Báo cáo khoa học: Ionizing radiation utilizes c-Jun N-terminal kinase for amplification of mitochondrial apoptotic cell death in human cervical cancer cells pptx

Ionizing radiation utilizes c-Jun N-terminal kinase
for amplification of mitochondrial apoptotic cell death
in human cervical cancer cells
Min-Jung Kim
1
, Kee-Ho Lee
2
and Su-Jae Lee
1
1 Laboratory of Molecular Biochemistry, Department of Chemistry, Hanyang University, Seoul, Korea
2 Division of Radiation Cancer Biology, Korea Institute of Radiological and Medical Sciences, Seoul, Korea
Exposure of cells to ionizing radiation results in the
simultaneous activation or down-regulation of multiple
signaling pathways, which play a critical role in con-
trolling cell death or cell survival after irradiation in a
cell-type-specific manner. The molecular mechanism by
which apoptotic cell death occurs in response to ioniz-
ing radiation has been widely explored but not pre-
cisely deciphered [1,2]. An improved understanding of
the mechanisms involved in radiation-induced apopto-
tic cell death may ultimately provide novel strategies
for intervention in specific signal transduction path-
ways to favorably alter therapeutic efficacy in the
treatment of human malignancies.
The Bcl-2 family proteins constitute critical control
points in the intrinsic apoptotic pathway. Pro-apopto-
tic members of the Bcl-2 family, such as Bax, Bak,
Keywords
Bax and Bak activation; Bcl-2
phosphorylation; Fas expression; ionizing
radiation; JNK

Correspondence
S J. Lee, Laboratory of Molecular
Biochemistry, Department of Chemistry,
Hanyang University, 17 Haengdang-Dong,
Seongdong-Ku, Seoul 133 791, Korea
Fax: +82 2 2299 0762
Tel: +82 2 2220 2557
E-mail:
(Received 24 October 2007, revised 20 Feb-
ruary 2008, accepted 27 February 2008)
doi:10.1111/j.1742-4658.2008.06363.x
Exposure of cells to ionizing radiation induces activation of multiple signal-
ing pathways that play a critical role in controlling cell death. However,
the basis for linkage between signaling pathways and the cell-death machin-
ery in response to ionizing radiation remains unclear. Here we demonstrate
that activation of c-Jun N-terminal kinase (JNK) is critical for amplifica-
tion of mitochondrial cell death in human cervical cancer cells. Exposure
of HeLa cells to radiation induced loss of mitochondrial membrane poten-
tial, release of cytochrome c and apoptosis inducing factor (AIF) from
mitochondria, and apoptotic cell death. Radiation also induced transcrip-
tional upregulation of Fas, caspase-8 activation, Bax and Bak activation,
and phosphorylation and downregulation of Bcl-2. Inhibition of caspase-8
attenuated Bax and Bak activation, but did not affect phosphorylation and
downregulation of Bcl-2. Expression of a mutant form of Bcl-2 (S70A-Bcl-
2) completely attenuated radiation-induced Bcl-2 downregulation. Interest-
ingly, inhibition of JNK clearly attenuated radiation-induced Bax and Bak
activation, and Bcl-2 phosphorylation as well as Fas expression. In addi-
tion, dominant-negative form of c-Jun inhibited radiation-induced Fas
expression and Bax and Bak activation. These results indicate that the
JNK–c-Jun pathway is required for the transcriptional upregulation of Fas

and subsequent activation of Bax and Bak, and that JNK, but not c-Jun,
is directly associated with phosphorylation and downregulation of Bcl-2 in
response to ionizing radiation. These results suggest that ionizing radiation
can utilize JNK for amplification of mitochondrial apoptotic cell death in
human cervical cancer cells.
Abbreviations
AIF, apoptosis inducing factor; DiOC
6
(3), 3,3¢-dihexyloxacarbolylanine; FACS, fluorescence activated cell sorting; FADD, Fas-associated death
domain; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; siRNA, small interfering RNA.
2096 FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS
Bid, Bad, Bok and Bim, induce the release of pro-
apoptotic mediators by causing mitochondrial dysfunc-
tion, and in turn, these activate the initiator caspase-9
[3,4]. These proteins are subdivided into ‘multidomain’
pro-apoptotic proteins (Bax or Bak) and ‘BH3-only’
proteins (Bid, Bim and Bok). BH3-only proteins,
which act as sensors of cellular stress, are activated by
transcriptional upregulation and ⁄ or post-translational
modification following an apoptotic stimulus [5]. Once
activated, these proteins induce the activation of Bax
and ⁄ or Bak. As a consequence, Bax and Bak form
oligomeric pores leading to the release of apoptogenic
factors from the mitochondria into the cytosol [6,7]. In
contrast, anti-apoptotic members of the Bcl-2 family,
such as Bcl-2, Bcl-xL, Bcl-w and Mcl–1 act primarily
to preserve the mitochondrial membrane potential and
suppress the release of apoptotic cell-death-activating
factors such as cytochrome c and apoptosis-inducing
factor [8,9]. The relative amounts or equilibrium

