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First published online 19 October 2004
doi: 10.1242/jcs.01486

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Contagious apoptosis facilitated by the HIV-1 envelope: fusion-induced cell-to-cell transmission of a lethal signal

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

View larger version (52K): [in a new window]   Fig. 1. Contagious apoptosis induced by syncytium formation. (A) Representative immunofluorescent images of contagious apoptosis. HeLa cells were left untreated (Co.) or were treated with a 3-hour pulse of STS (3 µM), leading to the acquisition of typical pre-apoptotic chromatin condensation (PACC). Syncytia were formed by overnight co-culture of untreated HeLa CD4 and HeLa Env cells (Syncytia, Co), or were obtained by co-culture of untreated HeLa CD4 cells and STS-pulsed HeLa Env cells, which were washed five times, either in the absence (Contagion) or in the presence (Contagion+zVAD) of the pan-caspase inhibitor Z-VAD.fmk (100 µM). HeLa Env and CD4 were pre-stained with CellTracker® Green and CellTracker® Red, respectively, and fixed before nuclear staining with Hoechst 33342. Note that, in contagious apoptosis, nuclei from previously untreated HeLa CD4 cells (red) show extensive chromatin condensation (arrows). (B) Transmission electron microscopy of control syncytia or syncytia generated with PACC+ Env cells. Note that contagious apoptosis is characterized by full-blown nuclear apoptosis with pyknosis of all nuclei (in the absence of Z-VAD). In the presence of Z-VAD, however, all nuclei showed PACC (partial chromatin condensation, irregular contours and rippled nuclear envelopes). N1, 2, 3 etc. indicate individual nuclei in each syncytium. (C) Quantification of PACC and nuclear apoptosis (NA), as determined by Hoechst 33342 staining, in HeLa Env induced by short-term exposure to STS alone (3h, STS), short-term STS exposure followed by washing and overnight culture (3h, STS+18h), control syncytia or contagion (induced as in A). Significance was calculated with the paired Student's t-test with respect to untreated controls. #P<0.005. (D) Independence of contagious apoptosis from the initial inducer. HeLa Env cells were primed to PACC by a 3-hour treatment with STS (3 µM), actinomycin D (ActD, 5 µM) or the HIV-1-derived apoptogenic peptide Vpr 52-96 (3 µM). Nuclear apoptosis mean ± s.d., n=3) affecting co-cultured HeLa CD4 cells was determined as above. (E) Contagious apoptosis induced by polyethylene glycol-mediated fusion. HeLa CD4 cells were left untreated or were pre-treated with STS (3 µM, for 3 hours followed by five washes), stained with CellTracker® Green and then co-cultured with CellTracker® Red-prestained cells, either without or after PEG-mediated fusion. Then, the percentage of CellTracker® Red-positive nuclei exhibiting chromatin condensation was measured as in A after 18 hours of co-culture. Statistical significance in D and E, #P<0.005, *P<0.05. (F) Signs of MMP in contagious apoptosis. Individual cells or syncytia generated in D were subjected to m quantitation with DiOC6(3), or were fixed, permeabilized and stained for the detection of activated Bak, the redistribution of cytochrome c, or the activation of caspase-3, as described in Materials and Methods. Significance was calculated with the paired Student's t-test with respect to untreated controls. *P<0.05; #P<0.005.

View larger version (30K): [in a new window]   Fig. 2. Mechanistic differences between contagious apoptosis and spontaneous syncytial cell death. (A) Kinetic analysis of nuclear condensation observed in control syncytia or contagious apoptosis. Cells were stained as in Fig. 1A and nuclear apoptosis was measured as immunofluorescence at the indicated times after cellular fusion. (B) Effect of contagion on the syncytial size. HeLa CD4 cells were cultured overnight with healthy-(Syncytia, Co.) or PACC+-HeLa Env cells in the absence (Contagion) or presence of zVAD.fmk (Contagion+zVAD). Cells were stained as in Fig. 1A and the number of nuclei per syncytium was determined (mean ± s.d., n=4). #P<0.005. (C) Differential effect of inhibitors on spontaneous syncytial death and contagious apoptosis. Cells were primed as in B and inhibitors were added prior to co-culture (roscovitine 10 µM, purvalanol 3 µM, rapamycin 1 µM, LY294002 10 µM, pifithrin- 10 µM and PDTC 50 µM). Nuclear pyknosis was measured as in A after 72 hours of co-culture of control syncytia, and overnight co-culture for contagious apoptosis. Results are expressed as inhibition of nuclear apoptosis as compared to untreated control cultures (mean ± s.d., n=3) #P<0.005.

View larger version (35K): [in a new window]   Fig. 3. Transcriptional silencing in contagious apoptosis, as determined by trans-complementation between a transactivator (Tat) and a promoter (LTR upstream of the ß-galactosidase gene) in syncytia. HeLa Env cells expressing Tat and HeLa CD4 cells transfected with the ß-galactosidase gene under the LTR promoter were co-cultured, followed by an in situ histochemical detection of ß-Gal using X-Gal as a substrate (A) or by enzymatic methods applied to cell extracts (B). Results (mean ± s.d. of triplicate samples) are representati ve of three independent experiments. #P<0.005.

View larger version (39K): [in a new window]   Fig. 4. DNA degradation and double strand breaks are associated with contagious apoptosis. (A) After the indicated regimes of STS/Z-VAD.fmk treatment, the frequency of comet assay-positive cells showing DNA double stranded breaks, was determined by microscopic observation (mean ± s.d., n=3; #P<0.005) in a minimum of 200 cells for each point. (B). Nuclear DNA from samples analyzed in A was subjected to pulse-field gel electrophoresis. Results are representative of three independent experiments.

