Mechanism of thymidylate synthase inhibition by methotrexate in human neoplastic cell lines and normal human myeloid progenitor cells

E. Chu, J. C. Drake, D. Boarman, J. Baram, C. J. Allegra

Research output: Contribution to journalArticlepeer-review

47 Scopus citations

Abstract

We have studied the roles of 5,10-methylenetetrahydrofolate (5,10-methylene-H4PteGlu) depletion and dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo thymidylate synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Using both a high pressure liquid chromatography system and a modification of the 5-fluoro-2'-deoxyuridine-5'-monophosphate radioenzymatic binding assay, we determined that the 5,10-methylene-H4PteGlu pool is 50-60% depleted in human MCF-7 breast cancer cells following exposure to 1 μm MTX for up to 21 h. Similar alterations in the 5,10-methylene-H4PteGlu pools were obtained when human promyelocytic HL-60 leukemia cells and normal human myeloid precursor cells were incubated with 1 μm MTX. The H2PteGlu pools within the MCF-7 cells increased significantly after 15 min of 1 μm MTX exposure, reaching maximal levels by 60 min. Thymidylate synthesis, as measured by labeled deoxyuridine incorporation into DNA, decreased to less than 20% of control activity within 30 min of 1 μm MTX exposure. The inhibition of thymidylate synthesis coincided temporally with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of thymidylate synthase. Furthermore, inhibition of this pathway was associated in a log-linear fashion with the intracellular level of dihydrofolate. These studies provide further evidence that depletion of the thymidylate synthase substrate 5,10-methylene-H4PteGlu is inadequate to account completely for diminished thymidylate synthesis resulting from MTX treatment. Our findings suggest that acute inhibition of de novo thymidylate synthesis is a multifactorial process consisting of partial substrate depletion and direct enzymatic inhibition by H2PteGlu polyglutamates.

Original languageEnglish (US)
Pages (from-to)8470-8478
Number of pages9
JournalJournal of Biological Chemistry
Volume265
Issue number15
StatePublished - 1990
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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