Mitotic Catastrophe Constitutes a Special Case of Apoptosis Whose Suppression Entails Aneuploidy

Mitotic catastrophe constitutes a special case of apoptosis whose suppression entails aneuploidy

Maria Castedo1, Jean-Luc Perfettini1, Thomas Roumier1, Alexander Valent2, Hana Raslova3, Kenichi Yakushijin4, David Horne4, Jean Feunteun1, Gilbert Lenoir1, Rene´ Medema5, William Vainchenker3 and Guido Kroemer*,1

1CNRS-UMR 8125, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France; 2Laboratoire de Ge´nomique Cellulaire des Cancers, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France; 3INSERM U362, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France; 4Department of Chemistry, Oregon State University, Covallis, OR 97331, USA; 5Division of Molecular Biology H8, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

A conflict in cell cycle progression or DNA damage can Introduction lead to mitotic catastrophe when the DNA structure checkpoints are inactivated, for instance when the Cancer occurs as a stochastic, relatively rare event that checkpoint kinase Chk2 is inhibited. Here we show that is frequently linked to aneuploidy, and chromosomal in such conditions, cells die during the metaphase of the instability; especially in neoplastic lesions with an cell cycle, as a result of caspase activation and subsequent unfavorable prognosis (Slovak et al., 2000; Choma mitochondrial damage. Molecular ordering of these et al., 2001; Jallepalli and Lengaue, 2001; Shih et al., phenomena reveals that mitotic catastrophe occurs in a 2001; Lingle et al., 2002; Masuda and Takahashi, p53-independent manner and involves a primary activation 2002a, b; Tort et al., 2002). The exact history of the of caspase-2, upstream of cytochrome c release, followed cytogenetic catastrophe leading to chromosomal in- by caspase-3 activation and chromatin condensation. stability has not been elucidated. However, it appears Suppression of caspase-2 by RNA interference or intellectually appealing that a violation of the principle pseudosubstrate inhibitors as well as blockade of the ‘better dead than wrong’ (the suppression of pro- mitochondrial membrane permeabilization prevent the grammed cell death) might participate in the generation mitotic catastrophe and allow cells to further proceed of aneuploid cells (Knudson, 2001). the cell cycle beyond the metaphase, leading to asym- Apoptosis is a morphologically defined type of metric cell division. Heterokarya generated by the fusion programmed cell death involving cytoplasmic shrinkage, of nonsynchronized cells can be driven to divide into three chromatin condensation (pyknosis) and nuclear or more daughter cells when Chk2 and caspases are fragmentation (karyorrhexis) with chromatinolysis simultaneously inhibited. Such multipolar divisions, re- (Kerr et al., 1972). Apoptosis is characterized by two sulting from suppressed mitotic catastrophe, lead to the major biochemical events, namely the activation asymmetric distribution of cytoplasm (anisocytosis), DNA of caspases and the permeabilization of the outer (anisokaryosis) and chromosomes (aneuploidy). Similarly, mitochondrial membrane, with the consequent release in a model of DNA damage-induced mitotic catastrophe, of multiple death effectors from the mitochondrial suppression of apoptosis leads to the generation of intermembrane space (Brenner and Kroemer, 2000; aneuploid cells. Our findings delineate a molecular path- Ferri and Kroemer, 2001; Ravagnan et al., 2002). way through which DNA damage, failure to arrest the cell Mitochondrial membrane