The cellular inhibitor of apoptosis protein (cIAP)-2 plays an important role in the protection against apoptosis by inhibiting the endogenous IAP inhibitor Smac, thus allowing other members of the IAP family, such as XIAP to block caspases. Additionally, cIAP2 functions as a ubiquitin ligase and mediates survival/proliferative signaling through NF-κB. cIAP2 is overexpressed in many human cancers and is believed to play an oncogenic role. This led to the development of small molecule IAP antagonists aimed at eliciting apoptosis in cancer cells. However, the loss of cIAP2 is also associated with multiple myeloma, in which constitutively active NF-κB signaling contributes to pathogenesis of the disease and suggests that cIAP2 may also perform a tumour suppressive function. We demonstrate a novel role for cIAP2 in maintaining p53 levels in mammary epithelial cells that express wildtype p53. Downregulation of cIAP2 resulted in activation of IKKs, which led to increased Mdm2-mediated degradation of p53. cIAP2 depletion also led to increased phosphorylation of ERK1/2. Reduction of p53 levels, in combination with survival signaling provided by NF-κB and MEK-ERK pathways were associated with increased colony formation in vitro and increased DMBA-induced adenocarcinomas in cIAP2-null mice. Treatment of cells with IAP antagonists resulted in significant cytotoxicity only in p53-mutant MDA-MB-231 cells, which was associated with autocrine production of TNF-α. We propose that the transcription of TNF-α is potentiated by gain-of-function mutation in p53 since downregulation of mutant p53 in MDA-MB-231 cells decreased TNF-α mRNA. Downregulation of cIAPs in p53-mutant cells resulted in a decrease in nuclear IKK-α, which may result in decreased IKK-α-mediated survival signaling. In contrast, cIAP downregulation in p53-wildtype cells resulted in no change in nuclear IKK-α, degradation of the corepressor SMRT and cell survival. We show that the effects of cIAP2 downregulation are context-dependent. Downregulation of cIAP2 in p53-wildtype cells results in a decrease in p53 and an increase in survival and proliferative signaling. These results suggest a tumour suppressor function for cIAPs that may account for cIAP mutation-associated cancers such as multiple myeloma. Moreover, our data also defines gain-of-function p53 mutation as a possible contributor to IAP antagonist sensitivity.

This work details the design and generation of a monomer specific p53 rabbit polyclonal antibody. The technique involved careful analysis of published Nuclear Magnetic Resonance(NMR)and X-Ray structures to select an epitope buried in the wildtype tetramer of p53,while exposed in the monomer. The antibody was validated with indirect Enzyme Linked Immunosorbant Assay(ELISA),competition ELISA and western blot. Following these experiments, the antibody, denoted as α-tet,was employed in immunofluorescence (IF) experiments of cancer cell lines DU145 (prostate carcinoma, p53 P223L and V274F) and A549 (lung carcinoma,p53 wildtype)to determine the localization of monomeric p53. Monomeric p53 was confined to the nucleolus of DU-145 cells. Additional staining of the Golgi apparatus in both cell lines was also observed. However, upon investigation of a p53 null cell line, SKOV-3,the Golgi staining was determined to be a result of cross reactivity with another protein. Nevertheless, the presence of nucleolar monomeric p53 in DU-145 cells indicates that monomerization of p53 does occur in this cell line.

This work details the design and generation of a monomer specific p53 rabbit polyclonal antibody. The technique involved careful analysis of published Nuclear Magnetic Resonance(NMR)and X-Ray structures to select an epitope buried in the wildtype tetramer of p53,while exposed in the monomer. The antibody was validated with indirect Enzyme Linked Immunosorbant Assay(ELISA),competition ELISA and western blot. Following these experiments, the antibody, denoted as α-tet,was employed in immunofluorescence (IF) experiments of cancer cell lines DU145 (prostate carcinoma, p53 P223L and V274F) and A549 (lung carcinoma,p53 wildtype)to determine the localization of monomeric p53. Monomeric p53 was confined to the nucleolus of DU-145 cells. Additional staining of the Golgi apparatus in both cell lines was also observed. However, upon investigation of a p53 null cell line, SKOV-3,the Golgi staining was determined to be a result of cross reactivity with another protein. Nevertheless, the presence of nucleolar monomeric p53 in DU-145 cells indicates that monomerization of p53 does occur in this cell line.

