Restoration of the Tumor Suppressor P53 by Downregulating Cyclin B1 in Human Papillomavirus 16/18-Infected Cancer Cells

Oncogene (2010) 29, 5591–5603 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Restoration of the tumor suppressor p53 by downregulating cyclin B1 in human papillomavirus 16/18-infected cancer cells

NN Kreis1,4, M Sanhaji1,4, A Kra¨mer1, K Sommer2,FRo¨del3, K Strebhardt1 and J Yuan1

1Department of Gynecology and Obstetrics, School of Medicine, JW Goethe University, Frankfurt, Germany; 2Department of Traumatology and Reconstructive Surgery, School of Medicine, JW Goethe University, Frankfurt, Germany and 3Department of Radiotherapy and Oncology, School of Medicine, JW Goethe University, Frankfurt, Germany

Abrogation of functional p53 is responsible for malignant therefore strongly associated with an increased suscep- cell transformation and the maintenance of malignant tibility to cancer in humans (Vogelstein et al., 2000). state of human papillomavirus-infected cancer cells. Thus, In response to oncogene activation, DNA damage and restoration of p53 has been regarded as an important other stress signals, p53 functions as the guardian of the strategy for molecular intervention combating papilloma- genome by inducing cell-cycle arrest, apoptosis, differ- virus-associated malignancies. We show here that depleting entiation and senescence, dependent on many intrinsic cyclin B1 stabilizes and reactivates p53 in papillomavirus- and extrinsic factors in cells (Vousden and Lu, 2002; infected cervical cancer cell lines HeLa and CaSki. HeLa Aylon and Oren, 2007; Brown et al., 2009). Inactivation cells depleted of cyclin B1 exhibit mitotic defects in spindle of p53 in tumors occurs through two general mechani- formation and chromosome alignment. Downregulation of sms, either by point mutations in p53 itself or by partial cyclin B1 increases p14 alternative reading frame of p16, abrogation of signaling pathways or effectors regulating the positive regulator of p53, and decreases phosphorylation p53 activity (Brown et al., 2009), such as the viral of Ser315 in p53. Whereas RO-3306, a selective inhibitor of protein E6 of human papillomavirus type 16/18 (HPV cyclin-dependent kinase 1 (Cdk1), suppresses this phosphory- 16/18 E6) in cervical cancer. The HPV E6 protein lation at Ser315 of p53, ZM447439, targeting Aurora A/B complexes with cellular proteins E6-AP and p53 and kinases, shows no effect. Further analyses in HeLa cells facilitates p53 degradation by the ubiquitin-dependent and HCT116 p53(À/À) cells suggest that the Ser315 proteolytic system (Scheffner et al., 1990, 1993). The phosphorylation by Cdk1 regulates negatively the protein function of p53 is abrogated in HPV-positive cancer stability and the function of p53. Moreover, increased cells, which is responsible for malignant cell transforma- p53 in HeLa cells is functional by showing its increased tion and the maintenance of the malignant state of downstream effectors p21, mouse double minute 2 and cancer cells (Butz et al., 1995). Thus, restoration of p53 Bax. Restoration of p53 and silencing cyclin B1 render in virus-infected cells can be an important strategy for cervical carcinoma cells more susceptible to DNA damage preventing and treating cancers associated with HPV 16/18 agent camptothecin. Taken together, targeting cyclin B1 infection. might be an attractive strategy for preventing and treating Small molecules specifically targeting the activity papillomavirus-associated cancer by reactivating p53 and of Cdk have been becoming a promising intervention by reducing the Cdk1 activity. for potential antitumor therapy and have been Oncogene (2010) 29, 5591–5603; doi:10.1038/onc.2010.290; intensively studied in clinical trials (Dickson and published online 26 July 2010 Schwartz, 2009). In line with these observations, we Keywords: targeting cyclin B1; p53 stabilization; HPV found that downregulation of cyclin B1, the regulatory 16/18; p14ARF and Ser315 phosphorylation of p53 subunit of cyclin-dependent kinase 1 (Cdk1), specifically suppresses the activity of Cdk1, leading further to a strong proliferation inhibition and apoptosis induction in various cancer cells (Yuan et al., 2004, 2006; Androic Introduction et al., 2008). Intriguingly, it has been revealed that Cdk inhibitors efficiently induce p53 either by stabilizing p53 The p53 tumor suppressor protein functions as a major by modulating its sensitivity to its negative regulator defense against cancer and loss or mutation of p53 is mouse double minute 2 (MDM2, but also used to denote human homolog) (Lu et al., 2001) or through the Correspondence: Dr J Yuan, Department of Gynecology and transcriptional inhibition of MDM2 (Demidenko and Obstetrics, School of Medicine, JW Goethe University, Theodor- Blagosklonny, 2004). We were wondering whether the Stern-Kai 7, 60590 Frankfurt, Germany. protein stability and function of the tumor suppressor E-mail: [email protected] p53 is effected by depleting cyclin B1, consequently 4These authors contributed equally to this work. Received 23 December 2009; revised 11 February 2010; accepted 13 June suppressing the activity of Cdk1, in HPV 16/18-infected 2010; published online 26 July 2010 cervical carcinoma cells. Stabilization of p53 by targeting cyclin B1 NN Kreis et al 5592 Results in HeLa GFP and HeLa 776-6 cells were comparable (Figure 1b). Tumor suppressor p53 is increased in HeLa and CaSki To exclude the possibility that upregulation of p53 is cells with reduced cyclin B1 only to be found in HeLa 776-6 clone because of the In previous work, we reported the generation of cervical artificial integration of shRNA cassettes in HeLa carcinoma HeLa clones stably expressing small hairpin genome, we analyzed other HeLa 776 clones, in which RNA (shRNA) against cyclin B1 (Yuan et al., 2006). HeLa cells were infected with the HPV 18 encoding the oncogenic E6 protein, which inactivates the tumor suppressor p53 by directly binding and promoting its degradation (Scheffner et al., 1990). To study the relationship between downregulation of cyclin B1 and p53, we selected one of the clones, HeLa 776-6, which holds the shRNA expression cassette targeting cyclin B1 (Supplementary Figure 1A), has 18% of cyclin B1 mRNA (Supplementary Figure 1B) and exhibits a strong reduction of cyclin B1 protein level (Supple- mentary Figure 1C, upper row) with a decreased activity of Cdk1 over control HeLa cells (Yuan et al., 2006). In addition, HeLa 776-6 cells in prometaphase showed a strongly reduced phospho-histone H3 signal, compared with control cells (middle row, Supple- mentary Figure 1C and 1D). To address whether p53 is affected in HPV-infected HeLa cells with reduced cyclin B1, HeLa 776-6 cells were synchronized to either the G1/S boundary by double thymidine block or to prometaphase by thymidine/nocodazole treatment for western blot analysis. Compared with control HeLa or HeLa green fluorescent protein (GFP) cells, the protein level of p53 was indeed enhanced in HeLa 776-6 cells, and in particular, in prometaphase cells (Figure 1a, upper panel). Further quantification revealed that p53 protein level in prometaphase cells increased eightfold over that in control cells (Figure 1a, lower panel). Yet, the p53 mRNA levels

