Biochemical and cell cycle perturbations in methotrexate-treated cells.

The effects of methotrexate (MTX) in the presence or absence of exogenous thymidine (dThd, 10(-5) M) or hypoxanthine (Hx, 10(-4) M) on cell cycle kinetics and deoxyribonucleoside triphosphate pools (dNTP) were studied in cultured human leukemic T-cells (CCRF-CEM). MTX cytotoxicity was found to increase linearly with drug dose for MTX concentrations between 10(-9) M and 10(-7) M. No further increase in cytoxicity was observed with much higher MTX concentrations (10(-7) M-10(-4) M). A similar dose-response relationship was found for both MTX-induced inhibition of DNA synthesis and changes in dTTP and dGTP pools but not for either MTX-induced inhibition of purine synthesis or changes in dATP and dCTP pools. Exogenous dThd reduced MTX cytotoxicity, at all MTX concentrations examined, but Hx reduced cytotoxicity only at MTX concentrations less than 6 X 10(-8) M and potentiated toxicity with higher MTX concentrations. This potentiation of cytotoxicity was accompanied by substantial elevation of dATP pools. In all instances where dThd or Hx reduced MTX cytotoxicity, a concomitant increase in both dTTP and dGTP levels and in the rate of DNA synthesis was observed. These results suggest a close correlation between MTX-induced alterations of dNTP and inhibition of DNA synthesis and subsequent MTX cytotoxicity. The possible modulation of MTX cytotoxicity by purines is discussed.