CELL CYCLE CHECKPOINTS AND CHEMOSENSITIVITY OF HUMAN CANCER CELLS

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This research project focuses on cell cycle checkpoints and the roles these systems play in determining chemosensitivity. We also aim to utilize our emerging knowledge of these checkpoint systems to design new therapeutic stratagems for cancer treatment. We are elucidating the checkpoints that respond to DNA damage to arrest cells in G1 and G2 phases. We are also defining defects in these systems in cancer cells. We aim to trace these systems from the point where DNA damage or unreplicated DNA is sensed by the cell to the response elements that interact with the cyclin-dependent kinases to arrest cell cycle progression or in some cases induce apoptosis. Our results suggest that p53 mutations i lymphoma cell lines confer decreased sensitivity to DNA damaging agents. but not antimitotic agents. Decreased sensitivity is related to an evasion of p53-mediated apoptosis, although other factors can also contribute to the outcome. We have been exploring domains in the p53 regulated gene product, Waf1/Cip1, that interact with cyclin E/Cdk2 and PCNA. Our results suggest two regions in the amino terminus of Waf1/Cip1 are involved in cyclin E/Cdk2 interaction and/or inhibition of this kinase while the carboxy-terminal region of Waf1/Cip1 is involved in PCNA interaction. The G2 checkpoint appears to protect cells from DNA damage by extending the time available for DNA repair. We have found that pentoxifylline preferentially abrogates the G2 checkpoint in cells with disrupted p53 and such cells can be preferentially killed by a combination of a DNA damaging agent and pentoxifylline. We are presently exploring the mechanisms underlying these activities with a special focus on the formation and activation of the G2 cyclin-dependent kinases. We are also investigating several G2 checkpoint abrogators that might be clinically useful in combination protocols to preferentially kill mutant p53 tumors. In conjunction with several other laboratories we have found that the majority of cell lines in the NCI cell screen have disrupted p53 function. We have also found these p53 disrupted cells tend to be less sensitive to a wide variety of currently used chemotherapeutic agents. A notable exception in this analysis was the microtubule agents, whose activity appeared independent of p53 status. Searching the 45,000 compound data base revealed a number of agents which might be preferentially active in cells with disrupted p53. These agents will undergo detailed analysis in isogeneic cell lines in which p53 function has been disrupted by transfection with either the HPV-E6 gene or a dominant negative mutant p53 gene.