The long term research objectives of this proposal are: a) to precisely define how the activity of DNA topoisomerase II (topo II) is regulated by phosphorylation in cultured cells, and b) to identify the specific phosphorylation sites which influence cell growth and differentiation. Preliminary data strongly suggest that topo II is phosphorylated at multiple sites. Increased topo II phosphorylation is associated with elevated enzymatic activity in cell-free systems; however, in phorbol ester-treated HL-60 cells it is associated with reduced activity. Based on these observations and other phosphorylation/activity relationships, we form the hypothesis that phosphorylation of certain topo II sites renders this enzyme less active, while the phosphorylation of other sites renders it more active. To scrutinize this hypothesis, and to define the biological significance of topo II phosphorylation in cultured cells, we propose to: a) map the multiple phosphorylation sites of topo II; b) identify the specific topo II sites modulated by protein kinase activators and inhibitors, define their impact on the catalytic (strand passing) activity of the enzyme; and c) identify specific topo II determinants which, when phosphorylated, influence the ability of the enzyme to ligate DNA in the presence of topo II-reactive antitumor agents. At the end of this project, it is anticipated that the regulation of cell growth and differentiation will be further clarified and the interactions of topo II and protein kinases will be documented.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29CA062184-03
Application #
2103252
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Project Start
1993-04-01
Project End
1998-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
3
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
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Salti, G I; Das Gupta, T K; Constantinou, A I (2000) A novel use for the comet assay: detection of topoisomerase II inhibitors. Anticancer Res 20:3189-93
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Moon, R C; Constantinou, A I (1997) Dietary retinoids and carotenoids in rodent models of mammary tumorigenesis. Breast Cancer Res Treat 46:181-9
Ganapathi, R; Constantinou, A; Kamath, N et al. (1996) Resistance to etoposide in human leukemia HL-60 cells: reduction in drug-induced DNA cleavage associated with hypophosphorylation of topoisomerase II phosphopeptides. Mol Pharmacol 50:243-8

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