Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the HIV-1 envelope
2002; Springer Nature; Volume: 21; Issue: 15 Linguagem: Inglês
10.1093/emboj/cdf391
ISSN1460-2075
AutoresMaria Castedo, Thomas Roumier, Julià Blanco, Karine F. Ferri, Jordi Barretina, Lionel Tintignac, Karine Andréau, Jean‐Luc Perfettini, Alessandra Amendola, Roberta Nardacci, Philip R. LeDuc, Donald E. Ingber, Sabine Druillennec, B P Roques, Serge A. Leibovitch, Montserrat Vilella-Bach, Jie Chen, José A. Esté, Nazanine Modjtahedi, Mauro Piacentini, Guido Kroemer,
Tópico(s)Nuclear Structure and Function
ResumoArticle1 August 2002free access Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the HIV-1 envelope Maria Castedo Maria Castedo Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Thomas Roumier Thomas Roumier Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Julià Blanco Julià Blanco Institut de Recerca de la SIDA-Caixa, Laboratori de Retrovirologia, Hospital Universitari Germans Trias i Pujol, Universitat Autónoma de Barcelona, Ctra Canyet s/n, 08916 Badalona, Catalonia, Spain Search for more papers by this author Karine F. Ferri Karine F. Ferri Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Jordi Barretina Jordi Barretina Institut de Recerca de la SIDA-Caixa, Laboratori de Retrovirologia, Hospital Universitari Germans Trias i Pujol, Universitat Autónoma de Barcelona, Ctra Canyet s/n, 08916 Badalona, Catalonia, Spain Search for more papers by this author Lionel A. Tintignac Lionel A. Tintignac Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Karine Andreau Karine Andreau Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Jean-Luc Perfettini Jean-Luc Perfettini Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Alessandra Amendola Alessandra Amendola Istituto Nazionale Malattie Infettive ‘L. Spallanzani’, Rome, 00149 Italy Search for more papers by this author Roberta Nardacci Roberta Nardacci Istituto Nazionale Malattie Infettive ‘L. Spallanzani’, Rome, 00149 Italy Search for more papers by this author Philip Leduc Philip Leduc Department of Surgery and Pathology, Children's Hospital and Harvard Medical School, Enders 1007, 300 Longwood Avenue, Boston, MA, 02115 USA Search for more papers by this author Donald E. Ingber Donald E. Ingber Department of Surgery and Pathology, Children's Hospital and Harvard Medical School, Enders 1007, 300 Longwood Avenue, Boston, MA, 02115 USA Search for more papers by this author Sabine Druillennec Sabine Druillennec Unité de Pharmacochimie Moléculaire et Structurale, INSERM U266–CNRS UMR860, Université René Descartes (Paris V), F-75005 Paris, France Search for more papers by this author Bernard Roques Bernard Roques Unité de Pharmacochimie Moléculaire et Structurale, INSERM U266–CNRS UMR860, Université René Descartes (Paris V), F-75005 Paris, France Search for more papers by this author Serge A. Leibovitch Serge A. Leibovitch Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Montserrat Vilella-Bach Montserrat Vilella-Bach Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801 USA Search for more papers by this author Jie Chen Jie Chen Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801 USA Search for more papers by this author José A. Este José A. Este Institut de Recerca de la SIDA-Caixa, Laboratori de Retrovirologia, Hospital Universitari Germans Trias i Pujol, Universitat Autónoma de Barcelona, Ctra Canyet s/n, 08916 Badalona, Catalonia, Spain Search for more papers by this author Nazanine Modjtahedi Nazanine Modjtahedi Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Mauro Piacentini Mauro Piacentini Istituto Nazionale Malattie Infettive ‘L. Spallanzani’, Rome, 00149 Italy Department of Biology, University of Rome Tor Vergata, Rome, 00133 Italy Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Maria Castedo Maria Castedo Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Thomas Roumier Thomas Roumier Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Julià