Resistance to multiple novel antifolates is mediated via defective drug transport resulting from clustered mutations in the reduced folate carrier gene in human leukaemia cell lines

Rothem, Lilah; Ifergan, Ilan; Kaufman, Yotam; Priest, David G.; Jansen, Gerrit; Assaraf, Yehuda G.

doi:10.1042/bj20020801

Cited by 65 publications

(110 citation statements)

References 38 publications

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“…First, the efficacy of MTX was progressively reduced when it was combined with increasing concentrations of SSZ. In fact, the levels of loss of MTX efficacy at high concentrations of SSZ (Ͼ400-fold in the presence of 2.5 mM SSZ) were similar to those demonstrated in cells displaying loss of MTX efficacy due to decreased expression of the RFC protein (28,38,39,56). In this respect, the decreased expression of RFC (up to 8-fold) in CEM/SSZ cells (Figure 4) also could have contributed to a diminished efficacy of MTX, but data shown in Figures 1A and B did not reveal a difference between the MTX efficacy in CEM/SSZ cells and that in CEM cells.…”

Section: Discussionsupporting

confidence: 57%

“…The blots were reacted with a polyclonal antiserum (1:700) prepared in mice against a C-terminal human RFC peptide. Blots were then rinsed in the same buffer for 10 minutes at room temperature and reacted with horseradish peroxidase-conjugated goat anti-mouse IgG (1:40,000 dilution; Jackson ImmunoResearch, West Grove, PA) for 1 hour at room temperature (38). Following three 10-minute washes in TBS at room temperature, enhanced chemiluminescence detection was performed according to the manufacturer's instructions (Biological Industries, Kibbutz Beit Haemek, Israel).…”

Section: Methodsmentioning

confidence: 99%

“…Given their anionic nature, natural folates and MTX cannot traverse the plasma membrane; therefore, RFC-mediated cellular uptake should be regarded as the first critical step in any antiinflammatory/antiproliferative mode of action of MTX (33)(34)(35)(36). Consequently, diminished transport activity of RFC has been associated with loss of MTX efficacy and with folate deficiency (31,(37)(38)(39)(40).…”

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confidence: 99%

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Sulfasalazine is a potent inhibitor of the reduced folate carrier: Implications for combination therapies with methotrexate in rheumatoid arthritis

Jansen¹,

Heijden²,

Oerlemans³

et al. 2004

Arthritis & Rheumatism

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Objective. To investigate whether interactions of sulfasalazine (SSZ) with reduced folate carrier (RFC), the dominant cell membrane transporter for natural folates and methotrexate (MTX), may limit the efficacy of combination therapy with MTX and SSZ in patients with rheumatoid arthritis. Conclusion. At clinically relevant plasma concentrations, interactions of SSZ with RFC provide a biochemical rationale for 2 important clinical observations: 1) the onset of (sub)clinical folate deficiency during SSZ treatment, and 2) the lack of additivity/synergism of the combination of SSZ and MTX when these diseasemodifying antirheumatic drugs are administered simultaneously. Thus, when considering use of these drugs in combination therapies, the present results provide a rationale both for the use of folate supplementation and for spacing administration of these drugs over time.

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Section: Discussionsupporting

confidence: 57%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Sulfasalazine is a potent inhibitor of the reduced folate carrier: Implications for combination therapies with methotrexate in rheumatoid arthritis

Jansen¹,

Heijden²,

Oerlemans³

et al. 2004

Arthritis & Rheumatism

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“…The truncated protein alone is unable to transport MTX and is correctly considered non-functional. 13 The decreased affinity of RFC protein to MTX may be brought 15,17 In the present study, the influence of RFC-1 gene (G80A) polymorphism on treatment outcome with MTX of patients with RA was examined. The patients with RFC-1 AA genotype responded to the therapy more effectively than carriers of AG and GG genotypes.…”

