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Real Time Analysis of Tumor Necrosis Factor-related Apoptosis-inducing Ligand/Cycloheximide-induced Caspase Activities during Apoptosis Initiation

2008; Elsevier BV; Volume: 283; Issue: 31 Linguagem: Inglês

10.1074/jbc.m802889200

ISSN

1083-351X

Autores

Christian T. Hellwig, Barbara Köhler, Anna-Kaisa Lehtivarjo, Heiko Düßmann, Michael J. Courtney, Jochen H.M. Prehn, Markus Rehm,

Tópico(s)

Drug-Induced Hepatotoxicity and Protection

Resumo

Employing fluorescence resonance energy transfer (FRET) imaging, we previously demonstrated that effector caspase activation is often an all-or-none response independent of drug choice or dose administered. We here investigated the signaling dynamics during apoptosis initiation via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor pathway to investigate how variability in drug exposure can be translated into largely kinetically invariant cell death execution pathways. FRET-based microscopy demonstrated dose-dependent responses of caspase-8 activation and activity within individual living HeLa cells. Caspase-8 on average was activated 45-600 min after TRAIL/cycloheximide addition. Caspase-8-like activities persisted for 15-60 min before eventually inducing mitochondrial outer membrane permeabilization. Independent of the TRAIL concentrations used or the resulting caspase-8-like activities, mitochondrial outer membrane permeabilization was induced when 10% of the FRET substrate was cleaved. In contrast, in Bid-depleted cells, caspase-8-like activity persisted for hours without causing immediate cell death. Our findings provide detailed insight into the intracellular signaling kinetics during apoptosis initiation and describe a threshold mechanism controlling the induction of apoptosis execution. Employing fluorescence resonance energy transfer (FRET) imaging, we previously demonstrated that effector caspase activation is often an all-or-none response independent of drug choice or dose administered. We here investigated the signaling dynamics during apoptosis initiation via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor pathway to investigate how variability in drug exposure can be translated into largely kinetically invariant cell death execution pathways. FRET-based microscopy demonstrated dose-dependent responses of caspase-8 activation and activity within individual living HeLa cells. Caspase-8 on average was activated 45-600 min after TRAIL/cycloheximide addition. Caspase-8-like activities persisted for 15-60 min before eventually inducing mitochondrial outer membrane permeabilization. Independent of the TRAIL concentrations used or the resulting caspase-8-like activities, mitochondrial outer membrane permeabilization was induced when 10% of the FRET substrate was cleaved. In contrast, in Bid-depleted cells, caspase-8-like activity persisted for hours without causing immediate cell death. Our findings provide detailed insight into the intracellular signaling kinetics during apoptosis initiation and describe a threshold mechanism controlling the induction of apoptosis execution. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 2The abbreviations used are: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; CFP, cyan fluorescent protein; CHX, cycloheximide; DISC, death-inducing signaling complex; FRET, fluorescence resonance energy transfer; MOMP, mitochondrial outer membrane permeabilization; STS, staurosporine; TMRM, tetramethylrhodamine methylester; tBid, truncated Bid; YFP, yellow fluorescent protein; siRNA, small interfering RNA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; fmk, fluoromethylketone; AMC, 7-amido-4-methylcoumarin. is a potent cytotoxic ligand inducing apoptosis preferentially in tumor cells (1Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.P. Nicholl J.K. Sutherland G.R. Smith T.D. Rauch C. Smith C.A. Goodwin R.G. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2664) Google Scholar). New TRAIL-based treatment regimes for adjuvant chemotherapies therefore are currently being studied in phase I and II clinical trials (2Koschny R. Walczak H. Ganten T.M. J. Mol. Med. 2007; 85: 923-935Crossref PubMed Scopus (175) Google Scholar). TRAIL binding to its cognate death receptors TRAIL-R1 and -R2 induces receptor trimerization. At their cytoplasmic domains, TRAIL-R1 and -R2 recruit the adaptor protein Fas-associated death domain into the so-called death inducing signaling complex (DISC). Via interaction of their death effector domains, Fas-associated death domain recruits procaspase-8 and -10 to the DISC, resulting in activation and processing of these initiator proteases (3Falschlehner C. Emmerich C.H. Gerlach B. Walczak H. Int. J. Biochem. Cell Biol. 2007; 39: 1462-1475Crossref PubMed Scopus (384) Google Scholar). Although in some cell lines caspase-8/-10 can directly activate effector caspase-3 (type I signaling) (4Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar), the majority of cells require caspase-8/-10 to initiate apoptosis by cleaving the BH-3-only protein Bid (type II signaling). Truncated Bid (tBid) then translocates to mitochondria and induces Bax/Bak-dependent mitochondrial outer membrane permeabilization (MOMP) (5Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3798) Google Scholar, 6Ward M.W. Rehm M. Duessmann H. Kacmar S. Concannon C.G. Prehn J.H. J. Biol. Chem. 2006; 281: 5837-5844Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 7Yamada H. Tada-Oikawa S. Uchida A. Kawanishi S. Biochem. Biophys. Res. Commun. 1999; 265: 130-133Crossref PubMed Scopus (116) Google Scholar, 8Korsmeyer S.J. Wei M.C. Saito M. Weiler S. Oh K.J. Schlesinger P.H. Cell Death Differ. 2000; 7: 1166-1173Crossref PubMed Scopus (851) Google Scholar). MOMP results in the release of mitochondrial intermembrane space proteins, such as cytochrome c and Smac, from the mitochondria into the cytosol, mitochondrial depolarization, and subsequent apoptosis execution by effector caspases, such as caspase-3, -7, and -6 (9Goldstein J.C. Waterhouse N.J. Juin P. Evan G.I. Green D.R. Nat. Cell Biol. 2000; 2: 156-162Crossref PubMed Scopus (886) Google Scholar, 10Kumar S. Cell Death Differ. 2007; 14: 32-43Crossref PubMed Scopus (664) Google Scholar, 11Rehm M. Dussmann H. Prehn J.H. J. Cell Biol. 2003; 162: 1031-1043Crossref PubMed Scopus (125) Google Scholar). Independent of the choice of stimulus or the dose applied, the induction of MOMP and the subsequent execution of apoptosis by effector caspases were shown to be kinetically invariant all-or-none signaling processes that guarantee cell death over a wide range of key protein concentrations (9Goldstein J.C. Waterhouse N.J. Juin P. Evan G.I. Green D.R. Nat. Cell Biol. 2000; 2: 156-162Crossref PubMed Scopus (886) Google Scholar, 12Rehm M. Dussmann H. Janicke R.U. Tavare J.M. Kogel D. Prehn J.H. J. Biol. Chem. 2002; 277: 24506-24514Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 13Rehm M. Huber H.J. Dussmann H. Prehn J.H. EMBO J. 2006; 25: 4338-4349Crossref PubMed Scopus (169) Google Scholar). In the case of effector caspase activation, this switchlike response was shown to emanate as a systems property from feedback signaling loops in the caspase activation network (13Rehm M. Huber H.J. Dussmann H. Prehn J.H. EMBO J. 2006; 25: 4338-4349Crossref PubMed Scopus (169) Google Scholar, 14Legewie S. Bluthgen N. Herzel H. PLoS Comput. Biol. 2006; 2: e120Crossref PubMed Scopus (188) Google Scholar). In