title: Enzyme studies of methotrexate-resistant human leukemic cell (K562) subclones.
abstract: Five methotrexate (MTX)-resistant K562 cell subclones (K562/MTX-1 approximately -5) were established and were examined for mechanisms of drug resistance. Impairment of MTX-polyglutamate formation, with membrane transport alteration, in the resistant cells was demonstrated in the previous studies (Koizumi, S. (1988) Jpn. J. Cancer Res. 79, 1230). Further analysis of sensitivity of the cells to trimetrexate (TMQ), which is not polyglutamated and does not require the reduced folate transporter, but is a potent inhibitor of human DHFR, revealed a modest decrease in sensitivity to TMQ (2.4- to 15-fold). Enzyme studies showed that the dihydrofolate reductase (DHFR) activities of these resistant subclones were very similar to that of the parent cells. The number of binding sites of these subclones for MTX calculated from Scatchard analysis was increased up to 7-fold in the K562/MTX-1 and -4 subclones and up to 3-fold in the other 3 subclones as compared to the parent cells. KD values of MTX for the DHFR in the K562/MTX-1 and -4 subclones also appeared to be altered relative to the parent cell line. Further, thymidylate synthase (TS) activity of the resistant subclones was reduced to 50% in K562/MTX-1 and -4 cells, and to 11-25% in the other subclones as compared to the parent cell line. These findings suggest that antifolate resistance in the newly established K562/MTX subclones in multifactorial with polyglutamation and transport defects accounting for the majority of resistance to MTX, and that alteration in the binding affinity of DHFR for MTX and diminished levels of TS may contribute to the 'residual' drug resistance to TMQ and have importance with respect to MTX.
authors.affiliation: Department of Pediatrics, Kanazawa University School of Medicine, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Koizumi
authors.forename: C J
authors.initials: CJ
authors.lastname: Allegra
mesh_terms: D001665:Binding Sites; D001692:Biological Transport; D004351:Drug Resistance; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D008727:Methotrexate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013940:Thymidylate Synthase / Q000378:metabolism; D016597:Trimetrexate / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism
chemical_list: D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D016597:Trimetrexate; D008727:Methotrexate