Báo cáo khoa học: IFN-c enhances TRAIL-induced apoptosis through IRF-1 pptx

IFN-c enhances TRAIL-induced apoptosis through IRF-1 Sang-Youel Park 1 , Jae-Won Seol 1 , You-Jin Lee 1 , Jong-Hoo Cho 1 , Hyung-Sub Kang 1 , In-Shik Kim 1 , Soo-Hyun Park 1 , Tae-Hyoung Kim 2 , John H. Yim 3 , Moonil Kim 3 , Timothy R. Billiar 3 and Dai-Wu Seol 3 1 Bio-Safety Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk, South Korea; 2 Department of Biochemistry, Chosun University School of Medicine, Dong-Gu, Gwangju, South Korea; 3 Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Tumor n ecrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family and a potent inducer of apoptosis. TRAIL has been shown to effectively limit tumor growth in vivo without detectable cytotoxic side- effects. Interferon (IFN)-c often modulates the anticancer activities of TNF family members including TRAIL. How- ever, little is known about the mechanism. To explore the mechanism, A549, HeLa, LNCaP, Hep3B and HepG2 cells were pretreated with IFN-c, and then exposed to TRAIL. IFN-c pretreatment augmented TRAIL-induced apoptosi s in all these cell lines. A549 cells were selected and further characterized for IFN-c action in TRAIL-induced apopto- sis. Western blotting analyses revealed that IFN-c dramat- ically increased the protein levels of interferon regulatory factor (IRF)-1, but not TRAIL receptors (DR4 and DR5) and pro-apoptotic (FADD a nd Bax) and anti-apoptotic factors ( Bcl-2, Bcl-XL, cIAP-1, cIAP-2 and XIAP). T o elu- cidate the functional r ole of I RF-1 in IFN- c-enhanced TRAIL-induced apoptosis, IRF-1 was first overexpressed by using an adenoviral v ector AdIRF-1. IRF-1 overexpres- sion minimally increased apoptotic cell death, but signifi- cantly enhanced apoptotic cell death induced by TRAIL when infected cells were treated with TRAIL. In further experiments using an antisense oligonucleotide, a specific repression of IRF-1 e xpression abolished enhancer activity of IFN-c for TRAIL-induced apoptosis. T herefore, our data indicate that IFN-c enhances TRAIL-induced apoptosis through IRF-1. Keywords: a poptosis; IFN-c;IRF-1;TRAIL. Apoptosis is a n active cell death proc ess that i s genetically regulated. This process plays an important role in the development and homeostasis of multicellular organisms [1]. Among apoptosis-inducing proteins, the best character- ized are the ligand-type cytokine molecules of the TNF family. T NF famil y member proteins such as TNF-a,Fas ligand and TRAIL are type II transmembrane molecules that trigger the apoptotic signal cascade by ligating cognate receptors displayed on the cell surface [2,3]. Although TRAIL is a T NF family member [4,5], it has some notable differences when compared with TNF- a and FasL. For example, unlike Fas, TRAIL receptors DR4 and DR5 are widely expressed [4,5], thus most tissues and cell types are potential targets to TRAIL. Furthermore, TRAIL induces apoptosis in a wide variety of tumor cells but not in most normal cells. Recent preclinical studies demonstrated that repeated systemic administration of recombinant TRAIL protein effectively limited tumor growth without detectable toxicity [6,7]. Thus, considerable attention has been paid to TRAIL as a promising therapeutic to treat human cancers. The transcription factor interferon regulatory factor (IRF)-1 was identified as a regulator of the interferon (IFN)-c system [8]. Accumulated evidence shows that IRF-1 fun ctions as a tumor suppressor [9–17]. IRF-1 suppresses the transformed phenotype [9,14,15] and i s essential for DNA-damage-induced apoptosis in mitogen- activated T lymphocytes [11,12]. IFN-c has b een also shown to sensitize cells to various apoptotic stimuli including TNF family members [18–20]. Recently, several studies demonstrated IFN-c and TNF synergism in cancer cell apoptosis and necrosis [18,20,21] and recent studies have also shown that IFN synergistically induced TRAIL-mediated apoptosis [22–25]. H