Synthesis, binding and intracellular retention of methotrexate polyglutamates by cultured human breast cancer cells.

Synthesis, binding, and intracellular retention of methotrexate polyglutamates by cultured human breast cancer cells were investigated by gel filtration and high-pressure liquid chromatography to separate methotrexate from its metabolites. MCF-7, ZR-75-1, and MDA-231 human breast cancer cells were found to readily convert methotrexate to higher polyglutamates during a 24-hr incubation period, although at differing rates. Examination of that portion of intracellular methotrexate specifically bound to dihydrofolate reductase revealed that, with prolonged incubation, methotrexate polyglutamates become the predominant drug form bound to the enzyme. Similarly, methotrexate polyglutamates accumulated free in the cytosol and when cells were suspended in drug-free medium, were retained intracellularly both bound to dihydrofolate reductase and in the unbound fraction, indicating their slow passage through the cell membrane. Studies of methotrexate polyglutamate binding to purified bacterial dihydrofolate reductase revealed high affinity binding for compounds with up to 6 additional glutamyl residues. These studies demonstrate that methotrexate polyglutamates are readily formed in human breast cancer cells, bind intracellularly to dihydrofolate reductase, and are selectively retained both bound to the enzyme and free in the cell cytosol.