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 IC(50)s of these MTX‐resistant clones were 10 to 40μMMTX, 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 was 70–80% of that in parent cells. To investigate the formation of MTX‐polyglutamates (MTX‐PGs), resistant cells were incubated with (3)H‐MTX (1 or 1OμM) for 24 hr in the presence of thymidine and deoxyinosine to prevent cytotoxicity. MTX (‐Glu(1)) and the polyglutamate metabolites (MTX‐Glu(2), ‐Glu(3), ‐Glu(4) and ‐Glu(5)) were analyzed by a high‐pressure liquid chromatography (HPLC) technique. After a 24‐hr incubation with 10μM 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‐Glu(3), ‐Glu(4) and ‐Glu(5)) in the five MTX‐resistant K562 cell lines, while in the parent cells MTX‐Glu(3–5) 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.