GENOTOXICITY OF CISPLATIN, A PLEIOTROPIC TOXIN

顺铂（cis-diamminedichloropatinum（II））是一种金属配位络合物，通过与DNA相互作用启动的机制杀死细胞。 使用生物化学和遗传学方法，该项目的目标是继续我们的工作，探索这种化合物如何杀死细胞。 这项工作的实用价值源于使用这种化合物作为对睾丸具有器官特异性的抗癌药物，并且在较小程度上，卵巢。 虽然研究顺铂的抗癌活性不是我们的目标，但我们发现的任何基本机制都可能对设计新的选择性毒素有价值。 在以前的工作中，我们发现顺铂-DNA加合物的结构是不寻常的，因为它吸引某些细胞蛋白质。 我们已经假设，并在某些情况下证明，有这些相互作用的有害影响。 首先，细胞蛋白质与加合物的结合保护了加合物免受DNA修复酶的伤害;加合物持续存在并具有增强的杀死细胞的可能性。 第二，一些加合物结合蛋白是转录因子。 如果转录因子与加合物而不是其启动子相关，基因表达就会受到不利影响。 第三，加合物吸引错配修复蛋白，并可能破坏它们在基因重组中的作用。 拟议的工作分为三个领域。已经确定加合物作为体外转录因子的分子诱饵，我们计划实验来测试转录因子，特别是hUBF（调节rRNA合成），是否在体内被劫持。 我们的第二个目标是继续最近的工作，这些工作表明，特定重组基因的突变可以导致对顺铂的敏感性比同基因野生型细胞提高10^4倍。 我们的建议是继续揭示重组影响细胞对顺铂反应的机制，此外，通过使用遗传技术确定错配修复途径是否与重组途径相互作用，影响细胞存活。 最后，我们计划生化实验，以补充上述基因重组和错配修复的研究。 据推测，顺铂加合物将是纯化的重组酶的底物，并且控制重组的许多方面的错配修复蛋白可能会干扰事件的有序进展，否则会允许加合物的耐受性。总之，这些研究将提供一个重要的DNA损伤剂的毒性作用的机制细节。

Cisplatin (cis-diamminedichloroplatinum (II)) is a metal coordination complex that kills cells by mechanisms initiated by its interaction with DNA. Using biochemical and genetic approaches, the goal of this project is to continue our work probing the how this compound kills cells. The practical value of the work stems from the use of this compound as an anticancer drug with organotropic specificity for the testis, and to a lesser extent, ovary. While study of the anticancer activity of cisplatin is not our goal, any basic mechanisms we uncover could be of value in the design of new selective toxins. In previous work we discovered that the architecture of cisplatin-DNA adducts is unusual in that it attracts certain cellular proteins. We have postulated and in some cases demonstrated that there are deleterious effects of these interactions. First, the binding of cellular proteins to adducts shields the adducts from DNA repair enzymes; the adducts persist and have an enhanced likelihood of killing the cells. Second, some adduct binding proteins are transcription factors. If the transcription factor becomes associated with an adduct, and not its promoter, gene expression is adversely affected. Third, adducts attract mismatch repair proteins and possibly disrupt their role in genetic recombination. The proposed work is divided into three areas. Having established that adducts act as molecular decoys for transcription factors in vitro, we plan experiments to test whether a transcription factor, specifically hUBF (which regulates rRNA synthesis), is hijacked in vivo. Our second aim is to continue recent work that has shown that mutations in specific recombination genes can result in enhanced sensitivity to cisplatin by as much as 10^4 fold over an isogenic wild type cell. Our proposal is to continue to uncover the mechanism by which recombination affects cellular responses to cisplatin and, in addition, determine by using genetic techniques if the mismatch repair pathway interacts with recombination pathways in affecting cellular survival. Finally, we plan biochemical experiments to complement the aforementioned genetic studies on recombination and mismatch repair. It is hypothesized that cisplatin adducts will be substrates for purified recombination enzymes and that mismatch repair proteins, which control many aspects of recombination, may interfere with the orderly progress of events that would otherwise allow tolerance of the adducts. Taken together these studies will provide mechanistic detail on the toxic effects of an important DNA damaging agent.