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 Lee2 and Su-Jae Lee1 Laboratory of Molecular Biochemistry, Department of Chemistry, Hanyang University, Seoul, Korea Division of Radiation Cancer Biology, Korea Institute of Radiological and Medical Sciences, Seoul, Korea 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 2299 0762 Tel: +82 2220 2557 E-mail: sj0420@hanyang.ac.kr (Received 24 October 2007, revised 20 February 2008, accepted 27 February 2008) doi:10.1111/j.1742-4658.2008.06363.x Exposure of cells to ionizing radiation induces activation of multiple signaling pathways that play a critical role in controlling cell death However, the basis for linkage between signaling pathways and the cell-death machinery in response to ionizing radiation remains unclear Here we demonstrate that activation of c-Jun N-terminal kinase (JNK) is critical for amplification of mitochondrial cell death in human cervical cancer cells Exposure of HeLa cells to radiation induced loss of mitochondrial membrane potential, release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria, and apoptotic cell death Radiation also induced transcriptional 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-Bcl2) completely attenuated radiation-induced Bcl-2 downregulation Interestingly, inhibition of JNK clearly attenuated radiation-induced Bax and Bak activation, and Bcl-2 phosphorylation as well as Fas expression In addition, 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 Exposure of cells to ionizing radiation results in the simultaneous activation or down-regulation of multiple signaling pathways, which play a critical role in controlling 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 ionizing radiation has been widely explored but not precisely deciphered [1,2] An improved understanding of the mechanisms involved in radiation-induced apoptotic cell death may ultimately provide novel strategies for intervention in specific signal transduction pathways 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-apoptotic members of the Bcl-2 family, such as Bax, Bak, Abbreviations AIF, apoptosis inducing factor; DiOC6(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 M.-J Kim et al Bid, Bad, Bok and Bim, induce the release of proapoptotic mediators by causing mitochondrial dysfunction, 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 influences the susceptibility of cells to apoptotic cell death The function of Bcl-2 may be regulated by transcriptional control and ⁄ or by post-translational modification [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 phosphorylation is critical for taxol-induced apoptosis in many malignant cells, including leukemic, prostate and nasopharyngeal 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, docetaxel, vincristine and vinblastine [14] Recently, multiple kinases have been proposed to mediate the phosphorylation of Bcl-2 following a variety of stimuli These include paclitaxel-activated Raf-1 [15], paclitexel- or vincristine-induced protein kinase A [16], bryostatin-1induced mitochondrial localized PKC-a [17], or JNK ⁄ SAPK when overexpressed or activated by paclitexel [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, including tumor necrosis factor, UV irradiation, cytokine, Role of JNK in radiation-induced mitochondrial cell death ceramide, and chemotherapeutic drugs [19] In these studies, 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] 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 celldeath 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 Molecular 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 fluorescence activated cell sorting (FACS) analysis with Annexin V staining Figure 1A shows that there is a time-dependent increase in apoptotic cell death, reaching 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 radiation treatment, but we did not detect any changes in the expression of TNFR or DRs (Fig 1B) In addition, 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 radiation 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 coimmunoprecitation 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 FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS 2097 Role of JNK in radiation-induced mitochondrial cell death M.-J Kim et al Fig 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, statistically significant (B) Upregulation of the level of Fas protein by irradiation HeLa cells were treated with 10 Gy of c-radiation in the presence 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 immunoprecipitated 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 with radiation In addition, caspase-8 and -3 were activated 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 molecules from the mitochondria in radiation-treated HeLa 2098 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), coinciding with changes in the mitochondrial membrane potential These results indicate that radiationinduced apoptotic cell death occurs in a mitochondrial FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS M.