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

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 micron 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 micron MTX. The H2PteGlu pools within the MCF-7 cells increased significantly after 15 min of 1 micron 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 micron 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.

• Authors: E Chu, J C Drake, D Boarman, J Baram, C J Allegra
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 265 Issue 15 Pg. 8470-8 (May 25 1990) ISSN: 0021-9258 [Print] United States
• PMID: 2341391 (Publication Type: Journal Article)

Chemical References

• Folic Acid
• Thymidylate Synthase
• Methotrexate

Topics

• Breast Neoplasms
• Cell Line
• Female
• Folic Acid (metabolism)
• Hematopoietic Stem Cells (enzymology)
• Humans
• Kinetics
• Leukemia, Promyelocytic, Acute
• Methotrexate (pharmacology)
• Thymidylate Synthase (antagonists & inhibitors)
• Tumor Cells, Cultured (enzymology)