Functional Interactions of Cu-ATPase ATP7B with Cisplatin and the Role of ATP7B in the Resistance of Cells to the Drug
2009; Elsevier BV; Volume: 284; Issue: 12 Linguagem: Inglês
10.1074/jbc.m805145200
ISSN1083-351X
AutoresKaroline Leonhardt, Rolf Gebhardt, Joachim Mössner, Svetlana Lutsenko, Dominik Hüster,
Tópico(s)Metal complexes synthesis and properties
ResumoCisplatin is a widely used chemotherapeutic agent for treatment of ovarian, testicular, lung, and stomach cancers. The initial response to the drug is robust; however, tumor cells commonly develop resistance to cisplatin, which complicates treatment. Recently, overexpression of the Cu-ATPase ATP7B in ovary cells was linked to the increased cellular resistance to cisplatin; and the role for Cu-ATPases in the export of cisplatin from cells was proposed. Our results support functional interactions between cisplatin and ATP7B but argue against the active transport through the copper translocation pathway as a mechanism of drug resistance. In hepatocytes, we observed no correlation between the levels of endogenous ATP7B and the resistance of cells to cisplatin. Unlike copper, cisplatin does not induce trafficking of ATP7B in hepatoma cells, neither does it compete with copper in a transport assay. However, cisplatin binds to ATP7B and stimulates catalytic phosphorylation with EC50 similar to that of copper. Mutations of the first five N-terminal copper-binding sites of ATP7B do not inhibit the cisplatin-induced phosphorylation of ATP7B. In contrast, the deletion of the first four copper-binding sites abolishes the effect of cisplatin on the ATP7B activity. Thus, cisplatin binding to ATP7B and/or general changes in cellular copper homeostasis are likely contributors to the increased resistance to the drug. The link between changes in copper homeostasis and cisplatin resistance was confirmed by treating the Huh7 cells with copper chelator and increasing their resistance to cisplatin.cisplatin Cisplatin is a widely used chemotherapeutic agent for treatment of ovarian, testicular, lung, and stomach cancers. The initial response to the drug is robust; however, tumor cells commonly develop resistance to cisplatin, which complicates treatment. Recently, overexpression of the Cu-ATPase ATP7B in ovary cells was linked to the increased cellular resistance to cisplatin; and the role for Cu-ATPases in the export of cisplatin from cells was proposed. Our results support functional interactions between cisplatin and ATP7B but argue against the active transport through the copper translocation pathway as a mechanism of drug resistance. In hepatocytes, we observed no correlation between the levels of endogenous ATP7B and the resistance of cells to cisplatin. Unlike copper, cisplatin does not induce trafficking of ATP7B in hepatoma cells, neither does it compete with copper in a transport assay. However, cisplatin binds to ATP7B and stimulates catalytic phosphorylation with EC50 similar to that of copper. Mutations of the first five N-terminal copper-binding sites of ATP7B do not inhibit the cisplatin-induced phosphorylation of ATP7B. In contrast, the deletion of the first four copper-binding sites abolishes the effect of cisplatin on the ATP7B activity. Thus, cisplatin binding to ATP7B and/or general changes in cellular copper homeostasis are likely contributors to the increased resistance to the drug. The link between changes in copper homeostasis and cisplatin resistance was confirmed by treating the Huh7 cells with copper chelator and increasing their resistance to cisplatin.cisplatin Cisplatin, cis-diamminedichloroplatinum (DDP), 3The abbreviations used are: DDP, cis-diamminedichloroplatinum; BCS, bathocuproine disulfonate; DTT, dithiothreitol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TGN, trans-Golgi network; Cu-ATPase, copper-transporting ATPase; PBS, phosphate-buffered saline; MOPS, 3-morpholinopropanesulfonic acid; MBS, metal-binding site. is a common anti-tumor agent that is used to treat many types of cancer. It is especially prescribed for testicular, ovarian, bladder, liver, lung, and stomach cancers (1Barocas D.A. Clark P.E. Curr. Opin. Oncol. 