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 vitro

L. H. Matherly, D. W. Fry, I. David Goldman

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37 Citations (Scopus)

Abstract

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-formyl-tetrahydrofolate (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 synthetase-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.

Original languageEnglish (US)
Pages (from-to)2694-2699
Number of pages6
JournalCancer Research
Volume43
Issue number6
StatePublished - 1983
Externally publishedYes

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Ehrlich Tumor Carcinoma
Tetrahydrofolate Dehydrogenase
Leucovorin
Methotrexate
Energy Metabolism
Folic Acid Antagonists
Folic Acid
Enzymes
Tetrahydrofolates
In Vitro Techniques
Thymidylate Synthase
Competitive Binding
Azides
Buffers
Glucose

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

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title = "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 vitro",
abstract = "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-formyl-tetrahydrofolate (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 synthetase-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.",
author = "Matherly, {L. H.} and Fry, {D. W.} and Goldman, {I. David}",
year = "1983",
language = "English (US)",
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T1 - 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 vitro

AU - Matherly, L. H.

AU - Fry, D. W.

AU - Goldman, I. David

PY - 1983

Y1 - 1983

N2 - 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-formyl-tetrahydrofolate (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 synthetase-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.

AB - 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-formyl-tetrahydrofolate (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 synthetase-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.

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