p53 promotes maintenance of genetic information either by causing apoptosis of damaged cells, or by altering the cell cycle and repair pathways such that damage can be accurately repaired. The nematode Caenorhabditis elegans possesses only one p53 family member, CEP-1, that controls apoptosis and the cell cycle in response to genotoxic stress. Mutation in the meiotic gene him-5 increases nondisjunction of the X chromosome, resulting in increased frequencies of XO male and XXX Dpy progeny, and it affects the frequency of meiotic recombination on X. him-5 is allelic to the ORF D1086.4, which encodes a putative basic protein with no clear homologues or domain structure. The modest embryonic lethality (Emb) of him-5 mutants is dramatically increased by mutation of cep-1 but no change is seen in the proportion of XO male or XXX Dpy progeny. The synergistic effects of cep-1 and him-5 mutation are independent of CEP-1's DNA damage regulators and other meiotic mutants, and they do not involve deregulated apoptosis. cep-1; him-5 double mutants have abnormal chromatin morphology in diakinesis-arrested oocytes reminiscent of that seen in double strand break (DSB) repair mutants. This phenotype depends on the presence of SPO-11-induced meiotic DSBs, suggesting CEP-1 and HIM-5 function together to promote accurate recombination during meiosis. In support of this hypothesis, cep-1; him-5 show a significant reduction in crossover frequency between autosomal markers compared to wild-type or either single mutant alone, suggesting they function together to promote meiotic crossing over. The X chromosome nondisjunction in both him-5 and cep-1; him-5 is a result of failure of DSB formation and subsequent chiasma formation on the X. However, the embryonic lethality phenotype of him-5 and cep-1; him-5 is caused by a defect either downstream or in parallel to meiotic DSB formation. The diakinesis chromatin phenotype of cep-1; him-5 suggests this defect may be in meiotic DSB repair. This is confirmed by the fact that cep-1; him-5 animals show more persistent meiotic DSB-associated RAD-51 foci staining compared to wild-type, suggesting CEP-1 and HIM-5 may function in efficient resolution of SPO-11-induced DSBs during meiosis. A role for CEP-1 in promoting accurate repair of DSBs during meiosis may be related to p53's function in promoting faithful meiotic recombination in mammalian cells. HIM-5's role in DSB formation and repair suggests another mechanistic link between these recombination steps. Meiotic recombination is vital for genome stability, and characterization of the role of CEP-1 and HIM-5 will increase our understanding of the p53 family and genetic redundancy at multiple steps in this process.

p53 promotes maintenance of genetic information either by causing apoptosis of damaged cells, or by altering the cell cycle and repair pathways such that damage can be accurately repaired. The nematode Caenorhabditis elegans possesses only one p53 family member, CEP-1, that controls apoptosis and the cell cycle in response to genotoxic stress. Mutation in the meiotic gene him-5 increases nondisjunction of the X chromosome, resulting in increased frequencies of XO male and XXX Dpy progeny, and it affects the frequency of meiotic recombination on X. him-5 is allelic to the ORF D1086.4, which encodes a putative basic protein with no clear homologues or domain structure. The modest embryonic lethality (Emb) of him-5 mutants is dramatically increased by mutation of cep-1 but no change is seen in the proportion of XO male or XXX Dpy progeny. The synergistic effects of cep-1 and him-5 mutation are independent of CEP-1's DNA damage regulators and other meiotic mutants, and they do not involve deregulated apoptosis. cep-1; him-5 double mutants have abnormal chromatin morphology in diakinesis-arrested oocytes reminiscent of that seen in double strand break (DSB) repair mutants. This phenotype depends on the presence of SPO-11-induced meiotic DSBs, suggesting CEP-1 and HIM-5 function together to promote accurate recombination during meiosis. In support of this hypothesis, cep-1; him-5 show a significant reduction in crossover frequency between autosomal markers compared to wild-type or either single mutant alone, suggesting they function together to promote meiotic crossing over. The X chromosome nondisjunction in both him-5 and cep-1; him-5 is a result of failure of DSB formation and subsequent chiasma formation on the X. However, the embryonic lethality phenotype of him-5 and cep-1; him-5 is caused by a defect either downstream or in parallel to meiotic DSB formation. The diakinesis chromatin phenotype of cep-1; him-5 suggests this defect may be in meiotic DSB repair. This is confirmed by the fact that cep-1; him-5 animals show more persistent meiotic DSB-associated RAD-51 foci staining compared to wild-type, suggesting CEP-1 and HIM-5 may function in efficient resolution of SPO-11-induced DSBs during meiosis. A role for CEP-1 in promoting accurate repair of DSBs during meiosis may be related to p53's function in promoting faithful meiotic recombination in mammalian cells. HIM-5's role in DSB formation and repair suggests another mechanistic link between these recombination steps. Meiotic recombination is vital for genome stability, and characterization of the role of CEP-1 and HIM-5 will increase our understanding of the p53 family and genetic redundancy at multiple steps in this process.

