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(2010) 29, 3477–3489 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE - and p21-dependent premature APC/C–Cdh1 activation in G2 is part of the long-term response to genotoxic stress

L Wiebusch and C Hagemeier

Children’s Hospital, Laboratory for Molecular Biology, Charite´-Universita¨tsmedizin Berlin, Berlin, Germany

The long-term cellular response to DNA damage is tumor-suppressor -53 (p53)-dependent transcrip- controlled by the tumor suppressor p53. It results in cell- tional activation of p21Waf1/Cip1 (p21) (Di Leonardo et al., cycle arrest followed by DNA repair and, depending on the 1994; Waldman et al., 1995; Bunz et al., 1998), a potent degree of damage inflicted, premature senescence or inhibitor of CDK activity (Harper et al., 1993; Besson apoptotic cell death. Here we show that in normal diploid et al., 2008). Cells arrested terminally after p21 fibroblasts the -promoting com- induction acquire a senescence-like phenotype, also plex or cyclosome (APC/C)–Cdh1 becomes prematurely called premature senescence. Although DNA damage- activated in G2 as part of the sustained long-term but not induced typically also depends on p53, p53 the rapid short-term response to genotoxic stress and target other than p21 are involved (Yu and Zhang, results in the degradation of numerous APC/C substrates. 2005). Thus, p21 has an antiapoptotic function Using HCT116 somatic knockout cells we show that (Polyak et al., 1996; Janicke et al., 2007) and is actively mechanistically premature APC/C activation depends on repressed in apoptotic cells (Allan and Fried, 1999; p53 and its transcriptional target p21 that mediates the Seoane et al., 2002). Both, DNA damage-induced signal through downregulation of the APC/C inhibitor apoptosis and senescence are considered to be important Emi1. Cdc14B is dispensable in this setting but might physiological mechanisms to withdraw pre-neoplastic function redundantly. Our data suggest an unexpected role cells from proliferation (Halazonetis et al., 2008). for the APC/C in executing a part of the p53-dependent In addition, the outcome of cytotoxic cancer therapy DNA damage response that leads to premature senescence. crucially depends on the ability of tumor cells to Oncogene (2010) 29, 3477–3489; doi:10.1038/onc.2010.99; elicit either an apoptotic or senescence response published online 12 April 2010 (Schmitt, 2003). As a , p53 has multiple regulatory Keywords: APC/C; p53; p21; Emi1; senescence; apop- functions. Besides its role as an activator of apoptosis tosis and senescence-inducing genes, p53 also acts as a direct transcriptional repressor of several cell-cycle-promoting factors (St Clair et al., 2004; Banerjee et al., 2009). Introduction Moreover, through p21-dependent inhibition of CDK activity, p53 indirectly induces the formation of During their lifetime, cells are exposed to a variety of repressive pocket protein– complexes, thereby lead- agents that cause damage to their DNA. An integral ing to sustained transcriptional downregulation of many part of the cellular response to DNA damage is the key enzymes of and replication (Lo¨hr et al., ability to arrest DNA synthesis and cell-cycle progres- 2003; Jackson et al., 2005; Spurgers et al., 2006; sion. This arrest can be rapidly induced by executing a Kidokoro et al., 2008). This negative effect on mRNA cascade of (de-)phosphorylation events that lead to an synthesis is complemented at the level of protein inactivation of -dependent kinases (CDKs) and production by another class of p53 target genes, the replication factors. This immediate response is only miR-34 family (He et al., 2007; Hermeking, 2007). transient and relieved when DNA damage has been Given the broad influence of p53 on transcription and repaired. However, in the case of more severe damage, translation, it is surprising that at the level of protein the transient cell-cycle arrest gives way to either degradation, a p53-driven mechanism of regulation apoptotic cell death or a delayed but long-lasting, is yet unknown. Such a mechanism might be particularly sometimes irreversible arrest. This long-term arrest can important for the long-term establishment of a stress- be established in G1 and and relies on the induced G2 arrest because in this cell-cycle phase cells have already accumulated significant amounts of mitotic Correspondence: Dr L Wiebusch or Dr C Hagemeier, Children’s enzymes. In principle, the anaphase-promoting complex Hospital, Laboratory for Molecular Biology, Charite´-Universita¨tsmedizin or cyclosome (APC/C) would represent an attractive Berlin, Ziegelstrasse 5-9, Berlin D-10117, Germany. target for such a p53-dependent regulation, as this E-mails: [email protected] or [email protected] ubiquitin ligase functions as a master regulator for the Received 8 August 2009; revised 22 February 2010; accepted 2 March stability of several rate-limiting controlling 2010; published online 12 April 2010 replication and (Peters, 2006). p53/p21-dependent activation of APC/C–Cdh1 in G2 L Wiebusch and C Hagemeier 3478 Indeed, two reports have shown that in response to In addition, after 2–3 days the arrested cells developed DNA damage the APC/C, which is normally kept features of such as b-galactosidase inactive from the late G1 to early M-phase, is prema- (SA-b-gal) activity and increase in both cell and nuclear turely activated