permeabilization (MMP) cycle and inhibition of apoptosis can favor the occurrence and caspase activation are closely intertwined of cytogenetic abnormalities that are likely to participate alterations because caspases (e.g. caspase-8 stimulated in oncogenesis. by death receptors or caspase-2 stimulated by DNA Oncogene (2004) 23, 4362–4370. doi:10.1038/sj.onc.1207572 damage) (Read et al., 2002) can stimulate MMP (Guo Published online 29 March 2004 et al., 2002), while MMP results into the release of several caspase activators. Such caspase activators Keywords: aneuploidy; cancer; caspases; Chk-2; pro- include cytochrome c, which triggers, via the activation grammed cell death of the Apaf-1/caspase-9-containing apoptosome, the activation of caspase-3, one of the principal death effectors in late apoptosis (Wang, 2002). In molecular terms thus, both MMP and caspase-activation, together *Correspondence: G Kroemer, CNRS-UMR 8125, Institut Gustave or alternatively, constitute the ‘checkpoint’ of the Roussy, Pavillon de Recherche 1, 39 rue Camille-Desmoulins, F-94805 Villejuif, France; E-mail: [email protected] apoptotic process (Green and Kroemer, 1998; Green Received 27 October 2003; revised 6 January 2004; accepted 27 January and Reed, 1998; Kroemer and Reed, 2000; Penninger 2004; Published online 29 March 2004 and Kroemer, 1998). Mitotic catastrophe M Castedo et al 4363 Some forms of cell death are thought to be carbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk), fundamentally different from apoptosis (‘type 1 cell all hallmarks of apoptosis, including the mitochondrial death’). This applies to necrosis (‘oncosis’) and cytochrome c release, were suppressed, while cells autophagic death (‘type 2 cell death’) (Leist and successfully completed the metaphase and entered into Jaattela, 2001; Lockshin and Zakeri, 2002), as well the telophase (Figure 1) and subsequent stages of the cell as to mitotic catastrophe. It has been argued that cycle (see below). This is at difference with many other mitotic catastrophe would be fundamentally different examples of apoptosis, in which the release of cyto- from apoptosis (Roninson et al., 2001) because, in chrome c occurs in a Z-VAD.fmk-refractory manner, some model systems, manipulations that disable the upstream of the activation of caspases (Penninger and apoptotic machinery (such as overexpression of Bcl-2 in Kroemer, 2003; Zamzami and Kroemer, 2003). For etoposide-treated HeLa cells (Lock and Stribinskiene, instance, the release of cytochrome c is independent 1996) or MRD1 overexpression in irradiated tumor from caspase activation in aging syncytia dying from cells (Ruth and Roninson, 2000)) reduce apoptosis, p53-dependent apoptosis before mitosis (Ferri et al., while they enhance the frequency of catastrophic 2000a). Thus, in mitotic catastrophe, at least one mitoses. Moreover, the fact that caspase inhibitors caspase must be activated at the pre-mitochondrial such as Z-VAD.fmk can fail to prevent the appearance level. of dying giant multinucleated cells (induced for instance by treatment with spindle poisons) has been used to conclude that mitotic catastrophe would be Mitotic catastrophe is accompanied by caspase-2 unrelated to apoptosis (Nabha et al., 2002). activation upstream of mitochondria Nonetheless, caspase-3activation has been observed in at least one model of mitotic catastrophe. Caspase-2, which may act upstream of MMP (Guo et al., Using a novel model of mitotic catastrophe, induced 2002; Read et al., 2002), was proteolytically activated in by the inhibition of checkpoint