Stabilization Of P53 In Response Research Articles

RING finger and WD repeat domain 3 regulates proliferation and metastasis through the Wnt/β-catenin signalling pathways in hepatocellular carcinoma

BACKGROUNDHepatocellular carcinoma (HCC) exhibits high invasiveness and mortality rates, and the molecular mechanisms of HCC have gained increasing research interest. The abnormal DNA damage response has long been recognized as one of the important factors for tumor occurrence and development. Recent studies have shown the potential of the protein RING finger and WD repeat domain 3 (RFWD3) that positively regulates p53 stability in response to DNA damage as a therapeutic target in cancers. AIMTo investigate the relationship between HCC and RFWD3 in vitro and in vivo and explored the underlying molecular signalling transduction pathways. METHODS RFWD3 gene expression was analyzed in HCC tissues and adjacent normal tissues. Lentivirus was used to stably knockdown RFWD3 expression in HCC cell lines. After verifying the silencing efficiency, Celigo/cell cycle/apoptosis and MTT assays were used to evaluate cell proliferation and apoptosis. Subsequently, cell migration and invasion were assessed by wound healing and transwell assays. In addition, transduced cells were implanted subcutaneously and injected into the tail vein of nude mice to observe tumor growth and metastasis. Next, we used lentiviral-mediated rescue of RFWD3 shRNA to verify the phenotype. Finally, the microarray, ingenuity pathway analysis, and western blot analysis were used to analyze the regulatory network underlying HCC. RESULTSCompared with adjacent tissues, RFWD3 expression levels were significantly higher in clinical HCC tissues and correlated with tumor size and TNM stage (P < 0.05), which indicated a poor prognosis state. RFWD3 silencing in BEL-7404 and HCC-LM3 cells increased apoptosis, decreased growth, and inhibited the migration in shRNAi cells compared with those in shCtrl cells (P < 0.05). Furthermore, the in vitro results were supported by the findings of the in vivo experiments with the reduction of tumor cell invasion and migration. Moreover, the rescue of RFWD3 shRNAi resulted in the resumption of invasion and metastasis in HCC cell lines. Finally, gene expression profiling and subsequent experimental verification revealed that RFWD3 might influence the proliferation and metastasis of HCC via the Wnt/β-catenin signalling pathway.CONCLUSIONWe provide evidence for the expression and function of RFWD3 in HCC. RFWD3 affects the prognosis, proliferation, invasion, and metastasis of HCC by regulating the Wnt/β-catenin signalling pathway.

Neuroblastoma Cell Lines Are Refractory to Genotoxic Drug-Mediated Induction of Ligands for NK Cell-Activating Receptors.

Neuroblastoma (NB), the most common extracranial solid tumor of childhood, causes death in almost 15% of children affected by cancer. Treatment of neuroblastoma is based on the combination of chemotherapy with other therapeutic interventions such as surgery, radiotherapy, use of differentiating agents, and immunotherapy. In particular, adoptive NK cell transfer is a new immune-therapeutic approach whose efficacy may be boosted by several anticancer agents able to induce the expression of ligands for NK cell-activating receptors, thus rendering cancer cells more susceptible to NK cell-mediated lysis. Here, we show that chemotherapeutic drugs commonly used for the treatment of NB such as cisplatin, topotecan, irinotecan, and etoposide are unable to induce the expression of activating ligands in a panel of NB cell lines. Consistently, cisplatin-treated NB cell lines were not more susceptible to NK cells than untreated cells. The refractoriness of NB cell lines to these drugs has been partially associated with the abnormal status of genes for ATM, ATR, Chk1, and Chk2, the major transducers of the DNA damage response (DDR), triggered by several anticancer agents and promoting different antitumor mechanisms including the expression of ligands for NK cell-activating receptors. Moreover, both the impaired production of reactive oxygen species (ROS) in some NB cell lines and the transient p53 stabilization in response to our genotoxic drugs under our experimental conditions could contribute to inefficient induction of activating ligands. These data suggest that further investigations, exploiting molecular strategies aimed to potentiate the NK cell-mediated immunotherapy of NB, are warranted.

Ubiquitin-specific Peptidase 42 (USP42) Functions to Deubiquitylate Histones and Regulate Transcriptional Activity

Ubiquitin-specific peptidase 42 (USP42) is a deubiquitylating enzyme that can target p53 and contribute to the stabilization of p53 in response to stress. We now show that USP42 can also regulate transcription independently of p53. USP42 co-localized with RNA polymerase II (RNA Pol II) in nuclear foci, bound to histone H2B, and deubiquitylated H2B. Depletion of USP42 increased H2B ubiquitylation at a model promoter and decreased both basal and induced transcription from a number of promoters. These results are consistent with a role for USP42 in regulating transcription by deubiquitylating histones.

