Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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We recently
demonstrated expression of a novel, glioma-specific
Cl current in glial-derived
tumor cells (gliomas), including stable cell lines such as STTG1,
derived from a human anaplastic astrocytoma. We used STTG1 cells to
study whether glioma Cl
channel (GCC) activity is regulated during cell cycle progression. Cells were arrested in defined stages of cell cycle
(G0,
G1,
G1/S, S, and M phases) using serum
starvation, mevastatin, hydroxyurea, demecolcine, and cytosine
-D-arabinofuranoside. Cell
cycle arrest was confirmed by measuring
[3H]thymidine
incorporation and by DNA flow cytometry. Using whole cell patch-clamp
recordings, we demonstrate differential changes in GCC activity after
cell proliferation and cell cycle progression was selectively altered;
specifically, channel expression was low in serum-starved,
G0-arrested cells, increased
significantly in early G1,
decreased during S phase, and increased after arrest in M phase.
Although the link between the cell cycle and GCC activity is not yet
clear, we speculate that GCCs are linked to the cytoskeleton and that
cytoskeletal rearrangements associated with cell division lead to the
observed changes in channel activity. Consistent with this hypothesis,
we demonstrate the activation of GCC by disruption of F-actin using
cytochalasin D or osmotic cell swelling.
ion channels; flow cytometry; proliferation; chlorotoxin; glioblastoma; astrocytoma
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INTRODUCTION |
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RECENT STUDIES SUGGEST THAT changes in ion channel
activity may be associated with cell proliferation (10, 21, 22, 24, 38), differentiation (26), migration (25), and biological activation
(13). Possible sites of involvement of ion channels include
transduction of mitogen-stimulated protein synthesis (18), rearrangements in cytoskeletal F-actin (4), generation of
Ca2+ signals (7), and pH
regulation (22). In a number of cell types, increased ion permeability
is one of the earliest events after mitogen stimulation (11), and, in
particular, K+ and
Cl channels have been
implicated in the proliferative response. For example, in lymphocytes,
K+ channels are involved in volume
regulation, control of cellular toxicity, and the immune response (23);
application of K+ channel blockers
such as tetraethylammonium, 4-aminopyridine, and quinine to lymphocytes
suppresses proliferation in a concentration-dependent manner (10).
Blockade of voltage-activated K+
channels also leads to a dose-dependent decrease in the proliferation of melanoma cells (21), breast cancer cells (38), and several glial
cell types including Schwann cells (6), retinal glial cells (24), and
astrocytes (22). More recently,
Cl
channels have been
implicated in the proliferative response of Schwann cells (37), B
lymphocytes (9), and glioma cells (35), suggesting that the link
between channel activity and proliferation also extends to this class
of channels.
Cell division can be operationally divided into defined stages of the
cell cycle (see also Fig. 6). Interestingly, the activity of some ion
channels has been shown to vary during the cell cycle. For example, in
mouse oocytes, a large-conductance (241 pS), voltage-activated K+ channel is active in
G1 and M phases but is inactive
during the G1/S transition (8).
Similarly, in lymphocytes, the delayed rectifier
K+ channel is thought to be
required for cell cycle progression as a signal for progression from
G0 to
G1, a transition that relies on
membrane depolarization and requires a transmembrane flux of Ca2+ (23). In these cells,
Cl permeability varies with
cell cycle, being low in G0 and S
phases and increases in G1/S (3).
Transient changes in ion membrane permeability and cell ion content are
generally assumed to play a key role in the transition from the
quiescent state G0 or early G1 phase to the S phase of DNA
replication (23, 27). Although the exact role that channel activity
plays in cell cycle progression is not clear, it has been proposed that
changes in channel activity result in both long-term changes in gene
expression and short-term modulation of preexisting channel proteins
(23).
