Molecular Oncology and Human Genetics

George S. Brush, Ph.D.
Craig N. Giroux, Ph.D.
Miriam L. Greenberg, Ph.D.
Hyeong-Reh C. Kim, Ph.D.
Raymond R. Mattingly, Ph.D.
Michael A. Tainsky, Ph.D.
Jeffrey W. Taub, M.D.
Hai-Young Wu, Ph.D.
Gen Sheng Wu, Ph.D.
Youming Xie, Ph.D.
Fayth K. Yoshimura, Ph.D.

Members of the ATM family of protein kinases are central regulators of the DNA damage response in eukaryotic cells, serving to directly and indirectly control downstream targets that mediate cell cycle delay, apoptosis, and DNA repair. A defect in any one of these three processes greatly increases the risk of cancer development. One of the main goals of our research program is to define the molecular mechanisms underlying the function of ATM and related enzymes such as ATR. Because the DNA damage response pathways have been highly conserved through evolution, we have elected to study the experimentally tractable budding yeast Saccharomyces cerevisiae. A principal homologue of ATM and ATR in yeast is Mec1, required for multiple checkpoint-associated cell cycle delay responses and certain types of DNA repair. We are employing both genetic and biochemical methods to further understand 1) how the Mec1 protein kinase is activated and 2) the consequences of this activation. Our recent studies have explored the role of Mec1 during meiosis and have led to a new project aimed at defining the means by which a meiotic cell commits to DNA replication and prevents DNA re-replication. We anticipate that these studies will provide fundamental insight into mechanisms that preserve genomic integrity during gamete development.

Selected Publications
Brush, G.S., Morrow, D.M., Hieter, P., and Kelly, T.J. The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast. Proc. Natl. Acad. Sci. USA 93: 15075-15080, 1996

Brush, G.S. and Kelly, T.J. Phosphorylation of the replication protein A large subunit in the Saccharomyces cerevisiae checkpoint response. Nucleic Acids Res. 28: 3725-3732, 2000

Brush, G.S., Clifford, D.M., Marinco, S.M., and Bartrand, A.J. Replication protein A is sequentially phosphorylated during meiosis. Nucleic Acids Res. 29: 4808-4817, 2001.

Clifford, D.M., Marinco, S.M., and Brush, G.S. The meiosis-specific protein kinase Ime2 directs phosphorylation of replication protein A.J. Biol. Chem. 279: 6163-6170, 2004.

Bartrand, A.J., Iyasu, D., and Brush, G.S.  DNA stimulates Mec1-mediated phosphorylation of replication protein A. J. Biol. Chem. 279: 26762-26767, 2004

Clifford, D.M., Stark, K.E., Gardner, K.E., Hoffmann-Benning, S., and Brush, G.S.  Mechanistic insight into the Cdc28-related protein kinase Ime2 through analysis of replication protein A phosphorylation. Cell Cycle 4, 1826-1833, 2005.

Bartrand, A.J., Iyasu, D., Marinco, S.M., and Brush, G.S. Evidence of meiotic crossover control in Saccharomyces cerevisiae through Mec1-mediated phosphorylation of replication protein A. Genetics172: 27-39, 2006.

Sawarynski, K.E., Kaplun, A., Tzivion, G., and Brush, G.S. Distinct activities of the related protein kinases Cdk1 and Ime2. Biochem. Biophys. Acta1773: 450-456, 2007.

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Craig N. Giroux, Ph.D.
Molecular Oncology and Human Genetics
Ph.D., Massachusetts Institute of Technology, 1979

In our laboratory, we consider the molecular origins of cancer as a fundamental problem in cellular gene - environment interactions: both genetic and environmental risk factors interact to determine neoplasia. The human genome is subject to continuous environmental challenge. This environmental challenge combines with genetic susceptibility factors to determine the phenotype and health of an individual and to predispose individuals to specific diseases. We are a systems biology oriented research group that integrates experimental laboratory studies with computational analysis and genetic network modeling. For both our laboratory studies and computational modeling, we employ yeast and mammalian cell systems. Comparison of the yeast and human genomes confirms that the genetic pathways mediating the cellular responses to environmental stress are remarkably conserved. We have exploited this conservation to develop a yeast functional genomics model for the cellular response to carcinogens. Our system provides mechanism-based risk assessment by profiling the carcinogen response at three levels: genetic sensitivity to identify susceptibility genes (cellular phenotype level), gene expression microarrays to identify biomarkers (genetic network level), and high throughput, quantitative cell based assays to screen for therapeutic agents (gene and pathway target level). Oxidative stress, a known mechanism that is implicated in chemical carcinogenesis, has been used to establish our experimental system. Yeast and mammalian cells exhibit a highly reproducible graded pattern of dose dependent responses to oxidant exposure: both protective responses (adaptation, cell cycle checkpoint arrest) and adverse responses (programmed cell death, necrosis). In one project in our lab, we are using molecular profiling technologies and computational modeling to identify gene network signatures, novel biomarkers of the protective and adverse cellular responses to drug and chemical exposure. In a second project in collaboration with the Karmanos oncogenomics group, we are using genetic network analysis of molecular profiling data to model the systems behavior of breast cancer progression for distinct tumor clinical subtypes. In a third project, we are developing high throughput screening systems to identify druggable gene targets that modulate cell survival and death pathways in response to chemical or chemotherapeutic challenge.

