Role of transcription factor activator protein (AP1) in epidermal growth factor-mediated protection against apoptosis induced by a DNA-damaging agent Kenji Takeuchi, Yu-ichiro Motoda and Fumiaki Ito Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan Keywords activator protein (AP1); adriamycin; Bcl-XL; epidermal growth factor; MAP kinase Correspondence K Takeuchi, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan Fax: +81 72 866 3117 Tel +81 72 866 3118 E-mail: takeuchi@pharm.setsunan.ac.jp (Received 31 March 2006, revised June 2006, accepted 14 June 2006) doi:10.1111/j.1742-4658.2006.05377.x We investigated the survival signals of epidermal growth factor (EGF) in human gastric adenocarcinoma cell line TMK-1 Treatment of TMK-1 cells with adriamycin (ADR) caused apoptosis and apoptosis-related reactions such as the release of cytochrome c from mitochondria and the activation of caspase However, EGF treatment greatly reduced the ADR-induced apoptosis as well as these reactions We previously reported that hepatocyte growth factor transmitted protective signals against ADR-induced apoptosis by causing activation of the phosphatidylinositol-3¢-OH kinase (PtdIns3-K) ⁄ Akt signaling pathway in human epithelial cell line MKN74 [Takeuchi K & Ito F (2004) J Biol Chem 279, 892–900] However, PtdIns3K ⁄ Akt signaling did not mediate the antiapoptotic action of EGF in TMK-1 cells EGF increased the expression of the Bcl-XL protein, an antiapoptotic member of the Bcl-2 family, but not that of other anti (Bcl2) or proapoptotic (Bad and Bax) protein members Expression of the c-Fos and c-Jun, components of activator protein (AP1), which are known to regulate bcl-XL gene transcription, were increased in response to EGF Pretreatment of the cells with PD98059, an inhibitor of MAP kinase kinase, inhibited the EGF-induced c-Fos and c-Jun expression, AP1 DNA binding, Bcl-XL expression, and the resistance against ADR-induced apoptosis, suggesting that EGF transmitted the antiapoptotic signal in such a way that it activated AP1 via a MAP kinase signaling pathway TMK-1 cells stably transfected with TAM67, c-Jun dominant-negative mutant, did not display EGF-induced Bcl-XL expression or resistance against ADRinduced apoptosis These results indicate that AP1-mediated upregulation of Bcl-XL expression is critical for protection of TMK-1 cells against ADR-induced apoptosis Diverse chemotherapeutic drugs can kill tumor cells by activating apoptotic pathways The intracellular machinery responsible for apoptosis depends on a family of cysteine aspases (caspases), and action of the two main apoptotic pathways, the death receptor and mitochondria pathways, results in the activation of caspase and caspase 9, respectively Apoptotic triggers such as chemotherapeutic drugs activate the latter pathway, which requires disruption of the mitochond- rial membrane and release of cytochrome c from the mitochondria Cytochrome c functions with Apaf-1 to activate caspase 9, thereby activating a set of downstream caspases [1] Bcl-2 was originally identified in B-cell lymphomas [2] and is now known to belong to a growing family of apoptosis regulatory proteins, known as the Bcl-2 family, which may be either death antagonists (e.g Bcl-2 and Bcl-XL) or death agonists (e.g Bax and Bad) [3] Abbreviations ADR, adriamycin; AP1, activator protein 1; EGF, epidermal growth factor; EMSA, electrophoretic mobility shift assay; HRP, horseradish peroxidase; PMSF, phenylmethylsulfonyl uoride; PtdIns3-K, phosphatidylinositol-3Â-OH kinase FEBS Journal 273 (2006) 37433755 ê 2006 The Authors Journal compilation ª 2006 FEBS 3743 Role of AP1 in EGF-mediated cell protection K Takeuchi et al The permeability of the mitochondrial membrane to cytochrome c has been shown to be controlled by the opposing actions of these anti- and proapoptotic proteins [4] The balance between these two types of regulatory proteins has been reported to partly control cell fate Hence, overexpression of Bcl-2 [5] or Bcl-XL [6] has been shown to inhibit apoptosis; and that of Bad [7] or Bax [8], to induce cell death The development of the chemotherapy-resistant phenotype is a major cause of failure in the treatment of malignancies Drug resistance has been linked to a number of molecular changes in cellular transport and drug metabolism, mutations of the p53 tumor suppressor gene, and overexpression of oncogenes [9,10] In addition, several studies indicate that growth factors such as nerve growth factor, insulin-like growth factor, fibroblast growth factor, epidermal growth factor (EGF), and hepatocyte growth factor can suppress apoptosis of target cell populations; although the mechanisms involved are not fully understood [11–16] Growth factors are multifunctional cytokines involved in many biological processes including proliferation, differentiation, migration, and cell survival They bind and activate a specific tyrosine kinase receptor that is coupled to multiple intracellular signaling pathways [17,18] Activation of tyrosine kinase receptors is involved in cell survival through downstream signaling cascades such as the MAP kinase and phosphatidylinositol-3¢-OH kinase (PtdIns3-K) ⁄ Akt pathways These signals influence survival