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J. Biol. Chem., Vol. 277, Issue 3, 1837-1844, January 18, 2002
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From the Institute for Virus Research, Kyoto University, 53 Shogoin
Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
Received for publication, June 1, 2001, and in revised form, November 8, 2001
Polycyclic aromatic hydrocarbons (PAHs)
such as 3-methylcholanthrene (MC) cause untoward effects including
carcinogenesis. Here we investigated the effect of MC on apoptosis. MC
induced apoptosis, preceded by serine 15 phosphorylation and
accumulation of p53. MC failed to cause apoptosis in p53-deficient MG63
cells, whereas ectopic expression of p53 in MG63 cells restored the
response to MC. Therefore, MC-induced apoptosis was dependent on
p53. MC also activated p38 mitogen-activated protein kinase (MAPK) at 16-24 h. Accumulation of p53 and p53 phosphorylated at serine 15 was
not changed by SB203580, a specific inhibitor of p38 MAPK or
overexpression of a dominant negative mutant of p38 MAPK at 8 h
after MC treatment, whereas the accumulation was suppressed at 24 h. These results suggest that MC induces accumulation and phosphorylation of p53 via a p38 MAPK-independent (early) and p38
MAPK-dependent (late) pathway. SB203580 repressed
MC-induced apoptosis. MC induced p38 MAPK activation in p53 expressing
cells but not in p53-deficient cells, indicating that the p38 MAPK
activation was dependent on early p53 activation. The current study
shows that both p53 and p38 MAPK activation are required for MC-induced apoptosis and provides a novel model of a functional regulation between
p53 and p38 MAPK in chemical stress-induced apoptosis.
Polycyclic aromatic hydrocarbons
(PAHs)1 including MC and
2,3,7,8-tetrachlorodibenzo-p-dioxin are ubiquitous
environmental pollutants that have many untoward effects including
immune suppression, thymic involution, endocrine disruption, wasting
syndrome, birth defects, and carcinogenesis (1, 2). MC is a potent
carcinogen and induces tumors in mice and rats (3). Although most of
the biological effects of MC and other PAHs are considered to be
mediated by aryl hydrocarbon receptor (AhR) (1), the exact mechanism of
carcinogenesis by MC is still unknown. AhR is a ligand-activated member
of the basic helix-loop-helix-PAS (Per-Arnt-Sim) family of
transcription factors that regulates multiple genes including members
of the cytochrome P-450 (CYP1A1) family through a xenobiotic responsive element. The induced CYP1A1 acts on PAHs to
produce reactive oxygen species and metabolizes PAHs to genotoxic (DNA damaging) metabolites (4, 5). These reactive oxygen species and the
metabolites can cause oxidative DNA damage and form adducts with DNA,
starting the mutagenic chain of events responsible for tumor initiation
(1, 6). It is therefore important to understand the protective
mechanism against PAH-induced oxidative stress.
Oxidative stress elicits a wide variety of cellular events such as
apoptosis (7), cell cycle arrest (8), and induction of antioxidant
enzymes (9, 10). Oxidative stress also causes stress signal resulting
in activation of transcription factors such as p53 (8). The p53 tumor
suppressor protein plays an important role in the cellular response to
various cellular stresses (11). After DNA damage, p53 is phosphorylated
and acetylated at a number of sites. Phosphorylation of p53, especially
at serine 15, represents an early cellular response to a variety of
genotoxic stresses and promotes both accumulation and functional
activation of p53 (12). p53 phosphorylation is mediated by protein
kinases, including DNA-dependent protein kinase (DNA-PK),
ATM, ATR, Jun kinases (JNKs), extracellular signal-related protein
kinases (ERKs), and p38 MAPK (12-15). PAH treatment activates the MAPK
cascade including JNK and p38 MAPK (16). In addition, besides
phosphorylation and acetylation, DNA binding of p53 is also modulated
by the reduction and oxidation (redox) regulation mechanism (8, 17,
18).
Studies have shown that PAHs induce apoptosis and cell cycle arrest
(6). MC causes thymocyte apoptosis in C57BL/6 and DBA/2 mice (19) and
inhibits growth of normal ectocervical epithelial cells (20). However,
the mechanism of MC-induced apoptosis and cell cycle arrest remains to
be elucidated.
In this study, we investigated the mechanism of cellular response to
MC. We demonstrated that MC causes apoptosis in a
p53-dependent manner. We also showed that MC induces
accumulation and phosphorylation of p53 through a p38 MAPK-independent
(early) and p38 MAPK-dependent (late) pathway.
Cell Culture--
HepG2 hepatoma cells, MG63 osteosarcoma cells,
and their stable transfectants, HepG2-pCMV-neo, HepG2-p38(AGF),
MG63-pCMV-neo, and MG63-wtp53 were maintained in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin in 5% CO2 at
37 °C.
Generation of Stable Transfectants--
HepG2 and
MG63 cells were transfected with pCMV-Flag-p38(AGF), pCMV-Neo-BAM-wtp53
(pCMV-wtp53), or pCMV-Neo-BAM control vector (pCMV-Neo) (21) using
FuGENETM 6 (Roche Molecular Biochemicals) following the
manufacturer's instructions. Transfected cells were cultured in fresh
medium containing 1 mg/ml (HepG2) or 800 µg/ml (MG63) G418. The
stable transfectants were obtained by selection for G418 resistance and confirmed by Western blotting analysis. To avoid clonal variation, three randomly selected clones were tested.
