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Genes to Cells (2007) 12, 461-471. doi:10.1111/j.1365-2443.2007.01065.x
© 2007 Blackwell Publishing or its licensors
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Oxidative stress induces p53-dependent apoptosis in hepatoblastoma cell through its nuclear translocation

 Hideki Yamamoto1,2, Toshinori Ozaki1, Mitsuru Nakanishi1, Hironobu Kikuchi1, Kaori Yoshida1, Hiroshi Horie3, Hiroyuki Kuwano2 and Akira Nakagawara1,*

1 Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba 260-8717, Japan
2 Department of General Surgical Science (Surgery 1), Gunma University, Graduate School of Medicine, Maebashi 371-8511, Japan
3 Chiba Children's Hospital, Chiba 266-0007, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Hepatoblastoma (HBL) is the most common malignant liver tumor in children. Since tumor suppressor p53 is rarely mutated in HBL, it remains unknown whether p53 could contribute to the hepatocarcinogenesis. In the present study, we have found for the first time that, like neuroblastoma (NBL), wild-type p53 was abnormally accumulated in the cytoplasm of the human HBL-derived Huh6 cells. In accordance with this notion, immunohistochemical analysis demonstrated that p53 is largely expressed in cytoplasm of human primary HBLs. In response to the oxidative stress, Huh6 cells underwent apoptotic cell death in association with the nuclear translocation of p53 and the transactivation of its target gene implicated in apoptotic cell death. siRNA-mediated knockdown of the endogenous p53 conferred the resistance of Huh6 cells to oxidative stress. Intriguingly, histone deacetylase inhibitor (nicotinamide) treatment strongly inhibited the oxidative stress-induced nuclear translocation of p53 as well as the p53-dependent apoptosis in Huh6 cells. In contrast to the previous observations, the cytoplasmic anchor protein for p53 termed Parc had undetectable effect on the cytoplasmic retention of p53. Collectively, our present results suggest that the abnormal cytoplasmic localization of p53 might contribute at least in part to the development of HBL.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Hepatoblastoma (HBL) is one of the most frequent malignant liver tumors of childhood. Indeed, its incidence is higher than that of hepatocellular carcinoma (HCC) in children. HBL arises from the hepatic precursor cells and displays a morphological similarity to the immature hepatocytes of the developing liver. In a sharp contrast to HCC, which is associated with hepatitis virus infection (Llovet et al. 2003), it has been shown that the incidence of HBL is highly elevated in patients with familial adenomatous polyposis (FAP), which carry germ-line mutations in the APC (adenomatous polyposis coli) tumor suppressor gene (Hughes & Michels 1992; Nagase & Nakamura 1993). APC protein forms a cytoplasmic multiprotein complex involved in the Wnt signaling pathway, which regulates the stability of ß-catenin (Henderson & Fagotto 2002). Although APC is rarely mutated in sporadic HBL, accumulating evidence demonstrated that the frequent mutations or deletions of ß-catenin at hot-spot regions within the exon 3 encoding its degradation targeting box are detectable in HBL, suggesting that the abnormal nuclear accumulation of the stabilized ß-catenin which collaborates with Tcf/Lef complex plays a central role in the genesis of HBL (Koch et al. 1999). Consistent with this notion, Takayasu et al. (2001) revealed that ß-catenin mutation is significantly correlated with the up-regulation of its target genes, including cyclin D1 and fibronectin. However, Harada et al. (2002, 2004) described that ß-catenin mutation alone is not sufficient for the hepatocarcinogenesis, indicating that the additional mutations or epigenetic changes might be required for the genesis of HBL. The detailed molecular mechanism(s) behind the pathogenesis and development of HBL remains unknown.

The p53 tumor suppressor is a nuclear transcription factor, which has an ability to transactivate various p53-target genes implicated in the regulation of G1 cell cycle arrest and/or apoptosis such as p21WAF1, MDM2, Bax and NOXA (Prives & Hall 1999; Sionov & Haupt 1999; Vousden & Lu 2002). The importance of p53 in the tumorigenesis has been emphasized by the observations showing that p53 mutation is detected in more than half of all human tumors (Hollstein et al. 1991; Vogelstein et al. 2000). The tumor-suppressive activity of p53 is dependent on its sequence-specific transactivation function. Indeed, the vast majority of p53 mutations are found within its central sequence-specific DNA-binding domain. Under normal conditions, p53 is a short-lived protein whose expression levels are kept extremely low. MDM2 acts as an E3 ubiquitin protein ligase for p53, and promotes its ubiquitination followed by degradation by 26S proteasome (Haupt et al. 1997; Honda et al. 1997; Kubbutat et al. 1997). Recently, it has been demonstrated that, like MDM2, Pirh2 and COP1 target p53 for degradation by 26S proteasome in an ubiquitin-dependent manner (Leng et al. 2003; Dornan et al. 2004). In response to genotoxic stresses, p53 is induced to be accumulated in cell nucleus through its phosphorylation at multiple sites, including Ser-15, Ser-20 and Ser-46, and exerts its pro-apoptotic activity (Sionov & Haupt 1999; Vousden & Lu 2002). In addition to the NH2-terminal phosphorylation of p53, p300/CBP (CREB-binding protein) with the histone acetyltransferase (HAT) activity binds to the NH2-terminal region of p53, mediates the acetylation of its COOH-terminal region and thereby enhances its activity (Gu & Roeder 1997). Thus, the post-translational modifications of p53 enhance its transcriptional as well as pro-apoptotic ability.

In contrast to other human tumors, p53 is infrequently mutated in certain human tumors such as neuroblastoma (NBL) and HBL (Vogan et al. 1993; Chen et al. 1995; Ohnishi et al. 1996; Kusafuka et al. 1997), indicating that p53 plays no role in the genesis and development of these tumors. However, this viewpoint has been challenged by the observations that the wild-type p53 is abnormally accumulated in the cytoplasm of NBLs (Moll et al. 1995). These findings strongly suggest that the nuclear exclusion of wild-type p53 might represent one non-mutational mechanism of p53 inactivation. In addition to NBL, wild-type p53 is abnormally sequestered in the cytoplasm in certain human tumors, including breast and colon cancers (Moll et al. 1992; Bosari et al. 1995). Although the detailed molecular mechanism(s) of the cytoplasmic accumulation of wild-type p53 remains unclear, Nikolaev et al. (2003) described that Parc (p53-associated parkin-like cytoplasmic protein) interacts with p53 in cytoplasm and inhibits its nuclear translocation.

