The applicant's long term goal is to understand the molecular mechanism(s) of tumor cell killing by topoisomerase poisons. Many anticancer drugs have been shown to target DNA topoisomerases. The key molecular event common of all of these topoisomerase poisons is the stabilization of a reaction intermediate, the covalent topoisomerase-DNA complex, of then referred to as the cleavable complex. Despite the importance of this cleavage intermediate in tumor cell killing, the molecular mechanism underlying this process remains unclear. In the current application, it is proposed to study the cellular repair of this critical cleavage complex using the topoisomerase I (TOP1) poison camptothecin (CPT) as a model compound. In the preliminary studies, they have already demonstrated that TOP1 in this CPT-induced cleavable complex is rapidly multi-ubiquitinated and subsequently destroyed by 26S proteasome (TOP1 down-regulation). Surprisingly, many tumor cells are proficient in forming ubiquitin-TOP1 conjugates but defective in degradation of these conjugates. The failure of tumor cells to down-regulate TOP1 also correlated with persistent transcription arrest (lack of transcription recovery) and hypersensitivity to CPT. Based on these results, they propose that ubiquitin/proteasome-dependent down-regulation of TOP1 represents a new cellular """"""""repair"""""""" pathway to remove TOP1-mediated DNA damage and that many tumor cells are defective in this pathway resulting in hypersensitivity to CPT. In order to test this hypothesis, they plan to carry out the following experiments (1) To characterize the ubiquitin-hTOP1 conjugation reaction in yeast and human. The proposed studies will include identification of the specific lysine residue(s) on ubiquitin involved in forming multi-ubiquitin chains on TOP1, identification of the specific lysine residue(s) on human TOP1 modified by ubiquitin, and identification of specific E2 and/or E2/E3 responsible for TOP1 multi-ubiquitination. These experiments will provide the necessary information for testing the role of TOP1 multi-ubiquitination in CPT sensitivity/resistance. (2) To characterize the ubiquitin/proteasome pathway for CPT-induced TOP1 down-regulation in tumor cells. The applicant has already identified the defect to be downstream from the ubiquitin-TOP1 conjugation step. Experiments are designed to study deregulation of 26S proteasome in tumor cells.
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