kinases (Castedo et al., DBH-treated syncytia, before caspase-3was processed 2004), we investigated to which extent apoptosis might to its active form (Casp-3a) (Figure 2a). In contrast, in participate in the execution of mitotic catastrophe. control syncytia, which undergo apoptosis at the G2/M Unexpectedly, we found that mitotic catastrophe does boundary, caspase-3activation was detected as a involve hallmarks of apoptosis and that it relies on the primary event (Figure 2a). RNA interference (RNAi) activation of caspase-2, upstream of MMP. Prevention of caspase-2 (Lassus et al., 2002) (Figure 2b) resulted in of MMP and caspase activation avoids mitotic cata- the inhibition of mitotic catastrophe. Caspase-2-specific strophe, indicating that this form of cell death indeed RNAi prevented cytochrome c release, caspase-3activa- constitutes a special case of apoptosis. Importantly, the tion, and nuclear apoptosis in DBH-triggered mitotic suppression of mitotic catastrophe – by inhibition of catastrophe, but had no effects on spontaneous syncytial apoptosis – can favor asymmetric division and the apoptosis (Figure 2c). To further address the molecular generation of aneuploid cells. Based on these data, we order between MMP and caspase activation, the speculate how the inhibition of the apoptotic program precursors of syncytia were transfected with specific can participate in oncogenesis. inhibitors of p53(DN p53H175) (Vogelstein et al., 2000), caspases (baculovirus-derived p35) (Bump et al., 1995) or MMP (cytomegalovirus-derived vMIA) (Gold- macher et al., 1999; Vieira et al., 2001), and syncytia Results were generated either in the absence of DBH (which leads to spontaneous prophase-associated apoptosis) or Mitotic catastrophe shares the biochemical hallmarks of in the presence of DBH (which induces mitotic apoptosis catastrophe during the metaphase). As to be expected from our previous findings, a dominant-negative p53 Two different HeLa cell lines expressing the HIV-1 mutant efficiently prevented the spontaneous apoptosis envelope (Env) or the Env receptor CD4, respectively, of syncytia (Castedo et al., 2001, 2002). However, in the form syncytia upon coculture (Ferri et al., 2000b, c). same experimental conditions, p53inhibition had no Such syncytia can be driven into mitotic catastrophe significant effect on the DBH-triggered mitotic cata- (cell death during the metaphase) by inhibition of the strophe (Figure 2c). Both p35 and vMIA suppressed the cell cycle checkpoint kinase-2 (Chk2), for instance by mitotic catastrophe of DBH-treated syncytia (Figure 2c). addition of the chemical Chk2 inhibitor debromohyme- Intriguingly, p35 did inhibit MMP (as detectable by nialdisine (DBH). The mitotic catastrophe induced the cytochrome c release in Figure 1) in mitotic by DBH was associated with the mitochondrial release catastrophe, while it failed to inhibit MMP in normal, of cytochrome c, the activation of caspase-3, and prophase-associated syncytial apoptosis (Figure 2c), TUNEL-detectable DNA fragmentation (Figure 1). again confirming that the order between MMP and This indicates that mitotic catastrophe manifests the caspase activation is distinct in the two models. In biochemical hallmarks of apoptosis, namely the per- conclusion, mitotic catastrophe results into caspase-2 meabilization of the outer mitochondrial membrane, activation, which subsequently triggers MMP with caspase activation and DNA fragmentation. In the cytochrome c release, caspase-3activation, and nuclear presence of the pan-caspase inhibitor N-benzyloxy- apoptosis.

