From the Department of Cell Metabolism, National
Medical Centre, Institute of Haematology and Immunology,
Diószegi út 64, Budapest H-1113, the § Institute
of Enzymology, Hungarian Academy of Sciences, Budapest H-1113, and
the ¶ Department of Medical Biochemistry, Semmelweis University,
Budapest H-1088, Hungary
Received for publication, June 4, 2002, and in revised form, November 14, 2002
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ABSTRACT |
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Survival and proliferation of cells of a human
myelo-erythroid CD34+ leukemia cell line (TF-1) depend on the presence
of granulocyte-macrophage colony-stimulating factor or interleukin-3.
Upon hormone withdrawal these cells stop proliferating and undergo
apoptotic process. In this report we demonstrate that a controlled
increase in [Ca2+]i induces
hormone-independent survival and proliferation of TF-1 cells. We found
that moderate elevation of [Ca2+]i by the
addition of cyclopiasonic-acid protected TF1 cells from apoptosis.
Furthermore, a higher, but transient elevation of
[Ca2+]i by ionomycin treatment induced cell
proliferation. In both cases caspase-3 activity was reduced, and Bcl-2
was up-regulated. Higher elevation of [Ca2+]i by
ionomycin induced MEK-dependent biphasic ERK1/2 activation,
sufficient to move the cells from G0/G1 to S/M
phases. Meanwhile, activation of ERK1/2, phosphorylation of the
Elk-1 transcription factor, and, consequently, a substantial elevation of Egr-1 and c-Fos levels and AP-1 DNA binding were observed. Moderate
elevation of [Ca2+]i, on the other hand, caused a
delayed monophasic activation of ERK1/2 and Elk-1 that was accompanied
with only a small increase of Egr-1 and c-Fos levels and AP-1 DNA
binding. The specific MEK-1 kinase inhibitor, PD98059, inhibited all
the effects of increasing [Ca2+]i, indicating
that the MAPK/ERK pathway activation is essential for TF-1 cell
survival and proliferation. Based on these results we suggest that the
elevation of the [Ca2+]i may influence the
cytokine dependence of hemopoietic progenitors and may contribute to
pathological hematopoiesis.
The tight control of proliferation, survival, and apoptosis of
bone marrow progenitor cells has a major role in allowing normal hematopoiesis. A variety of cytokines, including
GM-CSF1 and IL-3, regulate
viability, proliferation, differentiation, and function of hemopoietic
cells (1). Hemopoietic cells undergo apoptotic cell death in the
absence of growth factors, whereas an escape from this regulation may
result in leukemogenesis.
The human GM-CSF/IL-3-dependent myelo-erythroid cell line,
TF-1, has been reported to undergo apoptosis upon hormone deprivation (2). Apoptosis of these cells can be suppressed by re-administration of
GM-CSF, IL-3, or IL-4 (3) or overexpression of Bcl-2 (4). In this
report we demonstrate that cytoplasmic free calcium
([Ca2+]i)-increasing agents can also suppress the
apoptotic process and result in long term cell survival.
A number of endogenous substances (adrenergic, purinergic agonists, and
cytokines) are able to evoke different [Ca2+]i
signals. To examine the effect of calcium signals on cell survival and
proliferation, we have applied various
[Ca2+]i-mobilizing agents. SERCA
inhibitors, e.g. cyclopiasonic acid (CPA) and thapsigargin
deplete sarco-endoplasmic reticulum Ca2+ stores, which in
turn induce capacitative influx of Ca2+ (5).
Ca2+ ionophores like A23187 and ionomycin mobilize
Ca2+ from nonspecific sources (6). In parallel with the
distinct mechanism of Ca2+ elevation, the level of
[Ca2+]i and its physiological effects may also
differ among these [Ca2+]i-increasing reagents.
We demonstrate that endogenous substances induce similar
[Ca2+]i increases as CPA. Thus the elevation of
[Ca2+]i caused by CPA is within the range of
physiological [Ca2+]i signals.
The alteration of intracellular Ca2+ concentration control
cellular processes as diverse as proliferation, development,
contraction, or secretion. However, when exceeding its normal spatial
and temporal boundaries, Ca2+ can result in cell death
through both necrosis and apoptosis (7).
The purpose of this study was to delineate how Ca2+ can
have multiple roles in cell survival and growth. Ca2+
functions directly through regulation of gene expression,
e.g. c-fos (8) or via
activating Ca2+-dependent enzymatic action,
e.g. calcium/calmodulin-dependent kinases II and
IV in neurons, calcineurin in lymphocytes, and in conjunction with
other signaling pathways such as those regulated through MAPK and PI3K
(for review see Ref. 7). Another possible mechanism to consider is the
activation of signaling pathways similar to those activated by GM-CSF.
GM-CSF induces several pathways, e.g. STAT-5, MAPK, PI3K,
protein kinase C, and phospholipase C In T lymphocytes and PC12 cells, elevated Ca2+ was reported
to stimulate MAPK activity (9, 10). Stimulation of the MAPK cascade
causes the phosphorylation and activation of additional downstream
targets, such as p90Rsk, CREB, and Elk-1 (11-13). Activated CREB and
Elk-1 can induce the transcription of immediate early genes such as
egr-1 and c-fos (14, 15) followed by other genes that control apoptotic processes (16, 17) or promote cytokine production (18), all modulating proliferation and survival.
In this report we show that agents (CPA and ionomycin) that generate
distinct elevation of [Ca2+]i from different
sources result in the activation of the MEK/ERK/Elk-1 pathway.
Ionomycin induces similar cellular responses as GM-CSF, the natural
growth factor of TF-1 cells. In both cases activation of the
MEK/ERK/Elk-1 displayed similar kinetics and resulted in cell survival
and proliferation. In contrast, CPA induces a delayed MEK/ERK/Elk-1
activation, sufficient only to trigger cell survival. These results
point to the importance of the kinetics of the Ca2+ signal.
