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

THE WILLIAMSLABORATORYPROJECT SCIENTISTSMarina Antoch, Ph.D.Pratima Karnik, Ph.D.Xavier Lee, Ph.D.RESEARCH ASSOCIATESRoger Slee, Ph.D.Patricia Stanhope-Baker, Ph.D.Maryam Zamanian-Daryoush, Ph.D.POSTDOCTORAL FELLOWSJoao Marques, Ph.D.Ikenna Okereke, M.D.Anthony Sadler, Ph.D.Aristobolo Silva, Ph.D.Mark Whitmore, Ph.D.LEAD TECHNOLOGISTPatricia Kessler, M.S.TECHNOLOGISTSJeanna M. Guenther, B.S.Die Wang, B.S.GRADUATE STUDENTSMichelle Holko, B.S.Wenliang Li, B.S.Carol Ann Sledz, B.S.COLLABORATORSRobert Silverman, Ph.D. 1Jun Qin, Ph.D. 21Dept. of Cancer Biology, CCF2Center for Structural Biology,CCFThe Department of Cancer BiologySignaling Innate Immunity andTumor SuppressionThe major theme of my laboratory isinvestigation of the molecular mechanismscontrolling cellular responses to extracellularstimuli. In particular, our work focuses on the rolethat potential tumor suppressor genes may play inregulating cell growth and differentiation andapoptosis. We use genetic approaches to understandingthe mechanisms of action of interferons(IFNs), the potent cell-growth-regulating cytokines,and characterizing molecular genetic events involvedin Wilms tumorigenesis.Molecular Mechanisms of Interferon ActionMammalian cells use complex, overlappingsignal transduction pathways to sense environmentalchanges. When cells are subjected to viral challenge,double-stranded RNA (dsRNA), produced as partof the viral replicative cycle, stimulates cellulardefense mechanisms, resulting in the production ofinterferon and the development of an antiviralstate. We are investigating the hypothesis thatspecific signaling pathways are activated in thiscellular response. Signaling by IFNs and othercytokines activates a cascade of kinase activities andprotein-protein interactions. We have identified thestress-activated kinase p38 mitogen-activatedprotein (MAP) kinase as a key player in the IFNresponse. PKR, an IFN-induced protein kinase thatis autophosphorylated when activated by dsRNA, isan essential component of the cellular responses toextracellular stimuli. Using genetic and biochemicalapproaches, we have characterized the dsRNAbindingdomains (dsRBD) of PKR and identifiedsingle amino acid residues that are essential fordsRNA binding. In collaboration with Dr. Jun Qin(Center for Structural Biology), we solved thestructure of the dsRBD and provided a model forthe activation of this kinase by dsRNA. Furtherstructural studies, using X-ray crystallography, areunder way.PKR is able to act as a signal transducer notonly for dsRNA but also for growth regulatorycytokines. Formation of the transcription factorNFκB is induced by dsRNA, IFN or tumor necrosisfactor via a PKR-dependent pathway. In cellsderived from mice in which the PKR gene has beenhomozygously deleted, the response of NFκBdependentgenes is deficient. PKR, a stressresponsivekinase, is required to activate p38 MAPkinase by a number of cellular stress stimuli,including those mediated by pattern recognition Tollreceptors. In PKR-deleted cells, there is a deficiencyin signaling via transcription factor Stat3 in responseto platelet-derived growth factor. PKR is alsosubject to functional regulation during the cell cycleand regulates cellular responses to apoptotic stimuli.In collaboration with Dr. R.H. Silverman,we have deleted the known antiviral genes (PKRand RNASEL) from the germ line of mice anddiscovered that IFN is still able to offer someprotection against viral infection. To elucidate thepathways involved in this response, we have usedgene chip technology and identified novel IFNregulatedgenes. These data, including functionalcategories, can be accessed at: http://www.lerner.ccf.org/labs/williams/. The roleof these genes in IFN-responses is presently beinginvestigated.Tumor Suppressor GenesTumor suppressor genes are implicated in theinitiation/progression of a number of cancers.Wilms tumor (WT), a pediatric nephroblastoma, isassociated with the deletion or mutation of theWT1 gene residing at band p13 on chromosome 11.The expression pattern of WT, supported bymutational analyses in human disease and mousemodels, suggests a role in development of thegenitourinary system, in tumors originating fromthese tissues, and in the etiology of some leukemiasand possibly breast cancer. We are investigating theupstream regulators and downstream targets ofWT1 using custom cDNA arrays produced in ourlaboratory to better understand its role in developmentand cancer.Leitner, W.W., Hwang, L.N., DeVeer, M.J., Zhou, A., Silverman, R.H., Williams, B.R., Dubensky, T.W.,Ying, H., and N.P. Restifo (2003) Alphavirus-based DNA vaccine breaks immunological tolerance byactivating innate antiviral pathways. Nat. Med. 9:33-39.Frevel, M.A., Bakheet, T., Silva, A.M., Hissong, J.G., Khabar, K.S., and B.R. Williams (2003) p38Mitogen-activated protein kinase-dependent and -independent signaling of mRNA stability of AU-richelement-containing transcripts. Mol. Cell. Biol. 23:425-436.Tebo, J., Der, S., Frevel, M., Khabar, K.S., Williams, B.R., and T.A. Hamilton (2003) Heterogeneity inControl of mRNA stability by AU rich elements. J. Biol. Chem. 2003 Jan 28 [epub ahead of print].Bryan R.G. Williams, Ph.D.,Hon. FRSNZKhabar, K.S., Siddiqui, Y.M., Al-Zoghaibi, F., Al-Haj, L., Dhalla, M., Zhou, A., Dong, B., Whitmore, M.,Paranjape, J., Al-Ahdal, M., Al-Mohanna, F., Williams, B.R., and R.H. Silverman (2003) RNase Lmediates transient control of interferon response through modulation of the double-stranded RNAdependent protein kinase PKR. J. Biol. Chem. 2003 Feb 11 [epub ahead of print].Espert, L., Degols, G., Gongora, C., Blondel, D., Williams, B.R., Silverman, R.H., and N. Mechti (2003)ISG20, a new interferon-induced RNase specific for single-stranded RNA, defines an alternative antiviralpathway against RNA genomic viruses. J. Biol. Chem. 2003 Feb 19278:16151-16158.62