Oncogene (2007) 26, 7175–7184 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE B2 suppresses mitotic failure and DNA re-replication in human somatic cells knocked down for both B1 and B2

S Bellanger1, A de Gramont2 and J Sobczak-The´ pot

Laboratoire de Biochimie Cellulaire, CNRS UMR 7098, Universite´ P. & M. Curie – Paris 6, Paris, France

Cyclin-dependent kinase 1 (CDK1) plays a crucial role in A-type (A1 and A2) and two B-type (B1 and B2) cyclins establishing and has also been shown to prevent are able to bind and activate CDK1. /CDK DNA re-replication. Cyclins B1 and B2 are two known complexes are necessary and sufficient to drive G2 cells activators of CDK1 operating during in human into (Furuno et al., 1999) and are required for cells. Little is known about the specific roles of each of timely nuclear envelope breakdown (NEB)(Gong et al., these cyclins in CDK1 activation, but cyclin B2 is thought 2007). However, the principal activator of CDK1 as a to play a minor role and to be unable to replace mitosis-promoting factor is . The cyclin B/CDK1 for mitosis completion. In our study, we found that severe activity is necessary to phosphorylate key (for reduction by separate RNA interference of either cyclin example, lamins, histones, condensins and microtubule- B1 or cyclin B2 levels results in little or no associated proteins), leading to the radical reorganization alteration of the and, more specifically, of of subcellular structures allowing metaphase to take mitosis progression. In contrast, simultaneous depletion place (Dessev et al., 1991; Peter et al., 1991; Blangy et al., of both B-type cyclins leads to massive accumulation of 4N 1995; Morgan, 1995; Kimura et al., 1998). Nevertheless, cells, mitotic failure, premature mitosis exit and DNA re- CDK1 appears to be dispensable for early mitotic events. replication. These defects can be corrected by the ectopic Indeed, other kinases, especially those from the Aurora expression of a cyclin B2 resistant to the short hairpin and Polo families are responsible for the initiation of RNA. Altogether, these data show that, in cycling human condensation, nucleoli disassembly and cells, cyclin B2 can compensate for the downregulation of rearrangements of the cytoskeleton leading to rounding cyclin B1 during mitosis. They also clearly implicate cyclins up of the cells. In addition, although it was widely B1 and B2 as crucial activators of CDK1 in its biological accepted that cyclin B/CDK1 is absolutely essential for function of DNA re-replication prevention. NEB at the end of prophase, it has recently been shown Oncogene (2007) 26, 7175–7184; doi:10.1038/sj.onc.1210539; that this process is only slightly delayed in small published online 28 May 2007 interfering RNA-mediated cyclin B knockdown cells (Gong et al., 2007). Keywords: cyclin; mitosis; RNA interference; replication; Once metaphase is established, the sequential destruc- cell cycle tion of cyclins A and B, mediated by the - promoting complex/cyclosome (APC/C), triggers an irreversible inactivation of the CDK1 activity, which is necessary for mitosis exit. It has recently been shown Introduction that chemical inhibition of CDK1 after metaphase arrest promotes a rapid mitosis exit and premature abnormal Cyclin-dependent kinase 1 (CDK1) is a pivotal mitotic (Seong et al., 2003; Niiya et al., 2005; kinase (Lew and Kornbluth, 1996; Morgan, 1997; Ohi Vassilev et al., 2006). and Gould, 1999) present in excess throughout the cell The two B-type cyclins are localized differently within cycle of proliferating cells. Nevertheless, its activity is the cell. Cyclin B1 is essentially cytoplasmic, partially tightly regulated and depends primarily on the binding colocalizes with microtubules during , and of a cyclin-activating subunit. In mammalian cells, two abruptly translocates into the nucleus upon mitosis entry (Jackman et al., 1995; Hagting et al., 1999), a process that is delayed in cyclin A2-depleted cells (Gong Correspondence: Professor J Sobczak-The´ pot, Laboratoire de Biochi- et al., 2007). Human cyclin B2 colocalizes with the Golgi mie Cellulaire, CNRS UMR 7098, Universite´ P. & M. Curie – Paris 6, apparatus and contributes to its fragmentation during Paris, 75005 France. E-mail: [email protected] mitosis (Jackman et al., 1995; Draviam et al., 2001). 1Current address: Institute of Medical Biology, 61 Biopolis Drive, This difference of localization is in favor of specific roles Proteos Building, Singapore 138673. for each cyclin B as CDK1-activating subunits. This 2Current address: Laboratory of Molecular and Cellular Biology, idea has been strengthened by the fact that mutant mice National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, MD 20892, USA. exhibit different phenotypes. Cyclin B1 is essential for Received 6 February 2007; revised 18 April 2007; accepted 19 April 2007; viability because nullizygous mice die in utero, whereas published online 28 May 2007 cyclin B2 nullizygous mice are viable (Brandeis et al., Cyclin B2 compensates for cyclin B1 knockdown S Bellanger et al 7176 1998). Moreover, microinjection experiments have line in which the control of pre-RC formation is well shown that cyclin B1 alone, but not cyclin B2, can drive documented (Zhu et al., 2004). Depletion of both B-type G0/G1 cells into mitosis (Draviam et al., 2001). These cyclins was assessed by real-time reverse transcription– data argue for a nonessential role of cyclin B2 in mitosis PCR (RT–PCR) analysis, 42 h after transfection progression and indicate that, at least under certain (Figure 1a). After normalization using the glyceralde- conditions, cyclin B1, but not cyclin B2, is sufficient to hyde 3-phosphate dehydrogenase (GAPDH) sequence activate CDK1 in all its cytoplasmic and nuclear as an internal control, we obtained a specific and functions. This has led to the idea that cyclin B2 could equivalent extinction of both cyclins B1 and B2 mRNAs not compensate for a lack of cyclin B1. However, (up to 80% of the signal displayed with the empty while cyclin B1 depletion by RNA interference in pSuper vector-transfected cells), whether the two pSuper cultured cells has been shown to inhibit proliferation vectors were transfected separately (pSup B1 or pSup by arresting cells in G2/M, (Yuan et al., 2004, 2006), the B2), or together (pSup B1 þ B2). Immunoprecipitations degree of cell growth inhibition is dependent on the cell followed by in vitro kinase assays in cells depleted of type and, surprisingly, human primary cells do not cyclins B1 and B2 showed a 90% reduction of enzymatic exhibit any defect, despite a quasi-complete inhibition activity for cyclin B1/CDK1 after B1 but not B2 of cyclin B1/CDK1 kinase activity (Yuan et al., 2004). depletion, and no significant change in cyclin A2- Besides its role in mitosis, CDK1 has been shown to dependent kinase activity (Supplementary Figure S3a). participate in the negative control of DNA re-replica- Accordingly, the percentage of cyclin B1-positive cells tion in many systems (Blow and Dutta, 2005). DNA was decreased from 60–65to 10–12% after cyclin B1 replication should occur only once per cell cycle. depletion (Supplementary Figure S3b). Figure 1b shows Inhibition of DNA re-replication involves disassembly that cyclins B1 and B2 protein levels were nearly of the pre-replication complex (pre-RC), thereby nega- undetectable by western blot, whereas the expression tively regulating re-firing of already used or useless of CDK1 was not altered. Altogether, these results show origins at the end of the (Blow and Dutta, 2005). that a near-complete, specific and equivalent efficiency Redundant mechanisms of regulation include CDK2 of inhibition is achieved with the shRNAs targeting and CDK1 inactivating phosphorylations of Cdt1 (Liu cyclins B1 and B2, leading to undetectable or barely et al., 2004; Sugimoto et al., 2004), origin recognition detectable protein levels. Similar results were obtained complexes (Li et al., 2004), cdc6 (Petersen et al., 1999) independent of the status, in HCT-116 p53 þ / þ and replication protein A (RPA), and inhibition of Cdt1 cells (Supplementary Figure S1) and HeLa cells (data by geminin (Wohlschlegel et al., 2000). But, although not shown). geminin is thought to be the main negative regulator in higher eukaryotes, CDK1 also plays a central role Cyclin B2 becomes essential to prevent 4N arrest and because conditional inactivation of CDK1 in yeast and polyploidization when cyclin B1 is downregulated human cells induces re-replication (Itzhaki et al., 1997). Flow cytometry analysis of HCT-116 p53À/À cells Here, we show that, although separate RNA inter- expressing the cyclin B1 shRNA shows only subtle ference-mediated inhibition of cyclin B1 or cyclin B2 differences in the cell-cycle profile, compared to cells expression in human cells has little detectable effect on transfected with the empty pSuper vector (Figure 1c). cell-cycle progression, simultaneous inhibition of both Indeed, we detected less than a 10% increase in the cyclins B1 and B2 leads to a massive accumulation of 4N percentage of G /M cells 42 h after transfection of cells, abnormal mitoses, precocious mitosis exit and 2 pSuper cyclin B1, compared to the empty pSuper vector, re-replication. All these phenotypes can be rescued by despite the near-complete extinction of cyclin B1. This re-expression of an ectopic cyclin B2 mutated in the result, very surprising regarding the literature because short hairpin (shRNA) recognition site. Altogether, most of the CDK1 activity in G /M is thought to be these data show for the first time that in cycling 2 mediated by the cyclin B1/CDK1 complex, was never- mammalian cultured cells, severe reduction of cyclin theless highly reproducible (more than 15independent B1 does not significantly affect mitosis, because of a experiments performed in HCT-116 p53À/À, HCT-116 compensatory effect of cyclin B2. They also clearly p53 þ / þ and HeLa cells). In good agreement with the implicate cyclins B1 and B2 in the negative control of re- literature, depletion of cyclin B2 has no effect on the replication in mammalian cells. cell-cycle profile (Figure 1c). In contrast, simultaneous depletion of both cyclins B1 and B2 leads to a significant Results accumulation of 4N and 8N cells and loss of 2N cells (Figure 1c, right panel), suggesting an unexpected capacity of cyclin B2 to compensate for the down- Specific downregulation of cyclin B1 and/or cyclin B2 regulation of cyclin B1 in proliferating cells. by RNA interference We designed two shRNAs against the human cyclins B1 and B2 mRNAs. The corresponding sequences were Cells depleted of both cyclins B1 and B2 re-replicate cloned into the pSuper vector and plasmid DNAs were their DNA transfected by electroporation, either separately with In cells depleted simultaneously of both B-type cyclins, the empty pSuper vector, or together in the HCT-116 we visualized the appearance of a population with an p53À/À cell line (human colorectal cancer cells), a cell increased ploidy, characterized by the presence of cells

