides and proteins. Curr. Opin. Chem. Biol. 3, 89-94. Hellerstrom C, Andersson A, Korsgren O, Jansson L, S. S (1898) Aspects of pancreatic islet transplantation in diabetes mellitus. Baillieres Clin Gastroenterol. 3, 851-863. Heuser M, Wolf B, Vollmar B, Menger MD (2000) Exocrine contamination of islolated islets of Langerhans deteriorates the process of revascularization after free transplantation. Transplantation 69, 756-761. Hughes SM, Moussavi-Harami F, Sauter SL, Davidson BL (2002) Viral-mediated gene transfer to mouse primary neural progenitor cells. Mol Ther 5, 16-22. Isner JM (2002) Myocardial gene therapy. Nature 415, 234-239. Ito M, Kedes L (1997) Two-step delivery of retroviruses to postmitotic, terminally differentiated cells. Hum Gene Ther 8, 57-63. Jansson L, Carlsson P-O (2002) Graft vascular functioin after transplantation of pancreatic islets. Diabetologia 45, 749763. Ju Q, Edelstein D, Brendel MD, Brandhorst D, Brandhorst H, Bretzel RG, Brownlee M (1998) Transduction of nondividing adult human pancreatic beta cells by an integrating lentiviral vector. Diabetologia. 41, 736-739. Kapturczak M, Zolotukhin S, Cross J, Pileggi A, Molano RD, Jorgensen M, Byrne B, Flotte TR, Ellis T, Inverardi L, Ricordi C, Nick H, Atkinson M, Agarwal A (2002) Transduction of human and mouse pancreatic islet cells using a bicistronic recombinant adeno-associated viral vector. Mol Ther 5, 154-160. Karvonen M, Viik-Kajander M, Moltchanova E, Libman I, LaPorte R, Tuomilehto J (2000) Incidence of childhood type 1 diabetes worldwide. Diabetes Mondiale (DiaMond) Project Group. Diabetes Care. 23, 1516-1526. Kawakami Y, Iwata H, Gu YJ, Miyamoto M, Murakami Y, Balamurugan AN, Imamura M, Inoue K (2001) Successful subcutaneous pancreatic islet transplantation using an angiogenic growth factor-releasing device. Pancreas 23, 375-381. Kay MA, Glorioso JC, Naldini L (2001) Viral vectors for gene therapy: the art of turning infectious agents into vehiches of therapeutics. Nat Med 7, 33-40. Khan TA, Sellke FW, Laham RJ (2003) Gene therapy progress and prospects: therapeutic angiogenesis for limb and myocardial ischemia. Gene Ther 10, 285-291.


Gene Therapy and Molecular Biology Vol 7, page 163 Kim I, Kim HG, Moon SO, Chae SW, So JN, Koh KN, Ahn BC, Koh GY (2000) Angiopoietin-1 induces endothelial cell sprouting through the activation of focal adhesion kinase and plasmin secretion. Circ Res 86, 952-959. Koransky ML, Robbins RC, Blau HM (2002) VEGF gene delivery for treatment of ischemic cardiovascular disease. Trends Cardiovasc Med 12, 108-114. Korsgren O, Jansson L, AA (1989) Effects of hyperglycemia on function of isolated mouse pancreatic islets transplanted under kidney capsule. Diabetes 38, 510-515. Kotin RM, Linden RM, Berns KI (1992) Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination. EMBO J. 11, 5071-5078. Kuo CH, Juang JH, Hsu BR, Lu WT, Yao NK (2002) In vitro and in vivo studies on the cryopreserved pancreatic islets. Transplant Proc 34, 2693-2695. Kusumanto YH, Hospers GA, Mulder NH, Tio RA (2003) Therapeutic angiogenesis with vascular endothelial growth factor in peripheral and coronary artery disease: a review. Int J Cardiovasc Intervent. 5, 27-34. Kuttler B, Hartmann A, Wanka H (2002) Long-term culture of islets abrogates cytokine-induced or lymphocyte-induced increase of antigen expression on beta cells. Transplantation 74, 440-445. Laing SP, Swerdlow AJ, Slater SD, Burden AC, Morris A, Waugh NR, Gatling W, Bingley PJ, Patterson CC (2003) Mortality from heart disease in a cohort of 23,000 patients with insulin-treated diabetes. Diabetologia 760-765. Lakey JR, Anderson TJ, Rajotte RV (2001) Novel approaches to cryopreservation of human pancreatic islets. Transplantation 72, 1005-1011. Lakey JRT, Young ATL, Pardue D, Calvin S, Albertson TE, Jacobson L, Cavanagh TJ (2001) Non-viral transfection of intact pancreatic islets. Cell Transplant 10, 697-708. Lechler RI, Garden OA, Turka LA (2003) The complementary roles of deletion and regulation in transplantation tolerance. Nat Rev Immunol 2, 147-158. Lechner A, Habener JF (2003) Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus. Am J Physiol Endocrinol Metab 284, E259-266. Ledley FD (1995) Nonviral gene therapy: the promise of genes as pharmaceutical products. Hum Gene Ther 6, 1129-1144. Leibowitz G, Beattie GM, Kafri T, Cirulli V, Lopez AD, Hayek A, Levine F (1999) Gene transfer to human pancreatic endocrine cells using viral vectors. Diabetes 48, 745-753. Li J, Zhang YP, Kirsner RS (2003) Angiogenesis in wound repair: angiogenic growth factors and the extracellular matrix. Microsc ResTech 60, 107-114. Linn T, Schneider K, Hammes HP, Preissner KT, Brandhorst D, Morgenstern E, Kiefer F, Bretzel RG (2003) Angiogenic capacity of endothelial cells in islets of Langerhans. FASEB J. 17, 881-883. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R (2001) Differentiation of embryonic stem cells to insulinsecreting structures similar to pancreatic islets. Science 292, 1389-1394. Mah C, Byrne BJ, Flotte TR (2002) Virus-based gene delivery systems. Clin Pharmacokinet 41, 901-911. Mahato RI, Henry J, Narang AS, Sabek O, Fraga D, Kotb M, Gaber AO (2003) Cationic lipid and polymer-based gene delivery to human pancreatic islets. Mol Ther 7, 89-100.

Mandriota SJ, Seghezzi G, Vassalli J-D, Ferrara N, Wasi S, Mazzieri R, Mignatti P, Pepper MS (1995) Vascular endothelial growth factor increases urokinase receptor expression in vascular endothelial cells. J Biol Chem 270, 9709-9716. Mattson G, Jansson L, Carlsson P-O (2002) Decreased vascular density in mouse pancreatic islets after transplantation. Diabetes 51, 1362-1366. Mendola JF, Goity C, Esmatjes E, Seanz A, Fernandez-Cruz L, Gomis R (1997) Cyclosporine does not inhibit the process of vascularization of pancreatic islet transplantation. Cell Transplant 6, 69-76 . Menger MD, Vajkoczy P, Leiderer R, Jager S, Messmer K (1992) Influence of experimental hyperglycemia on microvascular blood perfusion of pancreatic islet isografts. J Clin Invest 90, 1361-1369. Menger MD, Yamauchi J-I, Vollmar B (2001) Revascularization and microcirculation of freshly grafted islets of Langerhans. World J Surg 25, 509-515. Mercadier JJ, Logeart D (2002) Mycardial gene therapy. Arch. Mal. Coeur. Vaiss. 95, 197-203. Mi Z, Mai J, Lu X, Robbins PD (2000) Characterization of a class of cationic peptides able to facilitate efficient protein transduction in vitro and in vivo. Mol Ther 2, 339-347. Mingozzi F, Schuttrumpf J, Arruda VR, Liu Y, Liu YL, High KA, Xiao W, Herzog RW (2002) Improved hepatic gene transfer by using adeno-associated virus serotype 5 vector. J Virol 76, 10497-10502. Murphy NP, Keane SM, Ong KK, Ford-Adams M, Edge JA, Acerini CL, Dunger DB (2003) Randomized Cross-Over Trial of Insulin Glargine Plus Lispro or NPH Insulin Plus Regular Human Insulin in Adolescents With Type 1 Diabetes on Intensive Insulin Regimens. Diabetes Care 26, 799-804. Newgard CB (1994) Cellular engineering and gene therapy strategies for insulin replacement in diabetes. Diabetes 43, 341-350. O'Brien T, Karlsen AE, Andersen HU, Mandrup-Poulsen T, Nerup J (1999) Absence of toxicity associated with adenoviral-mediated transfer of the !-galactosidase reporter gene to neonatal rat islets in vitro. Diab Res & Clin Pract 44, 157-163. Otani A, Takagi H, Oh H, Koyama S, Matsumura M, Honda Y (1999) Expressions of angiopoietins and Tie2 in human choroidal neovascular membranes. Invest Ophthalmol Vis Sci 40, 1912-1920. Pepper MS, Matsumoto K, Nakamura T, Orci L, Montesano R (1992) Hepatocyte growth factor increases urokinase-type plasminogen activator (uPA) and u-PA receptor expression on Madin-Darby canine kidney epithelial cells. J Biol Chem 267, 20493-20496 . Pepper MS, Sappino A-P, Stocklin R, Montesano R, Orci L, Vassalli J-D (1993) Upregulation of urokinase receptor expression on migrating endothelial cells. J Cell Biol 122, 673-684 . Plank J, Wutte A, Brunner G, Siebenhofer A, Semlitsch B, Sommer R, Hirschberger S, Pieber TR (2002) A direct comparison of insulin aspart and insulin lispro in patients with type 1 diabetes. Diabetes Care. 25, 2053-2057. Qing K, Li W, Zhong L, Tan M, Hansen J, Weigel-Kelley KA, Chen L, Yode MC, Srivastava A (2003) Adeno-associated virus type 2-mediated gene transfer: role of cellular T-cell protein tyrosine phosphatase in transgene expression in established cell lines in vitro and transgenic mice in vivo. J Virol 77, 2741-2746.


Zhang et al: Angiogenic Gene Therapy for Improving Islet Graft Vascularization Rabinowitz JE, Rolling F, Li C, Conrath H, Xiao W, Xiao X, Samulski RJ (2002) Cross-packaging of a single adenoassociated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. J Virol 76, 791-801. Rabinowitz JE, Samulski J (1998) Adeno-associated virus expression systems for gene transfer. Curr. Opin. Biotechnol. 9, 470-475. Rasmussen HS, Rasmussen CS, Macko J (2002) VEGF gene therapy for coronary artery disease and peripheral vascular disease. Cardiovasc. Radiot. Med. 3, 114-117. Rehman KK, Bertera S, Bottino R, Balamurugan AN, Mai JC, Mi Z, Trucco M, Robbins PD (2003) Protection of islets by in situ peptide-mediated transduction of the I#B kinase inhibitor nemo-binding domain peptide. J Biol Chem 278, 9862-9868. Robbins PD, Ghivizzani SC (1998) Viral vectors for gene therapy. Pharmacol Ther. 80, 35-47. Rosen EM, Lamszuz K, Laterra J, Polverini PJ, Rubin JS, Goldberg ID (1997) HGF/SF in angiiogenesis. Ciba Found Symp 212, 215-226, . Ryan EA, Lakey JR, Paty BW, Imes S, Korbutt GS, Knetema NM, Bigam D, Rajotte RV, Shapiro AM (2002) Successful islet transplantation: continued insulin reserve provides longterm glycemic control. Diabetes 51, 2148-2157. Ryan EA, Lakey JR, Rajotte RV, Korbutt GS, Kin T, Imes S, Rabinovitch A, Elliott JF, Bigam D, Kneteman NM, Warnock GL, Larsen I, Shapiro AM (2001) Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 50, 710-179. Saksela O, Rifkin DB (1988) Cell-associated plasminogen activation: regulation and physiologic functions. Ann Rev Cell Biol 4, 93-126, . Saldeen J, Curiel DT, Eizirik DL, Andersson A, Strandell E, Buschard K, Welsh N (1996) Efficient gene transfer to dispersed human pancreatic islet cells in vitro using adenovirus-polylysine/DNA complexes or polycationic liposomes. Diabetes 45, 1197-1203. Schwarze SR, Hruska KA, Dowdy SF (2000) Protein transduction: unrestricted delivery into all cells? Trends Cell Biol 10, 290-295. Sengupta S, Gherardi E, Sellers LA, Wood JM, Sasisekharan R, Fan TP (2003) Hepatocyte growth factor/scatter factor can induce angiogenesis independently of vascular endothelial growth factor. Arterioscher Thromb Vasc Biol 23, 69-75. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV (2000) Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343, 230-238. Sigrist S, Mechine A, Leenders H, Calanda V, Laurence K (2002) Influence of VEGF on the viability of encapsulated pancreatic rat islets after transplantation in diabetic mice. Diabetes 51 (suppl. 2), A10. Soria B, Roche E, Berma G, Leon-Quinto T, Reig J, Martin F (2001) Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 49, 157-162. Srivastava A (1994) Parvovirus-based vectors for human gene therapy. Blood Cells 20, 531-536. Srivastava A (2002) Obstacles to human hematopoietic stem cell transduction by recombinant adeno-associated virus 2 vectors. J Cell Biochem (Suppl.) 38, 39-45.

Sylven C (2002) Angiogenic gene therapy. Drugs Today 38, 819-827. Thurston G, Suri C, Smith K, McClain J, Sato TN, Yancopoulos GD, McDonald DM (1999) Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science 286, 2511-2514. Tokuda H, Hatakeyama D, Akamatsu S, Tanabe K, Yoshida M, Shibata T, Kozawa O (2003) Involvement of MAP kinases in TGF-beta-stimulated vascular endothelial growth factor synthesis in osteoblasts. Arch Biochem Biophys 415, 117125. Vague P, Selam JL, Skeie S, De Leeuw I, Elte JW, Haahr H, Kristensen A, Draeger E (2003) Insulin detemir is associated with more predictable glycemic control and reduced risk of hypoglycemia than NPH insulin in patients with type 1 diabetes on a basal-bolus regimen with premeal insulin aspart. Diabetes Care 26, 590-596. Vajkoczy P, Vollmar B, Wolf B, Menger MD (1999) Effects of cyclosporine A on the process of vascularization of freely transplanted islets of Langerhans. J Mol Med 77, 111-114. Valle D, Santoro D, Bates P, Scarpa L (2001) Italian multicentre study of intensive therapy with insulin lispro in 1184 patients with Type 1 diabetes. Diabetes Nutr. Metab. 14, 126-132. Vasir B, Jonas JC, Steil GM, Hollister-Lock J, Hasenkamp W, Sharma A, S. B-W, Weir GC (2001) Gene expression of VEGF and its receptors Flk-1/KDR and Flt-1 in cultured and transplanted rat islets. Transplantation 71, 924-935. Vasir B, Reitz P, Xu G, Sharma A, Bonner-Weir S, Weir GC (2000) Effects of diabetes and hypoxia on gene markers of angiogenesis (HGF, cMet, uPA adn uPAR, TGF-", TGF-!, bFGF and vimentin) in cultured and transplanted rat islets. Diabetologia 43, 763-772. Vigna E, Naldini L (2000) Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy. J. Gene Med. 2, 308-316. Vizzardelli C, Molano RD, Pileggi A, Berney T, Cattan P, Fenjves ES, Peel A, Fraker C, Ricordi C, Inverardi L (2002) Neonatal porcine pancreatic cell clusters as a potential source for transplantation in humans: characterization of proliferation, apoptosis, xenoantigen expression and gene delivery with recombinant AAV. Xenotransplantation 9, 14-24 . Waldmann H (2002) Reprogramming the immune system. Immunol Rev 185, 227-235. Walsh CE (2003) Gene therapy Progress and Prospects: Gene therapy for the hemophilias. Gene Ther 10, 999-1003. Wang G, Slepushkin V, Zabner J, Keshavjee S, Johnston JC, Sauter SJ, Jolly DJ, Dubensky TWJ, Davidson BL, McCray PBJ (1999) Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect. J Clin Invest 104, R55-62. Watanabe H, Sumi S, Urushihata T, Kitamura Y, Iwasaki S, Xu G, Yano S, Nio Y, Tamura K (2000) Immunohistochemical studies on vascular endothelial growth factor and platelet endothelial cell adhesion molecule-1/CD-31 in islet transplantation. Pancreas 21, 165-173. Weidner KM, Hartmann G, Sachs M, Birchmeier W (1993) Properties and functions of scatter factor/hepatocyte growth factor and its receptor c-Met. Am J Respir Cell Mol Biol 8, 229-237, . Welsh M, Andersson A (1994) Transplantation of transfected pancreatic islets. Stimulation of beta cell DNA synthesis by the src oncogene. Transplantation 57, 297-299.


Gene Therapy and Molecular Biology Vol 7, page 165 Welsh N, Oberg C, Hellerstrom C, Welsh M (1990) Liposome mediated in vitro transfection of pancreatic islet cells. Biomed Biochim Acta 12, 1157-1164. Wu P, Xiao W, Conlon T, Hughes J, Agbandje-McKenna M, Ferkol T, Flotte T, Muzyczka N (2000) Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism. J Virol 74, 8635-8647. Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J (2000) Vascular-specific growth factors and blood vessel formation. Nature 407, 242-248. Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ (1995) Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem, 18997-19007. Zhang N, Richter A, Suriawinata J, Altomonte J, Meseck M, Dong H (2003) Elevated VEGF production in islet cells enhances islet graft vascularization and improves functional islet mass post transplantation. Diabetes 52 (suppl. 1), A14. Zhang N, Schroppel B, Chen D, Fu S, Hudkins KL, Zhang H, Murphy BM, Sung RS, Bromberg JS (2003) Adenovirus transduction induces expression of multiple chemokines and chemokine receptors in murine ! cells and pancreatic islets. Am. J. Transplantation. In press.

Zhang N, Suriawinata J, Meseck M, Woo SLC, Dong H (2002) Effective ex vivo transduction of murine islets by feline immunodeficiency virus vectors. Adv. Islet Cell Biol: From stem cell differentiation to clinical transplantation. Anaheim, CA., p67. Zhou X, Huang L (1994) DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action. Biochem Biophys Acta 1189, 195203.

Dr. Hengjiang Dong .



Zhang et al: Angiogenic Gene Therapy for Improving Islet Graft Vascularization


Gene Therapy and Molecular Biology Vol 7, page 167 Gene Ther Mol Biol Vol 7, 167-172, 2003

G-CSF Receptor-mediated STAT3 activation and granulocyte differentiation in 32D cells Research Article

Ruifang Xu1, Akihiro Kume1, Yutaka Hanazono1, Kant M. Matsuda1, Yasuji Ueda2, Mamoru Hasegawa2, Fumimaro Takaku1,3 and Keiya Ozawa1,3 1

Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Tochigi 329-0498, Japan, 2 DNAVEC Research Inc., 1-25-11 Kannondai, Tsukuba, Ibaraki 305-0856, Japan, 3 Division of Hematology, Department of Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Tochigi 329-0498, Japan

__________________________________________________________________________________ *Correspondence: Akihiro Kume, M.D., Ph.D.; Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Tochigi 329-0498, Japan; Phone: +81-285-58-7402; Fax: +81-285-44-8675; E-mail: Key words: STAT3, G-CSF receptor, granulocyte differentiation, estrogen binding domain, selective amplifier gene Received: 3 July 2003; Accepted: 20 August 2003; electronically published: August 2003

Summary Granulocyte colony-stimulating factor (G-CSF) receptor (GcR) mediates growth and differentiation signals in the granulocyte/monocyte lineage of hematopoietic cells. To investigate the differentiation signal via GcR, a conditional receptor activation system was constructed. Wild-type and mutant GcRs were controlled by fusion to a molecular switch derived from the hormone binding domain of the estrogen receptor (ER). GcR-associated signaling molecules were analyzed in 32D progenitor cells that possess a potential of granulocyte differentiation. While the wild-type GcR-ER fusion molecule induced a granulocyte differentiation in 32D cells, a substitution of phenylalanine for tyrosine 703 (Y703F) in GcR resulted in a differentiation block. The activation of the JAK1 and JAK2 kinases was indistinguishable between the cells expressing the wild-type fusion and the Y703F mutant, and phosphorylation of the STAT5 transcription factor was comparable, too. On the other hand, tyrosine phosphorylation of STAT3 was significantly decreased following activation of the Y703F mutant compared to the wild-type GcR fusion. The results suggested that tyrosine 703 was responsible, at least in part, for transmitting a differentiation signal via STAT3 in 32D. The fusion system with the estrogen binding domain provides a valuable tool to analyze mutant effector proteins in the natural cellular milieu while bypassing the endogenous counterparts. GcR-derived growth signal upon binding to estrogen (Mattioni et al, 1994). Besides the prototype SAG encoding a chimera of the full-length GcR and ER-HBD (GcRER), a series of derivative fusion receptors were constructed to attain altered ligand specificity and signal characteristics. The modifications include a deletion of the G-CSF binding site (!GcR) (Ito et al, 1997), replacement of the ER with a mutant specific for 4-hydroxytamoxifen (TmR) (Xu et al, 1999), and the substitution of phenylalanine for the most proximal tyrosine residue in the GcR cytoplasmic domain (Y703FGcR) (Matsuda et al, 1999a). The Y703F mutant is of particular interest because this amino acid substitution apparently led to a differentiation block in myeloid progenitor 32D cells (Matsuda et al, 1999a). To explore the mechanisms of granulocyte differentiation in 32D cells, we examined

I. Introduction Recent advances in stem cell biology, together with gene transfer technology, have led to the prospect of a new generation of cell therapy. However, many obstacles must be overcome before this vision becomes a reality. One major hurdle is to control transplanted cells in the recipient’s body, in particular, to expand the desired cell subsets so that they exhibit therapeutic benefit. We have developed a novel system for selective expansion of genetically modified cells to supplement current gene transfer vectors (Ito et al, 1997; Kume et al, 2002). In this system, the target cells are harnessed with a ‘selective amplifier gene (SAG)’ which encodes a fusion protein comprising the granulocyte colony-stimulating factor (GCSF) receptor (GcR) and the hormone binding domain (HBD) of the estrogen receptor (ER). The ER-HBD works as a molecular switch so that the fusion protein generates a


Xu et al: G-CSF receptor-mediated STAT3 activation JAK-STAT pathways involved in GcR signaling, and identified reduced STAT3 phosphorylation associated with the Y703F mutation.

III. Results A. Construction of conditionally activated G-CSF receptors Structures of the chimeric receptors used in this study are shown in Figure 1. The fusion protein system is based on the fact that ER-HBD functions as an estrogenspecific molecular switch to control heterologous effector proteins, in our case, GcR (Mattioni et al, 1994). GcR belongs to the type I cytokine receptor superfamily, and its cytoplasmic domain comprises functionally distinct subdomains: the membrane-proximal region is sufficient for mitogenic signaling, and the membrane-distal portion is essential for granulocyte maturation (Dong et al, 1993; Fukunaga et al, 1993; Avalos, 1996; Koay and Sartorelli, 1999). All of the four conserved tyrosine residues in the cytoplasmic domain of GcR (at positions 703, 728, 743 and 763 in the murine GcR) are in the membrane-distal region and phosphorylated upon G-CSF stimulation. Among these, the tyrosine at position 703 (Y703) was most prominently phosphorylated and involved in granulocyte differentiation (Yoshikawa et al, 1995). However, previous studies on functional domains of GcR were carried out with ectopically expressed wild-type and mutant molecules in receptor-negative cells. It may be more informative if mutant receptors are analyzed in the natural intracellular environment where the endogenous molecule functions. From this viewpoint, the ER-HBD fusion system provides a valuable experimental tool. Estrogen specifically activates the introduced GcRER (and its derivatives) without influencing the endogenous GcR in the same cell, and the downstream events can be studied independently.