in G2 (Sudo et al., 2001; Bassermann size (Supplementary Figure S1B–D). This is consistent et al., 2008). However, this activation followed DNA with doxorubicin-induced senescence in tumor cells damage with short-term kinetics (as early as 90 min), (Chang et al., 1999) and primary fibroblasts (Baus making a p53-driven mechanism involving de novo et al., 2003; Demidenko and Blagosklonny, 2008). protein synthesis unlikely. Moreover, except for polo- Both, the transient arrest in S and the prolonged like kinase-1 (Plk1), both reports failed to observe a block in G2 were reflected by the expected biochemical significant downregulation of APC/C substrates during changes to DNA damage checkpoint proteins. The early the first 8 h after DNA damage, suggesting that APC/C response was characterized by induction of H2A-X, activation has only a confined role during the early Chk1, Chk2 and p53 phosphorylation, and of p53 response to DNA damage. abundance (Figure 1b). Six hours after doxorubicin Here we show that APC/C activation in G2-arrested treatment, these changes progressively decreased cells is part of the p53- and p21-dependent long-term whereas expression of the long-term cell-cycle arrest response to genotoxic stress that leads to premature effector p21 increased, reaching a steady-state level at senescence. It relies on Cdh1, the G1-phase-specific around 24 h after DNA damage (Figure 1b and adaptor subunit (Wa¨sch et al., 2010), and goes hand in Supplementary Figure S3). Thus, the experimental hand with marked decline of Emi1 expression, the main system at hand faithfully allowed to differentiate inhibitor of the APC/C (Hsu et al., 2002). between the immediate transient and a delayed sustained Importantly, activation of APC/C–Cdh1 is required for cell-cycle response to genotoxic stress. timely destruction of a broad range of APC/C substrates after DNA damage. Premature activation of the APC/C in doxorubicin- arrested G2 cells We next examined whether the APC/C becomes activated under these conditions and if so, which Results kinetics would the activation follow. We did not observe significant APC/C activation early after DNA damage A senescence-like phenotype of doxorubicin-treated (Figure 1c), even when focusing on the first 90 min of fibroblasts in G2 doxorubicin treatment or DNA damage induction in G2 To investigate the cellular response to genotoxic stress at rather than in S-phase cells (Supplementary Figure S2). the level of APC/C regulation we used primary Instead, we found induction of APC/C activity that fibroblasts, as most transformed cell lines have acquired parallels the long-term DNA damage response begin- genetic alterations in DNA damage checkpoint path- ning between 6 and 12 h and, like p21 protein expres- ways (Bartek et al., 2007). In particular, we wanted to sion, reaching a maximum at around 24 h after address whether the silent APC/C of S/ G2 cells could be doxorubicin treatment (Figure 1c). Importantly, this activated in response to DNA damage. Therefore, we APC/C activation appeared to be functionally relevant. synchronized cells in S-phase before applying DNA- All bona fide APC/C substrates analyzed were found to damaging agents. To avoid checkpoint activation by be downregulated with kinetics similar to the observed chemical inhibitors of DNA replication (Nayak and APC/C activation (Figure 1b). This included Cdc20, the Das, 2002; Kurose et al., 2006), synchronization in G 1 M-phase-specific adaptor subunit of the APC/C, and was achieved by contact inhibition and replating of cells other substrates of the APC/C–Cdh1, suggesting that at lower density. Using this protocol, 17–19 h after premature APC/C activation in G2 was mediated by replating 70–80% of cells entered S-phase synchro- Cdh1, which remained stably expressed (Figure 1b) and nously (Figure 1a) that well suffices the requirements of co-immunoprecipitated with the APC/C (data not this study. shown). Expression of UbcH10 was also affected by Moderate DNA damage of these cells was induced the DNA damage response, whereas another APC/C- with 1 mM doxorubicin treatment for 2 h, causing a block interacting ubiquitin conjugase, UbcH5, was upregu- to DNA replication. After removal of the drug, cells lated. Possibly, UbcH5, which compared with UbcH10 resumed cell-cycle progression but failed to undergo cell is considered as the less APC/C-specific E2 enzyme division and instead accumulated 4n-DNA content (Summers et al., 2008), has a role in the DNA damage- between 6 and 18 h after treatment (Figure 1a). Markers dependent induction of APC/C activity. of mitotic entry as histone-H3–serine-10 phosphoryla- tion (Figure 1b) and chromatin condensation (Figure 1d) were not detectable, showing that doxor- Premature induction of APC/C activity in G2 depends on ubicin-treated cells were arrested in G2 rather than Cdh1 and is required for timely destruction of APC/C mitosis. We further ruled out a tetraploid G1 arrest as substrates we detected no bi- or micro-nucleated cells (Figure 1d) Many APC/C substrate-encoding genes are known to that would origin from aberrant mitosis (Andreassen underlie additional cell-cycle- and checkpoint-dependent et al., 2001a, b). The G2 arrest was remarkably stable regulatory mechanisms, that is, at the level of transcrip- lasting for at least 4 weeks (Supplementary Figure S1A). tion and mRNA stability. Consistent with this notion,