Oncogene Cyclin B2 compensates for cyclin B1 knockdown S Bellanger et al 7177

Figure 1 Cyclin B2 compensates for the downregulation of cyclin B1 to ensure proliferation. (a) HCT-116 p53À/À cells were electroporated with the indicated pSuper vector, and the mRNAs corresponding to cyclin B1 and/or B2 were analysed by real-time reverse transcription–PCR, 42 h after transfection. Relative enrichment is expressed in arbitrary units. (b) Western blot experiment showing extinction of cyclin B1 or/and cyclin B2 after expression of the shRNAs. Equal amounts of cellular extracts from HCT-116 p53À/À electroporated cells were blotted, 42 h after transfection with the indicated antibodies. CDK1 is used as a loading control. (c) Left panel: flow cytometry analysis of HCT-116 p53À/À cells electroporated by the indicated pSuper vectors, 42 h after transfection (same experiment as in (b)). Right panel: quantification of 2N, 4N and >4N cells using XL2 and WinMDI software. Bars represent standard error of the mean from three independent experiments. with a DNA index from 4N to more than 8N, and a clear peak of 8N cells (Figure 1c and Supplementary Figure S3b). This population was not detected in cyclin B2- depleted cells and represents only a very small percentage of cyclin B1-depleted cells. Interestingly, this phenotype was observed in both p53À/À (Figure 1c) and p53 þ / þ HCT-116 cells (Supplementary Figure S1). To determine whether these 8N cells resulted from the fusion between two 4N cells or DNA re-replication of 4N cells, we performed bromodeoxyuridine (BrdU) labeling to detect cells undergoing replication. Trans- fected cells were pulse-labeled with BrdU, 42 h after transfection, and immediately analysed by flow cyto- metry. As shown in Figure 2, cells with a DNA index between 4N and 8N were positive for BrdU labeling and thus re-replicate DNA after a first complete round of replication. This observation suggests that re-replication is the major mechanism to explain the presence of proliferating 4N cells in the cyclin B1/cyclin B2-depleted population.

Ectopic expression of cyclin B2 restores a quasi-normal Figure 2 Cells depleted of both cyclins B1 and B2 re-replicate cell cycle in cells depleted of cyclins B1 and B2 their DNA. Cells were transfected with the pSuper vectors as To confirm the ability of cyclin B2 to compensate for the indicated, and 42 h later pulse-labeled for 30 min with BrdU, downregulation of cyclin B1, cells depleted for cyclins harvested immediately after the pulse, and analysed by flow B1 and B2 were co-transfected with a pCDNA cyclin B2 cytometry for BrdU incorporation and propidium iodide labeling. expression vector mutated in the shRNA recognition The quadrant shows the limits for replicating cells (upper left) and re-replicating cells (upper right). Percentages of cells in the different site. As shown in Figure 3a, pCDNA cyclin B2-trans- states are indicated. BrdU, bromodeoxyuridine. fected cells exhibit approximately a two to four-fold increase of cyclin B2, compared to cells transfected with cells, the level of cyclin B2 becomes comparable to the an empty pCDNA (compare lanes 1 and 2 with 5and 6). endogenous level (compare lanes 1 and 2 with 7 and 8), When the cyclin B2 shRNA is co-expressed in these reflecting inhibition of endogenous but not exogenous