Selected Publications
Weiss, A., Delproposto, J, and Giroux, C.N. High-throughput phenotypic profiling of gene-environment interactions by quantitative growth curve analysis in Saccharomyces cerevisiae. Analytical Biochemistry 327: 23 - 34, 2004

Giroux, C.N., Ekins, S., Fan, J., Abdullah, I., Nikolsky, Y., Bugrim, A., and Nikolskaya, T. A genetic network approach to comparative toxicogenomics and risk assessment: the oxidative stress response. Toxicological Sciences 84(S1):282, 2005.

Ekins, S. and Giroux, C.N. Chapter 6: Systems Approaches for Pharmaceutical Research and Development. in Computer Applications in Pharmaceutical Research and Development, pages 139 – 165, ed. Sean Ekins, John Wiley and Sons, Hoboken, NJ, 2006.

Ekins, S. and Giroux, C.N. Mammalian Proteome and Toxicant Network Analysis. in Pathway Analysis for Drug Discovery, edited by Anton Yuryev, to be published by John Wiley and Sons, in press, 2007. 

Brigitte Ganter and Craig N. Giroux, "Emerging Applications of Network and Pathway Analysis in Drug Discovery and Development", to appear in Current Opinion in Drug Discovery and Development, in press 2008.

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Two NIH-funded projects in her laboratory utilize genetic, molecular, and biochemical approaches in the yeast model system to address fundamental questions with potential impact on the treatment of human disorders. I. The Role of Cardiolipin (CL) in Mitochondrial Biogenesis and Function. Cardiolipin is a structurally unique phospholipid present in the mitochondria of all eukaryotes tested, and is hypothesized to play a role in apoptosis and in doxorubicin-induced cardiotoxicity. The importance of CL is underscored by the finding that the life-threatening genetic disorder Barth syndrome, characterized by cardiomyopathy, skeletal myopathy, neutropenia, and growth retardation results from defective CL composition. Dr. Greenberg's laboratory has shown that CL plays a major role in mitochondrial biogenesis and function. Recent evidence indicates that CL is also required for essential cellular processes independent of its role in mitochondrial bioenergetics, as CL mutants have defects that are not characteristic of general mitochondrial dysfunction (petite) mutants. Specifically, CL mutants exhibit defective growth at elevated temperature, which is associated with perturbation of cell wall biogenesis and reduced life span. The long-term goal of the Greenberg laboratory is to elucidate 1) the role of CL in cellular functions, 2) the mechanisms that regulate CL synthesis, and 3) the mechanism underlying cellular dysfunction in Barth syndrome, and 3) the role of CL in doxorubicin-induced cardiotoxicity. II. Molecular Targets of Lithium (Li) and Valproate (VPA). Bipolar affective disorder (manic-depressive illness, BD) is a chronic, severe, debilitating illness affecting 1-2% of the population. Two drugs, lithium (Li) and valproate (VPA), are currently approved by the FDA for treatment of BD, and neither drug is completely effective. The molecular mechanisms underlying the therapeutic effects of these drugs have not been established. The Greenberg laboratory is exploiting the yeast model system to understand these mechanisms, as follows: 1) They have shown that human myo-inositol-3-phosphate (MIP) synthase, the rate limiting enzyme in inositol synthesis, is functional in yeast, and that valproate indirectly inhibits both human and yeast enzymes. These studies identify MIP synthase as a target of valproate, and establish inositol synthesis as a common target of both lithium and valproate. Functional conservation of MIP synthase in yeast and human cells strongly validates the yeast model to elucidate the therapeutic mechanisms of the drugs. Her current research seeks to 1) elucidate the mechanism of inhibition of MIP synthase by valproate, 2) determine how MIP synthase is regulated in human neuronal cells, and 3) identify the cellular processes that are perturbed by lithium and valproate mediated inositol depletion.

Selected Publications
JShi, Y., Azab, A.N., Thompson, M.N., and Greenberg, M.L.  Inositol phosphates and phosphoinositides in health and disease.  In: Biology of Inositols and Phosphoinositides, Majumder, A.L., and Biswas, B.B., eds., Springer Press,Subcellular Biochemistry 39:265-292, 2006.

Azab, A.N. and Greenberg, M.L.  The Lipid Connection to Bipolar Disorder.Future Neurology, 1:505-513, 2006.

