Mutations in the reduced folate carrier gene which confer dominant resistance to 5,10-dideazatetrahydrofolate

Abstract

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, 2594425952). 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, ≃20-fold, decrease in the influx K(m). Influx of methotrexate and 5,10-dideazatetrahydrofolate were minimally altered. Transfection of mutated rfc eDNA 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.