Beta Human Papillomavirus E6 Expression Inhibits Stabilization of p53 and Increases Tolerance of Genomic Instability

Infections with the beta genus of human papillomaviruses (β-HPVs) may contribute to the development of nonmelanoma skin cancers. However, β-HPV genomes are found at too low a copy number in tumors for the virus to be necessary for tumor maintenance. Instead, they are hypothesized to destabilize the host genome by allowing the persistence of mutations that can drive tumorigenesis independently of the viral genome. Supporting this premise is our previous finding that the expression of some β-HPV E6 proteins can attenuate p53 signaling in response to DNA damage. We show that β-HPV E6 proteins can prevent the stabilization of p53 in response to two types of genome-destabilizing events, aberrant mitosis and dysregulated centrosome duplication. The inability to stabilize p53 in response to these stimuli allows cells expressing HPV5, HPV8, or HPV38 E6 to remain proliferatively active, leading to further genome deterioration in a proportion of these cells. These phenotypes are lost by the introduction of a mutation into the p300 binding domain of HPV8 E6 or by the transfection of mutated p300 that is resistant to the degradation mediated by HPV5 or HPV8 E6. These findings expand the understanding of the role played by p300 in promoting the faithful resolution of mitotic figures as well as proper centrosome duplication. Finally, we describe a phenomenon by which binucleated cells are resolved via cytokinesis into two cells, each with one nucleus. These data support the hypothesis that β-HPV infections may promote tumorigenesis via genome destabilization. The work described in this report provides support for the hypothesis that β-HPV infections may contribute to nonmelanoma skin cancer by increasing the likelihood that tumorigenic mutations are introduced into the host cell's genome. We demonstrate that expression of the E6 proteins from some of these viruses increases the tolerance of two genome-destabilizing events, aberrant cell division and dysregulated centrosome duplication. Typically, these mutagenic occurrences elicit the stabilization of the tumor suppressor p53, which prevents further propagation of cells containing these errors. We show that the expression of β-HPV E6 restricts this stabilization of p53, leading not only to continued cellular proliferation but also to further accumulation of similar mutagenic events. Finally, in addition to supporting a role for β-HPV infections in certain skin cancers, we present studies with a mutated β-HPV E6 protein suggesting that the histone acetyltransferase p300 plays a role in promoting genome stability during replication.

Impact Of p53 Impaired Function On Outcomes Of Multiple Myeloma Patients Carrying Deleted TP53 and/Or Amplified MDM4

