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INTRODUCTION |
It has been expected that signal transduction pathways involving
specific protein kinases are involved in mediating apoptosis. Extracellular signal-regulated kinase
(ERK)1 is most strongly
stimulated by activation of protein-tyrosine kinase receptors (1) and
also activated by both Ras-dependent (2-4) and
Ras-independent signalings (5) in response to activation of G
protein-coupled receptors, and common intermediates in intracellular signaling cascades are involved in diverse cellular functions including
growth and differentiation (6). Activation of PKC by
12-O-tetradecanoylphorbol-13-acetate results in the
activation of Raf1 (7) and ERK (8-11) within minutes, suggesting the
involvement of PKC in the signaling pathway leading to ERK activation.
In addition, Ras functions as an essential transducer of various physiological signals leading to cell growth and proliferation in a
PKC-dependent or PKC-independent manner (12), and activated Ras also renders cells susceptible to apoptosis after depression of PKC
activity (13, 14).
SAPK/JNK and p38 kinase have been proposed to mediate apoptosis, but a
number of reports have challenged the notion that the activation of
SAPK/JNK and/or p38 kinase is sufficient to induce apoptosis (15-21),
and the integration and balance of SAPK/JNK and p38 pathways probably
contribute to commitment to apoptosis (22, 23). In addition, the
inhibition of ERK activity has been reported to correlate with the
activation of SAPK/JNK and p38 kinase as well as with the induction of
apoptosis in nerve growth factor-deprived PC12 pheochromocytoma cells
(22), Fas-induced Jurkat cells (15, 24), and UV-irradiated mouse
fibroblasts (25), and the balance between the activity of the stress
kinases to that of ERK has been proposed to determine cell fate (26), because the Raf1-ERK signaling pathway plays a pivotal role in suppressing apoptotic death (27, 28).
Apoptosis is induced in human gliomas by exposure to calphostin C, a
specific inhibitor of PKC, and calphostin C-induced apoptosis was
preceded by down-regulation of bcl-2 mRNA and protein
(29). Overproduction of Bcl-2 can render cells more resistant to
induction of apoptosis by a wide variety of stimuli (30), and Bcl-2
appears to work both upstream and downstream of the caspase cascade to prevent effects of caspases (31). To characterize the molecular mechanism that regulates calphostin C-induced apoptosis of gliomas, the
contributions of mitogen-activated protein kinase family members to
cell death caused by calphostin C-induced down-regulation of Bcl-2 are
explored using various inhibitors of caspases, SAPK/JNK, and p38 kinase
in this work.
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MATERIALS AND METHODS |
Glioma Cell Lines--
Two glioma cell lines (wild type
p53-positive U-87MG and mutant p53-positive T98G obtained from the
American Type Culture Collection, Rockville, MD) were maintained in
DMEM supplemented with 10% fetal calf serum and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin) in a humidified
atmosphere of 5% CO2 and 95% air at 37 °C. Test
exposures to 100 nM calphostin C were conducted in the
continuous presence of light to promote photoactivation of this compound.
Microscopic Analysis--
Cells were fixed with 4%
paraformaldehyde for 30 min after washing with ice-cold
phosphate-buffered saline and then stained with 2.5 µg/ml Hoechst
33258. The number of apoptotic cells was assessed by nuclei staining,
and nuclei that were fragmented or condensed were scored as apoptotic.
The viability of cells was confirmed by the trypan blue or propidium
iodide dye exclusion method.
Agarose Gel Electrophoresis--
DNA was prepared from cells as
described previously (29). The resulting DNA preparation was analyzed
by 1.5% agarose gel electrophoresis in TBE buffer (89 mM
Tris borate buffer, pH 8.0, 89 mM boric acid, 22 mM EDTA) containing 0.5 µg/ml ethidium bromide at 100 V/cm for 30 min. The DNA fragmentation pattern was examined on
photographs taken under UV illumination.
Immunoblot Analysis--
Cells were lysed on ice in lysis buffer
(20 mM Tris-HCl at pH 7.4, 150 mM NaCl, 0.5%
Nonidet P-40, 1 mM EDTA, 50 µg/ml leupeptin, 30 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride) and then centrifuged at 15,000 × g for 10 min. Equivalent
amounts of cell lysates were heated in 2× sample buffer at 95 °C
for 3 min and then electrophoresed in 12% SDS-polyacrylamide gel,
except for immunoblotting of PARP in which 8% SDS-polyacrylamide gel
was used, and transferred onto polyvinylidene difluoride membranes. The
membranes were incubated with primary antibody against Bcl-2 (6C8;
Pharmingen, San Diego, CA), Bax (Mobio, Nagoya, Japan), Bcl-x
(Transduction, Lexington, KY), p10 subunit of caspase-1 (C-20; Santa
Cruz Biotechnology, Inc., Santa Cruz, CA), p12 subunit of caspase-3
(K19; Santa Cruz Biotechnology), PARP (C-2-10; a kind gift from Dr. Guy
G. Poirier, CHUL Research Center, Quebec, Canada), or Raf1 (Santa Cruz
Biotechnology); washed; and blotted with species-specific biotinylated
secondary antibodies (Vector, Burlingame, CA) and then with horseradish
peroxidase-streptavidin (Vector). The membrane was then developed in
ECL reagent (Amersham Pharmacia Biotech) and exposed to x-ray film.
