From the Department of Physiology and Biophysics,
School of Medicine, Wright State University, Dayton, Ohio 45435 and
¶ Division of Hematology, Department of Medicine, University of
Cincinnati College of Medicine, Cincinnati, Ohio 45243
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ABSTRACT |
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Exposure of mammalian cells to UV light causes
initial changes in the cell membrane, induces phosphorylation and
clustering of growth factor/cytokine receptors, and activates the Jun
N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling
pathway leading to programmed cell death (apoptosis). In this study, we found that an early event in the cell membrane of myeloblastic leukemia
(ML-1) cells was the vigorous activation of the voltage-gated K+ channel by UV irradiation. The strong enhancement
by UV irradiation of K+ channel activity in the cell
membrane subsequently activated the JNK/SAPK signaling pathway and
resulted in myeloblastic leukemia cell apoptosis. Suppression of
UV-induced K+ channel activation with specific channel
blockers prevented UV-induced apoptosis through inhibition of
UV-induced activation of the proteins SEK (SPAK kinase) and JNK.
However, suppression of K+ channel activity could not
protect cells from etoposide-induced apoptosis, which bypasses the
membrane event. Elimination of extracellular Ca2+ had no
effect on the UV-induced and K+ channel-mediated JNK/SAPK
activation. Thus, we have identified a novel mechanism in which
activation of K+ channels by UV-irradiation upstream of SEK
and SAPK/JNK mediates UV-induced myeloblastic cell apoptosis.
Exposure of mammalian cells to UV irradiation causes programmed
cell death and cancers. Early responses to UV irradiation include the
activation of transcription factors, Ap-1 and NF- What upstream events of this signaling transduction are induced by UV
irradiation? Ras, a small G protein and transmitting signal from
membrane to cytoplasm, is activated by UV irradiation and mediates
UV-induced activation of JNK, Erk, and transcription factors (6,
11-14). Src, a nonreceptor tyrosine kinase, is stimulated by UV
irradiation (13). Exposure to UV light induces clustering and
internalization of cell surface receptors for epidermal growth factor,
tumor necrosis factor, and interleukin 1. Inhibition of clustering or
receptor down-regulation attenuates the response to UV (7). UV
irradiation induces ligand-independent activation of numerous receptor
tyrosine kinases such as epidermal growth factor, platelet-derived
growth factor and insulin receptors, and protein-tyrosine kinases at
the inner side of the plasma membrane (13, 15-18). Other
membrane-associated proteins, the protein-tyrosine phosphatases, can be
inhibited with UV irradiation by targeting an essential -SH group in
the tyrosine phosphatase. This results in inhibition of
dephosphorylation and enhancement of autophosphorylation of epidermal
growth factor receptor and platelet-derived growth factor receptor
(18). It has been demonstrated in enucleated cells that UV activation
of NF- Several recent studies have implied that potassium (K+)
plays an important role in the regulation of programmed cell death. A
bacterial pore-forming cytolysin, staphylococcal The question that naturally arises, then, is in regard to the existence
of a relationship between K+ channel activity and UV
irradiation-induced apoptosis in ML-1 cells. Because voltage-gated
K+ channel activity involved in cell proliferation (24, 25) is regulated by growth factors (26) and is associated with Src-tyrosine kinase (27), we propose that activation of the K+ channel
can mediate UV irradiation-induced apoptosis. To address this
hypothesis, we first observed the effects of UV-C light on the
K+ channel in myeloblastic leukemia cells (ML-1) by using
whole-cell and cell-attached patch recording techniques. Then, we
investigated the role of K+ channels in UV-induced cell
death. Finally, we examined the effect of K+ channel
activity on UV-stimulated JNK pathway. Our results show that UV
irradiation activated K+ channels at both whole-cell and
single-channel levels. Blockade of K+ channels with
4-aminopyridine (4-AP) almost completely prevented UV-induced apoptosis
and suppressed UV-stimulated JNK pathway, indicating that UV-activated
K+ channels do mediate apoptosis in myeloblastic leukemia cells.
Cell Culture--
ML-1 cells originally isolated from an acute
myeloblastic leukemia patient were received as a generous gift from Dr.
