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GENE THERAPY & MOLECULAR BIOLOGY FROM BASIC MECHANISMS TO CLINICAL APPLICATIONS

Volume 7 2003 Published by Gene Therapy Press


GENE THERAPY & MOLECULAR BIOLOGY FREE ACCESS www.gtmb.org

!!!!!!!!!!!!!!!!!!!!!!!! ! 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@regulon.org

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

!!!!!!!!!!!!!!!!!!!!!!!! ! 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: maria@cancer-therapy.org

!!!!!!!!!!!!!!!!!!!!!!!! ! 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 maria@cancer-therapy.org

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 www.gtmb.org Scope Gene Therapy and Molecular Biology, bridging various fields is one of the most rapid with free access at gtmb.org. 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 (www.gtmb.org) 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 maria@cancer-therapy.org 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)

1-14

Review Article

Dynamic histone acetylation and its involvement in transcription

Virginia A. Spencer and James R. Davie

15-23

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

25-35

Review Article

Strategy of sensitizing tumor cells with adenovirus-p53 transfection

Jekunen Antti, Miettinen Susanna, Mäenpää Johanna, Kairemo Kalevi

37-42

Review Article

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

Alka Arora, Pradeep Seth

43-59

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

61-68

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

69-73

Mini Review

Regulation of vascular endothelial growth factor by hypoxia

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

75-89

Review Article

Gene therapy antiproliferative strategies against cardiovascular disease.

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

91-98

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

99-102

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


103-111

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

113-133

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

135-151

Review Article

Gene therapy for vascular diseases

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

153-165

Review Article

Angiogenic gene therapy for improving islet graft vascularization.

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

167-172

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

173-179

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

181-209

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

211-219

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

221-228

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

229-238

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

239-243

Research Article

PRL-3 as a target for cancer therapy

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

245-254

Review Article

Protective effect of heat shock proteins: potential for gene therapy

David S. Latchman

255-272

Review Article

Lung cancer gene therapy

273-289

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


291-298

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: Davie@cc.umanitoba.ca 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

1


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.

2


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

dynamics

of

histone

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

3


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 .

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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-

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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

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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

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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.

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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.

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Virginia A. Spencer and James R. Davie

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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*

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

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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: kalevi.kairemo@onkologi.uas.lul.se 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

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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

16


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

(5)

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

(4)

(1)

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

(2)

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

(3)

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

Reference

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

18

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.

19


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)

111

In 0.63 0.43 0.95 0.13

90

Y 4.41 3.59 10.1 0.57

76

Br 1.29 0.96 2.35 0.23

211

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

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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

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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.

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Kairemo et al: Oligonucleotide radiotherapy

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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; email:antti.jekunen@aventis.com 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)

26


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)

27


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.

of

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

30


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

Phase

Combination

n

Treatment responses

Reference (first author year)

NSCLC

II

no

24

2 PR, 17 SD_

(Swisher et al, 1999)

Head & neck

II

no

17

1CR, 2 PR, 6 SD_

(Clayman et al, 1998)

NSCLC Heach & neck (_) on patients

II II

Cisplatin + vinorelbin Cisplatin +5-FU

25 11

13 PR* 9 PR*

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

(*) on measurable lesions

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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.

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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: pseth@aiims.aiims.ac.in 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

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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

3

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

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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.

39


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).

40


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

pCIBRU-TRE

D3

3 doses

D0P2 D3P2

vPE8 and vPE16

Acknowledgments 2 doses

3 doses

D0V2 D3V2

P4

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

References

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

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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

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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: jose_delafuente@yahoo.com 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.

43


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.

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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

4A4

V-ATPase E subunit

CD052508

1C5

Na+/K+ ATPase, alpha subunit

CD052509

2A9

NADH dehydrogenase

CD052510

1D6

NADH dehydrogenase subunit 5 (nad5)

CD052511

1A4

Aldehyde dehydrogenase

CD052512

1C11

Translation initiation factor 5A (eIF5A)

CD052489

1E6

Translation initiation factor 5C (eIF-5C)

CD052490

2D2

Initiate factor 5 (if5)

CD052491

1C8

Virilizer (vir)

CD052513

1C10

Hsp70

CD052514

1A10

Elongation factor 2

CD052492

4F7

Elongation factor 1alpha

CD052493

3F6

Hsp60

CD052515

CD052494

1D1

Nucleotide binding protein 1 (Nubp1)

CD052516

1D8

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

CD052517

1F6

Ribosomal protein S4 (RpS4)

2B8

Ribosomal protein S11 (RpS11)

NR

2F8

Laminin receptor 1 (ribosomal protein SA)

CD052496

2F10

Ribosomal protein L3 (RpL3)

NR

3A10

Ribosomal protein L7A (RpL7A)

CD052497

1D11

Putative membrane protein

CD052518

3G9

Ribosomal protein S8 (RpS8)

CD052495

1E7

Sterol carrier protein

CD052519

3G10

Ribosomal protein L27A (RpL27A)