Oncogene Mitotic catastrophe M Castedo et al 4364

Figure 1 Chk2 inhibition drives mitotic catastrophe with hallmarks of apoptosis. Syncytia exhibiting characteristics of the metaphase or of subsequent catastrophe (generated in the presence of the Chk2 inhibitor debromhymenialdesine, DBH) or of the telophase (in the presence of DBH þ Z-VAD.fmk) were subjected to staining with antibodies speciﬁc for cytochrome c (Cyt. c) and the 19 kDa subunit of active caspase-3a (Casp-3a) or TUNEL staining, 18 h after syncytium formation. Note that Cyt. c is found in a cytoplasmic punctuate (mitochondrial) localization during the normal metaphase, yet is diffusely distributed throughout the cell in mitotic catastrophe

Suppression of mitotic catastrophe by caspase inhibitors esis (Figure 4). Thus, the simultaneous abolition of a cell cycle checkpoint and of apoptosis favors the generation Using HeLa cells stably transduced with a histone H2B- of aneuploid cells. GFP fusion protein (Kanda et al., 1998), we followed the move of individual chromosomes in living cells by fluorescence videomicroscopy. Most untreated control Mitotic catastrophe induced by DNA damage depends on cells fused by polyethylene glycol (PEG) treatment caspase activation underwent apoptosis after nuclear congregation without entering the metaphase (Figure 3a). These cells thus 14-3-3sÀ/À HCT116 colon carcinoma cells treated with entered apoptosis before mitosis, much as this has been the DNA-damaging drug doxorubicin (Doxo) undergo observed in the HeLa Env/HeLa CD4 coculture model. mitotic catastrophe (Chan et al., 1999). Inhibitory In strict contrast, Chk2-inhibited syncytia manifested experiments involving a variety of caspase inhibitors the apoptotic chromatin condensation during the further confirmed that inhibition of caspases (with Z- metaphase and thus underwent mitotic catastrophe VAD.fmk) and in particular of caspase-2 (with Z- (Figure 3b). The simultaneous inhibition of both Chk- VDVAD.fmk) can prevent the mitotic catastrophe of 2 and caspases with DBH and Z-VAD.fmk resulted into 14-3-3sÀ/À cells (Figure 5). In contrast, neither Z- cell division (Figure 3c–e), frequently leading to the FA.fmk (which is structurally related to Z-VAD.fmk simultaneous generation of three or more daughter cells and Z-VDVAD.fmk, yet does not inhibit caspases) nor with different sizes of the nucleus (anisokaryosis) cyclic pifithrin-a did inhibit the mitotic catastrophe of (Figure 3c) and the cytoplasma (anisocytosis) 14-3-3sÀ/À cells (Figure 5). These data corroborate the (Figure 3d). As to be expected from a polyploid system results obtained in the syncytial model of mitotic with multiple centrosomes chromosomes asymmetrically catastrophe, placing caspases as downstream distributed during the metaphase into the daughter cells, effectors of this form of cell death. Importantly, the as demonstrated by fluorescent in situ hybridization combined effect of Doxo-inflicted DNA damage and (FISH) performed on cells in the late stage of cytokin- apoptosis inhibition, favored the generation of

Oncogene Mitotic catastrophe M Castedo et al 4365

Figure 2 Hierarchy of mitochondrial alterations and caspase activation in mitotic catastrophe. (a) Detection of caspase-2 and -3 activation in spontaneous apoptosis of syncytia and in mitotic catastrophe. Syncytia were cultured in the absence or presence of DBH during 17 or 24 h, followed by immunoblot detection of proteolytically processed caspase-2 (Casp-2a) and caspase-3(Casp-3a).( b) RNA interference of caspase-2. HeLa cells were transfected with double-stranded oligonucleotides designed to interfere with Casp-2a expression or a control oligonucleotide, and 48 h later the expression of procaspase-2 and -3was determined by immunoblot. GAPDH expression was monitored as a loading control. (c) Contribution of caspases and mitochondria to spontaneous syncytial apoptosis and to DBH-triggered mitotic catastrophe. Single cells previously transfected with the indicated cDNA (vector only, dominant-negative p53, the caspase inhibitor p35, the MMP inhibitor vMIA, or a truncated vMIA version that fails to be imported into mitochondria, D2–23) or siRNA (scrambled or Casp-2a-specific) were used to generate syncytia and fixed after 24 h of coculture to determine the percentage (X7s.e.m., n ¼ 3) of syncytia manifesting the mitochondrial release of cyt. c, the activation of Casp-3a, or nuclear apoptosis. This experiment was conducted in the absence of DBH (which leads to cell death during the prophase) or in the presence of DBH (which leads to metaphase-associated death). Asterisks indicate significant (Po0.01) inhibitory effects as compatible with controls. Arrows indicate ‘specific’ inhibitory effects observable in prophase- but not in metaphase-associated death or vice versa. Note that, at difference with mitotic catastrophe, spontaneous apoptosis can be inhibited by p53H175 and involves a p35-resistant, Casp-2a- independent cyt. c release. The results are mean values of three independent experiments yielding similar results

aneuploid 14-3-3sÀ/À cells (Figure 6), again indicating among these events has been established by using that the suppression of mitotic catastrophe can lead to inhibitors acting on all caspases (Z-VAD.fmk, transfec- chromosomal instability. tion with p35) or for caspase-2 (Z-VDVAD.fmk; RNAi) as well as an inhibitor of MMP (vMIA). Thus, mitotic catastrophe is functionally linked to biochemical hall- Discussion marks of apoptosis, in particular caspase activation and MMP (Figure 7). Inhibition of either of these phenom- The results contained in this paper outline a molecular ena allows for progression in the cell cycle, beyond the pathway linking mitotic catastrophe to the activation of metaphase. the apoptotic machinery. During the metaphase, when While mitotic catastrophe and apoptosis share some mitotic catastrophe induced by Chk2 inhibition is features, important differences between the two cell induced, the cells ﬁrst activate caspase-2 and subse- death modalities emerge. First, it appears that p53is not quently undergo MMP and (caspase-3-dependent) involved in mitotic catastrophe, based on the observa- chromatin condensation. The functional hierarchy tion that dominant negative p53and the chemical p53

