1 School of Biosciences, Cardiff University, Cardiff CF10 3US, United Kingdom, and 2 Guangdong Medical College, Zhanjiang, Guangdong, China 524023
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ABSTRACT |
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Patch-clamping and cell image
analysis techniques were used to study the expression of the
volume-activated Cl current,
ICl(vol), and regulatory volume decrease (RVD)
capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated a
Cl
current with a linear conductance, negligible
time-dependent inactivation, and a reversal potential close to the
Cl
equilibrium potential. The sequence of anion
permeability was I
> Br
> Cl
> gluconate. The Cl
channel
blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),
and ATP inhibited ICl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by a
double chemical-block (thymidine and hydroxyurea) technique. The
expression of ICl(vol) was cell cycle dependent,
being high in G1 phase, downregulated in S phase, but
increasing again in M phase. Hypotonic solution activated RVD, which
was cell cycle dependent and inhibited by the Cl
channel
blockers NPPB, tamoxifen, and ATP. The expression of ICl(vol) was closely correlated with the RVD
capacity in the cell cycle, suggesting a functional relationship.
Inhibition of ICl(vol) by NPPB (100 µM)
arrested cells in G0/G1. The data also suggest that expression of ICl(vol) and RVD capacity are
actively modulated during the cell cycle. The volume-activated
Cl
current associated with RVD may therefore play an
important role during the cell cycle progress.
ion channels; volume regulation; cancer cells
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INTRODUCTION |
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CL
channels whose activity displays cell cycle dependence have been
reported in ascidian embryos (2, 31), ascidian eggs (8), human B lymphocytes (3), and glioma
cells (30). A maxi-Cl
channel had a
substantially higher incidence in highly proliferating cells than in
quiescent cells (14), and a voltage-activated Cl
channel (29) was expressed specifically
in glioma cells but not in normal cells. Cl
channel
blockers inhibited cell proliferation in many types of cells, including
pulmonary artery endothelial cells (32), neuroblastoma cells (21), lymphocytes (24, 21), Schwann
cells (19), microglial cells (22), liver
cells (38), and cervical cancer cells (25),
although they enhanced cell proliferation in some cases (9,
37). These observations suggest an important role for
Cl
channels in the cell cycle and cell proliferation.
An apparently ubiquitous response to swelling in vertebrate cells is
the activation of a Cl current. The outflow of
Cl
through the Cl
channel and of
K+ through a separate channel leads to a decrease in cell
volume, named regulatory volume decrease (RVD). It has been reported
that a voltage-gated Cl
channel in ascidian embryos was
modulated by both the cell cycle clock and cell volume
(31). Inhibition of Cl
currents by channel
blockers arrested cells in G0/G1 phase
(24). Moreover, a volume-sensitive Cl
channel has been reported to be associated with human cervical carcinogenesis. The Cl
current can only be induced by
hypotonicity in human cervical cancer cells but not in normal cervical
epithelial cells (6, 25). It has also been reported that
the magnitude of a volume-activated Cl
current was lower
in differentiated cells than that in proliferating cells
(33). In liver cells, volume-sensitive currents could only
been activated in dividing cells but not in nondividing cells (38). All this evidence indicates that the
volume-activated Cl
current is associated with the cell
cycle and cell proliferation. However, the expression of the
volume-activated Cl
current in the cell cycle has not yet
been well defined. In this study, the expression of volume-activated
Cl
currents and its relation with RVD capacity during
cell cycle progression have been investigated, and the roles of the
current and RVD in the cell cycle and cell proliferation are discussed.
