(2004) 23, 6845–6853 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc ORIGINAL PAPERS Crosstalk of the mitotic spindle assembly checkpoint with to prevent

Celia Vogel1,2, Anne Kienitz1,2, Irmgard Hofmann1,RolfMu¨ ller1 and Holger Bastians1,*

1Institute for Molecular and Tumor Research (IMT), Philipps University Marburg, Emil-Mannkopff-Strasse 2, D-35037 Marburg, Germany

Treatment of cells with inhibitors results in mitotic spindle assembly checkpoint, which inhibits activation of the mitotic spindle assembly checkpoint, onset until all are properly leading to mitotic arrest before anaphase. Upon prolonged attached to the mitotoic and aligned treatment, however, cells can adapt and exit at the plate (for reviews, see Shah and aberrantly, resulting in the occurrence of tetraploid cells Cleveland, 2000; Wassmann and Benezra, 2001; Chan in G1. Those cells subsequently arrest in postmitotic G1 and Yen, 2003). The molecular machinery of the spindle due to the activation of a p53-dependent G1 checkpoint. checkpoint involves the of , including Failure of the G1 checkpoint leads to Cenp-E, Bub1, BubR1, Bub3, , and Mps1, and further polyploidization. Using HCT116 and isogenic which are recruited specifically to that are p53-deficient or spindle checkpoint compromised deriva- not attached to the mitotic spindle. Although the tives, we show here that not only p53but also a functional significance of various –protein interactions spindle assembly checkpoint is required for postmitotic G1 observed during spindle checkpoint activation is not checkpoint function. During transient mitotic arrest, p53 well understood, it is widely accepted that Mad2 stabilization and activation is triggered by a pathway represents the distal effector protein that becomes independent of ATM/ATR, Chk1 and Chk2. We further activated at kinetochores. Mad2 inhibits the anaphase- show that a prolonged spindle checkpoint-mediated promoting complex/cyclosome (APC/C) by direct bind- mitotic arrest is required for proper postmitotic G1 ing and this results in stabilization of proteins including checkpoint function. In addition, we demonstrate that securin and B, whose degradation is required for polyploid cells are inhibited to re-enter mitosis by an anaphase onset and (Li et al., 1997; Fang additional checkpoint acting in G2. Thus, during a normal et al., 1998; Gorbsky et al., 1998). Therefore, activation cycle, polyploidization and subsequent aneuploidiza- of the spindle checkpoint inhibits anaphase onset and tion is prevented by the function of the mitotic spindle mitotic exit. checkpoint, a p53-dependent G1 checkpoint and an In normal cells, prolonged treatment with spindle- additional G2 checkpoint. damaging agents like or taxol leads to Oncogene (2004) 23, 6845–6853. doi:10.1038/sj.onc.1207860 transient pre-anaphase arrest due to the activation of the Published online 2 August 2004 spindle checkpoint, followed by an aberrant exit from mitosis without sister segregation and cyto- Keywords: checkpoint; genomic instability; kinesis, a process known as mitotic slippage. However, cell cycle; mad2 the resulting tetraploid cells arrest in G1, comprising a novel postmitotic G1 checkpoint preventing entry into the and polyploidization (Meek, 2000; Margolis Introduction et al., 2003). Cells lacking either p53, or pRb arrest in mitosis normally in response to spindle damage, but During tumorigenesis, polyploidy frequently precedes after mitotic slippage these cells enter S phase and , which is associated with high malignancy endoreduplicate their DNA, resulting in polyploidiza- and poor prognosis (Lengauer et al., 1998; Duesberg tion despite their intact spindle checkpoint, indicating a and Li, 2003; Rajagopalan et al., 2003). Although the strict dependence of the postmitotic G1 checkpoint on molecular basis for polyploidy observed in human the p53–pRb pathway (Di Leonardo et al., 1997; Khan is largely unknown, it is conceivable that and Wahl, 1998; Lanni and Jacks, 1998; Casenghi et al., polyploidy can be generated by uncoupling DNA 1999; Stewart et al., 1999). Thus, the activation of p53 replication from completion of mitosis. During mitosis, protects normal cells from polyploidization. In addition, proper sister chromatid segregation is monitored by the using inhibitors of tetraploid cells can be generated without spindle damage or segregation defects. Those cells also activate p53, leading to an *Correspondence: H Bastians; E-mail: [email protected] 2These authors contributed equally to this work arrest in G1, suggesting that tetraploidization itself Received 13 January 2004; revised 15 March 2004; accepted 4 May 2004; rather than aberrant exit from mitosis is triggering the published online 2 August 2004 p53 response (Andreassen et al., 2001). Nevertheless, the Crosstalk of the spindle checkpoint with p53 C Vogel et al 6846 molecular mechanisms leading to p53 activation in nocodazole treatment (Figure 1a). We treated all cell response to mitotic failure are unknown. Interestingly, it lines with nocodazole for up to 48 h, determined the has been observed that HeLa cells overexpressing a DNA content and found, in agreement with previous dominant-negative mutant of BUB1 or HCT116 cells work, that p53-deficient cells endoreduplicate, becoming exhibiting reduced levels of Mad2 (HCT116-MAD2 þ /À octaploid upon aberrant exit from mitosis. Importantly, cells) escape from mitotic arrest and subsequently endo- all the three spindle checkpoint compromised cell lines