CANCER RESEARCH55. 6077-6083. December 5. 19951
Volume-sensitive Chloride Channels Associated with Human Cervical
Carcinogenesis'
Cheng-Yang
Chou,2 Meng-Ru Shen, and Sheng-Nan Wu
Department of Obstetrics and Gynecology. National Cheng Kung University Medical C'ollege, 138 Sheng-Li Road, Tainan, Taiwan 704 IC-Y. C'., M-R. SI, and Department of
Research and Education, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan 813 (S-N. WI, Republic of China
ABSTRACT
Previous controversy has risen from the purported equivalence of the
volume-sensitive chloride channels with P-glycoprotein. The aim of this
study was to investigate the association between expression of volume
sensitive
Cl
channels
and
the
process
of malignant
transformation
of
cervical epithelial cells. We studied the activations of volume-sensitive and
cAMP-mediated
chloride
currents
in various
human
cervical
squamous
cells that were representative of different stages of cervkal carcinogenesis,
i.e., normal cervical epithelium, low-grade cervical intraepithelial neopla
sia, carcinoma in situ, and invasive carcinoma using the whole-cell patch
clamp technique. The volume-sensitive chloride channels, however, were
significantly activated only in the four cervical cancer cell lines, primary
culture cells of carcinoma in situ, and invasive cancer of the cervix. The
expression of volume-sensitive chloride currents was independent of the
state of human papillomavirus positivity. When these cells were exposed to
hypotonic shock, the cells swelled, and outward rectified chloride currents
were observed. These effects were readily reversed by returning the cells
to isotonic medium. In addition, 4,4'-diisothiocyanatostilbene-2,2-disulfo
mc acid, 1,9-dideoxyforskolin, and verapamil reversibly abolished the
volume-sensitive Cl currents In contrast, none of the cells from normal
cervices
and
human
papillomavirus-immortalized
cell lines,
the in vitro
equivalent of low-grade cervical intraepithelial neoplasia, developed sub
stantial chloride currents on exposure to hypotonicity. cAMP-mediated
chloride currents were ubiquitously activated in all cervical squamous
cells, regardless of the stages of carcinogenesis. This is the first report
suggesting
an in vivo association
between
the
development
of volume
sensitive chloride currents and human carcinogenesis.
INTRODUCTION
Cervical cancer is the most frequent cause of disease and death
from malignant neoplasms among women in developing countries (1).
Despite intensive studies that have been carried out over the last
decades, the etiology for this disease is still largely unknown. A
subgroup of HPVs3 is associated with 70—90%of the human cervical
carcinoma (2). However, only a certain percentage of CINs develop
into cervical carcinoma after a long latency period, independent of
their association with HPVs (3, 4). Moreover, low prevalence of
cancer was found in women positive for HPVs. These observations
suggest that other etiological factors must be involved in the progres
sion to malignancy.
In many epithelial cells, chloride channels are essential for the
transport of salt and water across the membrane bilayer. Three distinct
chloride currents, regulated by cAMP, Ca2@, and cell volume, respec
tively, have been demonstrated in airway epithelial cells and in the
T84 colonic carcinoma cell line (5, 6). The cAMP-regulated current
has been associated with cystic fibrosis transmembrane regulator (7).
The cystic fibrosis transmembrane regulator is involved in ion trans
Received6/6/95;
accepted10/17/95.
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.
I Supported
233l-B-037-093
2 To
3 The
whom
in part
by Grants
NSC-85-2331-B-006-070
(to
C.
Y.
C.)
and
NSC-84-
(to S. N. W.) from the National Science Council, Taiwan.
requests
abbreviations
for
reprints
used
are:
should
HPV,
be
human
addressed.
papillomavirus;
Fax:
port across many epithelia including the lung airways, pancreas, and
sweat glands (5, 6, 8, 9). The calcium-activated C1 current is recti
fled in an outward direction (5), and, to date, little biochemical
information is available for this channel. Volume regulation is a
widespread process that enables cells to maintain their normal volume
in the face of changes in extracellular osmolarity. An increase in cell
volume activates membrane transport pathways that mediate an efflux
of cell osmolytes to effect RVD (10). In one common type of RVD,
KC1 leaves the cell via distinct K@ and Cl channels (1 1, 12). The
activation of volume-sensitive C1 channels has been demonstrated in
several nonexcitable cell types and NIH3T3 fibroblasts transfected
with the P-glycoprotein gene (reviewed in Ref. 13). Because most of
the epithelial cells reported to express volume-sensitive chloride chan
nels are either cancer cell lines (14—17)or transformed cell lines (18,
19), and several groups argue against the direct correlation between
P-glycoprotein and volume-sensitive chloride channels (20—22),we
hypothesize that the expression of volume-sensitive chloride channels
may develop during the process of tumorigenesis or transformation.
