ANTICANCER RESEARCH 34: 3875-3880 (2014)

Alterations in Expression During Radiation-Induced Mitotic Catastrophe in HeLa Hep2 Cells

THERES LINDGREN1, TORGNY STIGBRAND2, LENNART JOHANSSON3, KATRINE RIKLUND4 and DAVID ERIKSSON5

Departments of 1Clinical Microbiology, Immunology, Umeå University, Umeå, Sweden; 2Clinical Microbiology, Immunology, Umeå University, Umeå, Sweden; 3Radiation Sciences, Radiation Physics, Umeå University, Umeå, Sweden; 4Radiation Sciences, Diagnostic Radiology, Umeå University, Umeå, Sweden; 5Clinical Microbiology, Immunology, Umeå University, Umeå, Sweden

Abstract. Aim: To explore kinetic changes in the gene morphology (3, 4), multiple nuclei (5) and/or several expression profile during radiation-induced mitotic catastrophes. micronuclei (6). Two important mechanisms for the induction Materials and Methods: changes were of mitotic catastrophe have been proposed. Firstly, a mitotic measured in HPV-infected HeLa Hep2 tumor cells following catastrophe has been proposed to occur as a consequence of exposure to 5 Gy of ionizing radiation (60Co). Signaling DNA damage and deficient cell-cycle checkpoints. Checkpoint pathways were explored and correlated to the biological inactivation is frequently a consequence of mutation/ responses linked to mitotic catastrophe. Results: Following inactivation of p53. Furthermore, the p53 is frequently irradiation a transient G2-arrest was induced. Anaphase bridge functionally-inactivated by viruses including high risk human formation and hyperamplification was observed. papillomaviruses (HPVs). HPVs may contribute to malignant These phenotypical changes correlated well with the observed transformation causing cervical and other anogenital cancers as gene expression changes. with altered expression were well as a sub-population of head and neck cancers (7). The found to be involved in mitotic processes as well as G2- and second proposed mechanism for the induction of mitotic spindle assembly checkpoints. Also centrosome-associated genes catastrophe is centrosome amplification (8). are a displayed an increased expression. Conclusion: This study major microtubule organizing center and exert an important elucidates specific characteristics in the altered gene expression function by formation of bipolar spindles (9). Hyperamplification pattern induced by irradiation, which can be correlated to the of centrosomes may result in multipolar spindles causing events of mitotic catastrophe in HeLa Hep2 cells. Therapeutic abnormal segregation, and generate cells with strategies modulating these alterations might potentiate future multiple micronuclei or binucleated giant cells (10, 11). therapy and enhance tumor cell killing. An improved understanding of the signaling pathways involved in radiation-induced mitotic catastrophe may help Mitotic catastrophe is considered to be the major cell death increase the efficacy of . To address this mechanism by which solid tumors respond to clinical issue, we investigated the kinetic response to ionizing radiotherapy (1). Mitotic catastrophe occurs during or as a radiation of HeLa Hep2 cells and evaluated if typical cellular result of aberrant (2). Aberrant mitosis produces phenotypes specific for mitotic catastrophe correlated with atypical chromosome segregation and cell division and leads alterations in gene expression. to the formation of giant cells with aberrant nuclear Materials and Methods

Cell lines. HeLa Hep2 cells (CCL-23, American Type Culture This article is freely accessible online. Collection (ATCC), Manassas, Virginia, USA)), were grown in DMEM (VWR International, Radnor, Pennsylvania, USA)) (1% Correspondence to: David Eriksson, Department of Clinical penicillin, streptomycin (VWR), 1% L-glutamine (VWR), and 5% Microbiology, Immunology, Umeå University, SE-901 85 Umeå, Fetal calf serum (VWR)) at 37˚C, 5% CO2. Sweden. Tel: +46 907852671, Fax: +46 907852250, e-mail: [email protected] Irradiation. Cells were exposed to absorbed doses of 5 Gy using a Cobalt-60 (Co60) treatment unit (Alcyon II, Best Theratronics Ltd, Key Words: Radiation, checkpoint, cell death, mitotic Ontario, Canada). Dose rate was approximately 0.45 Gy/min in the catastrophe, gene expression. middle of the treatment period.

