Folate Transport and the Modulation of Antifolate Sensitivity in a Methotrexate-Resistant Human Breast Cancer Cell Line

Abstract

The mechanism of acquired methotrexate-resistance in an estrogen-receptor positive human breast cancer cell line (MTX(R)ZR-75-1) was studied. MTX(R) ZR-75-1 cells are 250-fold resistant to methotrexate when grown in the presence of 1 microM folinic acid and 2,400-fold resistant in the presence of 1 microM folic acid. This drug resistant cell line also showed collateral sensitivity (10-fold) to trimetrexate (TMQ), when grown in the presence of folinic acid. Using fluoresceinated methotrexate (F-MTX), FACS analysis indicated that there is no intracellular accumulation of methotrexate into MTX(R) ZR-75-1 cells, as determined by competition of F-MTX and methotrexate binding to dihydrofolate reductase. These characteristics strongly indicate that the mechanism of resistance involved down regulation of the reduced-folate transporter. To investigate this further, the transport kinetics of parental and MTX(R) ZR-75-1 cells were examined. Although the V(max) for methotrexate transport in wild-type (WT) ZR-75-1 breast cancer cells was 1-2 orders of magnitude lower than that in the well characterized leukemia cell lines, such as L1210 and CCRF-CEM cells, kinetic analysis indicated that transport of methotrexate into WT ZR-75-1 cells involved a mechanism that was similar if not identical to the reduced folate transporter. In contrast, no specific uptake of methotrexate was detected in MTX(R) ZR-75-1cells. Furthermore, neither cell line expressed detectable levels of folate binding protein, a binding protein with high affinity for folic acid as well as for reduced folates and antifolates. These results indicate that the level of expression of the reduced-folate carrier may be an important factor in determining the sensitivity of breast cancer cells as well as leukemia cells to antifolate compounds.