through several mechanisms including the regulation of Bcl-2 and its family members [13,19,20] Phosphorylation [21] or increased expression [19] of Bcl-2 family members is a mechanism responsible for this regulation Several reports on survival signaling have connected activation of the PtdIns3-K ⁄ Akt signaling with the survival of neurons, fibroblasts, and hematopoietic cells [22,23] Because Akt phosphorylates caspase 9, Bad [24], a proapoptotic member of the Bcl-2 family, and the forkhead transcription factor FKHR [25], a proapoptotic transcription factor, thereby inhibiting them, the PtdIns3K ⁄ Akt signaling pathway has emerged as the major mechanism by which growth factors promote cell survival [26] The transcription factor activator protein (AP1) comprises members of the Jun and Fos families AP1 has been implicated in the regulation of apoptosis and cell proliferation [27] Members of the Jun family, JunB and c-Jun, are suggested to play roles in triggering apoptosis and promoting proliferation of erythroid cells, respectively [28] Previous studies have shown that the bcl-X gene has consensus sequences for the binding of several transcription factors, including 3744 NF-jB, AP1, and GATA-1 [29,30] In response to a suitable signal such as growth factors, the expression of c-Fos, one of the Fos family proteins, is induced through MAP kinase activation, allowing transactivation of genes containing AP1-binding elements However, it is not known whether EGF is capable of protecting cells from apoptosis via this AP1 activation route In this study we found that EGF prevented apoptosis induced by the chemotherapeutic agent adriamycin (ADR; a DNA topoisomerase IIa inhibitor) in TMK-1 cells It inhibited ADR-induced cytochrome c release into the cytosol and caspase activation Because caspase is intimately associated with the initiation of apoptosis, EGF seems to exert its protective action against ADR-induced apoptosis by suppressing caspase activity via stabilization of the mitochondria membrane The protective action resulted from the activation of a MAP kinase-dependent pathway, thereby stimulating bcl-XL transcription We also show that Bcl-XL expression was increased by AP1 activation, possibly through the stimulated transcription of c-Fos and c-Jun This study defines a new EGFinduced cell-survival signal Results Initially, we evaluated the ability of EGF to rescue TMK-1 cells from apoptosis induced by the DNAdamaging agent ADR Pretreatment of the cells with 10 or 100 ngỈmL)1 EGF for 48 h markedly suppressed the cell death induced by 10 or 20 lm ADR, whereas ngỈmL)1 EGF pretreatment markedly suppressed the cell death induced by 10 lm, ADR but not that by 20 lm ADR (Fig 1A) To evaluate whether this ADRinduced cell death resulted from apoptosis, we looked for DNA fragmentation after exposing the cells to 5, 10 or 20 lm ADR for h As shown in Fig 1B, ADR induced DNA fragmentation in a dose-dependent manner, and EGF markedly protected the cells against this DNA fragmentation when present at 10 or 100 ngỈmL)1 The protective action of EGF against 20 lm ADR was time dependent, and the maximal protection required pretreatment with 10 or 100 ngỈmL)1 EGF for 48 h (data not shown) Therefore, cells were pretreated with 100 ngỈmL)1 EGF for 48 h in subsequent experiments Cytochrome c is released from mitochondria by apoptotic triggers such as chemotherapeutic drugs, and the released cytochrome c is able to activate caspase through the formation of an apoptosome comprising Apaf-1, dATP, and caspase [1] We then determined whether ADR induced cytochrome c release into the FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS K Takeuchi et al Role of AP1 in EGF-mediated cell protection A Fig EGF protects TMK-1 cells against apoptosis induced by ADR (A) TMK-1 cells were pretreated or not with 1, 10 or 100 ngỈmL)1 EGF for 48 h Cells were then treated with 10 or 20 lM ADR for h and incubated in ADR-free medium The phasecontrast photomicrographs shown were taken h after incubation of the cells in ADR-free medium Scale bar, 100 lm (B) Cells were treated with EGF and ADR as described in (A) Cells were harvested at h after incubation in ADR-free medium and used for the DNA fragmentation assay as described in Experimental procedures B cytosol and caspase activation, and, if so, whether pretreatment with EGF would inhibit these ADRinduced reactions The cytosol level of cytochrome c was increased in response to ADR, but its release was completely inhibited by EGF treatment (Fig 2A) We next treated TMK-1 cells with ADR and collected cell extracts at various time points for the immunoblot analysis of caspase Beginning at h post treatment with ADR, an increase in the amount of the cleaved form of caspase was seen in ADR-treated cells (Fig 2B) However, when cells were pretreated with EGF, the conversion to the active-form caspase was prevented To further verify this finding, we assessed the activity of caspase by conducting an in vitro fluorometric protease assay (Fig 2C) In agreement with the results obtained using the immunoblot analysis, the ADR-induced activation of caspase was greatly diminished by the EGF pretreatment The requirement of prolonged pretreatment with EGF for