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- W2021736277 abstract "Cisplatin activates multiple signal transduction pathways involved in coordinating cellular responses to stress. Here we demonstrate a requirement for extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family in mediating cisplatin-induced apoptosis of human cervical carcinoma HeLa cells. Cisplatin treatment resulted in dose- and time- dependent activation of ERK. That elevated ERK activity contributed to cell death by cisplatin was supported by several observations: 1) PD98059 and U0126, chemical inhibitors of the MEK/ERK signaling pathway, prevented apoptosis; 2) pretreatment of cells with TPA, an activator of the ERK pathway, enhanced their sensitivity to cisplatin; 3) suramin, a growth factor receptor antagonist that greatly suppressed ERK activation, likewise inhibited cisplatin-induced apoptosis; and, finally, 4) HeLa cell variants selected for cisplatin resistance showed reduced activation of ERK following cisplatin treatment. Cisplatin-induced apoptosis was associated with cytochrome c release and subsequent caspase-3 activation, both of which could be prevented by treatment with the MEK inhibitors. However, the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected HeLa cells against apoptosis without affecting ERK activation. Taken together, our findings suggest that ERK activation plays an active role in mediating cisplatin-induced apoptosis of HeLa cells and functions upstream of caspase activation to initiate the apoptotic signal. Cisplatin activates multiple signal transduction pathways involved in coordinating cellular responses to stress. Here we demonstrate a requirement for extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family in mediating cisplatin-induced apoptosis of human cervical carcinoma HeLa cells. Cisplatin treatment resulted in dose- and time- dependent activation of ERK. That elevated ERK activity contributed to cell death by cisplatin was supported by several observations: 1) PD98059 and U0126, chemical inhibitors of the MEK/ERK signaling pathway, prevented apoptosis; 2) pretreatment of cells with TPA, an activator of the ERK pathway, enhanced their sensitivity to cisplatin; 3) suramin, a growth factor receptor antagonist that greatly suppressed ERK activation, likewise inhibited cisplatin-induced apoptosis; and, finally, 4) HeLa cell variants selected for cisplatin resistance showed reduced activation of ERK following cisplatin treatment. Cisplatin-induced apoptosis was associated with cytochrome c release and subsequent caspase-3 activation, both of which could be prevented by treatment with the MEK inhibitors. However, the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected HeLa cells against apoptosis without affecting ERK activation. Taken together, our findings suggest that ERK activation plays an active role in mediating cisplatin-induced apoptosis of HeLa cells and functions upstream of caspase activation to initiate the apoptotic signal. cis-diamminedichloroplatinum c-Jun N-terminal kinase extracellular signal-regulated kinase mitogen-activated protein kinase mitogen-activated protein kinase/ERK kinase N-acetylcysteine dithiothreitol phenylmethylsulfonyl fluoride 4,6-diamidino-2-phenylindole poly(ADP-ribosyl) polymerase benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone 12-O-tetradecanoylphorbol-13-acetate short wave length ultraviolet radiation etoposide doxorubicin epidermal growth factor growth factor receptor glutathioneS-transferase 4-morpholinepropanesulfonic acid Cisplatin (cis-diamminedichloroplatinum; CDDP)1 is a potent inducer of growth arrest and/or apoptosis in most cell types and is among the most effective and widely used chemotherapeutic agents employed for treatment of human cancers. However, a major limitation of CDDP chemotherapy is serious drug resistance. Multiple mechanisms have been implicated in the development of CDDP resistance including reduced accumulation of the drug, increased levels of glutathione (GSH), enhanced expression of metallothionein, enhanced DNA repair, increased levels of Bcl-2-related anti-apoptotic genes, and alterations in signal transduction pathways involved in apoptosis (1Timmer-Bosscha H. Mulder N.H. de Vries E.G. Br. J. Cancer. 