Tài liệu Báo cáo khoa học: Role of transcription factor activator protein 1 (AP1) in epidermal growth factor-mediated protection against apoptosis induced by a DNA-damaging agent doc

Role of transcription factor activator protein 1 (AP1) in
epidermal growth factor-mediated protection against
apoptosis induced by a DNA-damaging agent
Kenji Takeuchi, Yu-ichiro Motoda and Fumiaki Ito
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan
Diverse chemotherapeutic drugs can kill tumor cells
by activating apoptotic pathways. The intracellular
machinery responsible for apoptosis depends on a fam-
ily of cysteine aspases (caspases), and action of the
two main apoptotic pathways, the death receptor and
mitochondria pathways, results in the activation of
caspase 8 and caspase 9, respectively. Apoptotic trig-
gers such as chemotherapeutic drugs activate the latter
pathway, which requires disruption of the mitochond-
rial membrane and release of cytochrome c from the
mitochondria. Cytochrome c functions with Apaf-1 to
activate caspase 9, thereby activating a set of down-
stream caspases [1].
Bcl-2 was originally identified in B-cell lymphomas
[2] and is now known to belong to a growing family of
apoptosis regulatory proteins, known as the Bcl-2 fam-
ily, which may be either death antagonists (e.g. Bcl-2
and Bcl-X
L
) or death agonists (e.g. Bax and Bad) [3].
Keywords
activator protein 1 (AP1); adriamycin; Bcl-X
L
;
epidermal growth factor; MAP kinase
Correspondence

K. Takeuchi, Department of Biochemistry,
Faculty of Pharmaceutical Sciences,
Setsunan University, Hirakata, Osaka
573-0101, Japan
Fax: +81 72 866 3117
Tel. +81 72 866 3118
E-mail:
(Received 31 March 2006, revised 3 June
2006, accepted 14 June 2006)
doi:10.1111/j.1742-4658.2006.05377.x
We investigated the survival signals of epidermal growth factor (EGF) in
human gastric adenocarcinoma cell line TMK-1. Treatment of TMK-1 cells
with adriamycin (ADR) caused apoptosis and apoptosis-related reactions
such as the release of cytochrome c from mitochondria and the activation
of caspase 9. However, EGF treatment greatly reduced the ADR-induced
apoptosis as well as these reactions. We previously reported that hepato-
cyte growth factor transmitted protective signals against ADR-induced
apoptosis by causing activation of the phosphatidylinositol-3¢ -OH kinase
(PtdIns3-K) ⁄ Akt signaling pathway in human epithelial cell line MKN74
[Takeuchi K & Ito F (2004) J Biol Chem 279, 892–900]. However, PtdIns3-
K ⁄ Akt signaling did not mediate the antiapoptotic action of EGF in
TMK-1 cells. EGF increased the expression of the Bcl-X
L
protein, an
antiapoptotic member of the Bcl-2 family, but not that of other anti (Bcl-
2) or proapoptotic (Bad and Bax) protein members. Expression of the
c-Fos and c-Jun, components of activator protein 1 (AP1), which are
known to regulate bcl-X
L
gene transcription, were increased in response to

EGF. Pretreatment of the cells with PD98059, an inhibitor of MAP kinase
kinase, inhibited the EGF-induced c-Fos and c-Jun expression, AP1 DNA
binding, Bcl-X
L
expression, and the resistance against ADR-induced apop-
tosis, suggesting that EGF transmitted the antiapoptotic signal in such a
way that it activated AP1 via a MAP kinase signaling pathway. TMK-1
cells stably transfected with TAM67, c-Jun dominant-negative mutant, did
not display EGF-induced Bcl-X
L
expression or resistance against ADR-
induced apoptosis. These results indicate that AP1-mediated upregulation
of Bcl-X
L
expression is critical for protection of TMK-1 cells against
ADR-induced apoptosis.
Abbreviations
ADR, adriamycin; AP1, activator protein 1; EGF, epidermal growth factor; EMSA, electrophoretic mobility shift assay; HRP, horseradish
peroxidase; PMSF, phenylmethylsulfonyl fluoride; PtdIns3-K, phosphatidylinositol-3¢-OH kinase.
FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS 3743
The permeability of the mitochondrial membrane to
cytochrome c has been shown to be controlled by the
opposing actions of these anti- and proapoptotic pro-
teins [4]. The balance between these two types of regu-
latory proteins has been reported to partly control cell
fate. Hence, overexpression of Bcl-2 [5] or Bcl-X
L
[6]
has been shown to inhibit apoptosis; and that of Bad
[7] or Bax [8], to induce cell death.

The development of the chemotherapy-resistant phe-
notype is a major cause of failure in the treatment of
malignancies. Drug resistance has been linked to a
number of molecular changes in cellular transport and
drug metabolism, mutations of the p53 tumor suppres-
sor gene, and overexpression of oncogenes [9,10]. In
addition, several studies indicate that growth factors
such as nerve growth factor, insulin-like growth factor,
fibroblast growth factor, epidermal growth factor
(EGF), and hepatocyte growth factor can suppress
apoptosis of target cell populations; although the
mechanisms involved are not fully understood [11–16].
Growth factors are multifunctional cytokines involved
in many biological processes including proliferation,
differentiation, migration, and cell survival. They bind
and activate a specific tyrosine kinase receptor that is
coupled to multiple intracellular signaling pathways
[17,18]. Activation of tyrosine kinase receptors is
involved in cell survival through downstream signaling
cascades such as the MAP kinase and phosphatidyl-
inositol-3¢-OH kinase (PtdIns3-K) ⁄ Akt pathways.
These signals influence survival through several mecha-
nisms including the regulation of Bcl-2 and its family
members [13,19,20]. Phosphorylation [21] or increased
expression [19] of Bcl-2 family members is a mechan-
ism responsible for this regulation. Several reports on
survival signaling have connected activation of the
PtdIns3-K ⁄ Akt signaling with the survival of neurons,
fibroblasts, and hematopoietic cells [22,23]. Because
Akt phosphorylates caspase 9, Bad [24], a proapop-

