The antitumor drug camptothecin (CPT) poisons DNA topoisomerase I (Top1p) by reversibly stabilizing a covalent enzyme-DNA reaction intermediate. During S-phase, collisions of advancing replication forks with these complexes produce the DNA lesions that signal cell cycle arrest and cell death. However, little is known of the nature of the lesions produced and the cellular processes involved in the recognition and repair of CPT-induced DNA damage. This project will investigate the function of a novel class of TAH genes in mediating cell sensitivity to Top1p poisons. Conditional tah mutants, isolated in a yeast genetic screen, are viable in the absence of DNA damage, yet exhibit a 3-log drop in viability when exposed to Top1p-induced DNA lesions at the nonpermissive temperature. The TAH genes appear to regulate cell cycle progression and/or cellular responses to S-phase induced DNA damage. Surprisingly, several tah mutations also enhance cell sensitivity to rapamycin, a macrocyclic lactone that inhibits the TOR kinase. TOR has emerged as a central regulator of cellular responses to mitogenic stimuli, survival signals and nutrient deprivation. The hypersensitivity of yeast tah mutants to CPT and rapamycin suggests similar functions dictate cellular responses to the inhibition of mTOR and poisoning of Toplp by CPT. In this proposal, the synthetic lethality induced by Top1p-DNA damage in yeast tah mutants will be exploited to clone human TAH homologues. The overall goal is to determine whether the mechanisms regulating cell sensitivity to Top1p poisons are conserved from yeast to humans. The related analysis of rapamycin will further define cytotoxic responses common to mTOR inhibition and CPT. This will be accomplished by assessing human TAH gene activity in isogenic untransformed and transformed IMR90 cell lines, using pseudotyped retrovirus. This proposal will also extend the characterization of a novel gene TAH11 that genetically interacts with SIC1, the CDK-cyclin inhibitor that regulates yeast cell cycle progression from G1 to S phase, by isolating extragenic mutants that suppress tah11 mutant cell sensitivity to Top1p poisons. Based on recent structures of drug-bound Top1p-DNA complexes, novel CPT analogs were designed to enhance covalent complex stability. To determine whether this translates into increased antitumor activity, these compounds will be evaluated in yeast tah mutants, hTAH expressing cell lines and human tumor xenograft models. These studies will decipher conserved pathways regulating cellular responses to Toplp poisons and mTOR inhibitors.
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