II. Materials and methods A. Plasmids and cells Bicistronic vector plasmids were constructed with the pMX retrovirus backbone and the encephalomyocarditis virus (EMCV)-derived internal ribosome entry site (IRES; nucleotides 259-833 of EMCV-R genome) (Duke et al, 1992; Onishi et al, 1996). pMX/!GcRER-IRES-CD8a encodes a fusion protein of !GcR and ER-HBD, and murine CD8a as a selectable marker (Fukunaga et al, 1991; Koike et al, 1987; Nakauchi et al, 1985). The Y703F mutation in the GcR part was introduced into this plasmid as previously described (pMX/!Y703FGcRER-IRESCD8a) (Matsuda et al, 1999a). The recombinant DNA experiments were carried out following the National Institutes of Health guidelines and approved by the Jichi Medical School Recombinant DNA Research Advisory Board. The murine myeloid progenitor line 32D and its derivatives were maintained in RPMI-1640 medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (Bioserum, Victoria, Australia) and 0.5% conditioned medium of C3H10T1/2 cells transfected with a murine IL-3 expression plasmid pBMG-hph-IL-3 (Valtieri et al, 1987; Matsuda et al, 1999a; Xu et al, 1999).

B. Immunoprecipitation and western blotting 32D cells were deprived of serum and IL-3 for 3 hours at a density of 5 x 105 cells/ml, and incubated in RPMI medium containing 1 mM Na3OV4 for an additional 1 hour at 1 x 107 cells/ml. After starvation, cells were stimulated with either 10-7 M E 2 (Sigma, St. Louis, MO) or 10-9 M recombinant human GCSF (provided by Chugai Pharmaceuticals, Tokyo, Japan) for given periods, then washed with ice-cold phosphate-buffered saline (PBS) containing 100 µM Na3OV4. Subsequently, cells were solubilized in lysis buffer (1% NP-40, 20 mM Tris-HCl [pH 7.4], 137 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin and 2 mM Na3OV4) on ice for 30 minutes, and centrifuged for 10 minutes. The soluble proteins were measured by Protein Assay (Bio-Rad, Hercules, CA). For immunoprecipitation, the cell lysate containing 1 mg of protein was incubated with one of the following antibodies for 8 hours at 4°C: anti-JAK1 (Upstate Biotechnology, Lake Placid, NY), anti-JAK2 (Upstate Biotechnology), anti-STAT3 (C-20; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-STAT5 (C17; Santa Cruz Biotechnology). The immune complexes were absorbed by protein G-Sepharose beads (Sigma) for 2 hours at 4°C. The beads were washed with the lysis buffer and boiled in sample buffer (60 mM Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate [SDS], 10% glycerol and 5% 2-mercaptoethanol) for 3 minutes. After centrifugation, the supernatants were subjected to SDS-7.5% polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride membranes (Immobilon-P; Millipore, Yonezawa, Japan). After blocking treatment with 5% bovine serum albumin (Fraction V; Roche Diagnostics, Mannheim, Germany), the membranes were incubated with an antiphosphotyrosine antibody (4G10; Upstate Biotechnology) for 1 hour at room temperature. Immunoreactive proteins were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Little Chalfont, UK). In some instances, membranes were stripped by incubation in denaturing buffer (62.5 mM Tris-HCl [pH 6.7], 2% SDS and 100 mM 2mercaptoethanol) for 30 minutes at 50°C and reprobed with another antibody.

Figure 1. Structures of the chimeric receptors involved in this study. GcRER is a fusion of the full-length murine granulocyte colony-stimulating factor (G-CSF) receptor (GcR) and the hormone binding domain (HBD) of rat estrogen receptor (ER). !GcRER is a derivative of GcRER deleted of the G-CSF binding site (amino acids 5-195). !Y703FGcRER carries a substitution of phenylalanine for a cytoplasmic tyrosine at position 703 (Y703F) in GcR. Ext, extracellular domain; G, G-CSF binding site; TM, transmembrane domain; Cyt, cytoplasmic domain; TA, transactivation domain; DNA, DNA binding domain; YYYY, conserved tyrosine residues in GcR cytoplasmic domain; FYYY, Y703F mutation in GcR.


Gene Therapy and Molecular Biology Vol 7, page 169 In our previous report, the biological response to the !GcRER- and !Y703FGcRER-mediated signal was evaluated in murine myeloid progenitor 32D cells (! designates a deletion of amino acids 5-195 required for GCSF binding; Matsuda et al, 1999a). Parental 32D cells are dependent on interleukin-3 (IL-3) for continuous growth, and switching from IL-3 to G-CSF makes the cells differentiate into morphologically mature neutrophils (Valtieri et al, 1987). By retrovirus-mediated gene transfer, stable clones expressing !GcRER (32D/!GcRER) or !Y703FGcRER (32D/!Y703FGcRER) were established and stimulated by estrogen. While estrogen-treated 32D/!GcRER cells underwent granulocyte differentiation indistinguishable from that seen in G-CSF-treated cells, 32D/!Y703FGcRER cells showed a distinct phenotype. Estrogen supported a longterm proliferation of 32D/!Y703FGcRER with myeloblastic appearance, indicating that the Y703F mutation abrogated the differentiation signal (Matsuda et al, 1999a). This observation prompted us to characterize signaling molecules downstream of GcR in more detail. Following ligand-induced homodimerization, GcR induces a wide array of intracellular signaling events (Avalos, 1996). Like many other cytokine receptors, GcR has no intrinsic kinase activity; instead, it recruits and activates other cytoplasmic kinases such as Janus kinases (JAKs), signal transducer and activation of transcription (STAT) proteins, Src family kinases and components of the mitogen-activated protein kinase pathway. The activation of JAKs is one of the earliest events in the GcR signaling cascade, followed by the tyrosine phosphorylation of STATs and GcR itself (Nicholson et al, 1994; Dong et al, 1995). Since the signal transduction for granulocyte differentiation has been ascribed to the JAKSTAT pathway, we focused on these molecules in !GcRER and !Y703FGcRER cells.

JAK1/JAK2 phosphorylation were comparable whether the cells were stimulated with G-CSF or estrogen. As shown in Figure 2, the levels of estrogen-induced JAK1/JAK2 phosphorylation in 32D/!Y703FGcRER cells were comparable to those seen in 32D/!GcRER cells. Reprobing of the blots with anti-JAK1 and anti-JAK2 antibodies showed that approximately equal amounts of the kinases were loaded on these lanes (not shown). Thus, we concluded that the Y703F mutation had little, if any, effect on the tyrosine phosphorylation of JAK1 and JAK2. Considering that JAK1 and JAK2 are constitutively associated with the membrane-proximal region of GcR which is sufficient to activate them (Nicholson et al, 1994; Dong et al, 1995; Avalos, 1996), it is conceivable that the kinases were not affected by the GcR mutation in the membrane-distal region.

C. Comparable STAT5 phosphorylation following fusion receptor activation Next, we investigated the activation of STAT proteins in 32D/!GcRER and 32D/!Y703FGcRER cells. It was shown that G-CSF-induced signaling involves STAT1, STAT3 and STAT5 (Tian et al, 1994; de Koning et al, 1996; Tian et al, 1996; Shimozaki et al, 1997; Dong et al, 1998; Chakraborty et al, 1999; Ward et al, 1999). Since the membrane-distal cytoplasmic region of GcR was not required for STAT1 activation (de Koning et al., 1996), we addressed whether the phosphorylation of STAT5 and STAT3 is affected by the Y703F mutation. Figure 3 shows the time course of STAT5 activation in 32D/!GcRER and 32D/!Y703FGcRER cells (upper panel). STAT5 was not tyrosine-phosphorylated in unstimulated 32D cells, and addition of 10-9 M G-CSF induced a rapid phosphorylation of this molecule through crosslinking of the endogenous GcR. On the other hand, 10-7 M of E2 induced a slower and less extensive phosphorylation of STAT5.

B. Estrogen-induced phosphorylation of JAK1 and JAK2 via fusion receptors First, we examined the tyrosine phosphorylation of JAK1 and JAK2. As shown in Figure 2, these kinases were not tyrosine-phosphorylated in resting 32D/!GcRER and 32D/!Y703FGcRER cells. Addition of G-CSF rapidly induced phosphorylation of JAK1 and JAK2; this event was induced by dimerization of the endogenous GcR, and maximal activation was observed within 10 minutes (data not shown). Similarly, 10-7 M 17"-estradiol (E2) induced tyrosine phosphorylation of JAK1 and JAK2 in these cells (Figure 2). The estrogen-induced activation of JAK1 and JAK2 was mediated by chimeric receptors, at a slower rate than the activation mediated by the endogenous GcR; the maximal phosphorylation was observed 60 minutes after E2 addition (time course not shown). The difference in kinetics of JAK1/JAK2 phosphorylation may be due to different mechanisms of receptor activation. While G-CSF directly crosslinks GcR at the extracellular domain, the activation of ER-HBD fusion receptors is a ligand-induced derepression that involves other proteins such as HSP90 (Mattioni et al, 1994). Nevertheless, the levels of

Figure 2. Tyrosine phosphorylation of JAK1 and JAK2. Serumand cytokine-starved 32D/!GcRER and 32D/!Y703FGcRER cells were harvested before (0â&#x20AC;&#x2122;) and after 60 minutes (60â&#x20AC;&#x2122;) of incubation with 10-7 M of estradiol (E2). Lysates from 32D/!GcRER and 32D/!Y703FGcRER cells were immunoprecipitated (IP) with either an anti-JAK1 (#JAK1; upper panel) or an anti-JAK2 (#JAK2; lower panel) antibody. Immunoblotting (IB) was carried out with an antiphosphotyrosine antibody (#PY).


Xu et al: G-CSF receptor-mediated STAT3 activation The estrogen-induced STAT5 activation was comparable in 32D/!GcRER and 32D/!Y703FGcRER cells at 60 minutes after stimulation, and reprobing of the blot with an anti-STAT5 antibody showed that approximately equal amounts of STAT5 were loaded (Figure 3, lower panel). The delay in STAT5 phosphorylation may be associated with a slower JAK1/JAK2 activation through estrogeninduced dimerization of the chimeric receptors. The reason for the reduced STAT5 phosphorylation in the E2stimulated cells is currently unknown; we speculate that the linking of ER-HBD to the C-terminal of GcR might hinder STAT proteins from freely accessing the membrane-distal region of the receptor. In any case, STAT5 appeared to be phosphorylated to the same extent in 32D/!GcRER and 32D/!Y703FGcRER cells. Others demonstrated that STAT5 was activated even when the membrane-distal region of GcR was deleted or the receptor tyrosine phosphorylation was abrogated (Shimozaki et al, 1997; Tian et al, 1996). Taken together with our observation that JAK1 and JAK2 were activated in both 32D/!GcRER and 32D/!Y703FGcRER cells (Figure 2), we concluded that the Y703F mutation did not affect the tyrosine phosphorylation of STAT5.

Repeated experiments constantly demonstrated a decreased STAT3 phosphorylation in 32D/!Y703FGcRER. Consistent with this observation, Tian et al showed that the G-CSF-induced STAT3 activation was greatly abrogated in UT-7epo cell transfectants by deleting a membrane-distal part including Y703 from GcR (Tian et al, 1996). We therefore concluded that Y703 in GcR was involved in STAT3 activation, and that the event is crucial to granulocyte differentiation in 32D cells.

IV. Discussion The phosphotyrosine residues in GcR create potential docking sites for the recruitment of signaling molecules such as STATs that contain a Src homology 2 (SH2) domain. STAT3 is recruited via the interaction of its SH2 domain with receptor tyrosine residues that are present in a tyrosine-X-X-glutamine (YXXQ) sequence (Stahl et al, 1995). Among four conserved tyrosine residues in the cytoplasmic region of GcR, only Y703 provides a YXXQ motif, accounting for the reduced STAT3 activation by the Y703F mutant. However, there was a residual level of STAT3 activation in !Y703FGcRER and other GcR mutants devoid of this motif, which suggested the presence of another STAT3 binding site in GcR or some bridging molecule (Avalos, 1996; Chakraborty et al, 1999). We observed a few additional phosphorylated proteins coimmunoprecipitated with STAT3 including a 130 kDa species (Figure 4, upper panel, arrowheads). These proteins are yet to be identified; at least they did not react with an antibody against GcR in a subsequent reprobing (data not shown).

D. Decrease in STAT3 Activation by Y703F G-CSF Receptor Mutant Finally, we addressed whether the Y703F mutation in GcR affects tyrosine phosphorylation of STAT3. After cytokine starvation, 32D/!GcRER and 32D/!Y703FGcRER clones were incubated with 10-7 M of E2 for 60 minutes. While estrogen induced a significant tyrosine phosphorylation of STAT3 in 32D/!GcRER, only a slight activation of STAT3 was detected in 32D/!Y703FGcRER clones (Figure 4, upper panel, arrow). Reprobing of the membrane with an anti-STAT3 antibody revealed an even loading of STAT3 in these lanes (Figure 4, lower panel).

Figure 3. Tyrosine phosphorylation of STAT5. Starved 32D/!GcRER and 32D/!Y703FGcRER cells were harvested before (0’) and after 10, 30, and 60 minutes (10’, 30’, 60’) of incubation with 10-9 M of G-CSF or 10-7 M of estradiol (E2). Lysates were immunoprecipitated (IP) with an anti-STAT5 antibody (#STAT5) and immunoblotted (IB) with an antiphosphotyrosine antibody (#PY; upper panel). The blot was reprobed with the anti-STAT5 antibody to confirm the equal loading of STAT5 (lower panel).

Figure 4. Tyrosine phosphorylation of STAT3. Starved 32D/!GcRER and 32D/!Y703FGcRER (clone 1 and clone 2) cells were harvested before (0’) and after 60 minutes (60’) of incubation with 10-7 M of estradiol (E2). Lysates were immunoprecipitated (IP) with an anti-STAT3 antibody (#STAT3) and immunoblotted (IB) with an anti-phosphotyrosine antibody (#PY; upper panel). The blot was reprobed with the anti-STAT3 antibody to confirm the equal loading of STAT3 (lower panel). Besides STAT3 (92 kDa, arrow), several phosphoproteins including a 130 kDa species (arrowheads) were coimmunoprecipitated.


Gene Therapy and Molecular Biology Vol 7, page 171 gene in a case of acute myeloid leukemia results in the overexpression of a novel G-CSF-R isoform. Blood 85, 902911. Dong F, Liu X, de Koning JP, Touw IP, Henninghausen L, Larner A and Grimley PM (1998) Stimulation of Stat5 by granulocyte colony-stimulating factor (G-CSF) is modulated by two distinct cytoplasmic regions of the G-CSF receptor. J Immunol 161, 6503-6509. Duke GM, Hoffman MA and Palmenberg AC (1992) Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation. J Virol 66, 1602-1609. Fukunaga R, Ishizaka-Ikeda E, Pan C-X, Seto Y and Nagata S (1991) Functional domains of the granulocyte colonystimulating factor receptor. EMBO J 10, 2855-2865. Fukunaga R, Ishizaka-Ikeda E and Nagata S (1993) Growth and differentiation signals mediated by different regions in the cytoplasmic domain of granulocyte colony-stimulating factor receptor. Cell 74, 1079-1087. Ito K, Ueda Y, Kokubun M, Urabe M, Inaba T, Mano H, Hamada H, Kitamura T, Mizoguchi H, Sakata T, Hasegawa M and Ozawa K (1997) Development of a novel selective amplifier gene for controllable expansion of transduced hematopoietic cells. Blood 90, 3884-3892. Koay DC and Sartorelli AC (1999) Functional differentiation signals mediated by distinct regions of the cytoplasmic domain of the granulocyte colony-stimulating factor receptor. Blood 93, 3774-3784. Koike S, Sakai M and Muramatsu M (1987) Molecular cloning and characterization of rat estrogen receptor cDNA. Nucleic Acids Res 15, 2499-2513. Kume A, Hanazono Y, Mizukami H, Okada T and Ozawa K (2002) Selective expansion of transduced cells for hematopoietic stem cell gene therapy. Int J Hematol 76, 299-304. Matsuda KM, Kume A, Ueda Y, Urabe M, Hasegawa M and Ozawa K (1999a) Development of a modified selective amplifier gene for hematopoietic stem cell gene therapy. Gene Ther 6, 1038-1044. Matsuda T, Nakamura T, Nakao K, Arai T, Katsuki M, Heike T and Yokota T (1999b) STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 18, 4261-4269. Mattioni T, Louvion J-F and Picard D (1994) Regulation of protein activities by fusion to steroid binding domains. Methods Cell Biol 43, 335-352. Nakauchi H, Nolan GP, Hsu C, Huang HS, Kavathas P and Herzenberg LA (1985) Molecular cloning of Lyt-2, a membrane glycoprotein marking a subset of mouse T lymphocytes: molecular homology to its human counterpart, Leu-2/T8, and to immunoglobulin variable regions. Proc Natl Acad Sci USA 82, 5126-5130. Nicholson SE, Oates AC, Harpur AG, Ziemiecki A, Wilks AF and Layton JE (1994) Tyrosine kinase JAK1 is associated with the granulocyte-colony-stimulating factor receptor and both become tyrosine-phosphorylated after receptor activation. Proc Natl Acad Sci USA 91, 2985-2988. Niwa H, Burdon T, Chambers I and Smith A (1998) Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 12, 2048-2060. Onishi M, Kinoshita S, Morikawa Y, Shibuya A, Phillips J, Lanier LL, Gorman DM, Nolan GP, Miyajima A and Kitamura T (1996) Applications of retrovirus-mediated expression cloning. Exp Hematol 24, 324-329. Shimozaki K, Nakajima K, Hirano T and Nagata S (1997) Involvement of STAT3 in the granulocyte colony-stimulating factor-induced differentiation of myeloid cells. J Biol Chem 272, 25184-25189.

A consensus has been reached that tyrosine phosphorylation of GcR and activation of STAT3 is crucial to granulocyte differentiation, but there remains some controversy over the relative contribution of each tyrosine residue depending on the cells used (Tian et al, 1994, 1996; de Koning et al, 1996; Shimozaki et al, 1997; Chakraborty et al, 1999; Ward et al, 1999). Previous reports employed either GcR-negative cells to examine the function of the receptor and associated molecules, or overexpression of dominant-negative forms of GcR to elucidate the mechanisms for growth and differentiation. By using ER-HBD fusion proteins to bypass endogenous GcR, we herein provided additional data suggesting the major involvement of Y703 in STAT3 activation. It is of particular note that the cells retained the expression of wild-type GcR and downstream signaling molecules, thereby rapidly undergoing granulocyte differentiation in response to G-CSF, indistinguishable from the parent 32D cells (Matsuda et al, 1999a). Contrary to its promoting function in myeloid cell differentiation, STAT3 was shown to play a central role in the maintenance of the pluripotent phenotype of embryonic stem cells (Matsuda et al, 1999b; Niwa et al, 1998). STAT3 appears to dictate widely divergent instructions such as differentiation and proliferation depending on the cell type. Thus, it is crucial to set up an appropriate venue to study the physiological molecular interaction involving a promiscuous molecule such as STAT3. The HBD fusion system provides a powerful tool to examine the behavior of mutated proteins controlled by specific ligands, in the exact milieu where the wild-type molecules coexist but remain unstimulated.

Acknowledgments We are grateful to Chugai Pharmaceuticals for providing G-CSF. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Health, Labor and Welfare, Japan

References Avalos BR (1996) Molecular analysis of the granulocyte colonystimulating factor receptor. Blood 88, 761-777. Chakraborty A, Dyer KF, Cascio M, Mietzner TA and Tweardy DJ (1999) Identification of a novel Stat3 recruitment and activation motif within the granulocyte colony-stimulating factor receptor. Blood 93, 15-24. de Koning JP, Dong F, Smith L, Schelen AM, Barge RMY, van der Plas DC, Hoefsloot LH, Lรถwenberg B and Touw IP (1996) The membrane-distal cytoplasmic region of human granulocyte colony-stimulating factor receptor is required for STAT3 but not STAT1 homodimer formation. Blood 87, 1335-1342. Dong F, van Buitenen C, Pouwels K, Hoefsloot LH, Lรถwenberg B and Touw IP (1993) Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation. Mol Cell Biol 13, 7774-7781. Dong F, van Paassen M, van Buitenen C, Hoefsloot LH, Lรถwenberg B and Touw IP (1995) A point mutation in the granulocyte colony-stimulating factor receptor (G-CSF-R)


Xu et al: G-CSF receptor-mediated STAT3 activation Stahl N, Farruggella TJ, Boulton TG, Zhong Z, Darnell JEJr and Yancopoulos GD (1995) Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors. Science 267, 1349-1353. Tian S-S, Lamb P, Seidel HM, Stein RB and Rosen J (1994) Rapid activation of the STAT3 transcription factor by granulocyte colony-stimulating factor. Blood 84, 1760-1764. Tian S-S, Tapley P, Sincich C, Stein RB, Rosen J and Lamb P (1996) Multiple signaling pathways induced by granulocyte colony-stimulating factor involving activation of JAKs, STAT5, and/or STAT3 are required for regulation of three distinct classes of immediate early genes. Blood 88, 44354444. Valtieri M, Tweardy DJ, Caracciolo D, Johnson K, Mavilio F, Altmann S, Santoli D and Rovera G (1987) Cytokinedependent granulocytic differentiation: regulation of proliferative and differentiative responses in a murine progenitor cell line. J Immunol 138, 3829-3835. Ward AC, Smith L, de Koning JP, van Aesch Y and Touw IP (1999) Multiple signals mediate proliferation, differentiation, and survival from the granulocyte colony-stimulating factor receptor in myeloid 32D cells. J Biol Chem 274, 1495614962. Xu R, Kume A, Matsuda KM, Ueda Y, Kodaira H, Ogasawara

Y, Urabe M, Kato I, Hasegawa M and Ozawa K (1999) A selective amplifier gene for tamoxifen-inducible expansion of hematopoietic cells. J Gene Med 1, 236-244. Yoshikawa A, Murakami H and Nagata S (1995) Distinct signal transduction through the tyrosine-containing domains of the granulocyte colony-stimulating factor receptor. EMBO J 14, 5288-5296.

Dr. Akihiro Kume


Gene Therapy and Molecular Biology Vol 7, page 173 Gene Ther Mol Biol Vol 7, 173-179, 2003.

Calcium induces apoptosis and necrosis in hematopoetic malignant cells: Evidence for caspase8 dependent and FADD-autonomous pathway Research Article

Christof J. Burek†, Malgorzata Burek†, Johannes Roth#, and Marek Los†¨ †

Institute of Experimental Dermatology, University of Münster, D-48149 Münster; #Institute of Molecular Medicine, University of Düsseldorf, D-40225 Düsseldorf, Germany; ¨ Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Canada.

__________________________________________________________________________________ *Correspondence: Marek Los, MD/PhD, Institute of Experimental Dermatology, University of Münster, Röntgenstrasse 21, D-48149 Münster, Germany; Phone: 49-251-83-52943; Fax: 49-251-83-56549; e-mail: Key Words: A23187, apoptosis, Bcl-2, caspase-8, FADD, necrosis Abbreviations: propidium iodide (PI), Fas-associated death domain protein (FADD), endoplasmic reticulum (ER), mitochondrial permeability transition (MPT), apoptosis-inducing factor (AIF)

Received: 1 September 2003; Accepted: 18 September 2003; electronically published: September 2003

Summary One of the killing mechanisms employed by Natural Killer (NK) cells and Lymphokine-Activated Killer (LAK) cells is the perforation of the cellular membrane that causes the increase of cytoplasmic calcium concentration and disturbs further the homeostasis of other ions. Cytoplasmic calcium influx, exceeding the tolerated physiologic threshold in cell signaling events, can induce either apoptosis or necrosis depending on its final concentration. Despite several years of intensive research and identification of some molecular targets of action like e.g. calpains, calcineurin or calreticulin, the exact mechanism of calcium-induced cell death is not known in detail. We show here that death pathways triggered by calcium rely on a novel, caspase-8-dependent and Bcl-2-inhibitable pathway that is FADD-adaptor molecule -independent. This is shown in a leukemic cell model. The experimental system employs either cells that lack the expression of casapase-8 or cells genetically modified to overexpress, Bcl-2, or a FADDdominant negative mutant (FADD-DN). applied to manipulate intracellular Ca2+ concentration and thus to mimic signaling events or to induce cell death (Errasfa and Stern, 1994; Nakamura, 1996). Several authors provide observations that various tumor cell lines exposed to A-23187 or ionomycin undergo either nonapoptotic degeneration (Duke et al, 1994; Kressel and Groscurth, 1994), or classical apoptosis (Ojcius et al, 1991; Ning and Murphy, 1993). Caspases (cysteine-dependent aspartases) are crucial apoptotic executioner proteases (Los et al, 1995; Herr and Debatin, 2001). They are members of the C14 protease family according to the Barrett and Rawlings classification (Los et al, 1999; Barrett and Rawlings, 2001). All caspases are characterized by a nearly absolute specificity for substrates containing aspartic acid in the P1 cleavage position and a cysteine in the active center of the enzyme (Stennicke et al, 2002). There are currently 12 known caspases in humans. Caspases-1, -4 and -5 mainly play a role in the regulation of inflammatory response, by proteolytic activation of inflammatory cytokines (Cassens et al, 2003). Caspases-2, -3, -6, -7, -8, -9 and -10 are

I. Introduction Calcium is one of the most versatile and powerful small molecules applied by a cell to regulate its biologic functions. It can either protect from or induce cell death, depending on concentration and cell type (Franklin and Johnson, 1992; Barros et al, 2002). Although the mechanism of calcium triggered death has been investigated for years, the exact mechanism(s) responsible for this process are not known in detail. Dying cells enter either apoptosis, necrosis or an intermediate form of cell death, depending on the death stimulus, its intensity and the level of intracellular ATP (Leist and Jaattela, 2001; Los et al, 2002). In accordance, calcium can induce both forms of cell death as well as an intermediate process, depending on available intracellular concentration and cell type (Gwag et al, 1999; Barros et al, 2002). Calciumrelated cell death is best described in neurones (Gwag et al, 1999; Xu et al, 2001), however, detailed studies in lymphatic tissue, from recent date are scarce. Calcium ionophores, such as ionomycin or A-23187 are frequently


Burek et al: Calcium induced cell death considered to be involved predominantly in apoptotic signalling (Sadowski-Debbing et al, 2002). In addition to the role in apoptosis and inflammation, an involvement of caspases in other processes, like cell cycle regulation, hematopoesis and signal transduction in the immune system have been proposed (Denis et al, 1998; Los et al, 2001). All caspases are synthesized as inactive zymogens that are activated through proteolytic cleavage. Among the caspase activation pathways, the best described ones are the death-receptor dependent signalling cascades, with FADD adaptor molecule and caspase-8 as the key players, and the mitochondria/apoptosome dependent pathway that relies on Apaf-1 and caspase-9 (Krammer, 2000; Walczak and Krammer, 2000; Zheng and Flavell, 2000; Renz et al, 2001). Both pathways are interconnected, thus amplification loops may take place (Sadowski-Debbing et al, 2002). The mitochondrial pathway is largely controlled by Bcl-2 family members. Bcl-2 family proteins exert its pro-and antiapoptotic action partially by influencing calcium homeostasis of mitochondria and endoplasmic reticulum (ER) (reviewed in Hajnoczky et al, 2003). The family comprises both antiapoptotic and proapoptotic proteins. All antiapoptotic family members (e.g. Bcl-2, Bcl-XL) share three or four Bcl-2 homology (BH) regions, and they localize to the cytoplasmic side of intracellular membranes (Bouillet and Strasser, 2002). The proapoptotic Bcl-2 family members can be further divided into two subgroups. Members of the first subgroup, best represented by Bax and Bak (reviewed in Bouillet and Strasser, 2002) have two or three BH regions and appear to be structurally similar to their prosurvival relatives (Suzuki et al, 2000). The second subgroup of proapoptotic Bcl-2-related proteins, (e.g. Bad, Bid, Bim) share only the short BH3 region (reviewed in Bouillet and Strasser, 2002). The exact mechanism of apoptosis regulation by Bcl-2 family members is not fully understood (Strasser et al, 2000). It is widely believed that Bcl-2 functions to preserve the mitochondrial membrane integrity, mitochondrial and ER calcium homeostasis and prevent the release of cytochrome c and other proapoptotic molecules from the mitochondria. BH3-only proteins appear to sense stimuli that cause cellular stress and initiate the death cascade. Proapoptotic Bax and Bak are essential for cell killing governed by BH3-only proteins, and this form of cell death is antagonized by overexpresion of Bcl-2 (reviewed in Hajnoczky et al, 2003; Marsden and Strasser, 2003). To gain insight into the mechanisms that govern calcium triggered cell death we have used a T-cellleukemia based model and calcium ionophores as modulators of intracellular Ca2+ level. We show here that the calcium activated apoptotic pathway rely on yet-to-bedefined, caspase-8-dependent and Bcl-2-inhibitable pathway. Interestingly, the pathway does not rely on FADD-adaptor molecule. Thus, we provide further evidences for an intrinsic (death receptor-independent) death pathway that relies on caspase-8.

II. Materials and methods A. Materials and cell culture All cell lines were grown in 5% CO2 at 37°C using a RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics (GIBCO, Eggenstein, Germany). A23187 was purchased from Sigma (Deisenhofen, Germany). The caspase inhibitor zVADfmk (benzyloxycarbonyl-Val-AlaAsp-fluoro-methylketone) was purchased from Enzyme Systems Products (Dublin, CA), and staurosporine from Roche Biochemicals (Mannheim, Germany). All other chemicals were from Merck KG (Darmstadt, Germany) or Roth (Karlsruhe, Germany). Stable transfectants of Jurkat cells overexpressing Bcl-2 and Jurkat clone that was deficient in caspase-8 were a kind gift of Dr. J. Blenis, (Harvard Medical School, Boston, Massachusetts, USA).

B. Cell extracts and immunoblotting The proteolytic processing of caspase-3 and caspase-8 was detected by immunoblotting. Briefly, 5 x 10 5 cells were seeded in 6-well plates and treated with the apoptotic stimuli. After the indicated time, cells were washed in cold PBS and lysed in 1% Triton X-100, 50 mM Tris-HCl, pH 7.6 and 150 mM NaCl containing 3 µg/ml aprotinin, 3 µg/ml leupeptin, 3 µg/ml pepstatin A and 2 mM phenylmethylsulfonyl fluoride (PMSF). Subsequently, the proteins were separated under reducing conditions by 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted to a polyvinylidene difluoride membrane (Amersham, Braunschweig, Germany). The equal loading of protein was controlled by measuring the protein concentration using the Bradford assay (BioRad, Munich, Germany). Membranes were blocked for 1 h with 5% non-fat dry milk powder in TBS and then incubated for 1 h with murine monoclonal antibodies directed against caspase-3 (Transduction Laboratory, Heidelberg, Germany). Membranes were washed four times with TBS/0.02% Triton X-100 and incubated with the respective peroxidase-conjugated affinity-purified secondary antibody for 1 h. Following extensive washing, the reaction was developed by enhanced chemiluminescent staining using ECL reagents (Amersham).

C. Fluorimetric assay of caspase activity DEVD-ase assay Cytosolic cell extracts were prepared by lysing cells in a buffer containing 0.5% NP-40, 20 mM HEPES pH 7.4, 84 mM KCl, 10 mM MgCl2, 0.2 mM EDTA, 0.2 mM EGTA, 1 mM DTT, 5 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin and 1 mM PMSF. Caspase activity was determined by the incubation of cell lysates with 50 µM of the fluorogenic substrate DEVD-AMC (N-acetyl-Asp-Glu-Val-Aspaminomethylcoumarin, Bachem, Heidelberg, Germany) in 200 µl buffer containing 50 mM HEPES pH 7.3, 100 mM NaCl, 10% sucrose, 0.1% CHAPS and 10 mM DTT. The release of aminomethylcoumarin was measured by fluorometry using an excitation wavelength of 360 nm and an emission wavelength of 475 nm.

D. Measurement of cell death and apoptosis Cell death was measured either by the detection of hypodiploid nuclei (Nicoletti method) (Renz et al, 2001) or by the uptake of propidium iodide (PI) (Stroh et al, 2002). Briefly, for the measurement of hypodiploid DNA, nuclei were prepared by lysing 104 cells in 100 µl of hypotonic lysis buffer (1% sodium citrate, 0.1% Triton X-100, and 50 µg/ml PI). The nuclei


Gene Therapy and Molecular Biology Vol 7, page 175 were subsequently analyzed by flow cytometry, using a FACScalibur (Becton Dickinson, Heidelberg, Germany) and CellQuest analysis software. To assess PI uptake, cells were harvested after the indicated times and incubated with PI (2 µg/ml). The uptake of PI into nonfixed cells was measured by flow cytometry, using the FSC/FL2 profile.

III. Results A. Calcium influx induces apoptotic and necrotic cell death in a dose dependent manner In order to get insight into the mechanism(s) of calcium induced cell death we have performed time-, and concentration- kinetic studies. Jurkat human T-leukemia cells were treated with increasing concentrations of the A23187 calcium ionophore. A23187 induces cell death in a dose dependent manner (Figure 1). Higher concentrations of intracellular calcium induce cell death with faster kinetics. At the concentration of 800 ng/ml A23187 induces a maximum cell death at 18 h, whereas lower concentrations of the ionophore show slower kinetics. The assessment of data obtained by the measurement of PI uptake and apoptosis-specific measurement by the detection of hypodiploid nuclei (“Nicoletti” method) indicates that higher concentrations induce not only apoptotic, but also necrosis in the experimental system (Figure 1C). Since contrary to necrosis the apoptotic cell death relies on caspases, we repeated the series of experiments employing the broadspectrum caspase inhibitor zVADfmk (Figure 2). Thus zVAD-fmk inhibitable cell death represents the apoptotic fraction. The zVADfmk based approach largely confirms the data obtained by the combination of the PI-uptake based- and the “Nicoletti” method (Figure 1C). Unlike the Nicoletti method that detects (lack of) the intactness of nuclear DNA (hypodiploidy), PI-uptake stains cells with permeable cell membranes (necrotic and late apoptotic cells). zVADfmk inhibits the proteolytic caspase activity and, therefore, it blocks the apoptotic fraction of cell death. The experiments involving the caspase inhibitor indicate the highest zVADfmk-independent (presumably necrotic) fraction of cell death upon the treatment with intermediate (200 ng/ml) concentrations of A23187 calcium ionophore (Figure 2C). These method-related differential results are explained in detail in the discussion-part of the paper.

B. Caspase-8 deficiency impairs calcium induced cell death The broad-spectrum caspase inhibitor zVADfmk was largely protective against calcium induced cell death. To examine further the role of caspases in the death mechanism triggered by calcium we have employed a Jurkat cell clone that lacks caspase-8 activity. Calcium induced cell death measured by PI uptake was significantly impaired in cells lacking caspase-8 activity (Figure 3A). The observed effect could be detected at several time points and it was most pronounced after 18 h.

Figure 1. Induction of cell death by calcium ionophore in Jurkat cells. (A and B) show parallel-, time-kinetic experiments evaluated either by PI-uptake, a method that unspecifically detects cell death (A), or by apoptosis-specific “Nicoletti” method that measures hypodiploid, apoptotic nuclei (B). The standard deviation of four independent experiments shown here did not exceeded 11 %. The percentage representation of both death modes, that occurred after 18 h are visualized in the panel (C).


Burek et al: Calcium induced cell death

Figure 2. Delineation of caspase-dependent (zVADfmkinhibitable) and caspase-independent components of calciuminduced cell death. Jurkat cells were treated with different concentrations of A23187 as indicated in A and B. The application of zVADfmk, the broad-spectrum caspase inhibitor has significantly, but only partially blocked cell death events (B). The panel (B) shows data from four independent experiments. The standard deviation did not exceeded 9 %. The â&#x20AC;&#x153;zVADfmkâ&#x20AC;? resistant cell death component is depicted in the panel (C). Cell death was measured by PI-uptake.

Figure 3. Caspase-8 activity deficiency protects from necrotic component, but not from the apoptotic constituent of calcium-induced cell death. Jurkat cells were induced to die by the addition of 200 ng/ml of A23187. Cell death was measured in parallel by PI-uptake (A), and by the assessment of nuclear hypodiploidy that corresponds to apoptotic cell death (B). To get the confirmation of the data, we conducted a kinetic study using increasing concentrations of the calcium ionophore A23187 (C). The cell death was measured by PI-uptake. The activation of caspase cascade was assessed by Western blot detection of caspase-3 cleavage (D).


Gene Therapy and Molecular Biology Vol 7, page 177 Interestingly, despite having a strong effect on cell death (Figure 3A), caspase-8 deficient cells were equally sensitive towards the apoptotic form of cell death (Figure 3B), measured by the â&#x20AC;&#x153;Nicolettiâ&#x20AC;? method. To further confirm the observation, the A23187 concentration kinetics at 18 h was performed (Figure 3C). Similarly as in Figure 3A, here the cell death was measured by PIuptake that cannot discriminate well between apoptosis and necrosis. Also this data fully confirmed the observations that caspase-8 deficiency significantly protects from the ionophore-triggered death. To get further insight into the death mechanisms induced by calcium influx we have examined caspase-3 cleavage (activation) by Western blot (Figure 3D). To our surprise a significant portion of caspase-3 was cleaved unspecificaly, yielding non-active proteolytic fragments. The subsequent enzymatic measurement of caspase-3 (DEVDase) activity fully confirmed the Western blot data, showing only a very moderate increase in activity (data not shown).

C. Calcium induced cell death is FADDindependent, and it is inhibitable by Bcl-2 Since caspase-8 deficiency was largely protective against calcium-induced cell death in our experimental system, we next tested the effect of FADD, the adaptor molecule that is necessary for caspase-8 recruitment to death receptors. In addition, we examined the possible involvement of apoptosome/mitochondrial death pathway employing Jurkat cells overexpressing Bcl-2 proteins. Cells overexpressing a mutated form of the FADD molecule, that lack the death effector domain required for the interaction with caspase-8, were as equally sensitive as the control Jurkat cell line (Figure 4A). Thus, although caspase-8 deficiency significantly impairs death triggered by calcium, the adaptor molecule FADD plays no role in the system. Whereas, Bcl-2 overexpression was fully protective against low concentrations (200 ng/ml) of the calcium ionophore A23187 (Figure 4B). Higher concentrations of A23187 (e.g. 400 ng/ml) partially overcame the Bcl-2 protective effect, but still about 50 % more of the Jurkat-Bcl-2 cells survived the forced calcium influx as compared to the control Jurkat clone.

IV. Discussion The presented study identifies a novel, caspase-8 dependent, calcium-triggered pathway involved in the propagation of cell death. The pathway differs significantly from the classical, death receptor-triggered apoptotic signaling cascades since it is FADDindependent. Caspase-8 requires adaptor molecules for its activation. This requirement can be fulfilled by the ERlocalized protein Bap31 that binds caspase-8 (Breckenridge et al, 2002; Ducret et al, 2003). The observed sensitivity towards overexpression of Bcl-2 may be indicative for the involvement of mitochondrial/apoptosome-dependent signaling events. The Bcl-2 sensitivity of the pathway can also be explained alternatively. It has been described previously (FoyouziYoussefi et al, 2000; Vanden Abeele et al, 2002) that some

Figure 4. The effect of FADD death receptor adaptor molecule and Bcl-2 on calcium triggered apopotosis. FADDnegative- and control (J16) cells were treated with A23187 (400 ng/ml) over different time points and cell death was measured by PI-uptake (A). To examine the effect of Bcl-2 on calcium induced death we have used a Jurkat cell clone that overexpress the protein. Time kinetics were done with two different concentrations of A23187. Bcl-2 almost completely inhibited cell death induced by 200 ng/ml of A23187 (B), and it was about 4050 % protective upon treatment with 400 ng/ml of the ionophore (C). Cell death was measured by PI-uptake.


Burek et al: Calcium induced cell death Barros LF, Castro J, and Bittner CX (2002) Ion movements in cell death: from protection to execution. Biol Res 35, 209214. Bouillet P, and Strasser A (2002) BH3-only proteins evolutionarily conserved proapoptotic Bcl-2 family members essential for initiating programmed cell death. J Cell Sci 115, 1567-1574. Breckenridge DG, Nguyen M, Kuppig S, Reth M, and Shore GC (2002) The procaspase-8 isoform, procaspase-8L, recruited to the BAP31 complex at the endoplasmic reticulum. Proc Natl Acad Sci U S A 99, 4331-4336. Cassens U, Lewinski G, Samraj AK, von Bernuth H, Baust H, Khazaie K, and Los M (2003) Viral modulation of cell death by inhibition of caspases. Arch Immunol Ther Exp 51, 1927. Denis F, Rheaume E, Aouad SM, Alam A, Sekaly RP, and Cohen LY (1998) The role of caspases in T cell development and the control of immune responses. Cell Mol Life Sci 54, 1005-1019. Ducret A, Nguyen M, Breckenridge DG, and Shore GC (2003) The resident endoplasmic reticulum protein, BAP31, associates with gamma-actin and myosin B heavy chain. Eur J Biochem 270, 342-349. Duke RC, Witter RZ, Nash PB, Young JD, and Ojcius DM (1994) Cytolysis mediated by ionophores and pore-forming agents: role of intracellular calcium in apoptosis. Faseb J 8, 237-246. Errasfa M, and Stern A (1994) Melittin inhibits epidermal growth factor-induced protein tyrosine phosphorylation: comparison with phorbol myristate acetate and calcium ionophore A23187. Biochim Biophys Acta 1222, 471-476. Foyouzi-Youssefi R, Arnaudeau S, Borner C, Kelley WL, Tschopp J, Lew DP, Demaurex N, and Krause KH (2000) Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum. Proc Natl Acad Sci U S A 97, 57235728. Franklin JL, and Johnson EM, Jr. (1992) Suppression of programmed neuronal death by sustained elevation of cytoplasmic calcium. Trends Neurosci 15, 501-508. Gwag BJ, Canzoniero LM, Sensi SL, Demaro JA, Koh JY, Goldberg MP, Jacquin M, and Choi DW (1999) Calcium ionophores can induce either apoptosis or necrosis in cultured cortical neurons. Neuroscience 90, 1339-1348. Hajnoczky G, Davies E, and Madesh M (2003) Calcium signaling and apoptosis. Biochem Biophys Res Commun 304, 445-454. Herr I, and Debatin KM (2001) Cellular stress response and apoptosis in cancer therapy. Blood 98, 2603-2614. Krammer PH (2000) CD95's deadly mission in the immune system. Nature 407, 789-795. Kressel M, and Groscurth P (1994) Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA. Cell Tissue Res 278, 549-556. Leist M, and Jaattela M (2001) Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol 2, 589-598. Lemasters JJ, Qian T, He L, Kim JS, Elmore SP, Cascio WE, and Brenner DA (2002) Role of mitochondrial inner membrane permeabilization in necrotic cell death, apoptosis, and autophagy. Antioxid Redox Signal 4, 769-781. Los M, Mozoluk M, Ferrari D, Stepczynska A, Stroh C, Renz A, Herceg Z, Wang Z-Q, and Schulze-Osthoff K (2002) Activation and caspase-mediated inhibition of PARP: a molecular switch between fibroblast necrosis and apoptosis in death receptor signaling. Mol Biol Cell 13, 978-988. Los M, Stroh C, Janicke RU, Engels IH, and Schulze Osthoff K (2001) Caspases: more than just killers? Trends Immunol 22, 31-34.

antiapoptotic Bcl-2 family members including Bcl-2 itself and Bcl-XL, protect cells from calcium by lowering the Ca2+-storage capacity of ER. Thus, the death stimuli that cause the release of calcium from ER will be less efficient in elevating the cytoplasmic calcium concentration and therefore, will less effectively activate the calciumdependent signaling pathways. The death inducted by the calcium ionophore A23187 was a mixture of necrosis and apoptosis. A critical factor that influences the form of cell death (apoptotic or necrotic) is the cellular ATP content. Stimuli that under normal condition induce apoptosis will cause classical necrotic cell death if the cellular concentration of ATP drops below 10-15 % of the normal level (Nieminen et al, 1994; Los et al, 2002). One of the mechanisms that cause severe ATP depletion is the uncoupling of phosphorylative oxidation and ATP production caused by mitochondrial permeability transition (MPT). MPT may be triggered by a rising Ca2+ level and the subsequent activation of the hypothetical permeability transition pore component cyclophilin D. Once the pH and electrical gradient across the inner mitochondrial membrane collapses the final enzyme of the mitochondrial respiratory chain, the F1F0ATPase, that normally converts ADP to ATP, reverses and consumes ATP while trying to restore the gradient. This mechanism is among the strongest depletors of cellular ATP, since it also consumes ATP produced by the compensatory, glycolytic pathway (reviewed in Lemasters et al, 2002; Hajnoczky et al, 2003). The above mechanism permits both necrotic- and apoptotic death. A strong increase of Ca2+ concentration would cause a significant portion of mitochondria to collapse, massive ATP depletion would follow, thus, cells would die by necrosis. A less pronounced rise of calcium concentration would result in a slow and asynchronous MPT occurrence. Affected mitochondria would release proapoptotic molecules like cytochrome c, AIF and endonuclease G. While the depletion of ATP would not be significant, the cell would have enough energy to die in an orderly, apoptotic fashion. This is exactly what we observed in our experimental system. While low concentrations of the calcium ionophore A23187 induce apoptosis, intermediate and higher concentrations of it cause substantial necrosis. In summary, we are presenting here evidence for a new caspase-8-dependent calcium-induced death pathway. Since it is FADD-independent, we hypothesize that the Bap31 ER-localized adaptor molecule is involved in the pathway. In addition to the ER-compartment, the mitochondrial death pathways are important mediators of death induced by an elevated cellular calcium level.

Acknowledgements This work was supported by grants from â&#x20AC;&#x153;Deutsche Krebschilfeâ&#x20AC;? (10-1893), DFG (Lo 823/1-1 and Lo 823/31), and by IZKF-Muenster, (E-8).

References Barrett AJ, and Rawlings ND (2001) Evolutionary lines of cysteine peptidases. Biol Chem 382, 727-733.


Gene Therapy and Molecular Biology Vol 7, page 179 Los M, van de Craen M, Penning CL, Schenk H, Westendorp M, Baeuerle PA, DrĂśge W, Krammer PH, Fiers W, and SchulzeOsthoff K ( 1995) Requirement of an ICE/Ced-3 protease for Fas/Apo-1-1mediated apoptosis. Nature 371, 81-83. Los M, Wesselborg S, and Schulze Osthoff K (1999) The role of caspases in development, immunity, and apoptotic signal transduction: lessons from knockout mice. Immunity 10, 629-639. Marsden VS, and Strasser A (2003) Control of Apoptosis in the Immune System: Bcl-2, BH3-Only Proteins and More. Annu Rev Immunol 21, 71-105. Nakamura J (1996) Calcium ionophore, A23187, alters the mode of cAMP formation in wild-type S49 murine lymphoma cells. Biochim Biophys Acta 1313, 6-10. Nieminen AL, Saylor AK, Herman B, and Lemasters JJ (1994) ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition. Am J Physiol 267, C67-74. Ning ZQ, and Murphy JJ (1993) Calcium ionophore-induced apoptosis of human B cells is preceded by the induced expression of early response genes. Eur J Immunol 23, 3369-3372. Ojcius DM, Zychlinsky A, Zheng LM, and Young JD (1991) Ionophore-induced apoptosis: role of DNA fragmentation and calcium fluxes. Exp Cell Res 197, 43-49. Renz A, Berdel WE, Kreuter M, Belka C, Schulze-Osthoff K, and Los M (2001) Rapid extracellular release of cytochrome c is specific for apoptosis and marks cell death in vivo. Blood 98, 1542-1548. Sadowski-Debbing K, Coy JF, Mier W, Hug H, and Los M (2002) Caspases â&#x20AC;&#x201C; their role in apoptosis and other physiological processes as revealed by knock-out studies. Arch Immunol Ther Exp 50, 19-34. Stennicke HR, Ryan CA, and Salvesen GS (2002) Reprieval from execution: the molecular basis of caspase inhibition. Trends Biochem Sci 27, 94-101. Strasser A, O'Connor L, and Dixit VM (2000) Apoptosis signaling. Annu Rev Biochem 69, 217-245.

Stroh C, Cassens U, Samraj AK, Sibrowski W, Schulze-Osthoff K, and Los M (2002) The role of caspases in cryoinjury: caspase inhibition strongly improves the recovery of cryopreserved hematopoietic and other cells. FASEB J 16, 1651-1653. Suzuki M, Youle RJ, and Tjandra N (2000) Structure of Bax: coregulation of dimer formation and intracellular localization. Cell 103, 645-654. Vanden Abeele F, Skryma R, Shuba Y, Van Coppenolle F, Slomianny C, Roudbaraki M, Mauroy B, Wuytack F, and Prevarskaya N (2002) Bcl-2-dependent modulation of Ca(2+) homeostasis and store-operated channels in prostate cancer cells. Cancer Cell 1, 169-179. Walczak H, and Krammer PH (2000) The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems. Exp Cell Res 256, 58-66. Xu K, Tavernarakis N, and Driscoll M (2001) Necrotic cell death in C. elegans requires the function of calreticulin and regulators of Ca(2+) release from the endoplasmic reticulum. Neuron 31, 957-971. Zheng TS, and Flavell RA (2000) Divinations and surprises: genetic analysis of caspase function in mice. Exp Cell Res 256, 67-73.

Marek Los, MD, PhD


Burek et al: Calcium induced cell death


Gene Therapy and Molecular Biology Vol 7, page 181 Gene Ther Mol Biol Vol 7, 181-209, 2003

The current status and future direction of fetal gene therapy Review Article

Anna L David1, Michael Themis2, Simon N Waddington2, Lisa Gregory2, Suzanne MK Buckley2, Megha Nivsarkar2, Terry Cook3, Donald Peebles1, Charles H Rodeck1, Charles Coutelle2 1

Department of Obstetrics and Gynaecology, Royal Free and University College London Medical School, London WC1E 6HX 2 Gene Therapy Research Group, Section of Cell and Molecular Biology, Division of Biomedical Sciences, Imperial College School of Medicine, London SW7 2AZ 3 Department of Histopathology, Imperial College School of Medicine, London W12 0HS

__________________________________________________________________________________ *Correspondence: Dr A.L. David, Room 212, 2 nd floor, Department of Obstetrics and Gynaecology, Royal Free and University College Medical School, 86-96 Chenies Mews, London, WC1E 6HX, UK. Telephone: +44-20-7679-6059; Fax: +44-20-7383-7429; e-mail: Key words: fetal gene therapy; adenovirus; retrovirus; lentivirus; adeno-associated virus; Sendai virus; liposome Abbreviations: Cystic fibrosis (CF), Cystic Fibrosis Transmembrane Regulator (CFTR), and ornithine transcarbamylase (OTC), lysosomal storage disorders (LSDs), cerebrospinal fluid (CSF), Duchenne muscular dystrophy (DMD), Spinal muscular atrophy (SMA), survival motor neuron gene 1 (SMN 1), adeno-associated viral (AAV), severe combined immunodeficiency disorders (SCID), recessive adenosine deaminase deficiency (ADA), bone marrow transplantation (BMT), dystrophic form of epidermolysis bullosa (DEB), congenital diaphragmatic hernia (CDH), Intrauterine growth restriction (IUGR) Received: 18 September 2003; Accepted: 29 October 2003; electronically published: November 2003

Summary Application of gene therapy in utero has been considered as a strategy for treatment or even prevention of early onset genetic disorders such as cystic fibrosis and Duchenne muscular dystrophy. Prenatal gene transfer may target rapidly expanding stem cell populations that are inaccessible after birth, permit induction of immune tolerance against vector and transgene and allow permanent gene transfer by use of integrating vector systems. Application of this therapy in the fetus must be safe, reliable and cost-effective. Recent developments in the understanding of genetic disease, vector design, and minimally invasive delivery techniques have brought fetal gene therapy closer to clinical practice. Prenatal studies in animal models are being pursued in parallel with adult studies of gene therapy, but they remain presently at the experimental stage. tolerance against vector and transgene, and thereby facilitate repeated treatment after birth. Finally, and most importantly for clinicians, fetal gene therapy would give a third choice to parents following prenatal diagnosis of inherited disease, where currently termination of pregnancy or acceptance of an affected child have been the only options. Application of this therapy in the fetus must be safe, reliable and cost-effective. Recent developments in the understanding of genetic disease, vector design, and minimally invasive delivery techniques have brought fetal gene therapy closer to clinical practice. Prenatal studies in animal models are being pursued in parallel with adult studies of gene therapy, but they remain presently at the experimental stage. This review explores the latest developments in the field of in utero gene therapy and their implications for its future clinical application.

I. Introduction Gene therapy uses the intracellular delivery of genetic material for the treatment of disease. A wide range of diseases including cancer, vascular and neurodegenerative disorders and inherited genetic diseases are being considered as targets for this therapy in adults. Application of gene therapy in utero has been considered as a strategy for treatment or even prevention of early onset genetic disorders such as cystic fibrosis and Duchenne muscular dystrophy (Coutelle et al, 1995). Gene transfer to the developing fetus may target rapidly expanding stem cell populations that are inaccessible after birth and may allow permanent gene transfer by use of integrating vector systems. The functionally immature fetal immune system may permit induction of immune 181

David et al: Current status and future direction of fetal gene therapy Table 1: Examples of candidate diseases for fetal gene therapy Disease Cystic fibrosis (CF) Metabolic disorders: Ornithine transcarbamylase deficiency Glycogen storage disorders: Pompe disease Sphingolipid storage disorders: Tay-Sachs disease Mucopolysaccharide storage disorders: Sly disease Muscular dystrophies: Duchenne Neurological disorders: Spinal muscular atrophy Haemophilias: Haemophilia B Haemoglobinopathies: "o-thalassemia Immunodeficiency disorders: X-linked severe combined immunodeficiency Skin disorders: Dystrophic epidermolysis bullosa Non-inherited perinatal diseases: Hypoxia-ischaemia Infectious diseases: Herpes simplex Placental disorder Severe pre-eclampsia

Therapeutic gene product CF transmembrane regulator

Target cells/organ airway and intestinal epithelial cells

Ornithine transcarbamylase



hepatocytes, myocytes and neurons


fibroblasts, neurons


hepatocytes, neurons



survival motor neuron protein

motor neurons

human factor IX clotting factor


"-globin chains of haemoglobin

haematopoietic precursor cells

#c cytokine receptor

haematopoietic precursor cells

type VII collagen


neurotrophic factors

cortical neurons

herpes DNA

oral mucosa

nitric oxide synthase


transduction. Phase I gene therapy trials directed towards pulmonary disease in CF have shown equivocal results and highlight the problems of present gene therapy approaches in adults (Bigger and Coutelle 2001). The lungs may already be severely damaged or obstructed, even in young adult patients, limiting delivery of gene therapy to the airway epithelium. Fluorocarbon liquids such as perflubron have recently been shown to improve distribution of adenoviral vectors and gene expression in normal and diseased adult lungs (Weiss et al, 1999a, 2001). Pretreatment of airways with detergents (Parsons et al, 1998) or the fatty acid sodium caprate (Gregory et al, 2002) or EGTA (Wang et al, 2000) also improves adenovirus-mediated airways transduction. A comparison of agents to modulate paracellular permeability showed that pretreatment of adult murine airways with sodium caprate had a good safety profile, and enhanced adenovirus-mediated gene transfer to the trachea more efficiently than sodium laurate, another fatty acid sodium salt or EGTA, a calcium chelator (Johnson et al, 2003). Immune responses to the vector, particularly in the case of adenoviral vectors, limit the dose that may be safely administered, and reduce the duration of expression. The CFTR gene has been proposed to play an important, albeit still unknown, physiological role in normal fetal development (Gaillard et al, 1994; Tizzano et al, 1994). Furthermore the cystic fibrosis disease process appears to begin during development of CF fetuses since by the mid-trimester a pro-inflammatory state exists in fetal CF airways (Hubeau et al, 2001) and there are abnormalities of the pancreas and small bowel (BouĂŠ et al,

II. The candidate diseases Fetal gene therapy has been proposed to be appropriate for life-threatening disorders, in which prenatal gene delivery maintains a clear advantage over cell transplantation or postnatal gene therapy and for which there are currently no satisfactory treatments available (Wilson and Wivel 1999). Some of the diseases that may be suitable for in utero treatment are listed in Table 1 and are discussed as examples for conditions with similar manifestations and/or target tissues.

A. Cystic fibrosis Cystic fibrosis (CF) appears to be an ideal candidate for treatment with in utero gene therapy. Firstly it is the most common lethal autosomal recessive disorder in Caucasians with an incidence of 1 in 2000 livebirths in Western Europe and North America. Several mutations of the Cystic Fibrosis Transmembrane Regulator (CFTR) gene encoding the CFTR protein have been identified and the resulting disease is characterized by abnormal electrolyte transport in the epithelia of the airways, the ducts of the sweat glands and exocrine pancreas, and the intestine. The main sites of CFTR expression in the nonCF human bronchi are the submucosal glands (Engelhardt et al, 1992). In vitro studies where normal and CF airway cells were mixed, suggest that as few as 6-10% of cells expressing normal CFTR are required to correct the chloride transport defect of an epithelial cell monolayer (Johnson et al, 1992); thus, successful gene therapy may require only relatively low level epithelial airway


Gene Therapy and Molecular Biology Vol 7, page 183 1986). Prenatal diagnosis is usually performed by detection of the CFTR mutation in placental tissue, fetal skin cells or blood after chorionic villus biopsy, amniocentesis or cordocentesis respectively. Submucosal gland development has been studied in the rhesus monkey fetus (Plopper et al, 1986) and although not characterized in the human fetal airways, submucosal gland progenitors have been identified in the human adult lung (Engelhardt et al, 1995). Gene transfer to a human fetal lung xenograft model in SCID mice was efficiently achieved using adenoviral vectors (Peault et al, 1994) and long-term expression in the surface epithelial and submucosal gland cells was observed up to 4 weeks and 9 months after administration of adeno-associated and lentiviral vectors respectively (Lim et al, 2002, 2003). The early disease manifestation and poor results from gene therapy treatment of adults with CF has led to research on in utero gene therapy for this disease in animal models. Despite the multiorgan manifestation of CF, first approaches are directed towards gene delivery to the fetal airways, which has been achieved by intra-amniotic application and, in larger animals by intratracheal injection (see chapter IV). Other genetic diseases which could benefit from progress achieved in pulmonary gene delivery are !1-antitrypsin deficiency (Stecenko and Brigham 2003) and surfactant protein B deficiency (Cole et al, 2003).

inflammatory response syndrome that lead to his death (Raper et al, 2002). Because of its early onset, severity and present difficulties in postnatal gene therapy, OTC deficiency is an interesting candidate for in utero gene application targeted to the fetal liver (see chapter IV). Prenatal diagnosis for OTC deficiency by detection of the genetic mutation in fetal DNA is available in families with a known congenital abnormality. In non-informative families, deficiency of OTC enzyme can be detected in the fetal liver after liver biopsy (Holzgreve and Golbus, 1986). Other serious genetic diseases that would primarily require hepatocyte directed gene transfer are amino acid disorders (e.g. phenylketonuria, tyrosinaemia), carbohydrate disorders (e.g. galactosaemia) and fatty acid oxidation disorders (e.g. long-chain acyl-CoA dehydrogenase deficiency) (Preece and Green 2002).

C. Storage disorders The lysosomal storage disorders (LSDs) are a group of congenital deficiencies of one or more lysosomal enzymes. In mucopolysaccharidosis type VII (MPS type VII) a deficiency of "-glucuronidase activity leads to accumulation of undegraded glycosaminoglycans in lysosomes. Clinically, patients develop hepatosplenomegaly, mental and growth retardation, hearing and vision defects, skeletal deformities and die of cardiac failure. Many of the LSDs present already during fetal life with hydrops fetalis and prenatal diagnosis can be performed by detection of "-glucuronidase deficiency in chorionic villi or fetal blood (Geipel et al, 2002). Although individually rare, as a group they occur in approximately 1 in 7500 live births and are one of the more prevalent groups of inherited diseases in humans (Wraith, 2002). Bone marrow transplantation and enzyme replacement therapy are being developed for many of the mucopolysaccharidoses. However, the short half-life of lysosomal enzymes in the circulation means that patients need biweekly parenteral administration which increases the risk of an immune response to the infused enzyme. In addition, systemically administered enzyme is unable to cross the blood-brain barrier and can therefore not be used to treat central nervous system disease manifestation. The LSDs are considered to be good candidates for gene therapy and the liver may be the ideal site for gene transfer. Newly synthesized lysosomal enzymes are secreted into the systemic circulation and are recaptured by distant cells. Based on the observed enzyme levels in patients with mild late-onset disease, the amount of enzyme needed to correct the deficiency may only be 110% of normal levels (Cheng and Smith, 2003). Gene transfer to naturally occurring animal models of MPS type VII has been investigated using adeno-associated virus (Daly et al, 1999), adenovirus (Kamata et al, 2003) and lentivirus (McCray Jr et al, 2001). Intravenous administration of retroviral vectors containing canine "glucuronidase to neonatal MPS type VII dogs prevented some bone and joint abnormalities, corneal clouding and heart valve defects that commonly occur in this animal

B. Metabolic disorders Inherited inborn errors of metabolism can affect a number of metabolic pathways. For example the urea cycle disorders are caused by defects in genes encoding enzymes or membrane transporters in ureagenesis. Their prevalence is approximately 1:30,000 births and ornithine transcarbamylase (OTC) deficiency is one of the most severe of these conditions (Summar and Tuchman, 2001). OTC deficiency is transmitted as a partially dominant Xlinked trait. In patients with partial OTC deficiency, such as hemizygous males and heterozygous females, the first clinical episode is delayed for months or years with less severe hyperammonemia. However, patients with complete OTC deficiency present with life-threatening hyperammonemia within one week of birth and despite medical therapy to reduce the ammonia levels, 50% of the children are dead by the age of 4, and of those surviving, the mean IQ is less than 50 (Maestri et al, 1999). Since the urea cycle is principally sited in the liver, gene therapy directed towards hepatocytes has the potential to correct the metabolic abnormality. Indeed the success of orthoptic liver transplantation in long-term treatment of this condition supports the concept (Lee and Goss, 2001). Adenoviral vectors have been shown to transiently correct OTC deficiency in the sparse fur murine model after neonatal and adult treatment (Stratford-Perricaudet et al, 1990; Ye et al, 1996). In a phase I human clinical trial in patients with partial OTC deficiency, adenoviral vectors expressing the human OTC窶田DNA were administered. There was evidence of dose-related toxicity to the adenovirus and the last patient treated suffered a systemic


David et al: Current status and future direction of fetal gene therapy model (Ponder et al, 2002). Some aspects of bone disease were not prevented however, which may be due to abnormal bone formation in utero. There was also concern that systemic gene therapy administration may not reach the brain even in neonatal dogs when the blood-brain barrier is still forming. The immature blood-brain and blood 窶田erebrospinal fluid (CSF) barrier is more permeable to small proteins than in mature brains and there is a developmentally regulated mechanism that selectively transfers some larger proteins from the blood to the CSF (Dziegielewska et al, 2001). Thus a prenatal gene transfer approach may be more effective and also applicable to other disorders that affect the brain, such as the glycosphingolipid lysosomal storage diseases (Gaucher and Tay-Sachs disease) (Jeyakumar et al, 2002).

18th-20th week of gestation (Vassilopoulos and Emery, 1977; Turkel et al, 1981) and presents clinically between 2-4 years of age, complicates postnatal gene therapy. Thus a prenatal approach to treatment might prevent the disease process. Prenatal gene transfer may offer advantages over neonatal or adult treatment. Efficient gene delivery to several affected muscles groups is technically difficult and the alternative may be efficient gene transfer to a large percentage of existing and rapidly expanding muscle cells in utero. Postnatal gene delivery is also complicated by the risk of cellular immune responses against the transgenic proteins as demonstrated in the dystrophin-deficient mdx mouse model by loss of transgenic dystrophin-expressing fibres following dystrophin gene transfer (Wells and Wells, 2000; Chamberlain 2002). In contrast in utero gene transfer may avoid the development of immune reactions to the vector or transgene product and enable repeat injection postnatally. Furthermore immune responses have been reported in several adenovirus-mediated gene transfer studies although it was not possible to determine the relative contribution of the immune response to the vector or transgene. In most DMD patients, there is a lack of dystrophin expression which could lead to a functional copy of the dystrophin protein being recognised as a foreign antigen. Gene transfer during fetal life could lead to immunological tolerance to the dystrophin or allow repeated injection post-natally. Similar conditions such as the congenital Emery-Dreifuss and Fukuyama muscular dystrophies (Emery, 2002) could also potentially be treated using a prenatal gene transfer approach.

D. Muscular dystrophies Duchenne muscular dystrophy (DMD) is the commonest form of muscular dystrophy, a group of congenital disorders characterised by muscle wasting and weakness. This X-linked recessive disease has an incidence of 1 in 3500 live male births. Affected boys are usually diagnosed aged 3-4 years and characteristically, skeletal muscle degeneration after repeated rounds of necrosis is followed by the onset of fibrosis that eventually leads to muscle weakness and death (Emery, 1993). Patients are usually confined to a wheelchair by age 11 years, and although improved nursing care and positive pressure ventilation to aid breathing allows some patients to reach the 3rd decade, respiratory or cardiac failure is the common cause of death (Simonds et al, 2000). Prenatal diagnosis is available for almost all muscular dystrophies including Duchenne (Emery, 2002). Current treatment includes supportive measures such as surgery for correction of contractures and prevention of respiratory infections. The disease is caused by mutations in the DMD gene that encodes the 427kDA protein dystrophin, associated with the sarcolemma in muscle. Skeletal and cardiac muscle biopsies from DMD patients are characterized by absent or abnormal dystrophin. Gene transfer into muscle cells has been explored using naturally occurring animal models of muscular dystrophy that involve mutations in the DMD gene (Wells and Wells, 2000). The large size of dystrophin cDNA (14kb) precludes insertion into conventional vectors with the exception of gutless adenovirus. Consequently the majority of viral constructs incorporate mini or microdystrophin cassettes based on a 6.3kb truncated dystrophin gene resulting from a large inframe deletion in the rod domain which was isolated from a Becker muscular dystrophy patient with very mild symptoms. Adenoviral transfer of minidystrophin results in good transduction of neonatal mdx mouse muscle with reduced degeneration and improved muscle mechanics (Deconinck et al, 1996; Vincent et al, 1993). In the neonatal and adult mdx mouse, injection of an adeno-associated virus containing a minidystrophin into the leg muscle led to normal myofiber histology and protected membrane integrity (Wang B et al, 2000). The early onset of this disease, which begins to be visible histologically by the

E. Neurological disorders Spinal muscular atrophy (SMA) is one of the most common inherited causes of childhood mortality, with an incidence of 1 in 10,000 live births. It is characterized by progressive degeneration of alpha motor neurons within the spinal cord and results in proximal, symmetrical limb and trunk muscle paralysis that leads to death (Crawford and Pardo, 1996). SMA is caused by homozygous loss or mutation in the survival motor neuron gene 1 (SMN 1) which is telomeric. Humans and primates also have a centromeric copy called the SMN 2 gene but this fails to provide sufficient full-length SMN protein to maintain motor neurons. Evidence from family studies and animal models of SMA suggest that the number of copies of the SMN 2 gene may modify the severity of the disease. Gene therapy strategy would have to provide and express a functional copy of the SMN gene in the relevant neuronal cells. Efficient expression of the SMN gene was demonstrated recently after adenovirus-mediated delivery of the SMN gene to human primary fibroblasts from SMA patients in vitro (DiDonato et al, 2003). Intraspinal or intramuscular application of a vector targeting neuronal cells will be required for in vivo therapy and other diseases requiring this targeting include amyotrophic lateral sclerosis. Immunohistochemical analysis of normal fetal tissue has demonstrated that the expression of SMN protein is relatively high in skeletal muscle, heart and brain and 184

Gene Therapy and Molecular Biology Vol 7, page 185 undergoes a marked drop in the postnatal period. In contrast, SMN protein is greatly reduced in all tissues from fetuses affected with SMA (Burlet et al, 1998). These observations suggest that SMN protein may be required during embryo-fetal development and as such, prenatal gene transfer may be more effective than adult treatment. Prenatal diagnosis is available using deletion analysis of the SMN 1 gene (Matthjis et al, 1998).

Mediterranean region, the Middle East, the Indian subcontinent and South-East Asia where gene frequencies reach 3-10% of the population (Weatherall and Clegg, 1996). "-thalassaemia is characterized by insufficient production of the "-globin peptide by erythroid cells which results in low levels of the major form of adult haemoglobin, HbA, made up of two !- and two "-globin chains. The excess !-globin chains then precipitate in the erythroid cells, impair their maturation and this leads to haemolysis and anaemia. Homozygotes or compound heterozygotes suffer with the most severe form of the disease, "-thalassaemia major. Similarly !-thalassaemia results in excess "-globin chains due to different degrees of !-globin chain deficiency. In the most severe form, !othalassaemia, all four !-globin chains are defective or absent which leads to hydrops fetalis and intrauterine death. Patients with thalassaemia require regular lifelong blood transfusions to survive although this leads to iron overload that affects the liver, heart and endocrine organs. Prevention of iron overload with iron-chelating therapy such as parenteral deferoxamine is the mainstay of current patient management. Therapies aimed to increase the production of fetal haemoglobin have had disappointing results (Olivieri and Weatherall, 1998). Allogeneic haematopoietic stem cell replacement offers the only definitive cure and has been successful in over 1000 patients worldwide (Olivieri, 1999). Outcomes depend on whether the patient has hepatomegaly, portal fibrosis and has effective chelating therapy before transplantation. The 3 year disease-free survival falls from over 90% to 60% in children with the above risk factors. Gene therapy approaches have aimed to stably introduce a regulated human globin gene into haemopoietic stem cells. Recently high expression of erythropoietin was found to improve the anaemia of "thalassaemia in a mouse model by induction of high levels of HbF synthesis (Johnston et al, 2003). Expression of transgenic globin sequences would need to be sustained, finely regulated and at high levels since haemoglobin synthesis represents 95% of all protein synthesis in reticulocytes. Initial attempts at gene therapy using the "globin gene and a minimal locus control region (LCR) incorporated into a retroviral vector showed low levels and short-term expression of "-globin after transplantation of transduced haematopoietic stem cells into lethally irradiated mice (Raftopoulos et al, 1997; Sadelain 2002). More recently lentiviral vectors containing the "-globin gene and larger LCR elements have been used to transfect bone marrow from "-thalassaemic mice. This was then transplanted into "o-thalassaemic heterozygote mice and resulted in therapeutically relevant levels of circulating haemoglobin (May et al, 2000). An advantage of prenatal gene therapy application in this context could be the access to rapidly dividing stem cell populations. Prenatal diagnosis for haemoglobinopathies can be done by assessment of globin-chain synthesis in fetal blood or by direct analysis of fetal DNA obtained by chorionic-villus sampling or amniocentesis. Sickle cell disease, another inherited disorder of haemoglobin may also be amenable to prenatal gene therapy. In this condition missense mutations in the "-

F. Haemophilias The haemophilias A and B are also particularly suitable for gene therapy in utero. Both are X-linked hereditary haemorrhagic disorders which occur in 1 in 10,000 and 1 in 25,000 males respectively and are caused by the absence or dysfunction of the respective human factor VIII (hFVIII) or IX (hFIX) clotting factors (Furie et al, 1994). Current treatment uses replacement therapy with hFVIII or hFIX. Unfortunately, a number of patients develop antibodies to therapy leading to ineffective treatment and occasional anaphylaxis (Lusher, 2000). Indeed, the complications of haemophilia treatment have in some cases been far worse than the diseases themselves, increasing their morbidity and mortality (Soucie et al, 2000). As the coagulation factors are required in the blood and can be secreted functionally from a variety of tissues, the actual site of production is not so important as long as therapeutic plasma levels are realized. Adult gene therapy strategies have therefore concentrated on application to the muscle or the liver. Successful delivery and expression of FIX has been achieved in adult animal models of haemophilia B following portal intravascular administration of adenoviral (Kay et al, 1994) and retroviral vectors (Kay et al, 1993). Sustained FIX expression was also observed after intramuscular injection of adult haemophiliac dogs with adeno-associated viral (AAV) vectors expressing canine FIX (Chao et al, 1999; Herzog et al, 1999) and after intravascular injection of adult haemophiliac mice with AAV vectors expressing hFIX (Snyder et al, 1999). These results have culminated in the first clinical trial in humans that shows promising results although only low level hFIX expression has so far been observed (Kay et al, 2000). Successful delivery and expression of therapeutic hFIX without formation of antibodies has been achieved following administration of retroviral vectors in neonatal animal models (Xu et al, 2003). Prenatal gene therapy could be applied to the fetus via a number of routes including muscle, peritoneal, hepatic, intravascular or skin application. More recently our group has demonstrated that in utero application can provide long-term postnatal correction of the haemophiliac phenotype in FIX deficient mice (Waddington et al, submitted). Prenatal diagnosis is available early in pregnancy (Ljung, 1999).