In the present study, we have found that wild-type p53 is abundantly expressed in human primary HBLs and HBL-derived Huh6 cells. In response to oxidative stress, p53 was induced to be translocated into cell nucleus of Huh6 cells, and Huh6 cells underwent apoptotic cell death. Furthermore, nicotinamide treatment abolished the oxidative stress-induced nuclear translocation of p53, thereby inhibiting the p53-dependent apoptotic cell death.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Cytoplasmic expression of p53 in human HBLs

As previously described (Vogan et al. 1993; Moll et al. 1995), p53 is rarely mutated in human primary NBLs, and predominantly expressed in cytoplasm. Similar to NBLs, it has been shown that p53 is infrequently mutated in human primary HBLs (Chen et al. 1995; Ohnishi et al. 1996); however, its subcellular localization remains unclear. Then, we sought to examine the subcellular localization of p53 in surgically resected specimens of primary HBLs by immunohistochemistry. As shown in Fig. 1A, p53 immunoreactivity was detectable largely in cytoplasm of tumor cells, suggesting that, like NBLs, p53 might lack its intact function due to its abnormal cytoplasmic localization in HBLs. To further confirm these observations, we examined the subcellular distribution of p53 in HBL-derived Huh6 (Doi 1976) and HCC-derived HepG2 cells (Aden et al. 1979). As described (Bressac et al. 1990; Hsu et al. 1993), HepG2 cells carry wild-type p53. Our sequence analysis revealed that p53 expressed in Huh6 cells has a wild-type structure (data not shown). Huh6 and HepG2 cells were biochemically fractionated into cytoplasmic and nuclear fractions, and subjected to Western blotting with the anti-p53 antibody. {alpha}-tubulin and Lamin B were used as cytoplasmic and nuclear markers, respectively. Under our experimental conditions, E-cadherin, which is one of the membrane marker, was detected in the cytoplasmic fraction (data not shown). As shown in Fig. 1B, p53 was undetectable in each fraction of HepG2 cells, whereas p53 was largely expressed in cytoplasm of Huh6 cells, which was consistent with our immunohistochemical analysis of primary HBLs. It is worth noting that p53 is constitutively phosphorylated at Ser-15 in Huh6 cells in the absence of DNA damage. To rule out a possibility that the subcellular localization of p53 could be regulated by active nuclear export in Huh6 cells, Huh6 cells were treated with the nuclear export inhibitor leptomycin B (LMB). As shown in Fig. 1C, LMB had undetectable effect on the subcellular distribution of p53 in Huh6 cells.


Figure 1
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  		Figure 1  Cytoplasmic localization of p53 in HBL cells. (A) Immunohistochemical analysis. Sections (4 µm thick) of two primary hepatoblastoma tissues (case 1 and 2) were stained with the anti-p53 antibody. Note the positive signals in the cytoplasm of most tumor cells. (B) p53 is abundantly expressed in cytoplasm of Huh6 cells. Huh6 and HepG2 cells were biochemically fractionated into cytoplasmic (C) and nuclear (N) fractions as described under Experimental procedures. Equal amounts of cytoplasmic and nuclear extracts were subjected to Western blotting with the anti-p53 or with the anti-phosphorylated form of p53 at Ser-15. {alpha}-tubulin and Lamin B were used for the cytoplasmic and nuclear markers, respectively. (C) Leptomycin B has undetectable effect on the subcellular localization of p53. Huh6 cells were treated with or without 20 ng/mL of Leptomycin B (LMB). Six hours after the treatment, cells were fractionated into cytoplasmic (C) and nuclear (N) fractions, and subjected to Western blotting with the indicated antibodies.

 
Induction of apoptosis by oxidative stress in HBL-derived and HCC-derived cell lines

As described (Lluis et al. 2005), oxidative stress induced apoptotic cell death in hepatocytes. To examine a possible effect of the oxidative stress on Huh6 and HepG2 cells, these cells were treated with H2O2, and their cell viability was assessed by MTT cell survival assay. As shown in Fig. 2 A,B, Huh6 and HepG2 cells underwent apoptosis in response to H2O2 in a dose-dependent and a time-dependent manner. Since p53 plays a central role in the DNA damage-induced apoptosis (Prives & Hall 1999; Sionov & Haupt 1999), we examined the changes in endogenous p53 protein levels following treatment with H2O2. As shown in Fig. 2C, p53 was expressed at low levels in HepG2 cells without H2O2. Following exposure to H2O2, p53 was induced to be accumulated in association with a remarkable increase in the amounts of p53 phosphorylated at Ser-15. RT-PCR analysis revealed that the transcription levels of pro-apoptotic NOXA, which is one of the p53-target genes, are elevated in response to H2O2 treatment. In contrast, the amounts of total p53 remained almost constant and p53 was constitutively phosphorylated at Ser-15 in Huh6 cells regardless of H2O2 treatment (Fig. 2D). Under our experimental conditions, however, the expression levels of NOXA were increased in Huh6 cells exposed to H2O2, suggesting that p53 might contribute to the oxidative stress-mediated apoptotic cell death in Huh6 cells.


