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Department of Medicine and the Cancer Center, University of California, San Diego, La Jolla, California (K.K., R.S., G.S., A.H., S.B.H); and Globomax, Inc., Hanover, Maryland (M.R.)
Received January 21, 2003; accepted April 30, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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). The
mechanisms by which DDP and CBDCA enter and exit from cells are not well
characterized. Cellular accumulation of these drugs is relatively slow
compared with many other classes of chemotherapeutic agents. Given their
degree of hydrophilicity and polarity, transporters are likely to be required
for both influx and efflux. Indeed, uptake is influenced by factors that
suggest the involvement of both active and passive transporter-mediated
processes (Gately and Howell,
1993).
One curious feature of cells with acquired DDP resistance is that they
exhibit cross-resistance to a wide variety of metalloids, including arsenite
(Naredi et al., 1995),
antimony (Naredi et al., 1995;
Chen et al., 1998), and cadmium
(Lee et al., 1995). Recently,
we reported that these cells are also cross-resistant to copper
(Katano et al., 2002a), and
that cells selected for acquired resistance to copper are cross-resistant to
DDP (Safaei and Howell, 2001).
Copper homeostasis is maintained by a complex system of transporters and
chaperones that serve to both protect Cu(I) against oxidation and to prevent
the production of toxic reactive oxygen species as the copper enters and is
distributed throughout the cell
(O'Halloran and Culotta,
2000). The central feature of this system is a group of proteins
with unique metal binding sequences that chelate copper into protective
pockets and exchange it through intimate protein-protein interactions such
that copper is virtually never free in the cell
(Pufahl et al., 1997;
Rae et al., 1999). The primary
uptake transporter for copper is hCTR1
(Zhou and Gitschier, 1997;
Moller et al., 2000), and its
function in mammals is essential for embryonic survival
(Kuo et al., 2001;
Lee et al., 2001). hCTR1
transfers copper to one of three known copper chaperones: Cox17, hCCS1, and
HAH1. Cox17 delivers copper to cytochrome c oxidase, hCCS1 transfers
copper to SOD1, and HAH1 hands it to one or another of the two P-type ATPases,
ATP7A and ATP7B, that are reported to sequester copper into the
trans-Golgi network where it is loaded onto copper-dependent enzymes
in the secretory pathway and subsequently exported from the cell
(Klomp et al., 1997).
Mutations that disable ATP7A cause Menkes disease, and mutations that disable
ATP7B cause Wilson's disease (Bull et al.,
1993; Tanzi et al.,
1993; Yamaguchi et al.,
1993). ATP7A and ATP7B both have structural features common to
other heavy metal P-type ATPases, including six metal binding sequence motifs
in the N-terminal cytoplasmic domain
(Solioz and Vulpe, 1996). Both
molecules undergo copper-induced redistribution from the trans-Golgi
network; ATP7A traffics to the plasma membrane
(Petris et al., 1996;
Cobbold et al., 2002), whereas
ATP7B becomes relocalized to vesicular structures
(Hung et al., 1997;
Schaefer et al., 1999;
Roelofsen et al., 2000;
Vanderwerf et al., 2001).
A direct link between copper transport and DDP resistance has been
identified by Komatsu et al.
(2000) who found that prostate
cancer cells selected for resistance to DDP expressed increased levels of
ATP7B and that cells transfected with ATP7B were resistant to both copper and
DDP. Increased levels of ATP7B mRNA or protein expression have been noted in
several major human malignancies, including ovarian cancer
(Nakayama et al., 2002),
gastric carcinoma (Ohbu et al.,
2003), breast cancer (Kanzaki
et al., 2002), and various malignant cell lines
(Nakayama et al., 2001). We
have noted that cells selected for DDP resistance frequently overexpress
either ATP7A or ATP7B (Katano et al.,
2002b). To further investigate the role of ATP7B in platinum drug
resistance, we molecularly engineered human carcinoma cell lines to
overexpress ATP7B and examined them for changes in their sensitivity to CBDCA.
