Abstract
We have demonstrated previously decreased melphalan accumulation in a human breast cancer cell line selected for resistance to melphalan (MelR MCF-7). Cross-resistance studies of MelR MCF-7 cells revealed that this cell line was 6.7-fold cross-resistant to methotrexate, but only 2-fold resistant to trimetrexate. Methotrexate transport studies in MelR MCF-7 cells showed a 2-fold decrease in initial methotrexate uptake and a 2-fold decrease in the V max for methotrexate uptake in the resistant cells. Methotrexate resistance in MelR MCF-7 cells was also associated with a decrease in non-effluxable methotrexate following incubation with radiolabeled drug for 24 hr. Characterization of intracellular methotrexate after accumulation for 24 hr demonstrated decreased levels of free methotrexate in MelR MCF-7 cells. Analysis of methotrexate polyglutamate formation in MelR MCF-7 cells indicated that the decrease in non-effluxable, non-protein-bound methotrexate was associated with a 3-fold decrease in higher order methotrexate polyglutamate formation. No difference was noted in folylpolyglutamate synthetase activity between the resistant and parental cell lines. Therefore, the observed decrease in methotrexate polyglutamate formation in MelR MCF-7 cells appeared to result from decreased availability of substrate. There was no evidence of any alteration in the amount of the catalytic activity of dihydrofolate reductase in MelR MCF-7 cells compared with parental MCF-7 (WT MCF-7) cells; moreover, the binding affinity of dihydrofolate reductase for methotrexate and the percentage of protein-bound methotrexate were similar in both cell lines. In addition, the total amounts of thymidylate synthase protein and thymidylate synthase catalytic activity in MelR MCF-7 cells were unchanged. Thus, acquired methotrexate resistance in MCF-7 cells selected for resistance to melphalan appears to result from down-regulation of methotrexate uptake.
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