Inhibition of Extracellular Signal-regulated Protein Kinase or c-Jun N-terminal Protein Kinase Cascade, Differentially Activated by Cisplatin, Sensitizes Human Ovarian Cancer Cell Line
1999; Elsevier BV; Volume: 274; Issue: 44 Linguagem: Inglês
10.1074/jbc.274.44.31648
ISSN1083-351X
AutoresJun Hayakawa, Masahide Ohmichi, Hirohisa Kurachi, Hiromasa Ikegami, Akiko Kimura, Tetsu Matsuoka, Hiroaki Jikihara, Dan Mercola, Yuji Murata,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoWe have studied the roles of c-Jun N-terminal protein kinase (JNK) and extracellular signal-regulated protein kinase (ERK) cascade in both the cisplatin-resistant Caov-3 and the cisplatin-sensitive A2780 human ovarian cancer cell lines. Treatment of both cells with cisplatin but not transplatin isomer activates JNK and ERK. Activation of JNK by cisplatin occurred at 30 min, reached a plateau at 3 h, and declined thereafter, whereas activation of ERK by cisplatin showed a biphasic pattern, indicating the different time frame. Activation of JNK by cisplatin was maximal at 1000 μm, whereas activation of ERK was maximal at 100 μm and was less at higher concentrations, indicating the different dose dependence. Cisplatin-induced JNK activation was neither extracellular and intracellular Ca2+- nor protein kinase C-dependent, whereas cisplatin-induced ERK activation was extracellular and intracellular Ca2+- dependent and protein kinase C-dependent. A mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, PD98059, had no effect on the cisplatin-induced JNK activity, suggesting an absence of cross-talk between the ERK and JNK cascades. We further examined the effect of each cascade on the viability following cisplatin treatment. Either exogenous expression of dominant negative c-Jun or the treatment by PD98059 induced sensitivity to cisplatin in both cells. Our findings suggest that cisplatin-induced DNA damage differentially activates JNK and ERK cascades and that inhibition of either of these cascades sensitizes ovarian cancer cells to cisplatin. We have studied the roles of c-Jun N-terminal protein kinase (JNK) and extracellular signal-regulated protein kinase (ERK) cascade in both the cisplatin-resistant Caov-3 and the cisplatin-sensitive A2780 human ovarian cancer cell lines. Treatment of both cells with cisplatin but not transplatin isomer activates JNK and ERK. Activation of JNK by cisplatin occurred at 30 min, reached a plateau at 3 h, and declined thereafter, whereas activation of ERK by cisplatin showed a biphasic pattern, indicating the different time frame. Activation of JNK by cisplatin was maximal at 1000 μm, whereas activation of ERK was maximal at 100 μm and was less at higher concentrations, indicating the different dose dependence. Cisplatin-induced JNK activation was neither extracellular and intracellular Ca2+- nor protein kinase C-dependent, whereas cisplatin-induced ERK activation was extracellular and intracellular Ca2+- dependent and protein kinase C-dependent. A mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, PD98059, had no effect on the cisplatin-induced JNK activity, suggesting an absence of cross-talk between the ERK and JNK cascades. We further examined the effect of each cascade on the viability following cisplatin treatment. Either exogenous expression of dominant negative c-Jun or the treatment by PD98059 induced sensitivity to cisplatin in both cells. Our findings suggest that cisplatin-induced DNA damage differentially activates JNK and ERK cascades and that inhibition of either of these cascades sensitizes ovarian cancer cells to cisplatin. mitogen-activated protein cis-diaminodichloroplatinum trans-diaminodichloroplatinum extracellular signal-regulated (protein) kinase c-Jun N-terminal protein kinase dominant negative c-Jun polyacrylamide gel electrophoresis protein kinase C phorbol-12-myristate, 13-acetate mitogen-activated protein kinase/extracellular signal-regulated kinase kinase epidermal growth factor Various cellular stimuli that control cell growth and differentiation cause a rapid increase in the enzymatic activity of a family of serine/threonine kinases known as the mitogen-activated protein (MAP)1 kinase family. The MAP kinase family has been classified into three subfamilies: extracellular signal-regulated protein kinases (ERKs), including ERK1 and ERK2 also known as p44MAPK and p42MAPK, respectively; stress-activated protein kinases, also termed c-Jun N-terminal protein kinases (JNKs), including JNK1 of 46 kDa and JNK2 of 55 kDa; and p38 kinase, a homolog of the yeast high osmolarity glycerol response-1 kinase (1Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (997) Google Scholar). ERKs phosphorylate and activate the transcription factor p62TCF/Elk-1, which forms a part of the ternary complex that regulates the transcriptional activity of the c-Fos promoter serum response element or SRE (2Gille H. Kortenjann M. Thomae O. Moomaw C. Slaughter C. Cobb M.H. Shaw P.E. EMBO J. 1995; 14: 951-962Crossref PubMed Scopus (585) Google Scholar, 3Marais R. Wynne J. Treisman R. Cell. 1993; 73: 381-393Abstract Full Text PDF PubMed Scopus (1104) Google Scholar). In contrast, JNKs phosphorylate two sites of the N-terminal transactivating domain of c-Jun (Ser-63 and Ser-73), ATF-2, and Elk-1, thereby increasing their transcriptional activity (4Gupta S. Campbell D. Dérijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1336) Google Scholar). Recent data suggest that JNK is activated in response to cellular stress induced by certain DNA-damaging agents, including UV-C (5Dérijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2953) Google Scholar, 6Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1708) Google Scholar, 7Adler V. Fuchs S., Y. Kim J. Kraft A. King M., P. Pelling J. Ronai Z. Cell Growth Differ. 1995; 6: 1437-1446PubMed Google Scholar), ionizing radiation (8Macgregor P.E. Abate C. Curran T. 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Chem. 1996; 271: 30950-30955Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar),N-methyl-N′-nitro-N-nitrosoguanidine (5Dérijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2953) Google Scholar), 1-β-d-arabinofuranosylcytosine (12Kharbanda S. Pandey P. Ren R. Mayer B. Zon L. Kufe D. J. Biol. Chem. 1995; 270: 30278-30281Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), and hydrogen peroxide (13Yu R. Jiao J. Duh J. Tan T.H. Kong A. Cancer Res. 1996; 56: 2954-2959PubMed Google Scholar). These observations suggest that the JNK cascade may mediate a physiological response to DNA damage such as induction of one or more DNA repair enzymes (10Potapova 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 (219) Google Scholar). However, the effect of certain DNA-damaging agents on ERK cascade remains unclear. In this study, we sought to determine whether JNK and/or ERK play a role in the cellular stress response to the chemotherapeutic agent cisplatin, which damages DNA through the formation of bifunctional platinum adducts using both Caov-3 human ovarian cancer cells, which are resistant to cisplatin, and A2780 human ovarian cancer cells, which are sensitive to cisplatin. Here, we provide evidence that cisplatin, but not transplatin, which does not readily damage DNA (14Sherman S.E. Gibson D. Wang A.H. Lippard S.J. Science. 