We report here data demonstrating that ATP7B renders cells resistant to CBCDA
as well as copper and DDP and that this is associated with parallel reductions
in the cellular accumulation of both copper and CBDCA.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Cells and Vectors. The human ovarian carcinoma 2008 and IGROV-1 cell
lines, and the human head and neck squamous carcinoma cell line UMSCC10b were
grown in drug-free RPMI 1640 medium plus 10% fetal calf serum and maintained
in humidified air containing 5% CO2 at 37°C. A pRC/CMV vector
containing the full-length ATP7B cDNA and expressing a G-418 resistance
marker, constructed as described previously
(Terada et al., 1998;
Harada et al., 2000), was
generously provided by Dr. Toshihiro Sugiyama. Cells were transfected with
pRc/CMV-ATP7B or empty vector with LipofectAMINE (Invitrogen, Carlsbad, CA)
according to the manufacturer's directions. Transfected cells were selected in
the presence of 500 µg/ml G-418, and all surviving clones were combined for
form a multiclonal population. The 2008/ATP7B cells were further engineered to
express GFP by infecting them with the amphotropic pMSCV-EGFP retrovirus
generously provided by Dr. Martin Hass
(Fink et al., 1998;
Norris et al., 1998). Seven
days after infection, the 5% of the population with the brightest green
fluorescence was isolated by flow activated cell sorting and grown to mass
culture in medium containing 500 µg/ml G-418 and 1 µg/ml puromycin.
Assays of Drug Sensitivity. Colony assays were performed using triplicate cultures of 200 cells/35-mm plate grown in 5 ml of medium containing different concentrations of DDP, CBDCA, or CuSO4 until visible colonies had formed (10–14 days). The dishes were rinsed twice with PBS, fixed with 100% methanol, and stained with a 0.1% crystal violet solution. A ChemiImager 400 instrument (Alpha Innotech, San Leandro, CA) was used for counting colonies of >50 cells. Enrichment assays were performed by preparing a population containing 10% 2008/ATP7B-GFP cells and 90% 2008/EV cells, seeding 105 cells into 100-mm culture dishes, incubating them together for 24 h, and then exposing them to graded concentrations of DDP, CBDCA, or copper for 1 h. Fresh non–drug-containing medium was then added and the cells grown for 5 days before being harvested by trypsinization and analyzed for the percentage of 2008/ATP7B-GFP cells in the surviving population by flow cytometry. Each assay was performed with triplicate cultures.
Cellular Pharmacokinetic Assays. Uptake and efflux measurements were
made using 35-mm dishes seeded with 106 cells each and incubated in
medium until they were 75 to 80% confluent. For cellular accumulation
experiments, the medium was replaced by 1 ml of fresh medium containing 2
µM 64CuSO4 or 50 µM[14C]CBDCA and the
cells were incubated at 37°C. Copper and CBDCA concentrations were
selected based on prior reports indicating that alterations in the cellular
pharmacokinetic parameters could be detected at these concentrations
(Shen et al., 2000;
Lee et al., 2002b). Efflux
rates were measured by exposing the cells to 2 µM 64CuSO4 or 50
µM [14C]CBDCA for 1 h, rinsing them with fresh medium, and
incubating them in drug-free medium at 37°C. At the requisite time points
in both types of experiments, the medium was poured off and the dishes were
quickly rinsed three times with ice-cold PBS after which the cells were
harvested using a plastic scraper and transferred to scintillation vials
containing 3 ml of scintillation solution (National Diagnostics, Atlanta, GA).
64Cu and [14C]CBDCA were quantified by scintillation
counting. Six separate dishes were used for each time point in each
experiment. Cells harvested from a separate group of six dishes were used to
measure protein content by the Bradford assay.
