Isolation of a Human cDNA That Complements a Mutant Hamster Cell Defective in Methotrexate Uptake

Williams, Frederick M.; Flintoff, Wayne F.

doi:10.1074/jbc.270.7.2987

Cited by 117 publications

(71 citation statements)

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“…From the published DNA sequences for a methotrexate transporter from hamster and human 25,30 two degenerated primers and primers with homology to the pMtx9 were selected for the PCR of the 5Ј and the 3Ј end, respectively. By Southern blot analysis with the radiolabeled cDNA of the clone pMtx9 obtained from Prof. W. Flintoff, Department of Microbiology and Immunology, University of Western Ontario, London, Canada, it was verified that the DNA fragments show homology to the published methotrexate carrier sequences.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

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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“…In the first approach, based on that described by Canfield and Levenson (34), the cDNA was digested at unique restriction endonuclease sites (ApaI, 254; NotI, 883; CpoI, 1374; StuI, 1570; numbering based on the human rfc cDNA reported in Ref. 16) predicted to be in intra-or extracellular loops. These were blunt-ended using either nuclease S1 or the Klenow fragment of DNA polymerase I, as appropriate to maintain reading frame, and treated with calf intestinal alkaline phosphatase.…”

Section: Chemicals-restrictionmentioning

confidence: 99%

“…Recently, the cytoplasmic location of the carboxyl-terminal tail has been confirmed for the human RFC (22). The mouse and hamster homologues (518 amino acids each) have significantly shorter carboxyl-terminal tails than the human RFC (15)(16)(17)(18)(19)(20), although all three RFCs share about 68% identical or similar amino acid residues. The human RFC is glycosylated at an asparagine residue in the first extracellular loop (9,22,23), and while the hamster RFC contains the conserved sequence in this loop (15), its glycosylation status has not been confirmed.…”

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Topological and Functional Analysis of the Human Reduced Folate Carrier by Hemagglutinin Epitope Insertion

Ferguson¹,

Flintoff

1999

Journal of Biological Chemistry

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The membrane topology of the human reduced folate carrier protein (591 amino acids) was assessed by single insertions of the hemagglutinin epitope into nine sites of the protein. Reduced folate carrier-deficient Chinese hamster ovary cells expressing each of these constructs were probed with anti-hemagglutinin epitope monoclonal antibodies to assess whether the insertion was exposed to the external environment or to the cytoplasm. The results are consistent with the 12-transmembrane topology predicted for this protein. The hemagglutinin epitope insertion mutants were also tested for their effects on the function of the reduced folate carrier. For these studies, each of the constructs had a carboxyl-terminal fusion of the enhanced green fluorescent protein to monitor and quantitate expression. Insertions into the external loop between transmembrane regions 7 and 8 (Pro-297), the cytoplasmic loop between transmembrane regions 6 and 7 (Ser-225), and near the cytoplasmic amino and carboxyl termini (Pro-20 and Gly-492, respectively) had minor effects on methotrexate binding and uptake. The insertion into the cytoplasmic loop between transmembrane regions 10 and 11 (Gln-385) greatly reduced both binding and uptake of methotrexate, whereas the insertion into the external loop between transmembrane regions 11 and 12 (Pro-427) selectively interfered with uptake but not binding.Mammalian cells require reduced folates for several biochemical pathways involving purine, pyrimidine, and amino acid metabolism. Since mammals cannot synthesize folates, these compounds must be obtained in the diet and imported by cells via either the glycosylphosphatidylinositol-linked folate receptor (1-5) or the reduced folate carrier (RFC), 1 an integral membrane protein (6 -9). A third, low pH-dependent component for folate transport has been reported (10, 11), but its involvement in folate delivery is not clearly understood. The RFC is the main route for transport of 5-methyltetrahydrofolate, the major circulating folate in the bloodstream, 5-formyltetrahydrofolate (folinic acid), and the folate analog, methotrexate (Mtx) (9,(12)(13)(14).The rfc gene has been cloned from hamster (15), human (16 -19), murine (20), and rat 2 sources. The identity of the rfc gene was verified by cDNA transfections that restored Mtx sensitivity to RFC-deficient cells (15)(16)(17)(18)(19)(20). The human RFC protein is 591 amino acids as deduced from its cDNA sequence, and the hydropathy profile predicts 12 transmembrane-spanning (TM) domains with the amino and carboxyl termini located in the cytoplasm. Recently, the cytoplasmic location of the carboxyl-terminal tail has been confirmed for the human RFC (22). The mouse and hamster homologues (518 amino acids each) have significantly shorter carboxyl-terminal tails than the human RFC (15-20), although all three RFCs share about 68% identical or similar amino acid residues. The human RFC is glycosylated at an asparagine residue in the first extracellular loop (9,22,23), and while the hamster RFC contains the conserv...

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Reduced folate carrier gene (RFC1) expression and anti-folate resistance in transfected and non-selected cell lines

et al. 1997

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Methotrexate transport deficiency due to decreased reduced folate carrier (RFC) activity has been observed in several cell lines selected for resistance to methotrexate (MTX). Since MTX resistance is multifactorial, however, it is difficult to quantify the relative importance of changes in RFC activity in selected cell lines and even more so to determine the relative contribution of naturally occurring RFC activity in the MTX sensitivity of non-selected cell lines. We examined the role of RFC in MTX resistance by studying a transport-deficient cell line transfected with the gene for human RFC, RFC1, and by correlating relative RFC1 expression with MTX and trimetrexate (TMTX) growth inhibition (GI 50) in a panel of cell lines used in the NCI Anticancer Drug Screen. Clones of transport-deficient, MTX-resistant ZR-75-1 human breast cancer cells (MTX R ZR-75-1) transfected with RFC1 were 250-fold more sensitive to MTX and 300-fold more resistant to TMTX than control cell clones, showing that restoration of RFC activity has a significant impact on MTX and TMTX cytotoxicity. We also surveyed 40 of the 60 cell lines in the NCI drug screen panel for RFC1 RNA levels by a quantitative RT-PCR assay. RFC1 RNA levels varied over a range of 15-fold, with only 1 cell line found to be null in expression. Using data from the 6-day drug exposure assay, RFC1 correlated positively with MTX and negatively with TMTX cytotoxicity. As predicted by transfection studies, the calculated difference between MTX and TMTX potency was even more strongly correlated with RFC1 RNA levels of the cell lines. In addition, compounds in the NCI Anticancer Drug Screen database with cytotoxicity profiles which correlated with RFC1 RNA levels or with the calculated difference in MTX-TMTX potency were examined for MTX uptake inhibition and cytotoxicity in the RFC1-transfected MTX R ZR-75-1 cell line. Overall, our data demonstrate the importance of RFC1 in MTX resistance both as a transgene and as a constitutively expressed gene in non-selected cell lines. Int.

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Isolation of a Human cDNA That Complements a Mutant Hamster Cell Defective in Methotrexate Uptake

Cited by 117 publications

References 36 publications

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

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

Topological and Functional Analysis of the Human Reduced Folate Carrier by Hemagglutinin Epitope Insertion

Reduced folate carrier gene (RFC1) expression and anti-folate resistance in transfected and non-selected cell lines

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