Taken together we report that the increase of
[Ca2+]i by activating the MAPK/ERK results in an
escape of CD34-positive myelo-erythroid leukemia cells from apoptosis
induced by hormone-deprivation. These results suggest that an elevation
of [Ca2+]i may rescue hemopoietic progenitor
cells from apoptosis induced by hormone deprivation in the bone
marrow. These effects may protect normal progenitor cells but may also
lead to the development of pathological hematopoiesis.
Chemicals and Reagents--
Unless otherwise specified, all
chemical reagents were purchased from Sigma. The tissue culture media
were purchased from Invitrogen, whereas GM-CSF, IL-3, IL-4, PD98059
(MEK1 inhibitor), and UO126 (MEK1/2 inhibitor) were purchased from New
England BioLabs.
Cell Line, Culture Conditions, and Stimulation--
The
factor-dependent human myelo-erythroid leukemia cell line,
TF-1 (3), was kindly provided by Dr. C. Braun, Heinrich Pette Institute
for experimental Virology and Immunology (Hamburg, Germany). Cells were
grown in RPMI medium without nucleosides, supplemented with 10% (v/v)
fetal calf serum and 2.5 ng/ml GM-CSF. In all experiments prior to
stimulation cells were maintained overnight in GM-CSF-free medium. The
addition of the inducers was considered as the t = 0 h of the experiments. Cell viability was determined by trypan
blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide incorporation (19) or FACS analysis (propidium iodide) assay (20).
Cell Cycle Analysis--
Cell cycle analysis by propidium iodide
staining was performed according to a previous study (21). Briefly,
106 cells were washed with phosphate-buffered saline, fixed
overnight in 70% ethanol, washed, and incubated with 100 µg/ml RNase
(Sigma) and 10 µg/ml propidium iodide in phosphate-buffered saline
for 30 min at 37 °C. Cells were analyzed for DNA content by a
FACSCalibur (BD Biosciences) flow cytometer. Data were analyzed by
ModFit software.
Preparation of Cell Extracts and Analysis by Western
Blotting--
Cells were lysed as described previously (2). 50 µg of
lysed cell protein was separated by SDS-PAGE gel and transferred to
PVDF membrane using the Mini-Protean II system (Bio-Rad). The primary
antibodies anti-(Egr-1), anti-(c-Fos), anti-(ERK1), anti-(PARP), anti-(Bcl-xl), anti-(Bax) rabbit polyclonal IgG and anti-(phospho-ERK), anti-(phospho-Elk-1), and anti-(Bcl-2) mouse monoclonal IgG were purchased from Santa Cruz Biotechnology. Anti-rabbit IgG horseradish peroxidase conjugate and anti-mouse IgG horseradish peroxidase conjugate were obtained from Jackson ImmunoResearch as secondary antibodies. Membranes stained at first, with monoclonal antibodies anti-(phospho-ERK), and anti-(phospho-Elk-1), were incubated in TBS/0.1% Tween 20 containing 0.5% H2O2 at
room temperature for 30 min, than washed three times with TBS/0.1%
Tween 20, and developed by the polyclonal anti-ERK1 antibody. The ECL
system was used for chemiluminescence detection (Amersham Biosciences).
For quantitative analysis of Western blots, image analyses of x-ray
films were performed using Bioscan version 1.0 software following
digitalization with a Hewlett Packard 5100C scanner.
Preparation of Nuclear Extracts and Electromobility Shift
Assays--
DNA-binding proteins were extracted, and the band-shift
assay was performed by applying 5 µg of protein and a radiolabeled consensus oligonucleotide probe as reported previously (22). Santa Cruz
Biotechnology produced the oligonucleotide probe (AP-1).
Electrophoretic Analysis of Oligonucleosomal DNA Degradation
(Apoptosis)--
In experiments addressing the onset of
apoptosis during GM-CSF withdrawal and after the re-administration of
GM-CSF, ionomycin, or CPA, TF-1 cells were seeded (5 × 105 cells/ml) into RPMI 1640/10% FCS for 16 h, than
supplemented with 2.5 ng/ml GM-CSF, 1 µM ionomycin, or
7.5 µM CPA. After various incubation times, total
cellular DNA was isolated from 5 × 106 cells, based
on the method described previously (23), and loaded into the dry wells
of 1.8% agarose gel containing 0.1 µg/ml ethidium bromide.
Caspase-3 Activity Determinations--
Cells were treated with
the test substances as described in the figure legends. At various time
points 3 × 106 cells were harvested and cell extracts
were prepared by the freeze-thawing method in the following lysis
puffer: 20 mM HEPES-NaOH, pH 7.4, 50 mM NaCl, 3 mM MgCl2, 10 mM
[Ca2+]i Measurements--
Cells
(0.5-1 × 106 cells/ml) were loaded for 30 min at
37 °C with 1.5 µM fura-2AM in fresh culture medium
containing 1% FCS. Than extracellular fura-2AM was removed by
centrifugation, and the cells were resuspended in fresh culture medium
containing 10% FCS. Cells were treated for various incubation times
with the test substances, 2.5 ng/ml GM-CSF, 1 µM
ionomycin, or 7.5 µM CPA. In certain experiments 7.5 µM CPA, 1 mM ATP, 1 unit/ml thrombin, 250 ng/ml trypsin, 5 units/ml erythropoietin, 5 ng/ml IL-4, 5 ng/ml IL-3,
and 10 ng/ml nerve growth factor were added to untreated cells.
Fluorescence was measured in a Hitachi F4000 fluorescence
spectrophotometer at 37 °C under gentle agitation, as described
previously (25). Cytoplasmic free calcium concentration was calculated
by using the method of Tsien et al. (26). The presence of
extracellular fura-2AM fluorescence was checked by the addition of EGTA
(0.5 mM), followed by CaCl2 (1 mM),
to the control cells. The leakage of fura-2AM proved to be small
during the experimental period. Nevertheless, this leakage was taken into consideration in the calibration procedure.
