Effect of methotrexate on thymidylate synthetase in cultured parenchymal cells isolated from regenerating rat liver.

The effect of methotrexate (MTX) on thymidylate synthetase activity during liver regeneration was examined with parenchymal cells isolated 22 and 44 hr after partial hepatectomy and cultured as a monolayer. The synthetase activity in these cells decreased with a half-life of 18 to 24 hr, but if MTX (1.5 X 10(-6) to 1.5 x 10(-5) M) was present in the culture media, this decline could be delayed for at least 48 hr. In contrast, thymidine kinase activity decreased at a rate which we unaffected by MTX. Dihydrofolate reductase was inhibited at all concentrations of MTX used to block the decrease in synthetase activity. Folic acid at 10(-4) M, although less effective than MTX, also delayed the decrease in synthetase activity. The addition of cycloheximide, puromycin, or antinomycin D to the culture media did not alter the response of the synthetase to MTX. The latter studies, coupled with those indicating that the rapid loss of synthetase activity in crude extracts could be prevented by MTX or, more effectively, by MTX plus deoxyuridine 5'-monophosphate, suggest that the primary effect of MTX on thymidylate synthetase in vivo is that of enzyme stabilization. Similar stabilizing effects were obtained in liver cell extracts with 10(-5) M deoxyuridine 5'-monophosphate in combination with 10(-4) M folate or 10(-4) M dihydrofolate.