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  • 1
    Keywords: CELLS ; CELL ; Germany ; human ; TOOL ; CDNA ; GENE ; PROTEIN ; DOMAIN ; tumour ; hepatocytes ; FREQUENCY ; polymorphism ; POLYMORPHISMS ; VARIANTS ; TRANSPORT ; IDENTIFICATION ; RAT-LIVER ; GLUTATHIONE ; resistance ; UP-REGULATION ; MEMBRANE ; MUTATION ; HEPATOCYTE CANALICULAR ISOFORM ; DRUG-RESISTANCE ; MULTIDRUG-RESISTANCE ; ATP-dependent transport ; SUBSTRATE-SPECIFICITY ; VARIANT ; FUNCTIONAL-CHARACTERIZATION ; ANION TRANSPORT ; basolateral membrane,cholestasis,hepatocellular transporters,MRP3,polymorphism,SNP ; BINDING CASSETTE ; CHRONIC CONJUGATED HYPERBILIRUBINEMIA ; DUBIN-JOHNSON-SYNDROME
    Abstract: The human multidrug resistance protein 3 (MRP3, symbol ABCC3) is an ATP-binding cassette transporter that mediates the efflux of organic anions, including lipophilic substances conjugated with glucuronate, sulphate or glutathione, across the basolateral membrane of polarized cells (e.g. hepatocytes) into blood. Genetic variants of MRP3 may affect the transport of these substances out of cells. The aims of this study were: (i) to identify MRP3 polymorphisms; (ii) to functionally characterize one relatively frequent MRP3 polymorphism; and (iii) to establish whether MRP3 transports bilirubin glucuronosides. Exonic nucleotide variants in the ABCC3 gene were identified by single-strand conformation polymorphism analysis. The 3890G〉A mutation, resulting in MRP3-Arg(1297) His, was introduced into the ABCC3 cDNA which was stably transfected into MDCKII cells. For the functional characterization of MRP3-Arg(1297) His in comparison with MRP3, ATP-dependent transport was analysed in isolated membrane vesicles. Two non- synonymous MRP3 variants were identified with an allele frequency of 0.003 for 1643T〉A (MRP3-Leu(148)Gln) and 0.08 for 3890G〉A (MRP3-Arg(1297) His). Because of the high, frequency of the 3890G〉A mutation, and because of the close proximity of Arg(1297) to the second nucleotide-binding domain, we pursued the functional characterization of the MRP3-Arg(1297)His polymorphic variant. MRP3-Arg(1297)His was correctly localized to the basolateral membrane of polarized MDCKII cells. We identified monoglucuronosyl bilirubin, bisgiucuronosyl bilirubin and leukotriene C-4 as substrates for both MRP3 and MRP3-Arg(1297)His. Dehydroepiandrosterone-3-sulphate and 17beta-glucuronosyl oestradiol were transported with similar kinetics by MRP3 and MRP3-Arg(1297) His. This experimental setup provides a useful tool to analyse the functional consequences of polymorphic variants of MRP3. Pharmacogenetics 14: 213-223 (C) 2004 Lippincott Williams Wilkins
    Type of Publication: Journal article published
    PubMed ID: 15083066
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  • 2
    Keywords: brain ; CELLS ; ENDOTHELIAL-CELLS ; EXPRESSION ; tumor ; CELL ; Germany ; human ; MICROSCOPY ; PROTEIN ; PROTEINS ; SAMPLE ; SAMPLES ; DRUG ; FAMILY ; MEMBER ; MEMBERS ; GLYCOPROTEIN ; NO ; GLUTATHIONE ; resistance ; MEMBRANE ; NUCLEOTIDES ; REGION ; REGIONS ; LOCALIZATION ; POLYMERASE-CHAIN-REACTION ; CHAIN-REACTION ; DRUG-RESISTANCE ; MULTIDRUG-RESISTANCE ; CONJUGATE EXPORT PUMP ; ORGANIC ANION TRANSPORTER ; CHAIN ; quantitative polymerase chain reaction ; ADULT ; polymerase chain reaction ; P-GLYCOPROTEIN ; TRANSPORTER ; NEURONS ; GLYCOPROTEINS ; BASOLATERAL HEPATOCYTE MEMBRANE ; blood-brain barrier ; CEREBROSPINAL FLUID BARRIER ; CULTURED RAT ASTROCYTES ; human brain ; MICROVESSEL ENDOTHELIAL-CELLS ; MRP4 CONFERS RESISTANCE ; multidrug resistance proteins (MRPs,symbol ABCC) ; NORMAL HUMAN-TISSUES ; organic anions
    Abstract: Multidrug resistance proteins (MRPs, symbol ABCC) are membrane glycoproteins that mediate the ATP-dependent export of organic anions, including cytotoxic and antiviral drugs, from cells. To identify MRP family members possibly involved in the intrinsic resistance of human brain to cytotoxic and antiviral drugs, we analyzed the expression and localization of MRP1-MRP6 in rapidly frozen perilesional samples of several regions of adult human brain obtained during neurosurgery. Quantitative polymerase chain reaction analysis showed expression of MRP1, MRP2, MRP3, MRP4, and MRP5 mRNA, whereas MRP6 mRNA was below detectability. However, immunofluorescence microscopy of cryosections from human brain showed no reactivity for the MRP2 or MRP3 proteins. The proteins MRP1, MRP4, and MRP5 were clearly localized by confocal laser scanning microscopy to the luminal side of brain capillary endothelial cells. The MRP4 and MRP5 proteins were also detected in astrocytes of the subcortical white matter. Notably, MRP5 protein was present in pyramidal neurons. MRP proteins may, thus, contribute to the cellular efflux of endogenous anionic glutathione or glucuronate conjugates (substrates for MRP1), cyclic nucleotides (substrates for MRP4 and MRP5), or glutathione (co-substrate for MRP1 and MRP4); in addition, they may play an important role in the resistance of the brain to several cytotoxic and antiviral drugs. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 15501592