To test this hypothesis, human cervical squamous cells representing
different stages of carcinogenesis were used. These include normal
epithelium, low-grade CIN, high-grade CIN (including CIS), and
invasive carcinoma. We demonstrate that cell swelling activates the
ATP-dependent Cl currents in all four cervical cancer cell lines,
primary culture cells of CIS, and invasive cancer of the cervix. There
is no apparent association between expression of volume-sensitive
Cl channels and HPV infection. On the contrary, hypotonicity was
not able to activate Cl currents significantly in HPV-immortalized
cells and normal cervical epithelial cells. These HPV-immortalized
cell lines, which have been reported to display alterations in growth
and differentiation closely analogous to those seen in low-grade CIN
but not able to induce tumor formation in nude mice (23), were used
in this study as representative cells of low-grade CIN. Because chlo
ride channels are indispensable for the viability of epithelial cells, we
also assessed the presence of other types of chloride channels in these
cells. Our data showed that cAMP-dependent chloride currents were
ubiquitously activated in all cervical squamous cells. It is proposed
that elevated expression of this Cl conductance, leading to RVD, is
associated with human cervical carcinogenesis.
MATERIALS
AND METHODS
Cell Culture Two HPV-positive cervical carcinoma cell lines (SiHa and
CaSki) and one HPV-negative cell line (HT-3) were obtained from American
Type Culture Collection (Rockville, MD). Another HPV-negative cervical
carcinoma
cell line (CX) was established
in this laboratory.4
HPV-immortal
ized cervical epithelial cell lines Z183A, Z172, and Z133 were kindly provided
by Dr. Tzer-Ming Chen (National Taiwan University, Taipei, Taiwan, China).
The cervical cancer cell lines and HPV-immortalized cell lines used here have
been characterized and described in detail (23, 24). Cervical carcinoma cell
lines were maintained
at 37°C in a CO@/O2:5%/95%
atmosphere
and were used
2—3
days after subculturing. The growth medium for cervical cancer cells was
DMEM(GIBCO,GrandIsland,NY) supplementedwith 10%FCS (GIBCO),
886-6-2766185.
CIN,
cervical
intraepithe
hal neoplasia; CIS, carcinoma in situ; RVD, regulatory volume decrease; DIDS, 4,4'diisothiocyanatostilbene-2,2-disulfonic
acid; V,,@, reversal potential.
4 C. Y.
Chou,
Y.
H. Chen,
C. C. Tzeng,
T. M.
Chen,
and Y. C. Cheng.
Establishment
and characterization of a human-papillomavirus negative, p53-mutation negative human
cervical cancer cell line, manuscript submitted for publication.
6077
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VOLUME-SENSITIVE
80 IU/ml
penicillin,
and
HPV-immortalized
NuSerum
cell
80 ,xg/ml
lines
IV (Collaborative
were
streptomycin
maintained
Research,
Bedford,
CHLORIDE
CHANNELS
(Sigma,
St. Louis,
MO).
in DMEM
containing
10%
two-step,
cervical
epithelial
transformation
zone
cells
were
MA) in place of FCS.
of the cervix
derived
uteri
from
obtained
the porno
from
surface
Tissues
were placed
into a “rinsesolution― of PBS, without
at 37°C for 10 to 12 mm, during
which
oil-driven
micromanipulator
was used to position
35- to 45-year-old
Ca2@or Mg2@(GIBCO), containing 20 g.tg/mlgentamycin for 3 mm and then
were placed in a caseinolytic unit/mI dispase (Becton Dickinson, Bedford,
MA) and HBSS (GIBCO) with 5 @xg/mi
gentamycin. The tissues were incu
bated for 18 h at 4°C.After incubation in dispase, the epidermis was placed
into 2 ml trypsin-EDTA
micropipette
puller (PB-7;
Narishige
Scientific
Instruments,
(MO-103;
Narishige
Scientific
Instruments,
To
kyo, Japan), which was mounted on the fixed stage of an inverted microscope,
and
women with no history of CIN and who underwent hysterectomy for benign
uterine diseases. The serum-free keratinocyte medium (Keratinocyte-SFM;
GIBCO) as well as the manufacturer's instructions were used for cell culturing.
