2002 Van Andel Research Institute Scientific Report by Van Andel Institute

Van Andel Research Institute Scientific Report 2002

Title page photo: HGF/SF-induced scattering of MDCK cells in vitro Shown are dog kidney窶電erived (MDCK) cells stained for DNA (using DAPI, green) and actin (phalloidinrhodamine, red). (Phalloidin is a protein that preferentially binds to actin and can be labeled with a fluorescent dye, here rhodamine.) Although DAPI is normally seen as blue, here the DAPI emission is placed in the green channel so that co-localization can be seen as yellow. These cells were treated with scatter factor (HGF/SF) to induce separation from their substrate and begin their migration/scattering. There is a large cluster of cells in the center and random, attenuated cells with longer cellular processes in the left center and upper right that are starting the scattering process. (Resau)

Van Andel Institute 333 Bostwick Avenue, N.E. Grand Rapids, Michigan 49503, U.S.A.

Contents Directorâ&#x20AC;&#x2122;s Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Laboratory Reports Laboratory of Cell Structure and Signal Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Arthur S. Alberts, Ph.D. Antibody Technology Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Brian Cao, M.D. Mass Spectrometry and Proteomics Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Gregory S. Cavey, B.S. Laboratory of Signal Regulation and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Sara A. Courtneidge, Ph.D. Developmental Cell Biology Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Nicholas S. Duesbery, Ph.D. Bioinformatics Core Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Kyle Furge, Ph.D. Laboratory of DNA and Protein Microarray Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Brian B. Haab, Ph.D. Laboratory of Cancer Pharmacogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Han-Mo Koo, Ph.D. Laboratory of Integrin Signaling and Tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Cindy K. Miranti, Ph.D. Analytical, Cellular, and Molecular Microscopy Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 James H. Resau, Ph.D. Laboratory of Germline Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Pamela J. Swiatek, Ph.D. Laboratory of Cancer Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Bin T. Teh, M.D., Ph.D. Laboratory of Molecular Oncology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 George F. Vande Woude, Ph.D. Tumor Metastasis and Angiogenesis Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Craig P. Webb, Ph.D.

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Laboratory of Chromosome Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Michael Weinreich, Ph.D. Laboratory of Cell Signaling and Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Bart O. Williams, Ph.D. Laboratory of Structural Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 H. Eric Xu, Ph.D. Laboratory of Developmental Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Nian Zhang, Ph.D.

Daniel Nathans Memorial Award . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Postdoctoral Fellowship Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Student Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

VARI Seminar Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Conference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

VARI Photos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Directorâ&#x20AC;&#x2122;s Introduction

Director’s Introduction having access to this technology was instrumental in our efforts to recruit Eric. The members of the MCSB (VARI, University of Michigan, Michigan State University, and Wayne State University) partnered with Northwestern University to secure the beamline. We also were successful in recruiting Greg Cavey to establish our proteomics core facility. Greg comes with glowing credentials, having been responsible for establishing a proteomics core at Pharmacia in Kalamazoo, Michigan. The proteomics core will provide us with powerful tools necessary for measuring infinitesimal quantities of protein that contribute to cancer. At this writing, we are eagerly awaiting the arrival of our mass spectrometer, and Greg already has a backlog of samples to work with once the equipment is in place. We are grateful to Jack and Nancy Batts of Grand Rapids for endowing this laboratory through a Charitable Remainder Trust, and also to the Wege Foundation for their gift. We are pleased to announce the recruitment of David Nadziejka, our science editor. David has 22 years of experience in science editing and writing, including eight years as the lead technical editor for biology and biomedical science at Argonne National Laboratory. Since 1996, David has been the recipient of three of the four national awards given in the field of technical communication. He will be a valuable asset to our investigators as they prepare grant proposals, manuscripts, site visit reports, and other scientific documents.

I began writing this introduction during our 4th annual scientific retreat, held at a lodge in the quiet beauty of upper Michigan, where all of our scientists presented overviews of their George F. Vande Woude research and their future plans. Such a retreat is intense but extremely valuable, because it provides a forum for promoting collaborations and for testing hypotheses before a lively audience. I am pleased with the progress that the Van Andel Research Institute (VARI) has made in a very short period of time on so many fronts. Some of the highlights of the past year are quite impressive to me, and I hope you will find them to be also. Each year we select a member of the scientific community to receive the Daniel Nathans Memorial Award, choosing a scientist who has emulated Dr. Nathans’ extraordinary contributions and special character. Our most recent recipient was Dr. Francis Collins, who received the award for leading this nation’s effort in determining the sequence of the human genome. His two lectures in Grand Rapids on October 2, 2001—one to the scientific/medical community and one to the general public—were exciting and visionary (see page 65). We are delighted to have recruited Eric Xu, who came to us from GlaxoSmithKline to head our Laboratory of Structural Sciences. Eric is a superb crystallographer whose work has focused on the crystal structures of nuclear hormone receptors; at our Institute, he will continue studying the structural basis for protein/DNA interactions, protein/ligand interactions, and multiprotein complexes. Our success in attracting Eric to VARI was leveraged by funding from the Michigan Life Sciences Corridor (MLSC) for the Michigan Center for Structural Biology (MCSB), which will build and operate a state-ofthe-art synchrotron beamline at Argonne National Laboratory. This technology has revolutionized our ability to solve the structure of macromolecules, proteins, and nucleic acids, and

We have continued to expand our archive tissue repository and to establish tissue acquisition agreements with area hospitals. We have established agreements with Spectrum Health (Grand Rapids), Pennock Health Services and Hastings Surgeons P.C. (Hastings), Hackley Hospital (Muskegon), Ferguson Clinic (formerly Ferguson Hospital; Grand Rapids), and Holland Community Hospital (Holland). We are working on agreements with St. Mary’s Mercy Medical Center (Grand Rapids), and Metropolitan Hospital (Grand Rapids). Jim Resau, Rick Hay, Craig Webb, Bin Teh, and Brian Haab have worked out the methodology and conditions for collecting, storing, and processing specimens for gene expression profiling (microarray analysis) 3

grant are myself, Rick Hay, James Resau, and Brian Cao. VARI will also receive funding for the core services it provides as part of the Core Technology Alliance (CTA; comprising VARI, the University of Michigan, Michigan State University, and Wayne State University). The CTA is important to Michiganâ&#x20AC;&#x2122;s research efforts in that it provides cutting-edge biomedical technology services in proteomics, genomics, bioinformatics, structural biology, and animal models to all scientists in the state. Given sufficient time, this will propel Michigan into a leadership role in biotechnology. Pam Swiatek from our Institute has been a leader in helping to establish the CTA cores and is director of the Animal Models Consortium in Michigan.

and proteomics analyses. These investigators, together with Kyle Furge and Bryon Campbellâ&#x20AC;&#x2122;s team, generated the bioinformatics software of relational databases needed for processing, comparing, and annotating the clinical and molecular information. Collectively, we are establishing a network for translation of this research into clinical application. Our collaborations with surgeons, oncologists, and pathologists in a community hospital setting are unique and will become a powerful asset to Western Michigan. We have upgraded our imaging facility with the purchase of a multiphoton microscope. This microscope has a special detector that will enable us to see fluorescently labeled cells deep within living tissues without introducing the damage usually associated with ultraviolet light. This will enable us to not only see cells and tissues, but to follow their biology over time (as they grow, migrate, and develop) and to quantify the changes. We also are developing a state-of-theart Acusome ultrasound facility for in vivo imaging of mouse development, as well as of tumor growth and metastases using molecular markers and newly developed image-contrast reagents. Bart Williams led an effort to purchase an instrument that allows us to quickly determine bone mineral density and body fat content in living mice. This machine, obtained from GE Lunar, will be valuable in examining mutant mice that are susceptible to osteoporosis and diabetes, as well as in determining how mouse models of human cancer respond to various treatments.

With Drs. Brian Ross and Alnawaz Rehemtulla at the University of Michigan, we have received an In Vivo Cellular Molecular Imaging Center (ICMIC) P50 grant for in vivo imaging of oncogene activities and metastases. This grant was awarded by the National Cancer Institute (NCI). VARI investigators on the grant are myself, Ilan and Galia Tsarfaty, Pam Swiatek, Bryn Eagleson, and Jim Resau. Ilan, a cancer biologist, and Galia, a radiologist, are visiting scientists from Tel Aviv University and the Chaim Sheba Medical Center, respectively; they have a major interest in imaging and were key to our success in obtaining this grant. We are making a significant commitment to the molecular imaging of cancer cells and tumors in animal models as a means to create a knowledge bridge between basic science and the clinical diagnosis and therapy of human pathological states.

In June 2002, the Michigan Economic Development Corporation announced plans to fund 18 of the 111 full proposals submitted for the FY2002 competition. These projects, designed to advance the research and commercialization of cutting-edge life sciences products, represent a $45 million commitment from the MLSC Fund. A multi-institutional collaboration to develop new approaches for imaging and treating prostate cancer, using the molecule Met as a diagnostic and therapeutic target, was funded by the MLSC for a three-year project. This effort involves three institutions within Michigan (VARI, MSU, and the Veterans Affairs Health Care System in Ann Arbor) and two in Washington State (Fred Hutchinson Cancer Research Center and the Gerald P. Murphy Cancer Foundation). VARI investigators on the

Craig Webb and Brian Haab have begun a collaborative research project with Dr. Anthony Schaeffer, chairman of the Department of Urology at Northwestern University. They will examine, by gene expression analysis and proteomics, prostatic fluids and tissue specimens from patients with various diseases of the prostate, including prostatitis, benign prostate hyperplasia, and prostate carcinoma. They will perform gene and protein profiling on these samples for different pathological states to identify useful clinical markers. Further, Brian Haab has established a collaboration with Dr. Jose Costa, director of anatomical pathology of the Yale School of Medicine, to identify markers in pancreatic cancer; the work is funded through NCIâ&#x20AC;&#x2122;s Early Detection Research Network (EDRN). 4

The VIII International Workshop on Multiple Endocrine Neoplasia (MEN) was organized by Bin Teh and held at VARI on June 16–18, 2002. The meeting was well attended, with over 180 researchers representing 18 countries (see page 85). VARI will host the Cancer Intervention 2002 meeting in Grand Rapids on October 2–6, 2002, at which we will explore progress on the fronts of cancer diagnosis, treatment, and prevention with the leading investigators in cancer intervention strategies. We are excited about bringing this cutting-edge science to the Grand Rapids medical community.

In a first for VARI and the Grand Rapids community, Han-mo Koo helped develop a clinical trial for treating pancreatic cancer, which stems from work we began at NCI. This clinical trial is headed by Dr. Marianne Lange, together with Drs. Tim O’Rourke and Alan Campbell, through the Grand Rapids Clinical Oncology Program (GRCOP). The trial is managed by the GRCOP under the direction of Connie Szczepanek. This trial is unique in that it is the first Phase II trial to be developed locally and is locally managed through the GRCOP as a consortium. The members of the consortium are Battle Creek Health System (Battle Creek), Hackley Hospital (Muskegon), Holland Community Hospital (Holland), Metropolitan Hospital (Grand Rapids), Munson Medical Center (Traverse City), Spectrum Health (Grand Rapids), St. Mary’s Mercy Medical Center (Grand Rapids), and Mecosta County General Hospital (Big Rapids).

This year we have implemented a memorandum of understanding with Michigan State University (MSU) that establishes a cooperative relationship to enhance graduate recruitment, education, and research training opportunities for selected students in doctoral programs in one or more of the biomedical and life sciences programs at MSU. Sara Courtneidge, Cindy Miranti, and Michael Weinreich have been appointed adjunct professors at MSU, and we anticipate graduate students from MSU training in VARI laboratories.

One of the most important and dramatic changes occurring in cancer diagnosis and treatment is that we are moving from phenomenological approaches to molecular-based medicine. This will revolutionize the detection and treatment of cancer and, for that matter, of all pathological states, because visualized changes will be converted into quantifiable measurements. For example, Bin Teh’s laboratory—using gene microchips generated at VARI by Brian Haab’s lab—has discovered, in retrospective studies, alterations in the expression of specific genes in kidney cancer specimens that correlate with poor prognosis. The identification of these genes and their correlation with the aggressiveness of the disease at the time of diagnosis is helpful in identifying new targets for drug development that may lead to improved therapies for this group of patients. Using this technology, and in collaboration with urologists and pathologists from hospitals in Grand Rapids, the University of Chicago, and the University of Tokushima (Japan), Teh’s lab has molecular evidence that kidney cancers can be divided into five classes based on gene expression profiles. Similarly, together with members of the DeVos Children’s Hospital of Grand Rapids, they have identified gene expression profiles that correlate with Wilms’ tumors, a rare childhood kidney cancer that is refractory to treatment. Bin Teh’s lab has also collaborated with Drs. Lawrence Einhorn and Richard Foster from Indiana University, looking into testicular cancer that is refractory to chemotherapy.

The visual documentation of science sometimes produces images that are not only scientifically important, but aesthetically pleasing as works of art. One example is the photograph on the back cover of this report, contributed by Art Alberts, that shows the effects of a gene that negatively regulates cell motility. I hope you enjoy the other examples of nature’s artwork displayed throughout this report. In conclusion, I wish to express my appreciation and gratitude to all who have helped get us this far in a few short years. We are especially indebted to Jay Van Andel and his family and to all of our benefactors. Our community has many individuals who believe in what we are doing and have generously contributed to our research through the Hope on the Hill Foundation. We are very grateful for all the donations from organizations, individuals, and employees, as well as for the proceeds from fundraising events. It is very moving and encouraging that so many are partnering with us to build a center of excellence for world-class scientists and worldclass medical science. This support, both moral and financial, will enhance our ability to develop new knowledge and make new discoveries about cancer that will lead to better diagnostic and therapeutic strategies for treating this dreaded disease. 5

Van Andel Research Institute Laboratory Reports

An mDia2 mutant that disrupts the actin cytoskeleton This photo shows the effects of the expression of an altered form of the Diaphanous-related formin (DRF) mDia2, a protein that controls cytoskeletal remodeling. The mutation, an alanine substitution at methionine 1041, disrupts the ability of mDia2 to regulate itself. mDia2 then assumes the â&#x20AC;&#x153;openâ&#x20AC;? conformation and cannot regulate the formation of actin fibers. Red marks the actin fiber network, green marks the expressed protein. This work shows that proper autoregulation of DRF proteins is crucial for normal function. Similar mutations have arisen in human DRF proteins that have led to inherited deafness, infertility, and defects in cell growth. (Wallar and Alberts)

Laboratory of Cell Structure and Signal Integration Arthur S. Alberts, Ph.D. Dr. Alberts received his Ph.D. in physiology and pharmacology at the University of California, San Diego, in 1993, where he studied with James Feramisco. From 1994 to 1997, he served as a Postdoctoral Fellow in Richard Treisman’s laboratory at the Imperial Cancer Research Fund in London, England. From 1997 through 1999, he was an Assistant Research Biochemist in the laboratory of Frank McCormick at the Cancer Research Institute, University of California, San Francisco. Dr. Alberts joined VARI as a Scientific Investigator in January 2000. Laboratory Members Staff Jun Peng, M.D. Stephen Matheson, Ph.D. Brad Wallar, Ph.D. Akiko Vankirk, M.S.

Students Nicole Neuman Dare Odomosu

Research Projects switch proteins: they are “on” when bound to the chemical GTP and “off” after they convert GTP to GDP. This on/off cycle is regulated by guanine-nucleotide exchange factors, or Rho GEFs.

ormal cells base growth decisions on the sum of positive and negative inputs derived from extracellular cues. These signals are processed by biochemical networks composed of thousands of interacting proteins and small chemicals that shuttle information from one to the other. If the system becomes unbalanced— due to the presence of viral factors or DNA damage, for example—the cells will arrest and/or undergo a form of programmed cell suicide (apoptosis) in order to protect surrounding cells or tissues. In some cases, the protection system is overridden and damaged cells continue to live. As an afflicted cell loses control and continues to divide unchecked, it may incur further mutations that lead to tumor formation and disease.

Rho GEFs are positive activators of the Rho proteins, inducing the Rho proteins to bind GTP. Once GTP is bound, Rho proteins can bind to target factors. These targets can act as Rho effectors and directly participate in signaling. Alternatively, the Rho proteins target other “switch” proteins that are part of a signaling network. The Rho proteins are from the same family of GTP-binding proteins as Ras. Ras is an oncogene mutated in many tumors. Mutant Ras protein is locked in a GTP-bound active state, but unlike Ras, similarly active GTP-bound Rho mutants are unable to transform cells. Though nontransforming, the Rho proteins are required for Ras transformation. The role that Rho proteins play in transformation is unclear, but recent work suggests that they may indirectly regulate the cell cycle. They do, however, have an important role in cancer metastases by regulating cell shape and mobility during the invasion of surrounding tissues. Oncogenic mutant Rho GEFs transform cells by essentially force-feeding GTP to Rho proteins. But unlike mutant Rho proteins locked with GTP, Rho GEFs allow cycling between GTP and GDP bound states. This cycling process appears to be key to their ability to transform cells. We are investigating this in more detail by comparing signals generated by activated Rho proteins and the oncogenic Rho

Our lab is interested in the intracellular signaling networks that regulate proliferation, cell shape, and motility, as well as how cells become hijacked by disease. Our approaches depend upon a combination of molecular and cell biological techniques to define signal transduction and transformation mechanisms. In particular, we focus on the biology of the single cell and its instantaneous response to growth factor stimulation. Understanding these mechanisms is crucial in the development of anticancer treatments, because each step in a pathway may eventually be exploited for drug and gene therapy targets. The Rho family of GTP-binding proteins are signaling factors involved in cell growth responses, including changes in gene expression, cell shape, and motility. They act as molecular 9

tion regulated during the cell cycle or in directed cell migration? And, does Rho binding affect other DRF-binding partners activity or subcellular targeting? We are also testing the hypothesis that the DRF mDia2 binds to Src near its GTPase binding domain and that this might activate downstream signaling by disrupting autoinhibition. We are addressing these questions through a variety of methods that include digital timelapse microscopy and targeted gene disruption of the murine DRF family members in collaboration with the VARIâ&#x20AC;&#x2122;s Laboratory of Germline Modification headed by Pam Swiatek.

GEFs. On a molecular level, we are analyzing the regulation of Rho GEFs by phosphorylation, by transcriptional regulation, and by direct binding to viral and cellular proteins. The Diaphanous-related formins (DRFs) bind to activated Rho proteins. These molecular scaffolds bridge multiple growth factorâ&#x20AC;&#x201C;regulated signaling proteins and regulators of the cytoskeleton. One of these is Src tyrosine kinase. Src is a protooncogene whose expression is amplified in most breast tumors and whose function is critical for the growth of breast cancer cells. We have found that the DRFs bridge Src and Rho proteins in growth factor signaling. Our observation establishes an important link between two parallel signaling networks that control proliferation in response to growth factor stimulation.

Molecular regulation of the Diaphanous-related formins Src WISH profilin IRSp53

Rho-GDP

The DRFs are controlled by intramolecular autoinhibition, as illustrated in Figure 1. The GTPase binding domain (GBD) associates with the carboxy-terminal DAD (Dia-autoregulatory domain). This interaction is disrupted by GTPbound Rho. We are characterizing subsequent molecular events that occur as a result of Rho binding by posing the following questions: Where does DRF activation occur in cells following growth factor stimulation? Is the interac-

Rho-GTP ON OFF

GBD

FH3 FH1

activation and targeting? Src?

actin nucleation

Figure 1. Molecular regulation of the Diaphanous-related formins

External Collaborators Pierre Chardin, Institut de Pharmacologie du CNRS, Valbonne, France Phillipe Chavrier, Marie Curie Institut, Paris, France Jeff Frost, University of Texas, Houston Gregg Gundersen, Columbia University, New York George Prendergast, Lankenau Institute for Medical Research, Wynnewood, Pennsylvania Fred Wittinghofer, Max-Planck-Institut, Dortmund, Germany

FH2

DAD

? Actin remodeling machinery?

Publications Collins, Colin, Stanislav Volik, David Kowbel, David Ginzinger, Bauke Ylstra, Thomas Cloutier, Trevor Hawkins, Paul Predki, Christopher Martin, Meredith Wernick, Wen-Lin Kuo, Arthur Alberts, and Joe W. Gray. 2001. Comprehensive genome sequence analysis of a breast cancer amplicon. Genome Research 11(6): 1034â&#x20AC;&#x201C;1042. Palazzo, Alexander F., Hazel L. Joseph, Ying-Jiun Chen, Denis L. Dujardin, Arthur S. Alberts, K. Kevin Pfister, Richard B. Vallee, and Gregg G. Gundersen. 2001. Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization. Current Biology 11(19): 1536â&#x20AC;&#x201C;1541.

From left to right, back row: Waller, Alberts, Matheson front row: Vankirk, Peng, Odomosu, Newman

Antibody Technology Laboratory Brian Cao, M.D. Dr. Cao obtained his M.D. from Beijing Medical University, People’s Republic of China, in 1986. On receiving a CDC Fellowship Award, he was a Visiting Scientist at the National Center for Infectious Diseases, Centers for Disease Control and Prevention (1991–1994). He next served as a Postdoctoral Fellow at Harvard (1994–1995) and Yale (1995–1996). From 1996 to 1999, Dr. Cao was a Scientist Associate in charge of the Monoclonal Antibody Production Laboratory at the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland. Dr. Cao joined VARI as a Special Program Investigator in June 1999. Laboratory Members Staff Huiying Zhang, Ph.D. Ping Zhao, M.S. Jessica Kalbfleisch, B.S.

Students Jennifer Edgar, B.S. Josie Clowney Paul Veldhouse

Research Projects Brian Haab’s microarray technology core facility and James Resau’s cellular/molecular imaging core facility at VARI, we are developing xenograft animal models for several human cancers, including glioblastoma and soft-tissue sarcoma, in order to understand the correlation between key growth factors (VEGF, HGF/SF, EGF, etc.) and their receptors, which stimulate tumor angiogenesis and metastasis. Moreover, we seek to evaluate the effect of mAbs on these growth factors or receptors, individually or in combination, for potential clinical immunotherapeutic application. In collaboration with the University of Michigan, we are currently using our antibodies to HGF/SF and Met for clinical nuclear imaging diagnostic applications. We are in the process of characterizing several radiolabeled mAbs that have high affinity to this ligand-receptor in animal models bearing human tumors. In addition, we are using several combined polyclonal and monoclonal antibodies to analyze quantitatively the expression levels of various growth factors and their receptors in human cancer tissues and the levels of their normal counterparts from clinical specimens. These experiments may identify potential diagnostic and prognostic indicators for these diseases.

he antibody technology facility produces, purifies, and characterizes monoclonal and polyclonal antibodies. Our laboratory works with investigators on a variety of research projects, including the identification and characterization of novel proteins; affinity purification for structural analysis; development of clinical immunodiagnostic methods and kits; and engineering and humanizing monoclonal antibodies that have potential application for clinical diagnosis, prognosis, and immunotherapy in cancers and infectious diseases.

Hepatocyte growth factor/scatter factor (HGF/SF)-Met, a ligand-receptor pair, plays important roles in tumorigenesis, angiogenesis, and metastasis. Tumors with an autocrine loop of HGF/SF-Met are highly malignant and have a poor prognosis. We have generated a panel of monoclonal antibodies (mAbs) to the ligand and found some of them with biologic neutralizing activity when they are used in combination. We have characterized their antitumor effect both in vitro and in vivo. A panel of mAbs raised against the Met receptor extracellular domain has been generated; two of these mAbs are under further characterization for clinical imaging/diagnostic application. Both anti-ligand and receptor mAbs have been patented. Angiogenesis contributes significantly to the progression of cancer and, as tumors grow, they begin to produce a wider array of angiogenesis molecules. In collaboration with

Anthrax is a zoonotic disease transmissible from animal to man that is caused by the Grampositive, spore-forming bacterium Bacillus

anthracis. The three proteins of the exotoxin secreted by the organism are protective antigen (PA), lethal factor (LF), and edema factor (EF). The PA63 fragment forms a heptameric complex on the cell surface that is capable of binding with the 90 kDa LF protein to form lethal toxin (LeTx). It is known that macrophages are particularly sensitive to LF: at low concentrations, LF stimulates TNF-Îą and IL-1Î˛; at high concentrations, LF causes the death of macrophages and the release of cytokines into the bloodstream. The symptoms of systemic anthrax are inducible by injection of LeTx alone in animal models; it is likely that death is caused by cytokine-induced shock. Because of its rarity, anthrax is not often included in differential diagnosis; in cases of inhalational anthrax, the diagnosis is rarely made until the patient is moribund. Antibiotic treatment should be initiated at the earliest stage of infection. By the time characteristic symptoms appear, the bacteria are already multiplying rapidly in the bloodstream and have produced massive amounts of toxin. Killing the bacteria cannot eliminate the toxin, and its effects result in death of the host despite antibiotic treatment at this point. Therefore, we hypothesize that a high-affinity neutralizing monoclonal antibody to LF would be a potentially useful reagent for the treatment of anthrax infection, in combination with the use of antibiotics.

mAb in vivo on a variety of animals models (including mouse, rat, and rabbit) to observe if it neutralizes LF in vivo and protects the animal from death caused by challenges with either purified LeTx or live bacteria. Meanwhile, we have also epitope-mapped this mAb using the phagedisplay technique, and a few synthetic small peptides based on the epitope mapping information are being tested to determine if they could be LF antagonists with biological functions similar to those of LF-neutralizing mAbs. Our overall goals in this project are to characterize the in vivo neutralizing activities of anti-LF mAbs and/or synthetic peptides as LF antagonists for their suitability as passive protection for animals, including their potential clinical application to treat anthrax infection.

In collaboration with the laboratories of Nicholas Duesbery and Han-Mo Koo at VARI, we have developed two panels of mAbs against protective antigen and lethal factor. Our major goal of producing these mAbs is to find those that have properties of specifically blocking the biological functions of LF and to further evaluate those antibodies as potential agents for the treatment of anthrax infection. We have characterized their biological neutralization properties to PA and LF by in vitro bioassays and found a particular anti-LF mAb that has strong neutralizing activity to LF: at a low molar ratio to LF, it shifts the effective concentration (EC50) over 500-fold in the macrophage cell line J774A.1 (Figure 1). Our results also showed that this antibody specifically recognizes the binding site of LF to PA and blocks their binding, preventing the formation of lethal toxin (Figure 2). We will further test this

Figure 1. Macrophage cell survival

Figure 2. Gel shifting assay. Lane 1: molecular weight marker 66 kDa; lane 2: PA63 alone; lane 3: PA63 plus anti-LF mAb; lane 4: LF alone; lane 5: LF plus mAb; lane 6: PA63 plus LF; lane 7: PA63, LF, and mAb.

External Collaborators Lonson Barr, Michigan State University, Lansing Milton Gross, Department of Veterans Affairs Medical Center – University of Michigan Medical Center, Ann Arbor Yi Ren, Royal Mary Hospital, Hong Kong University Wei-cheng You, Beijing Institute for Cancer Research, People’s Republic of China Dong-zheng Yu, Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Beijing, People’s Republic of China Publications Hay, Rick V., Brian Cao, R. Scot Skinner, Ling-Mei Wang, Yanli Su, James H. Resau, George F. Vande Woude, and Milton Gross. 2002. Radioimmunoscintigraphy of tumors autocrine for human Met and hepatocyte growth factor/scatter factor. Molecular Imaging 1(1): 56–62. Qian, Chao-Nan, Xiang Guo, Brian Cao, Eric J. Kort, Chong-Chou Lee, Jindong Chen, Ling-Mei Wang, Wei-Yuan Mai, Hua-Qing Min, Ming-Huang Hong, George F. Vande Woude, James H. Resau, and Bin T. Teh. 2002. Met protein expression level correlates with survival in patients with late-stage nasopharyngeal carcinoma. Cancer Research 62(2): 589–596.

From left to right: Zhao, Edgar, Kalbfleisch, Cao

Immunostaining of human hepatocyte growth factor/scatter factor (HGF/SF) Formalin-fixed S-114 cells (NIH 3T3 cells transformed with human HGF/SF and its receptor Met) are stained with a rabbit anti-HGF/SF polyclonal antibody with rhodamine conjugate (red) and a mouse mAb (7-2) with FITC conjugate (green). Both antibodies were produced by the Antibody Technology Lab at VARI. The black and white is a Nomarski-DIC (differential interference contrast) image of the cellular detail. The yellow image indicates co-localized staining of the green monoclonal and red polyclonal antibodies. (Hudson, Zhao, Resau, and Cao)

A) Clustering of 15 Wilms tumors and clinical information; B) Three-dimensional clustering The clustering of patients (using Pearsonâ&#x20AC;&#x2122;s correlation) is based on global gene expression profiles consisting of median polished data of 5,594 well-measured spots. The tumors approximately clustered into two main groups, with one group consisting of mostly tumors of high stage (stages III and IV) and the other consisting of mostly tumors of low stage (stages I and II). Two patients with high-stage tumors who died of cancer (Wilms 6, 15) and one patient who had recurrence (Wilms 11) were closely clustered together. One patient (Wilms 14), represented by the orange circle in panel B, who had stage II disease but developed recurrence and died of cancer, was clustered with high-stage tumors. (Takahashi and Teh) 16

Mass Spectrometry and Proteomics Laboratory Gregory S. Cavey, B.S. Mr. Cavey received his B.S. degree from Michigan State University in 1990. Prior to joining VARI, he was employed at Pharmacia in Kalamazoo, Michigan, for nearly 15 years. As part of a biotechnology development unit, he was a group leader for a protein characterization core laboratory. More recently as a research scientist in discovery research, Greg was principal in the establishment and applications of a state-of-the-art proteomics laboratory for drug discovery. He joined VARI as a Special Program Investigator in July 2002.

Research Projects niques. Once the proteins are displayed on 2D gels, quantitative data can be used to identify proteins of interest, followed by automated identification using mass spectrometry and database searching. Forthcoming will be the use of an isotopecoded affinity tagging (ICAT) approach that will allow quantitative measurement of proteins in control vs. experimental samples. This approach will require the set-up and use of a multidimensional liquid chromatography system for the separation of complex mixtures of proteins.

roteomics is fast becoming a major effort in most research institutions. The rapid development of technology is providing powerful new tools for probing the function of proteins and for extracting information from the Human Genome project. Since nearly all drug targets are proteins, there is clear incentive to apply proteomics as a complementary approach to genomics research. For proteomics, the most important technology is the coupling of quantitative analytical protein separation and modern mass spectrometers to identify and characterize proteins with unprecedented sensitivity and throughput. This laboratory will collaborate with VARI investigators in applying current proteomics technology toward their research goals and will build external collaborations to speed the development of new tools to meet the many challenges in cancer research.

Cell-mapping proteomics will be used to identify components of protein complexes under various conditions in order to help understand the regulatory mechanism(s) of a given pathway. In this approach, a nondenatured sample is affinity-purified using either antibodies, a known protein carrying an affinity tag, or immobilized small molecules. Binding partners are separated by 2D or SDS polyacrylamide gel electrophoresis (PAGE) and are identified using mass spectrometry and database searching.

Expression proteomics and cell-mapping proteomics will be pursued, as will characterization of posttranslational modifications such as phosphorylation. For expression proteomics, we will initially rely on two-dimensional (2D) gel electrophoresis to display differentially expressed proteins of a given disease state, genetic manipulation, or drug treatment. This may require implementing an array of sample isolation, solubilization, and fractionation tech-

Posttranslational modification of proteinsâ&#x20AC;&#x201D;in particular phosphorylationâ&#x20AC;&#x201D;is known to be a regulatory event in signal transduction. The proteomics lab will work with various investigators to map phosphorylation sites of proteins. The unique talents, research, and resources at VARI provide numerous opportunities for proteomics applications.

Laboratory of Signal Regulation and Cancer Sara A. Courtneidge, Ph.D. Dr. Courtneidge completed her Ph.D. at the National Institute for Medical Research in London. She began her career in the basic sciences in 1978 as a Postdoctoral Fellow in the laboratory of J. Michael Bishop at the University of California School of Medicine. She later joined her alma mater as a member of the scientific staff. In 1985 Dr. Courtneidge joined the European Molecular Biology Laboratory as Group Leader and in 1991 was appointed Senior Scientist with tenure. She joined Sugen in 1994 as Vice President of Research, later becoming Senior Vice President of Research and then Chief Scientist. Dr. Courtneidge was appointed Senior Scientific Investigator and Deputy Director of the Van Andel Research Institute in January 2001. Laboratory Members Staff Eduardo Azucena, Ph.D. Hasan Korkaya, Ph.D. Darren Seals, Ph.D. Rebecca Uzarski, Ph.D. Rebecca Cruz, M.S. Daniel Salinsky, M.S.

Students Erik Freiter, B.S. Lisa Maurer Therese Roth

Research Projects ur lab wants to understand at the molecular level how proliferation is controlled in normal cells and by what mechanisms these controls are subverted in tumor cells. We largely focus on a family of oncogenic tyrosine kinases, the Src family. The prototype of the family, vSrc, originally discovered as the transforming protein of Rous sarcoma virus, is a mutated and activated version of a normal cellular gene product, cSrc. The activity of all members of the Src family is normally under strict control; however, the enzymes are frequently activated or overexpressed, or both, in human tumors. In normal cells, Src family kinases have been implicated in signaling from many types of receptors, including receptor tyrosine kinases, as well as integrin receptors and G protein-coupled receptors. Signals generated by Src family kinases are thought to play a role in cell cycle entry, cytoskeletal rearrangements, cell migration, and cell division. In tumor cells, Src may play a role in growth factorâ&#x20AC;&#x201C;independent proliferation or in invasiveness. In addition, some evidence points to a role for Src family kinases in angiogenesis. Some of the current projects in the laboratory are outlined below.

photyrosine antibodies to screen tyrosine-phosphorylated cDNA expression libraries. Several potential Src substrates were identified, including Fish, which has five SH3 domains and a phox

Novel Src substrates

Src-transformed cells were stained to visualize Fish (green) and F-actin (red). The podosomes are visible as rings of intense F-actin staining. Much of Fish is also present in these podosomes.

We recently described a method for identifying tyrosine kinase substrates by using anti-phos18

The connection between Src and p53

homology (PX) domain. Fish is tyrosine phosphorylated in Src-transformed fibroblasts (suggesting that it is a target of Src in vivo) and in normal cells after treatment with several growth factors.

The tumor suppressor p53 is present at low levels in growing cells. Many DNA tumor viruses encode proteins that inactivate p53 by direct association or ubiquitination-mediated degradation, presumably to facilitate the entry of cells into cycle and therefore viral replication. We have recently shown that the product of one such DNA tumor virus, the SV40 large T antigen, bypasses the requirements for several signals emanating from growth factor receptors. In particular, cells that lack p53, or in which p53 has been inactivated by T antigen binding, no longer require Src family kinases for growth factor signaling. These findings suggest that Src kinases are required to overcome the inhibitory effects of p53. There are perhaps also implications for the use of signal transduction inhibitors in human cancers where negative regulators such as p53 are frequently mutated or absent. We are now investigating in more detail the way in which Src kinases impact p53 function.

We have recently found that in Src-transformed cells, Fish is localized to specialized regions of the plasma membrane called invadopodia or podosomes. These actin-rich protrusions from the plasma membrane are sites of matrix invasion and locomotion. We have also determined that the PX domain of Fish associates with phosphatidylinositol 3-phosphate and that this domain targets Fish to the podosomes. Furthermore, the fifth SH3 domain of Fish mediates its association with members of the ADAMs family of membrane metalloproteases, which in Src-transformed cells are also localized to podosomes. Our current research aims to probe the role of Fish in invasion in both Src-transformed cells and in human tumor cell lines. Other novel proteins identified in the substrate screen have also been shown to be tyrosine phosphorylated in Src-transformed cells and are being characterized.

Breast cancer Increased Src activity can be demonstrated in the majority of breast cancers, both estrogendependent and estrogen-independent, yet the role of Src in breast tumorigenesis has not been established. We have begun to characterize the role of Src in both EGF-stimulated and estrogen-stimulated signal transduction pathways in breast cancer cell lines.

Using a Src-selective inhibitor to probe its role in signaling pathways The use of small molecule inhibitors to study molecular components of cellular signal transduction pathways provides a complementary means of analysis to techniques such as antisense, dominant negative (interfering) mutants and constitutively activated mutants. We have recently identified and characterized a small molecule inhibitor, SU6656, which exhibits selectivity for Src and other members of the Src family. The use of SU6656 confirmed our previous findings that Src family kinases are required for both Myc induction and DNA synthesis in response to PDGF stimulation of NIH 3T3 fibroblasts. We are currently comparing both PDGF-stimulated gene expression and tyrosine phosphorylation events in untreated and SU6656treated cells to define which events require Src family kinases. SU6656 should prove a useful tool for further dissecting the role of Src kinases in this and other signal transduction pathways.

Novel members of the Src family We are particularly interested in the human kinase Frk. This kinase has a domain structure typical of Src family kinases and is probably regulated in a similar manner. However Frk lacks the amino-terminal myristylation sequences and instead has a nuclear localization sequence in its SH2 domain. Interestingly, Frk is predominantly expressed in epithelial cells and is overexpressed in a high proportion of human tumors and tumor cells lines, particularly those deriving from lung. We have begun an extensive characterization of Frk, including its substrate specificity, regulation, transforming ability, and function.

Publications Courtneidge, S.A. 2002. The role of Src in signal transduction pathways. Biochemical Society Transactions 30(2): 11â&#x20AC;&#x201C;17.

From left to right, back row: Cruz, Mauer, Courtneidge middle row: Korkaya, Freiter, Seals front row: Salinsky, Azucena, Uzarski, Roth

Developmental Cell Biology Laboratory Nicholas S. Duesbery, Ph.D. Dr. Duesbery received both his M.S. (1990) and Ph.D. (1996) degrees in zoology from the University of Toronto, Canada. Before his appointment at VARI, he served as a Postdoctoral Fellow in the laboratory of George Vande Woude in the Molecular Oncology Section of the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland (1996–1999). Dr. Duesbery joined VARI as a Scientific Investigator in April 1999. Laboratory Members Staff Xudong Liang, M.D. Arun Prasad Chopra, Ph.D. Sherri Boone, B.S.

Visiting Scientist Jean-François Bodart, Ph.D.

Students Jonathon Douglas Marie Graves Jeanine Myles

Research Projects in the presence of cycloheximide. The preexistence of cyclin B2 in immature oocytes may explain why amplification of MPF is observed even in the absence of protein synthesis. It has been proposed that X. laevis oocytes contain a precursor of MPF, called pre-MPF, that is activated by the injection of MPF. However, the mechanisms of autoamplification of MPF remain poorly understood. It has been shown that MPF can phosphorylate and activate its positive regulator Cdc25 in vitro and that this mechanism may play a role in the autoamplification of MPF. Still, even if Cdc25 phosphorylation by p34cdc2 is required for the autoamplifying activity of MPF, it is not sufficient. Numerous kinases have also been implicated in the MPF autocatalytic loop; among them, members of the polokinase family have been involved in p34cdc2 activation by phosphorylating Cdc25 and have been characterized in X. laevis.

ur research group focuses on cellular aspects of oogenesis, meiosis, and mitosis in a variety of vertebrate model organisms. Using biochemical and molecular approaches, we seek to identify regulatory mechanisms involved in egg cell formation, fertilization, and early embryonic development, as well as to ascertain the roles of these mechanisms in human health and disease. Our current understanding of meiotic maturation in amphibian oocytes is largely based on extensive analyses of cell cycle regulation in Xenopus laevis. Immature amphibian oocytes, arrested at prophase of meiosis I, resume meiosis in response to hormonal stimulation. The resumption of meiosis is followed by germinal vesicle breakdown (GVBD) and the outward appearance of a white spot. Oocytes subsequently arrest at metaphase II as mature oocytes or eggs in anticipation of fertilization. In 1971, Masui and Markert found that cytoplasm from an egg, when injected into oocytes, was capable of inducing meiotic maturation. They thus concluded that eggs contain an activity, which they called maturation promoting factor (MPF), that was sufficient to induce maturation. Subsequent purification showed that MPF is a complex of two subunits: a catalytic subunit, p34cdc2, and a regulatory subunit, cyclin B. Since that initial report, MPF has been shown to be a universal regulator of entry into meiotic and mitotic metaphase.

Mitogen-activated protein kinase (MAPK) is activated during meiotic maturation of X. laevis oocytes at the same time that maturation promoting factor is. Though it remains unclear whether MAPK is activated before MPF, interconnections exist between the MPF and MAPK pathways. MAPK kinase (MEK1) injection or constitutively active thiophosphorylated-MAPK injection into X. laevis oocytes can induce resumption of meiosis. The mechanisms by which MAPK activation induces meiotic maturation seem to involve Myt1 inhibition by p90rsk. Indeed, it has been shown that p90rsk, which is phosphorylated and activated by MAPK, is able

MPF in X. laevis oocytes possesses the ability to activate itself; injection of MPF from eggs can induce recipient oocytes to undergo GVBD

MAPK appeared not to be required for GVBD in X. tropicalis oocytes. However, maturation in the absence of MAPK activation was delayed, and meiotic spindles failed to form.

to bind to Myt1, to phosphorylate it, and thus to inactivate the protein. However, GVBD can occur even in the absence of MAPK activity, a fact that leads us to conclude that MAPK activity might not be required for meiosis I in X. laevis oocytes. Nevertheless, MAPK activity is required for reactivation of MPF and suppression of DNA replication between meiosis I and II.

Our results indicate that the biochemical regulation of oocyte maturation in both of these species is similar in most respects, with the notable exception that X. tropicalis oocytes do not mature when injected with MPF in the presence of protein synthesis inhibitors. We are currently using a comparative approach to characterize the proteins present in MPF complexes isolated from X. laevis and X. tropicalis oocytes in order to identify elements required for MPF autoamplification.

Despite the advantages of X. laevis as a model system, its amenability to genetic approaches is limited as a consequence of its pseudotetraploidy. The use of Xenopus tropicalis, a diploid member of the same genus, has been proposed as a way of circumventing this problem. In addition to its promise as a genetic model, X. tropicalis may also be useful as a comparative model to complement studies in X. laevis. Although X. tropicalis development superficially resembles that of X. laevis, it may not be assumed that this similarity holds at all levels, because these species evolutionarily diverged 30–100 million years ago.

In the course of our studies, we serendipitously identified anthrax lethal factor (LF), a component of the toxin produced by Bacillus anthracis, as a proteolytic inhibitor of the MAPK pathway. Specifically, LF was found to remove seven amino acids from the amino terminus of MAPK kinase 1 (MEK1), the loss of which resulted in its inactivation. Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin on tumor cells. LF was very effective in inhibiting MAPK activation in V12 H-ras–transformed NIH 3T3 cells. Treatment of transformed cells in vitro with LF caused them to revert to a nontransformed morphology and also inhibited their ability to form colonies in soft agar and to invade Matrigel, without markedly affecting cell proliferation. In vivo, LF inhibited the growth of ras-transformed cells implanted in athymic nude mice—in some cases causing tumor regression—at concentrations that produced no apparent animal toxicity. Unexpectedly, LF also greatly decreased tumor neovascularization. These results demonstrate that LF potently inhibits ras-mediated tumor growth and is a novel, potential antitumor therapeutic.

Consequently, we have compared the biochemical regulation of oocyte maturation in the two species, focusing on the regulation of MPF activation and MAPK activation in X. tropicalis. The time required for progesterone-induced maturation of X. tropicalis oocytes was shorter (GVBD50 = 148.8 ± 44 min) than that of X. laevis oocytes. The maturation of X. tropicalis oocytes was marked by the appearance of a white dot and then the formation of a dark ring coincident, respectively, with entry into meiosis I and the onset of anaphase I. As with X. laevis, X. tropicalis maturation required protein synthesis but not transcription. The activity of MPF during maturation first peaked at 0.67 GVBD50, transiently declined, and remained stable thereafter. Crude lysates and cytoplasmic extracts of mature X. tropicalis oocytes could induce immature oocytes to mature. X. tropicalis oocytes, however, appeared to lack stores of pre-MPF, because these extracts could not induce GVBD in the presence of protein synthesis inhibitors. MAPK activity increased in parallel with that of MPF but remained elevated after the first meiotic division. Whereas injection of constitutively active MEK2 triggered GVBD,

Current research efforts in our lab are designed to characterize the protein regions necessary for LF-MEK interaction, to identify regions of LF that are important for cleaving MEK, and to determine how this cleavage results in inactivation of MEK. Such information may allow us to develop drugs that would interfere with this interaction and ultimately block anthrax toxin activity.

External Collaborators Jiahuai Han, Scripps Research Institute, San Diego, California Stephen Leppla, National Institute of Dental and Craniofacial Research, Bethesda, Maryland Robert Liddington, Burnham Institute, La Jolla, California Angel Nebreda, European Molecular Biology Laboratory, Heidelberg, Germany David Waugh, National Cancer Institute, Bethesda, Maryland Publications Bodart, Jean-François, Arun P. Chopra, Xudong Liang, and Nicholas S. Duesbery. 2002. Anthrax, MEK, and cancer. Cell Cycle 1(1): 10–15. Bodart, Jean-François, Davina V. Gutierrez, James H. Resau, Bree D. Buckner, Angel R. Nebreda, and Nicholas S. Duesbery. 2002. Characterization of MPF and MAPK activities during meiotic maturation of Xenopus tropicalis oocytes. Developmental Biology 245: 348–361. Koo, Han-Mo, Nicholas S. Duesbery, and George F. Vande Woude. 2002. Anthrax toxins, mitogenactivated protein kinase pathway, and melanoma treatment. Directions in Science 1: 123–126. Koo, Han-Mo, Matt VanBrocklin, Mary Jane McWilliams, Stephan H. Leppla, Nicholas S. Duesbery, and George F. Vande Woude. 2002. Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase. Proceedings of the National Academy of Sciences USA 99(5): 3052–3057.

From left to right: Chopra, Bodart, Liang, Boone, Douglas, Duesbery

Bioinformatics Core Program Kyle A. Furge, Ph.D. Dr. Furge received his Ph.D. in biochemistry from the Vanderbilt University School of Medicine in 2000. Prior to obtaining his degree, he worked as a software engineer at YSI, Inc., where he wrote operating systems for embedded computer devices. He did his postdoctoral work in the laboratory of George Vande Woude and became a Bioinformatics Scientist at VARI in June 2001. Laboratory Members Staff Ed Dere, B.S., B.Eng.

Student Joe Crawley

Research Projects microarray technology allows the measurement of expression levels for tens of thousands of genes in a single experiment. To help determine gene expression values that change in a significant way between two sample groups (i.e., normal tissue versus tumor tissue), we have developed several programs to perform specialized statistical analysis on microarray data sets. Of special interest is a new technology we have developed to identify tumor cell chromosomal abnormalities from gene expression microarray data. This technique organizes genes by their genome mapping location and then scans for genomic regions that contain a disproportionate number of genes that show either increased or decreased expression (Figure 1). We have termed this analysis comparative genomic microarray analysis, or CGMA, as regional gene expression biases often indicate chromosomal losses or gains. We hope to develop this technology further to allow more in-depth analysis of chromosomal changes in cancer cells and to identify candidate genes whose expression changes most in regions of frequent cytogenetic change.

s high-throughput biotechnologies such as DNA sequencing, gene expression microarray, and genotyping become more accessible to researchers, analysis of the data produced by these technologies becomes increasingly difficult. A relatively new field, termed bioinformatics, has emerged to store, distribute, integrate, and analyze this flood of biological data. Bioinformatics is a field that encompasses aspects of several disciplines, including information technology, computer science, statistics, and molecular biology/genetics. The bioinformatics program at VARI focuses on using a computational approach to understand how cancer cells differ from normal cells at the molecular level. In addition, we assist in the analysis of large and small data sets that are generated both within VARI and as part of external collaborations.

Assembled DNA sequence information for humans and mice has recently become available. To allow investigators at VARI to take advantage of this knowledge, we have downloaded the Ensembl version of the public human sequence database. In addition, we have several subscriptions to the Celera human and mouse databases. As sequence annotations are constantly being updated by the European Bioinformatics Institute, the National Center for Biological Information, and other institutes, we collect the sequence information from the various sources and summarize and distribute the results. In addition, we constantly monitor public gene sequence databases to ensure that as more gene sequences have cellular functions attributed to them, this information is available to our researchers.

Because many types of data analysis are computationally intensive, we are developing an infrastructure (as part of a collaboration) that will allow more-sophisticated computational analysis. This infrastructure, called cluster or grid computing, distributes a large computational workloads over many low-cost computers. Following completion of the analysis, a monitoring computer collects all of the data from the smaller computers and assembles the results. This type of computing is beneficial as a relatively small group of low-cost computers can efficiently process a large computational workload.

We also have active areas of research in the analysis of DNA microarray data. The DNA

Figure 1. Identification of gene expression biases on chromosome 8. A sliding window algorithm was used to scan for regional gene expression biases on chromosome 8. Regions of amplification (red) or deletion (green) can be detected by finding regions of expression biases that pass a significance threshold (in this example, the 95% confidence interval). Here, the portions of the p-arm of chromosome 8 above 95% are lost and the portions of the q-arm are gained. The 8p loss and 8q gain was confirmed by comparative genomic hybridization.

External Collaborators Greg Wolffe, Grand Valley State University, Allendale, Michigan Publications Furge, Kyle A., Ramsi Haddad, Jeremy C. Miller, J. Schoumans, Brian B. Haab, Bin T. Teh, Lonson Barr, and Craig P. Webb. In press. Genomic profiling and cDNA microarray analysis of human colon adenocarcinoma and associated peritoneal metastasis reveals consistent cytogenetic and transcriptional aberrations associated with progression of multiple metastases. Applied Genomics and Proteomics. Furge, Kyle A., David Kiewlich, Phuong Le, My Nga Vo, Michel Faure, Anthony R. Howlett, Kenneth E. Lipson, George F. Vande Woude, and Craig P. Webb. 2001. Suppression of Rasmediated tumorigenicity and metastasis through inhibition of the Met receptor tyrosine kinase. Proceedings of the National Academy of Sciences U.S.A. 98(19): 10722-10727. Haddad, Ramsi, Kyle A. Furge, Jeremy C. Miller, Brian B. Haab, J. Schoumans, B.T. Teh, L. Barr, and Craig P. Webb. In press. Genomic profiling and cDNA microarray analysis of human colon adenocarcinoma and associated intraperitoneal metastases reveals consistent cytogenetic and transcriptional aberrations associated with progression of multiple metastases. Applied Genomics and Proteomics. Rhodes, Daniel R., Jeremy C. Miller, Brian B. Haab, and Kyle A. Furge. 2002. CIT: identification of differentially expressed clusters of genes from microarray data. Bioinformatics 18(1): 205â&#x20AC;&#x201C;206.

From left to right: Crawley, Furge, Dere

Serum protein profiling and marker identification using antibody microarrays A) Scanned image of an antibody microarray, in which 48 antibodies targeting serum proteins were each spotted eight times on the array. Two serum samplesâ&#x20AC;&#x201D;a test sample and a reference sampleâ&#x20AC;&#x201D;were each labeled with one of two different-colored fluorescent dyes and incubated on the array. The array was scanned for sample-specific and reference-specific fluorescence, which reveal the relative protein binding to each antibody from the test and the reference samples. B) Two-way hierarchical clustering of microarray data from 53 serum samples (horizontal axis) and antibody measurements from four replicate experiment sets (vertical axis). Each colored square represents one antibody measurement from one array. The color and intensity of each square represents the relative protein binding from the sample versus the reference, red representing higher from the sample and green, higher from the reference. The red branches of the dendrogram indicate serum samples from prostate cancer patients, and the blue branches indicate serum samples from the controls. ELISA measurements of various serum proteins cluster tightly with the microarray measurements from the respective antibodies, showing the accuracy of the microarray measurements. C) Proteins with significantly different serum levels between the prostate cancer samples and the controls. The software program CIT calculated p-values for each antibody in the data from panel B. In the cancer patients, von Willebrand factor was higher and the other proteins were lower; all varied independently of PSA (column 3). These proteins, together with other markers or clinical indicators, may be useful in the clinical evaluation of prostate cancer.

Laboratory of DNA and Protein Microarray Technology Brian B. Haab, Ph.D. Dr. Haab obtained his Ph.D. in chemistry from the University of California at Berkeley in 1998. He then served as a Postdoctoral Fellow in the laboratory of Patrick Brown in the Department of Biochemistry at Stanford University. Dr. Haab joined VARI as a Special Program Investigator in May 2000. Laboratory Members Research laboratory Heping Zhou, Ph.D. Kerri Kaledas, B.S. Mark Schotanus, B.S.

Core facility Ramsi Haddad, Ph.D. Peterson Haak, B.S. Joshua Kwekel, B.S. Paul Norton, B.S.

Student Daniel Diephouse

Research Projects tion of very-low-level proteins. These detection methods taken together allow us to profile the wide range of protein concentrations that are present in physiological samples.

he discovery of new disease markers is particularly necessary for diseases difficult to detect or diagnose at an early, curable stage. For example, the differentiation of malignant from benign disease and the early detection of pancreatic cancer are extremely difficult with current imaging and cytological methods. An improved screening tool, such as a reliable and specific serum assay, would both avoid unnecessary surgery and allow performance of needed procedures at a curative stage. The difficulties of high-throughput protein detection and quantification make the discovery of a new disease marker challenging.

We use these methods to acquire protein profiles of serum and other fluid samples from cancer patients and controls. The antibodies are chosen to target putative markers and proteins involved in functions that are modulated by cancer, such as immune system proteins, angiogenesis proteins, acute phase reactants, growth factors, cytokines, and coagulation proteins. The patterns of protein abundances in the serum samples are compared with clinical information to achieve two goals: 1) to define sets of proteins with potential diagnostic or prognostic information and 2) to gain insight into the relationship between circulating factors and states of disease progression. In a collaborative study with Bin S. Teh of the Baylor College of Medicine, we validated accurate and specific detection of multiple serum proteins using the microarray assay, and we identified five serum proteins (von Willebrand Factor, IgM, IgG, Îą1-antichymotrypsin, and villin) that statistically differentiated prostate cancer serum samples from control serum samples (see page 26). Four of these proteins had been reported previously as associated with prostate cancer, and each has implications for the host response to the cancer. Further insight into the alterations of the secretory activity of prostatic epithelial cells is being gathered in a project with Anthony Schaeffer and John Grayhack to study the protein profiles of prostatic fluid samples. In collaboration with Paul Lizardi and Jose Costa at Yale University, we are

Antibody microarray analysis of fluids from cancer patients A new tool that is potentially well suited to meet this challenge is the protein microarray. The microarray enables highly multiplexed detection in a low-volume, rapid, and sensitive assay. A robotic arrayer prints antibodies targeting putative serum markers and cancer-related genes on derivatized glass surfaces. Serum samples are incubated on the surfaces of the arrays, and individual serum proteins bind to the surfaces through specific antibodyâ&#x20AC;&#x201C;antigen interactions. We are developing and validating a variety of methods to detect bound proteins according to the concentration range of the proteins. A promising method for high-sensitivity detection of low-abundance proteins is rolling circle amplification (RCA), which we are developing and applying in a project with Paul Lizardi and Jose Costa at Yale University. We also perform direct labeling of serum proteins with Cy3 or Cy5 to detect higher-abundance proteins, and we are developing microspot ELISA for the detec28

with a new microarray database built by the Information Technology department at VARI, provides extensive informatics capability for microarray users. The core is also involved in technology and methods development. New arrays are being produced that comprise only sequences from verified and named genes, or sequences pertaining to particular projects. Focused gene sets allow replicate spotting of each sequence and better verification of the quality of each sequence. Arrays made from sets of 70-base oligonucleotides, which may provide reduced cross-reactivity with other genes relative to cDNA clones, are being validated and characterized to complement our existing cDNA arrays. In addition, we are implementing methods of signal amplification to allow detection of low quantities of RNA.

studying the protein alterations in the sera of pancreatic cancer patients. Microarray core facility The DNA microarray is widely regarded as a revolutionary technology in biological research. Since its introduction about 10 years ago, microarrays have grown in use and usefulness and have contributed to many significant discoveries. VARI’s microarray core facility makes this technology accessible and useful to its researchers and to external collaborators. We have acquired sets of 40,000 human cDNA clones, 15,000 mouse cDNA clones, and through our collaboration with the Core Technology Alliance of the Michigan Life Sciences Corridor, 20,000 rat cDNA clones. A high-throughput liquid-handling robot is used to prepare these DNA sequences for microarrays, and a robotic arrayer spots the DNAs at high density onto the surfaces of glass slides (about 20,000 spots in 2 × 4 cm). The high-density spotting of DNA sequences allows simultaneous hybridization assays on thousands of genes. Rigorous quality control at every level of the microarray production and use has allowed us to routinely generate high-quality data, and more than 1,000 microarray experiments will have been performed at VARI in the year 2002.

Applications of DNA microarrays include the study of the mRNA expression patterns in human tumor samples and the study of changes in cell-line gene expression after perturbations. The data are analyzed to identify genes that statistically correlate with other genes, phenotypes, clinical parameters, disease states, or perturbation states. A detailed analysis of gene expression programs can yield insight into the function and interrelationship of genes and can suggest strategies of intervention in disease. Many VARI researchers, as well as external researchers, are making use of VARI’s microarray facility for such studies.

