1 CNRS-UMR8125
2 Immunology Unit, Department of Clinical Biology, Institut Gustave Roussy, 39 rue Camille-Desmoulins, 94805 Villejuif, France
3 Institut André Lwoff, UPR-1983, Laboratoire Replication de l'ADN et Ultrastructure du Noyau, 7 rue Guy Moquet, 94801 Villejuif, France
* Author for correspondence (e-mail: kroemer{at}igr.fr)
Accepted 6 August 2004
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Summary |
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Key words: Mitochondria, Bcl-2, HIV-1, DNA double strand breaks
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Introduction |
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Undoubtedly, Env can induce cell death through a cornucopia of different mechanisms. The Env glycoprotein precursor protein (gp160) undergoes proteolytic maturation to gp41 (membrane inserted) and gp120 (membrane inserted or shed from the cell surface). Soluble gp120 can stimulate pro-apoptotic signal via an action on chemokine receptors (CXCR4 for lymphotropic Env variants, CCR5 for monocytotropic Env variants) (Cicala et al., 2000; Roggero et al., 2001
; Twu et al., 2002
), the p38 mitogen-activated protein kinase pathway (Kaul and Lipton, 1999
), pertussis toxin sensitive G proteins (Twu et al., 2002
) and/or a rapid cytosolic Ca2+ increase (Haughey and Mattson, 2002
). The membrane-bound gp120/gp41 complex expressed on the surface of HIV-1-infected cells can induce apoptosis via interaction with uninfected cells expressing the receptor (CD4) and the chemokine co-receptor. Although this interaction can signal for apoptosis via a transient cell-to-cell contact involving a hemifusion-like event (Blanco et al., 2003
), in most instances, this interaction induces cellular fusion (cytogamy) (Lifson et al., 1986
; Sodroski et al., 1986
; Sylwester et al., 1997
) followed by nuclear fusion (karyogamy) within the syncytium (Ferri et al., 2000b
). This nuclear fusion is the expression of an abortive entry into the mitotic prophase stimulated by the transient activation of the cyclin B-dependent kinase-1 (Cdk1) (Castedo et al., 2002b
), accompanied by the permeabilization of the nuclear envelope, the nuclear translocation of mammalian target of rapamycin (mTOR), the mTOR-mediated phosphorylation of p53 on serine 15 (p53S15P) (Castedo et al., 2001
), the p53-mediated transcription of pro-apoptotic proteins including Puma and Bax (Castedo et al., 2001
; Perfettini et al., 2004
), and finally, the Puma-triggered Bax/Bak-mediated mitochondrial membrane permeabilization (MMP) (Ferri et al., 2000a
). This last step is accompanied by the activation of Bak (and Bax) on the surface of mitochondria, associated with a conformational change leading to the exposure of the N terminus of Bak, which becomes immunodetectable (Perfettini et al., 2004
). This leads to the release of cytochrome c from the mitochondrial intermembrane space, and cytochrome c then activates the apoptosome caspase activation complex. Activated, proteolytically mature caspase-3 can then trigger the biochemical cascade culminating in DNA fragmentation and nuclear apoptosis, including advanced chromatin condensation (pyknosis) and formation of nuclear apoptotic bodies (karyorrhexis) (Budijardjo et al., 1999
; Roumier et al., 2003
; Wang, 2002
).
The above mentioned scenario of Env-elicited syncytial apoptosis describes a slow process in which an abortive advancement in cell cycle as well as the sequential activation of at least two transcription factors (NFB and p53) are obligate steps on the path to death. Thus, apoptosis affects approximately half of the syncytia generated by fusion of Env- and CD4-transfected HeLa cells after 2 days (Castedo et al., 2001
; Castedo et al., 2002b
; Ferri et al., 2000a
; Perfettini et al., 2004
). However, this phenomenon could be significantly accelerated (half life
6 hours post cytogamy) when one of the two fusion partners was primed for apoptosis using a variety of different inducers, and in this case, apoptosis may be considered as a contagious phenomenon, as shown in the present work. We provide an extensive characterization of this novel form of apoptosis propagated through cell fusion. We demonstrate that contagious apoptosis obeys mechanistic rules that are completely different from `classical' fusion-induced apoptosis, and we suggest that it may constitute a novel mechanism through which Env-expressing (that is HIV-1-infected) cells destroy CD4+ T cells.
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Materials and Methods |
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Preparation of cytoplasts
HeLa Env cells were enucleated as described previously (Castedo et al., 1996; Chipuk et al., 2003
). Briefly, cells were treated in 2 ml of supplemented Dulbecco's modified Eagle's medium (DMEM) containing cytochalasin B (15 µg/ml; Calbiochem) and DNase I (80 U; Roche). Cell suspension was adjusted to a final concentration of 5-10x106/ml and incubated at 37°C for 45 minutes before being layered onto a previously prepared discontinuous Ficoll® density gradient consisting in 3 ml of 100%, 1 ml of 90% and 3 ml of 55% Ficoll® in supplemented DMEM. 2 ml of cytochalasine-treated cell suspension was applied to a pre-equilibrated gradient and centrifuged for 1 hour in a pre-warmed Beckman SW41 rotor at 100,000 g. Cytoplasts were collected in the 90% Ficoll® layer, washed and resuspended in supplemented DMEM. Enucleation efficiency was determined 3 hours later by microscopic analysis of pre-stained cytoplasts with Hoechst 33342 (2 µM; Sigma-Aldrich). For apoptosis induction, cytoplasts were subjected to 3 hours exposure to 1 µM STS or 5 µM ActD, then washed five times and co-cultured overnight with HeLa CD4 cells.
Immunofluorescence and flow cytometry
Different cells were cultured on coverslips coated with poly-L-lysine (PAA), pre-stained for 45 minutes with 5-chloromethylfluorescein diacetate (CellTracker® Green CMFDA, 15 µM; Molecular Probes), 5- and 6-{[(4-chloromethyl)benzoyl]amino}tetramethylrhodamine (CellTracker® Red CMTMR, 15 µM; Molecular Probes), 7-amino-4-chloromethylcoumarin (CellTracker® Blue CMAC, 20 µM; Molecular Probes) or MitoTracker® Green (500 nM; Molecular Probes) and nuclei were stained with 2 µM Hoechst 33342 (Sigma-Aldrich) before being examined using fluorescence microscopy. TUNEL staining was performed with a detection kit from Roche. The mitochondrial transmembrane potential (m) was determined with 40 nM 3,3'dihexyloxacarbocyanine iodide [DiOC6(3); Molecular Probes] or tetramethylrhodamine methylester polychlorate (TMRM, 150 nM; Molecular Probes) (Castedo et al., 2002a
). Specific antibodies for activated Bak (Bak Ab-1; Oncogene Research Products), cytochrome c (BD Pharmigen), activated caspase-3 (Casp-3 a; Cell Signaling Technology), phosphorylated-histone H2AX (Ser139; Upstate Cell Signaling) and phospho-Chk2 (Thr68; Cell Signaling Technology) were used on paraformaldehyde-fixed (4% w:v) cells; all were detected by a goat anti-mouse or goat anti-rabbit IgG-conjugated Alexa® Fluor from Molecular Probes (Boya et al., 2003
). Cytofluorometric analyses were performed on FACS Vantage (Becton Dickinson) using 40 nM DiOC6(3) for
m quantification (Castedo et al., 2002a
; Zamzami et al., 2000
), 1 µg/ml propidium iodide (PI) for determination of cell viability (Zamzami et al., 1995a
) and an annexin V conjugated with fluorescein isothiocyanate kit (Bender Medsystems) for the assessment of phosphatidylserine (PS) exposure (Castedo et al., 1996
). DNA content was quantified using 20 µM Hoechst 33342 for 30 minutes at 37°C.
Electron microscopy
Cells were fixed for 1 hour at 4°C in 2.5% glutaraldehyde in phosphate buffer (pH 7.4), washed and fixed again in 2% osmium tetroxide before embedding in Epon resin. Electron microscopy was performed with a transmission electron microscope (model EM902; Carl Zeiss MicroImaging, Inc.), at 80 kV, on ultrathin sections (80 nm) stained with uranyl acetate and lead citrate.
DNA fragmentation assays
For pulse field gel electrophoresis, DNA was prepared from agarose plugs (2x106 cells), followed by electrophoresis in a Bio-Rad Laboratories' CHEF-DRII (1% agarose, TBE, 200 V, 24 hours, pulse wave 60 seconds, 120° angle). Comet assays were performed (using a kit from Trevigen) to detect double-stranded DNA breaks in HeLa Env cells or overnight in HeLa Env/CD4 syncytia (nonapoptotic adherent or apoptotic cells). Briefly, cells were immobilized in a bed of low melting point agarose, following a gentle cell lysis; cleaved DNA fragments migrated out of the cell under electrophoresis.
Determination of ß-galactosidase activity
HeLa Env and HeLa CD4 cells were stably transfected with Tat and the lacZ, respectively, under the control of the HIV-1 long terminal repeat (LTR) and selected in medium containing 500 mg/ml G418. For the determination of ß-galactosidase activity in situ, cells were fixed with a mixture of formaldehyde (0.37%) and glutaraldehyde (0.2%) in PBS solution for 5 minutes, then treated overnight with buffer containing 200 mM potassium ferrocyanide, 1 M MgCl2 and 50 mg/ml X-Gal (Promega), and examined by contrast phase microscopy. Alternatively, co-cultured cells were lysed and assayed for ß-galactosidase activity using the Enhanced ß-galactosidase Assay kit (CPRG; Gene Therapy Systems) as well a MRX II microplate reader (Dynex Technologies).
Contagious apoptosis in vivo
In vivo experiments were performed using 10x106 CellTracker® Red-pre-labeled Jurkat cells injected into the peritoneal cavity of athymic mice (nu/nu). Eight hours later, 25x106 HeLa Env or HeLa CD4 cells were injected and peritoneal cells were recovered after overnight incubation. Jurkat cells were analyzed by flow cytometric determination of m loss and phosphatidylserine exposure (Castedo et al., 2002a
).
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Results and Discussion |
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Mechanistic differences between spontaneous and contagious syncytial apoptosis
Syncytia formed by co-culture of normal HeLa Env and HeLa CD4 cells undergo apoptosis after a latency phase of more than 24 hours. In contrast, contagious apoptosis occurred much more rapidly, attaining approximately 50% of heterokarya within 4 hours (Fig. 2A). While spontaneously arising syncytia typically contained >10 nuclei, syncytia formed by co-incubation of STS-pretreated and control cells had mostly less than five nuclei (Fig. 2B). As detailed in the Introduction, syncytia arising from interactions between normal HeLa Env and HeLa CD4 cells spontaneously die through a complex signaling pathway that relies on the step-wise activation of a range of cellular regulators including cyclin B-dependent kinase-1 (Cdk1), mammalian target of rapamycin (mTOR), and p53 (Castedo et al., 2001; Castedo et al., 2002b
; Perfettini et al., 2004
). Pharmacological inhibitors of Cdk1 (roscovitine, purvalanol), mTOR (rapamycin, LY294002), or p53 (pifithrin
, PDTC), however, had no effect on contagious apoptosis (Fig. 2C), indicating that this type of apoptosis is not just an accelerated version of spontaneous syncytial apoptosis and rather obeys different principles.
Nuclear manifestations of contagious apoptosis are only partially caspase-dependent
When healthy HeLa Env cells expressing the Tat protein were fused with HeLa CD4 cells expressing a Tat-transactivatable ß-galactosidase reporter gene placed under the control of the LTR promoter, the resulting syncytia produced ß-galactosidase, as detectable by cytohistochemical methods in situ (Fig. 3A) or by enzymatic methods (Fig. 3B). No such ß-galactosidase production was observed when either of the two fusion partners was PACC+, and all syncytial nuclei evolved to nuclear apoptosis. Addition of Z-VAD.fmk, which prevented the development of full-blown apoptosis, did not restore the expression of ß-galactosidase (Fig. 3A,B), pointing to a caspase-independent blockade of nuclear function. PACC+ (STS-pretreated) Env cells showed partial chromatinolysis, as determined by comet assay (Fig. 4A), yet lacked large-scale DNA fragmentation detectable by pulse-field electrophoresis (Fig. 4B), and were TUNEL negative (Fig. 5A). In contrast, when these cells underwent the transition to full-blown apoptosis, either as individual cells or after fusion with normal cells, their DNA underwent fragmentation to 50 kbp pieces (Fig. 4b), and was TUNEL positive (Fig. 5A). Z-VAD.fmk prevented the advancement to nuclear apoptosis (Fig. 5A, see also Fig. 1C), correlating with a complete inhibition of
50 kbp fragmentation (Fig. 4B) and a suppression of TUNEL positivity, both in single cells and in contagious apoptosis (Fig. 5A). Importantly, Z-VAD.fmk, however, did not prevent the `contagion' of PACC from HeLa Env to HeLa CD4 nuclei occurring within syncytia, as detectable by chromatin staining (Fig. 1A) or electron microscopy (Fig. 1B). Indeed, when STS-primed, PACC+ HeLa Env cells were fused with untreated HeLa CD4 cells in the presence of Z-VAD.fmk, all the nuclei within the resulting syncytia exhibited PACC. Moreover, Z-VAD.fmk did not reduce the scores of comet assays (Fig. 4A). The comet assay measures the occurrence of DNA double strand breaks, which lead to so-called `DNA foci', that is the accumulation of DNA repair proteins and cell cycle regulators in the proximity of DNA lesions (Banath and Olive, 2003
; Castedo et al., 2004
; Celeste et al., 2003
). Accordingly, PACC correlated with the phosphorylation of histone H2AX (on serine 139) and that of Chk2 (on threonine 68), which accumulated in discrete nuclear speckles, detectable with phospho-neoepitope-specific antibodies (Fig. 5B). When contagion occurred within syncytia in the presence of Z-VAD.fmk, all nuclei exhibited such foci. To verify that the DNA foci were also present in nuclei from the `recipient' cells, HeLa CD4 cells were pre-stained with CellTracker Red® and the fate of the nuclei within syncytia was followed. Phosphorylation of H2AX and Chk2 was also apparent in these `recipient' nuclei. In summary, PACC involves caspase resistance, comet assay positivity, transcriptional silencing and DNA foci. PACC can be transmitted from donor to recipient nuclei, in a caspase-independent fashion.
