Artigo Acesso aberto Revisado por pares

Multipolar mitosis of tetraploid cells: inhibition by p53 and dependency on Mos

2010; Springer Nature; Volume: 29; Issue: 7 Linguagem: Inglês

10.1038/emboj.2010.11

ISSN

1460-2075

Autores

Ilio Vitale, Laura Senovilla, Mohamed Jèmaà, Mickaël Michaud, Lorenzo Galluzzi, Oliver Kepp, Lisa Nanty, Alfredo Criollo, Santiago Rello‐Varona, Gwenola Manic, Didier Métivier, Sonia Vivet, Nicolas Tajeddine, Nicholas Joza, Alexander Valent, Maria Castedo, Guido Kroemer,

Tópico(s)

DNA Repair Mechanisms

Resumo

Article25 February 2010free access Multipolar mitosis of tetraploid cells: inhibition by p53 and dependency on Mos Ilio Vitale Ilio Vitale INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Laura Senovilla Laura Senovilla INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Mohamed Jemaà Mohamed Jemaà INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Mickaël Michaud Mickaël Michaud INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Lorenzo Galluzzi Lorenzo Galluzzi INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Oliver Kepp Oliver Kepp INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Lisa Nanty Lisa Nanty INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Alfredo Criollo Alfredo Criollo INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Santiago Rello-Varona Santiago Rello-Varona INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Gwenola Manic Gwenola Manic INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Didier Métivier Didier Métivier INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Sonia Vivet Sonia Vivet INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Nicolas Tajeddine Nicolas Tajeddine INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Nicholas Joza Nicholas Joza INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Alexander Valent Alexander Valent Unité de Recherche Translationnelle, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Maria Castedo Maria Castedo INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, FranceThese authors share senior co-authorship Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, FranceThese authors share senior co-authorship Search for more papers by this author Ilio Vitale Ilio Vitale INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Laura Senovilla Laura Senovilla INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Mohamed Jemaà Mohamed Jemaà INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Mickaël Michaud Mickaël Michaud INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Lorenzo Galluzzi Lorenzo Galluzzi INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Oliver Kepp Oliver Kepp INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Lisa Nanty Lisa Nanty INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Alfredo Criollo Alfredo Criollo INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Santiago Rello-Varona Santiago Rello-Varona INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Gwenola Manic Gwenola Manic INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Didier Métivier Didier Métivier INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Sonia Vivet Sonia Vivet INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Nicolas Tajeddine Nicolas Tajeddine INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Nicholas Joza Nicholas Joza INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Alexander Valent Alexander Valent Unité de Recherche Translationnelle, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Maria Castedo Maria Castedo INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, FranceThese authors share senior co-authorship Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, FranceThese authors share senior co-authorship Search for more papers by this author Author Information Ilio Vitale1,2,3, Laura Senovilla1,2,3, Mohamed Jemaà1,2,3, Mickaël Michaud1,2,3, Lorenzo Galluzzi1,2,3, Oliver Kepp1,2,3, Lisa Nanty1,2,3, Alfredo Criollo1,2,3, Santiago Rello-Varona1,2,3, Gwenola Manic1,2,3, Didier Métivier1,2,3, Sonia Vivet1,2,3, Nicolas Tajeddine1,2,3, Nicholas Joza1,2,3, Alexander Valent4, Maria Castedo1,2,3 and Guido Kroemer 1,2,3 1INSERM, Villejuif, France 2Institut Gustave