between these pro- and anti-apoptotic proteins influ-
ences the susceptibility of cells to apoptotic cell death.
The function of Bcl-2 may be regulated by transcrip-
tional control and ⁄ or by post-translational modifica-
tion [10]. Regulation of Bcl-2 at the transcriptional
level seems to be a critical factor in the development
of cancer, as has been demonstrated by enhanced
expression of Bcl-2 in cancer tissues [11]. Recently, it
has been suggested that the anti-apoptotic function of
Bcl-2 is dependent on its phosphorylation status rather
than its expression level [12]. In agreement with these
findings, recent studies showed that Bcl-2 phosphoryla-
tion is critical for taxol-induced apoptosis in many
malignant cells, including leukemic, prostate and naso-
pharyngeal carcinoma cells [13]. Further studies have
shown that phosphorylation of Bcl-2 on residues of in
its loop domain, including Ser70 and Ser87, is critically
involved in the apoptotic process, and is induced by
microtubule-damaging agents such as paclitaxel, docet-
axel, vincristine and vinblastine [14]. Recently, multiple
kinases have been proposed to mediate the phosphory-
lation of Bcl-2 following a variety of stimuli. These
include paclitaxel-activated Raf-1 [15], paclitexel- or
vincristine-induced protein kinase A [16], bryostatin-1-
induced mitochondrial localized PKC-a [17], or
JNK ⁄ SAPK when overexpressed or activated by pac-
litexel [13,18].
The c-Jun N-terminal kinase (JNK) pathway is a
subgroup of MAP kinases activated primarily by
cytokines and exposure to environmental stress

[19,20]. Numerous reports have provided evidence
that JNK can function as a pro-apoptotic kinase in
response to a variety of different stimuli, includ-
ing tumor necrosis factor, UV irradiation, cytokine,
ceramide, and chemotherapeutic drugs [19]. In these
studies, the JNK pathway has been shown to acti-
vate caspases, and may also target other factors that
have been implicated in apoptosis regulation, includ-
ing p53, Bcl-2 and Bax [21]. However, direct linkage
between JNK signaling and the apoptotic cell-death
machinery, especially mitochondrial cell death,
remains unclear.
In the present study, we investigated the basis for
interaction between the signaling pathway and the cell-
death machinery in response to radiation. We showed
that JNK activation in response to radiation appeared
to be correlated with transcriptional upregulation of
Fas and subsequent Bax and Bak activation, and with
phosphorylation and downregulation of Bcl-2. Molecu-
lar dissection of the signaling pathways that regulate
the apoptotic cell-death machinery is critical for both
our understanding of cell-death events after ionizing
irradiation and development of molecular targets for
cancer treatment.
Results
To examine the kinetics of the apoptotic cell death
induced by ionizing radiation in human cervical cancer
cells, we treated HeLa cells with 10 Gy radiation, and
analyzed induction of apoptotic cell death by fluores-
cence activated cell sorting (FACS) analysis with

Annexin V staining. Figure 1A shows that there is a
time-dependent increase in apoptotic cell death, reach-
ing approximately 35% of cells after 72 h of treatment.
To determine whether death receptors are involved in
radiation-induced apoptosis, we examined expression
changes in death receptors such as the tumor necrosis
factor receptor (TNFR), death receptor (DR)4, DR5
and Fas in response to radiation treatment. As shown
in Fig. 1B, flow cytometric analysis clearly revealed
that the protein levels of Fas were increased by radia-
tion treatment, but we did not detect any changes in
the expression of TNFR or DRs (Fig. 1B). In addi-
tion, the protein synthesis inhibitor cyclohexamide
completely inhibited radiation-induced Fas expression
(Fig. 1B), indicating that the Fas protein level
increased as a result of de novo synthesis after radia-
tion treatment. Fas-mediated activation of caspase-8
depends upon its oligomerization, which is mediated
by association of the death effector domain (DED)
domains of the adaptor molecule, Fas-associated death
domain (FADD), and caspase-8. We performed co-
immunoprecitation assays to analyze the association
of FADD and caspase-8 in HeLa cells after radiation
treatment. As shown in Fig. 1C, interaction between
FADD and caspase-8 was increased in cells treated
M J. Kim et al. Role of JNK in radiation-induced mitochondrial cell death
FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS 2097
with radiation. In addition, caspase-8 and -3 were acti-
vated in response to radiation.
To determine whether the mitochondrial pathway is

involved in the induction of apoptotic cell death by
radiation, we examined changes in mitochondrial
membrane potential and release of pro-apoptotic mole-
cules from the mitochondria in radiation-treated HeLa
cells. Ionizing radiation significantly disrupted the
mitochondrial membrane potential (Fig. 2A). The
cytosolic cytochrome c and apoptosis inducing factor
(AIF) levels were markedly increased (Fig. 2B), coin-
ciding with changes in the mitochondrial membrane
potential. These results indicate that radiation-
induced apoptotic cell death occurs in a mitochondrial
Fig. 1. Ionizing radiation induces expression of Fas and activation of caspases in human cervical cancer cells. (A) Ionizing radiation-induced
apoptotic cell death. HeLa cells were treated with 10 Gy of c-radiation, and were harvested at 24, 48 and 72 h after irradiation. Cell death
was determined by flow cytometric analysis. The results from three independent experiments are shown as means ± SEM. *P < 0.05, sta-
tistically significant. (B) Upregulation of the level of Fas protein by irradiation. HeLa cells were treated with 10 Gy of c-radiation in the pres-
ence or absence of the protein synthesis inhibitor, cycloheximide. After 48 and 72 h, the protein levels for TNFR, DR4, DR5 and Fas were
determined by flow cytometric analysis using anti-TNFR, -DR4, -DR5 and -Fas serum. *P < 0.05, statistically significant. (C) Interaction
between FADD and caspase-8 after irradiation. HeLa cells were treated with 10 Gy of c-radiation. After 24, 48 and 72 h, proteins were im-
munoprecipitated using anti-FADD serum, and the immunocomplexes were separated by SDS–PAGE and probed using anti-caspase-8
serum. Western blot analysis was performed using anti-FADD, anti-caspase-8, anti-caspase-3, anti-poly(ADP-ribose) polymerase (PARP) and
anti-b-actin serum. b-actin was used as a loading control.
Role of JNK in radiation-induced mitochondrial cell death M J. Kim et al.
2098 FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS
dysfunction-dependent fashion. As it has been shown
that Bcl-2 family members are crucial to the mitochon-
drial apoptotic cell-death pathways [3], we investigated
whether radiation treatment induces changes in mem-
bers of the Bcl-2 family. We first analyzed activity-
related conformational changes in Bax and Bak by
flow cytometric analysis using antibodies recognizing