Oncogene p53/p21-dependent activation of APC/C–Cdh1 in G2 L Wiebusch and C Hagemeier 3479

untreated + doxorubicin

hours hours 36 36 30 30 24 24 18 18 12 12 2n 6 2n 6 4n 2 4n 2 0 0

untreated + doxorubicin 0 2 6 1218243036 2 6 12 18 24 30 36 hours untreated + doxorubicin p(S10)-H3 control 0 2 6 1218243036 2 6 12 18 24 30 36 hours γH2AX (metUbi)n- p(S317)-Chk1 Cyclin B1 Chk1

p(S517)Chk2 untreated + doxorubicin Chk2 50 μm

p(S15)-p53 Phase p53 contrast p21 Emi1

Cyclin A DAPI Cyclin B1

Securin UbcH10 Lamin A/C Plk1 Aurora A Aurora B 30 h 30 h Tpx2 Nek2A

Geminin Cdc6 Cdc20 Cdh1 APC3 UbcH5

β-Actin

Figure 1 Doxorubicin-induced long-term G2 arrest is accompanied by activation of the APC/C. Primary lung fibroblasts were first synchronized in G1 by density arrest and then re-plated at lower cell density to allow re-entry into the . Nineteen hours after re-plating when most cells have reached mid-to-late S-phase (0 h), they were treated for 2 h with 1 mM doxorubicin or left untreated. Between 0 and 36 h after doxorubicin treatment, the cells were harvested at regular intervals and analyzed for cell-cycle position (a, b, d), DNA damage checkpoint activation (b), APC/C activity (c) and expression of APC/C substrates and regulators (b). (a) Cellular DNA content was analyzed by flow cytometry of propidium iodide-stained cells. Shown are DNA histograms where the relative cell number is plotted against DNA content (n ¼ haploid number of ). (b) Whole-cell lysates were analyzed for the expression and/or the phosphorylation state of the indicated proteins by immunoblot analysis. (c) The APC/C was immunoprecipitated from cell lysates and tested for ubiquitin ligase activity using methylated ubiquitin (met-Ubi) and radioactively labeled cyclin-B1 as substrates. (d) Cell nuclei were analyzed by phase-contrast and fluorescence microscopy using 40,6-diamidino-2-phenylindole (DAPI) stain and Alexa-488-labeled lamin-A/C-specific antibodies. APC/C, anaphase-promoting complex or cyclosome.

Oncogene p53/p21-dependent activation of APC/C–Cdh1 in G2 L Wiebusch and C Hagemeier 3480

untreated + doxorubicin untreated + doxorubicin probe tRNA 0 2 6 12182430362 6 1218243036 hours probe tRNA 0 2 6 12182430362 6 1218243036 hours

Cyclin A Cyclin B1 Cyclin C Geminin L32

Tpx2 L32 GAPDH

L32 Securin Cdc20 UbcH10 Plk1 Aurora A untreated + doxorubicin

Aurora B probe tRNA 0 2 6 12 18 24 30 36 2 6 12 18 24 30 36 hours

Emi1 L32 * Plk1

L32

Figure 2 Emi1 and APC/C substrates are repressed at the mRNA level after DNA damage. RNA preparations from the experiment described in Figure 1 were analyzed by ribonuclease protection assay for mRNA expression of the indicated APC/C substrates (a, b) and of Emi1 (c). L32 and GAPDH were analyzed to control for equal loading. A radiolabeled probe was loaded to control for complete digestion of single-stranded RNA. In addition, a tRNA control served to detect nonspecific hybridization products as the one indicated by asterisk in panel c. APC/C, anaphase-promoting complex or cyclosome; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

we found that after doxorubicin treatment APC/C importance to the efficient and particularly the prompt targets were also downregulated at the mRNA level decline of cell-cycle-related gene products in doxorubi- (Figure 2). To analyze the extent to which the observed cin-treated S/ G2 cells. downregulation of APC/C substrates could be attrib- uted to an APC/C-dependent decrease in protein stability, we examined the DNA damage response in DNA damage efficiently downregulates the APC/C fibroblasts with stable and efficient Cdh1 knockdown inhibitor Emi1 (Figure 3c). In these cells, doxorubicin-dependent APC/ The APC/C is normally kept inactive during S and G2 C activation was completely prevented (Figure 3b), by binding to its pseudosubstrate inhibitor Emi1. In confirming our assumption that the APC/C becomes doxorubicin-treated S/ G2 cells Emi1 protein expression prematurely activated in its Cdh1-bound form. As was found to be drastically reduced with kinetics shown for cyclin-A and B, knockdown of Cdh1 did inversely correlated to p21 expression (Figure 1b). This not significantly influence the steady-state mRNA levels decline was detectable before an effect at the Emi1 of the APC/C targets (Figure 3d) that finally become mRNA levels (Figure 2c), suggesting that Emi1 is also downregulated after DNA damage irrespectively of targeted for protein degradation by a DNA damage- available APC/C activity. By contrast, lack of APC/C– induced mechanism. Indeed, Emi1 protein expression in Cdh1 activation led to a significant delay (6–12 h) in the doxorubicin-treated cells was stabilized by proteasome DNA damage-dependent decrease of cyclin-A and B inhibition (Supplementary Figure S4B). Surprisingly, protein levels (Figure 3c). Thus, in line with the in vitro- this rescue of Emi1 expression did not prevent APC/C measured increase in APC/C enzymatic activity, degra- activation (Supplementary Figures S4D and S5A) dation of APC/C targets in vivo is of apparent although Emi1 became efficiently bound to the APC/C

