Departments of 1Medicine and 2Pathology, Liver Diseases Unit, University of Manitoba, Winnipeg, Manitoba, Canada R3E 3P4
Submitted 3 December 2002 ; accepted in final form 13 March 2003
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ABSTRACT |
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cancer; regeneration; membrane potential; proliferation, differentiation, liver disease, hepatocellular carcinoma
Resting hepatocytes have a carefully regulated transmembrane electrical potential difference (PD) of approximately 35 mV (39). In previous studies (42), we documented that after a growth stimulus such as 70% partial hepatectomy, hepatocytes promptly depolarize to PD values of approximately 20 mV. This depolarized state remains in effect until hepatocyte proliferative activity wanes. Whether the PD changes observed represent a cause or effect of hepatocyte proliferation remains unclear. That nuclear translocation of cationic growth promoters does not occur in cells that maintain their hyperpolarized state argues in favor of the former (11). Also supportive are findings that maintenance of hepatocyte PD at resting levels interferes with hepatocyte proliferative activity (25).
Numerous channels, pumps, receptors and the agents influencing the activity of these sites contribute to the PD of hepatocytes (9). Recently, we identified specific GABA receptors on the surface of isolated rat and human hepatocytes that regulate chloride flux (7). Activation of these sites with GABAA receptor agonists caused prompt hepatocyte hyperpolarization and inhibition of proliferation, whereas exposure to GABAA receptor antagonists depolarized hepatocytes and enhanced proliferative activity (14, 24, 41). These data suggest that the GABAergic system is an important regulator of hepatocyte PD and proliferative activity.
The purpose of the present study was to test the hypothesis that malignant hepatocytes are permanently depolarized as a result of downregulated or absent GABAergic activity. As a corollary to this hypothesis, we proposed that by restoring hepatocyte PDs toward those of resting hepatocytes (via increasing GABAergic activity) the proliferative and malignant features of these cells would revert to those associated with nonmalignant hepatocytes.
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MATERIALS AND METHODS |
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Cell culture. Cells were grown in DMEM and supplemented with (in %) 10 cool calf serum, 1 penicillin (10,000 units/ml)/streptomycin (10,000 µg/ml), 1 Fungizone, and 0.011 sodium pyruvate in a humidified, 37°C incubator in an atmosphere of 95% air-5% CO2.
Cell PD determinations. A Leica DM IRB fluorescence microscope
equipped with a PDMI-2 Open Perfusion Micro-Incubator (Harvard Apparatus,
Saint-Laurent, PQ, Canada) was used to measure PDs as described by Loew
(19). The incubating
temperature was 37°C. Twenty-four-hour-cultured healthy human hepatocytes
and malignant hepatocytes were washed with NB buffer [(in mM) 130 NaCl, 5.5
KCl, 1.8 CaCl2, 1.0 MgCl2, 25 glucose and 20 HEPES
buffer adjusted to pH 7.4] three times and then incubated for 10 min with NTB
buffer [100 nM tetramethylrhodamine ethyl ester (TMRE) in NB buffer]. After
photographs of the fluorescent cells were obtained, they were washed with KB
buffer [(in mM) 130 KCl, 5.5 NaCl, 1.8 CaCl2, 1.0 MgCl2,
25 glucose, and 20 HEPES buffer adjusted to pH 7.4] three times and incubated
for 10 min with DTB buffer (100 nM TMRE and 1 µM valinomycin in KB buffer).
Photographs were then obtained of the depolarized cells. Fluorescence within
the cell was designated intracellular fluorescence (Fin) and
fluorescence outside the cell, as extracellular fluorescence
(Fout). To calculate PDs, the Nernst equation was used: PD =
(RT/ZF) n(Fin
dFout)/(Fout dFin), where PD is membrane
potential, Z is the charge of the permeable ion, F is Faraday's
constant, R is the ideal gas constant, and T is the absolute
temperature.
RT-PCR for
GABAA-3
receptor. Total RNA was extracted from 1 x 106 cells
by the commercially available TRIzol method (Invitrogen, Carlsbad, CA). RT
reactions (20 ul) consisted of the following: 1 µg RNA, 5x reaction
buffer (Clontech, Palo Alto, CA), 0.5 mM dNTP, 0.5 units RNase inhibitor, 20
pmol oligo(dT)18 primer, and 20 units Moloney murine leukemia virus reverse
transcriptase (MMLV RT). Reactions were incubated at 42°C for 60 min, and
terminated at 99°C for 5 min. Five microliters of the reactions were used
for the PCR reaction.
