Selective Knockdown of the Long Variant of Cellular FLICE Inhibitory Protein Augments Death Receptor-mediated Caspase-8 Activation and Apoptosis
2005; Elsevier BV; Volume: 280; Issue: 19 Linguagem: Inglês
10.1074/jbc.m413962200
ISSN1083-351X
AutoresDarcie Sharp, David A. Lawrence, Avi Ashkenazi,
Tópico(s)Mitochondrial Function and Pathology
ResumoDeath receptors trigger apoptosis by activating the apical cysteine proteases caspase-8 and -10 within a death-inducing signaling complex (DISC). c-FLIP (cellular FLICE inhibitory protein) is an enzymatically inactive relative of caspase-8 and -10 that binds to the DISC. Two major c-FLIP variants result from alternative mRNA splicing: a short, 26-kDa protein (c-FLIPS) and a long, 55-kDa form (c-FLIPL). The role of c-FLIPS as an inhibitor of death receptor-mediated apoptosis is well established; however, the function of c-FLIPL remains controversial. Although overexpression of transfected c-FLIPL inhibits apoptosis, ectopic expression at lower levels supports caspase-8 activation and cell death. Simultaneous ablation of both c-FLIP variants augments death receptor-mediated apoptosis, but the impact of selective depletion of c-FLIPL on caspase-8 activation and subsequent apoptosis is not well defined. To investigate this, we developed small interfering RNAs that specifically knock down expression of c-FLIPL in several cancer cell lines and studied their effect on apoptosis initiation by Apo2L/TRAIL (Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand). Knockdown of c-FLIPL augmented DISC recruitment, activation, processing, and release of caspase-8, thereby enhancing effector-caspase stimulation and apoptosis. Thus, endogenous c-FLIPL functions primarily as an inhibitor of death receptor-mediated apoptosis. Death receptors trigger apoptosis by activating the apical cysteine proteases caspase-8 and -10 within a death-inducing signaling complex (DISC). c-FLIP (cellular FLICE inhibitory protein) is an enzymatically inactive relative of caspase-8 and -10 that binds to the DISC. Two major c-FLIP variants result from alternative mRNA splicing: a short, 26-kDa protein (c-FLIPS) and a long, 55-kDa form (c-FLIPL). The role of c-FLIPS as an inhibitor of death receptor-mediated apoptosis is well established; however, the function of c-FLIPL remains controversial. Although overexpression of transfected c-FLIPL inhibits apoptosis, ectopic expression at lower levels supports caspase-8 activation and cell death. Simultaneous ablation of both c-FLIP variants augments death receptor-mediated apoptosis, but the impact of selective depletion of c-FLIPL on caspase-8 activation and subsequent apoptosis is not well defined. To investigate this, we developed small interfering RNAs that specifically knock down expression of c-FLIPL in several cancer cell lines and studied their effect on apoptosis initiation by Apo2L/TRAIL (Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand). Knockdown of c-FLIPL augmented DISC recruitment, activation, processing, and release of caspase-8, thereby enhancing effector-caspase stimulation and apoptosis. Thus, endogenous c-FLIPL functions primarily as an inhibitor of death receptor-mediated apoptosis. Apoptosis is essential for development, tissue homeostasis, and immune function (1Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4060) Google Scholar). The key mediators of apoptosis are caspases, a family of cysteine proteases that cleave a critical set of cellular proteins near specific aspartic acid residues (2Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar). Apoptosis-inducing members of the tumor necrosis factor superfamily, such as Fas ligand (FasL) 1The abbreviations used are: FasL, Fas ligand; DISC, death-inducing signaling complex; DED, death effector domain; siRNA, small interfering RNA; FBS, fetal bovine serum; PBS-T, phosphate-buffered saline containing 0.5% Tween 20; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone; IETD, Ile Glu Thr Asp; AFC, 7-amino-4-trifluoromethyl coumarin. and Apo2L/TRAIL, activate caspases through the cell extrinsic apoptosis signaling pathway by engaging their