-J Kim et al dysfunction-dependent fashion As it has been shown that Bcl-2 family members are crucial to the mitochondrial apoptotic cell-death pathways [3], we investigated whether radiation treatment induces changes in members of the Bcl-2 family We first analyzed activityrelated 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 redistribution 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 radiationinduced 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 phosphorylation 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 protein 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 phosphorylation 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 downregulation 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 degradation of the Bcl-2 protein, indicating proteasomedependent 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 Role of JNK in radiation-induced mitochondrial cell death 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 subsequent release of apoptotic molecules from mitochondria 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 activation of Bax and ⁄ or Bak and mitochondrial release of pro-apototic molecules into the cytosol [7] To investigate whether caspase-8 activation precedes radiation-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 timedependent 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 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 downregulation of Bcl-2 (Fig 3B) In addition, pretreatment with z-IETD-fmk effectively attenuated radiationinduced 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 radiationinduced 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 treatment with a p38MAPK inhibitor, SB203580, or an MEK inhibitor, PD98059, slightly enhanced radiationinduced cell death (Fig 4A) FACS analysis with Annexin V staining also clearly showed that radiationinduced apoptotic cell death was selectively inhibited by pretreatment with SP600125 Pretreatment with SP600125 also inhibited radiation-induced loss of mitochondrial membrane potential (Fig 4B), release of cytochrome c from mitochondria, and caspase activation (Fig 4C), as well as JNK1 activation (Fig 4C) These results indicate that JNK1 acts as an important mediator of the radiation-induced mitochondrial apoptotic 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 FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS 2099 Role of JNK in radiation-induced mitochondrial cell death M.-J Kim et al Fig cascade As shown in Fig 5A, pretreatment with the JNK-specific inhibitor SP600125, or expression of dominant-negative forms of JNK1, completely attenu2100 ated radiation-induced transcriptional upregulation of Fas and subsequent association of FADD with caspase-8 (Fig 5B) Moreover, radiation-induced FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS M.-J Kim et al Role of JNK in radiation-induced mitochondrial cell death Fig 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 DioC6(3) added during the last 30 of treatment After removal of the medium, the amount of retained DioC6(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 recognizing 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 transmembrane potential of these cells was determined by assaying the retention of DioC6(3) added during the last 30 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 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 lM) 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 lM) After 48 h, proteins were immunoprecipitated using anti-Bcl-2 serum, and the immunocomplexes were separated by SDS–PAGE and subjected to western blot analysis using anti-ubiquitin serum 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 FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS 2101 Role of JNK in radiation-induced mitochondrial cell death M.-J Kim et al Fig 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 lM) 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, antiphospho-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 cytometric analysis *P < 0.05, statistically significant a critical upstream event in activity-related modulations of Bax and Bak and subsequent mitochondrial dysfunction in response to radiation treatment As it has been shown that JNK activation leads to inactivation of anti-apoptotic functions of Bcl-2 through phosphorylation in response to certain death stimuli, we examined whether activated JNK plays a role in radiation-induced phosphorylation and downregulation of Bcl-2 As shown in Fig 5G, inhibition of JNK by treatment with the JNK-specific inhibitor SP600125 completely 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) 2102 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 phosphorylation 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 (supplementary 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 FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS M.-J Kim et al Role of JNK in radiation-induced mitochondrial cell death Fig 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 lM), 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 DiOC6(3) added during the last 30 of treatments *P < 0.05, statistically 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 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 celldeath 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] Consistent 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 apoptotic cell death FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS 2103 Role of JNK in radiation-induced mitochondrial cell death M.-J Kim et al Fig Radiation-induced transcriptional upregulation of Fas is dependent on the JNK–c-Jun pathway (A) The effect of JNK inhibition on radiation-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, statistically significant (B) The effect of JNK inhibition on the interaction between FADD and caspase-8 HeLa cells were treated with 10 Gy of cradiation 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, proteins 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-terminal epitopes of Bak or Bax *P < 0.05, statistically significant (E) Effect of JNK inhibition on radiation-induced Bax translocation to the mitochondria 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 Fas is a death receptor on the cell surface of a wide variety of cell types that mediates rapid apoptosis Although Fas is constitutively expressed in a variety of cell types, UV irradiation, viral infection and chemotherapeutic agents effectively increase Fas 2104 transcription [23] Recently, a role of Fas has also been suggested in ionizing radiation-induced apoptosis of various cell types [24] We have provided further evidence that JNK plays a critical role in radiation-induced transcriptional upregulation of Fas FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS M.