Title: Sustained Activation of JNK/p38 MAPK Pathways in Response to Cisplatin Leads to Fas Ligand Induction and Cell Death in Ovarian Carcinoma Cells
Abstract: The efficacy of cisplatin in cancer chemotherapy is limited by the development of resistance. Although the molecular mechanisms involved in chemoresistance are poorly understood, cellular response to cisplatin is known to involve activation of MAPK and other signal transduction pathways. An understanding of early signal transduction events in the response to cisplatin could be valuable for improving the efficacy of cancer therapy. We compared cisplatin-induced activation of three MAPKs, JNK, p38, and ERK, in a cisplatin-sensitive human ovarian carcinoma cell line (2008) and its resistant subclone (2008C13). The JNK and p38 pathways were activated differentially in response to cisplatin, with the cisplatin-sensitive cells showing prolonged activation (8–12 h) and the cisplatin-resistant cells showing only transient activation (1–3 h) of JNK and p38. In the sensitive cells, inhibition of cisplatin-induced JNK and p38 activation blocked cisplatin-induced apoptosis; persistent activation of JNK resulted in hyperphosphorylation of the c-Jun transcription factor, which in turn stimulated the transcription of an immediate downstream target, the death inducer Fas ligand (FasL). Sequestration of FasL by incubation with a neutralizing anti-FasL antibody inhibited cisplatin-induced apoptosis. In contrast, chemoresistance in 2008C13 cells was associated with failure to up-regulate FasL. Moreover, in these cells, selective stimulation of the JNK/p38 MAPK pathways by adenovirus-mediated delivery of recombinant MKK7 or MKK3 led to sensitization to apoptosis through reactivating FasL expression. Thus, the JNK > c-Jun > FasL > Fas pathway plays an important role in mediating cisplatin-induced apoptosis in ovarian cancer cells, and the duration of JNK activation is critical in determining whether cells survive or undergo apoptosis. The efficacy of cisplatin in cancer chemotherapy is limited by the development of resistance. Although the molecular mechanisms involved in chemoresistance are poorly understood, cellular response to cisplatin is known to involve activation of MAPK and other signal transduction pathways. An understanding of early signal transduction events in the response to cisplatin could be valuable for improving the efficacy of cancer therapy. We compared cisplatin-induced activation of three MAPKs, JNK, p38, and ERK, in a cisplatin-sensitive human ovarian carcinoma cell line (2008) and its resistant subclone (2008C13). The JNK and p38 pathways were activated differentially in response to cisplatin, with the cisplatin-sensitive cells showing prolonged activation (8–12 h) and the cisplatin-resistant cells showing only transient activation (1–3 h) of JNK and p38. In the sensitive cells, inhibition of cisplatin-induced JNK and p38 activation blocked cisplatin-induced apoptosis; persistent activation of JNK resulted in hyperphosphorylation of the c-Jun transcription factor, which in turn stimulated the transcription of an immediate downstream target, the death inducer Fas ligand (FasL). Sequestration of FasL by incubation with a neutralizing anti-FasL antibody inhibited cisplatin-induced apoptosis. In contrast, chemoresistance in 2008C13 cells was associated with failure to up-regulate FasL. Moreover, in these cells, selective stimulation of the JNK/p38 MAPK pathways by adenovirus-mediated delivery of recombinant MKK7 or MKK3 led to sensitization to apoptosis through reactivating FasL expression. Thus, the JNK > c-Jun > FasL > Fas pathway plays an important role in mediating cisplatin-induced apoptosis in ovarian cancer cells, and the duration of JNK activation is critical in determining whether cells survive or undergo apoptosis. Cisplatin (cis-diamminedichloroplatinum(II) (CDDP) 1The abbreviations used are: CDDP, cisplatin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MEK, MAPK/ERK kinase; FasL, Fas ligand; ATF-2, activating transcription factor-2; PARP, poly(ADP-ribose) polymerase; Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethyl ketone; Ad, adenovirus; GFP, green fluorescent protein; MKK, mitogen-activated protein kinase kinase; PBS, phosphate-buffered saline; GST, glutathione S-transferase; DAPI, 4,6-diamidino-2-phenylindole; HA, hemagglutinin; MEKK, MEK kinase.) is a platinum-based compound that forms intra- and interstrand adducts with DNA (1Reed J.C. Curr. Opin. Oncol. 