totic member of the Bcl-2 family, and the forkhead
transcription factor FKHR [25], a proapoptotic tran-
scription factor, thereby inhibiting them, the PtdIns3-
K ⁄ Akt signaling pathway has emerged as the major
mechanism by which growth factors promote cell sur-
vival [26].
The transcription factor activator protein 1 (AP1)
comprises members of the Jun and Fos families. AP1
has been implicated in the regulation of apoptosis and
cell proliferation [27]. Members of the Jun family,
JunB and c-Jun, are suggested to play roles in trigger-
ing apoptosis and promoting proliferation of erythroid
cells, respectively [28]. Previous studies have shown
that the bcl-X gene has consensus sequences for the
binding of several transcription factors, including
NF-jB, AP1, and GATA-1 [29,30]. In response to a
suitable signal such as growth factors, the expression
of c-Fos, one of the Fos family proteins, is induced
through MAP kinase activation, allowing transactiva-
tion of genes containing AP1-binding elements. How-
ever, it is not known whether EGF is capable of
protecting cells from apoptosis via this AP1 activation
route.
In this study we found that EGF prevented apopto-
sis induced by the chemotherapeutic agent adriamycin
(ADR; a DNA topoisomerase IIa inhibitor) in TMK-1
cells. It inhibited ADR-induced cytochrome c release
into the cytosol and caspase 9 activation. Because
caspase 9 is intimately associated with the initiation of
apoptosis, EGF seems to exert its protective action

against ADR-induced apoptosis by suppressing
caspase 9 activity via stabilization of the mitochondria
membrane. The protective action resulted from the
activation of a MAP kinase-dependent pathway,
thereby stimulating bcl-X
L
transcription. We also show
that Bcl-X
L
expression was increased by AP1 activa-
tion, possibly through the stimulated transcription of
c-Fos and c-Jun This study defines a new EGF-
induced cell-survival signal.
Results
Initially, we evaluated the ability of EGF to rescue
TMK-1 cells from apoptosis induced by the DNA-
damaging agent ADR. Pretreatment of the cells with
10 or 100 ngÆmL
)1
EGF for 48 h markedly suppressed
the cell death induced by 10 or 20 lm ADR, whereas
1ngÆmL
)1
EGF pretreatment markedly suppressed the
cell death induced by 10 lm, ADR but not that by
20 lm ADR (Fig. 1A). To evaluate whether this ADR-
induced cell death resulted from apoptosis, we looked
for DNA fragmentation after exposing the cells to 5,
10 or 20 lm ADR for 6 h. As shown in Fig. 1B, ADR
induced DNA fragmentation in a dose-dependent

manner, and EGF markedly protected the cells against
this DNA fragmentation when present at 10 or
100 ngÆmL
)1
. The protective action of EGF against
20 lm ADR was time dependent, and the maximal
protection required pretreatment with 10 or
100 ngÆmL
)1
EGF for 48 h (data not shown). There-
fore, cells were pretreated with 100 ngÆmL
)1
EGF for
48 h in subsequent experiments.
Cytochrome c is released from mitochondria by
apoptotic triggers such as chemotherapeutic drugs, and
the released cytochrome c is able to activate caspase 9
through the formation of an apoptosome comprising
Apaf-1, dATP, and caspase 9 [1]. We then determined
whether ADR induced cytochrome c release into the
Role of AP1 in EGF-mediated cell protection K. Takeuchi et al.
3744 FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS
cytosol and caspase 9 activation, and, if so, whether
pretreatment with EGF would inhibit these ADR-
induced reactions. The cytosol level of cytochrome c
was increased in response to ADR, but its release was
completely inhibited by EGF treatment (Fig. 2A). We
next treated TMK-1 cells with ADR and collected cell
extracts at various time points for the immunoblot
analysis of caspase 9. Beginning at 6 h post treatment

with ADR, an increase in the amount of the cleaved
form of caspase 9 was seen in ADR-treated cells
(Fig. 2B). However, when cells were pretreated with
EGF, the conversion to the active-form caspase 9 was
prevented. To further verify this finding, we assessed
the activity of caspase 9 by conducting an in vitro
fluorometric protease assay (Fig. 2C). In agreement
with the results obtained using the immunoblot analy-
sis, the ADR-induced activation of caspase 9 was
greatly diminished by the EGF pretreatment.
The requirement of prolonged pretreatment with
EGF for protection against ADR-induced apoptosis
suggests that maximal protection may have required
new protein synthesis. Several recent studies indicate
that certain growth factors can suppress apoptosis by
modulating the process of apoptosis [11–14]. Thus, we
determined the effect of EGF on the levels of key
antiapoptotic (i.e. Bcl-2 and Bcl-X
L
) and proapoptotic
(i.e. Bad and Bax) proteins. Cells were incubated in
the presence of EGF for several periods, and cell
lysates were prepared from these cells to determine the
expression of Bcl-X
L
, Bad, and Bax by immunoblot-
ting (Fig. 3A). EGF increased the expression of Bcl-
X
L
, but not that of Bad or Bax. As for Bcl-2, we were

unable to detect it in TMK-1 cells (data not shown).
To address the signaling pathway leading to the upreg-
ulation of Bcl-X
L
, we determined the time course
of Bcl-X
L
mRNA expression after EGF addition
(Fig. 3B). The Bcl-X
L
mRNA level increased in
response to EGF and reached its maximum 12 or 24 h
after the start of treatment with EGF, indicating that
EGF regulated the level of the Bcl-X
L
protein at the
transcriptional level. Next we determined the effect of
ADR on the expression of Bcl-X
L
(Fig. 3C). Cells were
treated with EGF for 48 h, and then exposed to ADR
for 2, 4, 6 or 8 h. ADR treatment decreased the level
of Bcl-X
L
, but this ADR-induced decrease was not
observed in EGF-pretreated cells. Northern blot analy-
sis revealed that the Bcl-X
L
mRNA level was
decreased by ADR in both EGF-treated and untreated