contrast, little is known about the intracellular signaling dynamics during apoptosis initiation leading to the kinetically invariant activation of MOMP. Especially, since it is conceivable that at both physiological and therapeutic conditions, cells can be exposed to TRAIL receptor ligands over a wide range of different concentrations, it is unclear how this variability can be translated into a clear cell death decision. Here, we address this question by using a fluorescence resonance energy transfer (FRET)-based approach to monitor the real time kinetics of caspase-8/-10 activation and activity via the TRAIL receptor pathway using single cell time lapse imaging. We found that TRAIL exposure induced dose-dependent kinetics of caspase-8/-10 activation and activity. We furthermore identified a threshold mechanism that permits us to integrate such differential caspase activities into an all-or-none decision of apoptosis execution. Importantly, we performed these analyses excluding the potential contribution of high abundant downstream effector caspases with similar substrate specificities. Materials—Embryo-tested mineral oil and cycloheximide (CHX) were from Sigma. TRAIL was from Leinco Technologies (St. Louis, MO). Tetramethylrhodamine methylester (TMRM) was from MobiTec (Göttingen). Staurosporine (STS) was from Alexis (San Diego, CA). The broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fmk was purchased from Bachem (St Helen's, UK). G418 was from Invitrogen. Molecular Cloning of the IETD FRET Probe—A DEVDase-responsive enhanced CFP-DEVD-Venus FRET cassette was obtained from pSCAT3 (15Takemoto K. Nagai T. Miyawaki A. Miura M. J. Cell Biol. 2003; 160: 235-243Crossref PubMed Scopus (257) Google Scholar) (a generous gift of Masayuki Miura, RIKEN Brain Research Institute, Wako, Saitama, Japan) by digestion with BamHI and HindIII. This was subcloned into pTK-RL (Promega, WI), digested at the same sites, and treated with shrimp alkaline phosphatase (U. S. Biologicals/Amersham Biosciences). The resulting plasmid was digested with SacI and KpnI to remove the DEVDase substrate cassette and ligated with excess annealed oligonucleotides, forming an IETDase substrate cassette. This IETD cassette was generated using the following complementary oligonucleotides 5′-G AGC GGA ATC GAG ACC GAT GGTAC-3′ and 5′-C ATC GGT CTC GAT TCC GCT CAGCT-3′. This generated a cassette consisting of a coding sequence for IETD flanked NH2-terminally by a flexible glycine-serine dipeptide and COOH-terminally by a KpnI site (encoding glycine-threonine) and a flexible serine-glycine-serine tripeptide. The oligonucleotide sequences immediately adjacent to the overhangs were designed to destroy the SacI site upon ligation with corresponding vector overhangs and to generate a diagnostic PvuII site to exclude multiple concatenated oligonucleotides. The resulting pTK-reverse SCAT8 vector was digested with BamHI and HindIII to obtain the enhanced CFP-IETD-Venus FRET cassette. This was ligated into pcDNA3.1 vector to generate pSCAT8. Cell Culture and Transfection—All cells were cultured in RPMI 1640 medium supplemented with penicillin (100 μg/ml), streptomycin (100 μg/ml), and 10% fetal calf serum (Sigma). Cells were transfected with 500 ng of pSCAT8 plasmid DNA, and 4 μl of Metafecten (Biontex Laboratories, Munich, Germany) per milliliter of serum-free medium at 37 °C for 4 h. For the generation of stable cell lines, cells were selected in the presence of 50 μg/ml G418 (Invitrogen) for 3-4 weeks, and fluorescent clones were enriched. Generation of Stable Bid-depleted Cells—Three different short hairpin RNA sequences specific for human Bid mRNA were designed using the Dharmacon siRNA design tool (available on the World Wide Web). The