Figure 1 p53 is increased in HeLa 776-6 cells. (a) Upper panel: HeLa, HeLa GFP and HeLa 776-6 cells were nonsynchronized (C), synchronized to the G1/S boundary with a double thymidine block (T) or to prometaphase with thymidine/nocodazole treatment (N) for western blot analyses with antibodies against p53. Lower panel: quantiﬁcation of p53 protein levels in mitotic HeLa, HeLa GFP and HeLa 776-6 cells, relative to its loading control b-actin. (b) The mRNA level of p53 in HeLa GFP (GFP) and HeLa 776-6 (776-6) cells. Quantitative PCR measurements were carried out as described in Materials and methods. The error bars represent standard deviations from the mean. (c) Cyclin B1-knockdown HeLa clones 776-6, 776-13 and 776-15 were treated as in (a), and cellular extracts were prepared for western blot analyses with corresponding antibodies. HeLa 776-1 cells, which contain a comparable level of cyclin B1 mRNA as HeLa cells, served as control. (d) Cervical cancer cell line CaSki, infected with HPV 16, was stably transfected with plasmids expressing shRNA cyclin B1 or control shRNA GFP. Extracts from control cells (C), the G1/S boundary (T) and prometaphase cells (N) were prepared for western blot analyses with indicated antibodies. (e) Lung cancer cell line A549 and breast cancer cell line MCF7, both contain wild- type p53, and metastatic breast cancer cell line MDA-MB-231, in which p53 is deﬁcient, were treated with control small interfering RNA (sicon) or siRNA against cyclin B1 (sicycB1) and synchronized to prometaphase. Cellular extracts were prepared for western blot analyses with antibodies as indicated. (f) HCT116 p53( þ / þ ) cells were treated with siRNA control (sicon) or siRNA cyclin B1 (sicycB1). After 24 h, cellular extracts were prepared for western blot analyses with antibodies as indicated. b-actin served as loading control.

Oncogene Stabilization of p53 by targeting cyclin B1 NN Kreis et al 5593 cyclin B1 levels were also downregulated in different an important downstream molecule of p53, was also degrees. Similar to HeLa 776-6 cells, HeLa 776-13 and increased in interphase and mitotic HeLa 776-6 cells HeLa 776-15 clone cells, in which mRNA cyclin B1 was (Figure 2a, third row, lanes 7 and 11), compared with reduced to 37 and 22%, respectively, exhibited also an that in HeLa cells (Figure 2a, third row, lanes 1 and 5). increased level of p53 (Figure 1c, third row, lanes 9 and To study the kinetics of p53 during mitosis, pro- 12). By contrast, HeLa 776-1 cells, which contain almost metaphase cells were released into a fresh medium and so much cyclin B1 mRNA as HeLa cells (data not the cellular extracts were prepared at indicated time shown), exhibited little change in p53 protein amount points for western blot analysis. Whereas in HeLa cells, (Figure 1c, third row, lane 3). These data indicate that p53 was kept at low levels through mitosis (Figure 2b, p53 is increased in HeLa cells with reduced Cdk1 left panel, middle row, lanes 1–3) and increased as activity by depleting its regulatory subunit cyclin B1. cells entered G1 phase (Figure 2b, left panel, middle We were next interested in whether depletion of cyclin row, lanes 4–8), HeLa 776-6 cells exhibited a high level B1 also reactivates p53 in other HPV-infected cancer cell of p53 during mitosis (Figure 2b, right panel, middle lines rather than HeLa cells. CaSki cells, infected with row, lanes 9–11) and reached even higher levels as cells HPV 16 encoding the E6 protein therefore with were in G1 phase (Figure 2b, right panel, middle row, nonfunctional p53, were transfected with constructs lanes 12–16). expressing shRNA cyclin B1 or control shRNA GFP As shown in Figure 1b, the p53 mRNA level in HeLa and treated with G418 for 6 weeks. Cells were then 776-6 cells was not increased, compared with control synchronized for western blot analysis. Interestingly, a HeLa cells. Thus, we were wondering whether the distinctive increase in p53 protein was observed in CaSki protein stability of p53 was effected in HeLa 776-6 cells. cells with reduced cyclin B1, but was not found in CaSki To address this issue, HeLa and HeLa 776-6 cells were cells transfected with control constructs expressing synchronized to prometaphase and released into a fresh shRNA GFP (Figure 1d, middle row, lanes 6 and 9). medium containing protein synthesis inhibitor cyclohexi- The data imply that downregulation of cyclin B1 mide, and the kinetics of p53 protein level were stabilizes the tumor suppressor p53 might be a general investigated by western blot analysis. An increase in phenomena in HPV-infected cancer cells. protein stability of p53 in HeLa 776-6 cells was Furthermore, lung cancer cell line A549 and breast articulately observed: the half-life time of p53 was cancer cell line MCF7, both contain functional wild- 25 min in mitotic HeLa and 45 min in mitotic HeLa 776-6 type p53, were depleted of cyclin B1 by transient cells, respectively (Figure 2c). The data imply that p53 transfection of small interfering RNA targeting cyclin is more stable in HeLa 776-6 cells with reduced B1. We observed an increase in p53 level in cells depleted Cdk1 activity, particularly in mitosis. In addition, p53 of cyclin B1 (Figure 1e, middle row, lanes 2 and 4), protein was strongly accumulated on the treatment of although the increase was not as strong as that in stable MG132, a proteosome inhibitor (Figure 2c, upper panel, clones. By contrast, metastatic breast cancer cell line middle row, lanes 7 and 14), suggesting that p53 is MDA-MB-231, in which p53 is deficient, showed no destabilized during mitosis by the proteosome-mediated alteration after depletion of cyclin B1 (Figure 1e, second degradation. row, lane 6). To further underline whether this increased p53 after The HPV 18 E6/E6-AP/p53 pathway is intact in depleting cyclin B1 was functional, HCT116 cells with HeLa 776-6 cells wild-type p53 were depleted of cyclin B1 by transient As p53 in HeLa cells is basically inactivated and transfection. After 24 h of transfection, cells were degraded by the HPV 18 E6/E6-AP pathway (Scheffner harvested and cellular extracts were prepared for et al., 1993), we were interested to know whether western blot analysis. Downregulation of cyclin B1 downregualtion of cyclin B1 impacted this pathway in increased the amount of p53, accompanied with HeLa 776-6 cells. HeLa, HeLa 776-6 and HeLa 776-13 enhanced levels of its downstream molecules MDM2, cells were synchronized and the cellular extracts were Bax and p21 (Figure 1f). The findings suggest that p53 is analyzed by western blot analysis. The expression levels increased in cells with reduced level of cyclin B1 and this of the oncoprotein E6 as well as the E3 ubiquitin ligase increased p53 is functional. E6-AP were scarcely changed, relative to HeLa cells (Supplementary Figure 2A). Moreover, the binding Increased p53 localizes mainly in the nucleus and is more affinity of p53 to the ligase E6-AP was not altered stabilized in HeLa 776-6 cells either (Supplementary Figure 2B), suggesting that the The increased amount of p53 was more predominant in HPV 18 E6/E6-AP/p53 pathway is not perturbed and mitotic cells than in interphase cells (Figure 1a). To other pathway(s) must be involved in the stabilization of explore subcellular localization of this increased p53, p53 in HeLa 776-6 cells with reduced Cdk1 activity. HeLa and HeLa 776-6 cells were synchronized and cytosolic and nuclear extracts were separated for western blot analysis. As depicted in Figure 2a, in- Defects in spindle formation and chromosome alignment creased p53 was mainly localized in the nucleus but in HeLa 776-6 cells also to be found in the cytoplasm in prometaphase We set then out to search for the explanations for the extracts of HeLa 776-6 cells (Figure 2a, second row, increased amount of p53 in HeLa 776-6 cells. It is well lanes 11 and 12). Moreover, the kinase inhibitor p21, known that p53 is stabilized in response to mitotic stresses,