1992; 66: 227-238Crossref PubMed Scopus (176) Google Scholar, 2Chu G. J. Biol. Chem. 1994; 269: 787-790Abstract Full Text PDF PubMed Google Scholar, 3Perez R.P. Eur. J. Cancer. 1998; 34: 1535-1542Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Apoptosis induced by CDDP is generally considered to be the result of its ability to damage DNA (4Eastman A. Cancer Cells. 1990; 2: 275-280PubMed Google Scholar), but the detailed mechanisms by which such DNA damage triggers cell death remain unclear. Understanding the molecular basis of CDDP-mediated apoptosis could lead to strategies resulting in improved therapeutic benefits. Proteins comprising the mitogen-activated protein kinase (MAPK) family constitute important mediators of signal transduction processes that serve to coordinate the cellular response to a variety of extracellular stimuli. Three major mammalian MAPK subfamilies have been described: the extracellular signal-regulated kinases (ERK), the c-Jun N-terminal kinases (JNK, also called stress-activated protein kinase), and the p38 kinases. Each MAPK is activated through a specific phosphorylation cascade. The ERK pathway plays a major role in regulating cell growth and differentiation, being highly induced in response to growth factors, cytokines, and phorbol esters (5Johnson G.L. Vaillancourt R.R. Curr. Opin. Cell Biol. 1994; 6: 230-238Crossref PubMed Scopus (308) Google Scholar, 6Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar, 7He H. Wang X. Gorospe M. Holbrook N.J. Trush M.A. Cell Growth Differ. 1999; 10: 307-315PubMed Google Scholar). It is also activated by some conditions of stress, particularly oxidant injury, and in such circumstances is believed to confer a survival advantage to cells (8Guyton K.Z. Liu Y. Gorospe M. Xu Q. Holbrook N.J. J. Biol. Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar, 9Aikawa R. Komuro I. Yamazaki T. Zou Y. Kudoh S. Tanaka M. Shiojima I. Hiroi Y. Yazaki Y. J. Clin. Invest. 1997; 100: 1813-1821Crossref PubMed Scopus (630) Google Scholar, 10Wang X. Martindale J.L. Liu Y. Holbrook N.J. Biochem. J. 1998; 333: 291-300Crossref PubMed Scopus (689) Google Scholar). In contrast, JNK and p38 are generally only weakly activated by growth factors, but are highly activated in response to a variety of stress signals including tumor necrosis factor, ionizing and short wave length ultraviolet irradiation (UVC), and hyperosmotic stress. Their activation is most frequently associated with induction of apoptosis (10Wang X. Martindale J.L. Liu Y. Holbrook N.J. Biochem. J. 1998; 333: 291-300Crossref PubMed Scopus (689) Google Scholar, 11Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5036) Google Scholar, 12Chen Y.R. Wang X. Templeton D. Davis R.J. Tan T.H. J. Biol. Chem. 1996; 271: 31929-31936Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 13Graves J.D. Draves K.E. Craxton A. Saklatvala J. Krebs E.G. Clark E.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13814-13818Crossref PubMed Scopus (158) Google Scholar, 14Brenner B. Koppenhoefer U. Weinstock C. Linderkamp O. Lang F. Gulbins E. J. Biol. Chem. 1997; 272: 22173-22181Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Many studies have demonstrated an activation of JNK in response to CDDP treatment, but how it influences cell survival is unclear. Although most of the reports published thus far have suggested a role for JNK in the induction of apoptosis by CDDP, several studies have suggested that JNK signaling plays a role in enhancing survival of CDDP-treated cells (15Zanke B.W. Boudreau K. Rubie E. Winnett E. Tibbles L.A. Zon L. Kyriakis J. Liu F.F. Woodgett J.R. Curr. Biol. 1996; 6: 606-613Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 16Sanchez-Perez I. Murguia J.R. Perona R. Oncogene. 