HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 16, 13153-13159, April 20, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/16/13153    most recent M007646200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Suk, K. Articles by Lee, M.-S. Search for Related Content PubMed PubMed Citation Articles by Suk, K. Articles by Lee, M.-S. Social Bookmarking                      What's this?

Interferon  (IFN) and Tumor Necrosis Factor  Synergism in ME-180 Cervical Cancer Cell Apoptosis and Necrosis

IFN INHIBITS CYTOPROTECTIVE NF-B THROUGH STAT1/IRF-1 PATHWAYS^*

Kyoungho Suk^§¶, Inik Chang, Yun-Hee Kim^**, Sunshin Kim^**, Ja Young Kim^**, Hocheol Kim^¶§, and Myung-Shik Lee^**

From the  Clinical Research Center, Samsung Biomedical Research Institute and ^** Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea and ^§ Graduate School of East-West Medical Science, Kyunghee University, Seoul, 130-701, Korea

Received for publication, August 22, 2000, and in revised form, December 15, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We investigated the molecular mechanism of the synergism between interferon  (IFN) and tumor necrosis factor  (TNF) documented in a variety of biological occasions such as tumor cell death and inflammatory responses. IFN/TNF synergistically induced apoptosis of ME-180 cervical cancer cells. IFN induced STAT1 phosphorylation and interferon regulatory factor 1 (IRF-1) expression. Transfection of phosphorylation-defective STAT1 inhibited IFN/TNF-induced apoptosis, whereas IRF-1 transfection induced susceptibility to TNF. Dominant-negative IB transfection sensitized ME-180 cells to TNF. IFN pretreatment attenuated TNF- or p65-induced NF-B reporter activity, whereas it did not inhibit p65 translocation or DNA binding of NF-B. IRF-1 transfection alone inhibited TNF-induced NF-B activity, which was reversed by coactivator p300 overexpression. Caspases were activated by IFN/TNF combination; however, caspase inhibition did not abrogate IFN/TNF-induced cell death. Instead, caspase inhibitors directed IFN/TNF-treated ME-180 cells to undergo necrosis, as demonstrated by Hoechst 33258/propidium iodide staining and electron microscopy. Taken together, our results indicate that IFN and TNF synergistically act to destroy ME-180 tumor cells by either apoptosis or necrosis, depending on caspase activation, and STAT1/IRF-1 pathways initiated by IFN play a critical role in IFN/TNF synergism by inhibiting cytoprotective NF-B. IFN/TNF synergism appears to activate cell death machinery independently of caspase activation, and caspase activation seems to merely determine the mode of cell death.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The pleiotropic proinflammatory cytokine TNF¹ 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 (1). TNF also kills various tumor cell lines in vitro and mediates anti-tumor effect in vivo (2). TNF exerts its biological effects by binding to two types of cell surface receptors with molecular masses of 55 kDa (p55) and 75 kDa (p75). TNF cytotoxicity is mostly mediated by p55 receptors (3). 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 (4). This, in turn, triggers the caspase cascade and ultimately results in apoptotic cell death.

In many cases, the anti-tumor effect of TNF was enhanced by IFN (5) or metabolic inhibitors such as cycloheximide and actinomycin D (6). Although these metabolic inhibitors are believed to block the synthesis of cytoprotective proteins, the effects of IFN might be mediated by the induction of new proteins that increase the sensitivity of target cells to TNF. IFN/TNF 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 (7-9). However, the molecular mechanism of the synergism between the two cytokines is not clearly understood. It has been reported that IFN increases the expression of TNF receptors (10). However, because the sensitivity of the cells to TNF is not simply correlated with the level of TNF receptor expression (11, 12), up-regulation of TNF 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 IFN/TNF. We also studied the role of caspase activation in ME-180 cell death by IFN/TNF synergism. Our results indicate that 1) IRF-1 induction after STAT1 activation by IFN plays a central role in synergistic tumor cell death by IFN/TNF, 2) IFN-induced IRF-1 inhibits cytoprotective NF-B transactivation, 3) IFN/TNF induces ME-180 cell death regardless of caspase activation, and caspase activation dictates only the mode of cell death between apoptosis and necrosis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Line and Reagents-- 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 mM glutamine, and penicillin-streptomycin (Life Technologies, Inc.). Recombinant human IFN was purchased from R&D Systems (Minneapolis, MN). Recombinant human TNF 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.