reduplicate, despite the fact that HCT116 cells possess also endoreduplicate (Figure 1b). Quantification of cell intact p53 (Taylor and McKeon, 1997; Michel et al., populations containing a DNA content 44 N revealed 2001). These observations strongly suggest a possible that these cell lines arrest in mitosis for a short time and functional interplay between the mitotic spindle check- endoreduplicate already 16 h after treatment. p53- point and the p53-dependent postmitotic G1 checkpoint. deficient cells, however, arrest in mitosis and endoredu- Here we investigated a functional interaction between plicate after 24h of treatment (Figure 1c). To confirm a the p53-dependent postmitotic G1 checkpoint and G1 arrest in HCT wild-type cells and S-phase entry of spindle checkpoint activation. We found that p53, p53-deficient and spindle checkpoint compromised cells, together with an intact spindle checkpoint, is required we evaluated the protein levels of , and to prevent endoreduplication upon mitotic failure. p53 is cdc2 following nocodazole treatment on Western blots. activated during spindle checkpoint-mediated mitotic In agreement with previous studies (Stewart et al., 1999), arrest and this arrest is required to ensure G1 wild-type cells arrested in G1 show decreased cyclin A checkpoint functionality. In spindle checkpoint com- and B levels, which are also accompanied with a promised cells, mitotic arrest is shortened, resulting in decrease of cdc2 levels (Figure 1d). In contrast, p53- subsequent endoreduplication. Moreover, extending the deficient cells show only transient downregulation of time of mitotic arrest in these cells inhibits endoredu- cyclin A and B upon mitotic exit, and strong reaccu- plication. In addition, we identified a spindle damage- mulation when cells enter S phase accompanied with triggered checkpoint mechanism, which arrests cells in continuous high-level expression of cdc2, indicating the G2, preventing polyploid cells to re-enter mitosis. Thus, cycling status of these cells (Figure 1d). Both spindle prevention of polyploidy requires a functional spindle checkpoint compromised cell lines exit mitosis and enter checkpoint, a p53-dependent G1 checkpoint and a novel the S phase earlier supported by high levels of cyclin A spindle damage-triggered G2 checkpoint. and B after incubation with nocodazole for up to 32 h. At later time points, however, these cells contain low levels of cyclin A, cyclin B and cdc2, suggesting a cell Results cycle arrest after prolonged nocodazole treatment Spindle checkpoint and p53-deficient cells endoreduplicate (Figure 1d). Consistent with this observation, we found upon prolonged spindle damage that the number of p53-deficient cells with a 44 N DNA content appear to accumulate continuously, while To investigate a functional interaction between the spindle checkpoint compromised cells reach a maximum mitotic spindle assembly checkpoint and postmitotic of cells with 44 N at 32 h (Figure 1c). To test whether p53-dependent G1 checkpoint activation, we took p53-deficient cells keep cycling and spindle checkpoint advantage of a panel of isogenic cells derived from compromised cells indeed arrest upon prolonged spindle HCT116 human colon cells. HCT116 cells poison treatment, we pulse-labelled cells with BrdU and are well characterized with respect to their intact DNA determined the time window of DNA replication damage checkpoint, normal p53 responses and an intact (Figure 1e). p53-deficient cells slip out of mitosis after spindle assembly checkpoint. We used HCT-p53À/À about 24h (data not shown) and continuously replicate, (Bunz et al., 1998), HCT-MAD2 þ /À (Michel et al., indicating endoreduplication for up to 72 h. However, 2001) and two independent HCT-MAD1 knockdown spindle checkpoint compromised cells replicate at a high clones (HCT-MAD1-kd-2-2 and HCT-MAD1-kd-2-31) rate for only up to 24h, reflecting early mitotic slippage in which siRNA targeting MAD1 is stably expressed and subsequent DNA replication. Upon prolonged from the pSUPER vector in HCT116 cells (A Kienitz, C spindle damage for up to 72 h, these cells indeed arrest Vogel, R Mu¨ ller, H Bastians, manuscript in prepara- in G1 and possibly in G2. Our data support the notion tion). HCT-MAD2 þ /À and HCT-MAD1-kd cells display that p53-deficient cells continue to replicate even after reduced Mad2 and Mad1 protein levels, respectively 72 h of nocodazole treatment, suggesting that a fraction (Michel et al., 2001; data not shown). After treatment of these cells continue to cycle in the presence of spindle with nocodazole to activate the spindle checkpoint for damage. Consistent with this observation, we found that 16 h HCT116 wild type, a control cell line containing a a fraction of p53-deficient but not of spindle checkpoint stably integrated pSUPER vector (HCT þ pSUPER) compromised cells contain DNA contents of 48 N after and p53-deficient derivatives accumulate in mitosis at a 48, 72 and 96 h of nocodazole treatment (Figure 1f). high rate, indicating an intact spindle assembly check- point and showing that p53 is not required for spindle Spindle damage activates p53 during the spindle checkpoint activation (Figure 1a). As expected, HCT- checkpoint-mediated mitotic arrest MAD2 þ /À and both HCT-MAD1 knockdown clones showed a compromised spindle checkpoint activation, The postmitotic G1 arrest in response to spindle damage as shown by strongly reduced after 16 h of and aberrant exit from mitosis is dependent on p53 and

Oncogene Crosstalk of the spindle checkpoint with p53 C Vogel et al 6847

Figure 1 p53 and a functional spindle assembly checkpoint are required for postmitotic G1 arrest upon prolonged treatment with microtubule inhibitors. (a) Spindle checkpoint status in HCT116 (wild type, wt), HCT-p53À/À, HCT-MAD2 þ /À and two HCT-MAD1 knockdown cell lines (HCT-MAD1-kd 2-31 and 2-2). The indicated cell lines were treated with 150 nM nocodazole for 16 h and the mitotic index was determined by MPM2-FACS analyses. (b) p53 and a functional spindle assembly checkpoint prevent endoreduplication in response to spindle damage. The indicated cell lines were treated with nocodazole (Noc) for up to 48 h and the DNA content was determined by DNA and FACS analyses. (c) Quantification of cells from the experiment shown in (b), displaying a DNA content of 44N in response to nocodazole treatment. (d) Determination of the cell cycle status in HCT116 wild-type, p53-deficient and spindle checkpoint compromised cells upon prolonged spindle damage. The indicated cell lines were treated with nocodazole for up to 48 h and the protein levels of cyclin A, cyclin B, cdc2 and were determined on Western blots. (e) Determination of the time window of endoreduplication in response to prolonged spindle damage. The indicated cell lines were treated with nocodazole for up to 72 h. At 60 min before harvesting, cells were pulse-labelled with BrdU and incorporation of BrdU indicating endoreduplication was determined by BrdU-FACS analyses. All graphs show mean values of at least three independent experiments. (f) Generation of hyper-octaploid cells in reponse to prolonged spindle damage. The indicated cell lines were treated with nocodazole for 48, 72 and 96 h and the DNA content was determined by FACS analyses expression of its target p21. We verified that in by the tetraploidy state itself rather than by mitotic HCT116 cells p53 and p21 properly accumulate upon defects (Andreassen et al., 2001). To determine whether nocodazole or taxol treatment (Figure 2a; data not p53 stabilization is triggered directly by spindle damage shown). It was suggested that p53 activation is triggered during mitosis or upon abnormal mitotic exit associated