respective "death" receptors: Fas and DR4 or DR5, leading to assembly of the DISC (3Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5169) Google Scholar). Upon ligand stimulation, the adaptor protein FADD (Fas-associated death domain) binds to the receptor through homophilic death domain interactions. FADD then recruits the apoptosis initiator proteases caspase-8 and -10, through homophilic death effector domain (DED) interactions. The proximity of caspase molecules in the DISC facilitates their dimerization and thereby stimulates their proteolytic activity (4Shi Y. Cell. 2004; 117: 855-858Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 5Boatright K.M. Renatus M. Scott F.L. Sperandio S. Shin H. Pedersen I.M. Ricci J.E. Edris W.A. Sutherlin D.P. Green D.R. Salvesen G.S. Mol. Cell. 2003; 11: 529-541Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar, 6Donepudi M. Mac Sweeney A. Briand C. Grutter M.G. Mol. Cell. 2003; 11: 543-549Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Activation of caspase-8 is followed by two self-processing events; the first cleaves a 10-kDa fragment (p10) from the full-length caspase, leaving an intermediate fragment (p43/41) at the DISC, and the second cleaves an 18-kDa fragment (p18) from the p43/41 intermediate, allowing formation of an active caspase-8 (p18/p10)2 heterotetramer, which is released into the cytosol. In turn, cytosolic caspase-8 catalyzes the cleavage and activation of downstream effector caspases, such as caspase-3 and -7, which execute the apoptotic death program. c-FLIP (cellular FLICE inhibitory protein) is structurally related to procaspase-8 and -10 but lacks enzymatic activity (7Krueger A. Baumann S. Krammer P.H. Kirchhoff S. Mol. Cell. Biol. 2001; 21: 8247-8254Crossref PubMed Scopus (491) Google Scholar, 8Thome M. Tschopp J. Nat. Rev. Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar). At least 10 splice variants of c-FLIP exist on the mRNA level, but often only two c-FLIP proteins are detected: a 26-kDa short form (c-FLIPS) and a 55-kDa long form (c-FLIPL) (9Scaffidi C. Schmitz I. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 1541-1548Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 10Shu H.B. Halpin D.R. Goeddel D.V. Immunity. 1997; 6: 751-763Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). c-FLIPS resembles its viral counterpart, v-FLIP, consisting of two DEDs and a short C-terminal tail but entirely lacking a caspase-like domain (11Irmler M. Thome M. Hahne M. Schneider P. Hofmann K. Steiner V. Bodmer J.-L. Schröter M. Burns K. Mattmann C. Rimoldi D. French L.E. Tschopp J. Nature. 1997; 388: 190-195Crossref PubMed Scopus (2231) Google Scholar, 12Thome M. Schneider P. Hofmann K. Fickenscher H. Meinl E. Neipel F. Mattmann C. Burns K. Bodmer J. Schroter M. Scaffidi C. Krammer P. Peter M. Tschopp J. Nature. 1997; 386: 517-521Crossref PubMed Scopus (1146) Google Scholar). In contrast, c-FLIPL contains two N-terminal DEDs and a C-terminal caspase-like domain, similar to caspase-8 and -10. However, c-FLIPL is proteolytically inactive, because it lacks a specific cysteine residue that is critical for caspase activity. Both c-FLIP variants are capable of binding to the Fas DISC (7Krueger A. Baumann S. Krammer P.H. Kirchhoff S. Mol. Cell. Biol. 2001; 21: 8247-8254Crossref PubMed Scopus (491) Google Scholar, 8Thome M. Tschopp J. Nat. Rev. Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar). The presence of c-FLIPS or c-FLIPL in the Fas DISC does not preclude caspase-8 recruitment; rather, DISC-associated caspase-8/c-FLIP complexes are formed (9Scaffidi C. Schmitz I. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 1541-1548Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 13Krueger A. Schmitz I. Baumann S. Krammer P.H. Kirchhoff S. J. Biol. Chem. 2001; 276: 20633-20640Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 14Micheau O. Thome M. Schneider P. Holler N. Tschopp J. Nicholson D.W. Briand C. Grutter M.G. J. Biol. Chem. 2002; 277: 45162-45171Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 15Chang D.W. Xing Z. Pan Y. Algeciras-Schimnich A. Barnhart B.C. Yaish-Ohad S. Peter M.E. Yang X. EMBO J. 2002; 21: 3704-3714Crossref PubMed Scopus (482) Google Scholar). Because c-FLIPS lacks caspase cleavage sites, it is not processed by apical caspases in the DISC. In contrast, c-FLIPL is cleaved between its large (p20) and small (p12) subunits by apical caspases in the DISC; however, a second cleavage between the large subunit and the N-terminal DEDs has not been detected, probably because of the lack of a conserved cleavage site (9Scaffidi C. Schmitz I. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 1541-1548Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar). Interaction of caspase-8 with c-FLIPS does not support caspase-8 activation; therefore, binding of c-FLIPS to the Fas DISC inhibits Fas-mediated apoptosis (7Krueger A. Baumann S. Krammer P.H. Kirchhoff S. Mol. Cell. Biol. 2001; 21: 8247-8254Crossref PubMed Scopus (491) Google Scholar, 8Thome M. Tschopp J. Nat. Rev. Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar). Indeed, ectopic overexpression of c-FLIPS inhibits apoptosis induction by FasL or Apo2L/TRAIL (7Krueger A. Baumann S. Krammer P.H. Kirchhoff S. Mol. Cell. Biol. 2001; 21: 8247-8254Crossref PubMed Scopus (491) Google Scholar, 8Thome M. Tschopp J. Nat. Rev. Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar, 16Burns T.F. El-Deiry W.S. J. Biol. Chem. 2001; 276: 37879-37886Abstract Full Text Full Text PDF PubMed Google Scholar). Conversely, it has been shown that ectopically expressed c-FLIPL at the Fas DISC can support caspase-8 activation (14Micheau O. Thome M. Schneider P. Holler N. Tschopp J. Nicholson D.W. Briand C. Grutter M.G. J. Biol. Chem. 2002; 277: 45162-45171Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 15Chang D.W. Xing Z. Pan Y. Algeciras-Schimnich A. Barnhart B.C. Yaish-Ohad S. Peter M.E. Yang X. EMBO J. 2002; 21: 3704-3714Crossref PubMed Scopus (482) Google Scholar). Furthermore, recent studies with purified components show that c-FLIPL activates caspase-8 or -10 through heterodimerization (17Boatright K.M. Deis C. Denault J.B. Sutherlin D.P. Salvesen G.S. Biochem. J. 2004; 382: 651-657Crossref PubMed Scopus (198) Google Scholar). However, caspase activity within heterodimers with c-FLIPL permits only partial processing of caspase-8, resulting in cleavage between the large and small subunit, but not between the DEDs and the large subunit, similar to the cleavage of c-FLIPL described above (13Krueger A. Schmitz I. Baumann S. Krammer P.H. Kirchhoff S. J. Biol. Chem. 2001; 276: 20633-20640Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar). The partial processing of active caspase-8 in caspase-8/c-FLIPL heterodimers retains active caspases-8 at the DISC, preventing its release into the cytosol (18Chang D.W. Xing Z. Capacio V.L. Peter M.E. Yang X. EMBO J. 2003; 22: 4132-4142Crossref PubMed Scopus (209) Google Scholar). Although the role of c-FLIPS as an inhibitor of death receptor-mediated apoptosis is well understood and resembles the activity of v-FLIP proteins (7Krueger A. Baumann S. Krammer P.H. Kirchhoff S. Mol. Cell. Biol. 2001; 21: 8247-8254Crossref PubMed Scopus (491) Google Scholar, 8Thome M. Tschopp J. Nat. Rev. Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar), the specific involvement of c-FLIPL in death receptor modulation remains controversial. 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To date, loss-of-function studies that specifically interrogate the role of c-FLIPL in modulating caspase-8 activation at the DISC have not been reported. To examine the involvement of endogenous c-FLIPL in caspase-8 activation, we developed siRNA oligonucleotides that selectively knock down the expression of either c-FLIPL or c-FLIPS in several cancer cell lines and studied the effect of these siRNAs on apoptosis initiation by Apo2L/TRAIL. Knockdown of c-FLIPL enhanced the recruitment, activation, and processing of caspase-8 by the DISC, leading to greater effector caspase stimulation and apoptosis. Thus, despite the ability of c-FLIPL to support caspase-8 activation upon heterodimerization, the dominant role of endogenous c-FLIPL in the modulation of death receptor-mediated