The objectives of the proposal are to focus on thymidylate synthase (TS) as a therapeutic target in the treatment of colon adenocarcinoma. Studies will examine ways of combining 5-fluorouracil (FUra) with potential modulators of reduced folate pools, in particular leucovorin ([6RS]LV), to maximize TS inhibition and FUra cytotoxicity, and identify sites distal to TS that may be modulated to increase the cytotoxic effects of TS inhibition. Xenografts of human colon adenocarcinomas, clonal cultured cell lines derived from these, and clonal mutants selected for deficiency in de novo (TS-) or salvage (TK-) pathways for dTMP biosynthesis will be utilized. Optimization of FUra-[6RS]LV in vivo will be based upon in vitro and in vivo studies. Experimental designs include the relationship between dose of [6RS]LV, schedule of administration, metabolism, TS inhibition, and expansion of pools of tetrahydrofolate polyglutamates (H4PteGlun) and 5,10-methylenetetrahydrofolates (CH2-H4PteGlun), which are important for stabilizing the covalent ternary complex formed between 5- flurordeoxyuridylate (FdUMP), TS and CH2-H4PteGlun. In vivo studies will determine intracellular events relative to plasma pharmacokinetics of reduced folates, and will evaluate other potential precursors of CH2- H4PteGlu. The influence of the inactive [6R] isomer of LV on the cellular pharmacology of active [6S]LV and cytotoxic action of FUra, and other potential modulators of CH2-H4PteGlun and H4PteGlun pools will initially be evaluated in vitro. These studies will be important in understanding critical factors that influence the modulation of FUra activity in tumors with different metabolic characteristics of [6RS]LV metabolism. In human tumor xenografts and cell lines, the level and activity of TS varies by >40-fold, and is higher in FUra-unresponsive tumors. Turnover and regulation of TS may therefore have therapeutic significance and will b examined. Lastly, it will be determined whether thymine-less death is associated with dNTP imbalance and induction of an endonuclease activity proposed as a general model for antimetabolite death. Using the TS- and TK- mutants, evaluation of possible """"""""nonclassical"""""""" salvage in which TS+ cells may rescue TS- cells, will be made. These studies will design therapy to optimize FUra-folate combinations, shown to have clinical efficacy, and will allow development and testing of strategies that may have synergistic therapeutic efficacy with therapy directed at the TS locus.
Showing the most recent 10 out of 12 publications