Oncogene p53/p21-dependent activation of APC/C–Cdh1 in G2 L Wiebusch and C Hagemeier 3481 Premature APC/C activation after g-irradiation mock Cdh1-KD GFP-KD To analyze whether premature APC/C activation is specific to chemical induction of DNA damage, we next h post h post h post used g-ray doses between 2 and 100 Gy on S-phase cells doxo doxo doxo 30 30 30 and analyzed their short- and long-term response in 24 24 24 comparison with doxorubicin treatment. Both agents 18 18 18 had very similar effects. Again, the initial response was 12 12 12 6 6 6 characterized by a reduction in DNA replication 2n 4n 0 2n 4n 0 2n 4n 0 (Figure 4a), ongoing DNA repair and activation of checkpoint kinases as indicated by histone-H2A-X and Chk1 phosphorylation, respectively (Figure 4c). These mock Cdh1-KD GFP-KD effects had ceased by 24 h when a G2 arrest with high

control 0 6 12 18 24 30 0 6 12 18 24 30 0 6 12 18 24 30 h post doxo p21 expression (Figures 4a and c) and no visible signs of apoptosis (data not shown) was established. Impor-

(met-Ubi)n- tantly, at these time points the APC/C became activated Cyclin B1 also in g-irradiated cells (Figure 4b). This activation was Cyclin B1 dose-dependent and accompanied by a decrease in Emi1 and APC/C–Cdh1 substrates as seen in doxorubicin- treated cells. Interestingly, activation of the APC/C and mock Cdh1-KD GFP-KD downregulation of Emi1 and APC/C substrates corre- 0 6 12 18 24 30 0 6 12 18 24 30 0 6 12 18 24 30 h post doxo lated even stronger with p21 rather than p53 expression, suggesting a possible link between p21 induction and p53 APC/C regulation. Thus, activation of the APC/C in p21 G2-arrested cells is a general long-term consequence of applied genotoxic stress and correlates well with p21 Cdh1 induction.

Emi1 Premature activation of the APC/C is p53-dependent Cyclin B1 Given the strong correlation between the DNA damage- induced premature APC/C activation and p21 expres- Cyclin A sion we reasoned that APC/C activation might mechan- istically occur in a p53-dependent manner. Short-hairpin GAPDH RNA-mediated silencing of p53 in primary fibroblasts abolished both p53 expression and, as would be mock Cdh1-KD GFP-KD

obe expected, induction of p21 after doxorubicin treatment pr tRNA 0 6 12 18 24 30 0 6 12 18 24 30 0 6 12 18 24 30 h post doxo (Figure 5c). In agreement with the literature (Bunz et al., Cyclin A 1998), p53-deficient cells could still respond to DNA Cyclin B1 damage with G2 arrest (Figure 5a), possibly by keeping Cyclin C Cdk1 inactive through -dependent phosphoryla- Cyclin D1 Cyclin D2 tion (Figure 5c). However, cells are impaired to Cyclin D3 maintain this arrest and underwent mitotic catastrophe after 2–3 days (Bunz et al., 1998; data not shown). p53-knockdown cells failed to activate the APC/C L32 after doxorubicin treatment (Figure 5b) showing that GAPDH APC/C induction after genotoxic stress depends indeed on p53 but is dispensable for installing the observed G2 Figure 3 The APC/C–Cdh1 is required for timely destruction of arrest. Consistent with this observation, APC/C sub- APC/C substrates in DNA damage-induced G2 arrest. Primary strates, including cyclin-B1 and Aurora-A proteins, were fibroblasts were stably transduced with lentiviral expression vectors not downregulated as in control cells but rather for Cdh1- or GFP-specific short-hairpin RNAs. The resulting accumulated to high levels (Figure 5c). Interestingly, knockdown cells (Cdh1-KD, GFP-KD) and mock-infected control cells were treated with doxorubicin (doxo) as detailed in the legend Emi1 downregulation was also prevented by the lack of to Figure 1. After doxorubicin treatment, the cells were harvested p53, suggesting that both Emi1 protein and APC/C at 6-h intervals and analyzed for cell-cycle distribution (a), APC/C activity are targeted in a p53-dependent manner. activity (b), protein (c) and mRNA expression (d) as described in the legends to Figures 1 and 2 respectively. APC/C, anaphase- promoting complex or cyclosome; GFP, green fluorescent protein. p21 but not Cdc14B is required for premature APC/C activation in HCT116 cells (Supplementary Figure S5B). Thus, Emi1 is down- To further mechanistically investigate the link between regulated after DNA damage and in addition mechan- p53 and the APC/C, we used the human colon isms independent of Emi1 availability contribute to carcinoma cell line HCT116. As HCT116 cells are activate the APC/C after genotoxic stress. genetically stable and contain wild-type (wt) p53, they