Effect of Intracellular Ca2+ Level on Cell Survival,
Proliferation, and Apoptosis--
TF-1 cells were synchronized
by GM-CSF hormone deprivation and thereafter treated with CPA (7.5 µM), ionomycin (1 µM), or GM-CSF (2.5 ng/ml). These concentrations were chosen after optimization, considering the viability of treated cells.
The effects of the above reagents on the cell growth were determined
(Fig. 1A). The GM-CSF deprived
(control) cells did not multiply, and within 72 h their cell
number gradually decreased to about 26% of the original. When GM-CSF
was re-administered after a leg period of about 24 h, the
hormone-deprived cells started to grow exponentially. Interestingly,
the addition of ionomycin also induced a gradual increase in the cell
number, producing a cell growth of about 250% in the first 48 h.
Addition of CPA did not increase significantly the cell number, but
prevented its decline, relative to the control cells.
As documented in Fig. 1B, the viability of the control
(hormone-deprived) cells decreased to about 45% within 72 h. In
contrast, the viability of the GM-CSF-, ionomycin-, or CPA-treated
cells did not change notably within this time frame. Measuring the
viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide incorporation or propidium iodide gave similar results
(data not shown).
To test how the traverse of the cell cycle was affected, the DNA
content of cells subjected to various treatments was analyzed. Samples
of GM-CSF-, ionomycin-, and CPA-treated cells were collected every
3 h during an interval of 33 h. The ratio of cells in the three cell cycle phases, G1/G0, S, and
G2/M was determined by flow cytometry analysis by using
propidium iodide staining (Fig. 2). These
experiments indicated that re-administration of GM-CSF forced the
majority of cells to enter S phase within 18 h (Fig. 2A) while ionomycin induced a moderate access of cells to S
phase (Fig. 2B). In contrast, CPA treatment caused cell
cycle arrest (Fig. 2C). These results, along with the
proliferation measurements, reveal that ionomycin, similarly to GM-CSF,
induces cell growth.
Both CPA and ionomycin induce store depletion followed by an activated
Ca2+ influx. Therefore, we tested whether store depletion
and/or an increased level of [Ca2+]i were
responsible for CPA-induced cell survival. To buffer changes in
[Ca2+]i, cells were loaded with a relatively high
concentration of BAPTA-AM (160 µM). High concentration of
the chelator was needed due to the inability of lower concentrations to
abolish CPA-induced increase in [Ca2+]i in the
presence of external Ca2+ (not shown). In another approach,
extracellular Ca2+ was chelated with 3 mM EGTA.
Both treatments caused cell death. BAPTA-AM caused a drastic drop of
cell viability, with cells dying within 12 h irrespective of the
particular treatment. EGTA decreased the cell number by 75.1 ± 2.1%, 43.9 ± 5.2%, 87.2 ± 3.2%, and 94.6 ± 2.8%
of control, GM-CSF-, CPA-, and ionomycin-treated cells, respectively,
within 24 h. CPA and ionomycin presumably caused more excessive
store depletion, which may account for the more rapid cell death
compared with control or GM-CSF-treated cells. Thus, sustained store
depletion induces cell death rather than survival, and Ca2+
influx appears to be crucial in CPA- and
ionomycin-dependent survival of TF-1 cells.
Because non-excitable cells generally do not endure high elevations of
[Ca2+]i, the signs of apoptosis were also
examined. As shown in Fig. 3A,
long term hormone deprivation induced a delayed increase of caspase-3
activity and the appearance of PARP cleavage product (Fig.
3B). A marked DNA ladder formation (Fig. 3C) also
appeared within 24 h. In contrast, GM-CSF, CPA, and ionomycin
treatment suppressed caspase-3 activity to the level observed in the
normally cultured cells (41.5 ± 3.65%) within 6 h.
Furthermore, no cleavage of PARP or DNA ladder formation was detected
even after 48 h (Fig. 3). These results, reinforced by the
viability measurement, show that the treatments described above
protected the cells from apoptosis.
As a summary, hormone-deprived cells stopped growing and lost their
viability within 72 h. GM-CSF, the natural growth hormone for TF-1
cells, suppressed apoptotic processes and induced cell proliferation.
Controlled increase of [Ca2+]i caused by
ionomycin and CPA protected against cell death, and ionomycin even
produced a significant cell growth. These findings suggest that both
[Ca2+]i increase and GM-CSF can induce survival
and proliferation.
Receptor-mediated and Ca2+-mobilizing Agents Generated
Intracellular Ca2+ Level Changes in TF-1 Cells--
The
receptor (cytokine, purinergic, and proteinase-activated
receptor)-mediated Ca signals were compared with the
[Ca2+]i increase induced by
Ca2+-mobilizing agents. The
[Ca2+]i of overnight hormone-deprived
TF-1 cells (control) were estimated by pre-loading the cells
with fura-2AM and measuring the intracellular calcium
dependent fluorescence. Fig. 4
(A-D) shows the alterations of
[Ca2+]i in control cells following the addition
of the purinergic receptor ligand (1 mM ATP),
proteinase-activated receptor ligands (1 unit/ml thrombin, 250 ng/ml
trypsin), or 7.5 µM CPA. The
[Ca2+]i of control cells increased from
102.0 ± 11.7 to 180.8 ± 32.5, 562.2 ± 89.1, 256.3 ± 32.1, and 236.0 ± 45.4 nM following the
addition of 1 mM ATP, 1 unit/ml thrombin, 250 ng/ml
trypsin, and 7.5 µM CPA, respectively. This initial
elevation of [Ca2+]i diminished within a few
minutes in the case of ATP and thrombin treatment, whereas trypsin- and
CPA-induced sustained elevation of [Ca2+]i
(167.1 ± 28.4 and 212.2 ± 34.8 nM,
respectively).
Certain agents, like IL-3, IL-4, erythropoietin, and nerve growth
factor, which induce Ca2+ fluxes in other cells, had no
effect on intracellular Ca2+ levels in TF-1 cells (data not
shown). Thus, based on these experiments, both endogenous and
pharmacological agents may modify [Ca2+]i in TF-1 cells.
In the following experiments, we applied CPA and ionomycin to induce
controlled increases in [Ca2+]i in TF-1 cells.
