2004 Van Andel Research Institute Scientific Report by Van Andel Institute

Van Andel Research Institute

Scientific Report 2004

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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

Van Andel Research Institute Scientific Report 2004

Title page photo: Spectral karyotyping (SKY) SKY is a powerful 24-color, whole-chromosome painting assay that allows the visualization of each chromosome as a different color. Five spectrally distinct dyes are used in combination to create the unique cocktail of probes. The probe mixture is hybridized to metaphase chromosomes on a slide, then visualized through a spectral interferogram cube that allows the measurement of the entire emission spectrum with a single exposure. The image is captured with a CCD (charge-coupled device) camera system and processed by computer software. The software (SKYView, Applied Spectral Imaging) can distinguish differences in color not discernible to the naked eye by assigning a numerical value to a pseudocolor and/or an RGB color. SKY can detect chromosome material of unknown origin, complex rearrangements, translocations, large deletions, duplications, aneuploidy, and more. In the mouse fibroblast cell line shown here, the genome was found to be tetraploid (four of each chromosome instead of the normal two), with gains and losses of certain chromosomes; for example, loss of one chromosome 7 and gain of two additional copies of chromosome 19. Also, about 14% of the metaphases had a derivative chromosome 4 with a translocation from chromosome 3. (Koeman and Swiatek)

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

Han-Mo Koo, Ph.D. 1963â&#x20AC;&#x201C;2004 A native of Korea, Han-Mo Koo joined the Van Andel Research Institute in June of 1999 as the principal investigator of the Laboratory of Cancer Pharmacogenetics, in which he established, among others, projects to study melanoma and pancreatic cancer. In May 2004, Han-Mo passed away following a six-month battle with cancer. We mourn and miss him as an exceptional scientist, a generous colleague, and a special friend.

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

Laboratory Reports Cell Structure and Signal Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Arthur S. Alberts, Ph.D. Antibody Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Brian Cao, M.D. Mass Spectrometry and Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Gregory S. Cavey, B.S. Signal Regulation and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Sara A. Courtneidge, Ph.D. Developmental Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Nicholas S. Duesbery, Ph.D. Vivarium and Transgenics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Bryn Eagleson, A.A. Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Kyle Furge, Ph.D. Cancer Immunodiagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Brian B. Haab, Ph.D. Cancer Pharmacogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Han-Mo Koo, Ph.D. Integrin Signaling and Tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Cindy K. Miranti, Ph.D. Analytical, Cellular, and Molecular Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 and Molecular Diagnostics James H. Resau, Ph.D. Germline Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Pamela J. Swiatek, Ph.D. Cancer Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Bin T. Teh, M.D., Ph.D.

Molecular Oncology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 George F. Vande Woude, Ph.D. Tumor Metastasis and Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Craig P. Webb, Ph.D. Chromosome Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Michael Weinreich, Ph.D. Cell Signaling and Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Bart O. Williams, Ph.D. Structural Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 H. Eric Xu, Ph.D. Mammalian Developmental Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Nian Zhang, Ph.D.

Daniel Nathans Memorial Award . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Postdoctoral Fellowship Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Student Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

VARI Seminar Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Recent VARI Photos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Directorâ&#x20AC;&#x2122;s Introduction

Directorâ&#x20AC;&#x2122;s Introduction I am pleased to present our Van Andel Research Institute (VARI) Scientific Report. It is only five years since we began our research with a group of pioneers in rented space across George F. Vande Woude the street at the Butterworth campus of Spectrum Health Hospital. In January of this year we celebrated our fourth year in our spectacular research facility. We have grown considerably during this time and it is my privilege to tell you in this report about some of our accomplishments.

This may lead to opportunities for identifying new cancer-fighting drug targets. VARI recently initiated its first drug development and discovery program in the Laboratory of Cell Structure and Signal Integration. The new study, funded by a two-year R21 grant from the National Institutes of Health, is an outgrowth of research conducted four years ago by Arthur Alberts and his team, in which they discovered a molecule called DAD. Their recent work shows that this molecule has the ability to attack cancer cells. This finding has placed DAD at the forefront of a new class of anti-cancer drugs and is the basis of the current project. Art found that DAD has effects similar to those of the anti-cancer drug Taxol, which is used to treat breast cancer. However, because DAD is a subunit of a normal protein and works differently, Art and his lab hope to develop new and effective therapies that lack the harsh side effects commonly associated with Taxol.

All of our laboratories have become solidly established during the past few years. This has resulted in a steady increase in workload for my office, so in late 2003 I appointed three Deputy Directors: Bin Tean Teh is our Deputy Director for Research Operations, James Resau is Deputy Director for Special Programs, and Rick Hay is Deputy Director for Clinical Programs. They have already been a great help in managing and directing our Institute and programs.

During embryonic development, primordial germ cells migrate to form the gonads. Identifying the genes that contribute to this process and how they function will lead to a better understanding of fertility, and might also contribute to our knowledge of how cancer cells migrate. The National Institute of Child Health and Human Development awarded a five-year R01 grant to Nian Zhang to characterize the mouse mutation atrichosis (at), which causes male and female sterility.

Over the past year our scientists have had significant success in obtaining funding from many different sources, including the National Cancer Institute as well as other Institutes at the National Institutes of Health (NIH). Kyle Furge received the first NIH grant awarded to VARI, an R33. The grant is funding a three-year study to develop algorithms to predict chromosomal changes in kidney cancer based on gene expression microarray analysis of tumor cells.

Researchers at VARI continue their groundbreaking work in developing new therapies for pancreatic cancer, the fourth highest cause of cancer deaths in the United States. Last year, the Institute was awarded a two-year grant from the Cancer Research and Prevention Foundation for a study related to the early detection of the disease. Brian Haab and scientists in the Laboratory of Cancer Immunodiagnostics are studying proteins that can be used as early indicators of pancreatic cancer, which has a 99% mortality rate. Their work could result in a simple test to screen for the disease.

Sara Courtneidge has received an NIH research project grant (R01) to fund a five-year biological and genetic analysis of a Src substrate termed Fish. Src has been implicated in the development, progression, and metastasis of a number of human tumor types, especially those of breast and colon. Sara and members of her Laboratory of Signal Regulation and Cancer are conducting research that will shed light on the mechanisms by which cells control growth, motility, and the production of growth factors.

A recent three-year grant from the Lance Armstrong Foundation has allowed Bin Teh to broaden his study of testicular cancer, the most common form of cancer among men between the 3

Osteoporosis Foundation, the Life Sciences Research Foundation, the American Brain Tumor Foundation, the Gerber Foundation, and the Multiple Myeloma Research Foundation.

ages of 15 and 35. Previously, Bin collaborated with Indiana University’s Dr. Larry Einhorn, one of our previous Nathans Award recipients, on a study to determine why 10% of testicular cancer patients are resistant to treatment for the disease. Through this research the scientists identified a set of genes that distinguishes between testicular cancer patients who relapse during the early stages of therapy and those who relapse two or more years after their initial treatment. The latter group is resistant to chemotherapy. The new study could allow the identification of the underlying mechanisms for chemoresistance in those patients so that additional intervention measures may be applied.

During the past year Nick Duesbery has extended studies on the discovery that anthrax tumor lethal factor (TLF) has potential as an antitumor agent in the treatment of not only melanoma (studies with HanMo Koo) but also certain sarcomas. This has opened new avenues for investigation and has led to a collaboration with Dr. Art Frankel and a partnership with Wake Forest University Medical School to produce TLF of a pharmaceutical grade suitable for preclinical studies and potential testing in humans. By spring of 2005 there should be a significant stock of TLF for testing.

In a new study on prostate cancer, our scientists hope to gain a clearer understanding of what makes this disease spread. Led by Cindy Miranti, the goal of this project is to determine what happens to prostate tumor cells when two known metastatic suppressor genes stop functioning. Funded by a three-year grant from the Department of Defense, the research will allow Cindy and her Laboratory of Integrin Signaling and Tumorigenesis to better understand how the loss of function in these genes leads to increased metastasis. Currently, metastatic prostate cancer is not treatable. Cindy’s research aims to identify new and effective therapies that will ultimately save lives.

Craig Webb is establishing a dedicated multiple myeloma laboratory at VARI. This new research effort will use integrated genomic and proteomic capabilities developed within the laboratory to study the molecular causes of multiple myeloma. Current work centers on identifying the genes and proteins that can predict the response of myeloma patients to existing therapies, as well as to identify novel treatments for the disease. The goals are to develop tests that accurately diagnose the disease in its earliest stages and identify the optimal treatments for patients with multiple myeloma. This research is being conducted in collaboration with several oncologists/hematologists from Grand Rapids, Detroit, and Toronto. The multiple myeloma lab is being supported in part through generous contributions from the McCarty Foundation and a fellowship to Jennifer Bromberg-White from the Multiple Myeloma Research Foundation.

The Department of Defense also funded a three-year grant for Bart Williams to test the hypothesis that alterations in β-catenin regulation directly contribute to prostate cancer progression and the propensity of prostate cancer to metastasize to the bone. Members of Bart’s laboratory, in collaboration with Dr. Wade Bushman at the University of Wisconsin, have created a mouse model in which up-regulation of β-catenin leads to the development of early-onset prostate cancer. Their current work is aimed at examining the androgen dependence of these tumors, their capacity to metastasize, and the genetic interaction of changes in Wnt signaling with other genetic changes observed in prostate cancer.

Brian Cao (Laboratory of Antibody Technology) and Rick Hay (Laboratory for Molecular Oncology) have collaborated in the development of two radiolabeled monoclonal antibodies to the Met receptor molecule for nuclear imaging studies. We are currently using these radiolabeled antibodies in animal models of human and canine prostate cancer. These antibodies could potentially be used for radioimmunotherapeutic applications as well.

Additional awards, including postdoctoral fellowships, have come from many other funding organizations, such as the Elsa U. Pardee Foundation, the Wenner-Gren Foundation, the March of Dimes, the American Cancer Society, the National Cancer Center, the National

Both Greg Cavey and Eric Xu, the most recent appointments to our staff, have their labo4

ratories in full operation. Greg (Laboratory of Mass Spectrometry and Proteomics) is supporting a number of the VARI labs with Q-tof (quadrupole-time of flight) mass spectrometry for many types of proteomic studies. Two additional instruments, a second Q-tof and an ion trap mass spectrometer, are soon to be operational. Eric’s Laboratory of Structural Sciences is busy with studies of nuclear hormone receptors and the Met tyrosine kinase receptor, two different classes of proteins that have been proven to be important therapeutic targets for cancer and metabolic diseases. The lab has generated crystals of a number of nuclear hormone receptors and of fragments of hepatocyte growth factor (a Met receptor ligand). They are using the Advanced Photon Source (APS) at Argonne National Laboratory near Chicago to obtain high-resolution diffractions from these crystals. Together with Michigan State University, the University of Michigan, and Northwestern University, VARI was a founding member of the Life Sciences Collaborative Access Team that operates the APS Sector 21.

under the direction of Rick Hay, our Deputy Director for Clinical Programs, has been very well received by the community. We have partnered with the DeVos Children’s Hospital in Grand Rapids in a funded pediatric training fellowship program focused on pediatric cancer and blood disorders. DeVos Children’s will recruit one fellow per year for the three-year training program. During the second year and a half of their fellowship, the physicians will work at VARI with our cancer biologists and geneticists, learning cutting-edge technology in cancer gene analysis and aiding our efforts toward new discoveries that will help treat childhood cancers. As you saw on p. iii of this Report, Han-Mo Koo passed away on May 3, 2004, after a sixmonth-long battle with cancer. He joined my laboratory at the National Cancer Institute in Frederick, Maryland, in 1993 as a postdoctoral fellow and came to Grand Rapids with me in 1999. He established the Laboratory of Cancer Pharmacogenetics, and his laboratory was making real progress in showing and understanding the sensitivity of melanoma tumors to inhibitors of the MEK/MAPK pathway. Cancer affects every one of us, and for those of us at VARI, losing beloved friends and colleagues to the disease, like Han-Mo Koo and Dwight Reed, Michelle Reed’s husband, is just terrible. Han-mo spent his life trying to understand and cure cancer, and this has added even more incentive to our search for a cure. We have renamed our seminar series the Han-Mo Koo Memorial Seminar Series.

We continue to actively integrate into the Grand Rapids medical community as part of our commitment to translation of our research into clinical practice. We are now a member of the Michigan Cancer Consortium (MCC), a partnership of 75 public, private, and voluntary organizations committed to reducing the human and economic impact of cancer in Michigan. MCC was formed and its priorities developed as a focused, coordinated initiative among member organizations that will collectively have an impact far greater than individual efforts. One MCC priority is to increase the number and diversity of participants enrolled in clinical cancer research.

In April 2003, VARI was invited to become a member of the Grand Rapids Clinical Oncology Program (GRCOP). GRCOP is one of the oldest of the 53 NCI community clinical oncology programs (CCOP) in the United States, and we have the unique distinction of being the first basic research institution to affiliate with a CCOP. We anticipate a growing collaboration with them, and we will try to become more involved with the clinical trials network, in addition to the ongoing pancreatic cancer Phase II clinical trial. That pancreatic clinical trial was initiated with GRCOP in 2002, with VARI’s Han-Mo Koo serving as the project’s Research Investigator. The trial has enrolled 21 patients to date and continues; I will substitute for Han-Mo as Research Investigator.

In 2003 we established the Tumor Tissue Donation Program. Through an informational effort directed at the lay public and collaborating healthcare providers, we emphasized the crucial importance of fresh tumor tissue samples to our studies, which aim to determine the causes of cancer, how to prevent it, and how to treat it. We have asked area physicians and hospitals to seek consent from patients undergoing cancer surgery so that we may obtain tumor tissue left over from the surgery for research use. This program, 5

Each year we select a member of the research community to receive the Daniel Nathans Memorial Award, choosing a scientist who has emulated Dr. Nathans’ extraordinary contributions and special character (see page 69). Our most recent recipient was Robert A. Weinberg, who received the award for his pioneering work in cancer research as discoverer of the first human oncogene and the first tumor suppressor gene. Dr. Weinberg visited Grand Rapids on March 8, 2004, to receive the Nathans Award, and he presented two talks, one for the technical audience on mechanisms of human tumor formation, which involved some of his current research on telomerases, and a second that evening for the public titled “How cancer begins.”

which has provided funding via grants for the state’s life sciences efforts. As part of that effort, the University of Michigan, Michigan State University, Wayne State University, and the Van Andel Research Institute joined together to form the Core Technology Alliance (CTA) to provide statewide, cutting-edge core technologies in genomics, proteomics, bioinformatics, structural biology, and animal models. Funding from the MLSC has enabled these services to grow and is stimulating and strengthening research in the life sciences, which helps to promote the development of a strong biotechnology industry in Michigan. It is anticipated that the core services offered through the CTA will become self-sustaining over time. A peer review in 2003 by the American Association for the Advancement of Science supported that premise and recommended the formation of a corporation to provide business and marketing management expertise for the core services. The CTA was incorporated as a 501(c)3 organization in March 2004 with the four institutions as founding members. In late 2003, to strengthen ties to other Michigan opportunities, Governor Jennifer Granholm added to the MLSC the automotive industry and bioterrorism initiatives and renamed it the Michigan Technology Tri-Corridor (MTTC). I was appointed to be one of the members of the MTTC Steering Committee. The MTTC is continuing to solicit and fund grant applications in the life sciences.

On May 1, 2004, in collaboration with Wayne State University, the University of Michigan, and the Henry Ford Health System, VARI sponsored a one-day symposium on prostate cancer research. Hosted by the Michigan Prostate Research Colloquium, this symposium, entitled “Basic and Clinical Advances in Prostate Cancer Research,” was designed to bring local science and medical researchers in Michigan together to foster interactions and collaborations. The highlights included the keynote lecture delivered by Dr. Carrie Rinker-Schaeffer from the University of Chicago, who spoke on prostate cancer metastasis, and two speakers from each participating institution who presented their work on prostate cancer. The symposium also provided an opportunity for local physicians to learn about the latest basic and clinical prostate cancer research being conducted in the state.

As always, we wish to express our gratitude for the generosity of the Van Andel family. In January 2004, we, as a community, mourned the passing of Betty Van Andel. We are privileged to be a part of turning the vision of Jay and Betty into reality.

In 1999, legislation was enacted to form the Michigan Life Sciences Corridor (MLSC),

Van Andel Research Institute Laboratory Reports

Sites of interaction between the small GTPase RhoB and one of its effectors, mDia2, as determined by fluorescence resonance energy transfer (FRET). RhoB and mDia2 were fused to fluorescent proteins, cyan (blue-green)- and yellow-fluorescent protein, respectively. The cell was illuminated with light at a wavelength that excites the RhoB fusion protein and causes it to emit fluorescence photons; these emitted photons can cause the mDia2 fusion protein to fluoresce in turn, but only when the proteins are in very close proximity. This allows us to visualize where and when the two proteins interact. In this case, RhoB and mDia2 interact on vesicles that are important for the intracellular movement of growth factor receptors. Our data suggests that RhoB and mDia2 act as a circuit that controls the extent to which cells respond to positive growth cues. (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 Art Alberts, Ph.D. Jun Peng, M.D. Kathryn Eisenmann, Ph.D. Lisa Alberts

Visiting Scientists Stephen Matheson, Ph.D. Brad Wallar, Ph.D.

Students Tim Stowe Dare Odumosu Dave Beversluis

Research Interests ur lab is interested in the intracellular signaling networks that regulate cell proliferation and movement and how those networks become disrupted during tumor formation. Normal cells base growth decisions upon the sum of positive and negative inputs derived from extracellular cues. These signals are processed by biochemical networks. If the networks become unbalanced—for example, by viral factors or DNA damage—the cells will arrest and/or undergo a form of programmed cell suicide in order to protect surrounding cells or tissues. In some cases, this protection system is overridden and damaged cells continue to live. As a damaged cell loses control and continues to divide unchecked, it may incur further genetic mutations that lead to tumor formation and metastasis. The objectives of the current research are to

Our focus is the Rho family of small GTPases, their effectors, and their role in cancer pathophysiology.

act with effector proteins in the downstream network. The activated GTPases control the effectors by modulating their activities, in many cases by disrupting an autoinhibited conformation. Most of what we know about Rho proteins is from expression experiments with activated variants that lack the ability to hydrolyze GTP to GDP. Activated RhoA-G14V, for example, induces the formation of actin stress fibers; an analogous version of Cdc42 promotes the formation of microspikes and filopodia, and Rac promotes dendritic networks that form lamellae. All Rho GTPases influence the organization of the actin and microtubule networks, where the myriad signals become integrated to influence adhesion, motility, proliferation, vesicle trafficking, transcription, and survival. Some Rho family members, in particular RhoC, are overexpressed in inflammatory breast cancer and malignant melanoma, and the ability of RhoC to drive invasion and metastasis in models of those cancers cannot be matched by the closely related RhoA. On the other hand, RhoB has been hypothesized to be a negative regulator of growth and proliferation and is potentially a tumor suppressor protein. Little is known about the specific roles of the different GTPases and their respective effectors in the progression to malignancy.

Rho small GTPases and malignancy Rho proteins are members of the Ras superfamily of small GTPases. Like other small GTPases, Rho proteins act as molecular switches that alternate between GTP- and GDP-bound states in which the activated (GTP-bound) proteins preferentially inter-

Diaphanous-related formins One class of GTPase effectors includes the Diaphanous-related formins (Drfs); there are three closely related Drfs in mammals: mDia1, mDia2, and mDia3. Drfs are recognized by active Rho GTPases through a loosely conserved N-termi-

• identify key rate-limiting mechanisms that control tumor cell growth or movement and determine if they represent useful targets for anti-cancer therapy; and • develop anti-cancer drugs by exploiting our understanding of these molecular mechanisms.

nal GTPase binding domain (GBD). The GBD negatively regulates actin remodeling activity associated with the protein. Rho binding to the Drf’s GBD disrupts its intramolecular interaction with the C-terminal Diaphanous-autoregulatory domain (DAD). In addition to the GBD and DAD regions, Drfs have two other conserved regions: the prolinerich formin homology-1 (FH1) domain and the FH2 domain, the latter having the ability to nucleate and elongate nonbranched actin filaments. There is also a loosely conserved FH3 domain, the role of which may be to act as a protein-protein interaction domain or to confer subcellular targeting capability.

autoregulatory partner, DAD. These truncations create an activated form of the Drf. Alternatively, cellular Drfs can be activated by isolated DAD-region fragments. The binding of DAD fragments disrupts autoinhibition and activates actin remodeling, characterized by the generation of thin fibers, gene expression, and microtubule stabilization identical to that from the expression of GBD-truncated Drfs. Activated Drfs can cooperate with another GTPase effector, Rho kinase (also lacking its GTPase binding domain), to form actin stress fibers that are reminiscent of the effects observed in cells expressing activated RhoA. These observations have led to the conclusion that mDia1 and mDia2 are effectors for Formin-mediated actin nucleation RhoA. In these experiments, however, the influence and filament elongation of GTPase over the Drf (and Rho kinase) protein is Formins nucleate actin in an FH2-dependent lost; both GBD truncation and DAD expression manner and appear to “surf” on the barbed end of mask the contribution of the GTPases to Drf-mediatelongating nonbranched microfilaments. The ed signaling. Thus, it is unclear how specific alternative mechanisms are shown in Fig. 1. GTPase-Drf pairs contribute to cytoskeletal remodelThe first possibility is that the FH2 domain from ing in response to extracellular stimuli. the yeast Drf Bni1p processively elongates filaTo fill this knowledge gap, we have taken ments by adding monomers to the barbed end, a molecular and genetic approaches. The first process accelerated by the actin-monomer-binding approach is gene targeting of the Drf gene family, protein called profilin. In contrast, experiments comprising Drf1, Drf2, and Drf3, which encode with the FH2 region and the yeast formin Cdc12p mDia1, mDia3, and mDia2, respectively. The secsuggest that the FH2 domain caps the barbed end ond approach uses fluorescence resonance energy to nucleate new filaments; the cap is then relieved transfer (FRET) techniques to determine the sites by profilin in a mechanism termed “gating.” and extent of Rho GTPase-formin interactions. An Drfs are autoregulated example is shown in Fig. 2, where Cdc42, fused to enhanced cyan fluorescent protein (ECFP), interIn cells, the Drf autoregulatory mechanism can acts with EYFP mDia2 (yellow). This approach be demonstrated experimentally by expressing verallows for the measurement of protein-protein sions lacking either the autoinhibitory GBD or its interactions in cells. The method depends on the ability of two fluorophores to act as donor-acceptor pairs as a measure of association (distance < 30 Å). One fluorophore (ECFP) is fused to the GTPase, and when excited at the appropriate wavelength, generates a photon at a wavelength that then excites the acceptor fluorophore (EYFP) fused to mDia2. We then measure the light emission from the acceptor as an indication of GTPase activation of the Drf. Figure 1. FH2-mediated actin nucleation: alternative mechanisms

Ongoing work has allowed us to determine that mDia2 can interact with RhoA, RhoB, and Cdc42 at specific sites in cells associated with different types of actin structures. It is not known where and when other Drfs become activated and by which GTPase. In future studies, we will examine where other Drfs interact with other Rho family GTPases in order to decipher their different contributions to cytoskeletal remodeling. Moreover, we will determine if these sites become disrupted as cells become malignant.

Figure 2. mDia2 is an effector for Cdc42 during remodeling of cortical actin. This figure shows the distribution of F-actin in the cell, the distribution of the labeled proteins ECFP-Cdc42 and EYFP-mDia2, and the FRET image showing the co-localization of mDia2 and Cdc42.

Recent Publications Joseph, Hazel L., Ying-Jing Chen, Alexander F. Palazzo, Arthur S. Alberts, K. Kevin Pfister, Richard B. Vallee, and Gregg G. Gundersen. In press. CDC42, dynein, and dynactin comprise a pathway for regulating MTOC reorientation independent of microtubule stabilization. Nature Cell Biology. Chen, Jindong, Weng-Onn Lui, Michele D. Vos, Geoffrey J. Clark, Masayuki Takahashi, Jacqueline Schoumans, Sok Kean Khoo, David Petillo, Todd Lavery, Jun Sugimura, Dewi Astuti, Chun Zhang, Susumu Kagawa, Eamonn R. Maher, Catharina Larsson, Arthur S. Alberts, Hiro-omi Kanayama, and Bin Tean Teh. 2003. The t(1;3) breakpoint-spanning genes LSAMP and NORE1 are involved in clear cell renal cell carcinomas. Cancer Cell 4(5): 405–413. Peng, Jun, Bradley J. Wallar, Akiko Flanders, Pamela J. Swiatek, and Arthur S. Alberts. 2003. Disruption of the Diaphanous-related formin Drf1 gene encoding mDia1 reveals a role for Drf3 as an effector for Cdc42. Current Biology 13(7): 534–545. Wallar, Bradley J., and Arthur S. Alberts. 2003. The formins: active scaffolds that remodel the cytoskeleton. Trends in Cell Biology 13(8): 435–446. Tominaga, Tomoko, Wenxiang Meng, Kazuya Togashi, Hiroko Urano, Arthur S. Alberts, and Makoto Tominaga. 2002. The Rho GTPase effector protein, mDia, inhibits the DNA binding ability of the transcription factor Pax6 and changes the pattern of neurite extension in cerebellar granule cells through its binding to Pax6. Journal of Biological Chemistry 277(49): 47686–47691.

From left to right, Peng, Wallar, Eisenmann, L. Alberts, Stropich, A. Alberts, Schoenherr

Laboratory of Antibody Technology 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 Ping Zhao, M.S. Tessa Gieske, B.S.

Visiting Scientist Mei Guo, M.S.

Student Yong-jun Jiao

Research Interests for malignant glioma are less than 15% and 5%, respectively. These tumors are also considered to be endothelial-rich tumors, and prognostic relevance can be assigned to the vascular changes themselves, with poor survival correlating with increasing vascular density. HGF/SF and VEGF have been shown to be major mediators in the angiogenesis of glioblastoma multiforme, and HGF/SF is characterized through two different mechanisms: by direct-induction blood vessel formation and by induction of VEGF expression. Our previous results showed that the growth of human glioblastoma multiforme xenografts expressing HGF/SF and its receptor Met is markedly inhibited by combined neutralizing mAbs to HGF/SF; similar results have been shown with neutralizing mAb to VEGF by other labs, but none of the results have led to tumor regression.

epatocyte growth factor/scatter factor (HGF/SF) is a multifunctional, heterodimeric polypeptide produced by mesenchymal cells that interacts with cells expressing the Met receptor. Met, the product of the c-met protooncogene, is a receptor protein tyrosine kinase of the same family as epidermal growth factor (EGF) receptors. The activation of Met by HGF/SF affects downstream signaling pathways (which include other protein kinases) responsible for cellular differentiation, motility, proliferation, organogenesis, angiogenesis, and apoptosis. Aberrant expression of the MetHGF/SF receptor-ligand complex—resulting either from mutations in the complex or in conjunction with mutations in other oncogenes—is associated with an invasive/metastatic phenotype in most solid human tumors. Met-HGF/SF and downstream kinases are therefore attractive targets for new anti-cancer agents.

As tumors grow, they begin to produce a wide array of angiogenic molecules; if one angiogenic molecule is blocked, tumors may switch to pathways using a different molecule. We are currently developing and characterizing anti-VEGF mAbs. In collaboration with the microarray technology and cellular/molecular imaging programs at VARI, we seek to understand the relationships between key growth factors (VEGF, HGF/SF, etc.) and their receptors that stimulate tumor angiogenesis and metastasis, and to evaluate the effect of combinations of mAbs to these growth factors for clinical immunotherapeutic potential.

We have generated a panel of monoclonal antibodies (mAbs) to HGF/SF. Some of these antibodies have biological neutralizing activity when used in combination, and we have characterized their antitumor effects both in vitro and in vivo. Using an in vivo xenograft model, we are particularly interested in studying glioblastoma multiforme. These tumors are the most frequent and malignant form of human brain tumors; they are highly invasive and, irrespective of their histological grade of malignancy, even low-grade tumors can be poorly demarcated and are rarely encapsulated. The 2- and 5-year survival rates 12

play. The ability to co-select antibodies and their genes allows the isolation of high-affinity, antigenspecific mAbs derived from either immunized animals or nonimmunized humans. A variety of procedures for selecting such antibodies from recombinant libraries have been described, and some useful antibodies have been produced using this approach. Over the past two years, we have closely followed the development of this technology for producing novel recombinant antibody-like molecules. We have constructed a human naive Fab library with a diversity of 2 × 109, and from it we have screened out some specific mAb fragments against a tumor cell membrane protein. A new scFv library is under construction.

The aberrant expression of the highly tumorigenic Met receptor kinase in two-thirds of localized prostate cancers, and evidently in all osseous metastases, suggests that Met provides a strong selection for metastatic development. In a collaborative investigation, we are using our two radiolabeled anti-Met-extracellular-domain mAbs, designated Met3 and Met5, to study mouse xenograft and orthotopic models of localized and metastatic prostate cancer, with a view toward clinical nuclear imaging diagnostics and radio-immunotherapeutic applications. Moreover, we will be soon testing these two radiolabeled mAbs on dog spontaneous prostate cancers and bone metastasis models. Over the past few years, we have established the technology of a phage-display peptide library for mAb epitope mapping. A random peptide library is constructed by genetically fusing oligonucleotides coding for polypeptides of a given length to the coding sequence of a bacteriophage coat protein, resulting in display of the fused protein on the surface of the phage and its genetic element residing within. Phage display has been used to create a physical linkage between a vast library of random peptide sequences and the DNA encoding each sequence, allowing rapid identification of peptide ligands for a variety of target molecules such as antibodies. A library of phages is exposed to a plate coated with mAb. Unbound phages are washed away, and specifically-bound phages are eluted by lowering the pH. The eluted pool of phage is amplified, and the process is repeated for two more rounds. Individual clones are isolated, screened by ELISA, and sequenced. We have successfully mapped epitopes of a variety of important mAbs such as anti-HGF/SF, antiMet, and anti–anthrax lethal factor. We are now exploring this technology with protein-protein interactions—for example, mapping of the HGF/SF-Met binding site(s) in an in vitro system—and several interesting peptides have been selected from the library as having potential Met antagonistic activity.

Functioning as an antibody production facility at the Van Andel Research Institute, this lab has extensive capabilities in the generation, characterization, scaled-up production, and purification of a variety of mAbs using comprehensive, cutting-edge technologies. The technologies and services available include antigen preparation and animal immunization; peptide design and coupling to protein carriers; consultation about protein expression and purification; DNA immunization (gene-gun technology); immunization with living or fixed cells; conventional antigen/adjuvant preparation; immunization of a wide range of antibody-producing models (including mice, rats, rabbits, human cells, and transgenic or knock-out mice); and in vitro immunization. Also available are conventional techniques of hybridoma production, including the generation of hybridomas from spleen cells of immunized mice and rats; hybridoma expansion and subcloning; cryopreservation of hybridomas that secrete mAbs; mAb isotyping; ELISA screening of hybridoma supernatants; consultation on other hybridoma screening techniques; production of bulk quantities of mAbs using high-density cell culture techniques; and many more. Over the past few years, this facility has generated more than 200 different mAbs, resulting in or contributing to the submission and funding of grant proposals, the filing of four patent applications, and the licensing of several of these antibodies. We have also established contract services with local biotechnological companies to generate, characterize, produce, and purify mAbs for their research and diagnostic kit development.

In collaboration with Nanjing Medical University of China, we have initiated the construction of a phage-display antibody fragment library. This technique involves the construction and use of human/animal, immunized/naive, Fab and scFv antibody gene repertoires by phage dis13

External Collaborators Milton Gross, Department of Veterans Affairs Medical Center/University of Michigan, Ann Arbor David Wenkert, Michigan State University, East Lansing Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington David Waters, Gerald P. Murphy Cancer Foundation, Seattle, Washington Wei-cheng You, Beijing Institute for Cancer Research, China Ji-you Li, Beijing Institute for Cancer Research, China Yi Ren, Hong Kong University, China Xiao-hong Guan, Nanjing Medical University, China Zhen-qing Feng, Nanjing Medical University, China Kang-lin Wan, Chinese Centers for Disease Control and Prevention, Beijing Recent Publications Hay, Rick V., Brian Cao, R. Scot Skinner, Ling-Mei Wang, Yanli Su, James H. Resau, Beatrice S. Knudsen, Margaret F. Gustafson, Han-Mo Koo, George F. Vande Woude, and Milton D. Gross. 2003. Radioimmunoscintigraphy of human Met-expressing tumor xenografts using Met3, a new monoclonal antibody. Clinical Cancer Research 9(10): 3839S–3844S. You, Xueke, Hsiao-Man Yu, Leona Cohen-Gould, Brian Cao, Marc Symons, George F. Vande Woude, and Beatrice S. Knudsen. 2003. Regulation of migration of primary prostate epithelial cells by secreted factors from prostate stromal cells. Experimental Cell Research 288(2): 246–256. Hay, Rick, Brian Cao, Ilan Tsarfaty, Galia Tsarfaty, James Resau, and George Vande Woude. 2002. Grappling with metastatic risk: bringing molecular imaging of Met expression toward clinical use. Journal of Cellular Biochemistry S39: 184–193.

From left to right: Gieske, Guo, Jiao, Zhao, Cao

Suppression of LSAMP, NORE1A, and Nore1 reexpression Reexpression and localization of EGFP-LASMP, -NORE1A, and -NORE1 fusion protein 2 h after microinjection or 24 h after lipid-mediated transfection of pEGFP-LSAMP, -NORE1A, and -Nore1 plasmids. Green shows the expressed protein; blue, the cell nucleus; and red, microtubules. (Chen, Alberts, and Teh)

Laboratory of Mass Spectrometry and Proteomics 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 a member of a biotechnology development unit, he was group leader for a protein characterization core laboratory. More recently as a research scientist in discovery research, he was principal in the establishment and application of a state-ofthe-art proteomics laboratory for drug discovery. Mr. Cavey joined VARI as a Special Program Investigator in July 2002. Laboratory Members Staff Veronica Mutchler, B.A.

Students Wendy Schroeder Marie Graves

Research Interests the resulting spectra are used to search protein or translated DNA databases. Identifications are made using the amino acid sequences derived from the mass spectrometry data. We have optimized all aspects of this analysis for sample recovery yields and high-sensitivity protein identification.

he Mass Spectrometry and Proteomics program works with many of the research labs at the Institute to help answer a wide range of biological questions. State-of-the-art mass spectrometers, used in combination with analytical protein separation and purification methods, are powerful tools for studying proteins in disease processes. Using mass spectrometry data and database search software, proteins can be identified and characterized with unprecedented sensitivity and throughput. Since proteomics is a relatively new scientific discipline, many of the analytical techniques are rapidly changing; therefore our mission involves using established protocols, improving them, and developing new approaches to expand the scope of biological challenges being addressed.

Recently, we have been evaluating newly developed software that allows the electrophoresis separation step to be eliminated from these analyses, giving the potential to identify more proteins from complex mixtures. With this software, affinity-purified protein complexes are compared to a control sample using a technique known as peptide differential display. The proteins are digested into peptides in solution rather than from gels and are analyzed by LC-MS. Peptides that are unique to the experimental sample relative to the control are used to identify proteins that are part of a protein complex.

Protein-protein interactions Evaluating samples representing different cellular conditions or disease states is a step toward understanding the role of a protein with an unknown function or understanding the regulatory mechanism of several proteins in a given pathway. In this approach, a known protein is affinity-purified from a nondenatured sample using antibodies, affinity tags such as FLAG or TAP, or immobilized small molecules. The purified protein and its binding partners are separated using two-dimensional (2D) electrophoresis gels or SDS-PAGE. After staining, the proteins are cut from the gel, digested into peptides using an enzyme such as trypsin, and then analyzed by nanoscale high-pressure liquid chromatography on line with a mass spectrometer (LC-MS). The mass spectrometer fragments the peptides and

Protein characterization Our laboratory supports investigators by characterizing proteins and their post-translational modifications. Proteins expressed and purified by investigators are analyzed by protein electrospray to confirm the average protein molecular weight before proceeding to laborintensive studies such as protein crystallization. Mapping the post-translational modifications of proteins such as phosphorylation is an important yet difficult undertaking in cancer research. Phosphorylation regulates many protein pathways, several of which could serve as potential drug targets for cancer therapy. In recent years, 16

2D gel electrophoresis, image analysis of stained proteins, and identification of proteins from gels using mass spectrometry. Due to the labor-intensive nature of 2D gels and the underrepresentation of many different classes of proteins (such as membrane proteins), the field of proteomics has been moving toward solution-based separations and direct mass spectrometry. Our first approach is to digest all proteins into peptides and label the C-terminus of experimental samples with 18O water to effect a mass shift. Experimental and control samples are then mixed and separated by multidimensional highpressure liquid chromatography using strongcation ion exchange and reverse-phase separation modes. Peptides that are differentially expressed in experimental and control samples according their 16O/18O ratio are identified using mass spectrometry and database searching.

mass spectrometry has emerged as a primary tool that helps investigators determine exactly which amino acids of a protein are modified. This undertaking is complicated by many factors, but principally because pathway regulation can occur when only 0.01% of the molecules of a given protein are phosphorylated. Thus, we are dealing with an extremely small number of molecules, in addition to the fact that the purification of phosphopeptides is always difficult. Our lab collaborates with investigators to map protein phosphorylation using a variety of techniques, including immobilized metal affinity purification following esterification, immunoaffinity purification of phosphoproteins and peptides, and phosphorylation-specific mass spectrometry detection. Protein expression As mass spectrometry instruments and protein separation methods develop, we hope to use proteomics research to identify and quantitate all the proteins expressed in a given cell or tissue, as a means of evaluating all of the physiological processes occurring within. This approach, termed systems biology, aims at understanding how all proteins interact to affect a biological outcome. Traditionally this has been done using

We intend to apply this or other mass spectrometryâ&#x20AC;&#x201C;based approaches in the discovery of biomarkers for early cancer detection, more specific diagnosis, and more accurate prognosis following drug treatment. We are collaborating with Craig Webbâ&#x20AC;&#x2122;s lab in developing expression analysis methods.

External Collaborators Gary Gibson, Henry Ford Hospital, Detroit, Michigan Peter Leopold, BioAnalyte Inc., Portland, Maine Brett Phinney, Michigan State University, East Lansing

From left to right: Mutchler, Schroeder, Graves, Cavey

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 a 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 joined VARI in January 2001 as a Distinguished Scientific Investigator. Laboratory Members Students Erik Freiter Lia Tesfay

Staff Eduardo Azucena, Ph.D. Paul Bromann, Ph.D. Hasan Korkaya, Ph.D. Ian Pass, Ph.D. Darren Seals, Ph.D. Rebecca Uzarski, Ph.D. Rebecca Cruz, M.S. Daniel Salinsky, M.S.

Research Interests The role of the Src substrate Fish in tumorigenesis

ur laboratory 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, integrin receptors, and G proteinâ&#x20AC;&#x201C;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. Furthermore, some evidence points to a role for Src family kinases in angiogenesis. Some of the current projects in the laboratory are outlined below.

Fish is an adaptor protein which has five SH3 domains and a phox homology (PX) domain. Fish is tyrosine phosphorylated in Srctransformed fibroblasts (suggesting that it is a target of Src in vivo) and in normal cells after treatment with several growth factors. 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. The PX domain of Fish associates with phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, and it is required to target Fish to podosomes. 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. We have begun to dissect the role of Fish in Src transformation. Our preliminary data (using RNA interference to reduce the level of Fish in cells by approximately 80%) suggest that Fish is required for

The role of Src family kinases in mitogenic signaling pathways

efficient formation of podosomes and for extracellular matrix degradation. Current experiments seek to define other Fish binding proteins, as well as to determine whether the ADAMs family plays a role in podosome formation and/or function.

We have previously shown that Src family kinases are required for both Myc induction and DNA synthesis in response to PDGF stimulation of NIH3T3 fibroblasts. We have also previously identified and characterized a small molecule inhibitor of Src family kinases called SU6656. We recently compared PDGF-stimulated gene expression in untreated and SU6656-treated cells. We determined that a discrete subset of PDGF-responsive genes requires Src family kinases. By using other inhibitors, we could show that these genes are for the most part distinct from the genes requiring MAP kinase or PI3-kinase activities for stimulation. We are currently determining which of these Src-specific events are required for the induction of Myc.

We have also found that the Fish protein is overexpressed in invasive breast cancer cell lines relative to noninvasive cells. This observation has prompted us to begin an analysis of Fish expression in human breast cancer tissues, with the aim of determining if it is a marker for invasive breast cancer. Characterization of the Src-like kinase Frk We are particularly interested in the human kinase Frk. This kinase has a domain structure typical of Src family kinases. 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 determined that Frk is negatively regulated by its tail tyrosine, but Csk is unlikely to be the responsible enzyme. Like other members of the Src superfamily, active forms of Frk can activate the MAP kinase pathway, but Frk is unusual in that it appears to act downstream of Ras. Furthermore, Frk is not found on cellular membranes, but rather is found in both the cytoplasm and the nucleus. We have now begun to characterize the transforming ability of Frk in epithelial cells.

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 fully established. We have begun our studies by characterizing the role of Src in estrogen-stimulated signal transduction pathways in breast cancer cell lines. We have shown that Src family kinase activity is required for estrogen to stimulate mitogenesis in MCF7 cells. Furthermore, inhibition of Src prevents both estrogen stimulation of Myc and MAP kinase activity. We are currently dissecting which Src signaling pathways are necessary for estrogen-stimulated growth, as well as how Src activity results in the activation of MAP kinase and in the production of Myc.