Assessment of Cytotoxicity by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium Bromide (MTT) Assay-- 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 IFN and 10 ng/ml for TNF. 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 Analysis of Apoptotic Cells-- 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 (13). Transmission electron microscopy was carried out essentially as previously described (13). 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 M phosphate, 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).

DNA Ploidy Analysis-- 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.

Assessment of Caspase Activity-- 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.

Western Blot Analysis-- Cells were lysed in triple-detergent lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 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).

Transient Transfection-- 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 IB (14) 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 -D-galactopyranoside; 1 mg/ml) in 4 mM potassium 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-B Reporter Assays-- NF-B 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-B-responsive reporter gene construct carrying two copies of B sequences linked to luciferase gene (IgG NF-B-luciferase, generously provided by Dr. G. D. Rosen, Stanford University, Stanford, CA) (15) 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-B p65 (16) or coactivator p300 expression plasmid (0.5 µg; kindly provided by Dr. Livingston, Harvard Medical School, Boston, MA) (17) along with NF-B-responsive reporter plasmid (0.5 µg) and pRL-TK (0.1 µg). Results were presented as means ± S.E. (n = 3).

Immunofluorescence Staining-- 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.

Electrophoretic Mobility Shift Assay-- Nuclear extracts were prepared from ME-180 cells treated with cytokines as previously described (18). Synthetic double-strand oligonucleotides of consensus NF-B binding sequence, GAT CCC AAC GGC AGG GGA (Promega), were end-labeled with [-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-B were included in the reaction. DNA-protein complexes were resolved by electrophoresis in a 5% nondenaturing polyacrylamide gel, dried, and visualized by autoradiography.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

IFN and TNF Synergistically Induced the Apoptosis of ME-180 Cells-- First we screened several tumor cell lines to assess their sensitivity to IFN/TNF-induced cytotoxicity (data not shown). Cytotoxic synergism between IFN and TNF 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. 1A). The cytokine cytotoxicity was dependent on the dose of IFN used. However, concentration higher than 100 units/ml did not further increase the cytotoxicity (Table I). The reduction of cell viability was due to apoptosis as demonstrated by Hoechst 33258 staining and DNA ploidy analysis. IFN/TNF treatment induced nuclear condensation and fragmentation (Fig. 1B) and led to the appearance of sub-diploid cells (Fig. 1C), which are hallmarks of apoptotic cells. DNA ploidy assays also indicated that the effect of IFN/TNF was not due to the growth arrest as was shown by the absence of decrease in the percentage of cells in the S phase.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   IFN/TNF synergistically induces ME-180 cell apoptosis. A combination of IFN (100 units/ml) and TNF (10 ng/ml), but not either cytokine alone, induced ME-180 cell death. Cell viability was assessed by MTT assays after treatment with the cytokines for 48 h (A). Induction of ME-180 cell death was due to apoptosis, as demonstrated by chromatin condensation in Hoechst 33258 staining (B) or the appearance of sub-G1 peak in flow cytometric analyses (C) at 24 h after cytokine treatment.

