Role of Methotrexate Polyglutamylation and Cellular Energy Metabolism in Inhibition of Methotrexate Binding to Dihydrofolate ReducÃ-aseby 5-FormyltetrahydrofoÃ-ate in Ehrlich Ascites Tumor Cells in Wiro1

5-Formyltetrahydrofolate was found to reverse the binding of methotrexate to dihydrofolate redactase in the Ehrlich ascites tumor in vitro. When cells pretreated with methotrexate were resuspended in methotrexate-free buffer containing 5-formyltetrahydrofolate (or 5-methyltetrahydrofolate), net dissociation of the antifolate from the enzyme was observed. Methotrexate associated with the enzyme under these conditions was below the enzyme binding capacity. However, glucose or azide in creased the fraction of dihydrofolate reducÃ-aseassociated with methotrexate and abolished the effect of tetrahydrofolates on this intracellular component. Addition of 5-fluoro-2'-deoxyuridine had no effect on this response to the reduced folate, thereby precluding a direct role for the thymidylate synthase-dependent generation of dihydrofolate in this dissociation of methotrexate from dihydrofolate reducÃ-ase. Enzyme-bound methotrexate could also be reduced by exposure to 5-formyltetrahydrofolate prior to uptake and efflux of free methotrexate. When cells were incubated under conditions which favored formation of metho trexate polyglutamate derivatives, subsequent treatment with 5formyltetrahydrofolate had no effect on the binding of the con jugated antifolate to dihydrofolate reductase. These findings support a role for dihydrofolate reductase as a locus for com petitive binding interactions between reduced folates and meth otrexate that may be a basis for the ability of 5-formyltetrahydro folate to prevent the biochemical effects of this antifolate. These data suggest that the presence of methotrexate polyglutamate derivatives and cellular energy metabolism may be critical deter minants of the responsiveness of methotrexate-treated cells to reduced folates and may play important roles in the selectivity of 5-formyltetrahydrofolate rescue.