1998; 16: 533-540Crossref PubMed Scopus (222) Google Scholar, 17Sanchez-Perez I. Perona R. FEBS Lett. 1999; 453: 151-158Crossref PubMed Scopus (90) Google Scholar, 18Potapova O. Haghighi A. Bost F. Liu C. Birrer M.J. Gjerset R. Mercola D. J. Biol. Chem. 1997; 272: 14041-14044Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 19Hayakawa J. Ohmichi M. Kurachi H. Ikegami H. Kimura A. Matsuoka T. Jikihara H. Mercola D. Murata Y. J. Biol. Chem. 1999; 274: 31648-31654Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). There is also mixed evidence for the role of ERK in influencing cell survival of CDDP-treated cells. For example, two recent studies have suggested that ERK activation is associated with enhanced survival of CDDP-treated cells (19Hayakawa J. Ohmichi M. Kurachi H. Ikegami H. Kimura A. Matsuoka T. Jikihara H. Mercola D. Murata Y. J. Biol. Chem. 1999; 274: 31648-31654Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 20Persons D.L. Yazlovitskaya E.M. Cui W. Pelling J.C. Clin. Cancer Res. 1999; 5: 1007-1014PubMed Google Scholar). However, elevated expression of Ras, an upstream component of the ERK signaling pathway, has been connected with enhanced sensitivity to CDDP (21Gao X.S. Asaumi J. Kawasaki S. Nishikawa K. Kuroda M. Takeda Y. Hiraki Y. Ihara M. Ohnishi T. Anticancer Res. 1995; 15: 1911-1914PubMed Google Scholar, 22Fokstuen T. Rabo Y.B. Zhou J.N. Karlson J. Platz A. Shoshan M.C. Hansson J. Linder S. Anticancer Res. 1997; 17: 2347-2352PubMed Google Scholar). The present study sought to examine the roles of the MAPK signaling pathways in regulating CDDP-induced apoptosis in HeLa cells. Although ERK, JNK, and p38 were all found to be activated in response to CDDP treatment, only ERK activity appears to be involved in regulating cell survival. Using a variety of strategies to manipulate ERK activity, we provide evidence that ERK is important in mediating CDDP-induced apoptosis through a cytochrome crelease-dependent mechanism. Cisplatin, 12-O-tetradecanoylphorbol-13-acetate (TPA), hydrogen peroxide, etoposide, doxorubicin, and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma. Anti-JNK1 polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The MEK1/2 inhibitor, PD98059, the p38 inhibitors (SB202190 and SB203580), suramin, and N-acetylcysteine were all obtained from CalBiochem (San Diego, CA). The monoclonal anti-PARP and anti-cytochrome c antibodies were purchased from PharMingen (San Diego, CA); U0126 and the anti-phospho-ERK and anti-phospho-JNK rabbit polyclonal antibodies were purchased from Promega (Madison, WI). Anti-phospho-p38 and anti-phospho-MEK1/2 (Ser217/221) antibodies were purchased from New England Biolabs, Inc. (Beverly, MA), and the anti-caspase-3 antibody was from Transduction Laboratories (Lexington, KY). The caspase inhibitor zVAD-fmk was purchased from Enzyme Systems Products (Livermore, CA). The cisplatin-resistant cell lines, HeLa-R1 and HeLa-R3, and parental control line HeLa-C were kindly provided by Dr. Gilbert Chu (Stanford University, Stanford, CA) (23Chu G. Chang E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3324-3328Crossref PubMed Scopus (140) Google Scholar). HeLa and A549 cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gemini BioProducts Inc., Calabasas, CA), 100 units of penicillin, and 100 μg of streptomycin/ml. They were cultured at 37 °C in a humidified chamber containing 5% CO2. For the induction of apoptosis, cells were plated in 60-mm dishes 1 day prior to cisplatin treatment. DAPI staining was performed as described previously (24Wang X. Gorospe M. Huang Y. Holbrook N.J. Oncogene. 1997; 15: 2991-2997Crossref PubMed Scopus (203) Google Scholar). In brief, prior to staining, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, then washed with PBS. DAPI was added to the fixed cells for 30 min, after which they were examined by fluorescence microscopy. Apoptotic cells were identified by condensation and fragmentation of nuclei. Percentage of apoptotic cells was calculated as the ratio of apoptotic cells to total cells counted × 100. A minimum of 400 cells were counted for each treatment. For immunoblot analysis, cells were harvested in 300 μl of lysis buffer (20 mm Hepes, pH 7.4, 2 mm EGTA, 50 mm β-glycerol phosphate, 1% Triton X-100, 10% glycerol, 1 mm dithiothreitol (DTT), 1 mm phenylsulfonyl fluoride (PMSF), 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm Na3VO4, and 