IFN/TNF Synergism Involved IFN-induced STAT1 Activation and IRF-1 Induction-- Based on our results that the combination of IFN and TNF, but not either cytokine alone, induced ME-180 cell death, we explored the possibility that IFN sensitizes ME-180 cells to TNF-mediated cytotoxicity. This was first tested by sequential treatment of ME-180 cells with the two cytokines. After IFN treatment, TNF alone was sufficient to induce a significant cytotoxicity in ME-180 cells (Table II). However, sequential treatment with TNF and then with IFN did not have the same effects, indicating that IFN confers susceptibility to TNF 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 IFN signaling, we investigated the involvement of STAT1/IRF-1-signaling pathways in IFN/TNF synergism on ME-180 cell apoptosis. IFN, but not TNF, 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 IFN/TNF-induced ME-180 cell death, indicating that IFN-induced STAT1 activation is critical for the induction of TNF susceptibility (Fig. 3A). We next asked whether IRF-1, a downstream mediator of STAT1, is responsible for the priming effects of IFN. Transfection of IRF-1 conferred TNF 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 TNF-induced apoptosis (Fig. 3, B and C).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   IFN activates STAT1 (A) and induces IRF-1 expression (B) in ME-180 cells. Western blot analyses demonstrated that treatment of ME-180 cells with IFN induced STAT1 expression (24-h treatment) as well as its phosphorylation (30-min treatment) (A). IFN also induced IRF-1 expression at 24 h after treatment, and the expression was further increased at 48 h after the treatment (B). However, TNF alone did not change the expression of either STAT1 or IRF-1. C, untreated control; I, IFN (100 units/ml); T, TNF (10 ng/ml); D, IFN plus TNF.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   A key role for STAT1/IRF-1 signaling in IFN/TNF synergism. A, transient transfection of phosphorylation-defective STAT1 dominant-negative mutant (DN STAT1) significantly inhibited IFN/TNF cytotoxicity, as demonstrated by counting blue cells co-expressing lacZ at 48 h after cytokine treatment (IFN, 100 units/ml; TNF, 10 ng/ml). B, transfection of IRF-1 cDNA (1 µg) induced susceptibility to TNF. In contrast to empty vector (pcDNA3) transfectants, treatment of IRF-1 transfectants with TNF 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 TNF treatment was set to 100%.

Inhibition of Cytoprotective NF-B Activity by IFN-- TNF is known to initiate both death and survival signals, and recent studies on TNF-induced survival signal suggested an important role of NF-B activation (19-22). Thus, we investigated how IFN induces susceptibility to TNF-induced cytotoxicity by examining the role of NF-B in ME-180 cell death and its possible regulation by IFN. Treatment of ME-180 cells with a proteasome inhibitor (MG-132), which is known to inhibit NF-B activation (23), rendered the cells sensitive to TNF-induced apoptosis (Fig. 4A), suggesting the cytoprotective role of NF-B. Also, upon the transfection of phosphorylation-defective dominant-negative mutant IB, TNF alone induced a significant cytotoxicity, further supporting the cytoprotective role of NF-B (Fig. 4B). NF-B reporter assays indicated that IFN pretreatment attenuated TNF-induced NF-B activity, suggesting that IFN synergizes with TNF for ME-180 cell apoptosis by inhibiting TNF-induced cytoprotective NF-B activity (Fig. 4C). IFN pretreatment, however, did not inhibit nuclear translocation of p65 (Fig. 5) or DNA binding of NF-B induced by TNF treatment (Fig. 6). Also, IFN did not inhibit TNF-induced degradation of IB (data not shown). However, IFN treatment did inhibit the NF-B reporter activity induced by transfection of p65 subunit of NF-B (Fig. 4D), indicating that IFN directly inhibited NF-B-mediated transactivation within the nuclei without affecting the nuclear translocation or DNA binding of NF-B. We next studied if IRF-1 mediates this inhibitory action of IFN on NF-B. Transfection of IRF-1 alone was sufficient to inhibit TNF-induced NF-B activity, indicating a central role of IRF-1 in the inhibition of NK-B transactivation by IFN (Fig. 7A). We also investigated the possible mechanism of interference between IRF-1 and NF-B. Transfection of p300 coactivator abrogated the inhibitory effect of IFN treatment (Fig. 7B) or IRF-1 transfection (Fig. 7C) on TNF-induced NF-B activity, suggesting the possibility of coactivator competition between IFN-induced IRF-1 and TNF-induced NF-B.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Inhibition of cytoprotective NF-B by IFN. A, inhibition of NF-B by proteasome inhibitor MG-132 sensitized ME-180 cells to TNF. ME-180 cells were treated with either MG-132 (0.5 µM) alone or in combination with TNF (10 ng/ml) for 48 h, and then cell viability was assessed by MTT assays. B, inhibition of NF-B by transfection of dominant-negative mutant IB (DN IB) also rendered ME-180 cells sensitive to TNF treatment. Viability of ME-180 cells co-transfected with dominant-negative IB and lacZ was significantly decreased by TNF treatment (24 h), in contrast to the cells co-transfected with an empty vector (pcDNA3) and lacZ. C and D, NF-B reporter assays revealed that pretreatment (24 h, 100 units/ml) of ME-180 cells with IFN inhibited TNF-induced NF-B activity (C). IFN treatment (48 h) also inhibited NF-B reporter activity induced by p65 transfection (NF-B p65) (D). Transiently transfected cells were treated with cytokines for the indicated time period before NF-B reporter assays (C and D).

