EFFECTS OF TOPOISOMERASE II-ACTIVE DRUGS IN MITOCHONDRIA

这些研究的总体目标是识别和表征 细胞毒性药物，通过干扰 线粒体DNA（mtDNA）结构与功能 在这方面我们 最近发现，萘啶酸和环丙沙星，两个4- 已知靶向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mtDNA选择性丢失 细胞 线粒体DNA的丢失与细胞凋亡的减少有关。 呼吸和细胞活力的丧失。 4- 4号染色体的DNA分析 喹诺酮处理的细胞显示存在位点特异性双- 线粒体DNA中的链断裂表明哺乳动物线粒体可能 含有DNA拓扑异构酶II酶，该酶对抑制剂敏感， 细菌II型拓扑异构酶DNA旋转酶。 我们建议 研究4-喹诺酮类药物的细胞毒性作用是否 哺乳动物细胞是通过抑制线粒体 拓扑异构酶II酶，导致mtDNA功能丧失。 具体而言，我们建议： 1. 拓扑异构酶II活性药物对线粒体DNA的作用 在细胞和分离的线粒体中。 药物的特性- mtDNA中的诱导断裂将被表征，以确定它们是否 由线粒体拓扑异构酶介导。 此外，我们将 研究这些药物对结构、拓扑结构和 mtDNA的更新。 2. 纯化和表征药物诱导的线粒体DNA切割活性， 酵母和哺乳动物细胞。 3. 分离和表征编码“推定”的cDNA序列， 使用人cDNA文库中的线粒体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 真核生物和原核生物拓扑异构酶II之间的差异。 4. 确定mtDNA是否是4-喹诺酮的主要细胞毒性靶点 药物（即，萘二甲酸和环丙沙星），使用缺乏 线粒体DNA（mtDNA -）或突变体，选择抗性 到环丙沙星。 mtDNA突变体也将用于鉴定 通过干扰功能发挥作用的其他细胞毒性药物，或 mtDNA的复制 这些研究应该提供有关该机制的重要信息 潜在的细胞毒性作用的4-喹诺酮类，以及有助于 我们对拓扑异构酶的作用的基本知识， mtDNA结构和功能的调节。

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.