BackgroundMultiple Myeloma (MM) is a genetically complex disease. In MM, prevalent chromosomal numerical and structural aberrations are used to cluster patients (pts) into subtypes, frequently displaying distinct clinical behaviors.Among the umpteen chromosomal aberrations described so far in MM, the TP53 deletion on chromosome (chr) 17p13 defines a pts group with a particularly poor prognosis, even if its prevalence at diagnosis is quite low. Overall, the TP53 tumor-suppressor gene is mutated or functionally inactivated in most human cancers. Tumors that retain wild-type p53 frequently harbor defects either in the pathways that allow for p53 stabilization in response to stress, or in the effectors of p53 apoptotic activity. One of the most potent inhibitor of p53 is MDM4, which is often amplified in several types of tumors. The MDM4 locus is located on chr1q32.1, a region frequently amplified in MM. AimAim of the present work was to investigate the possibility that both TP53 deletion (del) and MDM4 amplification (amp) might affect similar pathways, thus finally leading to a poor prognosis MM pts carrying at diagnosis at least one of them. Pts and methodsEighty-nine pts treated with bortezomib-thalidomide-dexamethasone (VTD) as induction therapy prior to, and as consolidation after, double ASCT were analyzed at diagnosis by means of unpaired analysis of copy number alterations (CNA) (Affymetrix 6.0 SNP array) and gene expression profile (GEP) (Affymetrix U133 Plus2.0 array); in twenty-one pts carrying MDM4 amplification and for whom samples were available, the p53 activity was explored both by analyzing the TP53 mutational rate by deep sequencing (Roche GS Junior 454) and by evaluating the p53 activity by immunoblotting assays of MDM4, p-p53 and p53. Genomic results were evaluated in the clinical context. ResultsThe CNA analysis showed a 482 Kb minimal deleted region on chr17p13, including TP53, in 8/89 pts (8,9%) and a 1.1 Mb minimal amplified region on chr1q32.1 including MDM4 in 27/89 pts (30,3%).The GEP analysis performed at diagnosis in TP53 del and MDM4 amp pts generated two lists, including genes either differentially expressed among pts carrying or not amplified MDM4 (5840 probes sets, corresponding to 3841 genes, p<0.05), or differentially expressed among pts carrying or not deleted TP53 (3552 probes sets, corresponding to 2467 genes, p<0.05). Biological processes affected by the genes included in the two lists showed, in both cases, an overall deregulation of pathways related to the cell cycle, the DNA damage repair and the cell adhesion and cytoskeleton remodeling.In order to verify whether the presence of MDM4 amp might be related to a decreased p53 function, we analyzed 21 pts carrying MDM4 amp for their p53 activity, as evaluated by p-p53 immunoblotting assays, and we showed the absence of p-p53 protein in 60% of them. Finally, we analyzed the TP53 mutational rate, as detected by deep sequencing of exons 4-11, in 21 pts carrying MDM4 amp and we showed the presence of point mutations in 15 of them, with mutated reads frequencies ranging from 1.03% to 16.9% (median coverage for each amplicon = 1000 reads).We lastly analyzed the prognostic relevance of p53 pathway impaired function; to this purpose, we stratified pts into two subgroups according to the presence of MDM4 amp and/or TP53 del (group A, 34 pts, or 38%) or the absence of both these abnormalities (group B, 55 pts, or 62%). Baseline clinical characteristics were homogeneous, except for a higher rate of IgA isotype in group A. On the contrary, groups A and B resulted clearly imbalanced with respect to the genomic background: indeed, the t(4,14) frequency, as well as the average number of CNAs were overall higher in group A as compared to group B (38% vs. 14% t(4;14) positive, p=0.0002 and 165 vs 103 CNAs, p = 0.03). Despite the initially slightly higher response rate after VTD induction therapy of group A, as compared to group B (38% vs 20% ³near complete response), the presence of TP53 del and/or MDM4 amp correlated with shorter median PFS (44.05 months vs. undefined, p=0.003) and OS (66.6 vs undefined, p=0.0006). Of note, the poorer impact associated with MDM4 amplification was retained also in the absence of TP53 deletion (PFS: 46.45 months vs undefined, p=0.009). ConclusionsThe results overall suggest that the involvement of the p53 pathway alteration in MM might be wider than expected, possibly due to the activation of negative regulators of p53. Disclosures:Zamagni:Celgene: Honoraria; Janssen-Cilag: Honoraria. Cavo:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract IA09: Nucleotide biosynthetic enzyme GMP Synthase is a relay of p53 stabilization in response to genomic stress

Abstract Nucleotide biosynthesis is fundamental to normal cell proliferation and to oncogenesis. An adequate nucleotide supply is crucial to support accurate DNA replication, DNA repair and transcription. The metabolism of cancer cells is characterized by elevated rates of de novo nucleotide biosynthesis that fuel rapid cycles of cell growth and division. Here we will discuss the role of the nucleotide biosynthetic enzyme guanosine 5′-monophosphate synthase (GMPS) in regulation of the p53 tumor suppressor pathway. In addition to its essential biosynthetic function, GMPS associates with the ubiquitin-specific protease 7 (USP7). Biochemical and genetic evidence established that GMPS is required for histone H2B deubiquitylation by USP7 in Drosophila and human cells. GMPS-USP7 is targeted to specific genomic loci to direct gene expression. For example, GMPS-USP7 enhances Polycomb-directed silencing of homeotic genes. We found that GMPS is also a crucial relay of p53 tumor suppressor stabilization, in response to genomic stress. Nuclear GMPS drives the transfer of MDM2-bound p53 to a GMPS-USP7-p53 deubiquitylation complex. GMPS is crucial for the deubiquitylation and stabilization of p53 by USP7. Normally, GMPS is sequestered in the cytoplasm, separate from the nuclear USP7 and p53. Genomic stress triggers the accumulation of GMPS in the nucleus, where it can stimulate p53 stabilization by USP7. Our results suggest that GMPS, a classic biosynthetic enzyme that enables cell growth and DNA replication, is also a crucial relay of cell proliferation restriction by p53. We suspect that the connection between nucleotide biosynthetic enzymes and transcription control might be important for the cellular defense against genomic instability or replicative stress involving deficiencies in the nucleotide pool. These findings show that cellular processes that are commonly assumed to involve separate molecular machineries can be surprisingly intimately connected. Citation Format: Peter Verrijzer. Nucleotide biosynthetic enzyme GMP Synthase is a relay of p53 stabilization in response to genomic stress. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA09.