following 19-nucleotide sequences were chosen and subjected to BLAST analysis to avoid significant homology to other human genes: Bid-1 sense (5′-AAGCTGTTCTGACAACAGC-3′), corresponding to nucleotides 78-97 downstream of the Bid mRNA start codon; Bid-2 sense (5′-AAGGAGAAGACCATGCTGG-3′), homologous to nucleotides 430-449; and Bid-3 sense (5′-AAGAATAGAGGCAGATTCT-3′, spanning nucleotides 210-229. The Bid-specific short hairpin RNA duplexes along with a scrambled control sequence were ligated into the pSilencer 2.1-U6 hygro vector (Ambion, Cambridgeshire, UK) via their BamHI and HindIII sites. To generate stable knockdown cell lines, HeLa cells were transfected with the different short hairpin RNA constructs using Metafectene (Biontex, Munich, Germany) according to the manufacturer's instructions. 24 h post-transfection, the cells were serially diluted and transferred to 96-well plates, and stable clones were selected using hygromycin B (160 μg/ml). Bid expression in HeLa cells stably expressing Bid siRNA was compared with the Bid expression in parental HeLa cells by Western blotting, and the clone with the strongest Bid depletion was selected for this study. After background subtraction, chemiluminescence intensities from n = 3 independent whole cell protein extracts were densitometrically measured using AlphaEase FC software (Alpha Innotech, San Leandro, CA). Adenoviral Infection—The generation of adenoviruses for XIAP (X-linked inhibitor of apoptosis protein) expression and control viruses and the infection procedure were described previously (13Rehm M. Huber H.J. Dussmann H. Prehn J.H. EMBO J. 2006; 25: 4338-4349Crossref PubMed Scopus (169) Google Scholar, 16Eberhardt O. Coelln R.V. Kugler S. Lindenau J. Rathke-Hartlieb S. Gerhardt E. Haid S. Isenmann S. Gravel C. Srinivasan A. Bahr M. Weller M. Dichgans J. Schulz J.B. J. Neurosci. 2000; 20: 9126-9134Crossref PubMed Google Scholar). In brief, parental and Bid-depleted HeLa cells expressing the SCAT8 FRET probe were grown on WillCo dishes (Willco BV, Amsterdam, The Netherlands). Cells were washed twice with phosphate-buffered saline and infected at a multiplicity of infection of 1000 in serum-free medium for 2 h and subsequently cultured in full growth medium. Experiments were carried out >24 h postinfection. Preparation of Whole Cell Extracts and Western Blotting—Cells were collected at 1000 rpm for 3 min and washed with phosphate-buffered saline. The cell pellet was resuspended in lysis buffer (62.5 mm Tris-HCl, pH 6.8, 10% (v/v) glycerin, 2% (w/v) SDS, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, and 5 μg/ml aprotinin) and heated at 95 °C for 20 min. Protein content was determined with the Pierce Micro-BCA protein assay (Pierce). An equal amount of protein (20 μg) was loaded onto SDS-polyacrylamide gels. Proteins were separated at 100 V for 2.5 h and then blotted to nitrocellulose membranes (Protean BA 83; 2 μm; Schleicher & Schuell) in transfer buffer (25 mm Tris, 192 mm glycine, 20% methanol (v/v), and 0.01% SDS) at 18 V for 60 min. The blots were blocked with 5% nonfat dry milk in TBST (15 mm Tris-HCl, pH 7.5, 200 mm NaCl, and 0.1% Tween 20) at room temperature for 1 h. Membranes were incubated with the following antibodies: a rabbit polyclonal caspase-3 antibody (Cell Signaling Technology, Danvers, MA), a mouse monoclonal caspase-8 antibody (Alexis Biochemicals, San Diego, CA), a mouse monoclonal green fluorescent protein antibody (Clontech), a goat polyclonal Bid antibody (R&D Systems, Abingdon, UK), a mouse monoclonal α-tubulin antibody (Sigma), and a mouse monoclonal β-actin antibody (Sigma). Membranes were washed with TBST three times for 5 min and incubated with anti-mouse or anti-rabbit peroxidase-conjugated secondary