Gadd45a Regulates B-Catenin Distribution and Maintains Cell–Cell Adhesion/ Contact

Oncogene (2007) 26, 6396–6405 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Gadd45a regulates b-catenin distribution and maintains cell–cell adhesion/ contact

JJi1, R Liu1, T Tong, Y Song, S Jin, M Wu and Q Zhan

State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, PR China

Gadd45a,a growth arrest and DNA-damage gene,plays 1999;Jin et al., 2000;Tong et al., 2005). Gadd45a important roles in the control of cell cycle checkpoints, also plays roles in negative regulation of cell malig- DNA repair and apoptosis. We show here that Gadd45a is nancy. Mouse embryonic fibroblasts (MEFs) derived involved in the control of cell contact inhibition and cell– from Gadd45a-null mice exhibited genomic instability, cell adhesion. Gadd45a can serve as an adapter to enhance single oncogene-mediated transformation, loss of nor- the interaction between b-catenin and Caveolin-1,and in mal cellular senescence, increased cellular proliferation, turn induces b-catenin translocation to cell membrane for centrosome amplification and reduced DNA repair maintaining cell–cell adhesion/contact inhibition. This is (Hollander et al., 1999;Smith et al., 2000). Gadd45a- coupled with reduction of b-catenin in cytoplasm and null mice have an increased frequency of tumorigenesis nucleus following Gadd45a induction,which is reflected by by ionizing radiation (IR), ultraviolet radiation (UVR) the downregulation of cyclin D1,one of the b-catenin and dimethylbenzanthracene (DMBA), although these targeted genes. Additionally,Gadd45a facilitates ultra- mice do not develop spontaneous tumors (Hollander violet radiation-induced degradation of cytoplasmic and et al., 1999, 2001;Hildesheim et al., 2002). Additionally, nuclear b-catenin in a p53-dependent manner via activa- Gadd45a is a key factor protecting the epidermis against tion of p38 kinase. These findings define a novel link that UVR-induced skin tumors (Li et al., 1996;Hildesheim connects Gadd45a to cell–cell adhesion and cell contact et al., 2002). It has been identified that high frequency inhibition,which might contribute to the role of Gadd45a mutations of Gadd45a gene in pancreatic and abnormal in inhibiting tumorigenesis. methylation of Gadd45a promoter in breast cancer, Oncogene (2007) 26, 6396–6405;doi:10.1038/sj.onc.1210469; additionally strengthening its link to cancer (Yamasawa published online 23 April 2007 et al., 2002;Wang et al., 2005). Most recently, there is evidence for its involvement in regulating matrix Keywords: Gadd45a;cell–cell adhesion;cell contact metalloproteinases (MMP) to inhibit the malignant cell inhibition; b-catenin invasion (Hildesheim et al., 2004). All these suggested that Gadd45a might play an important role in negative regulation of development of cell malignancy. However, the detailed mechanism(s) is still poorly understood. Introduction The loss of cell contact inhibition (CCI) and cell–cell adhesion (CCA) results in cell malignant transformation Gadd45a, an 18.4-kDa acidic protein, is induced by (Vizirianakis et al., 2002;Kudo et al., 2004;Salon et al., various kinds of DNA-damaging agents and/or a 2004;Kato et al., 2005). Cadherins are implicated in this number of stresses associated with growth arrest process and specifically locate at intercellular adhesion (Kastan et al., 1992;Carrier et al., 1994). The roles of junctions (Gottardi and Gumbiner, 2001). b-Catenin is Gadd45a in the fundamental cellular processes have also essential for CCA and CCI by linking cadherins to been founded in growth control, maintenance of the actin cytoskeleton through its interaction with the genomic stability, DNA repair, cell cycle control and intracellular tail of cadherins (Kemler, 1993;Gumbiner, apoptosis via its interactions with proliferating cell 2000;Dietrich et al., 2002). The loss of cadherin/ nuclear antigen (PCNA), p21Cip1/Waf1, Cdc2/cyclin B1 b-catenin complex function leads to the disruption of complex, MTK1 and core histone protein (Fornace CCI and CCA, which is important for cell malignancy et al., 1988;Smith et al., 1994;Carrier et al., 1999; and cell invasion (Vizirianakis et al., 2002;Kudo et al., Hollander et al., 1999;Wang et al., 1999;Zhan et al., 2004;Salon et al., 2004;Kato et al., 2005). Therefore, cadherins/b-catenin complexes, CCA complexes, are Correspondence: Dr Q Zhan, State Key Laboratory of Molecular important in suppressing tumor progression. b-Catenin Oncology, Cancer Institute, Chinese Academy of Medical Sciences & is a central player not only in CCA but also in Wnt- Peking Union Medical College, Beijing 100021, PR China. signaling pathway (Cadigan and Nusse, 1997). Nor- E-mail: [email protected] 1These two authors contributed equally to this work. mally, the level of ‘free’ b-catenin in the cytoplasm is Received 23 October 2006;revised 12 February 2007;accepted 13 March rapidly degraded through the adenomatous polyposis 2007;published online 23 April 2007 coli protein (APC)/Axin complex ubiquitin-proteasome Gadd45a induces b-catenin distribution JJiet al 6397 system after being phosphorylated by glycogen synthase for examining CCI of both MEFs. As shown in Figure kinase (GSK)-3b (Aberle et al., 1997;Kitagawa et al., 1a and b, S phase was significantly decreased in 1999;Morin, 1999). Conventional Wnt signaling causes Gadd45a þ / þ MEFs with the increasing culture b-catenin uncoupling from APC degradation pathway, density (Po0.05), but not in Gadd45aÀ/À MEFs then accumulating in cytoplasm and translocating (P>0.05), indicating that cells with disrupted endogen- into the nucleus, where it forms a complex with the ous Gadd45a have lost cell contact-growth inhibition transcription factor T-cell factor/lymphoid enhancer and keep active proliferating property regardless of high factor (TCF/LEF) and transactivates the expression of culture densities. CCA assay revealed that the homo- Wnt targets such as c-Myc, cyclin D1, MMP7 and typic cell adhesion of Gadd45a þ / þ MEFs was more MMP26 (Brabletz et al., 1999;Shtutman et al., 1999; significant than that seen in Gadd45aÀ/À MEFs Barker and Clevers, 2000;Peifer and Polakis, 2000; (Figure 1c) (Po0.05). These results suggest that Marchenko et al., 2002). While upregulation of c-Myc Gadd45a might be involved in maintaining CCI/CCA, and cyclin D1 stimulates cell proliferation (van de and contribute to the suppression of cell malignancy. Wetering et al., 2002), and induced MMPs leads to the tumor metastasis (Brabletz et al., 1999;Marchenko et al., 2002). So, altered subcellular localizations of Gadd45a upregulates b-catenin by enhancing its stability b-catenin predispose cells to more malignant. Next, the effect of Gadd45a on expression of CCA- and Recent studies have revealed some molecular switches CCI-associated proteins was examined. Gadd45a pro- that help dictate whether b-catenin interacts with tein has been reported to be more abundant at high adhesive or transcriptional complexes. One mechanism culture densities than at low densities (Zhang et al., is the phosphorylation of b-catenin at tyrosine residues, 2003). In agreement with this observation, the expres- which results in a loss of CCA and an increase of nuclear sion of Gadd45a protein was enhanced with the b-catenin (Behrens et al., 1993;Piedra et al., 2003). increased culture densities in Gadd45a þ / þ MEFs Caveolin-1, a principle component of caveolae mem- but not detected in Gadd45aÀ/À MEFs. Strikingly, we branes (Glenney and Soppet, 1992;Rothberg et al., found that b-catenin, a CCA- and CCI-associated 1992), is also involved in regulating this ‘switch’. protein, was obviously upregulated in Gadd45a þ / þ Caveolin-1 has been shown to reinforce CCA and MEFs but not increased and even decreased in inhibit b-catenin/LEF pathway, as the result of its Gadd45aÀ/À MEFs following increased cell densities recruiting b-catenin to caveolae membranes (Galbiati (Figure 2a). It had been reported that parallel reductions et al., 2000). Here, we have addressed that Gadd45a is or elevations of b-catenin and Caveolin-1 expression involved in regulating such ‘switch’. Gadd45a plays a exist in response to a variety of cellular stimuli role in maintaining CCA/CCI, probably through indu- and represent an important mechanism to modulate cing the accumulation of b-catenin in cell membrane. cell–cell-mediated signaling, contributing to the Following cells’ exposure to UVR, Gadd45a accelerates CCA and CCI (Galbiati et al., 2000). Consistent with the degradation of b-catenin in a p53-dependent fashion this, our results demonstrated that Caveolin-1 expressed and, in turn, decreases the b-catenin/LEF transcrip- in parallel with b-catenin in MEFs. Both b-catenin and tional activity. These findings provide insights into the Caveolin-1 were upregulated by Gadd45a with the role of Gadd45a in suppressing cell malignancy. increased cell densities (Figure 2a). To further confirm these results, a tetracycline- regulated (tet-off) HeLa Gadd45a inducible cell line Results was used (Jin et al., 2002;Tong et al., 2005). The results in Figure 2b demonstrated that Gadd45a protein was Gadd45a is involved in maintaining the CCI and CCA highly induced after withdrawal of tetracycline, and in Gadd45a has been demonstrated to be an important agreement with our previous observations, Gadd45a molecule in suppressing carcinogenesis, but the under- induction substantially increased the expression of lying mechanism(s) need to be further elucidated. Some b-catenin and Caveolin-1. It also should be noted here biological events are involved in the cell malignant that we seeded the equal amounts of HeLa cells into transformation such as genomic instability, loss of the plates, withdrew the tetracycline at different times normal cellular senescence, increased cellular prolifera- and harvested these cells at the same time to insure tion, obtaining of colony formation property in soft that the cells from different plates were at the similar agar, and loss of CCI/CCA. It has been found that culture density. Taken together, these data indicate that MEFs derived from Gadd45a-null mice exhibits genomic b-catenin and Caveolin-1 are upregulated by Gadd45a, instability, single oncogene-mediated transformation, which might contribute to Gadd45a function in main- and increased cellular proliferation (Hollander et al., taining CCA/CCI. 1999;Smith et al., 2000). However, the correlation Much effort was then taken to explore the mechanism between Gadd45a and loss of CCI/CCA is still of b-catenin upregulation by Gadd45a. Initially, the unknown. So, we examined the CCA and CCI in both expression of b-catenin transcripts was examined. How- wild-type MEFs and Gadd45a knockout MEFs. MEFs ever, it was not changed with Gadd45a induction (data were seeded at different densities from low to high not shown), thus excluding the possibility of Gadd45a- and harvested till the highest-density cells reached mediated transcriptional regulation. Therefore, the confluent. Then, cell cycle distributions were analysed increased protein level of b-catenin by Gadd45a might

