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, MTXA, with acquired resistance to methotrexate (MTX) due to the loss of mobility of the reduced folate carrier. We now report on the transfection of MTXA with a cDNA encoding the murine homolog of the human folate receptor isoform of KB cells to produce MTXA-TF1, which constitutively expresses high levels of FR-. MTXA-TF1 and L1210 cells were utilized to compare transport of methotrexate mediated by FR- 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-. Transport kinetics indicated both a 20-fold lower influx K and V for MTXA-TF1 as compared to L1210 cells. The two cell lines exhibited distinct influx properties. Methotrexate influx in MTXA-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 MTXA-TF1 but stimulated influx in L1210 cells. When the pH was decreased to 6.2, methotrexate influx was not altered in MTXA-TF1 but was reduced in L1210 cells. Probenecid and sulfobromophthalein inhibit methotrexate influx in both L1210 and MTXA-TF1 cell lines; however, inhibition in MTXA-TF1 could be accounted for on the basis of inhibition of methotrexate binding to FR-. The data indicate that the reduced folate carrier and FR- function independently and exhibit distinct properties. FR- 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.

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