The core facility provides training in the use and analysis of microarrays and access to the latest analysis tools. Support from VARI bioinformaticians Kyle Furge and Edward Dere, along External Collaborators

Phil Andrews, Samir Hanash, and Gil Omenn, University of Michigan, Ann Arbor Jose Costa and Paul Lizardi, Yale University School of Medicine, New Haven, Connecticut Yi Ren, University of Hong Kong Anthony Schaeffer and John Grayhack, Northwestern University, Evanston, Illinois Peter Schirmacher, University of Cologne, Germany Bin S. Teh, Baylor College of Medicine, Houston, Texas Cornelius Verweij, University of Amsterdam, The Netherlands Tim Zacharewski, Michigan State University, Lansing

Publications Haddad, Ramsi, Kyle A. Furge, Jeremy C. Miller, Brian B. Haab, J. Schoumans, Bin T. Teh, L. Barr, and Craig P. Webb. In press. Genomic profiling and cDNA microarray analysis of human colon adenocarcinoma and associated intraperitoneal metastases reveals consistent cytogenetic and transcriptional aberrations associated with progression of multiple metastases. Applied Genomics and Proteomics. Miller, Jeremy C., E. Brian Butler, Bin S. Teh, and Brian B. Haab. 2001. The application of protein microarrays to serum diagnostics: prostate cancer as a test case. Disease Markers 17(4): 225–234. Miller, Jeremy C., Heping Zhou, Joshua Kwekel, Robert Cavallo, Jocelyn Burke, E. Brian Butler, Bin S. Teh, and Brian B. Haab. In press. Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers. Proteomics. Rhodes, Daniel R., Jeremy C. Miller, Brian B. Haab, and Kyle A. Furge. 2002. CIT: identification of differentially expressed clusters of genes from microarray data. Bioinformatics 18(1): 205–206. Robinson, William H., Carla DiGennaro, Wolfgang Hueber, Brian B. Haab, Makoto Kamachi, Erik J. Dean, Sylvie Fournel, Derek Fong, Mark C. Genovese, Henry E. Neuman de Vegvar, Gunter Steiner, David L. Hirschberg, Sylviane Muller, Ger J. Pruijn, Walther J. van Venrooij, Josef S. Smolen, Patrick O. Brown, Lawrence Steinman, and Paul J. Utz. 2002. Autoantigen microarrays for multiplex characterization of autoantibody responses. Nature Medicine 8(3): 295–301.

From left to right, back row: Haak, Kwekel, Vreder, Diephouse, Schotanus front row: Norton, Zhou, Kaledas, Haddad, Haab

Laboratory of Cancer Pharmacogenetics Han-Mo Koo, Ph.D. Dr. Koo received his Ph.D. in microbiology and molecular genetics at Rutgers–The State University of New Jersey in 1993. He then served as a Senior Postdoctoral Fellow in the laboratory of George Vande Woude in the Molecular Oncology Section of the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland. In June 1999, Dr. Koo joined VARI as a Scientific Investigator. Laboratory Members Staff Kate Eisenmann, Ph.D. Matt VanBrocklin, M.S. Nancy Staffend, B.S.

Students Susan Kitchen Tracey Millard

Research Projects tumor regression without apparent side effects. These results indicate that the MAPK signaling pathway represents a tumor-specific survival signaling in melanoma and that inhibition of this pathway may be a useful and potentially selective strategy for treating this cancer.

dvances in our understanding of the molecular pathophysiology of human cancers open promising opportunities for the prevention of and intervention in cancer. Our laboratory is interested in studying mechanisms of drug actions, identifying novel therapeutic targets, and developing novel anticancer agents by means of molecular-targeting approaches.

Our current research focuses on molecular characterization of the MAPK pathway–associated survival signaling in melanoma cells. In particular, we are investigating the phosphorylation and inactivation of the pro-apoptotic protein Bad mediated by the 90 kDa ribosomal S6 kinase. The molecular mechanism by which the inhibition of MAPK signaling specifically triggers apoptosis in human melanoma cells should reveal additional molecular targets useful for prevention of and intervention in melanoma, as well as in other MAPK-associated cancers such as pancreatic, lung, colon, and breast carcinomas, as well as gliomas. Additionally, further validation studies are ongoing to clinically develop the MAPK signaling pathway as a therapeutic target for melanoma treatment.

Mitogen-activated protein kinase (MAPK) signaling pathways are highly conserved among all eukaryotes and are integral for the transduction of a variety of extracellular signals. Furthermore, constitutive activation of MAPK signaling (e.g., the Raf-MEK1/2-ERK1/2 pathway) contributes to many aspects of human cancers; hence, the pathway has been identified as a potential therapeutic target for cancer intervention. Typically, cancer cells exhibit a cytostatic (growth arrest) response to the disruption of MAPK signaling. However, we have recently demonstrated that interfering with the MAPK signaling pathway evokes a cytotoxic response (apoptosis) in human melanoma cells but not in normal melanocytes: either anthrax lethal toxin (which proteolytically cleaves MAPK kinases [MEKs]) or small-molecule MEK inhibitors (such as PD90859 and U0126) triggers an apoptotic response in human melanoma cells. Normal melanocytes treated with the same inhibitors, on the other hand, simply arrest in the G1 phase of the cell cycle. More importantly, in vivo treatment with anthrax lethal toxin of human melanoma xenograft tumors in athymic nude mice renders either significant or complete

Activating mutations in RAS oncogenes are the most frequent gain-of-function mutations detected in human cancers. Besides their welldocumented role in cellular transformation and tumorigenesis, we have previously shown that the RAS oncogenes play an important role in sensitizing tumor cells to deoxycytidine analogues such as 1-β-D-arabinofuranosylcytosine (Ara-C) and gemcitabine, as well as to topoisomerase (topo) II inhibitors, more prominently to etoposide. These results are supported by clinical findings that patients who have RAS onco31

the Spectrum Health Cancer Program. This summer, through this collaboration, we have initiated a Phase II trial to evaluate the gemcitabine + etoposide combination treatment for patients with locally advanced or metastatic pancreatic carcinomas, which display RAS oncogene activation in over 95% of the cases.

gene–positive acute myeloid leukemia show an increased remission rate, longer remission duration, and improved overall survival in response to a combination therapy of Ara-C plus topo II inhibitor. To translate our results into a clinical trial, we have established a collaboration with the Grand Rapids Clinical Oncology Program and External Collaborators

Thomas M. Aaberg, Jr., Associated Retinal Consultants, Grand Rapids, Michigan Alan Campbell, Spectrum Health Cancer Program, Grand Rapids, Michigan Marianne K. Lang, Timothy J. O’Rourke, and Connie Szczepanek, Grand Rapids Clinical Oncology Program, Michigan Won Kyu Lee, Kent Pathology Laboratory, Ltd., Grand Rapids, Michigan Judith S. Sebolt-Leopold, Pfizer Global Research & Development, Ann Arbor, Michigan Lilly Research Laboratories, a division of Eli Lilly and Company, Indianapolis, Indiana Publications Koo, Han-Mo, Nicholas S. Duesbery, and George F. Vande Woude. 2002. Anthrax toxins, mitogenactivated protein kinase pathway, and melanoma treatment. Directions in Science 1: 123–126. Koo, Han-Mo, Matt VanBrocklin, MaryJane McWilliams, Stephan H. Leppla, Nicholas S. Duesbery, and George F. Vande Woude. 2002. Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase. Proceedings of the National Academy of Sciences U.S.A. 99(5): 3052–3057.

From left to right: Kitchen, VanBrocklin, Millard, Staffend, Eisenmann, Koo

Laboratory of Integrin Signaling and Tumorigenesis Cindy K. Miranti, Ph.D. Dr. Miranti received her M.S. in microbiology from Colorado State University in 1982 and her Ph.D. in biochemistry from Harvard Medical School in 1995. She was a Postdoctoral Fellow from 1995 to 1997 in the laboratory of Joan Brugge at ARIAD Pharmaceuticals, Cambridge, Massachusetts, and from 1997 to 2000 in the Department of Cell Biology at Harvard Medical School. Dr. Miranti joined VARI as a Scientific Investigator in January 2000; she is also an Adjunct Assistant Professor in the Department of Physiology at Michigan State University. Laboratory Members Staff Suganthi Chinnaswamy, Ph.D. Andrew Putnam, Ph.D. Veronique Schultz Patacsil, B.S.

Students Heather Bill, B.S. Andrea Pearson

Research Projects regulation of integrin function, or alterations in integrin-dependent signal transduction pathways.

ur laboratory is interested in understanding the mechanisms by which integrin receptors interacting with the extracellular matrix regulate cell function in normal and tumorigenic processes. Alterations in integrin receptors and their downstream signaling targets are common events in tumorigenesis, leading to a disruption of normal cell function. Using tissueculture models, biochemistry, molecular genetics, and ultimately mouse models, we are defining the signaling pathways and molecular events involved in integrin-dependent adhesion and migration that are important for tumorigenesis in general and specifically for melanoma and prostate cancer.

How integrins activate growth factor receptors Recent work in our laboratory has focused on characterizing the interactions between integrins and receptor tyrosine kinase. Adhesion of epithelial cells to several different extracellular matrices induces ligand-independent activation of the epidermal growth factor receptor (EGFR) and the Met receptor. Overexpression or mutation of EGFR family members or the Met receptor are common events in many epithelial tumors. We have shown that by recruiting EGFR, integrins are able to activate a subset of integrin-induced signaling pathways (Figure 1). In the absence of EGFR activation, the ability of the cells to induce the Ras/Erk signaling pathway and Akt is severely impaired. However, not all

Role of integrins in tumorigenesis Integrins are a class of heterodimeric transmembrane receptors for which there are currently 24 known family members: 15 alpha and 9 beta subunits. Each subunit contains a short cytoplasmic region with no known enzymatic activity, but through protein-protein interactions, subunits are able to interact with actin-containing microfilaments and important signaling molecules. Thus, the engagement of the integrin receptor by extracellular matrix components induces changes in actin structures, as well as the induction of several signal transduction pathways. Both the loss and gain of different integrins contribute to tumorigenesis and metastasis in many tumor types. In addition to changes in integrin expression, other contributors to tumorigenesis are alterations in integrin ligands, altered

Figure 1. Integrin-induced activation of EGFR is required for a subset of integrin-regulated signaling pathways

and α3b1 integrins and a loss of typical epithelial structures. We are interested in understanding how α6β1 and α3β1 integrins contribute to latestage prostate carcinoma and how androgen may regulate this process.

integrin signaling pathways are dependent on EGFR (e.g., FAK, Src, and PKC). We further have demonstrated that integrinmediated adhesion of epithelial cells, including primary prostate epithelial cells, is sufficient to induce several G1 cell cycle events, including increases in cyclin D1, p21, cdk4 kinase activity, and Rb phosphorylation. This is dependent on integrin activation of EGFR, Erk, and PI-3K (Figure 2). However, adhesion alone was not

The integrins α6β1 and α3β1 are known interact with an integrin-associated protein called CD82 (or alternatively, KAI1). Loss of expression of CD82 correlates with prostate metastasis, and the loss of CD82 would be predicted to alter the function of α6β1 and α3β1 integrins. Using primary prostate epithelial cells, which express high levels of CD82, as well as several prostate tumor cell lines that do not, we are exploring the role of CD82 in regulating α6β1- and α3β1mediated cell adhesion, migration, and cell signaling. We are using molecular genetic approaches such as mutagenesis, siRNA, and mouse models to alter CD82 expression in prostate cells. Integrin regulation of melanoma progression The incidence of melanoma has been steadily increasing in the last 10 years. If caught at an early stage it is usually curable, but once it has become invasive, metastatic melanoma is virtually untreatable and progresses very rapidly. Induced expression of the αvβ3 integrin correlates with increased invasive capacity of melanomas, yet the mechanisms underlying this shift in expression and increased invasiveness are unknown. We have initiated studies to determine how expression of the αvβ3 integrin in normal melanocytes alters cell function and the integrindependent signaling pathways involved. The serine/threonine protein kinase family, PKC, is a family of 11 related kinases that can be separated into three major classes: classical, novel, and atypical. This kinase family has been implicated in differentiation, growth regulation, cell survival, cell adhesion, cell migration, and tumorigenesis, but the exact role of each of these kinases is largely unknown. In normal melanocytes, PKC is required for cell growth and survival; in tumor cells, however, stimulation of PKC activity can result in growth arrest and cell death. In addition, PKC plays an important role in regulating cell adhesion and migration. We are interested in understanding how changes in expression of different PKC isoforms can regulate melanoma proliferation, migration, and invasion.

Figure 2. Integrin-induced activation of EGFR, and subsequently Erk and PI-3K, is required for a entry into G1 of the cell cycle, but is not sufficient for entry into S phase

sufficient for induction of DNA synthesis, indicating that additional signals are required. We are currently attempting to define what steps in G1 are blocked. Interestingly, HGF-mediated induction of DNA synthesis through the Met receptor was also dependent on integrin activation of EGFR. These data indicate that integrin regulation of EGFR activation is a critical mediator of cell cycle regulation. Integrin-mediated regulation of EGFR may be one mechanism that tumor cells use to regulate cell growth in the absence of exogenous growth factor. We are also exploring the mechanisms by which integrins activate EGFR and how integrins cooperate with the Met receptor to regulate cellular signaling. Integrin signaling in prostate cancer The development of metastatic prostate cancer is slow and is accompanied by the loss of androgen sensitivity. In normal epithelial cells, the α6 integrin is usually found in association with β4 integrin (α6β4) and is specifically localized to desmosomal junctions. However, in prostate carcinoma, β4 integrins are often lost, and there is a concomitant increase in the α6β1

The adhesion of normal melanocytes to the extracellular matrix induces the formation of focal adhesion complexes and actin stress fibers (Figure 3), but in a highly metastatic melanoma cell line, these structures are absent. We are exploring the biochemical basis for this difference. We have found that the activity level of Rac (a small GTPase required for regulating actin structure) is elevated and that the inhibition of PKC blocks this activity. The levels of PKCa are elevated in these cells as well. We are exploring the effects on cell structure, adhesion, and migration of PKCα levels.

Figure 3. Focal adhesions (green spots) and stress fibers (red fibers) are absent in metastatic melanoma

External Collaborators Joan Brugge, Harvard Medical School, Boston, Massachusetts Beatrice Knudsen, Cornell University Medical College, New York, New York Senthil Muthuswamy, Cold Spring Harbor Laboratory, New York Benjamin Neel, Beth Israel Deaconess Medical Center, Harvard Institute of Medicine, Boston, Massachusetts David Shalloway, Cornell University, Ithaca, New York Sheila Thomas, Harvard Institute of Medicine, Boston, Massachusetts Publications Miranti, Cindy K. 2002. Application of cell adhesion to study signaling networks. In Methods in Cell Matrix Adhesion, J.C. Adams, ed. Methods in Cell Biology series, San Diego: Academic Press, pp. 359–383. Miranti, Cindy K., and Joan S. Brugge. 2002. Sensing the environment: a historical perspective on integrin signal transduction. Nature Cell Biology 4(4): E83–E90. Woodside, Darren G., A. Obergfell, Lijun Leng, Julie L. Wilsbacher, Cindy K. Miranti, Joan S. Brugge, Sanford J. Shattil, and Mark H. Ginsberg. 2001. Activation of Syk protein tyrosine kinase through interaction with integrin β cytoplasmic domains. Current Biology 11(22): 1799–1804.

Left to right: Pearson, Putnam, Bill, Patacsil, Miranti

Human breast ductal epithelium This tissue was stained with two antibodies. In red is c-Met (polyclonal antibody c28) and in green is c-neu (monoclonal antibody OCS). c-neu stains for the tyrosine kinase receptor Her2-neu (involved in cell signal processes) that is amplified in breast cancer in 10â&#x20AC;&#x201C;20% of primary cases. The protein c-Met is also a tyrosine kinase receptor that is activated by the ligand hepatocyte growth factor/scatter factor (HGF/SF); c-Met has been shown to be a prognostic marker for human breast cancer. Co-localized foci are colored yellow, while red and green label the Met and her2neu separately. The c-Met is evident on the lumenal and basal border; c-neu is less specific but may be increased in the lateral boundaries between cells. (Resau)

Analytical, Cellular, and Molecular Microscopy Laboratory James H. Resau, Ph.D. Dr. Resau received his Ph.D. from the University of Maryland School of Medicine in 1985. Between 1968 and 1994, he was in the U.S. Army (active duty and reserve assignments) and served in Vietnam. From 1985 until 1992, Dr. Resau was a faculty member of the University of Maryland, School of Medicine, Department of Pathology, and was a tenured Associate Professor from 1990–1992. Dr. Resau then went to the NCI to be Director of the Analytical, Cellular and Molecular Microscopy Laboratory in the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland (1992–1999). Dr. Resau joined VARI as a Special Program Investigator in June 1999. Laboratory Members Staff Bree Buckner, B.S., HTL (ASCP), QIHC Eric Hudson, B.S. J.C. Goolsby

Students Hien Dang Marie Graves Lateefah Gray Maketta Hassen

Brandon Leeser Matthew Main Christine Moore Jeanine Myles

Research Projects researcher access to research collaborations with the intention of facilitating translation of new diagnostic, treatment, and evaluation protocols. We plan to generate gene expression profiles (microarray), establish new tumor cell lines, and develop new diagnostic and therapeutic agents through this collaboration. Epidemiologic evaluations will also be greatly improved by the coordination of clinical information, diagnosis, and research results. The goal of this project is to develop genetic-based diagnostic classification of human disease. There is a Scientific Advisory Board for this project comprising members of VARI and the Spectrum Health pathology, surgical and medical oncology, and surgery departments. Tissue is collected with explicit written permission of the participating physicians and patients. Protocols for the use of the material in this archive require the approval of both the VARI and the Spectrum Health IRBs.

ur laboratory works closely with VARI investigators, as well as in collaboration with outside parties, to provide a number of microscopy needs. We have a special interest in the quantification of imagery. We have two confocal microscopes that enable us to visualize organelles and processes in cells and tissues such as receptor–ligand interactions and co-localization of proteins with organelles. We have studied the location of two gene-targeted proteins within a cell (i.e., GFP and RFP) with a DNA marker, DAPI, in three dimensions. We have integrated laser-capture microdissection instrumentation into the program, as well as paraffin and frozensection staining. We also provide histotechnology services, consultation on staining, and direction for the human tissue services.

