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Experimental Therapeutics

Copper-transporting P-Type Adenosine Triphosphatase (ATP7B) Is Associated with Cisplatin Resistance

Masaharu Komatsu, Tomoyuki Sumizawa, Masato Mutoh, Zhe-Sheng Chen, Kunihiko Terada, Tatsuhiko Furukawa, Xiao-Li Yang, Hui Gao, Naoyuki Miura, Toshihiro Sugiyama and Shin-ichi Akiyama
Masaharu Komatsu
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 [M. K., T. Sum., Z-S. C., T. F., H. G., S-i. A.]; Basic Research Laboratories, Toray Industries, Inc., Kanagawa 248-8555 [M. M.]; and Department of Biochemistry, Akita University School of Medicine, Akita 010-8543 [K. T., X-L. Y., N. M., T. Sug.], Japan
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Tomoyuki Sumizawa
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 [M. K., T. Sum., Z-S. C., T. F., H. G., S-i. A.]; Basic Research Laboratories, Toray Industries, Inc., Kanagawa 248-8555 [M. M.]; and Department of Biochemistry, Akita University School of Medicine, Akita 010-8543 [K. T., X-L. Y., N. M., T. Sug.], Japan
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Masato Mutoh
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 [M. K., T. Sum., Z-S. C., T. F., H. G., S-i. A.]; Basic Research Laboratories, Toray Industries, Inc., Kanagawa 248-8555 [M. M.]; and Department of Biochemistry, Akita University School of Medicine, Akita 010-8543 [K. T., X-L. Y., N. M., T. Sug.], Japan
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Zhe-Sheng Chen
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 [M. K., T. Sum., Z-S. C., T. F., H. G., S-i. A.]; Basic Research Laboratories, Toray Industries, Inc., Kanagawa 248-8555 [M. M.]; and Department of Biochemistry, Akita University School of Medicine, Akita 010-8543 [K. T., X-L. Y., N. M., T. Sug.], Japan
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Kunihiko Terada
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 [M. K., T. Sum., Z-S. C., T. F., H. G., S-i. A.]; Basic Research Laboratories, Toray Industries, Inc., Kanagawa 248-8555 [M. M.]; and Department of Biochemistry, Akita University School of Medicine, Akita 010-8543 [K. T., X-L. Y., N. M., T. Sug.], Japan
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Tatsuhiko Furukawa
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 [M. K., T. Sum., Z-S. C., T. F., H. G., S-i. A.]; Basic Research Laboratories, Toray Industries, Inc., Kanagawa 248-8555 [M. M.]; and Department of Biochemistry, Akita University School of Medicine, Akita 010-8543 [K. T., X-L. Y., N. M., T. Sug.], Japan
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Xiao-Li Yang
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 [M. K., T. Sum., Z-S. C., T. F., H. G., S-i. A.]; Basic Research Laboratories, Toray Industries, Inc., Kanagawa 248-8555 [M. M.]; and Department of Biochemistry, Akita University School of Medicine, Akita 010-8543 [K. T., X-L. Y., N. M., T. Sug.], Japan
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Hui Gao
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 [M. K., T. Sum., Z-S. C., T. F., H. G., S-i. A.]; Basic Research Laboratories, Toray Industries, Inc., Kanagawa 248-8555 [M. M.]; and Department of Biochemistry, Akita University School of Medicine, Akita 010-8543 [K. T., X-L. Y., N. M., T. Sug.], Japan
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Naoyuki Miura
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 [M. K., T. Sum., Z-S. C., T. F., H. G., S-i. A.]; Basic Research Laboratories, Toray Industries, Inc., Kanagawa 248-8555 [M. M.]; and Department of Biochemistry, Akita University School of Medicine, Akita 010-8543 [K. T., X-L. Y., N. M., T. Sug.], Japan
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Toshihiro Sugiyama
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 [M. K., T. Sum., Z-S. C., T. F., H. G., S-i. A.]; Basic Research Laboratories, Toray Industries, Inc., Kanagawa 248-8555 [M. M.]; and Department of Biochemistry, Akita University School of Medicine, Akita 010-8543 [K. T., X-L. Y., N. M., T. Sug.], Japan
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Shin-ichi Akiyama
Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 [M. K., T. Sum., Z-S. C., T. F., H. G., S-i. A.]; Basic Research Laboratories, Toray Industries, Inc., Kanagawa 248-8555 [M. M.]; and Department of Biochemistry, Akita University School of Medicine, Akita 010-8543 [K. T., X-L. Y., N. M., T. Sug.], Japan
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DOI:  Published March 2000
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Abstract