Figure 2
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  		Figure 2  Effect of H2O2 treatment on Huh6 and HepG2 cell lines. (A, B) MTT cell survival assays. Huh6 (filled boxes) and HepG2 cells (open boxes) were exposed to H2O2 at the indicated concentrations for 6 h. After the treatment with H2O2, their cell viability was assessed by MTT assays (A). Similarly, Huh6 and HepG2 cells were treated with 1 mM of H2O2 for the indicated time periods, and their cell viability was examined by MTT assays (B). (C, D) Western blot analysis. HepG2 (C) and Huh6 cells (D) were treated with 1 mM of H2O2 for the indicated periods of time. Thereafter, whole cell lysates were prepared, and subjected to Western blotting with the anti-p53 (1st panel) or with anti-phospho-p53 at Ser-15 (2nd panel). Expression of actin was used to control equal protein loading (3rd panel). Alternatively, total RNA was extracted from cells treated with H2O2, and analyzed by RT-PCR for the expression of NOXA (4th panel). GAPDH was used to normalize (5th panel).

 
p53 plays a critical role in the H2O2-mediated apoptosis in Huh6 cells

To examine whether p53 could play an important role in the regulation of H2O2-dependent apoptosis in Huh6 cells, Huh6 cells were stably transfected with the expression plasmid encoding siRNA against p53 or with its control plasmid. Two weeks after the selection with G418 (at a final concentration of 400 µg/mL), we finally established several p53-knockdown cell clones as well as control cell clones (Fig. 3). We then investigated their sensitivity to H2O2 by TUNEL staining. Five hours after the treatment with H2O2 at a final concentration of 1 mM, V-2, V-3, P-9 and P-10 cells were subjected to TUNEL staining to identify the apoptotic cells. Cell nuclei were stained with DAPI. As shown in Fig. 4, exposure of V-2 and V-3 cells to H2O2 resulted in a significant increase in a number of TUNEL-positive cells, whereas two to threefold decrease in a number of cells with apoptotic nuclei was observed in P-9 and P-10 cells in response to H2O2. Similar results were also obtained in the other cell clones (data not shown). These results strongly suggest that p53 contributes at least in part to the H2O2-mediated apoptotic cell death in Huh6 cells.


Figure 3
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  		Figure 3  siRNA-mediated knockdown of p53 in Huh6 cells. Huh6 cells were stably transfected with the empty plasmid (V1–V3) or with the expression plasmid encoding siRNA against p53 (P1–P13), and cultured in the presence of G418 (at a final concentration of 400 µg/mL) for 2 weeks. Whole cell lysates prepared from the indicated cell clones and the parental Huh6 cells (Control) were analyzed by Western blotting for the expression levels of the endogenous p53 and actin.

 

Figure 4
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  		Figure 4  p53 is required for the H2O2-induced apoptosis in Huh6 cells. TUNEL staining. The indicated cell clones were treated with H2O2 at a final concentration of 1 mM or left untreated. Five hours after the treatment with H2O2, apoptotic cells were identified by TUNEL assay as described under Experimental procedures. The cell nuclei were stained with DAPI (left panels). The number of TUNEL-positive cells was counted, and expressed as a percentage of the total number of cells examined (right panels). H2O2-induced apoptosis was significantly different in cells expressing siRNA against p53 as compared with control cells. P < 0.05.

 
H2O2-mediated nuclear translocation of p53

It is well documented that p53 is induced to be accumulated in cell nucleus in response to various DNA damaging agents, including cisplatin (CDDP) (Fritsche et al. 1993). In accordance with this notion, CDDP treatment stimulated the nuclear accumulation of p53 in Huh6 cells in a time-dependent manner, whereas the amounts of cytoplasmic p53 remained unchanged regardless of CDDP treatment (Fig. 5A). Additionally, Huh6 cells underwent apoptotic cell death in response to CDDP (Fig. 5B). Intriguingly, there existed an inverse relationship between the amounts of cytoplasmic and nuclear p53 in response to H2O2 (Fig. 5C). Indirect immunofluorescent staining indicated that p53 is largely expressed in cytoplasm of Huh6 cells, whereas p53 accumulates in cell nucleus in response to H2O2 (Fig. 5D). Thus, it is likely that the H2O2-mediated nuclear translocation of p53 might be one of the molecular mechanisms underlying the H2O2-dependent apoptosis in Huh6 cells.


Figure 5
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  		Figure 5  Nuclear translocation of p53 in response to H2O2. (A) CDDP-induced nuclear accumulation of p53. Huh6 cells were treated with 20 µM of CDDP. At the indicated time points after the treatment, cells were collected and fractionated into cytoplasmic and nuclear fractions, followed by Western blotting with the indicated antibodies. (B) CDDP-induced apoptotic cell death of Huh6 cells. At the indicated time periods after the exposure to CDDP (20 µM), Huh6 cells were subjected to MTT assay. (C) H2O2-mediated nuclear translocation of p53 in Huh6 cells. At the indicated time periods after the treatment with H2O2 (1 mM), Huh6 cells were fractionated into cytoplasmic and nuclear fractions, and subjected to Western blotting with the indicated antibodies. (D) Indirect immunofluorescence. Huh6 cells were treated with H2O2 (1 mM) or left untreated, and stained with anti-p53 antibody (red). Cells were costained with DAPI (blue) to reveal cell nucleus.

 
Parc has an undetectable effect on the cytoplasmic retention of p53

The nuclear localization of p53 is critical for its transcriptional activity as well as apoptosis-inducing function. Recently, Nikolaev et al. (2003) have found that a Parkin-like ubiquitin ligase termed Parc acts as cytoplasmic anchor protein to block nuclear localization of p53. To ask whether Parc could be involved in the cytoplasmic retention of p53 in Huh6 cells, we examined the interaction between p53 and Parc by immunoprecipitation experiments. Whole cell lysates prepared from Huh6 cells were immunoprecipitated with normal mouse serum (NMS) or with the anti-p53 antibody, and the immunoprecipitates were analyzed by Western blotting with the anti-Parc antibody. Consistent with the previous results (Nikolaev et al. 2003), the anti-p53 immunoprecipitates contained the endogenous Parc (Fig. 6A). We then examined a possible effect of Parc on the subcellular distribution of p53. For this purpose, Huh6 cells were transiently transfected with siRNA against Parc or with the control siRNA. Twenty-four hours after transfection, total RNA and cytoplasmic/nuclear fractions were prepared and subjected to RT-PCR and Western blotting with the anti-Parc antibody, respectively. As shown in Fig. 6B, siRNA against Parc successfully reduced the expression levels of the endogenous Parc. Unexpectedly, siRNA-mediated knockdown of the endogenous Parc had a negligible effect on the subcellular localization of p53 in Huh6 cells as examined by Western blotting (Fig. 6C).