Blanco Julià Blanco Institut de Recerca de la SIDA-Caixa, Laboratori de Retrovirologia, Hospital Universitari Germans Trias i Pujol, Universitat Autónoma de Barcelona, Ctra Canyet s/n, 08916 Badalona, Catalonia, Spain Search for more papers by this author Karine F. Ferri Karine F. Ferri Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Jordi Barretina Jordi Barretina Institut de Recerca de la SIDA-Caixa, Laboratori de Retrovirologia, Hospital Universitari Germans Trias i Pujol, Universitat Autónoma de Barcelona, Ctra Canyet s/n, 08916 Badalona, Catalonia, Spain Search for more papers by this author Lionel A. Tintignac Lionel A. Tintignac Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Karine Andreau Karine Andreau Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Jean-Luc Perfettini Jean-Luc Perfettini Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Alessandra Amendola Alessandra Amendola Istituto Nazionale Malattie Infettive ‘L. Spallanzani’, Rome, 00149 Italy Search for more papers by this author Roberta Nardacci Roberta Nardacci Istituto Nazionale Malattie Infettive ‘L. Spallanzani’, Rome, 00149 Italy Search for more papers by this author Philip Leduc Philip Leduc Department of Surgery and Pathology, Children's Hospital and Harvard Medical School, Enders 1007, 300 Longwood Avenue, Boston, MA, 02115 USA Search for more papers by this author Donald E. Ingber Donald E. Ingber Department of Surgery and Pathology, Children's Hospital and Harvard Medical School, Enders 1007, 300 Longwood Avenue, Boston, MA, 02115 USA Search for more papers by this author Sabine Druillennec Sabine Druillennec Unité de Pharmacochimie Moléculaire et Structurale, INSERM U266–CNRS UMR860, Université René Descartes (Paris V), F-75005 Paris, France Search for more papers by this author Bernard Roques Bernard Roques Unité de Pharmacochimie Moléculaire et Structurale, INSERM U266–CNRS UMR860, Université René Descartes (Paris V), F-75005 Paris, France Search for more papers by this author Serge A. Leibovitch Serge A. Leibovitch Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Montserrat Vilella-Bach Montserrat Vilella-Bach Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801 USA Search for more papers by this author Jie Chen Jie Chen Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801 USA Search for more papers by this author José A. Este José A. Este Institut de Recerca de la SIDA-Caixa, Laboratori de Retrovirologia, Hospital Universitari Germans Trias i Pujol, Universitat Autónoma de Barcelona, Ctra Canyet s/n, 08916 Badalona, Catalonia, Spain Search for more papers by this author Nazanine Modjtahedi Nazanine Modjtahedi Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Mauro Piacentini Mauro Piacentini Istituto Nazionale Malattie Infettive ‘L. Spallanzani’, Rome, 00149 Italy Department of Biology, University of Rome Tor Vergata, Rome, 00133 Italy Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France Search for more papers by this author Author Information Maria Castedo1, Thomas Roumier1, Julià Blanco2, Karine F. Ferri1, Jordi Barretina2, Lionel A. Tintignac1, Karine Andreau1, Jean-Luc Perfettini1, Alessandra Amendola3, Roberta Nardacci3, Philip Leduc4, Donald E. Ingber4, Sabine Druillennec5, Bernard Roques5, Serge A. Leibovitch1, Montserrat Vilella-Bach6, Jie Chen6, José A. Este2, Nazanine Modjtahedi1, Mauro Piacentini3,7 and Guido Kroemer 1 1Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France 2Institut de Recerca de la SIDA-Caixa, Laboratori de Retrovirologia, Hospital Universitari Germans Trias i Pujol, Universitat Autónoma de Barcelona, Ctra Canyet s/n, 08916 Badalona, Catalonia, Spain 3Istituto Nazionale Malattie Infettive ‘L. Spallanzani’, Rome, 00149 Italy 4Department of Surgery and Pathology, Children's Hospital and Harvard Medical School, Enders 1007, 300 Longwood Avenue, Boston, MA, 02115 USA 5Unité de Pharmacochimie Moléculaire et Structurale, INSERM