Interaction of methotrexate polyglutamates and dihydrofolate during leucovorin rescue in a human breast cancer cell line (MCF-7).
Previous investigations have suggested that high-dose methotrexate with leucovorin rescue is a potentially useful strategy for overcoming antifolate resistance. Interactions between methotrexate (MTX) and leucovorin and their respective metabolites appear to occur at multiple intracellular sites, including dihydrofolate reductase (MTX/MTX polyglutamates versus dihydrofolate) and other folate-dependent enzymes (MTX polyglutamates versus reduced folate substrates). The present studies were designed to test the ability of dihydrofolate to compete with methotrexate and methotrexate polyglutamates for dihydrofolate reductase activity using an intact human breast carcinoma cell line (MCF-7) as the model system. Exposure of the breast cells to methotrexate for 24 h resulted in a concentration-dependent formation of methotrexate polyglutamates that markedly exceeded the dihydrofolate reductase-binding capacity for up to 24 h after the removal of drug from the growth media. Under these conditions of dihydrofolate reductase inhibition, we found that tritium-labeled dihydrofolate was capable of competing with methotrexate and its metabolites for dihydrofolate reductase activity as evidenced by the appearance of tritium-labeled reduced folates in the treated cells. We found the interaction between dihydrofolate and methotrexate to be dependent on the exposure concentrations of both methotrexate and dihydrofolate. These studies provide direct evidence that competition during leucovorin rescue occurs at the level of dihydrofolate reductase between methotrexate polyglutamates and dihydrofolate polyglutamates in intact human cells.
Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20892.
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D001667:Binding, Competitive; D001943:Breast Neoplasms / Q000032:analysis / Q000201:enzymology / Q000378:metabolism*; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D006801:Humans; D002955:Leucovorin / Q000627:therapeutic use; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D013763:Tetrahydrofolates / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism
D010455:Peptides; D013763:Tetrahydrofolates; D011099:Polyglutamic Acid; C010920:dihydrofolate; C030371:5,6,7,8-tetrahydrofolic acid; C014085:methotrexate polyglutamate; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D002955:Leucovorin; D008727:Methotrexate