R. W. Craig (Dartmouth Medical School, NH). All myeloblastic
leukemia cells were grown in Roswell Park Memorial Institute (RPMI)
1640 culture medium containing 7.5% heat-inactivated fetal bovine
serum and 25 mM HEPES buffer. Cells were grown in
suspension culture in a humidified incubator at 5% CO2,
37 °C. FDC-P1 murine myeloid progenitor cells were maintained by
incubating at 37 °C (5% CO2) in RPMI 1640 medium
supplemented with 25 mM HEPES buffer, 0.0004% (v/v)
Patch Clamp Experiments--
Patch pipettes with a resistance of
3-4 M Apoptosis Induction--
Myeloblastic leukemia cells at a
concentration of 3 × 105 cells/ml were incubated with
complete culture medium. K+ channel blockers were added
into the culture medium to a final concentration of 2.0 mM.
For UV irradiation experiments, cells were placed in a tissue culture
hood at a distance of 60 inches from the UV-C light source and exposed
at an intensity of 40 mW/cm2 for 3 to 8 min (60 to 72 J/m2). For exposure to etoposide (an apoptosis inducer), a
stock solution of 10 mg/ml etoposide was added to the culture medium at
a final concentration of 20 µg/ml. After etoposide and UV treatments, cells were incubated at 37 °C in 5% CO2 for 15 to
24 h. Cell viability was measured using the trypan blue dye
exclusion method.
Apoptosis Detection Assays--
Cell apoptosis was detected by
DNA fragmentation and nuclear staining with ethidium bromide/acridine
orange. To determine internuclosomal DNA cleavage, myeloblastic cells
were washed twice with phosphate-buffered saline. Lysis buffer (200 mM Tris-HCl, pH 8.0, 100 mM EDTA, 1% SDS, and
100 µg/ml proteinase K) was added, and cells were then incubated for
4 h at 55 °C. The nuclear lysates were extracted twice with an
equal volume of phenol and then extracted with an equal volume of
phenol/chloroform/isoamyl alcohol (25:24:1). DNA was precipitated with
0.05 volumes of 5 M NaCl and 2.5 volumes of absolute
ethanol, incubated overnight at Immunoblotting and Kinase Assays--
SEK-1 (SPAK kinase 1)
activity was determined by measuring the level of SEK-1 phosphorylation
with Western blotting using monoclonal antibody (1:1000) against
phosphorylated SEK-1 (New England Biolabs, Beverly, MA). After proper
treatments, 1 × 106 cells (5 × 105
cells/ml) were lysed in 20 µl of Laemmli buffer. Western blotting was
performed using the same protocol as described below. Phospho-SEK-1 levels were quantified by measuring film densities with a densitometer.
JNK-1 and p38 activities were measured by an immunocomplex kinase assay
with GST-ATF-2 as the substrate (28, 29). Briefly, ML-1 cells (7 × 106 cells) were washed once with ice-cold
phosphate-buffered saline, then lysed with 1 ml of lysis buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 1.5 mM MgCl2, 2 mM EDTA, 10 mM sodium pyrophosphate, 25 mM
JNK-1 or p38 protein levels were determined by Western blotting.
Briefly, an equal volume of 2× Laemmli buffer was added to 20 µl of
immunocomplex and boiled for 5 min. After fractionation on a 12%
SDS-polyacrylamide gel electrophoresis gel, proteins were transferred
to a polyvinylidene difluoride membrane (Millipore) and incubated with
the same antibodies (1:5000) used for JNK-1 or p38
immunoprecipitations. The membranes were then incubated with goat
anti-rabbit immunoglobulin IgG conjugated with alkaline phosphatase
(1:10,000) (Santa Cruz Biotechnology). Secondary antibodies were
detected with a Phototope-Star Western blot detection kit (New England Biolabs).
UV Irradiation Stimulates K+ Channel
Activity--
Changes in cell membrane K+ channel activity
can mediate functional adaptation to a variety of chemical and physical
stresses through membrane voltage stabilization and maintenance of salt and water balance. We found that cytokine-mediated stimulation of
proliferation in myeloblastic ML-1 cells is associated with increases
in K+ channel activity. This channel activity is sensitive
to inhibition by 4-AP but less sensitive to inhibition by
Ba2+ and tetraethyleneammonium (TEA) (24). Using the
nystatin-perforated whole cell technique, the whole-cell current was
activated by depolarization of the membrane potential from a holding
potential of
To further confirm the effect of UV irradiation on single
K+ channel activity, the cell-attached patch clamp was
used. The single-channel current was recorded at a membrane potential
of Effect of Suppressing K+ Channel Activity on UV-induced
Apoptosis--
To determine whether UV-induced K+ channel
hyperactivity is a component of the cell signaling pathway mediating
UV-induced apoptosis, the effect of blocking K+ channel
activity with the K+ channel inhibitor 4-AP was determined
by measuring cell viability after UV irradiation in the presence or
absence of 4-AP. In the presence of 2 mM 4-AP, cell
viability was protected from UV irradiation (99.5 ± 0.5% for
control, 96.1 ± 0.7% for 4-AP-treated, 32.0 ± 6.7% for
UV-induced, and 92.6 ± 1.6% for UV plus 4-AP) (Fig.
2A). In contrast, 4-AP had no
protective effect on cells treated with another apoptosis-inducer,
etoposide (an inhibitor of topoisomerase II). With etoposide alone,
viability decreased to 59.4 ± 3.2%. This decline was
indistinguishable from the effect of etoposide measured in the presence
of 4-AP (61.5 ± 0.4%) (Fig. 2A).
The protective effect of 4-AP on UV-induced cells was a
time-dependent process. The addition of 4-AP before or at
the onset of UV irradiation completely prevented cell death from UV
irradiation. However, when 4-AP was added 5 s after the onset of
UV irradiation, 30% of the cells died (Fig. 2B). We also
found that four other types of myeloblastic cells (FDC-P1, U937, HL-60,
and Himeg-1) exhibited 4-AP-sensitive K+ channel activity
(data not shown). After exposure of these cells to UV irradiation,
their viabilities decreased to 57.7 ± 3.6%, 8.3 ± 0.6%,
27.0 ± 0.3%, and 31.0 ± 7.0%, respectively. The
suppression of K+ channel activity with 4-AP had the same
protective effect on UV-induced apoptosis in these cells as it did in
ML-1 cells (Fig. 2C).
The effects of blocking channel activity on UV- and etoposide-induced
cell death were evaluated using DNA fragmentation and nuclear staining
methods. Suppression of K+ channel activity with 4-AP
completely prevented UV-induced DNA fragmentation in ML-1 cells but did
not prevent etoposide-induced DNA fragmentation (Fig. 2D).
The suppression of UV-induced DNA fragmentation was also observed in
four other myeloid leukemia cell lines (Fig. 2E). The
protective effect of 4-AP against UV- and etoposide-induced apoptosis
was evaluated based on the extent of nuclear staining with ethidium
bromide/acridine orange. Exposure of cells to UV irradiation and
etoposide resulted in orange-stained nuclei indicating nuclear death
(Fig. 2F). Suppression of K+ channel activity
with 4-AP protected the cells against UV irradiation-induced nuclear
death, whereas it was ineffective in preventing etoposide-induced nuclear death. These results reveal that UV irradiation elicits K+ channel hyperactivity, which, in turn, mediates
apoptosis. The ability of etoposide to induce apoptosis despite the
presence of 4-AP is consistent with its known inhibition of
topoisomerase II activity at the level of the nucleus.
Effect of Suppressing K+ Activity on UV-activated JNK
Signaling Pathway--
Many cells respond to UV irradiation by
activating their JNK signaling pathway. To determine whether UV
irradiation-induced K+ channel hyperactivity is an
essential component in the UV-activated JNK signaling pathway, JNK-1
activity was measured after suppression of K+ channel
activity. JNK-1 was strongly activated after 5 min of UV irradiation
(Fig. 3A). In contrast,
activation of JNK-1 by UV irradiation was almost completely prevented
when K+ channel activity was suppressed with 2 mM 4-AP. In addition, UV-induced JNK activation was
partially inhibited, falling to 62 and 24% of its original activity
when K+ channel activity was suppressed with either 10 mM TEA or 5 mM Ba2+, respectively
(Fig. 3A). The fact that 4-AP was the most potent protective
agent is consistent with its rank as the most potent inhibitor of this
particular type of K+ channel activity in these cells (24,
26). The suppression by 4-AP of K+ channel activity and of
JNK-1 activation was dose-dependent and reached its maximum
inhibitory effect at 1 mM (Fig. 3B). The
suppressive effect of 4-AP on UV-induced JNK activity closely
corresponds to its dose-dependent inhibition of the
K+ current in ML-1 cells (26).