cells; however, the level in EGF-treated cells was
higher at any time point than that in the untreated
A
B
Fig. 1. EGF protects TMK-1 cells against
apoptosis induced by ADR. (A) TMK-1 cells
were pretreated or not with 1, 10 or
100 ngÆmL
)1
EGF for 48 h. Cells were then
treated with 10 or 20 l
M ADR for 2 h and
incubated in ADR-free medium. The phase-
contrast photomicrographs shown were
taken 4 h after incubation of the cells in
ADR-free medium. Scale bar, 100 lm. (B)
Cells were treated with EGF and ADR as
described in (A). Cells were harvested at 4 h
after incubation in ADR-free medium and
used for the DNA fragmentation assay as
described in Experimental procedures.
K. Takeuchi et al. Role of AP1 in EGF-mediated cell protection
FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS 3745
cells (Fig. 3D). Thus we hypothesized that an anti-
apoptosis pathway involving Bcl-X
L
is at least partly
responsible for the protection of TMK-1 cells by EGF.
To examine this hypothesis, we transfected TMK-1
cells with a bcl-X

L
expression vector (clone no. 29) or
with the empty bcl-X
L
expression vector (clone pc10)
and isolated each clone. When clone no. 29 and clone
pc10 cells were exposed to ADR and assayed for DNA
ladder formation, clone no. 29 cells were significantly
resistant to ADR compared with clone pc10 cells
(Fig. 3E). This result is consistent with the hypothesis
that Bcl-X
L
is involved in the cytoprotective action of
EGF toward TMK-1 cells.
To explore the possibility that EGF increased Bcl-
X
L
expression through the activation of MAP kinase,
we first tested the effect of the MAP kinase kinase
inhibitor PD98059 on EGF-induced Bcl-X
L
expression
(Fig. 4A). PD98059 inhibited EGF-induced expression
of Bcl-X
L
whether or not cells were treated with ADR.
Northern blot analysis revealed that increased expres-
sion of Bcl-X
L
mRNA seen in the presence of EGF

was suppressed by PD98059 (Fig. 4B). We next deter-
mined whether PD98059 actually blocked MAP kinase
activity. As shown in Fig. 4C, EGF-induced phos-
phorylation of MAP kinase was not detectable in
PD98059-pretreated cells. PD98059 has recently been
reported to also inhibit MEK5, the upstream regulator
of ERK5 [31]. Therefore we tested the effect of
PD98059 on ERK5 phosphorylation. Although ERK5
phosphorylation was stimulated in EGF-treated TMK-
1 cells, PD98059 did not inhibit the EGF-induced
phosphorylation (data not shown). Taken together,
these experiments indicate that EGF controlled Bcl-X
L
mRNA expression via MAP kinase activation. To
determine a causal link between the activation of MAP
kinase and the antiapoptotic action of EGF, we tested
the effect of PD98059 on the protective action of
EGF. Cells were preincubated with PD98059 for 2 h
before EGF treatment, which was followed by expo-
sure to ADR and post incubation as usual. PD98059
had no significant effect on cell viability in control or
ADR-treated cells, but it reduced the degree of EGF-
mediated protection against ADR (Fig. 4D).
Transcription factor AP1 is composed of members of
the Jun and Fos families, and an AP1-binding site is
found around position )270 in the 5¢-end of the bcl-X
gene [30]. Because MAP kinase has been shown to regu-
late the transcription of c-Fos, a member of the Fos
family, AP1 may be implicated in the transcription of
bcl-X

L
induced by EGF. We then studied the effects of
EGF on AP1 DNA binding in TMK-1 cells. The results
of an electrophoretic mobility shift assay (EMSA) of
nuclear extracts prepared from cells treated with EGF
revealed that AP1 DNA binding activity increased
within 1 h following EGF treatment and peaked at 3 h
following the treatment (Fig. 5A). We also examined
the effect of PD98059 on this binding activity and, as
expected, observed a decrease in AP1 DNA binding.
Supershift analysis using antibodies specific for all
known Fos and Jun family members revealed that c-
Fos, c-Jun, and JunD were present in the AP1 complex
(Fig. 5B). This result implicated c-Fos, c-Jun, and JunD
as important factors in the inhibition of apoptosis and
led us to further examine their expression in these cells
during the early events after EGF treatment. Immuno-
blot analysis of nuclear extracts of TMK-1 cells treated
with EGF revealed that both c-Fos and c-Jun protein
levels increased within 1 h following EGF treatment
and that the increase was suppressed by the PD98059
A
B
C
Fig. 2. EGF prevents ADR-induced cytochrome c release and
caspase 9 activation. (A) Cells were treated with 100 ngÆmL
)1
EGF
and 20 l
M ADR as described in Fig. 1. Cytosolic fractions were pre-

