Distinguishing between folate receptor-α-mediated transport and reduced folate carrier-mediated transport in L1210 leukemia cells

Abstract

L1210 leukemia cells transport reduced folates and methotrexate via a well defined reduced folate carrier system and, in the absence of low folate selective pressure, do not express an alternate endocytotic route mediated by cell surface folate receptors. This laboratory previously described an L1210 leukemia cell line, MTX(r)A, with acquired resistance to methotrexate (MTX) due to the loss of mobility of the reduced folate carrier. We now report on the transfection of MTX(r)A with a cDNA encoding the murine homolog of the human folate receptor isoform of KB cells to produce MTX(r)A-TF1, which constitutively expresses high levels of FR-α. MTX(r)A-TF1 and L1210 cells were utilized to compare transport of methotrexate mediated by FR-α and the reduced folate carrier, respectively. Methotrexate influx in the two lines was similar when the extracellular level was 0.1 μM, but as the methotrexate concentration increased, influx via the reduced folate carrier increased in comparison to influx mediated by FR-α. Transport kinetics indicated both a ~20-fold lower influx K(b) and V(max) for MTX(r)A-TF1 as compared to L1210 cells. The two cell lines exhibited distinct influx properties. Methotrexate influx in MTX(r)A-TF1 was markedly inhibited by 50 nM folic acid and metabolic poisons. In L1210 cells, 1.0 μM folic acid did not affect MTX influx, and metabolic poisons either had no effect on or increased methotrexate influx. Removal of extracellular chloride markedly inhibited transport in MTX(r)A-TF1 but stimulated influx in L1210 cells. When the pH was decreased to 6.2, methotrexate influx was not altered in MTX(r)A-TF1 but was reduced in L1210 cells. Probenecid and sulfobromophthalein inhibit methotrexate influx in both L1210 and MTX(r)A-TF1 cell lines; however, inhibition in MTX(r)A-TF1 could be accounted for on the basis of inhibition of methotrexate binding to FR-α. The data indicate that the reduced folate carrier and FR-α function independently and exhibit distinct properties. FR- α expressed at sufficient levels can mediate influx of MTX and folates into cells at rates comparable to the reduced folate carrier and hence has pharmacologic and physiologic importance.