5 mm NaF). The resulting lysates were resolved on a 4–12% NuPAGE gels (NOVEX, San Diego, CA) (30 μg/lane) and transferred onto polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were blocked with Tris-buffered saline with Tween 20 (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.1% Tween 20) containing 5% milk and then hybridized with different antibodies. Proteins were detected by using enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech). Cells were washed twice with phosphate-buffered saline, the pellets collected by centrifugation, and resuspended in 500 μl of buffer A (20 mm HEPES, pH 7.5, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 0.1 mm PMSF, and 5 μg/ml each aprotinin and leupeptin) containing 250 mm sucrose, and homogenized 25 strokes on ice with a Dounce homogenizer. Nuclei and unbroken cells were removed by centrifugation at 1,000 × g for 10 min at 4 °C, and the supernatants centrifuged again at 14,000 × gfor 20 min at 4 °C. The resulting supernatant was used as the soluble cytosolic fraction. Equal amounts of lysate were separated by NuPAGE gel (4–12%), transferred to polyvinylidene difluoride membranes, and subsequently probed with anti-cytochromec. JNK activity was measured by an immunocomplex kinase assay as described previously (10Wang X. Martindale J.L. Liu Y. Holbrook N.J. Biochem. J. 1998; 333: 291-300Crossref PubMed Scopus (689) Google Scholar). In brief, cells were lysed in 1 ml of lysis buffer (20 mm Hepes, pH 7.4, 2 mm EGTA, 50 mm β-glycerol phosphate, 1% Triton X-100, 10% glycerol, 1 mm DTT, 1 mmPMSF, 1 mm Na3VO4, 5 mmNaF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). Equal amounts of protein samples were immunoprecipitated at 4 °C for 4 h with 5 μl of anti-JNK1 antibody with the addition of 35 μl of 50% slurry protein A-Sepharose. The beads were washed three times each in lysis buffer and kinase assay buffer (20 mm MOPS, pH 7.2, 2 mm EGTA, 10 mm MgCl2, 1 mm dithiothreitol, and 0.1% Triton X-100). JNK kinase assays were performed using GST-c-Jun-(1–135) as a substrate. ERK and p38 MAPK activations were determined by Western blot analysis using anti-phospho-ERK and anti-phospho-p38 MAPK antibodies. CDDP treatment results in apoptosis of many different cell types. To examine the ability of CDDP to induce apoptosis in HeLa cells, cultures were treated with various doses of the agent for 24 h, after which they were stained with DAPI and examined microscopically. As shown in Fig. 1 A (left panel), CDDP caused apoptosis of HeLa cells in a dose-dependent manner, with a concentration of 30 μm CDDP resulting in death of greater than 90% of the cell population by 24 h of treatment. The kinetics of CDDP-induced apoptosis were examined using a 30 μm concentration (Fig. 1 A, right panel). Morphological alterations characteristic of apoptosis were apparent within 14 h of treatment, as was cleavage of poly(ADP-ribosyl) polymerase (PARP), a biochemical feature of apoptosis that can be detected by Western blot analysis (Fig. 1 B). The ERK signaling pathway has been shown to be activated in response to certain cellular stresses. To investigate whether CDDP treatment led to ERK activation, lysates obtained at various times from CDDP-treated cells were subjected to Western blot analysis using an anti-phospho-ERK antibody to detect phosphorylated (and therefore activated) ERK (Fig. 2 A). The same blots were subsequently stripped and reprobed with an antibody that recognizes ERK2 to verify equal amounts of the protein in the various samples. As shown in the upper panel, 20 and 30 μm CDDP, both of which resulted in significant apoptosis, led to strong activation of ERK. Activation became apparent at about 6 h following treatment with 30 μmCDDP and was sustained over the following 14-h period (Fig. 2 A, lower panel). Importantly, we did not detect any acute activation of ERK (within the first hour of treatment), which frequently occurs with growth factor stimulation or treatment with oxidants (8Guyton K.Z. Liu Y. Gorospe M. Xu Q. Holbrook N.J. J. Biol. Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar, 10Wang X. Martindale J.L. Liu Y. Holbrook N.J. Biochem. J. 1998; 333: 291-300Crossref PubMed Scopus (689) Google Scholar). MEK1/2, the kinases lying directly upstream of ERK, and which are responsible for ERK activation, were also phosphorylated by CDDP treatment over the same time frame as seen for ERK (Fig. 2 B). Two specific inhibitors of MEK1/2, PD98059 and U0126, have been developed, which are highly selective in their inhibition of the ERK pathway (25Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2593) Google Scholar, 26Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3256) Google Scholar, 27Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2751) Google Scholar). These were used, therefore, to evaluate whether ERK activation is required for CDDP-induced apoptosis. HeLa cells were pretreated with various doses of PD98059 or U0126 for 30 min prior to addition of 30 μm CDDP. The morphology of cells treated with CDDP ± the MEK inhibitors is shown in Fig. 3 A. Cells treated with CDDP alone displayed typical features of apoptosis; shrinkage of the cytoplasm, membrane blebbing, and condensation of nuclei (Fig. 3 A, upper panel). Interestingly, pretreatment of cells with either PD98059 or U0126 markedly suppressed these morphologic changes induced by CDDP (Fig. 3 A,upper panel). Staining of the cells with DAPI further confirmed these morphological findings (Fig. 3 A,lower panel). Quantitation of apoptotic cells, summarized in Fig. 3 B, demonstrated that the protective influence of the MEK inhibitors was dose-dependent and occurred with doses expected to suppress ERK activation. That PD98059 and U0126 do indeed prevent activation of ERK in response to CDDP treatment is shown in Fig. 3 C. CDDP induces apoptosis in a variety of cell types. To determine if the anti-apoptotic effect of MEK1/2 inhibitors against CDDP in HeLa cells also occurs in other cell types, A549 lung carcinoma cells were examined for their responsiveness to CDDP treatment. As seen with HeLa cells, CDDP treatment resulted in apoptosis of A549 cells (Fig. 4 A). Pretreatment of these cells with U0126 significantly reduced CDDP-induced apoptosis, although the inhibitory effect was not as great as that seen in HeLa cells (Fig. 4 A). To evaluate the role of the ERK pathway in the induction of apoptosis following other stresses, HeLa cells were pretreated with the 60 μm PD98059 prior to their exposure to several stimuli including UVC (30 J/m2), hydrogen peroxide (600 μm), etoposide (VP16; 50 μm), and doxorubicin (DOX; 2 μm). We found no inhibitory effect of PD98059 on apoptosis occurring in response to any of these treatments (Fig. 4 B). In fact, PD98059 pretreatment enhanced both H2O2- and DOX-induced apoptosis. That ERK was indeed activated in response to UVC, VP16, and DOX treatment and could be inhibited in the presence of the MEK inhibitor is shown in thebottom panel. We have previously reported such findings for H2O2 (10Wang X. Martindale J.L. Liu Y. Holbrook N.J. Biochem. J. 1998; 333: 291-300Crossref PubMed Scopus (689) Google Scholar). These results are consistent with our previous studies supporting a pro-survival role for ERK during oxidant injury (8Guyton K.Z. Liu Y. Gorospe M. Xu Q. Holbrook N.J. J. Biol. Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar, 10Wang X. Martindale J.L. Liu Y. Holbrook N.J. Biochem. J. 1998; 333: 291-300Crossref PubMed Scopus (689) Google Scholar). Thus, activation of the ERK pathway participates in the induction of apoptosis by CDDP, but not that occurring with other stresses. JNK and p38 activities increase upon CDDP treatment in other cell types. Accordingly, we examined their activities in HeLa cells following exposure to CDDP for various lengths of time. JNK activation was assessed both by measuring its kinase activity using an immunocomplex kinase assay with GST-c-Jun-(1–135) fusion protein as a substrate, and by examining its degree of phosphorylation by Western blot analysis with anti-phospho-JNK1/2 polyclonal antibody. The activation state of p38 was likewise determined based on its degree of phosphorylation. Total JNK and p38 protein levels were monitored using antibodies capable of recognizing both phosphorylated