Recent Publications Abram, Clare L., Darren F. Seals, Ian Pass, Daniel Salinsky, Lisa Maurer, Therese M. Roth, and Sara A. Courtneidge. 2003. The adaptor protein Fish associates with members of the ADAMs family and localizes to podosomes of Src-transformed cells. Journal of Biological Chemistry 278(19): 16844â&#x20AC;&#x201C;16851. Courtneidge, S.A. 2003. Isolation of novel Src substrates. Biochemical Society Transactions 31(Pt. 1): 25â&#x20AC;&#x201C;28. Scaife, Robin M., Sara A. Courtneidge, and Wallace Y. Langdon. 2003. The multi-adaptor protooncoprotein Cbl is a key regulator of Rac and actin assembly. Journal of Cell Science 116(Pt. 3): 463â&#x20AC;&#x201C;473.

Seals, Darren F., and Sara A. Courtneidge. 2003. The ADAMs family of metalloproteases: multidomain proteins with multiple functions. Genes and Development 17(1): 7â&#x20AC;&#x201C;30. Voytyuk, Olexandr, Johan Lennartsson, Akira Mogi, Georgina Caruana, Sara Courtneidge, Leonie K. Ashman, and Lars Ronnstrand. 2003. Src family kinases are involved in the differential signaling from two splice forms of c-Kit. Journal of Biological Chemistry 278(11): 9159â&#x20AC;&#x201C;9166.

From left to right, back: Bromann, Pass, Freiter, Azucena, Uzarski, Seals front row: Salinsky, Cruz, Tesfay, Korkaya

Laboratory of Developmental Cell Biology 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, under the supervision of Yoshio Masui. Before his appointment as a Scientific Investigator at VARI in April 1999, he was 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 Development Center, Maryland. Laboratory Members Staff Xudong Liang, M.D. John Young, M.Sc.

Research Interests a described generic mitogen-activated protein kinase (MAPK) binding site, or docking (D) domain, consisting of a basic amino acid center flanked by hydrophobic residues on one or both sides. This raises the possibility that LF may be a D-domain protease that targets both activators and substrates of MAPKs. However, using in vitro cleavage assays, we have been unable to detect LF-induced cleavage of CL-100, c-jun, or ATF-2, known substrates of MAPKs that contain a D domain. Thus, other regions of MEKs, in addition to the NH2-terminal cleavage site, must be required for LF substrate recognition.

ur research group uses biochemical and molecular approaches to elucidate regulatory mechanisms involved in human health and disease and in the early embryonic development of Xenopus species. One of our main projects is described in the following text. The lethal effects of Bacillus anthracis have been attributed to an exotoxin it produces that is composed of three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). PA binds to a cell surface receptor and, upon proteolytic activation to a 63-kDa fragment, forms a heptameric membrane channel that mediates the entry of three molecules of LF or EF into the cell. EF is an adenylate cyclase that together with PA forms a toxin referred to as edema toxin. LF is a Zn2+-metalloprotease that together with PA forms a toxin referred to as lethal toxin (LeTx). LeTx is the dominant virulence factor produced by B. anthracis and is the major cause of death in infected animals.

Using mutational analysis, we have identified a functionally conserved COOH-terminal region of MEKs that is essential for LF-mediated proteolysis of MEK. The presence of a conserved region distal to the cleavage site, which is necessary for binding and/or cleavage by LF, may explain in part the failure to identify physiological LF substrates other than MEKs. The fact that other MEK1-regulatory proteins (such as B-Raf, PAK, and the scaffolding protein MP-1) also interact with MEK in this region suggests that the region constitutes a key regulatory domain of MEK1 and perhaps of other MEKs.

To date, mitogen-activated protein kinase kinase (MEK) molecules are the only identified physiological substrates of LF. A comparison of the LF cleavage sites on MEKs 1–4, 6, and 7 reveals elements of homology. In all cases the cleavage site is preceded by a series of basic residues and followed immediately by an aliphatic residue. Also, with the exception of MEKs 3 and 4, the cleavage site is preceded by one or more proline residues. Synthesizing these results, the consensus site for LF cleavage fits the pattern B·B/P·B·P·(X)2–3·Al, where B represents a basic residue; P, proline; X, variable; and Al, aliphatic residue. This motif is similar to one in

This possibility may have functional implications for LF toxicity, since the presence of MEK-binding proteins may alter LF’s access to its substrates. Conversely, LF might decrease MEK activity by competitively displacing positive regulators of MEKs. However, by itself this mechanism seems insufficient to explain how LF inactivates MEKs, because we have observed that LF can inhibit the activity of constitutively 21

activated MEK1. Since phosphorylation under these conditions depends upon not only kinase activity but also substrate affinity, we reasoned that LF might inhibit MEK by either reducing its intrinsic kinase activity or decreasing its affinity for ERK. The latter seemed more likely given that MEK1 deletion mutants lacking the 32 NH2terminal residues are deficient in their ability to bind ERK and that mutations in the docking domain decrease the efficiency with which MEK1 activates ERK. However, we have found that as well as decreasing MEK’s affinity for its substrate, LF also decreases MEK’s intrinsic kinase activity. The latter result was not expected but is not without precedent. Based on homology to the A-helix of cAMP-dependent protein kinase and the observation that NH2-terminal deletions and activation lip substitutions synergize to activate MEK1, Ahn and colleagues have hypothesized that regions of the NH2 terminus form long-range interactions with the activation loop and that perturbation of the structure within this region promotes conformational changes in the loop that favor activation. By analogy, we predict that the NH2 terminus of MEK1 associates with its activation loop to promote its activity. The NH2-terminal structure may also promote protein stability, because we have noted that the long-term stability of MEKs is decreased in cells treated with PA and LF. The proximity of the D domain to the activation loop may coordinate MEK-ERK interaction and facilitate ERK phosphorylation and activation.

Figure 1. Lethal toxin inhibits growth of colorectal adenocarcinomas. The WiDr human colorectal adenocarcinoma cell line was used to generate xenograft tumors in nude mice. Control mice were treated with buffered saline only (representative tumor, left). A tumor from a mouse that received lethal toxin every second day by tail vein injection is shown at right.

Institute’s 60–cell line anti-neoplastic drug screen indicates that several tumor types, including melanomas and colorectal adenocarcinomas, are sensitive to growth inhibition by lethal toxin. We have shown that LeTx can inhibit the growth and vascularization of murine fibrosarcomas and, in collaboration with Han-Mo Koo’s group at VARI, we have demonstrated that human melanoma-derived cell lines undergo apoptosis following treatment with LeTx. We have continued this work, assessing the efficacy of LeTx against human colorectal carcinoma–derived cell lines in vitro as well as in vivo. Whereas LeTx in vitro induced cell cycle arrest and apoptosis of the HT-29 and WiDr cell lines, respectively, it had no effect upon proliferation of the SW-620 cell line. By contrast, the pathological malignancy and growth of each of these cell lines as xenografts in athymic nude mice was significantly reduced following intravenous treatment with LeTx (Fig. 1). Our results indicate that proteolytic inhibition of multiple MEK signaling pathways reduces the growth and malignancy of human colorectal adenocarcinoma.

Increased MEK activity has been associated with many aspects of tumorigenesis. The combined roles of multiple MEK pathways in tumorigenesis and the unique activity of LF in inhibiting each of these pathways suggests that LeTx may be an effective therapeutic agent for the treatment of cancer. Indeed, preliminary screening of LeTx against the National Cancer

External Collaborators Stephen Leppla, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland Robert Liddington, Burnham Institute, La Jolla, California Angel Nebreda, European Molecular Biology Laboratory, Heidelberg, Germany Art Frankel, Wake Forest University, Winston-Salem, North Carolina Jean-François Bodart, Université des sciences et technologies de Lille, France. Recent Publications Singh, Y., X. Liang, et al. In press. Pathogenesis of Bacillus anthracis: the role of anthrax toxins. Microbial Toxins. T. Proft., ed. Norfolk, U.K.: Horizon Scientific. Chopra, Arun P., Sherrie A. Boone, Xudong Liang, and Nicholas S. Duesbery. 2003. Anthrax lethal factor proteolysis and inactivation of MAPK kinase. Journal of Biological Chemistry 278(11): 9402–9406. Frankel, Arthur E., Han-Mo Koo, Stephan H. Leppla, Nicholas S. Duesbery, and George F. Vande Woude. 2003. Novel protein-targeted therapy of metastatic melanoma. Current Pharmaceutical Design 9(25): 2060–2066. Perdiguero, Eusebio, Marie-Jeanne Pillaire, Jean-Francois Bodart, Florian Hennersdorf, Morten Frödin, Nicholas S. Duesbery, Gema Alonso, and Angel R. Nebreda. 2003. Xp38γ/SAPK3 promotes meiotic G2/M transition in Xenopus oocytes and activates Cdc25C. EMBO Journal 22(21): 5746–5756. Frankel, Arthur E., Bayard L. Powell, Nicholas S. Duesbery, George F. Vande Woude, and Stephan H. Leppla. 2002. Anthrax fusion protein therapy of cancer. Current Protein and Peptide Science 3(4): 399–407.

From left to right: Duesbery, Young, Liang

Vivarium and Transgenics Special Program 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 NCI-FCRDC, where she continued to develop her skills in transgenic technology and managed the transgenic mouse colony. During this time Ms. Eagleson attended Frederick Community College and Hood College in Frederick, Maryland. In 1999, she joined VARI as the Vivarium Director and Transgenics Special Program Manager. Laboratory Members Managerial staff Jason Martin, RLATG

Technical staff Dawna Dylewski, B.S. Audra Guikema, B.S., L.V.T. Kristin Munski, B.S. Elissa Boguslawksi

Vivarium staff Mark Sheely, B.S. Jamie Bondsfield Daniel Levins Sylvia Marinelli Joe Orcasitas Rich Rasmussen Robert Rogers

Research Interests 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. Transgenic mice are produced by injecting small quantities of foreign DNA (the transgene) into a pronucleus of a one-cell fertilized egg. 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 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 also be controlled by genetic engineering. These transgenic mice are excellent models for studying the expression and function of the transgene in vivo.

he goal of the vivarium and the transgenics program is to develop, provide, and support high-quality mouse modeling services for the Van Andel Research Institute investigators, Michigan Life Science Corridor collaborators, and the greater research community. We use 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. Imaging equipment, such as the PIXImus mouse densitometer and the Acuson Sequoia 512 ultrasound machine, is available for noninvasive imaging of mice. The VetScan blood chemistry and hematology analyzers are now available for blood analysis. Also provided by the vivarium technical staff are an extensive xenograft model development and analysis service, rederivation, surgery, dissection, necropsy, breeding, and health-status monitoring. Transgenics Fertilized eggs contain two pronuclei, one that is derived from the egg and contains the

From left to right: Martin, Guikema, Marinelli, Eagleson, Orcasitas, Levins, Boguslawski, Rogers, Sheely, Dylewski, Bondsfield, Munski

Bioinformatics Special 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. Dr. Furge did his postdoctoral work in the laboratory of Dr. George Vande Woude and became a Bioinformatics Scientist at VARI in June of 2001. Laboratory Members Staff Karl Dykema, B.A.

Student Dechrisha Bates

Research Interests Microarray technology allows us to measure expression levels for tens of thousands of genes in a single experiment. To handle the amount of gene expression data such experiments produce, we have expertise in several robust data analysis and statistical packages, including GeneSpring and BioConductor. BioConductor is a international research collaboration having a goal of providing access to a wide range of powerful statistical and graphical methods for the analysis of genomic data. Details can be found at <http://www.bioconductor.org>.

s high-throughput biotechnologies such as DNA sequencing, gene expression microarrays, and genotyping become more available to researchers, analysis of the data produced by these technologies becomes increasingly difficult. Computational disciplines such as bioinformatics and computational biology have recently emerged to help develop methods that assist in the storage, distribution, integration, and analysis of these data sets. The bioinformatics program at VARI is currently focused on using computational approaches 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 the Institute and as part of larger international collaborations. Currently, we have a special focus on genomes, including DNA sequence data, gene expression microarray data, and cytogenetic data. The bioinformatics program at VARI is also part of the overall bioinformatics effort in the state of Michigan through the Michigan Center for Biological Information.

Of special interest to our group is integrating gene expression data with traditional cytogenetic data. The goal is to use computational approaches to identify candidate genes for which expression is altered the most within regions of frequent cytogenetic change. We are developing several types of algorithms that can both identify probable cytogenetic abnormalities from gene expression data and identify candidate genes within these abnormal regions (Fig. 1). Because many types of data analysis are computationally intensive, we are developing an infrastructure that will allow more-sophisticated computational analysis to be used. This infrastructureâ&#x20AC;&#x201D;termed cluster computing, or grid computingâ&#x20AC;&#x201D;distributes a large computational workload over many lowcost computers. Following 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.

To allow investigators at VARI to take advantage of the most recent DNA sequence information, we maintain a local mirror of the Ensembl version of the public human sequence database augmented with the JEMBOSS tool set. In addition, we support the use of the Informax data platform for sequence analysis. As sequence annotations are constantly being updated by the European Bioinformatics Institute, the National Center for Biological Information, and other institutes, we collect this information, summarize it, and distribute the results.

Figure 1. A sliding-window algorithm for regional gene expression biases. The top panel shows gene expression values of tumor tissue relative to adjacent noncancerous tissue in a chromosomal region. In the middle panel, an exhaustive sliding-window algorithm identifies regions of more-significant downward gene expression bias (blue) or more-significant upward bias (red); white indicates regions of less significance. The size of the sliding window is varied and is smallest at the top to largest at the bottom. The lower panel summarizes the results and indicates where the most significant regional expression bias occurs, in this case in the far right chromosomal region.

Recent Publications Gray, S.G., C.-N. Qian, K. Furge, X. Guo, and B.T. Teh. 2004. Microarray profiling of the effects of histone deacetylase inhibitors on gene expression in cancer cell lines. International Journal of Oncology 24(4): 773–795. Takahashi, Masayuki, Jun Sugimura, Ximing Yang, Nicholas J. Vogelzang, Bin S. Teh, Kyle A. Furge, and Bin T. Teh. 2003. Gene expression profiling of renal cell carcinoma and its implications in diagnosis, prognosis, and therapeutics. Advances in Cancer Research 89: 157–181. Takahashi, Masayuki, Ximing J. Yang, Jun Sugimura, Jesper Backdahl, Maria Tretiakova, Chao-Nan Qian, Steven G. Gray, Robert Knapp, John Anema, Richard Kahnoski, David Nicol, Nicholas J. Vogelzang, Kyle A. Furge, Hiroomi Kanayama, Susumu Kagawa, and Bin Tean Teh. 2003. Molecular subclassification of kidney cancer and the discovery of new diagnostic markers. Oncogene 22(43): 6810–6818. Yang, Ximing J., Jun Sugimura, Maria S. Tretiakova, Kyle Furge, Gregory Zagaja, Mitchell Sokoloff, Michael Pins, Raymond Bergan, David J. Grignon, Walter M. Stadler, Nicholas J. Vogelzang, and Bin Tean Teh. 2003. Gene expression profiling of renal medullary carcinoma. Cancer 100(5): 976–985. Crawley, Joseph J., and Furge, Kyle A. 2002. Identification of frequent cytogenetic aberrations in hepatocellular carcinoma using gene expression data. Genome Biology 3: 0075.1–0075.8. Furge, Kyle A., Ramsi Haddad, Jeremy Miller, Brian B. Haab, Jacqueline Schoumans, Bin T. Teh, Lonson L. Barr, and Craig P. Webb. 2002. Genomic profiling and cDNA microarray analysis of human colon adenocarcinoma and associated peritoneal metastases reveals consistent cytogenetic and transcriptional aberrations associated with progression of multiple metastases. Applied Genomics and Proteomics 1(2): 123–134.

Takahashi, Masayuki, Ximing J. Yang, Todd T. Lavery, Kyle A. Furge, Bart O. Williams, Maria Tretiakova, Anthony Montag, Nicholas J. Vogelzang, Gian G. Re, A. Julian Garvin, Stefan Söderhäll, Susumu Kagawa, Debra Hazel-Martin, Agneta Nordenskjöld, and Bin Tean Teh. 2002. Gene expression profiling of favorable histology Wilms tumors and its correlation with clinical features. Cancer Research 62(22): 6598–6605.

From left to right: Furge, Dykema

Laboratory of Cancer Immunodiagnostics 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 Staff Muthu Shanmugam, Ph.D. Randall Orchekowski, B.S. Sara Forrester, B.S.

Visiting scientist Harvey Nikkel, Ph.D.

Student Darren Hamelinck

Research Interests by printing multiple microarrays on a single microscope slide. This allows us to efficiently perform studies on the large sample sets that are necessary for clinical research. Future technology development plans include measuring the relative levels of post-translational modification (such as glycosylation or phosphorylation states) for the proteins captured by the arrays. The microarray format also provides unique opportunities to study the fundamental aspects of immunoassays, such as the relative merits of competitive and noncompetitive assays or the effects of various buffer additives or surfaces on protein stability and activity.

he Haab laboratory is developing and applying new technologies to meet the need for improved molecular diagnosis of cancer. Using antibody microarrays and related technologies, we are identifying changes of protein composition in the bodily fluids of cancer patients that can be used for new diagnostic tests and that give molecular information about the cancers. The research in our laboratory is broadly divided into three areas: 1) technology development; 2) pancreatic cancer studies; and 3) prostate cancer studies. Technology development We have been engaged in developing new antibody and protein microarray methods that enable parallel measurement of many different, specific proteins in biological samples. The experimental advantages of microarray technology include low consumption of reagents and samples, good reproducibility, high sensitivity, and the ability to efficiently run many samples. We have given great attention to establishing robust, reliable, and versatile protocols that are portable to other formats and to other laboratories. A recent collaboration with Paul Lizardi and others from Yale University resulted in the establishment of a new high-sensitivity detection method for antibody microarrays. This method, based on rolling-circle amplification (RCA), produces reproducible and significantly enhanced fluorescent signals from antibody microarrays, resulting in lower protein detection limits relative to previous methods.

Pancreatic cancer studies A major goal of the Haab laboratory is to develop serum biomarkers for the early and specific detection of pancreatic cancer. Most pancreatic cancers are detected at an advanced, untreatable stage, resulting in a five-year survival rate of less than 5%. Early detection, as would be enabled by a highly accurate blood test, would provide an excellent chance to improve survival rates. Until now, the development of a clinically useful blood test for early detection of pancreatic cancer has been elusive, despite the identification of many proteins with altered serum levels. A problem with the existing blood tests is that they are based on single proteins. Since there are significant molecular differences among tumors and among people, the blood level of any single protein is highly variable and usually not reliable for a clinical test. The antibody microarray method developed in our laboratory uses an alternative strategy: to develop blood tests based on the combined measurements of multiple proteins, which

We also have developed robust methods for efficient, high-throughput processing of samples 29

to develop tests that will meet the need for improved diagnostics. An approach similar to that described above for pancreatic cancer is being used in a collaborative study to identify the protein profiles that best characterize the patient groups. Mass spectrometryâ&#x20AC;&#x201C;based methods will be used to validate the relevant antibody microarray measurements, and candidate markers will be used in further clinical studies with our collaborators.

would better account for protein heterogeneity and would produce fewer false positive and false negative results. In collaboration with researchers at Evanston Northwestern Healthcare, Yale, and the Fred Hutchinson Cancer Research Center, we are measuring many proteins in the sera of pancreatic cancer patients and controls that are most likely to contribute to diagnostic signatures specific for particular disease states. The proteins chosen are those 1) with serum levels associated with pancreatic cancer; 2) with genes having altered expression in the tumor environment; and 3) with genes belonging to classes that are altered in cancerous or precancerous lesions. We have uncovered protein patterns in the serum of cancer patients that differ from those of healthy individuals or individuals with benign disease (Fig. 1). We want to define the precise patterns that best distinguish the groups and to obtain reproducible measurements from as many proteins as may contribute to a diagnostic signature. The protein profiles from clinical samples also allow additional levels of biological interpretation. For example, we are interested in characterizing the variation of particular classes of proteins, such as inflammation-related proteins, in relation to both benign and malignant disease. Such analyses could give valuable insights into disease development and progression mechanisms.

In another collaboration, a complementary approach uses protein microarrays to characterize the immune responses to prostate tumors. We are using microarrays of tumor-derived proteins, prepared from prostate cancer cell lines and separated by two-dimensional liquid chromatography, to measure and characterize these tumorreactive antibodies. In this way, tumor antigens that commonly elicit immune responses in cancer patients can be identified and characterized. These studies could result in new diagnostic tests and could give insight into the nature of the molecular alterations in the tumor. An additional complementary study aims to characterize changes in the disease-associated protein composition of prostatic fluid. These changes give a direct look at the metabolic activity of the secretory epithelial cells of the prostate, which are the main precursor cells of prostate cancer. The concentration of proteins secreted by prostate epithelial cells will be higher in the prostatic fluid than in the serum, making their detection easier. We are measuring the levels of cytokines, proteases, protease inhibitors, and other proteins in samples from prostate cancer patients, prostatitis patients, and controls. Of particular interest is the association of inflammation-related proteins with certain types of pancreatitis and prostate cancer, as these proteins are implicated in the pathologies of both diseases.