Oncogene Stabilization of p53 by targeting cyclin B1 NN Kreis et al 5594

Figure 2 Increased mitotic p53 is predominantly localized in nucleus and more stable in HeLa 776-6 cells. (a) HeLa and HeLa 776-6 cells were synchronized as indicated and cytosolic (Cy) and nuclear extracts (Nu) were separated for western blot analyses with antibodies as indicated. Calnexin and lamin B1 were taken as cytoplasm and nuclear extract control, respectively. (b) HeLa and HeLa 776-6 cells were synchronized to prometaphase and released into a fresh medium. Cells were harvested at indicated time points and extracts were prepared for western blot analyses with antibodies against cyclin B1, p53 and b-actin (loading control). (c) Upper panel: mitotic HeLa and HeLa 776-6 cells were released into a fresh medium containing cycloheximide (CHX, 25 mg/ml) or MG132 (10 mM) and cellular extracts were prepared as indicated for western blot analyses using antibodies targeting cyclin B1, p53 and b-actin (loading control). Lower panel: p53 levels were quantiﬁed, relative to loading control b-actin.

such as treatment with taxol or vinca alkaloids that disrupt were examined by a microscopy in about 300 mitotic cells. mitosis (Brown et al.,2009).AsCdk1isessentialforthe HeLa 776-6 cells definitely exhibited failures in spindle mitosis initiation and many events in mitosis, we asked formation, such as aster-like and hair-similar spindles which mitotic defects could be resulted from depletion of (Figure 3a, second and third rows, tubulin staining), cyclin B1 in HeLa 776-6 cells. HeLa and HeLa 776-6 cells chromosome misalignment (Figure 3a, second to last rows, were synchronized by double thymidine block and released 40,6-diamidino-2-phenylindol dihydrochloride staining) intofreshmediumfor10h.Cellswerethenfixedand and multipolar spindle (Figure 3a, fourth and last rows, stained for tubulin, the centrosome marker pericentrin and pericentrin staining). Further quantifications showed that DNA. Spindle formation and chromosome morphology 30.4% of HeLa 776-6 cells, relative to 7.9% of HeLa cells,

Oncogene Stabilization of p53 by targeting cyclin B1 NN Kreis et al 5595 showed defects in the spindle formation (Figure 3b). very well deﬁned (Brown et al., 2009). p53 is mainly In addition, improper chromosome alignment was 70.2% controlled by MDM2, an E3 ubiquitin ligase targeting in HeLa 776-6 cells, compared with 20.4% in HeLa cells p53 for ubiquitin-dependent degradation and function- (Figure 3c). Moreover, 10.5% of HeLa 776-6 cells showed ing as a crucial negative regulator for p53 (Lavin and multipolar spindle and 6.3% monopolar spindle (Figures Gueven, 2006). On the other hand, the tumor suppressor 3d and e). The data suggest that depletion of cyclin B1, p14ARF directly binds to MDM2, thereby stabilizing the regulatory subunit of mitotic kinase Cdk1, triggers and activating p53 (Stott et al., 1998). We investigated dramatic mitotic failures and cells with reduced cyclin B1 whether p14ARF was effected in HeLa cells with face enormous stress during mitotic progression, leading reduced Cdk1 activity by downregulating cyclin B1. possibly to the stabilization of p53. HeLa, HeLa GFP and HeLa 776-6 cells were synchronized for western blot analysis with speciﬁc p14ARF Increased p14 alternative reading frame of p16 (p14ARF) antibodies. The protein level of p14ARF was obviously in HeLa 776-6 cells elevated in extracts from HeLa 776-6 cells (Figure 4a, However, the molecular mechanisms, by which p53 is third row, lanes 7–9). Interestingly, while p14ARF was stabilized and activated by mitotic stresses, are not undetectable in prometaphase extracts from control

