Pattern of Mutations that Results in Loss of Reduced Folate Carrier Function under Antifolate Selective Pressure Augmented by Chemical Mutagenesis

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 transcriptionpolymerase 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.

Resistance to antifolates

“…The first TMD appears to be a favored locus of mutations in acquired resistance to antifolates, further supported by the mutations at this site in a variety of CCRF-CEM cell lines selected for resistance to antifolates in the absence of a mutagen (Jansen et al, 1998;Drori et al, 2000;Rothem et al, 2002). Beyond single point mutations within the open reading frame, a variety of other mutations have been detected in MTX-resistant cell lines including mutation of the ATG start codon, insertions and frameshifts, truncated proteins, deletions, mutations resulting in RFC instability, and loss of RFC alleles due to translocations (Wong et al, 1999;Zhao et al, 1999b;Ding et al, 2001;Rothem et al, 2002).…”

“…The study provided an important insight into the critical role that natural folates within cells can play in modulating the activity of antifolates, in particular those that require polyglutamation for activity. Figure 4 illustrates the spectrum of mutations that accompanied murine L1210 leukemia cell resistance to MTX due to impaired RFC function following selection with this drug with chemical mutagenesis (Zhao et al, 1999b). It can be seen that virtually all the mutations were located in, or at the boundaries of, TMDs.…”

Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells

“…9,11,14 A large number of RFC gene mutations leading to an antifolate-resistant phenotype have been described in rodent and human cell lines. [15][16][17][18][19][20][21] Although mutations have been identified throughout the RFC coding region, they appear to be disproportionately clustered within the first putative transmembrane domain (eg G44E, E45K, S46N, I48F). [16][17][18][19][20]22 Of particular interest is E45K, originally identified in MTX-resistant murine cells 19 and, subsequently, in separate MTX-resistant CCRF-CEM human T-cell ALL sublines from our own 23 and another laboratory.…”

Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function

“…The approach used was chemical mutagenesis followed by selective pressure with dihydrofolate reductase (DHFR) inhibitors, a technique applied in this and other laboratories (9,35,40). The agents used were MTX, a classical antifolate that forms polyglutamate derivatives in cells, and 5,8-dideaza-N ␣ -(-4-amino-4-deoxypteroyl)-N ␦ -hemiphthaloyl-1-ornithine (PT632), a novel antifolate with 10-fold higher affinity for DHFR and RFC that does not form polyglutamate derivatives (34) and has very low affinity for low-pH transporters in several tumor cell lines (32).…”