and unphosphorylated forms of the proteins. As shown in Fig. 5 A, both JNK and p38 were activated in response to CDDP treatment. Although p38 activation occurred over the same time frame seen for ERK, activation of JNK was somewhat delayed. To investigate the functional consequences of JNK activation, we examined the CDDP responsiveness of HeLa cells stably expressing a dominant negative mutant form of SEK1 (SEK1-DN), a kinase that contributes largely to JNK activation during conditions of stress. We have previously shown that these SEK1-DN-expressing cells show attenuated JNK activation and reduced apoptosis in response to H2O2 treatment (10Wang X. Martindale J.L. Liu Y. Holbrook N.J. Biochem. J. 1998; 333: 291-300Crossref PubMed Scopus (689) Google Scholar). As shown in Fig. 5 B, SEK1-DN-expressing HeLa cell lines did not differ from vector control HeLa cells in their sensitivity to CDDP. To investigate the influence of p38 activation on survival of CDDP-treated cells, we utilized the pharmacologic agents SB202190 and SB203580, which act as specific inhibitors of p38 activity. We have demonstrated that the concentrations of SB202190 and SB203580 used in the present study result in complete inhibition of p38 kinase activity in HeLa cells (10Wang X. Martindale J.L. Liu Y. Holbrook N.J. Biochem. J. 1998; 333: 291-300Crossref PubMed Scopus (689) Google Scholar). As shown in Fig. 5 C, treatment of HeLa cells with these agents during exposure to CDDP did not alter the outcome. Taken together, these results indicate that neither JNK nor p38 plays a role in regulating CDDP-induced apoptosis of HeLa cells. If the activation of ERK plays an important role in mediating apoptosis of CDDP-treated cells, then agents capable of stimulating ERK activity, when combined with CDDP treatment, should potentiate apoptosis. To address this possibility, cells were treated with CDDP in the presence of the phorbol ester TPA. Numerous studies have indicated that TPA is a strong activator of the ERK signaling pathway (7He H. Wang X. Gorospe M. Holbrook N.J. Trush M.A. Cell Growth Differ. 1999; 10: 307-315PubMed Google Scholar, 28Thomas S.M. DeMarco M. D'Arcangelo G. Halegoua S. Brugge J.S. Cell. 1992; 68: 1031-1040Abstract Full Text PDF PubMed Scopus (503) Google Scholar, 29Troppmair J. Bruder J.T. Munoz H. Lloyd P.A. Kyriakis J. Banerjee P. Avruch J. Rapp U.R. J. Biol. Chem. 1994; 269: 7030-7035Abstract Full Text PDF PubMed Google Scholar, 30El-Shemerly M.Y. Besser D. Nagasawa M. Nagamine Y. J. Biol. Chem. 1997; 272: 30599-30602Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) at concentrations that do not alter JNK activities in HeLa cells (31Kaneki M. Kharbanda S. Pandey P. Yoshida K. Takekawa M. Liou J.R. Stone R. Kufe D. Mol. Cell. Biol. 1999; 19: 461-470Crossref PubMed Scopus (66) Google Scholar, 32Zhou H. Lin A. Gu Z. Chen S. Park N.H. Chiu R. J. Biol. Chem. 2000; 275: 22868-22875Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). 2X. Wang, and N. J. Holbrook, unpublished observations.. Cells were pre-incubated with or without 50 nm TPA for 1 h, followed by addition of 20 μm CDDP. The cells were evaluated for apoptosis 12 and 24 h later. When administered alone, TPA was not toxic for HeLa cells (Fig. 6 A). However, TPA-pretreated cells were much more sensitive to CDDP. This effect was attenuated by co-treatment with the MEK inhibitor U0126. To confirm that the apoptosis-enhancing effect of TPA was related to its ability to activate ERK, Western blot analysis was used to assess ERK phosphorylation (Fig. 6 B). TPA treatment alone significantly increased the level of phosphorylated ERK, with near maximum ERK activation achieved with 6 h of TPA treatment. Addition of CDDP did not appreciably alter this level. Furthermore, Fig. 6 Bshows that significant PARP cleavage was observed at 12 h of CDDP treatment when cells were sensitized with TPA, earlier than observed for CDDP alone (Fig. 1 B). However, as seen for CDDP-treated cells, TPA-induced ERK activation and PARP cleavage was significantly inhibited by the presence of U0126. These results suggest that the ability of TPA to enhance CDDP-induced apoptosis of HeLa cells is mediated through activation of the ERK signaling pathway. Previous findings from our laboratory and others have provided evidence that growth factor receptors (GFR) are important in initiating the activation of the ERK signaling pathway in response to certain stresses (33Zanella C.L. Posada J. Tritton T.R. Mossman B.T. Cancer Res. 1996; 56: 5334-5338PubMed Google Scholar, 34Rao G.N. Oncogene. 