N-terminal epitopes of Bax or Bak. As shown in
Fig. 2C, ionizing irradiation resulted in activity-related
modulations of both Bax and Bak, seen as a shift of
the peak to the right in the resulting histogram. In
addition, exposure of cells to radiation caused redistri-
bution of Bax from the cytosol to the mitochondria
without altering the protein expression level of Bax
(Fig. 2D). Small interfering RNA (siRNA) targeting of
the Bax or Bak significantly attenuated radiation-
induced dissipation of the mitochondrial membrane
potential and cell death (Fig. 2E), suggesting that
activation of Bax and Bak plays a crucial role in the
radiation-induced mitochondrial apoptotic cell-death
pathway. We also observed downregulation of Bcl-2 in
a time-dependent manner (Fig. 2F). The levels of Bcl-2
started to diminish at 24 h, and gradually decreased
until 72 h after radiation treatment. However, the level
of Bcl-xL did not alter over the time course examined
in HeLa cells. In addition, we observed phosphoryla-
tion of Bcl-2 after ionizing irradiation by western blot
analysis using a phosphorylation-specific antibody
against phospho-Bcl-2 (Ser70). Bcl-2 phosphorylation
peaked at 48 h after irradiation, and was decreased at
72 h, coinciding with downregulation of the Bcl-2 pro-
tein level. We next examined the involvement of Bcl-2
phosphorylation in radiation-induced mitochondrial
cell death. To determine whether phosphorylation of
Bcl-2 is associated with downregulation of Bcl-2, a
mutant Bcl-2 (S70A-Bcl-2), in which Ser70 of Bcl-2 is
replaced by Ala, was expressed in HeLa cells before

irradiation. Expression of S70A-Bcl-2 completely
attenuated downregulation of Bcl-2 as well as phos-
phorylation in response to radiation treatment
(Fig. 2G). In addition, overexpression of the mutant
Bcl-2 effectively prevented radiation-induced loss of
mitochondrial membrane potential and apoptotic cell
death (Fig. 2H). To further determine whether down-
regulation of Bcl-2 depends on proteasome activity, we
pretreated cells with the proteasome inhibitors MG132
or lactacystin. As shown in Fig. 2I, the proteasome
inhibitors clearly attenuated radiation-induced degra-
dation of the Bcl-2 protein, indicating proteasome-
dependent downregulation of Bcl-2. In addition,
ubiquitination of Bcl-2 appeared to be increased by
treatment with MG132 after irradiation (Fig. 2J).
These observations suggest that the activity-related
modulation of the pro-apoptotic proteins Bax and Bak
and the phosphorylation- and proteasome-dependent
downregulation of Bcl-2 after radiation treatment are
required for the cell-death pathway, accompanied by
loss of the mitochondrial membrane potential and sub-
sequent release of apoptotic molecules from mitochon-
dria.
Caspase-8 has been reported to cleave Bid, a ‘BH3
only’ protein of the Bcl-2 family, in the presence of
apoptotic stimuli. The truncated Bid then triggers acti-
vation of Bax and ⁄ or Bak and mitochondrial release
of pro-apototic molecules into the cytosol [7]. To
investigate whether caspase-8 activation precedes radi-
ation-induced apoptotic conformational changes in

Bax and Bak, we performed western blot analysis to
analyze Bid cleavage after irradiation. Exposure of
HeLa cells to radiation caused Bid cleavage in a time-
dependent manner (Fig. 3A). We next examined
whether caspase-8 is involved in radiation-induced
activity-related modulations of the conformation of
Bax and Bak. Inhibition of caspase-8 by a specific
inhibitor, caspase 8 inhibitor (z-IETD-fmk), prevented
radiation-induced conformational changes of Bax and
Bak (Fig. 3C) and mitochondrial translocation of Bax
as well as Bid cleavage (Fig. 3B). However, the same
treatment did not affect phosphorylation and downre-
gulation of Bcl-2 (Fig. 3B). In addition, pretreatment
with z-IETD-fmk effectively attenuated radiation-
induced apoptotic cell death (Fig. 3D).
Mitogen-activated protein kinases (MAPKs) have
been implicated in the regulation of apoptotic cell
death in response to various stimuli. To investigate a
potential involvement of MAPK in ionizing radiation-
induced cell death, we employed specific chemical
inhibitors of MAPK. As shown in Fig. 4A, treatment
with a JNK-specific inhibitor, SP600125, effectively
attenuated radiation-induced cell death, while treat-
ment with a p38MAPK inhibitor, SB203580, or an
MEK inhibitor, PD98059, slightly enhanced radiation-
induced cell death (Fig. 4A). FACS analysis with
Annexin V staining also clearly showed that radiation-
induced apoptotic cell death was selectively inhibited
by pretreatment with SP600125. Pretreatment with
SP600125 also inhibited radiation-induced loss of mito-