-J Kim et al Inhibition of JNK completely attenuated radiationinduced transcriptional upregulation of Fas and the Fas-mediated downstream cell-death cascade, indicating 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 inhibition leads to suppression of radiation-induced Bcl-2 phosphorylation We provide further evidence that phosphorylation of Bcl-2 is correlated with downregulation of Bcl-2 in response to radiation treatment Pretreatment with MG132, a proteosome inhibitor, completely blocked radiation-induced Bcl-2 downregulation, and markedly enhanced Bcl-2 phosphorylation Furthermore, overexpression of a mutant form of Bcl-2 (S70A-Bcl-2), in which Ser70 of Bcl-2 is replaced by Ala, effectively inhibited radiationinduced downregulation of Bcl-2 These results suggest that phosphorylation of Bcl-2 might be associated 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 phosphorylation ⁄ inactivation of Bcl-2 in a JNK-dependent manner An improved understanding of the mechanisms 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, Role of JNK in radiation-induced mitochondrial cell death 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 supplemented with 10% fetal bovine serum All media were supplemented with 100 unitsỈmL)1 penicillin and 100 lgỈmL)1 streptomycin, and all cells were incubated at 37 °C in 5% CO2 Cells were transfected with the full-length cDNA of Bcl-2, flag-tagged dominant-negative-JNK1 or dominantnegative c-Jun cloned into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA) or with the control vector (pcDNA3.1 Zeo) using Lipofectamine PLUS reagent (Invitrogen) 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 transfection, cells were plated in 10 cm dishes at 50% confluency, 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 instructions 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 · 105cells per dish and treated with radiation the next day At the indicated 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 propidium iodide-positive cells were quantified using a FACScan FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS 2105 Role of JNK in radiation-induced mitochondrial cell death flow cytometer fitted with cellquestproTM software (Becton Dickinson, Franklin Lakes, NJ, USA) Irradiation Cells were plated in 60 or 100 mm dishes and incubated at 37 °C under humidified 5% CO2–95% air in culture 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 previously [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 separated 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 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 recommendations Measurement of mitochondrial membrane potential Mitochondrial membrane potential was determined by assaying the retention of the mitochondria-specific dye 3,3¢dihexylocarbocyanine iodide [DiOC6(3); Molecular Probes Inc., Eugene, OR, USA] Cells were loaded with 30 nm DiOC6(3) during the last 30 of phytosphingosine treatment After removal of the medium, the cells were washed twice with NaCl ⁄ Pi, and the relative amount of retained DiOC6(3) was measured by flow cytometric analysis Preparation of cytosolic and mitochondrial protein fractions Cells were collected and washed twice in ice-cold NaCl ⁄ Pi, resuspended in S-100 buffer (20 mm Hepes, pH 7.5, 10 mm KCl, 1.9 mm MgCl2, mm EGTA, mm EDTA, mixture of protease inhibitors) and incubated on ice for 20 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 to collect the 2106 M.-J Kim et al 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, mm 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 and incubated with antibodies recognizing N-terminal epitopes of Bak (AM03 TC100; Oncogene Research Products, San Diego, CA, USA) or Bax (clone 6A7; Pharmingen, Franklin Lakes, NJ, USA) diluted : 100 in NaCl ⁄ Pi containing digitonin (100 g mL)1) and 5% fetal bovine serum at °C for 30 Cells were washed three times with NaCl ⁄ Pi and then incubated with a FITC-conjugated anti-mouse serum diluted : 200 in NaCl ⁄ Pi at °C for 30 After washing three times with NaCl ⁄ Pi, 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 immunocomplexes were collected on protein A–Sepharose beads and resuspended in kinase assay mixture containing c-[32P]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, 106 cells in 100 lL NaCl ⁄ Pi containing 5% fetal bovine serum were incubated with specific Fas antibodies (Santa Cruz) at °C for 30 Samples were washed twice in NaCl ⁄ Pi, and FITC-conjugated anti-mouse secondary serum was added in 100 lL NaCl ⁄ Pi at °C for 30 Samples were washed twice with NaCl ⁄ Pi 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 considered significant at P < 0.05 FEBS Journal 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supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 2096–2108 ª 2008 The Authors Journal compilation ª 2008 FEBS ... Role of JNK in radiation- induced mitochondrial cell death M.-J Kim et al Fig Ionizing radiation induces expression of Fas and activation of caspases in human cervical cancer cells (A) Ionizing radiation- induced... 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. .. results indicate that JNK1 acts as an important mediator of the radiation- induced mitochondrial apoptotic cell death in human cervical cancer cells We next examined whether JNK is involved in radiation- induced