Reagents--
MC, Assay for Cytotoxicity and Apoptosis--
Cells, plated at a
density of 2 × 105 cells/well onto 6-well plates were
collected and counted at various times for up to 72 h. Cell
viability was measured by the trypan blue dye exclusion test.
Caspase-3(-like) protease activity was measured as previously described
(7), using a fluorometer (Spectra Fluor, Tecan, Salzburg, Austria).
After treatment with MC, the cells were treated with 50 µg/ml
propidium iodide (Calbiochem) and analyzed by flow cytometry (Becton
Dickinson FACSCalibur) using CELLQUEST software. The position of the
cells with sub-G1 DNA content is indicative of apoptosis (23).
Immunoblot Analysis--
Whole cell lysates were prepared using
cell lysis buffer (lysis buffer 1, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM Coimmunoprecipitation Assay--
The effect of MC on the
interaction of p38 MAPK with p53 in vivo was analyzed by
coimmunoprecipitation assay. Briefly, HepG2 cells were exposed to MC
for 24 h and then lysed with lysis buffer 1. The lysates were
immunoprecipitated using monoclonal anti-p53 (DO-1) or control antibody
and protein G-Sepharose (Sigma). The eluates from the beads were
analyzed by immunoblotting using a specific antibody against
phosphorylated p38 MAPK.
Transfection and Luciferase Assay--
HepG2 cells were
transfected with SRE3-luc containing three tandem repeats of serum
response element (SRE) and a control backbone vector, Fos-40-luc
reporter plasmids (0.05 µg) (24) using FuGENE 6. Transfected cells
were serum-starved for 48 h, pretreated with or without PD98059,
and then stimulated with 10% fetal calf serum for 24 h.
Luciferase gene expression, normalized by Renilla luciferase activity, was analyzed using an assay kit (Promega, Madison, WI) with a
luminometer. Serum response through SRE of the c-fos gene was reported to be mostly dependent on ERK (25).
Induction of Apoptosis and Activation of Caspase-3(-like) Protease
in HepG2 Cells by MC--
We first studied the effect of a
representative PAH, MC, on cell growth and viability in HepG2 cells.
Proliferation of HepG2 cells was completely suppressed by exposure to
10 µM MC (Fig. 1A). MC decreased cell
viability to almost 25% of that of the control by 72 h (Fig.
1B).
We next tested whether the cell death induced by MC was due to
apoptosis, using PI staining to quantitate the number of cells with a
subdiploid DNA content. As shown in Fig.
2A, at 72 h after MC
treatment, a significant number of cells (85.3%) had entered into the
terminal stages of apoptosis, localizing in the subdiploid DNA peak.
Because caspase-3(-like) protease plays an essential role in executing
apoptosis, we then examined the caspase-3(-like) protease activity in
HepG2 cells cultured with various concentrations of MC. As shown in
Fig. 2B, 5 µM MC caused a substantial
induction of caspase-3(-like) protease activity in HepG2 cells.
Pretreatment with Ac-DEVD-CHO, a caspase-3-specific inhibitor,
decreased the number of apoptotic cells by about 60-70% (Fig.
2C). Because MC is known as a high affinity ligand of AhR,
we next tested whether AhR is involved in the process, using ANF, an
antagonistic ligand of AhR (26). As shown in Fig. 2, B and
C, the MC-induced apoptosis and caspase-3(-like) protease
activation were suppressed by cotreatment with ANF, whereas ANF itself
did not cause apoptosis (data not shown).
Phosphorylation of p53 at Serine 15 by MC--
Because genotoxic
reagents such as CDDP induce p53 activation (8) and MC was reported to
cause DNA damage, we then examined the level of p53. The p53 protein
level was augmented at 1 h after treatment with MC (Fig.
3A, middle panel).
Because the phosphorylation of the serine 15 residue of p53 is known as
a very early step in the activation of p53 (12), we assessed the amount
of p53 phosphorylated at serine 15 after MC treatment, using an
antibody that specifically recognizes the phosphorylated serine 15 residue of p53. The phosphorylation at serine 15 was induced at about 1 h after treatment with MC and peaked at 24 h (Fig.
3A, upper panel). The kinetics of the induction
coincided with the accumulation of p53, consistent with a previous
report (12). As shown in Fig. 3B, induction of both p53 and
p53 phosphorylated at serine 15 after MC treatment was inhibited by the
addition of ANF, whereas ANF itself did not change the level of p53. We
next analyzed whether the accumulation of p53 induced by MC is due to
decreased p53 protein degradation using a calpain/proteasome inhibitor,
LLnL, which stabilizes p53 protein by inhibiting its degradation by the
proteasome (27). Western blot analysis of lysates prepared from LLnL-treated cells demonstrated equivalent amounts of p53 protein.