CD052498

1F3

Cyclin C (CycC)

CD052520

3D9

Alpha tubulin

CD052521

3C3

QM homolog (DQM) ribosomal protein

CD052499

2A7

Beta tubulin

CD052522

4D12

Proteasome/Signalosome subunit

CD052500

2A11

Notchless (Nle)

CD052523

4E7

Proteasome subunit

CD052501

2B2

Export factor binding protein 2 (Refbp2)

CD052524

4D11

Proteasome subunit

CD052502

2B7

G protein-coupled receptor

CD052525

3D10

Ribophorin I

CD052503

2B9

Ubiquitin-conjugating enzyme

CD052504

Succinate dehydrogenase B (SdhB)

CD052526

1B12 1D10

Ubiquitin

CD052505

2C12

V-ATPase D subunit Contains microsatellite sequence

CD052506

Beta-amyloid precursor protein (APP)

CD052527

1A9

2D1

CD052528

V-ATPase C subunit

CD052507

Fructose-1,6-bisphosphatase (fbp gene)

2D5

DNA repair protein Rad1 (Rad1)

CD052529

2D6

Identity to S. pombe dim1+, helicase protein 1

CD052530

1B2 EST clone

Predicted protein

GenBank accession number

45


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

Esterase

CD052531

4G5

Disulfide isomerase

CD052563

2F9

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

CD052532

4G8

Fumarate hydratase

CD052564

4G10

Rab3D (member of the Ras superfamily of small GTPases)

CD052565

Transmembrane G-proteinresponsive adenylyl cyclase

CD052533

4G11

Chloride channel

CD052566

2G8

Lysyl-tRNA synthetase

CD052534

4H4

Solute carrier protein

CD052567

2H11

Sodium- and chloride-dependent taurine transporter

CD052535

1B7

Mitochondrion

NR

1B8

Mitochondrion

NR

3C12

RNA polymerase III

CD052536

2E9

Mitochondrion

NR

CD052537

2G11

Mitochondrion

NR

3C6

Mitochondrion

NR

3G4

Mitochondrion

NR

4A2

Mitochondrion

NR

2F12

3E1

Beta-adaptin

3E2

Microtubule-associated protein, RP/EB family

CD052538

3E4

Myosin II regulatory light chain

CD052539

3E6

Unknown Zinc finger like protein

CD052540

3E10

Mannose binding lectin (rhea)

CD052541

3E12

Clathrin heavy chain (Chc)

CD052542

3F4

Identity to M. musculus adult male testis cDNA

CD052543

3F10

Identity to D. melanogaster Pelement somatic inhibitor (Psi)

3G11

4E9

Mitochondrion

NR

2A6

Mitochondrion

NR

4G7

NAD-dependent malate dehydrogenase

NR

3D4

Cytochrome c oxidase I (COI)

NR

1C2

Cytochrome c oxidase II (COII)

NR

CD052544

4D2

Cytochrome c oxidase III (COIII)

NR

Identity to D. melanogaster BM40 extracellular basement membrane protein

CD052545

1G4

Cytochrome b (cytb)

NR

2G9

16S ribosomal RNA

NR

4A8

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

CD052546

1F4

CD052568

4A10

Identity to D. melanogaster homeoprotein phtf

CD052547

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

2C7

CD052569

4A12

Amino acid transporter system A (ATA2)

CD052548

Unknown Contains microsatellite sequence

3B6

Calmodulin

CD052549

4B7

Alpha-tubulin

CD052550

Unknown Contains a microsatellite sequence

CD052570

4B2 4C9

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

CD052551

4G12

Unknown Contains microsatellite sequence

CD052571

4H2

CD052572

4C11

Intracellular receptor of activated protein kinase C1 (Rack1)

CD052552

Unknown Contains microsatellite sequence

4D6

Identity to D. melanogaster CG10395 cDNA

CD052553

4D7

Identity to D. melanogaster LD23959 cDNA

CD052554

4E6

Identity to D. melanogaster CG13597 cDNA

CD052555

4D8

Identity to H. sapiens hypothetical protein FLJ10342

CD052556

4E1

Pre-mRNA splicing factor

CD052557

4E3

Receptor signaling protein serine/threonine kinase

CD052558

4F8

Nucleotidase

CD052559

4F1

Block of proliferation 1 (Bop1)

CD052560

4G1

Identity to H. sapiens hypothetical protein MGC2404

CD052561

4G2

LRP/alpha-2-macroglobulin receptor

CD052562

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

Cell

32

36.78

31.88

Cellular component Mitochondria

17

15.54

16.83

Cell membrane

14

16.09

13.86

Nucleus

11

12.64

10.89

Extracellular

2

2.30

1.98

Unlocalized

2

2.30

1.98

Unknown

9

10.34

8.91

Biological process Cell growth or maintenance

61

70.11

60.40

Physiological process

8

9.20

7.92

Developmental process

5

5.75

4.95

Cell communication

2

2.30

1.98

Unknown

11

12.64

10.89

Molecular function Ligand binding or carrier

30

34.48

29.70

Enzyme

29

33.33

28.71

Transporter

9

10.34

8.91

Chaperone

2

2.30

1.98

Structural molecule

7

8.05

6.93

Unknown

10

11.49

9.90

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.