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METHODS |
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Cell Culture
The poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z) (5, 15) were grown in culture medium (RPMI 1640 medium with 10% fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin) at 37°C in a humidified atmosphere of 5% CO2 and were subcultured every other day. For electrophysiological and RVD experiments, cells, which had been cultured for 48 h and reached about 80% confluence, were harvested, resuspended in culture medium, plated onto 12-mm circular glass coverslips, and incubated for 2 h before experiments.Cell Synchronization
Mitotic detachment. Synchronized cells were obtained by the "mitotic shake-off" (sometimes called "mitotic detachment") method. Terasima and Tolmach (28) first made use of the fact that mammalian cells, grown as monolayer, are less well attached to the growing surface when they round up during mitosis (cells in other stages in a adherent cell line are well attached to the culture surface). Gentle agitation of the growth medium will thus remove the mitotic cells, which can then be used as a starting point for a synchronous population. This method can provide highly synchronous cultures of mitotic cells (10, 12, 13). CNE-2Z cells were incubated at a density of 1.5 × 104 cells/cm2 in culture medium in 175-cm2 plastic tissue culture flasks at 37°C using a humidified atmosphere of 5% CO2 for 24 h. After cell attachment and before the mitotic cells were harvested, the cultures were washed to remove unattached dead cells. The culture medium was removed and the cells were washed once with buffered saline solution (BSS) without Ca2+ and Mg2+. After the BSS solution was removed, 15 ml of culture medium were added to the flask. The flasks were shaken by hand for 2-3 min. Cell suspension was collected, pelleted at 80 g for 5 min, and resuspended in prewarmed culture medium. In this way, mitotic cells mainly in metaphase were harvested. The cell suspension was plated onto glass coverslips (for electrophysiological and RVD experiments) or into flasks (for flow cytometric analysis) and incubated at 37°C in the humidified 5% CO2 incubator. Current recordings, RVD experiments, or flow cytometric analyses were carried out at the indicated time points after incubation.
Double chemical block. Double-block technique (10) was used to synchronize cells in S phase. The cells were arrested at G1/S boundary by the inhibitors of DNA synthesis, thymidine and hydroxyurea, and then allowed to enter S phase by removing the inhibitors. The cells were incubated in 2 mM thymidine for 14 h, washed, and allowed to grow in normal medium for 10 h and were then blocked by hydroxyurea (2 mM) for 14 h . The cells were harvested, resuspended in medium with hydroxyurea (2 mM) or washed twice with normal medium (no inhibitors), and resuspended in the normal medium (to release cells from block). The suspended cells were plated onto coverslips or flasks and incubated at 37°C in the humidified 5% CO2 incubator. Current recordings, RVD experiments, or flow cytometric analysis were carried out at the indicated time points after incubation.
Electrophysiological Recordings
Whole cell currents of single CNE-2Z cells were recorded using the patch-clamp technique previously described (4) with a List EPC-7 patch-clamp amplifier (List Electronic, Darmstadt, Germany). The experiments were performed at room temperature (20-24°C). The patch-clamp pipettes were manufactured from standard wall borosilicate glass capillaries with an inner filament (Clark Electromedical Instruments, Kent, UK) on a two-stage vertical puller (PB-7, Narishige, Tokyo, Japan) and gave a resistance of 4-5 MThe permeability ratios
(PX/PCl) of various
anions (X) relative to that of Cl
were
calculated by using the modified Goldman-Hodgkin-Katz equation
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(1) |
Measurements of Cell Volume
Cell volume was monitored and measured during the patch-clamp and RVD experiments. Cell images were captured by a charge-coupled device digital camera (Mono CCD625, Leica, Germany) that was connected to the microscope (Leitz DMIL; Leica Mikroskopie und Systeme, Germany) with a method reported previously (34). The acquisition of the cell images was controlled by the Quantimet Q500MC image processor and analysis software (Leica). The cell volume was calculated from cell diameters (d) with the equation 4/3Cell Capacitance
Capacitance of pipettes was measured before breaking the patch, and capacitance of CNE-2Z cells was determined once the whole cell configuration was achieved. Capacitance reading was obtained by adjusting and minimizing the capacity transients in response to a 2-mV voltage step using the amplifier functions, following the instruction of the amplifier manual (EPC-7, List Electronic, Germany). The capacitance increased during the cell cycle progression, along with increase of cell size. The capacitance was 31.2 ± 2.0 pF in G1 phase (n = 15), 42.3 ± 3.8 pF in S phase (n = 12), and 48.1 ± 3.1 pF in M phase (n = 9). In this study, currents were normalized to cell capacitance.Cell Cycle Analysis
Cell preparation and analysis followed the methods described by Holmes and Al-Rubeai (13). Cells were fixed in 70% ethanol atSolutions and Chemicals
The pipette solution contained (in mM) 70 N-methyl-D-glucamine ClHypertonic bath solution was obtained by adding 140 mM D-mannitol into the isotonic bath solution, giving an osmolarity of 440 mosmol/l (47% hypertonicity).
The pH of the pipette and bath solutions was adjusted to 7.25 and 7.4, respectively, with Tris base. BSS solution without Ca2+ and Mg2+ used to wash cells contained (in mM) 125 NaCl, 5 KCl, 10 NaHCO3, 10 HEPES, 5 glucose, and 20 sucrose, pH to 7.4 with 3 M NaOH.
Cell cycle blockers thymidine and hydroxyurea were dissolved in
phosphate-buffered saline (PBS) and diluted to the final concentrations of 2 mM with culture medium. For Cl channel block
experiments, ATP was dissolved in distilled water, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in dimethyl sulfoxide (DMSO), and tamoxifen in methanol, at concentrations of 100, 30, and 50 mM, respectively, and diluted to final concentrations of 10 mM, 100 µM, and 30 µM, respectively, with hypotonic bath solutions.
The final concentration of methanol in the tamoxifen solution was
0.06% and DMSO in the NPPB solution was 0.33%. The pH of the final
solutions was adjusted to 7.4. All chemicals were purchased from Sigma
(Poole; Dorset, UK).
Statistics
Data are expressed as means ± SE (number of observations) and, where appropriate, have been analyzed using Student's t-test and ANOVA. All experiments were repeated at least three times. ![]() |
RESULTS |
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Hypotonic-Activated Cl Currents in
CNE-2Z Cells
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Typical current traces under isotonic and 47% hypotonic conditions are
shown in Fig. 1, B and C. The current exhibited
an almost linear current-voltage (I-V)
relationship; the outward and inward currents in response to ±80 mV
were 47 ± 4.3 and 43 ± 4.5 pA/pF (n = 30), respectively. Under the voltage steps of ±40, and ±80 mV, the
current exhibited negligible time-dependent inactivation. There was
still no obvious time-dependent inactivation at more depolarizing
potentials up to +120 mV (data not shown). The I-V
relationship demonstrated that the hypotonic-activated current reversed
at a voltage close to the calculated ECl (
0.9 mV), with a mean value of
2.4 ± 1.0 mV (n = 16;
Fig. 1D). In these experiments, there was no K+
present either in the electrode or bath solutions. The concentrations of Cl
inside and outside the cells were almost equal,
giving a value of
0.9 mV for ECl, very close
to the experimental reversal potential. The equilibrium potentials for
Na+ and Ca2+ were both predicted to be greater
than +200 mV. Thus the data strongly support the hypothesis that the
hypotonic-activated current was carried primarily by Cl
.
This was further confirmed by the anion substitution experiments.
Permeability of Volume-Sensitive Cl
Channel
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Pharmacology of the Volume-Sensitive Current
The results above suggest that hypotonic bath solution activated a Cl
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NPPB (100 µM) also reversibly inhibited both the inward and outward
components of the volume-sensitive currents (Fig. 3, C and
D); 51.1 ± 7.0% of the inward current at 80 mV step
and 51.1 ± 7.1% of outward current at
80 mV step were blocked
(n = 14; P < 0.01). The results
indicated that the effects of NPPB were voltage independent (Fig. 3G).
The effects of tamoxifen on the volume-activated Cl
currents varied greatly among cells. The blockage of the current by 30 µM tamoxifen ranged from 35 to 99%, giving a mean value of 69.3 ± 10% (at
80 mV; n = 12). The remaining current
could be inhibited by 10 mM ATP. The typical example of responses is
shown in Fig. 3E. Figure 3F represents the
relationships between voltages applied and the mean currents of
tamoxifen treatments. It seems that the blockage of outward components
was slightly stronger than that of inward current (Fig. 3G),
but the inhibition of inward current (65.0 ± 8.9%; at
80 mV)
was not significantly different from that of outward current (69.3 ± 10%; at
80 mV) (n = 12; P > 0.05). Of those blockers used, ATP was the most effective (Fig.
3G).
In these experiments, NPPB solution contained 0.33% DMSO, and tamoxifen solution contained 0.06% methanol. The effects of DMSO and methanol on the currents were tested. DMSO or methanol at these concentrations did not significantly affect the currents (data not shown).
Expression of Volume-Sensitive Cl
Current in the Cell Cycle
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The Cl currents activated by 47% hypotonic solution of
the shake-off cells sampled at 4 h of incubation, which
represented those of G1 phase (85% of cells in
G1 phase), were large. The currents showed the properties
of linear conductance and negligible time-dependent inactivation (Fig.
4A, X). The mean value was
63.2 ± 2.6 pA/pF at + 80 mV and
56.7 ± 2.6 pA/pF at
80 mV (n = 15; Fig. 4B, X). The currents
were inhibited by ATP, NPPB, and tamoxifen, similar to those of
unsynchronized cells.
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To confirm this finding and to validate the mitotic shake-off
technique, we used an alternative approach to isolate G1
cells: visual identification of cells as they emerged from M phase. M phase is characterized by the appearance of condensed chromosomes and
is clearly visible under phase contrast microscopy (Fig.
5). The nuclear material, which was
condensed during metaphase and divides into two in anaphase (Fig. 5, 0 and 5 min), becomes diffuse (Fig. 5, 8 min). The septum dividing the
cells becomes more visible (Fig. 5, 18 min), and as the two daughter
cells begin to separate their morphology alters to become more
spherical (Fig. 5, 25 min). The cells now enter G1 phase.
When these newborn G1 cells were exposed to 47% hypotonic
solution, the volume-sensitive Cl current was activated;
the outward and inward currents in response to ±80 mV voltage pulses
were 59.6 ± 4.8 and
50.1 ± 4.3 pA/pF (n = 7), respectively. These currents were not significantly different from
those of the shake-off cells sampled 4 h after reincubation (85%
of cells in G1 phase as demonstrated by flow cytometry).
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The hypotonic-activated Cl currents declined dramatically
in the shake-off cells sampled at 10 h of incubation, when 71% of cells had reached S phase. The peak current (30.1 ± 3.1 pA/pF at
+80 mV; n = 13) was much smaller than that of cells
sampled at 4 h (P < 0.01), although the
properties of the currents were similar. The results indicated that
expression of volume-sensitive channels of cells in S phase was
inhibited or the machinery for activation of the channels had been
depressed, compared with that of cells in G1 phase.
Downregulated expression of the volume-sensitive currents in S phase
was further demonstrated by the results obtained from the cells
synchronized by double chemical-block (by 2 mM thymidine and
hydroxyurea). As shown in Fig. 4A, Y and 4B, Y,
cells sampled at 4 h after having been released from the block
expressed a hypotonic-activated Cl chloride current with
properties similar to those of shake-off cells. The mean current was
29.6 ± 2.1 pA/pF at +80 mV (n = 12). It was not
significantly different from that of shake-off cells recorded at 8 h of incubation but smaller than that of shake-off cells detected at
4 h of incubation (G1 phase). Flow cytometry analysis
demonstrated that the double-block cells were distributed predominately
in S phase (86% of the population) at 4 h after release from the
block. Only 7% were in G0/G1 and 7% in
G2/M phases. As for the cells that had not yet been
released from the chemical block (arrested on the border of
G1/S), their hypotonic-activated Cl
currents
were similar to those of cells sampled at 4 h after having been
released from the block, with a mean value of 34.8 ± 1.9 pA/pF
(n = 13).
The chemicals (thymidine and hydroxyurea) used to arrest cells could
have direct effects on the volume-activated Cl currents.
However, the experiments of acute application of thymidine and
hydroxyurea excluded this possibility. Addition of these chemicals to
the bath, at the same concentration (2 mM) as for arresting cells, did
not significantly affect the volume-activated currents.
For the cells in M phase, selected by their condensed chromosomes when
viewed under phase-contrast microscopy, currents were also recorded.
Perfusing the cells with a 47% hypotonic solution activated a
Cl current, showing properties similar to that of the
shake-off cells and with a peak value of 48.4 ± 4.3 pA/pF at + 80 mV (n = 9; Figs. 4A, Z, 4B,
Z). The current was smaller than that of shake-off cells at 4 h (P < 0.01) but larger than that at 8 h and that
of cells released from the double chemical block (P < 0.05). Figure 4C summarizes the expression of the
volume-activated currents of the three groups of cells, shake-off cells
at 4 h (nominal G1 phase), chemical-block cells 4 h after release (nominal S phase), and M cells. Figure 4D
gives the cell distribution at different stages of the cell cycle for
the three groups in the parallel flow cytometric analysis. The data
indicate that the expression or the activation of volume-activated
Cl
channels was significantly modulated during the cell
cycle progress. Their activities were high in G1 phase,
downregulated in S phase, but increased in M phase.
The results also show that the volume-sensitive Cl
currents, recorded in all the stages, shared the similar properties of a linear conductance, negligible time-dependent inactivation, reversal
at the voltage close to Cl
equilibrium potential, and
inhibition by Cl
channel blockers NPPB, tamoxifen, and
ATP. The peak current was the only feature that varied as cells
progressed through the cell cycle.
Correlation Between Volume-Sensitive
Cl Currents and RVD
The relationship between the volume-sensitive Cl current
and RVD was analyzed by comparing current level and RVD capacity. As
shown in Fig. 6A,
Cl
channel blockers NPPB, tamoxifen, and ATP inhibited
both the volume-sensitive current and RVD. The percentage of RVD
inhibition was a function of current inhibition by NPPB, tamoxifen, and
ATP. The inhibition of RVD was linearly correlated with the blockage of
the volume-sensitive current (r = 0.98;
P < 0.01). Furthermore, RVD capacity was a function of
the volume-activated Cl
current. Figure 6B
shows the correlation between RVD capacity and volume-activated
Cl
currents. RVD was plotted against the corresponding
current (evoked at +80 mV step) of the G1 group (shake-off
cells at 4 h of incubation), S-group (chemical block cells at
4 h after release), and M cells (selected under microscope).
Fitting the data, by linear regression, resulted in a positive
correlation between the two factors (RVD and current), with a linear
correlation coefficient r = 0.99 (P < 0.01).
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Linking ICl,vol to the Cell Cycle
Block of the volume-activated Cl ![]() |
DISCUSSION |
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This study demonstrates that CNE-2Z cells respond to hypotonic
shock by activating a volume-sensitive current. Four lines of evidence
suggest that the current is carried by Cl. First, only
the Cl
flow was consistent with the current direction
under the applied voltages and the designated pipette and bath
solutions. Second, the membrane reversal potential for the current was
close to the equilibrium potential for Cl
. Third,
substitution of Cl
with I
,
Br
, or gluconate shifted the reversal potential,
resulting in a permeability sequence of I
> Br
> Cl
> gluconate, consistent
with the reported properties of the volume-activated Cl
current (24, 38). Fourth, the current was inhibited by the conventional Cl
channel blockers NPPB ( see Refs.
18 and 27), tamoxifen (27, 38, 39), and
extracellular ATP (11, 36).
The volume-activated Cl current has been reported in many
cell types and has the characteristics of ATP dependency, voltage sensitivity showing outward rectification and time-dependent
inactivation at strong depolarizing potentials (see Refs.
18 and 27). However, the degree of outward rectification
and time-dependent inactivation varies among experiments or cell types.
It has been reported that ionic strength difference across the plasma
membrane affected the time-dependent inactivation (38).
Decrease of the ionic strength difference led to a smaller degree of
time-dependent inactivation. In our experiments, the absence of
significant time-dependent inactivation may result from the
experimental conditions of equal ionic strength between bath and
pipette solutions. Similar currents have been recorded by our group in
pigmented and nonpigmented ciliary epithelial cells (4, 16,
35) under the same recording conditions. However, we cannot
exclude the possibility that lack of time-dependent inactivation and
outward rectification in the CNE-2Z cell and the differing properties
of the currents between cells may arise from differential expression of
different types of Cl
channels. It has been reported that
there were three types of volume-activated Cl
channels in
ciliary epithelial cells and that the expression of these channels was
different between the two cell types of the ciliary epithelium
(40). Antisense knockdown of MDR1 (35), ClC-3
(36), or pICln (4) gene expression inhibited
the volume-activated Cl
currents. All this evidence
suggests the presence of more than one volume-activated
Cl
channel, although the molecular identity of the
volume-activated Cl
channel(s) has not yet been
determined (7, 27).
In synchronized cells, the expression of the volume-activated
Cl current was actively modulated during the cell cycle.
The shake-off cells, sampled at 4 h after reincubation (85% cells
in G1 phase), expressed a high level of the current, but
the expression of the current in cells sampled at 10 h (71% cells
in S phase) decreased significantly. The results implied that the
expression of the volume-activated Cl
current was high in
G1 phase and then was downregulated in S phase. This was
verified further by the results obtained from the double DNA block
studies. Cells blocked by thymidine and hydroxyurea and sampled 4 h after having been released from the chemical block possessed low
levels of the volume-activated Cl
current. Both thymidine
and hydroxyurea block DNA synthesis (a feature of S phase) and thereby
prevent cells from progressing into S phase, arresting cells at the
G1/S border (10). Once released from the
block, cells will progress into the S phase. Data showed that 86% of
cells were in S phase sampled at 4 h after released from the
block. The volume-activated Cl
current was downregulated
while cells progressed through the G1/S border and into the
S phase. The downregulation of expression of a Cl
channel
in S phase (30) has also been reported. As the cells progressed into M phase, the expression of the volume-activated Cl
current was upregulated again.
The cell cycle dependent expression of the volume-activated
Cl current suggests that the current may play an
important role in cell cycle progress and cell proliferation. It has
been reported that inhibition of the current by Cl
channel blockers suppressed cell proliferation and arrested cells in
G0/G1 phase (24). Our data confirm
this finding. Cells, when exposed to 100 µM NPPB, which blocks around
50% of the volume-activated Cl
current, were arrested in
G0/G1 phase. How does the current affect the
cell cycle progress and cell proliferation? Our results demonstrate that, as with the volume-activated Cl
current, RVD
capacity was also cell cycle dependent, and expression of the
volume-activated Cl
current was closely correlated to the
RVD capacity during the cell cycle. Both the current and RVD were high
in G1, low in S, and upregulated in M phase. Furthermore,
the blockage of the current and RVD was positively correlated. This
suggests that the volume-activated Cl
current may
regulate the cell cycle progress and proliferation via alterations in RVD.
The eukaryotic cell cycle consists of four separate phases:
G1, S, G2, and M phases. The nondividing cells
exit the cell cycle at G1 phase into either a quiescent
(G0) state or a terminally differentiated state. The
restriction point, which controls progression from G1 to S,
divides the G1 phase into two subphases, the G1 postmitotic phase (G1pm) and the G1
presynthesis phase (G1ps). The high level of the
volume-activated Cl channel activity and RVD in
G1 may ensure the concentration of critical factors needed
for controlling progress through the restriction point, as suggested by
Nilius (17). Having passed through the restriction point,
cells can progress into the next phase. However, mature cells must grow
before they divide to maintain normal cell size. It is thought that the
purpose of the G1ps period is to allow cells to grow so
that, later in the cycle, the cell can focus its energy on other
cellular processes such as DNA replication in S phase and
reorganization of the cellular infrastructure during mitosis
(1). It has been demonstrated that the length of
G1ps phase varies greatly. After having passed the
restriction point, larger cells may proceed almost immediately to the S
phase, whereas smaller cells may linger in G1ps for up to
10 h before beginning the transition to the S phase
(1). We have observed a remarkable increase in cell size
in late G1 phase (data not shown) and the downregulation of
the volume-activated Cl
current and RVD at the
G1/S border and S phase. The lower level of the current and
RVD capacity may facilitate cell growth and help the maintenance of
large cell size.
In this study we have also observed the upregulation of the expression
of the volume-activated Cl current and RVD in M phase. It
is known that one important cell cycle checkpoint that maintains
integrity of the genome occurs toward the end of mitosis. This
checkpoint monitors the alignment of chromosomes on the mitotic
spindle, thus ensuring that a complete set of chromosomes is
distributed accurately to the daughter cells.
The volume-activated Cl current may not only affect the
cell cycle progression via RVD but also interfere with the cell cycle or cell proliferation via undefined mechanisms. The Cl
currents can influence the intracellular pH or the pH in various organelles. The cell proliferation can also be affected by
intracellular pH (19a). Thus it has been
postulated that the volume-activated Cl
current may
affect cell proliferation by changing cellular pH (17).
In conclusion, this study has demonstrated that CNE-2Z cells express
volume-activated Cl currents. The expression of these
currents was actively modulated during the cell cycle and closely
correlated to the RVD capacity. The results suggest that the current
may play an important role in the cell cycle progression and
proliferation, but how the change of the current expression affects the
cell cycle progression remains to be elucidated.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Wellcome Trust (056909/299/Z) and the Education Ministry of China (GJ9901).
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. J. C. Jacob, School of Biosciences (BIOSI-2), Cardiff Univ., Museum Ave., Cardiff CF10 3US, UK (E-mail: jacob{at}cardiff.ac.uk).
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.
June 26, 2002;10.1152/ajpcell.00182.2002
Received 19 April 2002; accepted in final form 14 June 2002.
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