Oncogene Crosstalk of the spindle checkpoint with p53 C Vogel et al 6848

Figure 1 (Continued)

with tetraploidy in G1, we determined the timepoint of Spindle damage-induced p53 activation does not require p53 stabilization. We isolated equal amounts of mitotic components of the DNA damage pathway HCT116 cells treated for 8 and 16 h with either To address the question of how p53 is activated during nocodazole or taxol by shakeoff and determined the level of p53 protein by Western blotting (Figure 2b). In mitosis, we investigated a possible involvement of ATM, both experiments, we detected a time-dependent accu- ATR, Chk1 and Chk2, checkpoint kinases that are mulation of p53 protein in mitotic cells, indicating involved in p53 activation upon DNA damage. First, we that stabilization of p53 is initiated during the used caffeine as a known inhibitor of the ATM and spindle checkpoint-mediated mitotic arrest. Further, ATR kinases (Blasina et al., 1999; Hall-Jackson et al., on Northern blots we quantified the mRNA of p21Cip1 1999) and found no significant effect on p53 stabiliza- (p21) as an example of a p53 target gene and found tion, induction of p21 or postmitotic G1 arrest after an initial induction of p21 mRNA in mitotic cells (our spindle damage. However, caffeine at the concentration unpublished results). Thus, after spindle damage, used inhibits p53 accumulation and activation of p53 induced by nocodazole or taxol, p53 activation is and abolishes cell cycle arrest after DNA damage triggered during spindle checkpoint-mediated mitotic induced by adriamycin (Figure 3a, left panel; data not arrest. Similar results were obtained using monastrol as shown). This suggests that p53 is activated in response a spindle checkpoint-activating agent (Mayer et al., to spindle damage independent of ATM/ATR. To test 1999), indicating that mitotic p53 activation is not for an involvement of Chk1, we used UCN-01 and dependent on the nature of the mitotic damage but Go¨ 6976 as established inhibitors for Chk1 kinase rather on the prolonged spindle checkpoint-mediated activity (Graves et al., 2000; Kohn et al., 2003). mitotic arrest. In agreement with this, we could not Pretreatment of HCT116 cells with UCN-01 or observe any p53 accumulation during a normal mitosis Go¨ 6976 followed by incubation with adriamycin or when the spindle assembly checkpoint is activated (data nocodazole had no effect on p53 accumulation, p21 not shown). induction and G1 arrest after spindle damage, while