antibodies (Jackson Laboratories) for 1 h. Blots were washed and developed using the enhanced chemiluminescence detection reagent (Amersham Biosciences). Chemiluminescence was detected at 12-bit dynamic range using a Fuji LAS 3000 CCD system (Fujifilm UK Ltd., Bedfordshire, UK). Determination of Caspase-3-like Protease Activity—After exposure to staurosporine, TRAIL/CHX, or vehicle, culture medium was aspirated, and cells were lysed in 200 μl of lysis buffer (10 mm HEPES, pH 7.4, 42 mm KCl, 5 mm MgCl2, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 0.5% CHAPS, and 1% protease inhibitor mixture (Sigma)). Fifty microliters of this extract were added to 150 μl of reaction buffer (25 mm HEPES, pH 7.5, 1 mm EDTA, 0.1% CHAPS, 10% sucrose, 3 mm dithiothreitol). The reaction buffer was supplemented with 10 μm Ac-DEVD-AMC, the preferred fluorigenic substrate for effector caspase-3 and -7. Production of fluorescent AMC was monitored over 120 min using a fluorescent plate reader (excitation 360 nm, emission 465 nm). Autofluorescence of blanks containing lysis buffer only were subtracted. Protein content was determined using the Pierce Coomassie (Bradford) protein assay kit (Pierce). Caspase activity was expressed as change in fluorescent units/h/μg of protein. Epifluorescence Microscopy and Digital Imaging—Cells were cultivated on 12-mm glass bottom dishes (Willco BV) in 1 ml of medium at least overnight to let them attach firmly. Cells were equilibrated with 30 nm TMRM in 200 μl of RPMI 1640 medium supplemented with penicillin (100 units/ml), streptomycin (100 μg/ml), and 10% fetal calf serum, buffered with HEPES (10 mm; pH 7.4), covered with mineral oil, and placed in a heated (37 °C) incubation chamber that was mounted on the microscope stage. Cells were treated on stage with 1 μm STS or 10-1000 ng/ml TRAIL plus 1 μg/ml CHX. Fluorescence was observed using an Axiovert 200 M inverted microscope equipped with a ×40 numerical aperture 1.3 oil immersion objective (Carl Zeiss, Jena, Germany), polychroic mirror, and filter wheels in the excitation and emission light path containing the appropriate filter sets (cyan fluorescent protein (CFP), excitation 436 ± 10 nm, emission 480 ± 20 nm; YFP, excitation 500 ± 10 nm, emission 535 ± 15 nm; FRET, excitation 436 ± 10 nm, emission 535 ± 30 nm; TMRM, excitation 530 ± 25 nm, emission 592.5 ± 22.5 nm; dichroic mirrors for CFP (FRET), YFP, and TMRM; Semrock (Rochester, NY)). Images were recorded using a back-illuminated, cooled electron multiplying CCD camera (Andor Ixon BV 887-DCS, Andor Technologies, Belfast, Northern Ireland). The imaging setup was controlled by MetaMorph 7.1r1 software (Molecular Devices Ltd., Wokingham, UK). Confocal Microscopy and Acceptor Bleaching—To confirm resonance energy transfer within the IETD FRET probe, the acceptor Venus was bleached, and the donor dequenching was analyzed. HeLa cells expressing the FRET probe were placed on the heated stage of an LSM 510 Meta confocal microscope equipped with a Plan-Apochromat ×63 numerical aperture 1.4 oil immersion differential interference contrast objective (Carl Zeiss, Jena, Germany). The emission spectra were recorded at 10.3-nm step size using the 405-nm laser line (attenuated to 1.0%), and the 405/514 multichroic beamsplitter and the Meta detector in the range of 442-614 nm. Following each cycle of bleach scans with the nonattenuated 514 argon laser line (laser was run at 50% of its maximal power), spectral images were recorded. Mitochondrial Membrane Potential (ΔΨM) and FRET Disruption—After background subtraction, the cellular TMRM fluorescence intensity was calculated for each cell. Caspase cleavage kinetics were detected at the single-cell level by FRET analysis, as described