Section: Discussionmentioning

confidence: 89%

Reduced folate carrier-1 80G>A polymorphism affects methotrexate treatment outcome in rheumatoid arthritis

et al. 2007

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The folate antagonist methotrexate (MTX) is a drug currently used in the treatment of rheumatoid arthritis (RA). MTX enters the cells through the reduced folate carrier (RFC-1) and is activated to polyglutamates. Previous studies have shown that RFC-1 expression may influence the efficacy of therapy with MTX. The studies suggest that G80A polymorphism in RFC-1 is associated with altered folate/antifolate levels and the subjects carrying homozygous mutant 80AA genotype tend to have higher plasma folate and MTX concentrations and higher erythrocyte polyglutamate levels compared with those with the wild type or heterozygous genotype. It is possible that this polymorphism might influence MTX treatment outcome in patients with RA. In the present study, we examined the association between RFC-1 G80A polymorphism and treatment outcome in patients with RA administered MTX. The study was carried out on 174 patients diagnosed with RA treated with MTX (7.5-15.0 mg weekly) plus low doses of methylprednisone. The RFC-1 80G4A polymorphism (resulting in a histidine-to-arginine substitution at codon 27 of RFC-1) was detected using a polymerase chain reactionrestriction fragment length polymorphism method. The probability of remission of RA symptoms was 3.32-fold higher in carriers of 80AA genotype as compared with patients with 80GG genotype (P ¼ 0.021, OR ¼ 3.32,). The frequency of A allele among MTX responders was 62.1, compared to 47.8% in a group of poor MTX responders (P ¼ 0.013, OR ¼ 1.78, 95% CI: 1.13-2.81). Moreover, the increase of aminotransferase activity was noted more frequently in carriers of 80AA genotype. The present data suggest that evaluation of RFC-1 gene 80G4A polymorphism may be a useful tool to optimize MTX therapy in patients with RA.

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“…Expression of the RFC/SLC19A1 gene was down-regulated more than 2-fold in the MTXresistant cells, thereby confirming the role of this influx transporter as an important determinant of antifolate resistance [18][19][20][21] . However, the major finding of the present investigation is that the dramatic loss of FPGS activity in the 7-OHMTX cells, presumably associated with decreased intracellular levels of folate, was accompanied by consistent alterations in the expression of genes capable of preserving intracellular pools of reduced folate and/or increase purine nucleotide biosynthesis.…”

Section: Corroboration Of Array Expression Levels By Qrt-pcrmentioning

confidence: 60%

Gene expression profiling of leukemia T-cells resistant to methotrexate and 7-hydroxymethotrexate reveals alterations that preserve intracellular levels of folate and nucleotide biosynthesis

Fotoohi¹,

Assaraf²,

Moshfegh³

et al. 2009

Biochemical Pharmacology

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International audiencetreatment of human T-cell leukemia cells with 7-hydroxymethotrexate, the major metabolite of methotrexate resulted in acquired resistance as a result of the complete loss of folypolyglutamate synthetase (FPGS) activity. This was in contradistinction to the major modality of antifolate resistance of impaired drug transport in leukemia cells exposed to methotrexate.. To identify the genes associated with methotrexate and 7-hydroxymethotrexate resistance, we herein explored the patterns of genome-wide expression profiles in these antifolte-resistant leukemia sublines. mRNA levels of the reduced folate carrier, the primary influx transporter of folates and antifolates, were down-regulated >2-fold in methotrexate -resistant cells. The dramatic loss of FPGS activity in 7-hydroxymethotrexate - resistant cells was associated with alterations in the expression of various genes aimed at preserving reduced folates and/or enhancing purine nucleotide biosynthesis e.g. methylene tetrahydrofolate reductase, glycinamide ribonucleotide formyltransferase, adenosine deaminase, cystathionine β synthase, as well as the ATP-dependent folate exporters / and /. The observed changes in gene expression were generally not paralleled by acquired DNA copy numbers alterations, suggesting transcriptional regulatory mechanisms. Interestingly, gene expression of DNA/RNA metabolism and transport genes were more profoundly altered in methotrexate -resistant subline, whereas in 7-hydroxymethotrexate -resistant cells, the most profoundly affected groups of genes were those encoding for proteins involved in metabolism and cellular proliferation. Thus, the present investigation provides evidence that 7-hydroxymethotrexate induces gene expression alterations and an antifolate resistance modality that are distinct from its parent drug methotrexate

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Resistance to multiple novel antifolates is mediated via defective drug transport resulting from clustered mutations in the reduced folate carrier gene in human leukaemia cell lines

Cited by 65 publications

References 38 publications

Sulfasalazine is a potent inhibitor of the reduced folate carrier: Implications for combination therapies with methotrexate in rheumatoid arthritis

Sulfasalazine is a potent inhibitor of the reduced folate carrier: Implications for combination therapies with methotrexate in rheumatoid arthritis

Reduced folate carrier-1 80G>A polymorphism affects methotrexate treatment outcome in rheumatoid arthritis

Gene expression profiling of leukemia T-cells resistant to methotrexate and 7-hydroxymethotrexate reveals alterations that preserve intracellular levels of folate and nucleotide biosynthesis

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