Elevation of Dihydrofolate Reductase, Thymidylate Synthetase, and Thymidine Kinase in Cultured Mammalian Cells after Exposure to Folate Antagonists
Folic Acid Antagonists
Roswell Park Memorial Institute 4265 human lymphoblasts were grown with three dihydrofolate reductase inhibitors: a 2,4-diaminopteridine, methotrexate; a 2,4-diaminoquinazoline, chlorasquin; and, a 2,4-diaminotriazine, triazinate. In the absence of inhibitor, dihydrofolate reductase activity increased to a peak at mid-log growth and then declined during the later growth stages. When cells were grown with 10(-8) M antifolate, cell growth was not affected, but dihydrofolate reductase activity (assayed at pH 7.0) remained at approximately initial levels throughout the growth cycle. This represented 60 to 70% less activity at the mid-log stage of growth, as compared to control cells. Dihydrofolate reductase activity in cells grown with 10(-8) M methotrexate, when assayed at pH 8.5, reached levels twice those in control cells. Enzyme activity in cells grown with 10(-8) M chlorasquin, when assayed at pH 8.5, was also higher than at pH 7.0, but it was not as high as that observed in methotrexate-treated cells. Activity in cells grown with 10(-8) M triazinate was approximately the same when assayed at either pH 7.0 or 8.5. At 10(-8) M, the three antifolates had no effect on the activities of thymidylate synthetase, thymidine kinase, serine trans-hydroxymethylase, 5,10-methylenetetrahydrofolate dehydrogenase, 10-formyltetrahydrofolate synthetase, and thymidylate kinase. However, when concentrations were used which completely inhibited growth (10(-7) to 10(-5) M methotrexate or chlorasuin; 10(-6) to 10(-5) M triazinate), dihydrofolate reductase was progressively inhibited, and there was a two- and a threefold elevation of thymidylate synthetase and thymidine kinase activity, respectively. Quantitatively, the elevation of either enzyme was similar over the range of growth-inhibitory concentrations studied. The activities of the other enzymes were unaffected. Methotrexate and chlorasquin inhibited thymidylate synthetase in a noncompetitive manner (with respect to 5,10-methylenetetrahydrofolate) with approximate Ki values of 4.5 X 10(-5) M and 4.9 X 10(-6) M, respectively. Triazinate, at 10(-3) M, had no significant effect on thymidylate synthetase activity. At 10(-3) M, the antifolates produced a negligible inhibition of thymidine kinase. Deoxyuridine 5'-monophosphate (10(-5) M) effectively protected thymidylate synthetase from heat inactivation in vitro. Dihydrofolate or 5,10-methylenetetrahydrofolate, at 10(-3) M, only partially protected thymidylate synthetase. Concentrations of methotrexate (10(-7) to 10(-6) M), chlorasquin (10(-7) M), and triazinate (10(-6) to 10(-5) M), which produced thymidylate synthetase elevation in vivo, did not protect the enzyme from heat inactivation in vitro. Methotrexate at 10(-5) M and chlorasquin at 10(-6) M gave slight protection. Thymidine kinase was stabilized only by thymidine.