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• W1966452132 abstract "Inorganic arsenic is an established human carcinogen, but its metabolism is incompletely defined. The ATP binding cassette protein, multidrug resistance protein (MRP1/ABCC1), transports conjugated organic anions (e.g. leukotriene C4) and also co-transports certain unmodified xenobiotics (e.g. vincristine) with glutathione (GSH). MRP1 also confers resistance to arsenic in association with GSH; however, the mechanism and the species of arsenic transported are unknown. Using membrane vesicles prepared from the MRP1-overexpressing lung cancer cell line, H69AR, we found that MRP1 transports arsenite (AsIII) only in the presence of GSH but does not transport arsenate (AsV) (with or without GSH). The non-reducing GSH analogs l-γ-glutamyl-l-α-aminobutyryl glycine and S-methyl GSH did not support AsIII transport, indicating that the free thiol group of GSH is required. GSH-dependent transport of AsIII was 2-fold higher at pH 6.5-7 than at a more basic pH, consistent with the formation and transport of the acid-stable arsenic triglutathione (As(GS)3). Immunoblot analysis of H69AR vesicles revealed the unexpected membrane association of GSH S-transferase P1-1 (GSTP1-1). Membrane vesicles from an MRP1-transfected HeLa cell line lacking membrane-associated GSTP1-1 did not transport AsIII even in the presence of GSH but did transport synthetic As(GS)3. The addition of exogenous GSTP1-1 to HeLa-MRP1 vesicles resulted in GSH-dependent AsIII transport. The apparent Km of As(GS)3 for MRP1 was 0.32 μm, suggesting a remarkably high relative affinity. As(GS)3 transport by MRP1 was osmotically sensitive and was inhibited by several conjugated organic anions (MRP1 substrates) as well as the metalloid antimonite (Ki 2.8 μm). As(GS)3 transport experiments using MRP1 mutants with substrate specificities differing from wild-type MRP1 suggested a commonality in the substrate binding pockets of As(GS)3 and leukotriene C4. Finally, human MRP2 also transported As(GS)3. In conclusion, MRP1 transports inorganic arsenic as a tri-GSH conjugate, and GSTP1-1 may have a synergistic role in this process. Inorganic arsenic is an established human carcinogen, but its metabolism is incompletely defined. The ATP binding cassette protein, multidrug resistance protein (MRP1/ABCC1), transports conjugated organic anions (e.g. leukotriene C4) and also co-transports certain unmodified xenobiotics (e.g. vincristine) with glutathione (GSH). MRP1 also confers resistance to arsenic in association with GSH; however, the mechanism and the species of arsenic transported are unknown. Using membrane vesicles prepared from the MRP1-overexpressing lung cancer cell line, H69AR, we found that MRP1 transports arsenite (AsIII) only in the presence of GSH but does not transport arsenate (AsV) (with or without GSH). The non-reducing GSH analogs l-γ-glutamyl-l-α-aminobutyryl glycine and S-methyl GSH did not support AsIII transport, indicating that the free thiol group of GSH is required. GSH-dependent transport of AsIII was 2-fold higher at pH 6.5-7 than at a more basic pH, consistent with the formation and transport of the acid-stable arsenic triglutathione (As(GS)3). Immunoblot analysis of H69AR vesicles revealed the unexpected membrane association of GSH S-transferase P1-1 (GSTP1-1). Membrane vesicles from an MRP1-transfected HeLa cell line lacking membrane-associated GSTP1-1 did not transport AsIII even in the presence of GSH but did transport synthetic As(GS)3. The addition of exogenous GSTP1-1 to HeLa-MRP1 vesicles resulted in GSH-dependent AsIII transport. The apparent Km of As(GS)3 for MRP1 was 0.32 μm, suggesting a remarkably high relative affinity. As(GS)3 transport by MRP1 was