View larger version (40K): [in this window] [in a new window]   Fig. 5.   No effects of IFN on nuclear translocation of p65 subunit of NF-B. As compared with untreated control (A), TNF treatment (45 min, 10 ng/ml) induced nuclear translocation of p65 (B), which was not affected by IFN pretreatment (24 h, 100 units/ml) (C).

View larger version (33K): [in this window] [in a new window]   Fig. 6.   No significant effects of IFN on DNA binding of NF-B protein. A, IFN pretreatment (100 units/ml, 24 h) did not significantly affect TNF-induced B sequence binding of NF-B 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 IFN and TNF as indicated, and then NF-B was detected by electrophoretic mobility shift assay. IFN at all concentrations tested did not significantly influence TNF-induced DNA binding of NF-B, indicating that the inability of IFN to inhibit TNF-induced DNA binding of NF-B was not due to the low dose of IFN used.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   IRF-1 mediates NF-B-inhibiting effects of IFN probably through coactivator competition. A, transfection of IRF-1 inhibited TNF-induced NF-B reporter activity in a manner similar to IFN pretreatment. B, transfection of coactivator p300 abrogated IFN-mediated inhibition of NF-B reporter activity. Transiently transfected cells were treated with cytokines for an indicated time period before NF-B reporter assays (IFN, 100 units/ml; TNF, 10 ng/ml). C, ME-180 cells were co-transfected with NF-B 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-B reporter activity. CBP, cAMP-response element-binding protein (CREB)-binding protein.

Inhibition of Caspases Directed ME-180 Cells to Undergo Necrotic Cell Death-- We next investigated whether the activation of caspases is involved in the IFN/TNF-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 IFN/TNF-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 IFN/TNF synergism despite the activation of multiple caspases (Fig. 9A). Instead, IFN/TNF 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, B and 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).

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Activation of caspase-3-like activity by IFN/TNF in ME-180 cells. IFN/TNF treatment (IFN, 100 units/ml; TNF, 10 ng/ml) induced cleavage of DEVD-AMC, indicating activation of caspase-3-like activity. Pretreatment of ME-180 cells with z-VAD-fmk before cytokine treatment completely inhibited the caspase activity (IFN, 100 units/ml; TNF, 10 ng/ml).

