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 (MTX(R) ZR-75-1) (K, H, Dixon et al,, J, Biol, Chem, , 269: 17-20, 1994), Using this murine cDNA as a probe, we have isolat ed two homologous overlapping partial cDNAs from a human testis cDNA l ibrary, In addition, using human cDNA as a probe, we have isolated a 2 0-kb human genomic fragment which contains RFC coding regions, Analysi s of the nucleotide sequence of these clones revealed that the human R FC gene, RFC1, is approximately 65% homologous to the murine and hamst er genes, Using a human genomic Pr plasmid clone containing RFC1, we m apped the location of RFC1 by fluorescence in situ hybridization to th e end of the long arm of chromosome 21 (21q22.2-q22.3). Fluorescence i n situ hybridization analysis also showed that two copies of RFC1 were present in MTX(R) ZR-75-1 cells, and showed no evidence of rearrangem ent of this gene, Northern blot analysis of MTX(R) ZR-75-1 cells demon strated a marked decrease in the level of the 3-kb RFC1 transcript rel ative to the parental cell line, and Western blot analysis using a pol yclonal antibody raised against a peptide generated from the RFC1 sequ ence showed decreased expression of an approximately M(r) 56,000 prote in in MTX(R) ZR-75-1 cells. Finally, MTX(R) ZR-75-1 cells transfected with an RFC1 gene showed increased MTX uptake, which was more sensitiv e to competition by folinic acid than by folic acid, Therefore, decrea sed RFC1 expression appears to be the molecular mechanism of decreased MTX uptake in this MTX-resistant cell line.