SEK is a specific protein kinase in the JNK signaling cascade that
phosphorylates and activates JNK (30, 31). To confirm the involvement
of UV-stimulated K+ channel hyperactivity in mediating
events upstream from SEK, the relationship between the phosphorylation
state of SEK and K+ channel activity was characterized. The
SEK-1 protein was strongly phosphorylated after a 5-min exposure to UV
irradiation (Fig. 3C). Suppression of UV-induced
K+ channel activity with either 4-AP or Ba2+
inhibited SEK-1 phosphorylation by 70 and 16%, respectively. These
results indicate that suppression of UV-induced K+ channel
activity specifically inhibits the early events in the cell membrane
upstream from JNK.
Another stress-activated MAP kinase, p38, is also activated in response
to UV irradiation (32, 33). To test for K+ channel-mediated
p38 activation in response to UV irradiation in ML-1 cells, the effect
of UV irradiation was measured on p38 activity in the presence and
absence of K+ channel blockers. Activation of p38 occurred
irrespective of the presence or absence of 4-AP (Fig. 3D).
Our results strongly suggest that UV-stimulated K+ channel
hyperactivity is an essential upstream component of the JNK signaling
pathway; however, p38 activation is not linked to stimulation of
K+ channel activity. The mechanism underlying UV-induced
p38 activation and apoptosis remains to be elucidated.
Effects of Osmotic Stress and Ca2+ Influx on JNK
Activity in K+ Channel-suppressed Cells--
To further
support the notion that 4-AP is not a nonspecific inhibitor of JNK
pathways, we examined the effect of 4-AP on JNK-1 activation induced by
hyperosmolarity in ML-1 cells. Hyperosmotic shock (600 mM
sorbitol) strongly activated JNK-1. 4-AP had no effect on JNK activity
(Fig. 4A). This result
indicates that 4-AP has a specific inhibitory effect on UV-induced JNK
activation. In addition, it suggests that the effect of 4-AP on
UV-induced JNK-1 activation is likely through blockage of
K+ channel activity and that activation of JNK-1 by
hyperosmotic shock in these cells may not be a K+ channel
activity-mediated process.
Recent studies have shown that an increase of Ca2+ influx
may be a component of the signaling mechanism for mediating UV-induced apoptosis. Accordingly, we examined whether UV irradiation could induce
JNK stimulation when extracellular Ca2+ concentration was
reduced by the addition of EGTA. Our results showed that at a very low
Ca2+ concentration (0.5 mM EGTA) or in a
nominally Ca2+-free medium (5 mM EGTA) JNK
activation still occurred in response to UV irradiation (Fig.
4B). In addition, suppression of K+ channel
activity with 4-AP inhibited UV-induced JNK activation in
Ca2+-free medium. Therefore, Ca2+ influx did
not play a significant role in the JNK signaling pathway mediating
UV-induced apoptosis in ML-1 cells.
