This laboratory previously described an
L1210 leukemia cell line (MTXrA) selected for resistance to
methotrexate by virtue of impaired transport due to a functional defect in the translocation process. We now report on the sequence analysis of cDNAs encoding the
reduced folate carrier from this line and identify a single mutation that results in the substitution of a
proline for an
alanine in a highly conserved transmembrane region of the
protein. Transfection of the parental
reduced folate carrier into MTXrA cells resulted in a cell line which exhibited a complete restoration of
methotrexate uptake and an enhanced sensitivity to
methotrexate. Northern analysis and specific [3H]MTX cell surface binding indicated that expression of the
reduced folate carrier was elevated approximately 5-fold in the transfectant compared to parental and MTXrA cells. The MTX influx properties of the transfectant cell line were identical to those of the well characterized
reduced folate carrier from parental L1210 cells in terms of: 1) patterns of sensitivity to competing folates, 2) sensitivity to the organic
anion sulfobromophthalein, 3) lack of energy dependence, and 4) capacity for trans-stimulation. We also provide new data which suggests that the nucleotide sequence 5' of the predicted ATG
initiation codon may encode additional
protein information in the form of a leader sequence. Finally, we demonstrate that the MTXrA line has both the mutant and the parental
reduced folate carrier alleles but that expression appears to be restricted to the mutant allele. Thus, the
methotrexate transport phenotype and resultant drug resistance in this cell line result from genetic/regulatory events at both alleles.