chondrial membrane potential (Fig 4B), release of
cytochrome c from mitochondria, and caspase activa-
tion (Fig. 4C), as well as JNK1 activation (Fig. 4C).
These results indicate that JNK1 acts as an important
mediator of the radiation-induced mitochondrial apop-
totic cell death in human cervical cancer cells.
We next examined whether JNK is involved in
radiation-induced expressional upregulation of Fas
and subsequent activation of the apoptotic cell-death
M J. Kim et al. Role of JNK in radiation-induced mitochondrial cell death
FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS 2099
cascade. As shown in Fig. 5A, pretreatment with the
JNK-specific inhibitor SP600125, or expression of
dominant-negative forms of JNK1, completely attenu-
ated radiation-induced transcriptional upregulation
of Fas and subsequent association of FADD with
caspase-8 (Fig. 5B). Moreover, radiation-induced
Fig. 2.
Role of JNK in radiation-induced mitochondrial cell death M J. Kim et al.
2100 FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS
caspase-8 activation and Bid cleavage were completely
attenuated by pretreatment with the JNK-specific
inhibitor SP600125 (Fig. 5C). In addition, inhibition of
JNK by pretreatment with SP600125 attenuated con-
formation changes in Bax and Bak (Fig. 5D) and the
mitochondrial translocation of Bax (Fig. 5E) induced
by radiation treatment. These results suggest that
JNK1-mediated transcriptional upregulation of Fas is
Fig. 2. Ionizing radiation induces apoptotic conformational changes in Bax and Bak and phosphorylation of Bcl-2. (A) Loss of mitochondrial
transmembrane potential by c-radiation treatment. The mitochondrial transmembrane potential of these cells was determined by assaying

the retention of DioC
6
(3) added during the last 30 min of treatment. After removal of the medium, the amount of retained DioC
6
(3) was
measured by flow cytometry. *P < 0.05, statistically significant. (B) Release of cytochrome c and AIF from mitochondria after c-irradiation. A
cytosolic fraction was obtained and was subjected to western blot analysis using anti-cytochrome c, anti-AIF and anti-a-tubulin serum.
a-tubulin was used as a cytosolic marker protein. (C) Radiation induces apoptotic conformational changes of Bax and Bak after irradiation
(10 Gy). Activity-related modulations of Bax and Bak activity were determined by flow cytometric analysis using specific antibodies recogniz-
ing N-terminal epitopes of Bak or Bax as described in Experimental procedures. *P < 0.05, statistically significant. (D) Radiation-induced Bax
translocation to the mitochondria. Mitochondrial fractionation was performed on HeLa cells treated with 10 Gy of c-radiation. After 24, 48
and 72 h, proteins were subjected to western blot analysis using anti-Bax and anti-HSP60 serum. HSP60 was used as a mitochondrial
marker protein. (E) Effect of Bax siRNA and Bak siRNA on radiation-induced loss of mitochondrial transmembrane potential and apoptotic cell
death. HeLa cells transfected with Bax siRNA and Bak siRNA were treated with 10 Gy of c-radiation. After 72 h, the mitochondrial trans-
membrane potential of these cells was determined by assaying the retention of DioC
6
(3) added during the last 30 min of treatment. After
removal of the medium, the amount of retained DioC
6
(3) were measured by flow cytometry. Apoptotic cell death was determined by flow
cytometric analysis. *P < 0.05, statistically significant. (F) Phosphorylation of Ser70 of Bcl-2 after irradiation. HeLa cells were treated with
10 Gy of c-radiation. After 24, 48 and 72 h, proteins were subjected to western blot analysis using anti-phospho-Bcl-2 (Ser70), anti-Bcl-2,
anti-Bcl-xL and anti-b-actin serum. b-actin was used as a loading control. (G) Effect of overexpression of an Ser70-specific mutant form of
Bcl-2 (S70A) on radiation-induced Bcl-2 phosphorylation. HeLa cells transfected with the Ser70-specific mutant form of Bcl-2 (S70A) were
treated with 10 Gy of c-radiation. After 48 h, proteins were subjected to western blot analysis using anti-Flag, anti-phospho-Bcl-2 (Ser70),
anti-Bcl-2 and anti-b-actin serum. b-actin was used as a loading control. (H) Effect of overexpression of the Ser70-specific mutant form of
Bcl-2 (S70A) on radiation-induced loss of mitochondrial transmembrane potential and apoptotic cell death. HeLa cells transfected with the
Ser70-specific mutant form of Bcl-2 (S70A) were treated with 10 Gy of c-radiation. After 72 h, the mitochondrial transmembrane potential of
these cells was determined by assaying the retention of DioC
6