apoptosis initiation appears to be inhibitory. Cell Lines, Antibodies, and Reagents—A549 and H460 human lung carcinoma cells (catalog numbers CCl-185 and HTB-177, respectively) were obtained from ATCC and cultured in 50% Dulbecco's modified Eagle's medium, 50% FK12 medium supplemented with 10% FBS, and antibiotics (penicillin/streptomycin). HeLa human cervical adenocarcinoma cells (catalog number CCL-2) and 293 human embryonic kidney cells (catalog number CRL-1573) also were obtained from ATCC and cultured in 100% Dulbecco's modified Eagle's medium supplemented with 10% FBS and antibiotics (penicillin/streptomycin). U2OS human osteosarcoma cells were a gift from Ellen Filvaroff (Genentech, Inc., South San Francisco, CA) and were maintained in McCoy's F12 medium supplemented with 10% FBS and antibiotics (penicillin/streptomycin). Anti-c-FLIP antibody (NF6) (catalog number APO-20A-070-C050) was purchased from Alexis Biochemicals, (San Diego, CA). Anti-caspase-8 antibodies (1C12 (catalog number 9746) and 5C7 (catalog number 05-447)) were from Cell Signaling Technology (Beverly, MA) and Upstate Cell Signaling Solutions (Lake Placid, NY), respectively. Anti-caspase-3 antibody (catalog number SA-320) was from BioMol (Plymouth Meeting, PA). Anti-actin antibody (catalog number 69100) was from ICN Biomedicals (Aurora, OH). Anti-Apo2L (2E11) was generated at Genentech, Inc. Anti-DR4 (3G1 and 4G7) and anti-DR5 (3H3 and 5C7) monoclonal antibodies were generated at Genentech, Inc. using receptor-Fc fusion proteins as antigens. Anti-DR4 (4G7) and anti-DR5 (5C7) monoclonal antibodies, used to immunoprecipitate the Apo2L/TRAIL DISC, were conjugated to agarose using the ImmunoPure Protein G IgG Plus orientation kit (catalog number 44990) from Pierce. The anti-DR4 (3G1) and anti-DR5 (3H3) monoclonal antibodies, used for immunodetection of DR4/5 in DISC immunoprecipitations, were biotinylated using EZ-link Sulfo-NHS-LC biotinulation kit (catalog number 21217) from Pierce. As secondary reagents, we used horseradish peroxidase-conjugated goat anti-mouse IgG1 (catalog number 559626) from BD PharMingen (San Diego, CA), horseradish peroxidase-conjugated goat anti-mouse IgG2b (catalog number 1090-02) from Southern Biotechnology Associates (Birmingham, AL), or horseradish peroxidase-conjugated streptavidin from Amersham Biosciences. As substrates for immunodetection we used either ECL from Amersham Biosciences or SuperSignal West Dura Extended Duration Substrate from Pierce. Nontagged soluble Apo2L/TRAIL was prepared as described (35Lawrence D. Shahrokh Z. Marsters S. Achilles K. Shih D. Mounho B. Hillan K. Totpal K. DeForge L. Schow P. Hooley J. Sherwood S. Pai R. Leung S. Khan L. Gliniak B. Bussiere J. Smith C. Strom S. Kelley S. Fox J. Thomas D. Ashkenazi A. Nat. Med. 2001; 7: 383-385Crossref PubMed Scopus (636) Google Scholar). FLAG-tagged Apo2L/TRAIL was prepared and cross-linked with anti-FLAG antibody M2 (Sigma) as described (36Kischkel F.C. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar). FLAG-tagged FasL was prepared by cloning amino acids 130–281 of human FasL into pCMV FLAG (Sigma), followed by transient expression in Chinese hamster ovary cells and purification by affinity chromatography. Immunodetection—The cell lysates were prepared as described (36Kischkel F.C. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar) and quantitated using the BCA protein assay from Pierce. Between 15 and 50 μg of protein/lane was loaded onto a 10% (c-FLIP, caspase-8) or 4–12% (caspase-3) SDS-polyacrylamide gel, electrophoresed, and electrotransferred onto nitrocellulose following manufacturer's instructions (Invitrogen). The membranes were rinsed in phosphate-buffered saline containing 0.5% Tween 20 (PBS-T) and blocked with 5% milk in PBS-T overnight. The concentration of all primary antibodies was 1–2 μg/ml, secondary antibodies were used at 1:10,000–1:40,000 dilution, and horseradish peroxidase-strepavidin was used at a 1:250 dilution. The membranes were incubated with primary antibodies for 1–3 h, washed three times for 10 min in PBS-T, incubated with secondary antibodies for 1 h, washed three times for 10 min in PBS-T, and exposed to either ECL or SuperSignal. Immunoprecipitation and DISC Analysis—These experiments were done as previously described for Apo2L/TRAIL-FLAG + anti-FLAG DISC analysis (36Kischkel F.C. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar). The DR4/5 DISC immunoprecipitation experiments also were performed as described, except that anti-DR4 (4G7) and anti-DR5 (5C7) monoclonal antibodies were directly conjugated to agarose for the immunoprecipitation. siRNA Preparation and Transfection—siRNA oligonucleotides against c-FLIPL and c-FLIPS were synthesized by Dharmacon Research, Inc. (Lafayette, CO) using their custom SMARTpool and siDE-SIGN technology. siCONTROL Non-Targeting siRNA pool control (catalog number D-001206-13-20) was also from Dharmacon Research, Inc. All of the siRNA oligonucleotides were resuspended to a concentration of 20 μm. The cells were seeded for transfection in medium without antibiotics 1 day before transfection so that they were 85–95% confluent on the day of transfection. For transfection, regular medium was replaced with low serum media (0–10% FBS, depending on cell type) without antibiotics. The cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen) at a ratio of 1:1 to 1:3 siRNA (μg):Lipofectamine (μl), with a final concentration of 80–120 nm siRNA. The cells were incubated with the siRNA-Lipofectamine 2000 complexes overnight, and then low serum medium was replaced with normal media (10% FBS) without antibiotics and incubated for a total of 48–60 h before further analysis. Apoptosis Assays—To analyze cells for the percentage of apoptosis, we determined the percentage of subdiploid nuclei. The cells were stained with propidium iodide after ethanol fixation and RNase treatment and then analyzed by flow cytometry as previously described (37Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. J. Immunol. Methods. 1991; 139: 271-279Crossref PubMed Scopus (4431) Google Scholar), using CellQuest Pro software with a FACSCalibur (BD Biosciences). Caspase Activity Assays in Whole Cell Extracts—The caspase-8 activity assay was performed using the BD ApoAlert caspase fluorescent assay kit (catalog number K2028-2), according to the manufacturer's instructions. siRNA-transfected cells were harvested, counted, and aliquoted at equal numbers for each treatment. The cells were treated with 100 ng/ml FLAG-tagged Apo2L/TRAIL for varying amounts of time at 37 °C and then lysed in manufacturer-provided cell lysis buffer. The cell supernatants were transferred to a 96-well plate and 2× reaction buffer/dithiothreitol and the fluorescent caspase-8 substrate (IETD-AFC) were added and incubated for 1–3 h at 37 °C before reading in a fluorometer at 400/505 nm. The caspase 3/7 assay was performed using Apo-One homogenous caspase-3/7 assay (Promega) according to the manufacturer's instructions. siRNA-transfected cells were harvested, counted, and aliquoted at equal numbers for each treatment. The cells were treated with FLAG-tagged Apo2L/TRAIL at varying doses for 4 h at 37 °C and then lysed in manufacturer-provided homogeneous caspase-3/7 reagent (containing the caspase-3/7 substrate Z-DEVD-R110). The lysates were incubated at room temperature for 1–3 h before reading in a fluorometer at 485/530 nm. Caspase Activity Assays at the Apo2L/TRAIL DISC—The caspase-8 activity assay was performed using the Caspase-Glo 8 assay kit from Promega (catalog number G8201), according to the manufacturer's instructions, with the following modifications: siRNA-transfected cells were harvested, counted, and aliquoted at equal numbers (1–2 × 107 cells) for each treatment. The cells were treated with 333 ng/ml FLAG-tagged Apo2L/TRAIL + anti-FLAG M2 antibody for varying amounts of time at 37 °C and then lysed. The cell lysates were transferred to Immunopure Immobilized protein A/G beads (Pierce; catalog number 20421) and immunoprecipitated overnight at 4 °C. Protein A/G beads were washed four times in lysis buffer, resuspended in 100 μl of phosphate-buffered saline (not eluted), and transferred to a white-walled 96-well plate. Equal volume (100 μl) Caspase-8 