Figure 6
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  		Figure 6  Parc has an undetectable effect on the subcellular localization of p53. (A) Parc is associated with p53 in Huh6 cells. Whole cell lysates prepared from Huh6 cells were immunoprecipitated with normal mouse serum (NMS) or with the anti-p53 antibody. The immunoprecipitates were analyzed by Western blotting with the anti-Parc antibody. Input corresponds to 5% of the whole cell lysates used in this assay. (B) siRNA-mediated knockdown of the endogenous Parc. Huh6 cells were transiently transfected with siRNA against Parc or with the control siRNA. Twenty-four hours after transfection, total RNA and cytoplasmic (C) nuclear (N) fractions were prepared and processed for RT-PCR and Western blotting with the anti-Parc antibody, respectively. (C) siRNA-mediated knockdown does not lead to the nuclear access of p53. Huh6 cells were transiently transfected with siRNA against Parc or with the control siRNA. Twenty-four hours after transfection, cells were fractionated into cytoplasmic (C) and nuclear fractions (N), and subjected to Western blotting with the indicated antibodies.

 
Nicotinamide treatment inhibits the H2O2-mediated nuclear translocation of p53 and apoptosis

Recently, it has been shown that the chemical modifications including acetylation regulate the subcellular localization of p53 (Kawaguchi et al. 2006). We then examined a possible effect of histone deacetylase inhibitor nicotinamide (Nico) on the H2O2-mediated nuclear translocation of p53 and p53-dependent apoptotic cell death. To this end, Huh6 cells were treated with the indicated combinations of drug. At the indicated time periods after the treatment, cells were biochemically fractionated into cytoplasmic and nuclear fractions, and subjected to Western blotting with the anti-p53 antibody. As shown in Fig. 7A, nicotinamide significantly inhibited the H2O2-mediated nuclear translocation of p53. Next, we sought to address whether nicotinamide could affect the H2O2-induced apoptosis. For this purpose, Huh6 cells were exposed to the indicated combinations of drug, and the number of TUNEL-positive cells was measured. As shown in Fig. 7B, nicotinamide treatment markedly inhibited the H2O2-induced apoptotic cell death as compared with H2O2 alone. Similar results were also obtained in MTT assays (Fig. 7C). Together, our results strongly suggest that the acetylation status might be critical for the H2O2-induced nuclear translocation of p53 and apoptosis in HBL cells.


Figure 7
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  		Figure 7  Deacetylase inhibitor nicotinamide inhibits the H2O2-mediated nuclear translocation of p53 and apoptotic cell death in Huh6 cells. (A) Huh6 cells were treated with H2O2 alone (1 mM) or with H2O2 (1 mM) plus nicotinamide (Nico, 5 mM). At the indicated time periods after the treatment with the drugs, cells were harvested and fractionated into cytoplasmic and nuclear fractions followed by Western blotting with the anti-p53 antibody. (B) TUNEL staining. Huh6 cells were exposed to the indicated combinations of the drug for 5 h. Apoptotic cells were identified by TUNEL assays. Cell nuclei were visualized by DAPI (left panels). The number of TUNEL-positive cells was scored, and expressed as a percentage of the total number of cells examined (right panels). H2O2-mediated apoptosis was significantly inhibited by nicotinamide. P < 0.02. (C) MTT cell survival assay. Huh6 cells were treated with the indicated combinations of the drug for 5 h or left untreated, and their viability was examined by MTT assays.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
p53 acts as a tumor suppressor by inducing cell cycle arrest and/or apoptotic cell death in tumor cells. It has been well documented that p53 mutation is found in over 50% of all human tumors, resulting in the loss of its pro-apoptotic function (Hollstein et al. 1991; Vogelstein et al. 2000). The pro-apoptotic function of p53 can be also abrogated by non-mutational mechanisms (Prives & Hall 1999). For example, p53 is infrequently mutated in human NBL; however, p53 is largely localized in cytoplasm, indicating that p53 has no role in the genesis and development of NBL (Vogan et al. 1993). Like NBL, extensive studies on the p53 status demonstrated the absence of mutations in HBL (Chen et al. 1995; Ohnishi et al. 1996). Although ß-catenin is frequently mutated in HBL (Koch et al. 1999; Takayasu et al. 2001), recent studies suggest that ß-catenin mutation alone is not sufficient for hepatocarcinogenesis (Harada et al. 2002; Harada et al. 2004). In the present study, we have found that p53 is exclusively expressed in cytoplasm of human primary HBL as well as HBL-derived Huh6 cells. In response to oxidative stress, p53 was translocated into cell nucleus, and exerted its pro-apoptotic function. Intriguingly, histone deacetylase inhibitor treatment strongly inhibited the oxidative stress-induced nuclear access of p53. Thus, it is likely that the cytoplasmic retention of p53, which might be mediated by acetylation, contributes at least in part to the genesis and development of HBL.

As mentioned above, the previous studies demonstrated that wild-type p53 is significantly accumulated in cytoplasm of human NBL (Moll et al. 1995; Ostermeyer et al. 1996). According to their results, the treatment of NBL-derived cells with p53 COOH-terminal peptide resulted in the relocalization of p53 to the cell nucleus, suggesting that the COOH-terminal region of p53 contributes to its cytoplasmic retention. As described (Stommel et al. 1999), p53 shuttles between cell nucleus and cytoplasm in its potential COOH-terminal nuclear export signal (NES) and its receptor Crm-1-dependent manner. Based on our present results, however, the nuclear export inhibitor LMB treatment had undetectable effect on the subcellular localization of p53 in Huh6 cells, suggesting that the NES-mediated efficient nuclear export is not involved in a significant cytoplasmic accumulation of p53 in Huh6 cells. Nikolaev et al. (2003) found that Parc prevents the nuclear translocation of p53 through the interaction with its lysine-rich COOH-terminal region which contains the nuclear localization signals (NLSs), and also suggested that their interaction might be regulated by post-translational modifications of p53 such as phosphorylation and/or acetylation. Unexpectedly, our present results revealed that Parc binds to p53 in Huh6 cells; however, the cytoplasmic retention of p53 in Huh6 cells is regulated in a Parc-independent manner. It is worth noting that the nicotinamide treatment of Huh6 cells leads to the inhibition of the oxidative stress-induced nuclear access of p53 as well as the pro-apoptotic activity of p53, indicating that the acetylation status of p53 plays an important role in the regulation of the cytoplasmic retention of p53.