1985; 230: 412-417Crossref PubMed Scopus (416) Google Scholar, 15Zamble D.B. Lippard S.J. Trends Biochem. Sci. 1995; 20: 435-439Abstract Full Text PDF PubMed Scopus (478) Google Scholar), activates both JNK and ERK with different kinetics. Moreover, inhibition of both the JNK cascade and ERK cascade markedly decreased the cell viability following treatment with cisplatin but not with transplatin. Thus, both JNK and ERK are activated by cisplatin-induced DNA damage and are required for cell survival following cisplatin treatment. Phorbol-12-myristate, 13-acetate (PMA) was purchased from Sigma. Sturosporine was purchased from Calbiochem. Hygromycin was purchased from Wako Pure Chemical Industries (Tokyo, Japan). ECL Western blotting detection reagents were obtained from Amersham Pharmacia Biotech. [γ-32P]ATP (3000 Ci/mmol) was obtained from NEN Life Science Products. Anti-phosphotyrosine (PY20) and mouse monoclonal anti-ERK antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Rabbit polyclonal anti-ERK1 antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). PD98059 and the stress-activated protein kinase/JNK assay kit, including N-terminal c-Jun fusion protein bound to glutathione-Sepharose beads and a phosphospecific c-Jun antibody, were obtained from New England Biolabs (Beverly, MA). The cell Titer 96 cell proliferation assay was obtained from Promega (Madison, WI). Human ovarian papillary adenocarcinoma cell line (Caov-3) was obtained from American Type Culture Collection (Manassas, VA). Human ovarian cancer A2780 cell line derived from a patient prior to treatment was kindly provided by Dr. T. Tsuruo (Institute of Molecular and Cellular Biosciences, Tokyo, Japan) and Drs. R. F. Ozols and T. C. Hamilton (NCI, National Institutes of Health, Bethesda, MD) (16Hamilton T.C. Winker M.A. Louie K.G. Batist G. Behrens B.C. Tsuruo T. Grotzinger K.R. McKoy W.M. Young R.C. Ozols R.F. Biochem. Pharmacol. 1985; 34: 2583-2586Crossref PubMed Scopus (547) Google Scholar, 17Louite K.G. Hamilton T.C. Winker M.A. Behrens B.C. Tsuruo T. 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After 3 weeks, several clones were isolated using cloning rings. Selected clones were then maintained in medium supplemented with hygromycin (100 μg/ml), and only low passage cells (p < 10) were used for the experiments described here. Cell viability (20Gjerset R. Turla S. Sobol R. Scalise J. Mercola D. Collins H. Isabella P. Mol. Carcinogen. 1995; 14: 275-285Crossref PubMed Scopus (100) Google Scholar) was assessed by the addition of cisplatin or transplatin for 1 h 1 day after seeding test cells into 96-well plates followed by a change of medium to fresh medium. The number of surviving cells was determined 5 days later by determination of A 590 nm of the dissolved formazan product after the addition of MTS for 1 h as described by the manufacturer (Promega). All experiments were carried out in quadruplicate, and the viability is expressed as the ratio of the number of viable cells with cisplatin or transplatin treatment to that without treatment. Cells were incubated in the absence of serum for 16 h and then treated with various agents. They were then washed twice with phosphate-buffered saline and lysed in ice-cold HNTG buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EDTA, 10 mm sodium pyrophosphate, 100 μm sodium orthovanadate, 100 mm NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mmphenylmethylsulfonyl fluoride) (21Ohmichi M. Matuoka K. Takenawa T. Saltiel A.R. J. Biol. Chem. 1994; 269: 1143-1148Abstract Full Text PDF PubMed Google Scholar). The extracts were centrifuged to remove cellular debris, and the protein content of the supernatants was determined using the Bio-Rad protein assay reagent (Bio-Rad). 