Isolation of a gene encoding a human reduced folate carrier (RFC1) and analysis of its expression in transport-deficient, methotrexate-resistant human breast cancer cells

Our laboratory has previously reported the isolation of a murine cDNA which restores reduced folate carrier (RFC) activity and methotrexate (MTX) sensitivity to a MTX-resistant, transport-deficient human breast Cancer cell line (MTXR ZR-75-1) (K. H. Dixon et al., J. Biol. Chem., 269: 17-20, 1994). Using this murine cDNA as a probe, we have isolated two homologous overlapping partial cDNAs from a human testis cDNA library. In addition, using human cDNA as a probe, we have isolated a 20-kb human genomic fragment which contains RFC coding regions. Analysis of the nucleotide sequence of these clones revealed that the human RFC gene, RFC1, is approximately 65% homologous to the murine and hamster genes. Using a human genomic P1 plasmid clone containing RFC1, we mapped the location of RFC1 by fluorescence in situ hybridization to the end of the long arm of chromosome 21 (21q22.2-q22.3). Fluorescence in situ hybridization analysis also showed that two copies of RFC1 were present in MTXR ZR-75-1 cells, and showed no evidence of rearrangement of this gene. Northern blot analysis of MTXR ZR-75-1 cells demonstrated a marked decrease in the level of the 3-kb RFC1 transcript relative to the parental cell line, and Western blot analysis using a polyclonal antibody raised against a peptide generated from the RFC1 sequence showed decreased expression of an approximately M(r) 56,000 protein in MTXR ZR-75-1 cells. Finally, MTXR ZR-75-1 cells transfected with an RFC1 gene showed increased MTX uptake, which was more sensitive to competition by folinic acid than by folic acid. Therefore, decreased RFC1 expression appears to be the molecular mechanism of decreased MTX uptake in this MTX-resistant cell line.