Clinical resistance to chemotherapeutic drugs is a major impediment to the successful treatment of human neoplasia. Though a great deal of research on model cell lines and tumors has allowed an understanding of the determinants of response and the mechanisms of resistance to a variety of useful drugs, there is need for more detailed information on the extent and impact of genetic variation in drug response in human tumor cells of a relevant histological origin. Efforts in this grant have focused upon the molecular and genetic determinants of response to fluoropyrimidine antimetabolites in colonic tumor cell lines. Fluoropyrimidines such as 5-fluoro-2'-deoxyuridine (FdUrd) inhibit cell growth through formation of 5-fluoro-2-deoxyuridylate (FdUMP), which forms an inhibitory ternary complex with TS and N5,N10-methylenetetrahydrofolic acid; inhibition of TS results in thymidylate deprivation and cell death. In this application, it is proposed to investigate a possible link between TS expression and genes involved in mismatch repair in human colon tumor cell lines. The kinetics of FdUMP binding to TS, which occurs in a negatively cooperative fashion will also be examined. Amino acid substitutions at residue 33 enhance negative cooperativity, generating a reduced affinity for FdUMP and relative resistance to fluoropyrimidines. The overall goal of the study is to gather mechanistic information on the role of TS gene expression and protein structure in the response of colon tumor cells to fluoropyrimidines. Such an in-depth knowledge of the molecular, biochemical, and genetic factors that govern drug response is critical to improving the efficacy of antimetabolite chemotherapy in the treatment of neoplastic disease.
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