Pattern of mutations that results in loss of reduced folate carrier function under antifolate selective pressure augmented by chemical mutagenesis

Chemical mutagenesis with N-methyl-N-nitrosourea was employed to study the pattern of mutations in the reduced folate carrier (RFC1) that results in t ransport-related methotrexate resistance and to identify amino acid residue s that ave critical to carrier structure and/or function. Thirty-four metho trexate transport-defective L1210 leukemia cell lines were isolated with fo lic acid as the sole folate source under antifolate selective pressure. The RFC1 mRNA levels were comparable with, or not substantially decreased, in most of these cell lines relative to wild-type L1210 cells. The molecular b asis for the transport defects was investigated by sequencing multiple RFC1 cDNA clones isolated from these mutants by reverse transcription-polymeras e chain reaction, which encompassed the entire coding region. The mutations identified were further confirmed either by direct sequencing or, when app licable, by restriction analysis of total reverse transcription-polymerase chain reaction products. The majority of mutations(21) led to single amino acid substitutions that were in, or near, 9 of 12 predicted transmembrane d omains, with the highest frequencies in the first, fifth, and eighth. There were no mutations in the sixth, ninth, and twelfth transmembrane domains. Glycine, serine, and arginine were the most frequently mutated residues. Th ese data suggest that several transmembrane domains, rather than the amino- and carboxyl-termini, and the large intracellular loop between the sixth a nd seventh transmembrane domains play key roles as sites for RFC1 inactivat ion because ol:single point mutations. This panel of mutated cell lines off ers an important resource for studies on RFC1 structure-function and for th e evaluation of transport-related cross-resistance patterns with new-genera tion antifolate inhibitors of tetrahydrofolate cofactor-dependent enzymes.