The accumulation of cisplatin is decreased in many cisplatin-resistant cell lines, and an active efflux pump for cisplatin exists in some of them, but it has not yet been identified. In this study, we transfected the copper-transporting P-type ATPase cDNA (ATP7B) into human epidermoid carcinoma KB-3-1 cells. The transfectant, KB/WD cell line, which overexpressed the P-type ATPase, ATP7B, was resistant to both cisplatin (8.9-fold) and copper (2.0-fold). The accumulation of cisplatin in KB/WD cells was lower than in mock-transfected KB/CV cells, and the efflux of cisplatin from KB/WD cells was enhanced compared with KB/CV cells. KB/WD cells were sensitive to other heavy metals, such as antimony, arsenate, arsenite, cadmium, and cobalt. ATP7B was overexpressed in cisplatin-resistant prostate carcinoma PC-5 cells but not in the parental PC-3 cells and the revertant PC-5R cells. ATP7B may be involved in cisplatin resistance in some tumors.

INTRODUCTION

Cisplatin 3 is a neutral, square planar platinum(II) complex containing two chloride ligands oriented in a cis configuration. It has become one of the most effective chemotherapeutic agents for treating many malignancies, particularly for head and neck, testicular, ovarian, bladder, esophageal, and small cell lung cancers (1) . However, intrinsic or acquired resistance to cisplatin reduces its efficacy (2) . The mechanisms of resistance include inactivation of the drug by thiol compounds, decreased cisplatin accumulation, accelerated DNA repair, and an increase in MT (3 , 4) . The accumulation of cisplatin is frequently decreased in cisplatin-resistant cell lines, and an active efflux system for cisplatin exists in some of them (4, 5, 6, 7, 8, 9, 10, 11, 12) . ATP binding cassette transmembrane transporters, MRP and cMOAT, have been reported to be associated with cisplatin resistance (13, 14, 15, 16) . Although the accumulation of cisplatin was reduced, the transporters were not detected in cisplatin-resistant KCP-4 cells derived from human epidermoid carcinoma KB-3-1 cells (9) . These findings suggest that an unknown pump is involved in cisplatin resistance.

Copper, an essential trace element, is an integral component of many enzymes, which use its unique redox properties for their biological function (17) . However, excess accumulation of copper in the body is toxic. Thus, excess toxic levels of copper ions in the cells are excreted to the extracellular environment by an energy-dependent efflux system (18) . Wilson’s disease is an inherited autosomal recessive disorder of copper transport, which is manifested by chronic liver disease and/or neurological impairment and frequently by kidney malfunction. It is characterized by defective biliary copper excretion and a disturbance in the incorporation of copper into CPN (19) , resulting in excessive copper accumulation, primarily in the liver and also in the brain and kidney (20) . In addition, it is believed to be caused by mutations in the gene (ATP7B) that encodes a copper transporting P-type ATPase, and various mutations in this gene have been reported in patients with Wilson’s disease (21, 22, 23) . The ATP7B product, a protein of 1465 amino acids (ATP7B), is expressed predominantly in liver, kidney, and placenta in humans (24) . P-type ATPases, defined as those forming a covalent phosphorylated intermediate in their reaction cycle, transport a variety of cations across membranes (25) . Their general features include a TGEA/S motif (phosphatase domain), a DKTGT/S motif (phosphorylation domain), a TGDN motif (ATP-binding domain), and a MXGDGXNDXP sequence that connects the ATP-binding domain to the transmembrane segment (24 , 26) . Among P-type ATPases, the ATP7B is a member of a class of heavy metal-transporting, P-type ATPases that pump copper, cadmium, zinc, silver, or lead (27, 28, 29, 30) . ATP7B contains several unique features including eight transmembrane segments, six copper-binding motifs (GMTCXXC) at its NH2 terminus, a CPC motif in an intramembranous region, and a SEHPL motif (24) .