1999; 11: 68-75Crossref PubMed Scopus (342) Google Scholar, 2Kelland L.R. Farreell N.P. Platimium-based Drugs in Cancer Therapy. Humana Press Inc., Totowa, NJ2000Crossref Google Scholar). CDDP has a broad spectrum of anti-tumor activity and is widely used in the treatment of solid tumors (3Lippert B. Cisplatin: Chemistry and Biochemistry of a Leading Anticancer Drug. Wiley-VCH, New York1999Crossref Scopus (714) Google Scholar). However, one of the major limitations in its efficacy is that many tumors either are inherently resistant or acquire resistance after an initial response (1Reed J.C. Curr. Opin. Oncol. 1999; 11: 68-75Crossref PubMed Scopus (342) Google Scholar, 2Kelland L.R. Farreell N.P. Platimium-based Drugs in Cancer Therapy. Humana Press Inc., Totowa, NJ2000Crossref Google Scholar, 3Lippert B. Cisplatin: Chemistry and Biochemistry of a Leading Anticancer Drug. Wiley-VCH, New York1999Crossref Scopus (714) Google Scholar, 4Andrews P.A. Howell S.B. Cancer Cells. 1990; 2: 35-43PubMed Google Scholar). The molecular mechanisms that underlie this chemoresistance are largely unknown. Possible mechanisms of acquired resistance to CDDP include decreased platinum accumulation, elevated drug inactivation by metallothionine and glutathione, and enhanced DNA repair activity (5Kelley S.L. Basu A. Teicher B.A. Hacker M.P. Hamer D.H. Lazo J.S. Science. 1988; 241: 1813-1815Crossref PubMed Scopus (610) Google Scholar, 6Cohen S.M. Lippard S.J. Prog. Nucleic Acids Res. Mol. Biol. 2001; 67: 93-130Crossref PubMed Scopus (555) Google Scholar). Increased expression of anti-apoptotic genes and mutations in the intrinsic apoptotic pathway may contribute to the inability of cells to detect DNA damage or to induce apoptosis (1Reed J.C. Curr. Opin. Oncol. 1999; 11: 68-75Crossref PubMed Scopus (342) Google Scholar, 7Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1363) Google Scholar, 8Herr I. Debatin K.M. Blood. 2001; 98: 2603-2614Crossref PubMed Scopus (688) Google Scholar, 9Niedner H. Christen R. Lin X. Kondo A. Howell S.B. Mol. Pharmacol. 2001; 60: 1153-1160Crossref PubMed Scopus (121) Google Scholar). Because of the reactivity of CDDP and the complexity of the cellular response to DNA damage, CDDP-induced apoptotic signaling likely involves several pathways. Elucidation of the details of these signaling pathways is important because they may explain why tumor cells exposed to cisplatin often lose sensitivity to this agent and become resistant to apoptotic signals. Genotoxic stress induces multiple signal transduction pathways, among which are the MAPK pathways. These pathways are parallel cascades of structurally related serine/threonine kinases that play pivotal roles in transducing various extracellular signals to the nucleus. The MAPK signaling cascades regulate a variety of cellular activities, including cell growth, differentiation, survival, and death (10Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4420) Google Scholar, 11Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar). In mammals, MAPKs are divided into three major groups, ERKs, JNKs/stress-activated protein kinases, and p38, based on their degree of homology, biological activities, and phosphorylation motifs (12Cobb M.H. Prog. Biophys. Mol. Biol. 1999; 71: 479-500Crossref PubMed Scopus (762) Google Scholar). Even though these signaling systems are built from evolutionarily related protein kinases, they produce distinct biological responses. The biological effects of MAPK signaling are executed by phosphorylation of downstream substrates, most notably a number of signal-responsive transcription factors. The broad range of these substrates indicates that MAPKs have pivotal roles in cellular signal transduction and suggests that the extent and duration of MAPK activation play key roles in controlling cell