In vitro and Intact Cellular Inhibition of Cyclin-dependent Kinase 2-Cyclin E as a Potential Target for Anti-cancer Therapeutics
Goethals, Andrea J.
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In normal cells, transitions between the G1, S, G2 and M phases of the cell cycle are carefully regulated by the sequential activation of cyclin-dependent protein kinases (CDKs) and phosphatases. These enzymes serve as key checkpoint controls for reversible regulation of normal proliferating cells. In tumor cells, checkpoint control mechanisms are defective at one or more levels, including disregulation of CDK activity. This results in disregulation of cellular proliferation. Overexpression of cyclins, the regulatory proteins that bind to and activate CDKs, has been observed in various tumors. In addition, increased levels of CDK enzymatic activity have also been reported in clinical samples. These results suggest that agents which induce cell cycle arrest in cancer cells may have therapeutic value. Based on the importance of CDKs in control of proliferation, this study focused on CDKs, specifically CDK2 and its partner, cyclin E. Purified kinase-cyclin complexes were used to characterize the enzymatic characteristics of CDK2-cyclin E in vitro. Several agents were identified as CDK inhibitors, including butyrolactone I and other research compounds. Four compounds were used to determine the mechanisms of inhibition, as well as peptide substrate and ATP competition in vitro. In addition, the ability of these compounds to induce cell cycle arrest in a human carcinoma cell line was examined using proliferation assays and flow cytometry analysis.