300 μg of protein from the lysate samples was used for immunoprecipitation by treatment with ERK1 rabbit polyclonal antibody at 4 °C for 2 h. The immunoprecipitated products were washed once in HNTG buffer; twice in 0.5 m LiCl, 0.1 m Tris, pH 8.0; and once in kinase assay buffer (25 mm HEPES, pH 7.2–7.4, 10 mm MgCl2, 10 mm MnCl2, and 1 mm dithiothreitol), and the samples were resuspended in 30 μl of kinase assay buffer containing 10 μg of myelin basic protein and 40 μm [γ-32P]ATP (1 μCi) as described previously (22Ohmichi M. Koike K. Kimura A. Masuhara K. Ikegami H. Ikebuchi Y. Kanzaki T. Touhara K. Sakaue M. Kobayashi Y. Akabane M. Miyake A. Murata Y. Endocrinology. 1997; 138: 5275-5281Crossref PubMed Scopus (44) Google Scholar). The kinase reaction was allowed to proceed at room temperature for 5 min and stopped by the addition of Laemmli SDS sample buffer (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206597) Google Scholar). Reaction products were resolved by 15% SDS-PAGE. For analysis of tyrosine phosphorylation of ERK, cells were grown in 60-mm dishes. After treatment, the cells were washed, and then 100 μl of 1% SDS was added. Lysates were heated for 5 min at 100 °C and diluted 1:10 with ice-cold HNTG buffer, followed by incubation with anti-ERK2 monoclonal antibody. Immune complexes were precipitated with protein A-Sepharose, and the isolated proteins were analyzed by electrophoresis on 8% SDS-PAGE. Transfer to nitrocellulose, Western analysis with anti-phosphotyrosine antiserum, and washing were performed as described elsewhere (21Ohmichi M. Matuoka K. Takenawa T. Saltiel A.R. J. Biol. Chem. 1994; 269: 1143-1148Abstract Full Text PDF PubMed Google Scholar). Cells were incubated in the absence of serum for 16 h and then treated with various materials. They were then washed twice with phosphate-buffered saline and lysed in ice-cold lysis buffer (20 mm HEPES, pH 7.4, 150 mm NaCl, 1% Triton X-100, 1.5 mmMgCl2, 1 mm EDTA, 1 mm EGTA, 2.5 mm sodium pyrophosphate, 1 mmβ-glycerolphosphate, 1 mm sodium orthovanadate, 1 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride). The extracts were centrifuged to remove cellular debris, and the protein content of the supernatants was determined using the Bio-Rad protein assay reagent. 250 μg of protein from the lysate samples was incubated at 4 °C overnight with the N-terminal c-Jun-(1–89)-glutathione S-transferase fusion protein bound to glutathione-Sepharose beads in order to selectively precipitate JNK from cell lysates. c-Jun-(1–89) contains a high affinity binding site for JNK, close to the N terminus, which is the two phosphorylation sites at Ser63 and Ser73 (5Dérijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2953) Google Scholar, 24Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2411) Google Scholar, 25Dai T. Rubie E. Franklin C.C. Kraft A. Gillespie D.A. Avruch J. Kyriakis J.M. Woodgett J.R. Oncogene. 1995; 10: 849-855PubMed Google Scholar, 26Kallunki T. Su B. Tsigelny I. Sluss H.K. Dérijard B. Moore G. Davis R. Karin M. Genes Dev. 1994; 8: 2996-3007Crossref PubMed Scopus (593) Google Scholar). After selectively precipitating JNK using the c-Jun fusion protein beads, the beads were washed to remove nonspecifically bound proteins, and then the kinase reaction was carried out in the presence of cold ATP, and samples were resolved on 12% SDS-gel electrophoresis followed by Western blotting with a phosphospecific c-Jun antibody. This antibody specifically recognizes JNK-induced phosphorylation of c-Jun at Ser63, a site important for c-Jun-dependent transcriptional activity (5Dérijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2953) Google Scholar, 24Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2411) Google Scholar, 25Dai T. Rubie E. Franklin C.C. Kraft A. Gillespie D.A. Avruch J. Kyriakis J.M. Woodgett J.R. Oncogene. 