Loading of Cells with Caspase-1 and -3 Inhibitor
Peptides--
The osmotic lysis of pinosome method as described by
Moore et al. (32) was used to enhance uptake of the
inhibitor peptides into the cells. Briefly, cells were incubated for
8-10 min at 37 °C with 500 µl of a prewarmed hypertonic solution
of 0.5 M sucrose, 10% (v/v) polyethylene glycol 1000, 10 mM HEPES (pH 7.0) in DMEM containing 100 µM
Ac-YVAD.cho or 100 µM Ac-DEVD.cho. Hypotonic medium (60%
DMEM, 40% distilled water) was added to a final volume of 15 ml, and
incubation at 37 °C continued for 10 min. The hypotonic medium was
changed into DMEM, 10% fetal calf serum. Calphostin C was added to the
culture medium, and the mixture was incubated for the indicated times,
followed by agarose electrophoresis.
Shc Phosphorylation--
Cells were lysed on ice in lysis buffer
(50 mM HEPES at pH 7.5, 0.5% Triton X-100, 100 mM NaF, 10 mM sodium pyrophosphate, 4 mM EDTA, 2 mM Na3VO4, 2 mM Na2MoO4, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml phenylmethylsulfonyl fluoride). After
centrifugation at 15,000 × g for 10 min at 4 °C,
supernatants were absorbed with Affi-Gel protein A (Bio-Rad)-normal
rabbit serum complex for 90 min at 4 °C and then incubated with
Affi-Gel protein A-polyclonal anti-human Shc antibody (Upstate
Biotechnology, Inc., Lake Placid, NY) complex, which had been treated
in advance with 20 mM dimethyl pimelimidate in 0.2 M sodium borate for cross-linking. The immunoprecipitates were boiled for 5 min in 2× sample buffer, subjected to 10%
SDS-polyacrylamide gel electrophoresis, and transferred to
polyvinylidene difluoride membrane, which was then blocked with 1%
gelatin in phosphate-buffered saline containing 0.05% Tween 20 overnight and probed with mouse monoclonal anti-phosphotyrosine
antibody (Upstate Biotechnology) for 1 h. The secondary antibody
was horseradish peroxidase-conjugated anti-mouse IgG (Dako, Glostrup,
Denmark), and proteins were detected using the ECL system (Amersham
Pharmacia Biotech). The membranes were also immunologically reprobed
with anti-human Shc antibody after stripping each of the primary
antibodies off the membranes.
In Gel MBP Kinase Assay for ERK as Well as SAPK/JNK and p38
Kinase Assay--
Cells were lysed on ice in lysis buffer (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
-mercaptoethanol, 1 mM Na3VO4, 1 µg/ml leupeptin, 2 µg/ml phenylmethylsulfonyl fluoride). After centrifugation at 15,000 × g for 10 min at 4 °C and
boiling in 2× sample buffer for 5 min, cell lysates for in gel MBP
kinase assay for ERK were electrophoresed in 11% SDS-polyacrylamide
gel containing 0.5 mg/ml MBP fragment. SDS was removed from the gel using 20% propanol. The gel was washed in 50 mM Tris-HCl
at pH 8.0, 5 mM
-mercaptoethanol and thereafter
denatured with 6 M guanidine; renatured in 50 mM Tris-HCl at pH 8.0 containing 5 mM
-mercaptoethanol and 0.04% Tween 40; and incubated with kinase buffer (48 mM HEPES, pH 8.0, 25 µCi of
[
-32P]ATP, 30 µM ATP, 6 mM
MgCl2, 2.4 mM dithiothreitol, 0.12 mM EGTA) at room temperature for 1 h. After washing
with 5% trichloroacetic acid and 1% pyrophosphate solution, the gel
was dried, and kinase activity was visualized using FUJIX Bio-Imaging
Analyzer BAS2000. Cell lysates for SAPK/JNK and p38 kinase assay were
subjected to 12% SDS-polyacrylamide gel electrophoresis and
transferred to polyvinylidene difluoride membrane. The membranes were
blocked with Tris-buffered saline containing 0.1% Tween 20 and 5%
nonfat dry milk for 1-3 h and separately incubated with
anti-phosphospecific SAPK/JNK (Thr183/Tyr185)
antibody (New England Biolabs, Beverly, MA) and anti-phosphospecific p38 kinase (Tyr182) antibody (New England Biolabs)
overnight at 4 °C. The secondary antibody was horseradish
peroxidase-conjugated anti-rabbit IgG, and proteins were detected using
the ECL system (Amersham Pharmacia Biotech). Cell lysates were also
separately probed with anti-ERK1/ERK2 antibody (Zymed
Laboratories Inc., San Francisco, CA), anti-SAPK/JNK1 antibody
(Santa Cruz Biotechnology), or anti-p38 kinase antibody (New England Biolabs).
