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-XL expression, and the resistance ag
Trang 1epidermal 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-XL) 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: takeuchi@pharm.setsunan.ac.jp
(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-XL 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-XLgene 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-XL 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-XL expression or resistance against ADR-induced apoptosis These results indicate that AP1-mediated upregulation
of Bcl-XL 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.
Trang 2The 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-XL [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 promottrigger-ing 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-XLtranscription We also show that Bcl-XL 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)1EGF for 48 h markedly suppressed the cell death induced by 10 or 20 lm ADR, whereas
1 ngÆmL)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
Trang 3cytosol 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-XL) 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-XL, Bad, and Bax by immunoblot-ting (Fig 3A) EGF increased the expression of
Bcl-XL, 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-XL, we determined the time course
of Bcl-XL mRNA expression after EGF addition (Fig 3B) The Bcl-XL 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-XL protein at the transcriptional level Next we determined the effect of ADR on the expression of Bcl-XL(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-XL, but this ADR-induced decrease was not observed in EGF-pretreated cells Northern blot analy-sis revealed that the Bcl-XL 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)1EGF 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.
Trang 4cells (Fig 3D) Thus we hypothesized that an
anti-apoptosis pathway involving Bcl-XL 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-XL expression vector (clone no 29) or
with the empty bcl-XL 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-XL is involved in the cytoprotective action of
EGF toward TMK-1 cells
To explore the possibility that EGF increased
Bcl-XL expression through the activation of MAP kinase,
we first tested the effect of the MAP kinase kinase
inhibitor PD98059 on EGF-induced Bcl-XL expression (Fig 4A) PD98059 inhibited EGF-induced expression
of Bcl-XLwhether or not cells were treated with ADR Northern blot analysis revealed that increased expres-sion of Bcl-XL 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-XL 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-XLinduced 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)1EGF
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.
Trang 5pretreatment (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-XL, 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-XLmRNA 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-XL(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-XLmRNA Total RNA was isolated at the indicated times after the addition of 100 ngÆmL)1EGF 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-XL 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-XLmRNA 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.
Trang 6results for Puro2 cells, the EGF-induced increase in
the Bcl-XL 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 l M ) 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-XL 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-XLmRNA 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.
Trang 7apoptosis 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-XL, and its translational upregulation [19,21] We showed that among Bcl-2 family members, only the level of Bcl-XLwas 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-XL 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.
Trang 8vector encoding bcl-XLremained viable in the presence
of ADR (Fig 3E), EGF appears to transmit the
survi-val signal through the upregulation of Bcl-XL by
acti-vating the MAP kinase cascade
Bcl-XL belongs to the subfamily of antiapoptotic Bcl-2 family members that share several antiapoptotic features with Bcl-2 Bcl-XL 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-XLmRNA 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.
Trang 9balance 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-XL 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
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-XL 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-XLexpression
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-XL via the transcriptional factor AP1 Rodeck et al [52] reported that Bcl-XL steady-state mRNA expression was downregulated by blockade of EGF receptors in human keratinocytes However, nei-ther theirs or onei-ther reports defined the EGF-induced signaling pathway leading to Bcl-XL 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-XS⁄ 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);
Trang 10and 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)1of 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
37C 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⁄ Pi 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· 105cells⁄ 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)1p-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 Na4P2O7) The lysates were then incubated on
ice for 30 min and clarified by centrifugation at 12 000 g for
10 min at 4C 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-XL, 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)1of EGF for
48 h and thereafter with 20 lm ADR for defined times They were then washed twice with NaCl⁄ Pi, and scraped into ice-cold NaCl⁄ Pi 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 MgCl2, 1 mm 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 4C (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⁄ Pi, cells were lyzed at 4C by incubating them for 10 min in hypotonic buffer (10 mm Tris⁄ HCl, pH 7.8, containing 10 mm NaCl, 1.5 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm PMSF,
2 lgÆmL)1 leupeptin, 2 lgÆmL)1aprotinin, and 0.3% Noni-det P-40) After centrifugation at 4C (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 MgCl2, 20% glycerol, 0.5 mm dithiothreitol, 0.5 mm PMSF,
2 lgÆmL)1 leupeptin, and 2 lgÆmL)1aprotinin) at 4C for
30 min, and the supernatants were then collected after cen-trifugation at 4C (15 500 g) for 5 min (Kubota AF-2724A)
Northern blot analysis Cells were treated with 100 ngÆmL)1EGF, 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 bromideÆmL)1 and