We recently reported the expression of whole cell
Cl currents with unique
biophysical and pharmacological properties that selectively
characterizes glioma-derived cells (35). The goal of the present work
was to determine whether astrocytoma
Cl
current activity varies
as a function of cell cycle. Our results suggest a marked and
differential upregulation of
Cl
currents after cell
cycle arrest, with highest glioma
Cl
channel (GCC) activity
in early G1 and lowest activity in
G0/G1 and S phases. We also show that GCC can be activated by disruption of
F-actin filaments. Taken together, we speculate that changes in GCC
activity during the cell cycle result from rearrangements of the cells
cytoskeleton.
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METHODS |
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Cell culture. The STTG1 cell line (American Type Culture Collection, Rockville, MD) was grown in Dulbecco's modified Eagle's medium (DMEM, GIBCO) plus 10% fetal calf serum (FCS, HyClone) at 37°C in a humidified 10% CO2-90% air atmosphere. Cells attaining nearly confluent growth were harvested and plated onto uncoated 75-cm2 flasks or uncoated 12-mm circular glass coverslips for electrophysiology and were used 36-72 h after plating, unless otherwise noted. Viable cell counts were determined by trypan blue exclusion.
Electrophysiology.
Current and voltage recordings were obtained using standard whole cell
patch-clamp methods with an Axopatch-1D amplifier (Axon Instruments).
Patch pipettes were made from thin-walled borosilicate glass (WPI,
TW150F-40, 1.5 mm OD, 1.2 mm ID) and were filled with a solution
containing (in mM) 145 KCl, 1 MgCl2, 0.2 CaCl2, 10 ethylene
glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid [HEPES; pH adjusted to 7.4 with
tris(hydroxymethyl)aminomethane]. Pipettes typically had resistances
between 4 and 7 M
. Cells were continuously superperfused with a
standard bath solution containing (in mM) 125 NaCl, 5 KCl, 1.2 MgCl2, 1.0 CaCl2, 1.6 Na2HPO4,
0.4 NaH2PO4,
32.5 HEPES (acid), and 10.5 glucose (pH adjusted to 7.4 with NaOH).
Osmolarity was measured with a vapor pressure osmometer (Wescor, Logan,
UT) and ranged between 308 and 313 mosM. Calculated equilibrium
potentials for K+,
Na+, and
Cl
under the imposed ionic
gradients in control solution were as follows:
EK =
83.4
mV, ENa = +62.6
mV, and ECl = +2.8 mV.
Proliferation assay. Proliferation was studied quantitatively by determining incorporation of [3H]thymidine, as we have previously described (35). In brief, cells were plated and, after 1 day in culture, were treated for 48 h in the continuous presence or absence of the agent of interest. The growth effects of these agents were tested by the dilution of concentrated stock solutions into the medium. Cells were incubated with 1 µCi/ml radiolabeled thymidine ([methyl-3H]thymidine) for the final 4 h (at 37°C). Culture dishes were rinsed three times with ice-cold phosphate-buffered saline (PBS) and solublized with 0.3 N NaOH for 30 min at 37°C. One aliquot (50 µl) was used for cell protein determination using the bicinchoninic assay (Pierce, Rockford, IL). The remaining cell suspension was mixed with Ultima Gold, and radioactivity was determined with a scintillation counter. The results were expressed as counts per minute per milligram of protein.
DNA flow cytometric analysis. Cells from sister cultures were plated in parallel to those described above for proliferation assays at a density of 106 cells/well. Cells were incubated in the different drugs 48 h after seeding and were harvested by trypsinization after a 48-h incubation period. Cells were rinsed three times with cold PBS, fixed in 70% ethanol for 1 h at 4°C, rinsed with PBS, and incubated with propidium iodide (Boehringer Mannheim) for 1 h at 4°C in the dark. Flow cytometric analysis for cell cycle distribution was done on a FACScan cytofluorometer (Becton Dickinson) using CellFit software, which analyzes cells for their different DNA content based on propidium iodide staining. The SOBR model, which fits Gaussian distribution to the fluorescence peaks, was used to calculate the percentage of cells falling in the G0/G1, S, and G2+M populations.
