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Experimental Therapeutics |
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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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS AND METHODS |
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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 x 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 x g for 10 min at 4°C. The
supernatant fraction was saved as postnuclear supernatant and was
centrifuged at 23,400 x 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 x 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 x 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 x g at
4°C. The interface was collected, diluted 1:5 in buffer B, and
centrifuged for 45 min at 100,000 x 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.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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).
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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)
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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).
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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 8/16/99. Accepted 1/ 5/00.
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K. Katano, A. Kondo, R. Safaei, A. Holzer, G. Samimi, M. Mishima, Y.-M. Kuo, M. Rochdi, and S. B. Howell Acquisition of Resistance to Cisplatin Is Accompanied by Changes in the Cellular Pharmacology of Copper Cancer Res., November 15, 2002; 62(22): 6559 - 6565. [Abstract] [Full Text] [PDF]
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X. Lin, T. Okuda, A. Holzer, and S. B. Howell The Copper Transporter CTR1 Regulates Cisplatin Uptake in Saccharomyces cerevisiae Mol. Pharmacol., November 1, 2002; 62(5): 1154 - 1159. [Abstract] [Full Text] [PDF]
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J. L. Nitiss A copper connection to the uptake of platinum anticancer drugs PNAS, October 29, 2002; 99(22): 13963 - 13965. [Full Text] [PDF]
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S. Ishida, J. Lee, D. J. Thiele, and I. Herskowitz From the Cover: Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals PNAS, October 29, 2002; 99(22): 14298 - 14302. [Abstract] [Full Text] [PDF]
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H. Niedner, R. Christen, X. Lin, A. Kondo, and S. B. Howell Identification of Genes That Mediate Sensitivity to Cisplatin Mol. Pharmacol., December 1, 2001; 60(6): 1153 - 1160. [Abstract] [Full Text] [PDF]
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) 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.