Figure 3 Mitotic defects in HeLa 776-6 cells. HeLa and HeLa 776-6 cells were synchronized to mitosis and stained for tubulin, pericentrin and 40,6-diamidino-2-phenylindol dihydrochloride (DAPI). Mitotic phenotypes were analyzed by microscopy in about 300 mitotic cells. (a) The representatives of mitotic defects in HeLa 776-6 cells: spindle malformation and chromosome misalignment (second and third panels) and multipolar spindles (fourth and lower panels) in HeLa 776-6 cells, compared with control HeLa cells (upper panel). Bar: 5 mm. Quantiﬁcations of mitotic defects in HeLa and HeLa 776-6 cells, including spindle malformation (b), chromosome misalignment (c). Multi or monopolar spindles (d, e).

Oncogene Stabilization of p53 by targeting cyclin B1 NN Kreis et al 5596

Figure 3 Continued.

HeLa or HeLa GFP cells (Figure 4a, third row, lanes 3 cyclin B (Bischoff et al., 1990). However, the function of and 6), a considerable part of p14ARF still remained p53 regulation by Cdk1 has not been clarified. As the in mitosis of HeLa 776-6 cells (Figure 4a, third row, Cdk1 activity is strongly suppressed by stably depleting lane 9). Increased p14ARF was mainly localized in the cyclin B1 in HeLa 776-6 cells (Yuan et al., 2006), we nucleus in mitotic HeLa 776-6 cells (Figure 4b, second reasoned that phosphorylation of Ser315 in p53 could be row, lane 4). Moreover, compared with HeLa cells, inhibited. To test this issue, HeLa and HeLa 776-6 cells MDM2, the negative regulator and also a downstream were synchronized to prometaphase and released into a effector of p53, was apparently increased in mitotic fresh medium. Cells were harvested at indicated time HeLa 776-6 cells as well, possibly induced by increased points for western blot analysis using specific phospho- p53 (Figure 4b, third row). Further treatment with antibodies against Ser315 in p53. The phosphorylation nocodazole, which is a microtubule-destabilizing agent signal of Ser315 in p53 was indeed reduced in HeLa 776-6 and severed as a mitotic stress inducer, kept the protein cells, relative to control HeLa cells (Figure 5a, second levels of p53, p14ARF and MDM2 high throughout the row, lanes 6 and 12). This reduction of phosphorylation treatment in HeLa 776-6 cells, compared with HeLa cells was also found in HeLa 776-13 and HeLa 776-15 cells (Figure 4c). Moreover, while silencing of p14ARF reduced with reduced Cdk1 activity (Figure 5b, middle row, about 40% of the p53 protein level, control small interfering lanes 9 and 12), in contrast to HeLa 776-1 cells RNA barely affected the p53 level in HeLa 776-6 cells (Figure 5b, middle row, lane 3), which contain a (Figure 4d). Altogether, the data suggest that the high comparable level of cyclin B1 mRNA similar to control level of p14ARF in HeLa 776-6 cells, possibly induced HeLa cells. Moreover, phosphorylation of Ser315 is by mitotic stress because of the depletion of cyclin B1 also decreased in CaSki cells with reduced cyclin B1 in HeLa 776-6 cells, might bind to MDM2 thereby (Figure 5c). As overexpression of Aurora A phospho- interfering with its function in p53, leading further to rylates Ser315 in p53 (Katayama et al., 2004), we also stabilization of p53 in HeLa 776-6 cells. examined the Aurora A level in HeLa 776-6 cells. As shown in Figure 5d, HeLa 776-6 cells showed a reduced level of Aurora A, compared with HeLa cells. To further Reduced phosphorylation at Ser315 of p53 stabilizes explain this issue, mitotic cells were incubated with the the protein p53 in HeLa 776-6 cells Cdk1 inhibitor RO-3306 or Aurora A/B kinase inhibitor Furthermore, many postmodifications control the turn- ZM447439. Although phosphorylation at Ser315 decreased over of p53. It has been described that Aurora A on treatment with the Cdk1 inhibitor, almost no change phosphorylates the residue Ser315 in p53 and this was observed after incubation with ZM447439 (Figure 5e, phosphorylation is supposed to induce MDM2- middle row). These results strengthen the notion that mediated destabilization and inhibition of p53 (Katayama Ser315 of p53 is mainly phosphorylated by Cdk1 rather et al., 2004). Intriguingly, it has been published for than Aurora A/B kinases in mitosis, at least in the almost 20 years that Ser315 in p53 is targeted by Cdk1/ system we used.