Immunohistochemical staining. The cultures were fixed for at least 20 min at 4°C in 4% paraformaldehyde and then washed in PBS. Cells were incubated with rhodamine-conjugated phalloidin (Molecular Probes) for 30 min at room temperature. Coverslips were washed in PBS, mounted in Fluoromount (Fisher) on glass slides, and viewed under an epifluorescence microscope using standard procedures.
Data analysis. For all experiments, mean values, SD, and SE were computed from raw values entered into a spreadsheet (Excel, Microsoft). These data were exported to a scientific graphing and data analysis program (ORIGIN, MicroCal). Data were graphed as means ± SE. All statistical analysis was obtained using analysis of variance (ANOVA) test for multiple comparisons, and P values given represent Bonferroni-corrected values.
Drugs used.
Mevastatin was purchased from Biomol (Plymouth Meeting, PA). Unless
otherwise noted, cytosine
-D-arabinofuranoside (Ara-C), hydroxyurea, demecolcine, and all other chemicals were purchased from
Sigma. Ara-C, hydroxyurea, and demecolcine were diluted in distilled
H2O. Mevastatin was diluted in
ethanol (final concentration 0.08%).
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RESULTS |
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Whole cell voltage-clamp recordings were obtained from the human
anaplastic astrocytoma cell line, STTG1. Most of the STTG1 cells (85%)
were glial fibrillary acidic protein-positive (GFAP). Unsynchronized
cells were used as the "control" cell population and varied in
morphology from flat, polygonal cells with short or no processes to
elongated, bipolar-shaped cells (see Fig.
3A). Recordings from these control
cells consistently showed time- and voltage-dependent outward currents
in all (n = 843, Fig.
1A) cells. We previously showed that these outward currents were mediated by the inward movement of
Cl through a
glioma-specific class of Cl
channels (35). The resting potential, determined as the entrance potential with the KCl-containing pipette solution, was
14.1 ± 0.56 (SE) mV (n = 843). A
representative example of whole cell recordings from an STTG1 human
astrocytoma cell in response to depolarizing voltage steps is displayed
in Fig. 1A. Recordings were obtained
by stepping the cells from a holding potential of 0 mV to a series of
test potentials between
105 and 195 mV in 25-mV increments.
Potentials >45 mV resulted in fast activating, noninactivating
outward currents. Cells showed large outward transients on termination
of voltage steps (Fig. 1A). The
current-voltage (I-V) relationship
plotting peak current amplitude as a function of voltage showed
pronounced voltage dependence and outward rectification for both the
steady-state (Fig. 1B, ×) and
outward transient currents (Fig. 1B,
*). Cells from all studied STTG1 cells displayed such outwardly
rectifying currents. We recently published a comprehensive biophysical
and pharmacological characterization of these currents in STTG1 cells,
suggesting that the underlying channels, which we have since termed
glioma Cl
channels (GCCs),
are unique in some of their biophysical and pharmacological features
(35).
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Dependence of Cl channel
expression on cell cycle.
To test the hypothesis that GCC activity may change during cell cycle
progression, STTG1 cells were arrested in defined stages of cell cycle.
Methods used for cell cycle arrest were
1) serum starvation (to 0.1% FCS),
a procedure commonly used to synchronize and arrest the cells in early
G0/G1
(15); 2) treatment with mevastatin
(2.5 µM), which arrests cells in early
G1 (32);
3) treatment with hydroxyurea (1 mM), which arrests cells at the G1/S boundary (32);
4) treatment with Ara-C (10 µM),
which arrests cells in S phase; and
5) treatment with demecolcine (0.05 µM), which arrests cells in M phase (33). The concentrations of each of these reagents were established empirically as the lowest required concentration to yield the desired effect. These are in good agreement with concentrations used by others (32).