Li, G., Chen, S., Thompson, M.N., and Greenberg, M.L. New insights into the regulation of cardiolipin biosynthesis in yeast: Implications for Barth syndrome.Biochim Biophys Acta, 1771:432-41, 2007.

Azab, A.N., He, Q., Ju, S., Li, G., and Greenberg, M.L.  Glycogen synthase kinase-3 is required for optimal de novo synthesis of inositol,Mol. Microbiol. 63:1248-1258, 2007.

Azab, A.N. and Greenberg, M.L.  Anticonvulsant Efficacy of Valproate-like Carboxylic Acids - A Potential Target for Anti-bipolar Therapy. Bipolar Disorders, 9:197-205, 2007.

Zhong, Q., Li, G., Gvozdenovic-Jeremic, J., and Greenberg, M.L.  Up-regulation of the cell integrity pathway inSaccharomyces cerevisiae suppresses temperature sensitivity of thepgs1D mutant. J. Biol. Chem., 282:15946–15953, 2007.

Amigues, E., Greenberg, M.L., Ju, S., Chen, Y., and Migaud, M.E. Synthesis of spirocyclophospho-glucoses and glucitols. Tetrahedron, 63:10042- 63:10042-10053, 2007. 

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The long-term objective of our research is to unveil how growth factors and extracellular matrix regulate intracellular signal transduction pathways critical for breast and prostate cancer progression. Our particular interests include roles of tumor-produced platelet-derived growth factor in the mediation of tumor-stromal interactions critical for tumor cell invasion, angiogenesis and metastasis. We are also investigating the role of tissue inhibitor of metalloproteinase (TIMP)-1, a natural inhibitor of matrix degrading enzymes, in the regulation of cell survival and polarization of human breast epithelium.

Selected Publications
Yu, J-H., Deuel, T., and Kim, H-R. C.  Platelet-derived growth factor (PDGF) -inducedreceptor-alpha activates JNK-1 and antagonizes PDGF receptor-beta phenotypic transformation. J. Biol. Chem. 275: 19076-19882, 2000.
                                                                                                                                                          
Lin, H-M., Lee, Y. J., Li, G., Pestell, R. G., and Kim, H.-R. C.  Bcl-2 induces cyclin D1 promoter activity in human breast epithelial cells independent of cell anchorage.  Cell Death & Differentiation 8: 44-50, 2001.
 
Moon, B.-K.,  Lee, Y. J.,  Battle, P., Jessup, J. M., Raz, A. and Kim, H.-R. C.  Galectin-3 inhibits nitric oxide induced cell death in human breast carcinoma cells:  Implication of galectin-3 function during metastasis.  Am. J. Pathology 159: 1055-1060, 2001.

Yu, F., Finley, RL Jr., Raz, A., Kim, H.-R. C.   Galectin-3 translocates to the perinuclear membranes and inhibits cytochrome c release from the mitochondria:  A role for synexin in galectin-3 translocation. J. Biol. Chem.  277: 15819-15827, 2002.

Lin, H-M., Pestell, R. G., Raz, A. and Kim, H.-R. C.  Galectin-3 enhances cyclin D1 promoter activity through SP1 and a cAMP-responsive element in human breast epithelial cells.  Oncogene 21: 8001-8010, 2002.

Yu, J., Ustach, C. and Kim, H.-R. C.  Platelet-derived growth factor signaling and human cancer.  Journal of Biochemistry and Molecular Biology 36: 49-59, 2003

Liu, X-W, Bernardo, M.M., Fridman, R. and Kim, H.-R. C.  Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Protects Human Breast Epithelial Cells against Intrinsic Apoptotic Cell Death via the FAK/PI 3-kinase and MAPK Signaling Pathway Independent of Matrix Metalloproteinase Inhibition.  J. Biol. Chem. 278: 40364-40372, 2003  

Yu, J. Liu, X-W, and  Kim, H.-R. C.  Platelet-Derived Growth Factor (PDGF) Receptor-a-activated c-Jun NH2-terminal Kinase-1 is critical for PDGF-induced p21WAF1/CIP1 promoter activity independent of p53.   J. Biol. Chem. 278: 49582-49588, 2003

Ustach, C. V., Taube, M. E.,  Hurst, N., Bagat, S., Bonfil, D., Cher, M., Schuger, L and Kim, H.-R. C.  A potential oncogenic activity of PDGF D in prostate cancer progression.  Cancer Research, 64: 1722-1729, 2004.
        
Liu, X-W., Taube, M.E., Jung, K-K., Zhong, D.,Lee, Y. J., Roshy, S., Sloane, S., Fridman, R and Kim, H.-R. C. Tissue Inhibitor of Metalloproteinase-1 protects human breast epithelial cells from extrinsic cell death:  A potential oncogenic activity of TIMP-1.  Cancer Research, 65: 898-906, 2005.
 