functions. The ERK pathway, which is induced in response to mitogenic stimuli such as peptide growth factors, cytokines, and phorbol esters, involves ERK1 and ERK2, the participation of Raf-1 and Ras oncoproteins, and the activation of MEK1/2 (12Cobb M.H. Prog. Biophys. Mol. Biol. 1999; 71: 479-500Crossref PubMed Scopus (762) Google Scholar). Once activated, ERK phosphorylates several substrates, including Elk-1 (13Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1165) Google Scholar). The ERK pathway plays a major role in regulating cell proliferation and differentiation (12Cobb M.H. Prog. Biophys. Mol. Biol. 1999; 71: 479-500Crossref PubMed Scopus (762) Google Scholar) and provides a protective effect against apoptosis (14Holmstrom T.H. Schmitz I. Soderstrom T.S. Poukkula M. Johnson V.L. Chow S.C. Krammer P.H. Eriksson J.E. EMBO J. 2000; 19: 5418-5428Crossref PubMed Scopus (168) Google Scholar). On the other hand, the signaling cascades involving JNK and p38 are key mediators of stress signals and seem to be responsible mainly for protective responses, stress-dependent apoptosis, and inflammatory responses. These cascades can be stimulated by various stresses such as UV and γ-irradiation, osmotic stress, and heat shock; pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-1β and chemotherapeutic drugs (10Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4420) Google Scholar, 12Cobb M.H. Prog. Biophys. Mol. Biol. 1999; 71: 479-500Crossref PubMed Scopus (762) Google Scholar). To understand the molecular basis for the failure of CDDP-based chemotherapy, we compared the cellular responses of the human ovarian carcinoma cell line 2008 and its resistant subclone 2008C13 (15Andrews P.A. Velury S. Mann S.C. Howell S.B. Cancer Res. 1988; 48: 68-73PubMed Google Scholar) after treatment with a platinum-based anticancer agent. We found that differences in the duration of the activation of MAPK pathways correlated with CDDP-induced apoptosis. A strong sustained activation of both pathways seemed to be a required priming step for CDDP-induced apoptosis; this activation of both JNK and p38 MAPK in CDDP-sensitive cells correlated with up-regulation of the Fas ligand (FasL), an immediate downstream target of JNK, and was accompanied by the induction of caspase activity and apoptosis. The failure of cisplatin to elicit such a response in the resistant variant indicates that impaired FasL expression could contribute to the development of chemoresistance. Reduction of cisplatin-induced apoptosis by the expression of dominant-negative c-Jun lacking JNK phosphoacceptor sites or by the use of either a small drug inhibitor of JNK/p38 or a neutralizing anti-FasL antibody further underlines the critical role of c-Jun-dependent FasL expression signaling in the induction of apoptosis by genotoxic agents. Reagents—Cisplatin (Platinol-AQ cisplatin injection) was obtained from Bristol-Myers Squibb Co. Polyclonal antibodies to p38, phospho-p38 (Thr180/Tyr182), ATF-2, phospho-ATF-2 (Thr71), JNK, phospho-JNK (Thr183/Tyr185), c-Jun, phospho-c-Jun (Ser73), ERK, and phospho-ERK (Thr202/Tyr204) were purchased from Cell Signaling (Beverly, MA). The anti-JNK1 monoclonal antibody (clone 333.8), anti-human PARP antibody, anti-caspase-8 antibody, anti-human cytochrome c monoclonal antibody, neutralizing anti-human FasL antibody (NOK-2), and isotype-matched control antibody were obtained from Pharmingen. The anti-Fas monoclonal antibody (CH-11) was purchased from Medical & Biological Laboratories (Watertown, MA). The anti-capase-3/CPP32 antibody was purchased from Transduction Laboratories (Lexington, KY). Anti-β-actin monoclonal antibodies were obtained from Sigma. The caspase inhibitor Z-VAD-fmk and the JNK and p38 kinase inhibitor SB202190 were purchased from Alexis Biochemicals (San Diego, CA). CDDP-sensitive (2008) and CDDP-resistant (2008C13) ovarian cancer cells were kindly provided by Drs. S. B. Howell (University of California at San Diego, La Jolla, CA), S. G. Chaney (University of North Carolina, Chapel Hill, NC), and Z. H. Siddik (M. D. Anderson Cancer Center). The 2008 cell line, established from a patient with serous cystadenocarcinoma of the ovary, and its resistant subclone 2008C13, derived from 2008 cells by in vitro exposure to CDDP, have been characterized by Howell and co-workers (15Andrews P.A. Velury S. Mann S.C. Howell S.B. Cancer Res. 1988; 48: 68-73PubMed Google Scholar) and Chaney and co-workers (16Delmastro D.A. Li J. Vaisman A. Solle M. Chaney S.G. Cancer Chemother. Pharmacol. 