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DNA flow cytometric analysis. DNA flow cytometric analysis was used to confirm that the cells chosen for physiological evaluation were arrested at the predicted state of the cell cycle by the different reagents (5). Experiments were performed on sister cultures in parallel and electrophysiological recordings. In the absence of any reagents, most of the unsynchronized cells were in G0/G1 phase of cell cycle (60.6%), with a subpopulation of cells dividing so that 20.2% of the cells were in S phase and 19.2% of the cells were in G2+M phases. By contrast, with each of the arrest agents, we observed a selective accumulation of cells in defined stages of the cell cycle; the relative percentages of cells in the various cell cycle phases under the experimental conditions are given in Table 1. Because flow cytometry only distinguishes three groups of phases of cell cycle, namely, G0+G1, S, and G2+M, arrests by serum starvation (G0/G1) and hydroxyurea (G1/S) are virtually indistinguishable, since no clear G0/G1 distinction is possible. However, a large literature exists that suggests that serum starvation arrests at an earlier cell cycle stage than hydroxyurea.
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Effect of cell cycle on Cl channel
expression and cellular morphology in astrocytoma cells.
Patch-clamp recordings were obtained from sister cultures of STTG1
astrocytoma cells that had been incubated for 44-52 h in the
agents described above, and GCC conductances were determined to allow
comparison of GCC expression between these cell populations. Because
cell shape and cellular surface area change during cell division (31),
conductances were normalized to cell size as conductance densities to
allow for an independent comparison. Representative phase images of
cells treated with cell cycle arrest agents are shown in Fig.
3A, and
mean conductance densities are plotted in Fig.
3B. Marked changes in cell morphology
and GCC conductance density values were observed. Conductance densities were highest in early G1, lowest
in S phase, and intermediate in M phase. Table
2 lists mean values of resting potential,
cell capacitance, peak current amplitude, and conductance densities for
each condition. Conductance density did not differ significantly (P = 0.46) when serum-starved cells
(442 ± 58 pS/pF, n = 19) and control cells (497 ± 55 pS/pF, n = 40) were compared. Serum starvation changed cell morphology to a more
triangular, flat cell body with elongated, bipolar processes, and cell
resting potential was consistently more hyperpolarized (
30.1 mV
compared with
13.6 mV for control cells). A second population of
cells was allowed to progress to early
G1 phase and arrested with
mevastatin. Cell processes were almost exclusively bipolar (Fig.
3A), and conductance density was
significantly (P < 0.05) higher with
values of 798 ± 100 pS/pF (n = 19). Arrest at the G1/S transition
with hydroxyurea (Fig. 3B) led to a
small decrease in GCC conductance density (590 ± 78 pS/pF,
n = 21) that was not significantly
different from control or serum-starved cells and a change to more
polygonal cellular morphology. Conductance density was lowest after
arrest in S phase by Ara-C (318 ± 44 pS/pF,
n = 20, P < 0.01; Fig.
3B). Arrest by demecolcine in M
phase yielded conductance density values of 595 ± 71 pS/pF
(n = 21, P < 0.01). Cells treated with
demecolcine were visibly arrested in the process of cell division, had
rounded cell bodies, and had retracted all processes (Fig.
3A). In all cell cycle arrest
conditions, both steady-state and transient components of GCC currents
were affected about equally (Table 2).
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Changes in astrocytoma morphology and GCC activity are associated
with cytoskeletal changes.
Cell proliferation involves dramatic rearrangements of a cell's
cytoskeleton. It is now evident that ion channels including Cl channels can be
functionally linked to the cytoskeleton, and disruptions of these
contacts have been shown to lead to changes in the functional state of
ion channels (20). Consequently, we explored whether the observed
changes in GCC amplitudes after cell cycle arrest could be explained by
possible changes in the GCC-cytoskeleton relationship. Similar studies
in nontumor astrocytes (16) suggest that rearrangements in the cell
cytoskeleton can activate
Cl
channels. To establish
that GCC channels are linked to the cytoskeleton, we experimentally
disrupted the cytoskeleton by swelling cells with hyposmotic solution.