Oncogene Cyclin B2 compensates for cyclin B1 knockdown S Bellanger et al 7178 The very weak effect of cyclin B1 depletion on the cell cycle was confirmed, because 50% of cyclin B1-depleted cells timely entered and completed mitosis without any problem detectable by video microscopy. Arrows indicate two normal mitoses in each experiment (Figure 4a). However, an additional 7% of cells lacking cyclin B1, exhibited strong mitotic defects. They rounded up normally, indicating that they had entered mitosis, but appeared to be arrested in this state for at least 2 h before flattening out without division (not shown). These observations suggest that a minimal threshold level of cyclin B1 is sufficient to complete a normal mitosis, but that under this threshold value, mitosis is impaired. Furthermore, this phenotype was exacerbated in cells depleted in both cyclins B1 and B2. Only 25% of these cells rounded up during the 24 h of observation. Among these cells, 60% (which represents 15% of the total population) eventually flattened out on the plate without division after a delay of about 2 h (arrows indicate two of these abnormal mitoses), whereas only 40% (10% of the total population) exited mitosis timely compared to pSuper-transfected cells, giving birth to two apparently normal daughter cells. The latter cells presumably correspond to the minor fraction of untransfected cells. The average length of abortive mitoses was approximately two to three-fold longer than the average length of normal mitoses in both pSup B1- and pSup B1 þ B2-transfected cells. Abortive mitoses were detected neither in the pSup Ctrl population nor in the pSup B2 population (Figure 4b). Altogether, these data indicate that cyclin B2 becomes critical for mitosis completion when cyclin B1 is Figure 3 Ectopic expression of cyclin B2 restores a quasi-normal severely downregulated, demonstrating an unexpected cell cycle in cells downregulated for cyclins B1 and B2. HCT-116 capacity of cyclin B2 to compensate for cyclin B1 p53À/À cells were transfected with the indicated pSuper vectors. downregulation. Cells were co-transfected either with an empty pCDNA3 vector or with the cyclin B2 pCDNA3 vector mutated in the shRNA recognition site. (a) Levels of cyclins B1, B2 and CDK1 were analysed by western blot, 42 h after transfection. (b) Flow Cells depleted of cyclins B1 and B2 perform chromosome cytometry analysis of the DNA content. condensation but exit mitosis prematurely In the cyclins B1 and B2 codepleted cell culture, we attempted to better characterize cells arrested with a cyclin B2. Flow cytometry analysis of these cells reveals round morphology, which is frequently interpreted as a that both accumulation of 4N cells and re-replication metaphase arrest. To visualize chromatin condensation observed in cyclin B1- and B2-depleted cells can be and establishment of a metaphasic plate after depletion almost completely rescued by the expression of exogen- of both cyclins, cells were recorded after co-transfection ous cyclin B2 (Figure 3b). with a GFP-histone H2B expression vector. As shown in Figure 5a upper panel, whereas control cells exhibit a normal metaphase plate characterized by chromosome Mitosis is impaired in cells depleted of both cyclins B1 and alignment in the center of the cell, cyclin B1- and B2- B2 but not in cells depleted of cyclin B1 alone depleted cells exhibit DNA condensation typical of late To more precisely characterize the phenotypes asso- prophase, but never align their even after ciated with depletion of the two B-type cyclins, we a 2-h period of arrest in this state. This long arrest is performed videomicroscopy with HCT-116 p53À/À then immediately followed by mitosis exit without transfected cells between 24 and 48 h after transfection division (as shown in Figure 4a) or, alternatively, (Figure 4a and b). In this experiment, we monitored the abnormal cytokinesis characterized by a complete rounding up of the cells typical of mitosis entry, furrow ingression and the appearance of linked daugh- followed by division and flattening out of the two ter cells (Figure 5a, lower panel). However, DNA, even daughter cells. During this period, about 60% of cyclin if it begins to segregate between these daughter cells, B2-depleted cells entered and completed mitosis nor- seems to be trapped in the midzone. This phenomenon is mally within o 60 min (values similar to those obtained readily comparable to the phenotype described after after transfection with the empty pSuper vector). CDK1 chemical inhibition by BMI-1026 or RO-3306

Oncogene Cyclin B2 compensates for cyclin B1 knockdown S Bellanger et al 7179

Figure 4 Mitosis is impaired in cells depleted of both cyclins B1 and B2, but not in cells depleted of cyclin B1 alone. (a) Time-lapse video microscopy of HCT-116 p53À/À cells electroporated with the indicated pSuper vectors. (b) Upper panel: quantification of the video microscopy experiments. About 300 cells were visible for each experiment; the percentages of normal, abortive mitoses and interphasic cells were reported on the pies. Abortive mitoses were scored as mitotic cells that flatten out and reform an interphasic nucleus without division. Lower panel: mean value of mitosis length calculated from 70 to 200 mitoses is shown.