1997; 39: 245-253PubMed Google Scholar). Wild-type c-jun and c-jun–/– 3T3 fibroblasts were a gift from Drs. E. F. Wagner (Research Institute for Molecular Pathology, Vienna, Austria) and M. Karin (University of California at San Diego). Cell Culture and Adenoviral Infection—The CDDP-sensitive human ovarian carcinoma cell line 2008 and its resistant variant 2008C13 were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (Invitrogen) and 1% penicillin/streptomycin. Wild-type c-jun and c-jun–/– 3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented as described above. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Recombinant adenoviral vectors expressing green fluorescent protein (Ad-GFP) and activated mutants of MKK7 and MKK3 (Ad-MKK7D and Ad-MKK3bE) were constructed as previously described (17Wang Y. Su B. Sah V.P. Brown J.H. Han J. Chien K.R. J. Biol. Chem. 1998; 273: 5423-5426Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Cells were infected with adenoviruses at a multiplicity of infection of 50 plaque-forming units/cell for 5 h and then incubated for another 30 h to allow expression of the protein of interest as described (17Wang Y. Su B. Sah V.P. Brown J.H. Han J. Chien K.R. J. Biol. Chem. 1998; 273: 5423-5426Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Immunoblot Analysis—Cells in log-phase growth were treated or not treated with CDDP at 20 μm (2008 and 2008C13 cells) or 100 μm (wild-type c-jun and c-jun–/– 3T3 cells) for 1 h, after which they were washed, and fresh medium was added. At various times after CDDP exposure (1 min and 1, 3, 5, 8, and 12 h), the cells were collected and lysed in lysis buffer (25 mm HEPES, pH 7.7, 400 mm NaCl, 0.5% Triton X-100, 1.5 mm MgCl2, 2 mm EDTA, 2 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, protease inhibitors (10 μg/ml leupeptin, 2 μg/ml pepstatin, 50 μg/ml antipain, 2 μg/ml aprotinin, 20 μg/ml chymostatin, and 2 μg/ml benzamidine), and phosphatase inhibitors (50 mm NaF, 0.1 mm Na3VO4, and 20 mm β-glycerophosphate)). For PARP and caspase immunoblotting, cell lysates were prepared using radioimmune precipitation assay lysis buffer (50 mm Tris, pH 7.5, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 1 μg/ml pepstatin, and 2 μg/ml leupeptin). Aliquots of cell lysates (70 μg of protein) were resolved by 10–12% SDS-PAGE, transferred onto polyvinylidene difluoride membrane (Immobilon, Millipore Corp., Bedford, MA) or Hy-bond-P membrane (Amersham Biosciences), and probed with the appropriate primary antibodies. Reactions were visualized with a suitable secondary antibody conjugated with horseradish peroxidase (Bio-Rad) using enhanced chemiluminescence reagents (Amersham Biosciences). Drug Uptake and Adduct Level Assays—Cells were treated with 20 μm CDDP, after which cells were washed with phosphate-buffered saline (PBS), and fresh medium was added. Cell pellets were made immediately after 1 min and 1 h of CDDP exposure. After 1 h of drug exposure, cells were washed and cultured in drug-free medium for an additional 4 h. For protein analysis, cells were digested overnight in 0.2 n NaOH at 55–60 °C. Intracellular platinum levels were determined by solubilizing the cell pellet in Hyamine hydroxide and analyzed by flameless atomic absorption spectrophotometry using conditions previously described (detection limit = 100 pg of platinum) (18Siddik Z.H. Boxall F.E. Harrap K.R. Anal. Biochem. 1987; 163: 21-26Crossref PubMed Scopus (56) Google Scholar, 19Yoshida M. Khokhar A.R. Siddik Z.H. Cancer Res. 1994; 54: 3468-3473PubMed Google Scholar). For platinum adduct levels, cell pellets were lysed in extraction buffer (10 mm Tris, pH 8.0, 100 mm EDTA, 20 μg/ml RNase, and 0.5% SDS) overnight at 37 °C and then treated with proteinase K (100 μg/ml) for 3 h at 50 °C, and the DNA was extracted in phenol/chloroform. The amount of platinum bound to DNA was determined by flameless atomic absorption spectrophotometry. Immunocomplex Kinase Assays—Cells were serum-starved in 0.1% serum for 12–16 h before CDDP treatment. Whole cell extracts were prepared and treated as previously described (20Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1710) Google Scholar). Briefly, endogenous JNK1 was immunoprecipitated from 300 μg of cell lysate with the anti-JNK1 monoclonal antibody (clone 333.8) and protein A-agarose beads for 4 h at 4 °C. The precipitates were washed twice with lysis buffer and twice with kinase buffer (25 mm Hepes, pH 7.6, 20 mm MgCl2,20mm β-glycerophosphate, 0.1 mm sodium orthovanadate, 2 mm DTT). JNK kinase activity was measured using 2 μg of glutathione S-transferase (GST)-c-Jun-(1–79) as the substrate, and the reaction was initiated by the addition of 10 μm ATP and 10 μCi of [γ-32P]ATP (5000 Ci/mmol; ICN Biomedicals, Aurora, OH). After the cells were incubated for 30 min at 30 °C, the reactions were stopped with Laemmli sample buffer. The proteins were resolved by 12% SDS-PAGE and visualized by autoradiography. Cell Proliferation Assay—Cell proliferation was assessed in 96-well plates after cells had been treated with CDDP for 1 h, washed to remove the drug, and left to proliferate for the indicated times after the addition of fresh medium. The number of surviving cells was measured by nucleic acid staining with the CyQUANT cell proliferation assay kit (Molecular Probes, Inc., Eugene, OR) 4–5 days after seeding. The assay was conducted according to the manufacturer's instructions. The samples were analyzed on a Fluoroskan Ascent CF microplate fluorometer (ThermoLabSystems, Helsinki, Finland). All experiments were carried out in quadruplicate, and the proliferation rate was expressed as the ratio of the number of proliferating cells treated with CDDP to the number of proliferating cells not treated with CDDP. Flow Cytometry Analysis—To measure DNA content (apoptotic nuclei), cells were harvested; washed with PBS; fixed in 1% paraformaldehyde; stained with a solution containing 15 μg/ml propidium iodide, 0.5% Tween 20, and 0.1% RNase A; and incubated for 30 min at 24 °C. Cells were sorted using a FACScan (BD Biosciences) and analyzed with CELLQuest Version 3.3 software. Data were plotted on a logarithmic scale. Detection of Fas and FasL mRNA Expression by Reverse Transcriptase-PCR—Total RNA was isolated from the 2008 and 2008C13 cell lines using the RNeasy minikit (QIAGEN Inc., Valencia, CA) according to the instructions of the manufacturer. The reverse transcriptase assay was performed with 2 μg of total RNA using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's recommendations. A reaction without reverse transcriptase was performed in parallel to ensure the absence of genomic DNA contamination. PCR amplification was carried out in a final volume of 50 μl containing 5 μl of cDNA, 5 μl of 10× PCR buffer (10 mm Tris-HCl, pH 9, 50 mm KCl, and 0.1% Triton X-100), 0.5 μl of dNTP (10 μm), 3 μl of MgCl2 (25 mm), and 2.5 units of AmpliTaq Gold (PerkinElmer Life Sciences). PCR conditions were as follows: an initial denaturation step at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, either 57 °C (for Fas) or 61 °C (for FasL and β-actin) for 30 s, and 72 °C for 30 s. After a final extension at 72 °C for 5 min, PCR products were resolved on 1.2% agarose gels and visualized by ethidium bromide transillumination under UV light. Primer sequences were as follows: Fas, 5′-ATT TCT GCC ACT GCA GCC CTC AGG-3′ (forward) and 5′-TCC AGT TCG CTG GGC AGA CTT CTC-3′ (reverse); and FasL, 5′-ATG TTT CAG CTC TTC CAC CTA CAG A-3′ (forward) and 5′-CCA GAG AGA GCT CAG ATA CGT TGA C-3′ (reverse). These sequences span nucleotides 76–706 of Fas cDNA and nucleotides 365–856 of FasL cDNA and yield PCR products of 630 and 492 bp, respectively (21Eichhorst S.T. Muller M. Li-Weber M. Schulze-Bergkamen H. Angel P. Krammer P.H. Mol. Cell. Biol. 2000; 20: 7826-7837Crossref PubMed Scopus (112) Google Scholar). Each reverse-transcribed mRNA product was internally controlled by β-actin PCR using primers 