In collaboration with George Vande Woude and Rick Hay of VARI, we have developed the Tissue Collection Initiative between our Institute and the Spectrum Health System in Grand Rapids. We have expanded the program to include the Holland, Pennock, and Hackley hospitals. This program provides for the collection and characterization of fresh-frozen surgical tissues that will allow investigators to create a working repository for a wide range of projects. Surgically removed human tumors and normal tissue will be evaluated in institutional review board (IRB)–approved basic and translational research projects. This collection will at the same time provide to the physician

Directly related to this archive is the paraffinblock repository called SPIN (Shared Pathology Tissue and Informatics Network), a project that involves the same hospitals in west Michigan. This archive stores and catalogs paraffin blocks that are older than five years and ordinarily would be destroyed. Currently the archive holds approximately 150,000 tissue samples/paraffin blocks. They are not directly linked to any personal identifiers or names and there is limited demographic information available. Of the 150,000 samples or blocks, clinical and demographic data is available 37

with traditional surgical pathology diagnostic procedures to determine prognosis via an objective and quantifiable method(s). These toolsâ&#x20AC;&#x201D;and, more importantly, the processâ&#x20AC;&#x201D;will have application to many types of human disease. We have recently obtained NIH funding for a major effort in multiphoton imaging of developmental and carcinogenic events in GFP-expressing transgenic mice. George Vande Woude, Ilan Tsarfaty, and I will evaluate the role of Met and HGF/SF in branching morphogenesis, carcinogenesis, and therapy of cancer-related compounds. Other collaborations within VARI involve Met and HGF-SF in cells and tissues; the role of Met and HGF-SF in prostate and breast carcinogenesis; the location of gene-targeted proteins in rodents; evaluation of monoclonal antibodies as diagnostic reagents; application of human tissues to molecular-based studies; and the cellular and subcellular localization and quantification of proteins. We also have ongoing collaborations with scientists in other countries. We have in development a molecular imaging project with scientists at Tel Aviv University in Israel, and we have an international breast cancer project to evaluate Her2/neu and Met/HGF interactions with scientists in Germany. In addition to our research program, we have a long-standing interest in science education. Together with Grand Valley State University and Grand Rapids Community College, we have received NIH funding as part of the Bridges to the Baccalaureate program to support the recruitment and graduation of women and minorities into science, mathematics, and research careers. Dr. Resau is a co-investigator and site coordinator for the Bridges program.

for about 20%. The material from future years will be available with digital information on age, sex, and diagnosis. The current samples were collected by the hospitals with nondigital database technology, and the information is slowly being transcribed. These samples will be used in cellular and molecular protocols approved by our IRB. The samples and demographics are identified with basic information (such as diagnosis, age, sex, etc.) in a webbased, interactive format for determination of prognosis, diagnosis, and therapy. In the first six months of operation of SPIN there have been 12 users registered who have submitted 83 requests for searches and 33 subsequent tissue requests. We have provided tissue and histopathology services for 12 VARI investigators and have generated over 72,000 microscopic images and related image files for the collaborations. A major part of both the tissue initiative and SPIN programs will be to annotate and update information on each specimen. Our own research interest is in the retrospective review of archival tissues from individual samples with known clinical outcomes. We identify and quantify the location of particular proteins and examine the relationship between their pattern of expression and the prognosis of disease progression. We have submitted the results from our U.S. Army Breast Cancerâ&#x20AC;&#x201C;funded analysis of Met and Her2neu in human breast cancer and have nearly completed a major study in collaboration with investigators in Chicago on the same problem. The advantage of archival material is that new markers of prognosis can be evaluated. We can measure up to four proteins simultaneously, and we are developing methods to combine confocal microscopy, gene expression, and laser-capture microdissection External Collaborators Stephan Baldus, University of Cologne, Germany

Maria Birchenall-Roberts, Francis Ruscetti, Jerrold Ward, and George Pavlakis, National Cancer Institute, Frederick, Maryland Ruth Heilmann, University of Chicago, Illinois Iafa Keydar and Ilan Tsarfaty, Tel Aviv University, Israel Justin McCormick, Michigan State University, Lansing Toshio Mura, National Cancer Institute, Bethesda, Maryland John Sacci, University of Maryland, Baltimore Duane Smoot, Howard University, Washington, D.C.

Publications Albright, Craig D., Philip M. Grimley, Raymond T. Jones, and James H. Resau. 2002. Differential effects of TPA and retinoic acid on cell-cell communication in human bronchial epithelial cells. Experimental and Molecular Pathology 72(1): 62–67. Kino, Tomoshige, Roland H. Stauber, James H. Resau, George N. Pavlakis, and George P. Chrousos. 2001. Pathologic human GR mutant has a transdominant negative effect on the wild-type GR by inhibiting its translocation into the nucleus: importance of the ligand-binding domain for intracellular GR trafficking. Journal of Clinical Endocrinology and Metabolism 86(11): 5600–5608. Kort, Eric, Bryon Campbell, and James H. Resau. In press. A shared pathology informatics network. Computer and Programs in Biomedicine. Miura, Koichi, Kerry M. Jacques, Stacey Stauffer, Atsutaka Kubosaki, Kejin Zhu, Dianne Snow Hirsch, James Resau, Yi Zheng, and Paul A. Randazzo. 2002. ARAP1: a point of convergence for Arf and Rho signaling. Molecular Cell 9(1): 109–119. Miura, Koichi, Shoko Miyazawa, Shuichi Furuta, Junji Mitsushita, Keiju Kamijo, Hiroshi Ishida, Toru Miki, Kazumi Suzukawa, James Resau, Terry D. Copeland, and Tohru Kamata. 2001. The Sos1-Rac1 signaling: possible involvement of a vacuolar H+-ATPase E subunit. Journal of Biological Chemistry 276(49): 46276–46283. Qian, Chao-Nan, Xiang Guo, Brian Cao, Eric J. Kort, Chong-Chou Lee, Jindong Chen, Ling-Mei Wang, Wei-Yuan Mai, Hua-Qing Min, Ming-Huang Hong, George F. Vande Woude, James H. Resau, and Bin T. Teh. 2002. Met protein expression level correlates with survival in patients with late-stage nasopharyngeal carcinoma. Cancer Research 62(2): 589–596.

From left to right, back row: Hassen, Graves, Moore, Buckner, Resau, Leeser, Hudson front row: Myles, Goolsby

Laboratory of Germline Modification Pamela J. Swiatek, Ph.D. Dr. Swiatek received her M.S. (1984) and Ph.D. (1988) degrees in pathology from Indiana University. From 1988 to 1990, she was a Postdoctoral Fellow at the Tampa Bay Research Institute. From 1990 to 1994, she was a Postdoctoral Fellow at the Roche Institute of Molecular Biology in the laboratory of Tom Gridley. From 1994 to 2000, Dr. Swiatek was a Research Scientist and Director of the Transgenic Core Facility at the Wadsworth Center in Albany, New York, and an Assistant Professor in the Department of Biomedical Sciences at the State University of New York at Albany. She joined VARI as a Special Program Investigator, Laboratory of Germline Modification, in August 2000. Laboratory Members Staff Kathy Davidson, B.S. Kelly Sisson, B.S.

Student Cassandra Van Dunk

Bryn Eagleson, A.A. Bryn Eagleson began her career in laboratory animal services in 1981 with Litton Bionetics at the National Cancer Instituteâ&#x20AC;&#x2122;s Frederick Cancer Research and Development Center (NCI-FCRDC) in Maryland. In 1983, she joined the Johnson & Johnson Biotechnology Center in San Diego, California. In 1988, she returned to the NCI-FCRDC, where she continued to develop her skills in transgenic technology and managed the transgenic mouse colony. During this time she attended Frederick Community College and Hood College in Frederick, Maryland. In 1999, Bryn joined VARI as the Vivarium Director and Transgenic Core Manager. Managerial Staff Jason Martin, RLATG

Technical Staff Dawna Dylewski, B.S. Audra Guikema, B.S., L.V.T. Lori Ruff, B.S., RALAT Kristen Van Noord, B.S., RALAT

Vivarium Staff Shawn Ballard, A.S., B.A., RALAT Ben Buckrey, B.S. Elissa Boguslawski

Research Projects us of a one-cell fertilized egg. Fertilized eggs contain two pronuclei, one that is derived from the egg and contains the maternal genetic material and one derived from the sperm that contains the paternal genetic material. As development proceeds, these two pronuclei fuse, the genetic material mixes, and the cell proceeds to divide and develop into an embryo. DNA microinjected into a pronucleus randomly integrates into the mouse genome and will theoretically be present in every cell of the resulting organism. Expression of the transgene is controlled by genetic elements called promoters that are genetically engineered into the transgenic DNA. Depending on the selection of the promoter, the transgene can be expressed in every cell of the mouse or in specific cell populations such as neurons, skin cells, or blood cells. Temporal expression of the transgene during development can

he Germline Modification Laboratory provides transgenic and gene-targeting technology services to develop mouse models of human disease. These well-established and powerful techniques are used to insert specific genetic changes into the mouse genome in order to study the effect of these mutations in the complex biological environment of a living organism. These changes can include the introduction of a gene into a random site in the genome (transgenics), introduction of a gene into a specific site in the genome (gene knock-in), or the inactivation of a gene already present in the genome (gene knockout). Since these mutations are introduced into the reproductive cells known as the germline, they can be used to study the developmental aspects of gene function associated with inherited genetic diseases.

Transgenic mice are produced by injecting small quantities of foreign DNA into a pronucle40

Life Science Corridor clients will be assisted in the design and implementation of transgenic and gene-targeting experiments and, if necessary, trained in these techniques. New stem cell lines can be derived, and spectral karyotypic (SKY) analysis of mouse chromosomes—using highquality, 24-color fluorescent in situ hybridization paints—can aid in the detection of subtle and complex chromosomal rearrangements in ES cells. Upon production of the genetically modified mice, our lab will assist in developing breeding schemes and provide for the complete analysis of the mutant mice.

also be controlled by genetic engineering. These transgenic mice are excellent models for studying the expression and function of the transgene in the biological environment of the living mouse. Gene-targeting mutations are introduced into the mouse by genetic manipulation of pluripotent embryonic stem (ES) cells. ES cells, which are derived from 3.5-day-old embryos called blastocysts, have the potential to contribute to all tissues of a developing mouse. Genomic DNA containing the gene of interest is isolated, mutated, and inserted into ES cells. The mutated gene integrates into the genomes of the ES cells and, by a process called homologous recombination, replaces one of the two wild-type copies of the gene in the cells. These genetically modified cells, containing one mutant copy of the gene, are injected into wild-type blastocysts where they integrate into the developing embryo. These embryos, containing a mixture of wild-type and mutant ES cells, develop into offspring called chimeras. Offspring of chimeras that inherit the mutated gene are called heterozygotes, because they possess one copy of the mutated gene. The heterozygous mice are bred together, or intercrossed, to produce mice that completely lack the normal gene; these homozygous mice have two copies of the mutant gene and are called genetargeted or gene “knock-out” mice. A related technology, gene knock-in, employs similar methods to insert functional genes into specific locations in the mouse genome. Ultimately, gene-targeted mice can be observed for abnormalities associated with the inserted genetic change, and they provide powerful research tools for studying gene function in living organisms.

The vivarium utilizes two Topaz Technologies software products, Granite and Scion, for integrated management of the vivarium finances, the mouse breeding colony, and the Institutional Animal Care and Use Committee (IACUC) protocols and records. The efficiency of mutant mouse production and analysis is enhanced using the Autogen 9600, a robotic, high-throughput DNA purification machine. Imaging equipment, such as the PIXImus Mouse Densitometer and the Acuson Sequoia 512 ultrasound machine, is available for noninvasive imaging of mice. Mouse strains are archived using sperm cryopreservation and reconstituted using in vitro fertilization techniques. Additional services provided by the vivarium technical staff include an extensive xenograft model development and analysis service, rederivation, surgery, dissection, necropsy, breeding, and health-status monitoring. In summary, the goal of the germline modification laboratory is to develop, provide, and support high-quality mouse modeling technology services for the Van Andel Research Institute investigators, Michigan Life Science Corridor collaborators, and the greater research community.

The Germline Modification Laboratory is a full-service lab that functions at the level of service, research, and teaching. VARI and Michigan

External Collaborators Narayanan Parameswaran, Bill Smith, and Bill Spielman, Michigan State University, Lansing Douglas Ashley Monk, Michigan State University, Lansing Gary Litman, University of South Florida, Tampa Dan Rosen, Wadsworth Center, New York State Department of Health, Albany

Publications Su, Ting, Qing-Yu Zhang, Jianhua Zhang, Pamela J. Swiatek, and Xinxin Ding. 2002. Expression of the rat CYP2A3 gene in transgenic mice. Drug Metabolism and Disposition 30(5): 548â&#x20AC;&#x201C;552.

From left to right: Swiatek, Van Dunk, Sisson, Davidson

From left to right: Dylewski, Ruff, Eagleson, Martin, Boguslawski, Buckrey, Guikema, Van Noord, Ballard

Laboratory of Cancer Genetics Bin T. Teh, M.D., Ph.D. Dr. Teh obtained his M.D. from the University of Queensland, Australia, in 1992, and his Ph.D. from the Karolinska Institute, Sweden, in 1997. Before joining the Van Andel Research Institute, he was an Associate Professor of medical genetics at the Karolinska Institute. Dr. Teh joined VARI as a Senior Scientific Investigator in January 2000. Laboratory Members Staff Miles Chao-Nan Qian, M.D., Ph.D. Libing Song, M.D.. Ph.D. Jun Sugimura, M.D., Ph.D. Jindong Chen, Ph.D. Sok Kean Khoo, Ph.D. David Petillo, Ph.D. Chun Zhang, Ph.D. Eric Kort, M.S. Olga Motorna, B.S. Jason Yuhas, B.S.

Visiting Scientists Charlotta Lindvall, M.D., Ph.D. Joe Chien-Chung Chou, M.D. Carola Haven, M.D. Kanthimathi M.S., Ph.D. Vivve Howell, M.S. Jacqueline Schoumans, M.S.

Students Katherine Kahnoski Todd Lavery Casey Madura Grace Miguel Radoslav Nickolov Sarah Scolon

Research Projects tumors, we have studied the gene expression profiles of renal cell carcinoma and correlated them with clinical outcomes. We have also characterized the biological profiles of kidney tumors having different histopathologies, including pediatric kidney tumors (Wilms tumor). We confirm our microarray findings by both real-time polymerase chain reaction (RT-PCR) and immunohistochemical studies on kidney cancer tissue arrays. Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China and Southeast Asia. We are undertaking studies to identify cancer-related genes in NPC cell lines and primary tumors and to correlate their expression with clinical parameters. In endocrine or hormone-secreting tumors, we continue to work on multiple endocrine neoplasia type 1 and have found new mutations in new MEN1 families. We are currently focusing on the mapping of the gene for familial acromegaly, a hereditary condition characterized by tumors in the pituitary glands that secrete growth hormone.

ancer formation is a multistep process that results from genetic instability in the cells. At the molecular level it is characterized by multiple alterations in genes that play key regulatory roles in various cellular functions. Our laboratory is interested in identifying and studying these genetic alterations in both hereditary cancers and their sporadic counterparts. Currently we are focusing on three types of tumors: kidney tumors, nasopharyngeal carcinoma, and endocrine (hormone-secreting) tumors. We have close and extensive collaborations with researchers and clinicians at hospitals and universities in this country and overseas.

We have initiated a program to study hereditary kidney cancer and to date we have identified several families with the disease. Both cytogenetic and molecular studies have been performed to elucidate this cancerâ&#x20AC;&#x2122;s genetic basis. We have identified a family with a chromosome 3 translocation and currently we are trying to clone the breakpoint genes. We also have mapped the gene for Birt-Hogg-DubĂŠ syndrome, a hereditary cancer, to chromosome 17. This autosomal dominant disease is characterized by skin and kidney tumors and cysts in the lung. In addition, we have been working on another hereditary disease, hyperparathyroidismâ&#x20AC;&#x201C;jaw tumor syndrome, which is characterized by parathyroid tumors, jaw tumors, and kidney cysts and tumors. In sporadic kidney

In addition to carrying out these research projects, our laboratory has provided core sequencing and cytogenetic services to the Institute. To date over 15,000 sequences have been performed. We have also performed cytogenetics studies including FISH, conventional CGH, and SKY in collaboration with internal and external researchers. 43

External Collaborators We have extensive collaborations with researchers and clinicians from this country and overseas. Publications Dwight, T., S. Kytola, B.T. Teh, G. Theodosopoulos, A.L. Richardson, J. Philips, S. Twigg, L. Delbridge, D.J. Marsh, A.E. Nelson, C. Larsson, and B.G. Robinson. 2002. Genetic analysis of lithium-associated parathyroid tumors. European Journal of Endocrinology 146(5): 619–627. Dwight, T., A.E. Nelson, D.J. Marsh, B.T. Teh, C. Larsson, and B.G. Robinson. In press. Parathyroid tumorigenesis in association with primary hyperparathyroidism. Current Opinion in Endocrinology and Diabetes. Dwight, T., A.E. Nelson, G. Theodosopoulos, A.L. Richardson, D.L. Learoyd, J. Philips, L. Delbridge, J. Zedenius, B.T. Teh, C. Larsson, D. Marsh, and B.G. Robinson. In press. Independent genetic events associated with the development of multiple parathyroid tumors in patients with primary hyperparathyroidism. American Journal of Pathology. Haddad, Ramsi, Kyle A. Furge, Jeremy C. Miller, Brian B. Haab, J. Schoumans, B.T. Teh, L. Barr, and Craig P. Webb. In press. Genomic profiling and cDNA microarray analysis of human colon adenocarcinoma and associated intraperitoneal metastases reveals consistent cytogenetic and transcriptional aberrations associated with progression of multiple metastases. Applied Genomics and Proteomics. Lui, Weng-Onn, Jindong Chen, Sven Glasker, Bernhad U. Bender, Casey Madura, Sok Kean Khoo, Eric Kort, Catharina Larsson, Harmut P.H. Neumann, and Bin T. Teh. 2002. Selective loss of chromosome 11 in pheochromocytomas associated with the VHL syndrome. Oncogene 21(7): 1117–1122. Perrier, N.D., A. Villablanca, C. Larsson, M. Wong, B.T. Teh, and O.H. Clark. In press. Genetic screening for MEN1 in “familial isolated hyperparathyroidism.” World Journal of Surgery. Qian, Chao-Nan, Xiang Guo, Brian Cao, Eric Kort, Chong-Chou Lee, Jindong Chen, Ling-Mei Wang, Wei-Yuan Mai, Hua-Qing Min, Ming-Huang Hong, George F. Vande Woude, James H. Resau, and Bin T. Teh. 2002. Met protein expression level correlates with survival in patients with late-stage nasopharyngeal carcinoma. Cancer Research 62(2): 589–596. Takahashi, M., R. Kahnoski, D. Gross, D. Nicol, and B.T. Teh. 2002. Familial adult renal neoplasia. Journal of Medical Genetics 39(1): 1–5. Villablanca, Andrea, Filip Farnebo, Bin T. Teh, Lars-Ove Farnebo, Anders Höög, and Catharina Larsson. 2002. Genetic and clinical characterization of sporadic cystic parathyroid tumours. Clinical Endocrinology 56(2): 261–269.

