FOLATE-DEPLETION INDUCED BY METHOTREXATE AFFECTS METHIONINE SYNTHASE ACTIVITY AND ITS SUSCEPTIBILITY TO INACTIVATION BY NITROUS-OXIDE

We compared the effects of methotrexate (MTX) and nitrous oxide on the methionine (Met) synthase system in two variants of a human glioma ce ll line. The cells were protected from cytotoxic effect of MTX by addi ng thymidine and hypoxanthine to the cell culture medium. MTX(0-1 mu M ) was associated with a dose-and time-dependent reduction in 5-methylt etrahydrofolate (5-methyl-THF) in both cell lines. Already after 3 hr of exposure, 5-methyl-THF was reduced by 50% and after additional 48 h r, the level was undetectable. In addition to reduction in folate leve l, homocysteine (Hcy) remethylation in intact cells was markedly inhib ited as judged by an increased export of Hey from the cells, and Met s ynthase activity in cell extracts and level of cellular methylcobalami n (CH(3)Cbl) declined. MTX reduced Hey remethylation and CH(3)Cbl leve l more efficiently than nitrous oxide. In both cell variants, the inac tivation of Met synthase by nitrous oxide was almost completely preven ted in cells pre-exposed to MTX. This indicates that there is no catal ytic turnover in cells exposed to MTX, and emphasizes the importance o f the sequence of administration for synergistic effect of this drug c ombination. In conclusion, our data show that MTX through depletion of 5-methyl-THF reduces both the Met synthase activity and the cellular CH(3)Cbl level. Moreover, the effect of MTX on the Hey remethylation i s more pronounced than the inhibition caused by nitrous oxide. These o bservations should be taken into account in studies on MTX pharmacodyn amics.