5′-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3′ (forward) and 5′-CTA GAA TTT GCG GTC GAC GAT GGA GGG-3′ (reverse), covering region 2199–3065 of β-actin cDNA and yielding a 867-bp PCR product (21Eichhorst S.T. Muller M. Li-Weber M. Schulze-Bergkamen H. Angel P. Krammer P.H. Mol. Cell. Biol. 2000; 20: 7826-7837Crossref PubMed Scopus (112) Google Scholar). The FasL and Fas reverse transcriptase-PCR products were subsequently confirmed by direct sequencing. Propidium Iodide and 4,6-Diamidino-2-phenylindole (DAPI) Staining—To detect apoptosis, nuclear staining was performed using 5 μg/ml DAPI, and cells were analyzed with a fluorescence microscope (magnification ×400 for nuclear analysis and ×100 for morphologic analysis). Apoptotic cells were identified by morphology and by condensation and fragmentation of their nuclei. The percentage of apoptotic cells was calculated as the ratio of apoptotic cells to total cells counted, multiplied by 100. Three separate experiments were conducted, and at least 300 cells were counted for each experiment. Transfection and Immunofluorescence Staining—Expression vectors for hemagglutinin (HA) epitope-tagged wild-type c-Jun (pSRα-HA-c-Jun) and dominant-negative c-Jun (pSRα-HA-c-Jun(S63A/S73A)) were a gift from Dr. M. Karin. Liposome-mediated transfection was performed using LipofectAMINE Plus (Invitrogen). Briefly, the 2008 ovarian carcinoma cells were grown on chamber slides and transfected with vectors containing the HA epitope tag. After transfection, the cells were washed with PBS and fixed in methanol for 10 min at –20 °C, after which they were air-dried, washed three times with PBS, blocked in 1.5% bovine serum albumin in PBS (PBS/bovine serum albumin) for 1 h at room temperature, and then immunostained with a monoclonal antibody to HA (1:50 dilution in PBS/bovine serum albumin) for 1 h at room temperature. After three washes with PBS, transfected cells were visualized by incubation with a fluorescein isothiocyanate-conjugated rabbit anti-mouse antibody (1:40 dilution in PBS/bovine serum albumin; Dako, Carpinteria, CA) for 45 min at 37 °C. To visualize the nuclei of transfected cells, we included DAPI (5 μg/ml) in the wash after the incubation with the secondary antibody. Cells were examined and photographed with an Olympus microscope equipped for epifluorescence with the appropriate filters. Transfected cells were scored blindly for apoptosis. Detection of Cytochrome c Release—Cytosol extracts were prepared from the 2008 and 2008C13 cells essentially as previously described (22Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Crossref PubMed Scopus (1107) Google Scholar). Briefly, after cisplatin treatment and incubation for 6, 12, 18, and 24 h, the cells were collected by centrifugation. The cell pellet was washed twice with cold PBS and resuspended in ice-cold buffer A (20 mm HEPES, pH 7.5, 1.5 mm MgCl2, 10 mm KCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 μg/ml pepstatin A) containing 250 mm sucrose. The cells were homogenized with 25 strokes of a Dounce homogenizer with a type B pestle. Nuclei and intact cells were cleared by centrifugation at 1000 × g for 10 min at 4 °C. The supernatant was centrifuged at 14,000 × g for 20 min at 4 °C to pellet the mitochondrial fraction. An aliquot of the resulting supernatant was used as the soluble cytosolic fraction. The mitochondrial pellet was washed once and then suspended in buffer A. Protein extracts (equal amounts in the mitochondrial and cytosolic fractions) were subjected to Western blot analysis with a monoclonal antibody to cytochrome c. CDDP-induced Apoptosis in Ovarian Carcinoma Cells— CDDP-sensitive 2008 cells and CDDP-resistant 2008C13 cells were exposed to CDDP for 1 h, after which the drug was washed out to mimic in vivo chemotherapy. The time course for the induction of apoptosis was determined by microscopic examination of DAPI-stained cells (Fig. 1). In the chemosensitive 2008 cells, exposure to 20 μm CDDP resulted in morphologic alterations characteristic of apoptosis, including membrane blebbing, nuclear condensation and fragmentation (Fig. 1A), and DNA laddering (data not shown). The number