From left to right: Teh, Qian, Chen, Yuhas, Motorna, Khoo, Song, Petillo, Zhang, Sugimura

Laboratory of Molecular Oncology George F. Vande Woude, Ph.D. Dr. Vande Woude received his M.S. (1962) and Ph.D. (1964) from Rutgers University. From 1964–1972, he served first as a postdoctoral research associate, then as a research virologist for the U.S. Department of Agriculture at Plum Island Animal Disease Center. In 1972, he joined the National Cancer Institute as Head of the Human Tumor Studies and Virus Tumor Biochemistry sections and, in 1980, was appointed Chief of the Laboratory of Molecular Oncology. In 1983, he became Director of the Advanced Bioscience Laboratories-Basic Research Program at the National Cancer Institute’s Frederick Cancer Research and Development Center, a position he held until 1998. From 1995, Dr. Vande Woude first served as Special Advisor to the Director, and then as Director for the Division of Basic Sciences at the National Cancer Institute. In 1999, he was recruited to the Directorship of the Van Andel Research Institute in Grand Rapids, Michigan. Laboratory Members Staff Rick Hay, M.D., Ph.D. Chong-Feng Gao, Ph.D. Chong-Chou Lee, Ph.D. Ling-Mei Wang, Ph.D. Yu-Wen Zhang, Ph.D. Dafna Kaufman, M.S. Meg Gustafson, B.A. Yanli Su, A.M.A.T. Mary Beth Bruch

Visiting Scientists & Staff Nariyoshi Shinomiya, M.D., Ph.D. Galia Tsarfaty, M.D. Ilan Tsarfaty, Ph.D.

Students Daphna Atias Marketta Hassen Yasser Jimenez Jason Johnson Nathan Lanning Adi Laser, B.S. Kofi Obeng Dan Wohns

Research Projects esearch conducted in the Laboratory of Molecular Oncology uses a broad range of approaches to elucidate the molecular basis of cancer and to develop new agents for the diagnosis and therapy of cancer. We are primarily interested in the expression and activities of the receptor tyrosine kinase known as Met, its interactions with the ligand hepatocyte growth factor/scatter factor (HGF/SF), and the intracellular events influenced by Met activation. Aberrant expression of this receptor–ligand pair confers an invasive/metastatic phenotype in model systems of cancer. Inappropriate HGF/SF-Met expression occurs in most types of human solid tumors and is associated with poor clinical prognosis.

efforts are designed to use Stat3β to identify the downstream targets of Stat3 that influence anchorage-independent growth.

Therapeutics In collaboration with David Wenkert, we have been testing novel derivatives of the antitumor agent geldanamycin for suppression of Met activity in cultured tumor cells. We have observed that the plasmin-inhibitory activity of geldanamycin and its derivatives extend over a concentration range of almost nine logs. The most potent compounds inhibited plasmin activity at IC50’s of 5–30 fM, in stark contrast to the nanomolar concentrations required for the destabilizing effects of geldanamycin on HSP90. Three of the derivatives are more potent than geldanamycin: 17-[di-(2chloroethyl)amino]-17-demethoxygeldanamycin, 17-amino-17-demethoxygeldanamycin, and the most potent, 7′-bromogeldanoxazinone.

Biochemistry and molecular biology The transcription factor Stat3, implicated in cell transformation induced by many oncogenes, is also a downstream signaling molecule activated by HGF/SF-Met signaling. In collaboration with Richard Jove, we have utilized Stat3β, a dominant negative form of Stat3, to show that Stat3 activity is critical both for HGF/SFMet–mediated cell growth in soft agar and for tumor growth in athymic nude mice. Our current

In vivo imaging In collaboration with Brian Cao, Beatrice Knudsen, and Milton Gross, we are developing monoclonal antibodies raised against components of the Met-HGF/SF receptor–ligand pair as potential diagnostic and therapeutic agents. We 45

From these observations we conclude that antiMet monoclonal antibodiesâ&#x20AC;&#x201D;Met3 in particularâ&#x20AC;&#x201D; are robust reagents for detecting human Metexpressing cells. They are worthy of further evaluation as potential diagnostic and therapeutic agents for human cancers, including prostate cancer.

have undertaken a detailed characterization of the single anti-Met monoclonal antibody designated Met3. By immunofluorescence we have shown that Met3 binds with high avidity to cultured tumor cells expressing human Met. We have also shown that Met3 binds to cultured normal prostate epithelial cells and to prostate epithelium in human tissue sections. We have confirmed by FACS analysis that Met3 binds to cells of the human prostate carcinoma lines PC3 and DU145, which are known to express Met.

In collaboration with Brian Ross, our visiting scientists Ilan and Galia Tsarfaty are leading an effort to develop a noninvasive tumor molecular imaging program. In order to study the metabolic effects of Met-HGF/SF signaling in vivo, we recently demonstrated functional molecular imaging of Met receptor activity. DA3 mammary adenocarcinoma cells were injected into the mammary glands of mice, forming tumors expressing high levels of Met. We showed that Met activation in vivo by HGF/SF alters the hemodynamics of normal and malignant Met-expressing tissues. Organs and tumors expressing high levels of Met showed the greatest alteration in blood oxygenation levels as measured by BOLD-MRI (blood oxygenation levelâ&#x20AC;&#x201C;dependent MRI). Met-expressing tumors showed a 30% change in signal by BOLD-MRI, while no significant alteration was observed in tumors or organs that do not express Met. The extent of MRI signal alteration correlated with the dose of HGF/SF administered.

We have examined the ability of Met3 to image human tumors of different tissue origins. Tumor xenografts were raised subcutaneously in hind limbs of athymic nude mice, and animals were injected intravenously with [125I]Met3. Total-body gamma camera images were acquired and analyzed. The autocrine Met-expressing tumors S-114 (3T3 murine cells transformed with human Met and HGF/SF) and SK-LMS-1 (human leiomyosarcoma) and the paracrine Met-expressing human prostate carcinoma PC-3 were all readily imaged with [125I]Met3; peak tumor-to-control hind limb asymmetry was observed at about 3 days postinjection (Figure 1).

In autocrine tumors, the hemodynamic changes in the tumors are greatly enhanced compared with those in tumors that do not express HGF/SF. Moreover, the kidneys and livers of mice bearing autocrine tumors demonstrate increased hemodynamic activity that is proportional to the tumor size. These results indicate that functional molecular imaging of Met expression can serve as a powerful tool for understanding the metabolic activities affected by its signal transduction, and this approach could be used to understand the different molecular mechanisms of receptor activation. Moreover, functional molecular imaging of Met expression may be useful for the detection, analysis, and prognosis of a wide spectrum of human solid tumors. Human pathology collaborative studies In collaboration with Beatrice Knudsen, we have shown an overall 52% Met positivity among primary prostate cancers in a cohort of 90

Figure 1. Leiomyosarcoma in mouse thigh imaged with radioactive anti-Met monoclonal antibody

patients and confirmed that Met expression was present in all of 41 prostate cancer metastases to bone within this cohort (Figure 2). In collaboration with Nadia Harbeck and Ernest Lengyel, we have conducted a retrospective pilot study using breast cancer tissue obtained from about 40 patients to investigate whether Met-HGF/SF and HER-2/neu are coexpressed in HER-2-positive tumors. We correlated the results of immunohistochemical evaluation of tissue samples in our two laboratories with other clinicopathological data. We found that neither Met nor HER-2 expression in primary tumors correlated with established prognostic factors such as age, lymph node involvement, estrogen receptor expression, progesterone receptor expression, tumor size, or tumor grading. However, Met overexpression alone identified high-risk patients independent of HER-2 expression. Median disease-free survival associated with c-Met overexpressing tumors was 8 months, compared to 53 months in remaining

Figure 2. Met expression (arrow, brown) by prostate cancer metastasis in human bone

patients (p = 0.031; RR 3.1). We conclude that Met overexpression is associated with significantly diminished disease-free survival (DFS) and, in the majority of cases, this is independent of HER-2 overexpression.

External Collaborators Michael Clague, University of Liverpool, United Kingdom Milton Gross, Department of Veterans Affairs Healthcare System and University of Michigan, Ann Arbor Nadia Harbeck and Ernest Lengyel, Technische Universit채t, Munich, Germany Ruth Heimann and Samuel Hellman, University of Chicago, Illinois Richard Jove, H. Lee Moffitt Cancer and Research Institute, Tampa, Florida Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Len Neckers and Bert Zbar, National Cancer Institute, Bethesda, Maryland Brian Ross, University of Michigan, Ann Arbor Peter Schirmacher, University of Cologne, Germany David Waters, Gerald P. Murphy Cancer Foundation, Seattle, Washington David Wenkert, Michigan State University, East Lansing Robert Wondergem, East Tennessee State University, Johnson City

Publications Collazo, Carmen M., George S. Yap, Gregory D. Sempowski, Kimberly C. Lusby, Lino Tessarollo, George F. Vande Woude, Alan Sher, and Gregory A. Taylor. 2001. Inactivation of LRG-47 and IRG-47 reveals a family of interferon g–inducible genes with essential, pathogen-specific roles in resistance to infection. Journal of Experimental Medicine 194(2): 181–188. Furge, Kyle A., David Kiewlich, Phuong Le, My Nga Vo, Michel Faure, Anthony R. Howlett, Kenneth E. Lipson, George F. Vande Woude, and Craig P. Webb. 2001. Suppression of Rasmediated tumorigenicity and metastasis through inhibition of the Met receptor tyrosine kinase. Proceedings of the National Academy of Sciences U.S.A. 98(19): 10722–10727. Hay, Rick V., Brian Cao, R. Scot Skinner, Ling-Mei Wang, Yanli Su, James H. Resau, George F. Vande Woude, and Milton Gross. 2002. Radioimmunoscintigraphy of tumors autocrine for human Met and hepatocyte growth factor/scatter factor. Molecular Imaging 1(1): 56–62. Knudsen, Beatrice S., Glenn A. Gmyrek, J. Inra, D.S. Scherr, E. Darracott Vaughan, D.M. Nanus, M.W. Kattan, W.L. Gerald, and George F. Vande Woude. In press. High expression of the Met receptor in prostate cancer metastasis to bone. Urology. Koo, Han-Mo, Matt VanBrocklin, MaryJane McWilliams, Stephan H. Leppla, Nicholas S. Duesbery, and George F. Vande Woude. 2002. Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase. Proceedings of the National Academy of Sciences U.S.A. 99(5): 3052–3057. Qian, Chao-Nan, Xiang Guo, Brian Cao, Eric J. Kort, Chong-Chou Lee, Jindong Chen, Ling-Mei Wang, Wei-Yuan Mai, Hua-Qing Min, Ming-Huang Hong, George F. Vande Woude, James H. Resau, and Bin T. Teh. 2002. Met protein expression level correlates with survival in patients with late-stage nasopharyngeal carcinoma. Cancer Research 62(2): 589–596. Webb, Craig P., and George F. Vande Woude. 2002. Met gene. In Wiley Encyclopedia of Molecular Medicine, Haig H. Kazazian, ed. New York: Wiley, pp. 2049–2051. Zhang, Yu-Wen, Ling-Mei Wang, R. Jove, and George F. Vande Woude. 2002. Requirement of Stat3 signaling for HGF/SF-Met-mediated tumorigenesis. Oncogene 21(2): 217–226.

From left to right: Kaufman, Gao, Zhang, I. Tsarfaty, Shinomiya, Hay, G. Tsarfaty, Su, Vande Woude, Bruch, Gustafson

Tumor Metastasis and Angiogenesis Laboratory Craig P. Webb, Ph.D. Dr. Webb received his Ph.D. in cell biology from the University of East Anglia, England, in 1995. He then served as a Postdoctoral Fellow in the laboratory of George Vande Woude in the Molecular Oncology Section of the Advanced BioScience Laboratoriesâ&#x20AC;&#x201C;Basic Research Program at the National Cancer Instituteâ&#x20AC;&#x201C;Frederick Cancer Research and Development Center, Maryland (1995â&#x20AC;&#x201C;1999). Dr. Webb joined VARI as a Scientific Investigator in October 1999. Laboratory Members Staff Jeremy Miller, Ph.D. David Monsma, Ph.D. Emily Eugster, M.S.

Guest worker Lonson Barr, D.O.

Students Kelly Ballast, B.S. Donald Chaffee Meghan Sheehan

Research Projects Of particular importance, we are beginning to determine the factors that contribute to metastatic dormancy, a frequent occurrence in which individual metastatic cells within secondary tissues fail to progress to macroscopic disease, but instead lie dormant until a later time. Very little is known about the factors that contribute to this phenomenon, yet this aspect of metastasis likely accounts for the minimal residual disease and metastatic relapse observed in many patients. We are now using a combination of some state-of-the-art technologies, including laser capture microdissection, proteomics, and gene chip arrays, to integrate the genomic and proteomic events that occur during tumor-host interactions throughout the metastatic process in mouse models and human patient material. In this fashion, we have identified a number of candidate genes and proteins that appear to play prominent roles in metastasis to specific secondary sites. Our lab has now begun to systematically validate these targets for their precise functional roles. These types of studies will continue to generate essential information about the factors that regulate metastatic progression and will identify diagnostic/therapeutic targets for the future. In collaboration with local clinicians and some industrial partners, we are striving toward the early diagnosis and treatment of malignant disease.

umor metastasis, the process by which cancer spreads throughout a host to secondary tissues, accounts for the majority of cancer-related mortalities. The active recruitment of tumor vasculature, generally termed angiogenesis, is integral to both tumor growth and metastasis. Our laboratory focuses on identifying the key cellular and molecular determinants of metastatic progression in order to improve our conceptual understanding of the process, with the goal of developing diagnostics and therapeutics that target this most damaging aspect of cancer. Our lab currently utilizes various systems to study metastasis and angiogenesis both in vitro and in vivo. For example, we have previously described murine cell lines that display various metastatic propensities after ectopic expression of effector domain mutants of the ras oncogene. These mutants are particularly useful, because they differentially activate signaling pathways downstream of Ras and hence can be used to dissect the pathways and subsequent genetic/epigenetic events that mediate metastasis in this experimental setting. In tandem with our fluorescent imaging capabilities, we are now able to follow individual tumor cells as they undergo the various stages of metastatic spread. We are using these imaging strategies with other mouse models to follow the progression of metastases, such as metastasis to the liver after the formation of primary pancreatic carcinomas.

External Collaborators Lonson Barr, Donald Kim, Martin Luchtefeld, and Thomas Monroe, Spectrum Health, Grand Rapids, Michigan Yihai Cao, Karolinska Institute, Stockholm, Sweden Ann Chambers, University of Western Ontario, London, Canada Samir Hanash and Gil Omenn, University of Michigan, Ann Arbor Robert Hoffman, Anticancer Inc., San Diego, California Beatrice Knudsen, Cornell University, Ithaca, New York Ken Lipson, SUGEN, Inc., South San Francisco, California Martin McMahon, University of San Francisco, California Anthony Schaeffer, Northwestern University, Evanston, Illinois Bin S. Teh, Baylor College of Medicine, Waco, Texas Annette Thelen, Michigan State University, Lansing Affymetrix, Santa Clara, California Micromass, Beverly, Massachusetts Pharmacia (SUGEN Inc), California Publications Furge, Kyle A., David Kiewlich, Phuong Le, My Nga Vo, Michel Faure, Anthony R. Howlett, Kenneth E. Lipson, George F. Vande Woude, and Craig P. Webb. 2001. Suppression of Ras-mediated tumorigenicity and metastasis through inhibition of the Met receptor tyrosine kinase. Proceedings of the National Academy of Sciences U.S.A. 98(19): 10722–10727. Haddad, Ramsi, Kyle A. Furge, Jeremy C. Miller, Brian B. Haab, J. Schoumans, Bin T. Teh, Lonson Barr, and Craig P. Webb. In press. Genomic profiling and cDNA microarray analysis of human colon adenocarcinoma and associated intraperitoneal metastases reveals consistent cytogenetic and transcriptional aberrations associated with progression of multiple metastases. Applied Genomics and Proteomics. Haddad, Ramsi, and Craig P. Webb. 2001. Hepatocyte growth factor expression in human cancer and therapy with specific inhibitors. Anticancer Research 21(6B): 4243–4252. Webb, Craig P., and George F. Vande Woude. 2002. Met gene. In Wiley Encyclopedia of Molecular Medicine, Haig H. Kazazian, ed. New York: Wiley, pp. 2049–2051.

From left to right: Webb, Monsma, Miller, Sheehan, Eugster, Chaffee, Ballast, Barr

Before

After

Captured

Normal Epithelia

Colon Adenocarcinoma

Laser capture of normal colon crypts and adjacent adenocarcinoma Laser capture microdissection of normal colon crypts and adjacent adenocarcinoma of the colon, within a single histopathological section from a patient with metastatic colon cancer. Following capture, the small amounts of RNA and protein obtained can be applied to a global genomic and proteomic analysis using Affymetrix gene chips, cDNA microarrays, and mass spectrometry. In this fashion, genes and proteins that are expressed within the different subcompartments of a heterogeneous tumor (epithelia, endothelia, stroma, inflammatory cells, etc.) can be analyzed. This technology provides an excellent means for identifying the key genes and proteins that are associated with tumor progression and will likely yield targets for the future diagnosis and treatment of cancer. (Webb and Monsma)

Immunofluorescent analysis of blood vasculature in the eye of an Lrp5-deficient mouse Mutations that inactivate the gene encoding the low-density lipoprotein receptorâ&#x20AC;&#x201C;related protein 5 (Lrp5) cause the human syndrome osteoporosis pseudoglioma (OPG). Patients suffering from this syndrome develop earlyonset osteoporosis and have vision problems due to inappropriate vascularization of the eye. We have found that Lrp5-deficient mice model both the osteoporotic and eye problems seen in humans. Shown is an immunofluorescence analysis of a paraffin-embedded eye section from an Lrp5-deficient mouse stained with antibodies to Factor VIII (red) and CD31 (green), two molecules associated with endothelial cells. The prominent vascularization is absent in normal mice of this age. (Williams)

Laboratory of Chromosome Replication Michael Weinreich, Ph.D. Dr. Weinreich received his Ph.D. in biochemistry from the University of Wisconsin–Madison in 1993. He then served as a Postdoctoral Fellow in the laboratory of Bruce Stillman, director of the Cold Spring Harbor Laboratory, New York, from 1993 to 2000. Dr. Weinreich joined VARI as a Scientific Investigator in March 2000. Laboratory Members Staff Andrei Blokhin, Ph.D. Don Pappas, Ph.D. Carrie Gabrielse, B.S. Marleah Russo, B.S.

Student Ashley Mynsberge

Research Projects merases, and the activation of replication by cyclin-dependent kinases and the Cdc7 protein kinase.

ne critical step after the commitment to cell division is chromosome replication. Our laboratory is interested in understanding how the initiation of DNA replication occurs at the molecular level and how initiation events at each chromosomal origin are restricted to once per cell cycle. As cells exit mitosis and enter the G1 phase, they assemble a “pre-replicative complex” (pre-RC) at multiple replication origins. Additional lesswell-defined complexes formed at the origin in G1 are then activated to form bidirectional replication forks within a very short time (S-phase). Restricting the assembly of pre-RCs to G1 is a key regulatory event insuring that replication origins become competent only after completion of the previous cell cycle. In Saccharomyces cerevisiae, cyclin-dependent kinases inhibit pre-RC formation throughout the cell cycle, but as their levels fall during exit from mitosis, pre-RCs are able to form.