Oncogene Gadd45a induces b-catenin distribution JJiet al 6398 abMEF cell lines MEF cell lines Gadd45a +/+ Gadd45a −/− Gadd45a +/+ Gadd45a −/− Cell density G0-G1:31.88% G0-G1:41.30% G2-M: 15.89% G2-M: 10.86% 100% * S: 52.23% S: 47.94% Cell density 80% Counts Counts 60% 0 300 0 350 40% 0 1000 0 1000 DNA Content DNA Content 20% G0-G1:34.98% G0-G1:43.53% G2-M: 16.63% G2-M: 10.31% 0% S: 48.38% S: 46.15% ( S G2-M G0-G1) Counts Counts c Cell-cell adhesion

0 300 0 400 80% Gadd45a+/+ * 0DNA Content 1000 0DNA Content 1000 Gadd45a−/− 60% G0-G1:64.26% G0-G1:49.88% G2-M: 9.83% G2-M: 5.30% * S: 25.91% S: 44.83% 40%

Counts Counts 20% The index of The index Cell-cell adhesion

0 450 0 400 0% 0 1000 0 1000 60minutes 120minutes DNA Content DNA Content Figure 1 Gadd45a maintains CCA and CCI. (a) Cell cycle analysis of Gadd45a þ / þ and Gadd45aÀ/À MEFs at different densities by ﬂow cytometry. Both MEFs were seeded at different densities and harvested with trypsin for cell cycle analysis after monolayer of cells at high densities formed. (b) Histogram of all cell cycle phases for both MEFs at different densities. Data are presented as the mean7s.d. (: P>0.05;*: Po0.05). (c) Both MEFs were seeded into 24-well plates and ﬁxed after monolayer formed. Then homotypic MEFs were added into every well and incubated for 60 and 120 min for calculating CCA index. Data are presented as the mean7s.d. (*: Po0.05).

a MEF cell linesb Hela be due to its increased protein stability. As shown in Gadd45a +/+ Gadd45a −/− Gadd45a-inducible cell line Figure 2c, following addition of CHX, a translation- Cell density Tet-off 0 6 12 24 36 48(h) blocking agent, the degradation of b-catenin was more Gadd45a Gadd45a quickly in Gadd45a / MEFs than Gadd45a / β-catenin β-catenin À À þ þ MEFs. These results indicate that b-catenin stability is Gaveolin-1 Caveolin-1 enhanced by Gadd45a, resulting in the upregulation of Actin Actin b-catenin.

c MEF cell lines Gadd45a +/+ Gadd45a −/− CHX CHX Gadd45a enhances b-catenin stability by mediating its translocation to cell membrane

0 2 4 6 9(h) DMSO 0 2 4 6 9(h) DMSO β-catenin Given the classical model that b-catenin is phosphory- Caveolin-1 lated by GSK-3b and then undergoes ubiquitinated Actin degradation, we examined whether the increased stability of b-catenin by Gadd45a is correlated with GSK-3b. Figure 2 Gadd45a increases the b-catenin protein level by However, the interaction between b-catenin and GSK- enhancing its stability. (a) Gadd45a þ / þ and Gadd45aÀ/À MEFs were grown in 100-mm dishes and seeded at different densities 3b was similar in both MEFs (data not shown), sugges- (1 Â 104,2Â 104,4Â 104,8Â 104,1Â 105 and 2 Â 105). Cells were ting that b-catenin stability enhanced by Gadd45a might harvested till the highest density cells reached conﬂuent. Cell lysates not be related to GSK-3b. b-catenin plays dual roles in were prepared for analysing the expression of Gadd45a, b-catenin, stabilizing adhesive complexes when it localizes at the Caveolin-1. Actin was used as the loading control. (b) HeLa cell membrane and in modulating gene transcription Gad45a-inducible cell line was grown in DMEM in the presence of tetracycline at a concentration of 2 mg/ml. Then the tetracycline was when it localizes at the nucleus. Therefore, the b-catenin withdrawn and HeLa cells were cultured for different times in subcellular distributions greatly determine its biological DMEM without tetracycline till harvested. Cell lysates were roles. Since Gadd45a maintained CCA/CCI probably subjected to immunoblotting analysis with antibodies against through b-catenin, the immunoﬂuorescent staining Gadd45a, b-catenin, Caveolin-1 and actin. (c) Both exponentially growing MEFs were treated with 25 mg/ml CHX for the indicated assay was performed to examine b-catenin distribution. times. The immunoblotting analyses were conducted with anti- In both MEFs at low cell density, b-catenin was loca- bodies to b-catenin and Caveolin-1 and actin. lized in a punctate distribution over the entire cell

Oncogene Gadd45a induces b-catenin distribution JJiet al 6399 MEF cell lines acHela Gadd45a +/+ Gadd45a −/− Cell density Gadd45a-inducible cell line Nucleus ane

Membr CytoplasmaNucleus β -catenin Tet-off 0 48 0 48 0 48(h) Gadd45a Caveolin-1 β-catenin Caveolin-1

Gadd N-cadherin 45a PCNA Actin Nucleus

MEF cell lines Gadd45a +/+ Gadd45a −/− b Hela d Gadd45a-inducible cell line Cyclin D1 Tet-off 0 48(h) Nucleus Actin

e Hela β -catenin Gadd45a-inducible cell line Tet-off 0 6 12 24 36 48(h) Caveolin-1 Cyclin D1 Actin

Figure 3 Gadd45a induces the accumulation of b-catenin in membrane, and the reduction in cytoplasma and nucleus. (a and b) Subcellular distributions of b-catenin and Caveolin-1 were examined by immunofluorescence in Gadd45a þ / þ and À/À MEFs at low and high culture densities (a) and in HeLa Gadd45a inducible cell line after removal of tetracycline (b). Green fluorescence represents b-catenin and red fluorescence represents Caveolin-1. DAPI is included. In addition, Gadd45a induction was also examined by immunofluorescent assays. (c) Membrane, cytosol and nucleus were isolated from the HeLa Gadd45a inducible cell line after tetracycline withdrawal and subjected to IB with indicated antibodies. (d) Both MEFs were seeded at different densities and harvested till cells with highest density reached confluent. (e) HeLa Gad45a inducible cell line was grown in DMEM in the presence of tetracycline. Then the tetracycline was withdrawn and HeLa cells were cultured for different times in DMEM till harvested. (d and e) Cell lysates were prepared for analysing the expression of cyclin D1 and actin. surface and within the interior of the cell. When et al., 1999), its expression was examined. As shown Gadd45a þ / þ MEFs grew at the high density, where in Figure 3d, cyclin D1 was downregulated with Gadd45a was highly induced (Figure 3a), b-catenin the increased culture densities in both MEFs, while obviously redistributed and localized to membrane/ more significant reduction was seen in Gadd45a þ / þ areas of cell–cell contact. However, it remained un- MEFs. Additionally, cyclin D1 was also reduced in changed in Gadd45a À/À MEFs (Figure 3a) at the high HeLa Gadd45a-inducible cells following Gadd45a density. In HeLa Gadd45a inducible cell line, the induction (Figure 3e). These results go along with the localization of b-catenin in membrane was also more reduced b-catenin distribution in nucleus with Gadd45a obviously in most cells following the removal of expression. It was also worth noting that the Caveolin-1 tetracycline (Figure 3b). To further confirm the effect distribution was similar and it colocalized in the of Gadd45a on b-catenin distribution, the immuno- membrane with b-catenin following Gadd45a induction blotting analyses were carried out using the proteins (Figure 3a–c). isolated from cell membrane, cytoplasm and nucleus in HeLa Gadd45a inducible cells. The results of Figure 3c demonstrated that Gadd45a induction increased the Gadd45a-mediated b-catenin translocation is associated accumulation of b-catenin in the cell membrane. These with Caveolin-1 results suggest that enhanced b-catenin stability might Caveolin-1 has been implicated in recruiting proteins be due to b-catenin translocation to cell membrane by such as b-catenin to caveolae membranes (Galbiati Gadd45a, thus escaping from cytoplasmic degradation et al., 2000). The results presented in Figures 2 and 3 by APC complex. showed that both b-catenin and Caveolin-1 proteins Accompanied with the increased b-catenin accumula- were upregulated and colocalized in cell membrane tion in cell membrane, b-catenin distribution was following Gadd45a induction. Therefore, b-catenin reduced in cytoplasm and nucleus following Gadd45a accumulation in the membrane might be associated induction (Figure 3a–c). Given that cyclin D1 is trans- with Caveolin-1. As shown in Figure 4a, glutathione S- criptionally induced by b-catenin in nucleus (Shtutman transferase (GST)-pull down results demonstrated that

Oncogene Gadd45a induces b-catenin distribution JJiet al 6400 Gadd45a interacted with both b-catenin and Caveolin-1. through increasing the interaction between b-catenin Meanwhile, b-catenin was co-immunoprecipitated with and Caveolin-1 as an adaptor. more Caveolin-1 in HeLa Gadd45a-inducible cells after Gadd45a induction (Figure 4b). More interest- ingly, the interaction between b-catenin and Gadd45a Gadd45a facilitates the b-catenin degradation after UVR was enhanced after removal of tetracycline. These treatment in a p53-dependent manner results indicate that there are physical interactions After UVR, Gadd45a is identiﬁed to be essential for existing among the Gadd45a, b-catenin and Caveolin-1 negative regulation of cytoplasmic b-catenin in the proteins, and that b-catenin is capable of interacting keratinocytes by not only blocking the DNp63a expres- with more Caveolin-1 and Gadd45a following Gadd45a sion but also through association with the compo- induction. Owing to Caveolin-1’s role in recruiting nents of APC complex such as GSK-3b (Hildesheim b-catenin to cell membrane, Gadd45a might induce et al., 2004). Similarly, we found that b-catenin the translocation of b-catenin to membrane partially was substantially downregulated after UVR treatment in Gadd45a þ / þ MEFs, while a slight reduction of b-catenin was seen in Gadd45aÀ/À MEFs (Figure 5a). a b Hela We also excluded GSK-3b role in Gadd45a-mediated Gadd45a-inducible cell line degradation of b-catenin in MEFs, because the interaction between Gadd45a and GSK-3b remained same GST GST-Gadd45a Tet-off 0 48(h) Gadd45a β-catenin after UVR treatment (data not shown). Thus, additional mechanisms might exist in Gadd45a-mediated degrada- β-catenin Caveolin-1 tion of b-catenin in MEFs. Since p53 targets b-catenin

Caveolin-1 -catenim Gadd45a

β degradation (Ratovitski et al., 2001;Patturajan et al., IB: Actin IB: Actin

IP: IP: 2002) and Gadd45a contributes to p53 stabilization in µ µ (10 l lysates) (10 l lysates) response to DNA damage (Hildesheim et al., 2002;Jin Figure 4 The Gadd45a-mediated translocation of b-catenin is et al., 2003), b-catenin reduction might be correlated associated with Caveolin-1. (a) Total cellular proteins were with p53 induction in MEFs after UVR treatment. extracted from exponentially growing Gadd45a þ / þ MEFs for As showing in Figure 5a, p53 was signiﬁcantly induced GST-pulldown assay. GST-pulldown assay was performed with GST alone and GST-Gadd45a. GST-complexes were subjected to in both MEFs but exhibited much higher levels in immunoblotting assay using antibodies to b-catenin and Caveolin- Gadd45a þ / þ cells, and there was likely a negative 1. (b) HeLa Gadd45a inducible cells were harvested at 48 h after correlation between reduction of b-catenin and induc- tetracycline withdrawal. Co-immunoprecipitation with b-catenin tion of p53. This phenomenon was ensured by the antibody was performed and the precipitates were then subjected to immunoblotting analysis with antibodies to b-catenin, Caveolin-1 immuno-blotting analysis using the proteins isolated and Gadd45a. (a and b) Actin levels in 10 ml total material were from cell membrane, cytoplasm and nucleus of both used to input control. MEFs following UVR treatment (Figure 5b). To further

a MEF cell lines b MEF cell lines +/+ −/− Gadd45a Membrane Cytoplasma Nucleus +/+ − − +/+ − − +/+ − − 0 250 400 0 250 400 (J/m2) / / / Gadd45a − + − + − + − + − + − + UV Gadd45a β-catenin β-catenin p53 p53 Cyclin D1 Actin Actin d MEF cell lines c Gadd45a +/+ Gadd45a −/− −−− −−− HCT116 cell lines +++ +++ UV − + − −−+ − + − −−+ SB202190 +/+ −/− p53 −−+ −−+ −−+ −−+ DMSO 0 250 400 0 250 400 (J/m2) p53 p53 β-catenin β-catenin Actin Actin

Figure 5 Gadd45a facilitates the b-catenin degradation in a p53-dependent manner via p38 MAPK activation after UVR treatment. (a) Both exponentially growing MEFs were exposed to UVB at different doses (0, 250 and 400 J mÀ2) and cultured for 6 h. Then cell lysates of both MEFs were prepared for analysing the expression of Gadd45a, b-catenin, cyclin D1, p53 and actin. (b) Membrane, cytosol and nucleus were isolated from both MEFs after UVR treatment and subjected to immunoblotting with indicated antibodies. (c) After exposure to UVR, both exponentially growing HCT116 p53 þ / þ and À/À cells were cultured for 6 h. Cell lysates of both cell lines were then subjected to immunoblotting analysis with the antibodies to p53, b-catenin and actin. (d) Both Gadd45a þ / þ and À/À MEFs were treated with UVR in the presence of 10 mM SB202190 and followed by analysing the expression of b-catenin, p53 and actin.

Oncogene Gadd45a induces b-catenin distribution JJiet al 6401 verify the role of p53 in b-catenin degradation, we a MEF cell lines examined the b-catenin expression after UVR in Gadd45a+/+ Gadd45a−/− HCT116 p53 þ / þ and p53À/À cells, and observed a Cell density significant b-catenin degradation in the p53 þ / þ cells Gadd45a but not in p53À/À cells (Figure 5c). All these results p53 suggest that p53 likely acts as an important regulator in Actin the process of b-catenin degradation. p53 is normally subjected to phosphorylation by p38 MAPK, which b Hela leads to its increased stability and activation. Addition- Gadd45a-inducible cell line ally, Gadd45a contributes to p53 stability via p38 in Tet-off 0 6 12 2436 48(h) response to UVR (Hildesheim et al., 2002;Jin et al., Gadd45a 2003). Therefore, the role of Gadd45a in promoting p53 degradation of b-catenin after UVR might be correlated with p53 induction via p38 activation. The results Actin of Figure 5d showed that SB202190 (a p38-specific Figure 6 Gadd45a induces b-catenin accumulation is p53- inhibitor) inhibited significantly the induction of p53 independent. (a) Gadd45a þ / þ and Gadd45aÀ/À MEFs were after UVR and the degradation of b-catenin was greatly seeded at different densities and harvested till cells with highest disturbed following the suppressed p53 induction. These density reached confluent. Cell lysates were prepared for analysing the expression of Gadd45a, p53 and actin. (b) HeLa Gad45a- results indicate that Gadd45a-mediated b-catenin inducible cell line was grown in DMEM in the presence of degradation might require p38-activated p53 induction. tetracycline. Following the withdrawal of tetracycline, HeLa cells In addition, the results of Figure 5b showed that the were cultured for different times in DMEM till harvested. Cell downregulation of b-catenin after UVR was reflected lysates were subjected to immunoblotting analysis with antibodies mainly by its decreased cytoplasmic and nuclear against Gadd45a, p53 and actin. distribution, while b-catenin in cell membrane still kept the similar level. These suggest that Gadd45a induction most compelling finding on the biological function of following UVR does not disturb the role of b-catenin in Gadd45a is that its defect is closely associated with CCA/CCI, but substantially affect the transcriptional tumorigenesis and the development of cell malignancy. activity of b-catenin, which was further reflected by However, little is known about how Gadd45a contri- downregulation of oncogenic cyclin D1 expression butes to the suppression of malignant-cell development. (Figure 5a). In the current study, we demonstrate that Gadd45a is greatly involved in maintaining CCA/CCI, which might contribute to its tumor suppressive function. Gadd45a upregulating b-catenin is p53-independent Our initial observations showed that Gadd45a’s func- Since Gadd45a targets b-catenin degradation via p38- tions in maintaining the CCA/CCI might be correlated activated p53 induction, we initially thought that its with b-catenin. Gadd45a induction upregulated function in upregulating b-catenin might be p53-depen- b-catenin and the disruption of endogenous Gadd45a dent. Thus, p53 expression in both Gadd45a þ / þ and resulted in b-catenin downregulation. b-Catenin is a Gadd445aÀ/À MEFs at different culture densities was central player in CCA and its downregulation can lead analysed. To our surprise, the results showed that there to the disruption of CCA/CCI and cell malignancy were no obvious changes of p53 expression in both cell (Vizirianakis et al., 2002;Kudo et al., 2004). In addition, lines (Figure 6a). In addition, p53 expression was also b-catenin is a key component of the Wnt signal trans- examined in HeLa Gadd45a-inducible cell line. The duction pathway and activation of Wnt signaling leads results in Figure 6b demonstrated that Gadd45a protein to the nuclear translocation of b-catenin, which induces was highly induced, but p53 expression remained at gene transcription such as cyclin D1 and contributes to similar levels after withdrawal of tetracycline. Taken cell malignancy (Cadigan and Nusse, 1997). Interest- together, these observations suggest that b-catenin can ingly, following Gadd45a induction b-catenin accumu- be upregulated by Gadd45a regardless of p53 status. lation mainly occurred in membrane, coupled with significant reduction in the cytoplasm and nucleus, which was reflected by decreased expression of cyclin D1. The Discussion accumulation of b-catenin in membrane by Gadd45a bridges the cadherins and actin cytoskeleton to conform Gadd45a gene was initially isolated from Chinese the more stable CCA complex, which is important to Hamaster ovarian cells based on its inducible expression maintain CCA/CCI and to suppress the tumor metas- after UVR treatment. Subsequently, it was found to be tasis. The reduction of b-catenin in the cytoplasm and induced by a wide spectrum of DNA damaging agents nucleus is expected to reduce the transcription of Wnt and growth-arrest signals. Gadd45a has been found to target genes, which can lead to the suppression of cell play important roles in the control of cell cycle check- malignance and metastasis. All these can greatly account point, apoptosis and DNA repair (Fornace et al., 1988; for tumor suppressor function of Gadd45a. Smith et al., 1994;Carrier et al., 1999;Hollander et al., The b-catenin subcellular distributions greatly deter- 1999;Wang et al., 1999;Zhan et al., 1999;Jin et al., mine its biological roles in either maintenance of CCA/ 2000;Tong et al., 2005;Zhan, 2005). Currently, the CCI or oncogenic transcriptional regulation. Gadd45a

Oncogene Gadd45a induces b-catenin distribution JJiet al 6402 might be involved in regulating this ‘switch’ partially found that the reduction of b-catenin in MEFs after UV through Caveolin-1. Caveolin-1 stabilizes b-catenin by was significantly correlated with p38-mediated p53 blocking the interaction between b-catenin and GSK-3b, activation. This finding is supported by the previous and recruits b-catenin to plasma membrane (Galbiati report that p53 can induce b-catenin degradation via et al., 2000). In our investigations, Caveolin-1 and blocking DNp63a expression (Ratovitski et al., 2001; b-catenin were really parallelly induced and colocalized Patturajan et al., 2002). The employment of a p38 in the plasma membrane after Gadd45a induction. inhibitor-SB202190 confirmed this result for SB202190 Moreover, b-catenin interacted with more Caveolin-1 inhibiting significantly the p53 induction and b-catenin following Gadd45a induction. Then Gadd45a likely degradation after DNA damage. As a consequence, the mediates the cell membrane translocation of b-catenin degradation of b-catenin by Gadd45a after UVR via Caveolin-1. Our findings exclude the involvement of treatment was coupled with interruption of b-catenin GSK-3b in Gadd45a-mediated b-catenin upregulation transcriptional activity, as reflected by oncogenic cyclin and translocation to membrane since the interaction D1 downregulation. This might be one of the major between b-catenin and GSK-3b was not affected after reasons why Gadd45a-null mice have an increased Gadd45a induction (data not shown). Therefore, it is frequency of UVR-induced tumors than the Gadd45a- reasonably interpreted that b-catenin reduction in wild mice. cytoplasma and nucleus might be due to Caveolin-1 However, the role of Gadd45a in regulating CCA/ recruiting b-catenin to plasma membrane. In addition, CCI is likely independent of cellular p53 function. This we also found that the interaction of b-catenin and was demonstrated by several lines of evidence, including Gadd45a was enhanced following Gadd45a induction, the observations that induction of Gadd45a enhanced which coupled with the increased interaction of the cell membrane accumulation of b-catenin in HeLa b-catenin and Caveolin-1. Meanwhile, a physical inter- cell cells, where p53 protein is inactivated by the pre- action was seen to exist between Gadd45a and Caveolin- sence of HPVE6. In addition, expression of p53 protein 1. These findings further suggest that accumulation of was seen to remain at similar levels with different culture b-catenin in cell membrane is likely achieved by densities in both MEFs, where b-catenin degradation Gadd45 acting as an adapter that facilitates an effec- was confirmed to relate to the Gadd45a expression. tive interaction between b-catenin and Caveolin-1. Such In summary, we have shown that Gadd45a protein is Gadd45a-mediated interaction between b-catenin and involved in regulating b-catenin activity through two Caveolin-1 might represent an important mechanism to different ways, including p53-independent and -depen- modulate CCA/CCI, the loss of which contributes to the dent manners. First, Gadd45a induces the accumula- cell malignancy. tion of b-catenin in cell membrane to maintain the The interactions between Gadd45a and different CCA/CCI, probably through its enhanced interaction important cellular proteins (GSK-3b, PP2A or Caveo- between b-catenin and Caveolin-1. This is followed lin-1/b-catenin) are of importance in inhibiting cell by the reduction of b-catenin in cytoplasma and transformation. These interactions, such as Gadd45a/ nucleus. Second, Gadd45a accelerates the degradation GSK-3b or Gadd45a/Caveolin-1, might work together of b-catenin in a p53-dependent fashion after UVR to prevent cells from tumorigenesis and development of treatment via p38 MAPK. These findings have defined a malignancy. The Gadd45a interaction with GSK-3b novel link that connects Gadd45a to CCA/CCI, further pathway may result in activation of APC and causes supporting the role of Gadd45a in negative regulation of degradation of cytosol b-catenin. This action inhibits the carcinogenesis and tumor malignancy. b-catenin translocation to nucleus since nuclear b-catenin can act as an oncogenic transcription factor. Similarly, the Gadd45a interaction with caveolin-1/b-catenin Materials and methods causes the accumulation of b-catenin in membrane, which in turn, reduces the cytoplasmic and nuclear Cell culture and treatment b-catenin, these actions also inhibit the nuclear trans- Gadd45a þ / þ and À/À MEFs were kindly provided by Professor Albert J Fornace and M Christine Hollander and location of b-catenin. More importantly, the recruitment grown at 371C in a humidified atmosphere with 5% CO2. Cell of b-catenin to membrane leads to increased formation cultures were maintained in Dulbecco’s modification of Eagle’s of E-cadherin/caveolin-1/b-catenin complexes, which are medium (DMEM) with 10% fetal bovine serum (FBS). The critical for maintenance of CCA/CCI. Disruption of maintaining of Gadd45a tet-off inducible HeLa cell line and these interactions may result in the development of cell the induction of Gadd45a were performed as described malignancy. previously (Jin et al., 2002). Gadd45a has previously been reported to regulate For UV treatment, exponentially growing cells were rinsed negatively the cytoplasmic b-catenin after UVR treat- with PBS and irradiated with UVB at doses between 250 and 2 ment, probably through Gadd45a-suppressed expres- 400 J m for the indicated time. After cell exposure, fresh sion of DNp63a and Gadd45a association with GSK-3b medium was added and the cells were cultured for 6 h till harvest. p38 inhibitor (10 mM SB202190) was used before (Hildesheim et al., 2004). However, in the current study, 2 days in response to UVR and cells were cultivated for 6 h in it appears that GSK-3b is not mainly involved in the presence of the inhibitor till harvested. CHX (25 mg/ml, Gadd45a-accelerated b-catenin degradation as we did Sigma-Aldrich, St Louis, MO, USA) was added to exponen- not observe the increased interaction between Gadd45a tially growing cells to inhibit protein synthesis and incubated and GSK-3b after UVR (data not shown). Indeed, we at 371C for different times till harvested.

Oncogene Gadd45a induces b-catenin distribution JJiet al 6403 Plasmid clones and antibodies Flow cytometry GST-Gadd45a was constructed by inserting Gadd45a full- Cells were removed from plates with trypsin and pelleted with length cDNA into the XhoI site of the pGEX-5X-1 vector centrifuge 1000 g for 5 min. After washing three times with (Tong et al., 2005). Antibodies of Gadd45a, b-catenin, PBS, cell pellets were resuspended with 10% formaldehyde and Caveolin-1, cyclin D1, GSK-3b, N-cadherin and PCNA were incubated for 10 min. Next, cells were pelleted again and commercially procured from Santa Cruz (Santa Cruz, CA, washed. At last, cells were resuspended in PBS containing 3% USA). p53 and actin antibodies were from Sigma-Aldrich. serum and 70% ethanol. After at least 1 h at 41C, the cells were stained with propidium iodide and DNA content was Protein preparation and western blotting assay measured by flow cytometry. For whole-cell protein extraction, cells were lysed in 1 Â PBS, 1% Nonidet P-40, 2 mg/ml aprotinin, 2 mg/ml leupeptin and Cell–cell adhesion assay 50 mg/ml phenylmethylsulfonyl fluoride (PMSF). Cell lysates This assay was performed as described previously (Richard 1 were kept on ice for 40 min and centrifuged at 14 000 g at 4 C et al., 1995;Leali et al., 2001), with minor modifications. for 30 min. Then supernatants were collected as total protein. Briefly, MEF Gadd45a þ / þ and Gadd45aÀ/À cells were For preparation of membrane, cytoplasmic and nuclear seeded in 24-well plates at about 50 000 cells/cm2. After 24– protein, cells were collected and suspended in cold buffer 1 48 h, cell monolayers were fixed with 3% glutaraldehyde in M M M M M (1 m KCl, 5 m NaCl, 3 m MgCl2,50m Hepes, 1 m PBS for 2 h at 4 C. Fixation was stopped with 0.1 M glycine, dithiothreitol (DTT), 0.5 mg/ml Leupeptin, 20 mM PMSF). and cells were washed extensively with PBS. Then, 400 ml After freezing at liquid nitrogen and thawing at room homotypic cells (105 cells/ml) were added to monolayers in temperature for three times, the mixture was centrifuged at serum-free medium. After 60 and/or 120 min of incubation at 1 5000 g for 10 min at 4 C. The pellets were nucleus and cells 371C, unattached cells were collected by gentle washing with unlysed. The supernatant (cytosol protein and membrane PBS, and the washed cells were counted. The cell number of protein) were immediately centrifuged at 14 000 g for 10 min. CCA was calculated using the following formula: CCA Then the supernatant (cytoplasmic protein) was collected and index ¼ (total number of added cellsÀnumber of cells being insoluble pellets were centrifuged at 14 000 Â g for 10 min washed)/total number of added cells. again after being resuspended with cold buffer 2 (1 mM KCl, 5mM NaCl, 3 mM MgCl2,50mM Hepes, 1 mM DTT, 0.5 ug/ml Leupeptin, 20 mM PMSF, 1 mM ethylene glycol tetraacetic acid Immunofluorescent staining (EGTA)). The pellets (membrane protein) were dissolved in Cells were placed in dishes containing a glass coverslip. After buffer 3 (0.5 mg/ml leupeptin, 20 mM PMSF, 50 mM Tris-HCl 24–48 h, cells grown on glass coverslips were fixed with (pH 7.0)). The extraction of nuclear protein from the pellets of methanol for 10 min at room temperature and washed with nucleus and cells unlysed was performed as described PBS. Next, cells were subjected to fixation with cold methanol previously (Fan et al., 2002). at À201C. After incubation with associated antibody for 1 h at Cellular protein extracted was boiled and separated by 12% room temperature, plates were washed and incubated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis FITC-conjugated goat anti-mouse immunoglobulin (Ig)G or (SDS–PAGE) and transferred to nitrocellulose membranes. with TRITC-conjugated goat anti-rabbit IgG. After being Then, membranes were incubated with the indicated primary washed with PBS, cells were stained with DAPI and examined antibodies and anti-mouse or anti-rabbit secondary antibodies under a fluorescence microscope. conjugated to horseradish peroxidase for enhanced chemilu- minescence (Amersham, Arlington, Height, IL, USA) detec- Statistics tion of the signals. All experiments were performed and repeated at least three times. Data were presented as the mean7s.d. (standard GST-pulldown and immunoprecipitation deviation) and analysed with SPSS software. The differences The expression and extraction of GST-Gadd45a fusion protein of S phase of cell cycle in different culture densities and was performed as described previously (Tong et al., 2005). the difference of CCAI in MEFs were analysed by t-test. Next, the glutathione-agarose beads (Sigma-Aldrich) were A P-value of o0.05 was considered as significant. mixed with supernatant solution of lysed bacteria at 41C overnight. Then glutathione-agarose bead-conjugated GST fusion proteins were incubated with 1 mg lysates at 41C for Acknowledgements 12 h. Then the mixtures were centrifuged and 10 ml supernatant was reserved to analyse actin as control. After being washed We thank Professor Albert J Fornace of University of Harvard with lysis buffer for 3–5 times gently, the glutathione-agarose and M Christine Hollander of NIH for the immortalized beads incubated was boiled in SDS–PAGE loading buffer and Gadd45aÀ/À and matching þ / þ MEF cells and Professor analysed with the indicated antibodies. Bert Vogelstein of Johns Hopkins for HCT 116 p53 À/À and For immunoprecipitation, cellular lysates were incubated matching þ / þ cells. This work was supported in part by the with 10 ml of antibody and 20 ml of protein A/G agarose beads National Fundamental Research Program of China (Santa Cruz) at 41C for 8 h. The agarose beads immunocom- (2002CB513101) and the National Natural Science Founda- plexes were treated as described in GST-pulldown assay. tion of China (30225018 and 30400074).

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