We provide evidence for a novel mechanism of UV
irradiation-induced apoptosis in myeloblastic leukemia cells. An
important early component of the signaling process mediating UV-induced apoptosis is strong activation of cell membrane K+
channels. There is growing evidence showing that K+ channel
activities are probably involved in programmed cell death. Various
investigations have shown that K+ channel activity can be
affected by apoptosis inducers, including reactive oxygen species
(34-37), Fas ligand and tumor necrosis factor (38, 39), and anticancer
drugs (40, 41). The K+ channel blocker 4-AP inhibits the
shrinkage of human eosinophils undergoing apoptosis induced by cytokine
withdrawal (42), and a combination of two K+ channel
blockers, TEA and 4-AP, inhibited interleukin 1b release from
lipopolysaccharide-stimulated monocytes (43). Neurons undergoing apoptosis exhibited an up-regulation of outward K+
currents. This enhancement of outward K+ current, induced
by serum deprivation and staurosporine, can be prevented by the
K+ channel blocker TEA and by increasing the extracellular
K+ concentration. It has also been observed that the
K+ channel opener cromakalim induces neuronal apoptosis
(44). Thus, it appears that the activation of K+ channels
is responsible for K+ efflux and the consequent membrane
hyperpolarization and decrease in cell volume, thereby activating a
particular signaling system leading to 1 The stimulation of K+ channel activity could result in the
quick loss of intracellular K+. The loss of intracellular
K+ activates interleukin 1 UV-induced apoptosis in ML-1 cells is dependent on stimulation of
SEK/JNK and p38 pathways in ML-1 cells. We found that activation of
4-AP-sensitive K+ channel activity occurs upstream from the
stimulation of the SEK/JNK pathway and that p38 stimulation is a
component of the cell signaling systems responsible for UV-induced
apoptosis. Activity of p38, however, resides in a signaling pathway
parallel to that of SEK/JNK, as shown by the observation that
inhibition of K+ channel activity with 4-AP has no effect
on p38 activity. This result suggests that p38 stimulation may not be
linked to activation of this type of K+ channel activity.
Our finding that UV-induced K+ channel hyperactivity
precedes SEK and JNK stimulation documents for the first time a role
for membrane ion channels in mediating radiation- and cytokine-induced
signal transduction and apoptosis. This process, as well, has been
shown to account for serum deprivation-induced neuronal cell apoptosis
and occurs in the absence of Ca2+ influx across the cell
membrane (44). Some studies have shown that JNK activation is dependent
on extracellular Ca2+ influx (30, 31). Such an increase in
Ca2+ influx could occur as a consequence of UV-induced
stimulation of K+ channel activity, which increases the
electrical driving force for Ca2+ influx through membrane
hyperpolarization (26, 50). This appears to be true in lymphocytes and
neuronal cells where JNK activation and apoptosis are
Ca2+-dependent (50-53). There are other recent
studies, however, suggesting that the activation of JNK is
Ca2+-independent (49). Our result reveal that
Ca2+ influx does not appear to be a component of the JNK
signaling pathway mediating UV-induced apoptosis in ML-1 cells.
INTRODUCTION
Top
Abstract
Introduction
References
B (1, 2), and of
immediate early genes, c-fos and c-jun (3, 4).
This activation, which is known as the UV response, is mediated by
activation of intracellular signaling pathways that are shared with
growth factors. Erk1/2,
JNK/SAPK1, and p38 are three
mitogen-activated protein kinase pathways that can be activated by UV
irradiation (5, 6). The degree of activation, however, varies. UV
irradiation strongly increases JNK/SAPK activity but only modestly
increases Erk1/2 activity in opposed to growth factors such
as epidermal growth factor (7-10).
B and JNK does not require a nuclear signal (19). These two
observations, the full response of enucleated cells to UV irradiation
and the involvement of membrane-associated proteins in the UV response,
strongly suggest that important UV-induced cell events probably occur
through initiation of conformational changes in the plasma membrane.
-toxin, which selectively permeabilizes plasma membranes for monovalent ions, appeared to induce apoptosis (20). In contrast, diminishing the normal
K+ electrochemical gradient completely nullified the
ability of the anti-Fas antibody to induce apoptosis in Jurkat cells
(21). Apoptotic cells and shrunken cells have a much lower
intracellular K+ concentration compared with normal cells
(22-23). Furthermore, both DNA autodigestion and nuclease activity of
thymocytes are suppressed by an increase in extracellular
K+ concentration in a dose-dependent manner.
The complete inhibition can be reached at a concentration of 150 mM extracellular K+, close to the intracellular
K+ concentration found in normal cells (23).
MATERIALS AND METHODS
-mercaptoethanol, 10% fetal bovine serum, and 10% conditioned medium from WEHI-3b cells as a source of interleukin 3.
when filled with 150 mM KCl solution were
manufactured with a two-stage puller (PP-83, Narishige). For whole cell
K+ current recording, the nystatin perforated patch
technique was used. The pipette tip was filled with a solution
containing 140 mM KCl, 0.05 mM
CaCl2, 2 mM MgCl2, 2 mM
ATP, 0.05 mM GTP, 1 mM EGTA, and 10 HEPES
(titrated with KOH to pH 7.2). The remainder of the pipette was
back-filled with the same pipette solution with the addition of 200 µg/ml nystatin. The bath solution was composed of 140 mM
NaCl, 2 mM KCl, 1 mM CaCl2, 10 mM HEPES, pH 7.4. Voltage-clamp experiments were carried
out by using an Axopatch 200A patch-clamp amplifier, and data were
collected and analyzed with pCLAMP software (Axon Instruments, Inc).