(Seong et al., 2003; Niiya et al., 2005; Vassilev et al., accumulate in mitosis (Supplementary Figure S2a). 2006). However, in our case, the chromosomes even- To confirm this observation, all cells present on the tually collapse, giving rise to a single tetraploid nucleus. plate were harvested and analysed by flow cytometry for We therefore conclude that cells depleted of both cyclins their DNA content and for the presence of the mitosis- B1 and B2 transiently arrest in late prophase/early specific MPM2 epitope (Figure 5b). We counted 50% with condensed chromosomes, but even- of prometaphase-arrested MPM-2-positive cells after tually exit this state to directly enter interphase with transfection with the empty pSuper vector, and a similar an increased ploidy. Depending on the cell analysed, value (40%) for cells expressing the shRNA against mitosis exit can occur with (as shown in Figure 4a) or cyclin B2. The percentage of mitotic cells was decreased without abnormal cytokinesis. to 25% in the population expressing the shRNA against cyclin B1, in agreement with our observation that these Cells depleted of cyclins B1 and B2 do not accumulate cells entered mitosis less frequently. Only 9% of mitotic in prometaphase in the presence of nocodazole MPM2-positive cells were detected in the population We reasoned that if cells depleted of the two cyclins B1 expressing both cyclin B1 and B2 shRNAs, presumably and B2 exited mitosis before they reached metaphase, representing weakly or untransfected cells. they would not respond to activation of the spindle The lack of prometaphasic cells in the nocodazole- checkpoint. To address this issue, cells were cultured in treated culture depleted of both B-type cyclins was the presence of the microtubule depolymerizing agent, further confirmed by western blot, where an antibody nocodazole, from 24 to 42 h after transfection, to against the Ser-10 phosphorylated histone H3 (which activate the spindle checkpoint and specifically arrest labels cells from prophase to metaphase, Hendzel et al., them in prometaphase. Time-lapse microscopy revealed 1997) revealed a weak signal in cyclin B1- and B2- that only cells depleted of both cyclins B1 and B2 do not depleted cells, compared to the control cells or cells

Oncogene Cyclin B2 compensates for cyclin B1 knockdown S Bellanger et al 7180

Figure 5 Cells depleted of both cyclins B1 and B2 show mitotic defects and do not respond to nocodazole. (a) Time lapse video microscopy of HCT-116 p53À/À cells electroporated with the pSuper Ctrl vector (upper panel) or the pSuper B1 and pSuper B2 vectors (lower panel) together with an expression vector for GFP-histone H2B to stain chromosomes. (b) Cells were electroporated with the indicated pSuper vectors and treated with nocodazole from 24 to 42 h post-transfection. All cells were then fixed and labeled with propidium iodide for DNA content and with an anti-MPM2 (mitosis-specific) antibody, and then analysed by flow cytometry. Percentage of metaphase cells defined by a 4N DNA content and MPM2 positivity are indicated. (c) In an independent experiment, equal amounts of extracts from cells treated with nocodazole as described in (b) were blotted with an antibody against phosphorylated histone H3, which is typical of mitotic cells.

depleted of a single cyclin B (Figure 5c). This implies are detected and most of them are defective. Cells round that the majority of the cells depleted of both cyclins B1 up and condensate their chromosomes, but exit mitosis and B2 do not accumulate in prometaphase in the prematurely without establishing a metaphase. They presence of nocodazole. Therefore, the vast majority of then flatten out without division, giving birth to pseudo- 4N cells observed after cyclin B1 and B2 depletion are G1 4N cells. In addition, these cells re-replicate their interphasic. genome, independent of p53. These phenotypes can be Taken together, these experiments show that cells almost completely rescued by the ectopic expression of lacking cyclins B1 and B2 can enter early mitosis until a an shRNA-resistant cyclin B2, demonstrating that these round step, which, in this case, does not correspond to a cells depend on cyclin B2 for a successful mitosis and bona fide prometaphasic state, but rather to a late prevention of DNA re-replication. In addition, we have prophase state. They then prematurely exit this early observed that shRNA-mediated downregulation of mitosis stage, giving rise to interphasic cells with cyclin B1 alone, albeit to barely detectable levels, does increased ploidy. not significantly disturb mitosis initiation and comple- tion, except in a minority of cells (o10%), which exit mitosis prematurely as described above for cells Discussion knocked down for both cyclins B1 and B2. This result suggests that cyclin B1 expression must be very severely We have shown that shRNA-mediated downregulation reduced to levels below a threshold to impair efficiently of both cyclins B1 and B2 in human HCT-116 cells leads mitosis progression. Only a minority of cells are prob- to a drastic phenotype of mitotic failure. Few mitoses ably under that threshold and thus exhibit a mitotic