Oncogene Mitotic catastrophe M Castedo et al 4366

Figure 3 Multipolar division of syncytia subjected to the simultaneous inhibition of Chk2 and apoptosis. HeLa cells stably transfected with a histone H2B-GFP construct were fused with PEG, followed by ﬂuorescence videomicroscopy (a, b, c) or phase contrast videomicroscopy (d). Cells underwent apoptosis after fusion (a), entered mitotic catastrophe (in the presence of DBH, b), or divided (in the presence of DBH plus Z-VAD.fmk, c, d) into several (more than two) cells. Dividing syncytia manifested an asymmetric distribution of the cytoplasma and DNA, as demonstrated by tubulin/DNA staining (e). Representative cells stained for tubulin and DNA were photographed during the last step of cytokinesis (lower panel in c)

inhibitor piﬁthrin-a fail to affect the system (Figures 2 mitotic catastrophe (Burns et al., 2003). Second, mitotic and 5). This behavior may be expected from a mode of catastrophe appears to be special in the sense that cell death that occurs during mitosis and thus must be caspase-2 is activated at the pre-mitochondrial level, largely independent from the de novo transcription of which is not the case in many other models of apoptosis proapoptotic genes. Chk2 inhibitors, which favor the including spontaneous syncytial apoptosis (Figure 2). induction of mitotic catastrophe, had no effect on the Indeed, caspase-2 has been shown to be particularly status of p53S15 phosphorylation, in accordance with abundant in the nucleus (Paroni et al., 2002), to be the notion that this phosphorylation can be mediated by activated by DNA damage in an Apaf-1-independent other kinases (Ahn et al., 2003) such as mTOR in manner (Lassus et al., 2002; Read et al., 2002; syncytia (Castedo et al., 2002). This is also in line with Robertson et al., 2002), and to induce MMP directly reports that mitotic catastrophe induced by DNA (Guo et al., 2002). Caspase-2 has been previously shown damage and Chk2 inactivation in Drosophila embryos to interact with cyclin D3(Mendelsohn et al., 2002), is p53-independent (Takada et al., 2003) and that a gene which is not expressed during mitosis (and even in upregulated by p53, Snk/Plk2 can actually prevent catastrophic mitosis, not shown) and thus does not

Oncogene Mitotic catastrophe M Castedo et al 4367

Figure 4 Asymmetric division of syncytia subjected to the simultaneous inhibition of Chk2 and apoptosis. As compared to diploid control cells (left panel), syncytia generated in the presence of DBH and Z-VAD.fmk manifest an asymmetric distribution of individual chromosomes (chromosome 18 detected by FISH) to the multiple metaphase equators. Two representative examples are shown (middle and right panels). Note that these HeLa sublines are normally diploid (499%) with regard to chromosome 18 and that asymmetric distribution of chromosome 18 is clearly discernible in 470% of multipolar divisions