G. Haematopoietic diseases 1. The thalassaemias The thalassaemias are inherited anaemias caused by over 200 mutations and globally are the commonest monogenic disorders. They are most prevalent in the


David et al: Current status and future direction of fetal gene therapy globin gene lead to haemoglobin polymerization causing the red blood cells to become deformed or ‘sickled’. The ability of gene therapy to correct the pathophysiology has been demonstrated in a study in transgenic sickle Hb mouse models. Bone marrow transduced with lentiviral vectors containing a "A globin gene variant that prevents haemoglobin polymerization was transplanted into two mouse sickle cell disease models resulting in therapeutic correction of the disease (Pawliuk et al, 2001).

bone marrow and PEG-ADA treatment was continued in all patients during and after treatment which made it difficult to evaluate immune function (Blaese et al, 1995; Bordignon et al, 1995; Kohn et al, 1995). Some patients showed long term persistence of the transduced cells although at low level. A more recent trial was performed in two infants with nonmyeloablative conditioning using busulfan and without concurrent PEG-ADA treatment. Both patients showed sustained engraftment of genetically corrected haematopoietic stem cells with differentiation into multiple lineages and improvement in their clinical condition (Aiuti et al, 2002). In a similar way Xl-SCID has been treated using autologous transplantation of CD34+ bone marrow transduced ex vivo with retroviral vectors containing the #c gene. Fifteen patients have now been treated and effective immune reconstitution has been achieved in thirteen patients (Friedmann, 2003). Unfortunately because of a serious adverse event in two of the patients, all gene therapy trials involving retroviral vectors in haematopoietic stem cells were initially halted in the US (Gansbacher and European Society of Gene Therapy 2003) (see VI Ethical and safety issues) and have now been restricted to case by case reviewed permission (Friedmann, 2003). Nevertheless this study has shown the ability of gene therapy to cure such conditions. Because of the survival advantage of genetically corrected cells and the ineffective immune response in SCID patients, it is unlikely that prenatal gene transfer would provide a particular benefit over postnatal treatment of this condition.

2. Immunodeficiency disorders The greatest success of gene therapy so far has been in the treatment of congenital severe combined immunodeficiency disorders (SCID). These represent the most severe form of primary immunodeficiencies and they occur in approximately 1 in 75,000 births. The most common types of SCID are X-linked (Xl-SCID) and the autosomal recessive adenosine deaminase deficiency (ADA) found in 50% and 15% of sufferers respectively. In both conditions the genetic defect causes a profound block in T cell differentiation which leads to absent T cell and humoral responses. Xl-SCID is due to a deficiency of the #c chain, an essential component of cytokine receptors which is necessary for T cell and natural killer cell development. In ADA deficiency there is selective accumulation of the toxic metabolite deoxyATP in T cells. Clinically the patients present with chronic diarrhoea and failure to thrive with recurrent respiratory and opportunitstic infections leading to death within the first year of life (Cavazzana-Calvo et al, 2001). Histocompatible bone marrow transplantation (BMT) has been used to treat both conditions with some success. Survival after transplantation with HLA-identical bone marrow is over 90% but matched sibling donors are usually not available. Haploidentical BMT with T-cell depletion is commonly performed instead, with survival rates of up to 78% although many patients require lifelong immunoglobulin replacement therapy because of inadequate humoral activity (Buckley RH et al, 1999). In utero haematopoietic stem cell transplantation has been achieved in fetuses with Xl-SCID by ultrasound guided intraperitoneal or intravenous injection (Flake et al, 1996; Touraine 1992; Wengler et al, 1996; Westgren et al, 2002). A selective T-cell and natural killer cell reconstitution can be achieved but B cell engraftment has not been detected. In ADA deficiency, a long-circulating form of bovine ADA conjugated with polyethylene glycol (PEG-ADA) has been used to correct the metabolic abnormalities and prevent life-threatening opportunistic infections. The strategy for gene therapy of SCID is based on the concept that genetically corrected autologous T-cell precursors should have a selective survival advantage over non-corrected cells. In addition, patients are unable to mount an effective immune response to the transgene which has proved to be a major problem in gene therapy treatment of other genetic diseases. In ADA-SCID, clinical trials have used infusion of autologous peripheral T-cells, CD34+ bone marrow or umbilical cord blood cells transduced with a retroviral vector containing ADA cDNA. The earlier trials did not use conditioning of the

H. Skin disorders Fetal gene delivery into the amniotic cavity may have unique benefits for treatment of inherited skin disorders. Epidermolysis bullosa is a group of inherited blistering diseases characterized by epidermal-dermal separation resulting from mutations that affect the function of critical components of the basement membrane zone. The dystrophic form of epidermolysis bullosa (DEB) is due to mutations in COL7A1, the gene encoding type VII collagen and has a prevalence of up to 2.4 per 100,000 population (Horn and Tidman, 2002). The clinical presentation varies from a mild dominantly inherited disease characterized by skin and oral blisters and nail dystrophy to a severe recessive subtype in which patients suffer from contractures, severe dental caries, dysphagia, anal fissures and squamous cell carcinoma. Current therapy involves management of the disease manifestations with proper wound care, surgical release of skin contractures, balloon dilatation of oesophageal strictures and graft skin therapy (Pai and Marinkovich, 2002). Easy accessibility and visualization of skin make it an attractive target for gene therapy. Gene delivery can be in vivo by direct introduction to the skin by injection, electroporation or a ‘gene gun’. Alternatively a skin sample could be removed from the patient, and epidermal keratinocytes cultured and transduced ex vivo to insert genetic material and the genetically engineered cells


Gene Therapy and Molecular Biology Vol 7, page 187 returned in the form of a skin graft (Uitto and Pulkkinen, 2000). Preliminary studies show keratinocytes and fibroblasts from patients with DEB can be successfully transduced using lentiviral vectors containing the COL7A1 transgene in vitro resulting in long-term expression and synthesis of type VII collagen (Chen et al, 2002). In a canine animal model of DEB, transduction of keratinocytes with a retrovirus containing the collagen type VII cDNA corrected the observable defects in in vitro reconstructed skin (Baldeschi et al, 2003). A non-viral gene transfer approach has been used for junctional epidermolysis bullosa (JEB) in which there is severe laminin-5 deficiency. Integration of an attB-containing laminin 5 "3 expression plasmid using $C31 integrase into human keratinocytes from JEB patients produced skin tissue with no histological evidence of subepidermal blistering when regenerated on SCID mice (Ortiz-Urda et al, 2003). Epidermolysis bullosa however, is a generalized disorder affecting the entire skin and the extracutaneous tissues. Prenatal therapy delivered into the amniotic fluid would bathe the entire skin surface and reach the gastrointestinal system by fetal swallowing. Injection into the amniotic cavity can be performed safely at relatively early gestation, but the timing of intra-amniotic delivery will be important from developmental considerations. Even at 20 weeks gestation, the fetal epidermis is incompletely keratinized and this would aid gene tranfer. However there is a high rate of apotosis in fetal keratinocytes and therefore the ideal strategy would be to target stem cells (Haake and Cooklis, 1997). Prenatal diagnosis for epidermolysis bullosa can now be performed with a 98% success rate in at risk families, paving the way for preliminary studies into prenatal treatment (Pfendner et al, 2003). Disorders of defective keratinisation such as harlequin ichthyosis, an autosomal recessive severe and usually fatal congenital ichthyosis (Akiyama, 1998), may also be amenable to prenatal gene transfer.

hypoplasia. Temporary occlusion of the trachea with an expandable balloon for treatment of CDH results in impressive expansion of the hypoplastic lung with tracheal fluid. However â&#x20AC;&#x2DC;pluggingâ&#x20AC;&#x2122; has yet to be shown to improve outcome in the long term (Harrison et al, 1998). Studies suggest that pulmonary hypoplasia in CDH begins during embryogenesis as an abnormality in growth factor signalling and actually precedes the development of the anatomical defect (Jesudason 2002). Prenatal gene therapy could be envisaged in the future to enhance antenatal lung growth and maturation by the targeted delivery of growth factors at specific times during lung development.

J. Infectious disease Infectious diseases with pathogens such as Group B streptococcus, human immunodeficiency virus, hepatitis B virus and herpes simplex virus are a major cause of neonatal morbidity and mortality. Transmission of these diseases from mother to infant often occurs shortly before, during, or after birth by early rupture of the amniotic membranes or direct contact with infectious secretions during labor and delivery. Delivery by caesarean section to prevent such contact, and antibiotic and maternal antiviral treatments have been used with some success, particularly in the prevention of vertical HIV transmission. Immunisation of the fetus with DNA vaccines in late pregnancy has been proposed as an alternative approach to prevent neonatal infection (Gerdts et al, 2000; Sarzotti et al, 1996; Watts et al, 1999). The mucosal surfaces of the eyes, respiratory and gastrointestinal tract are the primary site of entry for infectious agents during birth and the neonatal period. Thus intra-amniotic or intra-oral delivery of antigen would probably provide the best disease protection. Studies in the fetal mouse (Sarzotti et al, 1996), sheep (Gerdts, et al, 2000) and baboon (Watts et al, 1999) have shown that fetal immunisation can induce active immunity in the newborn. In particular, in the fetal sheep, intra-oral administration of hepatitis B surface antigen DNA resulted in a higher protective antibody titre than an intramuscular injection of the recombinant protein vaccine (Gerdts, et al, 2003). The timing of such an intervention is crucial since exposure of the fetus to the antigen before immune competence is reached may result in tolerance. In addition a single in utero injection may not be sufficient to maintain immunity. At present there is no clinical indication for such a prenatal immunization strategy.

I. Perinatal disease Pulmonary hypoplasia is another important cause of neonatal morbidity and mortality. In this condition, the fetal lungs fail to develop resulting in respiratory insufficiency at birth. Current neonatal management is supportive and involves surfactant replacement, careful mechanical ventilation avoiding barotrauma and treatment of pulmonary hypertension. Pulmonary hypoplasia can occur when there is reduced or no liquor surrounding the fetus (oligo or anhydramnios) prior to 22 weeks gestation, most commonly because of preterm premature rupture of the membranes (PPROM). Serial amnioinfusion has been used for the prevention of pulmonary hypoplasia with some success but has a high complication rate (Tan et al, 2003). Space occupying lesions that compress the lungs within the chest cavity also result in pulmonary hypoplasia. Examples of such conditions include pleural effusion associated with congenital cardiac defects and congenital diaphragmatic hernia (CDH) in which the bowel herniates through the diaphragmatic defect. Fetal interventions such as drainage of pleural effusions can be used to treat the underlying cause of the pulmonary

K. Placental disorders Pre-eclampsia/eclampsia is one of the leading causes of maternal and fetal morbidity and mortality. The underlying defect is believed to be inadequate deep placentation that fails to transform the spiral arteries into uteroplacental vessels and thus limits placental blood flow (Brosens et al, 2002). Secondary damage such as fibrin deposition and thrombosis then limit placental perfusion further and there is also widespread activation of the maternal vascular endothelium leading to decreased formation of vasodilators such as nitric oxide (Walker, 2000). Gene therapy could be used to improve uteroplacental perfusion by for example, temporary expression of nitric oxide synthase or placental growth factor. This 187

David et al: Current status and future direction of fetal gene therapy could prolong the pregnancy until fetal maturity was attained and reduce the likelihood of long-term complications in the mother and fetus. Intrauterine growth restriction (IUGR) affects up to 8% of all pregnancies. It commonly occurs in pregnancies complicated by pre-eclampsia but can also arise in normotensive pregnancy. As well as leading to neonatal problems, the long-term consequences are serious since IUGR infants exhibit higher rates of coronary heart disease, type 2-diabetes, hypertension and stroke as adults (Barker et al, 1993). Abnormalities in placental development are believed to adversely affect placental function and deprive the fetus of the nutrients required for optimal growth. Transport of amino acids and essential fatty acids across the placenta is altered in IUGR fetuses and impaired oxygenation and acid base balance may be seen in severe cases (Pardi et al, 2002). Prenatal gene therapy could target placental transport mechanisms and increase the availability of essential nutrients to the fetus.

galactosidase gene (lacZ). These allow tracking of the transduced cells and to define tissue expression by biochemical staining assays. Alternatively, use of vectors carrying therapeutic genes allows the assessment of potentially curative levels of the expressed protein and, in animal models of disease, even the observation of phenotype correction. The hFIX gene for instance, can be used both as a marker gene, allowing the analysis of blood levels of the hFIX protein over time in non-haemophiliac animals, and to study the correction of the blood clotting parameters in animal models of haemophilia. Postnatal readministration of hFIX protein or the hFIX vector to fetally treated animals can be used to examine whether immune tolerance has been achieved.

1. Retrovirus Vectors that are able to integrate into the host genome such as retroviruses, lentiviruses and to a lesser extent adeno-associated viruses, may offer the possibility of permanent gene delivery. Although only fairly low virus titres can be produced, virus gene transfer may be improved by complexing vectors with cationic agents, (Themis et al, 1998) or by the administration of retrovirus producer cells in vivo to allow localised gene delivery close to the site of cell transfer (Douar et al, 1997; Russel et al, 1995). Retroviruses require dividing cells for gene transfer (Miller DG et al, 1990) which suggests that they may be better suited for use in fetal tissues where cells are rapidly dividing rather than in adult applications. Other problems include reports of premature promoter shutdown (Palmer et al, 1991; Challita and Kohn 1994) leading to transcriptional shutoff. Human serum can almost completely inactivate some retroviral particles (Welsh et al, 1975) which limits their use in vivo although increased resistance to serum inactivation can be achieved by generating retroviruses from particular human packaging cells (Cosset et al, 1995) or by pseudotyping, which replaces the natural envelope of the retrovirus with a heterologous envelope (Engelst채dter et al, 2001). A particular problem with in utero application is that amniotic fluid has also been shown in vitro to have a mild inhibitory effect on retrovirus infection (Douar et al, 1996). A further difficulty is the relatively short half-life of the retroviral particles in vivo which may hinder transduction because fetal cell division is nonsynchronized and only those cells undergoing cell division at the time of infection will become transduced. Retroviruses were used in the first successful gene therapy trial, where bone marrow stem cells transduced ex vivo with retroviral vectors expressing the correct cDNA were delivered to infants suffering from an X-linked form of severe combined immunodeficiency (SCID) (Cavazzana-Calvo et al, 2000). The infants were able to leave protective isolation, discontinue treatment and appear to be developing normally (Hacein-Bey-Abina et al, 2002). However two of the fifteen patients treated for X-linked SCID have developed leukemia which has been shown to involve insertional mutagenesis. An expanded clonal population of T-cells was demonstrated to be

III. Vectors for in utero gene delivery The development of efficient vector systems is crucial for the success of gene therapy. The ideal vector for fetal somatic gene therapy would introduce a transcriptionally regulated therapeutic gene into all organs relevant to the genetic disorder by a single safe application. Although none of the present vector systems meet all these criteria, many of them have characteristics that may be beneficial to the fetal approach.

A. Non-viral vectors Cationic liposome/DNA complexes have the advantage of being relatively non-toxic and nonimmunogenic but are still very inefficient in vivo. Another drawback with these vehicles is that the DNA introduced as plasmid molecules remains episomal and will be lost over time following cell division. This is a particular disadvantage in the fetus where cell populations are rapidly dividing. However, short term transgene expression has been shown to be a promising approach to maintain a patent ductus arteriosus prior to surgery for congenital heart defects in neonates (Mason et al, 1999). Liposomes containing plasmid expressing a decoy RNA designed to sequester fibronectin mRNA binding protein were delivered to the ductus arteriorus in fetal sheep at 90 days of gestation, prior to the onset of intimal cushion formation at 100 days of gestation. Fibronectin synthesis was inhibited resulting in a 60% reduction in intimal thickness and increased ductal patency at term. More recently, non-viral systems have been developed that integrate into the host genome and could thus in principle provide long term gene expression, but these vectors are still at an early stage of experimental design (Olivares et al, 2002).

B. Viral vectors Studies of in utero gene therapy have therefore concentrated on viral vectors, many of which have been designed to deliver reporter genes such as the "-


Gene Therapy and Molecular Biology Vol 7, page 189 carrying the transgene inserted at 11p13 in the region of LMO2, an oncogene frequently overexpressed in T cell leukemias (Marshall 2002). Insertional mutagenesis is an acknowleged potential complication with retroviral mediated gene transfer because gene integration occurs randomly into the genome. This is the first report of malignant change in humans following retroviral gene therapy and only one example has been found in extensive animal studies using this vector (Li et al, 2002). Investigations are ongoing to determine whether any other factor contributed to the development of insertional mutagenesis and clonal expansion in these particular patients (Friedmann 2003).

the wild type virus is predominantly at an apparently specific functionally unimportant location on human chromosome 19 reducing the theoretical risk of insertional mutagenesis; however recombinant vector appears to integrate at low levels and non-specifically (Monahan and Samulski, 2000). AAV vectors have a limited capacity for the insertion of foreign genes that is about 4.7kb, although recently 'split AAV vectors' have been designed where large genes are split between two AAV genomes to increase AAV packaging capacity. After concatemerisation of these genomes in the host cell mRNA, splicing allows the removal of intervening ITR sequences and restoration of the split coding sequence to yield wild-type functional protein (Sun et al, 2000). Because the extent of AAV integration is still in question, this vector system may not give the permanent gene expression ideal for in utero gene therapy without repeat treatment, although long term transgene expression after intraperitoneal delivery in mice has recently been reported (Lipshutz et al, 2003). Some caution has also been expressed as AAV integration appears to induce chromosome deletions (Nakai et al, 2003).

2. Lentivirus Because of the limitation of infection to dividing cells by retroviruses, alternative vectors such as lentiviruses have been developed to circumvent this restriction. Significant progress has been made in recent years in the development of lentiviral vectors, a retroviral sub-group based on the Human Immunodeficiency Virus (HIV) (Trono, 2000) or Equine Infectious Anaemia Virus (EIAV) (Mitrophanous et al, 1999). HIV vectors are capable of transferring genes into nondividing cells such as neurons (Naldini et al, 1996) and quiescent haematopoietic progenitor cells, (Case et al, 1999) which will be particularly useful for these tissue targets. Lentiviral vectors integrate into the genome randomly and are therefore theoretically able to cause insertional mutagenesis. Lentiviruses can be made more stable by pseudotyping which allows virus titres to be improved by ultracentrifugation. This offers the opportunity of infecting a greater number of cells in vivo and different envelopes allow targeted gene transfer to specific tissues, for example to the nervous system (Mazarakis et al, 2001) and airways (Kobinger et al, 2001). Both the EIAV vector, a vector derived from non-primate animal lentiviruses, (Mitrophanous et al, 1999) and Feline Immunodeficiency Virus (FIV) (Wang, et al, 1999) have been developed in an attempt to create vectors for use in human treatment which are not associated with any human pathology. Our recent work has shown that high level sustained transgene expression can be achieved in a variety of tissues using the EAIV vector in fetal mice after intravascular administration (Figure 1) (Waddington et al, 2003).

4. Adenovirus Adenoviral vectors have been used as attractive vectors for proof of principle studies in fetal gene therapy since they have continually achieved highly efficient gene transfer in vivo. The adenoviral coding sequences necessary for viral replication are deleted, rendering them replication defective. They are relatively stable and can be obtained at high titre making systemic administration in humans and large animal models feasible. The adenovirus genome replicates outside the chromosome, which avoids the risk of insertional mutagenesis but results in only transient gene expression. Their broad host range and tropism to most cells of the human body, including the respiratory epithelium has made them very useful in initial pathfinder studies on vector delivery and transgene expression. They are particularly useful for exploring different technical approaches to fetal gene therapy. Factors that determine the kinetics of transgene expression include vector elimination, since adenovirus is not an integrating vector, and promoter shutdown. Adenoviral vectors are also highly immunogenic. Major concerns about the safety of adenoviral vectors were raised following the death of Jesse Gelsinger from a systemic inflammatory response to a first generation adenovirus vector used for a phase I clinical trial towards gene therapy of the inherited metabolic disorder, ornithine transcarbamylase deficiency (Lehrman, 1999). Even fetal administration of adenoviral vectors has been associated with an immune response (McCray, et al, 1995) particularly after postnatal repeat exposure to the vector (Iwamoto et al, 1999). Attempts to reduce the immunogenicity and toxicity of the vector and to increase its insert capacity have led to the generation of the so called â&#x20AC;&#x2DC;gutless vectorsâ&#x20AC;&#x2122; in which essentially all adenoviral coding sequences have been eliminated (Chen et al, 1997; Schiedner, et al, 1998).

3. Adeno-associated viral vectors Adeno-associated virus (AAV) is also a promising novel vector system. It is a common human parvovirus that is not associated with any human pathology. AAV naturally requires co-infection with adenovirus as a helper virus, but the latest AAV vectors circumvent the need for adenovirus and therefore make the production of pure AAV particles easier (Xiao et al, 1998). AAV is also able to infect non-dividing cells and to achieve long-lasting gene correction in vitro and in vivo (Herzog et al, 1999; Wang et al, 1999; Kay et al, 2000). The basis for longterm transgene expression is not quite clear. Integration of


David et al: Current status and future direction of fetal gene therapy

Figure 1. Upper panel. Representative sections of fetal livers harvested at 72h, 7, 14, 28, 79, 168 days and 1 year after yolk sac injection of high titre titre EAIV SMART2Z (equine infectious anaemia virus vector expressing the "-galactosidase gene driven by the CMV promoter) lentiviral vector (n=1, 1, 3, 1 and 1, respectively). Uniform hepatocyte staining is observed after 72 h followed by the emergence of clusters of "-galactosidase-stained hepatocytes to day 79. Macroscopic appearance of liver sections (top row, x 10). Microscopic analyses (bottom row, x 400). Age matched noninfected control livers of 3 day old and 1-year-old animals are shown in the lower panel. Lower panel. Representative sections of fetal tissues harvested at 72 h, 7, 14, 79 days and 1 year after yolk sac injection of high titre EAIV SMART2Z lentiviral vector (nâ&#x20AC;Ś1, 1, 3 and 1, respectively). High-level staining is observed after 72 h and 79 days in brain, 7, 14 and 79 days in heart and 14 and 79 days in skeletal muscle. Low-level expression is shown in lung and kidney at 79 days postinjection. Macroscopic appearance of tissues (left columns, x 10). Microscopic analysis (right column, x 400). (Waddington et al 2003). Republished with permission from Nature Publishing Group.


Gene Therapy and Molecular Biology Vol 7, page 191 of intercellular propagation. In these vectors, genes encoding surface glycoproteins including the haemaglutinin-neuraminidase (HN) protein or the fusion (F) protein, which are responsible for cell binding and infection, have been deleted from the viral genome (Inoue et al, 2003). Injection of F-deficient Sendai virus vector into the fetal mouse via various routes including intravascular, intra-amniotic, intra-muscular, intra-peritoneal and intra-spinal resulted in expression of marker gene in gut wall, lung, muscle, peritoneal mesothelia and dorsal route ganglia respectively. Further optimisation will be needed to develop these first generation constructs into clinically applicable vectors (Waddington et al, submitted).