View larger version (58K): [in a new window]   Fig. 5. Contagious apoptosis is associated with caspase-resistant foci of DNA lesions. (A) DNA fragmentation occurring during contagious apoptosis. Cells and syncytia generated as in Fig. 1 were subjected to simultaneous Hoechst 33342 and TUNEL staining. Note that only cells with full-blown chromatin condensation (but not those with PACC) are TUNEL-positive. (B) Representative images of cells stained with antibodies specifically recognizing phosphorylated H2AX (Ser 139, H2AXP) or Chk2 (Thr68, Chk2P) and counterstained with Hoechst 33342. (C,D) Transmission of H2AX and Chk2 phosphorylation to nuclei from HeLa CD4 cells. Prior to co-culture, healthy HeLa CD4 were stained with CellTracker® Red. After fusion with either untreated HeLa Env cells (Co.), STS-pulsed HeLa Env cells in the absence (Contagion), or presence of Z-VAD.fmk, the resulting syncytia were fixed, permeabilized and stained for the detection of Chk2P (C) or H2AXP (D), revealed with an anti-IgG Alexa® Fluor Green conjugate. Nuclei were counterstained with Hoechst 33342. This experiment was repeated twice, with similar results.

View larger version (56K): [in a new window]   Fig. 6. Nuclei and mitochondrial DNA are dispensable for contagious apoptosis. (A) Representative immunofluorescence of contagious apoptosis triggered by enucleated HeLa Env cells. HeLa Env cytoplasts were pretreated or not with 1 µM STS, washed, and co-cultured overnight with healthy HeLa CD4. HeLa Env cytoplasts and normal HeLa CD4 were pre-stained with CellTracker® Green and CellTracker® Red, respectively, and syncytia were stained with the chromatin-specific dye Hoechst 33342. (B) Quantitative analysis of the data obtained as in A. HeLa Env cytoplasts were pretreated or not with STS (1 µM) or ActD (5 µM) and co-cultured with healthy CD4 cells in the presence or absence of zVAD.fmk. The phenotypic characteristics (mean ± s.d., n=3; *P<0.05; #P<0.005) of syncytia with PACC, i.e. nuclear apoptosis (NA) or low mitochondrial transmembrane potential (m) were determined by staining with Hoechst 33342 or DiOC6(3), respectively. (C) Representative images of syncytia formed by fusion of normal (+)HeLa Env cells mtDNA-depleted (°). HeLa Env or CD4 were pre-stained with MitoTracker® Green (MTG), as indicated, and m was assessed by TMRM staining. The yellow color indicates an overlap between the green (MTG) and red (TMRM) staining, observable in control syncytia, but not in contagious apoptosis in which the m is lost. (D) Quantitative analysis of data obtained in C. Nuclear apoptosis and loss of m were determined as previously. Values are means of two experiments ± s.d.

View larger version (20K): [in a new window]   Fig. 7. Contagious nuclear apoptosis is suppressed by Bcl-2 and vMIA overexpression. Either HeLa Env or CD4 were transiently transfected with a vector expressing the neomycin resistance cassette only (Neo), Bcl-2, vMIA or p35. 24 hours after transfection, the HeLa Env cells were subjected to the standard STS pulse (3 h, STS), washed and cultured alone for 18 hours (3 h STS + 18 h) or co-cultured with untreated HeLa CD4 cells (contagion). The frequency of PACC, NA and m loss was assessed as in Fig. 6B, and results are given as means of three independent experiments. P values were calculated with the paired Student's t-test with respect to Neo controls untreated controls, #P<0.005.

View larger version (24K): [in a new window]   Fig. 8. CD4+ T cells undergo contagious apoptosis both in vitro and in vivo. (A) Contagious, fusion-induced apoptosis occurring in T-lymphoid cell lines in vitro. Jurkat, CEM and U937 cell lines were pre-stained with CellTracker® Red or CellTracker® Green and co-cultured overnight with HeLa Env at a ration of 2:1. Env cells were pretreated for 3 hours in presence or absence of STS (3 µM) or ActD (5 µM). Loss of m, PS exposure and plasma membrane permeability were assessed by cytofluorometric analysis, while gating on the CellTracker® pre-labeled population. Data are means of three experiments ± s.d. Significance was calculated with the paired Student's t-test with respect to untreated controls. *P<0.05; #P<0.005. (B) HeLa Env cells triggered contagious apoptosis of primary human T cells. Freshly drawn peripheral blood lymphocytes (PBL) or T lymphoblasts (obtained by 5days stimulation with PHA plus IL-2) were pre-stained with CellTracker® Red or CellTracker® Green, co-cultured overnight with CellTracker® Blue-pre-labeled HeLa Env cells and double-stained-positive cells were subjected to FACS analysis for m loss and nuclear apoptosis. Prior to co-culture HeLa Env were given a r 3-hour incubation with STS or ActD (mean ± s.d., n=2, *P<0.05; #P<0.005), and the co-culture was performed in the presence or absence of 100 µM zVAD.fmk or 50 nM fusion inhibitor C34 peptide, as indicated. (C) HeLa Env cells trigger contagious apoptosis of Jurkat cells in vivo. CellTracker® Red-pre-labeled Jurkat cells were injected into the peritoneal cavity of athymic mice (nu/nu). HeLa Env were left untreated or pulsed with STS (3 µM), washed five times, and injected 8 hours after the Jurkat cells. After overnight incubation, peritoneal cells were recovered, and Jurkat cells (CellTracker® Red+) were analyzed cytofluorometrically to determine the m dissipation and PS exposure. Results are means for a minimum of five animals per group. *P<0.05; #P<0.005.