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Vol. 56, Issue 1, 68-76, July 1999
Departments of Medicine and Molecular Pharmacology, and the Albert
Einstein Comprehensive Cancer Center, Albert Einstein College of
Medicine, Bronx, New York
Chemical mutagenesis with
N-methyl-N-nitrosourea was employed to
study the pattern of mutations in the reduced folate carrier (RFC1)
that results in transport-related methotrexate resistance and to
identify amino acid residues that are critical to carrier structure
and/or function. Thirty-four methotrexate transport-defective L1210
leukemia cell lines were isolated with folic 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 basis for the
transport defects was investigated by sequencing multiple RFC1 cDNA
clones isolated from these mutants by reverse transcription-polymerase
chain reaction, which encompassed the entire coding region. The
mutations identified were further confirmed either by direct sequencing
or, when applicable, 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 domains, 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. These data
suggest that several transmembrane domains, rather than the amino- and
carboxyl-termini, and the large intracellular loop between the sixth
and seventh transmembrane domains play key roles as sites for
RFC1 inactivation because of single point mutations. This panel of
mutated cell lines offers an important resource for studies on RFC1
structure-function and for the evaluation of transport-related
cross-resistance patterns with new-generation antifolate inhibitors of
tetrahydrofolate cofactor-dependent enzymes.
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