In contrast, MC induced an increase in the level of phosphorylation of
p53 at serine 15 (Fig. 3A, upper panel), whereas
there was barely any detectable p53 phosphorylated at serine 15 in
lysates from untreated cells (Fig. 3C). These results
indicated that phosphorylation at serine 15 results in the accumulation
of p53 via inhibition of the protein degradation.
p53 Overexpression Restores Apoptosis Induction in MC-treated MG63
Cells--
To determine whether p53 mediates the apoptosis evoked by
MC, we used p53-deficient human osteocarcinoma MG63 cells to examine the effect of MC on cell growth and viability. MC did not suppress cell
growth and viability (Fig. 4,
A and B). In contrast, overexpression of
wild-type p53 caused a significant induction of caspase-3(-like) protease activity (Fig. 5A)
and apoptosis (Fig. 5B).
MC Activates p38 MAPK and ERK, but Not JNK--
Because p53 is
phosphorylated by various protein kinases, we first monitored the
activation of three members of the MAPK family, p38 MAPK, JNK, and ERK,
by Western blot analysis with antibodies that recognize the activated
phosphorylated forms of the three kinases. As shown in Fig.
6, we found that exposure to MC induced the phosphorylation of p38 MAPK and ERK but not JNK. MC-induced phosphorylation of p38 MAPK and ERK occurred at about 16 and 24 h,
respectively (Fig. 6, A and D). As shown in Fig.
6B, phosphorylation of p38 MAPK was inhibited by the
addition of ANF. As expected, ANF itself did not change the
phosphorylation of p38 MAPK.
Inhibition of p38 MAPK Reduced p53 Activation and
Apoptosis in Response to MC--
We then tested the involvement of p38
MAPK in the activation of p53 by MC. Pretreatment with 1-10
µM SB203580, a specific inhibitor of p38 MAPK, markedly
inhibited the accumulation of p53 and the phosphorylation of p53 at
serine 15 (Fig. 7A) at 24 h. Next, whether p38 MAPK is involved in the early p53 activation, we
pretreated with up to 20 µM SB203580. Pretreatment with
20 µM SB203580 failed to suppress the accumulation of
either p53 or p53 phosphorylated at 8 h after MC treatment (Fig.
7B), suggesting that MC induces accumulation and
phosphorylation of p53 via p38 MAPK-independent (early) and p38
MAPK-dependent (late) pathways.
To further test the role of p38 MAPK in p53 phosphorylation, we used a
dominant negative mutant of p38 MAPK (p38(AGF)). Overexpression of
p38(AGF) specifically blocked anisomycin and MC-induced p38 MAPK
activity (Fig. 7C). Overexpression of p38(AGF) abrogated the
accumulation of both p53 and phosphorylated p53 at 24 h following MC treatment (Fig. 7D). In addition, p38 MAPK was
co-precipitated with p53 in response to MC treatment (Fig.
7E). Pretreatment with 500 nM wortmannin, which
effectively inhibits DNA-PK or ATM, inhibited p53 phosphorylation at
serine 15 at 4 h after 10-30 µM etoposide (positive
control) and 2-6 h after 10 µM MC treatment (Fig.
7F).
We then asked whether p38 MAPK regulates p53-mediated apoptosis. We
analyzed caspase-3 activation by Western blot analysis and the number
of apoptotic cells by flow cytometric analysis. Pretreatment with 1-10
µM SB203580 inhibited MC-induced caspase-3 activation and
apoptosis by about 40-70% (Fig. 8,
A and B). Pretreatment with PD98059 did not
inhibit MC-induced apoptosis (Fig. 8C), whereas pretreatment
with PD98059 clearly inhibited serum-induced c-fos gene
activation (positive control). As in Fig. 2, B and
C, caspase-3 activation was inhibited by the addition of ANF
(Fig. 8A). These results collectively showed that p38 MAPK
plays a role in MC-induced p53 activation and apoptosis.
MC-induced Activation of p38 MAPK Was Dependent on Early p53
Activation--
We then asked whether the p38 MAPK activation was
dependent on early p53 activation. We examined activation of p38 MAPK
in the MG63 cells transfected with either wild type p53 (MG63-wtp53) or
control expression vectors (MG63-pCMV-neo). As shown in Fig. 9, MC induced activation of p38 MAPK in
the MG63 cells expressing wild type p53 but not in control cells.
We here first demonstrated the mechanism of apoptosis by MC. We
showed that MC treatment leads to apoptosis preceded by the activation
of caspase-3(-like) protease in HepG2 cells. This is quite similar to
the action of another genotoxic PAH, benzo(a)pyrene (28). MC
also caused p53 induction and phosphorylation at the serine 15 residue
of p53 in HepG2 cells. In the p53-deficient cell line MG63, MC did not
induce apoptosis (Fig. 4). In contrast, MC induced caspase-3(-like)
protease activity and apoptosis in MG63 cells transfected with wild
type p53 (Fig. 5). Hence, these results suggest that p53 mediates
MC-induced caspase-3-dependent apoptosis. Phosphorylation
of serine 15, a key phosphorylation target during the p53 activation
process, is critical for p53-dependent transactivation and
represents an early cellular response to a variety of genotoxic
stresses (12). Mutation at serine 15 impaired the apoptotic
activity of p53 (29), suggesting a pivotal role for serine 15 phosphorylation in the induction of apoptosis. Previous reports showed
that MC induces DNA single strand breaks in CHO-KI cells (30) and DNA
rearrangements in Balb/c 3T3 cells (31). Metabolites of PAHs
such as MC and benzo(a)pyrene are capable of binding to
cellular macromolecules including DNA (32). Therefore, p53 activation
and phosphorylation of serine 15 seems to be a downstream response to
MC-induced DNA damage. The present results show that p53 plays
important roles in host defense against MC-induced genotoxicity by
mediating the induction of apoptosis. p53-deficient mice are extremely
susceptible to radiation-induced tumorigenesis that is associated with
the accumulation of DNA damage (33). MC itself inactivates the p53 gene
in Syrian hamster embryo fibroblasts (34). Loss of p53 suppressor
function by mutation is a universal step in the development of human
cancer (35). Therefore, it could be hypothesized that the potent
carcinogenicity of MC is partly due to the induction of p53 mutation by
its potent genotoxicity, resulting in the accumulation of DNA damage,
which should have been eliminated by p53.