1995; 10: 849-855PubMed Google Scholar, 26Kallunki T. Su B. Tsigelny I. Sluss H.K. Dérijard B. Moore G. Davis R. Karin M. Genes Dev. 1994; 8: 2996-3007Crossref PubMed Scopus (593) Google Scholar). To evaluate whether JNK is activated by cisplatin in Caov-3 or A2780 human ovarian cancer cell, cultured cells were exposed to cisplatin for the indicated times (Fig.1 A) and at the indicated concentrations for 3 h (Fig. 1 B). Cell lysates were incubated with glutathione S-transferase-c-Jun fusion protein, followed by precipitation and Western analysis using anti-phospho-c-Jun antibody. The activation of JNK by cisplatin in Caov-3 cells was detectable at 1 h, reached a broad plateau from 3 through 24 h, and declined thereafter (Fig. 1 A,upper panel). The activation of JNK by cisplatin in A2780 cells was also detected at 1 h, reached a plateau at 3 h, and declined thereafter (Fig. 1 A, lower panel). Cisplatin induced the activation of JNK in a dose-dependent manner in Caov-3 (Fig. 1 B,upper panel) and A2780 cells (Fig. 1 B,lower panel). It is known that cisplatin but not transplatin forms covalent cross-links between the N-7 position of adjacent guanine or adenine-guanine residues (14Sherman S.E. Gibson D. Wang A.H. Lippard S.J. Science. 1985; 230: 412-417Crossref PubMed Scopus (416) Google Scholar, 15Zamble D.B. Lippard S.J. Trends Biochem. Sci. 1995; 20: 435-439Abstract Full Text PDF PubMed Scopus (478) Google Scholar). The treatment by transplatin at the same concentrations had no apparent effect on JNK activation, whereas cisplatin induced JNK activation in Caov-3 (Fig.1 C) and A2780 (data not shown) cells. These results indicate that only the DNA-damaging cisplatin isomer activates JNK activity in both types of cells. We next examined the effect of cisplatin on the activation of ERK, which is a member of the MAP kinase family. Cultured cells were exposed to cisplatin for the indicated times (Fig.2 A) and at the indicated concentrations for 30 min (Fig. 2 B). Cell lysates were immunoprecipitated with anti-ERK antibody and examined for ERK activity by assaying the incorporation of 32P into myelin basic protein. The cisplatin-dependent increase in ERK activity displayed a biphasic time course; the activity reached a maximum at 30 min, rapidly declined, increased again after 3 h of cisplatin stimulation, and declined thereafter in Caov-3 (Fig. 2 A,upper panel) and A2780 (Fig. 2 A,lower panel) cells. Cisplatin-induced activation of ERK was maximal at 100 μm and declined at higher concentrations in Caov-3 (Fig. 2 B) and A2780 (data not shown) cells. Treatment by transplatin had no effect on ERK activation, whereas in parallel experiments cisplatin induced strong ERK activation in Caov-3 (Fig. 2 C) and A2780 (data not shown) cells. Mitogenic stimuli activate ERK by increasing tyrosine and serine or threonine phosphorylation of the protein due to the activity of dual specificity MEK (27Kosako H. Gotoh Y. Matsuda S. Ishikawa W. Nishida E. EMBO J. 1992; 11: 2903-2908Crossref PubMed Scopus (146) Google Scholar). Therefore, the cisplatin-dependent tyrosine phosphorylation of the predominant form of ERK was evaluated by antiphosphotyrosine Western analysis using the anti-ERK immunoprecipitates. The Caov-3 cells were treated with cisplatin, transplatin, or EGF followed by lysis and evaluation of tyrosine phosphorylation of ERK (Fig. 2 D). Both cisplatin and EGF produced an increase in tyrosine phosphorylation of ERK, whereas transplatin had no effect. These results indicate that only the DNA-damaging cisplatin isomer again activates ERK activity and tyrosine phosphorylation. We examined an upstream mediator in the cascade of the cisplatin-induced activation of ERK. Treatment with 3 mmEGTA for 15 min to eliminate extracellular Ca2+ and intracellular Ca2+ (28William R.H. Ruth C.D. Shelton E.