(MCF-7) during Leucovorin Rescue in a Human Breast Cancer Cell Line Interaction of Methotrexate Polyglutamates and Dihydrofolate

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 leu covorin and their respective metabolites appear to occur at multiple intracellular sites, including dihydrofolate reducÃ-ase(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 poly glutamates 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 reducÃ-aseinhibition, we found that tritium-labeled dihydrofolate was capable of competing with meth otrexate and its metabolites for dihydrofolate reducÃ-aseactivity as evi denced 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 reducÃ-ase between methotrexate polyglutamates and dihydrofolate polyglutamates in intact human cells.