Prostate cancer studies Prostate cancer is the second leading cause of cancer death in U.S. men (over 28,000 deaths annually in the United States). The prostate-specific antigen (PSA) test has improved the outlook for prostate cancer patients, but the limitations of the test are significant. Our laboratory is taking multiple approaches to better characterize the protein alterations associated with prostate cancer and External Collaborators

Phil Andrews, University of Michigan, Ann Arbor Randall Brand, Evanston Northwestern Healthcare, Illinois Carlos Cordon-Costa, Memorial Sloan Kettering Cancer Center, New York City Jose Costa and Paul Lizardi, Yale University School of Medicine, New Haven, Connecticut Ziding Feng, Fred Hutchinson Cancer Research Center, Seattle, Washington Samir Hanash and Gil Omenn, University of Michigan, Ann Arbor Jorge Marrero, University of Michigan Hospital, Ann Arbor Alan Partin, Johns Hopkins University, Baltimore, Maryland 30

Figure 1. Identification of pancreatic cancerâ&#x20AC;&#x201C;specific serum protein profiles. The levels of many proteins in the sera of pancreatic cancer patients and controls were measured by antibody microarrays detected by RCA. The antibody measurements that together best distinguish the sample groups were clustered. Each column contains measurements from a single sample, with the labels color-coded according to patient class: blue represents pancreatitis; red, pancreatic cancer; pink, other gastrointestinal cancer; green, normal; and tan, other benign gastrointestinal disease. Each row contains measurements from a single antibody, and the color of each square indicates the level of protein binding: red = high, black = medium, and green = low. The columns and rows are ordered by similarity, the most similar being adjacent. The pancreatic cancer cases clearly segregate from both the normal and the pancreatitis samples, giving an early indication of the ability to specifically identify pancreatic cancer based on a serum protein profile.

Jorge Pisarello, Spectrum Health, Grand Rapids, Michigan Anthony Schaeffer and John Grayhack, Northwestern University, Evanston, Illinois Peter Schirmacher, University of Cologne, Germany Bin S. Teh and E. Brian Butler, Baylor College of Medicine, Houston, Texas Cornelius Verweij, University of Amsterdam, The Netherlands Recent Publications Qui, J., J. Madoz-Gurpide, D.E. Misek, R. Kuick, D.E. Brenner, G. Michailidis, B.B. Haab, G.S. Omenn, and S.M. Hanash. In press. Development of natural protein microarrays for diagnosing cancer based on an antibody response to tumor antigens. Journal of Proteome Research. Zhou, H., K. Bouwman, M. Schotanus, C. Verweij, J.A. Marrero, D. Dillon, J. Costa, P. Lizardi, and B.B. Haab. In press. Two-color, rolling circle amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements. Genome Biology. Haab, B.B., and H. Zhou. 2004. Multiplexed protein analysis using spotted antibody microarrays. Methods in Molecular Biology 278: 33–46. Bouwman, Kerri, Ji Qiu, Heping Zhou, Mark Schotanus, Leslie A. Mangold, Robert Vogt, Erik Erlandson, John Trenkle, Alan W. Partin, David Misek, Gilbert S. Omenn, Brian B. Haab, and Samir Hanash. 2003. Microarrays of tumor cell–derived proteins uncover a distinct pattern of prostate cancer serum immunoreactivity. Proteomics 3(11): 2200–2207. Haab, Brian B. 2003. Methods and applications of antibody microarrays in cancer research. Proteomics 3(11): 2116–2122. Miller, Jeremy C., Heping Zhou, Joshua Kwekel, Robert Cavallo, Jocelyn Burke, E. Brian Butler, Bin S. Teh, and Brian B. Haab. 2003. Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers. Proteomics 3(1): 56–63. Furge, Kyle A., Ramsi Haddad, Jeremy Miller, Brian B. Haab, Jacqueline Schoumans, Bin T. Teh, Lonson L. Barr, and Craig P. Webb. 2002. Genomic profiling and cDNA microarray analysis of human colon adenocarcinoma and associated peritoneal metastases reveals consistent cytogenetic and transcriptional aberrations associated with progression of multiple metastases. Applied Genomics and Proteomics 1(2): 123–134. Guo, X., W.-O. Lui, C.-N. Qian, J.D. Chen, S.G. Gray, D. Rhodes, B. Haab, E. Stanbridge, H. Wang, M.-H. Hong, H.-Q. Min, C. Larsson, and B.T. Teh. 2002. Identifying cancer-related genes in nasopharyngeal carcinoma cell lines using DNA and mRNA expression profiling analyses. International Journal of Oncology 21(6): 1197–1204.

From left to right: Shanmugam, Nikkel, Haab, Orchekowski, Hamelinck, Forrester

Laboratory of Cancer Pharmacogenetics Han-Mo Koo, Ph.D. Dr. Koo received his Ph.D. in microbiology and molecular genetics at Rutgersâ&#x20AC;&#x201C;The State University of New Jersey in 1993. In June 1999, he joined VARI as a Scientific Investigator. Dr. Koo passed away after a battle with cancer in May 2004, as this Report was going to press. Laboratory Members Staff Paula Davidson, M.S. Matt VanBrocklin, M.S. Susan Kitchen, B.S.

Students Stephanie Ellison, B.S.

Research Interests

been collected prior to and throughout the study for further proteomic analysis, with an emphasis on uncovering a better tumor marker, as well as developing novel therapeutic targets.

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-(Î˛-D-arabinofuranosyl)cytosine (Ara-C) and gemcitabine, as well as topoisomerase (topo) II inhibitors, namely etoposide. These results are supported by clinical findings that patients who have RAS oncogeneâ&#x20AC;&#x201C;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 the Spectrum Health Cancer Program. We have initiated a Phase II clinical trial to evaluate gemcitabine/etoposide combination treatment for patients with locally advanced or metastatic pancreatic carcinomas, over 95% of which display RAS oncogene activation. This trial was initiated in October 2002, and currently 19 patients are enrolled. Although not truly indicative, serum protein CA 19.9 is currently the best tumor marker for this and other GI cancers. Serum samples have

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 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. Both anthrax lethal toxin (which proteolytically cleaves MAPK kinases [MEKs]) and small-molecule MEK inhibitors (such as PD90859 and U0126) can trigger 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 provides either significant or complete tumor regression without apparent side effects. These results indicate that the MAPK signaling pathway represents 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. Additionally, we are employing RNAi to evaluate the contribution of other Bcl-2 family members in the survival of melanoma cells. The molecular mechanism by which the inhibition of MAPK signaling specifically triggers apoptosis in human

melanoma cells should reveal additional molecular targets useful for the prevention of and intervention in melanoma and other MAPK-associated cancers such 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. Note: Work begun by Dr. Koo will be completed under the direction of George Vande Woude. Inquiries about the work should be directed to Dr. Vande Woude.

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 David J. Waters, Purdue University, West Lafayette, Indiana Lilly Research Laboratories, a division of Eli Lilly and Company, Indianapolis, Indiana Recent Publications Eisenmann, Kathryn M., Matthew W. VanBrocklin, Nancy A. Staffend, Susan M. Kitchen, and HanMo Koo. 2003. Mitogen-activated protein kinase pathway–dependent tumor-specific survival signaling in melanoma cells through inactivation of the proapototic protein Bad. Cancer Research 63(23): 8330–8337. 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: Davidson, Kitchen, VanBrocklin

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 in the laboratory of Joan Brugge at ARIAD Pharmaceuticals, Cambridge, Mass., from 1995 to 1997 and in the Department of Cell Biology at Harvard Medical School from 1997 to 2000. 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. Mathew Edick, Ph.D. Robert Long, B.A. Veronique Schultz Patacsil, B.S.

Students Beverly Illian

Research Interests activating EGFR, integrins stimulate a subset of integrin-induced signaling pathways (Fig. 1). In the absence of EGFR activation, the ability of the integrins to induce the Ras/Erk signaling pathway and the PI-3K/Akt pathway is severely impaired. However, not all integrin signaling pathways depend on EGFR; for instance, integrin-mediated activation of FAK, Src, or PKC is not dependent on EGFR. Future experiments will be aimed at determining how integrin activation of ErbB2 or Met contributes to integrin signaling.

ur laboratory is interested in understanding the mechanisms by which integrin receptors, interacting with the extracellular matrix, regulate cell processes involved in the development of cancer. Using tissue culture models, biochemistry, molecular genetics, and mouse models, we are defining the cellular and molecular events involved in integrin-dependent adhesion and downstream signaling that are important for melanoma and prostate tumorigenesis and metastasis. Integrins are a class of heterodimeric transmembrane receptors that includes 15 alpha and 9 beta subunits. Each subunit contains a short cytoplasmic region that has no known enzymatic activity, but through protein-protein interactions it is able to interact with actin-containing microfilaments and specific signaling molecules. Consequently, the engagement of integrin receptors by extracellular matrix components induces actin microfilament rearrangement and activates many signal transduction pathways.

We have further demonstrated that integrinmediated adhesion of primary prostate epithelial cells is sufficient to induce several G1 cell cycle events, including increases in the levels of cyclin

Integrin and growth factor receptor cross-talk Our recent work has focused on characterizing the interactions between integrins and receptor tyrosine kinases. Adhesion of epithelial cells to several different extracellular matrices induces ligand-independent activation of the epidermal growth factor receptors EGFR and ErbB2 and of the HGF/SF receptor, Met. Overexpression of EGFR, ErbB2, or Met occurs in prostate cancer. We have demonstrated that by recruiting and

Figure 1. Integrin-induced activation of EGFR is required for a subset of integrin-regulated signaling pathways. EGFR is required for integrin activation of Cbl, PLCÎł, Erk, and Akt. EGFR is not required for integrin activation of PKC, Src, or FAK.

α6β1 and α3β1 both interact with an integrin-associated protein called CD82 or KAI1. CD82/KAI1 is a metastasis suppressor gene; loss of its expression correlates with prostate cancer metastasis. We predict that the loss of CD82/KAI1 alters the function of α6β1 and α3β1. Using primary prostate epithelial cells, which express high levels of CD82/KAI1, as well as several prostate tumor cell lines that do not, we are exploring the role of CD82/KAI1 in regulating α6β1- and α3β1-mediated cell adhesion, migration, and integrin signaling. We have found that overexpression of CD82 in tumor cells suppresses laminin-specific migration (Fig. 2) and invasion; integrin-induced EGFR and Met receptor activation; and Src and Lyn activation. We are currently determining how EGFR, Met, Src, or Lyn contributes to integrin-mediated migration and invasion. In reciprocal experiments, we are inhibiting the expression of CD82 in primary cells using siRNA and mouse models.

D1, p21, cdk4 kinase activity, and Rb phosphorylation. The induction of these cell cycle events is dependent on integrin activation of EGFR, Erk, and PI-3K. However, adhesion alone is not sufficient to induce DNA synthesis; while some G1 cell cycle events were activated by integrin-dependent EGFR activation, not all were. Adhesion failed to induce p27 degradation; c-myc, cdk2, or ATF-2 activation; or cyclin A synthesis. All are required for entry into S phase. Treatment of cells with EGF—the EGFR ligand—or overexpression of EGFR (as is observed in many tumors) was sufficient to restore these additional G1 events and induce DNA synthesis. These data indicate that integrin-mediated activation of EGFR is a critical regulator of the cell cycle. Furthermore, integrinmediated regulation of EGFR, especially in EGFR-overexpressing tumors, may be one mechanism by which tumor cells induce cell growth. We have also found that integrin-induced activation of EGFR in primary prostate epithelial cells is required for their survival in the absence of growth factors. Increased cell survival and resistance to cell death is often a prerequisite for tumorigenesis. Overexpression of growth factor receptors in tumor cells may help promote integrin-mediated cell survival. We are exploring the mechanism by which EGFR mediates integrindependent survival of prostate cells. We are also exploring the mechanisms by which integrins activate EGFR and ErbB2. Integrin activation of EGFR and ErbB2 is ligand independent, requires only the cytoplasmic domain of EGFR, and stimulates the phosphorylation of only a subset of sites on EGFR. Erb2 activation is dependent on EGFR, suggesting that integrins induce the formation of an EGFR/ErbB2 heterodimer. Integrin activation of Src or FAK is not required, but integrin activation of a phosphatase may be involved.

Figure 2. CD82 reexpression in tumor cells inhibits cell migration on laminin. Migration of CD82-expressing tumor cells was completely inhibited when cells were attached to laminin. Migration on collagen was partially inhibited.

Integrin signaling in prostate cancer

Integrin regulation of melanoma progression via PKC

Prostate cancer is the second leading cause of cancer death in U.S. men. Prostate cancer deaths are due to the development of metastatic disease, which is difficult to control. During prostate cancer progression there is a shift in the expression of laminin-specific integrins: β4 integrins are lost, and there is a concomitant increase in α6β1 and α3β1.

The incidence of melanoma has been steadily increasing in the last 10 years. If diagnosed at an early stage it is usually curable, but once it has become invasive, metastatic melanoma is virtually untreatable and progresses very rapidly. Metastasis and invasion by tumor cells require the activity of integrins. Therefore, an understanding of how integrins mediate metastasis and 36

invasion will help our understanding of melanoma progression.

forms can regulate melanoma proliferation, migration, and invasion.

The serine/threonine protein kinase family, PKC, comprises 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 kinase in the various cell functions is largely unknown. In normal melanocytes, PKC is required for cell growth and survival, while in tumor cells, 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 the expression of different PKC iso-

Adhesion of normal melanocytes to the extracellular matrix induces the formation of focal adhesion complexes and actin stress fibers. However, in a highly invasive, metastatic melanoma cell line, these structures are absent. The levels of PKCα are elevated in these cells. We have found that the activity of Rac, a small GTPase that regulates actin structure, is elevated and that inhibition of PKC blocks Rac activity. Overexpression of PKCα in immortalized normal melanocytes is sufficient to confer an invasive phenotype in vitro. We are exploring the blocking of PKCα expression with siRNA and the resulting effects on actin structures, migration, and invasion.

External Collaborators Joan Brugge, Harvard Medical School, Boston, Massachusetts Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Senthil Muthuswamy, Cold Spring Harbor Laboratory, New York Recent Publications Lee, C.C., A.J. Putnam, C.K. Miranti, M. Gustafson, L.M. Wang, G.F. Vande Woude, and C.F. Gao. In press. Overexpression of sprouty 2 inhibits HGF/SF-mediated cell growth, invasion, migration and cytokinesis. Oncogene. Bromberg-White, Jennifer L., Craig P. Webb, Veronique S. Patacsil, Cindy K. Miranti, Bart O. Williams, and Sheri L. Holmen. 2004. Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector. Journal of Virology 78(9): 4914–4916.

Left to right: Chinnaswamy, Patacsil, Illian, Long, Miranti, Edick

Transcription factor expression in human cells This is a confocal, fluorescent image of a unique transcription factorâ&#x20AC;&#x2122;s expression in human cells grown on cover slips. The blue stain labels the DNA of the cell and defines the cell nucleus; only the nucleus is seen in this image. The nucleus is the site of transcription, an activity in which the DNA genetic message is read and processed. A well-characterized transcription factor complex is labeled with red stain. The green stain marks the novel protein believed to be a component of the transcription complex. The green pixels are all within the nucleus and co-localize with many of the red pixels. The green pixels occupy a unique globular, peri-nuclear space. This is morphological correlation of a functional association between the novel protein and the transcription complex. Analysis indicates that 19.41% of the red pixels are co-localized with green pixels. This quantification is consistent with the novel green protein forming a portion of the transcription complex. (Resau; quantification by Kort. This work is part of a collaboration between Dr. Resau and Dr. O. Rosen of Harvard University.)

Laboratory of Analytical, Cellular, and Molecular Microscopy and Laboratory of Molecular Diagnostics James H. Resau, Ph.D. Dr. Resau received his Ph.D. from the University of Maryland School of Medicine in 1985. He has been involved in clinical and basic science imaging and pathologyrelated research since 1972. 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 tenured faculty member at the University of Maryland School of Medicine, Department of Pathology. He retired from the University of Maryland and joined the National Cancer Institute. Dr. Resau was the 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). He joined VARI as a Special Program Senior Investigator in June 1999, and in 2003 he was promoted to Deputy Director. In 2004, Dr. Resau assumed the direction of the Laboratory of Microarray Technology to consolidate the imaging and quantification of clinical samples in a CLIA-type research laboratory program. Laboratory Members Staff Robert Sigler, D.V.M., Ph.D. Eric Kort, M.S. Bree Berghuis, B.S., HTL (ASCP), QIHC

Students Pete Haak, B.S. Eric Hudson, B.S. Paul Norton, B.S. J.C. Goolsby

Brandon Leeser Christine Moore Amy Percival

Rebecca Roe Huang Tran

Research Interests Our laboratory is primarily responsible for the archived clinical histopathology program called SPIN (Frostbite). This program allows investigators to use existing clinical samples to assess the expression of proteins in human disease samples. In addition, we use these blocks to prepare a wide variety of tissue microarrays for research purposes. Currently the archive holds approximately 150,000 tissue samples/paraffin blocks. They are not directly linked to any personal identifiers or names and meet HIPAA/CLIA regulations. The material from future years will be available with digital information on age, sex, and diagnosis and linked to image files. These samples will be used in cellular and molecular protocols approved by our Institutional Review Board (IRB). The samples and demographics are identified with basic information in a webbased, interactive format for determination of diagnosis, prognosis, and therapy. In the first years of operation for SPIN there have been 24 users registered who have submitted 508 requests for searches and 75 subsequent tissue requests.

ur laboratories are responsible for producing and interpreting images and cDNA microarrays as they relate to the diagnosis and characterization of disease, injury, and differentiation. Although primarily we study cancer, we work with a variety of diseases. The ACMM lab works closely with VARI investigators, as well as with collaborators from outside the Institute, to provide support for a variety of imaging and pathology projects. We have a special interest in the quantification of imagery. We have two confocal microscopes that enable us to visualize organelles and proteins in cells or tissues. We have studied the location of two gene-targeted proteins (GFP and RFP) within a cell in three dimensions, using them with a nuclear (DAPI) marker. We have integrated laser-capture microdissection instrumentation into our imaging program, as well as paraffin- and frozen-section staining. We also provide histotechnology services, histopathology consultation, immunostaining expertise, and direction of the human tissue services. The MD lab produces highquality cDNA microarrays in accordance with CLIA protocols for use in the study of tissues, lesions, and organisms.

In collaboration with George Vande Woude and Rick Hay of VARI, we have augmented this program with the collection of freshly frozen human 39

mine an effective and accurate screen for quality of analysis. We are evaluating a series of cases to determine the features that define a “quality specimen.”

tissues from specific West Michigan hospitals. This HIPAA-approved program involves the active collaboration of surgeons and pathologists. This material is used primarily in cDNA and Affymetrix gene expression studies and discovery. Surgically removed human tumors and normal tissue are evaluated in IRB-approved basic and translational research projects. This collection will at the same time provide the participating physicians with access to research collaborations, with the intention of facilitating the translation of research results into clinical practice. We plan to generate gene expression profiles (microarray), establish new tumor cell lines, and develop new diagnostic and therapeutic agents through these collaborations. 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 genetics-based diagnostic classification of human disease. There is a Scientific Advisory Board for this project comprising members of VARI and of the Spectrum Health pathology, surgical and medical oncology, and surgery departments. Tissue is collected with explicit written permission of the participating patients and physicians. Protocols for the use of the material in this archive require the approval of both the VARI and the Spectrum Health IRBs.

We also focus on quantification of images and the development of objective measureable data from images. Kort et al.’s Cytometry paper in 2003 was our first report of a software program to quantify scattering. This is important in the development of drugs and interventions to control the branching, differentiation, and metastatic processes that are crucial in both normal and pathophysiology. We have recently obtained NIH funding for a major effort in multiphoton imaging of developmental and carcinogenic events in GFP-expressing transgenic mice in collaboration with George Vande Woude, Ilan Tsarfaty, and Rick Hay. This project and others will evaluate the role of Met and HGF/SF in branching morphogenesis, carcinogenesis, and therapy. Other collaborations within VARI involve Met and HGF/SF in cells and tissues; the location of gene-targeted proteins in rodents; evaluation of monoclonal antibodies as diagnostic reagents; and the cellular and subcellular localization and quantification of proteins. 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 of women and minorities into science careers. Dr. Resau is a co-investigator and site coordinator for the Bridges program. We have established an innovative program for public school students in Grand Rapids and have had a mentorship program funded by Pfizer for the past four years. Twelve high school students have trained in the laboratory and are now in baccalaureate programs. This program has recently been recognized by the Grand Rapids public school system and will be developed as a “school-within-aschool” program at Creston High School. This is a partnership between GRAPCEP, GVSU, and VARI and is directed by Davenport University.

