The role of methotrexate and dihydrofolate polyglutamates in the enhancement of fluorouracil action by methotrexate

The effect of the methotrexate (MTX)-5-fluorouracil (FUra) combination on L1210 cell viability was highly sequence dependent in that a 1.4 log (25-fold) increase in cytotoxicity was observed when the MTX exposure preceded FUra, as compared to the reverse sequence. The formation of ternary complexes of thymidylate synthetase has been examined as a basis for the interaction of FUra and MTX in L1210 cells. L1210 cells converted 39% of the total intracellular MTX into MTX-poly-γ-glutamates within 4 hours of a 1 μM MTX exposure. MTX-diglutamate (2,4-diamino,N¹⁰-methylpteroyldiglutamate) and MTX-triglutamate were the predominant metabolites. In contrast to the ternary complexes formed with MTX and MTX-diglutamate, the 5-fluorodeoxy-uridylate (FdUMP)-7,8-dihydropteroylpentaglutamate-enzyme and the FdUMP-5,10-methylenetetrahydropteroylpentaglutamate-enzyme complexes were stable to polyacrylamide gel electrophoresis under nondenaturing conditions. MTX-diglutamate enhanced the extent of tight-binding inhibition of thymidylate synthetase activity by FdUMP in the presence of saturating 5,10-methylenetetrahydropteroylpentaglutamate, suggesting that MTX-diglutamate did not antagonize the formation of FdUMP-5,10-methylenetetrahydropteroylpentaglutamate enzyme complex. We propose that the sequence-dependent effect of MTX plus FUra on L1210 cell viability results from MTX and MTX polyglutamate inhibition of dihydrofolate reductase, and consequently a trapping of intracellular folates as dihydrofolate polyglutamates that could increase the extent of FdUMP binding to thymidylate synthetase.