U266–CNRS UMR860, Université René Descartes (Paris V), F-75005 Paris, France 6Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801 USA 7Department of Biology, University of Rome Tor Vergata, Rome, 00133 Italy *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4070-4080https://doi.org/10.1093/emboj/cdf391 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Syncytia arising from the fusion of cells expressing the HIV-1-encoded Env gene with cells expressing the CD4/CXCR4 complex undergo apoptosis following the nuclear translocation of mammalian target of rapamycin (mTOR), mTOR-mediated phosphorylation of p53 on Ser15 (p53S15), p53-dependent upregulation of Bax and activation of the mitochondrial death pathway. p53S15 phosphorylation is only detected in syncytia in which nuclear fusion (karyogamy) has occurred. Karyogamy is secondary to a transient upregulation of cyclin B and a mitotic prophase-like dismantling of the nuclear envelope. Inhibition of cyclin-dependent kinase-1 (Cdk1) prevents karyogamy, mTOR activation, p53S15 phosphorylation and apoptosis. Neutralization of p53 fails to prevent karyogamy, yet suppresses apoptosis. Peripheral blood mononuclear cells from HIV-1-infected patients exhibit an increase in cyclin B and mTOR expression, correlating with p53S15 phosphorylation and viral load. Cdk1 inhibition prevents the death of syncytia elicited by HIV-1 infection of primary CD4 lymphoblasts. Thus, HIV-1 elicits a pro-apoptotic signal transduction pathway relying on the sequential action of cyclin B–Cdk1, mTOR and p53. Introduction Fusion of somatic cells, resulting in the formation of syncytia, is a normal process involved in the generation of myotubes, osteoclasts and the syncytiotrophoblast (Anderson, 2000). In contrast, non-physiological heterokaryon formation ultimately results in cell death, as this has been shown by overexpression of fusogenic viral glycoproteins, thereby enforcing syncytium formation (Bateman et al., 2000; Scheller and Jassoy, 2001). This applies also to the fusion of cells expressing Env, the envelope glycoprotein complex (gp120/gp41) encoded by the HIV type 1 (HIV-1) genome, with cells expressing the Env (co-)receptor, a combination of CD4 with either CXCR4 or CCR5. Syncytium formation resulting from the Env–CD4 interaction leads to apoptosis, a process thought to participate in the AIDS-associated depletion of CD4+ T lymphocytes (Lifson et al., 1986; Sodroski et al., 1986; Blaak et al., 2000; Maas et al., 2000; Mohri et al., 2001). Indeed, it appears that Env is (one of) the functionally most important apoptogenic HIV-1 product(s) (Gougeon and Montagnier, 1999; Badley et al., 2000; Selliah and Finkel, 2001). The Env complex is expressed on the surface of HIV-1-infected cells as well as on HIV-1 virions. Within this complex, gp120 mediates the primary interaction with the receptor/co-receptor complex, while gp41 participates in membrane interactions eventually leading to fusion (Eckert and Kim, 2001). The interaction between Env and the co-receptor can also mediate pro-apoptotic signals, the exact nature of which is a matter of debate. Env-elicited apoptosis may involve caspases, caspase-independent effectors, inactivation of survival signals mediated by focal adhesion complexes and calcineurin activation, as well as mitochondrial membrane permeabilization (Berndt et al., 1998; Cicala et al., 2000; Roggero et al., 2001; Vlahakis et al., 2001; Sasaki et al., 2002). Syncytial apoptosis induced by the Env–CD4 interaction involves a precise sequence of events: (i) activation of the mammalian target of rapamycin (mTOR), a serine/threonine kinase of the phosphatidyl inositol kinase family; (ii) mTOR-mediated phosphorylation of p53 on serine 15 (p53S15); (iii) p53-dependent upregulation of the expression of Bax, which undergoes a conformational change and translocates from the cytosol to mitochondria; (iv) Bax-mediated permeabilization of mitochondrial