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、阿马西林、 表鬼臼毒素和椭圆素(拓扑异构酶II活性药物)。 这项提案将侧重于两个领域的研究。第一个区域将 涉及主要药物诱因的鉴定和特征 人类染色体中的拓扑异构酶II裂解位点。第二个领域 研究的重点将是拓扑异构酶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。具体来说，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 将对裂解进行克隆和测序，以确定它们是否包含共同的 代表体内药物靶点的序列或结构 行动。 2.HeLa宫颈组织中拓扑异构酶I和II的磷酸化研究 癌细胞和HSF细胞。我们最近发现，拓扑异构酶I和 II是HeLa细胞中丝氨酸磷酸化的产物。为了进一步阐明 拓扑异构酶I和II磷酸化的调节，我们建议做 人HeLa宫颈癌和原发HSF的以下研究 单元格： 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和拓扑异构酶中不同丝氨酸位置的磷酸化 将研究Ii蛋白作为生长和细胞周期的函数。 穿越。 C)含有拓扑异构酶I和II多肽的区域 将确定主要的磷酸化位点。 D)细胞酶活性、含量和药物敏感性的变化 将被测量并与以下水平和位置的变化相关联 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。纯化的拓扑异构酶的体外研究 I和II已经表明，磷酸化调节两种催化 这些酶的功能和对抗肿瘤药物的敏感性提示 这种磷酸化可能是调节药物作用的重要因素。 在活体内。进一步阐明药物中磷酸化的重要性 行动我们将： A)直接确定拓扑异构酶I的磷酸化形式和 II参与药物诱导的DNA切割。 B)确定具有拓扑异构酶活性的抗肿瘤药物是否会引起 拓扑异构酶的磷酸化和酶降解。实验将会 来调查这些事件是否与细胞毒性有关 拓扑异构酶I、II类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.