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8837-8841Crossref PubMed Scopus (73) Google Scholar) attenuated the cisplatin-induced activation of ERK in Caov-3 (Fig.3 A, lanes 2 and 8) and A2780 (Fig. 3 B,left panel, lanes 2 and3) cells. Moreover, the treatment with 1 μmPMA for 24 h to down-regulate protein kinase C or 1 μm staurosporine for 10 min attenuated both cisplatin- and PMA-induced activation of ERK in Caov-3 (Fig. 3 A,lanes 2–7) and A2780 (Fig. 3 B,right panel, lanes 2–5) cells. Thus, cisplatin-induced ERK activation is extracellular Ca2+- and intracellular Ca2+-dependent and is also protein kinase C-dependent. We next examined an upstream mediator in the cascade of the cisplatin-induced activation of JNK. Treatment with 3 mm EGTA for 15 min to eliminate extracellular Ca2+ and intracellular Ca2+ had no effect on the cisplatin-induced activation of JNK in Caov-3 and A2780 cells (Fig.4, lanes 2,4, 6, and 8). Moreover, treatment with 1 μm staurosporine for 10 min to inhibit protein kinase C had no effect on cisplatin-induced activation of JNK in Caov-3 and A2780 cells (Fig. 4, lanes 2, 3,6, and 7). Thus, cisplatin-induced JNK activation is neither extracellular and intracellular Ca2+-dependent nor protein kinase C-dependent. These results suggest that the mechanism of cisplatin-induced activation of JNK is different from that of cisplatin-induced activation of ERK.Figure 4The role of Ca2+ and PKC in the activation of JNK by cisplatin . Cells were grown in 100-mm dishes. Extracellular and intracellular Ca2+ was chelated by a 15-min incubation with 3 mm EGTA (lane 3), or PKC was inhibited by a 10-min incubation with 1 μm staurosporine (lane 4). The cells were treated with 1000 μm cisplatin for 3 h (lanes 2–4 and 6–8), and the activity of JNK was measured as described in the legend of Fig. 1. The experiments were repeated three times with essentially identical results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm that cisplatin differentially activates JNK and ERK, the effect of a MEK inhibitor, PD98059, on the activation was tested in Caov-3 (Fig. 5) and A2780 (data not shown). This compound is relatively specific for MEK with no inhibitory activity against a number of other serine/threonine and tyrosine kinases (29Dudley 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 (2586) Google Scholar, 30Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1996; 270: 13585-13588Abstract Full Text Full Text PDF Scopus (895) Google Scholar, 31Lazar D.F. Wiese R.J. Brady M.J. Mastick C.C. Waters S.B. Yamauchi K. Pessin J.E. Cuartrecasas P. Saltiel A.R. J. Biol. Chem. 1996; 270: 20801-20807Abstract Full Text Full Text PDF Scopus (330) Google Scholar). Although the MEK inhibitor (100 μm) largely repressed the ERK activation induced by cisplatin for 30 min (Fig. 5 A) or 3 h (data not shown), this compound had no apparent effect on cisplatin-induced JNK activation (Fig. 5 B), suggesting that the activation of JNK by cisplatin is independent of the activation of ERK. To rule out the possibility that the second phase of ERK activation is caused by JNK activation, we examined whether ERK is activated by cisplatin in clonal lines of Caov-3 cells, which stably expressed a dominant negative inhibitor (32Smeal T. Binétruy B. Mercola D. Birrer M. Karin M. Nature. 1991; 354: 494-496Crossref PubMed Scopus (697) Google Scholar, 33Smeal T. Binétruy B. Mercola D. Bardwick-Grover A. Heidecker G. Rapp U. Karin M. Mol. Cell. Biol. 1992; 12: 3507-3513Crossref PubMed Scopus (304) Google Scholar) of the JNK cascade, dnJun. (Fig. 5 C). The dnJun mutant cannot be phosphorylated at the N-terminal serine residues due to substitution of serines 63 and 73 by alanine, thereby blocking the enhanced transactivation promoted by JNK-dependent phosphorylation of these sites (32Smeal T. Binétruy B. Mercola D. Birrer M. Karin M. Nature. 