EFFECTS OF TOPOISOMERASE II-ACTIVE DRUGS IN MITOCHONDRIA

这些研究的总体目标是确定和描述 细胞毒性药物，通过干扰细胞的调节而起作用 线粒体DNA的结构和功能。在这方面，我们 最近发现奈利地酸和环丙沙星两种4- 已知针对II型细菌的喹诺酮类药物 拓扑异构酶DNA旋转酶，导致哺乳动物线粒体DNA选择性丢失 细胞。线粒体DNA的丢失与细胞数量的减少有关 呼吸和细胞活力的丧失。4-甲氧基丁酸脱氧核糖核酸的分析 喹诺酮类药物处理的细胞显示存在位点特异性的双- 线粒体DNA链断裂表明哺乳动物线粒体可能 含有对抑制物敏感的DNA拓扑异构酶II酶 细菌II型拓扑异构酶DNA旋转酶。我们建议 探讨4-喹诺酮类药物对人卵巢癌细胞的细胞毒作用 哺乳动物细胞是通过对线粒体的抑制来调节的 拓扑异构酶II酶，导致线粒体DNA功能丧失。 具体来说，我们建议： 1.表征拓扑异构酶II活性药物对线粒体DNA的影响 在细胞和分离的线粒体中。药物的性质是- 将对线粒体DNA的诱导断裂进行表征，以确定它们是否 由线粒体拓扑异构酶介导。此外，我们还将 研究这些药物对细胞结构、拓扑结构和细胞周期的影响。 线粒体DNA周转率。 2.药物诱导的线粒体DNA裂解活性的纯化和鉴定 酵母和哺乳动物细胞。 3.分离和鉴定编码“推定”基因的cDNAs序列 人DNA文库中线粒体DNA拓扑异构酶II酶 含有保守序列的通用拓扑异构酶II探针 在真核或原核拓扑异构酶II酶之间。 4.确定线粒体DNA是否是4-喹诺酮类药物的主要细胞毒性靶点 使用缺乏突变体的药物(如奈利地酸和环丙沙星) 选择抗性的线粒体DNA(mtDNA-)或突变体 给环丙沙星。线粒体DNA突变体(S)也将用于鉴定 其他细胞毒性药物，通过干扰功能或 线粒体DNA的复制。 这些研究应提供有关该机制的重要信息。 4-喹诺酮类药物的细胞毒性作用及其贡献 根据我们关于拓扑异构酶在细胞周期中作用的基本知识 线粒体DNA结构和功能的调控。

The overall goal of these studies is to identify and characterize cytotoxic drugs that act by interfering with the regulation of mitochondrial DNA (mtDNA) structure and function. In this regard, we have recently found that nalidixic acid and ciprofloxacin, two 4- quinolone drugs that are known to target at the bacterial type II topoisomerase DNA gyrase, cause a selective loss of mtDNA from mammalian cells. The loss of mtDNA is associated with a decrease in cellular respiration and a loss in cell viability. Analysis of the DNA from 4- quinolone-treated cells revealed the presence of site-specific double- strand breaks in the mtDNA suggesting that mammalian mitochondria may contain a DNA topoisomerase II enzyme that is sensitive to inhibitors of the bacterial type II topoisomerase DNA gyrase. We propose to investigate whether the cytotoxic effects of the 4-quinolone drugs on mammalian cells are mediated through inhibition of a mitochondrial topoisomerase II enzyme, resulting in the loss of mtDNA function. Specifically we propose to: 1. Characterize the effects of topoisomerase II active drugs on mtDNA in cells and in isolated mitochondria. The properties of the drug- induced breaks in mtDNA will be characterized to determine if they are mediated by a mitochondrial topoisomerase. In addition, we will investigate the effects of these drugs on the structure, topology and turnover of mtDNA. 2. Purify and characterize the drug-induced mtDNA cleavage activity from yeast and mammalian cells. 3. Isolate and characterize the cDNA sequences encoding the "putative" mitochondrial DNA topoisomerase II enzyme from a human cDNA library using universal topoisomerase II probes that contain sequences conserved between eukaryotic or prokaryotic topoisomerase II enzymes. 4. Determine if mtDNA is the primary cytotoxic target of the 4-quinolone drugs (i.e., nalidixic acid and ciprofloxacin) using mutants lacking mitochondrial DNA (mtDNA -) or mutants that are selected for resistance to ciprofloxacin. The mtDNA-mutant(s) will also be used to identify other cytotoxic drugs that act by interfering with the function or replication of mtDNA. These studies should provide important information about the mechanism underlying the cytotoxic effects of 4-quinolones as well as contribute to our basic knowledge regarding the role of topoisomerases in the regulation of mtDNA structure and function.