In the last three calendar years, under our histology and pathology service program, we have processed 925 requests for histopathologic services that required 8,650 blocks and more than 48,000 glass slides. We have prepared over 107,000 digital images of this material and all are available on network servers to expedite their use by the research staff and external collaborators. We have provided tissue and histopathology services to 12 VARI investigators and have generated over 72,000 microscopic images and related files for the collaborations. The MD lab is preparing gene expression data from cells and tissues and is correlating that with histology, tissue volume, and nuclear density to deterExternal Collaborators

Greg Taylor, University of North Carolina, Chapel Hill Ernst Lengyel, University of Chicago, Illinois Eric Arnoys, Calvin College, Grand Rapids, Michigan Lonson Barr, Michigan State University, East Lansing Maria Roberts, National Cancer Institute, Frederick, Maryland

Christine Hughes, Harvard University, Cambridge, Massachusetts O. Orit Rosen, Harvard University, Cambridge, Massachusetts Josh Webster, Michigan State University, East Lansing Matti Koeppel, Michigan State University, East Lansing Stephan Baldus, University of Cologne, Germany D. Cowen, University of North Carolina, Chapel Hill Nadia Harbeck, Ludwig-Maximilians-Universität, Munich, Germany Kristina Lindemann, Munich, Germany Ilan Tsarfaty, Tel Aviv University, Israel Iafa Keydar, Tel Aviv University, Israel John Sacci, University of Maryland, Baltimore John Ubels, Calvin College, Grand Rapids, Michigan Recent Publications Hay, Rick V., Brian Cao, R. Scot Skinner, Ling-Mei Wang, Yanli Su, James H. Resau, Beatrice S. Knudsen, Margaret F. Gustafson, Han-Mo Koo, George F. Vande Woude, and Milton D. Gross. 2003. Radioimmunoscintigraphy of human Met-expressing tumor xenografts using Met3, a new monoclonal antibody. Clinical Cancer Research 9(10): 3839S–3844S. Kort, Eric J., Bryon Campbell, and James H. Resau. 2003. A human tissue and data resource: an overview of opportunities, challenges, and development of a provider/researcher partnership model. Computer Methods and Programs in Biomedicine 70(2): 137–150. Kort, Eric J., Ashley Jones, Michael Daumbach, Eric A. Hudson, Bree Buckner, and James H. Resau. 2003. Quantifying cell scattering: the blob algorithm revisited. Cytometry 51A(2): 119–126. Hay, Rick, Brian Cao, Ilan Tsarfaty, Galia Tsarfaty, James Resau, and George Vande Woude. 2002. Grappling with metastatic risk: bringing molecular imaging of Met expression toward clinical use. Journal of Cellular Biochemistry S39: 184–193.

Along window, top to bottom: Leeser, Resau, Goolsby, Haak, Norton, Hudson; left, top to bottom: Sigler, Buckner

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, N.Y., and an Assistant Professor in the Department of Biomedical Sciences at the State University of New York at Albany. Dr. Swiatek joined VARI as a Special Program Investigator in August 2000. Dr. Swiatek has been the chair of the Institutional Animal Care and Use Committee since 2002 and is an Adjunct Assistant Professor in the College of Veterinary Medicine at Michigan State University. Laboratory Members Staff Kellie Sisson, B.S. Julie Koeman, B.S. Juraj Zahatnansky, B.S.

Student Olga Motornao, B.S.

Research Interests the genome of the ES cell and, by a process called homologous recombination, replaces one of the two wild-type copies of the gene in the cells. Clones are identified, isolated, and cryopreserved, and genomic DNA is extracted from each clone and delivered to the client for analysis. Correctly targeted ES cell clones are thawed, established into tissue culture by a process called expansion, and cryopreserved in liquid nitrogen. Gene-targeting mutations are introduced into the mouse by microinjection of the pluripotent ES cell clones into 3.5-day-old mouse embryos called blastocysts. These embryos, containing a mixture of wild-type and mutant ES cells, develop into mice called chimeras. The 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 knock-out mice. Once we have the genetically modified mice, our lab assists in developing breeding schemes and provides complete analysis of the mutants. The efficiency of mutant mouse production and analysis is enhanced by the Autogen 9600, a robotic, high-throughput DNA isolation machine. Tail biopsies from genetically engineered mice are processed in a 96-well plate format and the DNA samples are delivered to the client.

he germline modification laboratory is a full-service lab that functions at the levels of service, research, and teaching to develop, analyze, and archive mouse models of human disease. Mouse models are produced using gene targeting technology, a well-established and powerful method for inserting specific genetic changes into the mouse genome. The resulting mice can be used to study the effects of these changes in the complex biological environment of a living organism. The genetic changes can include the 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 “knock-out”). 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. VARI and Michigan Life Science Corridor clients are assisted in the design and implementation of gene-targeting experiments and, if necessary, are trained in these techniques. The gene-targeting service has three major parts: DNA electroporation, clone expansion and cryopreservation, and microinjection. Gene targeting procedures are initiated by mutating the genomic DNA of interest and inserting it into embryonic stem (ES) cells using the electroporation technique. The mutated gene integrates into 42

chromosomal rearrangements. FISH analysis can also be performed on metaphase spreads as described above or on interphase nuclei derived from tissue touch preparations. This type of analysis aids in visualizing specific chromosomal regions of interest. Specialized techniques are available upon request. Finally, the germline modification lab provides cryopreservation services for archiving valuable mouse strains. Mouse embryos or sperm can be cryopreserved in liquid nitrogen and the frozen strains reconstituted using in vitro fertilization techniques.

In addition to the traditional gene-targeting technologies, the germline modification lab can produce mouse models in which the gene of interest is inactivated in a target organ or cell line instead of in the entire animal. These types of mouse models, known as conditional knockouts, are particularly useful in studying genes that, if missing, cause the mouse to die as an embryo. The lab also has the capability to produce mutant embryos that have a wild-type placenta using what is called tetraploid embryo technology. This technique is useful when the gene-targeted mutation prevents implantation of the mouse embryo in the uterus. We also assist in the development of ES or fibroblast cell lines from mutant embryos, which allows for in vitro studies of the gene mutation.

The VARI germline modification lab directs the Michigan Animal Model Consortium (MAMC) of the Core Technology Alliance Corp. This consortium, composed of labs located at VARI and the University of Michigan, receives funding from the Michigan Economic Development Corporation to provide mouse modeling services to Michigan researchers studying human diseases. The services provided include mouse and rat transgenics, TVA transgenics, gene targeting, xenograft models, cytogenetics, embryology, histology, veterinary pathology, sperm and embryo cryopreservation, rederivation, and mouse breeding. These services are described more completely on the MAMC website, <http://www.mlscmamc.org/>.

The germline modification lab has developed, in conjunction with the Laboratory of Cancer Genetics, technical expertise in mouse cytogenetics. Mouse cell lines derived from tumors, fibroblasts, or ES cells can be grown in tissue culture, growth-arrested, fixed, and spread onto glass slides. Spectral karyotyping (SKY) analysis of mouse metaphase chromosome spreads on the slide, using high-quality, 24-color fluorescent in situ hybridization (FISH) paints, can aid in the detection of subtle and complex

Recent Publications Zhuo, Xiaoliang, Jun Gu, Melissa J. Behr, Pam J. Swiatek, Huadong Cui, Qing-Yu Zhang, Yingiu Xie, Doris N. Collins, and Xinxin Ding. 2004. Targeted disruption of the olfactory mucosa-specific Cyp2g1 gene: impact on acetaminophen toxicity in the lateral nasal gland and tissue-selective effects on Cyp2a5 expression. Journal of Pharmacology and Experimental Therapeutics 308(2): 719–728. Peng, Jun, Bradley J. Wallar, Akiko Flanders, Pamela J. Swiatek, and Arthur S. Alberts. 2003. Disruption of the Diaphanous-related formin Drf1 gene encoding mDia1 reveals a role for Drf3 as an effector for Cdc42. Current Biology 13(7): 534–545. Wu, Lin, Jun Gu, Yan Weng, Kerri Kluetzman, Pam Swiatek, Melissa Behr, Qing-Yu Zhang, Xiaoliang Zhou, Qiang Xie, and Xinxin Ding. 2003. Conditional knockout of the mouse NADPHcytochrome p450 reductase gene. Genesis 36(4): 177–181.

From left to right: Zahatnansky, Koeman, Swiatek, Motornao (front), Sisson (back)

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. He became Deputy Director for Research Operations in the fall of 2003. Laboratory Members Staff Miles Chao-Nan Qian, M.D., Ph.D. Jun Sugimura, M.D., Ph.D. Min-Han Tan, M.D., MRCP Peng-Fei Wang, M.D. Jindong Chen, Ph.D.

Sok Kean Khoo, Ph.D. David Petillo, Ph.D. Chun Zhang, Ph.D. Eric Kort, M.S. Jon Ditlev, B.S. Katherine Kahnoski, B.S. Jared Knol, B.S.

Students Mark Batten, B.S. Timothy Yaw Bediako, B.S. Visiting Scientist Jens Alms

Research Interests papillary RCC. We have developed a monoclonal antibody for HRPT2 and are currently testing its utility for western blotting and immunohistochemical staining. Currently, our focus is on the functional roles of these novel genes.

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, with a major focus on kidney tumors.

In terms of sporadic kidney tumors, we have continued to perform gene expression profiles using both the spot microarray and Affymetrix GeneChip systems. To date, a total of 350 cases have been completed. We have identified the molecular signatures for different subtypes of tumors (molecular diagnosis), for prognostication, and for the prediction of drug response. Currently, we are focusing on data mining and validation by immunohistochemical staining.

In our studies of hereditary kidney tumors, we have reported the identification of two breakpoint genes, NORE1 and LSAMP, in a family with multiple cases of clear cell renal cell carcinoma. We have further demonstrated that both genes behave like tumor suppressors and have lower expression in sporadic renal cell carcinoma. As part of an international consortium, we have been involved in cloning the gene for hyperparathyroidismâ&#x20AC;&#x201C;jaw tumor syndrome, HRPT2, which is characterized by parathyroid tumors, jaw tumors, and kidney cysts and tumors; the kidney involvement includes adult Wilms tumors, mixed epithelial and stromal tumors, and

In addition to carrying out these research projects, our laboratory has provided core sequencing and cytogenetic services to the Institute. To date over 40,000 sequences have been performed. We have also performed cytogenetics studies including FISH, conventional CGH, and SKY in collaboration with internal and external researchers.

External Collaborators We have extensive collaborations with researchers and clinicians in the United States and overseas. Recent Publications Sugimura, J., X.J. Yang, M.S. Tretiakova, M. Takahashi, B. Funton, C.-N. Qian, T. Fujioka, N.J. Volgelzang, and B.T. Teh. In press. Gene expression profiling of mesoblastic nephroma and Wilms tumorsâ&#x20AC;&#x201D;comparison and clinical implications. Urology. 45

Duh, Fuh-Mei, Matthew Fivash, Michele Moody, Maria Li Lung, Xiang Gue, Eric Stanbridge, Michael Dean, Michael Voevoda, Li-Fu Hu, Vladimir Kashuba, Eugene R. Zabarovsky, Chao-Nan Qian, et al. 2004. Characterization of a new SNP c767A/T (Arg222Trp) in the candidate TSG FUS2 on human chromosome 3p21.3: prevalence in Asian populations and analysis of association with nasopharyngeal cancer. Molecular and Cellular Probes 18(1): 39–44. Gray, S.G., C.-N. Qian, K. Furge, X. Guo, and B.T. Teh. 2004. Microarray profiling of the effects of histone deacetylase inhibitors on gene expression in cancer cell lines. International Journal of Oncology 24(4): 773–795. Schoumans, J., B.-M. Anderlid, E. Blennow, B.T. Teh, and M. Nordenskjöld. 2004. The performance of CGH array for the detection of cryptic constitutional chromosome imbalances. Journal of Medical Genetics 41(3): 198–202. Tretiakova, M.S., S. Sahoo, M. Takahashi, N.J. Vogelzang, T. Krausz, B.T. Teh, and X.J. Yang. 2004. Expression of alpha-methylacyl-CoA racemase in papillary renal cell carcinoma. American Journal of Surgical Pathology 28(1): 69–76. Villablanca, A., A. Calender, L. Forsberg, A.W.K. Tso, J. Chen, D. Petillo, A. Höög, C. Bauters, K. Kahnoski, C. Lo, A. Richardson, L. Delbridge, A. Meyrier, et al. 2004. Germline and de novo mutations in the HRPT2 tumour suppressor gene in familial isolated hyperparathyroidism (FIHP). Journal of Medical Genetics 41(3): e32. Warner, J., J. Cardinal, M. Epstein, D. Singh, A. Sweet, J. Burgess, S. Stranks, P. Hill, D. Perry-Keene, D. Learoyd, B. Robinson, P. Birdsey, et al. 2004. Genetic testing in familial isolated hyperparathyroidism: unexpected results and their implications. Journal of Medical Genetics 41(3): 155–160. Yang, X.J., J. Sugimura, M.S. Tretiakova, K. Furge, G. Zagaja, M. Sokoloff, M. Ping, R. Bergan, et al. 2004. Gene expression profiling of renal medullary carcinoma. Cancer 100(5): 976–985. Chandrasekharappa, S.C., and B.T. Teh. 2003. Functional studies of the MEN1 gene. Journal of Internal Medicine 253(6): 606–615. Chen, Jindong, Weng-Onn Liu, Michele D. Vos, Geoffrey J. Clark, Masayuki Takahashi, Jacqueline Schoumans, Sok Kean Khoo, David Petillo, Todd Lavery, Jun Sugimura, Dewi Astuti, Chun Zhang, Susumu Kagawa, et al. 2003. The t(1;3) breakpoint-spanning genes LSAMP and NORE1 are involved in clear cell renal cell carcinomas. Cancer Cell 4(5): 405–413. Chen, J.D., C. Morrison, C. Zhang, K. Kahnoski, J.D. Carpten, and B.T. Teh. 2003. Hyperparathyroidismjaw tumour syndrome. Journal of Internal Medicine 253(6): 634–642. Gray, S.G., A. Iglesias, B.T. Teh, and F. Dangond. 2003. Identification of a novel splice variant of histone deacetylase 3. Gene Expression 11(1): 13–21. Howell, V.M., C.J. Haven, K. Kahnoski, S.K. Khoo, D. Petillo, J. Chen, G.J. Fleuren, B.G. Robinson, L.W. Delbridge, J. Philips, A.E. Nelson, U. Krause, et al. 2003. HPRT2 mutations are associated with malignancy in sporadic parathyroid tumours. Journal of Medical Genetics 40(9): 657–663. Kahnoski, K., S.K. Khoo, N. Nassif, J. Chen, G. Lobo, E. Segelov, and B.T. Teh. 2003. Alterations of the Birt-Hogg-Dubé gene (BHD) in sporadic colorectal tumors. Journal of Medical Genetics 40(7): 511–514. Khoo, Sok Kean, Katherine Kahnoski, Jun Sugimura, David Petillo, Jindong Chen, Ken Schockley, John Ludlow, Robert Knapp, Sophie Giraud, Stephane Richard, Magnus Nordenskjöld, and Bin Tean Teh. 2003. Inactivation of BHD in sporadic renal tumors. Cancer Research 63(15): 4583–4587. Lindvall, Charlotta, Mi Hou, Toshi Komurasaki, Chengyun Zheng, Marie Hendriksson, John M. Sedivy, Magnus Bjorkholm, Bin Tean Teh, Magnus Nordenskjöld, and Dawei Xu. 2003. Molecular characterization of hTERT-immortalized human fibroblasts by gene expression profiling: activation of the epiregulin gene. Cancer Research 63(8): 1743–1747. Qian, Chao-Nan, Masayuki Takahashi, Richard J. Kahnoski, and Bin Tean Teh. 2003. Effect of sildenafil citrate on an orthotopic prostate cancer growth and metastasis model. Journal of Urology 170(3): 994–997. Takahashi, Masayuki, Jun Sugimura, Ximing Yang, Nicholas Vogelzang, Bin S. Teh, Kyle Furge, and Bin T. Teh. 2003. Gene expression profiling of renal cell carcinoma and its implications in diagnosis, prognosis, and therapeutics. Advances in Cancer Research 89: 157–181. Takahashi, Masayuki, Ximing J. Yang, Jun Sugimura, Jesper Backdahl, Maria Tretiakova, Chao-Nan Qian, Steven G. Gray, Robert Knapp, John Anema, Richard Kahnoski, David Nicol, Nicholas J. Vogelzang, et al. 2003. Molecular sub-classification of kidney tumors and the discovery of new diagnostic markers. Oncogene 22(43): 6810–6818. 46

Tso, A.W.K., R. Rong, C.Y. Lo, K.C.B. Tan, S.C. Tiu, N.M.S. Wat, J.Y. Xu, A. Villablanca, C. Larsson, B.T. Teh, and K.S.L. Lam. 2003. Multiple endocrine neoplasia type 1 (MEN1): genetic and clinical analysis in Southern Chinese. Clinical Endocrinology 59(1): 129–135. Webb, Craig, Sarah Scollon, Jeremy Miller, and Bin Tean Teh. 2003. Gene expression profiling of endocrine tumors by microarray analysis. Current Opinion in Endocrinology and Diabetes 10(3): 162–167. Carpten, J.D., C.M. Robbins,A. Villablanca, L. Forsberg, S. Presciuttini, J. Bailey-Wilson, W.F. Simonds, E.M. Gillanders, A.M. Kennedy, J.D. Chen, S.K. Agarwal, R. Sood, et al. 2002. HRPT2, encoding parafibromin, is mutated in hyperparathyroidism-jaw tumor syndrome. Nature Genetics 32(4): 676–680. Dwight, Trisha, Anne E. Nelson, George Theodosopoulos, Anne Louise Richardson, Diana L. Learoyd, Jeanette Philips, Leigh Delbridge, Jan Zedenius, Bin T. Teh, Catharina Larsson, Deborah J. Marsh, and Bruce G. Robinson. 2002. Independent genetic events associated with the development of multiple parathyroid tumors in patients with primary hyperparathyroidism. American Journal of Pathology 161(4): 1299–1307. Guo, X., W.-O. Lui, C.-N. Qian, J.D. Chen, S.G. Gray, D. Rhodes, B. Haab, E. Stanbridge, H. Wang, M.-H. Hong, H.-Q. Min, C. Larsson, and B.T. Teh. 2002. Identifying cancer-related genes in nasopharyngeal carcinoma cell lines using DNA and mRNA expression profiling analyses. International Journal of Oncology 21(6): 1197–1204. Furge, Kyle A., Ramsi Haddad, Jeremy Miller, Brian B. Haab, Jacqueline Schoumans, Bin T. Teh, Lonson L. Barr, and Craig P. Webb. 2002. Genomic profiling and cDNA microarray analysis of human colon adenocarcinoma and associated peritoneal metastases reveals consistent cytogenetic and transcriptional aberrations associated with progression of multiple metastases. Applied Genomics and Proteomics 1(2): 123–134. Khoo, S.K., S. Giraud, K. Kahnoski, J. Chen, O. Motorna, R. Nickolov, O. Binet, D. Lambert, J. Friedel, R. Lévy, S. Ferlicot, P. Wolkenstein, et al. 2002. Clinical and genetic studies of Birt-Hogg-Dubé syndrome. Journal of Medical Genetics 39(12): 906–912. Perrier, N.D., Andrea Villablanca, C. Larsson, M. Wong, B.T. Teh, and O.H. Clark. 2002. Genetic screening for MEN1 in “familial isolated hyperparathyroidism.” World Journal of Surgery 26(8): 907–913. Takahashi, Masayuki, Ximing J. Yang, Todd T. Lavery, Kyle A. Furge, Bart O. Williams, Maria Tretiakova, Anthony Montag, Nicholas J. Vogelzang, Gian G. Re, A. Julian Garvin, Stefan Söderhäll, Susumu Kagawa, et al. 2002. Gene expression profiling of favorable histology Wilms tumors and its correlation with clinical features. Cancer Research 62(22): 6598–6605. von Horn, H., V. Hwa, R.G. Rosenfeld, K. Hall, B.T. Teh, M. Tally, T.J. Ekström, and S.G. Gray. 2002. Altered expression of low affinity insulin-like growth factor binding protein related proteins in hepatoblastoma. International Journal of Molecular Medicine 9(6): 645–649.

Standing, left to right: Qian, Kort, Tan, Ditlev, Petillo, Alms, Knol seated, left to right: Chen, Zhang, Teh, Khoo, Wang

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 1964â&#x20AC;&#x201C;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 Instituteâ&#x20AC;&#x2122;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 George Vande Woude, Ph.D. Rick Hay, Ph.D., M.D. Yu-Wen Zhang, M.D., Ph.D. Chongfeng Gao, Ph.D. Carrie Graveel, Ph.D. Sharon Moshkovitz, Ph.D.

