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[Cancer Research 66, 3051-3061, March 15, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Inhibition of Lymphotoxin-ß Receptor–Mediated Cell Death by Survivin-{Delta}Ex3

Ren-In You1, Mei-Chieh Chen2, Hsei-Wei Wang1, Yang-Chieh Chou1, Chi-Hung Lin1 and Shie-Liang Hsieh1,3,4

1 Institute and Department of Microbiology and Immunology, National Yang-Ming University; 2 Department of Microbiology and Immunology, Taipei Medical University; 3 Immunology Research Center, Taipei Veterans General Hospital; 4 Genomics Research Center, Academia Sinica, Taipei, Taiwan

Requests for reprints: Shie-Liang Hsieh, Institute of Microbiology and Immunology, National Yang-Ming University, Shih-Pai, Taipei 11221, Taiwan. Phone: 886-2-28267161; Fax: 886-2-28212880; E-mail: slhsieh{at}ym.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TNFSF14/LIGHT is a member of the tumor necrosis factor superfamily that binds to lymphotoxin-ß receptor (LTßR) to induce cell death via caspase-dependent and caspase-independent pathways. It has been shown that cellular inhibitor of apoptosis protein-1 inhibits cell death by binding to LTßR-TRAF2/TRAF3 complexes and caspases. In this study, we found that both Kaposi's sarcoma–associated herpesvirus K7 (KSHV-K7), a viral inhibitor of apoptosis protein, and the structurally related protein survivin-{Delta}Ex3 could inhibit LTßR-mediated caspase-3 activation. However, only survivin-{Delta}Ex3 could protect cells from LTßR-mediated cell death. The differential protective effects of survivin-{Delta}Ex3 and KSHV-K7 can be attributed to the fact that survivin-{Delta}Ex3, but not KSHV-K7, is able to maintain mitochondrial membrane potential and inhibit second mitochondria-derived activator of caspase/DIABLO release. Moreover, survivin-{Delta}Ex3 is able to inhibit production of reactive oxygen species and can translocate from nucleus to cytosol to associate with apoptosis signal-regulating kinase 1 after activation of LTßR. Furthermore, survivin-{Delta}Ex3 protects LTßR-mediated cell death in caspase-3-deficient MCF-7 cells. Thus, survivin-{Delta}Ex3 is able to regulate both caspase-dependent and caspase-independent pathways, whereas inhibition of caspase-independent pathway is both sufficient and necessary for its protective effect on LTßR-mediated cell death. (Cancer Res 2006; 66(6): 3051-61)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LIGHT [homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator (HVEM), a receptor expressed by T lymphocytes], a member of tumor necrosis factor (TNF) superfamily, exists as either a 29-kDa type II transmembrane protein and/or a soluble protein following metalloproteinase cleavage from the cell membrane (1). LIGHT is able to interact with lymphotoxin-ß receptor (LTßR) to induce cell death (2–4) or with HVEM to modulate immune cell functions (5–7). Moreover, LIGHT interacts with decoy receptor 3 (8), a soluble receptor overexpressed by tumor cells (9) and activated dendritic cells (10). LIGHT enhances IFN-{gamma} secretion by activated T cells (11), and IFN-{gamma} potentiates LIGHT-mediated cell death in MDA-MB-231 human breast cancer cells (7, 12) as well as in p53-deficient HT-29 human adenocarcinoma cells (13–15) and Hep3BT2 human hepatoma cells (16, 17). LIGHT also induces cell death in caspase-3-deficient breast cancer cells (MCF-7; ref. 7). Moreover, overexpression of Bcl-2 further enhances LTßR-mediated cell death (16). These observations indicate the complexity of signaling pathways initiated by LTßR engagement.

After engagement with LIGHT, LTßR self-aggregates and subsequently activates multiple signaling pathways, involving, for example, transcription factor nuclear factor-{kappa}B and c-Jun NH2-terminal kinase (3, 18, 19). Cross-linking of LTßR recruits TNF receptor–associated factors, a family of six RING finger proteins that bind directly to the cytosolic domain of LTßR (14, 20). Moreover, we have shown that cross-linking of LTßR is able to recruit TRAF3 and TRAF5 to activate the apoptosis signal-regulating kinase 1 (ASK1) to induce caspase-3-independent cell death (17). ASK1 is regulated by phosphorylation, oligomerization (21), and protein-protein interactions. In the normal reducing environment, ASK1 activity is inhibited via binding to thioredoxin (22), glutaredoxin (23), 14-3-3 (24), heat shock protein 72 (25), Raf-1 (26), protein serine/threonine phosphatase 5 (27), and glutathione S-transferases (28). After direct or indirect oxidative stress (i.e., H2O2, TNF-{alpha}, glucose/serum deprivation, or heat shock), these proteins are oxidized and form intramolecular disulfide bonds, which results in a conformational change and dissociation from ASK1. Unbound ASK1 then self-oligomerizes, becomes activated via either autophosphorylation and/or cross-phosphorylation, and subsequently phosphorylates downstream kinases to initiate an apoptotic program (29).

To further identify the proteins associated with LTßR after engagement with LIGHT, the LTßR-LIGHT complex was purified and analyzed by mass spectrometry. It is interesting to note that several proteins, such as TRAF2, TRAF3, second mitochondria-derived activator of caspase (Smac), and cellular inhibitor of apoptosis protein-1 (cIAP1), are also coprecipitated with LTßR-LIGHT complex (30). Although IAPs are able to inhibit death domain–containing receptors, such as TNFRI and Fas (31), their roles in the regulation of cell death pathways initiated by LTßR, which lacks death domain in the cytoplasm, have not been tested.

Recently, a novel viral antiapoptotic protein vIAP encoded by open reading frame of Kaposi's sarcoma–associated herpesvirus K7 (KSHV-K7) was identified (32). KSHV-K7 inhibits apoptosis by binding to Bcl-2 and caspase-3 via its putative BH2 and baculovirus IAP repeat (BIR) domains, respectively, thus preventing caspase-3 activation (32). KSHV-K7 is structurally related to survivin-{Delta}Ex3, a variant of human survivin that lacks exon 3 and has an additional frame shift in exon 4 (33). Both KSHV-K7 and survivin-{Delta}Ex3 have antiapoptotic properties and contain (a) a mitochondrial targeting sequence, (b) a BIR domain, and (c) a putative BH2-like domain (32). However, it is still not known whether survivin-{Delta}Ex3 has an antiapoptotic effect similar to that of KSHV-K7.

Here, we report that both survivin-{Delta}Ex3 and KSHV-K7 inhibit caspase-3 activity, but only survivin-{Delta}Ex3 can prevent LTßR-mediated cell death by inhibiting ASK1 activity, preventing Bcl-2 cleavage, and maintaining mitochondrial membrane potential ({Delta}{Psi}m). This reveals a novel mechanism used by survivin-{Delta}Ex3 to inhibit cell death via the targeting of multiple proteins involved in LTßR-mediated signaling pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Human hepatoma cells (Hep3BT2), human cervical carcinoma cells (HeLa), and human embryonic kidney cells (HEK293) were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Life Technologies) at 37°C in 5% (v/v) CO2.