Inhibition of SAPK/JNK and p38 Kinase--
Cells were plated
24 h before transfection at a density of 105/18-mm
cover glass. The cells were co-transfected with pcDL-SR
-SAPK-VPF plasmid expressing dominant-negative SAPK/JNK or pcDL-SR
-WT-SAPK plasmid expressing wild-type SAPK/JNK as a control, together with pEGFP-Vector (CLONTECH) using FuGENETM6
transfection reagent (Boehringer Mannheim) by following the manufacturer's instructions and/or were then treated with 100 µM SB203580 (33), a specific inhibitor of p38 kinase, 15 min prior to exposure to calphostin C for 8 h in U-87MG cells and for 6 h in T98G cells. The cells, after washing three times with phosphate-buffered saline, were incubated for 3 min with 10 µM Hoechst 33258 or 10 µM propidium iodide
and analyzed under an Olympus AX-80 fluorescence microscope.
Dominant-negative and wild-type SAPK/JNK expression plasmids were
obtained from Prof. E. Nishida (Kyoto University, Kyoto, Japan).
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RESULTS |
Calphostin C-induced Cellular Responses and DNA
Fragmentation--
Glioma cells exposed to 100 nM
calphostin C showed an apoptotic morphology: cell shrinkage and surface
blebbing by phase contrast images, and condensation or fragmentation of
nuclei by Hoechst 33258 stain (Figs. 1
and 11). Mutant p53-positive T98G cells appeared to become more quickly
apoptotic than wild type p53-positive U-87MG cells, suggesting that
calphostin C-induced apoptosis is p53-independent. Unambiguous
electrophoretic patterns of laddered oligonucleosomal DNA fragments
were observed as early as 8 h in U-87MG cells and 6 h in T98G
cells following exposure to 100 nM calphostin C, as shown
representatively in Fig. 2,
lanes 2 and 7, respectively.

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Fig. 1.
Representative photomicrographs of phase
contrast images and Hoechst 33258 stain of two glioma cell lines
(U-87MG and T98G) treated with 100 nM calphostin C for
8 h in U-87MG cells and for 6 h in T98G cells in the absence
or the presence of 100 µM
z-VAD.fmk, 100 µM Ac-DEVD.cho, or
100 µM Ac-YVAD.cho. Phase
contrast was ×400; Hoechst 33258 stain was ×200
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Fig. 2.
Representative agarose gel electrophoresis
showing the induction of oligonucleosomal DNA fragmentation by
calphostin C. Human glioma cells (U-87MG and T98G) were exposed to
100 nM calphostin C in the absence or the presence of 100 µM Ac-YVAD.cho, 100 µM Ac-DEVD.cho, or 100 µM z-VAD.fmk in U-87MG cells for 8 h
(lanes 2-5) and in T98G cells for 6 h
(lanes 7-10). The formation of oligonucleosomal fragments
was determined by agarose gel electrophoresis. Similar results were
achieved in three separate experiments with comparable outcomes.
Lanes 1 and 6, control;
lanes 2 and 7, calphostin C alone;
lanes 3 and 8, calphostin C plus
Ac-YVAD.cho; lanes 4 and 9, calphostin
C plus Ac-DEVD.cho; lanes 5 and 10,
calphostin C plus z-VAD.fmk; lane M, DNA size
markers.
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Calphostin C-induced Down-regulation of Bcl-2 and
Bcl-xL and Activation of Caspase-3--
Bcl-2 was shown as
a single 26-kDa peptide by immunoblot analysis, Bax as a single 21-kDa
peptide, and Bcl-xL as a doublet of peptides with an
apparent molecular mass of 29-31 kDa. The 19-kDa Bcl-xS
was not detected. Bcl-2 and Bcl-xL were down-regulated nearly synchronously as early as 2-4 h after treatment with 100 nM calphostin C in both U-87MG and T98G cells. In contrast,
Bax levels in both U-87MG and T98G cells were not changed during
treatment with calphostin C (Fig. 3).

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Fig. 3.
Immunoblot analysis showing Bcl-2 and Bax
proteins as a single band (thick arrows) and
Bcl-xL protein as a doublet (thin
arrows). Glioma cells (U-87MG and T98G) were exposed to
100 nM calphostin C for 2-12 h, and steady-state level of
each protein was monitored by immunoblot analysis. Results are
representative of three separate experiments with comparable
outcomes.
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There was no evidence of caspase-1 activation following 100 nM calphostin C treatment, since immunoreactive active
10-kDa caspase-1 fragment was not detected in both U-87MG and T98G
cells during treatment with calphostin C for 12 h (Fig.
4). The levels of 45-kDa procaspase-1
remained unchanged during calphostin C treatment for the first 6 h
and thereafter were decreased in both U-87MG and T98G cells, but active
caspase-1 fragment was not formed, probably due to nonspecific
proteolysis. Immunoreactive 32-kDa procaspase-3 was degraded into
active 12-kDa caspase-3 fragment as early as 8 h in U-87MG cells
and 4 h in T98G cells after treatment with calphostin C,
persisting for at least 12 h in both U-87MG and T98G cells (Fig.
4). The generation of a COOH-terminal 85-kDa PARP apoptotic fragment
(34, 35) was shown to be associated with the generation of active
12-kDa caspase-3 fragment (Fig. 4). These data are consistent with
activation of caspase-3 in calphostin C-treated gliomas.

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Fig. 4.
Immunoblot analysis showing activation of not
caspase-1 but caspase-3 associated with cleavage of PARP.
Formation of active 10-kDa caspase-1 fragment from 45-kDa procaspase-1
was not detected during treatment with 100 nM calphostin C
for 12 h, suggesting no activation of caspase-1, whereas 32-kDa
procaspase-3 was gradually degraded into a 12-kDa active caspase-3
fragment in both U-87MG and T98G cells, indicating activation of
caspase-3. Cleavage of 116-kDa PARP into an 85-kDa apoptotic fragment
was monitored by immunoblot analysis. Similar results were achieved in
three separate experiments.
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Two tetrapeptide derivatives, Ac-YVAD.cho and Ac-DEVD.cho, which are
relatively selective inhibitors of caspases-1 and -3, respectively (36,
37), were added to the culture medium 30 min prior to the exposure to
calphostin C to preferentially block one or the other group of cysteine
proteases. The induction of oligonucleosomal DNA laddering by
calphostin C could be completely blocked by treatment with 100 µM Ac-DEVD.cho in both U-87MG and T98G cells but not by
treatment with 100 µM Ac-YVAD.cho (Fig. 2), confirming
the activation of caspase-3 but not caspase-1. These findings again
suggest that calphostin C-induced apoptosis proceeds independently of
caspase-1 and may depend on caspase-3 or similar proteases. The
measurements of activities of caspase-1 and -3 using fluorescent
substrates also indicate similar
results.2
Calphostin C-induced Shc Phosphorylation, Raf-1 Activation, and ERK
Phosphorylation--
The anti-human Shc antibody specifically
recognized 46-, 52-, and 66-kDa isoforms of Shc as shown in Fig.
5. When the Shc immunoprecipitates were
immunoblotted with anti-phosphotyrosine antibody, 46-, 52-, and 66-kDa
Shc isoforms were shown to be tyrosine-phosphorylated, and the 52-kDa
isoform was the major phosphorylated species (Fig. 5). The
phosphorylation levels of Shc were increased as early as 2 h after
treatment with 100 nM calphostin C and persisting for at
least 6 h in both U-87MG and T98G cells, whereas the levels of
immunoreactive Shc were not changed during the increase in the Shc
phosphorylation, suggesting Shc activation. The activation of Raf1 was
determined by the presence of Raf1 with reduced gel electrophoretic
mobility due to phosphorylation (27, 38, 39). As shown in Fig.
6, immunoreactive Raf1 doublet in control
cells became a singlet 2 to 8 h after treatment with 100 nM calphostin C in both U-87MG and T98G cells, suggesting a
marked decrease in phosphorylation of Raf1. Phosphorylation of ERK was
estimated by an in gel MBP kinase assay. Two isoforms of ERK1 and ERK2
were shown to be phosphorylated to a similar degree prior to treatment with calphostin C, and the phosphorylation of ERK1 and ERK2 begins to
rapidly diminish as early as 2 h after treatment with calphostin C
in both U-87MG and T98G cells and thereafter continuously decreased, as
shown representatively in Fig. 7. On the
contrary, the levels of immunoreactive ERK1 and ERK2 were not changed
during treatment with calphostin C for 8 h and were slightly
decreased by treatment for 12 h in both U-87 MG and T98G cells as
shown in Fig. 7.

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Fig. 5.
Analysis of Shc phosphorylation showing a
gradual increase in tyrosine phosphorylation of 46-, 52-, and 66-kDa
isoforms of Shc proteins as early as 2 h after treatment with 100 nM calphostin C in both U-87MG (A) and
T98G (B) cells. The Shc phosphorylation persists
during the treatment for at least 6 h and then declines. Cell
lysates were immunoprecipitated with anti-human Shc antibody, resolved
by 12% SDS-polyacrylamide gel electrophoresis, and immunologically
probed with anti-phosphotyrosine antibody. The membranes were reprobed
with anti-human Shc antibody, showing no detectable changes in levels
of 46-, 52-, and 66-kDa isoforms of Shc proteins in both U-87MG
(A') and T98G (B') cells during the increase in
the phosphorylation of Shc proteins. The results are from
representative study performed three times with comparable
outcomes.
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Fig. 6.
Analysis of Raf1 activation shows that
immunoreactive Raf1 with reduced mobility (upper
arrow) is barely detectable as early as 2 h after
treatment with 100 nM calphostin C and persisting during
the treatment for at least 8 h in both U-87MG and T98G cells,
suggesting the decrease in phosphorylation of Raf1 by calphostin
C. Cell lysates were subjected to 12% SDS-polyacrylamide gel
electrophoresis and probed with anti-Raf1 antibody by immunoblotting.