1991; 354: 494-496Crossref PubMed Scopus (697) Google Scholar, 33Smeal T. Binétruy B. Mercola D. Bardwick-Grover A. Heidecker G. Rapp U. Karin M. Mol. Cell. Biol. 1992; 12: 3507-3513Crossref PubMed Scopus (304) Google Scholar). Thus, dnJun blocks c-Jun phosphorylation-dependent events of the JNK cascade (18Potapova O. Fakrai H. Baird S. Mercola D. Cancer Res. 1996; 56: 2800-2806Google Scholar,32Smeal T. Binétruy B. Mercola D. Birrer M. Karin M. Nature. 1991; 354: 494-496Crossref PubMed Scopus (697) Google Scholar, 33Smeal T. Binétruy B. Mercola D. Bardwick-Grover A. Heidecker G. Rapp U. Karin M. Mol. Cell. Biol. 1992; 12: 3507-3513Crossref PubMed Scopus (304) Google Scholar). 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The ERK activity induced by cisplatin for 3 h in dnJun-expressing cells appeared to be similar to that in an empty vector-expressing cells (Fig. 5 C), supporting the differential activation of ERK and JNK cascades. The effect of cisplatin treatment on the viability of a representative dnJun-expressing clonal line was compared with that of an empty vector-expressing control line (Fig.6 A). The viability of the control Caov-3 cells remained unaffected by increasing concentrations of cisplatin to >100 μm. Extended titrations revealed IC50 values of 380 and 412 μm for the parental cells and empty vector-expressing control lines, respectively (Table I). In contrast, the dnJun-expressing cells exhibited an IC50 as low as 50 μm or over 7.6-fold more sensitive to cisplatin than the control cells (Fig. 6 A, Table I). Transplatin had no discernible effect on the dnJun-expressing line at concentrations where the viability following treatment with cisplatin was less than 20% (Fig. 6 B). In extended titrations, no significant effect was observed with transplatin even at 250 μm, indicating that the requirement for sensitization by dnJun depends upon the stereospecific DNA-binding properties of cisplatin, consistent with the results in the activation of JNK. Expression of wild-type c-Jun did not affect the sensitivity to cisplatin, compared with the control line (Fig. 6 A). Thus, the sensitization to cisplatin observed in the dnJun-expressing cells appeared to be due to the interference with the activation of c-Jun by JNK.Table ISensitization of CA-OV3 or A2780 to cisplatin-induced cytotoxicityControlaParental and empty vector cells were analyzed in parallel and with equal concentrations of cisplatin and transplatin in the range 0–1000 μm all in quadruplicate. Transplatin had no effect on viability of any cell.dnJun-expressing IC50Cisplatin sensitizationbSensitization is defined by the ratio of IC50 values for the parental cells to IC50 values of the dnJun-expressing cells. (parental IC50/dnJun IC50)IC50μmμmCaov-3Parental380 ± 2550 ± 27.60Empty vector pLHCX412 ± 33A2780Parental84 ± 420 ± 24.20IC50 values were determined by direct titration of viability with cisplatin as described under "Experimental Procedures."a Parental and empty vector cells were analyzed in parallel and with equal concentrations of cisplatin and transplatin in the range 0–1000 μm all in quadruplicate. Transplatin had no effect on viability of any cell.b Sensitization is defined by the ratio of IC50 values for the parental cells to IC50 values of the dnJun-expressing cells. Open table in a new tab IC50 values were determined by direct titration of viability with cisplatin as described under "Experimental Procedures." We next examined whether the ERK cascade is also required for the cell viability following cisplatin treatment of Caov-3 cells. The cells pretreated with PD98059 exhibited an IC50as low as 39 μm, or over 9.7-fold more sensitive to cisplatin than the untreated cells (Fig.7 A). Transplatin had no discernible effect (Fig. 7 B), indicating that the requirements for sensitization by PD98059 also depends upon the stereospeci
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