Plasmids and transfection. The hemagglutinin (HA)–tagged expression constructs of ASK1 and catalytically inactive ASK1 (ASK1-KE) were kindly provided by Dr. Wen-Chen Yeh (Toronto University, Toronto, Ontario, Canada). HA-tagged Smac was a kind gift from Dr. Lih-Ling Lin (Wyeth Co., Boston, MA). The dominant-negative TRAF3 mutant was provided by Dr. Bharat B. Aggarwal (M.D. Anderson Cancer Center, Houston, TX). Survivin-{Delta}Ex3 and KSHV-K7 cDNA were subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) or pFLAG-CMV2 (Eastman Kodak Co., Rochester, NY). Deletion mutants survivin-{Delta}Ex3 ({Delta}BIR; deletion of amino acids 38-43), survivin-{Delta}Ex3 ({Delta}NLS; deletion of amino acids 81-98), and survivin-{Delta}Ex3 ({Delta}BH2; deletion of amino acids 101-107) with HA tag were subcloned into pCR3.1 (Invitrogen). To pull down these mutants with anti-Myc and anti-FLAG monoclonal antibodies (mAb), the cDNA of these deletion mutants were subcloned into pcDNA3.1 (Invitrogen) to get Myc tag and to pFLAG-CMV2 to get FLAG tag, respectively. For DNA transfection, cells were plated at a density of 3 x 105/mL, grown for 16 hours, and then transfected using calcium phosphate method or TransFast (Promega, Madison, WI).

Antibodies and other reagents. Expression of ASK1-HA and Smac-HA was detected using an anti-HA mAb (clone 3F10; Roche Molecular Biochemicals, Indianapolis, IN) or anti-human ASK1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). c-Myc-tagged TRAF3DN, survivin-{Delta}Ex3, and KSHV-K7 were detected using an anti-Myc tag polyclonal antibody (Upstate Biotechnology, Lake Placid, NY) or anti-FLAG antibody (Sigma, St. Louis, MO). Anti-FLAG (M2) affinity beads were obtained from Sigma, and anti-CPP32 was purchased from R&D Systems (Minneapolis, MN). Recombinant human IFN-{gamma} was purchased from Roche Molecular Biochemicals. LIGHT, LIGHT-R228E, and antihuman LTßR mAb were generated as described previously (17). All other chemical reagents, unless specified, were purchased from Sigma.

Generation of survivin-{Delta}Ex3 and KSHV-K7 stable transfectants. Survivin-{Delta}Ex3 and KSHV-K7 constructs were transfected into Hep3BT2 cells using TransFast. Stable transfectants were selected with G418 (800 µg/mL geneticin; Sigma) followed by immunoblot analysis with anti-Myc tag polyclonal antibody to confirm the expression of survivin-{Delta}Ex3 and KSHV-K7.

Determination of cell death. Cell death induced by LIGHT/IFN-{gamma} treatment was detected using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were seeded in 96-well flat-bottomed plates at a density of 5 x 103 per well. After treatment with LIGHT/IFN-{gamma}, 5 mg/mL MTT (10 µL) was added to each well and incubated at 37°C for 4 hours. Cells were then lysed by the addition of 50 µL of 10% (w/v) SDS in 0.4 N HCl per well and incubated at 37°C for another 16 hours. The absorbance of each sample was determined by measuring the absorbance at 570 versus 650 nm using an ELISA reader (RainBow, Tecan, Crailsham, Germany; ref. 16). Unless specified, the concentration of LIGHT and IFN-{gamma} used for cell death assay is 100 ng/mL and 100 units/mL, respectively.

Analysis of caspase-3 activity. Cells were treated with LIGHT/IFN-{gamma} for 12 hours under different conditions. Stimulated cells were collected by centrifugation, washed twice with ice-cold PBS, and resuspended in lysis buffer [50 mmol/L PIPES-NaOH (pH 7.0), 50 mmol/L KCl, 5 mmol/L EGTA, 2 mmol/L MgCl2, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 µg/mL leupeptin, 1 µg/mL pepstatin A] as described previously (16). Cell lysates (50 µg) were diluted with 500 µL ICE standard buffer [100 mmol/L HEPES-KOH (pH 7.5), 10% sucrose, 0.1% CHAPS, 10 mmol/L DTT, 0.1 mg/mL ovalbumin] and incubated at 30°C for 60 minutes with 20 µmol/L fluorescent substrate. Fluorescence intensity was measured using a fluorescence spectrophotometer (Hitachi, Tokyo, Japan) at an excitation wavelength of 325 nm and emission wavelength of 392 nm.

Analysis of {Delta}{Psi}m. Mitochondrial permeability transition was determined by staining cells with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Molecular Probes, Eugene, OR) as described previously (34). Cells (1 x 106) were incubated with JC-1 at 10 µg/mL in medium for 30 minutes at 37°C with moderate shaking. Cells were then centrifuged at 300 x g, 4°C for 5 minutes, washed twice with ice-cold PBS, and finally resuspended in PBS. JC-1 aggregates were detected at 585 nm (FL-2), and JC-1 monomers were detected at 530 nm (FL-1). The percentage of cells with red or green fluorescence was calculated by gate analysis on a FACSCalibur using CellQuest software (Becton Dickinson, Piscataway, NJ). For confocal laser scanning microscopy analysis of mitochondrial function, the JC-1-treated cells were excited at 488 nm, and emission was recorded simultaneously at 527 and 590 nm into independent detectors.

Confocal microscopy. Cells were cultured in chamber slides overnight. After treatment with LIGHT/IFN-{gamma}, cells were fixed with 4% (v/v) paraformaldehyde in PBS for 15 minutes and washed twice with PBS. Cells were then permeabilized with 1% (v/v) Triton X-100 in PBS for 5 minutes. Fixed cells were preincubated for 30 minutes in PBS containing 5% (v/v) bovine serum albumin at room temperature (32). For cytochrome c staining, cells were incubated with monoclonal anti–cytochrome c IgG (clone 6H2.B4 at 1:500; BD PharMingen, San Diego, CA) followed by anti-mouse IgG conjugated with FITC (1:1,000; The Jackson Laboratory, West Grove, PA). Stable cell lines were grown in six-well chamber slides overnight and treated with apoptotic agents. After treatment, cells were fixed and incubated with monoclonal anti-Myc tag antibody (clone 4A6 at 1:300; Upstate Biotechnology) followed by FITC-conjugated anti-mouse IgG (1:1,000; The Jackson Laboratory). For Smac/DIABLO and ASK1 staining, cells were incubated with polyclonal anti-Smac/DIABLO antibody (1:300; R&D Systems) or polyclonal anti-ASK1 antibody (1:100; Santa Cruz Biotechnology) followed by Cy3-conjugated donkey anti-rabbit antibody (1:500; The Jackson Laboratory) or Cy5-conjugated goat anti-rabbit antibody (1:200; The Jackson Laboratory). Cells were then washed 3 x 5 minutes in PBS and then counterstained with 1 mg/mL Hoechst 33342 (Molecular Probes) diluted 1:5,000 to visualize nuclei. Control slides were stained with secondary antibody alone. The distribution of mitochondria was visualized by transfection with pDsRed2-Mito (Clontech, Palo Alto, CA).