Alterations of [Ca2+]i were measured for 2 h
after the addition of the above reagents to estimate the long term
effect of CPA and ionomycin. We found that CPA induced a moderate,
transient increase in [Ca2+]i, with a maximum of
228.2 ± 40.6 nM, whereas ionomycin produced a higher
[Ca2+]i reaching 1397.5 ± 160.2 nM. In both cases [Ca2+]i approached
the control level within 1 h. No detectable changes of
[Ca2+]i within the time period examined were
observed in the control cells or in cells treated with GM-CSF (Table
I).
All in all, CPA- and ionomycin-induced [Ca2+]i
alterations were comparable with the effect of endogenous substances. CPA and ionomycin induced controlled [Ca2+]i
elevation, which was down-regulated within 1 h. GM-CSF, on the
other hand, did not increase [Ca2+]i in these cells.
Effect of [Ca2+]i on the Proteins of the
Bcl-2 Family--
To identify some of the possible mechanisms allowing
hormone-deprived cells to avoid apoptosis, changes in the well-known apoptotic protein family, Bcl-2 were examined. Synchronization by
overnight hormone withdrawal was taken as the starting point of all
investigations (t = 0 h). Whole cell extracts were
analyzed by Western blotting for anti-apoptotic (Bcl-2 and Bcl-xl) and pro-apoptotic (Bax) proteins. The level of Bcl-xl doubled in 24 h
after re-administration of GM-CSF, increasing to 2.5-fold by 48 h.
Unlike GM-CSF, ionomycin treatment induced only a minor increase in
Bcl-xl expression (1.5-fold) by 24 h, without further augmentation. Bcl-xl expression remained unaltered in CPA-treated cells
(Fig. 5A). In contrast,
GM-CSF, ionomycin, and CPA invariably triggered a comparable expression
of Bcl-2, each doubling its level. However, the rate of expression
induced by CPA was slower, reaching the level detected in
GM-CSF-treated cells with a 24-h delay (Fig. 5B). None of
these treatments yielded any change in the level of the pro-apoptotic
protein, Bax (Fig. 5C). The finding that the anti-apoptotic
proteins dominated the apoptotic proteins correlated with the viability
measurements (Fig. 1B), suggesting that the regulation of
the Bcl-2 family plays a major role in the survival of TF-1 cells.
Meanwhile, among the gradually dying control cells (Fig.
1B), a moderate decrease in the level of Bcl-xl and Bcl-2
(Fig. 5, A and B) was seen, accompanied by a
small increase of Bax (Fig. 5C).
To summarize, controlled elevation of [Ca2+]i
induced the up-regulation of the Bcl-2 anti-apoptotic protein without the alteration of other proteins of the Bcl-2 family. This Bcl-2 up-regulation enabled TF-1 cells to evade apoptosis.
Level and Time Course of ERK1/2 Phosphorylation Induced
by a Variety of Agents--
The activation of the MAPK pathway is one
of the major routes leading to cell survival and/or proliferation.
Specific monoclonal anti-pERK antibodies enabled us to follow the
kinetics and level of ERK1/2 kinase phosphorylation. As shown in Fig.
6A, in a good correlation with
the [Ca2+]i signal, a rapid activation of ERK1/2
was observed 5 min following treatment with the
[Ca2+]i-increasing agents. Thus, CPA resulted in
a minor stimulation, whereas ionomycin concentration above 500 nM induced marked phosphorylation. Below this ionomycin
concentration no activation of ERK1/2 was detected. Elk-1 activation
showed a correlation with ERK1/2 stimulation. As revealed, a threshold
of ERK1/2 phosphorylation has to be reached for Elk-1 activation. This
finding explains the activation of Elk-1 only above 500 nM
ionomycin and the failure of activation by CPA.
In terms of the physiological outcome, the kinetics of signaling
is probably as critical as the extent of ERK1/2 phosphorylation. To
better characterize the activation processes, the time course of ERK1/2
phosphorylation was studied.
Two phases of phosphorylation were observed in the case of GM-CSF,
ionomycin, and CPA. The first appeared 5 min after the exposure to
these agents, and the second followed between 2 and 3 h (Fig.
6B). At 5 min, CPA elicited only a moderate activation, while ionomycin and GM-CSF caused 13- and 28-fold elevations, respectively. In all cases, phosphorylation returned to control levels
within 1 h. Intriguingly, a second 11-fold elevation appeared at
2 h in the case of ionomycin, whereas GM-CSF and CPA induced only
a 7-fold increase later, at 3 h. Elk-1 phosphorylation followed ERK1/2 phosphorylation when ERK1/2 activation reached the critical level, described above (Fig. 6C). As expected, Elk-1
phosphorylation did not take place in control cells. Elk-1
phosphorylation followed similar kinetics as ERK1/2 in treated cells.
GM-CSF induced a 40-fold elevation of Elk-1 phosphorylation at 5 min,
followed by a 4-fold second peak 3 h later. Ionomycin induced
phosphorylation at the same time intervals as GM-CSF, causing 9- and
4-fold increases, respectively. As expected, CPA induced only the
second peak of 9-fold phosphorylation at 3 h, where the
phosphorylation of ERKs surpassed the threshold levels. No change in
phosphorylation level was detected in control cells within 6 h
(data not shown).
To test the effect of extracellular Ca2+ on ERK1/2 and
Elk-1 activation, cells were pre-treated for 5 min with 3 mM EGTA before the addition of CPA, ionomycin, or GM-CSF.
Samples were taken when phosphorylation of ERK1/2 and Elk-1 reached its
maximum level. Fig. 6D shows that EGTA pretreatment
abolished completely the phosphorylation of ERK1/2 and Elk-1 induced by
the [Ca2+]i-increasing agents, CPA or ionomycin,
but did not affect the receptor-mediated signal induced by GM-CSF.
In summary, GM-CSF induces two phases of activation, an extensive
initial ERK1/2 and Elk-1 activation, followed by a smaller second peak.