47


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 (%)

4D8

Identity to H. sapiens hypothetical protein FLJ10342 with unknown function

40 a

7a

4F8

Nucleotidase

50 a

17 a

1C10 b

Hsp70

17 a

0a

3F6

b

Hsp60

4D6

Identity to D. melanogaster CG10395 cDNA with unknown function

61

11

4E6

Identity to D. melanogaster CG13597 cDNA with unknown function

20

ND

3E1

Beta-adaptin

27

5

4G11

Chloride channel

38

30

17 clones b

Ribosomal proteins

15 a

0a

2C12

Beta-amyloid precursor protein (APP)

-8

c c

4F1

Block of proliferation Bop1

-39

3E10

Mannose binding lectin

-48 a, c

3C12

b

RNA polymerase III

-104

2F9 b

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

1A9, 1B2, 4A4 b

ATPase

a, c

-57 a, c

ND ND ND ND

ND

a

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)

LQHMTDFAIQFNKNSFGVIPSTPLAIHTPLMPNQSIDVSLPLNTLGPVMK MQPMTNFAIQLNKNSFGLVPASPMQ-AAPLPPNQSIEVSMALGTNGPIQR LQHMTDFAIQFNKNSFGVIPSTPLAIHTPLMPNQSIDVSLPLNTLGPVMK MQAMTGFAVQFNKNSFGLTPAQPLQLQIPLQPNFPADASLQLGTNGPVQK LQHMTDFAIQFNKNSFGLIPATPLQIHTPLMPNQSIDVSLPLNTNGPVQK

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

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

MEPLNNLQVAVKNNIDVFYFSCLIPLNVLFVEDGKMERQVFLATWKDIPN MEPLNNLQVAVKNNIDIFYFACLVHGNVLFAEDGQLDKRVFLNTWKEIPA MEPLNNLQVAVKNNIDVFYFSCLIPLNVLFVEDGKMERQVFLATWKDIPN MDPLTNLQVAIKNNVDVFYFSCLVPMHVLSTEDGLMDKRVFLATWKDIPA

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

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

ENELQFQIKECHLNADTVSSKLQNNNVYTIAKRNVEGQDMLYQSLKLTNG ANELQYTLSGVIGTTDGIASKMTTNNIFTIAKRNVEGQDMLYQSLKLTNN ENELQFQIKECHLNADTVSSKLQNNNVYTIAKRNVEGQDMLYQSLKLTNG QNEVQYTLDNVNLTADQVSQKLQNNNIFTIAKRNVDGQDMLYQSLKLTNG ENELQFTIKEVHLTADTVSSKLQNNNIFTIAKRNVEGQDMLYQSLKLTNG

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

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

IWILAELRIQPGNPNYTLSLKCRAPEVSQYIYQVYDSILKNIWVLLELKLQPGNPEATLSLKSRSVEVANIIFAAYEAIIRSP IWILAELRIQPGNPNYTLSLKCRAPEVSQYIYQVYDSILKNIWVLAELKIQPGNPRITLSLKTRAPEVAAGVQQTYELILHSIWILAELKIQPGNPNYTLSLKCRAPEVAQYIYQVYDSILKS

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)

DTPSLETPQAARLRRRQLIRQLLERDKTPLAILFMAAVVGTLVGLAA-VA DLKEGVCLSALWFNH--------EQ----------CCWTSNETTFAERDK DLKEGICLPWFWFNH--------EQ----------CCWQSNNVTFEDRNN DLKEGICPQAFWLNK--------EQ----------CCWASNDTFFKG-DD DLKTGVCADRFWLDH--------EH----------CCWSSNDTFYKD-DD DLKHGICPPAFWFNR--------EQ----------CCYPAKQSVFEE-GN AFRSGICANFFWLGR-------------------------N-MCCVDCRE DLKFGICPQAFWLNR--------EQ----------CCWSSNETSFDS-GN

49


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

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

DLKEGICLSALWYNH--------EQ----------CCWGSNETTFEERDK GFKLLLTSNLMLDGK----------------------------------NWKTGHCQRNWLLNKS-------------------FCCNGVVNEVTSTSN DLK GIC AFWLNR EQ CCW SN T F D

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)