2008; 20: 307-314Crossref PubMed Scopus (52) Google Scholar, 2Ferraldeschi R. Baka S. Jyoti B. Faivre-Finn C. Thatcher N. Lorigan P. Drugs. 2007; 67: 2135-2152Crossref PubMed Scopus (45) Google Scholar, 3Kelland L. Nat. Rev. Cancer. 2007; 7: 573-584Crossref PubMed Scopus (3826) Google Scholar, 4Moehler M. Galle P.R. Gockel I. Junginger T. Schmidberger H. Best Pract. Res. Clin. Gastroenterol. 2007; 21: 965-981Crossref PubMed Scopus (34) Google Scholar, 5Yuan J.N. Chao Y. Lee W.P. Li C.P. Lee R.C. Chang F.Y. Yen S.H. Lee S.D. Whang-Peng J. Med. Oncol. 2008; 25: 201-206Crossref PubMed Scopus (30) Google Scholar). DDP mediates its cytotoxic effects by binding to DNA, forming the intrastrand cross-links and thus causing an inhibition of DNA synthesis and repair with eventual cell death (6Jamieson E.R. Lippard S.J. Chem. Rev. 1999; 99: 2467-2498Crossref PubMed Scopus (2681) Google Scholar, 7Wong E. Giandomenico C.M. Chem. Rev. 1999; 99: 2451-2466Crossref PubMed Scopus (1787) Google Scholar). The initial tumor response to the treatment with DDP is robust; however, the efficacy of treatment decreases with longer and repetitive therapy cycles. The resistance arises rapidly and is sufficient to cause a failure of DDP therapy (8Andrews P.A. Jones J.A. Varki N.M. Howell S.B. Cancer Commun. 1990; 2: 93-100Crossref PubMed Scopus (122) Google Scholar). The mechanisms by which cells develop resistance to DDP are not fully understood. Detoxification of DDP, enhanced repair and tolerance of DNA adducts, inhibition of apoptosis, impaired uptake and increased efflux of the drug may contribute to the acquired resistance of cells (9Akiyama S. Chen Z.S. Sumizawa T. Furukawa T. Anticancer Drug Des. 1999; 14: 143-151PubMed Google Scholar, 10Mukai M. Kanzaki A. Chen Z.S. Miyashita H. Sumizawa T. Furukawa T. Haraguchi M. Takebayashi Y. Takamatsu H. Akiyama S. Oncol. Rep. 2002; 9: 839-844PubMed Google Scholar, 11Jordan P. Carmo-Fonseca M. Cell Mol. Life Sci. 2000; 57: 1229-1235Crossref PubMed Scopus (319) Google Scholar, 12Siddik Z.H. Oncogene. 2003; 22: 7265-7279Crossref PubMed Scopus (2739) Google Scholar, 13Brozovic A. Fritz G. Christmann M. Zisowsky J. Jaehde U. Osmak M. Kaina B. Int. J. Cancer. 2004; 112: 974-985Crossref PubMed Scopus (124) Google Scholar). Recently, an unexpected connection was discovered between the resistance of cells to DDP and cellular copper metabolism (14Safaei R. Cancer Lett. 2006; 234: 34-39Crossref PubMed Scopus (141) Google Scholar, 15Samimi G. Safaei R. Katano K. Holzer A.K. Rochdi M. Tomioka M. Goodman M. Howell S.B. Clin. Cancer Res. 2004; 10: 4661-4669Crossref PubMed Scopus (277) Google Scholar, 16Kuo M.T. Chen H.H. Song I.S. Savaraj N. Ishikawa T. Cancer Metastasis Rev. 2007; 26: 71-83Crossref PubMed Scopus (230) Google Scholar). Either down-regulation of CTR1 (49Zisowsky J. Koegel S. Leyers S. Devarakonda K. Kassack M.U. Osmak M. Jaehde U. Biochem. Pharmacol. 2007; 73: 298-307Crossref PubMed Scopus (118) Google Scholar), a transporter responsible for the uptake of copper, and/or up-regulation of the copper-transporting ATPases (Cu-ATPases) responsible for copper efflux were found to increase cells resistance to DDP, although correlation between the levels of CTR1 and resistance were not always observed (17Yoshizawa K. Nozaki S. Kitahara H. Ohara T. Kato K. Kawashiri S. Yamamoto E. Oncol. Rep. 2007; 18: 987-991PubMed Google Scholar). The DDP-resistant cells were shown to have a lower copper content (18Katano K. Kondo A. Safaei R. Holzer A. Samimi G. Mishima M. Kuo Y.M. Rochdi M. Howell S.B. Cancer Res. 2002; 62: 6559-6565PubMed Google Scholar), and a cross-resistance was detected between cells selected for resistance to either copper or DDP (19Safaei R. Howell S.B. Crit. Rev. Oncol. Hematol. 