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Nuclei and mitochondrial DNA are dispensable for contagious apoptosis, which however is suppressed by Bcl-2
To determine which cellular component actually transmits contagious apoptosis, HeLa Env cells were enucleated and the resulting cytoplasts were then treated with a sublethal STS dose, washed extensively, and fused with healthy HeLa CD4 cells. The nuclei of these latter cells readily advanced to PACC (in the presence of Z-VAD.fmk) or full-blown apoptosis (in the absence of Z-VAD.fmk) (Fig. 6A). Similar results were obtained when cytoplasts were pretreated with either STS or ActD. (Fig. 6B). Thus, the stimulus responsible for the transmission of the lethal signal is generated in cytoplasts and, as a consequence, must be cytoplasmic (non-nuclear). HeLa Env cells lacking mitochondrial DNA (° cells) driven into PACC also stimulated the advancement of HeLa CD4 nuclei to nuclear apoptosis (Fig. 6C,D), indicating that neither nuclear nor mitochondrial DNA were required for the transmission of the lethal signal. In a further series of experiments, we introduced the prominent apoptosis inhibitor Bcl-2 into either donor (STS-pretreated HeLa Env) or acceptor (non-treated HeLa CD4) cells, prior to cell fusion. Although Bcl-2 failed to prevent the occurrence of PACC in Env cells treated with STS, such cells returned to a normal nuclear morphology after overnight culture (Fig. 7), suggesting that PACC is actually a reversible phenomenon. If present in donor or acceptor cells, Bcl-2 also abolished the transmission of the apoptotic signal to the acceptor nuclei, showing that both donor and acceptor nuclei were morphologically normal 18 hours after fusion. Very similar results were found when Bcl-2 was replaced by the cytomegalovirus-encoded vMIA, which also acts on mitochondria to inhibit apoptosis (Goldmacher et al., 1999
; Poncet et al., 2004
). The behavior of Bcl-2 and vMIA was in marked contrast to that of the baculovirus caspase inhibitor p35, which did not reverse the transmission of PACC (Fig. 7). Thus, a Bcl-2/vMIA-inhibited checkpoint determines the transmission of the apoptotic signal in this particular experimental system.
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Contagious apoptosis as a mechanism of Env-mediated bystander killing
Two T-lymphoid cell lines (Jurkat and CEM) fuse with HeLa Env cells, while U937 do not fuse with HeLa Env and instead undergo transient interactions that involve transfer of plasma membrane lipids through a hemifusion-like process (Blanco et al., 2003). STS-pulsed HeLa Env cells with PACC did induce killing of Jurkat or CEM cells, yet had no major apoptosis-inducing effect on U737 cells, which is in accord with the notion that fusion (rather than hemifusion) is required for cell induction (Fig. 8A). CD4+ lymphocytes (either resting or PHA/IL-2-activated lymphoblasts) from healthy donors could fuse with healthy Env-expressing cells, yet failed to undergo nuclear apoptosis within a 18-hour culture period. However, when Env cells were driven into PACC (either with STS or with ActD), they readily induced the destruction of CD4+ lymphocytes and lymphoblasts (Fig. 8B). This effect was abolished by the use of the fusion inhibitor C34 (Fig. 8B) and was not observed when the lymphocytes were incubated with PACC+ HeLa CD4 cells (not shown). To demonstrate that contagious apoptosis can occur in vivo, CellTracker Red® -pre-labeled Jurkat cells were injected into the peritoneal cavity of athymic (nu/nu) mice. The instillation of STS-primed (but not untreated) HeLa Env cells (but not that of HeLa CD4 cells) induced a loss of
m in a considerable number of the Jurkat cells (Fig. 8C), which is a sign of imminent apoptosis (Zamzami et al., 1995b
). In conclusion, CD4+ T cells can undergo contagious apoptosis when exposed to dying Env-expressing cells, both in vitro and in vivo.