Oncogene Stabilization of p53 by targeting cyclin B1 NN Kreis et al 5597

Figure 5 Inhibited phosphorylation of Ser315 in p53 in HeLa 776-6 cells is attributed to the reduced activity of Cdk1. (a) HeLa and HeLa 776-6 cells were nonsynchronized (C), synchronized to the G1/S boundary (T) or to prometaphase (N). Cytosolic (Cy) and nuclear extracts (Nu) were prepared for western blot analyses with antibodies against p53 and phospho-p53 (S315). Calnexin and lamin B1 served as cytoplasm and nuclear extract marker, respectively. (b) HeLa stable clones were treated as in (a) and whole cellular extracts were prepared for western blot analyses with antibodies as indicated. HeLa 776-1 cells, containing comparable cyclin B1 mRNA as HeLa cells, were taken as control. b-actin served as loading control (con). The p53 level was also used as Figure 4 Increased tumor suppressor p14ARF and E3 ligase control for Figure 1c. (c) Cervical cancer cell line CaSki, infected MDM2 in HeLa 776-6 cells. (a) HeLa, HeLa GFP and HeLa 776-6 with HPV 16, was stably transfected with plasmids expressing cells were nontreated (C), synchronized to the G1/S boundary (T) shRNA cyclin B1 (shcycB1) or control shRNA GFP (shGFP). or prometaphase (N) and extracts were prepared for western blot Cells were nonsynchronized (C), synchronized to the G1/S analyses with antibodies as indicated. (b) Cells were synchronized boundary (T) or to prometaphase (N) for western blot analyses to prometaphase and cytosolic (Cy) and nuclear extracts (Nu) were with indicated antibodies. The p53 level as control was from separated for western blot analyses. (c) HeLa and HeLa 776-6 cells Figure 1d. (d) HeLa and HeLa 776-6 cells were synchronized and were treated with nocodazole (Noc) for indicated time points and cellular extracts were prepared for western blot analyses with lysates were prepared for western blot analyses with indicated antibodies as indicated. b-actin served as loading control. (e) Cdk1 antibodies. (d) HeLa 776-6 cells were treated with small interfering inhibitor RO-3306 inhibited Ser315 phosphorylation. HeLa cells RNA control (sicon) or siRNA against p14ARF (sip14ARF) and were synchronized to prometaphase and released into fresh synchronized to prometaphase. Cellular lysates were prepared for medium containing MG132 (10 mM) for 1 h to halt cells in western blot analyses with antibodies against p53 and p14ARF. metaphase, followed by adding either dimethyl sulfoxide (con), b-actin served as loading control. The ratio of p53 to actin is shown. Cdk1 inhibitor RO-3306, 2 mM or 5 mM Aurora A/B inhibitor ZM447439 for further 1 h. Cells were then harvested for western blot analyses with antibodies as indicated. b-actin served as loading control.

Oncogene Stabilization of p53 by targeting cyclin B1 NN Kreis et al 5598 Substitution of Ser315 by alanine stabilizes the respectively (Figure 6b). This experiment was further protein p53 performed in HCT116 p53(À/À) cells and similar results To test whether phosphorylation of Ser315 is directly were obtained (data not shown), indicating that associated with the p53 protein stability, Ser315 was phosphorylation of Ser315 in p53 is directly associated substituted either by alanine or aspartic acid in Flag- with the protein turnover of p53. tagged full-length p53. HeLa cells were transfected with As Ser315 locates within the nuclear localization wild-type p53 or its mutants and synchronized to sequence of p53, we investigated next the subcellular prometaphase. Cells were then released into a fresh localization of wild-type p53 and its mutants. HeLa 776-6 medium containing protein synthesis inhibitor cyclohex- cells were transfected with Flag-tagged wild-type p53 imide and harvested at indicated time points for western and its variants and synchronized to the G1/S boundary blot analysis. We found that p53 S315A was the most and released for 10 h. Cellular extracts from mitotic cells stable protein, compared with wild-type p53 and p53 were prepared for western blot analysis. In parallel, S315D (Figure 6a, upper panel). Further quantiﬁcations mitotic cells were ﬁxed and stained for Flag-tagged p53. showed that the half-life time of p53 S315D, wild-type The nuclear portion of p53 S315A was obviously more p53 and p53 S315A was about 1.25, 1.75 and 3 h, than that of wild-type p53 and p53 S315D (Figure 6c),

Figure 6 p53 S315A was more stable than wild-type p53 and p53 S315D. (a) HeLa cells were transfected with Flag-tagged wild-type (WT) p53 and its mutants p53 S315A and p53 S315D. Cells were then synchronized to prometaphase and released into a fresh medium with cycloheximide (CHX, 25 mg/ml) for indicated time periods. Cellular extracts were prepared for western blot analysis with Flag antibodies. b-actin served as loading control. (b) Quantiﬁcation of p53 protein, relative to loading control b-actin. The half-life time for WT p53 and its mutants is marked with lines. (c) HeLa 776-6 cells were transfected with WT p53, p53 S315A or p53 S315D and synchronized to prometaphase. Cytosolic (Cy) and nuclear extracts (Nu) were prepared for western blot analyses with Flag antibodies. Lamin B1 served as nuclear extract control and b-actin as loading control. (d) HeLa 776-6 cells were transfected as in (c), synchronized with a double thymidine block and released into fresh medium. After 10 h, cells were stained for Flag-tag, tubulin and DNA. Bar: 5 mm.