Ustach C. and Kim, H.-R. C.  Platelet-Derived Growth Factor D is activated by Urokinase Plasminogen Activator in Prostate Carcinoma Cells.  Mol. Cell. Biol, 25(14):6279-6288, 2005.

Taube, M.E., Liu, X-W, Fridman, R. and Kim, H.-R. C. TIMP-1 regulation of cell cycle in human breast epithelial cells via stabilization of p27KIP1 protein.  Oncogene 25: 3041-3048, 2006.

Jung, K-K., Liu, X-W., Chirco, R., Fridman, R., Kim, H.-R. C.  Identification of CD63 as a Tissue Inhibitor of Metalloproteinase-1 interacting cell surface protein.  The EMBO J., 25:3934-3942, 2006.

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Raymond R. Mattingly, Ph.D.
Molecular Oncology and Human Genetics
Ph.D., University of Virginia, 1993

Pathways of intracellular signal transduction are a principal characteristic of eukaryotic cells.  Signals are relayed through a variety of components that include receptors for hormones and neurotransmitters, the timed molecular switches of the GTPase superfamily, low molecular weight second messengers, and the many protein kinases.  Signalling is often directed to the nucleus and the control of transcription factors.  Integration and feedback-regulation of signal transduction provides exquisite control of complex, differentiated cells.  It is this control that underlies phenomena such as cell proliferation and programmed cell death (apoptosis) and executes processes that include memory and development.  Derangement of signal transduction produces diseases such as cancer.  Our studies focus on the physiological roles and pharmacological significance of small GTPases of the Ras superfamily.  We are currently pursuing three related investigations:  1. We have discovered a new mechanism for Ras activation by an exchange factor called Ras-GRF1.  Heterotrimeric G-protein beta/gamma-subunits and an increase in phosphorylation of Ras-GRF1 on serine residues mediate this pathway.  2. Type 1 Neurofibromatosis (NF1) is a common genetic disorder that is characterized by abnormal proliferation of neuroectodermal tissues.  Nearly all patients have benign neurofibromas, and there is increased risk of neurofibrosarcomas and other malignant tumours.  Current therapy for this disease is limited.  We have developed several collaborative projects with the goal of the identification of relatively non-toxic and mechanistically specific drugs for NF1 treatment.  3. Early stages of cancer exhibit hyperproliferation of epithelium coupled to increased survival of the epithelial cells without their normal attachments.  The latter property is a suppression of the normal process of detachment-induced cell death, or anoikis.  We are investigating the hypothesis that the Rac effector protein kinase termed PAK1 may play a key role in early breast carcinoma through its potential actions to co-ordinate cytoskeletal rearrangement, to increase intra- and peri-cellular proteolysis, and to suppress anoikis. 

Selected Publications

R.E. Menard, A.P. Jovanovski & R.R. Mattingly.  Active p21-activated protein kinase I (PAKI) rescues MCF10A breast epithelial cells from undergoing anoikis.  Neoplasia 7: 638-645, 2005.

R.R. Mattingly, J.A. Kraniak, J.T. Dilworth, P. Mathieu, B. Bealmear, J.E. Nowak, J.A. Benjamins, M.A. Tainsky & J.J. Reiners, Jr.  The MEK inhibitor PD184352/CI-1040 selectively induces apoptosis in malignant schwannoma cell lines. J. Pharmacol. Exp. Ther. 316: 456-465, 2006.

J.T. Dilworth, J.M. Kraniak, J.W. Wojtkowiak, R.A. Gibbs, R.F. Borch, M.A. Tainsky, J.J. Reiners Jr & R.R. Mattingly.  Molecular targets for emerging anti-tumor therapies in Neurofibromatosis type 1. Biochem. Pharmacol.  72: 1485-1492, 2006.
 
H. Yang & R.R. Mattingly. The Ras-GRF1 exchange factor coordinates activation of H-Ras and Rac1 to control neuronal morphology. Mol. Biol. Cell 17: 2177-2189, 2006.

M.K. Clark, S.A. Reigard, J. Wojtkowiak, R. Chirco, P. Mathieu, J.J. Reiners Jr, R.R. Mattingly, R.F. Borch & R.A. Gibbs.  Synthesis, biochemical, and cellular evaluation of farnesyl monophosphate prodrugs as farnesyltransferase inhibitors.  Accepted for publication in J Med Chem, 2007.