Similar results were achieved in three separate experiments with
comparable outcomes. , no treatment; +, treatment.
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Fig. 7.
Analysis of activities of ERK in U-87MG and
T98G cells by in gel MBP kinase assay, showing a rapid decline in
levels of phosphorylation of ERK1 and ERK2 as early as 2 h after
stimulation with 100 nM calphostin C and a continuous
decrease during the treatment for 4-12 h in U-87MG
(A) and T98G (B) cells. Cell
lysates were electrophoresed in 11% SDS-polyacrylamide gel containing
0.5 mg/ml MBP. The gel was incubated with a kinase buffer containing 25 µCi of [ -32P]ATP to measure kinase activity. Cell
lysates were also immunologically probed with anti-ERK1/ERK2 antibody,
showing no changes in levels of ERK1 and ERK2 during the treatment with
calphostin C for 8 h and thereafter slight decreases in levels of
ERK1 and ERK2 in U-87MG (A') and T98G (B') cells.
Results are representative of three independent experiments.
P, phosphorylation.
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Calphostin C-induced Activation of SAPK/JNK and p38
Kinase--
The activities of SAPK/JNK and p38 kinase were analyzed by
Western blotting using anti-phosphospecific SAPK/JNK
(Thr183/Tyr185) antibody and
anti-phosphospecific p38 kinase (Tyr182) antibody,
respectively. SAPK/JNK was shown as a doublet, and p38 kinase was shown
as a singlet. Phospho-SAPK/JNK1 and -SAPK/JNK2 (Thr183/Tyr185) as well as phospho-p38 kinase
(Tyr182) were increased gradually as early as 2 h
after treatment with calphostin C in both U-87MG and T98G cells. The
phosphorylation of SAPK/JNK1 and SAPK/JNK2 as well as p38 kinase
reached a peak at 8 or 12 h in U-87MG cells and at 6 or 8 h
in T98G cells (Figs. 8 and
9). SAPK/JNK2 was sometimes slightly more
phosphorylated than SAPK/JNK1. On the other hand, the levels of
immunoreactive SAPK/JNK1 and SAPK/JNK2 as well as p38 kinase were not
changed during the increase in phosphorylation of SAPK/JNK and p38
kinase in both U-87MG and T98G cells. Therefore, SAPK/JNK and p38
kinase are activated during treatment with calphostin C for 12 h
in U-87MG and T98G cells.

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Fig. 8.
Representative analysis of SAPK/JNK activity
by Western blot using anti-phosphospecific SAPK/JNK
(Thr183/Tyr185) antibody, showing a gradual
increase in levels of phosphospecific SAPK/JNK1 and SAPK/JNK2
(Thr183/Tyr185) isoforms in U-87MG
(A) and T98G (B) cells as early as
2 h after treatment with 100 nM calphostin C. The
phosphospecific SAPK/JNK reaches a peak at 8 h in U-87MG cells and
at 6 h in T98G cells after treatment with calphostin C. Immunoreactive SAPK/JNK1 and SAPK/JNK2 were also probed by
immunoblotting with anti-SAPK/JNK1 antibody, showing no significant
changes in levels during the treatment with calphostin C for 6 h
in both U-87MG and T98G cells and a slight decrease in SAPK/JNK2 after
treatment for more than 8 h in U-87MG (A') and T98G
(B') cells. Similar results were achieved in three separate
experiments with comparable outcomes. P-JNK, phosphospecific
SAPK/JNK.
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Fig. 9.
Analysis of p38 kinase activity using
anti-phosphospecific p38 kinase (Tyr182) antibody in U-87MG
(A) and T98G (B) cells, showing a
gradual increase in levels of phosphorylation of p38 kinase at
Tyr182 as early as 2 h after treatment with 100 nM calphostin C in both U-87MG and T98G cells. The
phosphorylation of p38 kinase reaches the peak at 8 or 12 h in
U-87MG cell and at 6 or 8 h in T98G cell after treatment with
calphostin C. Immunoreactive p38 kinase probed immunologically with
anti-p38 kinase antibody shows no significant changes in levels during
the increase in phosphorylation of p38 kinase in both U-87MG
(A') and T98G (B') cells. Results are
representative of three separate experiments with comparable outcomes.
P-p38, phospho-specific p38 kinase.
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Calphostin C-induced Activation of SAPK/JNK and p38 Kinase Is
Inhibited by z-VAD.fmk or Ac-DEVD.cmk--
To analyze the relationship
between caspases and SAPK/JNK or p38 kinase, we used two caspase
inhibitors with different specificities: 100 µM z-VAD.fmk
(a broad spectrum caspase inhibitor) and 100 µM
Ac-DEVD.cho. They were added to the culture medium 30 min prior to the
addition of 100 nM calphostin C. The COOH-terminal 85-kDa PARP fragment generated by calphostin C treatment for 8 h in
U-87MG and for 6 h in T98G cells was not detected in the presence
of z-VAD.fmk or Ac-DEVD.cho (Fig.