FDKGVAWLQNQRMGALVHTADNYPLLLTVAFLCSAVLAMFGYFLVRKYAP CPQWKSWAELILGQ--AEGPGSYIMNYFMYIYWALSFAFLAVCLVKVFAP CPEWRSWSQLVLGR--SEGAFPYILNYFMYVMWALLFSLLAVLLVRNFAP CKQWYRWPEMFDSGMDKDGAGFYLLSYLLYVMWSVLFATLAVMLVRTFAP CKAWTKWPWMLNYYN-SSSFLFLFLEWIFYIGWAVAMSTLAVLFVKIFAP CSTWKTWPEIFGLD--RNGTGPYIVAYIWYVLWALLFASLSASLVRMFAP CGEYYSWGEFFLGR---DNHVVAFVDFVMYVSFSTMAAVTAAYLCKTYAP CSQWYAWSEIFTSS--REGFGAYVISYFFYIMWAMLFALLAASLVRMFAP CPQWKTWAELIIGQ--AEGPGSYIMNYIMYIFWALSFAFLAVSLVKVFAP ----------------------YFQAFAAFAGCNVFFATCAAALCAFIAP LLLKRQEFECEAQG-LWIAWKGHVSPFIIFMLLSVLFALISTLLVKYVAP C W W EL EG YIL YIMYILWALLFA LA LVK FAP

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)

EAGGSGIPEIEGALE---DQRPVRWWRVLPVKFFGGLGTLGGGMVLGREG YACGSGIPEIKTILSGF-IIRGYLGKWTLMIKTITLVLAVASGLSLGKEG YACGSGIPEIKTILSGF-IIRGYLGKWTLIIKTMTLVLAVSSGLSLGKEG YACGSGIPEIKTILSGF-IIRGYLGKWTLTIKSVCLVLAVGAGLSLGKEG YACGSGIPEIKCILSGF-VIRGYLGKWTFIIKSVGLILSSASGLSLGKEG YACGSGIPEIKTILSGF-IIRGYLGKWTLLIKSVGLMLSVSAGLTLGKEG YASGGGIAEVKTIVSGH-HVKRYLGGWTLITKVVGMCFSTGSGLTVGKEG YACGSGIPEIKTILSGF-IIRSYLGKWTLIIKSVGIMLSVSAGLSLGKEG YACGSGIPEIKTILSGF-IIRGYLGKWTLMIKTITLVLAVASGLSLGKEG AAAGSGIPEVKAYLNG-IDAHSILAPSTLLVKIFGSILGVSAGFVVGKEG MATGSGISEIKVWVSGFEYNKEFLGLLTLVIKSVALPLAISSGLSVGKEG YACGSGIPEIKTILSGF IIRGYLGKWTLIIKSVGLVLAVSSGLSLGKEG

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)

PTVQIGGNIGRMV----------LDIFRLKG--DEARHTLLATGAAAGLA PLVHVACCCGNIF----------SYLFPKYSKNEAKKREVLSAASAAGVS PLIHVACCCGNIL----------CHLFTKYRKNEAKRREVLSAAAAAGVS PLVHVACCIGNIF----------SYLFPKYGKNEAKKREILSAAAAAGVS PMVHLACCIGNIF----------SYLFPKYGLNEAKKREILSASAAAGVS PMVHIASCIGNIF----------SHVFPKYGRNEAKKREILSAAAAAGVS PFVHIGACVGGII----------SGALPSYQQ-EAKERELITAGAGGGMA PMVHIASCIGNIL----------SYLFPKYGRNEAKKREILSAAAAAGVS PLVHVACCCGNIF----------SYLFPKYSTNEAKKREVLSAASAAGVS PMVHTGACIANLLGQGGSRKYHLTWKWLKYFKNDRDRRDLITCGAAAGVA PSVHYATCCGYLL----------TKWLLRDTLTYSTQYEYLTAASGAGVA PLVHIA CIGNIL SYLFPKY KNEAKKREILSAAAAAGVS

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)

AAFNAPLAGILFIIEEMRPQ--FRYTLISIKAVFIGVIMSTIMYRIFNHE VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFAALVAAFVLRSINPFGN VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFAALVAAFTLRSINPFGN VAFGAPIGGVLFSLEEVSYY--XPLKTLWRSFFCALVAASVLRSINPFGN VAFGAPIGGVLFSLEEASYY--FPLKTMWRSFFCALVAGIILRFVNPFGS VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFCALIAAFVLRSLTPFGN VAFGAPVGGVIFALEDVSTS--YNFKALMAALICGVTAVLLQSRVDLWHT VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFCALIAAFILRSINPFGN VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFAALVAAFVLRSINPFGN AAFRAPVGGVLFALEEIASW--WRSALLWRTFFTTAIVAMVLRSLIQFCR VAFGAPIGGVLFGLEEIASANRFNSSTLWKSYYVALVAITTLKYIDPFRN VAFGAPIGGVLFSLEEVSYY FPLKTLWRSFF ALVAA VLRSINPFGN

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)

VA----------LIDVGKLSDAPL SR----------LVLFYVEYHTPW SR----------LVLFYVEFHAPW DH----------LVMFYVEYDFPW NQ----------TSLFHVDYMMKW EH----------SVLFFVEYNKPW GR----------IVQFSVNYQHNW EH----------SVLFYVEYNKPW SR----------LVLFYVEYHTPW GGNCGLFGQGGLIMFDVNSGVSNY GR----------VILFNVTYDRDW LVLFYVEY PW

