BIOCHEMICAL AND MOLECULAR STUDIES ON DT-DIAPHORASE

我们以前的工作表明，抗肿瘤醌类如AZQ和MC是 通过DTD生物活化为遗传毒性和细胞毒性代谢物。我们还 显示人非小细胞肺癌（NSCLC） 相对于小细胞肺癌（SCLC）和正常人的DTD活性 肺。本申请中提出的实验代表了一种组合的 生物化学和分子方法，以醌的研究为中心 活化，细胞酶学和调节DTD活性， 合理设计新的治疗策略以靶向富含DTD的肿瘤 如NSCLC。 具体来说，我们建议比较大鼠和人类DTD的能力， 生物活性抗肿瘤醌类如AZQ、丝裂霉素C及其类似物 DNA反应性和细胞毒性物质。烷基化和交联 将检查DNA，并在DTD介导后在DNA中形成交联 醌的还原将被分离和表征。丝裂霉素C是 通过DTD以pH依赖性方式代谢，我们建议阐明 这种pH依赖性的机制。由于丝裂霉素C诱导 在较低的pH值下DNA损伤更大，丝裂霉素C的pH依赖性 将在富含DT-黄递酶的肿瘤细胞中检查诱导的细胞毒性 线我们已经证明，与非小细胞肺癌相比， SCLC和正常肺。我们希望检验这样一个假设： 有效的生物激活的DTD将是有效的药物治疗 的NSCLC。我们的数据还表明，酶以外的DTD参与 生物还原剂对肿瘤细胞的细胞毒性增加， 缺氧使用亚细胞系统和纯化的 酶将被用来定义潜在的代谢机制， 抗肿瘤醌类化合物对缺氧细胞的毒性。由于DTD扮演一个 在调节肿瘤细胞对生物还原剂的敏感性中的关键作用 了解这种酶在肿瘤中的调节机制是至关重要的。 细胞系统我们将研究控制细胞凋亡的分子机制。 DTD在人NSCLC中的表达，并研究突变的作用， 在人肿瘤细胞中调节DTD活性的缺失。

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.