(3) added during the last 30 min of treatment. After removal of the medium,
the amount of retained DioC
6
(3) were measured by flow cytometry. Apoptotic cell death was determined by flow cytometric analysis.
*P < 0.05, statistically significant. (I) Effect of the proteasome inhibitors MG132 and lactacystin on radiation-induced Bcl-2 degradation. HeLa
cells were treated with 10 Gy of c-radiation with or without MG132 (10 l
M) or lactacystin (20 lM). After 48 h, proteins were subjected to
western blot analysis using anti-Bcl-2 and anti-b-actin serum. b-actin was used as a loading control. (J) Verification of Bcl-2 ubiquitination by
c-radiation treatment. HeLa cells were treated with 10 Gy of c-radiation with or without MG132 (10 l
M). After 48 h, proteins were immuno-
precipitated using anti-Bcl-2 serum, and the immunocomplexes were separated by SDS–PAGE and subjected to western blot analysis using
anti-ubiquitin serum.
M J. Kim et al. Role of JNK in radiation-induced mitochondrial cell death
FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS 2101
a critical upstream event in activity-related modula-
tions of Bax and Bak and subsequent mitochondrial
dysfunction in response to radiation treatment. As it
has been shown that JNK activation leads to inactiva-
tion of anti-apoptotic functions of Bcl-2 through phos-
phorylation in response to certain death stimuli, we
examined whether activated JNK plays a role in radia-
tion-induced phosphorylation and downregulation of
Bcl-2. As shown in Fig. 5G, inhibition of JNK by treat-
ment with the JNK-specific inhibitor SP600125 com-
pletely attenuated radiation-induced phosphorylation
and downregulation of Bcl-2. To determine whether
Bcl-2 phosphorylation is directly caused by activated
JNK in response to radiation, we performed a JNK
kinase assay in vitro using GST–Bcl-2 as a substrate.
Phosphorylation of GST–Bcl-2 by activated JNK was

dramatically increased after irradiation (Fig. 5H).
However, phosphorylation of Bcl-2 by activated JNK
did not occur when GST–S70A-Bcl-2 was used as a
substrate. These results imply that activated JNK
might mediate downregulation of Bcl-2 by direct phos-
phorylation of Ser70 of Bcl-2 in response to ionizing
radiation in human cervical cancer cells.
Western blot analysis also showed that c-Jun in
HeLa cells was activated by radiation treatment: the
levels of phosphorylated c-Jun were markedly
increased under the same conditions (supplementary
Fig. S1A). Ectopic expression of dominant-negative
forms of c-Jun completely inhibited the Fas expression
(Fig. 1B), caspase-8 activation and Bid cleavage (sup-
plementary Fig. S1C) induced by radiation treatment,
suggesting that transcriptional upregulation of Fas in
response to radiation is dependent on the JNK–c-Jun
signaling pathway in human cervical cancer cells.
Fig. 3. Radiation-induced Bax and Bak activations are dependent on caspase-8 activation. (A) Activation of Bid after irradiation. HeLa cells
were treated with 10 Gy of c-radiation. After 24, 48 and 72 h, proteins were subjected to western blot analysis using anti-Bid and anti-b-actin
serum. b-actin was used as a loading control. (B) Effect of the caspase-8-specific inhibitor, z-IETD-fmk, on radiation-induced Bax
translocation to the mitochondria. HeLa cells pretreated with z-IETD-fmk (20 l
M) were treated with 10 Gy of c-radiation. After 48 h, the
mitochondrial fraction and total cell extract were subjected to western blot analysis using anti-caspase-8, anti-Bid, anti-Bax, anti-HSP60, anti-
phospho-Bcl-2, anti-Bcl-2 and anti-b-actin serum. HSP60 and b-actin were used as a mitochondrial marker protein and a loading control,
respectively. (C) Effect of z-IETD-fmk on the radiation-induced apoptotic conformation of Bax and Bak. HeLa cells pretreated with z-IETD-fmk
were treated with 10 Gy of c-radiation. After 48 h, activity-related modulations of Bax and Bak were determined by flow cytometric analysis
using specific antibodies recognizing N-terminal epitopes of Bak or Bax. (D) Effect of z-IETD-fmk on radiation-induced apoptotic cell death.
HeLa cells pretreated with z-IETD-fmk were treated with 10 Gy of c-radiation. After 48 and 72 h, cell death was determined by flow cyto-
metric analysis. *P < 0.05, statistically significant.

Role of JNK in radiation-induced mitochondrial cell death M J. Kim et al.
2102 FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS
Discussion
Ionizing radiation is one of the most commonly used
treatments for a wide variety of tumors. Intracellular
signaling molecules and apoptotic factors seem to play
an important role in determining the radiation
response of tumor cells. However, the basis of the link
between the signaling pathway and the apoptotic cell-
death machinery in response to ionizing radiation
remains largely unclear. The aim of our investigation
was to elucidate the molecular mechanisms of the
mitochondrial dysfunction-mediated apoptotic cell
death triggered by ionizing radiation in human cervical
cancer cells. We suggest that ionizing radiation utilizes
the JNK signaling pathway to amplify mitochondrial
dysfunction and subsequent apoptotic cell death.
Many reports have provided evidence that JNK can
function as a pro-apoptotic kinase in response to a
variety of different stimuli [22]. The JNK pathway has
been shown to activate caspases and may also target
other factors that have been implicated in apoptosis
regulation, including p53, Bcl-2 and Bax [21]. Consis-
tent with these findings, we found that JNK plays an
important role in radiation-induced apoptotic cell
death in human cervical cancer cells. Inhibition of
JNK effectively protected cells from radiation-induced
loss of mitochondrial membrane potential and apopto-
tic cell death.
Fig. 4. JNK activation is required for mitochondrial apoptotic cell death in response to ionizing radiation treatment. (A) Effect of inhibition of