Induction of ASAP (MAP9) contributes to p53 stabilization in response to DNA damage

p53 is a key tumor suppressor that controls DNA damage response and genomic integrity. In response to genotoxic stress, p53 is stabilized and activated, resulting in controlled activation of genes involved in cell cycle arrest, DNA repair and/or apoptosis. ASAP is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. We show here that following double-strand break DNA formation, ASAP directly interacts with and stabilizes p53 by enhancing its p300-mediated acetylation and blocking its MDM2-mediated ubiquitination and degradation, leading to an increase of p53 transcriptional activity. Upon DNA damage, ASAP is transiently accumulated before being degraded upon persistent damage. This work links the p53 response with the cytoskeleton and confirms that the DNA-damaging signaling pathway is coordinated by centrosomal proteins. We reveal the existence of a new pathway through which ASAP signals the DNA damage response by regulating the p300-MDM2-p53 loop. These results point out ASAP as a possible target for the design of drugs to sensitize radio-resistant tumors.

Increased aldehyde reductase expression mediates acquired radioresistance of laryngeal cancer cells via modulating p53

The main obstacle to cure tumors by radiotherapy has been ascribed to tumor radioresistance. To determine the mechanisms underlying resistance to irradiation, it is essential to compare proteins differentially expressed from radiotherapy-sensitive and -resistant cancer cells. Aldehyde reductase (AKR1A1) was recently identified as increased in radioresistant laryngeal cancer cells by comparative proteomics approach. Here, we provide the mechanism of AKR1A1-mediated radioresistance via p53 regulation in laryngeal cancer cells. AKR1A1 induction was correlated with the radioresistant phenotype of laryngeal cancer HEp-2 cells. AKR1A1 depletion with siRNA significantly enhanced radiation sensitivity of radioresistant HEp-2 cells by promoting radiation-induced cell death and accelerated radiation-mediated inhibition of cell proliferation, without affecting either the PI3K-Akt or MAPK-ERK pathways. Intriguingly, AKR1A1 depletion induced phosphorylation of p53 at serine 15 and G2/M transition in response to irradiation. We further found that AKR1A1 interacted with p53 and this interaction was dramatically increased in the irradiated radioresistant cells compared with the control cells. AKR1A1 expression also regulated p53 stability in response to irradiation. Furthermore, AKR1A1 depletion only sensitized HCT116 cells expressing p53 to irradiation and not p53-deficient cells. Therefore, our data suggest that radiation-inducible AKR1A1 contributes to acquired radioresistance of laryngeal cancer cells by suppressing p53 activation through inhibitory interaction.

Axl Receptor Tyrosine Kinase Signaling Pathway and the p53 Tumor Suppressor Protein Exist In A Novel Regulatory Loop In B-Cell Chronic Lymphocytic Leukemia Cells