For the cell-attached single channel patch clamp, solutions were 1) KCl
bath solution containing 140 mM KCl, 2 mM
MgCl2, 0.5 mM CaCl2, 1 mM EGTA, and 10 mM HEPES, pH 7.4; and 2)
pipette solution containing 140 mM KCl, 2 mM
MgCl2, 1 mM CaCl2, 10 mM HEPES, pH 7.4. Single-channel currents were recorded
with an Axopatch 200A amplifier (Axon Instruments, Inc.) and filtered
with a 4-pole low pass filter at 2 kHz and digitized at 22 kHz by a
pulse-code modulator (A. R. Vetter, Rebersburg, PA). The pCLAMP
program was used to analyze the single-channel data. Channel activity
was determined as NPo, where N represents
number of channels in the patch, and Po represents the probability of an open channel. All experiments were performed at
room temperature (21 to 23 °C).
20 °C, and centrifuged at
13,000 × g for 10 min at 4 °C. The DNA pellet was
dried and dissolved in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) containing 20 µg/ml RNase A and incubated for
1 h at 37 °C. The DNA was extracted with an equal volume of
phenol/chloroform/isoamyl alcohol (25:24:1). DNA samples were analyzed
by electrophoresis on 1.5% agarose gels, and the results were
visualized by staining with 1 µg/ml ethidium bromide.
PstI-digested
DNA was used as a molecular weight marker. Nuclear staining with ethidium bromide and acridine orange was done by
adding 2 µl of dye mixture containing 100 µg/ml each acridine orange and ethidium bromide to 25 µl of cell suspension. Cell populations were scored according to color using a UV-fluorescence microscope (Nikon). Nuclei staining green have not lost membrane integrity. In contrast, myeloblastic cells in which the nuclei stained
orange have lost membrane integrity. Apoptotic cells can be
distinguished from nonapoptotic cells on the basis of the absence or
presence of nuclear condensation/fragmentation.
-glycerophosphate, 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride,
10 µg/ml leupeptin). Cell lysates were incubated on ice for 10 min
and then were precleared by centrifugation at 13,000 × g for 25 min. JNK-1 or p38 proteins were immunoprecipitated
with 0.5 µg of rabbit polyclonal antibody against JNK-1 or p38 (Santa
Cruz Biotechnology, Santa Cruz, CA) and protein A-Sepharose beads
(Sigma). The immunocomplex was washed three times with lysis buffer and
twice with kinase buffer (20 mM HEPES, pH 7.6, 20 mM MgCl2, 25 mM
-glycerophosphate, 100 mM sodium orthovanadate, and 2 mM dithiothreitol) and resuspended in 50 µl of kinase
buffer. One µg of GST-ATF-2 (Santa Cruz Biotechnology) was added to
30 µl of the immunocomplex. The kinase reaction was initiated by
adding 2 µl of ATP mixture (20 µM ATP and 10 µCi of
[
-32P]ATP (Amersham Pharmacia Biotech). The reaction
proceeded at room temperature for 5 min before it was terminated by
adding 30 µl of 2× Laemmli buffer. Phosphorylation of ATF-2 was
visualized by autoradiography after SDS- polyacrylamide gel
electrophoresis. ATF-2 phosphorylation was quantified by densitometry.
RESULTS
60 to +80 mV in 20-mV increments. Upon exposure of ML-1
cells to UV-C light for 1 min, the amplitude of the K+
current increased markedly. UV-evoked K+ current was
sensitive to 4-AP and was completely blocked by 2 mM 4-AP
(Fig. 1, A and B).
The time course showed that the amplitude of the K+ current
doubled within 1 min after exposure to UV light and reached the maximal
amplitude within 5 min. In the presence of 4-AP, UV stimulation
of K+ currents was blocked, following the time course shown
(Fig. 1C).
