Resistance to Methotrexate in Thymidylate Synthetase-deficient Mutants of Cultured Mouse Mammary Tumor FM3A Cells1

Growth inhibition by methotrexate (MTX) of a cultured mouse mammary carcinoma FM3A line and its mutants deficient in thymidine kinase and thymidylate synthetase was studied un der a variety of conditions. In medium containing 10 ¡IMthy midine, the thymidylate synthetase-deficient mutants were slightly more resistant to MTX than the wild-type line and the thymidine kinase-deficient mutant. The addition of both 10 ¡J.M thymidine and 50 fiM hypoxanthine to the medium completely eliminated the inhibition by MTX of the wild-type and thymidyl ate synthetase-deficient mutants but had no effect on inhibition of the thymidine kinase-deficient mutant. Thus, addition of either thymidine or purine alone was not sufficient to protect FM3A cells against the growth inhibitory effect of MTX. In contrast, addition of a small amount of 5-methyltetrahydrofolate to medium containing 10 ¡IMthymidine caused an increase of several orders of magnitude in the resistance of thymidylate synthetase-deficient mutants to MTX but did not affect that of the wild-type line. Wild-type cells became almost as resistant to MTX as did the mutant cells by addition of 1 fiM 5-fluorodeoxyuridine as well as a small amount of 5-methyltetrahydrofolate with thymidine. These results show directly that thymidylate synthetase is essential in determining the cytotoxicity of MTX by modulating the intracellular telrahydrofolate pool.