Qian Xie, Ph.D. Dafna Kaufman, M.Sc. Meg Gustafson, B.A. Nathan Lanning, B.S. Yanli Su, A.M.A.T. Mary Beth Bruch

Visiting scientists Nariyoshi Shinomiya, M.D., Ph.D. Galia Tsarfaty, M.D. Ilan Tsarfaty, Ph.D. Students Adi Laser, B.S. Marketta Hassen

Research Interests ative and invasive phenotypes in response to HGF/SF, in a range from highly invasive to highly proliferative. Possessing both activities may be the most crucial aspect of malignant progression. Tumor cells must either possess both phenotypes or be able to shift from proliferative to invasive and back again in order to generate metastatic colonies.

he Laboratory of Molecular Oncology seeks to develop a better understanding of the molecular basis of cancer, with special emphasis on testing and establishing new agents for diagnosis and treatment. Our research focuses on the activities of the Met receptor tyrosine kinase following interaction with its ligand, hepatocyte growth factor/scatter factor (HGF/SF), and the in vitro and in vivo biochemical, biological, and physiological consequences of Met kinase activation. Met and HGF/SF receptor-ligand signaling is required for normal development and homeostasis in animal models. However, abnormal expression of Met occurs in most types of human cancer and is associated with poor clinical outcomes. Because of this, Met is emerging as an important diagnostic and therapeutic target in cancer.

While most tumor cells we study possess both phenotypes, we can find cells with predominantly one phenotype among brain tumor cells. We characterize these phenotypic populations in vitro using invasion, branching, and soft agar assays, and in vivo using tumorigenicity and metastasis assays. The results are striking. We observe functional partitioning of proliferative and invasive cells in both in vitro and in vivo assays as well as in preferential signaling pathways. These studies support the hypothesis that HGF/SF-Met signaling can facilitate both phenotypes (malignant progression). They also suggest that Met-HGF/SF signaling in cancer mimics its role in normal development, whereby (as shown by Carmen Birchmeierâ&#x20AC;&#x2122;s lab) cells from the epithelial dermomyotome lip undergo an epithelial-mesenchymal transition and migrate to form limb muscles and other tissues.

HGF/SF-Met in proliferation, invasion, and angiogenesis We are studying the role of HGF/SF-Met in tumor proliferation, invasion, metastasis, and angiogenesis. Hepatocyte growth factor and scatter factor were discovered independently as a mitogen for hepatocytes and a motility factor for canine kidney cells, respectively. However, most tumor cells expressing Met display both prolifer48

Crucial questions surround the role of these two phenotypes in cancer. For example, are the highly invasive cells responsible for tumor micrometastasis? and, most importantly, how do we therapeutically target both pathways? We have found that certain geldanamycin drugs can inhibit invasion even at femtomolar levels by preventing the activity of a protease commonly found on the surface of invading cells. This may lead to an innovative therapy that suppresses tumor progression without serious side effects. Once invasive and metastatic cells arrive at new sites, they must establish new blood vessels, as Folkman has stated for many years. HGF/SF is a potent angiogenic factor, and we are studying how HGF/SF-Met signaling influences tumor angiogenesis. We have shown that HGF/SF signaling operates as a major angiogenic switch, turning on vascular endothelial growth factor (VEGF) and turning off thrombospondin-1 (TSP-1) expression and angiogenesis inhibition in tumor cells. HGF/SF also stimulates the proliferation and migration of vascular endothelial cells, and consequently it can promote angiogenesis by influencing the tumor environment.

we have generated mice bearing selected single mutations and introduced them into the mouse germline copy of Met. These mice develop several types of tumors, including carcinomas, sarcomas, and lymphomas, and the mutations give rise to differences in the patterns of tumors that develop. The differences in tumor types and latency may be due to signaling differences triggered by the specific mutations. We are currently looking for variations in downstream signaling among the several types of tumors. Other pathological phenotypes are observed among the mouse strains carrying different mutations, indicating that one gene can give rise to multiple pathological phenotypes. Strategies for molecular imaging and therapy In collaboration with Milton Gross at the Department of Veterans Affairs Healthcare System (DVAHS) in Ann Arbor, Michigan, we are developing radiolabeled monoclonal antibodies (mAbs) against the Met receptor as potential clinical imaging agents. Two mAbs recognizing different epitopes in the Met extracellular domain generate nuclear images of Met-expressing human tumor xenografts in immunocompromised mice. We have shown a correlation between the apparent avidity of the xenografts for radiolabeled anti-Met mAb and the level of Met expressed in vitro. Our current efforts are devoted to developing these anti-Met mAbs for clinical testing.

HGF/SF-Met activation and tumor development in animal models We have established a strain of immunocompromised transgenic mice that express human (hu) HGF/SF in several tissues, including liver, brain, lung, and kidney. These mice are robust hosts for the growth of Met-expressing human tumor cells (xenografts), which grow significantly more rapidly in the huHGF/SF mice than in nontransgenic hosts. This mouse strain should be a useful model for examining the role of Met in human tumor xenografts.

In a collaboration involving VARI, DVAHS, Michigan State University, the Fred Hutchinson Cancer Research Center, and the Gerald P. Murphy Cancer Foundation, we are developing Met-directed strategies for imaging and treating metastatic prostate cancer. We have shown that anti-Met mAbs can be used to image human and canine prostate cancer xenografts in mice, and we are preparing to test one of these antiMet mAbs as a diagnostic agent for dogs with spontaneously occurring prostate cancer. Here, as in humans, the tumors metastasize to bone. One major question we wish to answer is how tumor imaging and therapy will be affected by anti-Met mAb reactivity with normal Metexpressing tissues such as liver and kidney.

To understand the relationship between tumorigenesis and inherited activating Met mutations in mice, we collaborated with Laura Schmidt and Bert Zbar at the National Cancer Institute (NCI) to characterize mutant forms of Met that had been shown to be tumorigenic in vitro and in vivo. The observation that activating mutations in Met occur as inherited and as sporadic mutations in human renal papillary carcinomas and other human cancers is compelling evidence that Met is an important oncogene in human cancer. To study how these activating mutations are involved in tumor development,

Our program in molecular imaging of Met oncogene activationâ&#x20AC;&#x201D;a collaborative effort 49

of sedated live animals) starting at the single cell level. Also, using contrast ultrasound and MRI, we have shown that exposing Met-expressing tumor xenografts in host mice to intravenously administered HGF/SF results in profound changes in the hemodynamic parameters and the blood oxygen levels near the tumor. The pattern of response in normal Met-expressing tissues such as liver and kidney is significantly different from that in tumors, most likely reflecting the abnormal tumor vasculature.

involving visiting scientists Ilan Tsarfaty (of the University of Tel Aviv) and Galia Tsarfaty (of Sheba Medical Center) and our colleagues Brian Ross and Alnawaz Rehemtulla (University of Michigan)—is supported by an NIH center grant. We use fluorescence-based strategies and noninvasive functional imaging technology (confocal laser microscopy, ultrasound, and MRI) to evaluate interactions between Met and HGF/SF as they occur in vitro and in vivo. With fluorescent Met derivatives, we can visualize spatial and temporal aspects of Met-HGF/SF interactions and signaling in transfected cells. Using timelapse confocal microscopy, we visualize the distribution of Met and its association with other proteins in the plasma membrane. When MetGFP constructs are used as transgenes, mice develop subcutaneous tumors. The development and progression of these tumors can be studied by intravital confocal microscopy (microscopy

Thus, we are studying the role of MetHGF/SF signaling at the biological, biochemical, and molecular levels in vitro. We use animal models of human cancer to address the relationships between Met-HGF/SF signaling, tumor development, and malignant progression. We are also developing strategies for molecular imaging, as well as therapies that inhibit Met-HGF/SF function or expression in in vivo animal models.

External Collaborators David Wenkert, Michigan State University, East Lansing Milton Gross, Department of Veterans Affairs Healthcare System, Ann Arbor, Michigan Nadia Harbeck and Ernest Lengyel, Technische Universität, Munich, Germany Richard Jove, H. Lee Moffitt Cancer and Research Institute, Tampa, Florida Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Brian Ross and Alnawaz Rehemtulla, University of Michigan, Ann Arbor Laura Schmidt and Bert Zbar, National Cancer Institute, Bethesda, Maryland Yuehai Shen, Michigan State University, East Lansing Olga Volpert, Northwestern University, Evanston, Illinois David Waters, Gerald P. Murphy Cancer Foundation, West Lafayette, Indiana Robert Wondergem, East Tennessee State University, Johnson City Recent Publications Lee, C.C., A.J. Putnam, C.K. Miranti, M. Gustafson, L.M. Wang, G.F. Vande Woude, and C.F. Gao. In press. Overexpression of sprouty-2 inhibits HGF/SF-mediated cell growth, invasion, migration, and cytokinesis. Oncogene. Fan, Jianqing, Paul Tam, George Vande Woude, and Yi Ren. 2004. Normalization and analysis of cDNA microarrays using within-array replications applied to neuroblastoma cell response to a cytokine. Proceedings of the National Academy of Sciences U.S.A. 101(5): 1135–1140. Birchmeier, Carmen, Walter Birchmeier, Ermanno Gherardi, and George F. Vande Woude. 2003. Met, metastasis, motility, and more. Nature Reviews Molecular Cell Biology 4(12): 915–925. Frankel, Arthur E., Han-Mo Koo, Stephan H. Leppla, Nicholas S. Duesbery, and George F. Vande Woude. 2003. Novel protein-targeted therapy of metastatic melanoma. Current Pharmaceutical Design 9(25): 2060–2066.

Hammond, Dean E., Stephanie Carter, John McCullough, Sylvie Urbé, George Vande Woude, and Michael J. Clague. 2003. Endosomal dynamics of Met determine signaling output. Molecular Biology of the Cell 14(4): 1346–1354. Hay, Rick V., Brian Cao, R. Scot Skinner, Ling-Mei Wang, Yanli Su, James H. Resau, Beatrice S. Knudsen, Margaret F. Gustafson, Han-Mo Koo, George F. Vande Woude, and Milton D. Gross. 2003. Radioimmunoscintigraphy of human Met-expressing tumor xenografts using Met3, a new monoclonal antibody. Clinical Cancer Research 9(10): 3839S–3844S. You, Xueke, Hsiao-Man Yu, Leona Cohen-Gould, Brian Cao, Marc Symons, George F. Vande Woude, and Beatrice S. Knudsen. 2003. Regulation of migration of primary prostate epithelial cells by secreted factors from prostate stromal cells. Experimental Cell Research 288(2): 246–256. Zhang, Yu-Wen, Yanli Su, Olga V. Volpert, and George F. Vande Woude. 2003. Hepatocyte growth factor/scatter factor mediates angiogenesis through positive VEGF and negative thrombospondin1 regulation. Proceedings of the National Academy of Sciences U.S.A. 100(22): 12718–12723. Zhang, Yu-Wen, and George F. Vande Woude. 2003. HGF/SF-met signaling in the control of branching morphogenesis and invasion. Journal of Cellular Biochemistry 88(2): 408–417. Frankel, Arthur E., Bayard L. Powell, Nicholas S. Duesbery, George F. Vande Woude, and Stephan H. Leppla. 2002. Anthrax fusion protein therapy of cancer. Current Protein and Peptide Science 3(4): 399–407. Hay, Rick, Brian Cao, Ilan Tsarfaty, Galia Tsarfaty, James Resau, and George Vande Woude. 2002. Grappling with metastatic risk: bringing molecular imaging of Met expression toward clinical use. Journal of Cellular Biochemistry S39: 184–193. Knudsen, Beatrice S., Glenn A. Gmyrek, Jennifer Inra, Douglas S. Scherr, E. Darracott Vaughan, David M. Nanus, Michael W. Kattan, William L. Gerald, and George F. Vande Woude. 2002. High expression of the Met receptor in prostate cancer metastasis to bone. Urology 60(6): 1113–1117. Shinomiya, Nariyoshi, and George F. Vande Woude. 2003. Suppression of Met expression: a possible cancer treatment. Commentary on “Reduced c-Met expression by an adenovirus expressing a c-Met ribozyme inhibits tumorigenic growth and lymph node metastases of PC3-LN4 prostate tumor cells in an orthotopic nude mouse model” (Kim et al.). Clinical Cancer Research 14: 5161–5170. Fan, Jianqing, Paul Tam, George F. Vande Woude, and Yi Ren. 2004. Normalization and significant analysis of cDNA micro-arrays using within-array replications applied to neuroblastoma cell response to MIF. Proceedings of the National Academy of Sciences U.S.A. 101: 1135–1140.

Back row, standing: Zhang, G. Tsarfaty, Su, Gustafson, Laser, Shinomiya, I. Tsarfaty, Gao; middle row, seated: Xie, Vande Woude, Moshkovitz; front row: Graveel, Bruch, Kaufman

Laboratory of Tumor Metastasis and Angiogenesis Craig P. Webb, Ph.D. Dr. Webb received his Ph.D. in cell biology from the University of East Anglia, England, in 1995. After receiving his degree, Dr. Webb 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 Jennifer Bromberg-White, Ph.D. Jeremy Miller, Ph.D. David Monsma, Ph.D. Emily Eugster, M.S.

Sujata Srikanth, M.Phil. Stephanie Benton, B.Sc. Erika Briegel, B.A. Meghan Sheehan, B.S.

Sabbatical John Ubels, Ph.D.

Research Interests in pathological samples and, moreover, predict the likelihood of metastatic relapse well in advance of clinical presentation.

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 addition to improving our conceptual understanding of the metastatic process, these studies may lead to the development of diagnostic and therapeutic strategies that target this most lethal aspect of cancer.

In addition, we are using laser capture microdissection in conjunction with genomic and proteomic technologies to identify key tumor-host interactions during metastatic progression, with particular emphasis on identifying the molecular factors that contribute to metastatic dormancy in the liver. Through these analyses, a number of candidate genes are now being pursued as potential targets for the future treatment of metastatic disease. For this purpose, we have developed a novel retroviral system for the delivery of small interfering RNA (siRNA) molecules that target candidate genes. Using this high-throughput approach, we are knocking out the expression of several potential mediators of the metastatic phenotype in human tumor cell lines and assessing the effect on their metastatic propensity in orthotopic xenograft murine models. Collectively, we are striving towards the early and accurate diagnosis of malignant disease and its successful treatment.

Our laboratory currently uses both in vitro and in vivo systems to study metastasis and angiogenesis. Working closely with several clinical collaborators, we have collected normal and tumor tissues together with blood plasma and urine from a number of patients presenting with widespread metastatic disease. Using a variety of molecular technologies, including single nucleotide polymorphism (SNP) chips, gene expression arrays, and proteomic analyses, we have identified genomic and proteomic correlates of metastatic disease that may be future biomarkers or molecular targets for accurate diagnosis and treatment. Using our proprietary informatics solution (described below), we have identified several genes that distinguish normal from abnormal colon tissue and predict the metastatic outcome of patients with colorectal cancer. The potential diagnostic applications of these data are currently being pursued in a larger cohort of colorectal cancer patients; our findings to date suggest we can accurately diagnose colon cancer

Multiple myeloma The most recent statistics from the American Cancer Society predict that approximately 15,000 new cases of multiple myeloma will be diagnosed within the United States this year. Some 40,000 Americans are living with multiple myeloma, and over 11,000 deaths are predicted per year, usually within three years of diagnosis. The patients endure prolonged pain associated with the spread of this cancer to multiple sites 52

correlates of therapeutic response and disease progression. We believe that these studies could be used to determine optimal treatments for patients with multiple myeloma in the future.

within bone, leading to bone wasting, fractures, and spinal compressions. At present, treatment options for this highly aggressive and devastating cancer are extremely limited, predominantly due to our lack of understanding of its underlying causes. Recent studies show that the incidence of multiple myeloma is rapidly increasing, likely due to an aging population and unknown environmental factors. Despite these facts, multiple myeloma research is underfunded at both the national and state levels. Without in-depth study of the molecular causes of multiple myeloma, it is unlikely that significant advances in the diagnosis and treatment of this disease will soon be forthcoming.

XenoBase XenoBase (patent pending) is a fully integrated genomic/proteomic/medical informatics database with associated analysis and annotation tools (Fig. 1); it was designed in the Laboratory of Tumor Metastasis and Angiogenesis at the Van Andel Research Institute. Raw data generated from a variety of platforms (comparative genomic hybridization, Affymetrix SNP chips, cDNA microarrays, Affymetrix GeneChips, 2D-gel/mass spectrometry) can be associated with specimens and subjects of interest (clinical samples, animal models, cell lines), and comparative analysis can be performed on data across platforms and species. Moreover, XenoBase allows for direct correlation between subject, sample and experimental parameters, and molecular data (Fig. 2). Literature-based and gene ontology annotation software have been incorporated, along with specific metrics for biomarker and target discovery. Currently available therapeutics that specifically target molecular aberrations of interest can also readily be identified. Thus, XenoBase represents an integrated system for basic biomolecular research, clinical diagnostics, and/or new pharmacogenomic strategies for the future.

At the end of 2002, with the generous support of the McCarty Foundation and Ralph Hauenstein, we initiated the development of a dedicated multiple myeloma research laboratory (MMRL). Our specific goal for this laboratory is to use our unique integrated approach to identify optimal treatments for patients. In collaboration with Keith Stewart, Director of the McLaughlin Centre for Molecular Medicine at the Princess Margaret Hospital, Toronto, we have performed genomic and proteomic analyses on a panel of 20 human multiple myeloma cell lines that display varying responses to the drug melphalan. Using a combination of gene expression profiling and proteomic analysis, we have identified a handful of candidate genes that appear to mediate melphalan resistance. Using shRNA and pharmaceutical agents, HL-7 we are attempting to reverse the (Clinical Data) Human Subject drug-resistant phenotype and restore • Birthdate • Sex Animal BioStore / the cytotoxic response to melphalan. • Condition (eg. Mouse) SCION • Images In addition, we have recently initiat• Treatments Frostbite ed a larger collaborative effort with Cell Line several local hematologists, oncoloSample gists, pathologists, and other medical • Tracking & Storage • Images • Detailed Information specialists to collect and process bone marrow aspirates/core biopsies, Internal or external data blood plasma, and urine from conDNA Genotype (SNP) Molecular Data senting patients having either mono• Protocols RNA Gene Expression • Virtual Notebook clonal gammopathy of undetermined • QC (MIAME) • Sample Usage Proteomics significance (MGUS) or multiple (2D Gels, mass spectrometry) Common ID (Unigene/Homologene) myeloma. Through the collection of Normalization Discovery detailed clinical information such as Analysis ➤ Biomarkers • Standard statistics treatment response (efficacy and tox• Annotation ➤ Targets • Functional predicting icity), coupled with single nucleotide • Filtering ➤ Diagnostic polymorphism, gene expression, and Expression builder proteomic analysis of collected samples, we aim to identify molecular Figure 1. Schematic of XenoBase 53

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Figure 2. Screen capture of the XenoBase integrated informatics database. This screen shows the hierarchical clustering of gene expression data collected from a series of human colorectal tumors and adjacent normal tissue. Two major sample groups were identified as abnormal (tumor samples labeled in red at top) and normal (blue labels) tissue. A single sample of “normal” tissue (green label) was found to have an abnormal gene expression signature. Further analysis of the histopathology of this tissue (insert) provided a diagnosis of ulcerative colitis, thereby explaining its abnormal genotype.

External Collaborators Alan Campbell, Spectrum Health Oncology, Grand Rapids, Michigan Jack O’Donnell, Hematology Consultants of West Michigan, Grand Rapids Angela Tiberio, Spectrum Health Information Technology, Grand Rapids, Michigan Michael Warzynski, Spectrum Health, Flow Cytometry Lab, Grand Rapids, Michigan Pamela Kidd, Spectrum Health Pathology, Grand Rapids, Michigan Martin Luchtefeld, Digestive Disease Institute, Grand Rapids, Michigan Tom Monroe, Digestive Disease Institute, Grand Rapids, Michigan Pamela Grady, Digestive Disease Institute, Grand Rapids, Michigan Richard McNamara, West Michigan Heart, Grand Rapids, Michigan David Langholz, West Michigan Heart, Grand Rapids, Michigan Linda Pool, Spectrum Health, Grand Rapids, Michigan Deborah Ritz-Holland, Spectrum Health, Grand Rapids, Michigan Leon Oostendorp, Spectrum Health, Grand Rapids, Michigan Tony Foster, Spectrum Health, Grand Rapids, Michigan Eric Lester, Oncology Care Associates, St. Joseph, Michigan Lonson Barr, Michigan State University, Grand Rapids, Michigan Martin McMahon, University of California, San Francisco Harvey Pass, Karmanos Cancer Institute, Detroit, Michigan Keith Stewart, Princess Margaret Hospital, Toronto, Canada Allen Shoemaker, Medical Education and Research Center, Grand Rapids, Michigan Alan Davis, Michigan State University, East Lansing Annette Thelen, Michigan State University, East Lansing Samir Hanash, University of Michigan, Ann Arbor Gilbert Omenn, University of Michigan, Ann Arbor James Baker, University of Michigan, Ann Arbor

Recent Publications Bromberg-White, Jennifer L., Craig P. Webb, Veronique S. Patacsil, Cindy K. Miranti, Bart O. Williams, and Sheri L. Holmen. 2004. Delivery of short hairpin RNA sequences using a replication-competent avian retroviral vector. Journal of Virology 78(9): 4914–4916. Miller, J., J.L. Bromberg-White, K. DuBois, E. Eugster, S. Srikanth, R. Haddad, C. DeLeeuw, and C.P. Webb. 2003. Identification of metastatic gene targets in a murine model of ras-mediated fibrosarcomas. Applied Genomics and Proteomics 2(4): 253–265. Webb, Craig, Sarah Scollon, Jeremy Miller, and Bin Tean Teh. 2003. Gene expression profiling of endocrine tumors by microarray analysis. Current Opinion in Endocrinology and Diabetes 10(3): 162–167. Furge, Kyle A., Ramsi Haddad, Jeremy Miller, Brian B. Haab, Jacqueline Schoumans, Bin T. Teh, Lonson L. Barr, and Craig P. Webb. 2002. Genomic profiling and cDNA microarray analysis of human colon adenocarcinoma and associated peritoneal metastases reveals consistent cytogenetic and transcriptional aberrations associated with progression of multiple metastases. Applied Genomics and Proteomics 1(2): 123–134.