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Michael A. Tainsky, Ph.D.
Molecular Oncology and Human Genetics
Ph.D., Cornell University, 1977

The research focus of the lab is based on our research experience in inherited cancer and genomic instability.  Finding the Cancer Early.  We have developed technology for a new biochip-based test for cancer that can predict cancer before there are symptoms.  Protein microarrays containing thousands of proteins are used in screening tests for breast and ovarian cancer.  Understanding Cells with Defective, Mutant Cancer Genes.  We discovered over 20 years ago that cells with defects in cancer predisposing genes grow abnormally in the laboratory. Using genomic profiling we have identified 3 genetic pathways that contribute to immortalization of cancer cells.  These pathways are being studied to develop new therapies for the early interference in cancer formation in patients genetically predisposed to the disease. 

Selected Publications
Draghici, S., Chatterjee, M. Tainsky, M.A.  Epitomics: Serum screening for the early detection of cancer on microarrays using complex panels of tumor antigens, Expert Reviews in Molecular Diagnostics, 5:735-743, 2005.

Tang, L., Roberts, P.C., Kraniak, J.M., Li, Q., and Tainsky, M.A. STAT1 expression is not sufficient to regulate interferon-signaling pathway in cellular immortalization, J Interferon & Cytokine Res. 26: 14-26, 2006.

Fridman, A.L., Tang, L., Kulaeva, O.I., Ye, B., Li, Q., Nahhas, F., Land, S.J., Roberts, P.C., Abrams, J., and Tainsky, M.A. Expression Profiling Identifies Three Pathways Altered in Cellular Immortalization:  Interferon, Cell Cycle and Cytoskeleton. J Gerontol A Biol Sci Med Sci. 61:879-89, 2006.

Chatterjee, M., Mohapatra, S., Wang, X., Nowak, J., Nahhas, A.F., Lu, K., Witkin, S.S, Fishman, D., Munkarah, A., Morris, R., Levin, K.N., Tromp, G., Abrams, J., Draghici, S., and Tainsky, M.A. Diagnostic markers of ovarian cancer by high-throughput antigen cloning and detection on arrays. Cancer Research, 66: 1181-90, 2006.

Xu, J., Zhou, J.Y., Tainsky, M.A., Wu, G.S. Evidence that Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Induction by 5-Aza-2'-Deoxycytidine Sensitizes Human Breast Cancer Cells to Adriamycin. Cancer Res. 67:1203-11, 2007.

Munkarah, A., Chatterjee, M., Tainsky, M.A. Update on Ovarian Cancer Screening. Current Opinion Obstet Gynecol. 10:22-6, 2007.

Fridman, A.L., Rosati, R., Li, Q., and Tainsky, M.A. Epigenetic and functional analysis of IGFBP3 and IGFBPrP1 in cellular immortalization, Biochemical and Biophysical Research Communications, 357:785-791, 2007.

Separovica, D. Hanada, K. Maitah, MYA. Nagy, B. Hang, I, Tainsky, MA. Kraniak, JM. Bielawski, J. Sphingomyelin synthase 1 suppresses ceramide production and apoptosis post-photodamage, Biochemical and Biophysical Research Communications, 358: 196-202, 2007.

Nahhas, F. Dryden, S.C., Abrams, J., and Tainsky, M.A. Phosphorylation of SIRT2 Deacetylase Does not Regulate its Interaction with HDAC6 and Tubulin, Molecular and Cellular Biochemistry, In Press. E-Pub online, May 22, 2007.

Tainsky, MA,  Chatterjee, M, Levin, NK, Draghici, S, and Abrams, J. Multianalyte Tests for the Early Detection of Cancer: Speedbumps and Barriers, Biomarkers Insights, In Press, 2007.

Li, Q., Tang, L., Roberts, P.C., Kraniak, J.M., Fridman, A., Kulaeva, O., Tehrani, O., and Tainsky, M.A.  Interferon Regulatory Factors Inhibit Growth and Induce Senescence in Immortal Li-Fraumeni Fibroblasts. In Press, 2007.

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Dr. Taub’s research has focused on studying the molecular epidemiology and pharmacology of childhood leukemia, the most common form of cancer in children and the leading cause of death from disease of American children.  Using a unique source of blood samples collected at birth and used for genetic screening (“Guthrie cards”), his lab is screening for the presence of preleukemic clones (by PCR-based minimal residual disease assays) present in the blood samples of children who subsequently developed leukemia.  This would confirm a prenatal origin of leukemia and potential in utero exposures being linked to the development of leukemia. These studies are also examining factors including differences in folate metabolism which may account for the 2-3-fold higher incidence of acute lymphoblastic leukemia (ALL) in Caucasian compared to African American children in the United States. Another project in the laboratory, is studying the biology of acute myeloid leukemia (AML) in children with Down syndrome, who have a 20-fold greater risk of developing leukemia compared to children without Down syndrome, and have the highest cure rate (80-100%) of any subgroup of AML patients.  These studies are utilizing both primary clinical samples of patients diagnosed with AML as well as clinically relevant leukemia cell lines, to examine the role of both chromosome 21-localized and non-chromosome 21-localized genes in both leukemogenesis and the metabolism of chemotherapy drugs.  Findings from these studies are also being applied to developing new leukemia therapies in clinical trials for children without Down syndrome.