pared at the indicated times after the ADR addition, separated by
15% SDS ⁄ PAGE, and analyzed by immunoblotting with anti-cyto-
chrome c. The blots were reprobed with a b-actin antibody to dem-
onstrate equal loading. Similar results were obtained from three
separate experiments. (B) Cells were treated with EGF and ADR as
described in (A), harvested at the indicated times after the addition
of ADR, and used for immunoblot analysis of pro-caspase 9 and
caspase 9. (C) Cells were treated with EGF and ADR as described
in (A). Lysates were prepared at the indicated times after the ADR
addition and analyzed for caspase 9 activity by using a fluorometric
substrate-based assay. Each point is the mean of triplicate sam-
ples, and the bar represents the standard deviation. Similar results
were obtained from three separate experiments.
Role of AP1 in EGF-mediated cell protection K. Takeuchi et al.
3746 FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS
pretreatment (Fig. 5C). By contrast, JunD expression
was not induced by EGF treatment (data not shown).
To determine if the AP1 site was responsible for the
EGF-stimulated expression of Bcl-X
L
, we transfected
TMK-1 cells with a vector directing the expression of
TAM67, a dominant-negative form of c-Jun, and isola-
ted TAM1 and TAM2 cells, in either of which TAM67
was detected (Fig. 6A). Because TAM67 lacks the
transactivation domain of c-Jun (amino acids 1–122),
but retains the DNA binding and leucine-zipper region
of c-Jun, it should function as a dominant-negative
mutant of c-Jun to block wild-type c-Jun binding to
the AP1 site [32]. As shown in Fig. 6B, EGF induced

the expression of Bcl-X
L
mRNA in control Puro2 cells,
which had been transfected with an empty vector, but
not in TAM1 cells. Furthermore, compared with the
A
B
C
D
E
Fig. 3. Effect of ADR on expression of Bcl-2 family proteins in EGF-treated cells. (A) Cells were treated with EGF for the indicated times,
and total cell protein was extracted from the cells. Aliquots of the protein (20 lg per lane) were electrophoresed on 12.5% SDS ⁄ PAGE gels,
after which the separated proteins were immunoblotted with anti-Bcl-X
L
(upper), anti-Bad (middle), or anti-Bax (lower), as described in Experi-
mental procedures. The blots were reprobed with a b-actin antibody to demonstrate equal loading. Relative signal intensities represent the
ratio of the densitometrically measured Bcl-X
L
, Bad, or Bax signals to the b-actin signal in each sample relative to controls shown as 1.
Experiments were repeated three times, with similar results each time. (B) The upper panel shows the result of northern blot analysis of
Bcl-X
L
mRNA. Total RNA was isolated at the indicated times after the addition of 100 ngÆmL
)1
EGF. The lower panel shows 18S and 28S
rRNA to ensure equal loading of samples. (C) Cells were treated with EGF for 48 h and then with ADR for 2 h. They were then incubated
for an additional 2, 4 or 6 h in ADR-free medium. Total cell proteins were immunoblotted with anti-Bcl-X
L
. Times after the addition of ADR
are indicated. Experiments were repeated three times, and similar results were obtained in each experiment. (D) Cells were treated in the

presence or absence of EGF for 48 h and exposed to ADR for 2 h. They were then incubated in ADR-free medium and harvested for nor-
thern blot analysis of Bcl-X
L
mRNA. Times after the addition of ADR are indicated. (E) Clone no. 29 and pc10 cells were exposed to ADR
for the indicated times and used for the DNA fragmentation assay as described in Experimental procedures.
K. Takeuchi et al. Role of AP1 in EGF-mediated cell protection
FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS 3747
results for Puro2 cells, the EGF-induced increase in
the Bcl-X
L
protein expression was significantly smaller
in TAM1 cells (Fig. 6C, upper). Moreover, EGF pre-
vented the conversion to active-form caspase 9 in
response to ADR in Puro2 cells, but not in TAM1
(Fig. 6C, lower). Finally, we evaluated the ability of
EGF to rescue TMK-1 cells, Puro2 cells, and TAM1
cells from apoptosis induced by ADR. EGF sup-
pressed ADR-induced cytotoxicity in both TMK-1 and
Puro2 cells, but not in the two dominant-negative
mutant cells, TAM1 and TAM2 (Fig. 6D).
Discussion
Diverse chemotherapeutic drugs can kill tumor cells
by activating apoptotic pathways. The resistance to
A
B
C
D
Fig. 4. Protective action of EGF against ADR-induced apoptosis is MAP kinase-dependent. (A) Cells were pretreated with PD98059 (50 lM)
for 90 min and thereafter treated with EGF for 48 h. They were then exposed to 20 l
M ADR for 2 h, incubated for 4 h in ADR-free medium,

and harvested for immunoblot analysis of Bcl-X
L
. The blot was thereafter reprobed with a b-actin antibody to demonstrate equal loading. Rel-
ative signal intensities represent the ratio of the Bcl-X
L
signal to the b-actin signal in each sample relative to controls shown as 1. (B) Cells
were treated with PD98059 and then with EGF for the indicated times, after which northern blot analysis of Bcl-X
L
mRNA was carried out.
The lower panel shows 18S and 28S rRNA to demonstrate equal loading of samples. Relative signal intensities represent the ratio of the
Bcl-X
L
mRNA signal to the 18S rRNA signal. (C) Cells were treated with PD98059 and then with EGF for the indicated times. Phosphorylated
MAP kinase was detected by use of anti-(phospho-MAP kinase). (D) Cells were treated with PD98059, EGF, and ADR as described in (A).
The phase-contrast photomicrographs were taken 4 h after incubation in ADR-free medium. Scale bar, 100 lm.
Role of AP1 in EGF-mediated cell protection K. Takeuchi et al.
3748 FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS
apoptosis can be acquired by cancer cells through a
variety of strategies and is a major cause of failure in
the treatment of malignancies. Several studies inclu-
ding ours indicate that growth factors confer on cancer
cells resistance to apoptosis [11–14,33]. For example,
EGF prevents cell death induced by several chemo-
therapeutic agents including ADR and paclitaxel in
human cancer cells [15,16].
We showed that EGF protected TMK-1 cells from
apoptosis induced by ADR (Fig. 1). Previous studies
have shown that the activation of PtdIns3-K and its
downstream effector Akt were associated with the
antiapoptotic signaling of various growth factors

