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Spindle Checkpoint Function Is Required for Mitotic Catastrophe Induced by DNA-Damaging Agents

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Spindle Checkpoint Function Is Required for Mitotic Catastrophe Induced by DNA-Damaging Agents Oncogene (2004) 23, 6548–6558 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc Spindle checkpoint function is required for mitotic catastrophe induced by DNA-damaging agents Masayuki Nitta1,2,3, Osamu Kobayashi1,2,3, Shinobu Honda1, Toru Hirota1, Shinji Kuninaka1, Tomotoshi Marumoto1, Yukitaka Ushio2 and Hideyuki Saya*,1 1Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, University of Kumamoto, 1-1-1 Honjo, Kumamoto 860-8556, Japan; 2Department of Neurosurgery, Graduate School of Medical Sciences, University of Kumamoto, Kumamoto, Japan Mitotic catastrophe is an important mechanism for the ments such as chemotherapy and radiation. Most cancer induction of cell death in cancer cells by antineoplastic cells are deficient in G1 checkpoint function and agents that damage DNA. This process is facilitated by therefore fail to arrest in G1 phase on exposure to geno- defects in the G1 and G2 checkpoints of the cell cycle that toxic agents. Instead, they accumulate temporarily in are apparent in most cancer cells and which allow the cells G2 phase. However, given that the G2 checkpoint is to enter mitosis with DNA damage. We have now also partially impaired in cancer cells, they are unable characterized the dynamics of mitotic catastrophe induced to maintain G2 arrest and eventually die as they enter by DNA-damaging agents in p53-deficient cancer cells. mitosis (Fingert et al., 1986; Chan et al., 1999). This Cells that entered mitosis with DNA damage transiently process is known as mitotic catastrophe or mitotic death arrested at metaphase for more than 10 h without (Chan et al., 1999; Roninson et al., 2001). segregation of chromosomes and subsequently died Downregulation or inhibition of G2 checkpoint directly from metaphase. In those metaphase arrested proteins, including p53, p21, and 14-3-3, induces G2 precatastrophic cells, anaphase-promoting complex ap- checkpoint abrogation and subsequent catastrophic peared to be inactivated and BubR1 was persistently death in response to DNA damage (Bunz et al., 1998; localized at kinetochores, suggesting that spindle check- Chan et al., 1999, 2000). A new strategy has therefore point is activated after the DNA damage. Furthermore, been developed to sensitize cancer cells to the effects suppression of spindle checkpoint function by BubR1 or of genotoxic agents with the use of small molecules that Mad2RNA interference in the DNA damaged cells led inhibit the induction of G2 arrest by DNA damage to escape from catastrophic death and to subsequent (Sausville et al., 2001). The induction of mitotic abnormal mitosis. Dysfunction of the spindle checkpoint catastrophe is thus an important goal of cancer in p53-deficient cancer cells is thus likely a critical factor therapies using DNA-damaging agents. However, mito- in resistance to DNA-damaging therapeutic agents. tic catastrophe has been characterized mostly by Oncogene (2004) 23, 6548–6558. doi:10.1038/sj.onc.1207873 morphological observations, with the underlying mole- Published online 28 June 2004 cular mechanism remaining largely unknown in human cells. Keywords: mitotic catastrophe; spindle checkpoint; The spindle checkpoint inhibits the activity of the DNA damage; p53; time lapse; BubR1 anaphase-promoting complex (APC) and blocks the transition from metaphase to anaphase until all chromosomes are aligned at the metaphase plate and achieve bipolar attachment to the mitotic spindle, thus Introduction ensuring accurate sister chromatid segregation during cell division. Impairment of spindle checkpoint function Cell cycle checkpoints prevent transition from one phase results in premature mitosis and missegregation of of the cell cycle to the next until all processes of the chromosomes, leading to aneuploidy (Michel et al., present phase are completed. Defects in the checkpoint 2001). The signal activating spindle checkpoint is functions result in gene mutations and chromosome supposed to be generated from kinetochores that either damages, which contribute to the development and fail to bind spindle microtubules or are not experiencing progression of tumors. However, loss of checkpoint the tension that normally results from bipolar attach- function in some cancer cells is considered to be ment to the spindle. Several proteins that localize to associated with their sensitivity to antineoplastic treat- kinetochores, including Mad1, Mad2, BubR1 (Mad3), Bub1, Bub3, and Mps1, contribute to generation of the spindle checkpoint signal. Recent observations have *Correspondence: H Saya; E-mail: [email protected] 3These authors contributed equally to this work shown that cells having spindle checkpoint abnormal- Received 6 February 2004; revised 26 April 2004; accepted 26 April 2004; ities are insensitive to Taxol, a spindle poison (O’Con- published online 28 June 2004 nor et al., 2002; Anand et al., 2003). However, in case of Mitotic catastrophe in cancer cells M Nitta et al 6549 