Abstract 799 Background:Axl is a constitutively active receptor tyrosine kinase (RTK) in CLL B-cells. We have shown that Axl is physically associated with multiple non-receptor cellular kinases and regulates their activities in CLL B-cells including Lyn kinase (a member of Src-family kinases reported to be overexpressed in CLL), PI3K, Syk and phospholipase γ2 (PLCγ2). These aspects likely relate to drug resistance and survival. Importantly, inhibition of Axl by pharmacological inhibitor (R428) induces massive cell death suggesting its critical role in CLL B-cell survival. We hypothesized that in addition to its role in regulating cellular kinases, Axl might modulate the tumor suppressor p53 to make CLL B-cells more resistant to conventional therapeutic agents. This is based on our unpublished findings that CLL B-cells from patients who have undergone treatment without any reduction in tumor burden expressed increased levels of Axl vs. their pre-therapy levels. In addition, we have detected higher levels of Axl in 17p-deleted CLL patients. Thus to understand the mechanism of Axl RTK-mediated regulation of CLL B-cell growth and survival, we interrogated the functional association between Axl and the HDM2 (human homolog of mouse MDM2)/p53 axis. Methods:We cloned the entire (∼2 kb) 3’-untranslated region (UTR) of the Axl transcript into pMIR-Report vector downstream of the fire-fly luciferase gene. Human embryonic kidney cells (HEK293) were transfected with a full length Axl gene containing the first 500 nucleotides of its 3’-UTR or the pMIR-Report plasmid with or without miR-34a. Cell lysates were examined for the expression of Axl by Western blot or performed luciferase assay. Purified CLL B-cells were also treated with various kinase inhibitors including, Wortmannin (pan-PI3K-inhibitor), PP1 (Src-inhibitor) and SKI-606 (targets both Src and Axl) to detect signaling pathway components downstream of Axl involved in regulating HDM2 function. Results:CLL B-cells from majority of CLL patients (n=15) showed constitutive levels of Axl phosphorylation which we found was associated with the phosphorylation level of HDM2 at Ser-166. HDM2 has an important function in controlling p53 stability in response to genotoxic stress. Recent studies have shown that HDM2 can be phosphorylated on Ser-166 by AKT, MAPKs, and other kinases and this phosphorylation regulates HDM2 nuclearcytoplasmic shuttling under certain conditions. However, we found that HDM2 phosphorylation in CLL is independent of PI3K/AKT, p70S6K, and Erk1/2 activation. Interestingly, we found that inhibition of Axl and/or Src kinases (using SKI-606 and PP1) inhibited HDM2 phosphorylation suggesting that Axl and Src kinases are critically involved in regulating HDM2 affinity towards p53 in CLL B-cells. We also identified a complementary binding sequence of miR-34a microRNA on the first 100 nucleotides of Axl 3’-UTR, suggesting a possible post-translational regulation of Axl. Interestingly, miR-34 family transcription was reported to be regulated by p53, and miR-34a was found differentially expressed in CLL versus normal CD5+ B-cells and is linked to response to therapy in CLL. Indeed, exogenous expression of the Axl gene containing the first 500 nucleotides of the Axl 3’-UTR in HEK293 cells exhibited a dose-dependent down regulation of the Axl protein upon co-transfection with miR-34a using Western blot analysis. Furthermore, activation of p53 in CLL B-cells upon treatment with the DNA-damage agent doxorubicin showed a 4–5 fold decrease of Axl protein by flow cytometry. Activation of p53 by doxorubicin was verified by detecting increased expression of p53 and its acetylation at lysine-382 by Western blot. In addition, luciferase assay data using the Axl-3’-UTR reporter plasmid DNA and miR-34a revealed a dose-dependent inhibition of the luciferase activity. Further studies are underway to fully dissect these novel regulatory signaling pathways. Conclusions: Together, these findings suggest that the two functionally distinct cell growth regulatory pathways can modulate CLL B-cell survival in response to various stimuli. This study has established a link between Axl RTK cell-survival signaling pathway and the tumor suppressor p53-mediated apoptosis-signaling pathway in CLL. These first of a kind observations also underscore that the relative balance of p53 status and Axl levels in tumor cells could relate to CLL therapeutic outcome. Disclosures:Kay:Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.

Role of Pirh2 in Mediating the Regulation of p53 and c-Myc

Ubiquitylation is fundamental for the regulation of the stability and function of p53 and c-Myc. The E3 ligase Pirh2 has been reported to polyubiquitylate p53 and to mediate its proteasomal degradation. Here, using Pirh2 deficient mice, we report that Pirh2 is important for the in vivo regulation of p53 stability in response to DNA damage. We also demonstrate that c-Myc is a novel interacting protein for Pirh2 and that Pirh2 mediates its polyubiquitylation and proteolysis. Pirh2 mutant mice display elevated levels of c-Myc and are predisposed for plasma cell hyperplasia and tumorigenesis. Consistent with the role p53 plays in suppressing c-Myc-induced oncogenesis, its deficiency exacerbates tumorigenesis of Pirh2−/− mice. We also report that low expression of human PIRH2 in lung, ovarian, and breast cancers correlates with decreased patients' survival. Collectively, our data reveal the in vivo roles of Pirh2 in the regulation of p53 and c-Myc stability and support its role as a tumor suppressor.

Reconstitution of human MCF‐7 breast cancer cells with caspase‐3 does not sensitize them to action of CDK inhibitors

Human MCF-7 breast cancer cells are resistant to pro-apoptotic stimuli due to caspase-3 inactivation. On the other hand, they should be sensitive to agents like selective pharmacological inhibitors of cyclin-dependent kinases (CDKs) that (re)activate p53 tumor suppressor protein because they harbor intact p53 pathways. In this study we examined whether reconstitution of caspase-3 in MCF-7 cells sensitizes them to inhibitors of CDKs, by analyzing the effects of roscovitine (ROSC) and olomoucine (OLO), two closely related selective pharmacological CDK inhibitors, on both mother MCF-7 cells and a secondary mutant line, MCF-7.3.28 that stably expresses human caspase-3. The results show that ROSC is, as expected, much more potent than OLO. Surprisingly; however, ROSC and OLO reduced proliferation of parental MCF-7 cells more strongly than caspase-3-proficient counterparts. Both inhibitors arrest human breast cancer cells at the G(2)-phase of the cell cycle. Analysis of cell-cycle regulators by immunoblotting revealed that ROSC strongly induces p53 protein activity by inducing its phosphorylation at Ser46 in the MCF-7 cells lacking caspase-3, but not in caspase-3-proficient cells. Furthermore, reconstitution of caspase-3 in MCF-7 cells neither elevates the mitochondrial apoptosis rate nor significantly increases caspases activity upon ROSC treatment. However, the stabilization of p53 in response to DNA damaging agents is the same in both caspase negative and positive MCF-7 cells. Cytotoxic agents induce caspase-3-dependent apoptosis in caspase-3-proficient cells. These results indicate that reconstitution of MCF-7 cancer cells with caspase-3 sensitize them to the action of DNA damaging agents but not to ATP-like pharmacological inhibitors of CDKs.

