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• W2006354480 abstract "We investigated the molecular mechanism of the synergism between interferon γ (IFNγ) and tumor necrosis factor α (TNFα) documented in a variety of biological occasions such as tumor cell death and inflammatory responses. IFNγ/TNFα synergistically induced apoptosis of ME-180 cervical cancer cells. IFNγ induced STAT1 phosphorylation and interferon regulatory factor 1 (IRF-1) expression. Transfection of phosphorylation-defective STAT1 inhibited IFNγ/TNFα-induced apoptosis, whereas IRF-1 transfection induced susceptibility to TNFα. Dominant-negative IκBα transfection sensitized ME-180 cells to TNFα. IFNγ pretreatment attenuated TNFα- or p65-induced NF-κB reporter activity, whereas it did not inhibit p65 translocation or DNA binding of NF-κB. IRF-1 transfection alone inhibited TNFα-induced NF-κB activity, which was reversed by coactivator p300 overexpression. Caspases were activated by IFNγ/TNFα combination; however, caspase inhibition did not abrogate IFNγ/TNFα-induced cell death. Instead, caspase inhibitors directed IFNγ/TNFα-treated ME-180 cells to undergo necrosis, as demonstrated by Hoechst 33258/propidium iodide staining and electron microscopy. Taken together, our results indicate that IFNγ and TNFα synergistically act to destroy ME-180 tumor cells by either apoptosis or necrosis, depending on caspase activation, and STAT1/IRF-1 pathways initiated by IFNγ play a critical role in IFNγ/TNFα synergism by inhibiting cytoprotective NF-κB. IFNγ/TNFα synergism appears to activate cell death machinery independently of caspase activation, and caspase activation seems to merely determine the mode of cell death. We investigated the molecular mechanism of the synergism between interferon γ (IFNγ) and tumor necrosis factor α (TNFα) documented in a variety of biological occasions such as tumor cell death and inflammatory responses. IFNγ/TNFα synergistically induced apoptosis of ME-180 cervical cancer cells. IFNγ induced STAT1 phosphorylation and interferon regulatory factor 1 (IRF-1) expression. Transfection of phosphorylation-defective STAT1 inhibited IFNγ/TNFα-induced apoptosis, whereas IRF-1 transfection induced susceptibility to TNFα. Dominant-negative IκBα transfection sensitized ME-180 cells to TNFα. IFNγ pretreatment attenuated TNFα- or p65-induced NF-κB reporter activity, whereas it did not inhibit p65 translocation or DNA binding of NF-κB. IRF-1 transfection alone inhibited TNFα-induced NF-κB activity, which was reversed by coactivator p300 overexpression. Caspases were activated by IFNγ/TNFα combination; however, caspase inhibition did not abrogate IFNγ/TNFα-induced cell death. Instead, caspase inhibitors directed IFNγ/TNFα-treated ME-180 cells to undergo necrosis, as demonstrated by Hoechst 33258/propidium iodide staining and electron microscopy. Taken together, our results indicate that IFNγ and TNFα synergistically act to destroy ME-180 tumor cells by either apoptosis or necrosis, depending on caspase activation, and STAT1/IRF-1 pathways initiated by IFNγ play a critical role in IFNγ/TNFα synergism by inhibiting cytoprotective NF-κB. IFNγ/TNFα synergism appears to activate cell death machinery independently of caspase activation, and caspase activation seems to merely determine the mode of cell death. The pleiotropic proinflammatory cytokine TNFα1 exerts a wide variety of biological activities such as induction of septic shock, activation of local inflammatory responses, and fever generation as an endogenous pyrogen (1Fiers W. FEBS Lett... 1991; 285: 199-212Google Scholar). TNFα also kills various tumor cell lines in vitro and mediates anti-tumor effect in vivo (2Beyaert R. Fiers W. FEBS Lett... 1994; 340: 9-16Google Scholar). TNFα exerts its biological effects by binding to two types of cell surface receptors with molecular masses of 55 kDa (p55) and 75 kDa (p75). TNFα cytotoxicity is mostly mediated by p55 receptors (3Schulze-Osthoff K. Ferrari D. Los M. Wesselborg S. Peter M.E. Eur. J. Biochem... 1998; 254: 439-459Google Scholar). After the ligation of p55 receptors, a canonical apoptotic signal transduction pathway is initiated. The cytoplasmic death domain of p55 receptor interacts with the death domain of intracellular adapter molecules such as TRADD (TNF receptor-associated death domain protein) and FADD (Fas-associated death domain protein), which leads to the activation of initiator caspases (4Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell.. 1996; 84: 299-308Google Scholar). This, in turn, triggers the caspase cascade and ultimately results in apoptotic cell death. In many cases, the anti-tumor effect of TNFα was enhanced by IFNγ (5Sugarman B.J. Aggarwal B.B. Hass P.E. Figari I.S. Palladino Jr., M.A. Shepard H.M. Science.. 1985; 230: 943-945Google Scholar) or metabolic inhibitors such as cycloheximide and actinomycin D (6Kirstein M. Fiers W. Baglioni C. J. Immunol... 1986; 137: 2277-2280Google Scholar). Although these metabolic inhibitors are believed to block the synthesis of cytoprotective proteins, the effects of IFNγ might be mediated by the induction of new proteins that increase the sensitivity of target cells to TNFα. IFNγ/TNFα synergism also has been reported in biological responses other than tumor cell killing. For instance, the two cytokines synergistically up-regulated the expression of numerous genes, including ICAM-1 (intercellular adhesion molecule 1), IP-10, and major histocompatibility complex class I heavy chain (7Ohmori Y. Hamilton T.A. J. Immunol... 