previously (12Rehm M. Dussmann H. Janicke R.U. Tavare J.M. Kogel D. Prehn J.H. J. Biol. Chem. 2002; 277: 24506-24514Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Images were processed using MetaMorph 7.1r1 software (Molecular Devices Ltd., Wokingham, UK). CFP/YFP and FRET/YFP emission ratio traces were obtained by dividing the fluorescence intensity values in the CFP and FRET channels by the YFP emission of single cells after background subtraction. The ratiometric readout automatically corrects for nonspecific changes in fluorescence intensities that affect all channels in parallel, such as slight drifts in optical focus or cellular movements. Statistics—Data are given as means ± S.D. or S.E. For statistical comparison, Student's t test or analysis of variance and subsequent Tukey's test were used for normal distributed data. Otherwise, Mann-Whitney U test or Kruskal-Wallis H test were used. p values smaller than 0.05 were considered to be statistically significant. Kruskal-Wallis H tests returning statistical significances were subsequently followed up with Bonferroni adjusted Mann-Whitney U tests. Acceptor Bleaching Confirms Resonance Energy Transfer in a New IETDase FRET Probe—Previously, recombinant FRET probes based on green fluorescent protein variants were successfully employed to analyze effector caspase activity in single living cells (12Rehm M. Dussmann H. Janicke R.U. Tavare J.M. Kogel D. Prehn J.H. J. Biol. Chem. 2002; 277: 24506-24514Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 13Rehm M. Huber H.J. Dussmann H. Prehn J.H. EMBO J. 2006; 25: 4338-4349Crossref PubMed Scopus (169) Google Scholar, 15Takemoto K. Nagai T. Miyawaki A. Miura M. J. Cell Biol. 2003; 160: 235-243Crossref PubMed Scopus (257) Google Scholar, 17Tyas L. Brophy V.A. Pope A. Rivett A.J. Tavare J.M. EMBO Rep. 2000; 1: 266-270Crossref PubMed Scopus (226) Google Scholar). Here, we developed a new FRET probe based on CFP and Venus, an enhanced yellow fluorescent protein, which are interconnected by a short linker containing a preferred caspase-8/-10 recognition site IETD (18Garcia-Calvo M. Peterson E.P. Leiting B. Ruel R. Nicholson D.W. Thornberry N.A. J. Biol. Chem. 1998; 273: 32608-32613Abstract Full Text Full Text PDF PubMed Scopus (849) Google Scholar, 19McStay G.P. Salvesen G.S. Green D.R. Cell Death Differ. 2008; 15: 322-331Crossref PubMed Scopus (258) Google Scholar). Upon CFP excitation, energy is transferred to the acceptor fluorophore Venus (Fig. 1A). Cleavage of the linker disrupts the resonance energy transfer as the distance between donor and acceptor increases and results in enhanced CFP emission (Fig. 1B). The probe was generated from a previously described FRET probe template for effector caspases, which provided a very high signal/noise ratio upon proteolytic cleavage (15Takemoto K. Nagai T. Miyawaki A. Miura M. J. Cell Biol. 2003; 160: 235-243Crossref PubMed Scopus (257) Google Scholar). This high signal/noise ratio facilitates the detection of low caspase activities as expected in the case of caspase-8/-10 during apoptosis initiation. Efficient resonance energy transfer was observed when expressing the IETD FRET probe in HeLa cervical cancer cells; upon photobleaching the acceptor fluorophore Venus in individual cells, CFP emission significantly increased, as observed in cellular fluorescence emission profiles (Fig. 1, C-F). Bid Depletion Can Prevent Apoptosis Execution but Not IETD Probe Cleavage following TRAIL Exposure—Biochemically, it has been shown that besides caspase-8 and -10, downstream effector caspase-3 and -6 can cleave IETD recognition sites as well (19McStay G.P. Salvesen G.S. Green D.R. Cell Death Differ. 2008; 15: 322-331Crossref PubMed Scopus (258) Google Scholar). Similar overlapping specificities were reported for synthetic caspase inhibitors, such as IETD- and DEVD-fmk, effectively preventing the selective inhibition of individual caspases (18Garcia-Calvo M. Peterson E.P. Leiting B. Ruel R. Nicholson D.W. Thornberry N.A. J. Biol. Chem. 1998; 273: 32608-32613Abstract Full Text Full Text PDF PubMed Scopus (849) Google Scholar, 19McStay G.P. Salvesen G.S. Green D.R. Cell Death Differ. 2008; 15: 322-331Crossref PubMed Scopus (258) Google Scholar). Using the above described new IETD FRET probe, we therefore investigated whether we could establish a model system that enabled us to temporally separate the downstream execution phase from caspase-8/-10-dependent signaling during the initiation phase of TRAIL-induced apoptosis. HeLa cells are type II signaling cells and consequently depend on the mitochondrial pathway for the activation of effector caspases during death receptor-induced apoptosis (20Engels I.H. Stepczynska A. Stroh C. Lauber K. Berg C. Schwenzer R. Wajant H. Janicke R.U. Porter A.G. Belka C. Gregor M. Schulze-Osthoff K. Wesselborg S. Oncogene. 2000; 19: 4563-4573Crossref PubMed Scopus (232) Google Scholar). We generated HeLa cells stably depleted of Bid expression by siRNA transfection to impair MOMP and subsequent effector caspase activation (Fig. 2A). Bid expression was reduced to ∼4.5 ± 3.0% of the wild type expression level as densitometrically quantified from Western blots. To investigate whether Bid depletion was sufficient to impair effector caspase activation during death receptor-induced apoptosis, we next biochemically characterized the response of parental and Bid-depleted HeLa cells to TRAIL/CHX (hereafter referred to as TRAIL) and intrinsic apoptosis stimulus STS. TRAIL was administered in combination with 1 μg/ml CHX to suppress the activation of translation-dependent survival signaling in response to TRAIL exposure (3Falschlehner C. Emmerich C.H. Gerlach B. Walczak H. Int. J. Biochem. Cell Biol. 2007; 39: 1462-1475Crossref PubMed Scopus (384) Google Scholar). In both parental and Bid-depleted cells, procaspase-8 was processed into active subunits with apparently identical kinetics in response to TRAIL (Fig. 2B). Although parental cells can activate caspase-3, Bid-depleted HeLa cells did not show any reduction in procaspase-3, and active subunits were only detected in minute amounts (Fig. 2, B and C). To analyze whether the observed caspase-3 processing results in caspase-3-like activity, we employed fluorigenic DEVD-AMC substrate assays. In these assays, Bid depleted HeLa cells exhibited massively reduced cleavage rates following death receptor stimulation with TRAIL, suggesting that downstream apoptosis execution is significantly inhibited in these cells (Fig. 2D). In contrast, Bid depletion did not influence the activation of caspase-3 or caspase-8 (Fig. 2E), which is largely activated downstream of caspase-3 during STS-induced apoptosis (20Engels I.H. Stepczynska A. Stroh C. Lauber K. Berg C. Schwenzer R. Wajant H. Janicke R.U. Porter A.G. Belka C. Gregor M. Schulze-Osthoff K. Wesselborg S. Oncogene. 2000; 19: 4563-4573Crossref PubMed Scopus (232) Google Scholar); nor was caspase-3 like activity significantly affected in fluorigenic assays (Fig. 2F). Next, we tested whether the IETD FRET probe can still be cleaved in Bid-depleted cells during TRAIL-induced apoptosis. Following TRAIL exposure, the full-length probe was cleaved into fragments corresponding to the molecular sizes of CFP linker and Venus, as shown by Western blotting (Fig. 2G). The addition of caspase inhibitor benzyloxycarbonyl-VAD-fmk blocked FRET substrate cleavage, showing this to be a caspase-dependent process (Fig. 2G). Taken together, these results show that