Measurement of Basal Copper Content. Cultures were quickly rinsed three times with ice-cold PBS, and cells were harvested into 15 ml of ice-cold PBS using a rubber policeman. After centrifugation at 3000 rpm for 10 min, the cells were resuspended in PBS, an aliquot was used for protein assay, and the remainder was digested in 68% nitric acid. Cell lysates were heated for 2 h at 65°C diluted to 5% nitric acid and assayed for platinum and copper content. An inductively coupled plasma optical emission spectroscopy apparatus (model 3000DV; PerkinElmer Life Sciences) at the Analytical Facility at the Scripps Institute of Oceanography was used for copper and platinum assays.
Pharmacokinetic Analysis. Mean data were fitted using a two-compartment pharmacokinetic model assuming a first order disposition process using WinNonlin Professional 3.1 (Pharsight, Mountain View, CA).
Western Blotting. Cells were rinsed twice with PBS, scraped from the
dish in PBS, and centrifuged for 10 min at 2500 rpm. Cells were lysed in 0.25%
Nonidet P-40 in 100 mM Tris HCl, pH 8, supplemented with 1 mM
p-amidinophenylmethylsulfonyl fluoride hydrochloride and 1 mM
-amino-n-caproic acid (Sigma-Aldrich) at 4°C and for 30
min. Postnuclear fractions were obtained by centrifugation of cell lysates for
10 min at 600g. Samples containing 50 to 100 µg of protein were
electrophoresed on 4 to 10% SDS polyacrylamide gels and then blotted onto
nitrocellulose filters using a Bio-Rad Mini Transblot apparatus (Bio-Rad).
Blots were incubated for 1 h with 5% fat-free dry milk in TBS at room
temperature and then overnight in primary antibody against ATP7B from Dr.
Jonathan D. Gitlin mixed with 5% milk in TBS at 4°C at a dilution of
1:3000. Blots were washed three times for 15 min each at room temperature with
0.025% Tween 20 in TBS. The secondary antibody was diluted 1:1000 in 5%
fat-free dry milk in TBS and added to blots for 1 h at room temperature. Blots
were washed again at room temperature three times for 15 min each in 0.025%
Tween 20 in TBS. The extent of specific staining was quantified by
chemiluminescence using the ECL kit from Amersham Biosciences Inc. The
membrane was immediately exposed to Fuji medical X-ray film (Super RX;
Fujifilm, Kanagawa, Japan). A ChemiImager 400 instrument (Alpha Innotech) was
used for determining the relative density of protein bands.
Statistics. Tests of significance used Student's t test; p values of <0.05 were considered significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The sensitivity of the empty vector- and ATP7B vector-transfected populations to DDP, CBDCA, and CuSO4 was tested in clonogenic assays using a 1-h drug exposure. The concentration-survival curves are shown in Fig. 2, and the IC50 values determined from these curves are summarized in Table 1. The expression of ATP7B conferred moderate but significant degrees of resistance to all three compounds in all three ATP7B-expressing cell populations. Of note is the fact that ATP7B expression conferred a greater degree of resistance to DDP than to copper in each of the three pairs of cell lines tested.
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The effect of low level ATP7B expression on sensitivity to DDP, CBDCA, and
copper was further examined in the 2008/EV and 2008/ATP7B cell pair using an
assay capable of detecting enrichment for ATP7B-expressing cells in the
population that survives drug exposure. The 2008/ATP7B cells were engineered
to express GFP by infection with a viral vector. The 2008/EV and
2008/ATP7B-GFP cells were then mixed to form a population containing 10%
2008/ATP7B-GFP cells as determined before drug exposure by flow cytometric
analysis. The mixed population was then exposed to increasing concentration of
DDP, CBDCA, or copper for 1 h and then cultured for 5 days before the fraction
of 2008/ATP7B-GFP cells was again determined by flow cytometry. This assay has
the advantage that both the control and ATP7B-expressing cells were exposed to
identical conditions in the same culture throughout the experiment
(Torrance et al., 2001). The
results of these experiments, presented in
Fig. 3, demonstrated that in
the absence of drug exposure, the fraction of 2008/ATP7B cells did not change
significantly during the 5 days of culture. However, all three drugs produced
very substantial degrees of enrichment for ATP7B-expressing cells, even at the
lowest concentration of drug tested. These results confirm that the level
ATP7B expression present in the population of 2008/ATP7B cells was sufficient
to confer a biologically important degree of resistance.
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ATP7B Alters Copper and CBDCA Steady-State Accumulation. Studies of
the cellular pharmacology of DDP at physiologically relevant concentrations
are problematic because of the lack of a readily available radiolabeled form
of the drug and the limited sensitivity of alternative analytic approaches.
Thus, we focused on the effect of ATP7B expression on the cellular
accumulation of copper, which could be measured by using 64Cu, and
CBDCA, for which 14C-labeled drug was available. Studies of the
accumulation of copper and CBDCA were carried out in the 2008/EV and
2008/ATP7B pair of cell lines. The steady-state level of copper in the cell is
quite sensitive to the activity of ATP7B
(La Fontaine et al., 1998).
Figure 4A shows the levels of
copper in the 2008/EV and 2008/ATP7B cells when grown in the tissue culture
medium to which no copper had been added, and
Fig. 4B shows the levels when
grown in medium containing 2 µM copper for 24 h. Steady-state accumulation
was reached for both copper by 24 h (data not shown). The basal content of
copper in the 2008/EV cells was 1.3-fold higher than that in the 2008/ATP7B
cells (p < 0.05). The copper content of the 2008/EV cells was
1.9-fold higher than that of the 2008/ATP7B cells when grown in the
copper-supplemented medium (p = 0.00002).
Figure 4C shows the level of
CBDCA in 2008/EV and 2008/ATP7B cells exposed to 50 µM CBDCA for 24 h, at
which time steady state had been achieved. The CBCDA content was 2.8-fold
higher in the 2008/EV than in the 2008/ATP7B cells (p = 0.00005).
Thus, the increased expression of ATP7B produced a reduction in the
accumulation of both copper and CBDCA under steady-state conditions.
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ATP7B Alters Copper and CBDCA Uptake and Efflux. The decreased steady-state accumulation of copper and DDP in the 2008/ATP7B cells must reflect either reduced influx, decreased intracellular binding, increased efflux, or some combination of all three effects. Figure 5 compares the time course of copper and CBDCA accumulation in the 2008/EV and 2008/ATP7B cells over the first hour of exposure. For both copper and CBDCA the extent of accumulation over the first hour was substantially less in the 2008/ATP7B cells than in the 2008/EV cells. Thus, expression of ATP7B markedly reduced the rate of accumulation of copper and CBDCA over this time in a parallel manner.
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Accumulation at 1 h can still reflect differences in intracellular binding and efflux as well as initial influx rate. To determine the relative contribution of changes in influx, ideally one would like to measure the initial influx rate over a very short period. However, copper and CBDCA both enter cells relatively slowly, and the shortest period over which the influx of copper and CBDCA could be accurately measured was 5 min. Over this period, the average rate of accumulation of copper in the 2008/EV cells was 56 ± 7 (S.E.M.) pmol/mg of protein/min, whereas in the 2008/ATP7B cells the rate was reduced by 37% to 36 ± 6 (S.E.M.) (p = 0.0007). The rate of accumulation of CBDCA was 113 ± 11 (S.E.M.) pmol/mg of protein/min in the 2008/EV cells, whereas it was reduced by 61% to 44 ± 10 (S.E.M.) pmol/mg of protein/min in the 2008/ATP7B cells (p = 0.0009). Thus, when measured over as short an interval as possible, increased expression of ATP7B resulted in a clear decrease in cellular accumulation for both copper and CBDCA.