Because adenoviruses provide highly efficient gene transfer yet transient expression, novel hybrid vectors have been developed to take advantage of adenovirus infectivity and the permanent nature of integrative vectors such as retroviruses and lentiviruses (Murphy et al, 2002; Kubo and Mitani, 2003). Hybrid vectors may offer efficient gene expression to fetal organs such as the lung in which it has so far proved difficult to achieve high level gene transfer with integrating vectors.

5. Sendai virus Recently, the negative strand RNA cytoplasmically replicating Sendai virus, a member of the paramyxovirus family was developed as a gene transfer vector. Early vectors still capable of self-propagation, were found to provide very high levels of marker gene expression in a wide range of tissues including bronchial epithelium (Yonemitsu et al, 2000), skeletal muscle (Shiotani et al, 2001) and vascular endothelium (Masaki et al, 2001). Second generation vectors, although still capable of intracytoplasmic replication of the RNA genome, are incapable

IV. Fetal gene therapy studies Since the initial attempts in the early 1990s, in utero gene therapy has been investigated in a range of different animals using a variety of techniques. The possible routes of administration are illustrated in Figure 2.

Figure 2. Routes of administration of gene therapy to the fetus. Routes in italics have not yet been applied in a fetal animal model using ultrasound guided injection.


David et al: Current status and future direction of fetal gene therapy late gestation. The guinea pig has the same placental structure as humans but they are not commonly used in prenatal gene therapy studies because of the small fetal size and lack of transgenic models of disease. There are unfortunately few large animal models of human genetic disease available for testing of gene therapy. Efforts to produce transgenic domestic animals are continuing particularly in the pig, sheep and cow (Piedrahita 2000). There are however, some dog models including mucopolysaccharidosis type VII, Duchenne muscular dystrophy and haemophilia B, which are useful for investigating the therapeutic effect of gene therapy. The dog is also a suitable model for minimally invasive delivery techniques and studies on prenatal gene transfer have used ultrasound guided intraperitoneal or yolk sac injection through the exposed uterus (Lutzko et al, 1999; Meertens et al, 2002).

A. Animal models Small animals are the most commonly used because they offer a number of advantages. Transgenic mouse models exist for many genetic diseases such as cystic fibrosis and haemophilia and this allows the therapeutic effect of the gene therapy to be studied. Small animals are also cheaper to maintain and have short breeding cycles with large litters which permit studies over several generations e.g. on germline transmission. However, their size precludes their use for the development of minimally invasive techniques for gene therapy delivery as required in human application. Studies in large animals have mainly used sheep, since they are well established as an animal model relevant to human fetal physiology, have a good tolerance to in utero manipulations and a consistent gestation period of 145 days, which is approximately half that of the human. There are some differences between ovine and human biology (Newnham and Kelly 1993). In late gestation the fetal growth rate in sheep is over double that in humans (Fowden, 1995) and the placental weight declines from 90 days gestation while it remains static in the human (Barcroft and Barron 1946). However the major difference is in the structure of the placenta. In sheep the synepitheliochorial placenta consists of six tissue layers, three from the mother and three from the fetus, and it is the most complete barrier possible (Benirschke and Kaufmann 1990). The maternofetal interdigitations (placentomes) are spread throughout the uterine cavity and may be difficult to avoid during ultrasound-guided uterine interventions. In humans, there is only a single discoid placenta and there is extensive invasion of the endometrium by the trophoblast that removes the three maternal tissue barriers and results in a hemomonochorial placenta at term. Probably as a result of these structural differences, #-globulin does not pass from the mother to the fetus in the sheep, but is able to cross the placenta in humans. Nonhuman primates are close physiologically to humans with menstrual cycles of similar length and hormonal control, comparable cellular and endocrine processes of implantation, and similar timetables of prenatal development. The placental structure in some nonhuman primates is also the same, for example in the rhesus monkey the placenta is hemomonochorial and bidiscoidal (Benirschke and Kaufmann 1990). For this reason they are used as an animal model in studies of teratology, developmental biology, infertility and contraception (Hendrickx and Peterson 1997). Ultrasound guided injection techniques as used in fetal medicine have also been applied extensively in the fetal nonhuman primate with comparable results (Tarantal, 1990). However nonhuman primates are more costly than sheep and are difficult to maintain. The rabbit has been studied in some prenatal gene therapy studies. Minimally-invasive percutaneous ultrasound guided injection and fetoscopic procedures are also being developed (Brandt et al, 1997; Papadopulos et al, 1999). Because of the small size of the fetus and litter number however, technically this is only possible from

B. Application routes in fetal medicine Invasive surgical techniques such as maternal laparotomy or hysterotomy must be performed to access the fetus in small animal models, but have also been applied in large animal studies such as in the sheep (Tran et al, 2000; Vincent et al, 1995). Surgery carries a high morbidity from wound infection and haemorrhage and the risk of mortality is significant. Minimally invasive procedures with fibreoptic telescopes are currently in use in fetal medicine and are being adapted for application of gene therapy in large animal fetuses. Fetoscopy was developed in the late 1970s for examination of 2nd trimester fetuses and for fetal blood sampling (Rodeck, 1980). The morbidity from fetoscopy is significant however, because of the relatively larger diameter of the puncture site in the fetal membranes which leads to premature rupture of the membranes and preterm labour and its associated problems. With the improvement in ultrasound technology in the 1990s, more detailed anatomical survey of the fetus could be performed and fetal blood sampling by ultrasound guided injection became routine practice. Operative fetoscopy has recently re-emerged for use together with ultrasound in endoscopic fetal surgery for conditions such as twin reversed-arterialperfusion sequence (Quintero et al, 1994), severe feto-fetal transfusion syndrome (Ville et al, 1997) and congenital diaphragmatic hernia (Harrison et al, 1998). Percutaneous ultrasound-guided injection is the least invasive technique for accessing the fetus and is used frequently in the clinical setting. Coelocentesis uses ultrasound to guide a needle into the extraembryonic coelom in the early first trimester. It has a success rate of >95% at 6-11 weeks of gestation, and has been suggested as a possible technique for stem cell engraftment in early gestation (Wilson and Wivel 1999). It may be of little use, however for in utero gene therapy because of the limited transfer from the extraembryonic coelom via the amniotic membrane to the amniotic cavity (Jauniaux and Gulbis 2000). Studies on the risk of miscarriage in ongoing pregnancies beyond the 1st trimester following coelocentesis gave controversial results (Makrydimas et al, 1997; Ross et al, 1997; Santolaya-Forgas et al, 1998).


Gene Therapy and Molecular Biology Vol 7, page 193 Amniocentesis is mainly used clinically for prenatal diagnosis. Although it is one of the safest intrauterine procedures, intra-amniotic application of vectors may be only of limited use in fetal gene therapy because of vector dilution by the large volume of amniotic fluid, although it would be the ideal application route for in utero gene therapy of skin diseases. Accessing the systemic circulation has greater potential. In fetal medicine, fetal blood can be obtained in the second trimester under ultrasound guidance either from the placental cord insertion, the fetal heart or more safely from the intrahepatic umbilical vein (Chinnaiya et al, 1998). The procedure has a good success rate clinically, is low risk and is used commonly for rapid karyotyping or fetal blood transfusion (Nicolini et al, 1990). From 12 weeks of gestation ultrasound-guided intracardiac puncture for fetal blood sampling has been performed on patients undergoing surgical termination of pregnancy (Jauniaux et al, 1999). Similarly, radiolabelled fetal liver cells were successfully injected into the heart of 13 week old fetuses under ultrasound guidance (Westgren et al, 1997) prior to prostaglandin termination of pregnancy. No fetal heart rate abnormalities were detected and all fetuses were alive at least 6 hours after the procedure. Intraperitoneal injection has been applied for in utero stem cell transplantation in humans from 14 weeks of gestation (Touraine 1999; Muench et al, 2001) and is an alternative route for blood transfusion before 18 weeks of gestation (Rodeck and Deans 1999). Ultrasound guided intramuscular injection has been used to deliver corticosteroid therapy for maturation of preterm infant lungs and vitamin K to the fetus (Larsen et al, 1978; Ljubic et al, 1999).

was seen in about 30% of fetal hepatocytes, and hFIX expression in fetal and neonatal plasma by ELISA analysis reached therapeutic levels within a week of delivery in two animals. In early gestation, delivery of adenoviral vectors into the umbilical vein of fetal sheep at 60 days of gestation via hysterotomy resulted in widespread transduction of fetal tissues (Yang et al, 1999). Our group has attempted ultrasound-guided umbilical vein injection of adenoviral vectors in fetal sheep at the earlier time of 53 days of gestation but this was unsuccessful due to procedurerelated mortality (David et al, 2003a). Ultrasound-guided intracardiac injection has been used to deliver adenoviral vectors to the late gestation fetal rabbit (Wang et al, 1998). Transgene expression was observed in up to 40% of fetal hepatocytes and was transient as expected. A fetal immune response to the vector and transgene was detected. Unfortunately the procedure also had a 25-40% mortality rate, comparable to other studies on fetal blood sampling in rabbits (Moise et al, 1992). Although technically straightforward, ultrasound-guided intracardiac delivery of adenoviral vectors to fetal sheep in early gestation resulted in 100% mortality due to haemorrhage (David et al, 2003a).

D. Alternative routes for targeting the fetal circulation and liver Due to the peculiarities of the fetal anatomy, vector delivery via the umbilical vein or yolk sac vessels will preferentially target the liver, which is an important organ for treatment of many genetic diseases. However in early pregnancy this not been technically possible and alternative approaches to reach the liver and the circulation have been tried.

C. Direct targeting of the fetal circulation Delivery of vectors to the systemic fetal circulation appears to be a highly effective route for targeting gene therapy to a range of fetal tissues and particularly to the liver for treatment of diseases such as the haemophilias and the metabolic and storage disorders. This can be accomplished in small animals such as the mouse by intracardiac injection (Christensen et al, 2000; Wang et al, 1998) or by injection into the yolk sac vessels (Schachtner et al, 1996). Indeed, yolk sac vessel injection of adenoviral vectors containing the hFIX gene into fetal mice resulted in therapeutic levels of hFIX expression (Waddington et al, 2002). Long-term transgene expression was observed in the liver, heart, brain and muscle up to a year after delivery of lentiviral vectors containing the "galactosidase gene into yolk sac vessels of fetal mice (Waddington et al, 2003) and was then used to achieve correction of the haemophilic phenotype in factor IX deffcient mice (Waddington, submitted). In larger animals such as in the sheep, intravascular delivery can be achieved by injection via the umbilical vein (Yang et al, 1999). Adenoviral vectors containing the lacZ or hFIX genes were delivered into the umbilical vein of late gestation fetal sheep using ultrasound-guided percutaneous injection from 102 days gestation (term = 145 days) (Themis et al, 1999). Positive lacZ expression

1. Intrahepatic injection Fetal intrahepatic injection has been performed in mice using adenoviral vectors (Lipshutz et al, 1999a, b, 2000; Mitchell et al, 2000), adeno-associated vectors (Mitchell et al, 2000; Sabatino et al, 2002) and lentiviral vectors (MacKenzie et al, 2002). In these studies, high levels of transgene expression in fetal hepatocytes were observed as well as gene transfer to other organs such as the heart, spleen, lung, intestine and brain suggesting haematogenic spread. Ultrasound guided intrahepatic injection has been performed in a few large animal models. In the late gestation fetal rabbit, X-gal staining of the fetal hepatocytes was seen 2 days after ultrasound guided intrahepatic injection of adenoviral vectors containing the "-galactosidase gene in late-gestation fetal rabbits (Baumgartner et al, 1999). Similarly, strong expression of transgenic enhanced green fluorescent protein was observed in hepatocytes one month after ultrasoundguided intrahepatic delivery of adeno-associated viral vectors to the late-gestation rhesus monkey (Lai et al, 2002). Ultrasound guided intrahepatic injection in early gestation sheep fetuses has also been performed with fetal survival rates of 81% (David et al, 2003a). Only low level 193

David et al: Current status and future direction of fetal gene therapy hepatocyte transduction however was observed after adenoviral and retroviral mediated gene transfer into fetal sheep (David et al, 2003a) and primates (Tarantal et al, 2001b).

such as hFIX in the treatment of haemophilias. In the fetal mouse, injection of adenoviral vectors containing the "galactosidase gene into the shoulder or hindlimb musculature resulted in persistent muscle and liver transgene expression for 16 and 8 weeks respectively after injection (Yang et al, 1999). Intramuscular injection of lentiviral vectors led to transduction of myocytes and cardiomyocytes indicating systemic spread of the virus from the site of injection (MacKenzie et al, 2002). Our group successfully achieved in vivo expression of hFIX after injection of adenovirus and AAV hFIX vectors in adult and fetal mice (Schneider et al, 2002). A recent study using EIAV lentivirus containing the lacZ gene combined intrathoracic, supracostal, intraperitoneal and intramuscular injection of three limbs and a single flank in the fetal mouse. This resulted in widespread gene expression in all injected muscles and also the diaphragm and heart which are the essential muscle groups to be reached for successful gene therapy of DMD (Gregory et al, 2003). Finally, delivery of adenoviral vectors into the hindlimb musculature by ultrasound guided injection has been explored in one study in the early gestation fetal sheep. Fetal survival was 91% and therapeutic levels of hFIX were also obtained after injection of adenovirus hFIX vector (Figure 3).

2. Intraperitoneal injection Intraperitoneal injection has also been used for successful gene transfer to multiple tissues including the liver in fetal mice (Lipshutz et al, 1999b, c) rats (Hatzoglou et al, 1990, 1995) and sheep (Tran et al, 2000). Persistent peritoneal expression was observed 18 months after intraperitoneal injection of adeno-associated virus serotype 2 (AAV2) vectors containing the luciferase gene in fetal mice (Lipshutz et al, 2001). Recent studies in the fetal mouse have shown that transgene expression could be increased by intraperitoneal injection of AAV5 serotype vectors rather than AAV2 serotype vectors and by changing from the elongation factor 1! or CMV promoter to the woodchuck hepatitis virus posttranscriptional regulatory element (Lipshutz et al, 2003). In large animal models, retroviral vectors containing the !-L-iduronidase gene were delivered by ultrasound guided injection after exteriorisation of the uterus into the peritoneal cavity or yolk sac of mid-gestation fetal dogs with canine !-L-iduronidase deficiency (mucopolysaccharidosis type 1). Low level tissue transduction was observed but expression of the transgene did not persist beyond the neonatal period (Meertens et al, 2002). In early gestation fetal primates, ultrasound guided intraperitoneal injection of Moloney murine leukemia virus amphotrophic and vesicular stomatitis virus-G protein (VSV-G) pseudotyped retrovirus and VSV-G pseudotyped HIV-1 lentiviral vectors resulted in only low level tissue transduction (Tarantal et al, 2001b). In contrast long-term transduction of hematopoietic stem cells in the bone marrow and blood could be demonstrated 5 years following delivery of retroviral vectors into the peritoneal cavity of early gestation fetal sheep at laparotomy (Porada et al, 1998). Delivery of adenoviral vectors containing the hFIX gene to early gestation fetal sheep by ultrasound guided intraperitoneal injection had good fetal survival of 77% and therapeutic hFIX production was achieved, albeit transiently (Figure 3) (David et al, 2003a). Immunohistochemical analysis after delivery of adenoviral vectors containing the lacZ gene showed positive transgene expression on the surface of the umbilical cord, in the fetal small bowel serosa and in the hepatocytes beneath the fetal liver capsule following intraperitoneal injection (Figure 4 A-C). The intraperitoneal route also gave the most comprehensive spread of vector to fetal tissues as determined by PCR analysis but no vector was detectable by sensitive PCR analysis in the germline of lambs born after each route of administration (David et al, 2003a).

Figure 3. Time course of transgene expression after ultrasound guided intraperitoneal, intramuscular, intrahepatic or intraamniotic delivery of an adenoviral vector containing the human factor IX gene to early gestation sheep fetuses. Concentrations of human factor IX in fetal or lamb plasma were determined by ELISA analysis. Fetal samples were collected at post mortem (David et al 2003a). Republished with permission from Mary Ann Liebert Inc, Publishers.

E. Intramuscular injection The main aim of intramuscular injection is to target the muscle for treatment of muscular dystrophies but this route may also be used for ectopic production of proteins 194

Gene Therapy and Molecular Biology Vol 7, page 195 Immunohistochemistry for "-galactosidase showed strong staining of the hindlimb musculature and occasional positively stained hepatocytes after injection of adenovirus lacZ vector. PCR analysis of vector presence in fetal tissues confirmed that broad haematogenic spread of vector had occurred (David et al, 2003a).

F. Targeting the fetal airways 1. Intra-amniotic injection Intra-amniotic application has been investigated extensively in small animal models. Adenoviral vectors expressing the lacZ gene have been delivered to the fetal rat (Sekhon and Larson, 1995), mouse (Holzinger et al, 1995; Sekhon and Larson, 1995; Douar et al, 1997; Larson et al, 1997; Larson et al, 2000a; Mitchell et al, 2000) and guinea pig (Senoo et al, 2000) while adeno-associated viral vectors have been applied to the fetal mouse (Mitchell et al, 2000). In general, transgene expression is maximal in those tissues in contact with the amniotic fluid, namely the amniotic membranes and the fetal skin with less transduction of the gut and the mucosae. Indeed, therapeutic plasma concentrations of hFIX were achieved in fetal mice after intra-amniotic injection of adenoviral vectors carrying the hFIX gene (Schneider et al, 1999) and the transgenic protein remained detectable after birth. Intra-amniotic delivery of retroviral producer cells to the fetal mouse resulted in only low level transduction of the amniotic membranes and fetal skin and no airways or gut transduction (Douar et al, 1997). In larger animals such as the fetal sheep, ultrasound guided intra-amniotic injection of an amphotropic retroviral producer cell line encoding the lacZ gene resulted in inefficient tissue transduction (Galan et al, 2002). Amniotic fluid was found to have an inhibitory effect on retroviral mediated tissue transduction, and this effect increased as gestational age progressed (Bennett et al, 2001). Better results have been obtained with adenoviral vectors. Low level transgene expression was seen in the fetal oesophagus and trachea after injection of adenoviral lacZ vectors at laparotomy in late gestation fetal sheep (Holzinger et al, 1995). Attempts to deliver adenoviral vectors into the amniotic cavity of fetal sheep using catheters placed at laparotomy had high mortality (Iwamoto et al, 1999). Ultrasound-guided intra-amniotic delivery of adenoviral vectors containing the lacZ or hFIX genes has been achieved in the early gestation fetal sheep (33 - 39 days of gestation, term = 145 days) equivalent to 8 â&#x20AC;&#x201C; 10 weeks gestation in humans with 86% fetal survival (David et al, 2003a). Therapeutic plasma concentrations of hFIX were detectable up to 11 days after injection (Figure 3) and immunohistochemical analysis showed positive expression of "-galactosidase in the fetal skin and nasal cavities (Figure 4 D-F). This suggests that transduction of keratinocytes in utero may be able to deliver proteins to the circulation as well as to treat hereditary skin disease such as epidermolysis bullosa. Gene transfer to the fetal airways is important for in utero treatment of cystic fibrosis. However, no significant airway or gastrointestinal tissue transduction was seen after ultrasound-guided intra-

Figure 4A-C. Expression of "-galactosidase by immunohistochemistry 2 days after intraperitoneal or intraamniotic delivery of an adenoviral vector containing the "galactosidase gene to early gestation fetal sheep. Original magnifications are as indicated. Intraperitoneal injection at 52 days of gestation, positive staining is seen in (A) fetal small bowel serosa, (B) surface of umbilical cord and (C) fetal subcapsular hepatocytes.


David et al: Current status and future direction of fetal gene therapy amniotic delivery of adenoviral vectors to early gestation fetal sheep (David et al, 2003a). Similarly ultrasoundguided intra-amniotic injection of adenoviral vectors in mid-trimester rhesus macaque fetuses resulted in significant transgene spread to tissues coming into contact with amniotic fluid but low level transgene expression in the fetal airways and intestine (Larson et al, 2000b). Similar findings were observed in fetal rabbits (Boyle et al, 2001). Low levels of airway transduction are probably due to dilution of the vector by the relatively larger volume of the amniotic fluid as well as the lack of fetal breathing movements or fetal swallowing at this early gestation. It may be possible to enhance fetal breathing movements in later gestation using agents such as theophylline (Moss and Scarpelli, 1981) that lead to an intake of amniotic fluid to the lungs against the continuous outflow of tracheal fluid (Badalian et al, 1993; Kalache et al, 2000). Indeed increased intake of marker dye and some enhancement of adenovirus mediated marker gene expression was observed in mouse fetuses after theophylline administration. However other still unknown factors appear to influence the level of gene transfer to the fetal airways more effectively (Buckley, in preparation). Recent work in our laboratory aimed to reproduce the iconoclastic report by Larson et al, (1997) that the CFphenotype in CFTR-knockout mice can be cured by shortterm prenatal expression of CFTR from an adenovirus vector, could not substantiate this claim (Buckley et al, 2003). We are, therefore, constructing integrating expression vector systems under tissue specific promoter control to achieve long-term postnatal CFTR-gene expression after in utero gene delivery.

2. Direct lung parenchymal injection Direct injection of the lung parenchyma has been attempted to access the fetal airways but with poor results. In mid-gestation fetal primates, ultrasound guided injection of lentiviral vectors into the lung resulted in low level transgene expression in the fetal airways (Tarantal et al, 2001a). However, in the mid-gestation sheep fetus, ultrasound-guided delivery of an adenoviral vector to the lung parenchyma elicited only localized gene transfer and no spread within the airways could be detected (unpublished results).

3. Tracheal injection Direct instillation of vector into the trachea has been more successful. Placement of catheters in the tracheae of fetal sheep can be performed by highly invasive techniques at laparotomy (McCray et al, 1995; Pitt et al, 1995; Vincent et al, 1995) or fetoscopically (Sylvester et al, 1997; Yang et al, 1999). Low level transduction of the proximal airways can be achieved using adenoviral or retroviral vectors, and occlusion of the trachea with a balloon improves distal airway transduction. These techniques however, carry a significant morbidity and mortality. Recently a percutaneous transthoracic route of injection of the fetal trachea has been developed in midgestation sheep using ultrasound guidance to target the

Figure 4 D-F. Intra-amniotic injection at 33 days of gestation, positive staining is seen in (D) surface of umbilical cord, (E) fetal nasal cavity and (F) fetal skin (David AL et al 2003a). Republished with permission from Mary Ann Liebert Inc, Publishers


Gene Therapy and Molecular Biology Vol 7, page 197 fetal airways as illustrated in Figure 5 (David et al, 2003b). Using this technique we achieved good transgene expression in the fetal trachea and airways following intratracheal delivery of an adenovirus containing the "galactosidase gene (Peebles et al, 2003). Transgene expression was enhanced by pretreatment of the fetal airways with sodium caprate, a fatty acid that opens the tight junctions between airways epithelial cells. This allows the vector to reach the basolateral surface where the coxsackie-adenovirus receptor (CAR receptor) responsible for binding adenovirus is located. Further enhancement of transgene expression was achieved by complexing the adenoviral vector with DEAE dextran, a polycation that neutralizes the negative charge on the vector, improving vector binding to the CAR receptor (Figure 6 and Figure 7).

Instillation of perflubron, an inert fluorocarbon, resulted in a redistribution of expression from the upper to the peripheral airways and is most likely due to flushing of the vector solution further down the airways by the water immiscible perflubron (Weiss et al, 1999b). These results show proof of principle for the relatively safe and minimally invasive in utero delivery of a gene therapy vector to the fetal airways that resulted in levels of transgene expression in the airway epithelia that may be relevant to a therapeutic application in cystic fibrosis gene therapy.

G. Targeting the fetal gut Intrapharyngeal delivery has been attempted once in fetal rabbits at laparotomy to target the fetal gastrointestinal system as a model for the treatment of meconium ileus due to cystic fibrosis (Wu et al, 1999). Gene transfer to the small bowel enterocytes was achieved but there was significant maternal and fetal loss related to anaesthesia and the invasive surgery used. Ultrasoundguided injection of barium into the fetal stomach of rabbits has been performed successfully (Brandt et al, 1997) and this technique could be extended to deliver gene to the fetal gut. Gene delivery to the gut of fetal mice has been observed after intra-amniotic vector application and was most likely a result of fetal swallowing (Douar et al, 1997).

H. Delivery to the placenta Targeting the placenta could be used in the treatment of placental disorders such as pre-eclampsia or intrauterine growth restriction. Low level gene transfer to the placenta has been achieved using angiographically guided injection of non-viral vectors into the uterine artery (Heikkil채 et al, 2001). The intraplacental route has been attempted in mice, rats, guinea pigs and rabbits. Somatic gene transfer to the fetal heart and liver was achieved in some studies using mice (Woo et al, 1997; T체rkay et al, 1999), but others have found little or no fetal gene transfer in mice and guinea pigs (Senoo et al, 2000) or rats (Xing et al, 2000). Commonly, the placenta showed the most transfection, but maternal tissues also demonstrated transgene expression, which although not unexpected, is undesirable in therapy aimed at the fetus.

V. Development of the fetal immune system A major restriction in adult gene therapy is the immune response to vector and/or transgene. In utero application, on the other hand, aims to circumvent this by treatment before maturity of the functional immune system and this depends critically on the time at which fetal tolerance might be induced. The human immune system develops progressively through the first trimester and is not fully functional until 1-2 years after birth (Riley, 1998). Lymphoid cells appear first in the fetal liver from 8 weeks of gestation, with B lymphocytes and natural killer cells predominating over T cells (Pahal et al, 2000). T lymphocytes increase in number in the fetal liver and circulation from 12 weeks of gestation.

Figure 5 : (A) Ultrasonogram and (B) diagram of sheep fetus at 114 days of gestation in longitudinal section. A 20 Gauge spinal needle is inserted into the fetal thorax between the 3rd and 4th rib, penetrates the lung parenchyma and enters the fetal trachea just proximal to the carina (David et al 2003b). Republished with permission from S Karger AG, Basel.


David et al: Current status and future direction of fetal gene therapy

Figure 6: Na-caprate stimulation of DEAE dextran complexed adenovirus mediated airway transduction. Panel 1: Examples of staining in the peripheral lungs after virus alone (a) and DEAE complexed virus (b) and of the trachea after Na-caprate pre-treatment and uncomplexed virus administration (c) in fetal sheep injected between 102 and 109 days of gestation. Panel 2a: Na-caprate pre-treatment followed by DEAE dextran complexed virus in a 108 day sheep. Widespread gene expression was seen in the small (a), medium (b) and large (c) airways and also the main bronchi (d) and trachea (e). Panel 2b: Similar results were observed in a fetus injected at 81 days of gestation. Expression was seen in the airways (a & b) and trachea (c) Panel 3 : Na-caprate pre-treatment followed by DEAE dextran complexed virus followed by perflubron. Staining of the peripheral airways in transverse sections (a & b) and longitudinal section showing gene expression was limited to the terminal branches of the bronchial tree (c). Some staining of the bronchioles (d) and trachea (e) was also observed, although less than in the absence of perflubron. Scale bar = 5mm in all cases. (Peebles et al 2003).


Gene Therapy and Molecular Biology Vol 7, page 199 Although they are not capable of producing a definitive cytotoxic response until 18 weeks of gestation (Mackenzie and Maclean, 1980) natural killer cells and some T cell lines may provide a limited immune response earlier in gestation (Miyagawa et al, 1992; Phillips et al, 1992). The fetal lamb is able to produce detectable circulating antibodies in response to some antigenic stimuli from 66 days of gestation (Silverstein et al, 1963) and to reject skin grafts after 77 days of gestation (Silverstein et al, 1964). This would suggest a 'window of opportunity' in the first third to half of pregnancy during which time introduction of foreign genetic material may not produce an immune response. No humoral immune response to the transgene was observed in early gestation fetal sheep, although antibodies to the adenoviral vector were detected for each route of injection (David et al, 2003a). Similarly, umbilical vein injection of adenoviral vectors into fetal sheep at 60 days of gestation via hysterotomy resulted in widespread transduction of fetal tissues with no humoral immune response to the adenoviral vector (Yang et al, 1999). Expression of a foreign antigen during early fetal development may also result in its recognition as â&#x20AC;&#x153;selfâ&#x20AC;? where exposure of the fetus to foreign antigen is maintained (Billingham et al, 1956; Binns, 1967) thus allowing development of tolerance. Evidence to support induced tolerance has been reported after in utero intraperitoneal delivery of retroviral vectors in fetal sheep (Tran et al, 2001). Induction of tolerance to transgene in adults although possible, is expensive, therefore, prenatal induction of tolerance may provide an excellent alternative. For example, a single injection of adenovirus expressing the factor IX gene into the fetal mouse was shown to provide long term, albeit diminishing expression over five months. Furthermore, 56% of these adult mice remained tolerant to repeated challenges with hFIX protein (Figure 8). In contrast, a group of mice which received adenovirus for the first time as adults developed high levels of anti-hFIX antibodies (Waddington et al, 2002). This provides proof of principle that gene therapy applicaton in utero may allow induction of immune tolerance. However the paradigm of self/non-self immune tolerance and sensitisation has been recently challenged by the hypothesis of Matzinger (2002). This suggests that immunity arises as a consequence of cellular alarm signals from distressed or injured cells stimulating antigen presenting cells. A recent study examined the idea that the fetus is particularly susceptible to induction of tolerance; the study concluded that, rather than being due to ignorance, timing-based tolerance or properties of naĂŻve T cells in early life, tolerance induction in fetus may arise from differences in fetal antigen presentation; this remains to be identified (Anderson, et al, 2001).

reactions or preterm labour on the fetus as well as on the mother. Furthermore, many parents decide to terminate an affected pregnancy, and therefore the option of in utero treatment must be at least as safe for the mother, and should also reliably treat the disease (Coutelle and Rodeck, 2002). There is a theoretical risk that the therapeutic gene product or vector that is required at a certain stage during fetal development could cause oncogenesis. In addition, insertion of vector sequences may cause developmental aberrations to occur. While one of the aims of prenatal gene therapy is to achieve immune tolerance to the transgene and delivery system, vectors must be designed to be sufficiently different to the wild type so that the immune system remains able to mount an effective immune response against wild-type virus infection. The problem of insertional mutagenesis as a potential risk of retroviral gene therapy has been debated for some years. This serious adverse event has now been identified in a trial of gene therapy for X-linked severe combined immunodeficiency syndrome in which CD34+ haemopoietic stem cells were transduced ex vivo with the #c gene using retroviral vectors. Two patients out of fifteen treated developed acute lymphoblastic leukemia (ALL) three years after successful gene therapy treatment. Analysis of the lymphocytes showed that the transgene had been inserted adjacent to an oncogene, LMO2, the product of which has been implicated in the pathogenesis of ALL (Juengst, 2003). Further work is needed to address this issue and to devise strategies to determine and possibly direct integration sites. Germline transmission is another risk that raises ethical concerns. Fetal somatic gene therapy does not aim to modify the genetic content of the germ-line but inadvertent gene transfer to the germ-line could occur. Compartmentalisation of the primordial germ cells in the gonads is complete by 7 weeks of gestation in humans and it is unlikely therefore that any therapy applied after this time would result in germ-line transduction. Examination of germ cells after delivery of retroviral vectors (Porada et al, 1998; Tran et al, 2000) or adenoviral vectors to early gestation fetal sheep has not shown any detectable transmission (David et al, 2003a). Following intravascular administration of adenoviral vectors to late gestation fetal sheep, vector DNA was detectable by PCR in the gonads, but extensive investigation by RT-PCR could not detect any gene expression. A similar risk of germline transduction occurs with AAV that can integrate into the genome. No AAV sequences were detectable in the germline tissues of fetal mice receiving injection of AAV vectors via the intraperitoneal route nor the tissues of their progeny (Lipshutz et al, 2001). Many of these issues are not confined to in utero or even adult gene therapy and concerns regarding germ-line transmission can be raised in particular for chemotherapy and infertility treatment (Schneider and Coutelle 1999).Finally there is the concern that fetal gene therapy research poses special challenges to informed consent (Burger and Wilfond 2000).

VI. Ethical and safety issues There are various ethical issues in relation to in utero gene therapy that need to be addressed before such therapy could be applied clinically (Fletcher and Richter, 1996; Recombinant DNA Advisory Committee 2000). One major concern is that fetal gene therapy has potential adverse effects such as injury, infection, severe immune 199

David et al: Current status and future direction of fetal gene therapy

Figure 7: Na-caprate stimulation of DEAE dextran complexed adenovirus mediated "-galactosidase expression. Panel 1: Na-caprate pre-treatment followed by DEAE dextran complexed virus. Widespread X-gal staining (a-c) and immunohistochemical localisation (d-f) of "-galactosidase expression in the trachea (a & d), bronchial epithelium (e) and airway epithelium (b,c & f). Panel 2: Na-caprate pretreatment followed by DEAE dextran complexed virus followed by perflubron. X-gal staining (a-c) and immunohistochemical localisation (d-f) of "-galactosidase expression in the peripheral airways. All fetuses were injected between 102 and 116 days. Scale bar = 5mm in all cases. (Peebles D et al 2003).


Gene Therapy and Molecular Biology Vol 7, page 201

Figure 8: Durability of expression and tolerance of exogenous and expressed hFIX. Prenatal and adult mice were injected intravenously with adenoviral vectors expressing the hFIX gene (AdhFIX) and repeatedly rechallenged, as adults, with intraperitoneal hFIX protein then intravenous AdhFIX while hFIX concentrations were measured. The y axis shows blood hFIX concentrations (Âľg/ml) after in utero or adult injection of AdhFIX (Phase I), repeated injection of hFIX protein to the adult mice (Phase II) and repeated injection of AdhFIX to the adult mice (Phase III). The x-axis shows the experimental time course in days. Arrows indicate injection points. Groups I and II are mice initially injected in utero with AdhFIX at days 15 and 17 of gestation, respectively. Group III contains mice initially injected intravenously with AdhFIX as adults. Group IV did not receive prior injection of AdhFIX. Group V received neither prior injections of AdhFIX or hFIX protein. A line representing a therapeutic threshold of 40 ng/ml hFIX is included. Points are meanÂąS.D. (Waddington et al 2002). Reprinted with permission from the American Society of Hematology.


David et al: Current status and future direction of fetal gene therapy The decision to participate in a fetal gene therapy trial would occur close to the time of prenatal diagnosis of the condition. The parents may hear information in a highly biased way and not consider the risk to future pregnancies. It will be important to ensure that parents are adequately counselled and understand these issues before agreeing to take part in any future research. The general public remains concerned that ethical discussion about issues such as gene therapy, cloning and the Human Genome Project are falling behind the technology (Brown, 2000). It is therefore important to provide adequate information which will allow the public to understand the risks and benefits of these novel techniques and to enable an educated involvement in the decision-making process along with health professionals. This will also help individuals to give informed consent as these procedures become used in clinical practice.

Successful induction of "-galactosidase in a rabbit model. Am J Obstet Gynecol 181, 848-852. Benirschke K, Kaufmann P ( 1990) Placental types. Pathology of the human placenta. Springer-Verlag, New York. Bennett M, Galan H, Owens G, Dewey R, Banks R, Hobbins J, Accurso F, Schaack J (2001) In utero gene delivery by intraamniotic injection of a retroviral vector producer cell line in a nonhuman primate model. Hum Gene Ther 12, 1857-1865. Bigger B, Coutelle C (2001) Perspectives on gene therapy for cystic fibrosis airway disease. Biodrugs 15, 615-634. Billingham RE, Brent L, Medawar PB (1956) Quantitative studies on tissue transplantation immunity III Actively acquired tolerance. Phil Trans R Soc (London) B239, 357369. Binns R (1967) Bone marrow and lymphoid cell injection of the pig fetus resulting in transplantation tolerance or immunity, and immunoglobulin production. Nature 214, 179-180. Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M, Shearer G, Chang L, Chiang Y, Tolstoshev P, Greenblatt JJ, Rosenberg SA, Klein H, Berger M, Mullen CA, Ramsey WJ, Muul L, Morgan RA, French Anderson W (1995) T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years. Science 270, 475-480. Bordignon C, Notarangelo LD, Nobili N, Ferrari G, Casorati G, Panina P, Mazzolari E, Maggioni D, Rossi C, Servida P, Ugazio AG, Mavilio F (1995) Gene therapy in peripheral blood lymphocytes and bone marrow for ADAimmunodeficient patients. Science 270, 470-475. BouĂŠ A, Muller F, Nezelof C, Oury JF, Duchatel F, Dumez Y, Aubry MC, Boue J (1986) Prenatal diagnosis in 200 pregnancies with a 1-in-4 risk of cystic fibrosis. Hum Genet 74, 288-297. Boyle MP, Enke RA, Adams RJ, Guggino WB, Zeitlin PL (2001) In utero AAV-mediated gene transfer to rabbit pulmonary epithelium. Mol Ther 4, 115-121. Brandt ML, Moise KJJ, Eckert JW, Johnson L, Waltrip T, Saade G, Wu Y, Finegold MJ (1997) Transuterine puncture of the fetal stomach provides access to the small bowel in the rabbit. J Invest Surg 10, 41-46. Brosens JJ, Pijnenborg R, Brosens IA ( 2002) The myometrial junctional zone spiral arteries in normal and abnormal pregnancies: a review of the literature. Am J Obstet Gynecol 187, 1416-1423. Brown P (2000) Regulations not keeping up with developments in genetics, says poll. BMJ 321, 1369. Buckley RH, Schiff SE, Schiff RI, Markert L, Williams LW, Roberts JL, Myers LA, Ward FE (1999) Hematopoietic stemcell transplantation for the treatment of severe combined immunodeficiency. New Engl J Med 340, 508-516. Buckley SMK, Waddington SN, Jezzard S, Themis M, Colledge WH, Coutelle C (2003) Intra-amniotic application of CFTRexpressing adenovirus does not reverse cystic fibrosis phenotype in inbred Cftr-knockout mice. Mol Ther 7, S200. Burger IM, Wilfond BS (2000) Limitations of informed consent for in utero gene transfer research: implications for investigators and institutional review boards. Hum Gene Ther 11, 1057-1063. Burlet P, Huber C, Bertrandy S, Ludosky MA, Zwaenepoel I, Clermont O, Roume J, Delezoide AL, Cartaud J, Munnich A, Lefebvre S ( 1998) The distribution of SMN protein complex in human fetal tissues and its alteration in spinal muscular atrophy. Hum Mol Genet 7, 1927-1933. Case SS, Price MA, Jordan CT, Yu XJ, Wang L, Bauer G, Haas DL, Xu D, Stripecke R, Naldini L, Kohn DB, Crooks GM (1999) Stable transduction of quiescent CD34(+)CD38(-) human hematopoietic cells by HIV-1-based lentiviral vectors. Proc Natl Acad Sci 96, 2988-2993.

VII. Conclusions Fetal gene therapy offers the potential for obstetricians and gene therapists not only to diagnose but also to treat inherited genetic disease. However, for the treatment to be acceptable, it must offer advantages over postnatal gene therapy, be safe for both mother and fetus, and preferably avoid germ-line transmission. Currently, in utero gene therapy remains an experimental procedure. But in the future, better understanding of the development of genetic disease in the fetus, and improvements in vector design and targeting of fetal tissues should allow this technology to move into clinical practice.

References Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A, Morecki S, Andolfi G, Tabucchi A, Carlucci F, Marinello E, Cattaneo F, Vai S, Servida P, Miniero R, Roncarolo MG, Bordignon C (2002) Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 296, 2410-2413. Akiyama M (1998) Severe congenital ichthyosis of the neonate. Int J Dermatol 37, 722-728. Anderson CC, Carroll JM, Gallucci S, Ridge JP, Cheever AW, Matzinger P (2001) Testing time-, ignorance-, and dangerbased models of tolerance. J Immunol 166, 3663-3671. Badalian SS, Chao CR, Fox HE, Timor TI (1993) Fetal breathing-related nasal fluid flow velocity in uncomplicated pregnancies. Am J Obstet Gynecol 169, 563-567. Badawi N, Kurinczuk JJ, Keogh JM, Alessandri LM, O'Sullivan F, Burton PR, Pemberton PJ, Stanley FJ (1998) Intrapartum risk factors for newborn encephalopathy: the Western Australian case-control study. BMJ 317, 1549-1553. Baldeschi C, Gache Y, Rattenholl A, Bouille P, Danos O, Ortonne JP, Bruckener-Tuderman L, Meneguzzi G (2003). Genetic correction of canine dystrophic epidermolysis bullosa mediated by retroviral vectors. Hum Mol Genet 12, 1897-1905. Barcroft J, Barron (1946) Observations upon the form and relations of the maternal and fetal vessels in the placenta of the sheep. Anat Rec 94, 569-595. Barker DJ, Gluckman PD, Godfrey KM, Harding JE, Owens JA, Robinson JS (1993) Fetal nutrition and cardiovascular disease in adult life. Lancet 341, 938-941. Baumgartner TL, Baumgartner BJ, Hudon L, Moise KJ (1999) Ultrasonographically guided direct gene transfer in utero:


Gene Therapy and Molecular Biology Vol 7, page 203 Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, Selz F, Hue C, Certain S, Casanova J, Bousso P, Le Deist F, Fischer A (2000) Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288, 669-672. Cavazzana-Calvo M, Hacein-Bey S, Yates F, De Villartay JP, Le Deist F, Fischer A (2001) Gene therapy of severe combined immunodeficiencies. J Gene Med 3, 201-206. Challita PM, Kohn DB (1994) Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo. Proc Natl Acad Sci 91, 2567-2571. Chamberlain JS (2002) Gene therapy of muscular dystrophy. Hum Mol Genet 11, 2355-2362. Chao H, Samulski RJ, Bellinger D, Monahan PE, Nichols T, Walsh CE (1999) Persistent expression of canine factor IX in hemophilia B canines. Gene Ther 6, 1695-1704. Chen HH, Mack LM, Kelly R, Ontell M, Kochanek S, Clemens PR (1997) Persistence in muscle of an adenoviral vector that lacks all viral genes. Proc Natl Acad Sci 94, 1645-1650. Chen M, Kasahara N, Keene DR, Chan L, Hoeffler WK, Finlay D, Barcova M, Cannon PM, Mazurek C, Woodley DT (2002) Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa. Nat Genet 32, 670-675. Cheng SH, Smith AE (2003) Gene therapy progress and prospects: gene therapy of lysosomal storage disorders. Gene Ther 10, 1275-1281. Chinnaiya A, Venkat A, Dawn C, Chee WY, Choo KB, Gole LA, Meng CT (1998) Intraheptatic vein fetal blood sampling: current role in prenatal diagnosis. J Obstet Gynaecol Res 24, 239-246. Christensen G, Minamisawa S, Gruber PJ, Wang Y, Chien KR (2000) High-efficiency, long-term cardiac expression of foreign genes in living mouse embryos and neonates. Circulation 101, 178-184. Cole FS, Hamvas A, Nogee LM (2003) Genetic disorders of neonatal respiratory function. Ped Res 50, 157-162. Cosset FL, Takeuchi Y, Battini JL, Weiss RA, Collins MK (1995) High-titer packaging cells producing recombinant retroviruses resistant to human serum. J Virol 69, 74307436. Coutelle C, Douar A-M, Colledge WH, Froster U (1995) The challenge of fetal gene therapy. Nat Med 1, 864-866. Coutelle C, Rodeck C (2002) On the scientific and ethical issues of fetal somatic gene therapy. Gene Ther 9, 670-673. Crawford TO, Pardo CA (1996) The neurobiology of childhood spinal muscular atrophy. Neurobiol Dis 3, 97-110. Daly TM, Vogler C, Levy B, Haskins ME, Sands MS (1999) Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease. Proc Natl Acad Sci 96, 2296-2300. David AL, Cook T, Waddington S, Peebles D, Nivsarkar M, Knapton H, Miah M, Dahse T, Noakes D, Schneider H, Rodeck C, Coutelle C, Themis M (2003a) Ultrasound guided percutaneous delivery of adenoviral vectors encoding "galactosidase and human factor IX genes to early gestation fetal sheep in utero. Hum Gene Ther 14, 353-364. David AL, Peebles DM, Gregory L, Themis M, Cook T, Coutelle C, Rodeck C, (2003b) Percutaneous ultrasound-guided injection of the trachea in fetal sheep: a novel technique to target the fetal airways. Fetal Diagn Ther 18, 385-390. Deconinck N, Ragot T, Marechal G, Perricaudet M, Gillis JM (1996) Functional protection of dystrophic mouse (mdx) muscles after adenovirus-mediated transfer of a dystrophin minigene. Proc Natl Acad Sci 93, 3570-3574. DiDonato CJ, Parks RJ, Kothary R (2003) Development of a gene therapy strategy for the restoration of survival motor

neuron protein expression: implications for spinal muscular atrophy therapy. Hum Gene Ther 14, 179-188. Douar A-M, Adebakin S, Themis M, Pavirani A, Cook T, Coutelle C (1997) Foetal gene delivery in mice by intraamniotic administration of retroviral producer cells and adenovirus. Gene Ther 4, 883-890. Douar A-M, Themis M, Sandig V, Friedmann T, Coutelle C (1996) Effect of amniotic fluid on cationic lipid mediated transfection and viral infection. Gene Ther 3, 789-796. Dziegielewska KM, Ek J, Habgood MD, Saunders NR (2001) Development of the choroid plexus. Microsc Res Tech 52, 5-20. Emery AEH (1993) Duchenne muscular dystrophy. Oxford University Press. Emery AEH (2002) The muscular dystrophies. Lancet 359, 687695. Engelhardt JF, Schlossberg H, Yankaskas JR, Dudus L (1995) Progenitor cells of the adult human airway involved in submucosal gland development. Development 121, 20312046. Engelhardt JF, Yankaskas JR, Ernst SA, Yang Y, Marino CR, Boucher RC, Cohn JA, Wilson JM (1992) Submucosal glands are the predominant site of CFTR expression in the human bronchus. Nat Genet 2, 240-248. Engelst채dter M, Buchholz CJ, Bobkova M, Steidl S, MergetMillitzer H, Willemsen RA, Stitz J, Cichutek K (2001) Targeted gene transfer to lymphocytes using murine leukaemia virus vectors pseudotyped with spleen necrosis virus envelope proteins. Gene Ther 8, 1202-1206. Flake AW, Roncarolo MG, Puck JM, Almeida-Porada G, Evans MI, Johnson MP, Abella EM, Harrison DD, Zanjani ED (1996) Treatment of X-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow. N Engl J Med 335, 1806-1810. Fletcher JC, Richter G (1996) Human fetal gene therapy: moral and ethical questions. Hum Gene Ther 7, 1605-1614. Fowden AL (1995) Nutrient requirements for normal fetal growth and metabolism. In: Hanson MA, Spencer JAD, Rodeck CH (Eds.), Fetus and Neonate: Physiology and Clinical Applications: Growth. Cambridge University Press, Cambridge, pp. 31-56. Friedmann T (2003) Gene therapy's new era: a balance of unequivocal benefit and unequivocal harm. Mol Ther 8, 5-7. Furie B, Limentani SA, Rosenfield CG (1994) A practical guide to the evaluation and treatment of hemophilia. Blood 84, 3-9. Gaillard D, Ruocco S, Lallemand A, Dalemans W, Hinnrasky J, Puchelle E (1994) Immunohistochemical localization of cystic fibrosis transmembrane conductance regulator in human fetal airway and digestive mucosa. Ped Res 36, 137143. Galan HL, Bennett ML, Tyson RW, Owens G, Ragnault TR, Accurso F, Robbins JC, Schaack J (2002) Inefficient transduction of sheep in utero after intra-amniotic injection of retroviral producer cells. Am J Obstet Gynecol 187, 469474. Gansbacher B, European Society of Gene Therapy (2003) Report of a second serious adverse event in a clinical trial of gene therapy for X-linked severe combined immune deficiency (X-SCID). Position of the European Society of Gene Therapy (ESGT). J Gene Med 5, 261-262. Geipel A, Berg C, Germer U, Krapp M, Kohl M, Gembruch U (2002) Mucopolysaccharidosis VII (Sly disease) as a cause of increased nuchal translucency and non-immune fetal hydrops: study of a family and technical approach to prenatal diagnosis in early and late pregnancy. Prenat Diagn 22, 493495.


David et al: Current status and future direction of fetal gene therapy Gerdts V, Babiuk LA, van Drunen Littel-van den Hurk, Griebel PJ (2000) Fetal immunization by a DNA vaccine delivered into the oral cavity. Nat Med 6, 929-932. Gerdts V, Snider M, Brownlie R, Babiuk LA, Griebel PJ (2003) Oral DNA vaccination in utero induces mucosal immunity and immune memory in the neonate. J Immunol 168, 18771885. Gregory L, Waddington SN, Holder M, Mitrophanous K, Buckley SMK, Bigger B, Ellard FM, Walmsley LE, Lawrence L, Cook T, Al-Allaf F, Kingsman S, Coutelle C, Themis M ( 2003) Highly efficient in utero gene transfer and persistent gene expression in heart and respiratory and limb musculature of mice demonstrate applicability of EIAV lentivirus for gene therapy of Duchenne/Becker Muscular Dystrophies. Hum Gene Ther submitted. Gregory LG, Harbottle RP, Lawrence L, Knapton HJ, Themis M, Coutelle C (2002) Enhancement of adenovirus-mediated gene transfer to the airways by DEAE dextran and sodium caprate in vivo. Mol Ther 7, 1-8. Haake AR, Cooklis M (1997) Incomplete differentiation of fetal keratinocytes in the skin equivalent leads to the default pathway of apoptosis. Exp Cell Res 231, 83-95. Hacein-Bey-Abina S, Le Deist F, Carlier F, Bouneaud C, Hue C, De Villartay JP, Thrasher AJ, Wulffraat N, Sorensen R, Dupuis-Girod S, Fischer A, Davies EG, Kuis W, Leiva L, Cavazzana-Calvo M (2002) Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 346, 1185-1193. Harrison MR, Mychaliska GB, Albanese CT, Jennings RW, Farrell JA, Hawgood S, Sandberg P, Levine AH, Lobo E, Filly RA (1998) Correction of congenital diaphragmatic hernia in utero IX: fetuses with poor prognosis (liver herniation and low lung-to-head ratio) can be saved by fetoscopic temporary tracheal occlusion. J Pediatr Surg 33, 1017-1022. Hatzoglou M, Lamers W, Bosch F, Wynshaw-Boris A, Clapp DW, Hanson RW (1990) Hepatic gene transfer in animals using retrovirus containing the promoter from the gene for phosphoenolpyruvate carboxykinase. J Biol Chem 265, 17285-17293. Hatzoglou M, Moorman A, Lamers W (1995) Persistent expression of genes transferred in the fetal rat liver via retroviruses. Somat Cell Mol Genet 21, 265-278. Heikkil채 A, Hiltunen MO, Turunen MP, Keski-Nisula L, Turunen A-M, R채s채nen H, Rissanen TT, Kosma V-M, Manninen H, Heinonen S, Yl채-Herttuala S (2001) Angiographically guided utero-placental gene transfer in rabbits with adenoviruses, plasmid/liposomes and plasmid/polyethyleneimine complexes. Gene Ther 8, 784788. Hendrickx AG, Peterson PE (1997) Perspectives on the use of the baboon in embryology and teratology research. Human Reprod Update 3, 575-592. Herzog RW, Yang EY, Couto LB, Hagstrom JN, Elwell D, Fields PA, Burton M, Bellinger DA, Read MS, Brinkhous DM, Podsakoff GM, Nichols TC, Kurtzman GJ, High KA (1999) Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adenoassociated viral vector. Nat Med 5, 56-63. Holzgreve W, Golbus MS (1986) Prenatal diagnosis of ornithine transcarbamylase deficiency utilizing fetal liver biopsy. Am JHuman Genet 36, 320-328. Holzinger A, Trapnell BC, Weaver TE, Whitsett JA, Iwamoto HS (1995) Intraamniotic administration of an adenoviral vector for gene transfer to fetal sheep and mouse tissues. Ped Res 38, 844-850.

Horn HM, Tidman MJ (2002) The clinical spectrum of dystrophic epidermolysis bullosa. Br J Dermatol 146, 267274. Hubeau C, Puchelle E, Gaillard D (2001) Distinct pattern of immune cell population in the lung of human fetuses with cystic fibrosis. J Allergy Clin Immunol 108, 524-529. Inoue M, Tokusumi Y, Ban H, Kanaya T, Tokusumi T, Nagai Y, Iida A, Hawegawa M (2003) Nontransmissible virus-like particle formation by F-deficient Sendai virus is temperature sensitive and reduced by mutations in M and HN proteins. J Virol 77, 3238-3246. Iwamoto HS, Trapnell BC, McConnell CJ, Daugherty C, Whitsett JA ( 1999) Pulmonary inflammation associated with repeated, prenatal exposure to an E1, E3-deleted adenoviral vector in sheep. Gene Ther 6, 98-106. Jauniaux E, Gulbis B (2000) Fluid compartments of the embryonic environment. Hum Reprod Update 6, 268-278. Jauniaux E, Gulbis B, Gerloo E (1999) Free amino acids in human fetal liver and fluids at 12 - 17 weeks of gestation. Hum Reprod 14, 1638-1641. Jesudason EC (2002) Challenging embryological theories on congenital diaphragmatic hernia: future therapeutic implications for paediatric surgery. Ann R Coll Surg Engl 84, 252-259. Jeyakumar M, Butters TD, Dwek RA, Platt FM (2002) Glycosphingolipid lysosomal storage diseases: therapy and pathogenesis. Neuropathol Appl Neurobiol 28, 343-357. Johnson LG, Olsen JC, Sarkadi B, Moore KL, Swanstrom R, Boucher RC (1992) Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis. Nat Genet 2, 21-25. Johnson LG, Vanhook MK, Coyne CB, Haykal-Coates N, Gavett SH (2003) Safety and efficiency of modulating paracellular permeability to enhance airway epithelial gene transfer in vivo. Hum Gene Ther 14, 729-747. Johnston J, Tazelaar J, Rivera VM, Clackson T, Gao GP, Wilson JM (2003) Regulated expression of erythropoietin from an AAV vector safely improves the anemia of beta-thalassemia in a mouse model. Mol Ther 7, 493-497. Juengst ET (2003) What next for human gene therapy? BMJ 326, 1410-1411. Kalache KD, Chaoui R, Marcks B, Nguyen-Dobinsky TN, Wernicke KD, Wauer R, Bollmann R (2000) Differentiation between human fetal breathing patterns by investigation of breathing-related tracheal fluid flow velocity using Doppler sonography. Prenat Diagn 20, 45-50. Kamata Y, Tanabe A, Kanaji A, Kosuga M, Fukuhara Y, Li XK, Suzuki S, Yamada M, Azuma N, Okuyama T (2003) Longterm normalization in the central nervous system, ocular manifestations, and skeletal deformities by a single systemic adenovirus injection into neonatal mice with mucopolysaccharidosis VII. Gene Ther 10, 406-414. Kay MA, Landen CN, Rothenberg SR, Taylor LA, Leland F, Wiehle S, Fang B, Bellinger D, Finegold M, Thompson AR, Read M, Brinkhous KM, Woo SLC (1994) In vivo hepatic gene therapy: Complete albeit transient correction of factor IX deficiency in hemophilia B dogs. Proc Natl Acad Sci 91, 2353-2357. Kay MA, Manno CS, Ragni MV, Larson PJ, Couto LB, McClelland A, Glader B, Chew AJ, Tai SJ, Herzog RW, Arruda V, Johnson F, Scallan C, Skarsgard E, Flake AW, High KA (2000) Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet 24, 257-261. Kay MA, Rothenberg SR, Landen CN, Bellinger DA, Leland F, Toman C, Finegold M, Thompson AR, Read MS, Brinkhous KM, Woo SLC (1993) In vivo gene therapy of hemophilia B:


Gene Therapy and Molecular Biology Vol 7, page 205 sustained partial correction in factor IX-deficient dogs. Science 262, 117-119. Kobinger GP, Weiner DJ, Yu QC, Wilson JM (2001) Filoviruspseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo. Nat Biotechnol 19, 225230. Kohn DB, Weiner CP, Nolta JA, Heiss IN, Lenarsky C, Crooks GM, Hanley ME, Annett G, Brooks JS, El-Khoureiy A, Lawrence K, Wells S, Moen RC, Bastian J, WilliamsHerman DE, Elder M, Wara D, Bowen T, Hershfield MS, Mullen CA, Blaese RM (1995) Engraftment of genemodified cells from umbilical cord blood in neonates with adenosine deaminase deficiency. Nat Med 1, 1017-1023. Kubo S, Mitani K (2003) A new hybrid system capable of efficient lentiviral vector production and stable gene transfer mediated by a single helper-dependent adenoviral vector. J Virol 77, 2964-2971. Lai L, Davison BB, Veazey RS, Fisher KJ, Baskin GB (2002) A preliminary evaluation of recombinant adeno-associated virus biodistribution in rhesus monkeys after intrahepatic inoculation in utero. Hum Gene Ther 13, 2027-2039. Larsen JF, Jacobsen B, Holm HH, Pedersen JF, Mantoni M (1978) Intrauterine injection of vitamin K before the delivery during anticoagulant therapy of the mother. Acta Obstet Gynecol Scand 57, 227-230. Larson JE, Delcarpio JB, Farberman MM, Morrow SL, Cohen JC (2000a) CFTR modulates lung secretory cell proliferation and differentiation. Am J Physiol 279, L333-L341. Larson JE, Morrow SL, Delcarpio JB, Bohm RP, Ratterree MS, Blanchard JL, Cohen JC (2000b) Gene transfer into the fetal primate: evidence for the secretion of transgene product. Mol Ther 2, 631-639. Larson JE, Morrow SL, Happel L, Sharp JF, Cohen JC (1997) Reversal of cystic fibrosis phenotype in mice by gene therapy in utero. Lancet 349, 619-620. Lee B, Goss J (2001) Long-term correction of urea cycle disorders. J Pediatr 138, S62-S71. Lehrman S ( 1999) Virus treatment questioned after gene therapy death. Nature 401, 517-518. Li Z, D端llmann J, Schiedlmeier B, Schmidt M, von Kalle C, Meyer J, Forster M, Stocking C, Wahlers A, Frank O, Ostertag W, K端hlcke K, Eckert H-G, Fehse B, Baum C (2002) Murine leukemia induced by retroviral gene marking. Science 296, 497. Lim F-Y, Kobinger GP, Weiner DJ, Radu A, Wilson JM, Crombleholme TM (2003) Human fetal trachea-SCID mouse xenografts: efficacy of vesicular stomatitis virus-G pseudotyped lentiviral-mediated gene transfer. J Pediatr Surg 38, 834-839. Lim F-Y, Martin B, Radu A, Crombleholme TM (2002) Adenoassociated virus (AAV)-mediated fetal gene transfer in respiratory epithelium and submucosal gland cells in human fetal tracheal organ culture. J Pediatr Surg 37, 1051-1057. Lipshutz GS, Flebbe-Rehwaldt L, Gaensler KML (1999a) Adenovirus-mediated gene transfer in the midgestation fetal mouse. J Pediatr Surg 84, 150-156. Lipshutz GS, Flebbe-Rehwaldt L, Gaensler KML (1999b) Adenovirus-mediated gene transfer to the peritoneum and hepatic parenchyma of fetal mice in utero. Surgery 126, 171-177. Lipshutz GS, Flebbe-Rehwaldt L, Gaensler KML (2000) Reexpression following readministration of an adenoviral vector in adult mice after initial in utero adenoviral administration. Mol Ther 2, 374-380. Lipshutz GS, Gruber CA, Cao Y, Hardy J, Contag CH, Gaensler KML (2001) In utero delivery of adeno-associated viral vectors: intraperitoneal gene transfer produces long-term expression. Mol Ther 3, 284-292.

Lipshutz GS, Sarkar R, Flebbe-Rehwaldt L, Kazazian H, Gaensler KML (1999c) Short-term correction of factor VIII deficiency in a murine model of hemophilia A after delivery of adenovirus murine factor VIII in utero. Proc Natl Acad Sci 96, 13324-13329. Lipshutz GS, Titre D, Brindle M, Bisconte AR, Contag CH, Gaensler KM (2003) Comparison of gene expression after intraperitoneal delivery of AAV2 or AAV5 in utero. Mol Ther 8, 90-98. Ljubic A, Cvetkovic M, Sulovic V, Radunovic N, Antonovic O, Vukolic D, Popovic B, Petkovic S (1999) New technique for artificial lung maturation. Direct intramuscular fetal corticosteroid therapy. Clin Exp Obstet Gynecol 26, 16-19. Ljung RCR (1999) Prenatal diagnosis of haemophilia. Haemophilia 5, 84-87. Lusher JM (2000) Inhibitors in young boys with haemophilia. Baillieres Best Pract Res Clin Haematol 13, 457-468. Lutzko C, Omori F, Abrams-Ogg AC, Shull R, Li L, Lau K, Ruedy C, Nanji S, Gartley C, Dobson H, Foster R, Kruth S, Dube ID (1999) Gene therapy for canine alpha-L-iduronidase deficiency: in utero adoptive transfer of genetically corrected hematopoietic progenitors results in engraftment but not amelioration of disease. Hum Gene Ther 10, 1521-1532. Mackenzie IZ, Maclean DA (1980) Pure fetal blood from the umbilical cord obtained at fetoscopy: experience with 125 consecutive cases. Am J Obstet Gynecol 138, 1214-1218. MacKenzie TC, Kobinger GP, Kootstra NA, Radu A, SenaEsteves M, Bouchard S, Wilson JM, Verma IM, Flake AW (2002) Efficient transduction of liver and muscle after in utero injection of lentiviral vectors with different pseudotypes. Mol Ther 6, 349-358. Maestri NE, Clissold D, Brusilow SW (1999) Neonatal onset ornithine transcarbamylase deficiency: A retrospective analysis. J Pediatr 134, 268-272. Makrydimas G, Georgiou I, Kranas V, Zikopoulos K, Lolis D (1997) Prenatal diagnosis of beta-thalassaemia by coelocentesis. Mol Hum Reprod 3, 729-731. Marshall E (2002) Gene therapy a suspect in leukemia-like disease. Science 298, 34-35. Masaki I, Yonemitsu Y, Komori K, Ueno H, Nakashima Y, Nakagawa K, Fukumura M, Kato A, Hasan MK, Nagai Y, Sugimachi K, Hasegawa M, Sueishi K (2001) Recombinant Sendai virus-mediated gene transfer to vasculature: a new class of efficient gene transfer vector to the vascular system. FASEB J 15, 1294-1296. Mason CA, Bigras JL, O'Blenes SB, Zhou B, McIntyre B, Nakamura N, Kaneda Y, Rabinovitch M (1999) Gene transfer in utero biologically engineers a patent ductus arteriosus in lambs by arresting fibronectin-dependent neointimal formation. Nat Med 5, 176-182. Matthjis G, Devriendt K, Fryns JP (1998) The prenatal diagnosis of spinal muscular atrophy. Prenat Diagn 18, 607-610. Matzinger P (2002) The danger model: a renewed sense of self. Science 296, 301-305. May C, Rivella S, Callegari J, Heller G, Gaensler KM, Luzzatto L, Sadelain M (2000) Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature 406, 86. Mazarakis ND, Azzouz M, Rohll JB, Ellard FM, Wilkes FJ, Olsen AL, Carter EE, Barber RD, Baban DF, Kingsman SM, Kingsman A, O'Malley K, Mitrophanous K (2001) Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery. Hum Mol Genet 10, 2109-2121. McCray Jr PB, Stein CS, Kang Y, Sauter SL, Townsend K, Staber P, Derksen TA, Martins I, Qian J, Davidson BL, McCray PB (2001) In vivo treatment of hemophilia A and


David et al: Current status and future direction of fetal gene therapy mucopolysaccharidosis type VII using nonprimate lentiviral vectors. Mol Ther 3, 850-856. McCray PB, Armstrong K, Zabner J, Miller DW, Koretzky GA, Couture L, Robillard JE, Smith AE, Welsh MJ (1995) Adenoviral-mediated gene transfer to fetal pulmonary epithelia in vitro and in vivo. J Clin Invest 95, 2620-2632. Meertens L, Zhao Y, Rosic-Kablar S, Li L, Chan K, Dobson H, Gartley C, Lutzko C, Hopwood J, Kohn D, Kruth S, Hough MR, Dube ID (2002) In utero injection of alpha-Liduronidase-carrying retrovirus in canine mucopolysaccharidosis type I: infection of multiple tissues and neonatal gene expression. Hum Gene Ther 13, 18091820. Miller DG, Adam MA, Miller AD (1990) Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 10, 42394242. Mitchell M, Jerebtsova M, Batshaw ML, Newman K, Ye X (2000) Long-term gene transfer to mouse fetuses with recombinant adenovirus and adeno-associated virus (AAV) vectors. Gene Ther 7, 1986-1992. Mitrophanous K, Yoon S, Rohll J, Patil D, Wilkes F, Kim V, Kingsman S, Kingsman A, Mazarakis N (1999) Stable gene transfer to the nervous system using a non-primate lentiviral vector. Gene Ther 6, 1808-1818. Miyagawa Y, Matsuoka T, Baba A, Nakamura T, Tsuno T, Tamura A, Agematsu K, Yabuhara A, Uehara Y, Kawai H (1992) Fetal liver T cell receptor gamma/delta+ T cells as cytotoxic T lymphocytes specific for maternal alloantigens. J Exp Med 176, 1-7. Moise KJ, Hesketh DE, Belfort MM, Saade G, Van den Veyer IB, Hudson KM, Rodkey LS (1992) Ultrasound-guided blood sampling of rabbit fetuses. Lab Anim Sci 42, 398-401. Monahan PE, Samulski RJ (2000) Adeno-associated virus vectors for gene therapy: more pros than cons? Mol Med Today 6, 433-440. Moss IR, Scarpelli EM (1981) Stimulatory effect of theophylline on regulation of fetal breathing movements. Ped Res 15, 870-873. Muench MO, Rai J, Barcena A, Leemhuis T, Farrell J, Humeau L, Maxwell-Wiggins JR, Capper J, Mychaliska GB, Albanese CT, Martin T, Tsukamoto A, Curnutte JT, Harrison MR (2001) Transplantation of a fetus with paternal Thy1(+)CD34(+) cells for chronic granulomatous disease. Bone Marrow Transplant 27, 355-364. Murphy SJ, Chong H, Bell S, Diaz RM, Vile RG (2002) Novel integrating adenoviral/retroviral hybrid vector for gene therapy. Hum Gene Ther 13, 745-760. Nakai H, Montini E, Fuess S, Storm TA, Grompe M, Kay MA (2003) AAV serotype 2 vectors preferentially integrate into active genes in mice. Nat Genet 34, 297-302. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263-267. Newnham JP, Kelly RW (1993) Ultrasound for research with fetal sheep. In: Neilson JP, Chambers SE (Eds.), Obstetric Ultrasound I. Oxford Medical Publications, pp. 203-222. Nicolini U, Nicolaidis P, Fisk NM, Tannirandorn Y, Rodeck C (1990) Fetal blood sampling from the intrahepatic vein: analysis of safety and clinical experience with 214 procedures. Obstet Gynaecol 76, 47-53. Olivares EC, Hollis RP, Chalberg TW, Meuse L, Kay MA, Calos MP (2002) Site specific genomic integration produces therapeutic Factor IX levels in mice. Nat Biotechnol 20, 1124-1128. Olivieri NF (1999) The beta-thalassaemias. N Engl J Med 341, 99-109.

Olivieri NF, Weatherall DJ (1998) The therapeutic reactivation of fetal haemoglobin. Hum Mol Genet 7, 1655-1658. Ortiz-Urda S, Thyagarajan B, Keene DR, Lin Q, Calos MP, Khavari PA (2003) _C31 Integrase-Mediated Nonviral Genetic Correction of Junctional Epidermolysis Bullosa. Hum Gene Ther 14, 923-928. Pahal GS, Jauniaux E, Kinnon C, Thrasher AJ, Rodeck C (2000) Normal development of human fetal hematopoiesis between eight and seventeen weeks' gestation. Am J Obstet Gynecol 183, 1029-1034. Pai S, Marinkovich MP (2002) Epidermolysis bullosa: new and emerging trends. Am J Clin Dermatol 3, 371-380. Palmer TD, Rosman GJ, Osborne WRA, Miller D (1991) Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes. Proc Natl Acad Sci 88, 1330-1334. Papadopulos NA, Dumitrascu I, Ordonez JL, Decaluwe H, Lerut TE, Barki G, Deprest JA (1999) Fetoscopy in the pregnant rabbit at midgestation. Fetal Diagn Ther 14, 118-121. Pardi G, Marconi AM, Cetin I (2002) Placental-fetal interrelationship in IUGR fetuses--a review. Placenta 23 suppl A, S136-S141. Parsons DW, Grubb BR, Johnson LG, Boucher RC (1998) Enhanced in vivo airway gene transfer via transient modification of host barrier properties with a surface-active agent. Hum Gene Ther 9, 2661-2672. Pawliuk R, Westerman KA, Fabry ME, Payen E, Tighe R, Bouhassira EE, Acharya SA, Ellis J, London IM, Eaves CJ, Humphries RK, Beuzard Y, Nagel RL, Leboulch P (2001) Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 294, 2368-2371. Peault B, Tirouvanziam R, Sombardier MN, Chen S, Perricaudet M, Gaillard D (1994) Gene transfer to human fetal pulmonary tissue developed in immunodeficient SCID mice. Hum Gene Ther 5, 1131-1137. Peebles D, Gregory LG, David A, Themis M, Waddington S, Knapton HJ, Miah M, Cook T, Lawrence L, Nivsarkar M, Rodeck C, Coutelle C (2003) Widespread and efficient marker gene expression in the airway epithelia of fetal sheep after minimally invasive tracheal application of recombinant adenovirus in utero. Gene Ther in press. Peebles DM, Wyatt JS (2002) Synergy between antenatal exposure to infection and intrapartum events in causation of perinatal brain injury at term. BJOG 109, 737-739. Pfendner EG, Nakano A, Pulkkinen L, Christiano AM, Uitto J (2003) Prenatal diagnosis for epidermolysis bullosa: a study of 144 consecutive pregnancies at risk. Prenat Diagn 23, 447-456. Phillips JH, Hori T, Nagler A, Bhat N, Spits H, Lanier LL (1992) Ontogeny of human natural killer (NK) cells: fetal NK cells mediate cytolytic function and express cytoplasmic CD3 epsilon, delta proteins. J Exp Med 175, 1055-1066. Piedrahita JA (2000) Targeted modification of the domestic animal genome. Theriogeniology 53, 105-116. Pitt BR, Schwarz MA, Pilewski JM, Nakayama D, Mueller GM, Robbins PD, Watkins SA, Albertine KH, Bland RD (1995) Retrovirus-mediated gene transfer in lungs of living fetal sheep. Gene Ther 2, 344-350. Plopper CG, Weir AJ, Nishio SJ, Cranz DL, St George JA (1986) Tracheal submucosal gland development in the rhesus monkey, Macaca mulatta: ultrastructure and histochemistry. Anat Embryol 174, 167-178. Ponder KP, Melniczek JR, Xu L, Weil MA, O'Malley TM, O'Donnell PA, Knox VW, Aguirre GD, Mazrier H, Ellinwood NM, Sleeper M, Maguire AM, Volk SW, Mango RL, Zweigle J, Wolfe JH, Haskins ME (2002) Therapeutic neonatal hepatic gene therapy in mucopolysaccharidosis VII dogs. Proc Natl Acad Sci 99, 13102-13107.


Gene Therapy and Molecular Biology Vol 7, page 207 Porada CD, Tran N, Eglitis M, Moen RC, Troutman L, Flake AW, Zhao Y, Anderson WF, Zanjani ED (1998) In utero gene therapy: transfer and long-term expression of the bacterial neor gene in sheep after direct injection of retroviral vectors into preim