Oncogene p53/p21-dependent activation of APC/C–Cdh1 in G2 L Wiebusch and C Hagemeier 3482 have been used to generate somatic knockouts of p53 p53-knockout (p53À/À) cells were arrested in G2 in and of genes functionally interacting with p53. Like response to doxorubicin treatment (Figure 6a) despite p53-knockdown fibroblasts both HCT116-wt and absence of p53 and p21 expression (Figure 6c). In wt cells establishment of the G2 arrest was also followed by induction of APC/C activity (Figure 6b), with the same late time kinetics as in primary fibroblasts. Premature APC/C activation was of a similar strength as the regular APC/C activation that occurred when untreated control cells divided and re-entered G1. By contrast, p53À/À cells were unable to activate the APC/C after DNA damage, although in untreated cells, G1-depen- dent APC/C activation was still preserved. After DNA damage Emi1 and APC/C substrates were repressed only in p53-positive cells (Figure 6c), whereas Emi1, + do Cdc20 and Aurora-A were reduced in G cells regardless xo 1 γ-IR of the p53 status. Thus, the main characteristics of DNA 2n 24 h untreat damage-dependent APC/C activation in G2 are con- + doxo 2 – 100 Gy 4n ed served between primary lung fibroblasts and HCT116 γ 4 h -IR cells, qualifying this transformed cell line as a suitable Gy experimental system for further analysis. 0 h untrea 2 – 100 To directly investigate the question which p53- ted dependent pathway leads to APC/C activation in G2, we took advantage of HCT116 lines with isogenic knockouts of genes acting downstream (p21, 14-3-3s) 100 100 from p53 in the DNA damage checkpoint. All cell lines 20 50 20 50 5 10 5 10 were able to respond to doxorubicin treatment with a 2 γ-IR (Gy) 2 γ-IR (Gy) + doxo untreated + doxo untreated G2 arrest albeit with varying efficiency (Figure 7a). Only

control 0444444442424242424242424 hours wt and 14-3-3sÀ/À cells showed induction of APC/C activity at 24 h after DNA damage (Figure 7b), implying (metUbi)n- that p21 is a crucial mediator of p53-dependent APC/C CyclinB1 activation in G2. This is consistent with the above CyclinB1 results showing close correlation between p21 induction and APC/C activity. Consequently, both p21 and p53 knockout had a strong negative effect on APC/C 100 100 20 50 20 50 substrate degradation (Figure 7c). Similarly, doxorubi- 5 10 5 10 2 γ-IR (Gy) 2 γ-IR (Gy) cin-induced downregulation of Emi1 was completely untreated + doxo untreated + doxo abolished in both p53- and p21-negative cells. This 0444444442424242424242424 hours further supports the view that Emi1 downregulation γH2A-X has a role in the p53-dependent induction of APC/C activity. p(S317)-Chk1 Recently, a Cdc14B-knockout HCT116 derivative has been presented (Berdougo et al., 2008). The same factor p53 was previously shown to be required for premature APC/C activation in doxorubicin-treated U2OS cells, by using an short interfering RNA-based knockdown p21-WAF1 approach (Bassermann et al., 2008). Surprisingly, Cdc14BÀ/À cells were still capable of activating the APC/C after doxorubicin treatment to the same extent Cdh1 as wt cells (Figure 7e). To rule out that the knockout clone we used had acquired compensatory mutations in

Emi1

Figure 4 g-Irradiation leads to premature APC/C activation in Cdc20 G2. Primary lung fibroblasts were synchronized in S-phase as described in Figure 1 and treated with either doxorubicin or g-irradiation at the indicated doses. Cells were harvested at early Cyclin A (4 h) and late (24 h) time points after DNA damage and analyzed for cell-cycle distribution (a), APC/C activity (b) and the β-Actin expression/phosphorylation state of the indicated proteins (c)as described in Figure 1. APC/C, anaphase-promoting complex or cyclosome.

Oncogene p53/p21-dependent activation of APC/C–Cdh1 in G2 L Wiebusch and C Hagemeier 3483 the APC/C activatory pathway, we further performed a level (Figure 7f), and this approach reproduced the knockdown experiment using the same short interfering results we achieved using the Cdc14BÀ/À cell line RNA that was used by Bassermann et al. and achieved (Figure 7g). In summary, this set of data shows that reproducibly 80% silencing of Cdc14B at the mRNA APC/C activation after genotoxic stress strictly depends on p21 and that Cdc14B is not a limiting factor for premature APC/C activation in HCT116 cells. mock + doxo

hours Depleting Emi1 from p53À/À cells is sufficient 72 to constitutively activate the APC/C in S/ G2 48 24 The inability of p53- and p21-deficient cells to activate 12 2n the APC/C in response to genotoxic stress goes hand in 4n 6 0 hand with impaired downregulation of Emi1 (Figures 5– p53-KD + doxo GFP-KD + doxo 7). This led us to examine whether Emi depletion would restore the induction of APC/C activity in p53-knockout

hours hours cells. Transfection of an Emi1-specific but not of a 72 72 nonspecific (NS) short interfering RNA led to efficient 48 48 24 24 silencing of Emi1 expression in HCT116-p53À/À cells 12 12 2n 2n (Figure 8c). Consistent with published data (Di Fiore 4n 6 4n 6 0 0 and Pines, 2007; Machida and Dutta, 2007) the Emi1 knockdown predisposes cells to over-replication of their + doxo + doxo + doxo cellular genome (Figure 8a). The remainder of cells with mock p53-KD GFP-KD a normal DNA content were still able to arrest in G2

control 0 6 12 24 48 0 6 12 24 48 0 6 12 24 48 hours after doxorubicin treatment (Figure 8a). Importantly, after Emi1 knockdown, G2-arrested, p53À/À cells (met-Ubi)n- showed the same level of APC/C activity as wt cells Cyclin B1 (Figure 8b). However, this was no de novo activation, as Cyclin B1 compared with S-phase cells at the beginning of doxorubicin treatment (0 h), APC/C activity was not