1995; 154: 5235-5244Google Scholar, 8Johnson D.R. Pober J.S. Mol. Cell. Biol... 1994; 14: 1322-1332Google Scholar, 9Collins T. Read M.A. Neish A.S. Whitley M.Z. Thanos D. Maniatis T. FASEB J... 1995; 9: 899-909Google Scholar). However, the molecular mechanism of the synergism between the two cytokines is not clearly understood. It has been reported that IFNγ increases the expression of TNFα receptors (10Ruggiero V. Tavernier J. Fiers W. Baglioni C. J. Immunol... 1986; 136: 2445-2450Google Scholar). However, because the sensitivity of the cells to TNFα is not simply correlated with the level of TNFα receptor expression (11Tsujimoto M. Feinman R. Vilcek J. J. Immunol... 1986; 137: 2272-2276Google Scholar, 12Aggarwal B.B. Eessalu T.E. J. Biol. Chem... 1987; 262: 10000-10007Google Scholar), up-regulation of TNFα receptor alone does not adequately explain the cytokine synergism in the anti-tumor action. In the current work, we utilized ME-180 human cervical cancer cells to investigate the molecular mechanism of synergistic anti-tumor effects of IFNγ/TNFα. We also studied the role of caspase activation in ME-180 cell death by IFNγ/TNFα synergism. Our results indicate that 1) IRF-1 induction after STAT1 activation by IFNγ plays a central role in synergistic tumor cell death by IFNγ/TNFα, 2) IFNγ-induced IRF-1 inhibits cytoprotective NF-κB transactivation, 3) IFNγ/TNFα induces ME-180 cell death regardless of caspase activation, and caspase activation dictates only the mode of cell death between apoptosis and necrosis. ME-180 cervical cancer cell line was obtained from ATCC (Manassas, VA) and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mmglutamine, and penicillin-streptomycin (Life Technologies, Inc.). Recombinant human IFNγ was purchased from R&D Systems (Minneapolis, MN). Recombinant human TNFα was generously provided by Dr. T. H. Lee (Yonsei University, Seoul, Korea). Caspase inhibitors (z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OCH3)-CH2-fluoromethyl ketone; BD-fmk,t-butoxycarbonyl-Asp(OCH3)-CH2F; z-DEVD-fmk, benzyloxycarbonyl-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-CH2-fluoromethyl ketone; z-IETD-fmk, benzyloxycarbonyl-Ile-Glu(OCH3)-Thr-Asp(OCH3)-CH2-fluoromethyl ketone) were purchased from Enzyme Systems (Livermore CA), and cathepsin B inhibitor FA (benzyloxycarbonyl-Phe-Ala-CH2-fluoromethyl ketone) and MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinal-H, also called Z-LLL) were from Calbiochem. All other chemicals were obtained from Sigma, unless stated otherwise. Cells (3 × 104/well) were seeded in 96-well plates and treated with various combinations of cytokines for the indicated time periods. The optimal concentrations of the cytokines for the cytotoxic action were 100 units/ml for IFNγ and 10 ng/ml for TNFα. In some experiments, cells were pretreated with caspase inhibitors or MG-132 for 1 h before cytokine treatment. After cytokine treatment, the medium was removed, and MTT (0.5 mg/ml) was added, followed by incubation at 37 °C for 2 h in CO2 incubator. After a brief centrifugation, supernatants were carefully removed, and Me2SO was added. After insoluble crystals were completely dissolved, absorbance at 540 nm was measured using a Thermomax microplate reader (Molecular Devices). Results were presented as means ± S.E. (n = 3). Morphological changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with 2.5 μg/ml bisbenzimide Hoechst 33258 fluorochrome (Calbiochem), followed by examination on a fluorescence microscope. In some experiments, cytokine-treated cells were double-stained with propidium iodide (PI, 2.5 μg/ml) and Hoechst 33258 (2.5 μg/ml) to distinguish apoptotic cells from necrotic cells. Intact blue nuclei, condensed/fragmented blue nuclei, condensed/fragmented pink nuclei, and intact pink nuclei were considered viable, early apoptotic, late apoptotic, and necrotic cells, respectively (13Shimizu S. Eguchi Y. Kamiike W. Itoh Y. Hasegawa J. Yamabe K. Otsuki Y. Matsuda H. Tsujimoto Y. Cancer Res... 1996; 56: 2161-2166Google Scholar). Transmission electron microscopy was carried out essentially as previously described (13Shimizu S. Eguchi Y. Kamiike W. Itoh Y. Hasegawa J. Yamabe K. Otsuki Y. Matsuda H. Tsujimoto Y. Cancer Res... 1996; 56: 2161-2166Google Scholar). In brief, cells were fixed in 4% glutaraldehyde, 1% paraformaldehyde, 0.2 m phosphate, pH 7.2, at 4 °C for 2 h. After two washes in 0.2 mphosphate, the cell pellet was post-fixed with 2% OsO4 in the same buffer for 30 min. The pellet was dehydrated in ethanol and then in 100% propylene oxide, followed by embedding overnight at 37 °C for another 3 days at 60 °C. Ultrafine sections were cut and examined on an electron microscope (Hitachi H7100, 75 kV). Cells were suspended in phosphate-buffered saline, 5 mm EDTA and fixed by adding 100% ethanol dropwise. RNase A (40 μg/ml) was added to resuspended cells, and the incubation was carried out at room temperature for 30 min. PI (50 μg/ml) was then added for flow cytometric analyses. Caspase-3- or -8-like activity was measured using a caspase assay kit (Pharmingen, San Diego, CA) according to the supplier's instruction. In brief, caspase-3 or -8 fluorogenic substrates (Ac-DEVD-AMC or Ac-IETD-AMC) were incubated with cytokine-treated cell lysates for 1 h at 37 °C, then AMC liberated from Ac-DEVD-AMC or Ac-IETD-AMC was measured using a fluorometric plate reader with an excitation wavelength of 380 nm and an emission wavelength of 420- 460 nm. Cells were lysed in triple-detergent lysis buffer (50 mm Tris-Cl, pH 8.0, 150 mmNaCl, 0.02% sodium azide, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mm phenylmethylsulfonyl fluoride). Protein concentration in cell lysates was determined using the Bio-Rad protein assay kit. An equal amount of protein for each sample was separated by 10 or 12% SDS-polyacrylamide gel electrophoresis and transferred to Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech). The membranes were blocked with 5% skim milk and sequentially incubated with primary antibodies (rabbit anti-human IRF-1, Santa Cruz; rabbit anti-human STAT1 and anti-human phospho-STAT1, New England Biolabs) and horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG, Amersham Pharmacia Biotech), followed by ECL detection (Amersham Pharmacia Biotech). ME-180 cells in 6-well plates were co-transfected with 1 μg of human STAT1 cDNA, dominant-negative mutant STAT1 cDNA (kindly provided by Dr. Hirano, Osaka University, Japan), human IRF-1 cDNA (kindly provided by Dr. Taniguchi, University of Tokyo), or phosphorylation-defective dominant-negative mutant IκBα (14Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol... 1995; 15: 2809-2818Google Scholar) together with 0.2 μg of lacZ gene (pCH110, Amersham Pharmacia Biotech) using LipofectAMINE reagent (Life Technologies, Inc.). 48 h after the transfection, cells were treated with cytokines. After another 48 h, the cells were fixed with 0.5% glutaraldehyde for 10 min at room temperature and stained with X-gal (5-bromo-4-chloro-3-indolyl β-d-galactopyranoside; 1 mg/ml) in 4 mmpotassium ferricyanide, 4 mm potassium ferrocyanide, 2 mm magnesium chloride at 37 °C for detection of blue cells. At least 200 blue cells were counted for each experiment, and transfection efficiency was 10–35%. Results were presented as means ± S.E. (n = 3). NF-κB reporter activity was measured using the dual-luciferase reporter assay system (Promega, Madison, WI). In brief, ME-180 cells in 12-well plates were co-transfected with 0.5 μg of NF-κB-responsive reporter gene construct carrying two copies of κB sequences linked to luciferase gene (IgGκ NF-κB-luciferase, generously provided by Dr. G. D. Rosen, Stanford University, Stanford, CA) (15Lee K.-Y. Chang W. Qiu D. Kao P.N. Rosen G.D. J. Biol. Chem... 1999; 274: 13451-13455Google Scholar) together with 0.1 μg of Renilla luciferase gene under hamster sarcoma virus thymidine kinase promoter (pRL-TK, Promega) using LipofectAMINE reagent (Life Technologies, Inc.). 24 h after the transfection, cells were treated with cytokines. After 5 h, activities of firefly luciferase and Renilla luciferase in transfected cells were measured sequentially from a single sample using the dual-luciferase reporter assay system (Promega). Results were presented as firefly luciferase activity normalized to Renilla luciferase activity. In some experiments, cells were co-transfected before cytokine treatment with NF-κB p65 (16Ballard D.W. Dixon E.P. Peffer N.J. Bogerd H. Doerre S. Stein B. Greene W.C. Proc. Natl. Acad. Sci. U. S. A... 1992; 89: 1875-1879Google Scholar) or coactivator p300 expression plasmid (0.5 μg; kindly provided by Dr. Livingston, Harvard Medical School, Boston, MA) (17Ekner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev... 1994; 8: 869-884Google Scholar) along with NF-κB-responsive reporter plasmid (0.5 μg) and pRL-TK (0.1 μg). Results were presented as means ± S.E. (n = 3). ME-180 cells seeded onto chamber slides (Lab-Tek, Nalge Nunc International, Naperville, IL) were fixed in 4% paraformaldehyde for 30 min at room temperature and then in cold methanol for 10 min at −20 °C. Fixed cells were permeabilized in 0.1% Triton X-100, 0.1% sodium citrate for 3 min at 4 °C and then sequentially incubated with mouse anti-p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), biotinylated anti-mouse IgG, and streptavidin-fluorescein isothiocyanate. Stained cells were examined on a fluorescent microscope. Nuclear extracts were prepared from ME-180 cells treated with cytokines as previously described (18Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res... 1989; 17: 6419Google Scholar). Synthetic double-strand oligonucleotides of consensus NF-κB binding sequence, GAT CCC AAC GGC AGG GGA (Promega), were end-labeled with [γ-32P]ATP using T4 polynucleotide kinase. Nuclear extract was incubated with the labeled probe in the presence of poly- (dI-dC) in a binding buffer containing 20 mm HEPES at room temperature for 30 min. For supershift assays, a total of 0.2 μg of antibodies against p65 or p50 subunit of NF-κB were included in the reaction. DNA-protein complexes were resolved by electrophoresis in a 5% nondenaturing polyacrylamide