osmotically sensitive and was inhibited by several conjugated organic anions (MRP1 substrates) as well as the metalloid antimonite (Ki 2.8 μm). As(GS)3 transport experiments using MRP1 mutants with substrate specificities differing from wild-type MRP1 suggested a commonality in the substrate binding pockets of As(GS)3 and leukotriene C4. Finally, human MRP2 also transported As(GS)3. In conclusion, MRP1 transports inorganic arsenic as a tri-GSH conjugate, and GSTP1-1 may have a synergistic role in this process. The 190-kDa MRP1 1The abbreviations used are: MRP, multidrug resistance protein; AsIII, arsenite; AsV, arsenate; As(GS)3, arsenic triglutathione; As2O3, arsenic trioxide; ABC, ATP binding cassette; CdII, cadmium chloride; E217βG, 17β-estradiol 17-(β-d-glucuronide); GST, glutathione S-transferase; LTC4, leukotriene C4; mAb, monoclonal antibody; SbIII, potassium antimony tartrate; WT, wild type; ArsAB, arsenic resistance onion translocating ATPase. (gene symbol ABCC1) is a member of the ATP binding cassette (ABC) superfamily of transport proteins and was originally isolated on the basis of its elevated expression in the multidrug resistant small cell lung cancer cell line, H69AR (1Cole S.P. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M. Deeley R.G. Science. 1992; 258: 1650-1654Google Scholar). In addition to its ability to confer resistance in tumor cells, MRP1 is expressed constitutively in many non-malignant tissues, with relatively high levels found in testes and lung (1Cole S.P. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M. Deeley R.G. Science. 1992; 258: 1650-1654Google Scholar, 2Flens M.J. Zaman G.J. van der Valk P. Izquierdo M.A. Schroeijers A.B. Scheffer G.L. van der Groep P. de Haas M. Meijer C.J. Scheper R.J. Am. J. Pathol. 1996; 148: 1237-1247Google Scholar). MRP1 is a primary active transporter of GSSG and GSH as well as glucuronate, GSH, and sulfate-conjugated organic anions of physiological and toxicological relevance (3Haimeur A. Conseil G. Deeley R.G. Cole S.P. Curr. Drug Metab. 2004; 5: 21-53Google Scholar). Endogenous substrates of MRP1 include the cysteinyl leukotriene LTC4, an important mediator of inflammatory response, and the conjugated steroids E217βG, estrone 3-sulfate, and dehydroepiandrosterone 3-sulfate (4Loe D.W. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 1996; 271: 9683-9689Google Scholar, 5Loe D.W. Almquist K.C. Deeley R.G. Cole S.P. J. Biol. Chem. 1996; 271: 9675-9682Google Scholar, 6Jedlitschky G. Leier I. Buchholz U. Barnouin K. Kurz G. Keppler D. Cancer Res. 1996; 56: 988-994Google Scholar, 7Zelcer N. Reid G. Wielinga P. Kuil A. van der Heijden I. Schuetz J.D. Borst P. Biochem. J. 2003; 371: 361-367Google Scholar, 8Qian Y.M. Song W.C. Cui H. Cole S.P. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Google Scholar). MRP1 and the related MRP2 (gene symbol ABCC2) have also been shown to transport various xenobiotics and are key components of the so-called Phase III elimination pathways of drug metabolism (9Leslie E.M. Deeley R.G. Cole S.P. Toxicology. 2001; 167: 3-23Google Scholar). Several studies show that MRP1 and MRP2 can act synergistically with the phase II conjugating glutathione S-transferases (GST) to confer resistance to the toxicities of some electrophilic drugs and carcinogens (9Leslie E.M. Deeley R.G. Cole S.P. Toxicology. 2001; 167: 3-23Google Scholar, 10Smitherman P.K. Townsend A.J. Kute T.E. Morrow C.S. J. Pharmacol. Exp. Ther. 2004; 308: 260-267Google Scholar, 11Depeille P. Cuq P. Mary S. Passagne I. Evrard A. Cupissol D. Vian L. Mol. Pharmacol. 2004; 65: 897-905Google Scholar). However, several substrates of MRP1 and MRP2, including most of the natural product drugs to which they confer resistance, are not conjugated to any significant extent in vivo, but their transport is stimulated by GSH. Current evidence suggests that at least some of these drugs are co-transported with GSH across the plasma membrane (12Renes J. de Vries E.G. Nienhuis E.F. Jansen P.L. Muller M. Br. J. Pharmacol. 1999; 126: 681-688Google Scholar, 13Loe D.W. Deeley R.G. Cole S.P. Cancer Res. 1998; 58: 5130-5136Google Scholar). The metalloid arsenic is an established multi-target human carcinogen and a major concern as a environmental pollutant (14Gomez-Caminero A. Howe P. Hughes M.F. Kenyon E. Lewis D.R. Moore M. Ng J. Aitio A. Becking G. Ng J. Environmental Health Criteria 224: Arsenic and Arsenic Compounds. 2nd Ed. International Program on Chemical Safety, World Health Organization, Geneva2001Google Scholar). In addition, arsenic-containing compounds (e.g. As2O3) are used in the treatment of several diseases including neoplasia and protozoal infections (15Borst P. Ouellette M. Annu. Rev. Microbiol. 1995; 49: 427-460Google Scholar, 16Shen Z.X. Chen G.Q. Ni J.H. Li X.S. Xiong S.M. Qiu Q.Y. Zhu J. Tang W. Sun G.L. Yang K.Q. Chen Y. Zhou L. Fang Z.W. Wang Y.T. Ma J. Zhang P. Zhang T.D. Chen S.J. Chen Z. Wang Z.Y. Blood. 1997; 89: 3354-3360Google Scholar). Thus, understanding the cellular mechanisms responsible for arsenic transport has both toxicological and pharmacological relevance. The ubiquitous nature of inorganic arsenic in the environment has led to the evolution of arsenic adaptation mechanisms in species ranging from bacteria to humans (17Rosen B.P. FEBS Lett. 2002; 529: 86-92Google Scholar). In bacteria, yeast, and protozoa, pathways of metalloid resistance have been extensively characterized, and it has been determined that arsenic is detoxified either by extrusion from cells or by sequestration within intra-cellular organelles as thiol conjugates (18Dey S. Dou D. Rosen B.P. J. Biol. Chem. 1994; 269: 25442-25446Google Scholar, 19Dey S. Ouellette M. Lightbody J. Papadopoulou B. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2192-2197Google Scholar, 20Ghosh M. Shen J. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5001-5006Google Scholar). In mammalian cell models, MRP1 has been shown to confer resistance to arsenite (AsIII) and arsenate (AsV) in a GSH-dependent manner (21Cole S.P. Sparks K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910Google Scholar, 22Vernhet L. Allain N. Bardiau C. Anger J.P. Fardel O. Toxicology. 2000; 142: 127-134Google Scholar, 23Zaman G.J. Lankelma J. van Tellingen O. Beijnen J. Dekker H. Paulusma C. Oude Elferink R.P. Baas F. Borst P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7690-7694Google Scholar, 24Liu J. Chen H. Miller D.S. Saavedra J.E. Keefer L.K. Johnson D.R. Klaassen C.D. Waalkes M.P. Mol. Pharmacol. 2001; 60: 302-309Google Scholar). However, arsenic transport by MRP1 has never been demonstrated directly, and the mechanism by which efflux occurs and the chemical nature of the transported species are still undefined. Exposure of cells expressing MRP1 to inorganic arsenic has been shown to result in the efflux of GSH and arsenic into the tissue culture media (23Zaman G.J. Lankelma J. van Tellingen O. Beijnen J. Dekker H. Paulusma C. Oude Elferink R.P. Baas F. Borst P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7690-7694Google Scholar). Arsenic-GSH conjugates have never been successfully isolated and identified in such systems, and this is possibly due to their unstable nature. Alternatively, as proposed by Salerno et al. (25Salerno M. Petroutsa M. Garnier-Suillerot A. J. Bioenerg. Biomembr. 2002; 34: 135-145Google Scholar), MRP1 could efflux arsenic in non-covalent association with free GSH. Convincing in vivo evidence indicates that MRP2 transports GSH-conjugated arsenic species into bile (26Kala S.V. Neely M.W. Kala G. Prater C.I. Atwood D.W. Rice J.S. Lieberman M.W. J. Biol. Chem. 2000; 275: 33404-33408Google Scholar, 27Gyurasics A. Varga F. Gregus Z. Biochem. Pharmacol. 1991; 42: 465-468Google Scholar). Although the substrate specificities of MRP1 and MRP2 are not identical, they are very similar. Thus, MRP1 could transport arsenic in a GSH-conjugated form. The major route of arsenic excretion is urinary (60-80% of total arsenic ingested), and very recently arsenic-GSH conjugates have been isolated from the urine of mice lacking the gene for γ-glutamyl transpeptidase, an enzyme responsible for GSH and GSH conjugate catabolism (28Kala S.V. Kala G. Prater C.I. Sartorelli A.C. Lieberman M.W. Chem. Res. Toxicol. 2004; 17: 243-249Google Scholar). Thus, γ-glutamyl transpeptidase is implicated in the processing of arsenic-GSH conjugates at the kidney (28Kala S.V. Kala G. Prater C.I. Sartorelli A.C. Lieberman M.W. Chem. Res. Toxicol. 2004; 17: 243-249Google Scholar). In the present study we have investigated the form of arsenic transported by MRP1 using membrane vesicles prepared from the MRP1 overexpressing H69AR and transfected HeLa cell lines. We found that MRP1 transports AsIII but only in the presence of GSH, and this transport is not supported by GSH analogs that lack a free thiol group. Unexpectedly, we found that the normally cytosolic- or nuclear-localized GSTP1-1 is associated with the membrane vesicle fraction of the H69AR cell line. We present evidence that the formation of As(GS)3 is necessary for AsIII efflux by MRP1 and that vesicle-associated GSTP1-1 is critical for this complex formation. Finally, the transport of As(GS)3 by MRP1 is extensively characterized. Materials—Carrier-free 73AsV was purchased from Los Alamos Meson Production Facility (Los Alamos, NM). ATP, AMP, GSH, GSSG, S-methyl GSH, sucrose, HEPES, KCl, MgCl2, purified GSTP1-1, and poly l-lysine were from Sigma-Aldrich. Creatine kinase, creatine phosphate, glutathione reductase (from yeast), NADPH, and protease inhibitor mixture tablets (Complete, mini EDTA free) were purchased from Roche Applied Science. Ophthalmic acid (l-γ-glutamyl-l-α-aminobutyryl glycine) was from Bachem (Torrance, CA). Antibodies and Cell Lines—The murine MRP1-specific monoclonal antibodies (mAb) QCRL-1 and QCRL-3 have been described previously (29Hipfner D.R. Gauldie S.D. Deeley R.G. Cole S.P. Cancer Res. 1994; 54: 5788-5792Google Scholar, 30Hipfner D.R. Mao Q. Qiu W. Leslie E.M. Gao M. Deeley R.G. Cole S.P. J. Biol. Chem. 1999; 274: 15420-15426Google Scholar). The rat MRP1-specific monoclonal antibody MRPr1 was from Alexis Biochemicals (San Diego, CA). The rabbit polyclonal antibody, MRP-1, was raised to a 15-amino acid peptide corresponding to a region in the NH2-proximal nucleotide binding domain of MRP1 (position 765-779), which includes the highly conserved “C” signature motif (30Hipfner D.R. Mao Q. Qiu W. Leslie E.M. Gao M. Deeley R.G. Cole S.P. J. Biol. Chem. 1999; 274: 15420-15426Google Scholar). This antibody cross-reacts with MRP2. The rabbit GSTP1-1-specific polyclonal antibody was from Calbiochem, and the murine GSTP1-1-specific mAb 353-10 was from Dako (Carpinteria, CA). Derivation and culture of the MRP1 and vector control-transfected HeLa cell lines and the human small cell lung cancer cell line H69 and its MRP1-overexpressing variant H69AR have been described previously (1Cole S.P. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M. Deeley R.G. Science. 