owever, little i s known about the synergy or enhancing molecular mech- anism of I FN-c on tumor cell apoptosis. Thus, w e investigated the role and regulation mechanism of IFN-c in T RAIL-induced apoptosis. In A549, HeLa, LNCaP, Hep3B and HepG2 cells, IFN-c-pretreatment augmented TRAIL-induced apoptosis. In A549 cells, IFN-c dramat- ically increased t he protein l evels of IRF-1. Overexpres- sion of IRF-1 protein by an adenoviral vector AdIRF-1 increased TRAIL-induced apoptosis upon expo sure of infected cells to TRAIL treatment. IFN-c-enhanced TRAIL-induced apoptosis was significantly blocked by antisense oligonucleotide that specifically suppresses IRF-1 protein expression. Therefore, our data indicate that IRF-1 is a key component in the IFN-c enhance- ment mechanism in TRAIL-induced apoptosis. Correspondence to D W. Seol, BST W1513 Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. Fax: +1 412 6241172, Tel.: +1 412 6246704, 2 E-mail: seold@pitt.edu Abbreviations: IFN, interferon; IRF, interferon regulatory factor; NK, natural killer; TRAIL, TNF-related apoptosis-inducing ligand; TNF, tumor necrosis factor. (Received 22 April 2004, revised 1 September 2004, accepted 7 September 2004) Eur. J. Biochem. 271, 4222–4228 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04362.x Materials and methods Cell culture A549 (a human lung carcinoma), HeLa (a human cervical carcinoma), LNCaP (a human prostate cancer cell line), Hep3B (a human hepatocellular carcinoma) and HepG2 (a human hepatocellular carcinoma) cells were obtained from ATCC and maintained in suggested culture medium supplemented w ith 10% (v/v) fetal bovine serum and antibiotics (100 lgÆmL )1 gentamycin and 100 lgÆmL )1 penicillin/streptomycin). Cell viability Cells grow n in 12-wells were pretre ated with human IFN-c (100 U ÆmL )1 ) (Roche Molecular Biochemicals, Mannheim, Germany). After 12 h, recombinant human TRAIL protein [26] was added to culture media directly and coincubated for an additional 3 h. Cell viability w as determined by thecrystal violet staining method as described [27], and cell m orphology was photographed under the microscope. Briefly, cells were stained for 10 min at room temperature w ith staining solution [0.5% (v/v) crystal violet in 30% (v/v) ethanol and 3% (v/v) formaldehyde], washed four times with water, and dried. Cells were lysed with 1% (w/v) SDS solution, and measured at 550 nm. Cell v iability was calculate d from relative dye intensity and compared with the controls. Western blotting To prepare whole cell lysates, cells were harvested, resus- pended in lysis buffer [25 m M HEPES (pH 7.4), 100 m M NaCl, 1 m M EDTA, 5 m M MgCl 2 ,0.1m M dithiothreitol, and protease inhibitor mixture] and sonicated. Proteins were separated on 12 or 15% (w/v) SDS gel and analyzed by Western blotting as described pre viously [28,29]. DR4 (AAP- 420), DR5 (AAP-430), caspase-3 (AAP-103) and caspase-8 (AAP-118) were probed with a ntibody obtained from Stressgen (Victoria, BC, Canada) and IRF-1 (sc-497) and IRF-2 (sc-498) from Santa Cruz (Santa Cruz, CA, USA). Adenoviral vectors E1- and E3-deleted AdIRF-1 was constructed through Cre-lox recombination as described previously [30]. Briefly, cDNA for IRF-1 or EGFP, driven by the CMV promoter and terminated by the SV40 poly(A) signal was inserted into theshuttlevectorpAdloxtocreatepAdlox-IRF-1or pAdlox-EGFP. Recombinant adenovirus was generated by cotransfection of appropriately digest ed pAdlox-IRF-1 or pAdlox-EGFP and Y5 helper virus DNA into the Ad packaging cell line CRE8 which expresses Cre recombinase. Recombinant adenoviruses were propagated on 293 cells and purified by cesium chloride density gradient centrifu- gation and subsequent dialysis. Adenoviral infection A549 cells were plated in six- or 12-well plates, a nd adenoviral infections were performed t he next day for 4 h with virus diluted in Opti-MEM I (Gibco 3 , Grand Island, NY, USA) to the desired multiplicity of infection (0–80). The infected cells were washed three times with phosphate- buffered s aline a nd maintained with the F-12K culture medium. A fter 24 h, infected ce lls were e xposed to recom- binant TRAIL protein for 3 h. Cell viability was determined by the crystal violet staining method [28], and morphology was photographed under the microscope. IRF-1 protein expressio n in AdIRF-1-infected cells was confirmed by Western blotting. Transfection of oligonucleotides A549 cells grown in six- or 12-well were transfected with 2 lg o f IRF-1 sense (S) or antisense (AS) phosphothioated oligonucleotide (S, 5¢-GCATCTCGGGCATCTTTC-3¢; AS, 5¢-GAAAGATGCCCGAGATGC-3 ¢) [ 31,32] using GenePorter transfection reagent (Gene Therapy Systems 4 , San Diego, C A, USA). After 6 h, the cells were exposed to IFN-c for 12 h and coincubated with TRAIL protein for an additional 3 h, then assayed for viability. For I RF-1 immunoblotting, t ransfected cells were tr eated with IFN-c for 2 h before the cells were lysed . Results Enhancement of TRAIL-induced apoptosis by IFN-c In some cell types, IFN-c induces cell death and has antitumor activities [33–35]. IFN-c has also been shown to increase susceptibility of target cells to Fas ligand- or TNF-a-induced apoptosis [18,20,36]. Generally, combina- tion therapies produce a better efficacy t han i ndividual therapies in cancer treatment. We previou sly observed that A549 cells (from human lung carcinoma) are relatively resistant to TRAIL. Thus, we selected A549 cells as our experimental model to determine wh ether IFN-c also enhances TRAIL-induced apoptosis. A549 cells were pretreated with IFN-c (100 UÆmL )1 ) for 12 h, and then e xposed to recombinant TRAIL protein [26] for an additional 3 h. The results of cell viability tests showed that TRAIL alone induced 20% cell death after a 3-h incubation, but 12-h IFN-c pretreatment increased TRAIL-induced cell death to more than 60% (Fig. 1A). IFN-c treatment alone did not induce cell death in this cell line. Cell death induced by TRAIL or TRAIL plus INF-c was completely b locked by a pan-caspase inhib- itor z-VAD-fmk o r a caspase-8 inhibitor z-IETD-fmk (Fig. 1 B), indicating that observed cell death is apoptotic cell death rather than necrotic cell death. Examination of cell m orphology also supported enhancer activity of IFN-c in TRAIL-induced apoptosis (Fig. 1C). Consis- tently, more caspase-8 was activated by cotreatment with IFN-c and TRAIL than by TRAIL a lone (Fig. 1D). Caspase-3 activation was also observed t o increase only slightly in response to treatment with IFN-c and TRAIL, compared with that observed for TRAIL alone (Fig. 1D). IFN-c was also observed to enhance TRAIL-induced apoptosis in other cell lines such as HeLa (a cervical carcinoma), LNCaP (a prostate cancer cell line), Hep3B (a hepatocellular c arcinoma) and Hep G2 (a hepatoc ellular carcinoma) (Fig. 1E), indicating that IFN-c acts in a bro ad range of tissues to enhance TRAIL-induced apoptosis. Ó FEBS 2004 IFN-c enhances TRAIL-induced apoptosis 1 (Eur. J. Biochem. 271) 4223 Stimulation of IRF-1 protein expression by IFN-c Generally, extra-cellular stimuli activate intracellular sign- aling cascades by stimulating the factors involved in the signaling cascades. Recently, we reported that TRAIL death-inducing signal transmits from activated receptors through c aspase-8, Bid, released cytochrome c , and execu- tioner caspases including caspase-3 [28]. It was suggested that modulation of any of these signaling components regulate TRAIL-induced apoptosis. Thus, we investigated whether IFN-c treatment regulates expression of these molecules. A549 cells were pretreated with IFN-c,further exposed to TRAIL and subjected to Western blotting analyses. IFN-c or IFN- c plus TRAIL treatment dramat- ically increased the protein levels of IRF-1, but not TRAIL receptors (DR4 and DR5) and IRF-2 ( Fig. 2). Treatment with TRAIL a lone did not affect IRF-1 expression, indicating that the increase o f