Mutations in the reduced folate carrier gene which confer dominant resistance to 5,10-dideazatetrahydrofolate.
L1210/D3 mouse leukemia cells are resistant to 5, 10-dideazatetrahydrofolate due to expansion of cellular folate pools which block polyglutamation of the drug (Tse, A., and Moran, R. G. (1998) J. Biol. Chem. 273, 25944-25952). These cells were found to have two point mutations in the reduced folate carrier (RFC), resulting in a replacement of isoleucine 48 by phenylalanine and of tryptophan 105 by glycine. Each mutation contributes to the resistance phenotype. Genomic DNA from resistant cells contained both the wild-type and mutant alleles, but wild-type message was not detected. Folic acid was a much better substrate, and 5-formyltetrahydrofolate was a poorer substrate for transport in L1210/D3 cells relative to L1210 cells. Enhanced transport of folic acid was due to a marked, approximately 20-fold, decrease in the influx Km. Influx of methotrexate and 5,10-dideazatetrahydrofolate were minimally altered. Transfection of mutated rfc cDNA into RFC-null L1210/A cells produced the substrate specificity and 5, 10-dideazatetrahydrofolate resistance observed in the L1210/D3 line. Transfection of the mutant cDNA into wild-type cells also conferred resistance to 5,10-dideazatetrahydrofolate. We conclude that the I48F and W105G mutations in RFC caused resistance to 5, 10-dideazatetrahydrofolate, that the region of the RFC protein near these two positions defines the substrate-binding site, that the wild-type allele was silenced during the multistep development of resistance, and that this mutant phenotype represents a genetically dominant trait.
Tse
A
A
Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, California 90033, USA.
Brigle
K
K
Taylor
S M
SM
Moran
R G
RG
D000818:Animals; D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics*; D002455:Cell Division / Q000235:genetics; D004252:DNA Mutational Analysis; D004351:Drug Resistance / Q000235:genetics*; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism / Q000493:pharmacokinetics*; D007700:Kinetics; D002955:Leucovorin / Q000378:metabolism; D007939:Leukemia L1210 / Q000235:genetics; D008727:Methotrexate / Q000378:metabolism; D051379:Mice; D010641:Phenotype; D017354:Point Mutation / Q000235:genetics; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface; D013763:Tetrahydrofolates / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011624:Pteroylpolyglutamic Acids; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D013763:Tetrahydrofolates; C045894:lometrexol; D005492:Folic Acid; D002955:Leucovorin; D008727:Methotrexate