CHARACTERIZATION OF CROSS-RESISTANCE TO METHOTREXATE IN A HUMAN BREAST-CANCER CELL-LINE SELECTED FOR RESISTANCE TO MELPHALAN

We have demonstrated previously decreased melphalan accumulation in a human breast cancer cell line selected for resistance to melphalan (Me lR 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 Me lR 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 res istant cells. Methotrexate resistance in MelR MCF-7 cells was also ass ociated with a decrease in non-effluxable methotrexate following incub ation with radiolabeled drug for 24 hr. Characterization of intracellu lar methotrexate after accumulation for 24 hr demonstrated decreased l evels of free methotrexate in MelR MCF-7 cells. Analysis of methotrexa te polyglutamate formation in MelR MCF-7 cells indicated that the decr ease in non-effluxable, non-protein-bound methotrexate was associated with a 3-fold decrease in higher order methotrexate polyglutamate form ation. No difference was noted in folylpolyglutamate synthetase activi ty between the resistant and parental cell lines. Therefore, the obser ved decrease in methotrexate polyglutamate formation in MelR MCF-7 cel ls appeared to result from decreased availability of substrate. There was no evidence of any alteration in the amount of the catalytic activ ity of dihydrofolate reductase in MelR MCF-7 cells compared with paren tal MCF-7 (WT MCF-7) cells; moreover, the binding affinity of dihydrof olate reductase for methotrexate and the percentage of protein-bound m ethotrexate were similar in both cell lines. In addition, the total am ounts of thymidylate synthase protein and thymidylate synthase catalyt ic activity in MelR MCF-7 cells were unchanged. Thus, acquired methotr exate resistance in MCF-7 cells selected for resistance to melphalan a ppears to result from down-regulation of methotrexate uptake.