Oncogene Crosstalk of the spindle checkpoint with p53 C Vogel et al 6849

Figure 2 p53 is stabilized and activated during spindle damage- induced mitotic arrest. (a) p53 is activated in response to spindle damage. HCT116-wild-type (wt) and HCT-p53À/À cells were treated with 150 nM nocodazole for up to 48 h and the protein levels of p53 and p21 were determined on Western blot. (b) p53 stabilization is triggered during mitotic arrest. HCT116 cells were treated with 150 nM nocodazole (Noc) or 100 nM taxol for 8 or 16 h and mitotic cells were isolated by shake-off. The mitotic index was determined by MPM2-FACS analyses and the level of endogenous p53 protein was determined by Western blotting Figure 3 p53 is stabilized in response to spindle damage independent of ATM, ATR, Chk1 and Chk2. (a) Left panel: HCT116 cells were incubated with DMSO or with 1.2 mM caffeine, UCN-01 abolishes G2 arrest in adriamycin-treated cells, followed by treatment with 350 nM adriamycin (ADR) for 14h or demonstrating an inactivation of Chk1 during p53- with 150 nM nocodazole (NOC) for 40 h. Protein levels of p53, p21 independent G2 arrest after DNA damage (Figure 3a, and actin were determined on Western blots. Right panel: HCT116 lower panel; data not shown). Chk2 is thought to be an cells were preincubated with 100 nM UCN-01, followed by treatment with adriamycin or nocodazole for 14and 40h, important upstream regulator of p53 (for a review, see respectively. Protein levels of p53, p21 and actin were determined McGowan, 2002). To test whether Chk2 is involved in on Western blots. Lower panel: Cell cycle distribution of cells used p53 activation in response to spindle damage, we used in the upper panels was determined by FACS analyses. (b) Chk2 is recently established HCT116 cells homozygously deleted not required for p53 activation after DNA damage and spindle for CHK2 (Jallepalli et al., 2003). Upon prolonged damage. Upper panel: HCT116 wild-type (wt) and HCT116- CHK2À/À cells were treated with 350 nM adriamycin (ADR) for 14h À/À nocodazole and adriamycin treatment, HCT-CHK2 and with nocodazole (NOC) for various times and the protein cells showed similar p53 and p21 accumulation as in levels of p53, p21 and actin were determined on Western blots. HCT116 wild-type cells (Figure 3b), suggesting that Lower panel: DNA content of HCT116 and HCT-CHK2À/À cells Chk2 is neither required for p53 activation after adria- after treatment with adriamycin (14h) and nocodazole (40h) was determined by FACS analyses mycin treatment nor upon prolonged spindle damage.

Prolonged mitotic arrest is required for proper postmitotic we tested whether a prolonged mitotic arrest, which G1 arrest upon mitotic failure occurs in response to spindle damage in wild-type cells but not in spindle checkpoint compromised cells, Given the fact that spindle checkpoint compromised is required to prevent endoreduplication (Figure 4b). cells endoreduplicate after mitotic failure and that the We incubated HCT116, HCT-MAD1-kd and HCT- postmitotic G1 arrest is dependent on p53, we investi- MAD2 þ /À cells with nocodazole for 8 h, isolated arrested gated whether an intact spindle assembly checkpoint is cells by mitotic shakeoff and treated them either with required for p53 accumulation upon spindle damage. DMSO as a control or with the inhibitor We compared the p53 levels of HCT116 control cells, ALLN in order to prolong the metaphase arrest HCT-MAD2 þ /À and HCT-MAD1-kd cells after spindle independent of the spindle checkpoint status of the damage on Western blots, and found no significant cells. After 4h, the proteasome inhibitor was washed differences in p53 stabilization in all the three cell lines off and aberrant exit from mitosis in the presence of (Figure 4a). Similar results were obtained after DNA nocodazole and further progression in G1 was mon- damage induced by adriamycin (data not shown). Next, itored by FACS analyses and determination of the

Oncogene Crosstalk of the spindle checkpoint with p53 C Vogel et al 6850

Figure 4 Prolonged mitotic arrest is required for efficient postmitotic G1 arrest. (a) Normal accumulation of p53 in spindle checkpoint compromised cells. HCT control cells (HCT þ pSUPER), HCT-MAD2 þ /À and HCT-MAD1-kd cells were treated with 150 nM nocodazole (Noc) for up to 48 h and the protein level of endogenous p53 was determined by Western blotting. (b) Prolonged mitotic arrest, mediated by an intact spindle assembly checkpoint, is required for preventing endoreduplication after spindle damage. HCT116-wild-type (wt), HCT-MAD2 þ /À and HCT-MAD1-kd cells were treated with 150 nM nocodazole and isolated by shake-off. Mitotic cells were either treated with DMSO (control) or with the proteasome inhibitor ALLN for additional 4h to arrest cells in mitosis independent of the spindle checkpoint activation, followed by washout of ALLN. Cells were harvested after 32 h in nocodazole and the proportion of cells containing a 44 N DNA content was calculated. The graph shows the mean values and standard error from three independent experiments