1996; 13: 713-719PubMed Google Scholar, 35Chen W. Martindale J.L. Holbrook N.J. Liu Y. Mol. Cell. Biol. 1998; 18: 5178-5188Crossref PubMed Scopus (140) Google Scholar). To test the possibility that growth factor receptors are involved in mediating CDDP-induced apoptosis in HeLa cells, we examined the ability of the broad spectrum growth factor receptor inhibitor suramin to prevent ERK activation and inhibit apoptosis of CDDP-treated cells. As shown, suramin markedly reduced the level of ERK phosphorylation occurring in response to CDDP treatment (Fig. 7 A) and significantly inhibited CDDP-induced apoptosis as assessed both by DAPI staining (Fig. 7 B) and PARP cleavage (Fig. 7 C). Given that a variety of oxidants have been found to activate ERK through growth factor signaling pathways, and the finding above that growth factor receptor signaling pathways appear to participate in ERK activation by CDDP, we investigated whether oxidative stress contributes to the apoptotic effects of CDDP. The influence of two antioxidants, N-acetylcysteine (NAC) and dimethyl sulfoximide (Me2SO), were tested. Treatment of cells with 1 mm NAC completely protected cells against CDDP-induced apoptosis. Me2SO likewise inhibited apoptosis in a dose-dependent manner (Fig. 8 A). It is important to note that Me2SO is a commonly employed solvent and indeed was used for preparation of our stock MEK1/2 inhibitor solutions. However, the concentrations used in MEK1/2 inhibitor solutions (0.1%) are lower than those shown here to inhibit ERK activation. In keeping with the ability of NAC to prevent apoptosis as assessed by DAPI staining, the antioxidant markedly inhibited ERK activation and completely prevented PARP cleavage (Fig. 8 B). Recent studies have indicated that cytochrome c participates in activating the cell death program (36Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4463) Google Scholar, 37Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 38Cai J. Yang J. Jones D.P. Biochim. Biophys. Acta. 1998; 1366: 139-149Crossref PubMed Scopus (639) Google Scholar). Cytochrome c normally resides in mitochondria, but is released into the cytoplasm following exposure of cells to certain stresses. In the cytoplasm it binds to Apaf-1, resulting in the activation of caspase-9 and downstream caspases such as caspase-3 (39Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar, 40Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6239) Google Scholar, 41Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1799) Google Scholar). Therefore, we sought to investigate whether cytochrome c release occurred in response to CDDP treatment, and if so, to determine whether it was dependent on ERK activation. Cells were treated with CDDP for different times in the presence or absence of PD98059 (60 μm) or U0126 (20 μm), after which cytosolic extracts were prepared as described under “Experimental Procedures,” and cytochromec protein levels were measured by immunoblotting. As shown in Fig. 9, cytochrome c levels in the cytoplasm increased in response to CDDP treatment, and this was correlated with cleavage of both caspase-3 and PARP. These processes were all markedly inhibited in the presence of the MEK inhibitors, particularly U0126, as was ERK activation (Fig. 3 C). These findings indicate that ERK acts" @default.
- W2021736277 created "2016-06-24" @default.
- W2021736277 creator A5006152899 @default.
- W2021736277 creator A5050400980 @default.
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- W2021736277 date "2000-12-01" @default.
- W2021736277 modified "2023-09-30" @default.
- W2021736277 title "Requirement for ERK Activation in Cisplatin-induced Apoptosis" @default.
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