Mitotic catastrophe constitutes a special case of apoptosis whose suppression entails aneuploidy Maria Castedo 1 , Jean-Luc Perfettini 1 , Thomas Roumier 1 , Alexander Valent 2 , Hana Raslova 3 , Kenichi Yakushijin 4 , David Horne 4 , Jean Feunteun 1 , Gilbert Lenoir 1 , Rene´ Medema 5 , William Vainchenker 3 and Guido Kroemer* ,1 1 CNRS-UMR 8125, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France; 2 Laboratoire de Ge´nomique Cellulaire des Cancers, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France; 3 INSERM U362, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France; 4 Department of Chemistry, Oregon State University, Covallis, OR 97331, USA; 5 Division of Molecular Biology H8, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands A conflict in cell cycle progression or DNA damage can lead to mitotic catastrophe when the DNA structure checkpoints are inactivated, for instance when the checkpoint kinase Chk2 is inhibited. Here we show that in such conditions, cells die during the metaphase of the cellcycle,asaresultofcaspaseactivationandsubsequent mitochondrial damage. Molecular ordering of these phenomena reveals that mitotic catastrophe occurs in a p53-independentmannerandinvolvesaprimaryactivation of caspase-2, upstream of cytochrome c release, followed by caspase-3 activation and chromatin condensation. Suppression of caspase-2 by RNA interference or pseudosubstrate inhibitors as well as blockade of the mitochondrial membrane permeabilization prevent the mitotic catastrophe and allow cells to further proceed the cell cycle beyond the metaphase, leading to asym- metric cell division. Heterokarya generated by the fusion ofnonsynchronizedcellscanbedriventodivideintothree or more daughter cells when Chk2 and caspases are simultaneously inhibited. Such multipolar divisions, re- sulting from suppressed mitotic catastrophe, lead to the asymmetricdistributionofcytoplasm(anisocytosis),DNA (anisokaryosis)andchromosomes(aneuploidy).Similarly, in a model of DNA damage-induced mitotic catastrophe, suppression of apoptosis leads to the generation of aneuploid cells. Our findings delineate a molecular path- waythroughwhichDNAdamage,failuretoarrestthecell cycleandinhibitionofapoptosiscanfavortheoccurrence of cytogenetic abnormalities that are likely to participate in oncogenesis. Oncogene (2004) 23, 4362–4370. doi:10.1038/sj.onc.1207572 Published online 29 March 2004 Keywords: aneuploidy; cancer; caspases; Chk-2; pro- grammed cell death Introduction Cancer occurs as a stochastic, relatively rare event that is frequently linked to aneuploidy, and chromosomal instability; especially in neoplastic lesions with an unfavorable prognosis (Slovak et al., 2000; Choma et al., 2001; Jallepalli and Lengaue, 2001; Shih et al., 2001; Lingle et al., 2002; Masuda and Takahashi, 2002a,b; Tort et al., 2002). The exact history of the cytogenetic catastrophe leading to chromosomal in- stability has not been elucidated. However, it appears intellectually appealing that a violation of the principle ‘better dead than wrong’ (the suppression of pro- grammed cell death) might participate in the generation of aneuploid cells (Knudson, 2001). Apoptosis is a morphologically defined type of programmed cell death involving cytoplasmic shrinkage, chromatin condensation (pyknosis) and nuclear fragmentation (karyorrhexis) with chromatinolysis (Kerr et al., 1972). Apoptosis is characterized by two major biochemical events, namely the activation of caspases and the permeabilization of the outer mitochondrial membrane, with the consequent release of multiple death effectors from the mitochondrial intermembrane space (Brenner and Kroemer, 2000; Ferri and Kroemer, 2001; Ravagnan et al., 2002). Mitochondrial membrane permeabilization (MMP) and caspase activation are closely intertwined alterations because caspases (e.g. caspase-8 stimulated by death receptors or caspase-2 stimulated by DNA damage) (Read et al., 2002) can stimulate MMP (Guo et al., 2002), while MMP results into the release of several caspase activators. Such caspase activators include cytochrome c, which triggers, via the activation 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 or alternatively, constitute the ‘checkpoint’ of the apoptotic process (Green and Kroemer, 1998; Green and Reed, 1998; Kroemer and Reed, 2000; Penninger and Kroemer, 1998). Received 27 October 2003; revised 6 January 2004; accepted 27 January 2004; Published online 29 March 2004 *Correspondence: G Kroemer, CNRS-UMR 8125, Institut Gustave Roussy, Pavillon de Recherche 1, 39 rue Camille-Desmoulins, F-94805 Villejuif, France; E-mail: kroemer@igr.fr Oncogene (2004) 23, 4362–4370 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc