Inhibition of the key enzyme thymidylate synthase (TS) is a major cause of the activity of 5-fluorouracil (5-FU) as a cancer chemotherapeutic agent. Newer methods of administration of 5-FU, such as combination with reduced folates and with inhibitors of ribonucleotide reductase, have recently improved the effectiveness of this drug against several adult carcinomas. Hence, there is a renewed urgency to efforts aimed at understanding the responses of normal and neoplastic stem cells to pharmacologically induced cellular deficiencies in the supply of thymidine nucleotides. This application proposes to study the biochemcial phenomena initiated by inhibition of TS that cause cell death in normal and neoplastic population. The progression of cells out of S-phase will be analyzed in cultured cells undergoing thymidine nucleotides. This application proposes to study the biochemical phenomena initiated by inhibition of TS that cause cell death in normal and neoplastic populations. The progression of cells out of S- phase will be analyzed in cultured cells undergoing thymidine nucleotide starvation using a series of biochemical techniques. The identity of the proteins accumulating in these cells and the appropriateness of the RNA species to the cell cycle phase will be examfined. The significance of discrete DNA fragments that can be detected in these cells will be pursued and related to the irreversible committment of cells to death. The generality of this phenomenon and its utility afor fine mapping the sequence of S-phase will be sgudied. The mechanism of the accumulation of TS and other proteins involved in S-phase specific events will be determined in cells in which TS is inhibited. The mechanism whereby hydroxyurea augments the cytotoxic activity of 5-FU will be studied. Initially, the hypothesis that inhibition of the accumulation of dUMP is the cause of this synergism will be investigated. The response of tumor cells to inhibition of TS by the folate analog CB 3717 will be compared with that following 5-FU. The role of the unnatural diastercomer of 5-formyltetrahydrofolate in limiting the activity of the natural isomer will be studied. Likewise, the cytotoxicity to 5-FU and folinic acid will be determined in a series of relevant human tumor cell lines and will be correlated with the stability of ternary complex in these cells. Finally, an explanation for the limited increase in the host toxicity of 5-FU when folinic acid is simultaneously administered will be sought on a biochemical level.
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