Oncogene Stabilization of p53 by targeting cyclin B1 NN Kreis et al 5599 which was further corroborated by immunofluorescence lane 7). From 36 to 48 h, while p53 levels were still high staining with Flag antibodies (Figure 6d), suggesting in HeLa cells, p53 went down in HeLa 776-6 cells Ser315 is directly involved in regulating the subcellular because of the apoptosis induction (Figure 8c, second localization of p53 and phosphorylation of Ser315 row). Further analysis of the caspase-3/7 activity within nuclear localization sequence facilitates the showed that more apoptosis took place in HeLa 776-6 release of p53 from the nucleus to cytoplasm. cells than in HeLa cells (Figure 8d). Altogether, these To investigate whether this Ser315 phosphorylation findings strongly suggest that increased p53 in HeLa by Cdk1 affects the function of p53 in tumor cells, 776-6 cells is functional and responds properly to DNA HCT116 p53(À/À) cells were cotransfected with wild- damage induced by either irradiation or camptothecin. type p53/pBabe-puro or its mutant/pBabe-puro con- Finally, it is well known that disruption of mitosis structs (Figure 7a). After selection transfected cells were alone can promote the sensitivity of cancer cells to challenged with camptothecin and stained for phos- chemotherapeutics, which we have also observed (Yuan phorylated histone gH2AX, a DNA damage marker, et al., 2006; Androic et al., 2008; Kreis et al., 2009). To and activated caspase-3, an apoptosis marker. As address the function of p53 in this scenario, HeLa cells expected, treatment with camptothecin induced a robust were depleted of either cyclin B1, p53 or both of them DNA damage in HCT116 p53(À/À) cells added back and then subjected to camptothecin. Depletion of cyclin with wild-type p53, p53 S315A or p53 S315D (gH2AX B1 triggered more apoptosis in HeLa cells with p53 than staining, Figure 7b and d). Interestingly, the activated in HeLa cells without p53 after exposure to camptothe- caspase-3 staining was much stronger in HCT116 p53 cin by showing increased levels of cleaved poly (ADP- S315A cells, compared with HCT116 cells with either ribose) polymerase and cytochrome C release, and a wild-type p53 or p53 S315D (activated capase-3 stain- decreased level of caspase-3 precursor (Supplementary ing, Figure 7b and c). These data shows that this Figure 3A). In line with these results, the caspase-3/7 phosphorylation of Ser315 by Cdk1 is associated with activity was the highest in HeLa cells depleted of cyclin apoptosis response of p53 in HCT116 cells. Further- B1 but with p53 (Supplementary Figure 3B). Moreover, more, to explore whether this phosphorylation also these data were further corroborated in HCT116 influenced the transcriptional activation of p53, p53( þ / þ ) and HCT116 p53(À/À) cells: silencing of HCT116 p53(À/À) cells were cotransfected with p21 cyclin B1 induced more cleavage of poly (ADP-ribose) promoter luciferase plasmids and wild-type p53 or its polymerase in HCT116 p53( þ / þ ) cells than in variant constructs. The transcriptional activity of p53 HCT116 p53(À/À) cells (Supplementary Figure 3C), S315D and p53 S315A was slightly reduced or increased, which was additionally supported by increased activity respectively, relative to wild-type p53 (Figure 7e). of caspase-3/7 with the same extracts (Supplementary Figure 3D). Taken together, these data strongly suggest that p53 strengthens the cytotoxicity of camptothecin in DNA damage response of HeLa 776-6 cells tumor cells with interruption of mitosis by depleting To study the functional significance of stabilized p53 in cyclin B1. cells, HeLa and HeLa 776-6 cells were treated with increasing doses of irradiation. Cells were then incubated with fresh medium for 1 h and extracts were prepared for western blot analysis with specific phospho- Discussion antibodies against Ser15 of p53, indicative of a DNA damage response of p53. The p53 protein levels were In more than 90% of cervical cancers and cancer- kept high on irradiation and responded to it by showing derived cell lines, the p53 pathway is disrupted by the stronger phosphorylation signals of Ser15 in p53 in HPV E6, which complexes with cellular proteins E6-AP HeLa 776-6 cells, relative to HeLa cells (Figure 8a, and p53 and facilitates p53 degradation by the second and third rows). ubiquitin-dependent proteolytic system (Scheffner Moreover, to underscore this finding, HeLa and HeLa et al., 1990). The abrogation of functional p53 is 776-6 cells were subjected to camptothecin for short- required for promoting transformation and maintaining term periods. Compared with control HeLa cells, the the malignant phenotype (Pathirana et al., 2009). Thus, levels of p53 were strongly enhanced in HeLa 776-6 cells restoration of p53 function by blocking this degradation upon treatment with camptothecin, (Figure 8b, second pathway has been reported as an attractive strategy to row). Along with increased p53, the phospho-signals of ensure an effective intervention (Hietanen et al., 2000; Ser15 in p53 were also more strong in HeLa 776-6 cells Rampias et al., 2009). Interestingly, beside the abroga- over that in control HeLa cells (Figure 8b, third row). tion of p53, HPV 18 infection increases Cdk1/cyclin To further study the long-term effect of this increased B1-associated activity by stabilizing cyclin B1 mRNA p53, HeLa 776-6 cells were treated with camptothecin as through the upregulation of HuR protein in HPV 18- indicated. At 12 h, DNA damage as well as apoptosis infected cervical lesions (Cho et al., 2006). Overexpres- were readily found in HeLa 776-6 cells, but not in HeLa sion of cyclin B1 has been reported in invasive cervical cells, by showing the appearance of phosphorylated cancer and has an important role in cervical carcino- histone gH2AX, a DNA damage marker, and cleaved genesis (Zhao et al., 2006). product of the poly (ADP-ribose) polymerase, an To rebalance these two important molecules in HPV apoptosis marker (Figure 8c, third and fourth rows, 16/18-infected cancer cells, we show here that depletion

Oncogene Stabilization of p53 by targeting cyclin B1 NN Kreis et al 5600

Figure 7 p53 S315A sensitizes cells to camptothecin by showing more apoptosis. (a) Working schedule. (b) HCT116 p53(À/À) cells were treated as described in (a) and stained for DNA damage marker gH2AX, apoptosis marker activated caspase-3 and DNA. The representatives are shown. Bar: 5 mm. (c) Percentages of activated caspase-3 were evaluated with about 400 cells from each group. The results are presented as mean±s.d. (d) Percentages of positive gH2AX, evaluated with about 400 cells from each group. The results are presented as mean±s.d. (e) HCT116 p53(À/À) cells were co-transfected with p21 promoter luciferase reporter gene plasmids and the Flag-tagged wild-type p53 or its variants. After 48 h of transfection, luciferase activities were measured. The results are presented as mean±s.d and statistically analyzed. *Po0.05. DAPI, 40,6-diamidino-2-phenylindol dihydrochloride; IF, immuno- ﬂuorescence; WT, wild type.

of cyclin B1 restores the p53 function in HPV 16/18- its downstream effectors, such as p21, MDM2 and Bax infected cervical cancer cell lines CaSki and HeLa. (Figures 1f, 2a, 4b and c). Furthermore, repression of Stabilization of p53 by depleting cyclin B1 possibly cyclin B1 and reactivation of p53 render HPV-infected overrides the threshold of HPV E6/E6-AP degrada- HeLa cells more susceptible to the DNA damage agent tion pathway in HPV-infected cervical cancer cells. camptothecin by showing strong apoptosis (Figures 8c Increased p53 is functional by showing an increase in and d). Therefore, downregulation of cyclin B1, inhibiting