Selected Publications

Taub JW, Huang X, Matherly LH, Stout ML, Buck SA, Massey GV, Becton DL, Chang MN, Weinstein HJ, Ravindranath Y:  Expression of chromosome 21-localized genes in acute myeloid leukemia: differences between Down Syndrome and non-Down Syndrome blast cells and relationship to in vitro sensitivity to cytosine arabinoside and daunorubicin.  Blood 94:1393-1400, 1999.

Taub JW, Konrad MA, Ge Y, Naber JM, Scott JS, Matherly LH, Ravindranath Y:  High frequency of leukemic clones in newborn screening blood samples of children with B-precursor acute lymphoblastic leukemia.  Blood 99:2992-2996, 2002.

Taub JW, Mundschau G, Ge Y, Poulik J, Qureshi F, Jensen TL, Matherly LH, James J, Weschler J, Crispino JD. Prenatal origin of GATA1 mutations may be an initiating step in the development of leukemia in Down syndrome. Blood 104:1588-1589, 2004.

Ge Y, Stout ML, Tatman DA, Jensen TL, Buck S, Thomas RL, Ravindranath Y, Matherly LH, Taub JW. Gata1, cytidine deaminase and the high cure rate of Down syndrome children with acute megakaryocytic leukemia. Journal of the National Cancer Institute 97:226-231, 2005.

Ge Y, Dombkowski AA, LaFiura KM, Tatman D, Yedidi RS, Stout ML, Buck SA, Massey G., Becton DL, Weinstein HJ, Ravindranath Y, Matherly LH, Taub JW. Differential gene expression, GATA1 target genes and the chemotherapy sensitivity of Down syndrome megakaryocytic leukemia. Blood 107: 1570-1581, 2006.

LaFiura KM, Bielawski DM, Posecion NC, Ostrea EM, Matherly LH, Taub JW, Ge Y. Ge Y, LaFiura KM, Dombkowski AA, Chen Q, Payton SG, Buck SA, Salagrama S, Diakiw AE, Matherly LH, Taub JW. The role of the proto-oncogene ETS2 in acute megakaryocytic leukemia biology and therapy. Leukemia 22:521-529, 2008.

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Complex biological events are dependent on the precision of temporal or spatial controls of gene expression.  The transcriptional precisio is determined by the architectural status of the chromosome. This is true in both prokaryotic and eukaryotic chromosomes.  Toward investigating the effects of chromosome architectural changes on transcription control, our research has focused on the DNA looping and the DNA supercoiling dynamic driven by the transcription processes.  We have successfully applied the abundant biophysical information of DNA conformations to the problem of transcription control.  We demonstrated that transcription process is the primary driving force for chromosome supercoiling dynamic.  A supercoiling-sensitive leu-500 promoter was activated by transcription activity of ilvIH promoter located 1.9 kb upstream. We have discovered that the cryptic leuO gene located within the 1.9 kb DNA sequence was responsible for the long-range promoter interaction.  This characterizes a promoter relay mechanism in which transcriptional activity from one promoter (i.e. pilvIH) can activate a distant promoter (i.e. pleu-500) via activation of an intermediate promoter (i.e. pleuO). The gene product of leuO, LeuO, plays a trans-acting role in this novel transcription coordination.  Data suggested that transcription elements involved in coordinated gene expression are responsive to DNA supercoiling driven by the adjacent transcription activities.  The striking DNA supercoiling responsiveness has led to the finding of a novel gene silencing and anti-silencing mechanism whereby expression of multiple genes in the gene cluster is controlled in a sequential manner.  Indeed, a heterochromatic nucleoprotein filament was found to be responsible for the bacterial gene silencing.  A bacterial boundary element-like activity is responsible for the anti-silencing mechanism.  The bacterial protein, LeuO, evidently exerted its boundary element-like activity by forming various DNA loops important in the transcriptional regulation.  We are currently investigating the protein-protein interaction between LeuO proteins and the interaction between LeuO and other protein factors.  The mechanisms under investigation are paradigmatic for the expression regulation of the eukaryotic genes.

Selected publications
Chen, C.-C., Ghole, M., Majumdar, A., Wang, Z. Chandana, S., and Wu, H.-Y.  “ LeuO-mediated transcriptional derepression” J. Biol. Chem. 278, 38094-38103, 2003.

Chen, C.-C.,  and Wu, H.-Y. “Transcription-driven DNA supercoiling and gene expression control” in the encyclopedia of DNA supercoiling and gene expression” in  Frontiers in Bioscience,  8, 430-439, 2003.