Detection of reactive oxygen species accumulation. Cells in six-well plates were cultured in phenol red–free medium and treated with cytokines for the indicated time periods. To detect reactive oxygen species (ROS) generation, cells were labeled with 5 µmol/L 2',7'-dichlorodihydrofluorescein diacetate (H2-DCF-DA; Molecular Probes) or dihydroethidium (Molecular Probes) at 37°C for 15 minutes. The stained cells were analyzed with a flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA) using CellQuest software (BD Biosciences; ref. 16).

Immunoblot analysis. Cell lysates were prepared by the addition of lysis buffer [50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 1% (v/v) NP40, 1 mmol/L PMSF, 2 µg/mL leupeptin, 2 µg/mL aprotonin]. Equal amounts of protein were subjected to electrophoresis, transferred onto nitrocellulose membrane (Hybond-C extra, Amersham Biosciences, Piscataway, NJ), and reacted with appropriate antibodies in PBS containing 5% nonfat dry milk, 0.02% (v/v) Tween 20. Blots were then incubated with horseradish peroxidase-conjugated secondary antibodies and then with enhanced chemiluminescence (ECL) reagents (Amersham Biosciences).

Kinase assay. To measure ASK1 activity, cell lysate (50-100 µg) was incubated with anti-ASK1 mAb followed by protein A-Sepharose CL 4B beads (Amersham Pharmacia, Piscataway, NJ) to pull down ASK1. The kinase activity was assayed at 30°C for 30 minutes with 2 µg myelin basic protein (MBP) as substrate in 30 µL solution containing 20 mmol/L Tris-HCl (pH 7.5)/10 mmol/L MgCl2/0.5 µCi [{gamma}-32P]ATP. Reactions were stopped by the addition of Laemmli sample buffer. Proteins were fractionated on 13% (w/v) SDS-PAGE followed by autoradiography and quantified using a densitometer (Amersham Biosciences; ref. 17).

Preparation of mitochondria, nuclei, and cytosol. Mitochondrial and cytosolic fractions were isolated as described (35) or by the cytosol/mitochondria fractionation kit (Calbiochem, San Diego, CA) to prevent contamination by other organelles. Cells were washed twice with ice-cold PBS and resuspended in ice-cold cytosol extraction buffer (250 mmol/L sucrose, 70 mmol/L KCl, 250 µg/mL digitonin, PIM [one tablet of Complete Mini protease inhibitor mixture (Roche Molecular Biochemicals) in 5 mL PBS containing 5 mmol/L EDTA], 2 mmol/L di-isopropyl fluorophosphate (DFP) in PBS) at a final concentration of 1 x 108/mL. After a 15-minute incubation on ice, when 80% to 90% cells had become trypan blue positive, the preparations were spun at 1,000 x g for 5 minutes. The cytosolic fraction was obtained by centrifugation of the 1,000 x g supernatant at 20,000 x g for 10 minutes at 4°C. To purify mitochondria, the pellets were resuspended in the same volume (as the cytosol extraction buffer) of ice-cold mitochondria lysis buffer [100 mmol/L NaCl, 10 mmol/L MgCl2, 2 mmol/L EGTA, 2 mmol/L EDTA, 1% (v/v) NP40, 10% (v/v) glycerol (v/v), PIM, 2 mmol/L DFP in 50 mmol/L Tris (pH 7.5)] and incubated for 10 minutes on ice followed by 10-minute centrifugation at 10,000 x g. The supernatants were taken as mitochondrial fractions. To prepare samples for Western blotting, either cytosolic or mitochondrial fraction (24 µL) was mixed with 8 µL of 4 x SDS sample buffer containing 8% (v/v) ß-mercaptoethanol, boiled for 5 minutes, and kept at –20°C until use. Western blotting was done as described above. The blots were probed with mAbs against cytochrome c (6H2.B4; Imgenex, San Diego, CA), cytochrome oxidase IV (COX IV; Molecular Probes), actin (Sigma), and Smac/DIABLO (R&D Systems). To prepare nuclear and cytosolic fractions, cell were washed with ice-cold PBS in 10 mmol/L HEPES (pH 7.9), 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 10 mmol/L KCl, 1 mmol/L DTT, and 0.5 mmol/L PMSF. After swelling on ice for 10 minutes, plasma membranes were disrupted by adding 0.1% (v/v) NP40 and mixing for 10 seconds. Cell breakage was examined under the microscope. The nuclei were pelleted by centrifugation at 800 x g for 45 seconds at 4°C, and the cytoplasmic fraction (supernatant) was recovered. The pellet was then washed thrice in 1 mL ice-cold sucrose buffer [0.32 mol/L sucrose, 3 mmol/L CaCl2, 2 mmol/L magnesium acetate, 0.1 mmol/L EDTA, 10 mmol/L Tris-HCl (pH 8.0), 1 mmol/L DTT, 0.5 mmol/L PMSF] and then centrifuged at 800 x g for 5 minutes at 4°C. The pellet was subsequently resuspended in 20 mmol/L HEPES (pH 7.9), 1 mmol/L EDTA, 1 mmol/L EGTA, 400 mmol/L NaCl, 1 mmol/L DTT, and 1 mmol/L PMSF. After rocking for 20 minutes at 4°C, samples were centrifuged at 10,000 x g for 15 minutes at 4°C to recover the nuclear fraction (supernatant).

