This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Matherly, L. H. Articles by Goldman, I. D. Search for Related Content PubMed PubMed Citation Articles by Matherly, L. H. Articles by Goldman, I. D.

Role of Methotrexate Polyglutamylation and Cellular Energy Metabolism in Inhibition of Methotrexate Binding to Dihydrofolate Reductase by 5-Formyltetrahydrofolate in Ehrlich Ascites Tumor Cells in Vitro¹

Departments of Medicine and Pharmacology, Medical College of Virginia, Richmond, Virginia 23298

5-Formyltetrahydrofolate was found to reverse the binding of methotrexate to dihydrofolate reductase 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 increased the fraction of dihydrofolate reductase 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 dihydrofolate reductase. 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 dihydrofolate reductase. These findings support a role for dihydrofolate reductase 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.

¹ Supported by Research Grant CA-16906 and Cancer Training Grant CA-09340 from the National Cancer Institute, NIH.

² To whom requests for reprints should be addressed.

Received 8/24/82. Accepted 1/26/83.

This article has been cited by other articles:

				T. Dervieux, D. Orentas Lein, J. Marcelletti, K. Pischel, K. Smith, M. Walsh, and R. Richerson HPLC Determination of Erythrocyte Methotrexate Polyglutamates after Low-Dose Methotrexate Therapy in Patients with Rheumatoid Arthritis Clin. Chem., October 1, 2003; 49(10): 1632 - 1641. [Abstract] [Full Text] [PDF]