MAPKs on radiation-induced apoptotic cell death. HeLa cells were treated with 10 Gy of c-radiation in the presence of the MEK-specific
inhibitor PD98059 (25 l
M), the p38 MAPK-specific inhibitor SB203580 (20 lM), or the JNK-specific inhibitor SP600125 (5 lM). After 72 h,
apoptotic cell death was determined by flow cytometric analysis. (B) Effect of inhibition of MAPKs on radiation-induced loss of mitochondrial
transmembrane potential. HeLa cells were treated with 10 Gy of c-radiation in the presence of the MEK-specific inhibitor PD98059, the p38
MAPK-specific inhibitor SB203580, or the JNK-specific inhibitor SP600125. After 72 h, the mitochondrial transmembrane potential of these
cells was determined by flow cytometry to assess the retention of DiOC
6
(3) added during the last 30 min of treatments. *P < 0.05, statisti-
cally significant. (C) Effect of JNK inhibition on radiation-induced cytochrome c release. HeLa cells were treated with 10 Gy of c-radiation in
the presence or absence of the JNK-specific inhibitor SP600125. After 48 h, the cytosolic fraction was obtained and was subjected to
western blot analysis using anti-cytochrome c and anti-a-tubulin serum. The total cell extract was subjected to western blot analysis using
anti-phospho-JNK, anti-JNK, anti-caspase-3 and anti-b-actin serum. a-tubulin and b-actin were used as a cytosolic marker protein and a
loading control, respectively.
M J. Kim et al. Role of JNK in radiation-induced mitochondrial cell death
FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS 2103
Fas is a death receptor on the cell surface of a
wide variety of cell types that mediates rapid apop-
tosis. Although Fas is constitutively expressed in a
variety of cell types, UV irradiation, viral infection
and chemotherapeutic agents effectively increase Fas
transcription [23]. Recently, a role of Fas has also
been suggested in ionizing radiation-induced apopto-
sis of various cell types [24]. We have provided fur-
ther evidence that JNK plays a critical role in
radiation-induced transcriptional upregulation of Fas.
Fig. 5. Radiation-induced transcriptional upregulation of Fas is dependent on the JNK–c-Jun pathway. (A) The effect of JNK inhibition on radi-
ation-induced Fas expression. HeLa cells pretreated with SP600125 or transfected with dominant-negative forms of JNK1 were treated with
10 Gy of c -radiation. After 48 h, the Fas protein level was determined by flow cytometric analysis using anti-Fas serum. *P < 0.05, statisti-
cally significant. (B) The effect of JNK inhibition on the interaction between FADD and caspase-8. HeLa cells were treated with 10 Gy of c-

radiation in the presence of SP600125. After 48 h, proteins were immunoprecipitated using anti-FADD serum, and immunocomplexes were
separated by SDS–PAGE and probed using anti-caspase-8 serum. Western blot analysis was performed using anti-FADD serum. (C) Effect
of JNK inhibition on caspase-8 activation. HeLa cells were treated with 10 Gy of c-radiation in the presence of SP600125. After 48 h, pro-
teins were subjected to western blot analysis using anti-caspase-8, anti-Bid and anti-b-actin serum. b-actin was used as a loading control. (D)
Effect of inhibition of JNK on Bax and Bak activation. HeLa cells were treated with 10 Gy of c-radiation in the presence of SP600125. After
48 h, activity-related modulations of Bax and Bak were determined by flow cytometric analysis using specific antibodies recognizing N-termi-
nal epitopes of Bak or Bax. * P < 0.05, statistically significant. (E) Effect of JNK inhibition on radiation-induced Bax translocation to the mito-
chondria. HeLa cells were treated with 10 Gy of c-radiation in the presence of SP600125. After 48 h, the mitochondrial fraction was
subjected to western blot analysis using anti-Bax and anti-HSP60 serum. HSP60 was used as a mitochondrial marker protein. (F) Effect of
JNK inhibition on radiation-induced Bcl-2 phosphorylation. HeLa cells were treated with 10 Gy of c-radiation in the presence of SP600125.
After 48 h, proteins were subjected to western blot analysis using anti-phospho-Bcl-2 (Ser70), anti-Bcl-2 and anti-b-actin serum. b-actin was
used as a loading control. (G) Direct phosphorylation of Bcl-2 by JNK. HeLa cells were treated with 10 Gy of c-radiation. After 24, 48 and
72 h, proteins were subjected to an immune complex kinase assay using anti-JNK serum. GST–Bcl-2 protein was used as a substrate. (H)
HeLa cells were treated with 10 Gy of c-radiation. After 48 h, proteins were subjected to an immune complex kinase assay using anti-JNK
serum. GST–Bcl-2 protein or GST–S70A-Bcl-2 protein were used as a substrate.
Role of JNK in radiation-induced mitochondrial cell death M J. Kim et al.
2104 FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS
Inhibition of JNK completely attenuated radiation-
induced transcriptional upregulation of Fas and the
Fas-mediated downstream cell-death cascade, indicat-
ing that induction of Fas expression in response to
radiation is JNK-dependent.
Recently, several reports have put forward the
hypothesis that the anti-apoptotic function of Bcl-2
is dependent on its phosphorylation status rather
than its expression level [12]. Consistent with these
findings, we observed a marked phosphorylation of
Bcl-2 after ionizing irradiation. Moreover, we found
that Bcl-2 phosphorylation in response to radiation
is closely associated with JNK activation, as its inhi-

