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 kinasefor amplification of mitochondrial apoptotic cell death in human cervical cancer cells Min-Jung Kim1, Kee-Ho Lee2and Su-Jae Lee1 1 Labo

Ionizing radiation utilizes c-Jun N-terminal kinase

for amplification of mitochondrial apoptotic cell death

in human cervical cancer cells

Min-Jung Kim1, Kee-Ho Lee2and Su-Jae Lee1

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: sj0420@hanyang.ac.kr

(Received 24 October 2007, revised 20

Feb-ruary 2008, accepted 27 FebFeb-ruary 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 controllsignal-ing 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.

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-translatranscrip-tional

modifica-tion [10] Regulamodifica-tion of Bcl-2 at the transcripmodifica-tional

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 activaradia-tion 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

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 abspres-ence 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.

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 irradiaphosphoryla-tion 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 activaactiva-tion (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

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.

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 DioC6(3) added during the last 30 min of treatment After removal of the medium, the amount of retained DioC6(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 DioC6(3) added during the last 30 min of treatment After removal of the medium, the amount of retained DioC6(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 l M ) 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.

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 funcinactiva-tions 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 caspase-8, Bid, Bax, 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.

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 l M ), or the JNK-specific inhibitor SP600125 (5 l M ) 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 DiOC6(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.

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.

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 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 37C in 5%

CO2 Cells were transfected with the full-length cDNA of Bcl-2, flag-tagged 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

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

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· 105cells per dish and trea-ted with radiation the next day At the indicatrea-ted time points, cells were harvested by trypsinization, and washed

in NaCl⁄ Pi The cells were labeled with Annexin V–FITC⁄ propidium iodide Annexin V-positive and propi-dium iodide-positive cells were quantified using a FACScan