Figure 5 Mitotic catastrophe in colon cancer cells in relation to apoptosis. Effect of apoptosis inhibitors on mitotic catastrophe. Wild-type HCT116 cells ( þ / þ ) and 14-3-3sÀ/À cells (À/À) were cultured for 72 h in the absence or presence of Doxo, followed by ethanol permeabilization and DAPI staining for the determination of subdiploid cells. The frequency (X7s.e.m., n ¼ 3) of subdiploid (apoptotic) cells was determined in the presence of the indicated inhibitors. This experiment has been repeated three times, with comparable results Figure 6 Suppression of mitotic catastrophe results into aneuploidy. 14-3-3sÀ/À cells normally manifest a diploid karyotype with 1 chromosome X (red), two chromsomes 8 (green), and two chromosomes 18 (blue), as determined by FISH (a). After explain caspase-2 activation during the metaphase. The treatment with Doxo plus Z-VAD.fmk, 7% of the 14-3-3sÀ/À cells molecular details accounting for caspase-2 activation manifest an aneuploid phenotype with supernumerary chromosomes (b), which is rare (p2%) after treatment of 14-3-3s þ / þ cells thus await further clarification. Third, the morphology with Doxo plus Z-VAD.fmk. No aneuploid cells were found of mitotic catastrophe is different from classical among surviving 14-3-3sÀ/À cells treated with doxorubicin alone (c) apoptosis. Chromatin condensation is not homogeneous but rather occurs in clumps, subsequent to metaphase (Figure 1). It is possible that this alteration, which is discernible in oversized syncytia, is overlooked in single designed to avoid the final separation of nuclei that, due cells, leading to an underestimation of the frequency of to DNA damage strand breaks and/or deficient DNA mitotic catastrophes in single cell cultures and tissue replication (which may also occur after illicit cell fusion sections. in vivo (DeWitt and Knight, 2002; Terada et al., 2002; The finding that inhibition of apoptosis by caspase Wurmser and Gage, 2002)) are prone to generate major inhibitors (p35, Z-VAD.fmk) or an MMP inhibitor genomic aberrations in the surviving daughter cells. As a (vMIA) can prevent mitotic catastrophe (Figure 2–6) proof of this concept, Z-VAD.fmk allows for the suggests a novel mechanism through which inhibition of separation of metaphase equators that, in heterokarya, apoptosis may contribute to oncogenesis. Indeed, contain uneven numbers of chromosomes and thus mitotic catastrophe may be viewed as a mechanism undergo asymmetric division (Figure 3). Moreover, the

Oncogene Mitotic catastrophe M Castedo et al 4368 vector only, p35 (gift from G Salvesen), vMIA (gift from V. Goldmacher) or p53DN plasmids (gift by T Soussi), was performed with Lipofectamine (InVitroGen) 24 h before coculture of HeLa CD4 and HeLa Env cells or 24 h before the addition of Doxo to HCT116 cell lines. Where indicated, transfections were carried out in the presence of the pDsRed- mito plasmid ((ClonTech), 30% of the quantity of the other plasmid) to identify transfected cells. RNAi to knock-down the caspase-2 gene was performed as described (Lassus et al., 2002).

Immunofluorescence, cytofluorometry, and videomicroscopy Rabbit antisera specific for Chk268P, Casp-3a (Cell Signaling Technology, MA, USA), was used on paraformaldehyde (4% w : v) and picric acid-fixed (0.19% v : v) cells (Daugas et al., 2000) and revealed with a goat anti-rabbit IgG conjugated to Alexa 568 (red) or Alexa 488 (green) fluorochromes from Molecular Probes. Cells were also stained for the detection of Chk2 (mAb), cyclin B, cytochrome c (mAb, BD Transduction Laboratories), b- tubulin (mAb, Sigma), revealed by anti- mouse IgG Alexa conjugates. TUNEL staining was performed Figure 7 Schematic representation of a pathway leading to with a detection kit from Roche. HCT116 cells were analysed aneuploidy. After activation of the DNA replication or DNA on the FACS after ethanol fixation and DAPI staining. HeLa damage checkpoints, cells usually arrest at the G2/M boundary and cells expressing H2B-GFP were cultured in a humidified eventually undergo apoptosis. In the advent that Chk2 is inhibited, chamber kept at a constant temperature of 371C and an cells instead proceed to the metaphase when they undergo mitotic atmosphere with 5% CO and were subjected to pulsed catastrophe. Blockade of primary caspase activation can avoid 2 apoptosis during the metaphase, thereby favoring asymmetric cell observations using a Zeiss equipment at the central imaging division with the consequent generation of aneuploid cells facility of the Jacques Monod Institute (http://imagerie.ijm.- jussieu.fr).