The oligonucleotide primers for PCR reaction were designed against human
GABAA receptor sequences by using an Oligo 5.0 program (National
Biosciences, Plymouth, MN). The sequences of human
GABAA-3 receptor oligonucleotide primers were as
follows: forward primer, 5'-AAGGGCTGGTTACCGGAGTGGA-3'; reverse
primer, 5'-CGAAGATGGGTGTTGATGG-3'. The PCR amplification was
carried out in 30 cycles of denaturation (94°C, 45 s), annealing
(57°C, 45 s) and elongation (72°C, 2 min) and with an additional 7-min
final extension at 72°C. Finally, 10 ul of the PCR products were run on 2%
agarose gels. The product length is 290 bp
(7).
SDS-gel electrophoresis and immunoblotting techniques. Cells were
harvested by scraping into a protease inhibitor mixture consisting of (in mM)
20 Tris (pH 7.4), 1 PMSF, 1 benzamidine, and 5 EDTA, with 100 µM leupeptin
and passed through a 26-gauge needle. Protein concentrations were measured by
using the Lowry protein assay
(20). Total protein extracts
(50 µg) were separated on 12% polyacrylamide-SDS gels and electroblotted to
nitrocellulose membranes as described previously
(7). Membranes were blocked
with 5% skim milk in Tris-buffered saline (0.02 M Tris-base, pH 7.6) for 1 h
at room temperature and incubated with rabbit anti-human
GABAA-3 receptor antibody (5.55 µg/ml)
(provided by Dr. W. Sieghart, University of Vienna, Austria) overnight at
4°C. Bands were detected with a horseradish peroxidase-labeled secondary
antibody-catalyzed chemiluminescence reaction (Amersham Pharmacia Biotech,
Burlington, ON, Canada) (7).
Controls included rat brain microsomal protein (Upstate Biotechnology, Lake
Place, NY), and membranes were incubated with secondary antibody but without
prior incubation with primary anti-GABA-
3 receptor antibody.
Cell proliferation. [3H]thymidine incorporation was
determined as described by Luk
(21). On the basis of
preliminary experiments designed to identify cell concentrations associated
with 6080% confluence at 48 h, 5 x 104 cells were
seeded into 6-well plates. After 2 days of culture to allow attachment to
plate bottoms, cells were incubated with 1 µCi [3H]thymidine for
2 h at 37C°. [3H]thymidine incorporated into cellular DNA was
precipitated by the addition of 10% TCA for 15 min at room temperature. Cells
were rinsed and resolublilized in a 0.3 M NaOH and 1% SDS solution and then
assayed for radioactivity in a -scintillation counter.
Bromodeoxyuridine (BrdU) incorporation was determined by seeding 5 x 103 cells into 96-well plates. At subconfluence, cells were incubated with serum-free DMEM. Twenty-four hours later, the medium was switched to DMEM containing BrdU for 2 h. Incorporation of BrdU was determined by using BrdU Labeling and Detection Kit III (Roche Diagnostics, Mississauga, ON, Canada) according to the manufacturer's instructions. Absorbance at 450490 nm was measured by using a microplate reader (Molecular Devices, Menlo Park, CA).
4-[3-(4-Lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) determinations were performed by using a commercially available kit (Boehringer-Mannheim, Laval, PQ, Canada). Briefly, 1 x 104 cells were seeded into 96-well plates. After 2 days of culture, one-tenth the volume of WST-1 (Boehringer-Mannheim) was added to each well, and the cells were incubated with WST-1 for 4 h in 37°C. Absorbance at A405A650 was determined by a microplate reader (Molecular Devices).
Colony formation in soft agar was conducted in 60-mm plates containing two
layers of media. The top layer contained DMEM supplemented with 15% cold calf
and 0.3% agarose, and the bottom layer contained DMEM supplemented with 15%
cold calf and 0.5% agarose. Chang cells and Chang cells transfected with
GABAA-3 or vector alone were harvested by
trypsinization and 0.5 x 104 cells were inoculated into the
top layer of agarose. Triplicate plates were incubated at 37°C under 5%
CO2, and the number of macroscopic colonies per plate were counted
after 3 wk of culture.
The number of cells undergoing mitoses was expressed as a percentage of 100 cells counted per high-power field (mitotic index). A total of six high-power fields were examined per cell population. The identity of the cell population was not known to the cytologist (E. Ravinsky) calculating the mitotic index.