View larger version (49K): [in this window] [in a new window]   Fig. 9.   Induction of necrotic death by IFN/TNF 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 (IFN, 100 units/ml; TNF, 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).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Here we present evidence that STAT1/IRF-1 pathways initiated by IFN play a central role in IFN/TNF synergism in the induction of ME-180 cell apoptosis. Transfection of dominant-negative STAT1 abolished IFN/TNF synergism, whereas transfection of IRF-1 sensitized ME-180 cells to TNF-induced apoptosis. Thus, STAT1 activation and IRF-1 induction by IFN appear to be important in IFN/TNF synergism in ME-180 cell apoptosis. However, dominant-negative STAT1 did not completely abolish cytotoxicity by IFN/TNF, and IRF-1 transfection could not be completely substituted for IFN. IFN induces STAT1 as well as IRF-1, and some cellular responses to IFN are reported to be mediated by both STAT1 and IRF-1 (24-25). Neither STAT1 or IRF-1 alone may not explain all of the priming effect of IFN in TNF-induced death. The role of IRF-1 in the induction of apoptosis by DNA damage or IFN has been previously suggested (26-28), which supports the proapoptotic action of IRF-1. Previous work in our laboratory also showed that IRF-1 plays a central role in IFN/TNF-induced apoptosis of pancreatic islet -cells in autoimmune diabetes.² Caspase induction has been suggested as a possible downstream event after IRF-1 induction in IFN-induced apoptosis (28). Although RNase protection assays revealed that the expression of caspase-1 and -4 was up-regulated by IFN 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 IFN/TNF-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 IFN/TNF cytotoxic synergism, our current work indicates that NF-B is one of the targets of STAT1/IRF-1 action. We demonstrated that IFN attenuated TNF-induced NF-B reporter activity in ME-180 cells. Also, the inhibition of NF-B either by transfection of dominant-negative IB "super repressor" or by treatment with a proteasome inhibitor (MG-132) rendered ME-180 cells sensitive to TNF-induced apoptosis. These results indicate that IFN sensitizes ME-180 cells to TNF-induced apoptosis by inhibiting NF-B-mediated activation of survival signals. Furthermore, this action of IFN was mediated by IRF-1. It has been previously reported that IRF-1 and NF-B interact in vitro as well as in vivo for the cooperative induction of inflammatory genes (29-31). In ME-180 cells, however, IRF-1 negatively influenced NF-B activity. IRF-1 does not seem to directly interact with NF-B because NF-B transcriptional activity was assessed using a reporter construct containing a B element but not an IRF-1 response element. Thus, in ME-180 cells, it is likely that IRF-1 indirectly affects NF-B transcriptional activity through the regulation of other factors modulating the transcriptional activity. We also demonstrated that IFN did not block the TNF-induced translocation of p65 from cytosol to nucleus or DNA binding of NF-B but yet inhibited NF-B reporter activity. These results suggest that IFN-induced IRF-1 inhibits the nuclear events of NF-B transactivation but not cytosolic events. Our work also showed that transfection of transcriptional coactivator p300 abolished the inhibition of NF-B reporter activity by IFN. Transcriptional activation by NF-B requires multiple coactivators (32). 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 (33). Thus, it is possible that IFN-induced IRF-1 competes with TNF-induced NF-B for the common coactivator(s) such as p300, and this competition may be responsible for the inhibition of NF-B transactivation. Then what are the target genes that are induced by NF-B 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-B (19). These are possible candidates for such target genes. Another puzzling point is what determines how IFN acts on NF-B. Previously, IFN has been shown to increase TNF-induced NF-B activation in enhancing the expression of multiple genes involved in the inflammatory responses (34). In sharp contrast, however, our work disclosed that IFN inhibited TNF-induced NF-B in ME-180 cells. This novel signaling pathway of synergism between IFN/TNF involving competition between IRF-1 and NF-B for p300 coactivator may not be generalized to other cell types, considering previous reports showing different signaling patterns in response to IFN/TNF (34). 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 IFN/TNF, whereas other cells may be activated by IFN/TNF to participate in inflammatory responses.

Because IFN is also known to activate the STAT1-signaling pathway, we investigated whether IFN also synergizes with TNF to destroy ME-180 cells. Our results indicated that IFN and TNF synergistically induced ME-180 cell death, and this was accompanied by the activation of STAT1 and inhibition of NF-B reporter activity by IFN in a manner similar to IFN.³ Thus, the cytotoxic priming role of IFN in IFN/TNF synergism presented in the current study does not seem to be restricted to IFN. 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 TNF.

Our results indicate that IFN/TNF induces death signaling in ME-180 cells regardless of caspase activation and activation of caspases determines the final mode of cell death (apoptosis versus necrosis). These results suggest that IFN/TNF-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 (35). 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 IFN/TNF-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 (36-38). Intracellular levels of ATP were reported to be a determinant of manifestation of cell death (apoptosis versus necrosis) (36). 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 (37, 38). 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 IFN/TNF synergism in the induction of ME-180 cell apoptosis: IFN synergized with TNF for apoptosis induction by activating STAT1/IRF-1 pathway. We also present evidence that NF-B activation is a survival signal in TNF-treated ME-180 cells, and IFN inhibits this survival mechanism, resulting in synergistic cytotoxicity with TNF. Moreover, the mode of ME-180 cell death by IFN/TNF synergism was dictated by caspase activation. The novel mechanism of IFN/TNF 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.