2005; 53: 13-23Crossref PubMed Scopus (238) Google Scholar, 20Safaei R. Katano K. Samimi G. Naerdemann W. Stevenson J.L. Rochdi M. Howell S.B. Cancer Chemother. Pharmacol. 2004; 53: 239-246Crossref PubMed Scopus (70) Google Scholar). The link between the Cu-ATPase ATP7B and cell response to DDP has been particularly well documented. ATP7B was found to be overexpressed in several solid tumors and in cultured cells; the increase in the ATP7B levels correlated with an increased resistance to DDP (18Katano K. Kondo A. Safaei R. Holzer A. Samimi G. Mishima M. Kuo Y.M. Rochdi M. Howell S.B. Cancer Res. 2002; 62: 6559-6565PubMed Google Scholar, 21Higashimoto M. Kanzaki A. Shimakawa T. Konno S. Naritaka Y. Nitta Y. Mori S. Shirata S. Yoshida A. Terada K. Sugiyama T. Ogawa K. Takebayashi Y. Int. J. Mol. Med. 2003; 11: 337-341PubMed Google Scholar, 22Kanzaki A. Toi M. Neamati N. Miyashita H. Oubu M. Nakayama K. Bando H. Ogawa K. Mutoh M. Mori S. Terada K. Sugiyama T. Fukumoto M. Takebayashi Y. Jpn. J. Cancer Res. 2002; 93: 70-77Crossref PubMed Scopus (49) Google Scholar, 23Ohbu M. Ogawa K. Konno S. Kanzaki A. Terada K. Sugiyama T. Takebayashi Y. Cancer Lett. 2003; 189: 33-38Crossref PubMed Scopus (51) Google Scholar, 24Komatsu M. Sumizawa T. Mutoh M. Chen Z.S. Terada K. Furukawa T. Yang X.L. Gao H. Miura N. Sugiyama T. Akiyama S. Cancer Res. 2000; 60: 1312-1316PubMed Google Scholar). In ovarian carcinoma cells, ATP7B was detected in vesicles resembling exosomes, prompting the suggestion that ATP7B facilitates DDP efflux (25Safaei R. Larson B.J. Cheng T.C. Gibson M.A. Otani S. Naerdemann W. Howell S.B. Mol. Cancer Ther. 2005; 4: 1595-1604Crossref PubMed Scopus (435) Google Scholar). The evidence was also provided for colocalization of the enhanced cyan fluorescent protein-tagged ATP7B and fluorescent DDP analogue in vesicles (26Katano K. Safaei R. Samimi G. Holzer A. Tomioka M. Goodman M. Howell S.B. Clin. Cancer Res. 2004; 10: 4578-4588Crossref PubMed Scopus (81) Google Scholar). These observations have raised significant interest and many questions. ATP7B is a P1-type-ATPase with high selectivity to Cu(I) (27Lutsenko S. Barnes N.L. Bartee M.Y. Dmitriev O.Y. Physiol. Rev. 2007; 87: 1011-1046Crossref PubMed Scopus (605) Google Scholar). Cu(I) stimulates catalytic activity of ATP7B, inducing the hydrolysis of ATP via formation of an acyl-phosphate intermediate, a step necessary for subsequent transport of copper across membranes (28Petris M.J. Voskoboinik I. Cater M. Smith K. Kim B.E. Llanos R.M. Strausak D. Camakaris J. Mercer J.F. J. Biol. Chem. 2002; 277: 46736-46742Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 29Tsivkovskii R. Eisses J.F. Kaplan J.H. Lutsenko S. J. Biol. Chem. 2002; 277: 976-983Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 30Cater M.A. La Fontaine S. Mercer J.F. Biochem. J. 2007; 401: 143-153Crossref PubMed Scopus (51) Google Scholar). Neither Cu(II) nor other divalent metals such as Zn2+ or Cd2+ stimulate the formation of phospho-intermediate (29Tsivkovskii R. Eisses J.F. Kaplan J.H. Lutsenko S. J. Biol. Chem. 2002; 277: 976-983Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Therefore, it is not apparent how ATP7B may coordinate and transport DDP or even platinum, a metal with electronic structure different from Cu(I). Recent studies provided the in vitro evidence for the DDP-dependent ATP-hydrolysis by ATP7B and the ATP7B-dependent transport of DDP at acidic pH (pH 4.6) (31Safaei R. Otani S. Larson B.J. Rasmussen M.L. Howell S.B. Mol. Pharmacol. 2008; 73: 461-468Crossref PubMed Scopus (93) Google Scholar). Because these conditions do not match the physiological conditions at which ATP7B operates, it remains uncertain whether the transport of DDP to vesicles represents the primary mechanism through which ATP7B mediates resistance of cells to DDP. ATP7B is highly expressed in hepatocytes. Given the poor response of hepatic tumors to chemotherapy and the poor prognosis of advanced hepatomas (32Wakamatsu T. Nakahashi Y. Hachimine D. Seki T. Okazaki K. Int. J. Oncol. 2007; 31: 1465-1472PubMed Google Scholar, 33Mathurin P. Rixe O. Carbonell N. Bernard B. Cluzel P. Bellin M.F. Khayat D. Opolon P. Poynard T. Aliment Pharmacol. Ther. 1998; 12: 111-126Crossref PubMed Scopus (169) Google Scholar), it was interesting and important to investigate the role of ATP7B in the sensitivity of hepatocytes and hepatoma cells to DDP. Hepatocytes, unlike ovary cells, express only one Cu-ATPase, ATP7B; this simplifies evaluation of the ATP7B contribution to DDP resistance. Contrary to expectation, in hepatoma cells and in primary hepatocytes, we did not observe a correlation between the levels of ATP7B and cell sensitivity to DDP, nor did we see the effect of DDP on ATP7B transport activity or trafficking. Instead, we demonstrate that DDP binds to ATP7B and induces formation of a phosphorylated catalytic intermediate. We found that the mode of DDP binding to ATP7B differs from that of copper and requires the presence of the N-terminal metal-binding domain. We propose a model of how the overexpression of ATP7B increases cell resistance to the drug. Reagents-The DDP solution (GRY-Pharma, Kirchzarten, Germany or Novaplus purchased through Ben Venue Laboratories, Bedford, OH) was obtained via the pharmacies of the University of Leipzig or the Oregon Health & Science University, respectively. It was kept as a 3.33 mm solution in 0.9% NaCl in the dark at room temperature. Other chemicals, unless otherwise specified, were provided by Sigma. Cell Lines-Sf9 cells (Invitrogen) were maintained at 27 °C in suspension cultures in SF900 II (Invitrogen). The human hepatoma cell lines HepG2 and Huh7 were cultured at 37 °C in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 2 mm l-glutamine, and 100 units/ml penicillin and streptomycin (Invitrogen). Mouse Strains-The generation of Atp7b-/- mice has been described previously (34Buiakova O.I. Xu J. Lutsenko S. Zeitlin S. Das K. Das S. Ross B.M. Mekios C. Scheinberg I.H. Gilliam T.C. Hum. Mol. Genet. 1999; 8: 1665-1671Crossref PubMed Scopus (171) Google Scholar). The Atp7b-/- mice were housed at the Medizinisch Experimentelles Zentrum (MEZ) of the University of Leipzig according to the university guidelines on the use of laboratory and experimental animals. The animals were euthanized by phenobarbital injection (intraperitoneal). Eight-week-old mice of either sex were used for the experiments. Primary Liver Cells-Hepatocytes were isolated according to Seglen et al. (35Seglen P.O. Exp. Cell Res. 1973; 82: 391-398Crossref PubMed Scopus (1036) Google Scholar) with modifications by Gebhardt et al. (36Gebhardt R. Jung W. Robenek H. Eur. J. Cell Biol. 1982; 29: 68-76PubMed Google Scholar). The isolated cells were centrifuged at room temperature at 37.5 × g for 2 min. The pellets were resuspended in culture medium Williams E (Biochrom AG, Berlin, Germany). The viability was determined by staining with trypan blue. The hepatocytes were plated on collagen-coated tissue culture dishes, prepared 1 day before hepatocyte isolation, and maintained in Williams E. For 24-well plates, hepatocytes were plated at a density 2.5 × 105 cells/dish. All of the cells were then grown in 5% CO2 at 37 °C for 2 h prior to treatment with DDP. Cytotoxicity Experiments-HepG2 and Huh7 cells were plated in 25-cm2 tissue culture flasks at a density of 7 × 105 cells (HepG2) or 3 × 105 cells (Huh7) in 5 ml of standard growth medium and left for 72 h in a CO2 incubator at 37 °C to form a monolayer. The cells were then treated with 0.1–100 μm DDP for 24 h. Copper deficiency was produced by incubating cells with 200 μm bathocuproine disulfonate (BCS) 48 h prior to and during treatment with DDP. The number of viable cells was determined by Casy TT® (Schärfe System, Reutlingen, Germany). The primary hepatocytes were cultured in a 24-well plate and then incubated with different concentration of DDP for 24 h. Drug-containing medium was then aspirated, and the cells were washed with PBS twice before the addition of MTT and then incubated with 200 μl of MTT solution (2 mg/ml PBS) for 2 h. MTT solution was removed, and 200 μl of Me2SO was added to each well to lyse cells. The percentage of viable cells after DDP treatment was determined photometrically by measuring the absorbance at 490 nm compared with untreated controls. ATP7B Expression in Hepatoma Cells-Huh7 and HepG2 cells were grown to >90% confluence and trypsinized in 0.05% trypsin/PBS. Then cells were centrifuged at 500 × g for 10 min at 4 °C. The cell pellets were resuspended in 1 mm 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.1% β-mercaptoethanol/PBS. The cells were homogenized and centrifuged for 10 min at 500 × g. The supernatant was subjected centrifugation for 30 min at 20,000 × g to sediment cell membranes. The membranes were resuspended in buffer containing 333 mm Tris, pH 6.8, 2.6 m urea, 3.3% SDS, and 0.1% β-mercaptoethanol. Protein concentration was determined (37Kruger N.J. Methods Mol. Biol. 1994; 32: 9-15PubMed Google Scholar), and Western blot analysis was carried out using antibody against the nucleotide-binding domain; staining with the anti-β-actin antibody was used as a protein loading control. Immunofluorescence Microscopy-HepG2 cells were grown in 10-cm2 cell culture dishes to >90% confluence before being trypsinized in 1 ml of trypsin solution (0.05% trypsin) and adjusted to 10 ml with the medium. In a 12-well tray, 16 μl of the 10 ml of cell suspension was seeded onto flamed glass coverslips and cultured in the appropriate growth media until the cells reached 80% confluence. The cells were then treated with either 200 μm BCS to decrease free copper levels in the medium, with 10–200 μm CuCl2 or with 10–200 μm DDP for 3 h. The cells were then fixed by immersion in acetone for 30 s at -20 °C. The cells were blocked overnight in 1% gelatin, 1% bovine serum albumin, 0.01% sodium azide in PBS at 4 °C and then incubated with the anti-ATP7B antibodies as in Ref. 38Bartee M.Y. Lutsenko S. Biometals. 2007; 20: 627-637Crossref PubMed Scopus (66) Google Scholar and trans-Golgi marker Syntaxin 6 (BD Sciences, San Jose, CA) in blocking solution for 1 h at room temperature (each antibody at 1:500 dilution). After washing four times with PBS for 1 h, the cells were treated with fluorescently labeled secondary antibodies (1:2000; Alexa Fluor 488 donkey anti-rat for ATP7B, Alexa Fluor 555 goat anti-mouse (Molecular Probes, Eugene, OR) for Syntaxin 6. The cells were then washed four times in PBS for 1 h, and then coverslips were mounted onto glass slides using mounting medium containing 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). The images were analyzed using a Zeiss Confocal Scanning microscope (Carl Zeiss, Göttingen, Germany). ATP7B Expression in Sf9 Insect Cells and Preparation of Membrane Fractions-The cells were infected with the recombinant virus encoding the wild-type ATP7B, the D1027A mutant (this mutation inactivates phosphorylation of invariant residue D1027), the ATP7B variant with the deletion of metal-binding sites 1–4 (ΔMBS1–4), and the ATP7B with CXXCto AXXA mutation of metal-binding sites 1–5, (mMBS1–5)) as previously described (29Tsivkovskii R. Eisses J.F. Kaplan J.H. Lutsenko S. J. Biol. Chem. 2002; 277: 976-983Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 39Huster D. Lutsenko S. J. Biol. Chem. 2003; 278: 32212-32218Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). To generate the CPC>SPS mutant, site-directed mutagenesis was performed using primers (forward) 5′-TGT GCA TTG CCA GCC CCA GCT CCC TGG GG-3′ and (reverse) 5′-CCC CAG GGA GCT GGG GCT GGC AAT GCA CA-3′ and using the wild-type ATP7B cDNA as a template; the baculovirus virus was generated as previously described (29Tsivkovskii R. Eisses J.F. Kaplan J.H. Lutsenko S. J. Biol. Chem. 2002; 277: 976-983Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). To obtain membrane preparations, the cells from 50 ml of culture were centrifuged at 500 × g for 10 min at 4 °C and resuspended in 5 ml of homogenizing buffer (25 mm imidazole, pH 7.4, 0.25 m sucrose, 1 mm dithiothreitol (DTT), and 1 mm 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride). One tablet of complete protease inhibitor mixture without EDTA (Roche) was added per 50 ml of buffer solution. The cells were homogenized 20 times in a manual glass homogenizer and then centrifuged for 10 min at 500 × g. The supernatant was subjected to an additional centrifugation for 30 min at 20,000 × g to sediment cell membranes. The pelleted cell membranes were resuspended in the homogenizing buffer. The membrane protein solution was stored frozen at -80 °C. Protein concentration was determined by the method of Lowry et al. (40Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Preparation of Vesicles for Copper Transport-The Sf9 insect cells were infected with recombinant virus or empty vector (used as a control) were harvested after 3 days. Cells from a 50-ml culture were pelleted, resuspended in 6 ml of ice-cold homogenizing buffer for vesicles: 50 mm Tris, pH 7.0, 50 mm mannitol, 2 mm EGTA, antipain, and leupeptin (each diluted 1:1000), phenylmethylsulfonyl fluoride (diluted 1:200). The cells were homogenized 20 times in a semi-automatic homogenizer (Schütt homgen plus, Göttingen, Germany) at 3000 rpm and then centrifuged for 10 min at 500 g at 4 °C. Subsequently, the supernatant was centrifuged for 1 h at 100,000 × g. The pelleted membranes were then resuspended in sterile filtered buffer (SMS; 50 mm sucrose, 100 mm potassium nitrate, 10 mm Hepes/Tris, pH 7.4). Vesicle formation was facilitated by vortexing and passing several times through a 25-gauge ⅝-inch needle. The vesicle solution was stored in liquid nitrogen. Protein concentration was determined by the method of Bradford (37Kruger N.J. Methods Mol. Biol. 1994; 32: 9-15PubMed Google Scholar). To confirm the ATP7B expression, the samples were analyzed by Western analysis using antibody against the nucleotide-binding domain of ATP7B (the region that is unaltered in all mutants). Cu64 Uptake in Vesicles-The transport assay has been carried as described by Gmaj et al. (41Gmaj P. Zurini M. Murer H. Carafoli E. Eur. J. Biochem. 1983; 136: 71-76Crossref PubMed Scopus (44) Google Scholar). The vesicle solution was thawed, diluted to 5 μg of protein/μl with sterile filtered SMS buffer and kept on ice. For the transport assay 20 μl of vesicle solution was preincubated at 37 °C for 1 min. Subsequently, the reaction was started at 37 °C by the addition of 80 μl of sterile filtered incubation solution (50 mm sucrose, 100 mm potassium nitrate, 10 mm Hepes/Tris pH 7.4, 12.5 mm magnesium nitrate, 12.5 mm DTT, 6.25 mm ATP, 2.5 μm copper chloride (final copper concentration, 2 μm), 6 μCi/ml Cu64 (Rotop Pharmaka, Radeberg, Germany), and 0–10 μm DDP). The reaction was stopped at different time intervals by the addition of 3 ml of ice-cold sterile filtered stopping solution (50 mm sucrose, 100 mm potassium chloride, 10 mm Hepes/Tris, pH 7.4, 0.5 mm EDTA) and filtered immediately through a 0.45-μm nitrocellulose vacuum filter system. The filter was washed with 3 ml of stopping solution before and after filtration, and its radioactivity was measured by the γ-Counter Cobra Quantum (PerkinElmer Life Sciences). The Effect of Copper or DDP on Phosphorylation of ATP7B with [γ-32P]ATP-75 μg of membrane protein was resuspended in 200 μl of phosphorylation buffer (20 mm bis-Tris propane, pH 6, 200 mm KCl, 5 mm MgCl2). 250 μm BCS was added and incubated on ice for 30 min; BCS was removed by centrifugation (5 min at 20,000 × g at 4 °C). The pellets were washed with phosphorylation buffer, resuspended in the same buffer containing 100 μm ascorbate and 100 μm tris(2-carboxyethyl)phosphine hydrochloride, and then incubated with of copper or DDP (0.5–10 μm) on ice for 10 min. In some cases, incubation with 2 μm DDP for 15 min was done prior to incubation with copper. To test the effect of BCS on phosphorylation, the metals were first removed by incubation with BCS as described above, then ATP7B was incubated for 10 min with either 10 μm copper (control) or DDP, then increasing concentrations of BCS (10–100 μm) were added for 30 min, and phosphorylation was measured. Radioactive [γ-32P]ATP (5 μCi; specific activity, 20 mCi/μmol; PerkinElmer Life Sciences) was added to a final concentration of 1 μm, and the reaction was incubated on ice for 4 min. In several experiments, 0–500 μm ADP was added on ice for 4 min prior to termination of the reaction. All of the additional treatments were done as described in the figure legends. The reaction was stopped by the addition of 50 μl of ice-cold stop solution (1 mm NaH2PO4 in 50% trichloroacetic acid) and then centrifuged for 10 min at 20,000 × g. The pellet was washed once with 1 ml of ice-cold water and centrifuged again for 5 min. The pellet was dissolved in 40 μl of sample buffer (5 mm Tris-PO4, pH 5.8, 6.7 m urea, 0.4 m DTT, 5% SDS, and bromphenol blue), and 30 μl were loaded on a 7% acidic gel (stacking gel: 5.5% acrylamide, 41.3 mm Tris, pH 5.8, 1% SDS, 5% ammonium persulfate, 5% N,N,N′,N′-tetramethylethylenediamine; separating gel: 7% acrylamide, 64.5 mm Tris-HCl, pH 6.8, 1% SDS, 6.25% ammonium persulfate, 6.25% N,N,N′,N′-tetramethylethylenediamine). After electrophoresis the gels were fixed in 10% acetic acid for 10 min and dried on blotting paper. The dried gels were exposed overnight either to screen for the Fuji BAS reader (Fuji, Düsseldorf, Germany) or Kodak BioMax MR film. The photon-stimulated-luminescence intensity of the bands was quantified using Aida Image Analyzer (Raytest, Straubenhardt, Germany). Papain Digestion and Western Blot Analysis of ATP7B-The membrane protein was solubilized in 1 mm MgCl2, 0.1% n-decyl-β-d-maltoside, 0.5 mm DTT, 10 mm cysteine, 20% glycerol, 50 mm MOPS·NaOH, pH 7 and centrifuged 5 min at 20,000 × g at 4 °C. The pellet was then resuspended and incubated in the buffer containing 500 μm BCS, 10 μm CuCl2, or 10 μm DDP at 30 °C for 1 h. Papain was activated in papain buffer (5 mm cysteine, 1 mm DTT, 50 mm MOPS·NaOH, pH 7) at room temperature for 30 min. ATP7B and papain were mixed 1:100–1:1000 at 30 °C for 2–200 min. The reaction was stopped by adding 1-trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane (E-64) up to a concentration of 2 mm. trichloroacetic acid was added to 2.5% immediately, and samples were centrifuged 5 min at 20,000 × g at 4 °C. Then the pellet was resuspended in 34 μl of loading buffer, and 30 μl was loaded onto 8–16% gradient gel (Pierce). The samples were transferred to polyvinylidene difluoride or nitrocellulose membrane (Millipore, Bedford, MA). The membranes were then incubated in 3% bovine serum albumin in PBS overnight at 4 °C. For the detection of ATP7B, anti-C-terminal domain antibody (generated against the recombinant protein corresponding to the last 92 residues of human ATP7B) was diluted in 2% bovine serum albumin in 0.1% PBS-T (1:3000) and incubated for 1.5 h at room temperature. Following extensive washing, the membranes were incubated with secondary antibody for 1 h at room temperature. The bands were visualized using Super Signal Chemiluminescent reagent (Pierce) and detected using x-ray films (Biomax O-Mat; Kodak). Statistical Analysis-The results are presented as the means ± S.D. Statistical differences were analyzed by Mann-Whitney-U test using SPSS software (SPSS Inc., Chicago, IL). The test of significance was two-tailed, and a p value of less than 0.05 was considered statistically significant. Hepatoma Cells HepG2 and Huh7 Show Different Sensitivity to DDP-Increased cellular ATP7B levels have been linked to changes in cell resistance to DDP and were interpreted as evidence for a major role of ATP7B in DDP efflux. To investigate the contribution of ATP7B to hepatoma cell response to DDP, the effect of DDP on cell survival was investigated using the HepG2 and Huh7 cells. These cells are both of human hepatic origin and have comparable levels of ATP7B (Fig. 1A). Therefore, if ATP7B plays a major role in DDP efflux, it was expected that both cell lines may have comparable sensitivity to the drug. However, the effect of DDP on cell viability was very different. The Huh7 cells showed a biphasic response with a sharp 50% decrease in cell survival and the half-maximum effect at 1.54 μm DDP (Fig. 1B). A further increase of the DDP concentration up to 100 μm induces a much smaller (additional 10%) decline in cell viability. In contrast, the HepG2 cells show monophasic decrease in cell viability with the EC50 for DDP equal to 16.15 μm. Higher DDP concentration in the medium induces larger decline in cell survival down to ∼20% at 100 μm (Fig. 1B). Primary Hepatocytes Lacking ATP7B Show Higher Resistance to DDP-The liver is the major detoxification organ, which mediates its function through multiple pathways. The individual contribution of these pathways may vary in cultured cell lines, thus producing distinct responses to DDP, as we observed for Huh7 and HepG2. Therefore, to examine the role of ATP7B more directly, we determined whether the elimination of endogenous ATP7B would render hepatocytes more sensitive to cisplatin. The primary hepatocytes were isolated from the control and Atp7b-/- mice, and their viability was compared in the presence of increasing concentration of DDP. Although in the absence of DDP the Atp7b-/- hepatocytes are generally less viable (as evidenced by the lower yield of cells during primary culture preparation), they showed higher resistance to cisplatin (EC50 = 135.2 μm) compared with control (EC50 = 57.8 μm) (Fig. 2). These results suggested that, in hepatocytes, ATP7B-mediated efflux did not play a major role in determining cell sensitivity to DDP. Trafficking of ATP7B in HepG2 Cells Is Not Regulated by DDP-One may argue that the inactivation of ATP7B in the Atp7b-/- liver induces significant remodeling of cell metabolism; this may involve up-regulation of other detoxification pathways and compensate for the loss of ATP7B function. Consequently,
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