Because GM-CSF did not promote a detectable calcium signal, it is
highly probable that in this case the induction of phosphorylation
proceeds through different pathways.
[Ca2+]i-increasing agents also induced two phases
of ERK1/2 activation, although in the case of CPA the first peak of
activation was insufficient for Elk-1 activation. Considering that both
ionomycin and GM-CSF induces not only cell survival but also
proliferation, the primary, or rather the biphasic, activation of the
ERK1/2-Elk-1 pathway may serve as a key signal for
proliferation. The experiments with EGTA pre-treatment
highlighted the fundamental role of Ca2+ influx in ERK1/2
phosphorylation induced by [Ca2+]i-increasing agents.
Effect of [Ca2+]i on the Expression of
c-Fos and Egr-1 Transcription Factors and on AP-1 DNA Binding--
We
have examined the expression of two transcription factors, c-Fos and
Egr-1, that can be stimulated in an Elk-1-dependent manner
and are thought to be involved in survival and proliferation of
hematopoietic cells. According to our experiments, in both GM-CSF- and
ionomycin-treated TF-1 cells mRNA (data not shown) and protein
levels (Fig. 7A) of Egr-1 and
c-Fos were elevated after 30 and 60 min, respectively. As discussed
above, although rapid and strong initial activation of ERK1/2 was
observed with GM-CSF and ionomycin, CPA induced only a faint
phosphorylation at the same time (Fig. 6B) and could not
promote Elk-1 phosphorylation (Fig. 6C). In accordance with
the notion that Elk-1 is the major stimulator of c-Fos and Egr-1
expression, none of these were expressed within 1 h of CPA
treatment. Accordingly, the activation observed by 3 h of CPA
treatment appeared strong enough to drive Elk-1 phosphorylation and
trigger the expression of the transcription factors examined (Fig.
7B). As expected, c-Fos and Egr-1 expression did not take
place in control cells.
We have examined whether the increased c-Fos expression is accompanied
by the increased DNA binding of AP-1. Our results provide evidence that
the elevated c-Fos expression manifests in the elevation of AP-1 DNA
binding (Fig. 7C).
Effect of MEK Inhibition on the Phosphorylation of
ERK1/2 and Elk-1 and on the Expression of c-Fos and Egr-1
Transcription Factors--
The specific MEK-1 inhibitor, PD98059,
abolished the ionomycin- and GM-CSF-induced ERK1/2 and Elk-1
phosphorylation as well as the following expression of Egr-1 and c-Fos
(Fig. 7A). Similarly, it suppressed the CPA-induced
phosphorylation of ERK1/2 and Elk-1 and the expression of the above
transcription factors. This difference was most apparent at 3 h,
where phosphorylation reaches its maximal level in CPA-treated cells
(Fig. 7B). In cells treated with
[Ca2+]i-increasing agents, the increase in the
AP-1 DNA binding level can also be blocked by PD98059 (Fig.
7C). Similar results were obtained by using another MEK1/2
inhibitor, UO126 (data not shown). Altogether, elevation of
[Ca2+]i is able to trigger c-Fos and Egr-1
expression via the MEK-ERK1/2-Elk-1 pathway.
Effect of MEK-1 Kinase Inhibition on
Ca2+-dependent Survival--
To elucidate the
role of the MAPK pathway in TF-1 cell survival and proliferation, the
long term effect of MEK-1 inhibition was investigated. Table
II shows that inhibition of MEK by
PD98059 significantly reduced the CPA- or ionomycin-induced Bcl-2
expression measured at 12 h after treatment. PD98059 slightly
reduced the weak Bcl-2 level in control cells also, demonstrating that
Bcl-2 expression is MEK/ERK-dependent. After 48 h,
inhibition by PD98059 caused a drastic drop in cell number and
viability that was more pronounced in cells treated with ionomycin than
CPA. This could be explained by the prominent elevation of
[Ca2+]i of ionomycin-treated cells. In contrast,
cell survival was not affected in GM-CSF-treated cells, only the rate
of proliferation was attenuated. This suggests that in GM-CSF-treated
cells the MAPK pathway is mainly critical for proliferation but not for survival, whereas Ca2+-induced survival principally goes
through the MAPK route.
In this report we have demonstrated that a controlled increase of
[Ca2+]i by CPA or ionomycin can induce cell
survival and the latter agent stimulates proliferation in a human
hemopoietic cell line, TF-1 (Fig. 1). Previous studies have implicated
that, in hormone-dependent hemopoietic mouse cell lines
(Baf3, FDCP.P2, and IC-2), calcium ionophores and SERCA pump inhibitors
may moderate hormone-deprivation induced cell death (27-30). However,
even in these cases, within 24 h cell viability fell
significantly. In contrast, we found that TF-1 cells exposed to CPA or
ionomycin showed no deterioration of cell viability for up to 72 h.
In search of the mechanisms that lead to cell survival, we have
examined the expression pattern of the Bcl-2 family. We have found that
both [Ca2+]i-raising treatments caused the
elevation of Bcl-2 level, without altering the Bcl-xl or Bax levels
(Fig. 5). It has been reported that ectopic expression of Bcl-2 in TF-1
cells prevents apoptosis without changes in morphology or phenotype in
the absence of GM-CSF (4). Thus, it is plausible that in TF-1 cells the elevation of Bcl-2 level induced by [Ca2+]i is
sufficient to repress the apoptotic processes without any changes of
Bcl-xl levels. This means that Bcl-xl is not essential in promoting
survival of TF-1 cells after hormone withdrawal.
The Bcl-2 gene is known to be regulated in an
MAPK-dependent manner (16, 17). We have shown here that
both CPA and ionomycin (Fig. 6) activate the MAPK pathway, suggesting
that the increase of Bcl-2 is a result of MAPK activation. Indeed, the
inhibition of MEK-1 by PD98059 decreased the level of Bcl-2 expression
measured at 12-h treatment. Longer exposure to PD98059 and
[Ca2+]i-increasing agents caused such a drastic
drop in cell number that Bcl-2 expression could not be tested. In
parallel to [Ca2+]i-raising agents, as shown in
an earlier investigation (2), re-administration of GM-CSF causes MAPK
activation and the augmentation of Bcl-2 with a raise in Bcl-xl levels.