[22,23]. One of the downstream targets of Akt is Bad,
a proapoptotic Bcl-2 family protein. Phosphorylated
Bad is sequestered in the cytoplasm, preventing it from
exerting its proapoptotic effect on mitochondria. Nerve
growth factor, insulin-like growth factor, and fibro-
blast growth factor have been reported to transmit sur-
vival signals through the phosphorylation of Bad [34].
Another target is caspase 9, phosphorylation of which
prevents the self-activation of caspase 9 [35]. Our pre-
vious report showed that hepatocyte growth factor
protects human gastric adenocarcinoma MKN74 cells
from ADR-induced apoptosis by blocking caspase 9
activity via the PtdIns-3K ⁄ Akt survival signaling path-
way [33]. In contrast to these results, in this study
PtdIns3-K ⁄ Akt signaling was not necessary for EGF-
induced protection of TMK-1 cells against apoptosis.
MAP kinase signaling provides an alternative path-
way by which some growth factors prevent apoptosis
[11,13,19]. Survival signaling connected with the acti-
vation of the MAP kinase cascade includes the
phosphorylation of Bcl-2 family members, the trans-
criptional upregulation of Bcl-X
L
, and its translational
upregulation [19,21]. We showed that among Bcl-2
family members, only the level of Bcl-X
L
was increased
in response to EGF. Further, when TMK-1 cells were
pretreated with the MAP kinase kinase inhibitor,

ADR-induced apoptosis as well as decreased Bcl-X
L
expression was observed even in the presence of EGF
(Fig. 4A,D). Because TMK-1 cells transfected with a
A
B
C
Fig. 5. MAP kinase is involved in the AP1 binding to DNA. (A) Cells
were pretreated with PD98059 (50 l
M) for 90 min and thereafter
treated with EGF for the indicated periods. Nuclear extracts of the
cells were then prepared and incubated with
32
P-labeled double-
stranded oligomer, 5¢-CGCTTGATGAGTCAGCCGGAA-3¢. Specific
binding was demonstrated by including a 100-fold molar excess of
homologous competitor oligonucleotide during the binding reaction
(100 · oligo). Complexes were separated by electrophoresis on a
nondenaturing gel and visualized by autoradiography. AP1 indicates
the migration position of the AP1 ⁄ oligonucleotide complex. Lane C
shows the migration of probe in the absence of added nuclear
extract. (B) Nuclear extracts were incubated with an appropriate
AP1 factor-specific antibody (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1,
or Fra-2) or a normal rabbit serum (nrs) and then with
32
P-labeled
double-stranded AP1 site oligomer as described in Experimental
procedures. Complexes were separated by electrophoresis on a
nondenaturing 4% acrylamide gel and visualized by autoradiogra-
phy. Arrowheads indicate the positions of the supershifted bands.

(C) Cells were treated with PD98059 and then with EGF for the
indicated times. Nuclear extracts were prepared from the cells and
used for immunoblot analysis of c-Fos (upper) and c-Jun (lower).
The blot was subsequently reprobed with an antibody to a-tubulin
to account for differences in loading between samples. Similar
results were obtained from three independent experiments.
K. Takeuchi et al. Role of AP1 in EGF-mediated cell protection
FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS 3749
vector encoding bcl-X
L
remained viable in the presence
of ADR (Fig. 3E), EGF appears to transmit the survi-
val signal through the upregulation of Bcl-X
L
by acti-
vating the MAP kinase cascade.
Bcl-X
L
belongs to the subfamily of antiapoptotic
Bcl-2 family members that share several antiapoptotic
features with Bcl-2. Bcl-X
L
is able to block chemo-
and irradiation therapy-induced cell death [36]. The
A
B
C
D
Fig. 6. Protective action of EGF against
ADR-induced apoptosis is AP1 dependent.

(A) TAM1 and TAM2 cells were cotransfect-
ed with TAM67 plus pBapePuro. Puro2 cells
were cotransfected with empty vector plus
pBapePuro. Immunoblotting of c-Jun and
TAM67 in total cell extracts was performed
as described in Experimental procedures. (B)
The upper panel shows the results of nor-
thern blot analysis of Bcl-X
L
mRNA in TAM1
and Puro2 cells. Total RNA was isolated at
the indicated times after the addition of
EGF. The lower panel shows 18S and 28S
rRNA to ensure the equal loading of sam-
ples. (C) Subconfluent TAM1 and Puro2 cells
were pretreated or not with EGF for 48 h,
incubated with or without 20 l
M ADR for
2 h, and then incubated for 0 or 4 h in fresh
drug-free medium. Cells were harvested,
and equal aliquots of total cell protein (20 lg
per lane) were analyzed for Bcl-X
L
and
caspase 9 by immunoblotting. Times after
the addition of ADR are indicated. (D) TMK-1,
Puro2, TAM1, and TAM2 cells were pre-
treated or not with EGF for 48 h. These cells
were then treated with 20 l
M ADR for 2 h

and subsequently incubated in ADR-free
medium. These phase-contrast photomicro-
graphs were taken 4 h after incubation in
ADR-free medium. Scale bar, 100 lm.
Role of AP1 in EGF-mediated cell protection K. Takeuchi et al.
3750 FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS
balance between antiapoptotic and proapoptotic Bcl-2
family members has been described as a primary event
in determining the susceptibility to apoptosis through
maintaining the integrity of the mitochondria and
inhibiting activation of the caspase cascade [37]. High
expression levels of antiapoptotic Bcl-2-related proteins
have been found in many tumors, and upregulation of
these proteins has been shown to be a key element in
tumor malignancy and drug resistance [36,37]. In this
study, we observed that the Bcl-X
L
level was increased
by EGF at the transcriptional level (Fig. 3B). The
bcl-X gene has consensus sequences for the binding of
several transcription factors, including NF-jB, AP1,
and GATA-1 [29,30]. In certain cell types, transcrip-
tion of the bcl-X gene is controlled by NF-jB [38,39].
The results of an EMSA revealed that EGF was not
able to increase NF-jB DNA-binding activity (data
not shown). It thus appears that transcription factor
NF-jB was not involved in the EGF-induced expres-
sion of the bcl-X gene in TMK-1 cells.
AP1 is composed of members of the Jun (c-Jun [40],
JunB [41], JunD [42]) and Fos (c-Fos [43], Fra-1 [44],