In this study, we transfected ATP7B cDNA into KB-3-1 cells to investigate whether the transfectants acquire resistance to cisplatin as well as to copper. We found that ATP7B conferred resistance to cisplatin.

MATERIALS AND METHODS

Cell Culture and Cell Lines

Human epidermoid KB carcinoma cells were obtained from Dr. M. M. Gottesman (National Cancer Institute, Bethesda, MD). They were subcloned twice, and a single recloned line, KB-3-1, was used as the parental cell line for the present study (31) . KB-3-1 cells were cultured in minimal essential medium (Nissui Seiyaku Co., Tokyo, Japan) containing 10% newborn calf serum, 1 mg/ml bactopeptone, 0.292 mg/ml glutamine, and 100 units/ml penicillin (MEM) under 5% CO2 at 37°C. Cisplatin-resistant KCP-4 cells were isolated from KB-3-1 cells by inducing mutagenesis with ethyl methanesulfonate and then incubating cells in selection media containing stepwise increasing concentrations (from 7 to 30μ m) of cisplatin (7) . PC-5 (P/CDP-5) cells were spontaneously isolated from a human prostate carcinoma PC-3 cell line and cultured as described (32 , 33) . The revertant KCP-4R and PC-5R cells were isolated from KCP-4 and PC-5 cells, respectively, by culturing in nonselective medium for at least 4 years.

Transfection of KB-3-1 Cells with ATP7B cDNA

Full-length human ATP7B cDNA was constructed as described (34) . To detect the expression of the ATP7B cDNA, the HA epitope (Boehringer Mannheim, Mannheim, Germany) was inserted into the constructed cDNA at the Eco47III site, corresponding to nucleotide number 4327 in wild-type ATP7B cDNA. The constructed cDNA was inserted into the mammalian expression vector pRc/CMV (Invitrogen, NV Leek, the Netherlands), which contains the bacterial neomycin phosphotransferase gene conferring resistance to G418.

KB-3-1 cells were transfected with pRc/CMV-ATP7B or empty vector with LipofectAMINE (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s directions for HeLa cells. The cells were first selected in the presence of 500 μg/ml G418 for 3 days and were then cultured in the presence of 250 μg/ml G418 for 6 days. In the case of ATP7B-transfected KB/WD cells, cells from G418-resistant colonies were further selected in media containing stepwise increasing concentrations (10, 30, and 50 μm) of CuCl2.

RT-PCR

Total cellular RNA was extracted by a single-step method using TRIzol (Life Technologies, Inc.). For RT-PCR, 10 μg of total RNA were used for cDNA synthesis using the Superscript Preamplification System (Life Technologies, Inc.), according to the manufacturer’s protocol. The 833-bp target in ATP7B was amplified by using the forward 22 mer, 5′-TCCTGGTGGCTATTGACGGTGT-3′ at positions 3539–3560 and reverse 24 mer, 5′-CATTCAGGCGCAGAGACCACTT-3′ at positions 4372–4349.

Preparation of Cytosol and Crude Membrane Fractions

Cells were washed and scraped with a rubber scraper into PBS containing 1% aprotinin (Sigma Chemical Co., St. Louis, MO) and centrifuged at 1000 × g for 3 min. The pellet was homogenized in hypotonic lysis buffer, 10 mm Tris-HCl (pH 8.0), containing 10 mm KCl, 1 mm MgCl2, and 1 mm p-amidinophenyl methanesulfonyl fluoride hydrochloride (Wako, Osaka, Japan), and centrifuged at 1000 × g for 10 min at 4°C. The supernatant fraction was saved as postnuclear supernatant and was centrifuged at 23,400 × g for 20 min at 4°C to separate the cytosol and crude membrane fractions.