It is well documented that p300/CBP-mediated acetylation of p53 at the COOH-terminal lysine residues including Lys-320, Lys-370, Lys-371, Lys-372, Lys-381 and Lys-382 enhances the transcriptional activity as well as stability of p53 (Brooks & Gu 2003). p300/CBP possess histone acetyl-transferase (HAT) activity (Ogryzko et al. 1996). Since the COOH-terminal lysine residues of p53 are tightly associated with the p300/CBP-mediated acetylation as well as the MDM2-mediated ubiquitination, it is possible that p53 acetylation catalyzed by p300/CBP reduces its ubiquitination levels by competition between acetylation and ubiquitination. Alternatively, Kawaguchi et al. (2006) described that the hyperacetylated forms of p53 are accumulated in cytoplasm. Histone deacetylases are divided into three classes including class I, II and III (de Ruijter et al. 2003). Among them, class III histone deacetylases are distinct from the remaining classes, and defined based on their similarity to the yeast silent information regulator 2 (Sir2). One of the human Sir2 homologues, SIRT2, was largely localized in cytoplasm of mammalian cultured cells, and its catalytic activity was significantly inhibited by nicotinamide (North et al. 2003). In addition, SIRT1, another member of the class III histone deacetylases, was sensitive to nicotinamide, and had an ability to deacetylate p53 at Lys-382 (Michishita et al. 2005). Although it remains unclear whether SIRT2 could deacetylate p53, it is likely that SIRT family member(s) might be involved in the regulation of the oxidative stress-induced nuclear translocation of p53 in Huh6 cells. However, the precise molecular mechanisms behind the cytoplasmic localization of p53 in HBL are still unknown.

According to our present findings, p53 was constitutively phosphorylated at Ser-15 and stabilized in cytoplasm of Huh6 cells. Accumulating evidence suggests that MDM2 binds to the NH2-treminal region of p53, acts as an E3 ubiquitin protein ligase for p53 and thereby promotes its proteasome-dependent proteolytic degradation (Chen et al. 1993; Haupt et al. 1997; Honda et al. 1997; Kubbutat et al. 1997). Previous studies demonstrated that the DNA damage-induced phosphorylation of p53 at Ser-15 mediated by ATM, ATR and/or DNA-PK disrupts the interaction between p53 and MDM2, resulting in the activation and stabilization of p53 (Shieh et al. 1997). Although the precise molecular mechanisms behind the constitutive phosphorylation of cytoplasmic p53 at Ser-15 remain unclear, it is likely that the significant accumulation of p53 in Huh6 cells without DNA damage might be due to the inhibition of the MDM2-mediated ubiquitination and proteasomal degradation of p53.

It has been shown that ß-catenin, which is a key component of the Wnt signaling pathway, is frequently mutated or deleted in HBLs (Koch et al. 1999; Takayasu et al. 2001). These alterations occur in the NH2-terminal region of ß-catenin, which is responsible for the GSK3ß-mediated phosphorylation (Aberle et al. 1997). Similar to the primary HBLs, HBL-derived Huh6 cells carry a mutant form of ß-catenin at Thr-41 (Koch et al. 1999). GSK3ß-mediated phosphorylation of ß-catenin is required for its ubiquitin-dependent proteolytic degradation by 26S proteasome (Aberle et al. 1997). In accordance with this notion, a large amount of ß-catenin was detectable in Huh6 cells in the absence of oxidative stress (data not shown). Intriguingly, it has been shown that DNA damage induces the nuclear accumulation of p53 as well as GSK3ß, and p53 forms a stable complex with GSK3ß (Watcharasit et al. 2002; Watcharasit et al. 2003). According to their results, p53 enhances the activity of GSK3ß through the direct interaction with GSK3ß in a phosphorylation-independent manner, and GSK3ß activates the p53-dependent apoptotic pathway in response to DNA damage. Of note, we found that GSK3ß was induced to be accumulated in cell nucleus in response to oxidative stress (data not shown). However, we have not yet explored the possible contribution of the functional interaction between p53 and GSK3ß to the oxidative stress-induced apoptotic response in Huh6 cells.

Taken together, our present findings suggest that, in addition to ß-catenin mutation, the abnormal cytoplasmic localization of p53 might be involved in the genesis and development of HBL, and the acetylation status of p53 plays an important role in the cytoplasmic retention of p53. Furthermore, the oxidative stress-induced apoptotic cell death in Huh6 cells was strongly associated with the active nuclear translocation of p53, which was distinct from the CDDP-mediated nuclear accumulation of p53. Thus, our present results provide a novel targeted approach to enhance the HBL cell cytotoxity.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell culture

Human hepatoblastoma Huh6 cells and human hepatocellular carcinoma HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and penicillin (100 IU/mL)/streptomycin (100 µg/mL). Cultures were maintained at 37 °C in a water-saturated atmosphere of 5% CO2 in air.

RNA preparation and RT-PCR

Total RNA was extracted from cells exposed to cisplatin or hydrogen peroxide using the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. cDNA was reverse transcribed from 1 µg of total RNA with random primers and SuperScript II (Invitrogen) as recommended by the supplier. Following the reverse transcription, the resultant cDNA was subjected to the PCR-based amplification. For PCR analysis, oligonucleotide sequences used were as follows: NOXA, 5'-GCAAGAATGGAAGACCCTTG-3' and 5'-GTGCTGAGTTGGCACTGAAA-3'; Parc, 5'-CGTCTCCTGAGCTTTGGTTC-3' and 5'-CCTCATCTTCCTGCTCCAAG-3'; GAPDH, 5'-ACCTGACCTGCCGTCTAGAA-3' and 5'-TCCACCACCCTGTTGCTGTA-3'. PCR products were resolved by 2% agarose gel electrophoresis, and visualized by ethidium bromide staining.

Western blot analysis

Cells were rinsed twice in ice-cold PBS and lyzed in SDS sample buffer containing 62.5 mM Tris–HCl, pH 6.8, 2% SDS, 2% ß-mercaptoethanol and 0.01% bromophenol blue, followed by a brief sonication. After centrifugation at 10 000 g for 10 min at 4 °C, the supernatant was transferred to a new tube. The protein concentration was measured by the Bradford protein assay (Bio-Rad laboratories, Hercules, CA), using bovine serum albumin as a standard. For Western analysis, equal amounts of protein were separated by 10% SDS-PAGE, and electro-transferred on to Immobilon-P membranes (Millipore, Bedford, MA). The membranes were blocked overnight at 4 °C with TBS-T (50 mM Tris–HCl, pH 8.0, 100 mM NaCl and 0.1% Tween 20) containing 5% non-fat dry milk, and then incubated for 1 h at room temperature with the monoclonal anti-p53 (DO-1; Oncogene Research Products, Cambridge, MA), polyclonal anti-phosphorylated p53 at Ser-15 (Cell Signaling Technology, Beverly, MA), polyclonal anti-Parc (Calbiochem, La Jolla), or with polyclonal anti-actin antibody (20–33; Sigma Chemical Co., St. Louis, MO). After washing with TBS-T, the membranes were incubated with a horseradish peroxidase-conjugated appropriate secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature. The chemiluminescence reaction was performed using the ECL reagent (Amersham Biosciences, Piscataway, NJ).

Subcellular fractionation

Cells were washed twice in ice-cold PBS and lyzed in lysis buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 0.5% NP-40) containing a protease inhibitor mix (Sigma Chemical Co.) for 10 min at 4 °C. Cell lysates were centrifuged at 10 000 g for 10 min at 4 °C to separate cytoplasmic fraction (supernatant). Insoluble materials were washed three times with the lysis buffer and nuclei were lyzed in 1x SDS sample buffer. The nuclear lysates were sonicated, centrifuged, and the supernatant was collected. The protein concentrations were determined by the Bradford protein assay (Bio-Rad Laboratories). The nuclear and cytoplasmic fractions were subjected to immunoblot analysis using the monoclonal anti-Lamin B (Ab-1; Oncogene Research Products) or monoclonal anti-{alpha}-tubulin antibody (Ab-2; NeoMarkers, Inc., Fremont, CA).

Indirect immunofluorescence

Huh6 cells on cover slips were fixed in ice-cold methanol for 5 min at room temperature and permeabilized with 0.2% Triton X-100 for 3 min at room temperature. After blocking with 3% bovine serum albumin in PBS, cover slips were incubated with anti-p53 antibody (DO-1) in PBS for 1 h at room temperature followed by incubation with rhodamine-conjugated secondary antibody (Invitrogen) in PBS for 1 h at room temperature. Cell nuclei were stained with DAPI.

Co-immunoprecipitation experiments

For co-immunoprecipitation experiments, cells were lyzed in lysis buffer (25 mM Tris–HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1% Triton X-100) supplemented with protease inhibitor mixture for 30 min at 4 °C, and clarified by centrifugation at 10 000 g for 15 min at 4 °C. Equal amounts of whole cell lysates were precleared by incubation with 30 µL of 50% slurry of protein G-Sepharose beads (Amersham Biosciences). After brief centrifugation, the supernatants were collected and incubated with the normal mouse serum (NMS), or with monoclonal anti-p53 antibody (DO-1; Oncogene Research Products) at 4 °C for 2 h. The immune complexes were precipitated with the protein G-Sepharose beads for 1 h at 4 °C, and the nonspecific bound proteins were removed by washing the beads with the lysis buffer three times at 4 °C. Immunoprecipitated proteins were subjected to 10% SDS-PAGE, and Western blot analysis was carried out using the polyclonal anti-Parc antibody.

siRNA-mediated knockdown of p53

Huh6 cells were transfected with 2 µg of the empty plasmid (pSUPER; OligoEngine, Seattle, WA) or with pSUPER expression plasmid encoding siRNA against p53 (pSUPER-siRNA-p53) by using FuGENE 6 transfection reagent as recommended by the manufacturer (Roche Molecular Biochemicals, Mannheim, Germany). Forty-eight hours after transfection, the transfected cells were put under a selection pressure of 400 µg/mL of G418 (Sigma Chemical Co.). Thereafter, the selection medium was replaced every 3 days. Two weeks after the selection in G418, drug-resistant clones were isolated and allow to proliferate in medium containing G418 (400 µg/mL).

Cell survival assay

Cell viability was determined by MTT assay and expressed as percent viable cells. In brief, Huh6 or HepG2 cells were seeded in 96-well microtiter plates (5 x 103 cells/well) with 100 µL of complete medium. The next day, the medium was changed and cells were exposed to the indicated concentrations of hydrogen peroxide. At the indicated time periods after the treatment with hydrogen peroxide, 10 µL of MTT labeling reagent were added to each well, and the cultures were incubated for 1 h at 37 °C. The absorbance readings for each well were carried out at a wavelength of 570 nm using the microplate reader (model 450; Bio-Rad Laboratories).

Apoptotic analysis

Apoptotic cells were detected by In Site Cell Death Detection Kit, TMR red (Roche Molecular Biochemicals). In brief, Huh6 cells were grown overnight on glass cover slips at 37 °C. Five hours after the treatment with hydrogen peroxide, cells were washed in PBS, fixed in 4% paraformaldehyde for 1 h at room temperature and then permeabilized with 0.1% Triton X-100 for 2 min on ice. The cells were subsequently incubated with TUNEL reaction mixture for 1 h at 37 °C in a humidified atmosphere in the dark. The cover slips were mounted onto microscope slides using the VECTASHIELD containing DAPI (Vector Laboratories, Burlingame, CA), and examined under a Fluoview laser scanning confocal microscope (Olympus, Tokyo, Japan).

Tumor tissues and immunohistochemistry

Hepatoblastoma tissues, which were collected at Gunma Children's Medical Center, were obtained at surgery, immediately frozen and stored at –80 °C until use. Sections from formalin-fixed, paraffin-embedded tumor samples were cut at 4-µm thickness and subjected to immunostaining. In brief, antigen retrieval was achieved by the treatment of deparaffinized sections with microwaves in 0.1 M citrate buffer (pH 6.0) for 20 min, followed by cooling at room temperature prior to incubation with the primary antibody. Tissue sections were incubated with monoclonal anti-p53 antibody (PAb240; Calbiochem) overnight at 4 °C and washed in PBS. The bound antibody was detected using the strepto-avidin-biotin complex method (Nichirei Corp., Tokyo, Japan) and visualized by diaminobenzidine tetrahydrochloride.


   Acknowledgements
 
We are grateful to Dr M. Kuroiwa for kindly providing the hepatoblastoma tissues and Ms Y. Nakamura for assistance with DNA sequencing. This work was supported in part by a Grant-in-Aid from the Ministry of Health, Labour and Welfare for Third Term Comprehensive Control Research for Cancer, a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science, and a Grant from Uehara Memorial Foundation.


   Footnotes
 
Communicated by: Takeo Kishimoto

* Correspondence: E-mail: akiranak{at}chiba-cc.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Aberle, H., Bauer, A., Stappert, J., Kispert, A. & Kemler, R. (1997) ß-catenin is a target for the ubiquitin-proteasome pathway. EMBO J. 16, 3797–3804.[CrossRef][Medline]

Aden, D.P., Fogel, A., Plotkin, S., Damjanov, I. & Knowles, B.B. (1979) Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature 282, 615–616.[CrossRef][Medline]

Bosari, S., Viale, G., Roncalli, M., Graziani, D., Borsani, G., Lee, A.K. & Coggi, G. (1995) p53 gene mutations, p53 protein accumulation and compartmentalization in colorectal adenocarcinoma. Am. J. Pathol. 147, 790–798.[Abstract]

Bressac, B., Galvin, K.M., Liang, J., Isselbacher, K.J., Wands, J.R. & Ozturk, M. (1990) Abnormal structure and expression of p53 gene in human hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA 87, 1973–1977.[Abstract/Free Full Text]

Brooks, C.L. & Gu, W. (2003) Ubiquitination, phosphorylation and acetylation: the molecular basis for p53 regulation. Curr. Opin. Cell Biol. 15, 164–171.[CrossRef][Medline]

Chen, T.C., Hsieh, L.L. & Kuo, T.T. (1995) Absence of p53 gene mutation and infrequent overexpression of p53 protein in hepatoblastoma. J. Pathol. 176, 243–247.[CrossRef][Medline]

Chen, J., Marechal, V. & Levine, A.J. (1993) Mapping of the p53 and mdm–2 interaction domains. Mol. Cell. Biol. 13, 4107–4114.[Abstract/Free Full Text]

Doi, I. (1976) Establishment of a cell line and its clonal sublines from a patient with hepatoblastoma. Gann 67, 1–10.[Medline]

Dornan, D., Wertz, I., Shimizu, H., Arnott, D., Frantz, G.D., Dowd, P., O’Rourke, K., Koeppen, H. & Dixit, V.M. (2004) The ubiquitin ligase COP1 is a critical negative regulator of p53. Nature 429, 86–92.[CrossRef][Medline]

Fritsche, M., Haessler, C. & Brandner, G. (1993) Induction of nuclear accumulation of the tumor-suppressor protein p53 by DNA-damaging agents. Oncogene 8, 307–318.[Medline]

Gu, W. & Roeder, R.G. (1997) Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595–606.[CrossRef][Medline]

Harada, N., Miyoshi, H., Murai, N., Oshima, H., Tamai, Y., Oshima, M. & Taketo, M.M. (2002) Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of ß-catenin. Cancer Res. 62, 1971–1977.[Abstract/Free Full Text]

Harada, N., Oshima, H., Katoh, M., Tamai, Y., Oshima, M. & Taketo, M.M. (2004) Hepatocarcinogenesis in mice with ß-catenin and Ha-ras gene mutations. Cancer Res. 64, 48–54.[Abstract/Free Full Text]

Haupt, Y., Maya, R., Kazaz, A. & Oren, M. (1997) Mdm2 promotes the rapid degradation of p53. Nature 387, 296–299.[CrossRef][Medline]

Henderson, B.R. & Fagotto, F. (2002) The ins and outs of APC and ß-catenin nuclear transport. EMBO Rep. 3, 834–839.[CrossRef][Medline]

Hollstein, M., Sidransky, D., Vogelstein, B. & Harris, C.C. (1991) p53 mutations in human cancers. Science 253, 49–53.[Abstract/Free Full Text]

Honda, R., Tanaka, H. & Yasuda, H. (1997) Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett. 420, 25–27.[CrossRef][Medline]

Hsu, I.C., Tokiwa, T., Bennett, W., Metcalf, R.A., Welsh, J.A., Sun, T. & Harris, C.C. (1993) p53 gene mutation and integrated hepatitis B viral DNA sequences in human liver cancer cell lines. Carcinogenesis 14, 987–992.[Abstract/Free Full Text]

Hughes, L.J. & Michels, V.V. (1992) Risk of hepatoblastoma in familial adenomatous polyposis. Am. J. Med. Genet. 43, 1023–1025.[CrossRef][Medline]

Kawaguchi, Y., Ito, A., Appella, E. & Yao, T.P. (2006) Charge modification at multiple C-terminal lysine residues regulates p53 oligomerization and its nucleus-cytoplasm trafficking. J. Biol. Chem. 281, 1394–1400.[Abstract/Free Full Text]

Koch, A., Denkhaus, D., Albrecht, S., Leuschner, I., von Schweinitz, D. & Pietsch, T. (1999) Childhood hepatoblastomas frequently carry a mutated degradation targeting box of the ß-catenin gene. Cancer Res. 59, 269–273.[Abstract/Free Full Text]