Heterochromatin silencing of p53 target genes by a small viral protein.

The transcription factor p53 guards against tumor and virus replication and is inactivated in almost all cancers. p53 activated transcription of target genes is thought to be synonymous with the stabilization of p53 in response to oncogenes and DNA damage. During adenovirus replication, the degradation of p53 by E1B-55k is considered essential for p53 inactivation, and is the basis for p53 selective viral cancer therapies. Here we reveal a dominant epigenetic mechanism that silences p53-activated transcription, irrespective of p53 phosphorylation and stabilization. We show that another adenoviral protein, E4-ORF3, inactivates p53 independently of E1B-55k by forming a nuclear structure that induces de novo H3K9me3 heterochromatin formation at p53 target promoters, preventing p53 DNA-binding. This suppressive nuclear web is highly selective in silencing p53 promoters and operates in the backdrop of global transcriptional changes that drive oncogenic replication. These findings are important for understanding how high levels of wild-type p53 might also be inactivated in cancer as well as the mechanisms that induce aberrant epigenetic silencing of tumor suppressor loci. Our study changes the longstanding definition of how p53 is inactivated in adenovirus infection and provides key insights that could enable the development of true p53 selective oncolytic viral therapies.

RFWD3–Mdm2 ubiquitin ligase complex positively regulates p53 stability in response to DNA damage

In unstressed cells, the tumor suppressor p53 is maintained at low levels by ubiquitin-mediated proteolysis mainly through Mdm2. In response to DNA damage, p53 is stabilized and becomes activated to turn on transcriptional programs that are essential for cell cycle arrest and apoptosis. Activation of p53 leads to accumulation of Mdm2 protein, a direct transcriptional target of p53. It is not understood how p53 is protected from degradation when Mdm2 is up-regulated. Here we report that p53 stabilization in the late phase after ionizing radiation correlates with active ubiquitination. We found that an E3 ubiquitin ligase RFWD3 (RNF201/FLJ10520) forms a complex with Mdm2 and p53 to synergistically ubiquitinate p53 and is required to stabilize p53 in the late response to DNA damage. This process is regulated by the DNA damage checkpoint, because RFWD3 is phosphorylated by ATM/ATR kinases and the phosphorylation mutant fails to stimulate p53 ubiquitination. In vitro experiments suggest that RFWD3 is a p53 E3 ubiquitin ligase and that RFWD3-Mdm2 complex restricts the polyubiquitination of p53 by Mdm2. Our study identifies RFWD3 as a positive regulator of p53 stability when the G(1) cell cycle checkpoint is activated and provides an explanation for how p53 is protected from degradation in the presence of high levels of Mdm2.

Role of polyamines in p53-dependent apoptosis of intestinal epithelial cells

Although p53 is known to play a critical role in the proliferation of gastrointestinal epithelia, the role of the Mdm2/p53 pathway in response to inducers of apoptosis in intestinal epithelial cells is unknown. Our data show that camptothecin (CPT)-induced apoptosis correlated with increased p53, p21Cip1, and Mdm2 protein levels, with a simultaneous increase in ATR Ser428, p53 Ser15 and Mdm2 Ser166 phosphorylation in IEC-6 cells. Increased p53 levels and its phosphorylation increased Bax protein, caspase-9, -3 activation and apoptosis. However, TNF-α/CHX-mediated apoptosis was independent of p53 protein levels and phosphorylation. The translation inhibitor, cycloheximide (CHX), prevented CPT-induced apoptosis. CHX completely prevented CPT-induced p53 phosphorylation and synthesis of p21Cip1, Bax and Bcl-xL proteins without altering p53 levels. The p53 activator, RITA, augmented CPT-induced apoptosis. The Mdm2 antagonist, Nutlin-3, significantly increased apoptosis, which was accompanied by increased p53, Mdm2 and p21Cip1 protein levels. The ATM/ATR kinase inhibitor, CGK733, blocked CPT-induced p53 Ser15 phosphorylation and protected cells from CPT-induced apoptosis. Inhibition of ornithine decarboxylase (ODC) with α-difluromethylornithine (DFMO) and subsequent depletion of intracellular polyamines increased p53 protein, Mdm2 Ser166 phosphorylation and conferred resistance to CPT-induced apoptosis. However, polyamine depletion had no effect on p53 phosphorylation. Nutlin-3 reversed the protective effect of DFMO and sensitized cells to CPT-induced apoptosis. These results suggest that p53 stabilization and accumulation in response to polyamine depletion predominantly modulate cell cycle checkpoints via p21Cip1 expression and inhibit transcription of target genes responsible for apoptosis. In contrast, phosphorylation and stabilization of p53 in response to DNA-damage lead to apoptosis, which indicates different roles of p53 during DNA damage and polyamine depletion.

Abstract A40: The enhancement of p53 stability via the inhibition of JNK-modulated ubiquitination with the activation of redox factor 1 (Ref-1) in response to non-genotoxic antioxidant selenomethionine (semet)

Abstract A40 p53 has been known to play an important role as an tumor suppressor gene for the maintaining of genome stability. In the field of cancer prevention study, p53 has been issued to clarify the mechanism of activation under the non-genotoxic cancer preventive agents containing antioxidant selenium. In previous our study, p53 has been reported to be the function of transcriptional activation activated without the genotoxicity through the redox modulation in response to selenomethionine (SeMet). In this study, we investigated that the mechanism of p53 protein stabilization enhanced by SeMet. Our data showed that the ubiquitinated p53 was decreased in the cells exhibiting the downregulation of JNK which is one of E3-ubiquitin ligases compared with in mock-treated cells suggesting that the JNK-modulated p53 ubiquitination might be inhibited by SeMet. To define the mechanism of the enhanced p53 stability in the presence of SeMet, redox factor 1 (Ref-1) which has been documented as one of the factors modulating the redox status in response to antioxidants was downregulated using siRNA. Our data showed the first time that the interaction of JNK and p53 was increased in Ref-1 siRNA-treated cells in the presence of SeMet implicating that the Ref-1 activated by SeMet might be involved in the inhibition of JNK-mediated p53 ubiquitination. Our study suggested that the enhancement of p53 stabilization in response to SeMet might provide an important clue for the cancer prevention. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A40.

P53 stabilization in response to DNA damage requires Akt/PKB and DNA-PK

The p53 protein is one of the major tumor suppressor proteins. In response to DNA damage, p53 is prevented from degradation and accumulates to high levels. Ionizing radiation leads to hypophosphorylation of the p53 ubiquitin ligase Mdm2 at sites where phosphorylation is critical for p53 degradation and to the phosphorylation and activation of Akt/PKB, a kinase that phosphorylates and inhibits GSK-3. GSK-3, which normally phosphorylates Mdm2, is inactivated in response to ionizing radiation. We show that p53 accumulates in lymphoblasts from patients with the hereditary disorder ataxia telangiectasia in response to ionizing radiation despite the absence of a functional ATM kinase. Also, knockdown of ATR did not prevent p53 accumulation in response to ionizing radiation. Instead, p53 stabilization in response to ionizing radiation depended on the inactivation of GSK-3 and the presence of Akt/PKB. Akt/PKB is a target of DNA-PK, a kinase that is activated after ionizing radiation. Correspondingly, down-regulation of DNA-PK prevented phosphorylation of Akt/PKB and GSK-3 after ionizing radiation and strongly reduced the accumulation of p53. We therefore propose a signaling cascade for the regulation of p53 in response to ionizing radiation that involves activation of DNA-PK and Akt/PKB and inactivation of GSK-3 and Mdm2.

Inactivation of the CDKN2A and the p53 tumour suppressor genes in external genital carcinomas and their precursors

p53 has been extensively studied in external genital carcinoma (EGC), and is frequently inactivated, but little is known about the role of the CDKN2A tumour suppressor gene in the oncogenesis of EGC. To investigate the role of CDKN2A and p53 in the pathogenesis of EGCs and their precursor lesions vulval intraepithelial neoplasia (VIN3), penile intraepithelial neoplasia and lichen sclerosus (LS). By means of CDKN2A and p53 mutation screening (single-strand conformational polymorphism analysis and sequencing), methylation analysis of alternative CDKN2A promoters (methylation-specific polymerase chain reaction) and p53 immununochemistry, we analysed eight invasive EGCs (five from vulva and three from penis) and 25 precancerous lesions (two undifferentiated VIN3 and 23 vulval/penile lesions of LS) from 33 patients. p53 mutations (mainly transversions) and CDKN2A mutations (including one hot spot) were present in 75% and 50% of invasive tumours, respectively, but were absent in all precancerous lesions. Remarkably, all CDKN2A-mutated tumours also harboured a p53 mutation. CDKN2A or p53 mutations were observed more frequently in LS-derived EGCs than in human papillomavirus-derived EGCs (P = 0.053). A positive anti-p53 staining, but without p53 mutations, was also detected in 30% of LS lesions, suggesting a p53 stabilization in response to inflammation and carcinogenic insult. Methylation of p16(INK4a) and p14(ARF) promoters was not a frequent mechanism of CDKN2A inactivation. Our study shows a high prevalence of co-inactivating mutations of p53 and/or CDKN2A genes in EGC, that seem to occur preferentially in LS-derived tumours and late in oncogenesis.

Ser46 Phosphorylation Regulates p53-Dependent Apoptosis and Replicative Senescence

Posttranslational modification such as phosphorylation of p53 plays important roles in activating p53 responses to various cellular and genotoxic stresses. Cell line studies have shown that phosphorylation of Ser46 is correlated with the activation of p53 apoptotic activity. To address the physiological roles of Ser46 phosphorylation, we employed homologous recombination and LoxP/Cre-mediated deletion to introduce Ser46 to Ala missense mutation into the human p53 knock-in (HUPKI) allele in mice (p53hkiS46A). p53 stabilization in response to various types of DNA damage is modestly reduced in p53hkiS46A embryonic stem (ES) cells, mouse embryonic fibroblasts (MEFs) and thymocytes. In addition, p53-dependent apoptosis is partially impaired in p53hkiS46A thymocytes and E1A/Ras-expressing mouse embryonic fibroblasts (MEFs) after DNA damage. Consistent with this finding, transcription of p53 target apoptotic genes is preferentially affected by S46A mutation after DNA damage. p53hkiS46A MEFs proliferate and reach senescence normally but can be spontaneously immortalized more easily than wild type MEFs. In addition, p53hkiS46A MEFs more readily escapes from Ras-induced senescence. Therefore, Ser46 phosphorylation activates p53-dependent apoptosis induced by DNA damage and cellular senescence induced by oncogenic stress.

ATR Activation Necessary But Not Sufficient for p53 Induction and Apoptosis in Hydroxyurea-Hypersensitive Myeloid Leukemia Cells

Hydroxyurea (HU) is a competitive inhibitor of ribonucleotide reductase that is used for the treatment of myeloproliferative disorders. HU inhibits DNA replication and induces apoptosis in a cell type-dependent manner, yet the relevant pathways that mediate apoptosis in response to this agent are not well characterized. In this study, we employed the human myeloid leukemia 1 (ML-1) cell line as a model to investigate the mechanisms of HU-induced apoptosis. Exposure of ML-1 cells to HU caused rapid cell death that was accompanied by hallmark features of apoptosis, including membrane blebbing, phosphatidylserine translocation, and caspase activation. HU-induced apoptosis required new protein synthesis, was induced by HU exposures as short as 15 min, and correlated with the accumulation of p53 and induction of the p53 target gene PUMA. p53 induction in ML-1 cells was ATR dependent and downregulation of p53 through RNAi delayed HU-induced apoptosis. HU did not induce p53 or induce apoptosis in Molt-3 leukemia cells, even though exposure to HU induced a comparable level of DNA damage and robustly activated the ATR pathway. The microtubule inhibitor nocodazole suppressed HU-induced p53 accumulation in ML-1 cells suggesting that a microtubule-dependent event contributes to p53 induction and apoptosis in this cell line. Our findings outline an HU-induced cell death pathway and suggest that activation of the ATR is necessary, but not sufficient, for stabilization of p53 in response to DNA replication stress.

Chk2 regulates transcription-independent p53-mediated apoptosis in response to DNA damage

The tumor suppressor protein p53 plays a central role in the induction of apoptosis in response to genotoxic stress. The protein kinase Chk2 is an important regulator of p53 function in mammalian cells exposed to ionizing radiation (IR). Cells derived from Chk2-deficient mice are resistant to the induction of apoptosis by IR, and this resistance has been thought to be a result of the defective transcriptional activation of p53 target genes. It was recently shown, however, that p53 itself and histone H1.2 translocate to mitochondria and thereby induces apoptosis in a transcription-independent manner in response to IR. We have now examined whether Chk2 also regulates the transcription-independent induction of apoptosis by p53 and histone H1.2. The reduced ability of IR to induce p53 stabilization in Chk2-deficient thymocytes was associated with a marked impairment of p53 and histone H1 translocation to mitochondria. These results suggest that Chk2 regulates the transcription-independent mechanism of p53-mediated apoptosis by inducing stabilization of p53 in response to IR.