Role of the Cellular Oxidation-Reduction State in Methotrexate Binding to Dihydrofolate Reductase and Dissociation Induced by Reduced Folates1

5-Formyltetrahydrofolate promotes the net dissociation of methotrexate bound to dihydrofolate reducÃ-ase in the Ehrlich ascites tumor (L. H. Matherly ef al., Cancer Res., 43: 26942699, 1983). Treatment of Ehrlich tumor cells with glucose or inhibitors of electron transfer stabilized the association of the antifolate with dihydrofolate reductase as reflected by a 2-fold increased fraction of dihydrofolate reductase-bound methotrex ate and an abolition of the 5-formyltetrahydrofolate-induced dis sociation of the inhibitor-enzyme complex. Glucose and azide were also found to increase the intracellular ratio of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to oxi dized nicotinamide adenine dinucleotide phosphate (NADPf) in the tumor approximately 8and 11-fold, respectively. However, other agents which enhanced the association between metho trexate and its target enzyme were less effective in increasing the intracellular level of NADPH relative to NADP+. Micromolar concentrations of NADPH promoted methotrexate binding to the purified Ehrlich tumor dihydrofolate reductase. Bound metho trexate could be dissociated from the purified enzyme by 5methyltetrahydrofolate but less readily by 5-formyltetrahydrofolate and only in the presence of reduced levels of NADPH relative to NADP+. The tetraglutamate derivative of 5-methyltetrahydrofolate was even more effective than the underivatized compound in dissociating methotrexate from dihydrofolate reductase. These findings suggest a critical role for the cellular oxidation-reduction state in determining the affinity of dihydrofolate reductase for methotrexate and thus the cellular sensitivity to the antifolate. In addition, the data are consistent with the possibility that dihydro folate reductase is a key locus for intracellular competitive inter actions between reduced folates and methotrexate during leucovorin rescue from the pharmacological effects of the antifolate.