IRF-1 by IFN-c plus TRAIL is mainly controlled by IFN-c. In parallel, we also examined other signaling components known to a ffect mainstream signaling of TRAIL-induced cell death. S imilar to T RAIL receptors, IFN-c treatment did not change the expression 0 30 60 90 120 A B D E C IFN-γ TRAIL IFN-γ TRAIL IFN-γ TRAIL IFN-γ TRAIL + –– – – – –– –– – – – –– – + + + + ++ + 0 30 60 90 120 ++ + None z-VAD-fmk z-IETD-fmk LNCaPHeLa Hep3B HepG2 0 30 60 90 120 Control TRAIL IFN- γ IFN- γ +TRAIL Procaspase-3 Procaspase 8 Active form Active form + ++ + Fig. 1. Effect of IFN-c on TRAIL-induced apoptosis. (A) A549 cells plated in 12-well were pretreated with IFN-c (100 UÆmL )1 ) for 12 h, and then coincubated with or witho ut reco mbinant TRAIL pro tein (100 ngÆmL )1 ) for an additional 3 h. Cell viability was determined by crystal violet staining method. Viability of control cells was set at 100%, and viability relative to the control was presented. The experiments were performed at triplicate, at least twice. The bar indicates standard error. (B) A549 cells plated in 12-well were pretreated with IFN-c (100 UÆmL )1 )for11hand incubated with z-VAD-fmk (100 l M ) or z-IETD-fmk (100 l M ) for an additional 1 h, and then coincubated with or without recombinant TRAIL protein (100 ngÆmL )1 ) for 3 h. Cell viability was determined as described in (A). (C) Cell morphology under the conditions as described in (A) was photographed. (D) A549 cells were pretreated with IFN-c (100 UÆmL )1 ) for 12 h, and then coincubated with or without recombinant TRAIL protein (100 ngÆmL )1 ) for 1 h. Whole cell lysates were prepared as described in Materials and methods and subjected to Western blotting analysis. (E) Cell viability of HeLa, LNCaP, Hep3B and HepG2 cells in response to media (control), IFN-c, TRAIL, or TRAIL plus IFN-c was determined as described in (A). 4224 S Y. Park et al. (Eur. J. Biochem. 271) Ó FEBS 2004 levels of other p ro-apoptotic proteins such as FADD and Bax (data n ot shown) and anti-apoptotic proteins such as Bcl-2, Bcl-XL, cIAP-1, cIAP-2 and XIAP (Fig. 2B). Our data suggest that IRF-1, a nuclear transcription factor, may play a role in mediating enhancer effects of IFN-c in TRAIL-induced A549 cell apoptosis. Enhancement of TRAIL-induced apoptosis by overexpression of IRF-1 protein IRF-1 has been shown to suppress tumor growth in vivo [10,17,37]. Thus, we h ypothesized that IRF-1 m ay directly mediate the enhancer effects of IFN-c in TRAIL-induced apoptosis. T o examine this possibility, we first over- expressed IRF-1 by taking advantage of AdIRF-1, an adenoviral vector expressing IRF-1. AdIRF-1 infected cells showed a minimal increase in baseline cell death throughout the experimental settings, whereas additional TRAIL treat- ment significantly increased cell death in AdIRF-1-infected cells (Fig. 3A). In contrast, AdEGFP infection did not significantly change cell d eath in response to T RAIL. Examination of cell morphology also supported the func- tional role of A dIRF-1 in TRAIL-induced cel l death (Fig. 3 B). To confirm IRF-1 protein expression by AdIRF-1 infection, infected cells were subjected to Western blotting analysis (Fig. 3C). I RF-1 protein was highly expressed by AdIRF-1 infection in contrast to the AdEGFP A TRAIL IFN-γ DR4 DR5 IRF-1 IRF-2 + ++ + Bcl-2 Bcl-XL XIAP cIAP-1 cIAP-2 TRAIL IFN-γ B + ++ + – –– –– – –– Fig. 2. Western blot analysis showing expression pattern of various proteins in A549 cells exposed to IFN-c and TRAIL protein. A549 cells were pretreated with IFN-c (100 UÆmL )1 ) for 12 h, and then coincu- bated with or without recombinant TRAIL protein (100 ngÆmL )1 )for 1 h. Whole cell lysates were prepared as de scribed in M aterials and methods and subjected to Western blotting analysis. B M OI +TRAIL -TRAIL AdIRF-1 20 80 A AdIRF-1 AdEGFP 10 20 8040 0 25 50 75 100 80 -TRAIL +TRAIL MOI 0 C IRF-1 AdEGFP AdIRF-1 MOI 80 10 20 40 80 0 NS Fig. 3. Effect of IRF-1 overexpression on TRAIL-induced