Immunoprecipitation. Cells were lysed in a Triton X-100–based lysis buffer [1% (v/v) Triton X-100, 10% (v/v) glycerol, 150 mmol/L NaCl, 20 mmol/L Tris (pH 7.5), 2 mmol/L EDTA, PIM] for 1 hour, the nuclear and cellular debris were cleared by centrifugation at 14,000 x g for 10 minutes, and the resulting lysate was precleared with {gamma}-binding beads for 1 hour at 4°C. FLAG M2 beads (10 µL) were then added to the cell lysate and incubated at 4°C for 3 hours. After binding, beads were washed five times with lysis buffer. Immune complexes bound to the beads were eluted with sample buffer, resolved on 7% or 15% (w/v) SDS-PAGE gels, transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA), probed with antibody, and detected with horseradish peroxidase–conjugated secondary antibody followed by ECL reagents.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of caspase-3 activity by survivin-{Delta}Ex3. KSHV-K7 has been reported to bind activated caspase-3, and lysates from HeLa cells overexpressing KSHV-K7 can inhibit recombinant caspase-3 activity (32). To determine whether survivin-{Delta}Ex3 has a similar inhibitory effect, we investigated caspase-3 activity in Hep3BT2 cells stably expressing survivin-{Delta}Ex3 or KSHV-K7. After LIGHT/IFN-{gamma} treatment, caspase-3 activity increased 4.2- and 7.1-fold, respectively, in wild-type Hep3BT2 cells and a Bcl-2-transfected stable clone (Fig. 1A ). In contrast, caspase-3 activity increased only 2.0-fold in the KSHV-K7 stable transfectant and was unchanged in the survivin-{Delta}Ex3 stable transfectant.


Figure 1
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  		Figure 1. Interaction of caspase-3 with KSHV-K7 and survivin-{Delta}Ex3. A, inhibition of caspase-3 activity in LIGHT/IFN-{gamma}-mediated cell death. Wild-type Hep3BT2 (WT) and transfectants stably expressing survivin-{Delta}Ex3, KSHV-K7, and Bcl-2 were incubated with IFN-{gamma} and LIGHT for 12 hours, and caspase-3 activities in cell lysates were determined using the fluorescent probe MCA-DEVD.APK (DNP). Columns, average of three independent experiments done in duplicate; bars, SD. *, P < 0.05, significantly different from untreated as determined by ANOVA. B, interaction between survivin-{Delta}Ex3, KSHV-K7, and activated caspase-3. HEK293 cells were treated with TNF-{alpha} (10 ng/mL) plus cycloheximide (1 µg/mL) for 8 hours, and cell lysates were then incubated with 1 µg recombinant FLAG-KSHV-K7 or FLAG-survivin-{Delta}Ex3 protein purified from pFLAG-KSHV-K7-transfected or pFLAG-survivin-{Delta}Ex3-transfected HEK293T cells followed by immunoprecipitation using anti-FLAG mAb. The presence of caspase-3 in the immunoprecipitates was determined using an anti-caspase-3 (CPP32) mAb. C, inhibition of activated caspase-3. Recombinant activated caspase-3 was incubated with Z-DEVD or transfected cell lysates in the presence of MCA-DEVD.APK (DNP) for 1 hour at 37°C. Caspase-3 activities were determined using a fluorescence spectrophotometer.

 
Because KSHV-K7 can interact with activated caspase-3 and inhibit its activity (32), we tested whether survivin-{Delta}Ex3 shares this ability. To address this question, HEK293 cells were treated with TNF-{alpha} (10 ng/mL) and cycloheximide (1 µg/mL) simultaneously for 8 hours to activate caspase-3. Cell lysates were then incubated with FALG-tagged survivin-{Delta}Ex3 or KSHV-K7 followed by immunoprecipitation to pull down survivin-{Delta}Ex3 or KSHV-K7. As shown in Fig. 1B, survivin-{Delta}Ex3 is able to pull down activated caspase-3. Moreover, survivin-{Delta}Ex3, like KSHV-K7, is able to inhibit the activity of recombinant active caspase-3 in an in vitro caspase activity assay (Fig. 1C).

Survivin-{Delta}Ex3 inhibits apoptosis via blocking specifically LTßR-mediated signaling. We next compared the protective effects of survivin-{Delta}Ex3, KSHV-K7, wild-type Bcl-2, and a caspase-3-resistant Bcl-2 mutant (Bcl-2 34E) on LIGHT/IFN-{gamma}-induced cell death (16). As shown in Fig. 2A , overexpression of Bcl-2 enhanced cell death, whereas Bcl-2 34E was resistant to LIGHT/IFN-{gamma}-induced cytotoxicity. Survivin-{Delta}Ex3 had a protective effect similar to that seen for Bcl-2 34E, whereas KSHV-K7 was unable to inhibit cell death. To further confirm that the protective effect was via inhibition of LTßR-mediated signaling, LIGHT was replaced by the LIGHT-R228E mutant, which interacts with LTßR but not HVEM (16). We found that LIGHT-R228E was as effective as wild-type LIGHT, indicating that the protective effect of survivin-{Delta}Ex3 does occur via inhibition of LTßR-mediated death signal (Fig. 2A). We further compared the protective effects of survivin-{Delta}Ex3 and KSHV-K7 in response to LIGHT/IFN-{gamma}. As observed before, survivin-{Delta}Ex3 not only inhibited IFN-{gamma}-mediated cell death but also increased the survival rate of Hep3BT2 cells, and the expression level of survivin-{Delta}Ex3 (Fig. 2B) correlated with its protective effect against LIGHT/IFN-{gamma}-mediated cell death (clone 9 versus clone 6; Fig. 2C). The protective effect of survivin-{Delta}Ex3 was comparable with that seen for Bcl-2 34E even in the presence of LIGHT concentrations up to 500 ng/mL (Fig. 2C). To further clarify the protective mechanism of survivin-{Delta}Ex3, caspase 3-deficient cell line MCF-7 was used (16) to test the protective effect of survivin-{Delta}Ex3 and KSHV-K7. As shown in Fig. 2D, overexpression of survivin-{Delta}Ex3 protects cells from LIGHT/IFN-{gamma}-mediated cell death efficiently (52.5% versus 27.3%). From these findings, we concluded that the protective effect of survivin-{Delta}Ex3 is mainly via inhibition of caspase-3-independent pathway.


Figure 2
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  		Figure 2. Inhibition of LTßR-mediated cell death by KSHV-K7 and survivin-{Delta}Ex3. A, wild-type Hep3BT2 cells and stable transfectants were incubated with LIGHT or LIGHT-R228E and IFN-{gamma} for 72 hours, and the survival rate was determined by MTT assay. *, P < 0.05, significantly different from untreated as determined by ANOVA. B, expression levels of transfected genes in stable clones were determined by Western blot analysis, with actin as a control. C, dose-dependent assays. Wild-type Hep3BT2 cells and stable transfectants were treated with various concentrations of LIGHT or LIGHT-R228E in the presence of IFN-{gamma} for 72 hours. Cell viability was determined by MTT assay, whereas the percentage of cell survival was determined by measurement of A280 for cells treated with cytokines versus cells cultured in medium alone. Points, average of three independent experiments done in triplicate; bars, SD. Data are percentages of the control values (no cytokine added). {blacksquare}, wild-type; {blacktriangleup}, survivin-{Delta}Ex3 #6; bullet, survivin-{Delta}Ex3 #9; {square}, KSHV-K7; {triangleup}, Bcl-2; {circ}, Bcl-2 34E. D, pCR3.1 or pCR3.1-survivin-{Delta}Ex3 (2 µg) was cotransfected with 0.4 µg pEGFP-N1 to MCF-7 cells for 24 hours followed by incubation with LIGHT (300 ng/mL) and IFN-{gamma} (100 units/mL) for 48 hours. Percentage of apoptotic cells was determined as described in (C).