Along wall, top to bottom: Ubels, Eugster, Srikanth, Sheehan, BrombergWhite; at left, from left to right: Webb, Benton, Monsma, Briegel

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 Don Pappas, Ph.D. Hongyu Liu, M.S. Aaron DeWard, B.S. Ryan Frisch, B.S. Carrie Gabrielse, B.S.

Students Aaron DeWard Victoria Hammond Kelli VanDussen

Research Interests binds to ORC and, together with Cdt1p, promotes loading of the MCM helicase at origins. ORC, Cdc6p, Cdt1p, and the MCM complex are together required to form the pre-RC. Additional proteins associate with the pre-RC during G1 to form a “pre-initiation complex”, and then this large complex of proteins is activated to form bi-directional replication forks by the two-subunit Cdc7p-Dbf4p protein kinase. The processes of origin unwinding and the recruitment of DNA polymerases are not well understood. We are focusing on how Cdc6p promotes preRC assembly and how the Cdc7p-Dbf4p kinase triggers initiation. 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. We have taken a genetic approach to identify genes that act together with CDC6 and have defined several previously unknown genes that influence DNA replication.

e are interested in how the initiation of chromosomal DNA replication occurs in budding yeast and in human cells. When cells reach a critical size in G1 phase and receive the appropriate growth signals, they commit to the process of cell division. The first essential step following commitment is the duplication of the genome, which occurs during S phase. Therefore, the transition from G1 into S phase (or from quiescence into the cell division cycle) is a highly regulated event. The initiation of DNA synthesis occurs at multiple replication origins throughout the genome during S phase, but each replication origin is activated only once per cell cycle. The timing and frequency of initiation is precisely controlled because errors in this process could cause genome amplification and instability. Since uncontrolled cell division and genomic instability are properties of many cancer cells, we are also investigating the aberrant regulation of initiation factors in the development of cancer. The initiation of DNA synthesis requires at least four steps (Fig. 1). The first is origin marking by ORC, a six-subunit protein complex that is required for replication initiation and that recognizes conserved DNA sequence elements in all origins. ORC then directs the assembly of a large macromolecular complex called the “pre-replicative complex,” or pre-RC. As cells exit mitosis, the Cdc6 protein

Figure 1. The yeast chromosome replication cycle

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Figure 2. A catalytic mutation in SIR2 suppresses the cdc64 temperature sensitivity. Serial tenfold dilutions of wild type (WT), cdc6-4, sir2-N345A, and cdc6-4 sir2-N345A cultures were spotted onto rich medium at 25 ˚C and 37 ˚C to assay for growth. The cdc6-4 strain is unable to grow at 37 ˚C but grows as WT following inactivation of Sir2 enzymatic function using the N345A mutation.

Dbf4p itself is phosphorylated in a MEC1and RAD53-dependent manner following inhibition of DNA replication, and this appears to inhibit Cdc7p-Dbf4p kinase activity. Mec1p is a homologue of the human ATM/ATR kinases that are key regulators of the response to DNA damage. Rad53p is the homologue of the human CHK2 checkpoint kinase. Genetic data also suggest that Cdc7p-Dbf4p kinase is required for recovery from replication-induced arrest or DNA damage. We have shown by a structure-function analysis that an amino-terminal BRCT domain within Dbf4p is required for the proper response to a variety of DNA-damaging agents. The BRCT domain is a phosphopeptide binding module that was first discovered in the BRCA1 breast cancer susceptibility gene, and it is present in a variety of proteins involved in the response to DNA damage. The Dbf4p BRCT domain is, however, not

For instance, we identified loss-of-function mutations in SIR2—which encodes a histone deacetylase required for the formation of transcriptionally “silent” heterochromatin—that suppress a cdc6 temperature-sensitive mutant (Fig. 2). We have further demonstrated that Sir2p is acting in a novel pathway to negatively regulate initiation at some (but not all) origins in yeast. The loss of Sir2p promotes MCM loading at origins when Cdc6p activity is compromised (Fig. 3), suggesting that Sir2p may directly influence chromatin structure at some origins or may regulate the expression or activity of a critical initiation protein. Another of our efforts is to understand how the Cdc7p-Dbf4p serine/threonine kinase triggers replication initiation in yeast and in human cells. Cdc7p kinase requires the Dbf4p subunit for

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activity, which is periodically degraded during the cell cycle. We have purified the human and yeast Cdc7p-Dbf4p kinases from baculovirus-infected Sf9 cells and have previously shown that the yeast protein phosphorylates subunits of the MCM complex and also DNA polymerase α-primase, the initiating polymerase. We are examining the substrate specificity of the Cdc7p-Dbf4p kinase in an effort to determine its essential targets for DNA replication initiation.

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Figure 3. Deletion of SIR2 restores Mcm2p loading at origins in the cdc6-4 mutant. Mcm2p chromatin immunoprecipitation (ChIP) assays were performed using cells arrested in G2/M with nocodazole and then released to a G1 arrest for 60, 90, or 120 min, to allow MCM protein loading at origins. The location of PCR primers shown in (A) were used to amplify origin and nearby nonorigin sequences for the ARS1, ARS315, and ARS501 origins following immunoprecipitation of Mcm2p. Data for the cdc6-4 permissive (C) and nonpermissive temperature (D) is shown. A representative input DNA PCR sample is shown for each origin examined.

NCI60 set of human tumor cell lines has indicated that both CDC7 and DBF4 exhibit increased expression in some of these cancer cell lines. Importantly, CDC7 protein abundance is also increased in a subset of these cell lines but is not detectable in normal tissues. We are currently examining how human CDC7-DBF4 kinase activity may influence normal and tumor cell growth.

required for its essential replication function. Determining how the DNA damage checkpoint pathway may alter the activity of Cdc7p-Dbf4p kinase or modulate DNA replication and repair is an ongoing and exciting area of research. Lastly, we are examining the abundance of CDC7 and DBF4 in normal and human cancer cell lines. Semiquantitative RT-PCR performed on the Recent Publications

Pappas, Donald L., Jr., Ryan Frisch, and Michael Weinreich. 2004. The NAD+-dependent Sir2p histone deacetylase is a negative regulator of chromosomal DNA replication. Genes and Development 18(7): 769–781. Weinreich, Michael, Madaleine A. Palacios DeBeer, and Catherine A. Fox. 2004. The activities of eukaryotic replication origins in chromatin. Biochemica et Biophysica Acta 1677(1–3): 142–157. Bose, M.E., K.H. McConnell, K.A. Gardner-Aukema, U. Müller, M. Weinreich, J.L. Keck, and C.A. Fox. 2004. The origin recognition complex and Sir4 protein recruit Sir1p to yeast silent chromatin through independent interactions requiring a common Sir1p domain. Molecular and Cellular Biology 24(2): 774–786.

From left to right: VanDussen, DeWard, Hammond, Frisch, Pappas, Weinreich

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 Charlotta Lindvall-Weinreich, M.D., Ph.D. Sheri L. Holmen, Ph.D. Troy A. Giambernardi, Ph.D. Cassandra Zylstra, B.S.

Students Holli Charbonneau Nicole Evans Jason Koning Aaron Massie Jose Toro

Research Interests We are currently performing experiments to directly test whether a Lrp5 deficiency causes changes in bone density due to aberrant signaling through ß-catenin. To do this, we have created mice carrying an osteoblast-specific deletion of Lrp5. These mice die within five weeks of birth due to profound deficiencies in bone development. A reciprocal experiment was also performed, in which mice were missing the Apc gene specifically in osteoblasts (and therefore were expressing elevated levels of ß-catenin). In this case, the mutant mice again died very shortly after birth. Their death was associated with a dramatic overgrowth of bone to the point where very little marrow cavity was present (Fig. 1).

y laboratory’s long-term interest is to understand how alterations in the Wnt signaling pathway cause human disease. Wnt signaling is an evolutionarily conserved process that has been adapted to function in the differentiation of most tissues within the body. Given its central role in growth and differentiation, it is not surprising that alterations in the pathway are among the most common events associated with human cancer. In addition, several other human diseases, including osteoporosis, have been linked to alterations in the regulation of Wnt signaling. Recently, my laboratory has focused on understanding the role of Wnt signaling in bone formation. Our interest is not only from the perspective of normal bone development, but also in trying to understand whether aberrant Wnt signaling plays any role in the predisposition of some common tumor types (for example, prostate or breast tumors) to metastasize to and grow in the bone. The long-term goal of this work is to provide insights that could be used in developing strategies to lessen the morbidity and mortality associated with skeletal metastasis.

Our current work on this project is aimed at addressing the molecular mechanisms that underlie the phenotypic observations. We have isolated osteoblasts from these mice and are analyzing them in tissue culture to determine their ability to produce and mineralize osteoid. Also, we are identifying potential downstream mediators of Wnt signaling in osteoblasts via microarray-based expression analysis. We are designing experiments to test the time frame during which activation of Wnt signaling can have effects on bone density. One approach is to use doxycycline-inducible systems to activate ß-catenin signaling in vivo. Another approach is to control the timing of ß-catenin induction in primary osteoblasts.

Wnt signaling in normal bone development Recently, several groups reported that mutations in the Wnt receptor, Lrp5, caused changes in bone density in human patients. We have characterized a mouse strain deficient for Lrp5 and shown that it recapitulates the low-bone-density phenotype seen in human patients deficient for Lrp5. We have furthered this study by showing that mice carrying mutations in both Lrp5 and the related Lrp6 protein have even more severe defects in bone density.

Wnt signaling in prostate development and cancer The activation of the Wnt signaling pathway has been shown in a significant percentage of 59

mechanisms that explain these findings. Interestingly, the development of precancerous hyperplasia in the mammary gland still occurs in Lrp5-deficient animals.

prostate carcinomas. In some cases, this is associated with activating mutations in the ß-catenin genes, while in others, a loss of APC has been demonstrated. Two hallmarks of advanced prostate cancer are the development of skeletal osteoblastic metastasis and the ability of the tumor cells to become independent of androgen for survival. The linkage between the activation of Wnt signaling and bone growth, and the fact that ßcatenin can bind to the androgen receptor and make it more susceptible to activation with steroid hormones other than DHT, make Wnt signaling an attractive candidate for explaining the phenotype of advanced prostate cancer.

In vitro studies of the Wnt receptor complex The reception of Wnt signals is regulated at many levels. The completion of the Human Genome Project has shown that there are 19 different genes that encode Wnt proteins, 9 that encode Frizzled proteins, and the Lrp5 and Lrp6 genes. In addition, there are several proteins that can inhibit Wnt signaling by binding to components of the receptor complex and interfering with normal signaling; these include Dickkopfs (Dkks) and Frizzled-related proteins (FRPs). One of the long-term goals of our laboratory is to understand how specificity is generated for the different signaling pathways.

We are trying to understand the role of the Wnt signaling pathway in normal prostate development. Analysis of mice carrying compound mutations in Lrp5 and Lrp6 revealed the presence of abnormal prostate lobes. We are furthering this analysis by creating mice with a prostate-specific deletion of ß-catenin. We have also created mice with a prostate-specific deletion of the Apc gene. Preliminary analysis shows that these mice develop early-onset prostate cancer. We are preparing to analyze mice at various ages to determine the ability of Apcdeficient prostate tumors to metastasize. We will also assess the ability of Apc-deficient prostate tumors to synergize with other genetic alterations to produce higher-grade prostate carcinoma. Our ultimate goal is to test whether loss of activation of ß-catenin signaling can lead to a mouse model of prostate cancer with skeletal metastases.

Wnt signaling in mammary development and cancer We are also examining the roles of Lrp5 and Lrp6 in normal mammary development. We have found that mice lacking these genes have delays in the normal development process. We are currently assessing the temporal and spatial organization of Lrp6 and Lrp5 expression during mammary development.

We are also addressing the relative roles of Lrp6 and Lrp5 in Wnt1-induced mammary carcinogenesis. We have found that a deficiency of Lrp5 dramatically inhibits the development of Wnt1-induced mammary tumors. In addition, the tumors that do develop are altered in their morphology. Current work aims to determine the

C 60

Figure 1. Disregulation of Wnt signaling specifically in osteoblasts results in dramatic changes in bone development. Shown in Panel A is a hematoxylin and eosin–stained section through a thoracic vertebra of a one-month-old normal mouse. Sections from the same region of bone in age-matched mice carrying an osteoblast-specific deletion of ß-catenin (B) and Apc (C) are shown. Osteoblastspecific deletion of either ß-catenin or Apc causes death within four weeks of birth. This lethality is associated with a dramatic reduction of bone in the ß-catenin mutants (B) and a vast overgrowth of bone in the absence of Apc (C).

External Collaborators Yi Li, Baylor Breast Center, Houston, TX Michael T. Lewis, Baylor Breast Center, Houston, TX Matthew Warman, Case Western Reserve University, Cleveland, OH Thomas Clemens, University of Alabama – Birmingham Mary Bouxsein, Beth Israel Deaconess Medical Center, Boston, Massachusetts Marie Faugere, University of Kentucky, Lexington Wade Bushman, University of Wisconsin – Madison Charles Turner, Indiana University – Purdue University, Indianapolis Kay Macleod, University of Chicago, Illinois Recent Publications Bromberg-White, Jennifer L., Craig P. Webb, Veronique S. Patacsil, Cindy K. Miranti, Bart O. Williams, and Sheri L. Holmen. 2004. Delivery of short hairpin RNA sequences using a replication-competent avian retroviral vector. Journal of Virology 78(9): 4914-4916. Holmen, Sheri L., Adrian Salic, Cassandra R. Zylstra, Marc W. Kirschner, and Bart O. Williams. 2002. A novel set of Wnt-Frizzled fusion proteins identifies receptor components that activate βcatenin-dependent signaling. Journal of Biological Chemistry 277(38): 34727–34735. Takahashi, Masayuki, Ximing J. Yang, Todd T. Lavery, Kyle A. Furge, Bart O. Williams, Maria Tretiakova, Anthony Montag, Nicholas J. Vogelzang, Gian G. Re, A. Julian Garvin, Stefan Söderhäll, Susumu Kagawa, Debra Hazel-Martin, Agneta Nordenskjöld, and Bin Tean Teh. 2002. Gene expression profiling of favorable histology Wilms tumors and its correlation with clinical features. Cancer Research 62(22): 6598–6605.

From left to right: back row, Williams, Charbonneau, Zylstra front row, Lindvall-Weinreich, Giambernardi, Holmen

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, he moved to GlaxoWellcome in 1996 as a research investigator in nuclear receptor drug discovery. Dr. Xu joined VARI as a Senior Scientific Investigator in July 2002. Laboratory Members Staff Yong Li, Ph.D. David Tolbert, Ph.D. Phuzile Ludidi, Ph.D. Jennifer Daugherty, B.S.

Amanda Kovach, B.S. Jennifer Kretschman, B.S. Kelly Suino, B.S.

Visiting scientist Ross Reynolds, Ph.D.

Research Interests binding domain (LBD) bound to diverse ligands, including fatty acids, the lipid-lowering drugs called fibrates, and a new generation of anti-diabetic drugs, the glitazones (Fig. 1). We have also determined crystal structures of these receptors bound to co-activators or co-repressors. These structures provide a framework for understanding the mechanisms of agonists and antagonists, as well as the recruitment of co-activators and co-repressors. These structures also have provided crucial insights for designing the next generation of PPAR medicines. Currently we are developing this project beyond the structures of the ligand-binding domains into the structures of large PPAR fragment/DNA complexes.

ur laboratory uses x-ray crystallography to study the structures and functions of key protein complexes that are important in basic biology, as well as in drug discovery relevant to human diseases such as cancer and diabetes. Currently we are focusing on nuclear hormone receptors and the Met tyrosine kinase receptor. The nuclear hormone receptor family comprises a large number of ligand-regulated and DNAbinding transcriptional factors, which include receptors for classic steroid hormones such as estrogen, progesterone, androgens, and glucocorticoids, as well as for proxisome proliferator activators, vitamin D, vitamin A, and thyroid hormones. One distinguishing fact about the classic receptors is that they are among the most successful targets in the history of drug discovery: every receptor has one or more cognate synthetic ligands currently being used as medicines. In the last two years, we have developed the following projects centering on the structural biology of nuclear receptors.

The human glucocorticoid receptor The human glucocorticoid receptor (GR) is a key regulator of energy metabolism and of homeo-

Peroxisome proliferator–activated receptors The peroxisome proliferator–activated receptors (PPARα, δ, and γ) are the key regulators of glucose and fatty acid homeostasis and, as such, are promising therapeutic targets for cardiovascular disease, diabetes, and cancer. Millions of patients have benefited from treatment with PPARγ medicines for type II diabetes; 2 of the top 100 drugs in 2003 were PPARγ ligands, with combined sales of over $3.0 billion. To understand the molecular basis of ligandmediated signaling by PPARs, we have determined crystal structures of each PPAR’s ligand-

Figure 1. Crystal structure of the PPAR ligandbinding domains. Each PPAR molecule is shown bound to its respective ligand. Each contains 13 α helices and 4 small β strands. The helices are arranged into a three-layer sandwich fold to create a cavity for ligand binding.

stasis of the immune system. GR is also a classic target of drug discovery because of its association with numerous pathological conditions. There are more than 10 GR ligands (including dexamethasone) that are currently used for treatment of such diverse conditions as asthma, allergy, autoimmune diseases, and cancer. At the molecular level, GR can function either as a transcription activator or a transcription repressor. Both functions are tightly regulated by small ligands that bind to the LBD. 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 LBD-LBD dimer interface (Fig. 2), 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. Currently we are crystallizing GR with various steroid or nonsteroid ligands. The information from these structures should provide a rational basis for designing new GR ligands that would reduce side effects relative to current GR drugs. In a collaborative effort, we are extending our studies to the structure of a large GR fragment bound to DNA.

Figure 2. Two views of the crystal structure of the human glucocorticoid receptor LBD bound to dexamethasone and a TIF2 co-activator.

mediate ligand-dependent receptor regulation; this domain has been the focus of intense structural studies. Crystal structures for more than half of the 48 human nuclear receptors have been determined. 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. They also provide many surprises in terms of the identity of ligands, the size and shape of the ligand-binding pockets, and the structural implications of the receptor signaling pathways. There is now only one receptor, constitutive androstanol receptor (CAR), for which the LBD structure remains unsolved. Currently we are studying the mouse CAR molecule in a collaborative effort.

The human androgen receptor The androgen receptor (AR) is the central molecule in the development and progression of prostate cancer, and as such it serves as the molecular target of anti-androgen therapy. However, the majority of prostate cancer patients develop resistance to such therapy, mostly due to mutations in AR that alter its three-dimensional structure, allowing it to escape repression by anti-androgen treatment. Such a hormone-independent prostate cancer is highly aggressive and is responsible for most deaths; currently there is no cure. The development of effective therapies requires detailed understanding of the structure and functions of the central molecule, i.e., the androgen receptor and its interactions with hormones and co-regulators. In this project, our goal is to determine the structures of the mutated AR proteins that alter the response to anti-hormone therapy. We are working on the crystal structure of the full-length AR/DNA complex.

The Met tyrosine kinase receptor Met is a tyrosine kinase receptor that is activated by hepatocyte growth factor/scatter factor (HGF/SF). Aberrant activation of the Met receptor has been linked to the development and metastasis of many types of solid tumors and has been correlated with poor clinical prognosis. HGF/SF has a modular structure with an N-terminal domain, four kringle domains, and an inactive serine protease domain. The structure of the N-terminal domain, which has a single kringle domain (NK1), has been determined; less is known about the structure of the Met extracellular domain. The molecular basis of the Met-HGF/SF interaction and the resulting activation of Met signaling remain poorly understood. In collaboration with George Vande Woude and Ermanno Gherardi, we are developing this project to solve the crystal structure of the Met receptor/HGF complex.