Roussy, Villejuif, France 3Faculté de Médecine, Université Paris-Sud XI, Villejuif, France 4Unité de Recherche Translationnelle, Institut Gustave Roussy, Villejuif, France *Corresponding author. INSERM, Institut Gustave Roussy, Pavillon de Recherche 1, rue Camille Desmoulins, Villejuif F-94805, France. Tel.: +33 1 4211 6046; Fax: +33 1 4211 6047; E-mail: [email protected] The EMBO Journal (2010)29:1272-1284https://doi.org/10.1038/emboj.2010.11 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Tetraploidy can constitute a metastable intermediate between normal diploidy and oncogenic aneuploidy. Here, we show that the absence of p53 is not only permissive for the survival but also for multipolar asymmetric divisions of tetraploid cells, which lead to the generation of aneuploid cells with a near-to-diploid chromosome content. Multipolar mitoses (which reduce the tetraploid genome to a sub-tetraploid state) are more frequent when p53 is downregulated and the product of the Mos oncogene is upregulated. Mos inhibits the coalescence of supernumerary centrosomes that allow for normal bipolar mitoses of tetraploid cells. In the absence of p53, Mos knockdown prevents multipolar mitoses and exerts genome-stabilizing effects. These results elucidate the mechanisms through which asymmetric cell division drives chromosomal instability in tetraploid cells. Introduction Tetraploid cells are detected in some precancerous lesions such as Barrett's oesophagus and cervical dysplasia, where their presence coexists with the loss of functional p53 (Heselmeyer et al, 1996; Maley et al, 2004). Owing to the increase in the number of chromosomes, perhaps coupled to changes in the geometry of the mitotic machinery (Storchova et al, 2006; Storchova and Kuffer, 2008), tetraploid cells frequently activate the DNA damage response and become genomically unstable. Thus, tetraploidy may be considered as a metastable state that links normal diploidy to cancer-associated aneuploidy (Storchova and Pellman, 2004; Fujiwara et al, 2005; Margolis, 2005). Numerous tumour suppressor genes including p53 (Margolis, 2005), BRCA1 (Schlegel et al, 2003), LATS2 (Aylon et al, 2006) and APC (Tighe et al, 2004) actively repress tetraploidy, meaning that their removal can either stimulate the spontaneous tetraploidization of cells or facilitate the survival of tetraploid cells generated upon cytokinesis or karyokinesis inhibition. The former has been shown for the knockdown of APC in cultured cells and for the conditional knockout of APC in small intestine epithelia in vivo (Caldwell et al, 2007). The latter has been demonstrated in cultured cancer cell lines that were depleted from p53 by gene knockout or RNA interference (Cross et al, 1995; Andreassen et al, 2001; Castedo et al, 2006a, 2006b), as well as in p53−/− primary mouse mammary epithelial cells (Fujiwara et al, 2005; Senovilla et al, 2009). Moreover, the expression of some oncogenes including Myc (Yin et al, 1999), Aurora-A (Wang et al, 2006) and human papillomavirus (HPV)-encoded E6 (Incassati et al, 2006) can stimulate tetraploidization. The mechanisms through which tetraploidy favours oncogenesis are complex and have not yet been entirely elucidated. One single tetraploid cell can undergo multipolar mitosis, which often leads to the generation of three or more daughter cells (Storchova and Pellman, 2004). This process causes the near-to-stochastic distribution of chromosomes and hence is lethal for most daughter cells. Nullisomy (the total absence of one particular chromosome) and polysomy (the presence of extra copies of one chromosome), indeed, result in major genetic defects involving the incorrect assembly of multiprotein complexes and fatal linkage disequilibria, which are rarely compatible with cell survival (Zhivotovsky and Kroemer, 2004; Roumier et al, 2005; Ganem et al, 2007). Moreover, during mitosis, the presence of more than two centrosomes in tetraploid cells can also favour merotelic chromosome attachments and hence chromosomal lagging, which may favour chromosome loss or asymmetric distribution among daughter cells, even when the division is bipolar (Ganem et al, 2009). Multipolar and asymmetric cell division, as they can result from tetraploidy (Levine et al, 1991; Ganem et al, 2007), are commonly observed in malignant lesions and have been suspected to contribute to oncogenesis for over a century (Boveri, 2008). Supernumerary centrosomes, as they are detected in malignant cells (Levine et al, 1991; D'Assoro et al, 2002), can be induced experimentally, and this reportedly suffices to trigger oncogenesis (Basto et al, 2008; Gergely and Basto, 2008). Moreover, ‘anisocytosis’ and ‘anisokaryosis’ (heterogeneity in cell size and nuclear size, respectively), which presumably result from asymmetric divisions, are well-established histological hallmarks of malignancy (Boveri, 2008; Holland and Cleveland, 2009). It is interesting to note that in some cancer types (e.g., non-small cell lung cancer), these morphological traits of malignancy correlate with the expression of one particular oncogene, Mos (Gorgoulis et al, 2001). Mos (also called c-Mos) is the first human oncogene cloned, and has been identified as the cellular homologue of the viral oncogene v-Mos, which is encoded by the Moloney murine sarcoma virus (Oskarsson et al, 1980; Watson et al, 1982). The p39Mos protein (hereafter referred to as Mos) has been shown to stimulate the transformation of murine fibroblasts in vitro (Okazaki and Sagata, 1995; Fukasawa and Vande Woude, 1997). Moreover, transfection-enforced overexpression of Mos reportedly inhibits mitotic progression (Wang et al, 1994) and causes the generation of binucleated cells due to the inhibition of cytokinesis (Okazaki et al, 1992; Fukasawa and Vande Woude, 1995). Apparently, Mos can stabilize another oncogene product, c-Fos, (Okazaki and Sagata, 1995) and enhance the expression of cyclins (Rhodes et al, 1997), thereby stimulating cell proliferation. The genetic invalidation of Mos has no obvious phenotypic consequences in mice (Colledge et al, 1994; Hashimoto et al, 1994). However, although Mos−/− male mice exhibit normal reproduction rates, Mos−/− female are nearly infertile (Colledge et al, 1994; Hashimoto et al, 1994), in line with the fact that Mos is strictly necessary for the first meiotic division of oocytes (Sagata et al, 1989a), and then exerts a critical checkpoint function during metaphase II (Sagata et al, 1989b). Both the meiosis-regulatory and the transforming effects of Mos require its serine–threonine kinase activity (Haccard et al, 1993; Okazaki and Sagata, 1995). Known Mos substrates include cyclin B2, tubulin and MEK1 (Roy et al, 1990; Zhou et al, 1991; Sagata, 1997), and the meiotic checkpoint function of Mos depends on its capacity to activate the mitogen-activated protein kinase (MAPK) pathway (Haccard et al, 1993). Thus, very little is known about the role of Mos in somatic cells and on the mechanisms by which Mos can act as an oncogene. Here, we developed a cellular model of aneuploidization in which p53−/− cells were driven into tetraploidy, which was followed by multipolar mitosis and re-acquisition of a near-to-diploid chromosome content. We found that Mos was upregulated in p53-deficient tetraploid cells and that it was strictly required for the occurrence of multipolar divisions, presumably because Mos acts as an inhibitor of centrosome coalescence. Results and discussion Generation of sub-tetraploid derivatives from p53-deficient tetraploid cells Shortly (2 days) after a 48 h-long treatment with the microtubule poison nocodazole or the cytokinesis inhibitor cytochalasin D, p53−/− human colon carcinoma HCT 116 cell cultures contained a higher fraction of polyploid cells (with a ⩾4n DNA content), yet a lower amount of dying and dead cells (exhibiting the dissipation of the mitochondrial transmembrane potential (ΔΨm) and the breakdown of plasma membranes, respectively) (Kroemer et al, 2007; Galluzzi et al, 2009) than their p53-proficient counterparts (Figure 1A and Supplementary Figure S1). Moreover, fluorescence-activated cell sorter (FACS)-purified cells with an ∼8n DNA content formed colonies more efficiently when they did not express p53 than when they did so (Figure 1B), in line with the notion that p53 deficiency is permissive for the generation and survival of tetraploid cells (Castedo et al, 2006b). Cultures derived from FACS-purified cells with an ∼8n DNA content were