 
Survivin-{Delta}Ex3 prevents Bcl-2 cleavage and maintains {Delta}{Psi}m after LIGHT/IFN-{gamma} treatment. Because survivin-{Delta}Ex3 and Bcl-2 34E had similar protective effects, we examined whether survivin-{Delta}Ex3 prevented Bcl-2 cleavage after LIGHT/IFN-{gamma} treatment. As shown in Fig. 3A , survivin-{Delta}Ex3, but not KSHV-K7, inhibited Bcl-2 cleavage. Moreover, its inhibitory effect was similar to that seen for the dominant-negative mutants of TRAF3 (TRAF3DN) and ASK1 (ASK-KE), which have been shown to inhibit LTßR-induced cell death (17). We then examined the ability of survivin-{Delta}Ex3 to maintain {Delta}{Psi}m. Hep3BT2 cells treated with LIGHT/IFN-{gamma} were incubated with fluorescent probe JC-1 to determine {Delta}{Psi}m. JC-1 emits red fluorescence (FL-2H) only when existing as a dimerism configuration in intact mitochondria. If the mitochondrial membrane breaks down, JC-1 converts to a monomer and emits green fluorescence (FL-1H). At 20 hours after LIGHT/IFN-{gamma} treatment, wild-type Hep3BT2 cells showed increased green fluorescence accompanied by a decrease in red fluorescence (Fig. 3B), indicating the breakdown of mitochondrial membranes. Although wild-type Bcl-2 was unable to maintain {Delta}{Psi}m, Bcl-2 34E was effective in preventing {Delta}{Psi}m breakdown. Furthermore, survivin-{Delta}Ex3 completely inhibited {Delta}{Psi}m decrease, whereas KSHV-K7 had only a partial protective effect (Fig. 3B). To further confirm this observation, cells were examined under confocal microscope (Fig. 3C). At 20 hours after LIGHT/IFN-{gamma} treatment, the number of cells with green fluorescence increased in cultures of wild-type Hep3BT2 cells and Bcl-2 stable transfectants. In contrast, red fluorescence still emitted from cells stably expressing survivin-{Delta}Ex3 and Bcl-2 34E. In addition to the weak and diffuse green fluorescence, the number and size of red aggregates in KSHV-K7 stable transfectants were reduced in comparison with survivin-{Delta}Ex3 and Bcl-2 34E stable transfectants. This further confirmed the differential effect abilities of survivin-{Delta}Ex3 and KSHV-K7 to maintain the integrity of mitochondrial membrane.


Figure 3
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  		Figure 3. Survivin-{Delta}Ex3 prevents Bcl-2 cleavage and maintains {Delta}{Psi}m of Hep3BT2 cells after LIGHT/IFN-{gamma} treatment. A, inhibition of Bcl-2 cleavage by survivin-{Delta}Ex3. Hep3BT2 Bcl-2 stable cells were transfected with survivin-{Delta}Ex3, KSHV-K7, TRAF3DN, or ASK1-KE for 18 hours followed by incubation with IFN-{gamma} and LIGHT for 48 hours. Cell lysates were fractionated on 13% SDS-PAGE, blotted onto nitrocellulose membrane, and probed with anti-Bcl-2 antibody. B and C, integrity of mitochondria was examined by incubation with JC-1 followed by flow cytometry (B) or observed using a confocal fluorescence microscope (C). Cells were incubated with DMEM alone (C1) or with LIGHT/IFN-{gamma} (C2-6) for 20 hours followed by incubation with JC-1 (10 µg/mL) for 30 minutes, before being subjected to flow cytometry analysis, or observed under a confocal microscope. Representative of three independent experiments.

 
Interaction of survivin-{Delta}Ex3 with Smac/DIABLO. We next examined the release of cytochrome c and Smac/DIABLO after LIGHT/IFN-{gamma} treatment. In wild-type Hep3BT2 cells, both cytochrome c and Smac/DIABLO were released from mitochondria to the cytosol. Wild-type Bcl-2 had no protective effect, whereas both survivin-{Delta}Ex3 and Bcl-2 34E effectively inhibited the release of cytochrome c and Smac/DIABLO from mitochondria (Fig. 4A ). Although KSHV-K7 inhibited cytochrome c release from mitochondria, it failed to block Smac/DIABLO release. To further confirm the localization of Smac/DIABLO, cells were examined using a confocal microscope after immunofluorescence staining. In wild-type cells (Fig. 4B, top), cytochrome c and Smac/DIABLO were retained in the mitochondria. After LIGHT/IFN-{gamma} treatment, the staining patterns of both cytochrome c and Smac/DIABLO became diffuse, indicating their release from mitochondria to the cytosol. In survivin-{Delta}Ex3 stable transfectants (Fig. 4B, middle), survivin-{Delta}Ex3 was translocated from the nucleus to the mitochondria and cytosol, whereas Smac/DIABLO was retained in the mitochondria and colocalized with survivin-{Delta}Ex3. In KSHV-K7 stable transfectants (Fig. 4B, bottom), KSHV-K7 was retained in the mitochondria, whereas Smac/DIABLO was translocated from mitochondria to the cytosol, after LIGHT/IFN-{gamma} treatment.