protection against ADR-induced apoptosis suggests that maximal protection may have required new protein synthesis Several recent studies indicate that certain growth factors can suppress apoptosis by modulating the process of apoptosis [11–14] Thus, we determined the effect of EGF on the levels of key antiapoptotic (i.e Bcl-2 and Bcl-XL) and proapoptotic (i.e Bad and Bax) proteins Cells were incubated in the presence of EGF for several periods, and cell lysates were prepared from these cells to determine the expression of Bcl-XL, Bad, and Bax by immunoblotting (Fig 3A) EGF increased the expression of BclXL, but not that of Bad or Bax As for Bcl-2, we were unable to detect it in TMK-1 cells (data not shown) To address the signaling pathway leading to the upregulation of Bcl-XL, we determined the time course of Bcl-XL mRNA expression after EGF addition (Fig 3B) The Bcl-XL mRNA level increased in response to EGF and reached its maximum 12 or 24 h after the start of treatment with EGF, indicating that EGF regulated the level of the Bcl-XL protein at the transcriptional level Next we determined the effect of ADR on the expression of Bcl-XL (Fig 3C) Cells were treated with EGF for 48 h, and then exposed to ADR for 2, 4, or h ADR treatment decreased the level of Bcl-XL, but this ADR-induced decrease was not observed in EGF-pretreated cells Northern blot analysis revealed that the Bcl-XL mRNA level was decreased by ADR in both EGF-treated and untreated cells; however, the level in EGF-treated cells was higher at any time point than that in the untreated FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS 3745 Role of AP1 in EGF-mediated cell protection K Takeuchi et al A B C Fig EGF prevents ADR-induced cytochrome c release and caspase activation (A) Cells were treated with 100 ngỈmL)1 EGF and 20 lM ADR as described in Fig Cytosolic fractions were prepared at the indicated times after the ADR addition, separated by 15% SDS ⁄ PAGE, and analyzed by immunoblotting with anti-cytochrome c The blots were reprobed with a b-actin antibody to demonstrate equal loading Similar results were obtained from three separate experiments (B) Cells were treated with EGF and ADR as described in (A), harvested at the indicated times after the addition of ADR, and used for immunoblot analysis of pro-caspase and caspase (C) Cells were treated with EGF and ADR as described in (A) Lysates were prepared at the indicated times after the ADR addition and analyzed for caspase activity by using a fluorometric substrate-based assay Each point is the mean of triplicate samples, and the bar represents the standard deviation Similar results were obtained from three separate experiments cells (Fig 3D) Thus we hypothesized that an antiapoptosis pathway involving Bcl-XL is at least partly responsible for the protection of TMK-1 cells by EGF To examine this hypothesis, we transfected TMK-1 cells with a bcl-XL expression vector (clone no 29) or with the empty bcl-XL expression vector (clone pc10) and isolated each clone When clone no 29 and clone pc10 cells were exposed to ADR and assayed for DNA ladder formation, clone no 29 cells were significantly resistant to ADR compared with clone pc10 cells (Fig 3E) This result is consistent with the hypothesis that Bcl-XL is involved in the cytoprotective action of EGF toward TMK-1 cells To explore the possibility that EGF increased BclXL expression through the activation of MAP kinase, we first tested the effect of the MAP kinase kinase 3746 inhibitor PD98059 on EGF-induced Bcl-XL expression (Fig 4A) PD98059 inhibited EGF-induced expression of Bcl-XL whether or not cells were treated with ADR Northern blot analysis revealed that increased expression of Bcl-XL mRNA seen in the presence of EGF was suppressed by PD98059 (Fig 4B) We next determined whether PD98059 actually blocked MAP kinase activity As shown in Fig 4C, EGF-induced phosphorylation of MAP kinase was not detectable in PD98059-pretreated cells PD98059 has recently been reported to also inhibit MEK5, the upstream regulator of ERK5 [31] Therefore we tested the effect of PD98059 on ERK5 phosphorylation Although ERK5 phosphorylation was stimulated in EGF-treated TMK1 cells, PD98059 did not inhibit the EGF-induced phosphorylation (data not shown) Taken together, these experiments indicate that EGF controlled Bcl-XL mRNA expression via MAP kinase activation To determine a causal link between the activation of MAP kinase and the antiapoptotic action of EGF, we tested the effect of PD98059 on the protective action of EGF Cells were preincubated with PD98059 for h before EGF treatment, which was followed by exposure to ADR and post incubation as usual PD98059 had no significant effect on cell viability in control or ADR-treated cells, but it reduced the degree of EGFmediated protection against ADR (Fig 4D) Transcription factor AP1 is composed of members of the Jun and Fos families, and an AP1-binding site is found around position )270 in the 5¢-end of the bcl-X gene [30] Because MAP kinase has been shown to regulate the transcription of c-Fos, a member of the Fos family, AP1 may be implicated in the transcription of bcl-XL induced by EGF We then studied the effects of EGF on AP1 DNA binding in