characterized by a 4n DNA content in the G1 phase of the cell cycle and by an 8n DNA content in the G2 and M phases, thereby exhibiting bona fide traits of tetraploidy. However, after several passages, p53−/− (but neither p21−/− nor Bax−/−) cultures progressively accumulated a population of cells with a ∼2n DNA content (Figure 1C and D). This was observed in six independent experiments in which the initial contamination with ∼2n cells (measured immediately after FACS purification) was undetectable. To understand the origin of such ∼2n population, we followed the fate of tetraploid cells 1 week after their generation by videomicroscopy, and found that p53−/− cells underwent multipolar (mostly tri- or tetrapolar) divisions—which are associated with Y- and X-shaped metaphases (Figure 2A)—much more frequently than p53+/+ control cells (Figure 2B and Supplementary Videos 1 and 2). These results suggest that cells with a ∼2n DNA content (hereafter referred to as ‘sub-tetraploid’) appearing at significant frequencies (⩾10%), as early as 15 days after tetraploidization, might result from a peculiar process of multipolar division. A significant percentage of daughter cells that originated from p53−/− tetraploid cells by multipolar mitosis could enter and terminate normal bipolar divisions (Figure 2C and D, and Supplementary Video 3), suggesting that such sub-tetraploid cells can give rise to a new lineage. The reduction of the chromosomal content was significantly more frequent among p53−/− tetraploid cells than among control ones (Figures 1 and 2), and, in another cell line, was exacerbated by pharmacological inhibition of p53 by cyclic pifithrin-α (Komarov et al, 1999) (Supplementary Figure S2). Thus, the tumour suppressor p53 reduces the probability of sub-tetraploidy. Figure 1.Effect of p53 on the survival and on the genomic stability of tetraploid HCT 116 cells. (A, B) The absence of p53 increases the clonogenic survival of freshly generated polyploid cells. Wild type (WT), p53−/−, Bax−/− and p21−/− diploid human colon carcinoma HCT 116 cells and HCT 116 cells stably transfected with a plasmid encoding the baculoviral inhibitor of caspases p35 (p35) were left untreated or treated with 100 nM nocodazole (Noco) for 48 h. After washing, cells were cultured for additional 48 h in drug-free culture medium, then stained with Hoechst 33342, followed by fluorescence-activated cell sorter (FACS) purification of diploid (2n, white and grey symbols for untreated and nocodazole-treated cells, respectively) or polyploid (>4n, black symbols) cell populations. In A, representative cell cycle distributions of WT and p53−/− cells from one out of three independent experiments are shown. X-axis = Hoechst 33342 fluorescence (DNA content); Y-axis = cell number per channel (counts). In B, the results of clonogenic survival assays carried out on such FACS-purified cells are reported. The upper part shows representative pictures of colonies formed by WT and p53−/− cells as observed upon crystal violet staining 10 days after FACS purification. Columns depict the survival fraction (mean±s.e.m., n=3 parallel wells, normalized for plating efficiency and to control diploid cells) of the diploid and polyploid cell populations with the indicated genotype and corresponding to the FACS-purified populations represented in A. Asterisks indicate statistically significant differences as compared to WT tetraploid cells (Student's t-test, P 4n, black symbols). Cells then were cultured for the indicated number of days and analyzed for DNA content. Representative cell cycle profiles of WT and p53−/− tetraploid cells are shown in C, whereas quantitative data are reported in D (mean±s.e.m., n=6 independent determinations). In C, the percentages of cells with a sub-tetraploid DNA content (indicating chromosomal loss) are indicated. Statistical significance resulting from the comparison to WT cells is highlighted by asterisks (Student's t-test, P 4n, black symbols) and monitored by fluorescence videomicroscopy for 48 h. In A snapshots taken at the indicated time points exemplify the appearance of tripolar and tetrapolar mitosis, thus exhibiting the representative Y- and X-shaped metaphase, respectively. Full-length movies proving the completion of cell division are available as Supplementary Videos S1 and S2. B reports the percentage of cell cycle arrest, apoptosis induction and various mitotic aberrations as quantified among 150 to 200 mitoses for each genotype (mean ± s.e.m., n=3 independent experiments). In C representative images captured at the indicated time points show that some daughter cells that originated from p53−/− tetraploid HCT 116 cells by multipolar mitosis can enter and terminate normal bipolar divisions (red arrows), whereas others undergo apoptosis (yellow arrows). A full-length movie that demonstrates the completion of a normal cell division by such a daughter cell is available as Supplementary Video S3. D reports the percentage of these cells (arisen from p53−/− tetraploid HCT 116 cells by multipolar mitosis) that underwent the indicated fate by the end of the experiment (as quantified among 150-200 daughter cells, mean±s.e.m., n=3 independent experiments). Note that only cells entering multipolar mitosis within the first 18 h of the assays were tracked for the subsequent 30 h. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint Sub-tetraploidy is linked to centrosome defects and aneuploidy Next, we generated multiple tetraploid clones from isogenic HCT 116 cells. As compared with wild type (WT), Bax−/−, p21−/− or apoptosis-resistant (owing to the expression of the caspase inhibitor p35 from Baculovirus) tetraploid cells, p53−/− tetraploid clones displayed some difficulties in maintaining a stable tetraploid genome. Four weeks after cloning, indeed, only one-third of p53−/− tetraploid clones still exhibited a clean tetraploid DNA content profile, whereas the other two-thirds accumulated viable sub-tetraploid populations (Figure 3A and B), first as rather broad shoulders (‘phase 1’) and later as sharper peaks (‘phase 2’) of sub-tetraploid cells. Such sharper peaks presumably arise in cultures that are dominated by one or few viable sub-clones with a comparable sub-tetraploid DNA content. Phase 1 and phase 2 unstable sub-tetraploid populations also arose after repeated transfection (approximately once every 5 days) with a small interfering RNA (siRNA) targeting p53 (Supplementary Figure S2). Chromosome counting confirmed that phase 2 unstable p53−/− tetraploid cells frequently contained a roughly diploid number of chromosomes (Figure 3C and D). Phase 1 unstable p53−/− tetraploid cells were characterized by a high frequency of aberrant mitoses that were either monopolar, bipolar characterized by lagging chromosomes or multipolar linked to supernumerary centrosomes. Multipolar mitoses were also more frequent among phase 2 unstable tetraploids as compared to WT or stable p53−/− tetraploids (Figure 3E). As compared to phase 1 cells, phase 2 cells proliferated more quickly, and among them the sub-tetraploid population had shorter duplication times and shorter mitoses than the tetraploid one (Supplementary Figure S3). This explains the outgrowth of sub-tetraploid cells over their tetraploid counterparts. When such sub-tetraploid cells were exposed to nocodazole, they could again tetraploidize and then revert once more to sub-tetraploidy, but this process of reversion was not accelerated (Supplementary Figure S4). Figure 3.Chromosome instability and centrosome amplification in p53−/− tetraploid HCT 116 clones. (A, B) p53 deficiency increases the percentage of unstable tetraploid clones. Tetraploid HCT 116 clones were generated from wild type (WT), p53−/−, Bax−/−, p21−/− and p35-expressing parental cells as described in Supplementary Data. Cell cycle distribution and apoptosis-related parameters were evaluated 4 weeks after cloning by multiparametric cytofluorometry upon staining with Hoechst 33342 (which measures DNA content), the mitochondrial transmembrane potential (ΔΨm)-sensitive dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)) and propidium iodide (PI, an exclusion dye that only stains dead cells). Representative plots are shown in A, the inserts therein showing positive controls for ΔΨm dissipation (as obtained by treating the cells for 30 min with 100 μM protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, FCCP) and plasma membrane permeabilization (as resulting from a 2 min-long incubation in 0.5% (w/v) saponin (Sapo)). Tetraploid clones were classified according to genomic instability (as evaluated by the quantification of viable sub-tetraploid populations) in stable (sub-tetraploid cells 10%). Unstable clones were subdivided into ‘phase 1’ and ‘phase 2’ clones, characterized by broad and sharp sub-tetraploid peaks, respectively. B reports the frequency of unstable clones generated from parental tetraploid cells of the indicated genotype. Frequency was calculated among 50–100 clones for each genotype (mean±s.e.m., n=3 independent series of clones). (C, D) Chromosome count in tetraploid clones. Representative examples of 4′,6 diamidino-2-phenylindole (DAPI)-stained metaphase spreads with the corresponding number of chromosomes are shown in C. For WT and p53−/− clones of the indicated type, D reports the percentage of cells containing 40–60, 61–80, 81–100 or >101 chromosomes, as quantified among 100 metaphases per condition (mean±s.e.m., n=4 different clones in independent assessments) (E) Centrosome amplification correlates with genomic instability. WT and p53−/− clones of the indicated class were cultured on glass coverslips and subjected to immunofluorescence detection of mitotic spindles (β-tubulin staining, green fluorescence) and centrosomes (γ-tubulin staining, red fluorescence). Nuclei were counterstained with Hoechst 33342 (emitting in blue). According to the number of centrosomes, interphase cells were divided in normal (1 or 2 centrosomes) and abnormal (more than 2 centrosomes, either congressed in a single pole or not), whereas metaphases were classified as monopolar (2 congressed centrosomes), normal and abnormal bipolar (2 centrosomes, the latter exhibiting the misalignment of one or more chromosomes), and multipolar (tripolar, tetrapolar or of higher-order polarity, characterized by 3, 4 or more centrosomes, respectively). Representative immunofluorescence microscopy images of each category are shown. The percentage of occurrence of each category is reported, as quantified among 150 to 200 cells for WT and p53−/− clones of the indicated type (mean±s.e.m., n=4 distinct clones). A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint Fluorescence in situ hybridization (FISH) carried out during interphase proved that a large portion of sub-tetraploid cells (which were FACS-purified from unstable p53−/− tetraploid clones) was aneuploid (Figure 4A-C). Thus, especially in phase 1 cultures, nullisomies (which are always lethal) were frequently detected (Figure 4C). Accordingly, most (>99%) of such phase 1 sub-tetraploid cells failed to form stable offspring in clonogenic assays and died (Figure 4D). In phase 2 cultures, the frequency of aneuploid cells was lower, and nullisomies were infrequent (Figure 4C), presumably because viable cells (which efficiently form clones, Figure 4D) had been positively selected. To further explore the behaviour of sub-tetraploid cells, we generated phase 1 and phase 2 clones from p53−/− tetraploid cells expressing histone H2B fused to the N-terminus of green fluorescent protein (H2B–GFP, which labels chromatin in green and hence allows for monitoring chromosome movement), and followed the fate of their sub-tetraploid derivatives after FACS purification (Figure 4E, F and Supplementary Figure S5, and Supplementary Videos 4, 5 and 6). Although phase 1 sub-tetraploid cells rarely (∼3%) engaged in subsequent cycles of division, phase 2 sub-tetraploid cells did so much more frequently (∼60%). Nonetheless, cell cycle blockade, mitosis without cytokinesis and cell death occurred more frequently among sub-tetraploid cells than among their diploid progenitors (Figure 4E, F and Supplementary Figure S5, and Supplementary Videos 4, 5 and 6). Figure 4.Characterization and fate of the sub-tetraploid offspring of p53−/− tetraploid cells. (A–C) An important fraction of sub-tetraploid cells are aneuploid. Sub-tetraploid cells derived from phase 1 and 2 unstable p53−/− tetraploid HCT 116 clones or the 2n population of the parental p53−/− diploid cell line were fluorescence-activated cell sorter (FACS)-purified as indicated in A. Thereafter, nuclei from the sorted po

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