Bid depletion efficiently inhibited apoptosis execution in response to TRAIL addition. Bid depletion, however, did not impair the processing of procaspase-8; nor did it prevent IETD FRET probe cleavage. TRAIL-induced Caspase-8-like Activities Can Persist for Hours without Causing Immediate Apoptotic Cell Death in Bid-depleted HeLa Cells—To analyze profiles of caspase-8-like activities in single living cells by time lapse imaging, we then expressed the IETD FRET probe in the Bid-depleted HeLa cells. Following administration of TRAIL, we observed that probe cleavage began individually in single cells rather than it being a synchronous response throughout the whole population in the field of view (Fig. 3A). Probe cleavage was detected as an intensity increase in the CFP/YFP emission ratio images (Fig. 3A) and a decrease in FRET/YFP emission ratio images (not shown). Previous studies in HeLa cells have shown a loss in ΔΨM in response to MOMP-induced cytochrome c release (9Goldstein J.C. Waterhouse N.J. Juin P. Evan G.I. Green D.R. Nat. Cell Biol. 2000; 2: 156-162Crossref PubMed Scopus (886) Google Scholar, 11Rehm M. Dussmann H. Prehn J.H. J. Cell Biol. 2003; 162: 1031-1043Crossref PubMed Scopus (125) Google Scholar, 21Dussmann H. Rehm M. Kogel D. Prehn J.H. J. Cell Sci. 2003; 116: 525-536Crossref PubMed Scopus (94) Google Scholar, 22Goldstein J.C. Munoz-Pinedo C. Ricci J.E. Adams S.R. Kelekar A. Schuler M. Tsien R.Y. Green D.R. Cell Death Differ. 2005; 12: 453-462Crossref PubMed Scopus (186) Google Scholar). Using TMRM as a ΔΨM-sensitive dye in parallel with the FRET probe, we found that in Bid-depleted cells, mitochondria either did not depolarize (Fig. 3A) or depolarized only at very late times during or after FRET probe cleavage (not shown). Importantly, classical apoptotic morphology, such as cellular shrinkage or membrane blebbing could not be observed as long as the cells did not depolarize. Corresponding to our biochemical analyses (Fig. 2), this indicates that MOMP-dependent mitochondrial depolarization and apoptosis execution are efficiently inhibited by Bid depletion. We next plotted the CFP/YFP and FRET/YFP emission ratios of individual Bid-depleted cells to graphically follow substrate cleavage over time. Probe cleavage began following an initial lag time, as observed by an increase in the CFP/YFP and a concomitant decrease in the FRET/YFP ratios (Fig. 3B). In response to 100 ng/ml TRAIL, probe cleavage on average began 56 ± 25 min after drug addition and lasted for 5.5 ± 2.8 h (data are mean ± S.D. from n = 20 cells analyzed). To exclude the possibility that type I-like direct activation of caspase-3 by caspase-8/-10 contributed to the activity measured (4Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar), in additional control experiments we employed adenoviral overexpression of XIAP, the most efficient intracellular inhibitor of downstream caspase-3, -7, and -9. XIAP overexpression did not affect IET-Dase activity in Bid-depleted cells in response to TRAIL (supplemental Fig. 1). These findings demonstrate that HeLa cells individually activate IETDases in response to TRAIL exposure and that this caspase-8-like activity can be a surprisingly stable response that can persist for hours without causing immediate apoptotic cell death. TRAIL-induced Profiles of Caspase-8-like Activities Upstream of MOMP in Parental HeLa Cells—We next analyzed caspase-8-like activities in parental HeLa cells following TRAIL exposure. In contrast to Bid-depleted cells, we observed that parental HeLa cells cleaving the FRET probe subsequently underwent cellular condensation and membrane blebbing characteristic of apoptotic cell death (Fig. 4A). Furt

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