Oncogene Stabilization of p53 by targeting cyclin B1 NN Kreis et al 5601 phosphorylated at least by five protein kinases, leading to an inhibitory or a stimulatory role in modulating p53 depending on the cellular context (Bischoff et al., 1990; Katayama et al., 2004; Pluquet et al., 2005; Radha- krishnan and Gartel, 2006). Of importance, Aurora A phosphorylates Ser315 of p53 and overexpression of Aurora A induces MDM2-mediated p53 ubiquitination and degradation (Katayama et al., 2004). Interestingly, it has been revealed for almost 20 years that Cdk1/cyclin B phosphorylates Ser315 (Bischoff et al.,1990),butonlythe in vitro function of this phosphorylation has been reported (Fuchs et al., 1995; Wang and Prives, 1995). The in vivo function of this phosphorylation by Cdk1/cyclin B1 remains undefined. In this work we show for the first time that phosphorylation at Ser315 of p53 is directly associated with the activity of Cdk1/cyclin B1 within cells, as depletion of cyclin B1 reduces strongly the phospho- signal of Ser315 in HeLa cells as well as in CaSki cells (Figures 5a–c). Moreover, specific small molecule inhibitor RO-3306 targeting Cdk1 strongly weakens this phospho-signal in cells, whereas the inhibitor ZM447439 against Aurora A/B shows no effect (Figure 5e), indicating Ser315 is mainly the target for Cdk1 in normal mitosis, when cells are not enforced to overexpress Aurora A as reported (Katayama et al., 2004). Moreover, it has been very often observed that phosphorylation by Cdk1 affects the localization and the function of its substrates (Sanhaji et al., 2010). This phosphorylation of p53 by Cdk1 indeed destabilizes the protein p53 (Figures 6a and b) by promoting its release from the nucleus to cytoplasm (Figures 6c and d), where it is degraded. Interestingly, HCT116 p53(À/À) cells transfected with p53 S315A, an unphosphorylable form of p53 by Cdk1, exhibited more strong apoptosis than cells with either wild-type p53 or its phospho-mimicking form p53 S315D after treatment with camptothecin (Figure 7b). Moreover, in contrast to previous data in vitro, we found that Figure 8 Increased p53 is functional and renders HeLa cells more phosphorylation of Ser315 in p53 can suppress its susceptible to camptothecin. (a) HeLa and HeLa 776-6 cells were transcriptional activity in vivo (Figure 7c), although in a subjected to irradiation at indicated dosage. After 1 h, extracts were prepared for western blot analyses with indicated antibodies. low extent. On the basis of these data, it is tempting to (b) HeLa or HeLa 776-6 cells were synchronized to prometaphase suggest that this phosphorylation at Ser315 by Cdk1 and then subjected to 10 mM camptothecin for indicated time. negatively regulates the protein stability and the Cellular lysates were prepared for western blot analyses with function of p53, possibly facilitating the smooth antibodies as indicated. (c) HeLa and HeLa 776-6 cells were treated progression of mitosis. with 5 mM of camptothecin for indicated time and cellular extracts were subjected to western blot analyses with indicated antibodies. The p14ARF tumor suppressor is a product of the (d) Activated caspase-3 was measured with the same extracts as in INK4a/ARF locus, a sequence that is frequently altered (c). The results were presented as mean±s.d. PARP, poly (ADP- in human cancer (Sharpless, 2005). p14ARF directly ribose) polymerase. binds to and interferes with the p53 negative regulator MDM2, thereby stabilizing and activating p53 (Stott et al., 1998). p14ARF transcription is induced by E2F1 (Gallagher et al., 2006) and stabilized by forming Cdk1 activity as well as stabilizing the functional p53, complex with nucleophosmin (B23), which restricts could be a novel strategy for treating cervical lesions p14ARF to the nucleolus (Korgaonkar et al., 2005). caused by HPV infection. We found that p14ARF protein level is increased in Phosphorylation has an essential role in activation mitotic HeLa 776-6 cells (Figures 4a–c), which could be and stabilization of the tumor suppressor p53 (Toledo associated with mitotic stresses such as aberrant mitotic and Wahl, 2006). The residue Ser315 locates within spindles and misaligned chromosomes induced by the nuclear localization signal (aa 305–322) of the depleting cyclin B1 (Figure 3). Moreover, depletion of C-terminal region of p53 and has been reported to be p14ARF in HeLa 776-6 cells decreased about 40% of