To visualize changes in the cytoskeleton, cells were stained with a
rhodamine conjugate of phalloidin. In control cells exposed to
normosmolar bath solution (310 mosM), the F-actin appeared in a
well-organized pattern of stress fibers that traversed the entire cell
and intersected with the cell membrane (Fig.
4A).
When the cells were swelled after change to a hypotonic bath solution
(200 mosM), the organized pattern of actin staining was disrupted and
stress fibers were greatly reduced (Fig.
4B). These data suggest that shape
and volume changes are indeed associated with cytoskeletal changes in
glioma cells and could be used as a model to study channel-cytoskeletal
interactions.
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DISCUSSION |
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GCC expression in STTG1 human anaplastic astrocytoma cells was altered if cell proliferation and cell cycle progression were selectively inhibited in defined phases of cell cycle by chemical reagents. STTG1 cells are well-differentiated, GFAP-positive cells widely used as an in vitro model for astrocytoma cell growth. These cells show other hallmarks of astrocytoma cells including responsiveness to epidermal growth factor (unpublished observations) and, as we have previously shown, consistent expression of GCC currents (35).
Striking changes in cellular morphology and GCC activity were associated with each phase of cell cycle. GCC conductance density was approximately twofold higher in G1-arrested cells than in quiescent cells and, overall, was lowest in cells arrested in S phase. Thus, if one views the cell cycle as a continuum, there appear to be cyclical changes in GCC activity (Fig. 6). These changes affected both steady-state and transient current components. Changes in whole cell GCC currents could have resulted from changes in channel expression, recruitment of quiescent channels, or changes in the biophysical properties of the underlying channels. Our electrophysiological approach cannot discriminate between these possibilities. Consequently, we do not at present understand the underlying mechanisms; however, examples exist in the literature to support each of these possibilities. For example, in mouse early embryos, a voltage-activated K+ channel is active throughout M and G1 phases and switches off during the G1/S transition (8). These changes in channel activity are accompanied by shifts in resting potential. K+ channels are also dependent on the cell cycle in breast cancer cells, where inhibition of K+ channels by channel blockers with different mechanisms of actions led to inhibition of proliferation in the G0/G1 phase of cell cycle (38). In HeLa cells (34) and neuroblastoma cells (1), the activation kinetics of the inwardly rectifying K+ channels and the resting potential itself were linked to the G1/S transition, and it is thought that the changes in channel biophysics were responsible for a depolarized resting potential that may be permissive for DNA synthesis. Because we observed reversible changes in GCC conductance of similar magnitude by experimental disruption of the cytoskeleton, we are compelled to speculate that conductance changes during the cell cycle were likewise due to changes in channel activity.
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Cl channels have also been
shown to be regulated by cell cycle in lymphocytes (3), cultured
myoblasts (14) and Boltenia embryos
(36). In Boltenia embryos and in mouse
early embryos, changes in current amplitude are thought to be due to
cyclical insertion of the channel protein into and removal from the
plasma membrane. The incidence and current amplitudes of the
large-conductance Cl
channel's cultured myoblasts were substantially higher in
proliferating vs. nonproliferating cells (14).
Our data do not distinguish between changes in ion channel numbers and
channel open probability. However, channels can be modulated on a
short-term basis by both changes in intracellular Ca2+ concentration and second
messenger systems. The primary physiological regulator of
Cl conductance in
lymphocyte cycling is probably intracellular
Ca2+ (2). In these cells,
cytosolic Ca2+ is low in
G0 and S and increases threefold
in G1 (30). We are currently
investigating whether such cyclical changes in
Ca2+ also occur in glioma cells.
Other regulatory mechanisms of ion channels can involve their
phosphorylation and dephosphorylation (36). These properties are
integral to the functioning of cyclins and cyclin-dependent kinases,
the operators of the cell-cycle clock (17). It is possible that the
channel's voltage sensitivity or activation properties are changed by
phosphorylation. Clearly, further studies are needed to address these
important issues.
The role of GCC channels in glioma cell function and their possible
relationship to the cell cycle is unclear. However,
Cl channels are commonly
implicated in cell volume regulation and water transport, and increased
Cl
conductances have been
demonstrated to be associated with volume changes in a number of cell
types (12, 19). Consistent with a possible involvement of GCCs in
volume regulation of glioma cells, GCC activity was enhanced in
response to osmotic swelling associated with hypotonic challenges (66%
osmolarity). Moreover, our experiments showing changes in GCC activity
after perturbation of the cytoskeleton led us to hypothesize that GCC
activity may be functionally linked to changes in cell shape, occurring
during cell swelling or cell migration. Any change in cell shape that results in a net gain or loss of
H2O requires movements of
Cl
and
K+ across the cell membrane. It is
thus conceivable that changes in GCC activity are associated with
H2O movements in conjunction with
cell shape changes.
A unique characteristic of glial tumors is their tendency to migrate
along anatomic pathways throughout the brain. To migrate, glioma cells
must be able to readily change cell shape, requiring significant
rearrangements in their cytoskeleton. Moreover, migrating glioma cells
have to squeeze through narrow extracellular spaces, probably requiring
them to transiently decrease their cell volume. As discussed above, any
decrease in cell volume requires extrusion of
H2O in conjunction with
Cl and
K+. It is thus conceivable that
GCC channels play an important role in facilitating volume and shape
changes associated with glioma migration. Such an involvement of
Cl
channels in shape
changes has been demonstrated for nontumor astrocytes in which the
transition from a flat to a stellate cell leads to the activation of
Cl
currents, a process that
can be prevented by stabilization of F-actin with phalloidin
(16). Indeed, a link of ion channels and particularly
Cl
channels to cytoskeletal
rearrangements has been documented for a number of other preparations
(20). On the basis of these findings, we hypothesize that glioma
Cl
channels may represent
an adaptive feature facilitating shape and volume changes related to
glioma cell proliferation and migration. Further studies are needed to
clarify the role of GCC in these processes.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Neurological Disorders and Stroke Grant R01-31234.
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FOOTNOTES |
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Present address of N. Ullrich: Interdepartmental Neuroscience Program, Yale University School of Medicine, New Haven, CT 06510.
Address for reprint requests: H. Sontheimer, Dept. of Neurobiology, University of Alabama at Birmingham, 1719 6th Ave S., CIRC Rm 545, Birmingham, AL 35294.
Received 6 November 1996; accepted in final form 27 May 1997.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arcangeli, A.,
L. Bianchi,
A. Becchetti,
L. Faravelli,
M. Coronnello,
E. Mini,
M. Olivotto,
and
E. Wanke.
A novel inward-rectifying K+ current with a cell-cycle dependence governs the resting potential of mammalian neuroblastoma cells.
J. Physiol. (Lond.)
489:
455-471,
1995[Abstract].
2.
Baran, I.
Calcium and cell cycle progression: possible effects of external perturbations on cell proliferation.
Biophys. J.
70:
1198-1213,
1996[Abstract].
3.
Bubien, J. K.,
K. L. Kirk,
T. A. Rado,
and
R. A. Frizzell.
Cell cycle dependence of chloride permeability in normal and cystic fibrosis lymphocytes.
Science
248:
1416-1419,
1990[Medline].
4.
Cantiello, H. F.
Role of the actin cytoskeleton on epithelial Na+ channel regulation.
Kidney Int.
48:
970-984,
1995[Medline].
5.
Carey, F. A.