gel, dried, and visualized by autoradiography. First we screened several tumor cell lines to assess their sensitivity to IFNγ/TNFα-induced cytotoxicity (data not shown). Cytotoxic synergism between IFNγ and TNFα was most evident in ME-180 cells. Although either cytokine alone exhibited no significant cytotoxicity, the combination of the two cytokines significantly reduced ME-180 cell viability (Fig.1 A). The cytokine cytotoxicity was dependent on the dose of IFNγ used. However, concentration higher than 100 units/ml did not further increase the cytotoxicity (TableI). The reduction of cell viability was due to apoptosis as demonstrated by Hoechst 33258 staining and DNA ploidy analysis. IFNγ/TNFα treatment induced nuclear condensation and fragmentation (Fig. 1 B) and led to the appearance of sub-diploid cells (Fig. 1 C), which are hallmarks of apoptotic cells. DNA ploidy assays also indicated that the effect of IFNγ/TNFα was not due to the growth arrest as was shown by the absence of decrease in the percentage of cells in the S phase.Table IDose response of cytokine cytotoxicityTreatments% Viability1-aME-180 cells were treated with increasing concentrations of IFNγ and TNFα (10 ng/ml) for 48 h, and then cell viability was assessed by MTT assays. Viability of untreated cells was set to 100%. Results are the means ± S.E. (n = 3). IFNγ alone at all concentrations tested did not have a significant cytotoxicity (data not shown). One hundred units/ml IFNγ and 10 ng/ml TNFα were the optimal concentrations for cytotoxic assays.None100IFNγ (1 unit/ml) + TNFα95.6 ± 3.6IFNγ (10 units/ml) + TNFα76.2 ± 2.5IFNγ (100 units/ml) + TNFα23.4 ± 3.1IFNγ (1000 units/ml) + TNFα24.7 ± 2.81-a ME-180 cells were treated with increasing concentrations of IFNγ and TNFα (10 ng/ml) for 48 h, and then cell viability was assessed by MTT assays. Viability of untreated cells was set to 100%. Results are the means ± S.E. (n = 3). IFNγ alone at all concentrations tested did not have a significant cytotoxicity (data not shown). One hundred units/ml IFNγ and 10 ng/ml TNFα were the optimal concentrations for cytotoxic assays. Open table in a new tab Based on our results that the combination of IFNγ and TNFα, but not either cytokine alone, induced ME-180 cell death, we explored the possibility that IFNγ sensitizes ME-180 cells to TNFα-mediated cytotoxicity. This was first tested by sequential treatment of ME-180 cells with the two cytokines. After IFNγ treatment, TNFα alone was sufficient to induce a significant cytotoxicity in ME-180 cells (Table II). However, sequential treatment with TNFα and then with IFNγ did not have the same effects, indicating that IFNγ confers susceptibility to TNFα on ME-180 cells through induction or up-regulation of certain genes in ME-180 cells. Because STAT1 and IRF-1 are known to be canonical intracellular signal-transducing molecules in IFNγ signaling, we investigated the involvement of STAT1/IRF-1-signaling pathways in IFNγ/TNFα synergism on ME-180 cell apoptosis. IFNγ, but not TNFα, induced phosphorylation of STAT1 and up-regulated IRF-1 expression in ME-180 cells (Fig. 2). Furthermore, the transfection of phosphorylation-defective dominant-negative mutant of STAT1 significantly inhibited IFNγ/TNFα-induced ME-180 cell death, indicating that IFNγ-induced STAT1 activation is critical for the induction of TNFα susceptibility (Fig. 3 A). We next asked whether IRF-1, a downstream mediator of STAT1, is responsible for the priming effects of IFNγ. Transfection of IRF-1 conferred TNFα susceptibility on ME-180 cells in a dose-dependent manner, indicating a central role for IRF-1 in the sensitization of ME-180 cells to TNFα-induced apoptosis (Fig. 3, B andC).Table IICytotoxic effects of sequential treatment of cytokinesTreatment2-aME-180 cells were treated with cytokines as indicated, either simultaneously or sequentially. Treatment with IFNγ (100 units/ml) for 24 h followed by TNFα (10 ng/ml) treatment for 48 h induced a significant cytotoxicity. However, sequential treatment with the two cytokines in a reverse order did not significantly affect ME-180 cell viability, indicating the priming role of IFNγ in TNFα-induced ME-180 cell death.% Viability2-bCell viability was assessed by MTT assays. Viability of untreated cells was set to 100%. Results are means ± S.E. (n = 3).None100IFNγ + TNFα (48 h)22.1 ± 3.1IFNγ (48 h)98.2 ± 3.4IFNγ (24 h) and then TNFα (48 h)52.3 ± 2.4TNFα (48 h)101.5 ± 2.9TNFα (24 h) and then IFNγ (48 h)90.3 ± 3.32-a ME-180 cells were treated with cytokines as indicated, either simultaneously or sequentially. Treatment with IFNγ (100 units/ml) for 24 h followed by TNFα (10 ng/ml) treatment for 48 h induced a significant cytotoxicity. However, sequential treatment with the two cytokines in a reverse order did not significantly affect ME-180 cell viability, indicating the priming role of IFNγ in TNFα-induced ME-180 cell death.2-b Cell viability was assessed by MTT assays. Viability of untreated cells was set to 100%. Results are means ± S.E. (n = 3). Open table in a new tab Figure 3A key role for STAT1/IRF-1 signaling in IFN γ/TNFα synergism. A, transient transfection of phosphorylation-defective STAT1 dominant-negative mutant (DN STAT1) significantly inhibited IFNγ/TNFα