Oncogene Cyclin B2 compensates for cyclin B1 knockdown S Bellanger et al 7181 phenotype, whereas the vast majority of cells, although mitosis and re-replicate their DNA in the presence of the they express cyclin B1 to nearly undetectable levels, drug. Because these cells do not reach metaphase, they accomplish timely mitosis. However, the latter cells now mostly depend on cyclin A2 destruction, a process that critically depend on cyclin B2 to perform mitosis, is nocodazole-insensitive, to exit mitosis. We thus although cyclin B2 by itself is nonessential. Altogether, anticipated that APC/C-mediated proteolysis occurs these results demonstrate an unforeseen capacity of during premature mitosis exit. Indeed, flow cytometry cyclin B2 to compensate for a cyclin B1 defect, and point experiments show that in the population depleted of to a critical role of cyclins B1 and B2 as CDK1 both B-type cyclins, cells complete two full rounds of activators in the negative control of re-replication in DNA replication without chromosome segregation, and mammalian cells. sequentially accumulate and degrade cyclin A2. 4N cells The critical role of cyclin B2 in supporting mitosis in that do not express cyclin B1 are able to degrade cyclin cyclin B1-depleted cells implies that cyclin B1/CDK1 A2 at the end of the first round of replication. Cyclin A2 and cyclin B2/CDK1 share a set of common substrates. is then re-synthesized during the second round of When targeted to the cytoplasm by replacing its replication and re-degraded at the end of this second N-terminal part by the N-terminus of cyclin B1, cyclin round (Supplementary Figure S1b). Moreover, we B2 acquires the broader properties of cyclin B1 for observed by western blot that cyclin A2 is not stabilized completing microtubule reorganization (Draviam et al., in cells depleted of cyclins B1 and B2 (Supplementary 2001), meaning that cyclin B2/CDK1 can recognize and Figure S1c). Altogether, these data suggest that APC/C- phosphorylate the cyclin B1/CDK1 cytoplasmic sub- mediated proteolysis of occurs normally after strates. Regarding nuclear substrates, it is not clear NEB in these cells depleted of cyclin B. In contrast, whether mammalian cyclin B2 can translocate into the the lack of activation of APC/C would have led to nucleus after G2 as does its avian ortholog (Gallant and stabilization, and therefore accumulation of its sub- Nigg, 1992), although it contains a cytoplasmic reten- strates. This was clearly not the case because we did not tion signal (CRS), which has been shown to be both observe any accumulation of either of the following necessary and sufficient to keep it in the cytoplasm proteins, Cdc20, Cdc6 and geminin (known APC/CCdh1 during interphase (Pines and Hunter, 1994). In any case, substrates degraded in late mitosis and early G1; data as cyclin A2 has recently been shown to promote NEB not shown). Therefore, the vast majority of 4N cells in cells depleted of cyclin B1 (Gong et al., 2007), cyclin observed after cyclin B1 and B2 depletion show the B2/CDK1 would thus have access to all nuclear and characteristics of interphasic G1 cells that would be able cytoplasmic substrates of cyclin B1/CDK1 after NEB, to re-replicate after cyclin A degradation. even if it were unable to translocate into the nucleus. Although the major negative control of re-replication Inhibition of CDK1 activity by the chemical inhibitor in yeast is attributed to the CDK1 homolog cdc2, a BMI-1026 in asynchronous cells leads to either a mitotic similar role of CDK1 in mammals is not so clear. arrest, characterized by accumulation of rounded cells, Several years ago, it was proposed that CDK1 took part or to precocious cleavage furrow ingression and in the negative control of premature S-phase entry in abnormal cytokinesis, depending on the drug concen- mammals, because CDK1 lacking human HT2–19 cells tration used. Under nocodazole treatment, BMI-1026 became polyploid (Itzhaki et al., 1997). However, more leads to mitosis exit and cell spreading without division. recently, despite the number of CDK1 inhibitors tested Depending on the nocodazole concentration, cells can in the literature, only RO-3306-mediated CDK1 inhibi- show membrane blebbing, which is assumed to represent tion was shown to lead to re-replication in HeLa cells a cortical activity of actomyosin contraction subsequent (Vassilev et al., 2006). Geminin is another possible key to formation of ectopic cleavage furrows (Seong et al., component that negatively regulates re-replication in 2003; Niiya et al., 2005). Our observation of precocious mammalian cells by inhibiting the replication factor mitosis exit and abnormal cytokinesis in proliferating Cdt1. Indeed, re-replication observed after geminin cells downregulated for cyclins B1 and B2 fully depletion in HCT-116 cells is well documented, but reproduces these phenotypes. In addition, in our appears different from the one we describe here. experiments, no prior arrest in metaphase was needed Geminin-depleted cells do not show the 8N peak we to observe the phenotype. This indicates that in the observed after B-type cyclin depletion, but show a more absence of both B-type cyclins, metaphase is clearly not diffuse re-replication distributed over a broad region a prerequisite to cytokinesis and furrowing. Further- with a DNA content between 4N and 8N (Zhu et al., more, cyclin B is probably necessary to inhibit mitosis 2004). This result indicates that the re-replication exit in the absence of a preliminary metaphase. observed after geminin depletion is a partial over- Importantly, we observed that, after depletion of a replication and not a full genome duplication. In single or the two cyclins B, cells in early mitosis always contrast, in the case of B-type cyclin depletion, the exhibited Mad2-positive kinetochores (Supplementary genome seems to be entirely re-replicated, which could Figure S2b), a hallmark of spindle checkpoint activa- be the result of one organized round of re-replication tion. This suggested that in the absence of both cyclins after the mitosis transient arrest. This hypothesis is B, cells exit mitosis independent of Mad2 signaling consistent with our preliminary observation that gemi- pathway. nin and Cdc6, two proteins involved in the assembly of We also observed that cells depleted of both B-type pre-RCs, are not deregulated in cyclin B-depleted cells. cyclins are resistant to nocodazole, that is, they exit In conclusion, when the two B-type cyclins are

Oncogene Cyclin B2 compensates for cyclin B1 knockdown S Bellanger et al 7182 compromised, cells exhibit a re-replication phenotype Flow cytometry analyses similar to the one observed in CDK1-null HT2–19 cells Forty-two hours after electroporation, cells were trypsinized, (Itzhaki et al., 1997). This fits well with a crucial role washed and fixed overnight in 70% ethanol/PBS at À201C, of cyclin B/CDK1 complexes in this phenotype, pro- rehydrated in PBS and stained for 1 h at 371C with propidium bably together with cyclin A/CDK1 complexes. Indeed, iodide (10 mg/ml) and RNAse A (10 mg/ml). A total of 20 000 cells were acquired by using an EPICS XL flow cytometer re-replication has been reported after cyclin A ablation (Beckman Coulter Inc., Fullerton, CA, USA) and XL2 in drosophila, and also after cyclin A knock down software. Statistical analyses were done with WinMDI soft- together with Emi-1 in mammals (Mihaylov et al., ware. 2002; Machida and Dutta, 2007). As cyclin A and B/ For BrdU labeling, cells were pulse-labeled with BrdU CDK1 complexes are activated in a temporal succes- (10 mM), ethanol-fixed, and treated with 1 ml HCl 2 N/0.5% sion, the data provided here, together with these two Triton X100 for 30 min at room temperature. After centrifuga- articles, lead to the idea that cyclin A would participate tion, the pellet was resuspended in 0.1 M Na2B4O7 (pH 8.7); in early re-replication inhibition in mammals con- cells were then washed with PBS containing 0.5% Tween 20/ trolled by CDK1, whereas cyclin B complexes would 1% bovine serum albumin (BSA). The pellet was resuspended inhibit re-replication later. In our experiments, in in pure anti-BrdU antibody coupled with fluorescein isothio- cyanate (556028; Becton Dickinson Biosciences, Franklin which cells are depleted of both B-type cyclins, Lakes, NJ, USA), and incubated for 30 min at room inhibition of re-replication would depend only on temperature. Cells were then washed twice in PBS/Tween/ cyclin A/CDK activity. Mitotic degradation of cyclin BSA and labeled for propidium iodide, as described above, A by APC/C in the context of cyclin B depletion, would before flow cytometry analysis (EPICS XL from Coulter). thus license re-replication. For the MPM-2 labeling, fixed cells were washed in PBS, In a more medical point of view, CDK inhibition resuspended in anti-MPM2 antibody (ab14581–50, 1/250; has been proposed to be used for proliferation Abcam, Cambridge, UK), and incubated for 30 min at room inhibition (Lee and Sicinski, 2006). In the light of temperature. Cells were then washed twice in PBS, and the our work and recently published data, it is most pellet was resuspended in the secondary antibody coupled with probable that pharmacological inhibition of CDK1 Alexa 488 (1/1000; A-11001; Molecular Probes, Invitrogen, Carlsbad, CA, USA). After a 1-hour incubation, cells were would not be sufficient to arrest cell proliferation at washed twice with PBS and stained with propidium iodide. mitosis and could have adverse effects such as cell polyploidization. Western blot experiments Forty-two hours after electroporation, cells were extracted in RIPA buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Materials and methods NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% desoxycho- late, 10 mM bglycerophosphate, 1 mM NaF, 0.1 mM NaVO , Cell culture, synchronization, electroporation and video 4 1mM DTT, and protease inhibitors), followed by sonication microscopy experiments (41C, 4 Â 30 s, 80%). Equal amounts of total proteins were HCT-116 cells were grown in 6- or 10-cm Petri dishes in denatured in Laemmli sample buffer, boiled and separated by McCoy’s 5A medium supplemented with 10% fetal calf serum. 12 or 14% SDS polyacrylamide gel. After electrophoretic Transient transfections were done by electroporation. Eighty transfer, nitrocellulose membranes (Hybond C; Amersham percent confluent cells were cultured for 4 h before transfection Biosciences, Little Chalfont, UK) were saturated in TNT in RPMI 1640 medium (Gibco, Invitrogen, Carlsbad, CA, buffer (25m M Tris–HCl (pH 7.6), 200 mM NaCl, 0.1% Tween USA) containing 0.1 mM dithiothreitol (DTT) but lacking calf 20) plus 5% milk, incubated with the appropriate first serum. Immediately before electroporation, the cells were antibodies (anti-cyclin B1 GNS1 (sc-245, 1/200), anti-cyclin trypsinized, harvested in phosphate-buffered saline (PBS), and 6 B2 (sc-5235, 1/200), anti-CDK1 (sc-54, 1/200), anti-ser10- resuspended in RPMI medium. About 6 Â 10 cells were phosphorylated histone H3 (1/1000; H-0412; Sigma)) over- pipetted into a 4-mm electroporation cuvette and DNA was night, and washed with TNT. Incubation with secondary added (15 mg of each pSuper vector, 20 mg of pCDNA3- antibodies coupled with peroxidase (anti-mouse (1/20 000; mutated cyclin B2 vector or 10 mg of pCDNA3 histone H2B- NA931V; GE Healthcare, Chalfont, St Giles, UK); anti-rabbit GFP vector). The mixture was incubated for 5min at room (1/20 000; NA934V; GE Healthcare); anti-goat (1/20 000; temperature. Electroporation was performed with a A9452; Sigma)) was done for 1 h, followed by washing in Pulser II electroporation system (300 V, 500 mF; Bio-Rad TNT. Membranes were revealed using the SuperSignal West Laboratories, Hercules, CA, USA). Cells were washed in Pico Chemiluminescent Substrate kit (Pierce Biotechnology, 10 ml of McCoy’s medium, and plated. The medium was Rockford, IL, USA). changed the day after electroporation, and cells were harvested 42 h after transfection for western blot, quantitative PCR or flow cytometry experiments. The efficiency of transfection was Quantitative real-time reverse transcription–PCR analyses routinely about 90%. For mitosis synchronization, cells were Total RNA (5 mg) was reverse-transcribed into cDNA by treated with nocodazole (50 ng/ml) from 24 to 42 h after MultiScribe reverse transcriptase, using the Reverse Transcrip- transfection. tion kit superscript II (Invitrogen, Carlsbad, CA, USA), as For videomicroscopy, cells were transferred to a thermo- recommended by the manufacturer. One-fiftieth of the stated chamber (7% CO2 at 371C) under the lens of the resulting synthesized single-stranded cDNA was used for each microscope, 24 h after electroporation, where they were PCR reaction in the presence of 3 mM specific primers and maintained for 24 h. Images were captured every 5min both Syber green PCR master mix (Applied Biosystems, Foster in phase contrast and fluorescence, using an Axiovert Zeiss City, CA, USA). Quantitative PCR was performed on an ABI microscope. Images were processed with Metamorph software Prism 7000 (Applied Biosystems), with cycling conditions of 20 and converted to Adobe Photoshop. at 501C, 100 at 951C and 40 Â (1500 at 951C, 10 at 601C). After

Oncogene Cyclin B2 compensates for cyclin B1 knockdown S Bellanger et al 7183 the last cycle, the temperature was progressively raised to USA), annealed and subcloned in the pSuper vector as described provide dissociation curves allowing for the assessment of the (Brummelkamp et al., 2002). An empty pSuper vector, a pSuper purity of the amplified product. Each PCR 96-well plate containing a sequence specific for mouse cyclin A2, or a contained serial dilutions of GAPDH, or 18S ribosomal DNAs pSuper against HPV-18 E7 protein, both inefficient in as housekeeping . Each PCR was performed in duplicate cells, served as controls (not shown). The pCDNA3-mutated and recorded on a standard curve providing a mean thres- cyclin B2 vector was constructed by directed mutagenesis. The hold cycle value that was translated into an arbitrary mutation is as follows: 50-TGAAACCTACTGCTTCTGT-30 DNA concentration. Cyclin B1 primers used are as follows: mutated to 50-TGAAGCCGACGGCATCGGT-30.Themuta- 50-AATAAGGAGGGAGCAGTGCG-30 and 50-GAAGAGC tion conserves the amino-acid sequence of cyclin B2, but the CAGCCTAGCCTCAG-30. Cyclin B2 primers used are as mRNA is not recognized by the shRNA against the cyclin follows: 50-GCGTTGGCATTATGGATCG-30 and 50-TCTT B2 wild-type sequence. CCGGGAAACTGGCTG-30.

Oligonucleotides used for constructions of shRNAs against Acknowledgements cyclins B1 and B2, and for the cyclin B2/shRNA-resistant vector Double-stranded shRNA corresponding to the cDNA We thank Malika Temal for her contribution to the early sequences of human cyclin B1 (50-GAACAGCUCUUG phase of this project, Franc¸oise Thierry and Moshe Yaniv for GGGACAU-30, nucleotides 220–238) and cyclin B2 (50- providing us a free access to the videomicroscope and flow UGAAACCUACUGCUUCUGU-30, nucleotides 284–302) cytometer. We also thank Stefan Geley for the histone H2B- were designed as recommended (Elbashir et al., 2001). The GFP expression vector and Katja Wassman for the anti-Mad2 cyclin B1 sequence was homologous to a validated Xenopus antiserum. This work was supported by grants from University cyclin B1 siRNA (Zhou et al., 2002). The cyclin B2 sequence Paris 6, CNRS, CNRS programme ‘Prote´ omique et Inge´ nierie was designed in the same region. No cross-reactive sequence des Prote´ ines’, contract PGP 03/0676 and IFR 83 ‘Biologie beside the genes of interest was found after a blast analysis Inte´ grative’. SB is a fellow from Association pour la Recherche against the . DNA oligonucleotides were sur le Cancer, and AdG was a fellow from Ministe` re de la purchased from QBiogene Research (Morgan Irvine, CA, Recherche et de la Technologie.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene