Role of methylenetetrahydrofolate depletion in methotrexate-mediated intracellular thymidylate synthesis inhibition in cultured L1210 cells.

The effect of low methotrexate levels on methylenetetrahydrofolate and four other reduced folate pools in cultured L1210 cells has been examined over a 48-h period. Media folate levels and methotrexate were used to alter intracellular levels of reduced folates, and the distribution among individual reduced folates, so that they could be evaluated in terms of their effects on thymidylate synthesis and cell proliferation. Over the media folate concentration range of 0.25-50 microM, growth rate and thymidylate synthesis remained essentially unchanged while total intracellular reduced folates, determined from the summation of the five individual pools measured, increased approximately 25-fold. The 5-methyltetrahydrofolate and 10-formyltetrahydrofolate pools accounted for over 90% of the total reduced folate at the highest media folate level, while low media folate resulted in a much more equal distribution among the five reduced folates examined. Methotrexate, over the concentration range of 0.25-30 nM, caused extensive growth and intracellular thymidylate synthesis inhibition at media folate levels used in RPMI 1640 media (2.5 microM) and lower. However, growth inhibition was much less at the highest media folate level used, and thymidylate synthesis was not inhibited to a statistically significant extent. Intracellular reduced folates also responded differently to methotrexate depending upon the level of media folate. Depletion of the thymidylate synthase substrate, methylenetetrahydrofolate, could not account for diminished growth or thymidylate synthesis inhibition, since at 0.25 and 2.5 microM media folate no depletion occurred in response to methotrexate and only slight depletion was observed at 50 microM media folate. Dihydrofolate showed a tendency to increase at each of the media folate levels used with the least increase at the highest folate level. However, the ratio of dihydrofolate to total reduced folates was quantitatively most consistent with thymidylate synthesis and growth inhibition results.