+ doxo + doxo + doxo further elevated. This shows that depletion of Emi1 in p53À/À cells resulted in constitutive APC/C activation mock p53-KD GFP-KD that was independent of the cell-cycle position and 0 6 12 24 48 72 0 6 12 24 48 72 0 6 12 24 48 72 persisted after DNA damage. Consistent with these data, the protein levels of APC/C substrates such as p53 cyclin-B1 and Cdc20 were strongly reduced both in cycling and G2-arrested, Emi1-knockdown cells p21 (Figure 8c). DNA over-replication, APC/C activation and destabilization of APC/C substrates were prevented by additional Cdh1 knockdown. These data emphasize Emi1 the importance of APC/C–Cdh1 activity for the destruction of pro-mitotic factors during DNA da- Cyclin B1 mage-induced G2 arrest.

Cyclin A Discussion Geminin In this report we have studied the APC/C under Aurora A conditions of an activated DNA damage checkpoint in the G2 phase of the cell cycle. We show that the APC/C Cdk1

pCdk1(Tyr15) Figure 5 Premature induction of APC/C activity after DNA Wee1 damage depends on the p53 tumor suppressor. Primary fibroblasts with a stable knockdown of p53 (p53-KD) and the indicated UbcH10 control fibroblasts (mock: mock-infected; GFP-KD: knockdown of green fluorescent protein) were synchronized and treated with doxorubicin (doxo) as described in Figure 1. Cells were UbcH5 harvested at 6, 12, 24, 48 and 72 h after doxorubicin treatment and analyzed for cell-cycle distribution (a), APC/C activity (b) and protein expression (c) as described in Figure 1. APC/C, GAPDH anaphase-promoting complex or cyclosome; p53, tumor-suppressor protein-53.

Oncogene p53/p21-dependent activation of APC/C–Cdh1 in G2 L Wiebusch and C Hagemeier 3484 HCT116-wt HCT116-p53-/-

untreated + doxorubicin untreated + doxorubicin

hours hours hours hours 36 36 36 36 30 30 30 30 24 24 24 24 18 18 18 18 12 12 12 12 6 6 6 6 2n 2n 2n 2n 4n 2 4n 2 4n 2 4n 2 0 0 0 0

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control 0 2 6 12 18 24 30 36 2 6 12 18 24 30 36 0 2 6 12 18 24 30 36 2 6 12 18 24 30 36 hours

(met-Ubi)n- Cyclin B1

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HCT116-wt HCT116-p53-/-

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p53

p21

* Emi1

Cdc20

Aurora A

GAPDH

Figure 6 p53-dependent APC/C activation in a transformed epithelial cell line. HCT116-wt cells and the isogenic p53-knockout strain (HCT116-p53À/À) were first synchronized in G1 by density arrest and then re-plated at lower cell density to allow reentry into the cell cycle. Fifteen hours after replating when most cells have reached mid-S-phase (0 h), they were treated with doxorubicin or left untreated. Between 0 and 36 h after doxorubicin treatment, cells were harvested at regular intervals and analyzed for cell-cycle distribution (a), APC/C activity (b) and protein expression (c) as described in Figure 1. A nonspecific protein band is indicated by asterisk. APC/C, anaphase-promoting complex or cyclosome; p53, tumor-suppressor protein-53; wt, wild type.

ubiquitin ligase activity can be prematurely activated in reported for X-irradiated HeLa cells (Sudo et al., 2001) G2 as part of a p53/p21-dependent long-term response and doxorubicin-treated U2OS cells (Bassermann et al., to DNA damage. Both, in primary fibroblasts and in 2008). Possibly, the chemical inhibitors of DNA HCT116 cells, this activation translates into a broad replication that were used in both studies of cell-cycle downregulation of APC/C substrates, thereby providing synchronization before induction of DNA damage an additional layer of p53-dependent gene repression particularly prime cells for rapid APC/C activation. In during the cellular response to genotoxic stress. Our support of this notion, exposure to such compounds has data are in good agreement with two recent studies that been reported to elicit a DNA damage-like stress also used HCT116 cells to show p21-dependent desta- response (Nayak and Das, 2002; Kurose et al., 2006). bilization of cyclin-B1 (Gillis et al., 2009; Lee et al., Alternatively, the different APC/C substrates used in the 2009), cyclin-A, Cdc20, Securin and Emi1 (Lee et al., in vitro ubiquitination assays, Cdc20 (Sudo et al., 2001), 2009) after induction of DNA damage. Plk1 (Bassermann et al., 2008) and cyclin-B1 (this We did not, however, find experimental support for study), might vary in their sensitivity to checkpoint- the rapid induction of APC/C activity that has been dependent changes in APC/C activity. With the exception