Wu, H.-Y. & Fang, M. “ DNA supercoiling & transcription control: A model from the study of suppression of the leu-500 mutation in Salmonlla  typhimurium topA- mutants.” Progress in Nucleic Acid Research and Molecular Biology,  vol. 73, pp 41- 66. Edited by Kivie Moldave.  Academic Press, 2003.

Chen, C.-C., Chou , M.-Y., Huang, C.-H., Majumdar, A., and Wu, H.-Y. “A cis-spreading nucleoprotein filament is responsible for the gene silencing activity found in the promoter relay mechanism” J. Biol. Chem. 280, 5101-5112, 2005.

Chen, C.-C.,  and Wu, H.-Y.  “LeuO protein delimits the transcriptionally active and repressive domains on the bacterial chromosome.”  J. Biol. Chem. 280, 15111-15121, 2005.

Chen, C.-C., and Wu, H.-Y. Genome organization: The effects of transcription-driven DNA supercoiling on gene expression regulation,  Chapter 28, pp. 458-469  In: Gene Expression and Regulation, (ed. J. Ma), a Current Scientific Frontiers Book, Higher Education Press & Springer, 2006.

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My research interests are to understand the cellular and molecular mechanisms underlying disruption of signaling pathways involved in development and treatment of human cancers, with a primary focus on the p53 pathway. p53 is a major tumor suppressor and is mutated in more than 50% of all tumors. p53 is activated by a variety of stress stimuli whereby it functions as a transcription factor to activate a number of downstream targets leading to inhibition of cell growth primarily through cell cycle arrest and apoptosis. Disruption of the p53 pathway has been implicated not only in the development of tumors but also in resistance of cancer cells to chemotherapy and radiotherapy. In a screen to identify genes that are involved in the p53 pathway, we identified several p53 responsive genes including MKP1 and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor KILLER/DR5. MKP1 is a member of the dual specificity protein phosphatase family that is able to dephosphorylate and inactivate mitogen-activated protein (MAP) kinases. Through MKP1, p53 may functionally interact with the MAPK pathway. My main research focus is to elucidate the role of MKP1 in the p53 pathway. We are currently investigating how p53 transcriptionally regulates MKP1 expression in response to a variety of stresses. We are also investigating the role of MKP1 in regulating the cell cycle and apoptosis. Another area of my research is to characterize several additional targets of p53. The roles of these new targets in p53-mediated biological responses are being evaluated using biological and biochemical approaches. The overall goal of my research is to better understand the p53 pathway and the mechanism by which this pathway is disrupted in human cancer. Ultimately, this information will help to design therapeutic strategies to restore the p53 tumor suppressor function in cancer.

Selected publications
Wu, G.S. and Ding, Z.  Caspase 9 is required for p53-dependent apoptosis and chemosensitivity in a human ovarian cancer cell line. Oncogene, 21:1-8, 2002.

Li, M., Zhou, J.Y., Ge, Y., Matherly, L. and Wu, G.S. The phosphatase MKP1 is a transcriptional target of p53 involved in cell cycle regulation. J Biol Chem, 278:41059-41068, 2003.

Zhou, J. Y., Liu, Y., and Wu, G.S. The role of mitogen-activated protein kinase phosphatase-1 in oxidative damage-induced cell death. Cancer Res 66:4888-4894, 2006.

Wang, Z., Xu, J., Zhou, J.Y., Liu, Y. and Wu, G.S. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is required for cisplatin resistance. Cancer Res 66:8870-8877, 2006.

Xu, J., Zhou, J.Y. and Wu, G.S. TNF-related apoptosis inducing ligand (TRAIL) is required for TNFa-mediated sensitization of human breast cancer cells to chemotherapy. Cancer Res 66:10092-10099, 2006.

Xu, J., Zhou, J.Y., Tainsky, MA and Wu, G.S. Evidence that tumor necrosis factor-related apoptosis-inducing ligand induction by 5-Aza-2'-deoxycytidine sensitizes human breast cancer cells to adriamycin. Cancer Res 67:1203-11, 2007.

Wang J, Zhou JY and Wu GS: ERK-dependent MKP-1-mediated cisplatin resistance in human ovarian cancer cells. Cancer Research Dec 15;67(24):11933-41, 2007.

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Youming Xie, Ph.D.
Molecular Oncology and Human Genetics
Ph.D., University of Texas, 1996

The focus of our research is to understand the molecular mechanisms underlying protein degradation mediated by the ubiquitin-proteasome system (UPS). UPS is the primary intracellular machine responsible for elimination of abnormal proteins and selective destruction of regulatory proteins involved in a wide range of cellular processes including cell cycle control, DNA transcription, replication and repair, and stress response. Dys-regulation of UPS is implicated in various human diseases including cancers. Moreover, UPS has now become an important target for anti-cancer therapy. We use both Saccharomyces cerevisiae and mammalian systems to define the pathways that control the degradation of several key regulatory proteins. In addition, we are studying the differential response of normal cells and cancer cells to the inhibitors of UPS.