membranes with reduction of the mitochondrial transmembrane potential (ΔΨm) and release of apoptogenic proteins from mitochondria, in particular apoptosis- inducing factor and cytochrome c; (v) cytochrome c-dependent caspase activation; and (vi) caspase-dependent nuclear chromatin condensation (Ferri et al., 2000a; Castedo et al., 2001). This sequence of events has been observed in Env-transfected HeLa cells co-cultured with CD4-transfected HeLa cells, in the absence of viral infection (Ferri et al., 2000a; Castedo et al., 2001), and has been confirmed for HIV-1-infected primary CD4+ lymphoblasts (Ferri et al., 2000a; Castedo et al., 2001; Genini et al., 2001; Petit et al., 2002). Intrigued by these premises, we employed a combination of proteomics and hypothesis-driven research with the aim of dissecting the molecular events occurring immediately upstream of mTOR activation. Recently, we discovered that the Env–CD4 interaction not only causes fusion of the cytoplasm but results, after a lag phase, in fusion of the nucleoplasm (karyogamy) (Ferri et al., 2000b). Here, we report that Env-elicited nuclear fusion results from the abortive initiation of mitosis, involving the transient activation of cyclin B-dependent kinase-1 (Cdk1). Importantly, our results reveal the existence of a novel, pro-apoptotic signal transduction pathway elicited by HIV-1, both in vivo, in HIV-1-infected patients, and in vitro, in HIV-1-infected lymphoblasts. This lethal pathway involves the sequential action of Cdk1, mTOR and p53. Results and discussion p53S15 phosphorylation correlates with karyogamy in syncytia elicited by HIV-1 Env HeLa cells stably transfected with a lymphotropic HIV-1 Env gene (HeLa Env) were fused by co-culture with CD4/CXCR4-expressing HeLa cells (HeLa CD4). Fusion events, which depend on the Env–CD4 interaction (Ferri et al., 2000a,b,c; Castedo et al., 2001), were monitored by means of two stable, non-toxic CellTracker fluorescent dyes with which HeLa Env (CellTracker Green) or HeLa CD4 cells (CellTracker Red) were pre-incubated. After several hours of co-culture, juxtaposed nuclei from both cell types (red or green) could be distinguished within a common cytoplasm (Figure 1A). Subsequently, nucleoplasm fusion (karyogamy) occurred, as detectable by the blending of the two CellTracker dyes (yellow) within the nucleus. Twenty-four hours post-fusion ∼50% of syncytia induced by the Env–CD4 interaction exhibited karyogamy, and this percentage was <10% for syncytia arising from PEG-mediated cellular fusion (data not shown). Karyogamy induced by the Env–CD4/CXCR4 interaction occurred before apoptotic nuclear chromatin condensation (detected with Hoechst 33342, Figure 1A) and DNA degradation (detected by TUNEL staining, Figure 1B). Apoptosis, in turn, was only found in karyogamic syncytia (Figure 1A and B). Similar results were obtained when the two fusion partners were labeled with CellTracker Red and CMAC (blue fluorescence, Figure 1C). Karyogamy (as defined by the purple blend of red and blue fluorescence) correlated with p53S15 phosphorylation (revealed in green), as indicated by triple staining experiments (Figure 1C and E) and kinetic analyses (Figure 1D). These data suggest that illicit nuclear fusion, linked to p53S15 phosphorylation, may be involved in syncytial apoptosis. Figure 1.Karyogamy correlating with p53S15 phosphorylation in HIV-1 Env-elicited syncytia. (A) Detection of nuclear fusion. HeLa Env and HeLa CD4 cells were stained with CellTracker Red and Green, respectively, followed by co-culture for 18 h. Representative fluorescence micrographs of Hoechst 33342-counterstained syncytia exhibiting cytoplasmic fusion without nuclear fusion (‘pre-karyogamy’), nuclear fusion without apoptosis (‘karyogamy’) and nuclear fusion with apoptosis are shown. Note that karyogamy correlates with a Hoechst 33342-detectable loss of internuclear boundaries (inserts). (B) DNA degradation