Isolation of Human cDNAs That Restore Methotrexate Sensitivity and Reduced Folate Carrier Activity in Methotrexate Transport-defective Chinese Hamster Ovary Cells

Wong, So C.; Proefke, Susan A.; Bhushan, Alok; Matherly, Larry H.

doi:10.1074/jbc.270.29.17468

Cited by 178 publications

(216 citation statements)

References 31 publications

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“…Resistance to antifolates R Zhao and ID Goldman (Dixon et al, 1994;Williams et al, 1994;Moscow et al, 1995;Prasad et al, 1995;Williams and Flintoff, 1995;Wong et al, 1995). The validity of this secondary structure has been strengthened by hemaglutatinin epitope insertion analysis (Ferguson and Flintoff, 1999) although there remains a question regarding the topology of TMDs 9-12 (Liu and Matherly, 2002).…”

Section: The Reduced Folate Carriermentioning

confidence: 99%

Resistance to antifolates

2003

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Section: The Reduced Folate Carriermentioning

confidence: 99%

Resistance to antifolates

2003

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“…Wildtype hRFC cDNA (KS43) 13 was excised from pcDNA3 and directionally ligated into pALTER-1 mutagenesis vector using KpnI and XbaI, the resultant construct designated pALT/RFC. Phosphorylated mutant sense oligonucleotide (5Ј-ACGGCCAGGGAAGAGCTTCA-3Ј) was annealed to alkaline denatured pALT/RFC, followed by mutant strand synthesis.…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

“…CEM/MTX R0 cells were transfected with either an empty-vector control, pcDNA3, the same vector containing wild-type hRFC cDNA, 13 or cDNA containing the E45K mutation. Logarithmically growing cells were harvested and resuspended in RPMI 1640 supplemented with 10% FCS (3 × 10 6 cells per 600 l medium) followed by addition of linearized plasmid DNA (50 or 100 g) and transferred to 0.4 cm electroporation cuvettes.…”

Section: Electroporationmentioning

confidence: 99%

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

Gifford

Haber

et al. 2002

Leukemia

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Collectively, the results demonstrate that expression of E45K hRFC leads to increased MTX resistance due to decreased membrane transport and, secondarily, from alterations in binding affinities and transport of folate substrates. However, despite these findings, we could find no evidence of this mutation in 121 childhood ALL samples, suggesting that it does not contribute to clinical MTX resistance in this disease.

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“…Different cDNAs and genomic clones for the reduced folate carrier, which has a high affinity for the reduced folates 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and methotrexate, but a low affinity for folate [20][21][22][23][24] have been also isolated from different immortal cell lines [25][26][27][28][29][30][31] but the clones were not characterized in detail.…”

mentioning

confidence: 99%

Cloning and functional characterization of the bile acid-sensitive methotrexate carrier from rat liver cells

Honscha¹,

Dötsch²,

Thomsen³

et al. 2000

Hepatology

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The antifolates methotrexate and trimetrexate are potent inhibitors of the dihydrofolate reductase, an enzyme that delivers by the metabolism of folate and its reduced forms the C1-groups for the synthesis of nucleotides and amino acids. Methotrexate, which has a comparatively moderate toxicity is one of the most important clinically used chemotherapeutics for the treatment of leukemias, such as acute lymphoblastic leukemia of children. It is also used in breast cancer, squamous tumors of the head and neck, lymphomas, and choriocarcinomas. 1,2 Uptake studies of methotrexate and the natural folic acid derivatives in different tumor and normal cells have shown that at least two different carrier systems mediate the uptake of these compounds. The 40 kd folate binding protein has a high affinity for folic acid but a low affinity for reduced folates. [3][4][5][6][7][8][9][10] The corresponding complementary DNAs (cDNAs) as well as genomic clones have been isolated from various sources, 11-19 but a detailed characterization of the transporter function is still missing.Different cDNAs and genomic clones for the reduced folate carrier, which has a high affinity for the reduced folates 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and methotrexate, but a low affinity for folate [20][21][22][23][24] have been also isolated from different immortal cell lines 25-31 but the clones were not characterized in detail.The expression and the abundance of the two carriers varied in different tumor and normal cells. From uptake studies it became evident that, first, in some cell systems both transport systems work in tandem 32 whereas two independently operating carrier systems were also described. 33,34 Second, tumor cells differ from normal cells in their transport properties. [35][36][37][38][39] Results from Chello et al. 40 showed, for example, a 30-fold higher affinity for methotrexate in the mouse leukemia cell line L1210 than in freshly isolated murine intestinal cells.Hepatocytes are exceptional with respect to folate transport. In contrast to most other cells, hepatocytes express a separate carrier system for 5-methyltetrahydrofolate, which is different from the reduced folate transporter. 41,42 We recently characterized the uptake of methotrexate into freshly isolated hepatocytes 43 and showed that this chemotherapeutic is taken up by a sodium-dependent active transport system, which is inhibited by the reduced folates, dihydrofolate, and tetrahydrofolate but not by folate itself. Moreover, substrates of two well-known organic anion transport systems in the liver, the Ntcp1 44,45 and the oatp1, 46 like the bile acids taurocholate and cholate as well as xenobiotics such as bromosulfophthalein, the loop diuretic bumetanide, and the mycotoxin ochratoxin A are potent inhibitors of the hepatocellular methotrexate carrier. Because some substrates were Abbreviations: cDNA, complementary DNA; RL-Mtx, rat liver methotrexate transporter; RACE-PCR, rapid amplification of cDNA ends polymerase chain reaction; DMEM, Dulbecco modi...

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Isolation of Human cDNAs That Restore Methotrexate Sensitivity and Reduced Folate Carrier Activity in Methotrexate Transport-defective Chinese Hamster Ovary Cells

Cited by 178 publications

References 31 publications

Resistance to antifolates

Resistance to antifolates

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

Cloning and functional characterization of the bile acid-sensitive methotrexate carrier from rat liver cells

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