bition leads to suppression of radiation-induced Bcl-2
phosphorylation. We provide further evidence that
phosphorylation of Bcl-2 is correlated with downre-
gulation of Bcl-2 in response to radiation treatment.
Pretreatment with MG132, a proteosome inhibitor,
completely blocked radiation-induced Bcl-2 downre-
gulation, and markedly enhanced Bcl-2 phosphoryla-
tion. Furthermore, overexpression of a mutant form
of Bcl-2 (S70A-Bcl-2), in which Ser70 of Bcl-2 is
replaced by Ala, effectively inhibited radiation-
induced downregulation of Bcl-2. These results
suggest that phosphorylation of Bcl-2 might be asso-
ciated with downregulation of Bcl-2 in response to
radiation treatment.
In summary, we demonstrate in the present study
that ionizing radiation can utilize the JNK signaling
pathway to amplify mitochondrial apoptotic cell
death in human cervical cancer cells. We show that
mitochondrial cell death in response to radiation is
induced by activation of Bax and Bak initiated by
transcription upregulation of Fas and by phosphory-
lation ⁄ inactivation of Bcl-2 in a JNK-dependent
manner. An improved understanding of the mecha-
nisms involved in radiation-induced apoptosis may
ultimately provide novel strategies for intervention in
specific signal transduction pathways to favorably
alter the therapeutic ratio in the treatment of human
malignancies.
Experimental procedures
Materials

Polyclonal antibody to caspase-3 and monoclonal antibodies
to poly(ADP-ribose) polymerase, Bax and cytochrome c
were obtained from Pharmingen (San Diego, CA, USA), and
polyclonal antibodies to caspase-8, TNFR, DR4, DR5, Bcl2,
Bcl-xL, Bid, c-Jun, ubiquitin, a-tubulin and the heat-shock
protein HSP60 were obtained from Santa Cruz (Santa
Cruz, CA, USA). Polyclonal antibodies to phospho-Bcl2,
phospho-c-Jun and phospho-JNK were obtained from
Cell Signaling Technology (Beverly, MA, USA). The
caspase-8 inhibitor z-IETD-fmk, the MEK inhibitor
PD98059, the p38 MAPK-specific inhibitor SB203580, the
JNK-specific inhibitor SP600125, and the PI3K-specific
inhibitor LY294002 were obtained from Calbiochem (San
Diego, CA, USA).
Cell culture and transfection
The human cervical carcinoma cell line (HeLa) was obtained
from the American Type Culture Collection (Rockville, MD,
USA). Cells were grown in RPMI-1640 medium supple-
mented with 10% fetal bovine serum. All media were supple-
mented with 100 unitsÆmL
)1
penicillin and 100 lgÆmL
)1
streptomycin, and all cells were incubated at 37 °Cin5%
CO
2
. Cells were transfected with the full-length cDNA of
Bcl-2, flag-tagged dominant-negative-JNK1 or dominant-
negative c-Jun cloned into the pcDNA3.1 plasmid (Invitro-
gen, Carlsbad, CA, USA) or with the control vector

(pcDNA3.1 Zeo) using Lipofectamine PLUS reagent (Invi-
trogen) according to the manufacturer’s recommendations.
Cells were analyzed 24 h after transfection.
Small interfering RNA (siRNA) transfection
siRNA targeting of Bax was performed using 23 bp siRNA
duplexes (AACATGGAGCTGCAGAGGATGAdTdT)
purchased from New England BioLabs (Beverly, MA,
USA). RNAi of Bak was performed using 21 bp siRNA
duplexes (including a two-deoxynucleotide overhang)
(GGAUUCAGCUAUUCUGGAAdTdT) purchased from
Ambion (Austin, TX, USA). A control siRNA
(CCACTACCTGAGCACCCAG) specific to the GFP
DNA sequence was used as a negative control. For trans-
fection, cells were plated in 10 cm dishes at 50% conflu-
ency, and siRNA duplexes (50 nm) were introduced into
the cells using Lipofectamine 2000 (Invitrogen) according
to the manufacturer’s recommendations.
Quantification of cell death
Apoptosis was investigated by Annexin V labeling using a
Sigma-Aldrich kit according to the manufacturer’s instruc-
tions. Annexin V–fluorescein isothiocyanate (FITC) is used
to quantitatively determine the percentage of cells within a
population that are actively undergoing apoptotic cell
death. For the cell-death assessment, the cells were plated
in 60 mm dish at a density of 2 · 10
5
cells per dish and trea-
ted with radiation the next day. At the indicated time
points, cells were harvested by trypsinization, and washed
in NaCl ⁄ P