TMK-1 cells The results of an electrophoretic mobility shift assay (EMSA) of nuclear extracts prepared from cells treated with EGF revealed that AP1 DNA binding activity increased within h following EGF treatment and peaked at h following the treatment (Fig 5A) We also examined the effect of PD98059 on this binding activity and, as expected, observed a decrease in AP1 DNA binding Supershift analysis using antibodies specific for all known Fos and Jun family members revealed that cFos, c-Jun, and JunD were present in the AP1 complex (Fig 5B) This result implicated c-Fos, c-Jun, and JunD as important factors in the inhibition of apoptosis and led us to further examine their expression in these cells during the early events after EGF treatment Immunoblot analysis of nuclear extracts of TMK-1 cells treated with EGF revealed that both c-Fos and c-Jun protein levels increased within h following EGF treatment and that the increase was suppressed by the PD98059 FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS K Takeuchi et al Role of AP1 in EGF-mediated cell protection A E B C D Fig Effect of ADR on expression of Bcl-2 family proteins in EGF-treated cells (A) Cells were treated with EGF for the indicated times, and total cell protein was extracted from the cells Aliquots of the protein (20 lg per lane) were electrophoresed on 12.5% SDS ⁄ PAGE gels, after which the separated proteins were immunoblotted with anti-Bcl-XL (upper), anti-Bad (middle), or anti-Bax (lower), as described in Experimental procedures The blots were reprobed with a b-actin antibody to demonstrate equal loading Relative signal intensities represent the ratio of the densitometrically measured Bcl-XL, Bad, or Bax signals to the b-actin signal in each sample relative to controls shown as Experiments were repeated three times, with similar results each time (B) The upper panel shows the result of northern blot analysis of Bcl-XL mRNA Total RNA was isolated at the indicated times after the addition of 100 ngỈmL)1 EGF The lower panel shows 18S and 28S rRNA to ensure equal loading of samples (C) Cells were treated with EGF for 48 h and then with ADR for h They were then incubated for an additional 2, or h in ADR-free medium Total cell proteins were immunoblotted with anti-Bcl-XL Times after the addition of ADR are indicated Experiments were repeated three times, and similar results were obtained in each experiment (D) Cells were treated in the presence or absence of EGF for 48 h and exposed to ADR for h They were then incubated in ADR-free medium and harvested for northern blot analysis of Bcl-XL mRNA Times after the addition of ADR are indicated (E) Clone no 29 and pc10 cells were exposed to ADR for the indicated times and used for the DNA fragmentation assay as described in Experimental procedures pretreatment (Fig 5C) By contrast, JunD expression was not induced by EGF treatment (data not shown) To determine if the AP1 site was responsible for the EGF-stimulated expression of Bcl-XL, we transfected TMK-1 cells with a vector directing the expression of TAM67, a dominant-negative form of c-Jun, and isolated TAM1 and TAM2 cells, in either of which TAM67 was detected (Fig 6A) Because TAM67 lacks the transactivation domain of c-Jun (amino acids 1–122), but retains the DNA binding and leucine-zipper region of c-Jun, it should function as a dominant-negative mutant of c-Jun to block wild-type c-Jun binding to the AP1 site [32] As shown in Fig 6B, EGF induced the expression of Bcl-XL mRNA in control Puro2 cells, which had been transfected with an empty vector, but not in TAM1 cells Furthermore, compared with the FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS 3747 Role of AP1 in EGF-mediated cell protection K Takeuchi et al A B C D Fig Protective action of EGF against ADR-induced apoptosis is MAP kinase-dependent (A) Cells were pretreated with PD98059 (50 lM) for 90 and thereafter treated with EGF for 48 h They were then exposed to 20 lM ADR for h, incubated for h in ADR-free medium, and harvested for immunoblot analysis of Bcl-XL The blot was thereafter reprobed with a b-actin antibody to demonstrate equal loading Relative signal intensities represent the ratio of the Bcl-XL signal to the b-actin signal in each sample relative to controls shown as (B) Cells were treated with PD98059 and then with EGF for the indicated times, after which northern blot analysis of Bcl-XL mRNA was carried out The lower panel shows 18S and 28S rRNA to demonstrate equal loading of samples Relative signal intensities represent the ratio of the Bcl-XL mRNA signal to the 18S rRNA signal (C) Cells were treated with PD98059 and then with EGF for the indicated times Phosphorylated MAP kinase was detected by use of anti-(phospho-MAP kinase) (D) Cells were treated with PD98059, EGF, and ADR as described in (A) The phase-contrast photomicrographs were taken h after incubation in ADR-free medium Scale bar, 100 lm results for Puro2 cells, the EGF-induced increase in the Bcl-XL protein expression was significantly smaller in TAM1 cells (Fig 6C, upper) Moreover, EGF prevented the conversion to active-form caspase in response to ADR in Puro2 cells, but not in TAM1 (Fig 6C, lower) Finally, we evaluated the ability