Role of thymidylate synthetase activity in development of methotrexate cytotoxicity.

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Abstract
Methotrexate (MTX) inhibition of the growth of mouse or human leukemia cells in culture was partially prevented by thymidine (dThd) or hypoxanthine. 5-Fluoro-2''-deoxyuridine (FdUrd) also decreased the growth-inhibitory potency of MTX in the presence of small concentrations of 5-formyltetrahydrofolate (citrovorum factor) and sufficient exogenous dTHd to support the synthesis of thymidylate nucleotides by salvage mechanisms. Citrovorum factor-induced reversal of MTX was several orders of magnitude more efficient in the presence of both FdUrd and dThd than in the presence of dThd alone or in the absence of both nucleosides. The presence of FdUrd (3 .mu.M) and dThd (5.6 .mu.M) completely prevented the lethality of 0.3 mM MTX to L1210 cells in culture medium supplemented with micromolar concentrations of citrovorum factor. This protection against the cytotoxic effects of MTX by dThd, hypoxanthine and FdUrd may have a common biochemical mechanism.sbd.inhibition of the de novo synthesis of thymidylate by a direct [FdUrd; inhibition of thymidylate synthetase (thymidylate synthase; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45)] or indirect (dThd and hypoxanthine; feedback inhibition by anabolites or ribonucleotide reductase and deoxycytidylate deaminase) effect. The resultant decreased rate of loss of reduced folates due to de novo thymidylate synthesis would allow a higher degree of inhibition of dihydrofolate reductase to be endured without damage to the cell.