In this study, we found that MC induces phosphorylation of p38 MAPK and
ERK but not JNK. Pretreatment with a p38 MAPK-specific inhibitor,
SB203580, but not PD98059 significantly inhibited the accumulation of
p53 at 24 h after MC treatment and apoptosis (Fig. 7, B
and C). In addition, expression of a dominant negative
mutant of p38 MAPK suppressed p53 accumulation, suggesting a role for p38 MAPK in MC-induced p53 phosphorylation (Fig. 7). Interestingly, we
showed that p38 MAPK was present in the imunoprecipitates obtained with
anti-p53 antibody from MC-treated HepG2 cells, consistent with a
previous report of direct interaction between p53 and p38 MAPK (36).
Accumulating evidences suggest a role of p38 MAPK in p53
phosphorylation. p38 MAPK phosphorylates p53 at serine 33 (37, 38) in
response to UV or osmotic shock. In this study, SB203580 or expression
of a dominant negative mutant of p38 MAPK also suppressed accumulation
of p53 phosphorylated at serine 15 at 24 h after MC treatment,
indicating the role of p38 MAPK in serine 15 phosphorylation. However,
it is controversial whether p38 MAPK directly phosphorylates serine 15. A recent report showed that p38 MAPK phosphorylates p53 at serine 15 in
response to UV irradiation (36) or resveratrol (39). Another report
showed that p38 MAPK is involved in integrated N-terminal
phosphorylation of p53 including serine 15, although p38 MAPK does not
directly phosphorylate serine 15 (40). Serine 15 can be phosphorylated in vitro by DNA-PK, ATM, and ATR, and in vivo in
response to IR and UV radiation (12). In our study, suppression of the
accumulation of p53 and p53 phosphorylated at serine 15 by SB203580 or
expression of a dominant negative mutant of p38 MAPK (data not shown)
was not detected at 8 h after MC treatment (Fig. 7B).
However, pretreatment with a selective inhibitor of DNA-PK or ATM,
wortmannin, inhibited the p53 phosphorylation at serine 15 by MC at
2-6 h (Fig. 7F). These results indicate that MC induces
accumulation of p53 and phosphorylated p53 at serine 15 in a p38
MAPK-independent (early) and p38 MAPK-dependent (late)
pathway. The mechanism of the serine 15 phosphorylation by p38 MAPK
should be investigated further. Recently, p53 phosphorylation at serine
46 was reported to be important for UV- or radiation-induced apoptosis
(41). The role of p53 phosphorylation at serine 46 in MC-induced
apoptosis needs to be analyzed. In addition, the mechanism of p38 MAPK
activation is currently unknown. We investigated whether p38 activation
is dependent on the early activation of p53, by examining p38 MAPK activation in the p53-deficient MG63 cells. MC did not activate p38
MAPK in p53-deficient MG63 cells, whereas it induced p38 MAPK activation in p53 overexpressing MG63 cells (Fig. 9). These results suggest the requirement of early p53 activation for p38 MAPK activation and the apoptosis, indicating a functional regulation (link) between p53 and p38 MAPK (Fig. 10).
ANF, a competitive inhibitor of AhR (26), blocked 1) the MC-induced
apoptosis, 2) the caspase-3(-like) protease activity, and 3) the
phosphorylation of p53 at serine 15. Therefore, MC-induced apoptosis
appears to be dependent on a functional AhR. Oxidative stress and
oxidized metabolites generated by AhR-regulated drug-metabolizing enzymes such as cytochrome P-450 (CYP1A1) may be responsible
for p38 MAPK activation. MC-induced p38 MAPK
activation and p53 accumulation were suppressed in thioredoxin
overexpressed cells.2
Thioredoxin may scavenge excess reactive oxygen species. Alternatively, thioredoxin may be involved in a negative feedback mechanism of p38
MAPK activation (42). Further work is necessary to elucidate the
molecular mechanism leading to the activation of p38 MAPK and serine 15 phosphorylation of p53 after MC treatment. It is also reported that
thioredoxin directly binds and inhibits the activity of apoptosis
signal-regulated protein kinase (ASK-1) (43). Therefore, it is possible
to speculate that oxidative stress or oxidized metabolites activate the
upstream signaling molecules such as ASK-1 (Fig. 10). To further
investigate the protective mechanism against MC-induced oxidative
stress and carcinogenesis, we are currently analyzing the effect of MC
in thioredoxin transgenic mice and the upstream pathway to p38 MAPK activation.
