BIOCHEMICAL AND MOLECULAR STUDIES ON DT-DIAPHORASE

我们以前的工作表明，AZQ和MC等抗肿瘤醌是 DTD对遗传毒性和细胞毒性代谢物的生物活性。我们还有 研究表明，人类非小细胞肺癌(NSCLC)已高度升高 小细胞肺癌(SCLC)和正常人的DTD活性 阿龙。本申请中提出的实验代表了一种组合 生物化学和分子方法，以对苯二酚的研究为中心 DTD活性的激活、细胞酶学和调节 合理设计针对富含DTD的肿瘤的新治疗策略 NSCLC等活动。 具体地说，我们建议比较大鼠和人类DTD的能力 AZQ、丝裂霉素C及其类似物的生物活性 对DNA活性物质和细胞毒性物质的影响。烷基化和交联化 检测DNA，并在DNA中形成DTD介导的交联物 将对苯二酚的还原进行分离和表征。丝裂霉素C是 DTD以pH依赖的方式代谢，我们建议澄清 这种pH依赖的潜在机制。自从丝裂霉素C诱导 低pH值下更大的DNA损伤，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测诱导的细胞毒性 台词。我们已经证明，与非小细胞肺癌患者相比，非小细胞肺癌患者的DTD显著升高 小细胞肺癌和正常肺。我们希望检验这样一种假设，即 DTD的高效生物活性将成为治疗的有效药物 非小细胞肺癌。我们的数据还表明，DTD以外的酶也参与了这一过程 在生物还原剂对肿瘤细胞毒性增加的情况下 缺氧。一种使用亚细胞系统和纯化的系统方法 酶将被用来定义潜在的代谢机制 抗肿瘤醌类化合物对低氧细胞的毒性。由于DTD扮演的是 调节肿瘤细胞对生物还原药物敏感性的关键作用 药物，了解这种酶是如何在肿瘤中调节的是至关重要的 细胞系统。我们将研究控制细胞生长的分子机制。 DTD在人非小细胞肺癌中的表达及其与基因突变的关系 人肿瘤细胞中DTD活性调节的缺失。

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.