10A), suggesting a marked
inhibition of caspase-3-like proteases by z-VAD.fmk or Ac-DEVD.cho (34, 35, 37, 40). In addition, oligonucleosomal DNA fragmentation induced by
100 nM calphostin C was not visualized in the presence of
z-VAD.fmk or Ac-DEVD.cho (Fig. 3, lanes 4,
5, 9, and 10). The pretreatment with
z-VAD.fmk or Ac-DEVD.cho had no stimulatory effect on calphostin
C-induced decrease in phosphorylation of ERK1 and ERK2 (Fig.
10B), whereas calphostin C-induced activation of SAPK/JNK
and p38 kinase was almost completely blocked by the pretreatment with
z-VAD.fmk or Ac-DEVD.cho (Fig. 10, C and D).

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Fig. 10.
Effects of z-VAD.fmk and Ac-DEVD.cho on
activities of ERK, SAPK/JNK, and p38 kinase as well as on cell
viability. z-VAD.fmk (100 µM) or Ac-DEVD.cho (100 µM) was added to the culture medium 30 min prior to the
addition of 100 nM calphostin C. After treatment with
calphostin C for the indicated times, U-87MG and T98G cells were
subjected to immunoblotting of PARP (A), in gel MBP kinase
assay for ERK (B), SAPK/JNK (C), p38 kinase assay
(D), and examination of cell viability (E). A
COOH-terminal 85-kDa PARP apoptotic fragment produced by calphostin C
treatment for 8 h in U-87MG cells (lane 2)
and for 6 h in T98G cells (lane 6) was not
detected by z-VAD.fmk (lanes 4 and 8,
respectively) or Ac-DEVD.cho (lanes 3 and
7, respectively), indicating the inhibition of
caspase-3-like proteases. The results are from a representative study
performed three times with comparable outcomes. Lane
1, no treatment in U-87MG cells; lane
5, no treatment in T98G cells; Cal. C,
calphostin C.
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Calphostin C-induced Cellular Responses Are Partly Inhibited, but
Cell Death Is Not Inhibited by z-VAD.fmk, Ac-DEVD.cho, Transfection of
Dominant Negative SAPK/JNK, or SB203580--
To dissect calphostin
C-induced apoptotic signaling pathways, we investigated the effects of
three caspase inhibitors (100 µM z-VAD.fmk, 100 µM Ac-DEVD.cho, and 100 µM Ac-YVAD.cho) on
calphostin C-induced cellular responses by adding to the culture medium
30 min prior to the treatment with calphostin C. z-VAD.fmk and
Ac-DEVD.cho demonstrated similar effects on calphostin C-mediated
cellular responses; they inhibited cell shrinkage, but surface blebs
still remained unchanged in number in both U-87MG and T98G cells as revealed by phase contrast images (Fig. 1, e, e',
g, and g'). They also blocked chromatin
condensation and fragmentation as revealed by Hoechst 33258 stain (Fig.
1, f, f', h, and h'), and this result was consistent with the inhibition of oligonucleosomal DNA
fragmentation by z-VAD.fmk or Ac-DEVD.cho as shown by agarose gel
electrophoresis (Fig. 2). In contrast, the pretreatment with Ac-YVAD.cho did not inhibit calphostin C-induced apoptotic
morphological features (Fig. 1, i and i') and
oligonucleosomal DNA fragmentation (Fig. 2). To examine the viability
of calphostin C-treated cells, we used the trypan blue dye exclusion
method. Two caspase inhibitors (100 µM z-VAD.fmk or 100 µM Ac-DEVD.cho), which inhibited calphostin C-induced
activation of SAPK/JNK and p38 kinase as well as cell shrinkage and
apoptotic nuclear changes but did not block calphostin C-induced
surface blebbing, could not prevent calphostin C-induced cell death in
both U-87MG and T98G cells (Fig. 10E). It is suggested, therefore, that the activation of SAPK/JNK and p38 kinase is
dispensable for z-VAD.fmk-resistant surface blebbing and cell death and
that both glioma cell lines succumb not to the nuclear damages but to
the surface blebbing events in the presence of z-VAD.fmk or Ac-DEVD.cho. Ac-YVAD.cmk did not inhibit the calphostin C-mediated apoptotic morphological features and cell death in both cell lines.
Transfection of dominant negative SAPK/JNK was achieved in about 75%
of U-87MG and T98G cells. Transfection of dominant negative SAPK/JNK or
treatment with SB203580 induced similar effects on calphostin
C-mediated cellular responses in both U-87MG and T98G cells as did
z-VAD.fmk or Ac-DEVD.cho: marked decrease in cell shrinkage and
chromatin condensation and fragmentation but maintenance of surface
blebbing (Fig. 11, A and
B). The marked decrease in calphostin C-induced apoptotic
nuclear damages by dominant negative SAPK/JNK or SB203580, as revealed
by Hoechst 33258 stain, suggested the inactivation of caspase-3, but
the calphostin C-induced PARP cleavage was unchanged by the inhibition
of SAPK/JNK or p38 kinase (Fig. 11C), although about 25% of
untransfected U-87MG and T98G cells still demonstrated the apoptotic
nuclear damage. However, calphostin C-induced cell death shown by the
propidium iodide dye exclusion method in Fig. 11, A and
B, was not inhibited by transfection of dominant negative
SAPK/JNK or SB203580.