assessed by TUNEL staining is restricted to cells exhibiting apoptotic chromatin condensation. (C) Coincidence of karyogamy and p53S15P phosphorylation. HeLa Env and HeLa CD4 cells were pre-stained with CellTracker Red or CMAC, respectively, then fused by co-culture, fixed, permeabilized and stained for p53S15P. Representative images are shown, showing that karyogamic cells lacking signs of apoptosis are p53S15P+. (D) Kinetics of karyogamy, p53S15 phosphorylation, and nuclear apoptosis [detected as in (C)]. (E) Quantitation of karyogamy among syncytia as a function of the phosphorylation status of p53S15, 18 h after co-culture. At this stage, p53S15P can be considered as a surrogate marker of karyogamy. Download figure Download PowerPoint Karyogamy results from an abortive entry into the mitotic prophase Quantitative immunoblots performed with a panel of ∼900 monoclonal antibodies revealed that a cluster of proteins accumulating at the G2/M boundary were overexpressed in HIV-1 Env-elicited syncytia, early after fusion, at 9 h (Figure 2A). This applies to AIM1 (aurora- and Ip1-like midbody-associated protein), IAK1 (IP1- and aurora-related kinase-1), PLK-1 (polo-like kinase-1), the serine-threonine kinase Nek2 and the topoisomerase IIα isoform and its binding protein (ToBP1). Among the cyclins, only cyclin B was altered, with a transient overexpression at 18 h (Figure 2A and B). Based on this information, we determined the relationship between karyogamy and cell cycle. Whereas individual nuclei from pre-karyogamic syncytia incorporated the DNA precursor BrdU, no signs of DNA synthesis could be detected in karyogamic nuclei (Figure 2C). Karyogamic nuclei stained for tubulin (Figure 2D) and exhibited a loss in lamin B staining, indicating the dissolution of the nuclear envelope (Figure 2E). Accordingly, a dextran–FITC conjugate (molecular weight 70 kDa) microinjected into the cytoplasm was excluded from nuclei of pre-karyogamic syncytia, yet did penetrate into karyogamic nuclei (Figure 2F). These features (arrest of DNA synthesis and annihilation of the barrier function of the nuclear envelope) suggest that karyogamy corresponds to the entry into the early mitotic prophase, before spindle formation and pronounced mitotic chromatin condensation occur. Cyclin B was found to accumulate in the cytosol (and sometimes in the nucleus) of syncytia before karyogamy, yet was absent from most karyogamic cells, with a peak increase ∼12 h after Env–CD4 co-cultures (Figure 2G). This suggests that the Cdk1, which is required for lamin phosphorylation/depolymerization and progression through mitosis (Smits and Medema, 2001), has been inactivated early after karyogamy. Accordingly, the inactivating phosphorylation of Cdk1 on Tyr15 (Kumagai and Dunphy, 1991) was found to be a relatively late event (Figure 2H). Immunoprecipitation of the cyclin B–Cdk1 complex confirmed a higher Cdk1 activity (measured on histone H1) in syncytia as compared with individual control cells, and this increase in kinase activity peaked at ∼12 h (Figure 2I). Figure 2.Phenotypic characterization of Env-induced karyogamy. (A) Cell cycle-relevant proteins whose expression level changes (by a factor of ≥2) upon syncytium formation as determined by quantitative immunoblots (X ± SEM, n = 3). (B) Examples of cell cycle-relevant proteins detectable in HeLa cells whose expression level varies by a factor <2. (C) 5-bromo-2′deoxyuridine (BrdU) incorporation into syncytia as a function of the karyogamy (KG) status. Sixteen-hour-old syncytia were incubated with BrdU for 2 h and then subjected to the immunodetection of BrdU into S-phase nuclei. (D) Tubulin staining of 18-h-old syncytia. Note that the tubulin network is excluded from the nucleus before karyogamy. After karyogamy, nuclei stain positively for tubulin. (E) Lamin B immunostaining of syncytia. Note that karyogamic syncytia exhibit the breakdown of lamin B. (F) Loss of nuclear envelope