	ACKNOWLEDGEMENTS

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.

	FOOTNOTES

^* This work was supported by National Research Laboratory Grants 2000-N-NL-01-C-232 from the Korea Institute of Science and Technology Evaluation and Planning and by Science Research Center Grants from Korea Science and Engineering Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by Brain Korea 21 project from the Ministry of Education, Korea.

These authors contributed equally to this work.

Recipient of Juvenile Diabetes Foundation International Research Grant 1-1999-760). To whom correspondence should be addressed. Tel.: 82-2-3410-3436; Fax: 82-2-3410-3849; E-mail: mslee@smc.samsung.co.kr.

Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M007646200

³ K. Suk, I. Chang, Y.-H. Kim, J. Y. Kim, and M.-S. Lee, unpublished data.

² K. Suk, I. Chang, Y.-H. Kim, S. Kim, J. Y. Kim, and M.-S. Lee, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: TNF, tumor necrosis factor; IFN, interferon; IRF, interferon regulatory factor; STAT, signal transducer and activator of transcription; PI, propidium iodide; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OCH₃)-CH₂-fluoromethyl ketone; BD-fmk, t-butoxycarbonyl-Asp(OCH₃)-CH₂-fluoromethyl ketone; z-DEVD-fmk, benzyloxycarbonyl-Asp(OCH₃)-Glu(OCH₃)-Val-Asp(OCH₃)-CH₂-fluoromethyl ketone; z-IETD-fmk, benzyloxycarbonyl-Ile-Glu(OCH₃)- Thr-Asp(Ome)-CH₂-fluoromethyl ketone; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Ac, acetyl; AMC, amidome-thylcoumarin.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				N. Kravchenko-Balasha, S. Mizrachy-Schwartz, S. Klein, and A. Levitzki Shift from Apoptotic to Necrotic Cell Death during Human Papillomavirus-induced Transformation of Keratinocytes J. Biol. Chem., April 24, 2009; 284(17): 11717 - 11727. [Abstract] [Full Text] [PDF]

				A. S. Kapeu, T. Luostarinen, E. Jellum, J. Dillner, M. Hakama, P. Koskela, P. Lenner, A. Love, E. Mahlamaki, S. Thoresen, et al. Is Smoking an Independent Risk Factor for Invasive Cervical Cancer? A Nested Case-Control Study Within Nordic Biobanks Am. J. Epidemiol., February 15, 2009; 169(4): 480 - 488. [Abstract] [Full Text] [PDF]

				M. Labeur, D. Refojo, B. Wolfel, J. Stalla, V. Vargas, M. Theodoropoulou, M. Buchfelder, M. Paez-Pereda, E. Arzt, and G. K Stalla Interferon-{gamma} inhibits cellular proliferation and ACTH production in corticotroph tumor cells through a novel janus kinases-signal transducer and activator of transcription 1/nuclear factor-kappa B inhibitory signaling pathway J. Endocrinol., November 1, 2008; 199(2): 177 - 189. [Abstract] [Full Text] [PDF]

				O. Tliba and Y. Amrani Airway Smooth Muscle Cell as an Inflammatory Cell: Lessons Learned from Interferon Signaling Pathways Proceedings of the ATS, January 1, 2008; 5(1): 106 - 112. [Abstract] [Full Text] [PDF]

				J. Park, K. Kim, E.-J. Lee, Y.-J. Seo, S.-N. Lim, K. Park, S. B. Rho, S.-H. Lee, and J.-H. Lee Elevated level of SUMOylated IRF-1 in tumor cells interferes with IRF-1-mediated apoptosis PNAS, October 23, 2007; 104(43): 17028 - 17033. [Abstract] [Full Text] [PDF]