Oncogene p53/p21-dependent activation of APC/C–Cdh1 in G2 L Wiebusch and C Hagemeier 3485

wt p53-/- 150 hrs hrs 24 24 100 6 6

2n 4n 0 2n 4n 0 50 p21-/- 14-3-3 σ-/- % Cdc14B mRNA expression

hrs hrs 24 24 wt flox 6 6 p53-/-

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wt p53-/- Cdc14Bflox Cdc14B-/- wt p53-/- p21-/- 14-3-3σ-/-

control 0 6 24 0 6 24 0 6 24 0 6 24 h post doxo

control 0624062406240624h post doxo

(met-Ubi)n- Cyclin B1 (met-Ubi)n- Cyclin B1 Cyclin B1

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σ wt p53-/- p21-/- 14-3-3 -/- 120 0 6 24 0 6 24 0 6 24 0624h post doxo 100

Emi1 80

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p21 % Cdc14B mRNA expression

A A

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+ NS-siRN untransfected

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(met-Ubi)n- Cyclin B1 Cyclin B1

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untransfected + NS-siRNA + Cdc14B siRNA

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p21

GAPDH

Figure 7 p21 but not Cdc14B is necessary for long-term APC/C activation after DNA damage. HCT116-wt cells and the indicated knockout/knockdown derivatives were synchronized and treated with doxorubicin as described in Figure 6. At 0, 6 and 24 h after doxorubicin treatment cells were harvested and analyzed for cell-cycle distribution (a), APC/C activity (b, e, g) and protein expression (c, h) as described in Figure 1. Cdc14B knockout and Cdc14B knockdown were confirmed by quantitative real-time PCR analysis of Cdc14B mRNA expression (d, f). APC/C, anaphase-promoting complex or cyclosome; wt, wild type. of Plk1, both studies surprisingly failed to observe that, in contrast to primary fibroblasts and HCT116 a significant decrease of APC/C substrates in vivo cells, in G2-arrested U2OS cells most APC/C substrates (Bassermann et al., 2008). Interestingly, we also found resist APC/C-dependent degradation even at late time

Oncogene p53/p21-dependent activation of APC/C–Cdh1 in G2 L Wiebusch and C Hagemeier 3486 0 hours points after DNA damage (Supplementary Figure S6). Therefore, it appears that in U2OS and HeLa cells, cell- pol yploidy: type-specific mechanisms might be in place that restrict 6% APC/C activity. Possibly, deubiquitinases like USP28 siRNAs: 22% (Zhang et al., 2006; Bassermann et al., 2008), lack of emi1+ APC/C-specific E2 enzymes, weakened APC/C-protea- 3% some interaction (Kob et al., 2009), a defect in the 3% emi1 downregulation of APC/C substrate mRNAs and/or the 4% NS moderate strength of premature APC/C induction (Supplementary Figure S6) could be responsible for 2n 5% - p53-/- 4n the low effectiveness of APC/C activity in these cells. NS 8n Remarkably, we found that APC/C activation in G2 - is associated with the induction of a senescence-like wt phenotype. Consistent with such a significant phenoty- pic change it is not surprising that the APC/C is 24 hours activated in its Cdh1 and not the Cdc20-bound form. First, the APC/C–Cdh1 has a much broader substrate polyploi specificity, including many crucial activators of cell dy: 13% division and DNA replication that are not targeted by siRNAs: 33% APC/C–Cdc20 (Peters, 2006; Wa¨sch et al., 2010). emi1+ Second, because p21 is a substrate of the APC/C– 8% cdh1 Cdc20 in mitosis (Amador et al., 2007) and of SCFSkp2 9% emi1 during interphase (Yu et al., 1998; Bornstein et al., 2003; 11% NS Wang et al., 2005), APC/C–Cdh1-dependent degrada- tion of Cdc20 (Pfleger and Kirschner, 2000) and Skp2 13% p53-/- 2n - (Bashir et al., 2004; Wei et al., 2004; Liu et al., 2007) 4n NS might reinforce the p53-dependent induction of p21 8n - expression after DNA damage. Given that p21 on the wt one hand leads to activation of APC/C–Cdh1 (this wt p53-/- study) and on the other hand inhibits CDK-dependent phosphorylation, which otherwise would protect Skp2 emi1 + : siRNAs from APC/C–Cdh1 targeting (Rodier et al., 2008), the - NS - NS emi1 cdh1 existence of a positive feedback loop between p21 control expression and APC/C–Cdh1 activation appears to be - 0 24024 24 0240240240 hours an attractive option and might be complemented and further enforced by p53/p21-dependent repression of (met-Ubi)n- Cyclin B1 Cdc20 transcription (Kidokoro et al., 2008). An additional regulatory circuitry might be formed Cyclin B1 between p21, the APC/C and Cdc6. Like Skp2, Cdc6 is withdrawn from APC/C–Cdh1-dependent turnover in w t p53-/- G1 by CDK2 phosphorylation (Mailand and Diffley, emi1 2005). p53/p21-mediated inhibition of CDK activity + : siRNAs after DNA damage abrogates this protection, thereby - NS - NS emi1 cdh1 preventing Cdc6-dependent replication licensing (Duursma and Agami, 2005). The circuitry is completed 024024024024024024hours by the finding that forced expression of Cdc6 can p53 interfere with p21-dependent inhibition of CDK2 Emi1 activity (Kan et al., 2008). Our finding of p21-dependent activation of APC/C–Cdh1 in G2 suggests that the same Cdh1

