Genomic organization of the gene and a related pseudogene for a human folate binding protein

Sadasivan, Easwara; Cedeno, Marisol M.; Rothenberg, Sheldon P.

doi:10.1016/0167-4781(92)90103-7

Cited by 18 publications

(12 citation statements)

References 8 publications

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“…This receptor has a high affinity for folic acid but a low affinity for reduced folates (Antony et al 1985;Elwood et al 1986;Kane et al 1986a,b;Henderson et al 1988;Deutsch et al 1989;Jansen et al 1989;Kane and Waxman 1989). The corresponding cDNAs as well as genomic clones have been isolated from various tissues but not from the liver (Elwood 1989;Lacey et al 1989;Sadasivan and Rothenberg 1989;Brigle et al 1991;Campbell et al 1991;Coney et al 1991;Ragoussis et al 1992;Sadasivan et al 1992Sadasivan et al , 1994. The second carrier system, named the reduced folate carrier, has a high affinity for the reduced folates 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and methotrexate but a low affinity for folate (Horne et al 1979;Price et al 1988;Fan et al 1991;Matherly et al 1991Matherly et al , 1992Matherly and Angeles 1994).…”

Section: Introductionmentioning

confidence: 99%

Characterization of the bile acid sensitive methotrexate carrier of rat liver cells

Honscha¹,

Petzinger²

1999

Naunyn-Schmiedeberg's Arch Pharmacol

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The chemotherapeutic agent methotrexate is widely used in tumor therapy for different forms of leukemia and for the therapy of arthritis. Methotrexate is eliminated from systemic blood circulation by the liver and its transport into hepatocytes is therefore described in detail in this paper. Methotrexate uptake is energy- and sodium-dependent. The Km and the Vmax are 23 microM and 36 pmol/mg protein min, respectively. The apparent activation energy (E(app)) of methotrexate uptake (5 microM [3H]methotrexate) is 53.73 kJ/mol, which indicates an energy-dependent carrier-mediated process. Although methotrexate is a folate derivative, folate itself does not inhibit methotrexate uptake, whereas the reduced folates, dihydrofolate and tetrahydrofolate are weak uncompetitive inhibitors. In contrast, the bile acids taurocholate and cholate are effective competitive inhibitors of methotrexate uptake into hepatocytes. Further strong inhibitors are the loop diuretic bumetanide, the mycotoxin ochratoxin A and bromosulfophthalein. Because tumor patients develop drug resistance during methotrexate therapy, the uptake of methotrexate was tested in different hepatoma cell lines. In HepG2-cells and Reuber hepatoma Fao-cells the transport was non-existent or very small. However, the hepatocytoma fusion cell line HPCT-1E3, a hybrid cell line between primary rat hepatocytes and rat Reuber Fao-cells, shows an intermediate transport activity with a threefold increase of the methotrexate uptake. These results indicate the presence of a bile acid sensitive methotrexate carrier in hepatocytes which is absent in dedifferentiated hepatoma cells. The carrier differs from previously described transporters for the uptake of organic anions.

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Section: Introductionmentioning

confidence: 99%

Characterization of the bile acid sensitive methotrexate carrier of rat liver cells

Honscha¹,

Petzinger²

1999

Naunyn-Schmiedeberg's Arch Pharmacol

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“…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][12][13][14][15][16][17][18][19] but a detailed characterization of the transporter function is still missing.…”

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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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“…Folate-binding protein has a high affinity for folic acid and a low affinity for reduced folates including the chemotherapeutic agent methotrexate (Mtx) 1 (1)(2)(3)(4)(5)(6)(7)(8). The cDNA encoding folate-binding protein has been cloned from various sources (9 -14), and its genomic structure has been determined (15)(16)(17).…”

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Structural Organization of the Reduced Folate Carrier Gene in Chinese Hamster Ovary Cells

Murray

Williams

Flintoff

1996

Journal of Biological Chemistry

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The reduced folate carrier gene (rfc) encodes a putative protein that is involved in the intracellular accumulation of folates. In this report, we describe the organization of the rfc gene from Chinese hamster ovary cells. The hamster rfc gene contains 7 exons and 6 introns, which span 15.3 kilobases. It codes for two alternatively spliced messenger RNAs, one that contains all 7 exons and one that lacks exon 2 but contains the remaining 6 exons. The transcriptional start of the gene has been mapped to six sites approximately 200 base pairs upstream of the putative ATG initiation codon. The promoter region has no TATA box-like sequence but contains a consensus Sp1 binding site. This is the first report of the genomic structure of the reduced folate carrier gene from any species.The intracellular accumulation of folic acid and reduced folates is essential for the survival of mammalian cells. Folates cannot be synthesized endogenously but are required as precursors for the biosynthesis of purines, pyrimidines, and amino acids.There are two known systems for the internalization of folic acid and its reduced forms. One system consists of a membrane-bound 40-kDa glycoprotein folate receptor termed folatebinding protein. Folate-binding protein has a high affinity for folic acid and a low affinity for reduced folates including the chemotherapeutic agent methotrexate (Mtx) 1 (1-8). The cDNA encoding folate-binding protein has been cloned from various sources (9 -14), and its genomic structure has been determined (15-17).The second system consists of a 36 -78-kDa glycoprotein termed the reduced folate carrier, which has a higher affinity for reduced folates including Mtx and a lower affinity for folic acid (18 -22). Several cDNAs that putatively encode the reduced folate carrier have recently been cloned from human (23-26), mouse (27), and hamster (28) cells.The availability of cDNA clones from various sources provides a powerful tool for examining folate transport processes. However, at present very little is known about the molecular details of folate transport.We have previously described the isolation of both a hamster genomic DNA cosmid clone (29) and a cDNA clone (pMtxT9) (28) that can, upon transfection, complement mutant Chinese hamster ovary (CHO) cell lines that are unable to take up folates. Mutant cells transfected with the cDNA are able to bind and take up Mtx and thus become sensitive to the cytotoxic action of the drug.In our continuing studies to understand the molecular nature of the reduced folate carrier gene we have localized the gene for hamster rfc to chromosomes 1 and Z1 at position q2-q3 in CHO cells (30). The present study describes the organization of the hamster rfc gene, the identification of two alternatively spliced mRNA isoforms, and the mapping of the transcriptional start site. EXPERIMENTAL PROCEDURESCell Lines, Cosmid Clones, and Plasmid Clones-The wild-type, Mtxsensitive (Pro Ϫ 3) and Mtx-resistant (Pro Ϫ 3 MtxRII 5-3) cell lines and their maintenance have been previously described (31-3...

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Genomic organization of the gene and a related pseudogene for a human folate binding protein

Cited by 18 publications

References 8 publications

Characterization of the bile acid sensitive methotrexate carrier of rat liver cells

Characterization of the bile acid sensitive methotrexate carrier of rat liver cells

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

Structural Organization of the Reduced Folate Carrier Gene in Chinese Hamster Ovary Cells

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