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.
Koizumi
S
S
Department of Pediatrics, Kanazawa University School of Medicine.
D004351:Drug Resistance; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism / Q000473:pathology; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000493:pharmacokinetics / Q000494:pharmacology*; D010452:Peptide Biosynthesis; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000096:biosynthesis*; D011687:Purines / Q000096:biosynthesis; D014407:Tumor Cells, Cultured / Q000187:drug effects
D011687:Purines; D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; C030985:purine; D008727:Methotrexate