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Fig. 1.
Activation of a 4-AP-sensitive K+
channel by UV irradiation. A, effect of UV-irradiation
on the 4-AP-sensitive K+ current. Whole-cell currents were
activated by exposure of ML-1 cells to UV light in the absence and
presence of 4-AP. The membrane potential was depolarized from a holding
potential of -60 mV to +80 mV at 20-mV increments. B,
current-voltage relationship of the 4-AP-sensitive K+
current activated by UV light. C, time course of
UV-activated K+ current in the absence and presence of
4-AP. Currents were normalized as
IUV/IC where
IUV and IC represent
amplitudes of the K+ current measured before and after UV
irradiation, respectively. D, single channel recording of
K+ channel in ML-1 cells. Outward current recorded as an
upward deflection was obtained from cell-attached patches at a membrane
potential of 60 mV. UV-C irradiation was directly applied to the
patch chamber to activate K+ channels in the same patch.
The bottom trace demonstrates that UV irradiation induced an
increase in K+ channel activity that was blocked by
application of 100 µM 4-AP in the patch pipette. Channel
activity (NPo) was plotted as a function of time in
the lower panel. E, statistics of K+
channel activity stimulated by UV irradiation and blocked by 100 µM 4-AP or by 20 mM TEA. Vertical
bars represent mean K+ channel activity
(horizontal bars represent S.E.). An asterisk
represents a significant difference (statistical tests: ANOVA and
Tukey, p < 0.001). Data were collected from seven
independent experiments.
60 mV in vivo (Fig. 1D). Exposure to UV-C
irradiation (~45 J/m2) strongly stimulated K+
channel activity (NPo). Activity increased from
9.6 ± 1.6% to 68.0 ± 5.6% within 30 s (Fig.
1E). In seven independent patches with 100 µM
4-AP in the patch pipette, UV irradiation failed to activate
K+ channel activity, and NPo remained
unchanged at 12.2 ± 3.5% (Figs. 1, D and
E). The addition of 20 mM TEA in the patch pipette reduced the UV-activated channel activity to 28.8 + 4.9% (n = 4, Fig. 1E). These results suggest that
an early effect of UV irradiation is the direct stimulation of cell
membrane K+ channel activity in ML-1 cells.
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Fig. 2.
Protection from UV irradiation-induced
apoptosis by suppressing K+ channel activity with 1 mM 4-AP in myeloblastic leukemia cells. A,
viability of ML-1 cells treated with UV irradiation or etoposide in the
presence or absence of 4-AP blockade. Cell viabilities were determined
24 h after UV irradiation or etoposide (Eto) induction.
An asterisk indicates a significant difference (statistical
tests: ANOVA and Tukey, p < 0.01, n = 12). B, time course of protection from UV
irradiation-induced death by the addition of 4-AP in the culture
medium. The inset shows an expanded time scale with 5 s/division. C, protection from UV irradiation by 4-AP
blockade in various myeloid cells. Cell viabilities were determined
24 h after UV irradiation. An asterisk indicates that
significant differences were found (statistical tests: ANOVA and Tukey,
p < 0.01, n = 9-12). D,
effect of 4-AP blockade on UV- and etoposide-induced DNA fragmentation
of ML-1 cells. Internuclosomal DNA cleavage was determined at 8 and
15 h after apoptotic inductions. PstI-digested -DNA
was used as a molecular weight marker. E, protection from
UV-induced DNA fragmentation by 4-AP blockade in various myeloid cells.
Internuclosomal DNA cleavage was analyzed 15 h after UV
irradiation. F, effect of UV irradiation and etoposide on
nuclear condensation/fragmentation in the presence and absence of 4-AP
blockade in ML-1 cells. Untreated ML-1 cells served as controls.
Assessment of damage was by the ethidium bromide/acridine orange
method. Photographs were taken at a magnification of 400×.
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Fig. 3.