50


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

PHAQGFIEVDQNVTTHHPIVREEKIVPNMQINGYENPTYKYFE PQAQGFVQVDQGALPASPEER---HLASMQVNGYENPTYKYFE PHAQGFVEVDQAVGAPVTPEE--RHVANMQINGYENPTYKYFE PHAQGFVEVDQ V P ER HVANMQINGYENPTYKYFE

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

Species

GenBank accession number

4F7 1A2

Elongation factor 1-alpha

95% 85%

Neacarus texanus

AAK12660 NP_031932

1A10

Elongation factor-2

88% 80%

Mastigoproctus giganteus Mus musculus

AAK12348 BAC26203

1C11

eIF-5A

65% 59%

Drosophila melanogaster Mus musculus

AAM68297 XP_203336

1F6 2C3

RpS4

79% 75%

Spodoptera frugiperda Mus musculus

AAL26580 AAH09100

2B8

RpS11

92% 80%

Dermacentor variabilis Mus musculus

AAO92287 XP_133477

2F8

Laminin receptor 1 (RpSA)

66% 73%

Anopheles gambiae Mus musculus

EAA00413 NP_035159

2F10

RpL3

70% 68%

Spodoptera frugiperda Mus musculus

AAL62468 AAH09655

3A10

RpL7A

55% 60%

Drosophila melanogaster Mus musculus

NP_511063 A30241

3D10

Ribophorin I

57% 50%

Drosophila melanogaster Mus musculus

AAN71150 BAC26679

3G9

RpS8

70% 71%

Spodoptera frugiperda Mus musculus

AAL62472 XP_134904

3G10

RpL27A

42%

Spodoptera frugiperda

AAK92158

Mus musculus

51


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

Mus musculus

XP_137118

4D11

Proteasome subunit

60% 55%

Drosophila melanogaster Mus musculus

NP_524115 NP_035315

4D12

Proteasome/Signalosome subunit

43% 56%

Anopheles gambiae Mus musculus

EAA11895 AAC33900

4E7

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)

50 ------------------------MAGACGKPHMSPASLPGKRRLEPDQE MTKKLALKRRGKDSEPTNEVVASSEASENEEEEEDLLQAVKDPGEDSTDD ----------------------------SVRPEKRRSEPELEPEPEPEPP ---------------------QENLLGSIENEGEDSSDSDGEYATDDDED ----------------------MGPKTLSKQPAKASSSTSKRTAGPTISK P S E A D D D 51 100 LQIQEPPLLSD-PDSSLSDSEESVFSGLEDSGSDSSEEDTEGVA----GS EGIDQEYHSDSSEELQFESDEEGNYLGRKQSSSAEEDEESSDEEDN---E LLCTSPLSHSTGSDSGVSDSEESVFSGLEDSGSDSSEDDDEGDEEGEDGA DVLSFESLNSDGEE---EDEEEDAGTTLEEVEREAEEDDDEEDAERKQRE QTEDSDDEGSSSAYSDLEDSEGADSSDSNDLSDTEASEDDYDDSQDEENT I E SS DS LEDSEES FSGLEDS SDSSEEDDEDDAE 101 150 SGDEDNHRAEETSEELAQAAPLCSRTEE--------------AGALAQDE EEESTDGEEVEDEEKDSKSKQTDDKPSGSGAASKKALTAELPKRDSSKPE LDDEGHSGIKKTTEEQVQASTPCPRTEM--------------ASARIGDE EQFESDDEPLPDDLKLGRIEDVLGTGEKKTRGLGVFPPVPKRKGKAAQDE KITLTGVEGKDLELRGKDQEAPVESGKRSAWHRQQEDAKEDRRTQVVEDE DET E E EEK A R E K A DE 151 200 YEE-DSSDEEDIRNTVGNVPLAWYDEFPHVGYDLDGKRIYKPLRTRDELD YQDSDTSDEEDIRNTVGNIPMHWYDEYKHIGYDWDAKKIIKPPQG-DQID YAE-DSSDEEDIRNTVGNVPLEWYDDFPHVGYDLDGRRIYKPLRTRDELD YAAGDTSDEEDIRNTVGNIPMHWYDEYKHVGYDWDAKKIIKAKKG-DAID YAF-DSSDEEDVRNTVGNIPLEWYEHYPHIGYDLEGKPILKPPRV-SDLD YAE DSSDEEDIRNTVGNIPL WYDEYPHVGYDLDGKKIIKP R DELD 201 250 QFLDKMDDPDFWRTVQDKMTGRDLRLTDEQVALVHRLQRGQFGDSGFNPY EFLRKIEDPDFWRTVKDPLTGQDVRLTDEDIALIKRIVSGRIPNKDHEEY QFLDKMDDPDYWRTVQDPMTGRDLRLTDEQVALVRRLQSGQFGDVGFNPY DFLQRMEDPNFWRTVTDPQTGQKVVLSDEDIGLIKRIMSGRNPDAEYDDY DFLRKMDDPNYWRTVKDKSTGQDVVLTDEDVDLIQRLQKGQFPSSTTDPY DFL KMDDPDFWRTV DPMTGQDVRLTDEDVALIKRLQSGQFPDS FDPY 251 300 EPAVDFFSGDIMIHPVTNRPADKRSFIPSLVEKEKVSRMVHAIKMGWIKP EPWIEWFTSEVEKMPIKNVPDHKRSFLPSVSEKKRVSRMVHALKMGWMKT EPAVDFFSGDVMIHPVTNRPADKRSFIPSLVEKEKVSRMVHAIKMGWIQP EPFIEWFTSEVEKMPIRNIPESKRSFLPSKAEKHKIGRYVHALKMGWMKT EPFEDIFSHETMIHPVTRHPPQKRSFVPSRIEKAMVSKMVHAIKMGWIKP EPFIDFFS EVMIHPVTN P KRSFIPSLVEK KVSRMVHAIKMGWIKP 301 350 RRPHD------PTPSFYDLWAQEDPNAVLG-RHKMHVPAPKLALPGHAES TEEVEREKQAKRGPKFYMLWETDTSREHMR-RIHDPVSAPKRDLPGHAES RRPRD------PTPSFYDLWAQEDPNAVLG-RHKMHVPAPKLALPGHAES MAEKRRLEAIRRQPKFYMLWTTDHGKEEMR-RIHDHVAAPKRMLPGHAES RVKKH------DPERFSLLWDKDDSTAGSNERMQRHIPAPKMKLPGHEES R KD PKFYMLW DD A L RI HVPAPKL LPGHAES 351 400 YNPPPEYLPTEEERSAW--MQQEPVERKLNFLPQKFPSLRTVPAYSRFIQ YNPPPEYLFDAKETKEWLKLKDEPHKRKLHFMPQKFKSLREVPAYSRYLR YNPPPEYLLSEEERLAW--EQQEPGERKLSFLPRKFPSLRAVPAYGRFIQ