We thank Dr. Roger J. Davis for providing
pCMV-Flag-p38(AGF), Dr. Toshio Nikaido and Dr. Takehiko Kamijo for
helpful discussions, Yoko Kanekiyo for her secretarial help, and
Shinichi Araya and Yoshimi Yamaguchi for their excellent technical help.
*
This study was supported by a grant-in-aid for scientific
research from the Ministry of Education, Culture, Sports, Science and
Technology and by a grant-in-aid of research for the future from the
Japan Society for the Promotion of Science.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.: 81-75-751-4026;
Fax: 81-75-761-5766; E-mail: hmasutan@virus.kyoto-u.ac.jp.
Published, JBC Papers in Press, November 12, 2001, DOI 10.1074/jbc.M105033200
2
Y.-W. Kwon, J. Yodoi, and H. Masutani,
unpublished observations.
The abbreviations used are:
PAH, polycyclic
aromatic hydrocarbons;
AhR, aryl hydrocarbon receptor;
MC, 3-methylcholanthrene;
ANF,
Mechanism of p53-dependent Apoptosis Induced by
3-Methylcholanthrene
INVOLVEMENT OF p53 PHOSPHORYLATION AND p38 MAPK*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-naphthoflavone (ANF), anisomycin, and
etoposide (Sigma) were dissolved in Me2SO.
cis-Diamminedichloroplatinum(II) (CDDP) was dissolved in 3 mM NaCl and 1 mM sodium phosphate (pH 7.4). 7-Amino-4-methylcoumarin (AMC) was purchased from the Peptide Institute
(Osaka, Japan). A caspase-3-specific inhibitor,
acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), a p38 MAPK-specific
inhibitor, SB203580, and a MEK1-specific inhibitor, PD98509, were
purchased from Calbiochem. The calpain/proteasome inhibitor,
N-acetyl-Leu-Leu-Norleu-al (LLnL), and the DNA-PK or ATM
inhibitor, wortmannin (Sigma), were dissolved in Me2SO. The
antibodies used for Western blotting were anti-p53 (DO-1, Santa Cruz),
anti-
-actin (AC-74, Sigma), anti-cleaved caspase-3, anti-p38 MAPK,
anti-ERK, anti-JNK, anti-phospho-p53-specific (serine 15),
anti-phospho-p38 MAPK-specific (Thr-180/Tyr-182), anti-phospho-ERK-specific (Thr-202/Thr-204), anti-phospho-JNK-specific (Thr-183/Thr-185), and anti-phospho-ATF-2-specific (Thr-71). Other than
the first two, all antibodies listed were from New England Biolabs. The
expression vector pCMV-Flag-38(AGF) (dominant negative mutant of p38
MAPK) was a generous gift from Dr. Roger J. Davis (Howard Hughes
Medical Institute, Worcester, MA) (22).
-glycerophosphate, and 1 mM
phenylmethylsulfonyl fluoride). Immunoblot analysis was performed as
described previously (7).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
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Fig. 1.
Time-dependent effect of MC on
cell growth and viability. HepG2 cells were treated with either
0.1% Me2SO (DMSO) or 10 µM MC.
Cells were harvested at the indicated time after treatment for analyses
of cell number (A) or cell viability (B). The
experiments were performed in triplicate, and similar results were
obtained from three independent experiments. The results are shown as
the mean ± S.D. of triplicate cultures. *, p < 0.05 when compared with Me2SO.
View larger version (29K):
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Fig. 2.
MC-induced apoptosis in HepG2 cells.
A, time course analysis for the appearance of apoptosis
induced by MC. HepG2 cells were treated with 10 µM MC for
0-72 h, stained with PI, and analyzed by flow cytometry to quantitate
cells with a subdiploid DNA content. B, activation of
caspase-3(-like) protease by MC. Cells were treated with different
concentrations of MC for 24 h. P.C., positive
control (1 µM AMC). *, p < 0.05 when
compared with Me2SO (DMSO). C,
inhibition of MC-induced apoptosis by caspase-3 inhibitor. HepG2 cells
were treated with 10 µM MC for 48 h in the presence
or absence of 50 µM Ac-DEVD-CHO, stained with PI, and
analyzed by flow cytometry to quantitate cells with a subdiploid DNA
content. Data shown are representative of three (A and
B) and two (C) independent results. The results
are shown as the mean ± SD. *, p < 0.05 when
compared with MC.
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Fig. 3.
Activation of p53 by MC. A,
expression of p53 and p53 phosphorylated at serine 15 at different time
points (0-24 h) in MC-treated HepG2 cells by Western blot analysis.
The membrane was probed either with a phospho-specific p53
(Ser15) antibody or with a p53-specific antibody. The
membranes were reprobed with an anti- -actin monoclonal antibody to
assess protein levels. B, inhibition of p53 accumulation by
ANF. HepG2 cells were treated with 10 µM MC for 24 h
in the presence or absence of 10 µM ANF. C,
involvement of protein stabilization in MC-induced p53 accumulation.
HepG2 cells were pretreated with 50 µM calpain/proteosome
inhibitor, LLnL, for 2 h, then treated with 10 µM MC
and processed as described in A.
View larger version (16K):
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Fig. 4.
Time-dependent effect of MC on
cell growth and viability in MG63 cells. MG63 cells were treated
with either 0.1% Me2SO (DMSO) or 10 µM MC. Cells were harvested at the indicated time after
treatment for analysis of cell number (A) and cell viability
(B).
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Fig. 5.
Induction of caspase-3(-like) protease
activity and apoptosis by MC in p53-deficient MG63 cells transfected
with wild type p53. A, overexpression of p53-induced
caspase-3 activation by MC in MG63 cells. Caspase-3(-like) protease
activity was measured after 10 µM MC treatment for
48 h in MG63 cells transfected with either the wild type p53
expression vector or the control vector. 2 µM
AMC, positive control. B, overexpression of
p53-induced apoptosis after MC treatment. MG63 cells, stably
transfected with indicated vectors, were treated with 10 µM MC for 48 h, stained with PI, and analyzed by
flow cytometry to quantitate cells with a subdiploid DNA content. This
is representative of two independent experiments. The results are shown
as the mean ± S.D. of triplicate cultures. *, p < 0.05 when compared with MG63-pCMV-neo.
View larger version (22K):
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Fig. 6.
MC induces p38 MAPK and ERK but not JNK in
HepG2 cells. A, analysis of activation of the MAPK
systems by MC in HepG2 cells. HepG2 cells were treated with
Me2SO (DMSO) or MC for the time designated
(top). Cell lysates were analyzed by immunoblotting with
antibodies to phospho-p38 MAPK and p38 MAPK (A), phospho-JNK
and JNK (C), or phospho-ERK and ERK (D).
p.c, positive control (50 ng/ml anisomycin). B,
inhibition of p38 MAPK phosphorylation by ANF. HepG2 cells were treated
with 10 µM MC for 24 h in the presence or absence of
10 µM ANF. Results are representative of two independent
experiments.
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Fig. 7.
Involvement of p38 MAPK in MC-induced p53
activation. A, inhibition of p53 accumulation by p38
MAPK inhibitor, SB203580. HepG2 cells were pretreated with 1-10
µM SB203580 for 30 min, treated with 10 µM
MC, and then cultured for 24 h. The membrane was first probed with
an anti-phospho-specific p53 (Ser15) antibody and then
stripped and probed with a anti-p53-specific antibody. B,
failure of inhibition of early p53 activation by p38 MAPK inhibitor.
HepG2 cells were treated with 20 µM SB203580 for testing
the involvement of p38 MAPK in the early p53 activation and then
culture for indicated time points. The membranes were also reprobed
with an anti- -actin monoclonal antibody to assess protein levels.
C, expression of a dominant negative mutant of p38 MAPK
expression vector blocks p38 MAPK activation. HepG2 stable
transfectants as indicated were exposed to anisomycin (100 ng/ml) or MC
(10 µM) for 24 h and then analyzed by Western
blotting using phospho-specific ATF-2 antibody. D,
expression of a dominant negative mutant of p38 MAPK expression vector
blocks p53 activation by MC. HepG2 stable transfectants as indicated
were exposed to MC (10 µM) for 24 h. 1,
2, and 3 indicate different subclones,
respectively. E, association of p53 with p38 MAPK. HepG2
cells were exposed to 10 µM MC for 24 h. Lysates
were prepared from these cells and immunoprecipitated using anti-p53
antibody or control antibody. The p53 immunoprecipitates were
immunodetected with anti-phospho-p38 MAPK antibody. IP,
immunoprecipitation. F, inhibition of MC-induced early p53
phosphorylation at serine 15 by DNA-PK or ATM inhibitor, wortmannin.
HepG2 cells were pretreated with wortmannin (500 nM)
for 30 min followed by treatment with etoposide for 4 h (50 ng/ml,
positive control) or MC for 2-6 h.
View larger version (19K):
[in a new window]
Fig. 8.
Suppression of MC-induced caspase-3
activation and apoptosis by pretreatment of cells with SB203580 but not
PD98059. A, inhibition of MC-induced caspase-3
activation by SB203580 or ANF. HepG2 cells pretreated with SB203580
(1-10 µM) or ANF (10 µM) were then treated
with 10 µM MC for 24 h. The cleavage of
caspase-3 from the inactive proform (32 kDa) to the active form (17 kDa) was detected by Western blotting using specific antibody to the
active form of caspase-3 (17 kDa). B, suppression of
MC-induced apoptosis by SB203580. HepG2 cells were pretreated with
1-10 µM SB203580 and then treated with 10 µM MC for 36 h, stained with PI, and analyzed by flow cytometry to quantitate cells
with a subdiploid DNA content. C, failure of suppression of
MC-induced apoptosis by PD98059. HepG2 cells were pretreated with the
indicated concentration of PD98059, then treated with 10 µM MC for 36 h, and assessed for apoptosis
induction. The data are representative of two separate experiments. *,
p < 0.05 when compared with MC. To confirm that
PD98059 is active, we tested the effect of PD98059 treatment on
serum-induced c-fos gene activation. HepG2 cells were
transfected with either a luciferase reporter plasmid containing three
tandem repeats of SRE (SRE3) or a control vector (Fos-40), together
with a pRL-TK (Renilla luciferase) as an internal control
for transfection efficiency. Transfected cells were serum-starved for
48 h, pretreated with or without PD98059, and then stimulated with
10% fetal calf serum for 24 h. Luciferase gene expression was
analyzed using an assay kit (Promega) with a luminometer.
View larger version (11K):
[in a new window]
Fig. 9.
MC-induced activation of p38 MAPK is
dependent on p53 activation. MG63 cells stably transfected with
control (MG63-pCMV-Neo) or wild type p53
expression vector (MG63-wtp53) were exposed to MC (10 µM) for 24 h. Cell lysates were analyzed by
immunoblotting with antibodies to phospho-p38 MAPK and p38 MAPK.
Results are representative of two independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
[in a new window]
Fig. 10.
A model of the MC-induced signaling pathway
leading to p53 activation and apoptosis. In the proposed model, MC
induces the activation of p53 via p38 MAPK-independent (early) and p38
MAPK-dependent (late) pathways, resulting in
caspase-3-dependent apoptosis.
ACKNOWLEDGEMENTS
FOOTNOTES
Recipient of a grant-in-aid from the Sasakawa Health Science Foundation.
ABBREVIATIONS
-naphthoflavone;
Ac-DEVD-CHO, acetyl-Asp-Glu-Val-Asp-aldehyde;
AMC, 7-amino-4-methylcoumarin;
ATM, ataxia telangiectasia mutated;
MAPK, mitogen-activated protein kinase;
DNA-PK, DNA-dependent protein kinase;
JNK, c-Jun N-terminal
kinase;
ERK, extracellular signal-regulated kinase;
CDDP, cis-diamminedichloroplatinum(II);
SRE, serum response
element;
PI, propidium iodide.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Nebert, D. W.,
Puga, A.,
and Vasiliou, V.
(1993)
Ann. N. Y. Acad. Sci.
685,
624-640
2.
Holsapple, M. P.,
Karras, J. G.,
Ledbetter, J. A.,
Schieven, G. L.,
Burchiel, S. W.,
Davila, D. R.,
Schatz, A. R.,
and Kaminski, N. E.
(1996)
Annu. Rev. Pharmacol. Toxicol.
36,
131-159
3.
Toth, B.
(1968)
Cancer Res.
28,
727-738
4.
Safe, S. H.
(1995)
Pharmacol. Ther.
67,
247-281
5.
Burczynski, M. E.,
and Penning, T. M.
(2000)
Cancer Res.
60,
908-915
6.
Nebert, D. W.,
Roe, A. L.,
Dieter, M. Z.,
Solis, W. A.,
Yang, Y.,
and Dalton, T. P.
(2000)
Biochem. Pharmacol.
59,
65-85
7.
Ueda, S.,
Nakamura, H.,
Masutani, H.,
Sasada, T.,
Yonehara, S.,
Takabayashi, A.,
Yamaoka, Y.,
and Yodoi, J.
(1998)
J. Immunol.
161,
6689-6695
8.
Ueno, M.,
Masutani, H.,
Arai, R. J.,
Yamauchi, A.,
Hirota, K.,
Sakai, T.,
Inamoto, T.,
Yamaoka, Y.,
Yodoi, J.,
and Nikaido, T.
(1999)
J. Biol. Chem.
274,
35809-35815
9.
Kim, Y. C.,
Masutani, H.,
Yamaguchi, Y.,
Itoh, K.,
Yamamoto, M.,
and Yodoi, J.
(2001)
J. Biol. Chem.
276,
18399-18406
10.
Masutani, H.,
Ueno, M.,
Ueda, S.,
and Yodoi, J.
(2000)
in
Antioxidant and Redox Regulation of Genes
(Sen, C. K.
, Sies, H.
, and Baeuerle, P. A., eds)
, pp. 297-311, Academic Press, San Diego
11.
Levine, A. J.
(1997)
Cell
88,
323-331
12.
Shieh, S. Y.,
Ikeda, M.,
Taya, Y.,
and Prives, C.
(1997)
Cell
91,
325-334
13.
Tibbetts, R. S.,
Brumbaugh, K. M.,
Williams, J. M.,
Sarkaria, J. N.,
Cliby, W. A.,
Shieh, S. Y.,
Taya, Y.,
Prives, C.,
and Abraham, R. T.
(1999)
Genes Dev.
13,
152-157
14.
Hu, M. C.,
Qiu, W. R.,
and Wang, Y. P.
(1997)
Oncogene
15,
2277-2287
15.
Huang, C., Ma, W. Y.,
Maxiner, A.,
Sun, Y.,
and Dong, Z.
(1999)
J. Biol. Chem.
274,
12229-12235
16.
Ng, D.,
Kokot, N.,
Hiura, T.,
Faris, M.,
Saxon, A.,
and Nel, A.
(1998)
J. Immunol.
161,
942-951
17.
Jayaraman, L.,
Murthy, K. G.,
Zhu, C.,
Curran, T.,
Xanthoudakis, S.,
and Prives, C.
(1997)
Genes Dev.
11,
558-570
18.
Gaiddon, C.,
Moorthy, N. C.,
and Prives, C.
(1999)
EMBO J.
18,
5609-5621
19.
Lutz, C. T.,
Browne, G.,
and Petzold, C. R.
(1998)
Toxicology
128,
151-167
20.
Rorke, E. A.,
Sizemore, N.,
Mukhtar, H.,
Couch, L. H.,
and Howard, P. C.
(1998)
Int. J. Oncol.
13,
557-563
21.
Yamamoto, M.,
Yoshida, M.,
Ono, K.,
Fujita, T.,
Ohtani-Fujita, N.,
Sakai, T.,
and Nikaido, T.
(1994)
Exp. Cell Res.
210,
94-101
22.
Raingeaud, J.,
Gupta, S.,
Rogers, J. S.,
Dickens, M.,
Han, J.,
Ulevitch, R. J.,
and Davis, R. J.
(1995)
J. Biol. Chem.
270,
7420-7426
23.
Haupt, Y.,
Rowan, S.,
Shaulian, E.,
Vousden, K. H.,
and Oren, M.
(1995)
Genes Dev.
9,
2170-2183
24.
Masutani, H.,
Magnaghi-Jaulin, L.,
Ait-Si-Ali, S.,
Groisman, R.,
Robin, P.,
and Harel-Bellan, A.
(1997)
Oncogene
15,
1661-1669
25.
Treisman, R.
(1995)
EMBO J.
14,
4905-4913
26.
Wilhelmsson, A.,
Whitelaw, M. L.,
Gustafsson, J. A.,
and Poellinger, L.
(1994)
J. Biol. Chem.
269,
19028-19033
27.
Maki, C. G.,
Huibregtse, J. M.,
and Howley, P. M.
(1996)
Cancer Res.
56,
2649-2654
28.
Salas, V. M.,
and Burchiel, S. W.
(1998)
Toxicol. Appl. Pharmacol.
151,
367-376
29.
Unger, T.,
Sionov, R. V.,
Moallem, E.,
Yee, C. L.,
Howley, P. M.,
Oren, M.,
and Haupt, Y.
(1999)
Oncogene
18,
3205-3212
30.
Park, J. K.,
Lee, J. S.,
Lee, H. H.,
Choi, I. S.,
and Park, S. D.
(1991)
Life Sci.
48,
1255-1261
31.
Honma, M.,
Mizusawa, H.,
Sasaki, K.,
Hayashi, M.,
Ohno, T.,
Tanaka, N.,
and Sofuni, T.
(1994)
Mutat. Res.
304,
167-179
32.
Harris, C. C.,
Genta, V. M.,
Frank, A. L.,
Kaufman, D. G.,
Barrett, L. A.,
McDowell, E. M.,
and Trump, B. F.
(1974)
Nature
252,
68-69
33.
Kemp, C. J.,
Wheldon, T.,
and Balmain, A.
(1994)
Nat. Genet.
8,
66-69
34.
Albor, A.,
Flessate, D. M.,
Soussi, T.,
and Notario, V.
(1994)
Cancer Res.
54,
4502-4507
35.
Harris, C. C.
(1996)
Environ. Health. Perspect.
3,
435-439
36.
She, Q. B.,
Chen, N.,
and Dong, Z.
(2000)
J. Biol. Chem.
275,
20444-20449
37.
Takekawa, M.,
Adachi, M.,
Nakahata, A.,
Nakayama, I.,
Itoh, F.,
Tsukuda, H.,
Taya, Y.,
and Imai, K.
(2000)
EMBO J.
19,
6517-6526
38.
Kishi, H.,
Nakagawa, K.,
Matsumoto, M.,
Suga, M.,
Ando, M.,
Taya, Y.,
and Yamaizumi, M.
(2001)
J. Biol. Chem.
276,
39115-39122
39.
She, Q. B.,
Bode, A. M., Ma, W. Y.,
Chen, N. Y.,
and Dong, Z.
(2001)
Cancer Res.
61,
1604-1610
40.
Bulavin, D. V.,
Saito, S.,
Hollander, M. C.,
Sakaguchi, K.,
Anderson, C. W.,
Appella, E.,
and Fornace, A. J., Jr.
(1999)
EMBO J.
18,
6845-6854
41.
Oda, K.,
Arakawa, H.,
Tanaka, T.,
Matsuda, K.,
Tanikawa, C.,
Mori, T.,
Nishimori, H.,
Tamai, K.,
Tokino, T.,
Nakamura, Y.,
and Taya, Y.
(2000)
Cell
102,
849-862
42.
Hashimoto, S.,
Matsumoto, K.,
Gon, Y.,
Furuichi, S.,
Maruoka, S.,
Takeshita, I.,
Hirota, K.,
Yodoi, J.,
and Horie, T.
(1999)
Biochem. Biophys. Res. Commun.
258,
443-447
43.
Saitoh, M.,
Nishitoh, H.,
Fujii, M.,
Takeda, K.,
Tobiume, K.,
Sawada, Y.,
Kawabata, M.,
Miyazono, K.,
and Ichijo, H.
(1998)
EMBO J.
17,
2596-2606
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