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  • 3
    Keywords: CANCER ; EXPRESSION ; tumor ; carcinoma ; Germany ; DEATH ; PROTEIN ; PROTEINS ; TISSUE ; TUMORS ; PATIENT ; MECHANISM ; FAMILY ; MALIGNANCIES ; resistance ; chemotherapy ; LOCALIZATION ; SUPERFAMILY ; Jun ; adenocarcinoma ; QUANTITATIVE-ANALYSIS ; drug resistance ; DRUG-RESISTANCE ; MULTIDRUG-RESISTANCE ; pancreatic cancer ; pancreatic carcinoma ; CONJUGATE EXPORT PUMP ; SUBSTRATE-SPECIFICITY ; ONCOLOGY-GROUP ; PHASE-II ; multidrug resistance ; MALIGNANCY ; PANCREATIC-CANCER ; DUCTAL ADENOCARCINOMA ; P-GLYCOPROTEIN ; TRANSPORTER ; BASOLATERAL HEPATOCYTE MEMBRANE ; multidrug resistance protein ; TISSUE SAMPLES ; MRP3 ; ABCC family ; CYCLIC-NUCLEOTIDES ; INDUCIBLE EXPRESSION ; MDR1 ; MRP ; NORMAL HUMAN TISSUES
    Abstract: Pancreatic ductal adenocarcinoma is among the top 10 causes of death from cancer in industrialized countries. In comparison with other gastrointestinal malignancies, pancreatic cancer is one of the tumors most resistant to chemotherapy. An important mechanism of tumor multidrug resistance is increased drug efflux mediated by several transporters of the ABC superfamily. Especially BCRP (ABCG2), MDR1 P-glycoprotein (ABCB1) and members of the MRP (ABCC) family are important in mediating drug resistance. The MRP family consists of 9 members (MRP1-MRP9) with MRP1-MRP6 being best characterized with respect to protein localization and substrate selectivity. Here, we quantified the mRNA expression of BCRP and of all MRP family members in normal human pancreas and pancreatic carcinoma and analyzed the mRNA level of the transporters most abundantly expressed in pancreatic tissue, BCRP, MRP1, MRP3, MRP4 and MRP5, in 37 tissue samples. In addition, we determined the localization of the 4 MRP proteins in normal human pancreas and in pancreatic carcinoma. The expression of BCRP, MRP1 and MRP4 mRNA did not correlate with tumor stage or grading. On the other hand, the expression of MRP3 mRNA was upregulated in pancreatic carcinoma samples and was correlated with tumor grading. The MRP5 mRNA level was significantly higher in pancreatic carcinoma tissue compared to normal pancreatic tissue. These data suggest that MRP3 and MRP5 are involved in drug resistance of pancreatic tumors and that quantitative analysis of their expression may contribute to predict the benefit of chemotherapy in patients with pancreatic cancer. © 2005 Wiley-Liss, Inc
    Type of Publication: Journal article published
    PubMed ID: 15688370
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  • 4
    Keywords: PEPTIDE ; SPECTRA ; CELLS ; INHIBITOR ; Germany ; human ; INFORMATION ; PROTEIN ; DRUG ; kidney ; FAMILY ; hepatocytes ; PRIMARY CULTURES ; TRANSPORT ; IDENTIFICATION ; GLUTATHIONE ; resistance ; MEMBRANE ; LINE ; POLYPEPTIDE ; MULTIDRUG-RESISTANCE PROTEIN ; HUMAN LIVER ; DRUG-RESISTANCE ; MULTIDRUG-RESISTANCE ; CONJUGATE EXPORT PUMP ; SUBSTRATE-SPECIFICITY ; HUMAN HEPATOCYTES ; multidrug resistance ; INHIBITORS ; RE ; secretion ; TRANSPORTER ; hepatobiliary transport ; NEED ; multidrug resistance protein ; TRANSCELLULAR TRANSPORT ; VECTORIAL TRANSPORT ; APICAL MEMBRANE ; OATP1B3 OATP8
    Abstract: Hepatobiliary elimination of many organic anions is initiated by OATP1B1 (OATP2, LST-1, OATP-C), OATP1B3 (OATP8), and OATP2B1 (OATP-B), which are the predominant uptake transporters of human hepatocytes. Thereafter, the unidirectional efflux pump ABCC2 (multidrug resistance protein 2) mediates the transport of organic anions, including glutathione conjugates and glucuronosides, into bile. In this study, we generated a Madin-Darby canine kidney (MDCKII) cell line stably expressing recombinant OATP1B1, OATP1B3, and OATP2B1 in the basolateral membrane and ABCC2 in the apical membrane. Double-transfected MDCKII cells stably expressing ABCC2 together with OATP1B1, OATP1B3, or OATP2B1 served as control cells. The quadruple-transfected cells exhibited high rates of vectorial transport of organic anions, including bromosulfophthalein, cholecystokinin peptide (CCK-8), and estrone 3-sulfate. The quadruple-transfected cells enabled the identification of substrates for uptake or vectorial transport that may be missed in studies with a double-transfected cell line, as exemplified by CCK-8, which is a substrate for OATP1B3 but not for OATP1B1 or OATP2B1. The broad substrate spectrum covered by the three hepatocellular OATP transporters enables representative analyses of the uptake of many organic anions into human hepatocytes. The broad spectrum of organic anions transported vectorially by the quadruple-transfected cells also provides valuable information on the substrate selectivity of ABCC2, without the need for studies in inside-out membrane vesicles containing the ABCC2 protein. The quadruple-transfected MDCKII-ABCC2/OATP1B1/1B3/2B1 cells may thus be useful for the identification of substrates and inhibitors, including drug candidates, undergoing uptake and secretion by human hepatocytes, under conditions that may be better defined than in primary cultures of human hepatocytes
    Type of Publication: Journal article published
    PubMed ID: 16046661
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  • 5
    Keywords: brain ; LUNG-CANCER ; TRANSPORT ; CONJUGATE ; DRUG-RESISTANCE ; P-GLYCOPROTEIN ; MICROVESSEL ENDOTHELIAL-CELLS ; CYCLIC-NUCLEOTIDES ; PRIMARY GLIAL CULTURES ; GLUTATHIONE SYSTEM
    Abstract: Multidrug resistance proteins (Mrps) are ATP-driven export pumps that mediate the export of organic anions from cells. So far only little information is available on expression and physiological functions of Mrps in brain. The expression of mRNAs of six Mrp paralogs in rat brain, as well as in rat cultures enriched for neurones, astrocytes, oligodendrocytes and microglial cells, was studied by qualitative and semiquantitative RT-PCR analysis. In adult rat brain as well as in neural cell cultures the mRNAs coding for Mrp1, Mrp3, Mrp4 and Mrp5 were detected. Semiquantitative analysis revealed that the mRNAs coding for Mrp1 and Mrp5 were more abundant in the four cell culture types than mRNAs of the other Mrps. mRNAs coding for Mrp3 and Mrp4 were found at significant levels in cultured astrocytes and microglial cells, whereas cultures of neurones and oligodendrocytes contained only marginal quantities of these mRNAs. Putative physiological functions of Mrps in brain cells are discussed.
    Type of Publication: Journal article published
    PubMed ID: 12153495
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  • 6
    Keywords: EXPRESSION ; IDENTIFICATION ; MULTIDRUG-RESISTANCE PROTEIN ; HUMAN LIVER ; DRUG-RESISTANCE ; CONJUGATE EXPORT PUMP ; DUBIN-JOHNSON-SYNDROME ; AFRICAN-AMERICANS ; CANALICULAR ISOFORM ; EUROPEAN-AMERICANS
    Abstract: The organic anion transporter SLC21A6 (also known as OATP2, OATP-C, or LST-1) is involved in the hepatocellular uptake of a variety of endogenous and xenobiotic substances and drugs. We analyzed 81 human liver samples by immunoblotting and found one with a strongly reduced amount of SLC21A6 protein suggesting mutations in the SLC21A6 gene. The SLC21A6 cDNA from this sample contained five base pair changes in one allele; three of the mutations resulted in amino acid substitutions designated SLC21A6-N130D, SLC21A6-P155T, and SLC21A6-L193R. The former two were polymorphisms (SLC21A6*1b and SLC21A6*4), whereas SLC21A6-L193R represents the first naturally occurring mutation identified in one allele of the SLC21A6 gene, which affects protein maturation and organic anion transport. We introduced each of the mutations into the SLC21A6 cDNA and established stably transfected MDCKII cells expressing the respective mutant SLC21A6 protein. Immunofluorescence microscopy and uptake measurements were used to study localization and transport properties of the mutated proteins. Both proteins carrying the polymorphisms were sorted to the lateral membrane like wild-type SLC21A6, but their transport properties for the substrates cholyltaurine and 17beta-glucuronosyl estradiol were altered. Importantly, most of the mutant protein SLC21A6-L193R was retained intracellularly, and this single amino acid exchange abolished transport function.
    Type of Publication: Journal article published
    PubMed ID: 12196548
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