It has been suggested that the pattern of chemoresistance found in many solid tumors may be due to their failure to efficiently activate programmed cell death (PCD) processes upon exposure to chemotherapeutic agents. Nevertheless, despite the fact that it is often difficult to activate PCD in these tumors, it is still possible to kill them selectively using certain chemotherapeutic drugs, suggesting that either these agents are particularly good at precipitating PCD in such tumors, or that these drugs are able to kill through mechanisms that don't require activation of PCD (i.e., by non-programmed cell death processess). Our studies suggest that both programmed and non-programmed forms of cell death may be important in mediating fluoropyrimidine cytotocicity. In this application we propose a model to define the relative roles of programmed- and non-programmed death processes in cells exposed to these drugs. Our objective is to evaluate that model by testing predictions about the consequences of modulating proteins involved in the formation of DNA lesions following thymidylate synthase inhibition (uracil-DNA-glycosylase), progression of cells though the cell cycle (p21/WAF1/Cip1) or the activation of programmed cell death processes (Bcl-X) in the HT29 human collorectal carinoma cell line.
Our specific aims are given below, in the form of hypotheses to be tested. H1: Inhibition of uracil excision-repair activity in FdUrd-treated HT29 cells will increase uracil incorporation into DNA, while decreasing overall cytotoxicity and DNA single- and double-strand break formation H2: Temporary inhibition of S-phase progression in FdUrd-treated HT20 cells (by conditional enforcement of a G1/S checkpoint using p21/WAF1/Cip1) will not affect formation of DNA single stand breaks in parental DNA, but will reduce or eliminate DNA double-strand break formation and cytoxicity. H3: Temporary expression of Bcl-XS (which antagonizes the anti-PCD effect of Bcl-XL) will increase FdUrd-induced cytotoxicity in HT29 cells and will change the pattern of FdUrd-induced DNA fragmentation to one that is characteristic of PCD, without affecting uracil incorporation into DNA or DNA single-strand break formation.
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