Cdc20

Cyclin B1 Figure 8 Emi1 depletion is sufficient to restore APC/C activation in p53-deficient cells. HCT116-wt and p53À/À cells were first Cdc6 transfected with the indicated short interfering RNAs and then, 3 days later, treated with doxorubicin as described in Figure 5. At 0 GAPDH and 24 h after doxorubicin treatment cells were harvested and analyzed for cell-cycle distribution (a), APC/C activity (b) and protein expression as described in Figure 1 (c). The percentages of polyploid cells (DNA content 44n) are indicated (a). APC/C, anaphase-promoting complex or cyclosome; p53, tumor-suppressor protein-53; wt, wild type.

Oncogene p53/p21-dependent activation of APC/C–Cdh1 in G2 L Wiebusch and C Hagemeier 3487 mechanisms controlling Cdc6 stability in G1 can be downregulation. Thus, it seems that p21 interferes with activated in G2-arrested cells and protect them from Emi1 expression at two levels: increased degradation over-replication. In fact, p21 is an essential factor for the removes the pre-existing Emi1 protein and transcrip- maintenance of normal DNA content in G2-arrested tional silencing inhibits its de novo synthesis. Currently, cells (Waldman et al., 1996). Tight control of Cdc6 we can only speculate as to how Emi1 is destabilized expression in DNA-damaged cells might be particularly after DNA damage. During mitotic entry of the important in the absence of Emi1 expression that is undisturbed cell cycle, Emi1 is marked for SCFbTrCP- known to trigger genome over-replication (Di Fiore and dependent degradation by Plk1-dependent phosphory- Pines, 2007; Machida and Dutta, 2007). lation (Hansen et al., 2004; Moshe et al., 2004). Whereas What links p21 to APC/C activation at the molecular Plk1 is inhibited by the DNA damage checkpoint level? So far Emi1 is the prime candidate as its (Smits et al., 2000) and can therefore not account downregulation during doxorubicin-induced G2 arrest for Emi1 destabilization, its homologues Plk2 and Plk3 does not only correlate with p21 induction but proved to are activated after genotoxic stress (Xie et al., 2001; be p53- and p21-dependent. In addition, we have shown Bahassi el et al., 2002; Matthew et al., 2007) and that Emi1 silencing led to constitutive APC/C activation can phosphorylate Emi1 in vitro (L Wiebusch and in p53-deficient cells. However, several lines of evidence C Hagemeier, unpublished observation). It is thus also suggest that Emi1 is not the only factor that can conceivable that after DNA damage Plk2 or Plk3 mediate p21-dependent APC/C activation. First, pre- substitute for Plk1 and trigger bTrCP-dependent degra- vention of Emi1 downregulation and APC/C–Emi1 dation of Emi1. Alternatively, one has to consider that dissociation by proteasome inhibition had no influence CDK activity in S/ G2 has an as yet unrecognized on APC/C activation (Supplementary Figures S4 and positive influence on Emi1 protein stability that is S5). This is reminiscent of Emi1 degradation not being compromised by the CDK-inhibitory function of p21. required for APC/C activation in mitosis (Di Fiore and We are currently exploring the possible mechanisms of Pines, 2007). Second, in U2OS cells APC/C activation p21-dependent Emi1 degradation to close the gap in by DNA damage occurs despite inefficient downregula- understanding how such important cell-cycle regulators tion of Emi1. Thus, in addition to Emi1 downregulation as p21 and the APC/C are interconnected. a functionally redundant mechanism must exist down- stream from p21 to safeguard APC/C activation after DNA damage. Although Cdc14B is not required for Materials and methods APC/C activation in HCT116 cells (Figure 7), it remains an excellent candidate to work in parallel with Emi1. In See Supplementary information for complete methods. that case Cdc14B regulation would not be required for APC/C activation when Emi1 downregulation is in place. Such a model (summarized in Supplementary Conflict of interest Figure S7) is supported by the finding that Cdc14B is a necessary factor for premature APC/C activation in The authors declare no conflict of interest. U2OS cells where Emi1 downregulation is not efficient (Bassermann et al., 2008). It appears that p21-induced Emi1 reduction can be rationalized by at least two distinct mechanisms. Lee Acknowledgements et al. (2009) provided evidence that the CDK-inhibitory function of p21 contributes to Emi1 repression through We thank Eli Berdougo, Philipp Haemmati, Christoph a -dependent inhibition of Emi1 Hanski, Hans-Ju¨rgen Heidebrecht, Prasad Jallepalli, Bert Vogelstein and Ralph Wa¨sch for the generous supply of transcription. Consistent with this notion, we also reagents. We acknowledge Matthias Truss for help with observed downregulation of Emi1 mRNA levels in our lentivirus production and Ralf Uecker for excellent technical system, but in addition found that disappearance of assistance. This work was supported by Grants HA1575/2-1 Emi1 protein precedes the decline of Emi1 mRNA. and WI2043/2-2 from the Deutsche Forschungsgemeinschaft Also, proteasomal inhibition was able to prevent Emi1 (DFG).

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

Oncogene