Mechanism of the cytotoxic synergism of fluoropyrimidines and folinic acid in mouse leukemic cells

We have investigated the mechanism by which reduced folates, such as folinic acid, enhance the cytotoxicity of fluoropyrimidines in L1210 mouse leukemic cells. Exposure of L1210 cells to folinic acid resulted in expansion of intracellular pools of 5,10-CH₂-H₄PteGlu(n), delayed the reappearance of catalytically active thymidylate synthase (TS) following 5-fluoro-2'-deoxyuridine exposure, and stabilized inhibited TS complexes over the same concentration range that augmented the cytotoxic effects of fluorodeoxyuridine and 5-fluorouracil. The data showed that, in intact L1210 cells, fluorodeoxyuridylate behaves as an inhibitor whose complexes with TS dissociate with a biologically significant rate. However, these complexes become functionally irreversible in cells incubated with high levels of folinic acid. It was also found that bound and total TS levels increased in cells treated with fluorodeoxyuridine to an extent that substantially exceeded the increase in protein content per cell under the same conditions. These results are in accord with the concept that folinic acid augments the effects of the fluoropyrimidines by expansion of cellular 5,10-CH₂-H₄PteGlu(n) pools with subsequent stabilization of ternary complexes among 5-fluoro-2'-deoxyuridine 5'-monophosphate, TS, and 5,10-CH₂-H₄PteGlu(n). In light of the accumulation of TS that occurs following exposure to fluoropyrimidines, this stabilization may be needed for efficient tumor cell killing by these agents.