Our long-term goal is to define the protein components of the replication complexes that form in G1, and particularly to understand how complex assembly is regulated. Cdc6p is a critical limiting factor for assembly of the pre-RC. We have previously shown that Cdc6p interacts with ORC and that its essential activity requires a functional ATP-binding domain. Cdc6p also couples replication initiation with progression through the remainder of the cell cycle. If initiation does not occur, a nonessential N-terminal domain of approximately 50 amino acids is required for preventing a “reductional mitosis,” in which the unreplicated chromosomes are randomly segregated. Cdc6p very likely regulates passage through mitosis by inhibiting cyclindependent kinases. We have taken a genetic approach to identifying additional factors that are important for initiation. Using a temperature-sensitive mutation in CDC6, we have isolated a number of dosagedependent and extragenic suppressors that restore growth at high temperature. These suppressors define several novel pathways influencing replication. For example, we isolated one class of extragenic suppressors that contained mutations in the silent information regulators SIR2, SIR3, and SIR4. The Sir proteins are required for the formation of heterochromatic regions at the silent mating-type loci and at telomeres. In addition, Sir2p suppresses recombination at the rDNA locus and promotes

Eukaryotic origins of replication have been precisely defined only in budding yeast. An initiator protein (ORC) has also been extensively characterized. ORC is a six-subunit complex that recognizes conserved sequence elements in all origins and is required for the initiation of replication. ORC is bound to origins throughout the cell cycle; however, in late mitosis, Cdc6p binds to ORC and promotes loading of the MCM complex (a helicase) at the origin. ORC, Cdc6p, and the MCM complex are required to form the pre-RC in vivo. Subsequent events occurring in G1 are much less understood, including the association of Cdc45p, the loading of DNA poly53

hypomutable and fail to respond normally to signals generated by stalled replication forks. Therefore, Cdc7p-Dbf4p is emerging as perhaps a more global regulator of chromosome maintenance and stability than previously thought.

increased life span in yeast and Caenorhabditis elegans. SIR2 encodes a histone-dependent deacetylase and has at least seven orthologues in human cells. No evidence has been reported that the Sir proteins influence replication globally, as our data suggest. We are testing whether the Sir proteins act directly at origins of replication and negatively regulate initiation events. If this is occurring, it could provide a mechanism for the establishment of transcriptional or developmental states that were coupled to replication of certain chromosomal domains.

For this reason we are studying this protein both in yeast and in human cells. We have generated wild-type human cDNA clones and have constructed baculoviruses for purification of both the human and yeast enzymes. We have raised monoclonal antibodies against human Cdc7 and are now raising antibodies against the Dbf4 subunit. We hope to gain valuable reagents for examining the regulation and localization of the human kinase, both during the normal cell cycle and during periods of genomic stress. The Dbf4 protein has two classical D-box motifs and also a KEN-box motif. Both of these sequences are known to promote polyubiquitylation and proteasome-dependent degradation of cyclins and other unstable proteins. We are examining if these sequences function similarly in the human and yeast kinases. We are most interested in determining the role of the Cdc7p-Dbf4p kinase during periods of DNA damage or replication-fork arrest. Both the human and yeast Dbf4 proteins contain a single BRCT domain at the amino terminus. BRCT domains (first defined as a tandem repeat at the C-terminus of BRCA1) are present in a large family of proteins involved in DNA repair. Published and unpublished work indicates that yeast Cdc7pDbf4p is an important target of the S-phase checkpoint. The S-phase checkpoint in yeast responds to stalled replication forks that occur through a variety of insults. Since abrogating checkpoints are thought to facilitate tumorigenesis, we are examining if the human Cdc7 kinase is similarly a target of checkpoint kinases following DNA damage. Also, we are taking a genetic approach in yeast to more accurately determine its effect on DNA repair and replication.

Figure 1. S. cerevisiae replication cycle

We are also studying the Cdc7p-Dbf4p kinase, which is a conserved, two-subunit serine/threonine protein kinase required for a late step in replication initiation. We are interested in understanding the regulation of Cdc7p-Dbf4p kinase activity and determining its critical in vivo substrates. Cdc7p subunit abundance is constant throughout the cell cycle, but the Dbf4p subunit is cyclically expressed and is degraded during mitosis. The Cdc7p-Dbf4p kinase is required for DNA replication, but it has less-well-defined roles in promoting error-prone DNA repair and progression through meiosis. In response to DNA damage, Dbf4p is phosphorylated in a checkpoint-dependent manner and this decreases Cdc7p-Dbf4p kinase activity. CDC7 mutants are

External Collaborators Catherine Fox, University of Wisconsinâ&#x20AC;&#x201C;Madison Chun Liang, Hong Kong University Publications Weinreich, Michael, Chun Liang, Hsu-Hsin Chen, and Bruce Stillman. 2001. Binding of cyclindependent kinases to ORC and Cdc6p regulates the chromosome replication cycle. Proceedings of the National Academy of Sciences U.S.A. 98(20): 11211â&#x20AC;&#x201C;11217.

From left to right: Mynsberge, Weinreich, Russo, Gabrielse, Pappas, Blokhin

Laboratory of Cell Signaling and Carcinogenesis Bart O. Williams, Ph.D. Dr. Williams received his Ph.D. in biology from Massachusetts Institute of Technology in 1996. For three years, he was a Postdoctoral Fellow at the National Institutes of Health in the laboratory of Harold Varmus, former Director of NIH. Dr. Williams joined VARI as a Scientific Investigator in July 1999. Laboratory Members Staff Troy Giambernardi, Ph.D. Sheri Holmen, Ph.D. Scott Robertson, B.S. Cassandra Zylstra, B.S.

Students Holli Charbonneau Jennifer Daugherty Jennifer Mieras

Research Projects These include Dickkopfs (Dkks) and Frizzledrelated proteins (FRPs). One of the long term goals of our laboratory is to understand how specificity is generated for the different signaling pathways. The following projects are currently being pursued in the laboratory.

ur laboratory is focused on understanding how alterations in the Wnt signaling pathway cause human disease. Alterations in the pathway are among the most common changes associated with human cancer and have also been linked to other disorders, including osteoporosis. A very complete overview of this pathway can be found on a Web site (http://www.stanford.edu/~rnusse/wntwindow.html) developed by Dr. Roel Nusse.

Analysis of Lrp5-deficient mice Recently, several laboratories have demonstrated that Lrp5 is required for the maintenance of normal bone density in humans. Consistent with the work of other laboratories, we have shown that Lrp5-deficient mice are viable and fertile but have decreased bone density and persistent vascularization of the lens. We have created Lrp5-deficient mice on several genetic

We are particularly interested in elucidating the mechanisms by which the secreted Wnt ligand activates the pathway by binding to the receptor complex at the plasma membrane. Wnt binds to a receptor complex that includes a member of the frizzled family and LRP5 or LRP6. The activation of this complex then inhibits the targeting of Î˛-catenin and plakoglobin for ubiquitin-dependent proteolysis. This inhibition results in the accumulation of these proteins in the cytosol, where they interact with the LEF/TCF family of DNA binding proteins and subsequently translocate to the nucleus where they alter gene expression. In other contexts, Wnt ligands can activate protein kinase C or Rho-dependent pathways. There are many levels of regulating the reception of Wnt signals. The completion of the human genome project has shown that there are 19 different genes that encode Wnt proteins, 9 encoding Frizzled proteins, and two LDL receptorâ&#x20AC;&#x201C;related proteins that function in Wnt signaling (LRP5 and LRP6). In addition, there are several proteins that can inhibit Wnt signaling by binding to components of the receptor complex and interfering with normal signaling (Figure 1).

Figure 1. Overview of the Wnt signaling pathway (Reprinted by permission from Nature Cell Biology 4(7): E172-E173, ÂŠ (2002) Macmillan Publishers Ltd.)

which alterations in this signaling pathway have been introduced into the mouse genome.

backgrounds to assess whether alleles that may modify bone density and lens vascularization (and perhaps Wnt signaling) can be identified in the mouse genome. In addition, we are assessing whether Lrp5 is required for mammary tumorigenesis in MMTV-Wnt1 transgenic mice.

An in vivo model is being developed for studying melanoma using a retroviral-based gene targeting system. We have generated a mouse strain expressing the avian leukosis virus receptor, TV-A, under the control of a promoter that is only active in melanocyte precursor cells. These cells can then be infected by avian leukosis virus A (AVL-A) which is harboring genes of interest that have been introduced into AVL-A vectors. This system allows multiple genes to be delivered to a single TV-A+ cell. We can then study the effects of multiple oncogenes and tumor suppressor genes on melanoma development and progression. In addition, this model will allow comparison of melanomas induced by different genetic changes. These can then be used to evaluate the efficacy of different therapeutic strategies.

Analysis of mice deficient for both Lrp5 and Lrp6 Mice carrying a mutated allele of Lrp6 (generously provided by Bill Skarnes) are being crossed to mice deficient for Lrp5. We are interested in potential phenotypes of Lrp6+/–;Lrp5–/– mice. One possibility is that these mice will have more severe defects in bone density and eye vascularization. In addition, we are determining the phenotype of mice homozygously deficient for both genes. We expect these mice will die very early in gestation. To further characterize the functions of these genes, we are creating mouse embryonic stem cell lines deficient for both genes and using them to generate chimeric mice for analysis (in collaboration with the VARI laboratory of Pamela Swiatek).

We are also developing a system to regulate the expression of an activated version of β-catenin in melanocytes. Given that mutations in the βcatenin gene have been identified in melanomas, we feel that this system will allow insight into the role of Wnt signaling in melanocyte differentiation and melanoma-genesis.

Wnt-Fz fusion constructs and specificity in the Wnt signaling pathway

The role of Wnt signaling in prostate cancer

We have recently published an analysis of fusion proteins between selected Wnt and Frizzled molecules in collaboration with Adrian Salic and Marc Kirschner of Harvard Medical School. We found that expression of several such fusion proteins with Lrp6 could significantly activate a Wnt/β-catenin–responsive reporter gene and stabilize cytoplasmic levels of β-catenin. We are continuing to utilize these constructs to address questions about Wnt signaling specificity.

We are developing a tetracycline-regulated system to control the expression of an activated version of β-catenin specifically in the prostate. The rationale for doing this is that up-regulation of β-catenin activity is observed in a significant percentage of prostate tumors. In addition, the βcatenin protein physically interacts with the androgen receptor and alters its activity. This work is done with in collaboration with Wade Bushman of the University of Wisconsin.

Expression of Wnt receptor components in tumorigenesis and development

The role of MMP8 in melanoma Expression of matrix metalloproteinase 8 (MMP8), previously thought to be restricted in expression to neutrophils, was recently detected in melanomas but not normal melanocytes. In addition, expression was also detected in neural crest cells during embryonic development. We are currently creating mice deficient in MMP8 to further define its role in development. We are also examining various stages of melanoma to identify the point in melanoma progression at which MMP8 is turned on.

We have developed probes for RT-PCR analysis of Wnt, Frizzled, Lrps, Dkks, Kremens, and FRPs. We are systematically examining the expression of these components in various tumor cell lines and in tissue samples. Mouse models for melanoma Our laboratory is also interested in the broad goal of improving mouse models of carcinogenesis. Given our interest in Wnt signaling, we are particularly focused on developing models in

External Collaborators Wade Bushman, University of Wisconsin – Madison J. Fred Hess, Merck Pharmaceuticals Marc Kirschner and Adrian Salic, Harvard Medical School, Boston, Massachusetts Publications Holmen, Sheri L., Adrian Salic, Cassandra R. Zylstra, Marc W. Kirschner, and Bart O. Williams. In press. A novel set of Wnt-Frizzled fusion proteins identifies receptor components that activate βcatenin-dependent signaling. Journal of Biological Chemistry Yang, Hong, Bart O. Williams, Phillip W. Hinds, T. Shane Shih, Tyler Jacks, Roderick T. Bronson, and David M. Livingston. 2002. Tumor suppression by a severely truncated species of the retinoblastoma protein. Molecular and Cellular Biology 22(9): 3103–3110.

From left to right, back row: Williams, Daugherty, Robertson, Zylstra middle row: Giambernardi, Holmen front row: Charbonneau, Mieras

Laboratory of Structural Sciences H. Eric Xu, Ph.D. Dr. Xu went to Duke University and the University of Texas Southwestern Medical Center, where he earned his Ph.D. in molecular biology and biochemistry. Following a postdoctoral fellowship with Carl Pabo at MIT, Dr. Xu moved to GlaxoWellcome in 1996, where he solved the crystal structures of a number of nuclear hormone receptors. Until recently he was a senior research investigator of nuclear receptor drug discovery at GlaxoSmithKline in Research Triangle Park, North Carolina. Dr. Xu joined VARI as a Senior Scientific Investigator in July 2002.

Research Projects and cardiovascular disease. Millions of people have benefited from treatment with novel PPARγ medicines for type II diabetes; two of the top 100 drugs in 2001 were PPARγ ligands, having combined sales of over $2.4 billion. This demonstrates that studying PPARs can have a major effect on human health and disease treatment. To understand the molecular basis of ligand-mediated signaling by PPARs, we have determined crystal structures of each PPAR’s LBD (Figure 1)

ur laboratory is using x-ray crystallography to study structures of key protein complexes that are important in various signaling pathways, as well as for drug discovery relevant to human diseases such as cancers and diabetes. Our major focus is on the superfamily of nuclear hormone receptors, which includes receptors for classic steroid hormones such as estrogen, progesterone, androgens, and glucocorticoids, as well as receptors for proxisome proliferator activators, vitamin D, vitamin A, and thyroid hormones. These receptors are DNA-binding and ligand-dependent transcriptional factors that regulate genes essential for a broad aspect of human physiology, ranging from development and differentiation to metabolic homeostasis. A typical nuclear receptor contains three major domains: an N-terminal activation function-1 domain (AF-1), a central DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD). In addition to its role in ligand recognition, the LBD also contains dimerization motifs and an activation function-2 domain that is highly dependent on the bound ligand. The LBD is thus the key to the ligand-dependent regulation of nuclear receptor signaling and as such has been the focus of intense structural studies.

Figure 1. Crystal structure of the PPAR LBDs and their ligand-binding pocket. Each PPAR contains 13 α-helices and 4 small β-strands. The helices are arranged into a three-layer sandwich fold to create a ligand-binding pocket (white surface). The ability of PPARs to recognize so many and such diverse ligands can be accounted by the enormous size of the PPAR pocket, which is over 1,300 Å3 and is much larger than the pocket seen in any other nuclear receptor.

One set of receptors we will study is that of the peroxisome proliferator–activated receptors, alpha, delta, and gamma, (PPARα, δ, and γ). As nuclear receptors, PPARs are regulated by the binding of small-molecule ligands. In response to ligand binding, PPARs affect a wide range of biological activities, including the regulation of lipid metabolism and of glucose homeostasis. Thus, PPARs are important therapeutic targets for common diseases such as diabetes, cancer,

bound to many diverse ligands, including fatty acids, the lipid-lowering drugs called fibrates, and a new generation of antidiabetic drugs, glitazones. These structures have provided a framework for understanding the mechanisms of ago-

nists and antagonists, as well as the recruitment of co-activators and co-repressors in gene activation and repression. Furthermore, these structures also serve as a molecular basis for understanding potency, selectivity, and binding mode of diverse ligands, information that has provided critical insights for designing the next generation of PPAR medicines. The other nuclear receptor we will study is the human glucocorticoid receptor (GR). GR plays key roles in the metabolism of lipids and carbohydrates, development of the central nervous system, and homeostasis of the immune system. GR is also associated with numerous pathological pathways and as such is an important drug target. In fact, GR has a rich history in drug discovery and human medicine. There are more than 10 GR ligands (including dexamethasone) that are currently used for treatment of such diverse medical conditions as asthma, allergies, autoimmune diseases, and cancer. At the molecular level, GR can function either as a transcription activator or a transcription repressor. Both of these functions of GR are tightly regulated by small ligands that bind to the GR ligand-binding domain. To explore the molecular mechanism of ligand binding and signaling of GR, we have determined a crystal structure of the GR LBD bound to dexamethasone and a co-activator motif from TIF2. The structure reveals a novel LBDâ&#x20AC;&#x201C;LBD dimer interface (Figure 2) that is crucial for GR-mediated transactivation but not transrepression, suggesting a novel role of LBD dimerization in the GR signaling pathways. The structure also contains an unexpected charge clamp responsible for sequence-specific binding of co-activators and a unique ligand-binding pocket to account for specific recognition of diverse GR ligands. The detailed molecular interactions between the receptor and dexamethasone should also facilitate the discovery of new gluco-

Figure 2. Crystal structure of the human GR LBD bound to dexamethasone and a TIF2 co-activator. This structure reveals a novel dimer configuration, a second charge clamp, and a unique GR pocket. The GR LBD dimer interface, composed of Î˛-loops and turns, is distinct from the helix-10 interface of the RXR/PPARÎł heterodimer or the RXR homodimer.

corticoid receptor molecules that would reduce the side effects of current GR drugs. Beside PPARs and GR, the human genome contains 44 other nuclear receptors. X-ray structures have now been solved for more than a dozen LBDs, bound to agonists and antagonists, co-activators and co-repressors, and in forms of monomers, homodimers, heterodimers, and tetramers. These structures have illustrated the details of ligand binding, the conformational changes induced by agonists and antagonists, the basis of dimerization, and the mechanism of coactivator and co-repressor binding. All of the nuclear receptors studied to date have broadly similar structures and mechanisms of activation, but functionally significant differences have arisen among the various receptors over the course of evolution. We can expect more surprises as structural work continues on the remaining nuclear receptors, and as crystallographers tackle higherorder complexes involving the LBD with the AF1 domain, the DBD, and other proteins and nucleic acids involved in gene transcription.

Laboratory of Developmental Genetics Nian Zhang, Ph.D. Dr. Zhang received his M.S. in entomology from Southwest Agricultural University, People’s Republic of China, in 1985 and his Ph.D. in molecular biology from the University of Edinburgh, Scotland, in 1992. From 1992 to 1996, he was a Postdoctoral Fellow at the Roche Institute of Molecular Biology. He next served as a Postdoctoral Fellow (1996) and a Research Associate (1997–1999) in the laboratory of Tom Gridley in mammalian developmental genetics at the Jackson Laboratory, Bar Harbor, Maine. Dr. Zhang joined VARI as a Scientific Investigator in December 1999. Laboratory Members Staff Jun Chen, M.D., Ph.D. Jihong Ma, Ph.D. Lan Wang, Ph.D. Liang Kang

Research Projects The second focus of our laboratory is germ cell development, particularly the mechanisms that govern germ cell migration, survival, spermatogonial stem cell renewal and differentiation, and their implications for human disease. It is unclear how spermatogonial stem cells are regenerated during the entire reproductive life in mammals. Previous studies on the nematode Caenorhabditis elegans have shown that the Notch/lin12-mediated signal transduction pathway is important for germ cells to remain in an undifferentiated state. Mutations that compromise this pathway force germ cells to enter meiosis earlier than normal. A constitutively activated signal prevents germ cells from entering meiosis, resulting in overproliferation of germ cells, a phenotype called “germ cell tumor.” Given the fact that some members in the Notch signaling pathway are expressed in the testis, we speculate that Notch signaling may play a similar role in spermatogonial differentiation in mammals. We will further examine the role Notch signaling may play during spermatogenesis using transgenic animals and conditional gene targeting.

e are interested in understanding the cellular and molecular mechanisms underlying pattern formation during embryonic development. We previously cloned and targeted the mouse Lunatic fringe (Lfng) gene, which plays an important role in embryo segmentation. Mice homozygous for the Lfng mutation suffer from severe malformation in their axial skeletons as a result of irregular somite formation during embryonic development. Lfng encodes a secreted signaling molecule essential for regulating the Notch signaling pathway in mice. We showed that Lfng expression was in response to a biological clock that oscillated once during the formation of each segment, and the failure of the Lfng mutants in responding to this clock resulted in the abnormal segmentation phenotype.

We want to understand how the rhythmic expression of Lfng is controlled. Using transgenic animals, we are analyzing regulatory elements that control Lfng expression, and we are also isolating genes that regulate Lfng expression. In addition, we are identifying proteins that interact with Lfng during embryo segmentation. Another approach that we are taking to understand somitogenesis is to examine a spontaneous mutant with a phenotype similar to that of the Lfng mutant. We plan to clone this mutation positionally and test if this mutation interacts with Lfng and other mutations that affect somitogenesis.

We are also studying a spontaneous mutation that causes sterility. Preliminary data suggest that this mutation arrests the development of spermatogenic cells at meiosis II. We are interested in identifying the gene that is disrupted by this mutation and understanding the role that this gene plays in meiosis.

External Collaborators Xiang Gao, Center of Model Animal Genetics, Nanjing University, Peopleâ&#x20AC;&#x2122;s Republic of China Douglas L. Pittman, Medical College of Ohio, Toledo

From left to right: Chen, Kang, Wang, Zhang

Daniel Nathans Memorial Award

Daniel Nathans Memorial Award The Daniel Nathans Memorial Award was established in memory of Dr. Daniel Nathans, a distinguished member of our scientific community and a founding member of VARI’s Board of Scientific Advisors. We established this award to recognize individuals who emulate Dan and his contributions to biomedical and cancer research. It is our way of thanking and honoring him for his help and guidance in bringing Jay and Betty Van Andel’s dream to reality. The Daniel Nathans Memorial Award was announced at our inaugural symposium, “Cancer & Molecular Genetics in the Twenty-First Century,” in September 2000.

Award Recipients 2000 2001

Richard D. Klausner, M.D. Francis S. Collins, M.D., Ph.D.

Francis S. Collins, October 2, 2001

Postdoctoral Fellowship Program

Postdoctoral Fellowship Program The Van Andel Research Institute provides postdoctoral training opportunities to Ph.D. scientists beginning their research careers. The fellowships help promising scientists advance their knowledge and research experience, while at the same time supporting the research endeavors of VARI. The fellowships are funded in three ways: 1) by the laboratories to which the fellow is assigned; 2) by the VARI Office of the Director; or 3) by outside agencies. Each postdoctoral fellow is assigned to a scientific investigator who oversees the progress and direction of research. Postdoctoral fellows who worked in VARI laboratories over the past year are listed below.

Troy Giambernardi University of Texas Health Science Center, San Antonio VARI mentor: Bart Williams

Eduardo Azucena Wayne State University, Detroit, Michigan VARI mentor: Sara Courtneidge Andrei Blokhin Institute of Biochemical Physics, Moscow, Russia VARI mentor: Michael Weinreich

Steven Gray Karolinska Institute, Stockholm, Sweden VARI mentor: Bin Teh

Jean-Franรงois Bodart University of Science and Technology, Lille, France VARI mentor: Nicholas Duesbery

Xiang Guo Sun Yat-Sen University of Medical Sciences, Guangzhou, China VARI mentor: Bin Teh

Jun Chen West China University of Medical Sciences, Chengdu, China VARI mentor: Nian Zhang

Sheri Holmen Mayo Graduate School, Rochester, Minnesota VARI mentor: Bart Williams Sok Kean Khoo Tokyo University of Fisheries, Japan VARI mentor: Bin Teh

Jindong Chen Karolinska Institute, Stockholm, Sweden VARI mentor: Bin Teh Suganthi Chinnaswamy Southern Illinois University, Carbondale VARI mentor: Cindy Miranti

Hasan Korkaya International Center for Genetic Engineering and Biotechnology, New Delhi, India VARI mentor: Sara Courtneidge

Arun Chopra Jiwaji University, Gwalior, India VARI mentor: Nicholas Duesbery

Jihong Ma Jiamusi Medical College, Jiamusi, China VARI mentor: Nian Zhang

Kathryn Eisenmann University of Minnesota, Minneapolis VARI mentor: Han-Mo Koo

Jeremy Miller University of Michigan, Ann Arbor VARI mentor: Craig Webb

Chongfeng Gao Tokyo Medical and Dental University, Japan VARI mentor: George Vande Woude

Donald Pappas, Jr. Louisiana State University, Baton Rouge VARI mentor: Michael Weinreich

Andrew Putnam University of Michigan, Ann Arbor VARI mentor: Cindy Miranti

Sridhar Venkataraman Michigan State University, Lansing VARI mentor: Michael Weinreich

Chao-Nan Qian Sun Yat-Sen University of Medical Sciences, Guangzhou, China VARI mentor: Bin Teh

Bradley Wallar University of Minnesota, Minneapolis VARI mentor: Arthur Alberts Chun Zhang Tokyo Medical and Dental University, Japan VARI mentor: Bin Teh

Libing Song Sun Yat-Sen University of Medical Sciences, Guangzhou, China VARI mentor: Bin Teh

Huiying Zhang Institute of Microbiology and Epidemiology, Beijing, China VARI mentor: Brian Cao

Jun Sugimura Iwate Medical University, Morioka, Japan VARI mentor: Bin Teh

Heping Zhou Fudan University, Shanghai, China VARI mentor: Brian Haab

Rebecca Uzarski Michigan State University, Lansing VARI mentor: Sara Courtneidge

Student Programs

Grand Rapids Area Pre-College Engineering Program The Grand Rapids Area Pre-College Engineering Program (GRAPCEP) is administered by Davenport College and jointly sponsored and funded by Pfizer, Inc., and VARI. The program is designed to provide selected high school students who have plans to major in science or genetic engineering in college the opportunity to work in a research laboratory. In addition to training in research methods, the students also learn workplace success skills such as teamwork and leadership. The three 2002 GRAPCEP students are Marie Graves Union High School Grace Miguel Central High School Jeanine Myles Ottawa Hills High School

Summer Student Internship Program The VARI summer student internships were established to provide college students with an opportunity to work with professional researchers in their fields of interest, to use state-of-the-art equipment and technology, and to learn invaluable people and presentation skills. At the completion of the 10week program, the students summarize their projects in oral presentations. From September 2001 through August 2002, VARI hosted 38 students from 13 colleges and universities, both in formal summer internships and in other student positions during the year.

Albion College, Albion, Michigan Cassandra Van Dunk

Grand Valley State University, Allendale, Michigan Heather Bill Jenn Daugherty Davina Gutierrez Katie Kahnoski Susan Kitchen Nate Lanning Adi Laser Brandon Leeser Therese Roth

Aquinas College, Grand Rapids, Michigan Donald Chaffee Holli Charbonneau Ashley Mynsberge Sarah Scollon Calvin College, Grand Rapids, Michigan Kelly Ballast Dan Diephouse Todd Lavery Andrea Pearson Meghan Sheehan

Harvard University, Cambridge, Massachusetts Christine Moore Hope College, Holland, Michigan Jason Johnson

Duke University, Durham, North Carolina Joe Crawley

Kenyon College, Gambier, Ohio Lisa Maurer

Grand Rapids Community College, Michigan Marketta Hassen Yasser Jimenez Kofi Obeng

Michigan State University, Lansing Erik Freiter Sara Kienzle Tony Kokx Casey Madura Tracey Millard

Michigan Technological University, Houghton Hien Dang Radoslav Nickolov

University of Chicago, Illinois Jon Douglas University of Michigan, Ann Arbor Daphna Atias Jennie Edgar

Spring Arbor University, Spring Arbor, Michigan Matthew Main Stanford University Dan Wohns

VARI Seminar Series

VARI Seminar Series 2001 September Phillippe Soriano, Hutchinson Cancer Research Center, Seattle, Washington “PDGF signaling in mouse development” October Francis Collins, National Institutes of Health, Bethesda, Maryland The Daniel Nathans Lecture: “Decrypting the genome: consequences of the Human Genome Project for medicine and society” November Tony Wynshaw-Boris, University of California, San Diego “Modeling human genetic diseases in the mouse” Mary Ann Handel, University of Tennessee, Knoxville “Genetic models for analysis of chromosome segregation and gametogenesis” Tayyaba Hasan, Harvard Medical School, Boston, Massachusetts “Therapeutic and diagnostic approaches using light-activatable chemicals” John Blenis, Harvard University, Cambridge, Massachusetts “Regulation of cell motility, size and proliferation by Ras and PI3-kinase signaling systems” Sandra Rempel, Henry Ford Hospital, Detroit, Michigan “SPARC modulates glioma growth, attachment, and migration in vitro and in vivo” December Gregg Gundersen, Columbia University, New York, New York “Regulation of microtubules by Rho and Cdc42 GTPases in migrating fibroblasts” Constantine Stratakis, National Institutes of Health, Bethesda, Maryland “Molecular genetics of adrenocortical tumors: Carney complex and PRKAR1A, a novel tumor suppressor gene”

2002 January Don Bottaro, EntreMed, Inc., Rockville, Maryland “Extracellular and intracellular regulation of hepatocyte growth factor signaling” John Young, University of Wisconsin – Madison “Retrovirus-receptor and anthrax toxin-receptor interactions” Robert Sigler, Esperion Therapeutics, Inc., Ann Arbor, Michigan “Role of the toxicologic pathologist in drug development” February Michael Sheets, University of Wisconsin – Madison “Control of early Xenopus development by regulated cell signaling” Michael Clague, University of Liverpool, United Kingdom “Linkages between tyrosine kinase receptor trafficking, generation of phosphophosphatidylinositol lipids, and cell signaling” Michael Brenan and Nick LaRusso, Mayo Clinic, Rochester, Minnesota “The cholangiopathies: from bedside to bench and hopefully back!” James Herman, John Hopkins University, Baltimore, Maryland “Promoter methylation in cancer: biology and clinical applications” Robert King, Bristol-Myers Squibb, Wilmington, Delaware “Regulation of hepatitis C virus negative-strand RNA replication by 5′ and 3′ untranslated regions” March Steven Frisch, Burnham Institute, La Jolla, California “Cell adhesion, apoptosis, and the epithelial phenotype” Sue Vande Woude, Colorado State University, Fort Collins “The biology of nondomestic feline lentiviruses” Peter Sicinski, Harvard University, Cambridge, Massachusetts “Cyclins in development and in breast cancer” Peter Vogt, Scripps Research Institute, LaJolla, California “The secret life of oncogenes”

April David Waters, Purdue University, West Lafayette, Indiana “The role of pet dogs with spontaneous bone and prostate cancers in the development of new cancer imaging and therapeutic agents” John Condeelis, Albert Einstein College of Medicine, Bronx, New York “Mechanisms of chemotaxis of carcinoma cells during metastasis from the primary tumor” Natalie Ahn, University of Colorado, Boulder “Regulation and function of the MAP kinase pathway” Benjamin Neel, Harvard School of Medicine, Boston, Massachusetts “Tyrosine phosphatases in health and disease” May George Klein, Karolinska Institute, Stockholm, Sweden “The role of epigenetic and genetic changes in tumor evolution” Louis Staudt, National Cancer Institute, Bethesda, Maryland “Molecular diagnosis of cancer by gene expression profiling” Olga Volpert, Northwestern University, Chicago, Illinois “The cross-talk between inducers and inhibitors of angiogenesis” Janet Rossant, Mount Sinai Hospital, New York, New York “Stem cells from the mammalian blastocyst” Bin Sing Teh, Baylor College of Medicine, Houston, Texas “Combined gene therapy and intensity-modulated radiotherapy (IMRT) for prostate cancer” Jim Woodgett, University of Toronto, Canada “Physiological functions and regulation of protein kinase B and GSK-3” June Judith Sebolt-Leopold, Pfizer, Ann Arbor, Michigan “The potential of MEK inhibitors for anticancer therapy” John Diffley, Cancer Research U.K., Hertfordshire, United Kingdom “DNA replication, genome stability, and cancer: lessons from budding yeast?” Steve Goff, Columbia University, New York, New York “Host gene products affecting the replication of mammalian retroviruses”

July Stan Korsmeyer, Dana-Farber Cancer Institute, Boston, Massachusetts “Mitochondrial gateway to apoptosis” Valerie Weaver, University of Pennsylvania, Philadelphia “Stromal-epithelial interactions and breast cancer progression: a structural perspective”

Conference

VIII International Workshop on Multiple Endocrine Neoplasia The Van Andel Research Institute hosted the VIII International Workshop in Grand Rapids, Michigan, on June 16–18, 2002. Over 180 researchers took part in the sessions. Hosted by Bin T. Teh of VARI, the workshop featured 29 speakers from around the world.

MEN Conference speaker list Sunita Argawal National Institutes of Health, U.S.A.

Irina Lubensky National Cancer Institute, U.S.A.

Douglas Ball John Hopkins University, U.S.A.

Eammon Maher University of Birmingham, U.K.

Albert Beckers, University of Liège, Belgium

Stephen Marx National Institutes of Health, U.S.A.

John Carpten National Institutes of Health, U.S.A.

Jeffrey F. Moley Washington University School of Medicine, U.S.A.

Judy Crabtree National Institutes of Health, U.S.A.

Lois Mulligan Queen’s University, Canada

Charis Eng The Ohio State University, U.S.A.

Patricia Niccoli-Sire Hospital of Timone, France

Nicholas Hayward Queensland Institute of Medical Research, Australia

Naganari Ohkura National Cancer Center Research Institute, Japan

Geoff Hendy McGill University, Canada

Bruce Robinson Sydney University, Australia

Wouter de Herder Erasmus Medical Centre, Netherlands

G. Romeo International Agency for Research on Cancer, France

Robert Jensen National Institutes of Health, U.S.A.

James Scheiman University of Michigan, U.S.A.

Sissy Jhiang The Ohio State University, U.S.A.

Joseph Shepherd University of Tasmania, Australia

Catharina Larsson Karolinska Institute, Sweden

Constantine Stratakis National Institutes of Health, U.S.A.

Rajesh V. Thakker University of Oxford, England

Masahide Takahashi Nagoya University School of Medicine, Japan

Yu Xiong University of North Carolina, U.S.A.

Bin T. Teh Van Andel Research Institute, U.S.A.

Zhengping Zhuang National Cancer Institute, U.S.A.

Session chairs Maria-Luisa Brandi University of Florence, Italy

Harmut HP Neumman Albert-Ludwigs University, Germany

Settara Chandrasekharappa National Institutes of Health, U.S.A.

Magnus Nordenskjold Karolinska Institute, Sweden

Sara Courtneidge Van Andel Research Institute, U.S.A.

Britt Skogseid University of Uppsala, Sweden

Christopher Ellison The Ohio State University, U.S.A.

Constantine Stratakis National Institutes of Health, U.S.A.

Charis Eng The Ohio State University, U.S.A.

Pam Swiatek Van Andel Research Institute, U.S.A.

Robert Gagel M.D. Anderson Cancer Center, U.S.A.

Norman Thompson University of Michigan, U.S.A.

Catharina Larsson Karolinska Institute, Sweden

George Vande Woude Van Andel Research Institute, U.S.A.

C.J. Lips University Hospital Utrecht, The Netherlands

Bart Williams Van Andel Research Institute, U.S.A.

Eammon Maher University of Birmingham, U.K.

Organization

Van Andel Research Institute Director

George Vande Woude, Ph.D.

Deputy Director

Associate Director for Research Administration

Sara Courtneidge, Ph.D.

Roberta Jones

Administrator to the Director

Science Editor

Michelle Reed

David E. Nadziejka

Left to right: Shellie Kraemer Shelly Novakowski Lynn Ritsema

Research Administration Group

Not shown: Carol Hallas Kaye Johnson

Van Andel Research Institute Boards

VARI Board of Trustees David L. Van Andel, Chairman and CEO Christian Helmus, M.D. Fritz M. Rottman, Ph.D. James B. Wyngaarden, M.D.

David L. Van Andel Board of Scientific Advisors The Board of Scientific Advisors advises the CEO and the Board of Trustees, providing recommendations and suggestions regarding the overall goals and scientific direction of VARI. The members are Michael S. Brown, M.D., Chairman Richard Axel, M.D. Joseph J. Goldstein, M.D. Richard D. Klausner, M.D. Phillip A. Sharp, Ph.D.

Scientific Advisory Board The Scientific Advisory Board advises the VARI Director, providing recommendations and suggestions specific to the ongoing research, especially in the areas of cancer, genomics, and genetics. It also coordinates and oversees the scientific review process for the Instituteâ&#x20AC;&#x2122;s research programs. The members are Alan Bernstein, Ph.D. Malcolm Brenner, M.D., Ph.D. Patrick O. Brown, M.D., Ph.D. Webster Cavenee, Ph.D. Tony Hunter, Ph.D. Frank McCormick, Ph.D. Davor Solter, M.D., Ph.D. Bruce Stillman, Ph.D.

Van Andel Institute Administrative Organization The organizational units listed below provide administrative support to both the Van Andel Research Institute and the Van Andel Education Institute.

Executive R. Jack Frick, Chief Financial Officer Ann Schoen

Purchasing Richard Disbrow, Manager David Clark Christian Kutchinski Amy Poplaski

Communications and Development Casey Wondergem, Vice President Sandra G. Katt Margo Pratt Tina Shelton

Facilities Samuel Pinto, Supervisor Jason Dawes Gerald Ladd Richard Ulrich

Information Technology Bryon Campbell Chief Information Officer David Drolett, Manager Michael Roe, Manager Kathleen Cerasoli Michael Foster Kenneth Hoekman Kimberlee Jeffries Candy Wilkerson

Security Kevin Denhof, Chief Kelley Herrick Glass Washing/ Media Preparation Melissa Donnelly Troy Lawson Contract Support Valeria Long, Librarian (Grand Valley State University) Jim Kidder, Safety Manager (Michigan State University) Stephen Burns, Housekeeping Tim Pospisil, Housekeeping Raymond Rupp, Housekeeping

Human Resources Linda Zarzecki, Manager Margie Hoving Pamela Murray Angela Plutschouw Grants and Contracts Carolyn Witt, Director Sara Oâ&#x20AC;&#x2122;Neal Finance Timothy Myers, Controller Matthew Blok, Asst. Controller Richard Herrick Keri Jackson Angela Lawrence Jamie VanPortfleet

VARI Photos 2001-2002

100

Back cover photo: Genetic ablation of the Drf1 gene gives rise to hypermotile cells The Drf1 gene was genetically ablated by homologous recombination. Drf1â&#x20AC;&#x201C; cells become hypermotile and produce large lamellipodia. These observations suggest that Drf1 genes participate in the regulation of motility and that they negatively regulate motility. (Peng, Swiatek, and Alberts)

The Van Andel Institute and/or its affiliated organizations (VARI and VAEI), through its responsible managers, recruits, hires, upgrades, trains, and promotes in all job titles without regard to race, color, religion, sex, national origin, age, disability status, or veteran status, except where an accommodation is unavailable and/or is a bone fide occupational qualification.

Van Andel Research Institute

Scientific Report 2002

333 Bostwick Avenue, N.E., Grand Rapids, MI 49503 Phone (616) 234-5000; Fax (616) 234-5001; Web site: www.vai.org

Cover photograph of the Van Andel Institute building, Grand Rapids, Michigan ÂŠ Jeff Goldberg/Esto