Quantitation of protein expression suppression of mitotic catastrophe can lead to the generation of aneuploid cells (Figure 4 and 6). Pending Protein samples were prepared from HeLa Env and HeLa confirmation in vivo, this mechanism might link disabled CD4 single cells mixed at a 1 : 1 ratio in lysis buffer (0 control) apoptosis to chromosomal instability and concomitant or from HeLa Env/CD4 syncytia obtained after the indicated period of coculture. Aliquots of protein extracts (40 mg/lane) tumorigenic transformation. were subjected to immunoblots using antibodies specific for Irrespective of these speculative considerations, our Casp-3a or caspase-2 (Cell Signaling Technology). results provide important new insights into how mitotic catastrophe is executed. It appears that, in mechanistic FISH terms, mitotic catastrophe – a mechanism through which cytogenetic aberrations are suppressed – consti- FISH of HeLa syncytia was performed as described (Raslova tutes a special case of apoptosis with upstream caspase-2 et al., 2003) with chromosome 18 satellite probe directly labeled with Rhodamine 123(Oncor) and counterstained with activation. In addition, the experimental system de- an mAb specific for b-tubulin (Sigma) revealed with an FITC- scribed in this work delineates the stepwise recapitula- conjugated anti-mouse IgG. FISH of HCT116 cells, which tion of aneuploidization in vitro, thereby providing an normally have a stable karyotype (Roschke et al., 2002), was experimental tool for the detailed dissection of the performed with a mixture of three commercial probes natural history of cancers. (Qbiogene, France) that detect the centromeric region of chromosome 8 (labeled with FITC – green color) chromosome 18 (labeled with Aqua – blue color) and chromosome X (labeled with rhodamine – red color). Cells were fixed directly Materials and methods on the slides with mixture of methanol : acetic acid (9:1) during 5 min, air-dried overnight and hybridized with a three-probe Cell lines, transfection, and RNAi mixture next day (Valent et al., 2001). For each cell category we surveyed 100–300 nuclei. Two different HeLa cells stably transfected with CD4 (HeLa CD4) or the Env gene of HIV-1 LAI (HeLa Env), respectively, were cocultured (Ferri et al., 2000a) in the absence or presence of DBH (standard dose 20 mM) (Barrios Sosa et al., 2000), and/ Abbreviations or Z-VAD.fmk (100 mM) or other caspase-inhibitors (from Casp-2a, activated caspase-2; Casp-3a, activated caspase-3; Enzyme Systems). Derivatives of the HCT116 cell line Cdk1, cyclin dependent kinase-1; Cyt. c, cytochrome c; DBH, (parental or 14-3-3sÀ/À) were a kind gift from B Vogelstein debromohymenialdisine; DN, dominant negative; Env, envel- and were treated with doxorubicin (doxo) (200 nM, Sigma), ope glycoprotein complex; GAPDH, glyceraldehyde-3-phos- while HeLa expressing a histone H2B-green fluorescent protein phate dehydrogenase; HIV, human immunodeficiency virus; (GFP) fusion construct (H2B-GFP) were a gift from Dr GM KG, karyogamy; MMP, mitochondrial membrane permeabi- Wahl (Kanda et al., 1998). Transfection with pcDNA3.1 lization; mTOR, mammalian target of rapamycin; p53S15P,

Oncogene Mitotic catastrophe M Castedo et al 4369 p53with phosphorylated serine 15; RNAi, RNA interference; (ImmunoGen, Cambridge, MA, USA), Dr. Thierry Soussi siRNA, small interfering RNA; SC, single cell; Syn, syncytia; (Institut Curie, Paris, France), Dr Guy Salvesen (Burnham Z-VAD.fmk, N-benzyloxycarbonyl-Val-Ala-Asp-ﬂuoromethyl- Institute, La Jolla, CA, USA) for cDNA constructs; Didier ketone. Me´ tivier, Nathanael Larochette (CNRS, Villejuif, France) for assistance, and the NIH AIDS reagents program (Bethesda, MD, USA) for cell lines. This work has been supported by a Acknowledgements special grant from LNC, as well as grants from ANRS, FRM, We thank Dr Bernd Vogelstein (Johns Hopkins Oncology European Commission (QLG1-CT-1999-00739 and Contract Center, Baltimore, MD for cell lines, Dr. Victor Goldmacher No. QLK3-CT-20002-01956) (to GK)

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