cytotoxicity, as demonstrated by counting blue cells co-expressing lacZ at 48 h after cytokine treatment (IFNγ, 100 units/ml; TNFα, 10 ng/ml).B, transfection of IRF-1 cDNA (1 μg) induced susceptibility to TNFα. In contrast to empty vector (pcDNA3) transfectants, treatment of IRF-1 transfectants with TNFα alone for 48 h significantly decreased the number of blue cells.C, the effects of IRF-1 were dependent upon the dose of IRF-1 cDNA (0.1, 0.5, 1, and 2 μg) used in the transient transfection. The number of blue cells upon transfection with an empty vector without TNFα treatment was set to 100%.View Large Image Figure ViewerDownload (PPT) TNFα is known to initiate both death and survival signals, and recent studies on TNFα-induced survival signal suggested an important role of NF-κB activation (19Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science.. 1998; 281: 1680-1683Google Scholar, 20Beg A.A. Baltimore D. Science.. 1996; 274: 782-784Google Scholar, 21Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science.. 1996; 274: 784-787Google Scholar, 22Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell.. 1996; 87: 565-576Google Scholar). Thus, we investigated how IFNγ induces susceptibility to TNFα-induced cytotoxicity by examining the role of NF-κB in ME-180 cell death and its possible regulation by IFNγ. Treatment of ME-180 cells with a proteasome inhibitor (MG-132), which is known to inhibit NF-κB activation (23Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell.. 1994; 78: 773-785Google Scholar), rendered the cells sensitive to TNFα-induced apoptosis (Fig.4 A), suggesting the cytoprotective role of NF-κB. Also, upon the transfection of phosphorylation-defective dominant-negative mutant IκBα, TNFα alone induced a significant cytotoxicity, further supporting the cytoprotective role of NF-κB (Fig. 4 B). NF-κB reporter assays indicated that IFNγ pretreatment attenuated TNFα-induced NF-κB activity, suggesting that IFNγ synergizes with TNFα for ME-180 cell apoptosis by inhibiting TNFα-induced cytoprotective NF-κB activity (Fig. 4 C). IFNγ pretreatment, however, did not inhibit nuclear translocation of p65 (Fig.5) or DNA binding of NF-κB induced by TNFα treatment (Fig. 6). Also, IFNγ did not inhibit TNFα-induced degradation of IκBα (data not shown). However, IFNγ treatment did inhibit the NF-κB reporter activity induced by transfection of p65 subunit of NF-κB (Fig.4 D), indicating that IFNγ directly inhibited NF-κB-mediated transactivation within the nuclei without affecting the nuclear translocation or DNA binding of NF-κB. We next studied if IRF-1 mediates this inhibitory action of IFNγ on NF-κB. Transfection of IRF-1 alone was sufficient to inhibit TNFα-induced NF-κB activity, indicating a central role of IRF-1 in the inhibition of NK-κB transactivation by IFNγ (Fig.7 A). We also investigated the possible mechanism of interference between IRF-1 and NF-κB. Transfection of p300 coactivator abrogated the inhibitory effect of IFNγ treatment (Fig. 7 B) or IRF-1 transfection (Fig.7 C) on TNFα-induced NF-κB activity, suggesting the possibility of coactivator competition between IFNγ-induced IRF-1 and TNFα-induced NF-κB.Figure 5No effects of IFN γ on nuclear translocation of p65 subunit of NF-κB. As compared with untreated control (A), TNFα treatment (45 min, 10 ng/ml) induced nuclear translocation of p65 (B), which was not affected by IFNγ pretreatment (24 h, 100 units/ml) (C).View Large Image Figure ViewerDownload (PPT)Figure 6No significant effects of IFN γ on DNA binding of NF-κB protein. A, IFNγ pretreatment (100 units/ml, 24 h) did not significantly affect TNFα-induced κB sequence binding of NF-κB proteins (lanes 4 and 5). The identity of DNA-complexed proteins was confirmed by supershift assays using antibodies (Ab) against p65 (lane 6), p50 (lane 7), or both (lane 8). B, ME-180 cells were similarly treated with increasing doses of IFNγ and TNFα as indicated, and then NF-κB was detected by electrophoretic mobility shift assay. IFNγ at all concentrations tested did not significantly influence TNFα-induced DNA binding of NF-κB, indicating that the inability of IFNγ to inhibit TNFα-induced DNA binding of NF-κB was not due to the low dose of IFNγ used.View Large Image Figure ViewerDownload (PPT)Figure 7IRF-1 mediates NF-κB-inhibiting effects of IFN γ probably through coactivator competition. A, transfection of IRF-1 inhibited TNFα-induced NF-κB reporter activity in a manner similar to IFNγ pretreatment. B, transfection of coactivator p300 abrogated IFNγ-mediated inhibition of NF-κB reporter activity. Transiently transfected cells were treated with cytokines for an indicated time period before NF-κB reporter assays (IFNγ, 100 units/ml; TNFα, 10 ng/ml). C, ME-180 cells were co-transfected with NF-κB reporter construct and the indicated plasmids, and then the luciferase activity was measured after 24 h. Co-transfection of coactivator p300 also abolished the IRF-1 transfection-mediated inhibition of NF-κB reporter activity. CBP, cAMP-response element-binding protein (CREB)-binding protein.View Large Image Figure ViewerDownload (PPT) We next investigated whether the activation of caspases is involved in the IFNγ/TNFα-induced apoptosis of ME-180 cells. Cytokine-induced apoptosis of ME-180 cells was accompanied by the activation of caspase-3-like activity, as demonstrated by the cleavage of Ac-DEVD-AMC in IFNγ/TNFα-treated cells (Fig.8). Cytokine treatment also induced the cleavage of Ac-IETD-AMC, indicating concurrent activation of caspase-8-like activity (data not shown). However, pretreatment with broad-spectrum caspase inhibitors such as z-VAD-fmk or BD-fmk failed to inhibit ME-180 cell death by IFNγ/TNFα synergism despite the activation of multiple caspases (Fig.9 A). Instead, IFNγ/TNFα in the presence of caspase inhibitors unexpectedly induced the necrosis of ME-180 cells, as judged by the swelling of dying cells on a light microscope (data not shown). Hoechst 33258/PI staining and electron microscopy confirmed the necrosis of the cells (Fig. 9, Band C). To study the involvement of individual caspases in the switching process from apoptosis to necrosis, cells were pretreated with inhibitors specific for individual caspases instead of z-VAD-fmk. Because we observed the activation of caspase-3 and -8 in the cytokine-treated ME-180 cells, we tested the effects of z-DEVD-fmk and z-IETD-fmk alone or in combination. The z-DEVD-fmk and z-IETD-fmk acted additively in conversion from apoptosis to necrosis, suggesting the involvement of multiple caspases in determining the mode of ME-180 cell death (Table III).Figure 9Induction of necrotic death by IFN γ/TNFα in the presence of caspase inhibitors. A, pretreatment of ME-180 cells with broad spectrum caspase inhibitors such as z-VAD-fmk or BD-fmk did not block the cytokine-induced cytotoxicity as measured by MTT assays at 48 h after the treatment (IFNγ, 100 units/ml; TNFα, 10 ng/ml). B and C, pretreatment with z-VAD-fmk switched the mode of cell death from apoptosis to necrosis as judged by Hoechst 33258/PI double-staining (B) and electron microscopy (C). In Hoechst 33258/PI double-staining, cells with blue intact nuclei were viable cells, whereas those with blue fragmented nuclei were early apoptotic cells. Cells with pink intact nuclei were necrotic cells, whereas cells with pink fragmented nuclei were late apoptotic cells. The values in the parentheses below the photographs represent the percentage of apoptotic (early or late) or necrotic cells out of the total 500 cells counted (B).View Large Image Figure ViewerDownload (PPT)Table IIIEffects of various caspase inhibitors on the conversion of ME-180 cell death from apoptosis to necrosisTreatment3-aME-180 cells were pretreated with caspase inhibitors indicated (100 μm) for 1 h before cytokine treatment for 48 h (IFNγ, 100 units/ml; TNFα, 10 ng/ml). Cathepsin B inhibitor, FA, benzyloxycarbonyl-Phe-Ala-CH2-fluoromethyl ketone (FA) was used as a negative control.% Viable cells3-bPercentage of viable, necrotic, or apoptotic cells was assessed by double-staining with Hoechst 33258 and PI.% Necrotic cells3-bPercentage of viable, necrotic, or apoptotic cells was assessed by double-staining with Hoechst 33258 and PI.% Apoptotic cells3-bPercentage of viable, necrotic, or apoptotic cells was assessed by double-staining with Hoechst 33258 and PI.None94.73.12.2IFNγ + TNFα14.78.676.7IFNγ + TNFα + z-VAD16.578.55.0IFNγ + TNFα + DEVD16.825.258.0IFNγ + TNFα + IETD20.434.545.1IFNγ + TNFα + DEVD + IETD27.655.217.2IFNγ + TNFα + FA11.210.578.33-a ME-180 cells were pretreated with caspase inhibitors indicated (100 μm) for 1 h before cytokine treatment for 48 h (IFNγ, 100 units/ml; TNFα, 10 ng/ml). Cathepsin B inhibitor, FA, benzyloxycarbonyl-Phe-Ala-CH2-fluoromethyl ketone (FA) was used as a negative control.3-b Percentage of viable, necrotic, or apoptotic cells was assessed by double-staining with Hoechst 33258 and PI. Open table in a new tab Here we present evidence that STAT1/IRF-1 pathways initiated by IFNγ play a central role in IFNγ/TNFα synergism in the induction of ME-180 cell apoptosis. Transfection of dominant-negative STAT1 abolished IFNγ/TNFα synergism, whereas transfection of IRF-1 sensitized ME-180 cells to TNFα-induced apoptosis. Thus, STAT1 activation and IRF-1 induction by IFNγ appear to be important in IFNγ/TNFα synergism in ME-180 cell apoptosis. However, dominant-negative STAT1 did not completely abolish cytotoxicity by IFNγ/TNFα, and IRF-1 transfection could not be completely substituted for IFNγ. IFNγ induces STAT1 as well as IRF-1, and some cellular responses to IFNγ are reported to be mediated by both STAT1 and IRF-1 (24Dong Y. Rohn W.M. Benveniste E.N. J. Immunol... 1999; 162: 4731-4739Google Scholar, 25Stark G.D. Kerr I.M. Williams B.R.G. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem... 1998; 67: 227-264Google Scholar). Neither STAT1 or IRF-1 alone may not explain all of the priming effect of IFNγ in TNFα-induced death. The role of IRF-1 in the induction of apoptosis by DNA damage or IFNγ has been previously suggested (26Tanaka N. Ishihara M. Kitagawa M. Harada H. Kimura T. Matsuyama T. Lamphier M.S. Aizawa S. Mak T.W. Taniguchi T. Cell.. 1994; 77: 829-839Google Scholar, 27Tamura T. Ishihara M. Lamphier M.S. Tanaka N. Oishi I. Aizawa S. Matsuyama T. Mak T.W. Taki S. Taniguchi T. Nature.. 1995; 376: 596-599Google Scholar, 28Kano A. Haruyama T. Akaike T. Watanabe Y. Biochem. Biophys. Res. Commun... 1999; 257: 672-677Google Scholar), which supports the proapoptotic action of IRF-1. Previous work in our laboratory also showed that IRF-1 plays a central role in IFNγ/TNFα-induced apoptosis of pancreatic islet β-cells in autoimmune diabetes. 2K. Suk, I. Chang, Y.-H. Kim, S. Kim, J. Y. Kim, and M.-S. Lee, submitted for publication. Caspase induction has been suggested as a possible downstream event after IRF-1 induction in IFNγ-induced apoptosis (28Kano A. Haruyama T. Akaike T. Watanabe Y. Biochem. Biophys. Res. Commun... 1999; 257: 672-677Google Scholar). Although RNase protection assays revealed that the expression of caspase-1 and -4 was up-regulated by IFNγ treatment in ME-180 cells (data not shown), there remains yet to be determined how the increases in the expression of these caspases mediate IRF-1 action. In IFNγ/TNFα-induced death of ME-180 cells, caspases seem to be involved in determining the mode of cell death rather than decision between death and survival (see below). Although further works are necessary to completely delineate the downstream signaling pathways after STAT1/IRF-1 in IFNγ/TNFα cytotoxic synergism, our current work indicates that NF-κB is one of the targets of STAT1/IRF-1 action. We demonstrated that IFNγ attenuated TNFα-induced NF-κB reporter activity in ME-180 cells. Also, the inhibition of NF-κB either by transfection of dominant-negative IκB “super repressor” or by treatment with a proteasome inhibitor (MG-132) rendered ME-180 cells sensitive to TNFα-induced apoptosis. These results indicate that IFNγ sensitizes ME-180 cells to TNFα-induced apoptosis by inhibiting NF-κB-mediated activation of survival signals. Furthermore, this action of IFNγ was mediated by IRF-1. It has been previously reported that IRF-1 and NF-κB interact in vitro as well as in vivo for the cooperative induction of inflammatory genes (29Neish A.S. Read M.A. Thanos D. Pine R. Maniatis T. Collins T. Mol. Cell. Biol... 1995; 15: 2558-2569Google Scholar, 30Drew P.D. Franzoso G. Becker K.G. Bours V. Carlson L.M. Siebenlist U. Ozato K. J. Interferon Cytokine Res... 1995; 15: 1037-1045Google Scholar, 31Saura M. Zaragoza C. Bao C. McMillan A. Lowenstein C.J. J. Mol. Biol... 1999; 289: 459-471Google Scholar). In ME-180 cells, however, IRF-1 negatively influenced NF-κB activity. IRF-1 does not seem to directly interact with NF-κB because NF-κB transcriptional activity was assessed using a reporter construct containing a κB element but not an IRF-1 response element. Thus, in ME-180 cells, it is likely that IRF-1 indirectly affects NF-κB transcriptional activity through the regulation of other factors modulating the transcriptional activity. We also demonstrated that IFNγ did not block the TNFα-induced translocation of p65 from cytosol to nucleus or DNA binding of NF-κB but yet inhibited NF-κB reporter activity. These results suggest that IFNγ-induced IRF-1 inhibits the nuclear events of NF-κB transactivation but not cytosolic events. Our work also showed that transfection of transcriptional coactivator p300 abolished the inhibition of NF-κB reporter activity by IFNγ. Transcriptional activation by NF-κB requires multiple coactivators (32Sheppard K.A. Rose D.W. Haque Z.K. Kurokawa R. McInerney E. Westin S. Thanos D. Rosenfeld M.G. Glass C.K. Collins T. Mol. Cell. Biol... 1999; 19: 6367-6378Google Scholar). It has been recently reported that the intracellular amount of the coactivator p300 is limited compared with other transcriptional factors and that competition for p300 may regulate transcriptional activity (33Hottiger M.O. Felzien L.K. Nabel G.J. EMBO J... 1998; 17: 3124-3134Google Scholar). Thus, it is possible that IFNγ-induced IRF-1 competes with TNFα-induced NF-κB for the common coactivator(s) such as p300, and this competition may be responsible for the inhibition of NF-κB transactivation. Then what are the target genes that are induced by NF-κB and are subject to the inhibitory action of IRF-1? Recently, a role of TNF receptor-associated factor 1 (TRAF2), TRAF2, c-IAP1 (inhibitor of apoptosis (IAP)) and cIAP2 was reported in anti-apoptosis mediated by NF-κB (19Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science.. 1998; 281: 1680-1683Google Scholar). These are possible candidates for such target genes. Another puzzling point is what determines how IFNγ acts on NF-κB. Previously, IFNγ has been shown to increase TNFα-induced NF-κB activation in enhancing the expression of multiple genes involved in the inflammatory responses (34Cheshire J.L. Baldwin Jr., A.S. Mol. Cell. Biol... 1997; 17: 6746-6754Google Scholar). In sharp contrast, however, our work disclosed that IFNγ inhibited TNFα-induced NF-κB in ME-180 cells. This novel signaling pathway of synergism between IFNγ/TNFα involving competition between IRF-1 and NF-κB for p300 coactivator may not be generalized to other cell types, considering previous reports showing different signaling patterns in response to IFNγ/TNFα (34Cheshire J.L. Baldwin Jr., A.S. Mol. Cell. Biol... 1997; 17: 6746-6754Google Scholar). The same stimulus seems to activate distinct signaling pathways depending on the cell types. Because of this discrepancy in signal transduction pathways, the final outcome would be different among different cell types. Some cells would undergo death by IFNγ/TNFα, whereas other cells may be activated by IFNγ/TNFα to participate in inflammatory responses. Because IFNα is also known to activate the STAT1-signaling pathway, we investigated whether IFNα also synergizes with TNFα to destroy ME-180 cells. Our results indicated that IFNα and TNFα synergistically induced ME-180 cell death, and this was accompanied by the activation of STAT1 and inhibition of NF-κB reporter activity by IFNα in a manner similar to IFNγ. 3K. Suk, I. Chang, Y.-H. Kim, J. Y. Kim, and M.-S. Lee, unpublished data. Thus, the cytotoxic priming role of IFNγ in IFNγ/TNFα synergism presented in the current study does not seem to be restricted to IFNγ. Rather, the STAT1/IRF-1-signaling pathway that can be initiated by either type I or type II interferon appears to be critical for the cytotoxic synergism with TNFα. Our results indicate that IFNγ/TNFα induces death signaling in ME-180 cells regardless of caspase activation and activation of caspases determines the final mode of cell death (apoptosisversus necrosis). These results suggest that IFNγ/TNFα-induced apoptotic and necrotic death signaling pathways have common signaling components, and the mode of cell death depends on distinct signaling events such as caspase activation. A similar dual pathway in cell death has been reported in L929 cells transfected with Fas cDNA (35Vercammen D. Brouckaert G. Denecker G. Van de Craen M. Declercq W. Fiers W. Vandenabeele P. J. Exp. Med... 1998; 188: 919-930Google Scholar). Ligation of Fas with anti-Fas antibody induced apoptosis of these cells. However, pretreatment with z-VAD, which inhibits activation of caspases, resulted in necrotic death. Moreover, necrosis of Fas-expressing L929 cells was inhibited by reactive oxygen intermediate (ROI) scavengers such as butylated hydroxyanisol, indicating the involvement of ROI generation in necrotic cell death pathway. Butylated hydroxyanisol, however, did not block IFNγ/TNFα-induced ME-180 cell death in the presence of caspase inhibitors (data not shown), suggesting distinct signal transduction between the two cell types. Nevertheless, dual pathways of death signaling appear to be present in the two cells, and it will be of great interest to see if this type of response could be found in other cell types exposed to similar or different death signals. Whether a cell undergoes apoptosis or necrosis by a given stimulus may be determined by intracellular milieu (36Eguchi Y. Shimizu S. Tsujimoto Y. Cancer Res... 1997; 57: 1835-1840Google Scholar, 37Kane D.J. Sarafian T.A. Anton R. Hahn H. Gralla E.B. Valentine J.S. Ord T. Bredesen D.E. Science.. 1993; 262: 1274-1277Google Scholar, 38Cory S. Annu. Rev. Immunol... 1995; 13: 513-543Google Scholar). Intracellular levels of ATP were reported to be a determinant of manifestation of cell death (apoptosis versus necrosis) (36Eguchi Y. Shimizu S. Tsujimoto Y. Cancer Res... 1997; 57: 1835-1840Google Scholar). Also, the fact that Bcl-2 blocks both apoptotic and necrotic cell death supports the presence of common signaling components between the two death-signaling pathways (37Kane D.J. Sarafian T.A. Anton R. Hahn H. Gralla E.B. Valentine J.S. Ord T. Bredesen D.E. Science.. 1993; 262: 1274-1277Google Scholar, 38Cory S. Annu. Rev. Immunol... 1995; 13: 513-543Google Scholar). Although our work cannot provide detailed biochemical mechanisms of cell death machinery in ME-180 cells, our studies point out the existence of common components between apoptotic and necrotic death signaling and the role of caspases in determining the type of cell death, which may help understand the general cell death mechanism. In conclusion, we report a novel signal transduction of IFNγ/TNFα synergism in the induction of ME-180 cell apoptosis: IFNγ synergized with TNFα for apoptosis induction by activating STAT1/IRF-1 pathway. We also present evidence that NF-κB activation is a survival signal in TNFα-treated ME-180 cells, and IFNγ inhibits this survival mechanism, resulting in synergistic cytotoxicity with TNFα. Moreover, the mode of ME-180 cell death by IFNγ/TNFα synergism was dictated by caspase activation. The novel mechanism of IFNγ/TNFα synergism presented here may also be applicable to other circumstances, where a similar cytokine synergism could be found such as autoimmune destruction of self tissues by cytokines. We thank Drs. Kye Young Lee, Tae H. Lee, Jae W. Lee, Minho Shong, Soo Young Lee, Il-Seon Park, and Young S. Ahn for insightful discussions and technical help. tumor necrosis factor interferon interferon regulatory factor signal transducer and activator of transcription propidium iodide benzyloxycarbonyl-Val-Ala-Asp(OCH3)-CH2-fluoromethyl ketone t-butoxycarbonyl-Asp(OCH3)-CH2-fluoromethyl ketone benzyloxycarbonyl-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-CH2-fluoromethyl ketone benzyloxycarbonyl-Ile-Glu(OCH3)- Thr-Asp(Ome)-CH2-fluoromethyl ketone 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide acetyl amidome-thylcoumarin" @default.
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• W2006354480 title "Interferon γ (IFNγ) and Tumor Necrosis Factor α Synergism in ME-180 Cervical Cancer Cell Apoptosis and Necrosis" @default.
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