Immature myeloid precursor cells were preferentially selected from normal human bone marrow by using immune rosette techniques that employed monoclonal antibodies against mature granulocytes, monocytes, T and B lymphocytes, and erythroid precursors (Mo5, M3, OKT3, B1, and EP1, respectively). We examined the formation, retention, and cytotoxic effects of methotrexate (MTX) polyglutamates (MTX-PGs) in these purified myeloid precursor cells. After 1- and 24-h exposures to MTX, with thymidine and deoxyinosine as rescue, the intracellular MTX-PG profile was examined by high-pressure liquid chromatography. Efflux patterns of MTX-PGs were also studied after additional 1- and 24-h incubations in drug-free media. Cytotoxic effects of retained MTX-PGs on bone marrow myeloid precursors were examined by colony formation in drug-free semisolid agar. Normal myeloid precursor cells converted MTX to MTX-PGs in a concentration- and time-dependent manner, preferentially retaining MTX-PGs with three to five glutamyl moieties. At low concentrations of MTX (1 microM), MTX-PG formation was insufficient to maintain saturation of the target enzyme dihydrofolate reductase after removal of drug from the incubation medium, and there was no decrease in myeloid colony formation. At higher concentrations of MTX (10 microM), formation of higher molecular weight polyglutamates was sufficient to allow for 24-h saturation of intracellular binding capacity after removal of extracellular drug and resulted in a 35% reduction in the formation of colony-forming units in culture. Comparison of MTX metabolism in normal bone marrow cells and the MTX-sensitive HL-60 human leukemia cell line showed twofold greater PG formation by these tumor cells after 24-h exposure to 1 or 10 microM MTX, and a marked (greater than 30-fold) increase in cytotoxicity for the HL-60 cells as compared with normal myeloid precursors, suggesting that the MTX polyglutamation may be important to its selective antitumor action.
S Koizumi, G A Curt, R L Fine, J D Griffin, B A Chabner