Our previous work has shown that antitumor quinones such as AZQ and MC are bioactivated to genotoxic and cytotoxic metabolites by DTD. We have also shown that human non small cell lung cancer (NSCLC) has highly elevated DTD activity relative to small cell lung cancer (SCLC) and normal human lung. The experiments proposed in this application represent a combined biochemical and molecular approach, centered on studies of quinone activation, cellular enzymology and regulation of DTD activity, for the rational design of new therapeutic strategies to target tumors rich in DTD activity such as NSCLC. Specifically, we propose to compare the ability of rat and human DTD to bioactivate antitumor quinones such as AZQ, mitomycin C and their analogs to DNA reactive and cytotoxic species. Both alkylation and crosslinking of DNA will be examined and the crosslink formed in DNA after DTD mediated reduction of quinones will be isolated and characterized. Mitomycin C is metabolized in a pH-dependent manner by DTD and we propose to elucidate the mechanisms underlying such pH-dependence. Since mitomycin C induced greater DNA damage at lower pH values, the pH-dependence of mitomycin C induced cytotoxicity will be examined in DT-diaphorase rich tumor cell lines. We have shown that DTD is highly elevated in NSCLC when compared to SCLC and normal lung. We wish to test the hypothesis that agents which are efficiently bioactivated by DTD will be effective agents for the therapy of NSCLC. Our data also suggests that enzymes other than DTD are involved in the increased cytotoxicity of bioreductive agents to tumor cells under hypoxia. A systematic approach using subcellular systems and purified enzymes will be used to define the metabolic mechanisms underlying the toxicity of antitumor quinones to hypoxic cells. Since DTD plays a critical role in modulating the sensitivity of tumor cells to bioreductive agents, It is critical to understand how this enzyme is regulated in tumor cell systems. We will investigate molecular mechanisms controlling the expression of DTD in human NSCLC and investigate the role of mutations and deletions in modulation of DTD activity in human tumor cells.
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