Figure 4
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  		Figure 4. Survivin-{Delta}Ex3 prevents the release of cytochrome c and Smac from Hep3BT2 cells after LIGHT/IFN-{gamma} treatment. A, mitochondrial integrity assay by subcellular fractionation. Cells were incubated with LIGHT/IFN-{gamma} for 20 hours followed by digitonin-based subcellular fractionation to separate the cell lysate into cytosol-enriched (left) and mitochondria-enriched (right) fractions and by Western blot analysis using antigen-specific antibodies. (Each fraction represents 5 x 106 cells.) Actin and COX IV served as references for the cytosol and mitochondria, respectively, as described in Materials and Methods. B, colocalization of survivin-{Delta}Ex3 and Smac. Hep3BT2 cells were incubated with LIGHT/IFN-{gamma} for 20 hours. Survivin-{Delta}Ex3 and KSHV-K7 were stained with anti-Myc tag, whereas cytochrome c (Cyt c) and Smac were stained with anti–cytochrome c and anti-Smac antibodies, respectively. Smac and anti-Myc signals were merged. The cell nucleus was stained with Hoechst 33342. C, association of survivin-{Delta}Ex3 with Smac. Hep3BT2 cells were cotransfected with FLAG-tagged survivin-{Delta}Ex3, KSHV-K7, or survivin in conjunction with HA-tagged Smac for 18 hours followed by incubation with LIGHT/IFN-{gamma} for another 20 hours. Cells were then harvested, and cell lysates were incubated with M2 anti-FLAG antibody for immunoprecipitation before blotting onto nitrocellulose membrane. Blots were probed with anti-FLAG or anti-HA mAb. D, translocation of survivin-{Delta}Ex3 to mitochondria. Hep3BT2 cells were cotransfected pDsRed2-Mito and either survivin-{Delta}Ex3 or KSHV-K7 for 18 hours followed by incubation with LIGHT/IFN-{gamma} for various time intervals. Survivin-{Delta}Ex3 and KSHV-K7 were stained with anti-Myc tag antibody; the cell nucleus was stained with Hoechst 33342. Colocalization in the merged images of green and red channels. E, BIR domain is essential for the interaction between survivin-{Delta}Ex3 and Smac. Hep3BT2 cells (1 x 106) were cotransfected with FLAG-tagged survivin-{Delta}Ex3 (2 µg/mL) or survivin-{Delta}Ex3 ({Delta}BIR; 2 µg/mL) in conjunction with HA-tagged Smac (2 µg/mL) for 18 hours followed by incubation with LIGHT/IFN-{gamma} for another 20 hours. Cells were then harvested, and cell lysates were incubated with M2 anti-FLAG antibody for immunoprecipitation before blotting onto nitrocellulose membrane. Blots were probed with anti-FLAG or anti-HA mAb. F, colocalization of Smac with survivin-{Delta}Ex3 but not survivin-{Delta}Ex3 ({Delta}BIR). Hep3BT2 cells (3 x 105) were cotransfected with pDsRed2-Mito, HA-tagged Smac, in conjunction with Myc-tagged survivin-{Delta}Ex3 or survivin-{Delta}Ex3 ({Delta}BIR) for 18 hours followed by incubation with LIGHT/IFN-{gamma} for 20 hours. Cells were incubated with anti-Myc antibody and FITC-conjugated anti-mouse IgG subsequently to detect survivin-{Delta}Ex3 and survivin-{Delta}Ex3 ({Delta}BIR) or stained with anti-Smac antibody and Cy5-conjugated goat anti-rabbit antibody subsequently. Colocalization in the merged images of green [FITC for survivin-{Delta}Ex3 or survivin-{Delta}Ex3 ({Delta}BIR)], red (DsRed for mitochondria), and blue (Cy5 for Smac/DIABLO) channels. Mt, mitochondrial fraction.

 
It had been shown that survivin binds to Smac and reduces Smac/DIABLO antagonism to X-linked IAP (XIAP) during Taxol-induced apoptosis (36); therefore, we asked whether survivin-{Delta}Ex3 could also interact with Smac/DIABLO. To address this, HA-tagged Smac/DIABLO was cotransfected with FLAG-tagged survivin, survivin-{Delta}Ex3, or KSHV-K7. After pull-down with an anti-FLAG mAb, Smac/DIABLO was coprecipitated with survivin and survivin-{Delta}Ex3 but not with KSHV-K7 (Fig. 4C). Similarly, survivin and survivin-{Delta}Ex3 were coprecipitated with Smac/DIABLO by an anti-HA antibody (Fig. 4C). Because Smac/DIABLO was not released into the cytosol in the presence of survivin-{Delta}Ex3 (Fig. 4A), we could not observe any interaction between these two molecules in the cytosolic fraction. These results showed that both survivin-{Delta}Ex3 and survivin interact with Smac in the mitochondria, whereas KSHV-K7 did not interact with Smac/DIABLO at all.

The translocation of survivin-{Delta}Ex3 from the nucleus to mitochondria after LIGHT/IFN-{gamma} treatment was further confirmed in survivin-{Delta}Ex3-overexpressing Hep3BT2 cells transfected with pDsRed2-Mito, a marker for mitochondria. As shown in Fig. 4D, survivin-{Delta}Ex3 is translocated from the nucleus and colocalizes with DsRed-Mito. This is in accord with the fact that survivin-{Delta}Ex3 has a mitochondrial localization signal (37) and further confirms that survivin-{Delta}Ex3 can enter mitochondria to interact with Smac/DIABLO. To further confirm whether BIR domain is responsible for the retention of Smac/DIABLO in the mitochondria, we investigated the localization of Smac/DIABLO in cells overexpressing wild-type survivin-{Delta}Ex3 and deletion mutant survivin-{Delta}Ex3 ({Delta}BIR) after LIGHT/IFN-{gamma} treatment. We found that survivin-{Delta}Ex3 ({Delta}BIR) was unable to interact with Smac/DIABLO (Fig. 4E). Compared with cells overexpressing wild-type survivin-{Delta}Ex3, less amount (0.6-fold) of Smac/DIABLO was retained in the mitochondria fraction, whereas certain amount of Smac/DIABLO was released to cytosol, in cells overexpressing survivin-{Delta}Ex3 ({Delta}BIR; Fig. 4E). Moreover, survivin-{Delta}Ex3 colocalized with Smac/DIABLO in the mitochondria (Fig. 4F, merged, white), whereas most of the survivin-{Delta}Ex3 ({Delta}BIR) does not (Fig. 4F, merged, green). This indicates that BIR domain is responsible for the interaction between survivin-{Delta}Ex3 and Smac/DIABLO and plays an essential role to retain Smac/DIABLO in the mitochondria after LIGHT/IFN-{gamma} treatment.

ASK1 activity is regulated by survivin-{Delta}Ex3. It has been shown that free radicals play essential role in LIGHT/IFN-{gamma}-mediated cell death, and activation of LTßR results in the recruitment of TRAF3 and TRAF5. This leads to activation of ASK1, which is involved in the caspase-3-independent pathway of LIGHT/IFN-{gamma} (17). Therefore, we firstly examined the effect of survivin-{Delta}Ex3 in the production of free radicals after LIGHT/IFN-{gamma} treatment. As shown in Fig. 5A , survivin-{Delta}Ex3 was more efficient than both Bcl-2 34E and KSHV-K7 in the inhibition of ROS production, because no obvious fluorescence shift was observed at 12 hours after the cross-linking of LTßR by 31G4D8 mAb. We further asked whether survivin-{Delta}Ex3 was able to regulate ASK1 activity. To test this hypothesis, HeLa cells were transfected with survivin-{Delta}Ex3, KSHV-K7, or a dominant-negative mutant of ASK1 (ASK1-KE) followed by incubation with LIGHT-R228E for 30 minutes. We found that ASK1 activity was suppressed by both ASK1-KE and survivin-{Delta}Ex3, but not by KSHV-K7, in an in vitro kinase assay (Fig. 5B). To further confirm this observation, the agonistic anti-LTßR mAb 31G4D8 was used to activate LTßR for kinetic studies. In wild-type Hep3BT2 cells, ASK1 activity peaked at 10 minutes and decreased to background at 60 minutes after LTßR activation. A similar pattern was observed in a KSHV-K7 stable transfectant. However, ASK1 activity was not up-regulated in a survivin-{Delta}Ex3 stable transfectant (Fig. 5C). Because ASK1 is regulated by association with cellular factors, we examined whether survivin-{Delta}Ex3 might be one of such factors. To address this question, Hep3BT2 cells were cotransfected with HA-tagged ASK1 and Myc-tagged survivin-{Delta}Ex3 for 18 hours and then incubated with LIGHT/IFN-{gamma}. Protein localization was then determined by confocal microscopy. As shown in Fig. 6A , some of the survivin-{Delta}Ex3 was translocated from the nucleus to the cytoplasm and partially colocalized with ASK1. After LIGHT/IFN-{gamma} treatment for 1 hour, survivin-{Delta}Ex3 had translocated from the nucleus to the cytoplasm (Fig. 6B) and was coprecipitated with ASK1 (Fig. 6C).