barrier function in karyogamy. Syncytia were microinjected into the cytoplasm with dextran-70 FITC conjugated, which is excluded from the nucleus of pre-karyogamic cells, yet penetrates into the nucleus of karyogamic syncytia. (G) Cyclin B accumulation in pre-karyogamic syncytia. Two phenotypes (12 h after fusion) are shown, namely pre-karyogamic (p53S15P+) cells exhibiting cyclin B in the cytoplasm or in the nucleus. In contrast, most karyogamic cells lack immunodetectable cyclin B. The kinetics of cyclin B accumulation is shown at different intervals. The few karyogamic (p53S15P+) cells positively staining for cyclin B demonstrate a diffuse (cytoplasmic + nuclear) staining pattern. (H) Kinetics of cyclin B accumulation and the phosphorylation of p53S15 or Cdk1T15. (I) Kinetics of Cdk1 activity. Cdk1 was immunoprecipitated at different intervals after syncytium formation and its capacity to phosphorylate histone H1 in vitro was determined (H1-P*) in the absence or presence of roscovitine (1 μM). Immunoblot detection to confirm equal loading of Cdk1 was also performed. Download figure Download PowerPoint Cdk1 is involved in karyogamy and syncytial apoptosis Addition of Cdk1 inhibitor roscovitine to HeLa Env/HeLa CD4 co-cultures prevented karyogamy, as well as p53S15 phosphorylation and all subsequent steps leading to apoptosis such as mitochondrial translocation of Bax, loss of the ΔΨm and apoptotic chromatin condensation (Figure 3A and B). Roscovitine allowed for the formation of larger syncytia, with more nuclei (Figure 3C). Roscovitine failed to inhibit apoptosis induced by staurosporine or by HIV-1-encoded viral protein R (Vpr), indicating that it does not act as a general apoptosis inhibitor (such as Bcl-2; Figure 3D). Chemically unrelated Cdk1 inhibitors such as purvalanol, olomoucine and N-9 isopropyl-olomoucine inhibited the Env-elicited p53S15 phosphorylation and apoptosis, much as roscovitine did (Figure 3E). These pharmacological data were corroborated at the genetic level, by transient transfection with a dominant-negative (DN) cyclin Cdk1 mutant (Figure 3F), which prevented karyogamy, p53S15 phosphorylation and apoptosis (Figure 3F and G). Moreover, microinjection of an anti-cyclin B antibody into 6-h-old syncytia inhibited karyogamy and p53S15 phosphorylation (Figure 3H). Injection of an active Cdk1–cyclin B complex into the cytoplasm of freshly formed syncytia resulted into accelerated nuclear fusion, and this effect was prevented by roscovitine (Figure 3I). This suggests that lamin phosphorylation/depolymerization induced by Cdk1 (Peter et al., 1990) is sufficient to induce karyogamy. Microinjection of Cdk1–cyclin B into syncytia also induced karyogamy and, after a lag phase, phosphorylation of p53S15 and apoptosis (Figure 3I). Figure 3.Involvement of Cdk1 in karyogamy and apoptosis. (A) Effects of the Cdk1 inhibitor roscovitine on karyogamy, p53S15 phosphorylation, Bax upregulation and signs of apoptosis. Values (X ± SEM, n = 3), obtained after 16 h of co-culture, are representative of seven independent experiments. Asterisks denote significant (P < 0.01, Student's t-test) differences compared to untreated control cultures. (B) Roscovitine (10 μM)-mediated inhibition of p53S15 phosphorylation, as determined by immunoblot detection. A 1:1 mixture of HeLa CD4/HeLa Env single cells (SC) or syncytia (18 h of co-culture) generated in the absence or presence of 10 μM roscovitine were subjected to immunoblotting analysis. GAPDH was detected to confirm equal protein loading (40 μg per lane). (C) Roscovitine-mediated increase in the syncytial size (determined by counting the number of Hoechst 33342-stained nuclei per syncytium, n = 100). (D) Failure of roscovitine to act as a general apoptosis inhibitor. HeLa cells stably transfected with Bcl-2 or the Neo resistance gene only were exposed for 6 h to 4 μM staurosporine (STS) or synthetic Vpr peptide, followed by the cytofluorometric