Glo reagent (Z-LETD-aminoluciferin) was added and incubated for 30 min to 2 h at room temperature before reading in a luminometer. Affinity Precipitation of Active Caspase-8 —siRNA-transfected cells were harvested, counted, and aliquoted at equal numbers (1–2 × 107 cells) for each treatment. The cells were treated with 333 ng/ml FLAG-tagged Apo2L/TRAIL + anti-FLAG M2 for varying amounts of time at 37 °C and then lysed. The cell lysates were incubated with 10 μm Biotin-IETD-FMK at 37 °C for 30 min to label active sites and transferred to immunopure immobilized streptavidin beads (Pierce; catalog number 20349) for precipitation overnight at 4 °C. Streptavidin beads were washed five times in lysis buffer and eluted in sample buffer for immunoblot analysis. Knockdown of Endogenous c-FLIPL Enhances Apoptosis Induction by Apo2L/TRAIL—Taking advantage of the different C-terminal ends of the c-FLIP variants, we designed siRNA oligonucleotides (38Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8186) Google Scholar) expected to knock down either c-FLIPL (P5), c-FLIPS (s1), or both variants together (P1, P3, P6). We transfected human A549 lung carcinoma cells with these siRNAs, incubated the cells for 48 h, and examined the levels of c-FLIP proteins by immunoblot analysis (Fig. 1A). The results confirmed the ability of oligonucleotides P5 and s1 to knock down expression of FLIPL and FLIPS, respectively, whereas P1 and P3 knocked down both c-FLIP variants, and P6 was ineffective at knocking down c-FLIPL. Apoptosis analysis (Fig. 1C) indicated that knockdown of c-FLIPL increased the level of spontaneous cell death to ∼10% as compared with transfection of the control siRNA, whereas knockdown of c-FLIPS had a minimal effect. Notably, depletion of both c-FLIP variants resulted in 33–35% of the cells undergoing spontaneous apoptosis. This observation suggests that the two c-FLIP variants may act cooperatively in A549 cells to block apoptosis signals that may spontaneously emanate from death receptors. We therefore focused our subsequent experiments on knocking down either c-FLIPL or FLIPS, but not both. Given the high degree of sequence homology between c-FLIP and caspase-8 and -10, we examined the effect of c-FLIP siRNA transfection and consequent protein reduction on caspase-8 and -10 protein levels. None of the c-FLIP siRNA oligonucleotides showed a significant effect on the amounts of caspase-8 and -10 (Fig. 1B). Transfection with the P1 and P3 siRNA oligonucleotides was associated with some degree of spontaneous processing of caspase-8, as indicated by the presence of a p43/41 band, consistent with increased apoptosis in cells with both c-FLIP variants reduced (Fig. 1C). These results verify that the c-FLIP siRNA oligonucleotides specifically knock down c-FLIPL, c-FLIPS, or both but not caspase-8 or -10. To assess the effect of c-FLIP knockdown on death receptor-induced apoptosis, we transfected the A549 cells with control or c-FLIP variant-specific siRNA, incubated them for 48 h, added increasing doses of Apo2L/TRAIL for another 4 h, and determined the resulting amount of apoptosis. c-FLIP variant knockdown was confirmed (Fig. 2A, inset). Knockdown of c-FLIPL modestly increased the level of apoptosis in the absence of added ligand and substantially enhanced apoptosis induction by Apo2L/TRAIL at all doses (Fig. 2A); depletion of c-FLIPS also augmented apoptosis sensitivity, particularly at higher doses of Apo2L/TRAIL. Next, we examined the effect of c-FLIP variant knockdown on the time course of Apo2L/TRAIL-induced apoptosis. To this end, we transfected A549 cells with control or c-FLIP variant-specific siRNA and treated with an intermediate dose of Apo2L/TRAIL (33 ng/ml) for increasing amounts of time. c-FLIP variant knockdown was confirmed (Fig. 2B, inset). c-FLIPL-depleted A549 cells were more sensitive to Apo2L/TRAIL and underwent Apo2L/TRAIL-induced apoptosis much earlier than control cells (Fig. 2B, 2 h). Knockdown of c-FLIPS also increased Apo2L/TRAIL-induced apoptosis as early as 2 h but not as robustly as c-FLIPL k
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