It is widely accepted that both CPA and ionomycin induce Ca store
depletion followed by Ca2+ influx. We demonstrated that
store depletion, induced by either an intracellular (BAPTA) or by an
extracellular Ca2+ chelator (EGTA) is toxic, cells die
rapidly. This is consistent with the notion that store depletion
induces cell death rather than survival. It has been demonstrated that
the release of Ca2+ from endoplasmic reticulum leads to
apoptosis in Chinese hamster ovary (31) and in SH-SY5Y neuronal cells
(32). As demonstrated in this work short incubation with EGTA blocked
Ca2+ influx and ERK1/2/Elk-1 activation induced by CPA or
ionomycin. As also shown, inhibition of ERK activation abolished cell
survival. Thus, the activated Ca2+ influx and moderate
increase in [Ca2+]i are involved in the
pro-survival mechanisms of [Ca2+]i-increasing
agents in TF-1 cells. Similar results were obtained in excitable
neuronal cells, where apoptosis could be blocked by a sustained
increase of steady state free Ca2+ (33).
It is worth mentioning that no changes in the level of expression of
the sarco-endoplasmic reticulum calcium ATPase and plasma membrane
Ca2+ ATPase proteins were observed after 24 h
of CPA or ionomycin treatments. Moreover, these cells do not display
any detectable Na/Ca exchanger
activity.2 Therefore, it is
unlikely that these Ca2+ transporters are involved in CPA
or ionomycin induced cell-survival.
Another finding of our investigation was that transient, high elevation
of [Ca2+]i can trigger the proliferation of
hemopoietic cells through the activation of ERK1/2. The time course of
ERK1/2 activation determines the fate of several cell responses,
including cell proliferation (34). We have determined the delicately
controlled time course of ERK1/2 activation in TF-1 cells in response
to GM-CSF, ionomycin, and CPA exposure (Fig. 6B). GM-CSF and
ionomycin prompted cells to enter S phase (Fig. 2, A and
B) and cell division (Fig. 1A) and caused a
biphasic ERK1/2 activation. With CPA there is only one significant peak
of ERK1/2 activation, which is sufficient to promote cell survival, but
falls short of making cells re-enter the cell cycle. This observation
correlates well with the recent proposal of Jones and Kazlauskas (35)
that growth factor-dependent mitogenesis requires two
distinct phases of signaling in NIH3T3 cells. The occurrence of the
double phases of ERK1/2 phosphorylation under our experimental
conditions may be a result of the transient activation of the MAPK
phosphatases, which are regulated by ERKs and/or calcium (36).
Interestingly, PD98059 was able to selectively inhibit the second peak
also,2 demonstrating that the activation of both peaks were
MEK-dependent.
We have shown that in TF-1 cells [Ca2+]i
elevation-induced Elk-1 activation is ERK1/2-dependent.
Besides ERK 1/2 Elk-1 is known to be phosphorylated on Ser-383
by SAPK/JNK and p38 MAPK (37). However, in our experiments Elk-1 is
activated by similar kinetics and only when ERK1/2 was sufficiently
activated (Fig. 6). Moreover, Elk-1 and ERK1/2 activation were
MEK-dependent (Fig. 7) pointing to the pivotal role of the
MEK/ERK1/2 in Ca2+-dependent Elk-1 activation.
The expression of c-Fos and Egr-1 proteins, reportedly involved in cell
proliferation, are regulated through Elk-1 via SRE and other pathways,
e.g. CRE and sis inducible element (38). Furthermore,
increase in nuclear Ca2+ has been reported to control
CRE-mediated transcription, whereas a rise of cytoplasmic
Ca2+ is believed to activate SRE-driven transcription (39).
We have found that c-Fos and Egr-1 expression, like Elk-1 activation, is induced by [Ca2+]i elevation and is
MEK-1-dependent (Fig. 7). This suggests that the expression
of these proteins is regulated mainly through Elk-1-dependent mechanisms, although the role of CREB
cannot be ruled out. Interestingly, GM-CSF induced only a minor
elevation of c-Fos, in comparison to ionomycin and CPA. This may be
explained by the involvement of Ca2+-dependent
intragenic regulatory elements involved in c-Fos regulation (40).
Because GM-CSF does not induce [Ca2+]i elevation,
it is unlikely that these regulatory elements become activated.
It has been reported that Egr-1 is important in regulating cell growth,
differentiation, and development (41), depending on the cell type. In
our investigation, elevation of Egr-1 expression correlated with cell
growth. The highest level of Egr-1 expression was in the case of GM-CSF
treatment, followed by that caused by ionomycin. CPA only resulted in a
low Egr-1 level, and no Egr-1 protein was detected in control cells.
The activation of the MEK-1/ERK1/2/Elk-1 pathway and the following
transcription of immediate early genes (c-Fos and Egr-1) could be
completely blocked by the MEK-1 inhibitor PD98059 in all treatments
applied here. This inhibition in the GM-CSF-treated cells caused an
extensive reduction of proliferation rate without any change in cell
viability, indicating that PD98059 is not toxic for TF-1 cells in the
concentration used. This implies that the MAPK/ERK pathway is central
for promoting proliferation in GM-CSF-treated cells, and activation of
other pathways is mostly involved in cell survival. In the case of
cells treated with [Ca2+]i-increasing agents,
PD98059 induced a drastic drop of cell number and viability, which was
most significant in ionomycin-treated cells. This can be attributed to
the toxicity of high elevation of [Ca2+]i caused
by ionomycin in the absence of MAPK/ERK activation.
It has been suggested (28, 29) that the limited suppression of
apoptosis in different hemopoietic mouse cells induced by
[Ca2+]i-increasing agents may be a consequence of
autocrine IL-4 production. In our experiments TF-1 cells survive
hormone deprivation in the presence of IL-4 (data not shown) in a good agreement with the results of Kitamura et al. (3). Despite this, no production of IL-4 was detected by enzyme-linked immunosorbent assay, neither was any increase in the DNA binding of nuclear factor of activated T cells,2 which is involved in the
induction of IL-4 expression (42). Nonetheless, the presence of
cytokine production cannot be ruled out.