apoptosis. (A) A549 cells were infected with AdEGFP or AdIRF-1 for 4 h, washed, and further cultured. Twenty-fo ur hours later, recombinant TRAIL protein (100 ngÆmL )1 ) was added to culture medium and incubated for 3 h. Cell viability w as determined by crystal violet staining method. Viability of control cells was set at 100%, and viability relative to the control was presented. The experiments were performed at triplicate, at least twice. The bar indicates standard error. (B) Cell morpholo gy under t he con ditions as d escribed i n (A) was photographed. ( C) A549 cells we re i nfec ted w ith AdEGFP or A dIRF-1 for 4 h, washed, and further cultured. Twenty-four hours later, whole cell lysates were prepared and subjected to Western blotting analysis for IRF-1 expression. The NS indicates a nonspecific protein band that was used to ensure equal protein loading. Ó FEBS 2004 IFN-c enhances TRAIL-induced apoptosis 1 (Eur. J. Biochem. 271) 4225 control vector that showed no expression. This result indicates that TRAIL-induced cell death is enh anced by IRF-1. Blockade of IFN-c-enhancement by IRF-1 suppression in TRAIL-induced apoptosis Although o verexpression of IRF-1 e nhanced TRAIL- induced apoptosis, the role of IRF-1 in mediating IFN-c enhancer activity in TRAIL-induced apoptosis is unclear. Therefore, to address this question, we used an antisense oligonucleotide that specifically suppresses IRF-1 protein expression. A549 cells were transfected with a sense or antisense oligonucleotide, and pretreated with IFN-c for 12 h, followed by TRAIL treatment for an additional 3 h . The sense oligonucleotide d id not affect TRAIL- induced apoptosis in IFN-c-pretreated cells. However, the antisense oligonucleotide almost completely p rotected IFN-c-pretreated cells from TRAIL-induced cell death (Fig. 4 A). Western blotting analysis revealed that IRF-1 protein expression was effectively suppressed by the antisense oligonucleotide (Fig. 4B). Therefore, our data demonstrate that IFN-c enhances TRAIL-induced apop- tosis through IRF-1, and IRF-1 is a key mediator in transmitting IF N-c enhancer signal in TRAIL-induced cell death. Discussion We have demonstrated that IRF-1 directly regulates IFN-c enhancement of TRAIL-induced apoptosis. Overexpression of IRF-1 protein by AdIRF-1 enhanced TRAIL-induced apoptosis, and a specific suppression of IRF-1 protein expression by an antisense oligonucleotide prevented enhancer activity of IFN-c in TRAIL-induced apoptosis. This is the first indication that IFN-c enhancement of TRAIL-induced apoptosis is regulated by IRF-1 protein. Other studies have demonstrated that IFN-c also synergizes Fas- and TNF receptor-mediated tumor cell d eath [10,18– 20,36]. Thus, IFN-c commonly enhances cell death induced by the three major death-inducing ligands of the TNF family. These results indicate that IFN-c regulation of death signaling pathway is commonly involved in TRAIL-, Fas ligand- and TNF-a-induced cell death. However, it is poorly understood how IRF-1 regulates IFN-c enhancement of apoptosis induced by these ligand molecules. As suggested [10,18–20,36], IFN-c or IFN- c-induced IRF-1 may inhibit activation of the transcription factor nuclear factor-kappa B which a ntagonizes activation of various apoptosis-inducing signals. IRF-1 was shown to play a c ritical role in DNA- damage-induced apoptosis in mature T lymphocytes [11,12], and regulate a cycline-dependent kinase inhibitor p 21 and lysyl 5 oxidase genes [13,38]. Thus, it is tempting to examine if p21-driven c ell cycle arrest is involved in this enhancer mechanism. In addition, as we reported recently [39], IRF-1 may regulate expression of cellular factors induced by TRAIL an d enhance TRAIL-induced apoptosis. However, which cellular factors are the targe ts of IRF-1 has yet to be determined. The protein level of FADD, Bax, Bcl-2, Bcl- XL, cIAP-1, cIAP-2 and XIAP that are known to act in death signaling pathways in TRAIL-induced apoptosis did not