mitotic index (data not shown). After 32 h of nocodazole HCT-p53À/À, HCT-MAD1-kd and HCT-MAD2 þ /À cell treatment, cells were harvested and the proportion of lines by MPM2-FACS analyses (Figure 5a). We found cells which had undergone endoreduplication was that a significant proportion of octaploid p53-deficient determined by FACS analyses. As predicted, ALLN cells (about 15% after 48 h) had entered mitosis after treatment kept all the three spindle checkpoint compro- endoreduplication, whereas octaploid HCT-MAD1-kd mised cell lines in a arrest state until they and HCT-MAD2 þ /À cells arrested in G2 (Figure 5b). were washed off (data not shown). Most importantly, This finding is in agreement with our observation that spindle checkpoint compromised cells forced to arrest in p53-deficient cells but not spindle checkpoint compro- mitosis showed a significant reduction of endoreduplica- mised cells continue to cycle in the presence of spindle tion (Figure 4b), suggesting that a prolonged mitotic damage (Figure 1e and f). This indicates the existence arrest mediated by normal activation of the spindle of an additional p53-dependent checkpoint mechanism assembly checkpoint is required for an efficient post- that prevents polyploid cells from re-enter mitosis. mitotic G1 arrest.

Spindle damage triggers a G2 checkpoint that prevents Discussion re-entry into mitosis Aneuploidy and polyploidy are frequent in human During tumorigenesis, polyploidy seems to be a frequent solid tumors and could be explained by cells gaining precursor of aneuploidy (Shackney et al., 1989), or losing whole chromosomes or large fractions which might occur through endoreduplication asso- thereof at high frequency, which is referred to as ciated with amplification and subsequent re- instability (CIN). To date, the genetic entry into mitosis (Borel et al., 2002; Meraldi et al., lesions causing CIN are largely unknown. As aneuploi- 2002). To investigate whether p53-deficient and spindle dy can be directly a consequence of missegregation of checkpoint compromised cells that have undergone chromosomes during mitosis, in spindle endoreduplication would re-enter mitosis, we deter- checkpoint have been discussed for causing CIN mined the proportion of octaploid mitotic cells in (Rajagopalan et al., 2003). In addition, it is observed

Oncogene Crosstalk of the spindle checkpoint with p53 C Vogel et al 6851 explain why polyploidy often precedes aneuploidy during tumorigenesis (Borel et al., 2002; Meraldi et al., 2002). In our work, we demonstrate that p53 is stabi- lized and activated during a spindle checkpoint-induced mitotic arrest, rather than upon aberrant exit from mitosis and generation of a tetraploid state. It is yet unclear how p53 is activated during mitotic arrest, but it has been shown previously that the phosphoryla- tion pattern on p53 is distinct after DNA damage and after spindle damage, suggesting a separate signalling pathway that activates p53 during mitotic arrest and after DNA damage, respectively (Stewart et al., 2001). Moreover, our data indicate that activation of p53 during mitosis occurs independent of components of the p53-dependent DNA damage checkpoint pathway, including ATM, ATR, Chk1 and Chk2. In fact, in a first attempt to identify signalling components of this novel pathway activating p53 after spindle damage, we used a panel of kinase inhibitors and found that the phosphatidylinositol-3-kinase inhibitor LY294002 strongly inhibits the accumulation of p53 associated with reduced p21 expression after spindle damage. This suggests a possible involvement of PI3-kinase or PI3-kinase-related kinase in this novel signalling pathway (our unpublished results).