Measurement of nuclear DNA content in histological and cytological specimens: principles and applications.
J. Pathol.
172:
307-312,
1994[Medline].
6.
Chiu, S. Y.,
and
G. F. Wilson.
The role of potassium channels in Schwann cell proliferation in Wallerian degeneration of explant rabbit sciatic nerves.
J. Physiol. (Lond.)
408:
199-222,
1989[Abstract].
7.
Cornell-Bell, A. H.,
S. M. Finkbeiner,
M. S. Cooper,
and
S. J. Smith.
Glutamate induced calcium waves in cultured astrocytes: long-range glial signaling.
Science
247:
470-473,
1990[Medline].
8.
Day, M. L.,
S. J. Pickering,
M. H. Johnson,
and
D. I. Cook.
Cell-cycle control of a large-conductance K+ channel in mouse early embryos.
Nature
365:
560-562,
1993[Medline].
9.
Deane, K. H.,
and
M. D. Mannie.
An alternative pathway of B cell activation: stilbene disulfonates interact with a Cl binding motif on AEN-related proteins to stimulate mitogenesis.
Eur. J. Immunol.
22:
1165-1171,
1992[Medline].
10.
DeCoursey, T. E.,
G. Chandy,
S. Gupta,
and
M. D. Cahalan.
Voltage-gated K+ channels in human T lymphocytes: a role in mitogenesis?
Nature
307:
465-468,
1984[Medline].
11.
Dubois, J. M.,
and
B. Rouzaire-Dubois.
Role of potassium channels in mitogenesis.
Prog. Biophys. Mol. Biol.
59:
1-21,
1993[Medline].
12.
Garber, S. S.
Outwardly rectifying chloride channels in lymphocytes.
J. Membr. Biol.
127:
49-56,
1992[Medline].
13.
Gardner, P.
Patch clamp studies of lymphocyte activation.
Annu. Rev. Immunol.
8:
231-252,
1990[Medline].
14.
Hurnak, O.,
and
J. Zachar.
Conductance-voltage relations in large-conductance chloride channels in proliferating L6 myoblasts.
Gen. Physiol. Biophys.
13:
171-192,
1994[Medline].
15.
Langan, T. J.,
M. C. Slater,
and
K. Kelly.
Novel relationships of growth factors to the G1/S transition in cultured astrocytes from rat forebrain.
Glia
10:
30-39,
1994[Medline].
16.
Lascola, C. D.,
and
R. P. Kraig.
Whole-cell chloride currents in rat astrocytes accompany changes in cell morphology.
J. Neurosci.
16:
2532-2545,
1996[Abstract].
17.
Leake, R.
The cell cycle and regulation of cancer cell growth.
Ann. NY Acad. Sci.
784:
252-262,
1996[Abstract].
18.
Lewis, R. S.,
and
M. D. Cahalan.
Potassium and calcium channels in lymphocytes.
Annu. Rev. Immunol.
13:
623-653,
1995[Medline].
19.
Lohr, J. W.,
and
L. A. Yohe.
Mechanisms of hypoosmotic volume regulation in glioma cells.
Brain Res.
667:
263-268,
1994[Medline].
20.
Mills, J. W.,
E. M. Schwiebert,
and
B. A. Stanton.
The cytoskeleton and membrane transport.
Curr. Opin. Nephrol. Hypertens.
3:
529-534,
1994[Medline].
21.
Nilius, B.,
and
W. Wohlrab.
Potassium channels and regulation of proliferation of human melanoma cells.
J. Physiol. (Lond.)
445:
537-548,
1992[Abstract].
22.
Pappas, C. A.,
N. Ullrich,
and
H. Sontheimer.
Reduction of glial proliferation by K+ channel blockers is mediated by changes in pHi.
Neuroreport
6:
193-196,
1994[Medline].
23.
Premack, B. A.,
and
P. Gardner.
Role of ion channels in lymphocytes.