				A. S. Gunnell, T. N. Tran, A. Torrang, P. W. Dickman, P. Sparen, J. Palmgren, and N. Ylitalo Synergy between Cigarette Smoking and Human Papillomavirus Type 16 in Cervical Cancer In situ Development Cancer Epidemiol. Biomarkers Prev., November 1, 2006; 15(11): 2141 - 2147. [Abstract] [Full Text] [PDF]

				J. M. Roda, R. Parihar, A. Lehman, A. Mani, S. Tridandapani, and W. E. Carson III Interleukin-21 Enhances NK Cell Activation in Response to Antibody-Coated Targets J. Immunol., July 1, 2006; 177(1): 120 - 129. [Abstract] [Full Text] [PDF]

				M. Liu, M. Mendicino, Q. Ning, A. Ghanekar, W. He, I. McGilvray, I. Shalev, D. Pivato, D. A. Clark, M. J. Phillips, et al. Cytokine-Induced Hepatic Apoptosis Is Dependent on FGL2/Fibroleukin: The Role of Sp1/Sp3 and STAT1/PU.1 Composite cis Elements. J. Immunol., June 1, 2006; 176(11): 7028 - 7038. [Abstract] [Full Text] [PDF]

				O. H. Kramer, D. Baus, S. K. Knauer, S. Stein, E. Jager, R. H. Stauber, M. Grez, E. Pfitzner, and T. Heinzel Acetylation of Stat1 modulates NF-{kappa}B activity. Genes & Dev., February 15, 2006; 20(4): 473 - 485. [Abstract] [Full Text] [PDF]

				A G Bais, I Beckmann, J Lindemans, P C Ewing, C J L M Meijer, P J F Snijders, and T J M Helmerhorst A shift to a peripheral Th2-type cytokine pattern during the carcinogenesis of cervical cancer becomes manifest in CIN III lesions J. Clin. Pathol., October 1, 2005; 58(10): 1096 - 1100. [Abstract] [Full Text] [PDF]

				K. B. Bouker, T. C. Skaar, R. B. Riggins, D. S. Harburger, D. R. Fernandez, A. Zwart, A. Wang, and R. Clarke Interferon regulatory factor-1 (IRF-1) exhibits tumor suppressor activities in breast cancer associated with caspase activation and induction of apoptosis Carcinogenesis, September 1, 2005; 26(9): 1527 - 1535. [Abstract] [Full Text] [PDF]

				H. S. Kim and M.-S. Lee Essential Role of STAT1 in Caspase-Independent Cell Death of Activated Macrophages through the p38 Mitogen-Activated Protein Kinase/STAT1/Reactive Oxygen Species Pathway Mol. Cell. Biol., August 1, 2005; 25(15): 6821 - 6833. [Abstract] [Full Text] [PDF]

				H. S. Kim, I. Chang, J. Y. Kim, K.-H. Choi, and M.-S. Lee Caspase-Mediated p65 Cleavage Promotes TRAIL-Induced Apoptosis Cancer Res., July 15, 2005; 65(14): 6111 - 6119. [Abstract] [Full Text] [PDF]

				M. Thomas, C. E. Finnegan, K. M.-A. Rogers, J. W. Purcell, A. Trimble, P. G. Johnston, and M. P. Boland STAT1: A Modulator of Chemotherapy-induced Apoptosis Cancer Res., November 15, 2004; 64(22): 8357 - 8364. [Abstract] [Full Text] [PDF]

				K. B. Bouker, T. C. Skaar, D. R. Fernandez, K. A. O'Brien, R. B. Riggins, D. Cao, and R. Clarke Interferon Regulatory Factor-1 Mediates the Proapoptotic but Not Cell Cycle Arrest Effects of the Steroidal Antiestrogen ICI 182,780 (Faslodex, Fulvestrant) Cancer Res., June 1, 2004; 64(11): 4030 - 4039. [Abstract] [Full Text] [PDF]

