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 to expand existing information and to assess its significance in the clinical setting. Thus, it is pertinent to examine the extent and impact of genetic variation on parameters of drug response in human tumor cells of relevant histological origin. The fluoropyrimidine anti-metabolite 5-fluoro-2'-deoxyuridine (FdUrd) inhibits cell growth through the formation of 5-fluoro-2'- deoxyurdylic acid (FdUMP), which binds to and inhibits thymidylate synthase (TS). In previous work, we have shown that response to FdUrd varies considerably among human colonic tumor cell lines. Of particular interest is cell line HCT116, which produces a variant form of TS exhibiting a reduced affinity for FdUMP; this alteration results from a T -> C mutation in the TS gene, which causes the replacement of a tyrosine by a histidine at residue 33 of the TS polypeptide. The Tyr33 - > His33 change confers relative resistance to the inhibitory effects of FdUrd in both mammalian and bacterial cells. In the present application, we propose to conduct further studies of this mutation and its role in response to FdUrd in colonic tumor cells. We will: (1) determine the origin and frequency of the Tyr33 -> His33 substitution among colon tumor cell lines; (2) compare the Tyr33 and His33 forms of human TS with regard to their kinetics of interactions with ligands; (3) characterize the effects of different amino acid substitutions at residue 33; (4) identify and study additional variations in TS structure and function among a variety of human colonic tumor cell lines. These experiments will provide information on the mechanism by which the Tyr33 -> His33 mutation perturbs the function of TS as a fluoropyrimidine target. In all, the study of TS and its function as a drug target will generate fundamental knowledge on the relationship between protein structure and function, promoting the development of improved strategies for the design and utilization of anti-neoplastic agents. Furthermore, these studies will result in a deeper understanding of the genetic factors that govern drug response, and may lead to the identification of markers that could be useful in the pre-clinical prediction of therapeutic response in many patients.
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