of apoptotic cells increased with time and accounted for 50–70% of the total cell population by 18–24 h. The CDDP-resistant 2008C13 cells, in contrast, had a markedly different apoptotic response to this “pulsed” exposure to CDDP (Fig. 1B). Immunoblot analysis revealed cleavage of the pro form of caspase-3 (32 kDa) to its active form (17 kDa), compatible with the induction of apoptosis, from 12 to 48 h after CDDP treatment in 2008 cells, but not in 2008C13 cells (Fig. 1C). PARP cleavage products also persisted from 12 to 48 h in CDDP-treated 2008 cells as detected by immunoblot analysis, whereas in the 2008C13 cell extracts, no PARP cleavage fragments were detected, which correlated with caspase-3 (Fig. 1C). Immunoblotting with an anti-β-actin antibody was used as a loading control. This finding reflected the resistance of 2008C13 cells to CDDP-induced apoptosis. To study the mechanism behind this CDDP resistance, we first studied drug uptake and DNA adduct formation in both the sensitive and resistant 2008 cells (Table I). As shown, drug uptake and DNA adduct formation were similar in the two cell lines at 1 min after CDDP treatment, whereas at 1 and 5 h after treatment, the differences between the CDDP-sensitive and CDDP-resistant cells were <2-fold, with the resistant cells showing a lower value of DNA adduct formation and drug uptake. Resistance to CDDP in 2008C13 cells is >2-fold (15Andrews P.A. Velury S. Mann S.C. Howell S.B. Cancer Res. 1988; 48: 68-73PubMed Google Scholar, 16Delmastro D.A. Li J. Vaisman A. Solle M. Chaney S.G. Cancer Chemother. Pharmacol. 1997; 39: 245-253PubMed Google Scholar), which prompted us to investigate additional mechanisms of resistance.Table ICDDP uptake and CDDP-DNA adduct formation in 2008 and 2008C13 cellsTimeaAfter initiation of CDDP treatment.1 min1 h5 hUptake (ng Pt/mg protein)200816.14 ± 1.9781.88 ± 1.6136.81 ± 0.942008C1315.84 ± 0.9844.59 ± 2.3126.90 ± 0.03DNA adducts (ng Pt/mg DNA)20086.89 ± 0.64147.03 ± 3.4174.22 ± 1.032008C136.06 ± 0.1369.29 ± 2.7655.12c ± 1.52a After initiation of CDDP treatment. Open table in a new tab Differential Activation of MAPK Pathways by CDDP in Sensitive Versus Resistant Cell Lines—Because activation of MAPKs and phosphorylation of c-Jun have been reported after treatment with chemotherapeutic drugs in other cell types (23Pandey P. Raingeaud J. Kaneki M. Weichselbaum R. Davis R.J. Kufe D. Kharbanda S. J. Biol. Chem. 1996; 271: 23775-23779Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 24Liu Z.G. Baskaran R. Lea-Chou E.T. Wood L.D. Chen Y. Karin M. Wang J.Y. Nature. 1996; 384: 273-276Crossref PubMed Scopus (347) Google Scholar, 25Zanke 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, 26Sanchez-Perez I. Murguia J.R. Perona R. Oncogene. 1998; 16: 533-540Crossref PubMed Scopus (222) Google Scholar, 27Potapova 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), we compared the activation of JNK between CDDP-sensitive and CDDP-resistant ovarian cancer cells after treatment with cisplatin. The activity of immunoprecipitated JNK was assayed using a GST-c-Jun fusion protein as substrate. CDDP treatment of the sensitive 2008 cells induced an increase in the ability of the JNKs to phosphorylate the GST-c-Jun substrate, beginning 1 h after treatment and persisting through the next 3–5 h (Fig. 2A). On the other hand, extracts from CDDP-treated resistant 2008C13 cells showed only transient JNK activity after 1 h of treatment, and this activity declined rapidly over the next 3–5 h (Fig. 2A). Next, we investigated the effect of CDDP on the phosphorylation of JNK and p38 as well as that of their respective target substrates, the c-Jun and ATF-2 transcription factors, over time. CDDP treatment of the 2008 cells, which resulted in significant apoptosis, led to sustained activation (from 1 min to 12 h after treatment) of JNK and p38, as assessed by their phosphorylation states using specific antibodies that recognize the phosphorylated (activated) forms of theses enzymes (Fig. 2B). p38 MAPK activation occurred over the same period as JNK activation. Although phosphorylation was detected very early (at 1 min), maximal phosphorylation of bo