In this work we have demonstrated that Ca2+ induces a
unique, MEK-dependent activation of ERK/Elk/c-Fos/AP-1
pathway in CD34-positive myelo-erythroid leukemia cells. Elevation of
Ca2+ is known to stimulate proliferation-associated
MAPKs in different non-hemopoietic cells. In vascular smooth
muscle a Ca2+-dependent isoform of protein
kinase C and calmodulin kinase II are upstream activators of
ERK1/2 (43). In rat fibroblast functional calcium-sensing receptors are
required for activation of signal-regulated kinase kinase and
MAPK in response to extracellular calcium (44). Calmodulin binding to
an IQ motif in Ras-guanine nucleotide releasing factor 2 exchange
factor in kidney epithelial cells stimulates GTP binding by Ras and
potentiates elevated Ca2+ concentration-induced ERK1/2
activation (45). Similar calmodulin-dependent Ras-GRF activation was suggested in the regulation of neuronal function by Ca2+ signals (46), but the role of a protein
kinase A-dependent activation of the small G-protein Rap-1
can not be ruled out (47). Moreover, the requirement of the
Ca2+-activated proline-rich tyrosine kinase 2 for the
activation of the MAPK cascade induced by Ca2+ in PC 12 cells (48) or in pulmonary vein endothelial cells (49) was also
reported. Any of the above mentioned pathways may be involved in the
Ca2+-induced ERK activation of hemopoietic cells. The exact
mechanism of MEK/ERK1/2 activation by [Ca2+]i
elevation in TF-1 cells needs further investigations.
The elevation of [Ca2+]i caused by CPA is within
the range of physiological calcium signals, as confirmed by ATP,
thrombin, or trypsin treatments of TF-1 cells (Fig. 4). ATP and
thrombin are known cellular modulators, and trypsin was found to be
expressed at low levels in vascular endothelial cells and leukocytes
(50) and overexpressed in certain cancer cells and tissues (51, 52). All these observations reinforce our hypothesis that increases in
[Ca2+]i similar to those caused by CPA or
ionomycin can occur in vivo.
In summary, we have shown that the elevation of
[Ca2+]i in a human hemopoietic cell line induced
the activation of the MEK/ERK/Elk/c-Fos/AP-1 pathway. This unique
activation led to hormone-independent survival and limited
proliferation. Moreover, we showed that the kinetics of calcium
signaling influenced the kinetics of ERK1/2 activation and cellular response.
Our results point toward a possible mechanism through which bone marrow
cells may escape from hormone-deprivation induced cell death. The
[Ca2+]i-induced, hormone-independent survival may
be leading to clone selection and leukemogenesis. Accordingly, a report
on a Phase I clinical trial of carboxyamidotriazole, a synthetic inhibitor of non-excitable calcium channels, suggested that
carboxyamidotriazole reversibly inhibits angiogenesis, tumor cell
proliferation, and metastatic potential of refractory solid tumors
(53). Other pharmacological agents, inhibiting receptor-linked
Ca2+ entry in vitro, might slow prostate cancer
proliferation in vivo (54). Thus, elevation of
[Ca2+]i may not only facilitate the development
of leukemia but in certain cases may play a role in solid tumor
development and progression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Because the major route
activated by GM-CSF is the MAPK pathway, our primary goal was to
determine whether all biological events are the result of this
activation or whether additional pathways are responsible.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 0.4% Nonidet P-40, 2 mM
phenylmethylsulfonyl fluoride, 100 µM
Na3V04, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 10 µg/ml antipain. The protein content of the samples
was determined using the Bradford dye-assay (reagent obtained from
Sigma), and aliquots containing 50 µg of protein were used in a
fluorometric caspase-3 enzyme activity assay, carried out in a
microplate reader (24). In a final volume of 100 µl, containing 10 mM HEPES-KOH, pH 7.5, 10% sucrose, 1 mM
dithiothreitol, 0.025% CHAPS, and 50 µM Ac-DEVD-AMC
(acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin) as substrate, the
liberation of AMC was followed in time (excitation at 360 nm and
emission at 460 mm). The fluorescence intensities were converted into
AMC concentrations using an AMC calibration curve. Activities were
standardized to the caspase-3 activity obtained in GM-CSF-deprived
cells (3.3 ± 0.5 µM ACM calculated from three
independent experiments).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
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Fig. 1.
Proliferation (A) and
viability (B) of GM-CSF-, ionomycin-, and CPA-treated
and control cells. TF-1 cells were synchronized by overnight
GM-CSF hormone deprivation and thereafter treated with 7.5 µM CPA, 1 µM ionomycin, 2.5 ng/ml GM-CSF.
Proliferation and cell viability was determined by Trypan-Blue
exclusion test at the times indicated. Results of cell growth were
normalized to the initial cell numbers (100%). Results represent the
mean ± S.E. of six independent experiments.
View larger version (17K):
[in a new window]
Fig. 2.
DNA profile alteration of GM-CSF
(A)-, ionomycin (B)-, and CPA
(C)-treated cells. Samples of 2 × 106 cells, treated as described under "Experimental
Procedures," were taken at every 3 h and fixed by 70% EtOH.
Propidium iodide staining and flow cytometry analyses were performed
using a FACSCalibur. Results were processed by ModFit software. The
figure shows one representative results from three independent
experiments.
View larger version (21K):
[in a new window]
Fig. 3.
Time course changes of caspase-3 activity
(A), PARP cleavage (B), and DNA
ladders (C) of GM-CSF-, ionomycin-, CPA-treated and
control cells. TF-1 cells were deprived from GM-CSF for 16 h
and than exposed to the test compounds and harvested at different time
points. At t = 0 h, cells were supplemented with
2.5 ng/ml GM-CSF, 7.5 µM CPA, 1 µM
ionomycin, or not supplemented. 50 µg of protein was used for the
fluorometric caspase-3 enzyme activity assay as described under
"Experimental Procedures." Results represent the mean ± S.E.
of three independent experiments normalized to the caspase-3 activity
of 16 h GM-CSF-deprived cells (A). Western blot
analyses of PARP cleavage, as described under "Experimental
Procedures." Samples were taken at the indicated time points
(B). 5 × 106 GM-CSF-deprived cells were
plated and treated with the above reagents. Total cellular DNA was
isolated from the cells at the indicated time points, and 1 µg of DNA
from each samples was analyzed on 1.8% agarose gel containing 0.1 µg/ml ethidium bromide (C).
View larger version (19K):
[in a new window]
Fig. 4.
Alteration of [Ca2+]i
of cells treated with ATP (A), thrombin
(B), trypsin (C), or CPA
(D). TF-1 cells were synchronized by overnight
GM-CSF hormone deprivation. Cells were preloaded with fura-2AM and
intracellular calcium-dependent fluorescence was measured.
Thereafter cells were treated with 1 mM ATP, 1 unit/ml
thrombin, 250 ng/ml trypsin, and 7.5 µM CPA at the times
indicated by a downward arrow. One representative result
from three independent experiments is shown.
Measurement of [Ca2+]i by fura-2AM measurements
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[in a new window]
Fig. 5.
Western blot analyses of Bcl-xl
(A) and Bcl-2 (B) anti-apoptotic
proteins and Bax (C) pro-apoptotic protein from whole
cell extracts of GM-CSF-, ionomycin-, CPA-treated and control
cells. Cells were supplemented with GM-CSF, ionomycin, or CPA or
not supplemented, and cellular extracts were made at the time points
indicated. 40 µg of total cellular protein was run on 12.5%
SDS-polyacrylamide gel, transferred to a PVDF filter, and analyzed by
immunoblotting using antibodies against Bcl-xl (A), Bcl-2
(B), and Bax (C) proteins. Results represent the
mean ± S.E. of three independent experiments normalized to the
protein levels of 16 h GM-CSF-deprived cells. Protein loading was
standardized to ERK1/2 levels.
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Fig. 6.
Western blot analyses of pERK1/2 and pElk-1 as
a function of ionomycin concentration (A) and time (B
and C). The effect of EGTA on p-Elk and p-ERK1/2 levels
(D). Representative blots of p-ERK1/2 and p-Elk1 levels
(A) are shown as a function of ionomycin or CPA treatment
after 5 min. Time course changes of ERK1/2 (B) and Elk-1
(C) phosphorylation of GM-CSF-, ionomycin-, CPA-treated and
control cells are represented. Western blot analyses of p-ERK1/2 and
p-Elk-1 levels in CPA-, ionomycin-, GM-CSF-treated and control cells in
the presence or absence of 3 mM EGTA are shown in
D. Protein loading was standardized to ERK1/2 protein levels
of the cellular extracts and given as the optical densities of the
bands obtained by Western blot analyses and determined by BioScan v1.0.
Results represent the mean ± S.E. of three independent
experiments.
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Fig. 7.
Effect of the MEK-1 inhibitor, PD98059, on
the protein expression of c-Fos and Egr-1, and activation of Elk-1 and
ERK1/2 of GM-CSF, ionomycin (A)- and CPA
(B)-treated cells, and AP-1 DNA binding activity of
the two latter (C). Cells were treated, and when
indicated were exposed 30 min previously to MEK-1 inhibitor, PD 98059, as described under "Experimental Procedures." 50 µg of total
cellular protein was separated by electrophoresis on 10% SDS -PAGE,
transferred to PVDF filter, and analyzed by immunoblotting using
monoclonal antibodies against p-ERK1/2, p-Elk-1, and polyclonal ERK1/2
protein. Phospho-protein levels were detected from cells 5 min
following exposure to drugs (A) or as indicated
(B). Nuclear extracts of ionomycin- or CPA-treated cells,
with or without inhibition by PD 98059, were quantified for AP-1 DNA
binding activity by means of electrophoretic mobility shift assay at
the times indicated. Specific DNA binding was evaluated with an excess
of unlabeled oligonucleotide probe for AP-1 (C). For more
details see "Experimental Procedures."
Effect of the MEK-1 inhibitor, PD98059, on cell proliferation,
viability, and Bcl-2 level
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank András Schaefer for the valuable discussions and for assistance in Northern blot technique and Judit Szelényi for measuring IL-4 levels. We also thank Balázs Sarkadi and Agnes Enyedi for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Hungarian Research Foundation (Grant OTKA T029291) by the Scientific Research Council, Ministry of Health (Grant ETT 206/2001), and by NRDP 1/024, Ministry of Education, Hungary.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.
To whom correspondence should be addressed. Tel./Fax:
36-1-372-4353; E-mail: magocsi@biomembrane.hu.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M205528200
2 Á. Apáti, J. Jánossy, A. Brózik, P. Bauer, and M. Magócsi, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor; AP-1, activator protein-1; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester; [Ca2+]i, cytoplasmic free calcium concentration; CPA, cyclopiazonic acid; CREB, cAMP response element binding protein; MAPK, mitogen-activated protein kinase; ERK1/2, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; Egr-1, early growth response protein-1; Elk-1, Ets domain protein-1; p90RSK, 90-kDa ribosomal S6 kinase; PD98059, 2'-amino-3-methoxyflavone (MEK-1 inhibitor); UO126, 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (MEK1/2 inhibitor); PI3K, phosphoinositide 3-kinase; SRE, serum response element; STAT-5, signal transducer and activator of transcription-5; IL-3, interleukin-3; FACS, fluorescence-activated cell sorting; PVDF, polyvinylidene difluoride; PARP, poly(ADP-ribose) polymerase; FCS, fetal calf serum; AMC, amido-4-methylcoumarin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; JNK, c-Jun N-terminal kinase; SERCA, sarco-endoplasmic reticulum Ca2+ ATPase.
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