Structural genomics of nuclear receptor ligand-binding domains The ligand-binding domain (LBD) of nuclear receptors contains key structural elements that 63

External Collaborators Ermanno Gherardi, University of Cambridge, United Kingdom Steve Kliewer, University of Texas Southwestern Medical Center, Dallas Mill Lambert and Tim Willson, GlaxoSmithKline Inc., Research Triangle Park, North Carolina Donald MacDonnell, Duke University, Durham, North Carolina Stoney Simmons, National Institutes of Health, Bethesda, Maryland Brad Thompson, University of Texas Medical Branch at Galveston Ming-Jer Tsai, Baylor College of Medicine, Houston, Texas Scott Thacher, Orphagen Pharmaceuticals, San Diego, California Recent Publications Agostini Maura, Mark Gurnell, David B. Savage, Emily M. Wood, Aaron G. Smith, Odelia Rajanayagam, Keith T. Garnes, Sidney H. Levinson, H. Eric Xu, John W.R. Schwabe, Timothy M. Willson, Stephen O’Rahilly, and V. Krishna Chatterjee. 2004. Tyrosine agonists reverse the molecular defects associated with dominant-negative mutations in human peroxisome proliferator-activated receptor γ. Endocrinology 145(4): 1527–1538. Stanley, T.B., L.M. Leesnitzer, V.G. Montana, C.M. Galardi, M.H. Lambert, H.E. Xu, L.B. Moore, S.G. Blanchard, and J.B. Stimmel. 2003. Subtype-specific effects of peroxisome proliferator–activated receptor ligands on co-repressor affinity. Biochemistry 42: 9278–9287. Li, Yong, Millard H. Lambert, and H. Eric Xu. 2003. Activation of nuclear receptors: a perspective from structural genomics. Structure 11(7): 741–746. Sznaidman, Marcos L., Curt D. Haffner, Patrick R. Maloney, Adam Fivush, Esther Chao, Donna Goreham, Michael L. Sierra, Christelle LeGrumelec, H. Eric Xu, Valerie G. Montana, Millard H. Lambert, Timothy M. Willson, et al. 2003. Novel selective small molecule agonists for peroxisome proliferator–activated receptor δ (PPARδ)—synthesis and biological activity. Bioorganic & Medicinal Chemistry Letters 13(9): 1517–1521. Xu, H.E., and M.H. Lambert. 2003. Structural insights into regulation of nuclear receptors by ligands. NURSA e-journal 1(1): ID# 3.06132003.06132001. Xu, H.E., and M.H. Lambert. 2003. Structural mechanisms of ligand-mediated signaling by nuclear receptors. In Handbook of Cell Signaling, R.A. Bradshaw and E.A. Dennis, eds. Vol. 3, Chap. 272. San Diego: Academic Press.

From left to right: Daugherty, Kretschman, Kovach, Suino, Reynolds, Tolbert, Xu, Li

Laboratory of Mammalian 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. Lan Wang, Ph.D. Liang Kang

Visiting scientist Dong Kong, Ph.D.

Research Interests important for the rapid degradation of Lfng mRNA, which ensures accurate oscillation.

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 of their axial skeleton 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. Our recent studies indicate that the cyclic expression of Lfng is controlled by a negative feedback loop. The signals transmitted through the loop are mediated by the components in the Notch signaling pathway. We found that that there is a binary switch that determines the “on” and “off” states of Lfng transcription. When Lfng is on, it modifies the Notch receptors, therefore activating the downstream effecter Hes7, a transcription repressor. We found that Hes7 can directly bind to the 5′ regulatory region of the Lfng gene and switch off the transcription of Lfng. When Lfng is down-regulated, it results in down-regulation of Hes7 and thus relieves the transcriptional repression of Hes7 on Lfng. We have also demonstrated that the 3′ untranslated region (UTR) is

Germ cell development The second focus of our laboratory is on germ cell development—particularly the mechanisms that govern germ cell migration, survival, spermatogonial stem cell renewal, and differentiation—and the 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 if germ cells are 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. We are also studying spontaneous mutations that cause sterility. In mice, primordial germ cells (PGCs) are differentiated from the epiblasts during an early embryonic stage. After their formation, they migrate through the dorsal mesen65

ing the candidate genes by transgenic rescue and direct sequencing.

tery and enter the genital ridge, where they collaborate with the somatic gonad cells to form the gonads. The PGCs proliferate during their migration. We are studying the spontaneous mutation atrichosis (at), which causes male and female sterility. Preliminary data suggest that this mutation affects fetal germ cell proliferation. We found that by 12.5 dpc, there already are significantly fewer germ cells in the gonads of atrichosis embryos relative to the wild type. We have mapped the atrichosis mutation to a 270-kb region on chromosome 10. We are now screen-

Another mutation we are studying is in the sks gene; this mutation affects normal meiosis in both sexes. Our results indicate that sks is required for the metaphase/anaphase transition in meiosis I. In the sks mutant, homologous chromosomes fail to separate; therefore, meiosis is stopped at MI. We have demonstrated that this failure is due to the inability of the sks mutant to degrade securin in the primary spermatocytes and possibly in the oocytes.

Recent Publications Mustonen, Tuija, Mark Tummers, Tadahisa Mikami, Nobuyuki Ito, Nian Zhang, Thomas Gridley, and Irma Thesleff. 2002. Lunatic fringe, FGF, and BMP regulate the Notch pathway during epithelial morphogenesis of teeth. Developmental Biology 248(2): 281â&#x20AC;&#x201C;293. Zhang, Nian, Christine R. Norton, and Thomas Gridley. 2002. Segmentation defects of Notch pathway mutants and absence of a synergistic phenotype in lunatic fringe/radical fringe double mutant mice. Genesis 33(1): 21â&#x20AC;&#x201C;28.

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

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.

2003 Award to Dr. Robert A. Weinberg The 2003 recipient of the Daniel Nathans Memorial Award was Robert A. Weinberg, who received the award for his pioneering work in cancer research as discoverer of the first human oncogene and the first tumor suppressor gene. He presented two talks on March 8, 2004, in Grand Rapids, a technical presentation on mechanisms of human tumor formation, which involved some of his current research on telomerases, and a talk to the lay public titled “How cancer begins.”

George Vande Woude and Robert Weinberg

Award Recipients 2000 2001 2002 2003

Richard D. Klausner, M.D. Francis S. Collins, M.D., Ph.D. Lawrence H. Einhorn, M.D. Robert A. Weinberg, Ph.D.

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 fellow is assigned to a scientific investigator who oversees the progress and direction of research. Fellows who worked in VARI laboratories in 2003 and early 2004 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 Paul Bromann Northwestern University, Evanston, Illinois VARI mentor: Sara Courtneidge

Carrie Graveel University of Wisconsin â&#x20AC;&#x201C; Madison VARI mentor: George Vande Woude

Jennifer Bromberg-White Pennsylvania State University College of Medicine, Hershey VARI mentor: Craig Webb

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

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

Xudong Liang Qinghai Medical University, Xining, China VARI mentor: Nicholas Duesbery

Suganthi Chinnaswamy Southern Illinois University, Carbondale VARI mentor: Cindy Miranti

Charlotta Lindvall Karolinska Institute, Stockholm, Sweden VARI mentor: Bart Williams

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

Phumzile Loudidi Cambridge University, England VARI mentor: Eric Xu

Mathew Edick University of Tennessee, Memphis VARI mentor: Cindy Miranti

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

Kathryn Eisenmann University of Minnesota, Minneapolis VARI mentor: Arthur Alberts

Ian Pass University of Dundee, Scotland VARI mentor: Sara Courtneidge

Chong-Feng Gao Tokyo Medical and Dental University, Japan VARI mentor: George Vande Woude

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

Muthu Shanmugam National University of Singapore, Singapore VARI mentor: Brian Haab

Peng Fei Wang Fourth Military Medical University, China VARI mentor: Bin Teh

Min-Han Tan National University of Singapore, Singapore VARI mentor: Bin Teh

Yu-Der Wen University of South Florida, Tampa VARI mentor: Eric Xu

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

Qian Xie Fudan University, Shanghai, China VARI mentor: George Vande Woude

Bradley Wallar University of Minnesota, Minneapolis VARI mentor: Arthur Alberts

Chun Zhang Tokyo Medical and Dental University, Japan VARI mentor: Bin Teh

Lan Wang Peking Union Medical College, Beijing, China VARI mentor: Nian Zhang

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 four 2003 GRAPCEP students were

Mary Beth Clifford Ottawa Hills High School

Karen Macy Creston High School

Jaynna DeLeeu Ottawa Hills High School

Huong Tran 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 technologies, and to learn invaluable people and presentation skills. At the completion of the 10week program, the students summarize their projects in an oral presentation. From May 2003 to February 2004, VARI hosted 32 students from 19 colleges and universities, both in formal summer internships under the Frederik and Lena Meijer Student Internship Program and in other student positions during the year.

Anderson University, Anderson, Indiana Rachel Wezeman Aquinas College, Grand Rapids, Michigan Kerry Lucas Calvin College, Grand Rapids, Michigan Erin Connelly Aaron DeWard Beverly Illian Jared Knol Jason Koning Aaron Massie Allison Shively Central Michigan University, Mount Pleasant Megan Senchuk Cornell University, Ithaca, New York Susan Kloet Cornerstone University, Grand Rapids, Michigan Juraj Zahatnansky Ferris State University, Big Rapids, Michigan Jon Rutkowski Grand Rapids Community College, Michigan Jennifer Kaufman Grand Valley State University, Allendale, Michigan Nicole Evans Harvard University, Cambridge, Massachusetts Christine Moore

Hope College, Holland, Michigan Wendy Schroeder Timothy Stowe Kalamazoo College, Kalamazoo, Michigan Laila Al-Duwaisan Michigan State University, East Lansing Meg Closs Stephanie Ellison Casey Madura Hilary Wade Northwestern University, Evanston, Illinois Colleen Bisch Trinity College, Dublin, Ireland Olga Motornao University of Bath, United Kingdom Victoria Hammond University of Chicago, Illinois Jonathan Douglas University of Michigan, Ann Arbor Daphna Atias Erika Briegel Amy White Natalie Wolters Vanderbilt University, Nashville, Tennessee John Zoerhoff

VARI Seminar Series

VARI Seminar Series 2003 January Shuichi Takayama, University of Michigan, Ann Arbor “Cell biology chips: technologies for cell sorting and subcellular signaling (EGF/integrin) studies” John Sacci, University of Maryland School of Medicine, Baltimore “Identifying gene expression in plasmodial liver-stage parasites: a needle in a haystack?” Jack Dixon, University of Michigan, Ann Arbor “Functional genomics: probing the intersection of bacterial pathogens with signal transduction pathways” Nita Maihle, Mayo Clinic, Rochester, Minnesota “Soluble ErbB/EGF receptors: form, function, and clinical utility”

February Thomas Sturgill, University of Virginia, Charlottesville “Regulation, function, and structure of MAPKAP kinases” David Baltimore, California Institute of Technology, Pasadena “NF-κ as a neuronal intracellular messenger” Zhenping Zhuang, National Institutes of Health, Bethesda, Maryland “Integration of tissue microdissection into genomics and proteomics: a new approach to study disease” March Richard Vile, Mayo Clinic, Rochester, Minnesota “Gene and immunotherapy for cancer: making the tumor a dangerous place to die” Marc Lippman, University of Michigan, Ann Arbor “Inducing apoptosis as a new approach to cancer therapy” George Prendergast, Thomas Jefferson University, Philadelphia, Pennsylvania “Novel mechanisms of cancer suppression and cell suicide” April Chung Lee, Northwestern University, Evanston, Illinois “TGF-β-based gene therapy for cancer” Fred Chang, Columbia University, New York City “Microtubules and formins in regulation of cell polarity” Max Wicha, University of Michigan, Ann Arbor “Stem cells in the normal and cancerous breast” 83

May Michael White, University of Texas Southwestern Medical Center, Dallas “Molecular linchpins in the tumorigenic platform” Maxwell Gottesman, Columbia University, New York City “AKAP 121 and cAMP-dependent mRNA translocation to mitochondria” Alfred Singer and Dinah Singer, National Cancer Institute, Bethesda, Maryland “Regulation of MHC class I gene expression” (D.S.) “How a bipotential cell in the thymus determines its appropriate cell fate” (A.S.) Larry Einhorn, Indiana University, Bloomington The Daniel Nathans Lecture: “Testicular cancer: a model for a curable neoplasm” “Curing cancer: unrealistic expectations versus promises fulfilled” (lay audience) June Stuart Yuspa, National Cancer Institute, Bethesda, Maryland “mtCLIC/CLIC4, a p53- and TNF-α-regulated chloride channel protein, is a novel molecular target involved in apoptosis, growth arrest, and cancer pathogenesis” James Baker, University of Michigan, Ann Arbor “Nanotherapeutics for cancer” Richard Gaynor, Eli Lilly, Indianapolis, Indiana “Regulation of the NK-κB pathway” Karen Antman, Columbia University, New York City “Translational studies in sarcomas, an understudied malignancy” July Norman Coleman, National Cancer Institute, Bethesda, Maryland “Radiation oncology in the 21st century: technical advancements and biological concepts” Alfred Brown, Yale University, New Haven, Connecticut “The university discovery engine: turning academic research into public benefit” Bruce Kemp, St. Vincent’s Institute of Medical Research, Melbourne, Australia “AMPK mediating the health benefits of exercise and diet” Jas Lang, Ohio State University, Columbus “Molecular genetics of head and neck cancer” August Evan Keller, University of Michigan, Ann Arbor “The biology of prostate cancer skeletal metastases” Mario Capecchi, University of Utah, Salt Lake City “Gene targeting into the 21st century: mouse models of human disease from cancer to psychiatric disorders”

September Rudy Castellani, Michigan State University, East Lansing “Strain variation in sporadic Creutzfeldt-Jakob disease and the role of 14-3-3 protein in diagnosis” Mary Ann Handel, University of Tennessee, Knoxville “Genetic models for analysis of chromosome segregation and gametogenesis” Susan Love, Susan Love Breast Cancer Research Foundation, Santa Barbara, California “The intraductal approach: a window to the breast” October Timothy Willson, GlaxoSmithKline, Research Triangle Park, North Carolina “Molecular mechanisms of nuclear receptor regulation by lipid metabolites” Christopher Leamon, Endocyte, Inc., West Lafayette, Indiana “Folate-targeted technology” Richard Fisher, University of Rochester, New York “Biology and mechanisms of non-Hodgkins lymphoma” November Richard Paules, National Institute of Environmental and Health Sciences, Research Triangle Park, North Carolina “The use of genomics in the quest for predicting the adverse health effects of environmental stressors” Brad Ozanne, Beatson Institute for Cancer Research, Glasgow, Scotland “AP-1 regulates a multigenic invasion program”

2004 January Karen Vousden, Beatson Institute for Cancer Research, Glasgow, Scotland “The p53 pathway as a therapeutic target” Patrick Brophy, University of Michigan, Ann Arbor “Ureteric budding: controlling two ends of the event” February Jeffrey Settleman, Harvard Medical School, Boston, Massachusetts “Rho GTPase signaling in development” Josef Prchal, Baylor College of Medicine, Houston, Texas “Tumors, hypoxia, and polycythemic disorders” March Robert Weinberg, Whitehead Institute for Biomedical Research, Cambridge, Massachusetts The Daniel Nathans Lecture: “Mechanisms of human tumor formation” “How cancer begins” (lay audience) Mike Caliguiri, Ohio State University, Columbus “Natural killer cells: biology and clinical implications” Morris Pollard, Walther Cancer Institute, Indianapolis, Indiana “Prevention of hormone-refractory prostate cancer in LW rats” James Basilion, Massachusetts General Hospital, Charlestown, Massachusetts “Noninvasive imaging of gene expression: imaging multiple targets simultaneously” David Frank, Harvard University, Cambridge, Massachusetts “STAT signal transduction in the pathogenesis and treatment of cancer” April Ernst-Robert Lengyel, University of Chicago, Illinois “Regulation of proteolysis by adhesion receptors” Kathleen Siminovitch, Mount Sinai Hospital, Toronto, Canada “The Wiskott Aldrich syndrome protein: forging the link between actin and T cell activation” Jose Cibelli, Michigan State University, East Lansing “Embryonic stem cells by parthenogenesis in mammals”

Organization

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 L. Goldstein, M.D. Tony Hunter, Ph.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. Joan Brugge, Ph.D. Webster Cavenee, Ph.D. Frank McCormick, Ph.D. Davor Solter, M.D., Ph.D. Bruce Stillman, Ph.D.

Van Andel Research Institute Office of the Director Director

George Vande Woude, Ph.D.

Deputy Director for Clinical Programs

Deputy Director for Special Programs

Rick Hay, M.D., Ph.D.

James H. Resau, Ph.D.

Deputy Director for Research Operations

Director for Research Administration

Bin T. Teh, M.D., Ph.D.

Roberta Jones

Van Andel Research Institute Administrator to the Director

Science Editor

Michelle Reed

David E. Nadziejka

Administration Group

From left to right: Carrigan, Johnson, Novakowski, Holman, Ritsema, Pyle

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 Casey Wondergem, Special Liasion to the Chairman Ann Schoen

Purchasing Richard Disbrow, Manager David Clark Chris Kutchinski Amy Poplaski

Communications and Development Patrick Kelly, Vice President, Development John Van Fossen, Director of External Affairs Dianna Davidson Tina Newhouse Margo Pratt

Facilities Samuel Pinto, Facilities Manager Jason Dawes Richard Sal Richard Ulrich Security Kevin Denhof, Chief Sabrina Antio Sandra Folino Emily Young

Information Technology Bryon Campbell, Ph.D., Chief Information Officer David Drolett, Manager Michael Roe, Manager Kathleen Cerasoli Michael Foster Kenneth Hoekman Kimberlee Jeffries Russell Vander Mey Candy Wilkerson

Glass Washing/ Media Preparation Heather Frazee Troy Lawson Nick Miltgen Contract Support Valeria Long, Librarian (Grand Valley State University) Jim Kidder, Safety Manager (Michigan State University) Terry Bruning Raymond Rupp Patty Sund

Human Resources Linda Zarzecki, Director Margie Hoving Pamela Murray Angela Plutschouw Grants and Contracts Carolyn W. Witt, Director Sara Oâ&#x20AC;&#x2122;Neal Rob Junge Finance Timothy Myers, Controller Stacie Bohr Richard Herrick Keri Jackson Angela Lawrence Kevin Tefelsky Jamie VanPortfleet 92

Van Andel Institute Van Andel Institute Board of Trustees David Van Andel, Chairman Peter C. Cook Ralph W. Hauenstein John C. Kennedy

Board of Scientific Advisors Michael S. Brown, M.D., Chairman Richard Axel, M.D. Joseph L. Goldstein, M.D. Tony Hunter, Ph.D. Phillip A. Sharp, Ph.D.

Van Andel Research Institute Board of Trustees David Van Andel, Chairman Christian Helmus, M.D. Fritz Rottman, Ph.D. James B. Wyngaarden, M.D.

Van Andel Education Institute Board of Trustees David Van Andel, Chairman Gordon Van Harn, Ph.D. Gordon Van Wylen, Sc.D.

Chief Executive Officer David Van Andel

Van Andel Research Institute Director George Vande Woude, Ph.D.

Van Andel Education Institute Director Gordon Van Harn, Ph.D.

Communications Vice President Casey Wondergem

Development Vice President Patrick Kelly

Chief Financial Officer R. Jack Frick

Van Andel Research Institute DIRECTOR â&#x20AC;&#x201C; George Vande Woude

SCIENTIFIC ADVISORY BOARD Alan Bernstein, Ph.D. Malcom Brenner, M.D., Ph.D. Patrick O. Brown, M.D., Ph.D. Joan Brugge, Ph.D. Webster Cavenee, Ph.D. Frank McCormick, Ph.D. Davor Solter, M.D., Ph.D. Bruce Stillman, Ph.D.

Deputy Directors Clinical Programs - Rick Hay Special Programs - James Resau Research Operations - Bin Teh

Director for Research Administration Roberta Jones

SPECIAL PROGRAMS

BASIC SCIENCE

Antibody Technology Cao Molecular Diagnostics Resau Cancer Cell Biology

Animal Models

Haab -- Protein Microarray Technology & Cancer Diagnostics Koo -- Cancer Pharmacogenetics Vande Woude -- Molecular Oncology Webb -- Tumor Metastasis & Angiogenesis

Duesbery -- Developmental Cell Biology Williams -- Cell Signaling & Carcinogenesis Zhang -- Mammalian Developmental Genetics

Signal Transduction

Cancer Genetics

Alberts -- Cell Structure & Signal Integration Courtneidge -- Signal Regulation & Cancer Miranti -- Integrin Signaling & Tumorigenesis

Teh -- Cancer Genetics

DNA Replication & Repair

Structural Biology

Weinreich -- Chromosome Replication

Xu -- Structural Sciences

Germline Modification Swiatek Transgenics and Vivarium Eagleson Cytogenetics Swiatek Teh Sequencing Teh Analytical, Cellular, & Molecular Microscopy Resau Mass Spectrometry & Proteomics Cavey Bioinformatics Furge

Recent VARI Photos

Back cover photo: Mouse spermatocyte cluster In mammalian testes, germ cells form clusters and they develop in synchrony. This photograph shows a cluster of primary spermatocytes at meiosis I in a mouse seminiferous tubule. Condensed chromosomes are stained red with anti-phospho-histone-3, and Îą-tubulin is stained green. (Zhang)

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, height, weight, marital status, disability, pregnancy, or veteran status, except where an accommodation is unavailable and/or it is a bone fide occupational qualification.

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