Impairment of methotrexate (MTX)-polyglutamate formation of MTX-resistant K562 cell lines.

We examined the mechanism of methotrexate (MTX) resistance in five K562 cell subclones resistant to MTX. Based on a clonogenic assay, the IC50S of these MTX-resistant clones were 10 to 40 microM MTX, indicating 2,000 to 5,000-fold resistance as compared to that of the parent cell line. The doubling times of these MTX-resistant K562 cell lines are longer (27-60 hr) than that of the parent K562 cell line (24 hr). One-hour MTX accumulation in the resistant cells cells was 70-80% of that in parent cells. To investigate the formation of MTX-polyglutamates (MTX-PGs), resistant cells were incubated with 3H-MTX (1 or 10 microM) for 24 hr in the presence of thymidine and deoxyinosine to prevent cytotoxicity. MTX (-Glu1) and the polyglutamate metabolites (MTX-Glu2, -Glu3, -Glu4 and -Glu5) were analyzed by a high-pressure liquid chromatography (HPLC) technique. After a 24-hr incubation with 10 microM MTX, the total concentration of intracellular MTX reached 39 to 89 nmol/g protein, only 20 to 40% of the MTX level of the parent K562 cells. The HPLC analysis revealed that less than 2% of intracellular MTX was in the form of high-molecular MTX-PGs (MTX-Glu3, -Glu4 and -Glu5) in the five MTX-resistant K562 cell lines, while in the parent cells MTX-Glu3-5 comprised 46% of the total intracellular MTX. These data indicate the possibility that impairment of MTX-PG formation, with transport alteration, may be a special mechanism for the high level of resistance to this agent in human leukemic cells.