assessment of the frequency of cells incorporating the vital dye propidium iodide (X ± SEM, n = 3). (E) Comparative assessment of different pharmacological Cdk1 inhibitors. HeLa Env/HeLa CD4 syncytia were generated in the presence of 10 μM roscovitine, 3 μM purvalanol, 60 μM olomoucine or 30 μM N-9-isopropylolomoucine, and the indicated parameters were scored 24 h after initiation of co-cultures. (F) Effect of DN Cdk1 on p53S15 phosphorylation, apoptosis and syncytial size. Cells were transiently transfected 24 h before fusion with vector only (Co.), wild-type (WT) or DN Cdk1 and the indicated parameters were determined 16 h after co-culture among cells strongly staining for Cdk1-WT or Cdk1-DN. (G) Representative immunostainings of syncytia transfected with either Cdk1-WT or Cdk1-DN [as in (F)] and stained with antibodies specific for Cdk1 (which also recognizes Cdk1-DN) and p53S15P. (H) Microinjection of a neutralizing cyclin B antibody into newly formed syncytia. A cyclin B-specific monoclonal antibody (or an anti-paxillin antibody serving as a negative control) were microinjected into the cytoplasm of 6-h-old syncytia, and the frequency of karyogamy or p53S15 phosphorylation was scored 12 h later. (I) Karyogamy triggered by microinjection of cyclin B–Cdk1. Six-hour-old syncytia were microinjected with an active cyclin B–Cdk1 complex or PBS (Co.) and cultured either in the absence or in the presence of 10 μM roscovitine for the indicated period, followed by determination of karyogamy, p53S15P and nuclear apoptosis. Download figure Download PowerPoint mTOR is activated downstream of Cdk1 One p53S15 kinase activated by HIV-1 infection has been identified as mTOR (Castedo et al., 2001). Accordingly, mTOR, which is normally found in the cytoplasm of individual HeLa cells, was detected in nuclei from karyogamic (p53S15 phosphorylation+) syncytia (Figure 4A). Cdk1 inhibition by roscovitine, which inhibits p53S15 phosphorylation and karyogamy (Figure 3A and B), completely suppressed the nuclear relocalization of mTOR, which stayed cytoplasmic (Figure 4A). A similar inhibition of the nuclear localization of mTOR was observed upon transfection with DN-CDK1 or treatment with olomoucine (data not shown). In view of the reported intimate relationship between mTOR and cell size control (Zhang et al., 2000; Bodine et al., 2001), we tested whether external constraint on cell size of HeLa Env/HeLa CD4 heterokarya would modulate syncytial apoptosis via an effect on the subcellular localization and activity of mTOR. Syncytia cultured on adhesive squares of 20 μm diameter (Ferri et al., 2000c) exhibited a reduced number of nuclei per syncytium (2.9 ± 0.3 versus 4.7 ± 1.2 on unpatterned control substrates, 18 h after co-culture, X ± SEM, n = 100, P < 0.001), as well as increased Bax translocation and apoptosis (Figure 4B). However, the reduction of syncytial size had no effect on cyclin B accumulation, karyogamy, nuclear accumulation of mTOR and p53S15 phosphorylation (Figure 4B). Culture on a substrate specifically designed to increase spreading via extension of cell processes that attach to squares of 5 μm in diameter, separated by 10 μm of non-adhesive zones [5/10 self-assembled monolayer (SAM)] (Figure 4B) (Chen et al., 1997; Ferri et al., 2000c) resulted in an increase in the average number of nuclei per syncytium (7.2 ± 2.8, P < 0.001). In spite of the inhibition of Bax translocation and apoptosis by the 5/10 SAM, there was no effect on the nuclear relocalization of mTOR (Figure 4B). Taken together, these data suggest that, downstream of cyclin B, mTOR is activated by a pathway that is not linked to cell size control. Figure 4.mTOR involvement in karyogamy and p53S15 phosphorylation. (A) mTOR acts downstream of Cdk1. Untreated 24-h-old syncytia (Co.) or syncytia cultured in the continuous presence of roscovitine were subjected to immu
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