Plasmid construction. The 1640-bp cDNA containing the
GABAA-3 receptor coding region was cut with
XbaI from plasmid pRK5-
3 and cloned into the pcDNA3.1/V5-His C
vector producing a plasmid referred to as pcDNA-
3. The cloned cDNA
fragment is under the transcriptional control of the immediate early gene of
the human cytomegalovirus promoter and the vector contains polyadenylation
signals and ampicillin- and zencin-resistant genes
(30). pcDNA-
3 plasmids
were isolated, purified, and sequenced to confirm that the cDNA of the
GABAA-
3 receptor was in frame with the
cytomegalovirus promoter.
Stable transfection. Briefly, 1 x 106 cells in a
3.5-cm dish were transfected with 1 µg of linearized pcDNA-3 or pcDNA
vector alone by using LipofectAMINE (GIBCO/BRL) according to manufacturer's
instructions. Stable transfected cells were established in the presence of
G418 (800 µg/ml), and resistant clones were isolated by using cloning
cylinders and were maintained under G418 selection (200 µg/ml). Clones were
analyzed individually by RT-PCR and Western blotting for levels of
GABAA-
3 receptor mRNA and protein expression,
respectively (7).
DNA analysis. 1 x 106 cells were stained with propidium iodide as described by Diez-Fernandez et al. (6). Emitted fluorescence was assayed in a FACScan flow cytometer (Becton-Dickinson). A double discriminator module was used to distinguish between single nuclei and nuclear aggregation.
Statistical analyses. ANOVA followed by paired Student's t-test for parametric data and a Wilcoxon rank sum test for nonparametric data were performed where appropriate. P values <0.05 were considered significant. All experiments were performed on at least three occasions, and each proliferation assay was documented in 35 wells/plates unless otherwise stated. The results provided represent the means ± SE for all data sets generated.
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RESULTS |
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GABAA receptor expression. The human
liver expresses two of the 15 known GABAA receptor subtypes
(3 and
) (7,
33). Of the two, only
3
has the capacity to form GABA-gated channels
(33). The results of RTPCR for
GABAA-
3 receptor mRNA expression are shown in
Fig. 2. In Chang, Hep
G2, and HuH-7 cells, expression was undetectable, whereas in
PLC/PRF/5 cells, expression was evident. Sequence analysis of the
GABAA-
3 receptor mRNA expressed in PLC/PRF/5 cells revealed a
nonrelevant, single mutation (G to A) at nt 1058. The absence of
GABAA-
3 receptor mRNA expression in the three
receptor-deficient malignant cell lines was confirmed at the protein level by
Western blot analyses. In the case of PLC/PRF/5 cells, a discordance existed
in that despite the presence of the transcript, GABAA-
3
receptor protein expression was absent by Western blot analysis
(Fig. 3).
GABAA-
receptor mRNA and protein expression were present in
all four malignant cell lines (Fig.
4).
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Transfection studies. To determine what impact restoration of
GABAA-3 receptor mRNA expression has on cellular PD and the
proliferative activity of receptor-deficient cells, Chang cells were
transfected with GABAA-
3 receptor cDNA in pcDNA 3.1/V5-His C
vector or vector alone. Restoration of GABAA-
3 receptor mRNA
and protein expression after transfection were confirmed by RT-PCR and Western
blot analyses, respectively, but remained undetectable in cells transfected
with vector alone (Figs. 5 and
6). After transfection, the PDs
of GABAA-
3 receptor cDNA transfected cells increased (became
more hyperpolarized) from a baseline PD of 7.5 ± 1.0 to a PD of
12.9 ± 0.4 mV (P < 0.0001), whereas the PDs of cells
transfected with vector alone remained unaltered.
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Proliferative activity. Synthesis (S) phase proliferative activity
was documented by [3H]thymidine and BrdU incorporation rates,
whereas mitosis (M) phase activity was documented by WST-1 activity, mitotic
index, and FACScan DNA analyses. In addition, cell doubling times and counts
over a 10-day culture period were calculated. As shown in
Table 1, S phase activity was
significantly decreased in GABAA-3 receptor cDNA-transfected
Chang cells compared with Chang cells transfected with vector alone. However,
an even greater decrease was observed in M phase activity. Of note, the
percentage of GABAA-
3 receptor cDNA-transfected cells
undergoing mitosis after GABAA-
3 receptor cDNA transfection
(34%) was similar to that reported for nonmalignant hepatocytes in culture
(6).