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Staining of mitotic cells. Anti-phospho-Ser/Thr-Pro Mitotic Protein Monoclonal 2 (MPM-2) antibody (Upstate Cell Signaling Solutions, Millipore, Watford, UK) was used to quantify mitotic cells as earlier described (3). Cells were fixed in PEM buffer (80 mmol/L piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES), 1 mmol/L (ethylene glycol tetraacetic acid) (EGTA), 4 mmol/L MgCl2*6H2O, 0.2% saponin, pH 7.0 (all from Sigma Aldrich, Gillingham, Dorset, UK) containing 2% paraformaldehyde (Sigma Aldrich). Incubation of primary MPM-2 antibody was followed by Alexa Fluor 488– conjugated goat anti-mouse antibody (Invitrogen, Paisley, UK). Cells were then suspended in propidium iodide (20 mmol/L Tris solution (pH 7.6), propidium iodide (PI) (50 μg/mL), NP40 (0.1%), and RNase (20 μg/mL) (all from Sigma-Aldrich). Data was collected using FACS (BD Biosciences, San Jose, CA, USA), and results were analyzed using CellQuest software (BD Biosciences). Fluorescence microscopy. All fluorescence stainings were examined Figure 1. Quantification of mitotic cells by FACS, using MPM-2, a by confocal laser scanning microscopy using a Leica SP2 confocal monoclonal antibody with specificity for mitosis-specific and cell microscope equipped with an argon and HeNe laser. cycle–regulated phosphoproteins. A temporary decrease in mitotic cells indicates that radiation induce a transient G2-arrest in HeLa Hep2 Staining of centrosomes and DNA. Cells were fixed at –20˚C in 95% cells. One representative experiment is presented. C=Control. methanol and 5% acetic acid and incubated with PI solution as described above. Antibodies specific for γ-tubulin were used to visualize centrosomes as earlier described (3). Cells were fixed in PEM buffer containing 2% paraformaldehyde followed by labeling with a monoclonal antibody recognizing γ-tubulin (clone GTU-88, This arrest is transient and the frequency of mitotic cells returns Sigma Aldrich). Alexa Fluor 488 conjugated goat anti-mouse to control levels approximately 13 h after exposure to irradiation. antibody (Invitrogen) was used as secondary antibody. Cells were counterstained with PI as described above. Induction of anaphase bridges. Pronounced formation of Extracting total RNA. Cells were harvested at 6, 12, 24, 48, 72 and anaphase bridges were seen approximately 15 h following 96 h following irradiation. RNA was extracted using the RNeasy irradiation. Checkpoint adaptation and entry into mitosis with Mini Kit (QIAGEN, Hamburg, Germany) according to the unrepaired DNA damages is probably the cause of the manufacturer’s instructions. Quality was determined using the RNA increased frequency of anaphase bridges observed especially 6000 Nano assay on the 2100 Bioanalyzer (Agilent Technologies, during the first mitosis following irradiation (Figure 2). Santa Clara, CA, USA). RNA integrity value (RIN) for all samples were in the range of 9.5-10. Centrosome hyperamplification. Irradiation induces RNA labeling and gene expression analysis. Total RNA was centrosome hyperamplification in HeLa Hep2 cells (Figure prepared using the Illumina Total Prep RNA Amplification Kit 3). We have earlier shown that this centrosome amplification (Ambion, Austin TX, USA) according to instructions. 250 ng of is dose-dependent and is most pronounced in 2 to 3 days total RNA was used. Quality was determined using the RNA 6000 after exposure to irradiation (3, 10). Nano assay. Expression profiling was done using HumanRef-8 Bead- Chips (Illumina, San Diego, CA, USA). Raw data were Transcriptional profile of irradiated HeLa Hep2 cells. We analyzed using GenomeStudio V 3.2.3 (Illumina). Data was investigated the temporal changes in gene expression normalized using the cubic spline method. Illumina custom error model was used to compute a false discovery rate. p-Values were following irradiation. The number of genes that were calculated using the GenomeStudio V 3.2.3 and applying the significantly altered at different time-points evaluated are Illumina custom error model. Cut-off was set to p-values<0.05 and presented in Table I. Significantly altered signaling pathways >2-fold change in expression. were explored using MetaCore. At 6 and 12 h, only a few genes was significantly altered. From 24 h to 96 h, several Biological functions and pathway analysis. Signaling pathways and genes displayed altered expression. A major part of these processes were explored using MetaCore (Genego Inc., St Joseph, genes were found to be involved in cell-cycle progression MI, USA). Normalized gene expression data was exported. Filters were set on p<0.05, and fold change 2 (positive and negative). and regulation, mitotic processes and the G2- and spindle assembly checkpoints. At 96 h pathways involving interferon Results and other cytokines were significantly changed. We chose to explore genes that were continuously altered Induction of G2-arrest. Irradiation induces a transient at 24, 48, 72, and 96 h in order to reduce the complexity of G2-arrest as seen in Figure 1. The results indicate an initial G2- interpretation. These time-points were chosen as most arrest and a consequent decrease in the fraction of mitotic cells. cellular events associated with mitotic catastrophe occur