Oncogene Stabilization of p53 by targeting cyclin B1 NN Kreis et al 5602 the p53 protein amount (Figure 4d), indicating that this purchased from Santa Cruz (Heidelberg, Germany). Mouse increased p14ARF is directly involved in the stability of monoclonal antibodies against p21 and gH2AX, rabbit poly- p53 in HeLa 776-6 cells. The molecular mechanism for clonal antibodies against poly (ADP-ribose) polymerase and increased p14ARF in HeLa 776-6 cells requires further rabbit polyclonal phospho-antibodies against p53 (S315 and investigations. S15) were obtained from Cell Signaling (Beverly, MA, USA). In addition, increased p53 in HeLa 776 clones with Mouse monoclonal antibodies against Flag and b-actin were from Sigma-Aldrich. Rabbit polyclonal antibodies against reduced cyclin B1 is more predominant in mitosis than phospho-histone H3 were from Upstate (Lake Placid, NY, in interphase (Figure 1a), which was also observed in USA). Mouse monoclonal antibodies against calnexin and p53 wild-type MCF7 and A549 cells in mitosis after lamin B1 were from BD Biosciences (Heidelberg, Germany) depleting cyclin B1 (Figure 1e). The half-life time of and Biozol, respectively. Western blots were quantified as p53 in mitotic HeLa 776-6 cells was at least 20 min described previously (Kreis et al., 2009). Cytosolic and nuclear longer than that in HeLa cells (Figure 2c). This might be extracts were prepared by using the Nuclear Complex Co-IP Kit attributed to enormous mitotic stresses induced by (Active Motif, Rixensart, Belgium). depleting cyclin B1 (Figure 3). Moreover, p53 is obviously degraded by proteosome during mitosis, as Construction of DNA plasmids, transient transfections p53 was strongly accumulated upon treatment with the and quantitative PCR proteosome inhibitor MG132 (Figure 2c), indicating The p53 complementary DNA was amplified by PCR from that p53 functions in mitosis and is then degraded pFC-p53 (Stratagene, Amsterdam, The Netherlands) and through possibly postmodifications, such as phospho- subcloned into the EcoRI/BamHI sites of 3xFlag-CMV 7.1 rylation at Ser315 by Cdk1. (Invitrogen, Karlsruhe, Germany). Point mutations of Ser315 Taken together, in this work we showd that down- in p53 were generated with Quick Change-site-directed muta- regulation of cyclin B1 stabilizes and restores the genesis Kit (Stratagene). All mutant constructs were confirmed function of the tumor suppressor p53 in HPV 18/16- by DNA sequencing. Transient transfection of plasmid was carried infected cervical cancer cells by means of triggering out as described (Kreis et al., 2009; Sanhaji et al., 2010). For mitotic stress, increasing its positive regulator p14ARF quantitative-PCR, total RNAs from HeLa, HeLa GFP and HeLa 776-6 cells were extracted and purified using RNeasy and reducing the Ser315 phosphorylation in p53. The Kit and RNase-free DNase Set (Qiagen, Hilden, Germany). restored functional p53 and disruption of mitosis by Complementary DNAs were prepared using High-Capacity silencing cyclin B1 render cervical cancer cells infected complementary DNA Reverse Transcription Kit (Applied with HPV 16/18 more susceptible to the DNA damage Biosystems, Darmstadt, Germany). Quantitative PCR analyses agent camptothecin. Awakening guardian p53 by were carried out with commercial primer pairs and probes for drugging its pathway has been considered as an human p53, human cyclin B1 and control GAPDH and with exceptionally attractive intervention in tumor cells TaqMan Fast Universal PCR Master Mix (Applied Biosystems), (Brown et al, 2009). Thus, downregulation of cyclin as described (Kreis et al., 2009). B1, specifically inhibiting the activity of Cdk1, could be a promising strategy for preventing and combating Indirect immunofluorescence papillomavirus-associated malignancies. Cells transfected with Flag-tagged p53 and its mutant constructs were fixed for 8 min at À20 1C with MeOH. The following primary antibodies were used for staining: poly- Materials and methods clonal rabbit antibodies against pericentrin (Cell Signaling), monoclonal rat antibodies against a-tubulin (Sigma-Aldrich), Cell culture, synchronization and cellular extract preparation monoclonal mouse antibodies against Flag-tag (Sigma- MCF7, MDA-MB-231, A549, HeLa, CaSki, HCT116 p53( þ / Aldrich) or p53 (Santa Cruz), monoclonal mouse antibodies þ ) and HCT116 p53(À/À) cells were cultured as instructed. against gH2AX (Cell Signaling) and polyclonal rabbit HeLa 776 clones with various levels of cyclin B1 were antibodies against activated caspase-3 (Cell Signaling). Fluor- generated as described previously (Yuan et al., 2006). Cell escein isothiocyanate goat anti-mouse and Cy3 donkey anti-rat synchronization to the G1/S boundary by double thymidine antibodies (Jackson Immunoresearch, West Grove, PA, USA) block and to prometaphase by thymidine/nocodazole treat- were used as secondary antibodies. DNA was stained using ment and cell lysis were carried out, as described (Kreis et al., 40,6-diamidino-2-phenylindol dihydrochloride (Roche). Slides 2009). Camptothecin, cycloheximide, RO-3306 and ZM447439 were examined using an Axio Imager 7.1 microscope (Zeiss, were from Fluka (Buchs, Switzerland), Sigma-Aldrich (Tauf- Go¨ttingen, Germany) and images were taken using an Axio kirchen, Germany), Roche Applied Science (Mannheim, Cam MRm camera (Zeiss). Germany) and Biozol (Eching, Germany), respectively. CaSki stable cell clones expressing shRNA cyclin B1/GFP were established as described (Kreis et al., 2009). Luciferase reporter assay and active caspase-3 measurement For reporter gene analysis HCT116 p53(À/À) cells were transfected with 0.5 mg of p21 promoter luciferase reporter Western blot analysis, cytosolic and nuclear extract separation gene plasmid and 1 mg of Flag-tagged wild-type p53 or its Western blot analysis was carried out as previously described variants. Luciferase activities were measured 48 h after (Kreis et al., 2009). Mouse monoclonal antibodies against transfection using the Luciferase Assay System (Promega, cyclin B1, Cdk1, Plk1, p53, p14ARF, MDM2, caspase-3, Mannheim, Germany). For active caspase-3 assays, HeLa and rabbit polyclonal antibodies against p53 and E6-AP, goat HeLa 776-6 cells were treated with 5 mM of camptothecin and antibodies against HPV E6 and Aurora A, goat anti-mouse/ the activity of caspase-3/7 was analyzed with Caspase-Glo 3/7 anti-rabbit and chicken anti-goat secondary antibodies were Assay (Promega), as instructed.

Oncogene Stabilization of p53 by targeting cyclin B1 NN Kreis et al 5603 Transient transfection with small interfering RNA, irradiation Conflict to interest and statistic analysis Transient transfection of small interfering RNA and irradiation The authors declare no conflict of interest. were carried out as described (Kreis et al., 2009). For assays in vitro, Student’s t-test was used to evaluate the significance of difference between HeLa and HeLa 776-6 cells. Difference Acknowledgements was considered as statistically significant, when Po0.05. This work is supported by the Deutsche Krebshilfe (#107594 and #108553). We are grateful to Dr B Vogelstein, Ludwig Abbreviations Center at Johns Hopkins, Howard Hughes Medical Institute, Baltimore, for HCT116 p53( þ / þ ) and p53(À/À) cell lines, p14ARF, p14 alternative reading frame of p16; Cdk1, cyclin- and Dr K Hoppe-Seyler, Deutsches Krebsforschungszentrum, dependent kinase 1; HPV 16/18, human papillomavirus type 16/18; Heidelberg, for CaSki cell line. We thank Ms Roth and Ms MDM2, mouse double minute 2; shRNA, small hairpin RNA. Zimmer for their excellent technical support.

References

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