Effects of suppressing K+ channel
activity on JNK and p38 kinase activities. A, protein
immunoblot analysis and kinase assay of JNK-1 activation induced by UV
irradiation. After incubation with 2 mM 4-AP, 10 mM TEA, or 5 mM Ba2+ in normal
culture medium at 37 °C, ML-1 cells were exposed to UV irradiation
for 5 min at room temperature. After an additional 10-min incubation at
room temperature, cells were collected, and JNK-1 activity was measured
by immunocomplex kinase assays. JNK-1 protein levels were analyzed by
Western blot as shown in the lower panel. B,
dose-dependent inhibition of UV-induced JNK-1 activity by
suppression of K+ channel activity with 4-AP. JNK-1 kinase
activity in ML-1 cells was measured after UV irradiation, and JNK-1
protein concentrations were measured by Western blot (lower
panel). Nonirradiated cells served as controls. C,
prevention of UV irradiation-induced SEK-1 phosphorylation by
suppression of K+ channel activity. Western blotting was
performed using the anti-phospho-SEK antibody. SEK-1 activity was
determined by measuring the level of SEK-1 phosphorylation with Western
blotting. Phospho-SEK-1 levels were quantified by densitometry. All of
the kinase activity data were repeated in three to four independent
experiments and were quantified on the basis of band density.
D, effect of suppressing K+ channel activity
with 4-AP on p38 kinase activity in response to UV irradiation. Kinase
activity of p38 was determined by immunocomplex kinase assays; protein
concentration of p38 is shown in the lower panel. JNK-1 and
P38 activities were measured by immunocomplex kinase assay with
GST-ATF-2 as the substrate. ATF-2 phosphorylation was quantified by
densitometry. JNK-1 or p38 protein levels were determined by Western
blotting. Data shown are representatives from three to four independent
experiments.
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Fig. 4.
Effects of hyperosmotic shock and
extracellular Ca2+ on JNK-1 in ML-1 cells.
A, effect of 4-AP on JNK activity induced by hyperosmotic
shock. Hyperosmolarity shock (Hi osm) was performed by
application of 600 mM sorbitol in culture medium for 10 min
in the presence and absence of 2 mM 4-AP. Cells were then
harvested and measured for JNK-1. Height of bars represents
mean values of JNK-1 activity and is quantified on the basis of band
density. B, effect of extracellular Ca2+ on
JNK-1 activation in response to UV irradiation. ML-1 cells were exposed
to UV irradiation in the presence (4th and 6th lanes from
the left) or absence of 4-AP (1st-3rd and fifth lanes from
the left). Extracellular Ca2+ was removed by preincubation
of cells with either 0.5 mM or 5 mM EGTA (3rd
to 6th lanes) 30 min before UV irradiation. JNK-1 activity
was measured under these different conditions by immunocomplex kinase
assays.
DISCUSSION
-converting enzyme
activation and apoptosis.
-converting enzyme (21, 43,
45). Some evidence suggests that interleukin 1
-converting enzyme can affect upstream events in the JNK pathway at the JNK level (46). UV-induced activation of interleukin 1
-converting enzyme and JNK-1
could occur subsequent to the stimulation of K+ channel
activity and the loss of intracellular K+. This mechanism
has been implicated in apoptosis in neuronal cells (47, 48).
Alternatively, cell shrinkage that occurs as a result of a quick fall
in intracellular K+ concentration, may trigger apoptosis.
Accordingly, suppression of K+ channel activity may prevent
a quick loss of intracellular K+ ions resulting from
UV-induced K+ channel hyperactivity. This possibility is
supported by recent findings that UV irradiation-induced JNK activation
can be mimicked by hypertonic stress in HeLa cells (7). In addition,
cytokine receptors can be activated by either UV irradiation or
hypertonic stress. It is speculated that cytokine receptor activation
induced by hypertonic stress occurs as a consequence of cell shrinkage.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. W. Putnam for assistance with fluorescent pictures and Dr. P. Reinack for critically reading the manuscript.
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FOOTNOTES |
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* This study was supported by National Institutes of Health Grants GM46834 and EY11653 (to L. L.) and CA59985 (to W. D.).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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
937-775-3858; Fax: 937-775-3769; E-mail: LUO.LU{at}WRIGHT.EDU.
The abbreviations used are: JNK/SAPK, Jun N-terminal kinase/stress-activated protein kinase; ML-1, myeloblastic leukemia cells; 4-AP, 4-aminopyridine; TEA, tetraethyleneammonium; GST, glutathione S-transferase.
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REFERENCES |
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