ROLE OF METHOTREXATE POLYGLUTAMYLATION AND CELLULAR-ENERGY METABOLISM IN INHIBITION OF METHOTREXATE BINDING TO DIHYDROFOLATE-REDUCTASE BY 5-FORMYLTETRAHYDROFOLATE IN EHRLICH ASCITES TUMOR-CELLS INVITRO

  • 1 January 1983
    • journal article
    • research article
    • Vol. 43  (6) , 2694-2699
Abstract
5-Formyltetrahydrofolate (leucovorin) reversed the binding of methotrexate to dihydrofolate reductase (DHFR) in the mouse 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 increased the fraction of DHFR associated 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 DHFR. 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 methotrexate polyglutamate derivatives, subsequent treatment with 5-formyltetrahydrofolate had no effect on the binding of the conjugated antifolate to DHFR. These findings support a role for DHFR as a locus for competitive binding interactions between reduced folates and methotrexate that may be a basis for the ability of 5-formyltetrahydrofolate 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 determinants of the responsiveness of methotrexate-treated cells to reduced folates and may play important roles in the selectivity of 5-formyltetrahydrofolate rescue during chemotherapy.