Fra-2 [45], FosB [46]) families. Jun and Fos proteins
dimerize via a series of leucine repeats (a leucine
zipper) and bind in a sequence-specific manner to a
heptad DNA sequence known as the 12-O-tetradeca-
noyl-13-phorbol acetate-responsive element [47]. The
regulatory mechanism of c-fos expression by extracellu-
lar signaling molecules has been studied in great detail.
Ligands such as growth factors bind to their specific
receptors and activate the MAP kinase cascade. MAP
kinase phosphorylates ternary complex factors such as
p62TCF or Elk-1 [48], which binds together with
serum response factor to the cis-acting regulatory ele-
ment of the c-fos gene, termed the serum response ele-
ment, resulting in the induction of c-fos transcription.
The expression of c-jun is also stimulated through the
MAP kinase cascade [49]. Our study showed that EGF
caused a substantial increase in AP1 DNA binding. In
addition, this increase was prevented by MAP kinase
kinase inhibitor PD98059 (Fig. 5A). The EMSA detec-
ted c-Fos, c-Jun, and JunD as members of the Jun and
Fos families in the AP1 complex. Because the expres-
sion of c-Fos and c-Jun, but not that of JunD, was
induced in response to EGF, AP1 must be activated
in EGF-treated TMK-1 cells, possibly through the
increased expression of c-Fos and c-Jun, via the MAP
kinase signaling pathway.
TAM67 retains the DNA binding and leucine-zipper
region of c-Jun, but it lacks the transactivation domain
of c-Jun (amino acids 1–122). It thus blocks the bind-
ing of wild-type c-Jun to the AP1 site and functions

as a dominant-negative mutant of c-Jun [50]. EGF
protected the cells from ADR-induced apoptosis
(Fig. 1) and induced the expression of Bcl-X
L
mRNA
(Fig. 3B); however, both of these EGF activities were
abolished by the introduction of TAM67 into TMK-1
cells. Therefore, transcription factor AP1 must play
critical roles in the EGF-induced protection against
apoptosis by increasing Bcl-X
L
expression.
Many studies have implicated PtdIns3-K ⁄ Akt signa-
ling in the inhibition of apoptosis of a variety of cells
through the increased phosphorylation of Bad and
caspase 9 or through the transcriptional activation of
NF-jB [33–35,51]. Surprisingly, in TMK-1 cells,
which we used in this study, EGF was not able to
activate the PtdIns3-K ⁄ Akt pathway, although it pro-
tected the cells from apoptosis induced by ADR.
Instead of activating this pathway, EGF stimulated
the MAP kinase pathway and upregulated the expres-
sion of Bcl-X
L
via the transcriptional factor AP1.
Rodeck et al. [52] reported that Bcl-X
L
steady-state
mRNA expression was downregulated by blockade of
EGF receptors in human keratinocytes. However, nei-

ther theirs or other reports defined the EGF-induced
signaling pathway leading to Bcl-X
L
expression. Thus,
our study defines a new EGF-induced cell survival
signal and indicates that there are some fungible
mechanisms by which EGF endows tumor cells with
resistance to anticancer drugs. In cases in which
tumor cells develop resistance against anticancer
drugs, we need to clarify the mechanisms responsible
for this resistance in these cells. Understanding the
molecular basis of resistance against apoptosis is thus
important for the development of an effective anti-
cancer therapy.
Experimental procedures
Materials
EGF (ultra-pure) from mouse submaxillary glands was
purchased from Toyobo Co., Ltd. (Osaka, Japan). Fetal
bovine serum came from GibcoBRL (Auckland, New Zeal-
and). Phenylmethylsulfonyl fluoride (PMSF), pepstatin A,
aprotinin, and leupeptin were obtained from Sigma (St
Louis, MO). RPMI-1640 medium was from Nissui Pharma-
ceutical Co., Ltd. (Tokyo, Japan). Antibodies used and
their sources were as follows: anti-Bad and anti-Bax, from
BD Transduction Laboratories (San Jose, CA); anti-
(caspase 9 p10) (H-83), anti-(Bcl-X
S ⁄ L
) (S-18), anti-(Bcl-2)
(N-19), and anti-(b-actin) (C-11) from Santa Cruz Biotech-
nology, Inc. (Santa Cruz, CA); anti-(ACTIVE MAP kinase),

from Promega (Madison, WI); anti-(a-tubulin) (B-5-1-2),
from Sigma; swine horseradish peroxidase (HRP)-linked
anti-rabbit Ig serum, from DAKO (Glostrup, Denmark);
K. Takeuchi et al. Role of AP1 in EGF-mediated cell protection
FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS 3751
and sheep HRP-linked anti-(mouse Ig) serum, from GE
Healthcare (Piscataway, NJ).
Cell cultures
Human gastric adenocarcinoma TMK-1 cells were cultured
to subconfluence in RPMI-1640 medium supplemented with
10% fetal bovine serum and used for all of the experi-
ments.
Treatment of cells with ADR
For most experiments, subconfluent cultures in 60- or
100-mm dishes were preincubated with or without
100 ngÆmL
)1
of EGF for 48 h and then treated with 20 lm
ADR for 2 h. After exposure to ADR, cultures were
washed twice to remove the drug and then incubated at
37 °C for defined times in RPMI-1640 medium supplemen-
ted with 5% fetal bovine serum. The cells were then harves-
ted for use in the DNA fragmentation assay (described
below) or for immunoblotting.
DNA fragmentation assay
The DNA fragmentation assay was performed as described
previously [53]. Briefly, after various times of treatment
with ADR, adherent cells and floating cells were harvested
by centrifugation and washed twice in NaCl ⁄ P
i

