Oncogene (2004) 23, 4362–4370 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc 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