Figure 5
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  		Figure 5. Survivin-{Delta}Ex3 inhibits free radical production and regulates ASK1 activity. A, inhibition of free radical production by survivin-{Delta}Ex3. After incubation with LIGHT/IFN-{gamma} for various time intervals, wild-type Hep3BT2, survivin-{Delta}Ex3, Bcl-2, and Bcl-2 34E stable transfectants were stained with 5 µmol/L H2-DCF-DA to determine their fluorescence intensities by flow cytometry. Shaded histograms, medium alone; open histograms, LIGHT/IFN-{gamma}. B, survivin-{Delta}Ex3 inhibited ASK1 in HeLa cells. HeLa cells were transfected with ASK1-KE, KSHV-K7, or survivin-{Delta}Ex3 for 18 hours followed by incubation with LIGHT-R228E (100 ng/mL) for 30 minutes. Cell lysates were then subjected to immunoprecipitation with anti-ASK1 antibody, and the ASK1 activities contained in the immunoprecipitates were determined by in vitro kinase assay using MBP as substrate. C, kinetic study of ASK1 activity by in vitro kinase assay. Wild-type Hep3BT2 cells (lanes 1-4), KSHV-K7 stable transfectant (lanes 5-8), and survivin-{Delta}Ex3 stable transfectant (lanes 9-12) were incubated with anti-LTßR antibody 31G4D8 (10 µg/mL) for various time intervals, and the ASK1 activities in the immunoprecipitates were determined by in vitro kinase assay.

 

Figure 6
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  		Figure 6. Translocation of survivin-{Delta}Ex3 from nucleus to cytosol and colocalization of survivin-{Delta}Ex3 with ASK1 after LIGHT/IFN-{gamma} treatment. A, translocation of survivin-{Delta}Ex3 from nucleus to cytosol. Hep3BT2 cells were transfected with Myc-tagged survivin-{Delta}Ex3 followed by treatment with LIGHT/IFN-{gamma} for various time intervals. Localization of survivin-{Delta}Ex3 was determined by confocal microscopy using anti-HA (for ASK1) and Cy3-conjugated donkey anti-rat secondary antibody (red) subsequently, whereas survivin-{Delta}Ex3 was detected by anti-Myc antibody and FITC-conjugated goat anti-mouse secondary antibody (green) subsequently. Merge, merged images from survivin-{Delta}Ex3 and ASK1 staining. The cell nucleus was stained with Hoechst 33342. Bar, 8 µm. B, subcellular fractionation of survivin-{Delta}Ex3 and ASK1. Hep3BT2 cells were transfected with Myc-tagged survivin-{Delta}Ex3 and treated with LIGHT/IFN-{gamma}. Cytosolic and nuclear fractions were prepared as described in Materials and Methods. Western blots of the two fractions (nuclear and cytosol) were probed with anti-FLAG (for survivin-{Delta}Ex3) or anti-HA (for ASK1) antibody. Anti-tubulin and anti–poly(ADP-ribose) polymerase (PARP) were used as controls for the cytoplasmic and nuclear fractions, respectively. C, association of survivin-{Delta}Ex3 with ASK1. Hep3BT2 cells were cotransfected with FALG-survivin-{Delta}Ex3 and HA-ASK1 for 18 hours and incubated with LIGHT/IFN-{gamma}. Following subcellular fractionation, the nuclear and cytoplasmic fractions were immunoprecipitated with antibody and blotted onto nitrocellulose membrane. The blot was probed with anti-FLAG and anti-HA antibody to detect the presence of survivin-{Delta}Ex3 and ASK1, respectively. D, mapping of interaction domain of survivin-{Delta}Ex3 with ASK-1. HeLa (1 x 106) cells were transfected with pCR3.1, pCR3.1-survivin-{Delta}Ex3, or its deletion mutants for 18 hours followed by immunoprecipitation using anti-HA to pull down survivin-{Delta}Ex3 and deletion mutants and incubated with anti-ASK-1 mAb (top). Alternatively, cell lysates were precipitated by anti-ASK-1 mAb followed by incubation with anti-HA mAb to detect survivin-{Delta}Ex3 and its mutants (bottom). E, HeLa cells were transfected with pCR3.1, pCR3.1-survivin-{Delta}Ex3, or pCR3.1-survivin-{Delta}Ex3 ({Delta}NLS) for 18 hours followed by incubation with LIGHT-R228E (100 ng/mL) for various time intervals, and the ASK1 activities in the immunoprecipitates were determined by in vitro kinase assay. Nu, nuclear fraction.

 
To further investigate whether survivin-{Delta}Ex3-ASK1 association correlated with the inhibition of ASK1 activity, we identified the domain of survivin-{Delta}Ex3 responsible for interaction with ASK1. Compared with wild-type and other two deletion clones [survivin-{Delta}Ex3 ({Delta}BIR) and survivin-{Delta}Ex3 ({Delta}BH2)], survivin-{Delta}Ex3 ({Delta}NLS) only had weak interaction with ASK1 (Fig. 6D) and was unable to inhibit ASK1 activity (Fig. 6E). This suggests that the NLS domain is responsible for survivin-{Delta}Ex3-ASK1 interaction and is crucial for inhibition of ASK1 activity. Based on all these findings, we concluded that survivin-{Delta}Ex3 is a potent inhibitor of LTßR-mediated cell death via the targeting of both caspase-dependent and caspase-independent pathways.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently, survivin and its alternatively spliced variants, including survivin-{Delta}Ex3, have been detected in many cancer tissues (38). Like survivin, survivin-{Delta}Ex3 exhibits a protective effect against methotrexate and CD95-induced cell death (33, 39). However, the mechanism of this effect has not been investigated. KSHV-K7, a protein that is structurally related to survivin-{Delta}Ex3, has been shown to block apoptosis induced by anti-Fas mAb (40), TNF-{alpha}, and Bax (32). The antiapoptotic function of KSHV-K7 is mediated via binding to Bcl-2 and activated caspase-3 (32). Before this study, it has not been determined whether survivin-{Delta}Ex3 and KSHV-K7 can prevent LTßR-mediated cell death.

Previously, we have shown that overexpression of Bcl-2 enhances LTßR-mediated cytotoxicity in Hep3BT2 cells. In contrast, the caspase-3-resistant mutant Bcl-2 34E could inhibit LTßR-mediated cell death, indicating that this process occurs mainly via a mitochondrial pathway. To further confirm this, we tested the protective effects of survivin-{Delta}Ex3 and KSHV-K7 in LTßR-mediated cell death. Although KSHV-K7 was able to bind caspase-3 and suppress its activity, it neither inhibited Bcl-2 cleavage nor protected cells from LTßR-mediated cell death (Fig. 2A). Therefore, the interaction between Bcl-2 and KSHV-K7 (32) did not prevent Bcl-2 cleavage after activation of LTßR, and this suggested that other caspases were also responsible for Bcl-2 cleavage. Because Bcl-2 34E was derived from the mutation of Asp34 to Glu34 to remove the caspase-3 recognition site (16), the as-yet-undefined caspase must recognize the same motif as caspase-3 to cleave Bcl-2.

In contrast to KSHV-K7, survivin-{Delta}Ex3 inhibited caspase-3 activity and prevented Bcl-2 cleavage. It also interacted with Smac/DIABLO and prevents cytochrome c release after activation of LTßR (Fig. 4A). Moreover, it associated with ASK1 to regulate its activity (Fig. 5). Therefore, survivin-{Delta}Ex3 was apparently more potent than KSHV-K7 in inhibiting the LTßR-mediated cytotoxic effect. The protective effect of survivin-{Delta}Ex3 was not only limited to LTßR-triggered signaling pathway but also effective in the context of TNF related–mediated apoptosis-inducing ligand–induced apoptosis in Hep3BT2 cells (data not shown). This suggests that survivin-{Delta}Ex3 is more effective than KSHV-K7 in blocking cell apoptosis.

As shown in Fig. 4A, overexpression of survivin-{Delta}Ex3 prevented the reduction of {Delta}{Psi}m and the translocation of cytochrome c and Smac/DIABLO that are associated with LTßR-induced cell death. It has been shown that survivin protects against caspase-independent cell death by blocking apoptosis-inducing factor release (41) and that survivin is able to reduce Smac/DIABLO antagonism to XIAP during Taxol-induced apoptosis (36). Smac/DIABLO is able to potentiate LIGHT-induced apoptosis through association with cIAP1, and survivin-{Delta}Ex3 apparently has a similar effect as survivin in regulating cell death via association with Smac/DIABLO. It has been speculated that heterodimerization of survivin and survivin-{Delta}Ex3 is required for their recruitment to mitochondria (37). Whether survivin-{Delta}Ex3 cooperates with survivin to protect Smac release from mitochondria will be the subject of further investigation.

It has been shown that the general caspase inhibitor z-VAD could not prevent LTßR-induced cell death (16); thus, the protective effect of survivin-{Delta}Ex3 must also occur via a caspase-independent pathway. This is supported by the observation that overexpression of survivin-{Delta}Ex3 inhibits cell death in caspase-3-deficient MCF-7 cells (Fig. 2D). We have shown that activation of LTßR leads to the recruitment of TRAF2, TRAF3, and TRAF5 to generate ROS, which in turn activates ASK1 to induce caspase-dependent and caspase-independent cell death (17). Moreover, it has been shown that the antiapoptotic effect of IAP occurs via binding to TAK1, a component of MAP3K, to induce TAK1-dependent JNK1 activation (42). Because survivin-{Delta}Ex3 also interacts with ASK1 to regulate enzyme activity, survivin-{Delta}Ex3 represents another example of the ability of IAPs to regulate caspase-independent pathways via interaction with a component of MAP3K (ASK1).

Feng et al. showed that KSHV-K7 interacts with the calcium-modulating cyclophilin ligand, a protein that regulates intracellular Ca2+ concentration. Moreover, overexpression of KSHV-K7 alters the kinetic release of Ca2+ and cell death induced by thapsigargin, an inhibitor of endoplasmic reticulum calcium pump. In addition to KSHV-K7, Bcl-2 (wild-type) is also able to inhibit thapsigargin-induced cell death (40). However, neither KSHV-K7 nor Bcl-2 (wild-type) is able to inhibit LIGHT/IFN-{gamma}-mediated cell death, suggesting LTßR-mediated cell death is not via calcium release to the cytosol.

In summary, we have shown that the protective effect of survivin-{Delta}Ex3 against LTßR-mediated cell death involves at least the targeting of ASK1, Bcl-2, Smac/DIABLO, and caspase-3 and the inhibition of free radical production (Fig. 7 ). As well as inhibiting caspase-3 activity, survivin-{Delta}Ex3 also interacts with ASK1 to regulate its activity. Moreover, survivin-{Delta}Ex3 inhibits Bcl-2 cleavage and the release of cytochrome c and Smac/DIABLO. This study shows that survivin-{Delta}Ex3 is a potent antiapoptotic protein, and its role in helping tumor survival clearly warrants further investigation in the future.


Figure 7
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  		Figure 7. Schematic diagram of the roles of survivin-{Delta}Ex3 and KSHV-K7 in LTßR-mediated cell death. Activation of LTßR induces ROS production, which in turn activates ASK1 to trigger an endogenous apoptotic pathway. Survivin-{Delta}Ex3 also prevents LTßR-mediated cell death via inhibition of ROS production and ASK1 activity. Moreover, survivin-{Delta}Ex3 prevents Bcl-2 cleavage and interacts with Smac/DIABLO to prevent its release from mitochondria. Caspase-3 activity is inhibited by both KSHV-K7 and survivin-{Delta}Ex3. Cell death induced by LIGHT/IFN-{gamma} is mainly via activation of caspase-independent pathway.

 

   Acknowledgments
 
Grant support: National Sciences Council, Taiwan grants NSC 93-3112-B-010-025, NSC 93-2320-B-010-040, NSC 93-2320-B-010-011, and NSC 94-2320-B-010-015, Taipei Veterans General Hospital grant VGH37113, National Research Program of Genomic Medicine (NRPGM), Taiwan grant 94IDI002, and Academia Sinica, Taiwan grant 94 M002-1.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Michael Karin, Wan-Wan Lin, and Wen-Chen Yeh for providing expression vectors, John Yu, Alice Lin-Tsing Yu, Li-Wen Lin, and Chia-Lin Ho for technical assistance, and Dr. Caroline Milner for critical review of this article.

Received 7/18/05. Revised 12/15/05. Accepted 1/11/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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