To examine the contribution of changes in efflux to the reduced accumulation the 2008/EV and 2008/ATP7B cells were loaded by exposure to either 2 µM copper or 50 µM CBDCA for 1 h. At each of multiple time points after removal of the drugs, six cultures were harvested using a rapid sampling technique. Figure 6 shows the efflux curves over the first hour after the end of drug exposure. For both copper and CBDCA, the efflux curves were characterized by an initial rapid phase, extending over approximately the first 5 min, followed by a much slower phase. Inspection of the curves indicates that increasing the level of ATP7B expression increased the efflux for both copper and CBDCA. The data were fitted to a two-compartment model, and the estimated half-lives for the initial and terminal phases of efflux are presented in Table 2. For copper, the initial phase of efflux was approximately 3-fold more rapid from the 2008/ATP7B than from the 2008/EV cells. Because the second phase of copper efflux from the 2008/EV cells was so slow, a half-life for this phase could not be accurately estimated but it was clearly shorter in the 2008/ATP7B cells. For CBDCA two-compartment modeling yielded similar half-lives for the initial phase of efflux from the 2008/EV and 2008/ATP7B cells, but efflux during the second phase was 3.9-fold faster from the 2008/ATP7B cells. Thus, increased expression of ATP7B increased at least the second phase of efflux for both copper and CBDCA in a parallel manner and to a substantial degree. It is important to note that the absolute efflux rates cannot be directly compared between copper and CBDCA because the extent of intracellular binding of these two metalloids is not known.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). For the 2008/EV and 2008/ATP7B cell pair, the results of
the colony formation assays were confirmed by enrichment assays that
demonstrated a large increase in the ATP7B-expressing cells in the population
that survived drug exposure. It is noteworthy that the magnitude of the change
in IC50 value was greater for DDP than for copper in each of the
three cell lines. The reason for this is not known, but it is consistent with
the results of our prior studies of cross-resistance between DDP and copper in
cells selected for resistance to either DDP
(Katano et al., 2002a) or
copper (Safaei and Howell,
2001), and the results reported by Komatsu et al.
(2000) in epidermoid cells
transfected with ATP7B. In all of these systems, relatively modest degrees of
resistance to copper were associated with significantly higher degrees of
resistance to DDP.
The cellular pharmacology studies in the 2008/EV and 2008/ATP7B pair of
cell lines provide evidence that the mechanism by which ATP7B expression
renders cells resistant to copper and CBDCA is through reduction in cellular
drug accumulation. The ATP7B-expressing cells exhibited reduced steady-state
levels of copper when grown in regular tissue culture medium, and when grown
in medium supplemented with 2 µM CuSO4. The same effect was
observed when the cells were exposed to CBDCA for 24 h, and the magnitude of
the effect on CBDCA steady-state levels was even greater than for copper. The
diminished steady-state levels must reflect either decreased influx, reduced
cellular binding, or enhanced efflux. Compared with many other types of
compounds, the influx of copper and CBDCA is relatively slow, making it
difficult to obtain accurate measurements of initial influx rate even with the
use of radioactive forms of the compounds. However, the time course of uptake
over the first hour of copper or CBDCA exposure suggests that measurements of
accumulation at 5 min provide a useful estimate of the initial influx rate,
and such measurements have been used by others for this purpose
(Lee et al., 2002a;
Puig et al., 2002). Given the
evidence that ATP7B functions primarily in copper export, it is of particular
interest that increased expression of ATP7B reduced the average initial
accumulation rate by 37% for copper and by fully 61% for CBDCA. This may be
explained in part by the fact that the initial efflux rate for both copper and
CBDCA is quite rapid. Two-compartment modeling yielded an estimate for the
initial efflux half-lives from the ATP7B-expressing cells that were short
enough that concurrent drug export may have operated to reduce the apparent
initial cellular accumulation rate. Alternatively, high levels of ATP7B may
sequester copper chaperones needed for optimal inwardly directed transfer of
copper. CTR1 is thought to hand copper to ATOX1 for further transfer to ATP7B
(Hamza et al., 2001). ATOX1
binds to ATP7B (Walker et al.,
2002) and high levels of ATP7B may limit the availability of ATOX1
for partnering with CTR1. We (Lin et al.,
2002) have confirmed the observation made by Ishida et al.