Preparation of Membrane Vesicles

As described previously (35) , membrane vesicles were prepared by a nitrogen cavitation method. Cell monolayers (109 cells) were washed once and scraped into PBS containing 1% aprotinin. The cells were washed in PBS, collected by centrifugation (4000 × g for 10 min) at 4°C, suspended in buffer A [0.01 m Tris-HCl (pH 7.5) containing 0.25 m sucrose and 0.2 mm CaCl2] and equilibrated at 4°C under a nitrogen pressure of 28 kg/cm2 for 15 min. EDTA was added to the lysed cell suspension to a final concentration of 1 mm. The lysed cell suspension was then diluted 1:4 with buffer B [0.01 m Tris-HCl (pH 7.5) and 0.25 m sucrose] and centrifuged at 1000 × g for 10 min at 4°C to remove nuclei and unlysed cells. The supernatant was layered onto a 35% sucrose cushion [0.01 m Tris-HCl (pH 7.5) containing 35% sucrose and 1 mm EDTA], and centrifuged for 30 min at 16,000 × g at 4°C. The interface was collected, diluted 1:5 in buffer B, and centrifuged for 45 min at 100,000 × g at 4°C. The vesicle pellet was resuspended in buffer B using a 25-gauge needle. Vesicles were stored at −80°C.

Immunoblotting

The cytosol, crude membrane, or membrane vesicles (50 or 100 μg of protein) were subjected to 6% SDS-PAGE under reducing conditions according to the method of Laemmli (36) . The samples were not heated prior to electrophoresis to reduce ATP7B aggregation. Transfer to PVDF membranes (Immobilon-P, Millipore, Bedford, MA) was performed electrophoretically for 30 min at 15 V (constant voltage) using a Transblot SD apparatus (Bio-Rad, Richmond, CA) as described by Kyhse-Anderson (37) . The membrane was blocked with 5% skimmed milk in buffer C [0.35 m NaCl, 10 mm Tris-HCl (pH 8.0), and 0.05% Tween 20] for 1 h at room temperature and then incubated overnight at 4°C with 2000-fold diluted polyclonal antibody against HA epitope (Santa Cruz Biotechnology, Santa Cruz, CA) or 500-fold diluted monoclonal antibody against the NH2-terminal region of ATP7B, which included the six copper-binding domains (amino acid number from 21 to 623; Ref. 38 ). The membrane was washed three times with buffer C and then incubated for 1 h with 1000-fold diluted horseradish peroxidase conjugated antirabbit IgG or antimouse IgG (Amersham, Buckinghamshire, United Kingdom) for detection of ATP7B. The PVDF membrane was rinsed once for 15 min and four times for 5 min with buffer C and then evenly coated using the ECL Western blotting detection system (Amersham, Buckinghamshire, United Kingdom) for 1 min. The membrane was immediately exposed to Fuji medical X-ray film (RX-U; Fujifilm, Kanagawa, Japan) in a film cassette at room temperature for various periods.

Cell Survival by MTT Assay

The MTT colorimetric assay was used to assess the sensitivity of the cells to agents in vitro as described (39) . Exponentially growing cells were trypsinized and harvested, and equal numbers of cells in 180 μl of MEM were inoculated into each well of a 96-well microplate. After incubating overnight, 20 μl of agent solution were added to the cultures, and they were incubated for 4 days. Surviving cells were determined as described (9) . The IC50 was measured as the concentration of agents that reduced the number of cells to 50% of that in control medium.

Cisplatin Accumulation and Efflux

To measure cisplatin accumulation, confluent KB/WD and KB/CV cells in 100-mm plastic dishes were incubated for 2 days in DMEM (Life Technologies, Inc.) containing 10% newborn bovine serum and then incubated for 2 h at 37°C with cisplatin at the indicated concentrations. Cells were washed three times with cold PBS and immediately harvested with a rubber scraper. The harvested cells were again washed three times with cold PBS. Cells were counted with a hemocytometer (Microcell counter F-300; Sysmex, Kobe, Japan) before the last wash.

To measure cisplatin efflux, confluent KB/WD and KB/CV cells were incubated for 10 min at 37°C with 500 μm cisplatin in glucose-free HBSS containing 1 mm 2,4-dinitrophenol. The cells were then incubated at 37°C for the indicated times in HBSS without cisplatin and harvested. Cells were counted with a hemocytometer.

Cell pellets were hydrolyzed in nitric acid, and the platinum content was determined by inductive coupled plasma mass spectrometry using a Model SPQ6500 apparatus (Seiko Instruments, Tokyo, Japan).

Statistical Analysis

Differences between groups were analyzed by Student’s t test. P < 0.05 was considered significant. Significance levels given are those for the two-tailed Student’s t test.

RESULTS

Expression of ATP7B in ATP7B cDNA Transfected Cells

We transfected ATP7B cDNA with an HA tag into KB-3-1 cells. The fragment of ATP7B mRNA containing the HA tag sequence was detected at 914 bp in one clone, KB/WD, by RT-PCR, and it was distinguished from an endogenously faintly expressed ATP7B mRNA fragment (833 bp; Fig. 1A ⇓ ). It was not detected in KB-3-1 cells transfected with empty vector (KB/CV cells). The expression of ATP7B was examined by immunoblotting using anti-HA antibody. A Mr 170,000 ATP7B with the HA tag was detected in KB/WD cells but not in KB/CV cells (Fig. 1B) ⇓ . The HA signal was also not present in parental KB-3-1 cells (data not shown).

Fig. 1.
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Fig. 1.

A, expression of ATP7B mRNA in KB/WD, KB/CV, and HepG2 cells was detected by RT-PCR. PCR was done using a set of sense (position 3539–3560) and antisense primers (position 4372–4349). Amplified fragment contained a HA tag sequence in the transfected ATP7B cDNA. B, immunoblot for ATP7B with HA tag in KB/WD cells. The crude membrane fractions (100 μg of protein) were subjected to 6% SDS-PAGE and transferred to a PVDF membrane. The blots were probed with rabbit anti-HA polyclonal antibody (1:2000), followed by horseradish peroxidase-conjugated antirabbit IgG (1: 2000). Bound antibody was detected by enhanced chemiluminescence.

Cross-Resistance to Heavy Metals

The sensitivity of the transfected cells to the heavy metals was measured by the MTT assay. Interestingly, in cells transfected with copper transporter cDNA (KB/WD), the level of resistance to cisplatin was higher than to copper. KB/WD cells were 8.9-fold more resistant to cisplatin than KB/CV cells (Fig. 2 ⇓ and Table 1 ⇓ ). KB/WD cells were 2.02- and 1.97-fold more resistant to CuCl2 and CuSO4 than KB/CV cells, respectively, but they were not resistant to arsenite, arsenate, antimony, CoCl2, or CdCl2 (Table 1) ⇓ .

Fig. 2.
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Fig. 2.

Sensitivity of KB/WD and KB/CV cells to copper and cisplatin. KB/WD (•) and KB/CV cells (○) were exposed to various concentrations of CuCl2 (A), CuSO4 (B), or cisplatin (C) and incubated for 4 days. The cytotoxic effect of the agents was examined using the MTT assay. Values are means of triplicate determinations; bars, SD.

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Table 1

Cross-resistance to heavy metals in KB/WD cells

Cisplatin Accumulation

To investigate whether ATP7B is involved in cisplatin resistance, the accumulation of cisplatin in KB/WD and KB/CV cells was examined. When the cells were incubated in medium containing 20, 40, and 80μ m cisplatin, the intracellular levels of platinum in KB/WD cells were approximately 56, 61, and 59% of those in KB/CV cells, respectively (Fig. 3) ⇓ .