3876 Lindgren et al: Radiation Induced Gene Expression in HeLa Hep2 Cells

Figure 2. Propidium iodide staining of DNA in control and irradiated HeLa Hep2 cells. Irradiated cells displayed an increased frequency of anaphase bridges.

Figure 3. Centrosomes visualized with a γ-tubulin antibody (green) and DNA stained with propidium iodide (red). Left: a mitotic cell with normal number of centrosomes; Right: an irradiated mitotic cell with hyperamplified number of centrosomes.

Table I. Differentially expressed genes at 6, 12, 24, 48, 72 and 96 h Table II. The 10 most significantly altered pathways at 24, 48, 72 and following irradiation. p≤0.05 and ≥2 fold change in expression. 96 h following irradiation. p-Values were calculated using MetaCore.

Time after Total number Up-regulated Down-regulated Pathway p-Value irradiation of genes Cell cycle: Chromosome condensation in prometaphase 1,87×10–24 6h 16 16 0 Cell cycle: Role of APC in cell cycle regulation 4,06×10–17 12h 79 79 0 Cell cycle: The metaphase checkpoint 1,86×10–16 24h 314 314 0 –13 48h 374 362 12 Cell cycle: Spindle assembly and chromosome separation 4,00×10 72h 325 319 6 Cell cycle: Initiation of mitosis 9,86×10–9 96h 329 328 1 Cell cycle: Nucleocytoplasmic transport of CDK/Cyclins 1,60×10–6 Reproduction: Progesterone-mediated oocyte maturation 5,65×10–6 Cell cycle: Cell cycle (generic schema) 9,22×10–6 ATP/ITP metabolism 1,50×10–5 during this time. The 10 most significantly changed DNA damage: ATM/ATR regulation of G2/M checkpoint 2,25×10–5 pathways obtained by MetaCore are displayed in Table II. Seven out of 10 pathways are related to the cell cycle and especially mitotic processes. The remaining pathways are related to DNA damage regulation of the G2/M checkpoint. Several of these genes were associated with centrosome A total of 44 genes were found to have an increased gene regulation, spindle assembly checkpoint and G2- to M-phase expression at all time-points (Table III). progression (Table IV).

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Table III. The 44 genes which had a continuously changed expression at Table IV. Genes associated with SAC, centrosomes and G2/M arrest. 24, 48, 72 and 96 h following irradiation. p≤0.05 and fold change ≥2. Fold changes at 24, 48, 72 and 96h following 5 Gy of irradiation are p-Values were calculated using the GenomeStudio software. presented.

Gene 24 h 48 h 72 h 96 h G2/M arrest 24h 48h 72h 96h

ACTC1 7.42 9.06 8.67 7.70 ANLN 3.70 3.35 2.75 1.85 ANLN 3.70 3.35 2.75 1.85 CDCA3 5.50 4.63 2.97 2.50 APOBEC3B 2.27 2.13 2.28 2.09 CDK1/CDC2 3.67 2.36 2.09 1.92 ASB9 3.76 6.57 4.31 3.53 CENPE 9.01 6.88 4.14 2.42 ASPM 5.44 4.22 3.70 2.24 CCNA2 5.95 5.68 4.02 3.13 ATOH8 2.98 3.68 4.66 3.60 CCNB1 3.41 2.20 1.88 2.08 AURKA 4.96 5.28 4.14 2.22 DLGAP5 5.36 5.93 4.55 2.53 BNC2 2.80 2.80 3.24 2.87 E2F7 2.76 2.52 2.11 1.92 BORA 4.42 3.30 2.50 2.59 MASTL 2.26 2.03 2.61 3.31 BUB1 4.77 4.24 3.16 2.44 NCAPG 4.13 3.79 3.06 2.31 CCNA2 5.95 5.68 4.02 3.13 NEK2 2.88 2.42 2.19 1.84 CCNB1 3.41 2.20 1.88 2.08 RPRM 3.71 2.07 2.83 2.07 CDCA3 5.50 4.63 2.97 2.50 SMC4 3.83 2.84 2.08 2.15 CDCA8 4.44 3.93 2.83 1.85 CDK1/CDC2 3.67 2.36 2.09 1.92 Centrosomes; Amplification/ 24h 48h 72h 96h CENPE 9.01 6.88 4.14 2.42 Separation/Maturation CEP152 3.04 2.60 2.15 2.74 CEP55 3.74 3.42 3.02 2.31 ASPM 5.44 4.22 3.70 2.24 DDX39A 3.08 2.47 2.36 2.03 AURKA 4.96 5.28 4.14 2.22 DLGAP5 5.36 5.93 4.55 2.53 BORA 4.42 3.30 2.50 2.59 E2F7 2.76 2.52 2.11 1.92 CEP152 3.04 2.60 2.15 2.74 HJURP 4.18 3.52 2.65 2.00 CEP55 3.74 3.42 3.02 2.31 HMMR 5.66 4.00 2.53 2.13 CCNA2 5.95 5.68 4.02 3.13 ID1 3.46 3.59 3.39 4.65 CCNB1 3.41 2.20 1.88 2.08 ID2 2.76 2.75 3.77 3.71 HJURP 4.18 3.52 2.65 2.00 INCENP 8.15 4.40 4.13 2.33 HMMR 5.66 4.00 2.53 2.13 KIF14 5.43 5.01 3.40 2.58 INCENP 8.15 4.40 4.13 2.33 KIF18A 4.30 2.74 2.58 1.95 NEK2 2.88 2.42 2.19 1.84 LAMA1 2.84 2.95 3.74 2.22 NUF2 4.84 3.31 2.77 3.44 MASTL 2.26 2.03 2.61 3.31 NUSAP1 2.84 2.18 2.06 1.49 MLLT11 2.12 2.68 3.46 2.75 SGOL1 3.50 3.01 2.18 2.19 MUC1 3.80 5.09 4.47 3.16 TTK/MPS1 5.14 4.26 3.45 2.74 NCAPG 4.13 3.79 3.06 2.31 NEK2 2.88 2.42 2.19 1.84 Spindle assembly checkpoint 24h 48h 72h 96h NUF2 4.84 3.31 2.77 3.44 NUSAP1 2.84 2.18 2.06 1.49 ASPM 5.44 4.22 3.70 2.24 RAD54L 3.78 2.66 2.82 3.06 BUB1 4.77 4.24 3.16 2.44 RPRM 3.71 2.07 2.83 2.07 CDCA8 4.44 3.93 2.83 1.85 RRM2 3.09 2.70 3.18 2.49 CEP55 3.74 3.42 3.02 2.31 SGOL1 3.50 3.01 2.18 2.19 KIF14 5.43 5.01 3.40 2.58 SMC4 3.83 2.84 2.08 2.15 KIF18A 4.30 2.74 2.58 1.95 TPX2 3.76 4.52 3.16 2.13 NUF2 3.48 3.31 2.77 3.44 TTK 5.14 4.26 3.45 2.74 SGOL1 3.50 3.01 2.18 2.19 WDR62 3.91 5.54 4.36 3.27 TPX2 3.76 4.52 3.16 2.13 TTK/MPS1 5.14 4.26 3.45 2.74

Discussion profile in HeLa Hep2 cells passing through a mitotic Mitotic catastrophe is considered the most common mode of catastrophe. HeLa Hep2 cells have wild type p53, but the cell death in solid tumors in which p53 function is impaired protein level is reduced by the human papilloma virus (12). In the present study we analyzed the gene expression protein E6 to give no apparent p53 response.

3878 Lindgren et al: Radiation Induced Gene Expression in HeLa Hep2 Cells

We observed a transient G2-arrest in irradiated HeLa Hep2 Several inhibitors are currently investigated in pre-clinical cells (Figure 1) followed by a re-entry into the cell cycle and clinical studies including aurora kinases (26, 27), PLK1 with unrepaired DNA damage, probably as a consequence of (11), (28) and kinesin family members (KIF11, an impaired G2/M checkpoint and adaptation (13). KIFC1) (21, 29). This study indicates that therapeutic Maintenance of the G2-arrest is dependent on p53 and its strategies combining these inhibitors with irradiation might downstream signaling (14). potentiate therapy and enhance tumor cell kill. Cells with impaired p53 function have a higher expression level of these downstream genes (15), which is in agreement Acknowledgements with our results and probably contribute to enhance This project was financed by the Swedish Cancer Research Council, checkpoint adaptation process. Checkpoint adaptation cause the Lions Foundation in Umeå, the county of Västerbotten and the premature mitotic entry of cells with unrepaired DNA University of Umeå. damage and induction of a prometaphase arrest (13). In agreement with this observation, one of the most significantly References affected signaling pathways induced by irradiation of HeLa Hep2 cells involved the prometaphase (Table II). We observed 1 Dewey WC, Ling CC and Meyn RE: Radiation-induced an increased expression of several genes implicated in the : relevance to radiotherapy. Int J Radiat Oncol Biol Phys 33(4): 781-796, 1995. regulation of the G2/M checkpoint including PLK1, CCNB, CDC25B and CDC25C (Table IV). All these mitosis- 2 Galluzzi L, Maiuri MC, Vitale I, Zischka H, Castedo M, Zitvogel promoting factors have been described to accumulate during L and Kroemer G: Cell death modalities: classification and pathophysiological implications. Cell Death Differ 14(7): 1237- G -arrest and consequently trigger checkpoint adaptation 2 1243, 2007. (13). Impaired checkpoints, checkpoint adaptation and 3 Eriksson D, Lofroth PO, Johansson L, Riklund KA and premature entry into mitosis are probably the explanations for Stigbrand T: Cell cycle disturbances and mitotic catastrophes in increased frequency of anaphase bridges and lagging HeLa Hep2 cells following 2.5 to 10 Gy of ionizing radiation. chromosomal material that we observed especially during the Clin Cancer Res 13(18 Pt 2): 5501s-5508s, 2007. first mitosis following irradiation (Figure 2). Anaphase 4 Castedo M, Perfettini JL, Roumier T, Andreau K, Medema R and bridges may be generated when broken , Kroemer G: Cell death by mitotic catastrophe: a molecular definition. Oncogene 23(16): 2825-2837, 2004. induced by irradiation, fuse. We and others have reported that 5 Erenpreisa J, Kalejs M, Ianzini F, Kosmacek EA, Mackey MA, hyperamplification of centrosomes occurs after exposure to Emzinsh D, Cragg MS, Ivanov A and Illidge TM: Segregation radiation (Figure 3) (8, 10, 16). In the present study multiple of genomes in polyploid tumour cells following mitotic genes involved in centrosome amplification were up-regulated catastrophe. Cell Biol Int 29(12): 1005-1011, 2005. following irradiation (Table IV). Genes known to be involved 6 Roninson IB, Broude EV and Chang BD: If not apoptosis, then in centrosome duplication like CDK2, CCNE, CCNA, AURKA what? Treatment-induced senescence and mitotic catastrophe in and CDC25 (17-19) were all up-regulated at multiple time- tumor cells. Drug Resist Updat 4(5): 303-313, 2001. points following radiation. (20). Furthermore, KIF11 and 7 zur Hausen H: Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2(5): 342-350, 2002. KIFC1 have crucial roles in the formation of bipolar spindles 8 Dodson H, Wheatley SP and Morrison CG: Involvement of (21, 22). Additionally, PLK1 is an important kinase that centrosome amplification in radiation-induced mitotic controls centrosome maturation. Reduced activity of PLK1 catastrophe. Cell Cycle 6(3): 364-370, 2007. results in functional defects of centrosomes and failure to 9 Loffler H, Lukas J, Bartek J and Kramer A: Structure meets form bipolar mitotic spindles (23). Genes significantly function--centrosomes, genome maintenance and the DNA changed and involved in the spindle assembly checkpoint damage response. Exp Cell Res 312(14): 2633-2640, 2006. (SAC) are presented in Table IV. SAC is important for DNA 10 Eriksson D, Blomberg J, Lindgren T, Lofroth PO, Johansson L, Riklund K and Stigbrand T: Iodine-131 induces mitotic damage induced mitotic catastrophe since this checkpoint catastrophes and activates apoptotic pathways in HeLa Hep2 prevents cells from entering anaphase until all chromosomes cells. Cancer Biother Radiopharm 23(5): 541-549, 2008. are properly attached to the bipolar mitotic spindles (17, 24). 11 Wang Y, Ji P, Liu J, Broaddus RR, Xue F and Zhang W: The spindle checkpoint proteins inhibit CDC20 and Centrosome-associated regulators of the G(2)/M checkpoint as consequently the onset of the anaphase (24). The results of targets for cancer therapy. Mol Cancer 8(8): 1476-4598, 2009. this study clearly demonstrate a relation between radiation- 12 Vakifahmetoglu H, Olsson M and Zhivotovsky B: Death through induced alterations in gene expressions and the subsequent a tragedy: mitotic catastrophe. Cell Death Differ 15(7): 1153- execution of the mitotic catastrophe. 1162, 2008. 13 Syljuasen RG: Checkpoint adaptation in human cells. Oncogene. Novel therapeutic strategies currently aim at centrosome 2007;26(40):5833-5839. and G2/M checkpoint associated regulators with the intention 14 Lukas J, Lukas C and Bartek J: Mammalian cell cycle to target and inactivate them, thus promoting mitotic checkpoints: signalling pathways and their organization in space catastrophe and consequently increasing cell death (25). and time. DNA Repair 3(8-9): 997-1007, 2004.

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15 Sur S, Pagliarini R, Bunz F, Rago C, Diaz LA Jr., Kinzler KW, 23 Barr FA, Sillje HH and Nigg EA: Polo-like kinases and the Vogelstein B and Papadopoulos N: A panel of isogenic human orchestration of cell division. Nat Rev Mol Cell Biol 5(6): 429- cancer cells suggests a therapeutic approach for cancers with 440, 2004. inactivated p53. Proc Natl Acad Sci USA 106(10): 3964-3969, 24 Musacchio A and Salmon ED: The spindle-assembly checkpoint 2009. in space and time. Nat Rev Mol Cell Biol 8(5): 379-393, 2007. 16 Kawamura K, Morita N, Domiki C, Fujikawa-Yamamoto K, 25 Kawabe T: G2 checkpoint abrogators as anticancer drugs. Mol Hashimoto M, Iwabuchi K and Suzuki K: Induction of Cancer Ther 3(4): 513-519, 2004. centrosome amplification in p53 siRNA-treated human fibroblast 26 Gautschi O, Heighway J, Mack PC, Purnell PR, Lara PN Jr. and cells by radiation exposure. Cancer Sci 97(4): 252-258, 2006. Gandara DR: Aurora kinases as anticancer drug targets. Clin 17 Fukasawa K: Oncogenes and tumour suppressors take on Cancer Res 14(6): 1639-1648, 2008. centrosomes. Nat Rev Cancer 7(12): 911-924, 2007. 27 Kitzen JJ, de Jonge MJ and Verweij J: Aurora kinase inhibitors. 18 Fisk HA and Winey M: The mouse Mps1p-like kinase regulates Crit Rev Oncol Hematol 73(2): 99-110, 2010. centrosome duplication. Cell 106(1): 95-104, 2001. 28 Mita AC, Mita MM, Nawrocki ST and Giles FJ: Survivin: key 19 Fisk HA, Mattison CP and Winey M: Human Mps1 protein regulator of mitosis and apoptosis and novel target for cancer kinase is required for centrosome duplication and normal mitotic therapeutics. Clin Cancer Res 14(16): 5000-5005, 2008. progression. Proc Natl Acad Sci USA 100(25): 14875-14880, 29 Rello-Varona S, Vitale I, Kepp O, Senovilla L, Jemaa M, 2003. Metivier D, Castedo M and Kroemer G: Preferential killing of 20 Zhou H, Kuang J, Zhong L, Kuo WL, Gray JW, Sahin A, tetraploid tumor cells by targeting the mitotic kinesin Eg5. Cell Brinkley BR and Sen S: Tumour amplified kinase STK15/BTAK Cycle 8(7): 1030-1035, 2009. induces centrosome amplification, aneuploidy and transformation. Nat Genet 20(2): 189-193, 1998. 21 Kwon M, Godinho SA, Chandhok NS, Ganem NJ, Azioune A, Thery M and Pellman D: Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes. Genes Dev 22(16): 2189-2203, 2008. 22 Kashina AS, Rogers GC and Scholey JM: The bimC family of Received April 23, 2014 kinesins: essential bipolar mitotic motors driving centrosome Revised May 28, 2014 separation. Biochim Biophys Acta 24(3): 257-271, 1997. Accepted June 3 2014

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