Comparative effects of folate antagonists versus enzymatic folate depletion on folate and thymidine enzymes in cultured mammalian cells
Folic Acid Antagonists
Folate and thymidine enzymes were monitored during lag, log, and stationary growth of RPMI 4265 human lymphoblasts. The activities of dihydrofolate reductase, thymidylate synthetase, thymidine kinase, thymidylate kinase, and methionine synthetase peaked during log phase, while those of serine transhydroxymethylase and 5,10-methylenetetrahydrofolate reductase were maximum during stationary phase. 10-Formyltetrahydrofolate synthetase had peaks of activity in log and in stationary phase, while the activity of 5,10-methylenetetrahydrofolate dehydrogenase did not change during the growth cycle. In cells whose growth was inhibited with folate antagonists (methotrexate, chlorasquin, or triazinate), dihydrofolate reductase was inhibited and thymidylate synthetase and thymidine kinase were elevated twofold and threefold respectively. Other enzymes were unaffected. Growth with noninhibitory concentration of each antifolate affected only dihydrofolate reductase activity (inhibition when assayed at pH 7.0; elevated activity in extracts from methotrexate- and chlorasquin-treated cells when assayed at pH 8.5). The folate-cleaving enzyme, carboxypeptidase G1, produced intracellular folate depletion. In the presence of 0.0025 units/ml, growth inhibition occurred after the cells reached midlog phase. The activities of dihydrofolate reductase, thymidylate synthetase, and thymidine kinase were significantly lower 24 hours before, and were undetectable when growth inhibition occurred. In contrast, the activities of other enzymes were not reduced until growth inhibition occurred. Use of 0.001 units/ml also produced an intracellular folate depletion, but did not significantly retard growth. In these cells, only the activity of dihydrofolate reductase was lower than in control cells.