Fig. 3.
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Fig. 3.

Accumulation of cisplatin in KB/CV and KB/WD cells. Intracellular levels of platinum were determined by inductive coupled plasma mass spectrometry. Columns, means of triplicate determinations; bars, SD. ∗, P < 0.05; ∗∗, P < 0.01.

Efflux of Cisplatin

We examined whether the decreased accumulation of cisplatin in KB/WD cells was attributable to enhanced active efflux or reduced uptake of cisplatin. When KB/WD and KB/CV cells were incubated with 500μ m cisplatin for 10 min at 37°C and then without cisplatin for 30 min, the percentage of cisplatin remaining in KB/WD cells was 81.9%. On the other hand, there was no efflux of cisplatin from KB/CV cells (Fig. 4A) ⇓ .

Fig. 4.
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Fig. 4.

A, efflux of cisplatin. The release of cisplatin from KB/WD (•) and KB/CV cells (○) in the presence of ATP is shown. Points, means of triplicate determinations; bars, SD. ∗, P < 0.03. B, effect of 2,4-dinitrophenol on accumulations of cisplatin. Intracellular levels of platinum in the presence (ATP−) or absence (ATP+) of 1 mm 2,4-dinitrophenol at 0 min in the efflux experiment (A). Data represent means of triplicate determinations; bars, SD. ∗, P < 0.05;∗∗ , P < 0.01.

2,4-Dinitrophenol is an uncoupler of oxidative phosphorylation. It allows electron transport to continue but prevents the phosphorylation of ADP to ATP. Viability of the cells was not decreased after a 60-min treatment with 1 mm 2,4-dinitrophenol in glucose-deficient HBSS. Accumulation of cisplatin at time 0 in the efflux study was∼ 1.6 times enhanced in KB/WD cells but not in KB/CV cells by 2,4-dinitrophenol. The accumulation of cisplatin in KB/WD cells in the presence of 2,4-dinitrophenol was nearly the same as that in KB/CV cells without 2,4-dinitrophenol (Fig. 4B) ⇓ . Thus, ATP7B expressed in KB/WD cells seems to transport cisplatin and be involved in resistance to it.

Expression of ATP7B in Cisplatin-resistant Cancer Cells

We analyzed the endogenous expression of ATP7B in three cisplatin-resistant human cell lines, prostate carcinoma PC-5, epidermoid carcinoma KCP-4, and ovarian cancer A2780/CP cells, and compared it with that in parental and revertant cells. Endogenous ATP7B with Mr 165,000 was strongly detected in PC-5 cells but not in PC-3 and PC-5R cells (Fig. 5) ⇓ . However, the protein was faintly detected in A2780/CP and KCP-4 cells as well as in parental and revertant cells (data not shown).

Fig. 5.
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Fig. 5.

Immunoblots for ATP7B. Membrane vesicles (Lanes 1–3, 100 μg of protein; Lane 4, 50 μg protein) from PC-3 (Lane 1), PC-5 (Lane 2), PC-5R (Lane 3), and HepG2 cells (Lane 4) were subjected to 6% SDS-PAGE and transferred to PVDF membrane. The blots were probed with mouse antihuman ATP7B monoclonal antibody (1:500), followed by horseradish peroxidase-conjugated antimouse IgG (1:2000). Bound antibody was detected by enhanced chemiluminescence.

DISCUSSION

Cisplatin is a platinum compound that is one of the most effective agents in cancer chemotherapy. We transfected full-length ATP7B cDNA into KB-3-1 cells to investigate whether the ATP7B confers not only copper but also cisplatin resistance to KB/WD cells. KB/WD cells were resistant to cisplatin and defective in accumulating cisplatin and actively effluxed it. Although ATP7B is a copper transporter, it also transported cisplatin. This is the first report that a member of the heavy metal-transporting, P-type ATPase family is involved in cisplatin resistance and transports cisplatin or its metabolite(s).