52


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

(325) YNPPPEYLFDEKELEEWNKLANQPWKRKRAYVPQKYNSLREVPGYTRYVK (321) YNPPAEYLFTEEEEAKWR--EQEPEERRINFLPAKYPCLRAVPAYERFIE (351) YNPPPEYLFTEEE W L QEP ERKL FLPQKFPSLR VPAYSRFI 401 450 (348) ERFERCLDLYLCPRQRKMRVNVDPEDLIPKLPRPRDLQPFPVCQALVYRG (396) ERFLRCLDLYLCPRAKRVKLNIDAEYLIPKLPSPRDLQPFPTVESMVYRG (349) ERFERCLDLYLCPRQRKMRVNVDPEDLIPKLPRPRDLQPFPTCQALVYRG (375) ERFLRCLDLYLAPRMRRSRVAVGAEYLIPKLPSPRDLQPFPTLQNLIYTG (369) ERFERCLDLYLCPRQRKMRVNVDAEDLIPQLPKPKDLQPFPSIQSIVYEG (401) ERFERCLDLYLCPRQRKMRVNVDAEDLIPKLPRPRDLQPFPTIQALVYRG 451 500 (398) HSDLVRCLSVSPGGQWLASGSDDGTLKLWEVATARCMKTVHVGGVVRSIA (446) HTDLVRSVSVEPKGEYLVSGSDDKTVKIWEIATGRCIRTIETDEVVRCVA (399) HSDLVRCLSVSPGGQWLVSGSDDGSLRLWEVATARCVRTVPVGGVVKSVA (425) HTSLIRCISVEPKGEYIVTGSDDMTVKIWEISTARCIRTIPTGDIVRSVA (419) HTDCVLCLSLEPAGQFFASXSEDGTVRIWELLTGXCLKKFQFEAPVKSVA (451) HTDLVRCLSVEPGGQWLVSGSDDGTVKIWEIATARCIRTI GGVVRSVA 501 550 (448) WNPNPTICLVAAAMDDAVLLLNPALGDRLLVGSTDQLLEAF----TPPEE (496) WCPNPKLSIIAVATGNRLLLVNPKVGDKVLVKKTDDLLAEAPSQDVIESE (449) WNPSPAVCLVAAAVEDSVLLLNPALGDRLVAGSTDQLLSAF----VPPEE (475) WCPNSKISLVAAASGKRVLLINPKVGDYMLVKKTDDLLTEAPRSDTVDSE (469) WCP--VVVPMKLCVDKTVSMLDAGVTDKLLPFTTGHRVVCPPRRVLGPGG (501) WCPNP I LVAAAVD VLLLNPAVGDKLLV STD LL P V P E 551 600 (494) PALQPARWLEVSEEEHQRGLRLRICHSKPVTQVTWHGRGDYLAVVLSSQE (546) RIKTAVQWSNAEADEQEKGVRVVITHFKPIRQVTWHGRGDYLATVMPEGA (495) PPLQPARWLEASEEERQVGLRLRICHGKPVTQVTWHGRGDYLAVVLATQG (525) RIRSAVQWGEVTEEEKKLGVRIVITHFREVRQVTWHGRGDYFATVMPDGA (517) GSGVGADVGLLSRVPLPGGASAGRSPPR-CGAGDVALEGRLLCHCHGRGT (551) AA W EVSEEE GLRL ITH KPV QVTWHGRGDYLA VL GA 601 650 (544) HTQVLLHQVSRRRSQSPFRRSHGQVQCVAFHPSRPFLLVASQRSIRIYHL (596) NRSALIHQLSKRRSQIPFSKSKGLIQFVLFHPVKPCFFVATQHNIRIYDL (545) HTQVLIHQLSRRRSQSPFRRSHGQVQRVAFHPARPFLLVASQRSVRLYHL (575) YRSVMIHQLSKRRSQVPFSKSKGLIQCVLFHPIKPCLFVATQRHIRVYDL (566) GHRACPSVVHAAVRRLPFSKAKGGVSRVLFHPLRPFLLVACQRTVRVYHL (601) H VLIHQLSKRRSQIPFSKSKG VQ VLFHPIRPFLLVASQRSIRIYHL 651 700 (594) LRQELTKKLMPNCKWVSSMAVHPAGDNIICGSYDSKLVWFDLDLSTKPYK (646) VKQELVKKLLTNSKWISGMSIHPKGDNLLVSTYDKKMLWFDLDLSTKPYQ (595) LRQELTKKLMPNCKWVSSLAVHPAGDNVICGSYDSKLVWFDLDLSTKPYR (625) VKQLMMKKLYPGCKWISSMAIHPKGDNLLIGTYEKRLMWFDLDLSTKPYQ (616) LKQELAKRLTSNCKWISCMGRPPPGDNLLIGTYEKRLMWFDLDLSTKPYQ (651) LKQEL KKLMPNCKWISSMAIHP GDNLLIGTYDKKLMWFDLDLSTKPYQ 701 750 (644) VLRHHKKALRAVAFHPRYPLFASGSDDGSVIVCHGMVYNDLLQNPLLVPV (696) TMRLHRNAVRSVAFHLRYPLFASGSDDQAVIVSHGMVYNDLLQNPLIVPL (645) MLRHHKKALRAVAFHPRYPLFASGSDDGSVIVCHGMVYNDLLQNPLLVPV (675) QLRIHNAAIRSVAFHPRYPLFASAGDDRSVIVSHGMVYNDLLQNPLIVPL (666) QLRIHNAAIRSVAFHPRYPLFASAGDDRSVIVSHGMVYNDLLQNPLIVPL (701) LRIHK AIRSVAFHPRYPLFASGSDD SVIVSHGMVYNDLLQNPLIVPL 751 790 (694) KVLKGHTLTRDLGVLDVAFHPTQPWVFSSGADGTIRLFS(746) KKLQTHEKRDEFGVLDVNWHPVQPWVFSTGADSTIRLYT(695) KVLKGHVLTRDLGVLDVIFHPTQPWVFSSGADGTVRLFT(725) RRLKNHAVVNDFSVFDVVFHPTQPWVFSSGADNTVRLYT(716) RRLKNHAISKGMGVLDCAFHPHQPWIVTAGADSTLRLFT(751) KRLK H LTRDLGVLDV FHPTQPWVFSSGAD TIRLFT

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.

53


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 3.6.1.34). 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).

54


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)

50 MLFFCPSCGNILIIEEDTNCHRFTCNTCPYISKIRRKISTKTFPRLKEVD MLLFCPGCGNGLIVEEGQRCHRFSCNTCPYVHNITRKVTNRKYPKLKEVD MLMFCPTCGNLLLVEESTDSLRFSCNTCPYICKIRRTISSRIYPTLKEVD MLLFCPTCANILIVEQGLECFRFACNTCPYVHNIKAKMSNRKYPRLKDVD MLLFCPTCGNILIVEEGTDCHRFSCNTCPYIHNIRRKISNRKYPRLKEVD 51 100 HVLGGKAAWENVDSTDAECPTCGHKRAYFMQIQTRSADEPMTTFYKCCNH DVLGGAAAWENVDSTAESCPKCEHPRAYFMQLQTRSADEPMTTFYKCCNA HVMGGSAAWENVDSTDAVCPSCSHNRAYFMQMQTRSADEPMTTFYKCCNQ DVLGGAAAWENVDSTEEKCPKCGHERAYFMQIQTRSADEPMTTFYKCCNQ HVLGGAAAWENVDSTDE CPKCGH RAYFMQIQTRSADEPMTTFYKCCNQ 101 ECNHTWRD QCGHRWRD TCGHNWRD LCGHQWRD CGHNWRD

B I. scapularis A. variegatum H. sapiens Consensus

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

MVDPEDEEVQLDEAMDEMAAYFRKEYTPKLLITTSDNPHRRTIKFCRELK MVQADDEEVQLDEAMDEMAAYFRKEYIPKLLITTSDNPHTRTIRFCRELK TVDPNDEEVAYDEATDEFASYFNKQTSPKILITTSDRPHGRTVRLCEQLS MVDP DEEVQLDEAMDEMAAYFRKEY PKLLITTSDNPH RTIRFCRELK