1992; 258: 1650-1654Google Scholar, 31Ito K. Olsen S.L. Qiu W. Deeley R.G. Cole S.P. J. Biol. Chem. 2001; 276: 15616-15624Google Scholar). The SV40-transformed human embryonic kidney (HEK293T) cell line was maintained in Dulbecco's modified Eagle's medium supplemented with 4 mml-glutamine and 10% fetal bovine serum. Chemical Synthesis of 73AsIII and 73As(GS)3—73AsIII was prepared from 73AsV with metabisulfite-thiosulfate reagent as previously described (32Reay P.F. Asher C.J. Anal. Biochem. 1977; 78: 557-560Google Scholar). Briefly, 40 μl of a reducing solution (0.1 mm NaAsO2, 66 mm Na2S2O5, 27 mm Na2S2O3, and 82 mm H2SO4) was added to 40 μl of carrier-free 73AsV (30 μCi) and incubated for 40 min at room temperature. The formation of 73AsIII was monitored by TLC on SigmaCell type 100 cellulose plates (Sigma) developed with an isopropanol:acetic acid: water (10:1:5) solvent system (33Styblo M. Thomas D.J. Biochem. Pharmacol. 1995; 49: 971-977Google Scholar). 73As(GS)3 was prepared as described previously by Delnomdedieu et al. (34Delnomdedieu M. Basti M.M. Otvos J.D. Thomas D.J. Chem. Biol. Interact. 1994; 90: 139-155Google Scholar) with modifications. 73AsIII (final concentration 2.5 μm) and GSH (final concentration 75 mm) were mixed under an argon atmosphere in HEPES/KCl buffer at a pH of ∼3 and incubated at 4 °C for >1 h. Formation of 73As(GS)3 was monitored by TLC as described above for 73AsV reduction, and all 73AsIII was consumed and converted to 73As(GS)3. Relative mobility (RF) values were determined for starting materials and products and were consistent with previously published values (33Styblo M. Thomas D.J. Biochem. Pharmacol. 1995; 49: 971-977Google Scholar). MRP1 and MRP2 Expression Vectors and Transfections in HEK293T Cells—The construction and expression of wild-type MRP1 (WTMRP1), wild-type MRP2 (WT-MRP2), and the MRP1 mutants K332L, D336K, K319D, and K347D in HEK293T cells have been described previously (31Ito K. Olsen S.L. Qiu W. Deeley R.G. Cole S.P. J. Biol. Chem. 2001; 276: 15616-15624Google Scholar, 35Haimeur A. Deeley R.G. Cole S.P. J. Biol. Chem. 2002; 277: 41326-41333Google Scholar, 36Haimeur A. Conseil G. Deeley R.G. Cole S.P. Mol. Pharmacol. 2004; 65: 1375-1385Google Scholar, 37Ito K. Oleschuk C.J. Westlake C. Vasa M.Z. Deeley R.G. Cole S.P. J. Biol. Chem. 2001; 276: 38108-38114Google Scholar). Membrane Vesicle Preparation and Immunoblotting—Plasma membrane vesicles from H69, H69AR, HeLa vector, HeLa-MRP1, and transfected HEK293T cells were prepared as described, with modifications (5Loe D.W. Almquist K.C. Deeley R.G. Cole S.P. J. Biol. Chem. 1996; 271: 9675-9682Google Scholar). Briefly, cells were homogenized in buffer containing 250 mm sucrose, 50 mm Tris, pH 7.5, 0.25 mm CaCl2, and protease inhibitor mixture tablets. Cells were disrupted by N2 cavitation (a 5-min equilibration at 200 p.s.i.) and then released to atmospheric pressure, and EDTA was added to 1 mm. The suspension was centrifuged at 800 × g at 4 °C for 10 min, and the supernatant was layered onto 10 ml of a 35% (w/w) sucrose, 50 mm Tris, pH 7.4, cushion. After centrifugation at 100,000 × g at 4 °C for 1 h, the interface was removed and placed in a 25 mm sucrose, 50 mm Tris, pH 7.4, solution and centrifuged at 100,000 × g at 4 °C for 30 min. The membranes were washed with Tris sucrose buffer (250 mm sucrose, 50 mm Tris, pH 7.4) and then resuspended by vigorous syringing with a 27-gauge needle. Protein concentrations were determined by a Bradford assay (Bio-Rad), and aliquots of membrane vesicles were stored at -80 °C. Relative levels of MRP1 protein in membrane vesicles (1 and/or 2 μg of protein) were determined by immunoblot analysis as described previously, with the human MRP1-specific mAb QCRL-1 (29Hipfner D.R. Gauldie S.D. Deeley R.G. Cole S.P. Cancer Res. 1994; 54: 5788-5792Google Scholar). Relative levels of GSTP1-1 protein in membrane vesicles (5 μg of protein) were determined by immunoblot analysis as for MRP1, with a rabbit polyclonal antibody specific for human GSTP1-1 at a dilution of 1:1000. Comparison of MRP1 and MRP2 protein expression levels was done using the polyclonal rabbit antibody MRP-1 at a dilution of 1:1000 as described previously (30Hipfner D.R. Mao Q. Qiu W. Leslie E.M. Gao M. Deeley R.G. Cole S.P. J. Biol. Chem. 1999; 274: 15420-15426Google Scholar). Where appropriate, relative levels of MRP1 and MRP2 expression were estimated by densitometric analysis using a ChemiImager™ 4000 (Alpha Innotech, San Leandro, CA). Equal loading of all protein was confirmed by Amido Black staining of the membranes. 73AsIII and 73As(GS)3Transport Studies—73As transport assays were carried out by a rapid filtration method as previously described (5Loe D.W. Almquist K.C. Deeley R.G. Cole S.P. J. Biol. Chem. 1996; 271: 9675-9682Google Scholar). Membrane vesicles (20 μg of protein per time point) were incubated at 37 °C at a final volume of 60 μl (single time point) or 300 μl (5 time points). Unless otherwise noted, the transport assay buffer used was HEPES (50 mm, pH 7.5), KCl (100 mm) and contained ATP or AMP (4 mm), MgCl2 (10 mm), creatine phosphate (10 mm), creatine kinase (100 μg/ml), glutathione reductase (5 μg/ml), NADPH (0.35 mm), with and without GSH (3 mm) and 73AsIII (30 nCi, 100 nm) or 73As(GS)3 (30 nCi, 100 nm). Conditions for synthesis of 73As(GS)3 resulted in the presence of 3 mm GSH in all transport reactions. At the indicated time points, 60 μl of transport reaction mix was removed and placed in 800 μl of HEPES (50 mm, pH 7.5), KCl (100 mm) buffer, filtered through glass fiber filters (type A/E; Pall Life Sciences, East Hills, NY), and washed twice, and radioactivity was quantitated by liquid scintillation counting. Transport in the presence of AMP was subtracted from transport in the presence of ATP and reported as ATP-dependent73AsIII or 73As(GS)3 transport. The effects of potential modulators of 73As(GS)3 or 73AsIII transport (MRP1-specific mAbs QCRL-1 and QCRL-3 (10 μg/ml), ophthalmic acid (1, 3, or 5 mm), S-methyl GSH (1, 3, or 5 mm), pH change (pH 6.5, 7, 7.5, or 8), sucrose (250-1000 mm), E217βG (25 μm), LTC4 (1 μm), GSSG (0.5 mm), AsV (1 or 10 μm), CdCl2 (1 or 10 μm), potassium antimony tartrate (SbIII) (1 or 10 μm)) were measured at a single time point of 3 min. The effect of exogenous purified GSTP1-1 (0.4 ng or 79 microunits/60-μl reaction) on 73AsIII uptake in the presence of GSH by HeLa-MRP1 vesicles was measured as above. Before use, the purified GSTP1-1 was dialyzed to remove dithiothreitol and EDTA (potential arsenic chelators) using a Slide-A-Lyzer MINI dialysis unit (Pierce) according to the manufacturer's instructions. Kinetic parameters were determined by measuring the initial rate of 73As(GS)3 uptake at 7 different substrate concentrations (50-2500 nm) at a single time point of 1 min. The mode of As(GS)3 transport inhibition by SbIII was examined by determining kinetic parameters in the presence and absence of 5 μm of this metalloid. Confocal Microscopy—Triple staining experiments for nuclei, MRP1 (as a plasma membrane marker), and GSTP1-1 were done using the H69AR and HeLa-MRP1 cell lines. H69AR and HeLa-MRP1 cells were seeded at 2 × 106 and 1 × 106 cells/well, respectively, in a 6-well plate on coverslips pretreated with poly l-lysine (Mr 70,000-150,000). Thirty-six hours later the cells were fixed with 4% paraformaldehyde for 10 min and then incubated in permeabilization buffer (0.2% Triton X-100 in phosphate-buffered saline) for 5 min, washed 3 times in blocking solution (0.1% Triton X-100, 1% bovine serum albumin in phosphate-buffered saline) over 15 min, and then incubated with the GSTP1-1-specific murine mAb 353-10 (1:50 dilution in blocking solution) and the MRP1-specific rat mAb MRPr1 (1:500 dilution) overnight. After washing with blocking solution 3 times over 1 h, highly cross-adsorbed Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) and Alexa Fluor 594 goat anti-rat IgG (H+L) (Molecular Probes, Eugene, OR) were added (1:500 dilution) with RNase A (10 μg/ml) and incubated in the dark for 30 min. Cells were washed with blocking solution changed 4 times over 1 h. Nuclei were then stained with Hoechst 33342 (2.5 μg/ml in phosphate-buffered saline) for 20 min, rinsed 5 times with phosphate-buffered saline, and coverslips were placed on slides with ProLong antifade mounting media (Molecular Probes). A Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss Inc., Thornwood, NY) with a Zeiss Plan-Apo 63× oil (numerical aperture = 1.4) objective lens was used to obtain the fluorescence images. The Hoechst 33342, Alexa 488, and Alexa 594 images were acquired consecutively using, respectively, the 364-, 488 (Ar-ion lasers)-, and 543-nm (HeNe laser) laser lines for excitation and the 385-nm long pass, 505-550-nm band pass, and 560-nm long pass filters for emission. The pinhole was adjusted so that the theoretical Z-resolution was ∼0.7 μm. Negative control experiments with Alexa Fluor 488- and/or 594-conjugated goat anti-mouse IgG (H+L) in the absence of primary antibodies were also performed. Negative control experiments in the presence of both secondary antibodies and individual primary antibodies were also conducted to ensure no cross-reactivity between antibodies. Uptake of AsIII (plus GSH) and As(GS)3by MRP1-enriched Membrane Vesicles—To determine whether AsIII was a substrate for MRP1, ATP-dependent uptake into membrane vesicles prepared from the human small cell lung cancer cell line H69 and its MRP1-overexpressing variant, H69AR, was determined. A time course of AsIII uptake by the H69 and H69AR membrane vesicles is shown in Fig. 1A. ATP-dependent uptake of AsIII by the MRP1-enriched H69AR vesicles was extremely low and similar to uptake observed with AMP or with the non-MRP1-containing H69 vesicles. However, in the presence of GSH (3 mm), ATP-dependent uptake of AsIII by the H69AR but not the H69 vesicles was observed. Uptake was maximal at 3 min, at which time it reached a level of 2.6 pmol mg-1. Similar experiments with AsV were also conducted, and no ATP-dependent transport was observed in the presence or absence of GSH (data not shown). Incubation of AsIII with GSH (as described under “Experimental Procedures”) resulted in the spontaneous formation of As(GS)3 as detected by TLC (73AsIII RF = 0.6; 73AsIII plus GSH RF = 0.01). Uptake of As(GS)3 by the H69 and H69AR membrane vesicles was also measured (Fig. 1A). Uptake of As(GS)3 by H69AR membrane vesicles was linear for 3 min, at which time it reached a level of 4.7 pmol mg-1 and then began to plateau. The maximum activity for As(GS)3 uptake was almost 2-fold greater than the maximum activity observed with AsIII (plus GSH). Results for the H69 vesicles were similar to that of the AsIII time course (not shown). Inhibition of As(GS)3Transport by the MRP1-specific mAb QCRL-3—Several MRP1-specific mAbs have been shown previously to inhibit transport of conjugated and unconjugated MRP1 substrates including LTC4, E217βG, aflatoxin B1-GS, vincristine, and NNAL-O-glucuronide (plus GSH) (4Loe D.W. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. 1996; 271: 9683-9689Google Scholar, 5Loe D.W. Almquist K.C. Deeley R.G. Cole S.P. J. Biol. Chem. 1996; 271: 9675-9682Google Scholar, 13Loe D.W. Deeley R.G. Cole S.P. Cancer Res. 1998; 58: 5130-5136Google Scholar, 30Hipfner D.R. Mao Q. Qiu W. Leslie E.M. Gao M. Deeley R.G. Cole S.P. J. Biol. Chem. 1999; 274: 15420-15426Google Scholar, 38Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P. J. Biol. Chem. 2001; 276: 27846-27854Google Scholar). When the mAb QCRL-3 (which recognizes a conformational-dependent epitope) was tested for its ability to inhibit ATP-dependent uptake of As(GS)3 by H69AR membrane vesicles, complete inhibition was observed at a concentration of 10 μg ml-1 (Fig. 1B). mAb QCRL-1, which recognizes a linear epitope in part of the linker region of MRP1 and does not inhibit transport of other MRP1 substrates (39Hipfner D.R. Almquist K.C. Stride B.D. Deeley R.G. Cole S.P. Cancer Res. 1996; 56: 3307-3314Google Scholar), had no effect on As(GS)3 transport. These results further confirm the MRP1 specificity of ATP-dependent transport of As(GS)3. Ophthalmic Acid and S-Methyl GSH Do Not Support MRP1-mediated AsIII Transport—In previous studies, it has been shown that ophthalmic acid (l-γ-glutamyl-l-α-aminobutyryl-glycine) and S-methyl GSH can substitute for GSH and support the transport of several GSH-dependent MRP1 substrates (8Qian Y.M. Song W.C. Cui H. Cole S.P. Deeley R.G. J. Biol. Chem. 2001; 276: 6404-6411Google Scholar, 13Loe D.W. Deeley R.G. Cole S.P. Cancer Res. 1998; 58: 5130-5136Google Scholar, 38Leslie E.M. Ito K. Upadhyaya P. Hecht S.S. Deeley R.G. Cole S.P. J. Biol. Chem. 2001; 276: 27846-27854Google Scholar, 40Leslie E.M. Bowers R.J. Deeley R.G. Cole S.P. J. Pharmacol. Exp. Ther. 2003; 304: 643-653Google Scholar). These findings indicate that the thiol group of GSH is not required for transport of these substrates and rules out the possibility that formation of a GSH conjugate is critical for transport to occur. In contrast with other MRP1 substrates, neither ophthalmic acid (Fig. 2A) nor S-methyl GSH (Fig. 2B) supported the uptake of AsIII into H69AR membrane vesicles, demonstrating that the free thiol group of GSH is required for AsIII transport by MRP1. GSH-dependent AsIII Uptake Is pH-dependent—The stability and formation rate of As(GS)3 are known to be greater at acidic pH (25Salerno M. Petroutsa M. Garnier-Suillerot A. J. Bioenerg. Biomembr. 2002; 34: 135-145Google Scholar, 34Delnomdedieu M. Basti M.M. Otvos J.D. Thomas D.J. Chem. Biol. Interact. 1994; 90: 139-155Google Scholar), whereas MRP1 transport of LTC4 has been shown to be relatively insensitive to changes in pH; specifically, LTC4 transport was unchanged between pH 6.5 and 7.5 and modestly increased above pH 8 (41Mao Q. Leslie E.M. Deeley R.G. Cole S.P. Biochim. Biophys. Acta. 1999; 1461: 69-82Google Scholar). To determine if transport of AsIII (plus GSH) is influenced by pH, transport was measured over a pH range of 6.5-8. Transport of AsIII (plus GSH) was similar at acidic (pH 6.5) and neutral pH (∼6 pmol mg-1 3 min-1), whereas uptake by vesicles at more basic pH (pH 7.5 and 8) was reduced by 50% (∼2.8 pmol mg-1 3 min-1) (Fig. 3). The increased AsIII transport activity observed at neutral and acidic pH is consistent with the enhanced formation and stability of As(GS)3 at acidic pH and supports the conclusion that this GSH conjugate is being formed before transport. GSTP1-1 Is Associated with the Plasma Membrane of H69 and H69AR Cell Lines—During optimization of AsIII (plus GSH) uptake assay, TLC analysis indicated that under the transport assay conditions used, As(GS)3 was formed from free AsIII and GSH. However, further experimentation showed that conjugate formation did not occur in the absence of membrane vesicles (data not shown). This suggested that the membrane vesicles contained a component that catalyzed the formation of As(GS)3. Immunoblot analysis revealed the pres" @default.
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• W1966452132 title "Arsenic Transport by the Human Multidrug Resistance Protein 1 (MRP1/ABCC1)" @default.
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