REGULATION OF TOPOISOMERASE I AND II PHOSPHORYLATION

这项研究的长期目标是识别和表征 调节拓扑异构酶细胞毒性作用的细胞事件- 活性抗肿瘤药物。 这些药物包括喜树碱 （拓扑异构酶I活性药物）和蒽环类，安吖啶， 表鬼白毒素和椭圆树碱（拓扑异构酶II活性药物）。 这项建议将侧重于两个研究领域。 第一个领域将 涉及主要药物诱导的 拓扑异构酶II切割位点。 第二区域 的研究将集中在拓扑异构酶I和II的调节 磷酸化及其在细胞毒作用中的潜在作用 拓扑异构酶活性药物。 具体而言，我们建议： 1. 药物诱导的拓扑异构酶II热点基因的克隆与鉴定 HeLa细胞染色体的分裂。 拓扑异构酶II的这些热点 将克隆并测序以确定它们是否含有共同的 代表药物的体内靶标的序列或结构 行动上 2. 宫颈癌HeLa细胞拓扑异构酶I和II磷酸化的研究 癌和HSF细胞。 我们最近发现拓扑异构酶I和 II是在HeLa细胞中磷酸化的丝氨酸。 为了进一步阐明 调节拓扑异构酶I和II磷酸化，我们建议这样做 在人HeLa宫颈癌和原发性HSF中的以下研究 细胞： a）拓扑异构酶I和II的磷酸化和去磷酸化 蛋白质将作为生长和细胞周期变化的函数进行测量。 B）拓扑异构酶I内不同丝氨酸位点的磷酸化，和 II蛋白将作为生长和细胞周期的函数进行研究 横穿 c）拓扑异构酶I和II多肽的含有拓扑异构酶I和II多肽的区域。 将鉴定磷酸化的主要位点。 d）酶活性、含量和药物敏感性的细胞变化 将被测量，并与水平和网站的变化相关， 拓扑异构酶磷酸化 3. 研究磷酸化在细胞毒作用中的作用 拓扑异构酶活性药物。 纯化拓扑异构酶的体外研究 I和II已经表明，磷酸化调节了两种催化 这些酶的功能和对抗肿瘤药物的敏感性表明， 磷酸化可能是调节药物作用的重要因素 in vivo. 为了进一步阐明磷酸化在药物治疗中的重要性， 我们会采取以下行动： a）直接确定拓扑异构酶I的磷酸化形式和拓扑异构酶I的磷酸化形式是否存在。 II参与药物诱导的DNA切割。 B）确定拓扑异构酶活性的抗肿瘤药物是否触发 拓扑异构酶磷酸化和酶降解。 实验将 研究这些事件是否与细胞毒性相关 拓扑异构酶I和II抗癌药物的作用机制。

The long-term goal of this research is to identify and characterize cellular events that modulate the cytotoxic action of topoisomerase- active antitumor drugs. These drugs include the camptothecins (topoisomerase I-active drugs) and the anthracyclines, amsacrines, epipodophyllotoxins, and ellipticines (topoisomerase II-active drugs). This proposal will focus on two areas of research. The first area will involve the identification and characterization of the major drug-induced topoisomerase II cleavage sites in human chromosomes. The second area of research will focus on the regulation of topoisomerase I and II phosphorylation and its potential role in the cytotoxic action of topoisomerase-active drugs. Specifically we propose to: 1. Clone and characterize the hotspots of drug-induced topoisomerase II cleavage in HeLa cell chromosomes. These hotspots of topoisomerase II cleavage will be cloned and sequenced to determine if they contain common sequences or structures that represent the in vivo targets for drug action. 2. Study the phosphorylation of topoisomerase I and II in HeLa cervical carcinoma and HSF cells. We have recently shown that topoisomerase I and II are serine phosphorylated in HeLa cells. To further elucidate the regulation of topoisomerase I and II phosphorylation, we propose to do the following studies in human HeLa cervical carcinoma and primary HSF cells: a) Phosphorylation and dephosphorylation of topoisomerase I and II proteins will be measured as a function of growth and cell cycle changes. b) Phosphorylation at different serine sites within topoisomerase I and II proteins will be studied as a function of growth and cell cycle traverse. c) Regions of the topoisomerase I and II polypeptides containing the major sites of phosphorylation will be identified. d) Cellular changes in enzyme activity, content and drug sensitivity will be measured and correlated with changes in the levels and sites of topoisomerase phosphorylation. 3. Study the role of phosphorylation in the cytotoxic action of topoisomerase-active drugs. In vitro studies with purified topoisomerase I and II have shown that phosphorylation modulates both catalytic function and sensitivity of these enzymes to antitumor drugs suggesting that phosphorylation may be an important factor modulating drug action in vivo. To further elucidate the importance of phosphorylation in drug action we will: a) Directly determine if the phosphorylated forms of topoisomerase I and II are involved in drug-induced DNA cleavage. b) Determine if topoisomerase-active antitumor drugs trigger changes in topoisomerase phosphorylation and enzyme degradation. Experiments will be done to investigate whether these events are linked to the cytotoxic mechanism of topoisomerase I and II anticancer drugs.