i
. The cells were labeled with Annexin
V–FITC ⁄ propidium iodide. Annexin V-positive and propi-
dium iodide-positive cells were quantified using a FACScan
M J. Kim et al. Role of JNK in radiation-induced mitochondrial cell death
FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS 2105
flow cytometer fitted with cellquestpro
TM
software (Bec-
ton Dickinson, Franklin Lakes, NJ, USA).
Irradiation
Cells were plated in 60 or 100 mm dishes and incu-
bated at 37 °C under humidified 5% CO
2
–95% air in cul-
ture medium until 70–80% confluent. Cells were then
exposed to c-rays using a 137Cs c-ray source (Atomic
Energy of Canada, Ltd, Mississauga, Canada) at a dose
rate of 3.81 Gy min
)1
.
Western blot analysis
Western blot analysis was performed as described previ-
ously [25]. Briefly, cell lysates were prepared by extracting
proteins with lysis buffer (40 mm Tris ⁄ HCl pH 8.0,
120 mm NaCl, 0.1% NP-40; Sigma, St Louis, MO, USA)
supplemented with protease inhibitors. Proteins were sep-
arated by SDS–PAGE and transferred to nitrocellulose
membrane. The membrane was blocked using 5% nonfat
dry milk in Tris-buffered saline, and then incubated with

primary antibodies for 1 h at room temperature. Blots
were developed using peroxidase-conjugated secondary
antibody, and proteins were visualized by enhanced
chemiluminescence procedures (Amersham, Arlington
Heights, IL, USA) according to the manufacturer’s rec-
ommendations.
Measurement of mitochondrial membrane
potential
Mitochondrial membrane potential was determined by
assaying the retention of the mitochondria-specific dye 3,3¢-
dihexylocarbocyanine iodide [DiOC
6
(3); Molecular Probes
Inc., Eugene, OR, USA]. Cells were loaded with 30 nm
DiOC
6
(3) during the last 30 min of phytosphingosine treat-
ment. After removal of the medium, the cells were washed
twice with NaCl ⁄ P
i
, and the relative amount of retained
DiOC
6
(3) was measured by flow cytometric analysis.
Preparation of cytosolic and mitochondrial
protein fractions
Cells were collected and washed twice in ice-cold
NaCl ⁄ P
i
, resuspended in S-100 buffer (20 mm Hepes,

pH 7.5, 10 mm KCl, 1.9 mm MgCl
2
,1mm EGTA, 1 mm
EDTA, mixture of protease inhibitors) and incubated on
ice for 20 min. The cells were then homogenized using a
Dounce glass homogenizer and a loose pestle (Wheaton,
Millville, NJ, USA) for 70 strokes. Cell homogenates
were centrifuged at 1000 g to remove unbroken cells,
nuclei and heavy membranes. The supernatant was
centrifuged again at 14 000 g for 30 min to collect the
mitochondria-rich fraction (the pellet) and the cytosolic
fraction (the supernatant). The mitochondria-rich fraction
was washed once with extraction buffer, followed by a
final resuspension in lysis buffer (150 mm NaCl, 50 mm
Tris ⁄ Cl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate,
1mm EGTA) containing protease inhibitors for western
blot analysis.
Flow cytometric analysis of Bax and Bak
activation
Bak- or Bax-associated conformational changes were
assessed as previously described [26]. Briefly, cells were
fixed with 0.25% paraformaldehyde for 5 min and incu-
bated with antibodies recognizing N-terminal epitopes of
Bak (AM03 TC100; Oncogene Research Products, San
Diego, CA, USA) or Bax (clone 6A7; Pharmingen, Frank-
lin Lakes, NJ, USA) diluted 1 : 100 in NaCl ⁄ P
i
containing
digitonin (100 g mL
)1

) and 5% fetal bovine serum at 4 °C
for 30 min. Cells were washed three times with NaCl ⁄ P
i
and then incubated with a FITC-conjugated anti-mouse
serum diluted 1 : 200 in NaCl ⁄ P
i
at 4 °C for 30 min. After
washing three times with NaCl ⁄ P
i
, cells (10 000 per sample)
were analyzed on a FACS-Calibur flow cytometer, using
FACScan flow cytometer.
Immune complex kinase assay
Cell lysates were incubated with primary antibody, and im-
munocomplexes were collected on protein A–Sepharose
beads and resuspended in kinase assay mixture containing
c-[
32
P]ATP and substrate. Proteins were separated by SDS–
PAGE, and bands were detected by autoradiography.
Immunofluorescence analysis of death receptor
expression
Indirect antibody staining and flow cytometric analyses
were performed as described previously [27]. Briefly, 10
6
cells in 100 lL NaCl ⁄ P
i
containing 5% fetal bovine
serum were incubated with specific Fas antibodies (Santa
Cruz) at 4 °C for 30 min. Samples were washed twice in

NaCl ⁄ P
i
, and FITC-conjugated anti-mouse secondary
serum was added in 100 lL NaCl ⁄ P
i
at 4 °C for 30 min.
Samples were washed twice with NaCl ⁄ P
i
and analyzed
on a FACS-Calibur flow cytometer, using cell quest
software.
Statistical analysis
Statistical analyses were performed using Student’s t-test.
The data were expressed as means ± SEM derived from at
least three independent experiments. Differences were con-
sidered significant at P < 0.05.
Role of JNK in radiation-induced mitochondrial cell death M J. Kim et al.
2106 FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS
Acknowledgements
This work was supported by the Korea Science and
Engineering Foundation (KOSEF) and the Ministry of
Science and Technology (MOST), Korean Govern-
ment, through its National Nuclear Technology Pro-
gram.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. c-Jun is involved in radiation-induced tran-
scriptional upregulation of Fas.
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Role of JNK in radiation-induced mitochondrial cell death M J. Kim et al.
2108 FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS