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Home , Cell cycle, Cyclin A, G1 phase, S phase

(2007) 26, 1949–1953 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc SHORT COMMUNICATION downregulates expression of the G1/S cycle phosphatase Cdc25A

K Rother1,2, R Kirschner1,KSa¨ nger1,LBo¨ hlig1,2,JMo¨ ssner1 and K Engeland1

1Medizinische Klinik und Poliklinik II, Max-Bu¨rger-Forschungszentrum, Universita¨t Leipzig, Leipzig, Germany and 2Interdisziplina¨res Zentrum fu¨r Klinische Forschung (IZKF) Leipzig, Max-Bu¨rger-Forschungszentrum, Universita¨t Leipzig, Leipzig, Germany

Overexpression of Cdc25A phosphatase is often observed Another important level of regulation is reversible in and results in poor prognosis. Cdc25A mainly . Initially, /Cdk complexes are dephosphorylates and thereby activates Cyclin-dependent kept in an inactive state by phosphorylation. The Myt1 2 and thus induces progression in the and mediate the inactivation of Cdk1 and from G1 to . Here, we demonstrate that the tumor Cdk2 by phosphorylation of two sites in their ATP suppressor p53 downregulates expression from the binding loop (Obaya and Sedivy, 2002). Conversely, Cdc25A . In a p53-inducible cell system, Cdc25A dual-specificity phosphatases catalyse cell cycle expression on the mRNA and level is down- progression by dephosphorylating the same residues, regulated upon p53 expression. Promoter-reporter assays thereby activating Cdks (Nilsson and Hoffmann, 2000). show that this regulation is dependent on the Cdc25A In human cells, three Cdc25 family members –Cdc25A, promoter. Mutant p53 fails to reduce Cdc25A transcrip- Cdc25B and Cdc25C –have been characterized (Sadhu tion. In contrast to p53, neither p63 nor can repress et al., 1990; Galaktionov and Beach, 1991; Nagata et al., Cdc25A . The Cdc25A promoter displays no 1991). They are able to dephosphorylate both phospho- p53 binding site, and p53 does not bind directly to the serines/threonines and phosphotyrosines on the same promoter DNA as shown by immunoprecipita- protein substrate. Cdc25B or Cdc25C regulate the tion assays. Previously, the contribution of p53 to G1/S transition from to through Cdk1 arrest has been mostly linked to activating the expression dephosphorylation (Millar et al., 1991; Strausfeld of the Cdk inhibitor p21WAF1/CIP1. By downregulating et al., 1994; Lammer et al., 1998). Cdc25A mainly Cdc25A expression, p53 may impair transition from G1 to activates Cdk2 and thereby induces progression from G1 S phase independently of p21WAF1/CIP1. Therefore, the data to S phase. Microinjection of antibodies against Cdc25A suggest that, as long as p53 is intact, Cdc25A transcrip- arrests cells before the S phase, whereas ectopic tional downregulation might play a role in cancer expression of the phosphatase shortens the prevention. and accelerates cell cycle progression (Hoffmann et al., Oncogene (2007) 26, 1949–1953. doi:10.1038/sj.onc.1209989; 1994; Jinno et al., 1994; Blomberg and Hoffmann, 1999; published online 25 September 2006 Sexl et al., 1999). Emphasizing its role, Cdc25A has been described as an oncogene as the protein can induce cell Keywords: p53; Cdc25A; G1 to S transition; repression; transformation and is overexpressed in a wide variety of tumor suppressor; transcription tumors (Galaktionov et al., 1995; Broggini et al., 2000; Cangi et al., 2000). Recent evidence suggests that Cdc25A may also play a role in regulating the G2/M transition (Mailand et al., 2002; Chen et al., 2003). Cell cycle progression is mainly controlled by A principle factor involved in both G1 and G2 growth and Cyclin-dependent kinases (Cdks), which succes- arrest as well as regulation is the tumor sively act in G1 to initiate the S phase and in G2 to suppressor protein p53. It can be activated by several initiate mitosis. Proper control of Cyclin/Cdk activity stress conditions, such as DNA damage, hypoxia, heat- ensures that the initiation of cell cycle events occurs only shock, expression of viral and cellular , as well after a successful completion of the previous cycle. as ionizing irradiation (Somasundaram, 2000). p53 Cyclin/Cdk activity is regulated on several different exerts its functions mainly as a levels, such as transcription of their , , and binds to a specific DNA sequence activating subcellular localization and binding of specific inhibitors transcription of target genes. One example of the (Obaya and Sedivy, 2002; Vermeulen et al., 2003). numerous p53 transcriptional target genes is the Cdk inhibitor p21WAF1/CIP1 (el-Deiry et al., 1993). p21WAF1/CIP1 Correspondence: K Engeland, Medizinische Klinik und Poliklinik II, binds to Cyclin/Cdk complexes, which leads to the Max-Bu¨ rger-Forschungszentrum, Universita¨ t Leipzig, Johannisallee inhibition of their kinase activity (Harper et al., 1995). 30, D-04103 Leipzig, Germany. WAF1/CIP1 E-mail:[email protected] Transactivation of and other p53 target Received 6 October 2005; revised 4 August 2006; accepted 7 August genes like GADD45 and 14-3-3s is involved in mediating 2006; published online 25 September 2006 cell cycle arrest by p53 (Kastan et al., 1992; el-Deiry Repression of Cdc25A by p53 K Rother et al 1950 et al., 1994; Hermeking et al., 1997; Bunz et al., 1998). In (Krause et al., 2000; Dietz et al., 2002). Induction of addition to activating transcription, p53 is also able to wild-type but not mutant p53 protein leads to an repress transcription from various promoters (Ho and approximately four-fold downregulation of Cdc25A Benchimol, 2003). We have previously shown that the mRNA from the cellular gene 9 h after the removal of tumor suppressor protein p53 plays an important role in doxycycline compared to the non-induced state reducing the concentration of active /Cdk1 (Figure 1a). As described earlier, selective induction of complex in the nucleus contributing to G2 arrest. To this wild-type p53 expression for 9 h neither leads to a end, p53 can repress transcription of , Cyclin relevant change in the cell cycle distribution nor to an B2 and Cdc25C genes (Krause et al., 2000, 2001; Manni elevated level of apoptotic cells (Krause et al., 2000; et al., 2001). Additionally, it has been shown that Dietz et al., 2002). Therefore, the observed reduction of transcriptional repression by p53 is important for the the Cdc25A mRNA level is not caused by cell cycle promotion of apoptosis (Ho and Benchimol, 2003). arrest or cells undergoing apoptosis. In order to investigate whether p53 affects mRNA With p63 and p73, two genes coding for levels of the cell cycle regulator Cdc25A, we employed homologous to p53 have been identified (Kaghad et al., the colorectal adenocarcinoma DLD-1 cell line. DLD-1 1997; Schmale and Bamberger, 1997; Osada et al., 1998; cells are negative for functional p53 protein, but the Yang et al., 1998). p53, p63 and p73 possess a similar DLD-1-tet-off-p53 cells were stably transfected with a domain structure and share substantial amino-acid system that permits (tet)-off-regulated expression of identity in their DNA-binding domains. Nevertheless, wild-type p53 (D53wt) or DNA-binding mutant p53 p63 and p73 proteins do not represent classical tumor R175H (D53mut) (Yu et al., 1999; Krause et al., 2000). suppressors as their genes are rarely found mutated or This system allows selective induction of p53 protein deleted in tumors (Melino et al., 2003). Furthermore, expression by removal of doxycycline from the cell unlike p53 knockouts, p63 and p73 knockouts do not culture media (Figure 1b, left panel). In order to analyse show enhanced cancer susceptibility but exhibit severe primary changes in mRNA expression levels of p53 developmental abnormalities (Mills et al., 1999; Yang target genes without influences by major changes in cell et al., 1999, 2000). p63 and p73 proteins are expressed in cycle distribution or by apoptosis induction, we had numerous splice variants making an analysis of their tested earlier several different time periods for p53 properties rather complex (Moll and Slade, 2004). induction. Nine hours after the removal of doxycycline However, due to the structural homologies, some of from the cell culture medium, the p53 protein has just the biological functions of p63 and p73 overlap with the reached a level sufficient to influence its target genes role of p53. For example, p63 and p73 can activate transcription of some p53-responsive genes, such as p21WAF1/CIP1 and Bax (Zhu et al., 1998; Katoh et al., 2000). Therefore, we investigated the effect of p53 homo- logues on Cdc25A mRNA levels. The p53 family members included in this study were p73a, TAp63a, TAp63g, TA*p63a, TA*p63g, DNp63a and DNp63g.We used the same DLD-1-tet-off system to selectively induce expression of six different splice variants of p63 and p73a protein (Dietz et al., 2002). Although the induction of protein expression of the p53 homologues are comparable to that of p53 (Figure 1b and data not shown), levels of Cdc25A mRNA only increased slightly after the induction of expression of all p53 homologues (Figure 1a and data not shown). Thus, in contrast to wild-type p53, the p73 and p63 subfamilies do not downregulate Cdc25A expression. However, as a con- trol, we had shown earlier in the same experimental system that inducing the expression of p73a, TAp63a and TAp63g increases the expression of mRNA from WAF1/CIP1 Figure 1 Cdc25A mRNA levels transcribed from the chromoso- the chromosomal p21 gene (Dietz et al., 2002). mal gene are downregulated upon expression of wild-type p53 but This suggests that at least these three p53 family not after expression of p73a or TAp63a. DLD-1 members are transcriptionally active, but do not cells with (tet)-off-regulated wild-type transgenes for p53, p73a or influence Cdc25A mRNA levels. TAp63a, respectively (Dietz et al., 2002), were expressed from these genes by the removal of doxycycline. (a) Relative Cdc25A mRNA As Cdc25A mRNA drops upon p53 induction we levels measured by real-time RT–PCR as described earlier (Krause were interested if the protein level of Cdc25A is also et al., 2000, 2001). Levels were standardized to GAPDH mRNA affected by an increase in wild-type p53. Western blot expression. Concentrations of mRNA from control DLD-1-tet-off analysis of total cell lysate prepared from DLD-1-tet- cells in a medium containing doxycycline were set as the 100% reference. One representative experiment is shown. (b) Western blot off-p53 cells, with or without the induction of wild- analyses of respective cell lysates document similar expression type p53 expression for 9 h, showed a significant levels of the different effector proteins after their induction. decrease in Cdc25A protein expression (Figure 2, upper

Oncogene Repression of Cdc25A by p53 K Rother et al 1951

Figure 2 Expression of Cdc25A protein is downregulated after p53 induction. Western blot analysis of Cdc25A expression from total cell lysates and nuclear extracts, prepared from DLD-1-tet- off-p53 cells before or after induction of wild-type p53 expression, was carried out as described (Dietz et al., 2002; Tscho¨ p et al., 2006). Samples were analysed with antibodies directed against Cdc25A (F-6, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Figure 3 Cdc25A promoter is downregulated by p53 but not by p53 (DO-1, 1:2000, Oncogene Merck, Darmstadt, Germany) or TAp63a or p73a. The Cdc25A-promoter-reporter Cdc25A-wt-luci b- (AC-1, 1:5000, Sigma-Aldrich, Taufkirchen, Germany). was co-transfected together with wild-type or mutant versions of Protein extracts from C33A cells untreated or transfected with a p53, TAp63a or p73a expression plasmids in SaOS-2 cells according plasmid expressing Cdc25A protein served as a control. to the manufacturer’s instructions. All experiments were standar- dized to Renilla luciferase activity as a control expressed from co- transfected pRL-null vector. Two hundred and fifty nanograms of Cdc25A-wt-luci plasmid were co-transfected with 25 ng of vector panel, lanes 3 and 4). When analysing nuclear extracts control or 25 ng of plasmids expressing wild-type or DNA-binding prepared from DLD-1-tet-off-p53 cells, an almost deficient mutant of p53, TAp63a or p73a. The vector control served complete loss of Cdc25A protein expression occurred as 100% standard. Firefly and Renilla luciferase activities were assayed with the Dual Luciferase Assay System (Promega, 9 h after p53 induction (Figure 2, upper panel, lanes 5 Mannheim, Germany) as suggested by the manufacturer. and 6). As the anti-Cdc25A mouse monoclonal antibody employed displayed several nonspecific bands, untreated C33A cells and C33A cells transfected with a plasmid expressing Cdc25A protein served as a control for the not shown). In control experiments, we found that p73a, size of Cdc25A protein (Figure 2, upper panel, lanes 1 p73b, TAp63g and TA*p63g are able to activate and 2). As a control for enhanced p53 expression, the transcription from a p21WAF1/CIP1 reporter (Dietz et al. blot was stripped and reprobed with an anti-p53 mouse (2002) and data not shown). This confirms that these monoclonal antibody. b-Actin served as a loading four p53 family members are transcriptionally active. control. However, this activity does not alter transcription from To elucidate whether the decrease in Cdc25A mRNA the Cdc25A promoter. is dependent on the function of p53 as a regulator of Expression of Cdc25A phosphatase is repressed by transcription, we analysed the regulation of the Cdc25A wild-type p53 at the transcriptional level. A DNA- promoter. To this end, we created a Cdc25A promoter binding mutant of p53 fails to repress Cdc25A promoter construct by cloning a fragment of 906 nucleotides activity. In order to examine if wild-type p53 can upstream of the translation start codon into a vector to actually bind to the Cdc25A promoter we performed drive the expression of a firefly luciferase reporter. The chromatin immunoprecipitation assays (ChIPs). Chro- Cdc25A-wt-luci construct was transiently co-transfected matin of DLD-1-tet-off-p53 cells before and after with expression plasmids for human wild-type p53, a selective induction of p53 protein expression was cross- DNA-binding mutant of p53 (V143A) or a vector linked and immunoprecipitations were performed using control in SaOS-2 cells, which are defective in p53 antibody directed against p53. Promoter fragments of function. Wild-type p53 clearly downregulates the p21WAF1/CIP1 and Cdc25A were amplified by PCR and a transcription from the Cdc25A promoter by approxi- representative agarose gel of the PCR products is shown mately 20-fold (Figure 3). Only a small reduction of in Figure 4. p53 bound to the p21WAF1/CIP1 promoter both reporter activity is seen upon expression of the p53 before and after selective induction of p53 protein mutant. expression. The presence of p53 before induction is due To test the Cdc25A promoter for responsiveness to to the leakage of the tet-off-system employed. The p73 and p63 we employed plasmids coding for wild-type absence of PCR amplification of the Cdc25A promoter p73a, p73b, TAp63a, TAp63g, TA*p63a, TA*p63g, after immunoprecipitation with anti-p53 antibody in- DNp63a and DNp63g. We compared them to the dicates that p53 does not bind to the Cdc25A promoter, respective DNA-binding deficient mutants. Neither neither before nor after selective induction of p53 p73a nor p73b nor any of the p63 splice variants tested protein expression. p53 likely represses Cdc25A indi- are able to change transcription from the Cdc25A rectly by transactivating one or more p53 target genes. promoter significantly when compared to their tran- Such a mechanism should be considered as no direct scriptionally inactive mutant forms (Figure 3 and data binding of p53 to the Cdc25A promoter could be

Oncogene Repression of Cdc25A by p53 K Rother et al 1952 In summary, our results reveal the cell cycle phos- phatase Cdc25A as a new p53 target gene. This suggests that the tumor suppressor p53 may impair G1/S transition and thereby contribute to the prevention of uncontrolled . Thus, downregulation of Cdc25A could play a role in the cancer prevention as long as p53 is intact. Transcriptional repression of Cdc25A is neither observed with p63 nor with p73. This emphasizes different properties of the p53 family members in cell cycle control and points out the Figure 4 p53 does not bind in vivo to the Cdc25A promoter. ChIPs importance of p53 in the prevention of cancer by with Cdc25A and p21WAF1/CIP1 promoters, before and after induction inducing cell cycle arrest by several independent path- of wild-type p53 expression in DLD-1 cells, were carried out as ways. described previously (Wasner et al., 2003a, b) using 1 Â 107 cells. Crosslinks were precipitated by 5 mg of anti-p53 mouse monoclonal antibody (DO-1, Oncogene) or without antibody (no Ab). The Acknowledgements p21WAF1/CIP1 promoter that has been shown to contain a p53 binding site served as a positive control. M denotes the marker lane with a 100 bp ladder (Fermentas, St Leon-Rot, Germany) giving the We thank Bert Vogelstein for providing plasmids and cells, 500 bp band as the most prominent. Ingrid Hoffmann for the Cdc25A cDNA and an antibody preparation, Jana Lorenz for expert technical assistance and Christine Engeland for critical reading of the paper. This work was supported by grants from the Interdisziplina¨ res Zentrum observed (Figure 4). Further experiments will elucidate fu¨ r Klinische Forschung (IZKF) Leipzig and the Deutsche the details of this mechanism. Forschungsgemeinschaft (to KE).

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