of EGF to rescue TMK-1 cells, Puro2 cells, and TAM1 cells from apoptosis induced by ADR EGF sup3748 pressed ADR-induced cytotoxicity in both TMK-1 and Puro2 cells, but not in the two dominant-negative mutant cells, TAM1 and TAM2 (Fig 6D) Discussion Diverse chemotherapeutic drugs can kill tumor cells by activating apoptotic pathways The resistance to FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS K Takeuchi et al Fig MAP kinase is involved in the AP1 binding to DNA (A) Cells were pretreated with PD98059 (50 lM) for 90 and thereafter treated with EGF for the indicated periods Nuclear extracts of the cells were then prepared and incubated with 32P-labeled doublestranded oligomer, 5¢-CGCTTGATGAGTCAGCCGGAA-3¢ Specific binding was demonstrated by including a 100-fold molar excess of homologous competitor oligonucleotide during the binding reaction (100 · oligo) Complexes were separated by electrophoresis on a nondenaturing gel and visualized by autoradiography AP1 indicates the migration position of the AP1 ⁄ oligonucleotide complex Lane C shows the migration of probe in the absence of added nuclear extract (B) Nuclear extracts were incubated with an appropriate AP1 factor-specific antibody (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1, or Fra-2) or a normal rabbit serum (nrs) and then with 32P-labeled double-stranded AP1 site oligomer as described in Experimental procedures Complexes were separated by electrophoresis on a nondenaturing 4% acrylamide gel and visualized by autoradiography Arrowheads indicate the positions of the supershifted bands (C) Cells were treated with PD98059 and then with EGF for the indicated times Nuclear extracts were prepared from the cells and used for immunoblot analysis of c-Fos (upper) and c-Jun (lower) The blot was subsequently reprobed with an antibody to a-tubulin to account for differences in loading between samples Similar results were obtained from three independent experiments apoptosis can be acquired by cancer cells through a variety of strategies and is a major cause of failure in the treatment of malignancies Several studies including ours indicate that growth factors confer on cancer cells resistance to apoptosis [11–14,33] For example, EGF prevents cell death induced by several chemotherapeutic agents including ADR and paclitaxel in human cancer cells [15,16] We showed that EGF protected TMK-1 cells from apoptosis induced by ADR (Fig 1) Previous studies have shown that the activation of PtdIns3-K and its downstream effector Akt were associated with the antiapoptotic signaling of various growth factors [22,23] One of the downstream targets of Akt is Bad, a proapoptotic Bcl-2 family protein Phosphorylated Bad is sequestered in the cytoplasm, preventing it from exerting its proapoptotic effect on mitochondria Nerve growth factor, insulin-like growth factor, and fibroblast growth factor have been reported to transmit survival signals through the phosphorylation of Bad [34] Another target is caspase 9, phosphorylation of which prevents the self-activation of caspase [35] Our previous report showed that hepatocyte growth factor protects human gastric adenocarcinoma MKN74 cells from ADR-induced apoptosis by blocking caspase activity via the PtdIns-3K ⁄ Akt survival signaling pathway [33] In contrast to these results, in this study PtdIns3-K ⁄ Akt signaling was not necessary for EGFinduced protection of TMK-1 cells against apoptosis Role of AP1 in EGF-mediated cell protection A B C MAP kinase signaling provides an alternative pathway by which some growth factors prevent apoptosis [11,13,19] Survival signaling connected with the activation of the MAP kinase cascade includes the phosphorylation of Bcl-2 family members, the transcriptional upregulation of Bcl-XL, and its translational upregulation [19,21] We showed that among Bcl-2 family members, only the level of Bcl-XL was increased in response to EGF Further, when TMK-1 cells were pretreated with the MAP kinase kinase inhibitor, ADR-induced apoptosis as well as decreased Bcl-XL expression was observed even in the presence of EGF (Fig 4A,D) Because TMK-1 cells transfected with a FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS 3749 Role of AP1 in EGF-mediated cell protection K Takeuchi et al A B C Fig Protective action of EGF against ADR-induced apoptosis is AP1 dependent (A) TAM1 and TAM2 cells were cotransfected with TAM67 plus pBapePuro Puro2 cells were cotransfected with empty vector plus pBapePuro Immunoblotting of c-Jun and TAM67 in total cell extracts was performed as described in Experimental procedures (B) The upper panel shows the results of northern blot analysis of Bcl-XL mRNA in TAM1 and Puro2 cells Total RNA was isolated at the indicated times after the addition of EGF The lower panel shows 18S and 28S rRNA to ensure the equal loading of samples (C) Subconfluent TAM1 and Puro2 cells were pretreated or not with EGF for 48 h, incubated with or without 20 lM ADR for h, and then incubated for or h in fresh drug-free medium Cells were harvested, and equal aliquots of total cell protein (20 lg per lane) were analyzed for Bcl-XL and caspase by immunoblotting Times after the addition of ADR are indicated (D) TMK-1, Puro2, TAM1, and