Selected publications
Wang, L., Mao, X., Ju, D. & Xie, Y. Rpn4 is a physiological substrate of the Ubr2 ubiquitin ligase. J. Biol. Chem. 279, 55218-55223, 2004.

Ju, D. & Xie, Y.  Proteasomal degradation of RPN4 via two distinct mechanisms: ubiquitin-dependent and -independent. J. Biol. Chem. 279, 23851-23854, 2004.

Ju, D. & Xie, Y.  Identification of the preferential ubiquitination site and ubiquitin- dependent degradation signal of Rpn4. J. Biol. Chem. 281, 10657-10662, 2006.

Ju, D., Wang, X., Xu, H. & Xie, Y.  The armadillo repeats of the Ufd4 ubiquitin ligase recognize ubiquitin-fusion proteins. FEBS Lett 581, 265-270, 2007.

Xu, H., Ju, D., Jarois, T. & Xie, Y. Diminished feedback regulation of proteasome expression and resistance to proteasome inhibitors in breast cancer cells. Breast Cancer Res Treat [Epub ahead of print: DOI 10.1007/s10549-007-9553-4], 2007.

Ju, D., Wang, X., Xu, H. and Xie, Y.  Genome-wide analysis identifies MYND-domain protein Mub1 as an essential factor for Rpn4 ubiquitylation. Mol. Cell. Biol. [published ahead of print on 10 December 2007, doi:10.1128/MCB.01787-07, 2007.

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Fayth K. Yoshimura, Ph.D.
Molecular Oncology and Human Genetics
Ph.D., Yale University, 1972

Dr. Yoshimura’s research interests include the study of tumorgenesis by murine retroviruses. To elucidate the mechanism by which murine retroviruses cause T cell tumors, her laboratory has studied the regulation of transcription of cellular oncogenes by retroviral sequences. She has identified binding sites for transcription factors in the long terminal repeat region of the virus that regulate transcription in a T cell specific manner. Some of these sites have been shown to be important for tumorigenesis by these viruses. Her more recent research includes studies of the ability of oncogenic murine retroviruses to induce apoptosis in thymic lymphocytes in vivo and other cell types in vitro. Her lab has shown that virus-induced apoptosis is dependent on both viral glycoprotein-receptor interactions and the production of high levels of unintegrated viral DNA during virus replication. Furthermore, she has demonstrated that apoptosis correlates with the ability of a retrovirus to produce thymic tumors. She is also interested in the induction of apoptosis in tumor cells by the soy product genistein, and determining whether genistein may be effective as a treatment for human T-cell leukemia and lymphoma. The methodology involved in her laboratory includes DNA footprinting, gel mobility shift assays, site-directed mutagenesis, transfection of mammalian cells, reporter gene assays, flow cytometry, assays for apoptosis, and immunoblotting. Questions her laboratory is currently pursuing include the 1). Identification of the pathway that is involved in apoptotic signaling by viral glycoprotein-receptor interactions; 2). Identification of regions of the viral receptor protein that are involved in apoptosis signaling; 3). Determination of the mechanism by which genistein induces apoptosis in T-cell; 4). Identification of the nuclear site where retrovirus DNA integrates into cellular chromosomes.

Selected Publications
Wilson, C. A., S. Laeeq, A. Ritzhaupt, W. Colon-Moran, and F. K. Yoshimura. Sequence analysis of porcine endogenous retrovirus long terminal repeats and identification of transcriptional regulatory regions. J. Virol. 77:142-149, 2003.

Baxa, D. M. and F. K. Yoshimura. Genistein reduces NF-?B in T-lymphoma cells via a caspase-mediated cleavage of I?B?. Biochem. Pharmacol. 66:1009-1018, 2003.

Nanua, S. and F. K. Yoshimura. Differential cell killing by lymphomagenic murine leukemia viruses occurs independently of p53 activation and mitochondrial damage. J. Virol. 78:5088-509, 2004.

Nanua, S. and F. K. Yoshimura. Mink epithelial cell killing by pathogenic murine leukemia viruses involves ER stress. J. Virol. 78:12071-12074, 2004.

Yoshimura, F. K. Genistein induces apoptosis via mitochondrial damage in acute lymphoblastic leukemia T-cell lines. Cancer Therapy 2:429-440, 2004.

Baxa, D. M., X. Luo, and F. K. Yoshimura. Genistein induces apoptosis in T lymphoma cells via mitochondrial damage. Nutr. Cancer 51:93-101, 2005.

Yoshimura, F. K. and X. Luo. Induction of endoplasmic reticulum stress by the envelope precursor polyprotein of a murine leukemia virus during the preleukemic period in mice. J. Virol. 81: 4374-4377, 2007.

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