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

Rongbao Zhao, Iraida G. Sharina, I. David Goldman

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)68-76
Number of pages9
JournalMolecular Pharmacology
Volume56
Issue number1
StatePublished - 1999

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Reduced Folate Carrier Protein
Folic Acid Antagonists
Mutagenesis
Mutation
Folic Acid
Methotrexate
Cell Line
Reverse Transcription
Methylnitrosourea
Leukemia L1210
Polymerase Chain Reaction
Coenzymes
Amino Acid Substitution
Point Mutation
Glycine
Serine
Arginine
Complementary DNA
Clone Cells
Amino Acids

ASJC Scopus subject areas

  • Pharmacology

Cite this

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abstract = "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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N2 - 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.

AB - 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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