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The results of DNA analyses by FACScan supported WST-1 and mitotic index
findings. Specifically, the proportion of Chang cells transfected with
GABAA-3 receptor cDNA in the G0/G1
phase of the cell cycle was decreased, whereas those in the G2/M
phase were increased relative to Chang cells transfected with vector alone
(Fig. 7).
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The doubling time of Chang cells transfected with vector was 31.5 ±
2.4 h (Table 1). In Chang cells
transfected with GABAA-3 receptor cDNA the doubling time
increased to 53.2 ± 3.6 h (P < 0.0005).
Over a 10-day period, Chang cells transfected with vector alone grew at
exponential rates (Fig. 8).
However, Chang cells transfected with GABAA-3 receptor cDNA
grew at a slower rate such that by day 5, cell counts in
GABAA-
3 receptor cDNA-transfected cells were only 50% that of
Chang cells transfected with vector alone and 30% on day 10 of
culture.
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As shown in Fig. 9, colony
formation in soft agar was significantly decreased in Chang cells transfected
with GABAA-3 receptor cDNA compared with cells transfected
with vector alone (P < 0.005).
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There were fewer abnormal mitoses (abnormal spindle patterns) in
GABAA-3 receptor cDNA-transfected cells (9%) compared with
Chang cells transfected with vector alone (18%). Chang cells transfected with
vector alone also had more multinucleated cells with macronucleoli compared
with those transfected with GABAA-
3 receptor cDNA.
Representative cells are shown in Fig.
10.
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Receptor augmentation. To determine whether further
hyperpolarization could be achieved in Chang cells transfected with
GABAA-3 receptor cDNA, these cells together with
nontransfected Chang cells and Chang cells transfected with vector alone were
exposed to 50 µM muscimol, a specific GABAA receptor agonist for
48 h. As shown in Fig. 11,
muscimol had little or no effect on the PDs of nontransfected Chang cells and
Chang cells transfected with vector alone but increased by
40% the PDs of
Chang cells transfected with GABAA-
3 receptor cDNA
(premuscimol: 12.0 ± 0.9 vs. postmuscimol: 16.4 ±
0.8, P < 0.01).
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To determine whether the addition of muscimol and/or other GABAA
receptor agonists might have therapeutic value in inhibiting the growth of
malignant hepatocytes with restored GABAA-3 receptor mRNA
expression, proliferative activity was documented in Chang cells transfected
with GABAA-
3 receptor cDNA or vector alone after exposure to
varying concentrations of muscimol (0100 µM). As shown in
Fig. 12, a dose-dependent
decrease in proliferative activity was documented in GABAA-
3
receptor cDNA-transfected cells, whereas in Chang cells transfected with
vector alone, proliferative activity remained essentially unaltered.
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DISCUSSION |
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Other investigators have reported
(23) that malignant cells are
significantly depolarized in situ and in cell culture systems. In the case of
thyroid cancer, PDs of malignant thyroid cells were 50% depolarized
compared with nonmalignant thyroid cells
(13). Similar extents of
depolarization have been documented in glioma, cervical, breast, and gastric
carcinoma cells (16,
23,
28,
35). The only previous study
describing PDs of malignant hepatocytes was reported by Binggeli and Cameron
(1), who documented PD values
of 19.8 ± 7.1 mV in rat hepatoma tissue compared with
37.1 ± 4.3 mV in normal rat liver. The higher PD values in that
study reflecting the authors' use of in situ determinations that are known to
be associated with higher PD readings than those obtained from isolated cells
or transformed cell lines as were employed in the present study
(27).
Whether the depolarized state of malignant cells represents a cause or effect of malignant transformation remains to be determined. In favor of the former are findings that hepatocytes derived from cirrhotic livers, which can be considered a potentially premalignant state, are depolarized compared with hepatocytes derived from healthy livers (3). More compelling, however, are data from the present study indicating that malignant features including [3H]thymidine and BrdU incorporation rates, WST-1 activity, mitotic indexes, doubling times, growth patterns, colony formation in soft agar, and the number of abnormal mitoses are significantly decreased and doubling times signifi-cantly prolonged when cells are hyperpolarized by augmenting GABAA receptor expression. Clearly, a prospective study wherein PDs and GABA receptor expression are documented during well-defined stages of malignant transformation is required to further support a causative role.