(2002) that loss of CTR1
function in Saccharomyces cerevisiae markedly reduces the uptake of
DDP and further demonstrated that CTR1 modulates the uptake of CBDCA,
oxaliplatin, and other DDP analogs as well as copper. Thus, if overexpression
of ATP7B indirectly impairs CTR1 transport capacity, it would be reasonable to
expect it to reduce DDP and CBDCA influx as well.
Increased expression of ATP7B was associated with more rapid efflux of both copper and CBDCA from the cell. For both compounds the efflux curve was characterized by two phases; the first phase was very much more rapid than the second. For both copper and CBDCA, efflux during the second phase was substantially faster from the 2008/ATP7B cells than from the 2008/EV cells. An accurate estimate of the magnitude of this change could not be developed for copper because the efflux half-life for the 2008/EV cells was so long. However, in the case of CBDCA this phase of efflux was 3.9-fold faster from the 2008/ATP7B cells. Interestingly, two-compartment modeling suggested that although increased expression of ATP7B produced a 2.9-fold increase in initial efflux of copper, it had little impact on the initial efflux of CBDCA. This may be related to differences in the intracellular binding of these two compounds and/or their positioning in cellular subcompartments relative to that of ATP7B. Although it is appropriate to compare the 2008/EV and 2008/ATP7B cells with respect to efflux of copper and CBDCA, the inability to assess free intracellular levels limits the validity of comparing the absolute rate of efflux of copper to that of CBDCA.
A number of questions about the transport of DDP and CBDCA by ATP7B remain
to be addressed. Given the ability of ATP7B to render cells resistant to DDP
as well as CBDCA, one might expect ATP7B to alter the initial cellular
accumulation and enhance the initial efflux of DDP and oxaliplatin as well as
that of CBDCA. Komatsu et al.
(2000) noted reduced
accumulation and retention of DDP in epidermoid cells transfected with ATP7B,
but detailed cellular pharmacokinetic studies of DDP have yet to be presented.
The export of copper is thought to proceed first through sequestration into
the trans-Golgi and subsequently via vesicle migration to the cell
surface (Walker et al., 2002).
It remains to be determined whether the platinum-containing drugs are
similarly sequestered into vesicles of the secretory pathway. ATP7B is found
predominantly in the trans-Golgi network or a closely related
endosomal compartment. However, it has been reported that copper induces
redistribution of ATP7B to other areas of the cell
(Hung et al., 1997;
Schaefer et al., 1999;
Roelofsen et al., 2000;
Vanderwerf et al., 2001).
Whether the platinum-containing drugs cause a similar redistribution is the
focus on ongoing studies. How large a change in ATP7B levels is required to
produce clinically significant degrees of resistance to DDP and CBDCA is a
remaining question of substantial clinical significance.
The demonstration that increased expression of ATP7B produces DDP and CBDCA
resistance in several different cell systems and that it does so by modulating
the cellular pharmacology of these drugs provides a solid foundation for
investigation of the expression of this transporter in human tumors known to
be intrinsically sensitive or resistant to the platinum-containing drugs.
Together with evidence that the major copper influx transporter CTR1 mediates
the uptake of DDP (Ishida et al.,
2002; Lin et al.,
2002), this suggests the unifying concept that DDP and CBDCA enter
the cell, are distributed to subcellular locations, and are exported from the
cell in part using the transporters and chaperones that evolved primarily to
mediate copper homeostasis.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: DDP, cisplatin; CBDCA, carboplatin; GFP, green fluorescent protein; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; CMV, cytomegalovirus.
Address correspondence to: Dr. Stephen B. Howell, Department of Medicine 0058, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0058. E-mail: showell{at}ucsd.edu
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, cells
transfected with empty vector; 
, cells transfected with ATP7B-expressing
vector. A to C, IGROV-1/EV compared with IGROV-1/ATP7B; D to F, 2008/EV
compared with 2008/ATP7B; G to I, UMSCC10b/EV compared with UMSCC10b/ATP7B.
Each data point represents the mean of three independent experiments each
performed with triplicate cultures; vertical bars are ± S.E.M.