DNA-damaging agents, it is largely unknown whether phosphorylation of histone H2AX on serine-139 (Ro- dysfunction of the spindle checkpoint correlates with the gakou et al., 1998), which then results in the formation ineffectiveness of these drugs aimed at inducing mitotic of g-H2AX complexes that can be detected by the catastrophe. antibodies (Rogakou et al., 1999). Given that DBSs We have now characterized the dynamics of mitotic are introduced in cells even in an ordinary cultured catastrophe induced by DNA damage in p53-deficient condition (Haber, 1999), the g-H2AX complexes were cancer cells. Most cells entering mitosis with DNA detected in a small population of cultured cells. damage arrested at metaphase and subsequently under- However, treatment with aphidicolin induced a marked went cell death. Furthermore, metaphase arrest prior increase in the number of g-H2AX-positive spots to the catastrophe was clearly shown to result from the apparent in the nuclei of both p53 þ / þ and p53À/À activation of the spindle checkpoint, and inhibition of HCT116 cells in G2 phase (Figure 1d, left and right checkpoint function using RNA interference allowed panels). In addition, multiple g-H2AX-positive spots cancer cells to escape mitotic catastrophe. Our findings were detected on the chromosomes of p53À/À cells that demonstrate that DNA damage can activate spindle subsequently entered mitosis (Figure 1d, right panel). checkpoint in cells with impaired G2 checkpoint Exposure to ionizing radiation at G2 phase, which function in a way where persistent kinetochore localiza- also induces DNA damage, showed effects similar to tion of BubR1 seems to have a critical role. The spindle those of aphidicolin treatment in p53 þ / þ and p53À/À checkpoint function is thus required for the induction of HCT116 cells. The cells were synchronized in G0 phase catastrophe in cancer cells treated with DNA-damaging by serum deprivation, returned to serum-containing antineoplastic agents. medium and, after 12 h, exposed to 20Gy of X- radiation. The p53À/À cells entered mitosis after a transient G2 delay and underwent cell death (see Supplemental Figure 1). Results Aphidicolin induced the expression of both p53 and p21 proteins in p53 þ / þ HCT116 cells, but no p53 and Loss of G2 checkpoint function results in mitotic cell death À/À after DNA damage very little p21 proteins in p53 cells (Figure 1e and f). Consistent with previous observations (Bunz et al., Cancer cells with impaired G2 checkpoint exhibit an 1998, 1999), these results suggest that the failure to increased sensitivity to DNA-damaging agents, to which induce sufficient expression of p21 in the p53-deficient they respond by undergoing mitotic death (Bunz et al., cells results in G2 checkpoint abrogation. To confirm 1999; Borel et al., 2002). We established a system to that the death of p53À/À HCT116 cells treated with investigate mitotic death in response to DNA damage. aphidicolin occurs during mitosis, we blocked the G2–M To avoid the effect of G1 checkpoint, we used transition in these cells by exposure to butyrolactone I, aphidicolin to induce DNA damage. This drug, at which specifically inhibits the activity of cyclin-depen- concentrations between 0.3 and 1.0 mg/ml, inhibits DNA dent kinase 1 (Cdk1), 24 h after the onset of aphidicolin polymerases a, d,ande, and thereby induces a treatment. Butyrolactone I (50 mM) induced G2 arrest prolonged stall in DNA replication that prevents its and significantly suppressed cell death (see Supplemen- completion (Borel et al., 2002). The resulting gaps in the tal Figure 2). These results thus indicate that cells having double-stranded DNA are recognized as DNA strand DNA damage with impaired G2 checkpoint undergo cell breaks (Toledo et al., 2000) and activate the DNA death after they enter mitosis. þ / þ damage-induced G2 checkpoint. We treated p53 and À/À p53 HCT116 human colon cancer cells with aphidi- DNA damage induces metaphase arrest and subsequent colin (0.5 mg/ml) and examined cell cycle distribution by death in p53-deficient cells flow cytometry. Both p53 þ / þ and p53À/À cells transiently arrested at S phase and subsequently entered G2 phase We next analysed the dynamics of mitotic catastrophe þ / þ (Figure 1a). The p53 cells arrested in G2 phase and triggered by aphidicolin-induced DNA damage. To did not enter mitosis, whereas the p53À/À cells were facilitate visualization of the kinetics of chromosomes À/À unable to maintain G2 arrest and entered mitosis (Figure in living cells, we used HeLa and p53 HCT116 cells 1a and b). Furthermore, less than 15% of p53 þ / þ cells that stably express green fluorescent protein (GFP)- treated with aphidicolin for 96 h underwent cell death tagged histone H2B. Time-lapse fluorescence and (Figure 1c). In contrast, B50% of p53À/À cells under- differential interference contrast (DIC) microscopy went cell death after entry into mitosis. Similar to p53À/À using GFP-HH2B-expressing HeLa cells revealed that HCT116 cells, HeLa cells, in which the p53 is cells undergo mitotic catastrophe in three phases functionally inactivated by human papilloma virus-E6, (Table 1, Figure 2a, Supplemental video 1).
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