We have suggested previously the existence of an active efflux pump for cisplatin in KCP-4 cells (7, 8, 9 ,, 40) . Reduced accumulation and enhanced energy-dependent efflux of cisplatin have also been observed in other cisplatin-resistant cells (5 , 6 , 11 , 12) . Drug resistance mediated by active efflux pumps is widespread from prokaryotes to eukaryotes. A multidrug resistance phenotype in mammalian cells is often correlated with overexpression of members of the ATP binding cassette transmembrane transporter superfamily, such as P-glycoprotein (41) , MRP (42) , or cMOAT (14) . P-glycoprotein is not associated with cisplatin resistance (43) . Ishikawa et al. (13) speculated that overexpression of MRP and an increased level of GSH in cisplatin-resistant human leukemia HL-60 cells are responsible for cisplatin resistance. However, an MRP-transfected cell line that expressed MRP showed no cross-resistance to cisplatin (44) . Koike et al. (15) reported that transfection of cMOAT antisense cDNA into HepG2 cells reduced cMOAT expression, as well as GSH levels, and the transfected cells were∼ 5-fold more sensitive to cisplatin than cells transfected with empty vector (15) . cMOAT cDNA transfected cells, LLC/cMOAT-1, had a 3-fold increased resistance to cisplatin (16) . These findings suggest that cMOAT is involved in low resistance to cisplatin. However, neither MRP nor cMOAT was detected in KCP-4 cells (9) . Thus, an unknown export pump for cisplatin that is different from MRP or cMOAT exists in KCP-4 cells (8 , 9 , 40) .

We became interested in the heavy-metal transporting P-type ATPase family, such as ATP7B. A peculiar feature of ATP7B is the presence of a large NH2-terminal domain, which contains six repeats of a putative copper-binding domain. Each domain consists of∼ 30 amino acids and contains one copy of a GMTCXXC motif. The two cysteine residues in each repeat are most likely involved in metal binding (27) . DiDonato et al. (45) examined the multiple metal binding abilities of the copper-binding domain of ATP7B. The binding ability of the copper-binding domain for at least Zn2+, Hg2+, Au3+, and Cd2+ was revealed by immobilized metal ion affinity chromatography or competition zinc blotting assays (45) . However, the binding site for cisplatin on ATP7B is thus far unknown.

The reason why the level of resistance to cisplatin was higher than to copper in KB/WD cells is thus far unclear. Normal copper transport in hepatocytes is described as follows. After copper is taken up into hepatocytes by a plasma membrane transporter and forms copper-ligand complexes, it is transported into the lumen of the Golgi apparatus and secretory vesicles by ATP7B (24) . Subsequently, apoCPN incorporates this copper and is secreted into blood as oxidase-active holoCPN. This protein is synthesized mainly in hepatocytes and secreted into plasma with six atoms of copper/molecule of holoCPN (19) . In our previous study (34) , ATP7B cDNA was introduced into the Long-Evans cinnamon rat, an animal model for Wilson’s disease, by recombinant adenovirus-mediated gene delivery. Subsequently, the transgene expression in the Long-Evans cinnamon rat liver was localized to the Golgi apparatus of hepatocytes. In addition, secretion of holoCPN was detected after introducing the ATP7B cDNA but not the adenovirus alone. These data indicate that ATP7B function in copper transport may be coupled with CPN synthesis. In this investigation, we transfected KB-3-1 cells with ATP7B cDNA alone without CPN cDNA. Endogenous CPN was scarcely expressed in KB/WD as well as KB-3-1 and KB/CV cells (data not shown), because KB cells were not derived from hepatocytes. These results suggest that resistance to copper involves both the ATP7B and CPN in KB/WD cells. Thus, the level of resistance of KB/WD cells to copper may have been lower than to cisplatin.

