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.
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