				S. Y. Park, I. Chang, J.-Y. Kim, S. W. Kang, S.-H. Park, K. Singh, and M.-S. Lee Resistance of Mitochondrial DNA-depleted Cells against Cell Death: ROLE OF MITOCHONDRIAL SUPEROXIDE DISMUTASE J. Biol. Chem., February 27, 2004; 279(9): 7512 - 7520. [Abstract] [Full Text] [PDF]

				B. Feldhaus, I. D. Dietzel, R. Heumann, and R. Berger Effects of Interferon-{gamma} and Tumor Necrosis Factor-{alpha} on Survival and Differentiation of Oligodendrocyte Progenitors Reproductive Sciences, February 1, 2004; 11(2): 89 - 96. [Abstract] [PDF]

				H. Lee, S. Cha, M.-S. Lee, G. J. Cho, W. S. Choi, and K. Suk Role of Antiproliferative B Cell Translocation Gene-1 as an Apoptotic Sensitizer in Activation-Induced Cell Death of Brain Microglia J. Immunol., December 1, 2003; 171(11): 5802 - 5811. [Abstract] [Full Text] [PDF]

				K. Chen, Y. Wei, G. C. Sharp, and H. Braley-Mullen Mechanisms of Spontaneous Resolution versus Fibrosis in Granulomatous Experimental Autoimmune Thyroiditis J. Immunol., December 1, 2003; 171(11): 6236 - 6243. [Abstract] [Full Text] [PDF]

				K. Suk, H. Lee, S. S. Kang, G. J. Cho, and W. S. Choi Flavonoid Baicalein Attenuates Activation-Induced Cell Death of Brain Microglia J. Pharmacol. Exp. Ther., May 1, 2003; 305(2): 638 - 645. [Abstract] [Full Text] [PDF]

				I. Chang, S. Kim, J. Y. Kim, N. Cho, Y.-H. Kim, H. S. Kim, M.-K. Lee, K.-W. Kim, and M.-S. Lee Nuclear Factor {kappa}B Protects Pancreatic {beta}-Cells From Tumor Necrosis Factor-{alpha}-Mediated Apoptosis Diabetes, May 1, 2003; 52(5): 1169 - 1175. [Abstract] [Full Text] [PDF]

				J. Sanceau, D. D. Boyd, M. Seiki, and B. Bauvois Interferons Inhibit Tumor Necrosis Factor-alpha -mediated Matrix Metalloproteinase-9 Activation via Interferon Regulatory Factor-1 Binding Competition with NF-kappa B J. Biol. Chem., September 13, 2002; 277(38): 35766 - 35775. [Abstract] [Full Text] [PDF]

				P. K. Baker, A. R. Pettitt, J. R. Slupsky, H. J. Chen, M. A. Glenn, M. Zuzel, and J. C. Cawley Response of hairy cells to IFN-alpha involves induction of apoptosis through autocrine TNF-alpha and protection by adhesion Blood, June 28, 2002; 100(2): 647 - 653. [Abstract] [Full Text] [PDF]

				V. T. Nguyen and E. N. Benveniste Critical Role of Tumor Necrosis Factor-alpha and NF-kappa B in Interferon-gamma -induced CD40 Expression in Microglia/Macrophages J. Biol. Chem., April 12, 2002; 277(16): 13796 - 13803. [Abstract] [Full Text] [PDF]

				J. Lee, J. Hur, P. Lee, J. Y. Kim, N. Cho, S. Y. Kim, H. Kim, M.-S. Lee, and K. Suk Dual Role of Inflammatory Stimuli in Activation-induced Cell Death of Mouse Microglial Cells. INITIATION OF TWO SEPARATE APOPTOTIC PATHWAYS VIA INDUCTION OF INTERFERON REGULATORY FACTOR-1 AND CASPASE-11 J. Biol. Chem., August 24, 2001; 276(35): 32956 - 32965. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 276/16/13153    most recent M007646200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Suk, K. Articles by Lee, M.-S. Search for Related Content PubMed PubMed Citation Articles by Suk, K. Articles by Lee, M.-S. Social Bookmarking                      What's this?