Public reporting burden for this collection of information Is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of Information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducdng this burden to Washington Headquarters Services, Directorate for Intforation Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Adington, VA 22202-4302, and to the Office of Management and Budget, AUTHOR(S)Vincent L. Cryns, M.D. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBERNorthwestern University Chicago, Illinois 60611 E-Mall:v-cryns@northwestern.edu SPONSORING I MONITORING AGENCY NAME(S) AND ADDRESS(ES)10. SPONSORING / MONITORING AGENCY REPORT NUMBER U.S. Army Medical Research and Materiel CommandFort Detrick, Maryland 21702-5012 SUPPLEMENTARY NOTES Original contains color plates:All DTIC reproductions will be in black and white. Abstract (Maximum 200 Words) (abstract should contain no proprietary or confidential information)Using a novel expression cloning strategy and secondary functional screen of a human prostate cancer cDNA library, we have identified one new, functionally relevant caspase substrate: the mismatch repair protein MLHI implicated in a variety of cancers including those of the prostate. We have demonstrated that human MLH1 is specifically cleaved by caspase-3 (but not by other caspases) at ASP418 in vitro. Furthermore, we have shown that MLH1 is rapidly proteolyzed by caspase-3 in prostate cancer cells induced to undergo apoptosis by treatment with TNF-related apoptotosis-inducing ligand (TRAIL) or the topoisomerase II inhibitor etoposide, which induces DNA double-strand breaks. Importantly, proteolysis of MLH1 by caspase-3 triggers its partial redistribution from the nucleus to the cytoplasm. In addition, a capase-3 cleavage-resistant D418E MLHII mutant inhibits etoposide-induced apoptosis but has little effect on TRAIL-induced apoptosis. These results indicate that MLHI is specifically targeted for proteolysis by caspase-3, and this proteolytic event promotes the execution of apoptotic signals initiated by DNA double-strand breaks. In this way, our results suggest a novel function of MLHI in the response to DNA double-strand breaks that is deregulated by caspase proteolysis. These experiments, then, may lead to novel treatments for prostate cancer that specifically target MLH1. IntroductionApoptosis or programmed cell death is a genetically regulated cellular suicide response that is activated in prostate cancer cells by androgen ablation and irradiation. Indeed, the tumoricidal activity of these treatment modalities stems from their ability to induce apoptosis in prostate cancer cells (1). Caspases are a conserved family of cysteine proteases that are universal effectors of programmed cell death (2, 3). However, t...