TAM2 cells were pretreated or not with EGF for 48 h These cells were then treated with 20 lM ADR for h and subsequently incubated in ADR-free medium These phase-contrast photomicrographs were taken h after incubation in ADR-free medium Scale bar, 100 lm D vector encoding bcl-XL remained viable in the presence of ADR (Fig 3E), EGF appears to transmit the survival signal through the upregulation of Bcl-XL by activating the MAP kinase cascade 3750 Bcl-XL belongs to the subfamily of antiapoptotic Bcl-2 family members that share several antiapoptotic features with Bcl-2 Bcl-XL is able to block chemoand irradiation therapy-induced cell death [36] The FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS K Takeuchi et al balance between antiapoptotic and proapoptotic Bcl-2 family members has been described as a primary event in determining the susceptibility to apoptosis through maintaining the integrity of the mitochondria and inhibiting activation of the caspase cascade [37] High expression levels of antiapoptotic Bcl-2-related proteins have been found in many tumors, and upregulation of these proteins has been shown to be a key element in tumor malignancy and drug resistance [36,37] In this study, we observed that the Bcl-XL level was increased by EGF at the transcriptional level (Fig 3B) The bcl-X gene has consensus sequences for the binding of several transcription factors, including NF-jB, AP1, and GATA-1 [29,30] In certain cell types, transcription of the bcl-X gene is controlled by NF-jB [38,39] The results of an EMSA revealed that EGF was not able to increase NF-jB DNA-binding activity (data not shown) It thus appears that transcription factor NF-jB was not involved in the EGF-induced expression of the bcl-X gene in TMK-1 cells AP1 is composed of members of the Jun (c-Jun [40], JunB [41], JunD [42]) and Fos (c-Fos [43], Fra-1 [44], Fra-2 [45], FosB [46]) families Jun and Fos proteins dimerize via a series of leucine repeats (a leucine zipper) and bind in a sequence-specific manner to a heptad DNA sequence known as the 12-O-tetradecanoyl-13-phorbol acetate-responsive element [47] The regulatory mechanism of c-fos expression by extracellular signaling molecules has been studied in great detail Ligands such as growth factors bind to their specific receptors and activate the MAP kinase cascade MAP kinase phosphorylates ternary complex factors such as p62TCF or Elk-1 [48], which binds together with serum response factor to the cis-acting regulatory element of the c-fos gene, termed the serum response element, resulting in the induction of c-fos transcription The expression of c-jun is also stimulated through the MAP kinase cascade [49] Our study showed that EGF caused a substantial increase in AP1 DNA binding In addition, this increase was prevented by MAP kinase kinase inhibitor PD98059 (Fig 5A) The EMSA detected c-Fos, c-Jun, and JunD as members of the Jun and Fos families in the AP1 complex Because the expression of c-Fos and c-Jun, but not that of JunD, was induced in response to EGF, AP1 must be activated in EGF-treated TMK-1 cells, possibly through the increased expression of c-Fos and c-Jun, via the MAP kinase signaling pathway TAM67 retains the DNA binding and leucine-zipper region of c-Jun, but it lacks the transactivation domain of c-Jun (amino acids 1–122) It thus blocks the binding of wild-type c-Jun to the AP1 site and functions as a dominant-negative mutant of c-Jun [50] EGF Role of AP1 in EGF-mediated cell protection protected the cells from ADR-induced apoptosis (Fig 1) and induced the expression of Bcl-XL mRNA (Fig 3B); however, both of these EGF activities were abolished by the introduction of TAM67 into TMK-1 cells Therefore, transcription factor AP1 must play critical roles in the EGF-induced protection against apoptosis by increasing Bcl-XL expression Many studies have implicated PtdIns3-K ⁄ Akt signaling in the inhibition of apoptosis of a variety of cells through the increased phosphorylation of Bad and caspase or through the transcriptional activation of NF-jB [33–35,51] Surprisingly, in TMK-1 cells, which we used in this study, EGF was not able to activate the PtdIns3-K ⁄ Akt pathway, although it protected the cells from apoptosis induced by ADR Instead of activating this pathway, EGF stimulated the MAP kinase pathway and upregulated the expression of Bcl-XL via the transcriptional factor AP1 Rodeck et al [52] reported that Bcl-XL steady-state mRNA expression was downregulated by blockade of EGF receptors in human keratinocytes However, neither theirs or other reports defined the EGF-induced signaling pathway leading to Bcl-XL expression Thus, our study defines a new EGF-induced cell survival signal and indicates that there are some fungible mechanisms by which EGF endows tumor cells with resistance to anticancer drugs In cases in which tumor cells develop resistance against anticancer drugs, we need to clarify the mechanisms responsible for this resistance in these cells Understanding the molecular basis of resistance against apoptosis is thus important for the development of an effective anticancer therapy Experimental procedures Materials EGF (ultra-pure) from mouse submaxillary glands was purchased from Toyobo