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Enzyme studies of methotrexate-resistant human leukemic cell (K562) subclones
1Department of Pediatrics, Kanazawa University School of Medicine, Japan.
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.
Insights
Methotrexate resistance in K562 cells involves multiple factors, including impaired drug metabolism and transport. Altered dihydrofolate reductase binding and reduced thymidylate synthase also contribute to antifolate resistance.
Area of Science:
- Pharmacology
- Cancer Biology
- Biochemistry
Background:
- Methotrexate (MTX) is a key antifolate chemotherapy agent.
- Drug resistance mechanisms are crucial for understanding treatment efficacy.
- Previous studies indicated impaired MTX-polyglutamate formation and altered membrane transport in resistant K562 cells.
Purpose of the Study:
- To investigate the multifactorial mechanisms of methotrexate resistance in K562 cell subclones.
- To analyze the role of dihydrofolate reductase (DHFR) activity and binding affinity.
- To assess the contribution of thymidylate synthase (TS) activity to drug resistance.
Main Methods:
- Establishment of five MTX-resistant K562 cell subclones.
- Sensitivity testing with trimetrexate (TMQ), a DHFR inhibitor.
- Enzyme activity assays for DHFR and TS.
- Scatchard analysis to determine MTX binding sites and affinity for DHFR.
Main Results:
- Resistant cells showed modest decreased sensitivity to TMQ.
- DHFR activity remained similar to parent cells, but MTX binding sites increased up to 7-fold.
- MTX binding affinity (KD) for DHFR was altered in resistant subclones.
- TS activity was reduced by 50-89% in resistant subclones.
Conclusions:
- MTX resistance in K562 cells is multifactorial, primarily due to polyglutamation and transport defects.
- Altered DHFR binding affinity and diminished TS levels contribute to residual resistance, particularly to TMQ.
- These mechanisms are important for understanding MTX resistance in cancer therapy.