At surgery, the incised cervical portios measuring approximately 30 X 10 mm
were placed into Keratinocyte-SFM medium containing 5 @xg/ml
gentamicin
for transportation.
vertical
CANCER
Tokyo, Japan). When the pipettes were filled with the internal solution, their
direct current resistance varied between 3 and 5 Mfl. A three-dimensional
Establishment of Primary Culture and Histopathological Diagnosis.
Normal
AND CERVICAL
time it was
was corrected
validate
the pipette
immediately
the experimental
near the cell. The liquid junctional
before
the pipette
conditions
used
was attached
in the present
potential
to the cell. To
study,
when
the
whole-cell recording mode was established, some cells were allowed to remain
stabilized for about 5 to 10 mm. In experiments designed to construct the
current versus voltage (I-V) relationships, either square command pulses with
a duration of 30 ms from the holding potential to various potentials or ramp
command
pulses
with a duration
of 100 ms from
—80 mV to +40
mV were
used. The square or ramp voltage-step command signals were generated at a
rate of 0.2—0.5 Hz by the use of a programmable
stimulator
(SMP
310;
Biologic Corp., Claix, France).
Data Recording and Analysis. The signals consisting of voltage and
aspirated with a 2-mi pipette every 2—3
mm to dissolve the cells. Following
incubation, the trypsin action was stopped by adding 10—13
ml soybean trypsin
inhibitor (GIBCO), at 10 mg/ml dissolved in PBS. The cells were spun at
current
600 X g for 10 mm at room temperature.
Taiwan, China) that was controlled by a 25-MHz Eagletak computer and
Snapshot software version 3.52 from HEM Data Corp. (Southfield, MI). These
records were also simultaneously stored on a digital tape record (model 1202;
35-mm
tissue
inverted
culture
microscope
dishes,
which
The primary
could
at a cell density
cells were seeded
be mounted
on the stage
of 1 X l0@ cells/dish
into
of an
in Keratinocyte
tracings
were monitored
on a storage
oscilloscope
(model
HM205—3;
Hameg Instruments, Anaheim, CA) and digitized in real time at the sampling
frequency
of 5—10 kHz with a PCL-818
interface
board
(Advantech
Corp.,
Biologic Corp.), the frequency response of which is direct current 20 kHz with
SFM supplemented with bovine pituitary extract (20—30,xg/ml) and recom
binant epidermal growth factor(l—5ng/ml). The dishes were incubated at 37°C a dynamic of 14bits. After the experiments, the stored data were then sent back
to a personal computer for analysis, graphing, and archiving. All values are
in a humidified
5% CO2 incubator and were fed with fresh medium twice a
week. For explant culture of cervical carcinoma and CIS cells, tissue fragments reported as mean ±SD. Student's paired t test was used for statistical analysis.
of about 10 mm3 were obtained from colposcopy-guided cervical biopsy to Differences between values were considered significant when P < 0.05.
minimize
the contamination
of normal
cervical
epithelium
Drug
at the Department
of Obstetrics and Gynecology, National Cheng Kung University (Tainan,
Taiwan, China). Each fragment was divided into smaller pieces with a sterile
scalpel and washed thoroughly with sterile HBSS to remove contaminating
RBCs. Washed fragments were then cultured in DMEM supplemented with
10% fetal bovine serum and antibiotics in a 35-mm Petri dish at 37°Cin a
humidified
5% CO2 incubator.
Once the cells began
to grow on the edges
of
the tissue fragments, they were fed with fresh medium twice a week. Within 2
weeks, the primary explants were ready to use. The histopathological diag
noses
of normal
confirmed
cervix,
CIS,
on hematoxylin
and
invasive
squamous
and eosin-stained
slides
tissue from the specimen of either hysterectomized
cell carcinoma
of adjacent
was
sections
of
cervix or conized cervix.
Furthermore, after electrophysiological analysis the cells were stained with
hematoxylin and eosin and cytokeratin to rule out the contamination of stromal
cells.
Detection
between
and
HPV
primers
Typing
positivity
of HPV
and
Genome.
activation
were used for HPV detection
purchased
from
Perkin
To investigate
of chloride
and typing.
Elmer/Cetus
Corp.
the correlation
channels,
two
sets
of
One set (MYO9 and MY 11),
(Roche
Molecular
Systems
Inc.,
Branchburg, NJ), was designed to amplify the Ll region from HPV types 6, 11,
16, 18, 31, and 33 and at least 25 other HPV types (25). The other set was the
sense
primer
pU-lM,
5'-TGTCAAAAACCGTTGTGTCC-3',
sense
primer
pU-31B, 5'-TGCTAAUCGGTGCTACCTG-3', and antisense primer pU-2R,
5'-GAGCTGTCGCTTAAUGCTC-3'. The pU-lMIpU-2R were used to am
plify the E6/E7
plification
genomic
described
region
of HPV6
of the E61E7 region
DNA
and I 1, while
of HPV
of each cell was amplified
(27). A positive
and negative
pU-31B/pU-2R
enabled
am
16, 18, 3 1, 33, 52b, and 58 (26). The
and type classified
control
were included
using
PCR as
in the PCR. The
DNA was tested against an internal control gene (r-JFN) to evaluate DNA
quality.
Electrophysiological Measurements. Cells were transferredto a record
ing chamber
@
that was mounted
on the stage of an inverted
microscope
(TMS-F;
Nikon, Tokyo, Japan). To monitor the changes of cell size, the microscope was
coupled to a video camera system with magnification up to X 1500.Cells were
bathed at room temperature (20—25°C)
and continuously superfused at a rate
of about
2 ml/min
with
Tyrode's
solution.
Membrane
potentials
or ionic
currents were recorded in current clamp or voltage clamp mode with patch
pipettes
in a whole-cell
configuration
(28, 29). The pipettes
were connected
to
the input stage of an Axopatch-l D amplifier (Axon Instruments, Burlingame,
CA). For the preparation of the patch pipettes, the Kimax glass capillaries
(Kimble Products, Vineland, NJ) were heated and pulled by gravity using a
and
Solutions.
All of the chemicals
were
purchased
from
Sigma
Chemical. The composition of normal Tyrode's solution (300 mOsm/liter) was
as follows (in mM):NaC1, 136.5; KC1,5.4; CaCl2, 1.8; MgCl2, 0.53; glucose,
5.5, and HEPES-NaC1 buffer 5 (pH 7.4). The composition of hypotomc
solution
(200 mOsm/liter)
was as follows
(in mM): NaCI, 86; KC1, 5.4; CaCI2,
I.8; MgCl2,0.53; glucose, 5.5; and HEPES-NaOH buffer 5 (pH 7.4). To record
C1 and block K@current, KC1inside the pipette solution was replaced with
equimolar
CsC1, and the pH was adjusted
to 7.2 with CsOH.
RESULTS
Volume-sensitive Chloride Currents in Cervical Cancer Cell
Lines. The whole-cell configuration of the patch clamp technique
was used to investigate the volume-sensitive Cl currents in two
HPV-positive (SiHa and CaSki) and two HPV-negative (HT-3 and
CX) cervical cancer cell lines. The membrane currents were evoked
by the ramp command pulses at the voltage range between —80mV
and +40 mV, with a duration of 100 ms before and after the addition
of extracellular hypotonic solution (200 mOsm/liter). These experi
ments were conducted with pipettes containing 3 nmi ATP and 0.1 m@i
GTP. As depicted in Fig. I, prior to the application of hypotonicity,
some background currents were observed in all cervical cancer cell
lines. Within 30—50s after these cancer cells were exposed to extra
cellular hypotonic solutions, a membrane current was significantly
activated (P < 0.01 for all four cell lines, Table 1) and reached
stationary values after 50—150s. These hypotonicity-induced effects
were readily reversible by returning the cancer cells to isotonic
medium. The current trace for the I-V relationship of hypotonicity
induced current appeared to be outwardly rectifying at the range of
—8OmVand +40 mV. In addition, notable relaxation of the current
was not observed during perfusion with hypotonic solution. In all
experiments,
for the CF current was relatively constant over
time.
Because of the symmetrical Cl concentrations in the bath and
pipette solutions, the reversal potential of the volume-induced current
was close to 0 mV. Increases in inward current at —80mV and in
outward current at +40 mV, with no change in current at 0 mV, could
also result from activation of a nonselective cation conductance or a
6078
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VOLUME-sENsmvE
CHLORIDE CHANNELS AND CERVICAL CANCER
and extracellular concentration of chloride in the CX cell line. When
the chloride concentration inside the pipette solution was kept at 140
mM and
the external
chloride
concentration
was
reduced
by the
replacement with sodium gluconate, the V@,,for hypotonicity-induced
current shifted to the more positive level. For instance, when the
external chloride concentration was reduced to 30 mr@iand 5 mt@i,the
Vrev values
were
significantly
changed
to 50 ± 5 mV and 78 ± 7 mV,
respectively (n = 6). This result indicates the strong dependence of
changes of ionic currents on the extracellular chloride concentration.
In other words, the more the external chloride concentration is re
duced, the more the driving force on the chloride ion through open
chloride channels is increased. A linear correlation was obtained
between the values of the logarithm of the external chloride concen
tration and those for reversal potential (Fig. 2).
To identify the possible nucleotide requirement for the hypotonic
ity-sensitive Cl currents, experiments were conducted with pipettes
containing 3 mM AlP and/or 0.1 mr@iGTP. The results showed that
ATP, instead of GTP, was required to elicit Cl current induced by
hypotonicity. No activation of Cl current was observed when AlP
was omitted from the intracellular solution (data not shown).
SIHa
Caski
Effect of DIDS, 1,9-Dideoxyforskolln, and Verapamil on the
HT-3
1 nA
25
msec
Fig. I . Volume-sensitive chloride currents in various cervical cancer cell lines. The
patch pipette contained the 140 msi CsCl internal solution. The membrane potential was
held at —40mV and the 100-ms ramp pulses from —80to +40 mV at a rate of 0.2 Hz
was applied. I, membrane current recorded at isotonic medium (300 mOsm/liter); 2,
membrane current recorded after perfusion with hypotonic medium (200 mOsm/liter).
tare Cells of CIS and Invasive Squamous Cell Carcinoma of the
Table I Comparison
inCell
of the C@ conductance activated by the hypotonic
different cervical squamous cells―shock
nS/pFn@'Primary
Control, nS/pF
Cervix. Membrane currents were also measured with the whole-cell
configuration on the nonconfluent cells of eight CIS and seven inva
sive squamous cell carcinoma samples plated in 35-mm Petri dishes.
Swollen.
cellsNormal
culture
0.0940N.S.CCIS
epithelium
0.2250<0.01Invasive
.03250<0.01Established
cancer
linesHVP-immortalized
cell
cellsZI83A
0.0540N.S.Z172
0.0430N.S.Z133
0.0640N.S.Cancer
linesSiHa cell
0.3030<0.01HT-3
130<0.01CaSki
0.0630<0.01CX
‘IThe
values
of
the
slope
1.10 ±0.07
0.21 ±0.01
0.60 ±0.06
1.07 ±
3.22 ±
4.23 ±
100
>
conductance
1.25 ±0.03
0.42 ±0.03
0.36 ±0.02
1.32 ±
0.46 ±
0.45 ±
0.40
1.40
0.40
0.21
2.40
3.20
2.03
2.05
±0.02
±0.01
±0.03
±0.01
at
+30
mV
are
corrected
E
w
±
a 0.1
±
±0.0330<0.01
for
the
series
[Cr]@=140
mM
80
60
4-'
0
40
us
a)
>
a,
resistance
and normalized for the membrane capacitance. Each value represents mean ±SD.
b Number of experiments.
( N.S.,
Hypotonic-induced
Currents. Fig. 3 is a representative recording
obtained for a CX cell. Clearly, 1 mt@iDIDS produced a remarkable
inhibition on the volume-sensitive Cl current. The inhibitory effect
of DIDS on the hypotonic-induced currents was rapid onset, dose
dependent, and reversible. The concentration required for the half
maximal inhibitory effect of DIDS was about 100 ,.LM.Interestingly,
l,9-dideoxyforskolin (100 p.M), which was not able to activate adeny
late cyclase activity, effectively and reversibly inhibited the volume
sensitive C1 currents in all cancer cell lines (Fig. 4A, a representative
recording for a CX cell). The percentage of inhibition of volume
sensitive C1 current in the CX cell line caused by 10 and 100 pM
l,9-dideoxyforskolin was 60 ±10% and 90 ±6%, respectively (Fig.
4B). Similarly, verapamil also abolished the volume-sensitive chlo
ride currents ofthe CX cell line in a dose-dependent manner (Fig. 4B).
These results, taken together with those from ion replacement studies,
suggest that hypotonicity activates a C1 channel and rule out the
possibility that hypotonicity-induced current is from cation flow or a
leak.
Hypotonicity Activated the Chloride Currents in Primary Cul
nonsignificant.
20
t@
,—@.
1
10
100
1000
[Cflo mM
leak. To further clarify whether this current is predominantly carried
by chloride ion, the external chloride concentration in hypotonic
solution was replaced with an equimolar concentration of sodium
gluconate. Fig. 2 shows the relationship between the reversal potential
Fig. 2. The reversal potential for the hypotonicity-induced current at various extracel
lular C1 concentrations (lCl10) ofthe CX cell line. Points, mean; bars, SD (n = 6). The
line was plotted semilogarithmically and was well fit by the linear regression analysis. Of
note,the abscissais shownin logarithmscale.[Cl I, intracellularC1 concentration.
6079
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VOLUME-SEN5FflVE
CHLORIDE
CHANNELS
AND CERVICAL
CANCER
nS/pF at +30 mV (Fig. 5, n
50 cells from 8 patients). Exposure to
the hypotonic solution induced a fast-activating and outward-rectify
ing current that was reversed between 0 and + 10 mV. In addition,
hypotonicity elicited a 7-fold increase in current to 1.5 ±0.3 nA and
—3.5±0.8 nA at +40 and —
80 mV, respectively. The slope con
ductance at +30 mV was increased by 16-fold to 3.22 ±0.22 nS/pF
(P < 0.01, Table 1). A similar current was also observed in invasive
cancer cells exposed to hypotonic solution (Fig. 5). In the invasive
cancer cells, the slope conductance at + 30 mV was increased from
0.60 ±0.06 nS/pF to 4.23 ±0.32 nS/pF by hypotonic activation
(n
50 from 7 patients; P < 0.01, Table I). Among the 15 CIS and
invasive cancer samples, 13 were positive for HPV DNA and most of
the HPV-positive samples had HPV 16 DNA (10/13). The cells from
the two HPV-negative samples developed volume-sensitive chloride
currents that were indistinguishable from those of HPV-positive cells.
These results, together with those from cervical cancer cell lines,
1 nA L
25 msec
Fig. 3. The volume-sensitive Cr current in the CX cell line was effectively and
reversibly inhibited by a Cl channel blocker, 1 mat DIDS. The patch pipette contained
the 140 mM CsC1 intemal solution. The membrane potential was held at —40mV, and the
100-ms ramp pulses from —80to +40 mV at a rate of 0.2 Hz was applied. 1. basal
membrane current recorded at isotonic medium (300 mOsm/liter); 2, current recorded
after perfusion with hypotonic medium (200 mOsm/liter); 3, current recorded after adding
1 inst D@S in the bath medium; 4, current recorded after washing out DIDS.
A
A
C's
2
ddFSK
U)
4-'
C
C
a,
—1
-2
U
a,
C
-3
-@
E
a,
0
ISO
.
HYPO
-4
Seconds
-50
- 1 00
50
0
mV
B
100
C
B
80
Invasive
Cancer
3
0
4-'
.D
60
2
C
0
40
1
20
—.
ddFSK
—0-—
Verapamil
0
0
1
1'O
Concentration
@
100
C
(pM)
Fig. 4. Effect of P-glycoprotein inhibitors (l,9-dideoxyforskolin and verapamil) on the
volume-sensitive C1 current of the CX cell line. The membrane currents were recorded
at the holding potential of +40 mV. A, time course of the inhibitory effect of 1,9dideoxyforskolin (100 @.LM)
on the hypotonicity-induced outward current. The sequences
including drug application and the replacement of the superfusion solution are shown
above A. B, dose-response relationships of P-glycoprotein inhibitors for the percentage of
inhibition of hypotonicity-induced current measured at the level of +40 mV. Points,
mean; bars, SD (n = 6). CJ4FSK, l,9-dideoxyforskolin.
—1
-2@
0
-3.
S
-50
-100
Fig. 5 summarizes the mean results obtained on CIS and cancer cells
challenged with hypotonic solution. In the isotonic bath, CIS has a
.
@
mV
HYPO
50
0
Fig.5. Effectof hypotonicityon current-voltage(I-V) relationshipsin the primary
culturecellsof CIS(A) and invasivecancerof the cervix(B). The membranepotential
was
held
at
—40
mV,
and
the
100-ms
ramp
pulses
from
—80
to
+40
mV
at
a rate
of
0.2
small background current averaging 0.3 ±0. 1 nA at +40 mV and
wasapplied.Points,mean;bars, SD (n = 50). ISO,isotonicmedium(300mOsm/
—0.2±0.1 nA at —80mV with a slope conductance of 0.21 ±0.01
liter);HYPO,hypotonicmedium(200mOsm/liter).
6080
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V0LuME-sENsmvE
A.
Normal
Cervical
B. HPV-immortalized
CHLORIDE CHANNELS AND CERVICAL CANCER
Epithelium
Cell
zi 33
zi 72
and Z183A) were used in this study as representative cells of low
grade CIN. Similar experiments using these HPV-immortalized cell
lines also failed to show activation of chloride currents by hypotonic
ity (Fig. 7B and Table 1). To eliminate the possibility that different
culture conditions might affect the expression of the volume-sensitive
chloride channel in squamous cells, cells from four cancer cell lines
were cultured in serum-free medium, and cells from three HPV
immortalized cell lines were cultured in medium used for cancer cell
lines for 2 or more days. Patch clamp analyses were carried out again,
and the results demonstrated that the expression of volume-sensitive
chloride channels in each cell type remained unchanged regardless of
the medium used.
cAMP-activated
Currents
in Normal Cervical Epithelium,
HPV-immortalized
Cell Line, and Cervical Cancer Cell Line. As
shown in Fig. 7, the whole-cell currents were low and typically
remained stable under the isotonic medium in unstimulated HPV
immortalized cells (Z172, 150 ±50 pA at +30 mV), cancer cell lines
(CX, 200 ±50 pA at +30 mV), and normal cervical epithelial cells
(100 ±20 pA at +30 mV, data not shown). In these cells, 10 pr@i
forskolin produced rapid and sustained increases in membrane cur
rents, of which the magnitude of the induced currents varied between
10 and 15 times basal values (normal epithelium, 2.3 ±0.5 nA; Zl72,
2.0 ±0.1 nA; CX, 2.4 ±0.3 nA at 30 mV). These forskolin-induced
currents were entirely reversible with washout of the bath medium.
The forskolin-stimulated I-V relation is essentially linearly over the
A
Z172
4
2
zi 83A
0
@n@I
-2
@
mA
-4
25msec
I
0
0
Control
pM
Forskolin
-6
Fig. 6. Nonsignificant activation of chloride currents by hypotonicity in normal cer
vical epithelial cell (A) and HPV-immortalized cells (B). The patch pipette contained the
140 mat CsCI intemal solution. The membrane potential was held at —40mV, and the
100-ms ramp pulses from —80to +40 mV at a rate of 0.2 Hz was applied. 1, membrane
-100
0
-50
50
mV
current recorded at isotonic medium (300 mOsm/liter); 2, membrane current recorded
B
after perfusion with hypotonic medium (200 mOsm/liter).
cx
4
suggest that the expression of volume-sensitive chloride channels in
CIS and in squamous carcinoma cells is independent of their associ
ation with HPV.
Hypotonicity Could Not Significantly Activate Volume-sensi
five Chloride Currents in Normal Cervical Epithelial Cells and
HPV-immortalized Cells. Fig. 6A shows the effect of hypotonicity
@
2
0
•—fr__o•___o_@_•__@___'7_
°
C
-2
on the membrane currents of normal cervical epithelial cells derived
from eight patients. In the isotonic bath, normal cervical epithelial
-4
I
pM
Forskolin
cells have a background current averaging 0.4 ±0.2 nA at +40 mV
0
Control
and —0.3 ± 0.1 nA at —80 mV with a slope conductance of
-6
1. 10 ±0.07 nS/pF at +30 mV (n = 40 from 8 patients). After
50
-50
0
-1 00
mV
exposure to the hypotonic medium, the normal cervical epithelial cells
were sizably swollen. However, there were no significant changes in
Fig. 7. cAMP-activated whole-cell currents in HPV-immortalized cell line (Z172; A)
and cervical cancer cell line (CX; B). Points, mean; bars, SD (n
6). The membrane
membrane currents or the slope conductance (0.45 ±0.19 nA at +40
potential was held at —40mV, and the 100-ms ramp pulses from —80to +40 mV at a
mV; —0.36±0. 15 nA at —80mV; 1.07 ±0.09 nS/pF at + 30 mV;
rate of 0.2 Hz was applied. The membrane current was recorded before and after
P > 0.05, Table 1). Three HPV-immortalized cell lines (Zl 33, Z172,
superfusion with 10 1.@M
forskolin.
6081
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.
VOLUME.5EN5ITIVE CHLORIDE CHANNELS AND CERVICAL CANCER
voltage range from —80to +40 mV, and the reversal potential was
near 0 mV for these cells, as expected for a C1 current (Fig. 1).
Prompt onset and reversibility of action were considered essential
criteria for identifying agonist-specific effects on cell currents. Fur
thermore, forskolin stimulated the whole-cell currents at —70mV
without changing currents at 0 mV, suggesting that forskolin in
creased membrane C1 conductance without altering conductances to
K@ or Nat. Similar currents evoked by 200 pr@idibutyl cAMP were
indistinguishable from those activated by 10 pr@iforskolin (data not
shown). In contrast, 1,9 dideoxyforskolin, an analogue that does not
activate adenylate cyclase, had no effect on the whole-cell currents in
forskolin-responsive cells (10 pr@i,n = 6 for normal cervical epithe
hum, Zl72 cell line, and CX cell line). Thus, the adenylate cyclase
activation and increased cellular cAMP level mediate the forskolin
action.
DISCUSSION
precise function of this channel in cervical squamous cancer cells is
currently under investigation.
Interestingly, our results show that cervical squamous cancer cells
developed volume-sensitive chloride currents that were indistinguish
able from those in HeLa cells, a cervical adenocarcinoma cell line, in
terms of the magnitude of currents, ATP dependency, and reversibly
inhibition by DIDS, l,9-dideoxyforskolin, and verapamil (15). It is
possible that such channels may be associated with carcinogenesis of
cervical adenocarcinoma.
Our results show that cAMP-dependent chloride currents are ex
pressed ubiquitously in all cervical tissues and cell lines studied here,
suggesting that these currents may be involved in ion transport across
cervical epithelia. These results demonstrated that not all of the Cl
channels are uniformly up-regulated during cervical carcinogenesis.
The role of cAMP-dependent currents might be especially important
in cervical cells that are not able to express volume-sensitive chloride
channels significantly.
The outward rectified, ATP-dependent, volume-sensitive chloride
currents were induced by hypotonicity in all of the CIS and invasive
carcinoma of uterine squamous cells investigated, but not in HPV
immortalized cells and normal cervix, suggesting that activation of
volume-sensitive chloride channels is associated with malignant trans
formation of human cervical squamous epithelium independent of
their association with HPV. This study may provide a model for better
understanding of the molecular carcinogenesis of human cervical
cancer. In addition, volume-sensitive chloride channels in cervical
cancer cells may provide a potential target for therapeutic intervention
of cervical carcinoma and the reversal of malignant progression in
human cervical carcinogenesis.
This study demonstrates that volume-sensitive chloride currents
were distinctly activated in cervical cancer cell lines as well as in
primary culture cells of CIS and invasive squamous cell carcinoma,
but not in HPV-immortalized cells and normal cervix. These results
strongly suggest that the activation of volume-activated chloride cur
rents is associated with malignant transformation of human cervical
squamous epithelium, independent of its association with HPV. Al
though activation of Cl channels in response to hypotonicity has
previously been demonstrated in several epithelial cells (14—19),
these studies emphasized the electrophysiological and pharmacolog
ical properties of these currents. Our present study addresses the
expression of Cl channels and the evolution of cervical carcinoma.
ACKNOWLEDGMENTS
This is the first report demonstrating the association of volume
sensitive chloride channels with human carcinogenesis.
We thank Professor Tien M. Kuo (M. D. Anderson Cancer Center, Houston,
It has been known for several years that HPV infection is associated
TX) for critical advice and Dr. Tzer-Ming Chen (National Taiwan University,
cells.
with cervical cancer. However, HPV infection alone is insufficient to Taipei, Taiwan, China) for providing the HPV-immortalized
initiate malignant transformation of the cervical epithelium (30—32),
suggesting that other etiological factors must be involved in the
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6083
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Volume-sensitive Chloride Channels Associated with Human
Cervical Carcinogenesis
Cheng-Yang Chou, Meng-Ru Shen and Sheng-Nan Wu
Cancer Res 1995;55:6077-6083.
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