An intact spindle checkpoint is required for proper postmitotic G1 checkpoint function Figure 5 Spindle damage activates a p53-dependent G2 check- point. (a) Analysis of different populations of p53-deficient cells In our work, we could show that not only intact p53 but upon spindle damage. HCT-p53À/À cells were treated with 150 nM also an intact spindle assembly checkpoint is required nocodazole for 32 h and analysed in MPM2-FACS, revealing tetra- for postmitotic G1 arrest and therefore for prevention and octaploid mitotic and nonmitotic cell populations. (b) After of polyploidy upon aberrant mitotic exit. Moreover, polyploidization, p53-deficient cells re-enter mitosis. The indicated we have demonstrated that a prolonged mitotic arrest cell lines were treated with 150 nM nocodazole for up to 48 h and the proportion of mitotic cells containing an octaploid DNA is critical for postmitotic G1 arrest, indicating the content was quantified by MPM2-FACS analyses. The graph time of mitotic arrest to be critical for proper p53 shows the mean values from three independent experiments activation. We did not find significant defects in p53 stabilization, neither after spindle nor upon DNA damage. However, our preliminary analysis of the that polyploidy often precedes aneuploidy during expression of two p53 target genes, bax and p21, tumorigenesis (Shackney et al., 1989). It is therefore of after spindle damage suggests that the transcriptional great interest to unravel the mechanisms underlying activation of p53 might be altered in spindle check- aneuploidy and polyploidy. point compromised cells after spindle damage (our unpublished results). Polyploidy upon aberrant exit from mitosis is prevented Thus, it is tempting to speculate that proper trans- by a p53-dependent postmitotic G1 checkpoint criptional activation of p53, which is essential for pre- venting polyploidy, occurs during a prolonged mitotic Prolonged treatment with spindle poisons leads to an arrest. aberrant exit from mitosis, resulting in tetraploidy due to of the mitotic spindle assembly check- p53 – a surveillance factor for mitotic defects? point. Although the nature of the mitotic checkpoint adaptation is unknown, previous studies have shown Although we and others have shown that p53 is clearly that cells exiting aberrantly from mitosis activate not required for spindle checkpoint activation and for subsequently a p53-dependent G1 checkpoint, which mitotic arrest, there are several lines of evidence that p53 arrests cells in the postmitotic (Di Leonardo might be an important surveillance factor for mitotic et al., 1997; Khan and Wahl, 1998; Lanni and Jacks, defects: (i) p53 is activated in response to various mitotic 1998; Casenghi et al., 1999; Stewart et al., 1999; Meek, defects (Morris et al., 2000; Andreassen et al., 2001), 2000; Margolis et al., 2003). Inactivation of p53 not only (ii) p53 is localized to during mitosis and leads to endoreduplication but also to centrosome is displaced upon spindle damage (Ciciarello et al., amplification, giving rise to and 2001), (iii) 53BP1, a p53 interacting protein impli- thus missegregation of the . This might possibly cated in the early response to DNA damage, is localized