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Fig. 11.
Effects of dominant negative SAPK/JNK and
SB203580 on calphostin C-induced apoptotic phenotype, cell viability,
and PARP cleavage. A, representative photomicrographs
of phase contrast images and fluorescence images of Hoechst 33258 stain, propidium iodide stain, and pEGFP-Vector transfection of T98G
cells. The arrows show phase contrast image as well as
fluorescence images of Hoechst 33258 stain and propidium iodide stain
of T98G cells transfected with pEGFP-Vector (magnification, ×400).
B, bar graphs showing percentage numbers of more than 100 U-87MG and T98G cells, demonstrating chromatin condensation or
fragmentation in Hoechst 33258 stain and positive propidium iodide
stain. C, calphostin C-induced PARP cleavage into the 85-kDa
apoptotic fragment is unchanged by dominant negative SAPK/JNK or
SB203580. U-87MG and T98G cells were treated with 100 nM
calphostin C, 100 nM calphostin C plus transfection with
pcDL-SR -SAPK-VPF, and 100 nM calphostin C plus SB203580
for 6 h.
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DISCUSSION |
Since treatment of cells with
12-O-tetradecanoylphorbol-13-acetate results in activation
of Raf1 (7) and ERK (8-11), the effect of calphostin C on the
signaling pathway leading to ERK activation was studied. In general,
ERK1 and ERK2 are most strongly stimulated by activation of
protein-tyrosine kinase receptors (13), and one established mechanism
of receptor protein-tyrosine kinase coupling involves activation of Ras
by translocation of the Grb2-Sos complex to the plasma membrane, as a
consequence of its binding to an autophosphorylation site in the
receptor protein-tyrosine kinase itself or to a substrate or adaptor
protein, such as Shc, phosphorylated by the receptor protein-tyrosine
kinase (41). In addition, ERK is activated by both
Ras-dependent (2-4) and Ras-independent (5) signaling
pathways in response to activation of G protein-coupled receptors. The
release of 
subunits of heterotrimeric G proteins following
ligand binding to Gi-coupled receptors leads to tyrosine
phosphorylation of Shc and increased functional association of Shc,
Grb2, and Sos in COS-7 cells (4). These data suggest that G
subunits can activate Ras using mechanisms that are similar to those
employed by receptor tyrosine kinases and that Shc is implicated as a
go-between that relays message from G
-coupled receptors to
protein-tyrosine kinase receptors. On the other hand, Ras-independent
signaling pathways involve the G
subunits of Go and
Gq-coupled receptors and are PKC-dependent (5).
In calphostin C-induced apoptosis, Raf1 and ERK in both U-87MG and T98G
cells were inactivated despite Shc activation prior to the onset of
apoptosis. In general, signal transduction from Shc leads to Ras
activation, which recruits Raf1 to the plasma membrane, where Raf1 is
activated by an intricate multistep process (42). PKC has been reported
to activate Raf1 (43-45), but whether PKC directly phosphorylates and
regulates Raf1 activity requires further investigation. A dominant
inhibitory form of Raf1 abrogates the protective effect of Bcl-2 on
cell survival (46). Bcl-2 is reported to target Raf1 to the outer
mitochondrial membrane (46), where Raf1 mediates the phosphorylation of
Bad. The phosphorylated Bad is sequestered in the cytosol bound to
14-3-3, and only unphosphorylated Bad heterodimerizes with Bcl-2 or
Bcl-xL to promote cell death (47). However, the cross-talk
between Bcl-2 and Raf1 may be little developed in calphostin C-induced
apoptosis because of the down-regulation of Bcl-2 and the inactivation
of Raf1, but abrogation of c-Raf expression is reported to induce
apoptosis in tumor cells (48). Direct and selective activation of the ERK pathway may actually suppress the effects of factors that mediate
apoptosis (22, 23), and Raf1-ERK signaling pathway plays a pivotal role
in suppressing apoptotic death probably due to rapid up-regulation of
bcl-2 and bcl-xL (27, 28), both suggesting that Raf1-ERK signaling is a survival pathway.
In addition to the inhibition of ERK, the activation of SAPK/JNK and
p38 kinase coincided with the onset of calphostin C-induced apoptosis,
as determined by apoptotic morphological features, suggesting that
activation of SAPK/JNK and p38 kinase appears to be required for
induction of calphostin C-induced apoptosis in glioma cells. The
cleavage of mitogen-activated protein kinase kinase kinase 1 into the
91-kDa fragment induced by caspase-3 (50) increases the kinase activity
of mitogen-activated protein kinase kinase kinase 1 to enhance SAPK/JNK
activation (51, 52). The inhibition of ERK below a basal threshold
level is reported to trigger apoptosis in HeLa cells with activation of
p38 kinase but not SAPK/JNK (49). The inhibition of ERK activity and
the concomitant activation of SAPK/JNK and p38 kinase has been reported in apoptosis of nerve growth factor-deprived PC12 cells (22), Fas-stimulated Jurkat cells (15, 24), and UV-irradiated mouse fibroblasts (25). Thus, it seems that the ability of a cell to die or
survive may be dictated by a critical balance between the ERK and the
SAPK/JNK and p38 pathway (26). However, the results as to the
involvement of SAPK/JNK and p38 kinase in apoptosis are conflicting so
far, depending upon variety of stimuli and cell types.
In the calphostin C-mediated apoptosis, the down-regulation of Bcl-2
and Bcl-xL induces cytochrome c release from
mitochondria2 to activate caspase-3. Here, we try to shed
light on the molecular details of the contribution of caspase-3 to the
activation of the SAPK/JNK and p38 signaling pathway using three
caspase inhibitors (z-VAD.fmk, Ac-DEVD.cho, and Ac-YVAD.cho).
Calphostin C-induced inhibition of ERK in glioma cells remained
unchanged by z-VAD.fmk or Ac-DEVD.cho. However, both of these
inhibitors inhibited calphostin C-induced activation of SAPK/JNK and
p38 kinase as well as apoptotic nuclear damage (chromatin
condensation and DNA fragmentation) and cell shrinkage but did not
block calphostin C-induced surface blebbing and cell death. In
addition, the induction of cell death by calphostin C was not changed
by transfection of dominant negative SAPK/JNK or treatment with
SB203580, suggesting that the activation of SAPK/JNK or p38 kinase is
dispensable for cell death induced by calphostin C. Calphostin
C-induced cell death in U937 cells was reported to be unaffected by
genetic manipulation of SAPK/JNK (53). Furthermore, the hypothesis that
stress-activated protein kinase/ERK kinase (SEK1)-mediated activation
of SAPK/JNK is required for the induction of cell death in response to
apoptotic inducers is invalidated by experiments involving cell lines
or animals in which the SEK1 gene is inactivated. Apoptosis does occur
in SEK
/
ES cells, SEK1
/
thymocytes, and
SEK1
/
splenic T cells in response to anisomycin, serum
depletion, UV and
-irradiation, sorbitol-mediated changes in
osmolarity, heat shock, anti-cancer drugs (etoposide, adriamycin, and
cisplatinum), CD3/CD28 ligation, and phorbol myristate
acetate/Ca2+ ionophore with similar kinetics and at similar
doses as in SEK+/+ and SEK1+/
cells (54).
In the calphostin C-mediated apoptosis, z-VAD.fmk and Ac-DEVD.cho
inhibit not only caspase-3 but also SAPK/JNK and p38 kinase, resulting
in inhibition of apoptotic nuclear damage and cell shrinkage but
maintenance of cell death and surface blebbing. Ac-YVAD.cho did not
block any calphostin C-mediated responses. It is suggested that
caspase-3-like proteases function upstream of SAPK/JNK and p38 kinase
in calphostin C-induced apoptosis, in which cells die without nuclear
damages. In caspase-independent commitment and death systems,
inhibition of caspase activation prevents acquisition of the
apoptotic phenotype but does not inhibit cell death. This applies
to a number of different models of apoptosis induced by overexpression
of bax (55), c-myc overexpression, and serum withdrawal or overexpression of bak (56), protonophore,
protoporphyrine IX, dexamethasone, ceramide, etoposide, or nitric oxide
(57-62) and cytotoxic T lymphocyte granule exocytosis (63). However, Bcl-2 maintains survival of cells that have been induced to die in a
caspase-independent manner with glucocorticoids, growth factor withdrawal, c-Myc, Bax, or Bak (55, 56, 61, 62). Therefore, the
down-regulation of Bcl-2 in calphostin C-induced apoptosis is an
important commitment event that determines whether or not a cell dies
even when it is independent of caspase activation. On the other hand,
the inhibition of SAPK/JNK and p38 kinase in calphostin C-induced
apoptosis did not inhibit PARP cleavage, namely activation of
caspase-3, but induced similar effects on the apoptotic phenotypes and
the cell death as did z-VAD.fmk and Ac-DEVD.cho. Since caspase-3
cleaves DNA fragmentation factor 45 (DFF45)/inhibitor of
caspase-activated DNase and inactivates its DFF40/caspase-activated
DNase-inhibitory effect, allowing DFF40/caspase-activated DNase to
enter the nucleus and degrade chromosomal DNA (64-66), the absence of
calphostin C-induced chromatin condensation or fragmentation by the
inhibition of SAPK/JNK or p38 kinase suggests that SAPK/JNK or p38
kinase is involved downstream of caspase-3 in the inactivation of
DFF45/inhibitor of caspase-activated DNase and/or in the translocation
of DFF40/caspase-activated DNase into the nucleus.