Co., Ltd (Osaka, Japan) Fetal bovine serum came from GibcoBRL (Auckland, New Zealand) Phenylmethylsulfonyl fluoride (PMSF), pepstatin A, aprotinin, and leupeptin were obtained from Sigma (St Louis, MO) RPMI-1640 medium was from Nissui Pharmaceutical Co., Ltd (Tokyo, Japan) Antibodies used and their sources were as follows: anti-Bad and anti-Bax, from BD Transduction Laboratories (San Jose, CA); anti(caspase p10) (H-83), anti-(Bcl-XS ⁄ L) (S-18), anti-(Bcl-2) (N-19), and anti-(b-actin) (C-11) from Santa Cruz Biotechnology, Inc (Santa Cruz, CA); anti-(ACTIVE MAP kinase), from Promega (Madison, WI); anti-(a-tubulin) (B-5-1-2), from Sigma; swine horseradish peroxidase (HRP)-linked anti-rabbit Ig serum, from DAKO (Glostrup, Denmark); FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS 3751 Role of AP1 in EGF-mediated cell protection K Takeuchi et al and sheep HRP-linked anti-(mouse Ig) serum, from GE Healthcare (Piscataway, NJ) Cell cultures Human gastric adenocarcinoma TMK-1 cells were cultured to subconfluence in RPMI-1640 medium supplemented with 10% fetal bovine serum and used for all of the experiments Treatment of cells with ADR For most experiments, subconfluent cultures in 60- or 100-mm dishes were preincubated with or without 100 ngỈmL)1 of EGF for 48 h and then treated with 20 lm ADR for h After exposure to ADR, cultures were washed twice to remove the drug and then incubated at 37 °C for defined times in RPMI-1640 medium supplemented with 5% fetal bovine serum The cells were then harvested for use in the DNA fragmentation assay (described below) or for immunoblotting DNA fragmentation assay The DNA fragmentation assay was performed as described previously [53] Briefly, after various times of treatment with ADR, adherent cells and floating cells were harvested by centrifugation and washed twice in NaCl ⁄ Pi DNA was extracted and purified from the pellet by use of IsoQuick (ORCA Research Inc., Bothell, WA), and it was dissolved in gel loading buffer and then analyzed by 2% agarose gel electrophoresis For visualization of ‘DNA ladders’, the electrophoresed gel was soaked in Tris-borate ⁄ EDTA solution containing lg ethidium bromidmL)1 Preparation of cellular lysates and immunoblotting Preparation of cellular lysates and immunoblotting were performed as described previously [32] Briefly, cells were seeded at a density of 3.0 · 105 cells ⁄ 60-mm dish and cultured for days The cells were washed with buffer A (25 mm Hepes ⁄ NaOH, pH 7.4, containing 135 mm NaCl) supplemented with a mixture of protease inhibitors (100 lgỈmL)1 PMSF, lgỈmL)1 leupeptin, lgỈmL)1 pepstatin A, and lgỈmL)1 p-toluenesulfonyl-l-arginine methyl ester) Subsequently, the cells were lysed with buffer B (20 mm Tris ⁄ HCl, pH 7.4, containing 137 mm NaCl, mm EGTA, mm EDTA, 0.1% Nonidet P-40, 0.1% Triton X-100, 100 lgỈmL)1 PMSF, lgỈmL)1 pepstatin A, lgỈmL)1 p-toluenesulfonyl-l-arginine methyl ester, lgỈmL)1 leupeptin, mm sodium orthovanadate, 50 mm sodium fluoride, and 30 mm Na4P2O7) The lysates were then incubated on ice for 30 and clarified by centrifugation at 12 000 g for 3752 10 at °C Total cellular lysates were resolved by SDS ⁄ PAGE and transferred to an Immobilon-P membrane (Millipore, Bedford, MA) The membranes were sequentially incubated, first with primary antibody for h and then with HRP-conjugated species-specific Ig for h; the samples were subsequently developed with ECL western blotting detection reagents (GE Healthcare) and exposed to autoradiography film (Fuji Medical X-ray film RX-U; Fuji Photo Film Co., Ltd., Tokyo, Japan) The relative amount of Bcl-XL, Bad, and Bax was estimated by measuring the optical density of the corresponding band with a densitometer (ATTO densitograph AE-6900; ATTO, Tokyo, Japan) Isolation of the cytosolic fraction Cells were pretreated or not with 100 ngỈmL)1 of EGF for 48 h and thereafter with 20 lm ADR for defined times They were then washed twice with NaCl ⁄ Pi, and scraped into icecold NaCl ⁄ Pi Cells were pelleted in microtubes and resuspended in 50 lL of ice-cold buffer C (20 mm Hepes ⁄ NaOH, pH 7.4, 10 mm KCl, 1.5 mm MgCl2, mm EDTA, mm EGTA, mm dithiothreitol, 0.1 mm PMSF) containing 250 mm sucrose The cells were lyzed by homogenization with a mini cordless grinder (Funakoshi Co., Ltd., Tokyo, Japan) for After centrifugation at 750 g for 10 (Kubota AF-2724A; Kubota, Tokyo, Japan), the supernatants were centrifuged at 105 000 g for 60 at °C (Hitachi S100AT3; Hitachi Koki Co., Ltd., Tokyo, Japan) The resulting supernatant was used as the cytosolic fraction Cytoplasmic and nuclear extracts After having been washed with ice-cold NaCl ⁄ Pi, cells were lyzed at °C by incubating them for 10 in hypotonic buffer (10 mm Tris ⁄ HCl, pH 7.8, containing 10 mm NaCl, 1.5 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm PMSF, lgỈmL)1 leupeptin, lgỈmL)1 aprotinin, and 0.3% Nonidet P-40) After centrifugation at °C (1500 g) for (Kubota AF-2724A), supernatants were collected as cytoplasmic extracts Nuclear extracts were prepared by resuspension of the crude nuclei in high-salt buffer (20 mm Tris ⁄ HCl, pH 7.8, containing 420 mm NaCl, 1.5 mm MgCl2, 20% glycerol, 0.5 mm dithiothreitol, 0.5 mm PMSF, lgỈmL)1 leupeptin, and lgỈmL)1 aprotinin) at °C for 30 min, and the supernatants were then collected after centrifugation at °C (15 500 g) for (Kubota AF-2724A) Northern blot analysis Cells were treated with 100 ngỈmL)1 EGF, and total RNA was obtained by use of Isogen (Nippon Gene, Tokyo, Japan) Fifteen micrograms of RNA was separated electrophoretically Equal loading of samples was determined by staining the gel in lg ethidium bromidmL)1 and FEBS Journal 273 (2006) 3743–3755 ª 2006 The Authors Journal compilation ª 2006 FEBS K Takeuchi et al visualizing the rRNA with UV light The RNA was transferred to a Hybond-N+ membrane (GE Healthcare) The blots were hybridized with human bcl-XL cDNA that had been labeled with [32P]dCTP by use of a Rediprime II DNA Labeling System (GE Healthcare) Caspase activity assay Caspase activity was examined according to the instruction manual of the Caspase ⁄ Mch6 Fluorometric Protease Assay kit (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan) Briefly, TMK-1 cells were pretreated or not with EGF for 48 h, stimulated with ADR for 0, 2, or h, and washed with buffer A containing a mixture of protease inhibitors Subsequently, the cells were resuspended in 50 lL of chilled Cell Lysis Buffer The lysates were incubated on ice for 10 min, after which 50 lL of · reaction buffer containing mm dithiothreitol was added to each sample The samples were incubated with LEHD-AFC Substrate (50 lm final concentration) at 37 °C for h and subsequently read in a fluorometer (VersaFluor; BioRad, Hercules, CA) equipped with a 340–380 nm excitation filter (EX 360 ⁄ 40) and 505–515 nm emission filter (EM 510 ⁄ 10) cDNA construct of and transfection with bcl-XL The primers for the human bcl-XL coding region were designed based on the NCBI nucleotide sequence database The EcoRI restriction site and hemagglutinin- conjugated upper primer was 5¢-GGAATTCCGCCACCATGCCATA CGATGTTCCAGATTACGCT-3¢ The XbaI restriction site-conjugated lower primer was 5¢-GCTCTAGAGCTCA TTTCCGACTGAAGA-3¢ Amplification of bcl-XL cDNA was performed from first-strand cDNA with these primers using PCR PCR products were digested with EcoRI and XbaI, and were cloned into pcDNA 3.1 mammalian expression vectors (Invitrogen, Carlsbad, CA) at EcoRI and XbaI sites The sequence of all clones was verified by DNA sequencing (ABI PRISM377 DNA sequencer; PerkinElmer Life Science, Wellesley, MA) Parental TMK-1 cells were transfected overnight with the bcl-XL vector or the empty pcDNA3.1 vector by using Lipofectamine (Invitrogen) Cells were selected in neomycin, and clones were isolated and screened for Bcl-XL expression Creation of TAM67-overexpressing cell line The dominant-negative c-Jun mutant TAM67 was composed of amino acids 123–331 of c-Jun TAM67 was created as an EcoRI fragment and then cloned into the EcoRI site of the expression vector pcDNA3.1 Plasmid DNA was prepared by standard techniques (QIAGEN Plasmid Midi Kit; Hilden, Germany) pBabePuro, a puromycin-resistant vector, was kindly provided by Dr K Shuai (University of Role of AP1 in EGF-mediated cell protection California, Los Angeles) TMK-1 cells were cotransfected with 8.5 lg of TAM67 and 1.5 lg of pBabePuro by using the Lipofectamine reagent, and the transfected cells were selected by exposure to 2.5 mg of puromycin (Sigma) per mL of medium for weeks Empty vector and pBabePuro were also used for cotransfection as a negative control Expression of the TAM67 protein was verified by immunoblot analysis using an anti-(c-Jun) (Oncogene Research Products, Boston, MA) EMSA AP1 (sense: 5¢-CGCTTGATGAGTCAGCCGGAA-3¢) and NF-jB (sense: 5¢-AGTTGAGGGGACTTTCCCAGGC-3¢) consensus double-stranded oligonucleotides were purchased from Promega These oligonucleotides were labeled at the 5¢-end with 32P by the T4 DNA polynucleotide kinase provided in the labeling kit from Promega and separated from free [32P]ATP by a MicroSpin G-25 column procedure (GE Healthcare) TMK-1 cells treated with EGF for different periods were harvested, and nuclear proteins were prepared from the cells as described above The protein concentrations were determined by using the Pierce Coomassie Plus protein assay kit (Pierce, Rockford, IL) For each binding reaction, 35 fmol of the 32P-labeled oligonucleotide (50,000–100 000 c.p.m.) was incubated with lg of the nuclear protein extract at room temperature for 20 in the binding buffer [4% glycerol, mm MgCl2, 0.5 mm EDTA, 0.5 mm dithiothreitol, 50 mm NaCl, 10 mm Tris ⁄ HCl, and 0.05 mgỈmL)1 of poly(dI-dC)Ỉpoly(dI-dC)] For supershift experiments, AP1 factor-specific antibodies were incubated with the binding reaction mixture for 30 at room temperature before addition of the 32 P-labeled oligonucleotide For competition experiments, 3.5 pmol of cold oligonucleotide was added at the same time After binding, protein–DNA complexes were resolved by electrophoresis on 4% native polyacrylamide gels for h at 350 V Gels were dried and 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A (19 94) Epidermal growth factor, phorbol esters, and aurintricarboxylic acid are survival... the MAP kinase cascade [49] Our study showed that EGF caused a substantial increase in AP1 DNA binding In addition, this increase was prevented by MAP kinase kinase inhibitor PD98059 (Fig 5A) The