Oncogene Crosstalk of the spindle checkpoint with p53 C Vogel et al 6852 at kinetochores during mitosis and upon spindle targeting human MAD1 (a detailed characterization of the damage, resembling the localization of spindle check- MAD1 knockdown cells will be described elsewhere). All cells point proteins (Jullien et al., 2002), (iv) Chk2, an were grown in RPMI 1640 medium supplemented with 10% important regulator of p53, is localized to centrosomes fetal calf serum, 1% glutamine and 100 mg/ml streptomycin 1 and interacts with polo-like kinase 1, which is involved and 100 U/ml penicillin (Gibco) at 37 C under 95% water and 5% CO atmosphere. When indicated cells were treated et al 2 in several aspects of mitotic regulation (Tsvetkov ., with 150 nM nocodazole (Sigma), 100 nM taxol (Sigma), 68 mM 2003). monastrol (Sigma) or 200 mM ALLN (Calbiochem). It is conceivable that p53 is activated in response to various mitotic defects including spindle damage, Western blotting centrosome malfunction, prolonged mitotic delay and tetraploidization to act as a second fail-safe mechanism For Western blot analyses, cells were lysed in ice-cold lysis in G1 that prevents endoreduplication after mitotic buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, failure. 5mM EGTA, 1% (v/v) Nonidet-P-40, 0.1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS) containing protease inhibitors (Complete, Roche, Germany). Cellular debris was removed by centrifugation and the supernatants were snap frozen and An additional checkpoint in G2 prevents aneuploidy upon stored at À801C. Proteins were resolved by electrophoresis on polyploidization SDS/12.5 or 14% polyacrylamide gels and transferred onto nitrocellulose membranes. Blocking of the membrane and As inactivation of p53 is frequent in tumor cells, one incubations were performed in TBS plus 5 and 2% would expect that the postmitotic G1 checkpoint is nonfat dry milk, respectively. Immunoblots were developed suppressed in tumors, which in turn would allow using enhanced chemiluminescence (SuperSignal, Pierce). The polyploidization and subsequent aneuploidization. We following were used: anti-p53 (DO-1; PAb1801, have shown that an additional checkpoint mechanism Oncogene, San Diego, CA, USA), anti-p21 (Oncogene, San exists that arrests cells in G2 in response to mitotic Diego, CA, USA), anti-cyclin A (Santa Cruz, CA, USA), anti- failure and polyploidization. Polyploid p53-deficient cells cyclin B (Santa Cruz, CA, USA), anti-cdc2 (Santa Cruz, CA, can re-enter mitosis, whereas polyploid spindle check- USA), anti-actin (Sigma) and secondary antibodies conjugated point compromised p53-proficient cells stably arrest to HRP (Jackson ImmunoResearch, PA, USA). in G1 and G2 upon prolonged spindle damage. Therefore, we suggest that this novel G2 checkpoint Determination of the DNA content also involves the function of p53. Moreover, in our Cells were fixed in 70% ethanol for at least 16 h, resuspended preliminary experiments by using caffeine and UCN-01 in PBS and treated with 1 mg/ml RNase for 16 h. DNA was as inhibitors for ATM/ATR and Chk1 kinases, respec- stained using 50 mg/ml propidium iodide and analysed on a tively, we observe a significant increase of p53-deficient FACS Calibur (Becton Dickinson). cells entering mitosis, suggesting that ATM/ATR and Chk1 together with p53 might be involved in the Determination of the mitotic index spindle damage activated G2 checkpoint (our unpub- To determine the mitotic index, fixed cells were incubated for lished results). 2 h on ice with 2.5 mg/ml anti-MPM2 antibodies (Upstate, UK) These results, together with our data showing a high diluted in PBS, 0.2% Triton-X-100 and 2% FCS. Cells rate of DNA synthesis and the generation of hyper- were washed twice and incubated for 1 h on ice with goat- octaploid DNA contents in p53-deficient cells in the anti-mouse-DTAF antibodies (1 : 100, Jackson Immuno- presence of spindle poisons, suggest that p53 might Research). Cells were treated with RNase, DNA was stained represent a key surveillance factor to prevent polyploidy with propidium iodide and cells were analysed on a FACS and most likely aneuploidy after mitotic failure. How- Calibur (Becton Dickinson). ever, p53-deficient cells are not polyploid or aneuploid per se (Bunz et al., 1998), suggesting that additional Determination of BrdU incorporation genes are required to prevent polyploidy and aneuploidy. To measure the incorporation of BrdU, cells were pulse- As we have shown here, spindle checkpoint genes labelled with 50 mM BrdU (Sigma) for 1 h. Cells were fixed in that are required to prevent polyploidy might be prime ethanol for 24h and rehydrated in PBS, 0.5% BSA. DNA was candidates. denatured with 2 M HCl in water for 25 min at RT. Cells were washed and incubated for 1 h with anti-BrdU antibodies (diluted 1 : 200 in PBS, 0.5% BSA, 0.5% Tween-20). Cells were then processed as described for MPM2 staining. Materials and methods

Cell culture Acknowledgements HCT116 (wild type for TP53, CHK2, MAD1 and MAD2), We thank Dr Bert Vogelstein, Dr Loren Michel and Dr Robert HCT116-p53À/À (Bunz et al., 1998) and HCT116-CHK2À/À cells Benezra for providing HCT116 and derivative cell lines. We (Jallepalli et al., 2003) were generously provided by Dr Bert are grateful to Dr Heike Krebber for suggestions and critically Vogelstein (Baltimore, MD, USA). HCT116-MAD2 þ /À cells reading the manuscript, and we thank the Developmental (Michel et al., 2001) were a kind gift from Dr Loren Michel Therapeutics Program of the National Cancer Institute for and Dr Robert Benezra (New York, NY, USA). HCT116- provinding UCN-01. This work was supported by grants from MAD1 knockdown cells were generated by stable the Deutsche Forschungsgemeinschaft (SFB397), the Deutsche of pSUPER (Brummelkamp et al., 2002) expressing siRNA Krebshilfe and the PE Kempkes Stiftung.

Oncogene Crosstalk of the spindle checkpoint with p53 C Vogel et al 6853 References

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