J. Clin. Immunol.
11:
225-238,
1991[Medline].
24.
Puro, D. G.,
F. Roberge,
and
C. C. Chan.
Retinal glial cell proliferation and ion channels: a possible link.
Invest. Ophathal. Vis. Sci.
30:
521-529,
1989[Abstract].
25.
Rakic, P.,
R. S. Cameron,
and
H. Komuro.
Recognition, adhesion, transmembrane signaling and cell motility in guided neuronal migration.
Curr. Opin. Neurobiol.
4:
63-69,
1994[Medline].
26.
Reuter, H.,
A. Bouron,
R. Neuhaus,
C. Becker,
and
B. Reber.
Inhibition of protein kinases in rat pheochromocytoma (pc12) cells promotes morphological differentiation and down-regulates ion channel expression.
Proc. R. Soc. Lond. B Biol. Sci.
249:
211-216,
1992[Medline].
27.
Rozengurt, E.,
and
S. Mendoza.
Monovalent ion fluxes and the control of cell proliferation in cultured fibroblasts.
Ann. NY Acad. Sci.
339:
175-190,
1980[Medline].
28.
Sampath, P.,
and
T. D. Pollard.
Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments.
Biochemistry
30:
1973-1980,
1991[Medline].
29.
Schwiebert, E. M.,
J. W. Mills,
and
B. A. Stanton.
Actin-based cytoskeleton regulates a chloride channel and cell volume in a renal cortical collecting duct cell line.
J. Biol. Chem.
269:
7081-7089,
1994
30.
Silver, R. B.
Calcium and cellular clocks orchestrate cell division.
Ann. NY Acad. Sci.
582:
207-221,
1990[Abstract].
31.
Sit, K. H.,
R. Paramanantham,
B. H. Bay,
and
K. P. Wong.
Reduced surface area in apoptotic rounding of human chang liver cells from serum deprivation.
Anat. Rec.
240:
456-468,
1994[Medline].
32.
Soma, M. R.,
R. Baetta,
S. Bergamaschi,
M. R. De Renzis,
C. Davegna,
F. Battaini,
R. Fumagalli,
and
S. Govoni.
PKC activity in rat C6 glioma cells: changes associated with cell cycle and simvastatin treatment.
Biochem. Biophys. Res. Commun.
200:
1143-1149,
1994[Medline].
33.
Takagi, K.,
Y. Isobe,
K. Yasukawa,
E. Okouchi,
and
Y. Suketa.
Nitric oxide blocks the cell cycle of mouse macrophage-like cells in the early G2+M phase.
FEBS Lett.
340:
159-162,
1994[Medline].
34.
Takahashi, A., H. Yamaguchi, and H. Miyamoto. Change in
density of K+ current of HeLa
cells during the cell cycle. Jpn. J. Physiol. 44, Suppl. 2:
S321-S324, 1994.
35.
Ullrich, N.,
and
H. Sontheimer.
Biophysical and pharmacological characterization of chloride currents in human astrocytoma cells.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1511-C1521,
1996
36.
Villaz, M.,
J. C. Cinniger,
and
W. J. Moody.
A voltage-gated chloride channel in ascidian embryos modulated by both the cell cycle clock and cell volume.
J. Physiol. (Lond.)
488:
689-699,
1995[Abstract].
37.
Wilson, G. F.,
and
S. Y. Chiu.
Mitogenic factors regulate ion channels in Schwann cells cultured from newborn rat sciatic nerve.
J. Physiol. (Lond.)
470:
501-520,
1993[Abstract].
38.
Woodfork, K. A.,
W. F. Wonderlin,
V. A. Peterson,
and
J. S. Strobl.
Inhibition of ATP-sensitive potassium channels causes reversible cell-cycle arrest of human breast cancer cells in tissue culture.
J. Cell. Physiol.
162:
163-171,
1995[Medline].