. DNA was
extracted and purified from the pellet by use of IsoQuick
(ORCA Research Inc., Bothell, WA), and it was dissolved
in gel loading buffer and then analyzed by 2% agarose gel
electrophoresis. For visualization of ‘DNA ladders’, the
electrophoresed gel was soaked in Tris-borate ⁄ EDTA solu-
tion containing 1 lg ethidium bromideÆmL
)1
.
Preparation of cellular lysates and
immunoblotting
Preparation of cellular lysates and immunoblotting were per-
formed as described previously [32]. Briefly, cells were seeded
at a density of 3.0 · 10
5
cells ⁄ 60-mm dish and cultured for
3 days. The cells were washed with buffer A (25 mm
Hepes ⁄ NaOH, pH 7.4, containing 135 mm NaCl) supple-
mented with a mixture of protease inhibitors (100 lgÆmL
)1
PMSF, 2 lgÆmL
)1
leupeptin, 1 lgÆmL
)1
pepstatin A, and
1 lgÆmL
)1
p-toluenesulfonyl-l-arginine methyl ester). Subse-
quently, the cells were lysed with buffer B (20 mm Tris ⁄ HCl,
pH 7.4, containing 137 mm NaCl, 2 mm EGTA, 5 mm

EDTA, 0.1% Nonidet P-40, 0.1% Triton X-100,
100 lgÆmL
)1
PMSF, 1 lgÆmL
)1
pepstatin A, 1 lgÆmL
)1
p-toluenesulfonyl-l-arginine methyl ester, 2 lgÆmL
)1
leupep-
tin, 1 mm sodium orthovanadate, 50 mm sodium fluoride,
and 30 mm Na
4
P
2
O
7
). The lysates were then incubated on
ice for 30 min and clarified by centrifugation at 12 000 g for
10 min at 4 °C. Total cellular lysates were resolved by
SDS ⁄ PAGE and transferred to an Immobilon-P membrane
(Millipore, Bedford, MA). The membranes were sequentially
incubated, first with primary antibody for 2 h and then with
HRP-conjugated species-specific Ig for 1 h; the samples were
subsequently developed with ECL western blotting detection
reagents (GE Healthcare) and exposed to autoradiography
film (Fuji Medical X-ray film RX-U; Fuji Photo Film Co.,
Ltd., Tokyo, Japan). The relative amount of Bcl-X
L
, Bad,

and Bax was estimated by measuring the optical density of
the corresponding band with a densitometer (ATTO densito-
graph AE-6900; ATTO, Tokyo, Japan).
Isolation of the cytosolic fraction
Cells were pretreated or not with 100 ngÆmL
)1
of EGF for
48 h and thereafter with 20 lm ADR for defined times. They
were then washed twice with NaCl ⁄ P
i
, and scraped into ice-
cold NaCl ⁄ P
i
. Cells were pelleted in microtubes and resus-
pended in 50 lL of ice-cold buffer C (20 mm Hepes ⁄ NaOH,
pH 7.4, 10 mm KCl, 1.5 mm MgCl
2
,1mm EDTA, 1 mm
EGTA, 1 mm dithiothreitol, 0.1 mm PMSF) containing
250 mm sucrose. The cells were lyzed by homogenization
with a mini cordless grinder (Funakoshi Co., Ltd., Tokyo,
Japan) for 1 min. After centrifugation at 750 g for 10 min
(Kubota AF-2724A; Kubota, Tokyo, Japan), the super-
natants were centrifuged at 105 000 g for 60 min at 4 °C
(Hitachi S100AT3; Hitachi Koki Co., Ltd., Tokyo, Japan).
The resulting supernatant was used as the cytosolic fraction.
Cytoplasmic and nuclear extracts
After having been washed with ice-cold NaCl ⁄ P
i
, cells were

lyzed at 4 °C by incubating them for 10 min in hypotonic
buffer (10 mm Tris ⁄ HCl, pH 7.8, containing 10 mm NaCl,
1.5 mm MgCl
2
, 0.5 mm dithiothreitol, 0.5 mm PMSF,
2 lgÆmL
)1
leupeptin, 2 lgÆmL
)1
aprotinin, and 0.3% Noni-
det P-40). After centrifugation at 4 °C (1500 g) for 5 min
(Kubota AF-2724A), supernatants were collected as cyto-
plasmic extracts. Nuclear extracts were prepared by resus-
pension of the crude nuclei in high-salt buffer (20 mm
Tris ⁄ HCl, pH 7.8, containing 420 mm NaCl, 1.5 mm MgCl
2
,
20% glycerol, 0.5 mm dithiothreitol, 0.5 mm PMSF,
2 lgÆmL
)1
leupeptin, and 2 lgÆmL
)1
aprotinin) at 4 °C for
30 min, and the supernatants were then collected after cen-
trifugation at 4 °C (15 500 g) for 5 min (Kubota AF-2724A).
Northern blot analysis
Cells were treated with 100 ngÆmL
)1
EGF, and total RNA
was obtained by use of Isogen (Nippon Gene, Tokyo,

