Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

THE ALMASANLABORATORYVISITING SCHOLARQuan Chen, Ph.D.Inst. of Zoology, Chinese Acad.of Sciences, BeijingPOSTDOCTORAL FELLOWSJohn Hissong, M.D., Ph.D.Suparna Mazumder, Ph.D.Subrata Ray, Ph.D.GRADUATE STUDENTSMeredith Crosby, A.B.Erica DuPree, M.S.Marcela Oancea, B.S.Dragos Plesca, M.S.COLLABORATORSGuy M. Chisolm, Ph.D. 1Daniel Lindner, M.D., Ph.D. 2Roger M. Macklis, M.D. 3Lily Ng, Ph.D. 4Gordan Srkalovic, M.D., Ph.D. 51Dept. of Cell Biology, CCF2Drug Development, TaussigCancer Center, CCF3Dept. of Radiation Oncology,CCF4Dept. of Chemistry, ClevelandState Univ., Cleveland, OH5Dept. of Hematology andMedical Oncology, CCFResponse of Tumor Cells to Genotoxic Stress:Mechanisms of Apoptosis andCell-Cycle ControlThe overall goal of our laboratory is to gaina better understanding of how mammaliancells respond to genotoxic stress elicited byionizing radiation (IR) and chemotherapeuticagents. These agents, commonly used in thetreatment of cancer, are thought to ultimatelykill tumor cells by triggeringapoptosis. Cytochrome c,caspases, and Bcl-2-familyproteins represent the basicregulators of apoptosis. We areinterested in the precisemechanism by which theseproteins interact to regulate thecommitment to cell death.ApoptosisWe have shown that IRinducedapoptosis is dependenton the activation of multiplecaspases, which proteolyticallycleave cellular proteins,including Bcl-2 and cyclin E. Wehave proposed a model forIR-induced apoptosis, activatedby cytochrome c through: (i) aninitial release into the cytoplasm of low levels ofcytochrome c, sufficient to induce caspaseactivation, and (ii) a late-stage depletion ofmitochondrial cytochrome c and loss ofmitochondrial functions. We have identifiedBcl-2 as a mitochondrial caspase target whichthus converts an anti-apoptotic protein to apro-apoptotic one. We are interested in the roleof Bcl-2 and the related Bcl-2 homology domain3 (BH3: Bik, PUMA, Noxa) and multi-domain(Bax, Bak) apoptotic proteins in the control ofcytochrome c release and the commitment toapoptosis. We have also found that the deathChen, Q., Gong, B., Mahmoud-Ahmed, A.S., Zhou, A., Hsi, E.D., Hussein, M., and A.Almasan (2001) Apo2L/TRAIL and Bcl-2-related proteins regulate type I interferoninducedapoptosis in multiple myeloma. Blood 98:2183-2192.Mazumder, S., Gong, B., Chen, Q., Drazba, J.A., Buchsbaum, J.C., and A. Almasan(2002) Proteolytic cleavage of cyclin E leads to inactivation of associated kinaseactivity and amplification of apoptosis in hematopoietic cells. Mol. Cell. Biol. 22:2398-2409.Chen, Q., Chai, Y.C., Mazumder, S., Jiang, C., Macklis, R.M., Chisolm, G.M., and A.Almasan (2003) The late increase in intracellular free radical oxygen species duringapoptosis is associated with cytochrome c release, caspase activation, and mitochondrialdysfunction. Cell Death Differ. 10:323-334.Almasan, A., and A. Ashkenazi (2003) Apo2L/TRAIL: apoptosis signaling, biology, andpotential for cancer therapy. Cytokine Growth Factor Rev. 14:337-348.Ray, S., and A. Almasan (2003) Apoptosis induction in prostate cancer cells and xenograftsby combined treatment with apo2L/TRAIL and CPT-11. Cancer Res. 63:4713-4723.The Department of Cancer BiologyAlexandru Almasan, Ph.D.ligand Apo2 ligand/TNF-related apoptosisinducingligand (Apo2L/TRAIL) is activated byIR or interferons and is responsible for cell deathin T-cell or multiple myeloma-derived tumorsthrough a receptor-mediated pathway. Currenttesting of this unique tumor-specific apoptoticactivator, alone or in combinationwith other therapeutics, invitro and in mouse xenografts,should pave the way for its usein clinical trials.Cyclin ECell-cycle control iscritical for all cellular functions,and its deregulation leads toapoptosis. The cell-cycle arrestor apoptosis induced by DNAdamagingagents such as IRlargely results from activationof the tumor suppressor p53,which transcriptionally inducesmany genes, including inhibitorsof the cyclin E/cyclindependentkinase (CDK)complexes. This in turnprevents phosphorylation of the retinoblastomatumor supressor protein (pRb), thus sequesteringthe E2F transcription activators, which controlexpression of S-phase genes. Cyclin E representsan essential regulator of pRb phosphorylation, aswell as of DNA replication. We have extendedour previous studies on the role of pRb inapoptosis to that of cyclin E. We found that,when overexpressed, cyclin E greatly acceleratesIR-induced apoptosis. We believe that cyclin Eexpression could affect cell death at two distinctlevels. First, cyclin E expression and associatedkinase activity increase following irradiation.Second, most cyclin E is proteolytically cleaved inall human tumor hematopoietic lineages. We haveidentified the site of cyclin E cleavage and foundthat a cyclin E proteolytic fragment is a potentinducer of apoptosis. We are determining itssubcellular localization and interactions withcellular proteins to address the role it plays inregulating apoptosis.To further elucidate the signals to apoptosisand cell-cycle control and discover novelradiation-induced genes, we are using microarraytechnologies. The knowledge gained from theabove studies may lead to a better understandingof signals leading to cell death followingtreatment with genotoxic agents, and ultimatelyimprove cancer therapy by enhancing theelimination of malignant cells.48