Oxaliplatin as an effective chemotherapeutic agent in FOLFOX regimens is using to treat colorectal cancer. In this study we investigate cytotoxicity of Oxaliplatin as single chemotherapeutic agent toHCT116P53+/- to identify molecular mechanism of Oxaliplatin action in induction of apoptosis pathway. Oxaliplatin exposure to HCT116P53+/- colorectal cell lines with deficiency of mismatch repair characteristic resulted to decrease the number of viable cells through apoptosis. Effective Oxaliplatin concentrations (IC50) which inhibit 50% of cell viability were determined using XTT method. Standard curve and time-dependent assay performed to confirm IC50 concentration. Western blot analysis demonstrated relocalization of Bax to mitochondria and induction of intrinsic apoptosis pathway resulted Oxaliplatin exposure. Inactivation of Bax in HCT116P53+/- will result significant reduction in number of viable cells following treatment with Oxaliplatin

b-catenin plays a dual role as a structural component of adherens junctions and as a transcriptional coactivator through its interactions with E-cadherin and TCF/LEF transcription factors, respectively. Normally, free b-catenin in cytoplasma is regulated by proteosome-dependent degradation system. In malignant tumor cell , deregulation of b-catenin degradation results in its aberrant accumulation, and leading to cancer. The goals of this study were to explore the reason of aberrant b-catenin accumulation in cervical carcinoma and evaluate the correlation between b-catenin¡BE-cadherin and p53 in different FIGO stage. Seventy paraffin embedded specimen with different FIGO stage were included in this study. Immunohistochemical staining was performed using anti-b-catenin polyclonal antibody and anti-p53 polyclonal antibody respectively and direct sequencing methods to analyze the mutation of CTNNB1 exon 13. The results showed 58 cases (82.8%) displayed cytoplasmic/nuclear b-catenin and no mutations in exon 13 of b-catenin gene, whereas no significant correlation between b-catenin expression level and tumor metastasis. However, b-catenin expression intensity had significant correlation with tumor size (p=0.008) and inversely correlated with E-cadherin (p=0.027) in different FIGO stage. The other way, the p53 staining intensity was significant correlated with b-catenin expression intensity (p=0.013) . Therefore, we suggest that mutations of CTNNB1 exon 13 may not be a reason for aberrant b-catenin accumulation in cervical carcinoma and aberrant p53 may play an important factor in accumulation of b-catenin.

Hepatocellular carcinoma (HCC) is one of the most common cancerous diseases worldwide. The annual occurrences exceed one million peoples affected. Currently, the treatment modalities for HCC include surgical resection, trans-arterial embolization (TAE) and chemotherapy. However, these modalities are not completely effective, underscoring the need for development of novel therapeutic approaches. PTEN, a tumor suppressor that antagonizes the PI3K pathway, is frequently mutated or deleted in various human cancers. Loss of PTEN occurs in 40-50% of surgical resected HCC samples and predicts poor prognosis for HCC patients, suggesting PTEN restoration may constitute a treatment alternative for HCC. Since PTEN increased ethanol-induced cytotoxicity in hepatoma cells, PTEN gene delivery may serve as an adjuvant therapy in conjunction with ethanol TAE for HCC. In the present study, we evaluated the efficacy of PTEN gene therapy and its combination with ethanol in a syngenic Novikoff hepatoma model by implantation of N1-S1 cells into livers of Sprague Dawley rats. Adenovirus encoding PTEN (Ad-PTEN) or green fluorescent protein (Ad-GFP) was generated for gene delivery studies. The optimal condition for adenovirus vectors to infect N1-S1 cells was determined at multiplicity of infection (MOI) of 100-200. Infection of N1-S1 cells with Ad-PTEN, but not Ad-GFP, increased PTEN levels and led to 40-50% inhibition of cell proliferation via cell cycle arrest. Besides, the half maximal -inhibitory concentration (IC50) for ethanol in N1-S1 cells was determined at 6%. Combination with PTEN gene delivery further augmented the cytotoxicity of ethanol in N1-S1 cells from 40% to 70% inhibition. To evaluate the prevention efficacy of PTEN gene delivery, N1-S1 cells were infected with adenovirus vectors then implanted into livers of Sprague-Dawley rats to induce Novikoff hepatoma. Injection of PBS- or Ad-GFP-treated N1-S1 cells led to large hepatoma (with an average size of 3-4 cm) with tumor incidence of 80-90%. In contrast, injection of Ad-PTEN-infected N1-S1 cells only induced one hepatoma (with size of 0.1 cm) in six rats, suggesting that pretreatment with PTEN gene delivery effectively abolished the tumorigenic potential of N1-S1 hepatoma cells in vivo. In summary, these results validate the feasibility of PTEN gene delivery as a new promising therapeutic strategy for the treatment of orthotopic hepatoma in immune-competent rats.

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