Japan). Fifteen micrograms of RNA was separated electro-
phoretically. Equal loading of samples was determined by
staining the gel in 1 lg ethidium bromide Æ mL
)1
and
Role of AP1 in EGF-mediated cell protection K. Takeuchi et al.
3752 FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS
visualizing the rRNA with UV light. The RNA was trans-
ferred to a Hybond-N
+
membrane (GE Healthcare). The
blots were hybridized with human bcl-X
L
cDNA that had
been labeled with [
32
P]dCTP by use of a Rediprime II
DNA Labeling System (GE Healthcare).
Caspase 9 activity assay
Caspase 9 activity was examined according to the instruc-
tion manual of the Caspase 9 ⁄ Mch6 Fluorometric Protease
Assay kit (Medical and Biological Laboratories Co., Ltd.,
Nagoya, Japan). Briefly, TMK-1 cells were pretreated or
not with EGF for 48 h, stimulated with ADR for 0, 2, 4 or
6 h, and washed with buffer A containing a mixture of pro-
tease inhibitors. Subsequently, the cells were resuspended in
50 lL of chilled Cell Lysis Buffer. The lysates were incuba-
ted on ice for 10 min, after which 50 lLof2· reaction
buffer containing 1 mm dithiothreitol was added to each
sample. The samples were incubated with LEHD-AFC Sub-

strate (50 lm final concentration) at 37 °C for 1 h and sub-
sequently read in a fluorometer (VersaFluor; BioRad,
Hercules, CA) equipped with a 340–380 nm excitation filter
(EX 360 ⁄ 40) and 505–515 nm emission filter (EM 510 ⁄ 10).
cDNA construct of and transfection with bcl-X
L
The primers for the human bcl-X
L
coding region were
designed based on the NCBI nucleotide sequence database.
The EcoRI restriction site and hemagglutinin- conjugated
upper primer was 5¢-GGAATTCCGCCACCATGCCATA
CGATGTTCCAGATTACGCT-3¢. The XbaI restriction
site-conjugated lower primer was 5¢ -GCTCTAGAGCTCA
TTTCCGACTGAAGA-3¢. Amplification of bcl-X
L
cDNA
was performed from first-strand cDNA with these primers
using PCR. PCR products were digested with EcoRI and
XbaI, and were cloned into pcDNA 3.1 mammalian expres-
sion vectors (Invitrogen, Carlsbad, CA) at EcoRI and XbaI
sites. The sequence of all clones was verified by DNA
sequencing (ABI PRISM377 DNA sequencer; PerkinElmer
Life Science, Wellesley, MA). Parental TMK-1 cells were
transfected overnight with the bcl-X
L
vector or the empty
pcDNA3.1 vector by using Lipofectamine (Invitrogen).
Cells were selected in neomycin, and clones were isolated
and screened for Bcl-X

L
expression.
Creation of TAM67-overexpressing cell line
The dominant-negative c-Jun mutant TAM67 was com-
posed of amino acids 123–331 of c-Jun TAM67 was created
as an EcoRI fragment and then cloned into the EcoRI site
of the expression vector pcDNA3.1. Plasmid DNA was pre-
pared by standard techniques (QIAGEN Plasmid Midi Kit;
Hilden, Germany). pBabePuro, a puromycin-resistant vec-
tor, was kindly provided by Dr K. Shuai (University of
California, Los Angeles). TMK-1 cells were cotransfected
with 8.5 lg of TAM67 and 1.5 lg of pBabePuro by using
the Lipofectamine reagent, and the transfected cells were
selected by exposure to 2.5 mg of puromycin (Sigma) per
mL of medium for 3 weeks. Empty vector and pBabePuro
were also used for cotransfection as a negative control.
Expression of the TAM67 protein was verified by immuno-
blot analysis using an anti-(c-Jun) (Oncogene Research Pro-
ducts, Boston, MA).
EMSA
AP1 (sense: 5¢-CGCTTGATGAGTCAGCCGGAA-3¢) and
NF-jB (sense: 5¢-AGTTGAGGGGACTTTCCCAGGC-3¢)
consensus double-stranded oligonucleotides were purchased
from Promega. These oligonucleotides were labeled at the
5¢-end with
32
P by the T4 DNA polynucleotide kinase pro-
vided in the labeling kit from Promega and separated from
free [
32

P]ATP by a MicroSpin G-25 column procedure (GE
Healthcare). TMK-1 cells treated with EGF for different
periods were harvested, and nuclear proteins were prepared
from the cells as described above. The protein concentra-
tions were determined by using the Pierce Coomassie Plus
protein assay kit (Pierce, Rockford, IL). For each binding
reaction, 35 fmol of the
32
P-labeled oligonucleotide
(50,000–100 000 c.p.m.) was incubated with 5 lg of the
nuclear protein extract at room temperature for 20 min in
the binding buffer [4% glycerol, 1 mm MgCl
2
, 0.5 mm
EDTA, 0.5 mm dithiothreitol, 50 mm NaCl, 10 mm
Tris ⁄ HCl, and 0.05 mgÆmL
)1
of poly(dI-dC)Æpoly(dI-dC)].
For supershift experiments, AP1 factor-specific antibodies
were incubated with the binding reaction mixture for
30 min at room temperature before addition of the
32
P-labeled oligonucleotide. For competition experiments,
3.5 pmol of cold oligonucleotide was added at the same
time. After binding, protein–DNA complexes were resolved
by electrophoresis on 4% native polyacrylamide gels for
2 h at 350 V. Gels were dried and subjected to autoradio-
graphy. AP1 factor-selective rabbit polyclonal antibodies
specifying c-Fos (H-125), FosB (H-75), Fra-1 (R-20), Fra-2
(Q-20), c-Jun (H-79), JunB (N-17), and JunD (329) were

from Santa Cruz Biotechnology.
Acknowledgements
We thank Dr E. Tahara for supplying TMK-1 cells, Dr
K. Shuai for providing pbabePuro, and J. Takino, M.
Tagawa, and K. Uchida for technical assistance. This
work was supported in part by a grant from the
program Grants-in-Aid for Young Scientists of the
Ministry of Education, Culture, Sports, Science, and
Technology (MEXT) and by funding from the
‘Research for the Future’ Program from the Japan
K. Takeuchi et al. Role of AP1 in EGF-mediated cell protection
FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS 3753
Society for the Promotion of Science (JSPS) and
MEXT.
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