DISTINGUISHING BETWEEN FOLATE RECEPTOR-ALPHA-MEDIATED TRANSPORT AND REDUCED FOLATE CARRIER-MEDIATED TRANSPORT IN L1210 LEUKEMIA-CELLS

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 rou te mediated by cell surface folate receptors. This laboratory previous ly described an L1210 leukemia cell line, MTX(r)A with acquired resist ance 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 cD NA 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-alpha. MTX(r)A-TF1 and L1210 cells were utilized to compa re transport of methotrexate mediated by FR-alpha and the reduced fola te carrier, respectively. Methotrexate influx in the two lines was sim ilar when the extracellular level was 0.1 mu M, but as the methotrexat e concentration increased, influx via the reduced folate carrier incre ased in comparison to influx mediated by FR-alpha. Transport kinetics indicated both a similar to 20-fold lower influx K-b and V-max for MTX (r)A-TF1 as compared to L1210 cells. The two cell Lines exhibited dist inct influx properties. Methotrexate influx in MTX(r)A-TF1 was markedl y inhibited by 50 mu M folic acid and metabolic poisons. In L1210 cell s, 1.0 mu M folic acid did not affect MTX influx, and metabolic poison s either had no effect on or increased methotrexate influx. Removal of extracellular chloride markedly inhibited transport in MTX(r)A-TF1 bu t 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 i nflux 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 meth otrexate binding to FR-alpha. The data indicate that the reduced folat e carrier and FR-alpha function independently and exhibit distinct pro perties. FR-alpha 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.