Kubbutat, M.H., Jones, S.N. & Vousden, K.H. (1997) Regulation of p53 stability by Mdm2. Nature 387, 299–303.[CrossRef][Medline]

Kusafuka, T., Fukuzawa, M., Oue, T., Komoto, Y., Yoneda, A. & Okada, A. (1997) Mutation analysis of p53 gene in childhood malignant solid tumors. J. Pediatr. Surg. 32, 1175–1180.[CrossRef][Medline]

Leng, R.P., Lin, Y., Ma, W., Wu, H., Lemmers, B., Chung, S., Parant, J.M., Lozano, G., Hakem, R. & Benchimol, S. (2003) Pirh2, a p53-induced ubiquitin-protein ligase, promotes p53 degradation. Cell 112, 779–791.[CrossRef][Medline]

Llovet, J.M., Burroughs, A. & Bruix, J. (2003) Hepatocellular carcinoma. Lancet 362, 1907–1917.[CrossRef][Medline]

Lluis, J.M., Morales, A., Biasco, C., Colell, A., Mari, M., Garcia-Ruiz, C. & Fernandez-Checa, J.C. (2005) Critical role of mitochondrial glutathione in the survival of hepatocytes during hypoxia. J. Biol. Chem. 280, 3224–3232.[Abstract/Free Full Text]

Michishita, E., Park, J.Y., Burneskis, J.M., Barrett, J.C. & Horikawa, I. (2005) Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins. Mol. Biol. Cell 16, 4623–4635.[Abstract/Free Full Text]

Moll, U.M., LaQuaglia, M., Benard, J. & Riou, G. (1995) Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors. Proc. Natl. Acad. Sci. USA 92, 4407–4411.[Abstract/Free Full Text]

Moll, U.M., Riou, G. & Levine, A.J. (1992) Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion. Proc. Natl. Acad. Sci. USA 89, 7262–7266.[Abstract/Free Full Text]

Nagase, H. & Nakamura, Y. (1993) Mutations of the APC (adenomatous polyposis coli) gene. Hum. Mutat. 2, 425–434.[CrossRef][Medline]

Nikolaev, A.Y., Li, M., Puskas, N., Qin, J. & Gu, W. (2003) Parc: a cytoplasmic anchor for p53. Cell 112, 29–40.[CrossRef][Medline]

North, B.J., Marshall, B.L., Borra, M.T., Denu, J.M. & Verdin, E. (2003) The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase. Mol. Cell 11, 437–444.[CrossRef][Medline]

Ogryzko, V.V., Schiltz, R.L., Russanova, V., Howard, B.H. & Nakatani, Y. (1996) The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 29, 953–959.

Ohnishi, H., Kawamura, M., Hanada, R., Kaneko, Y., Tsunoda, Y., Hongo, T., Bessho, F., Yokomori, K. & Hayashi, Y. (1996) Infrequent mutations of the TP53 gene and no amplification of the MDM2 gene in hepatoblastomas. Genes Chromosomes Cancer 15, 187–190.[CrossRef][Medline]

Ostermeyer, A.G., Runko, E., Winkfield, B., Ahn, B. & Moll, U.M. (1996) Cytoplasmically sequestered wild-type p53 protein in neuroblastoma is relocated to the nucleus by a C-terminal peptide. Proc. Natl. Acad. Sci. USA 93, 15190–15194.[Abstract/Free Full Text]

Prives, C. & Hall, P.A. (1999) The p53 pathway. J. Pathol. 187, 112–126.[CrossRef][Medline]

de Ruijter, A.J., van Gennip, A.H., Caron, H.N., Kemp, S. & van Kuilenburg, A.B. (2003) Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem. J. 370, 737–749.[CrossRef][Medline]

Shieh, S.Y., Ikeda, M., Taya, Y. & Prives, C. (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91, 325–334.[CrossRef][Medline]

Sionov, R.V. & Haupt, Y. (1999) The cellular response to p53: the decision between life and death. Oncogene 18, 6145–6157.[CrossRef][Medline]

Stommel, J.M., Marchenko, N.D., Jimenez, G.S., Moll, U.M., Hope, T.J. & Wahl, G.M. (1999) A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J. 18, 1660–1672.[CrossRef][Medline]

Takayasu, H., Horie, H., Hiyama, E., Matsunaga, T., Hayashi, Y., Watanabe, Y., Suita, S., Kaneko, M., Sasaki, F., Hashizume, K., Ozaki, T., Furuuchi, K., Tada, M., Ohnuma, N. & Nakagawara, A. (2001) Frequent deletions and mutations of the ß-catenin gene are associated with overexpression of cyclin D1 and fibronectin and poorly differentiated histology in childhood hepatoblastoma. Clin. Cancer Res. 7, 901–908.[Abstract/Free Full Text]

Vogan, K., Berstein, M., Leclerc, J.M., Brisson, L., Brossard, J., Brodeur, G.M., Pelletier, J. & Gros, P. (1993) Absence of p53 gene mutations in primary neuroblastomas. Cancer Res. 53, 5269–5273.[Abstract/Free Full Text]

Vogelstein, B., Lane, D. & Levine, A.J. (2000) Surfing the p53 network. Nature 408, 307–310.[CrossRef][Medline]

Vousden, K.H. & Lu, X. (2002) Live or let die: the cell's response to p53. Nat. Rev. Cancer 2, 594–604.[CrossRef][Medline]

Watcharasit, P., Bijur, G.N., Song, L., Zhu, J., Chen, X. & Jope, R.S. (2003) Glycogen synthase kinase-3ß (GSK3ß) binds to and promotes the actions of p53. J. Biol. Chem. 278, 48872–48879.[Abstract/Free Full Text]

Watcharasit, P., Bijur, G.N., Zmijewski, J.W., Song, L., Zmijewska, A., Chen, X., Johnson, G.V. & Jope, R.S. (2002) Direct, activating interaction between glycogen synthase kinase-3ß and p53 after DNA damage. Proc. Natl. Acad. Sci. USA 99, 7951–7955.[Abstract/Free Full Text]

Received: 20 October 2006
Accepted: 21 December 2006





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