I. scapularis A. variegatum H. sapiens Consensus

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

QSIPDAEFRWRNRSRIKKTVEQAVERGYSDIAIINEDRRHPSKFVVQFL QSIPNADFRWRNRSRIKKTVEQAIERGYSDIAVINEDRRHPNGLLLTHL TVIPNSHVYYRRGLALKKIIPQCIARDFTDLIVINEDRKTPNGLILSHL QSIPNA FRWRNRSRIKKTVEQAIERGYSDIAVINEDRRHPNGL L HL

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

1A9

TATATATATATATATATA

4G12

CACACACAGACACACTCACA ATATATATATATA

1F4

GCGCGCGCGTGTGCGTGTGTGTGTGTGTGT

2C7

GTGTGTGTGTGTGTGTGT

3B6

TATATATATATATATATATATATA

4H2

TGAAATGAAATGAAATGAAA

(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

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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.

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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

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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: greenbergerjs@msx.upmc.edu 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,

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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.

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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.

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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).

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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).

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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

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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.

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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: gilana@tx.technion.ac.il 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

of

VEGF

gene

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).

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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

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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

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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.

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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.

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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: vandres@ibv.csic.es 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.

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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.

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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

Ref.

Animal model

CDK2

Balloon angioplasty (rat)

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

CDC2

Balloon angioplasty (rat)

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

Cyclin B1

Balloon angioplasty (rat)

Morishita et al, 1994b

CDC2/PCNA

Graft arteriosclerosis (rabbit, rat)

Mann et al, 1995; Miniati et al, 2000

CDC2/PCNA

Balloon angioplasty (rat)

Morishita et al, 1993

CDK2

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

Ribozyme

PCNA

Stent (pig)

Frimerman et al, 1999

TGF-!1

Balloon angioplasty (rat)

Yamamoto et al, 2000

PDGF-A

Balloon angioplasty (rat)

Kotani et al, 2003

12-lipoxygenase

Balloon angioplasty (rat)

E2F

Balloon angioplasty (rat, pig)

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

E2F

Graft arteriosclerosis (rabbit, mouse, monkey)

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

AP-1

Balloon angioplasty (rat, rabbit, minipig)

p21Cip1

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;

p21Cip1

Graft arteriosclerosis (rabbit)

Bai et al, 1998

p27Kip1

Balloon angioplasty (rat, pig)

Chen et al, 1997; Tanner et al, 2000

pRb

Balloon angioplasty (rat, pig)

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

RB2/p130

Balloon angioplasty (rat)

Claudio et al, 1999

p53

Balloon angioplasty (rabbit, rat)

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

GAX

Balloon angioplasty (rat, rabbit)

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

GATA-6

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).

78

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

Design

Strategy

Disease

Outcome

Refs.

PREVENT I

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

PREVENT II

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

ITALICS

Randomized, placebo-controlled

PREVENT: ITALICS:

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.

79


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

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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)

protein

kinases

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.

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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.

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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.

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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: torusuzu-tky@umin.ac.jp 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;

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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,

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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.

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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.

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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).

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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.

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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

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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: nagy.habib@imperial.ac.uk 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

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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)

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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.

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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.

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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: triantafyllos.chavakis@med.uni-heidelberg.de 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

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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.

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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.

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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

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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.

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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.

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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: sanlioglu@akdeniz.edu.tr 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

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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

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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.

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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.

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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.

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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

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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 (www.wiley.co.uk/genmed/clinical) 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

USA USA

Arie Belldegrun Christopher J. Logothetis

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

I I/II

USA

Dov Kadmon

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

I

USA

Jonathan W. Simons

Selectively replicating adenovirus (CN706)

I

USA

Thomas A. Gardner

Suicide gene therapy (HSV-tk)

I

USA

Jae Ho Kim

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

I

USA USA USA USA USA USA

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)

I/II I I/II I/II II I

USA USA USA

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

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

I I II

USA USA

Eric J. Small Svend O. Freytag

II I

USA

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

127

I/II


Sanlioglu et al: Adenovirus mediated gene therapy for prostate carcinoma

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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).

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Dr. Salih Sanlioglu

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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: claunap@tin.it 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:

VEGF* !Anti-VSMC

migration & matrix remodelling: TIMPs

Vein Graft Failure

Gene Therapy Strategies !Anti-VSMC

proliferation:

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

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

!-Cytostatic:

!Re-endothelialization:

VEGF

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

!Anti-thrombotic:

none tested !Re-endothelialization: Ctype natriuretic peptide

!Re-endothelialization:

VEGF* !Anti-VSMC

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.

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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).

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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.

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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 http://www.wiley.co. 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.

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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: hengjiang.dong@mssm.edu. 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.

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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

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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

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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

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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.

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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).

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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.

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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


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Dr. Hengjiang Dong .

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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: kume@jichi.ac.jp 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

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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.

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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).

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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.

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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