During the selection of an ATP7B-expressing colony, we incubated the colonies in the medium containing copper to eliminate the colonies that express ATP7B at a low levels to obtain colonies of high expression. It is possible that secondary mechanisms of copper resistance distinct from ATP7B are involved in this transfectant as a result of this selection technique. Copper reportedly induced MT in human hepatocytes. MT and GSH are known to be important protective molecules against copper toxicity. GSH was also involved in the transport of copper out of hepatocytes via cMOAT (46) . Increased levels of MT, GSH, and cMOAT have been reported to be involved in cisplatin resistance (4) . Therefore, we examined the levels of these molecules in KB/WD cells and found that they were similar to those in KB/CV cells (data not shown). These findings suggest that MT, GSH, and cMOAT are not involved in cisplatin resistance in KB/WD cells.

Expression of the ATP7B was enhanced in human prostate cisplatin-resistant PC-5 cells compared with its parental PC-3 cells or its revertant PC-5R cells. The expression level of ATP7B mRNA in PC-5 cells was also higher than in PC-3 and PC-5R cells (data not shown). We have analyzed previously the sensitivity to cisplatin of PC-3, PC-5, and PC-5R cells by MTT assay (7) . The IC50s for PC-3, PC-5, and PC-5R cells were 5.1, 50, and 3.2 μm cisplatin, respectively. PC-5 cells were ∼9.8 times resistant to cisplatin compared with the parental PC-3 cells, and cisplatin resistance had completely reverted in PC-5R cells. These results suggest that the ATP7B might be, at least in part, involved in cisplatin resistance in PC-5 cells. KCP-4 cells scarcely expressed ATP7B, and the expression level of ATP7B in KCP-4 cells was similar to that in KB-3-1 and KCP-4R cells (data not shown). We presume that another unknown pump is expressed in KCP-4 cells.

We suppose that unknown heavy metal-transporting P-type ATPases such as a ATP7B may function to efflux cisplatin from some carcinoma cells. ATP7B may be involved in acquired cisplatin resistance of certain tumors and in intrinsic cisplatin resistance in some tumors such as hepatocytes. It is necessary to establish ATP7B and cisplatin resistance in clinical samples to clarify this.

Acknowledgments

We thank Etsuko Sudoh for excellent technical assistance and Hiromi Kakura for excellent secretarial assistance.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This study was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, and the Ministry of Health and Welfare, Japan.

  • ↵2 To whom requests for reprints should be addressed, at Department of Cancer Chemotherapy, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520. Fax: 81-99-265-9687; E-mail: akiyamas{at}khosp2.kufm.kagoshima-u.ac.jp

  • ↵3 The abbreviations used are: cisplatin, cis-diamminedichloroplatinum(II); MT, metallothionein; MRP, multidrug resistance associated protein; cMOAT, canalicular multispecific organic anion transporter; CPN, ceruloplasmin; HA, hemagglutinin; G418, geneticin; RT-PCR, reverse transcription-PCR; PVDF, polyvinylidene difluoride; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GSH, glutathione.

  • Received August 16, 1999.
  • Accepted January 5, 2000.
  • ©2000 American Association for Cancer Research.

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Copper-transporting P-Type Adenosine Triphosphatase (ATP7B) Is Associated with Cisplatin Resistance
Masaharu Komatsu, Tomoyuki Sumizawa, Masato Mutoh, Zhe-Sheng Chen, Kunihiko Terada, Tatsuhiko Furukawa, Xiao-Li Yang, Hui Gao, Naoyuki Miura, Toshihiro Sugiyama and Shin-ichi Akiyama
Cancer Res March 1 2000 (60) (5) 1312-1316;

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Copper-transporting P-Type Adenosine Triphosphatase (ATP7B) Is Associated with Cisplatin Resistance
Masaharu Komatsu, Tomoyuki Sumizawa, Masato Mutoh, Zhe-Sheng Chen, Kunihiko Terada, Tatsuhiko Furukawa, Xiao-Li Yang, Hui Gao, Naoyuki Miura, Toshihiro Sugiyama and Shin-ichi Akiyama
Cancer Res March 1 2000 (60) (5) 1312-1316;
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