change significantly in response to IFN-c. Thus, other cellular factors involved in death signaling pathways activated by TRAIL are now under investigation. We do not rule out the possibility that IRF-1 may transmit enhancer activity of INF-c via a protein–protein interaction in TRAIL-induced apoptosis. A s well docu- mented, p53, a tumor suppressor and transcription f actor, modulates cell physiology not only b y interacting with various cellular factors [40,41], but also by regulating transcription of the target genes [42–44]. Thus, this possi- bility is also under investigation in this laboratory. Importantly, a recent study demonstrated that TRAIL plays an essential role in the natural killer (NK) cell- mediated and IFN-c-dependent tumor surveillance in vivo [45,46]. IFN-c was shown to modulate TRAIL-mediated tumor s urveillance, not only by r egulating TRAIL expres- sion on NK cells, but also by sen sitizing tumor cells to TRAIL-induced cytotoxicity. Although the mechanism by which IFN-c sensitizes tumor cells to TRAIL-induced apoptosis was not elucidated in the report, our data suggest an active role of IRF-1 i n t he mechanism. Thus, our data sheds light on better un derstanding an in vivo tumor A 0 25 50 75 100 T RAIL Sense Antisense IFN-γ + – ––– – + ++ + + B Control Sense Antisense IFN-γ IRF-1 NS +++ Cell Viability Fig. 4. Effect of o ligonucleotides on IFN-c enhanced TRAIL-induced apoptosis. (A) Six hours after transfection of IRF-1 sense or antisense oligonucleotide, A549 cells were pretreated with IFN-c (100 UÆmL )1 ) for 12 h, and further exp osed to TRAIL protein (0 or 100 ngÆmL )1 )for 3 h. Cell viability w as determined by crystal violet staining method. Viability of control cells was set at 100%, and viability relative to the control was presented. The experiments were perfo rmed at triplic ate, at least twice. T he b ar indicates standard erro r. ( B) S ix hours a fter transfection of IRF-1 sense or antisense oligonucleotide, A549 c ells were pretreated with IFN-c (100 UÆmL )1 ) f or 2 h. Whole cell lysates were prepared and subjected to Western blotting analysis for IRF-1 expression. The NS indicates a nonspecific protein band that was used to ensure equal protein loading. 4226 S Y. Park et al. (Eur. J. Biochem. 271) Ó FEBS 2004 surveillance mechanism [45,46]. This result and our data also suggest a possible c ombination therapy o f IFN-c and TRAIL for cancer treatment in humans. Because combi- nation therapies produce a better prognosis than individu al therapies in cancer treatment, the combination of IFN-c and TRAIL may b e a very promising a nticancer therapy to treat human cancers. Furthermore, our data also suggest that in addition to IFN-c, IRF-1 may combine with TRAIL protein to induce effective tumor cell death. Acknowledgements This work was supported by the Competitive Medical Research Fund, the Department of D efense grant D AMD17-01-1-0607 and Vascular System Research Grant of KOSEF (D W.S.), and National Institutes of Health grants GM4410 0 a nd GM53789 (T.R.B.), and Bio-Safety Research Institute grant, Chonbuk National University in 2004 (S Y.P.). References 1. Nagata, S. (1997) Apoptosis by death factor. Cell 88, 355–365. 2. Cha, S.S., Kim, M.S., Choi, Y.H., Sung, B.J., Shin, N.K., Shin, H.C., Sung, Y.C. & Oh, B.H. (1999) 2.8 A ˚ resolution crystal structure of hum an TRAIL, a c ytokine with se lective a ntitumor activity. Immunity 11, 253–261. 3. Hymowitz, S.G., Christinger, H.W., Fuh, G., Ultsch, M., O’Connell, M., K elley, R.F., Ashkenazi, A. & de Vos, A.M. (1999) Triggering cell death: the crystal structure of Apo2L/TRAIL in a complex with death receptor 5. Mol. Cell 4, 563–571. 4. 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T herefore, our data indicate that IFN-c enhances TRAIL-induced apoptosis through. 1E), indicating that IFN-c acts in a bro ad range of tissues to enhance TRAIL-induced apoptosis. Ó FEBS 2004 IFN-c enhances TRAIL-induced apoptosis 1 (Eur.