Whereas PD determinations have been reported in various malignant cell
lines and tissues, to our knowledge, direct determinations of GABAA
receptor expression have hitherto not been described. Nonetheless, our finding
of absent GABAA-3 receptor mRNA expression was not
unexpected as in previous studies
(10,
12) we described the absence
and/or marked downregulation of the sodium-dependent GABA transporter system
in seven human HCC tissues and GABA-transporter expression tends to parallel
GABAA receptor activity.
Despite the lack of documentation of GABA receptor expression in malignant
cells and/or tissues, we and others
(2,
4,
15,
17,
34,
36,
40) have reported that
augmentation of GABAergic activity, by the administration of high
concentrations of GABA, GABA receptor agonists, or transient transfection with
GABAA receptor c-DNA has an inhibitory effect on cell proliferative
activity and in the case of malignant hepatocytes, -fetoprotein mRNA
expression. Results of the present study involving stable
GABAA-
3 receptor cDNA-transfections indicate the
same effect can be achieved by restoring GABAA receptor expression
in malignant hepatocytes.
Although the precise mechanism(s) whereby restoration of hepatocyte PD
toward those of resting values results in decreased proliferative activity has
yet to be elucidated, the findings of more pronounced inhibition of M phase
(decreased WST-1 expression and a lower mitotic index) relative to S phase
(decreased [3H]thymidine and BrdU incorporation) activity in
GABAA-3 receptor transfected cells suggests that
cell cycle arrest is occurring predominantly in the G2 phase of the
cell cycle. The G2/M increase on FACScan supports this
interpretation. Further studies are required to determine what impact cell PDs
have on the polar activities of centrosomal segregation, mitotic spindle
formation, and their respective migration toward the outer membranes of
dividing cells.
Not all malignant hepatocyte cell lines had absent
GABAA-3 receptor mRNA expression despite a
uniformly depolarized state. Indeed, mRNA expression in PLC/PRF/5 cells was
similar if not increased relative to healthy, human hepatocytes. That
sequencing of the transcript failed to identify mutations resulting in
functional changes at the cell membrane level suggests that either
posttranscriptional factors are responsible for the absence of
GABAA-
3 receptor expression at the protein level
or that GABAergic activity is intact and the depolarization in this cell line
reflects disturbances in other regulators of hepatocyte PD such as
volume-activated chloride channels, KATP channels or the
Na+/K+ ATPase pump
(38,
22,
31).
The GABAA- receptor is the only GABAA receptor
subtype that does not form GABA-gated chloride channels
(33). Thus unlike the
3 receptor subtype, the
receptor subtype has no
inherent electrogenic properties. Recent data suggest its role is in
regulating the distribution and display of other GABAA receptor
subtypes throughout the cell
(5).
Results of the present study raise interesting possibilities regarding new
approaches to the treatment of HCC. For example, if depolarization is an
essential feature of malignant cells, then interventions that result in tumor
hyperpolarization would be expected to have an inhibitory effect on tumor
growth. In the case of HCC, such interventions appear to require gene therapy
with the GABAA-3 receptor gene as the prevalence
of receptor-deficient or mutated cells appears to be high, all four malignant
hepatocyte cell lines in this study. In addition to the general problems
associated with gene therapy, including potential host toxicity and the need
to efficiently transfect the majority if not all malignant cells, there are
certain issues related to activation of GABAA receptors in the
liver that will need to be addressed. Specifically, GABA is rapidly cleared
from the systemic circulation and presently available GABAA
receptor agonists have only a transient effect on receptor activity
(8,
24,
37). Whether inhibition of
hepatic GABA metabolism, interference with GABA clearance by hepatocytes,
and/or manipulation of nonGABAergic electrogenic systems, such as increasing
Na+/K+ ATPase activity, can be exploited to achieve the
desired PD changes remains to be determined
(18,
26). It should also be noted
that augmentation of GABAergic activity in the liver inhibits healthy
hepatocyte proliferation, an important survival mechanism in patients with
advanced liver disease (14,
41).
In conclusion, the results of this study indicate that malignant
hepatocytes exist in a significantly depolarized state. This depolarized state
is associated with an absence of GABAA-3 receptor
expression. The results also indicate that increasing the PD of malignant
hepatocytes by transfecting receptor-deficient cells with
GABAA-
3 receptor cDNA, is associated with a loss
or attenuation of malignant features. The later findings raise the possibility
that if causative, interventions directed toward altering cell membrane
potentials, an approach we have termed electrogenics, would represent a new
therapeutic strategy in the treatment of hepatocellular carcinoma.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the Medical Research Council/Canadian Institutes of Health Research.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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