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Concluding remarks
When HIV-1 Env-expressing cells are exposed to a short-term stress (STS, ActD, Vpr) that does not induce acute apoptosis, yet commits cells to undergo apoptosis later (even after withdrawal of the inducer by extensive washing), such cells can transmit the induction of apoptosis to their healthy CD4+ fusion partner. We have termed this fusion-mediated transmission of an apoptotic signal as `contagious apoptosis' and characterized it to some detail. As an expression of commitment to apoptosis, the contagious fusion partner can show PACC with DNA double strand breaks, yet lack obvious signs of apoptosis, such as caspase-3 activation and mitochondrial changes indicative of MMP (Bak activation, cytochrome c release, m dissipation). Within hours, contagious apoptosis leads to the synchronized nuclear pyknosis in both the `donor' cell (that is the stressed, apoptosis prone cell) and in the `recipient' cell (that is the healthy, untreated fusion partner). This transmission of nuclear pyknosis correlates with the activation of caspase-3 and is fully inhibited by the broad-spectrum caspase inhibitor Z-VAD.fmk or the baculovirus-derived caspase inhibitor p35. Contagious apoptosis is accompanied by MMP, which, however, occurs independently from Z-VAD.fmk-inhibitable caspases (Fig. 1). Contagious apoptosis does not depend on cell cycle advancement or the action of the pro-apoptotic transcription factor p53 (Fig. 2), and instead correlates with an (caspase-independent) arrest of transcription (Fig. 3).
While massive chromatinolysis (measured by agarose gel electrophoresis in Fig. 4B and TUNEL staining in Fig. 5A) occurring in contagious apoptosis can be suppressed by Z-VAD.fmk, there are other, more subtle nuclear changes that occur in a caspase-independent fashion. Thus, STS-pulsed `donor' cells have DNA double strand breaks (as determined by comet assay in Fig. 4A or by assessing H2AXP and Chk2P foci) and transmit such DNA double strand breaks to `recipient' nuclei, even in the presence of Z-VAD.fmk (Fig. 5B-D). The morphological manifestation of DNA double strand breaks is PACC, that is a sub-apoptotic, partial, peripheral chromatin condensation affecting ruffled nuclei (Figs 1, 6). Thus, in the presence of Z-VAD.fmk, PACC appears to be contagious (Figs 1, 6), correlating with MMP (Fig. 1F).
Paradoxically however, DNA (including damaged DNA) is not required to transmit the lethal signal in contagious apoptosis, as demonstrated by removal of nuclear DNA (by acute enucleation) or removal of mitochondrial DNA (by suppression of mitochondrial DNA replication) from the `donor' Env-expressing cells (Fig. 6). However, suppression of MMP by Bcl-2 and vMIA could avoid the transmission of nuclear apoptosis and even that of PACC (observable in the presence of Z-VAD.fmk) (Fig. 7), pointing to the involvement of mitochondria (but not mitochondrial DNA, Fig. 6C,D) in the contagion of apoptosis. This latter observation indicates an interesting difference between caspase inhibition by Z-VAD.fmk and the effects of Bcl-2/vMIA. Z-VAD.fmk prevents the manifestation and transmission of nuclear apoptosis, yet has no effect on apoptosis-associated MMP or on the transmission of PACC. In contrast, Bcl-2 and vMIA, which inhibit MMP, do abrogate the transmission of PACC, pointing to an unexpected (and hitherto unexplained) cross talk between MMP and nuclear DNA lesions.
Irrespective of the detailed mechanisms of contagious apoptosis, our data indicate that this phenomenon could potentially affect CD4+ T cells in HIV-1 infection. Stressed, Env-expressing cells can kill CD4+ lymphoblastoid cell lines and primary T cells, as well as activated T lymphoblasts in vitro. Moreover, stressed Env+ cells can drive CD4+ cells into rapid apoptosis in vivo (Fig. 8). Although it has not been shown that this phenomenon also occurs in vivo, in HIV-1-infected individuals evolving toward AIDS, a plausible scenario would be that Env-expressing cells (which hence are HIV-1 infected) could transmit apoptosis to interacting CD4 cells. In conditions in which viral replication overwhelms the cellular defense response and triggers (pre-)apoptosis of the `donor' Env+ cells, fusion with `recipient' CD4+ cells would contribute to the depletion of CD4+ T lymphocytes by contagious apoptosis.
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Acknowledgments |
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