Putative P53 Response Elements Research Articles

The p53 Tumor Suppressor Network Is a Key Responder to Microenvironmental Components of Chronic Inflammatory Stress

Activation of the p53 network plays a central role in the inflammatory stress response associated with ulcerative colitis and may modulate cancer risk in patients afflicted with this chronic disease. Here, we describe the gene expression profiles associated with four microenvironmental components of the inflammatory response (NO*, H2O2, DNA replication arrest, and hypoxia) that result in p53 stabilization and activation. Isogenic HCT116 and HCT116 TP53-/- colon cancer cells were exposed to the NO* donor Sper/NO, H2O2, hypoxia, or hydroxyurea, and their mRNA was analyzed using oligonucleotide microarrays. Overall, 1,396 genes changed in a p53-dependent manner (P < 0.001), with the majority representing a "unique" profile for each condition. Only 14 genes were common to all four conditions. Included were eight known p53 target genes. Hierarchical sample clustering distinguished early (1 and 4 hours) from late responses (8, 12, and 24 hours), and each treatment was differentiated from the others. Overall, NO* and hypoxia stimulated similar transcriptional responses. Gene ontology analysis revealed cell cycle as a key feature of stress responses and confirmed the similarity between NO* and hypoxia. Cell cycle profiles analyzed by flow cytometry showed that NO* and hypoxia induced quiescent S-phase and G2-M arrest. Using a novel bioinformatic algorithm, we identified several putative p53-responsive elements among the genes induced in a p53-dependent manner, including four [KIAA0247, FLJ12484, p53CSV (HSPC132), and CNK (PLK3)] common to all exposures. In summary, the inflammatory stress response is a complex, integrated biological network in which p53 is a key molecular node regulating gene expression.

P53 tumor suppressor protein regulates the levels of huntingtin gene expression

The p53 protein is a transcription factor that integrates various cellular stress signals. The accumulation of the mutant huntingtin protein with an expanded polyglutamine tract plays a central role in the pathology of human Huntington's disease. We found that the huntingtin gene contains multiple putative p53-responsive elements and p53 binds to these elements both in vivo and in vitro. p53 activation in cultured human cells, either by a temperature-sensitive mutant p53 protein or by gamma-irradiation (gamma-irradiation), increases huntingtin mRNA and protein expression. Similarly, murine huntingtin also contains multiple putative p53-responsive elements and its expression is induced by p53 activation in cultured cells. Moreover, gamma-irradiation, which activates p53, increases huntingtin gene expression in the striatum and cortex of mouse brain, the major pathological sites for Huntington's disease, in p53+/+ but not the isogenic p53-/- mice. These results demonstrate that p53 protein can regulate huntingtin expression at transcriptional level, and suggest that a p53 stress response could be a modulator of the process of Huntington's disease.

Full-length p73α Represses Drug-induced Apoptosis in Small Cell Lung Carcinoma Cells

The p73 gene, a member of the p53 family, encodes several variants through differential splicing and use of alternative promoters. At the NH2 terminus, two different promoters generate the full-length and the DeltaN isoforms, with or without the transactivating domain. At the COOH terminus, seven isoforms generated through alternative splicing have been cloned. Previous studies have demonstrated that DeltaNp73 isoforms exert a dominant-negative effect on p73 by blocking their transactivation activity and hence the ability to induce apoptosis. Considerable efforts are made to identify the functional diversity of the COOH-terminal p73 variants. In this study, we found that p73alpha inhibited drug-induced apoptosis in small cell lung carcinoma cells, whereas p73beta promoted it. p73alpha prevented Bax activation, mitochondrial dysfunction, and caspase activation. In addition, p73alpha was also able to reduce apoptosis induced by the BH3-only protein PUMA (p53 up-regulated modulator of apoptosis). Furthermore, we discovered that p73alpha is able to inhibit the pro-apoptotic effect of p73beta, demonstrating the existence of equilibrium between these two p73 isoforms. In conclusion, the reported overexpression of p73alpha in certain tumor types, and our findings that p73alpha exerts anti-apoptotic functions, indicate a potential oncogenic activity for p73.

DOWN-REGULATION OF THE PROSTATE SPECIFIC ANTIGEN PROMOTER BY p53 IN HUMAN PROSTATE CANCER CELLS

The WT p53 gene appears to have a broad role in suppressing prostatic tumorigenesis. We identified the mechanisms responsible for the effect of p53 on prostate specific antigen (PSA) expression by prostate cancer cell lines in vitro and investigated the role of a putative p53 response element in the PSA promoter region in prostate cancer cells. LNCaP cells were used to determine the effect of doxorubicin on p53 and PSA expression. The putative p53 response element in the human PSA promoter was identified by transient gene expression with site direct mutagenesis assays using a PSA reporter vector. Quantitative PSA secretions were assessed using enzyme-linked immunosorbent assays. Enzyme-linked immunosorbent and immunoblot assays indicated that doxorubicin treatment increased p53 expression but inhibited PSA levels in LNCaP cells. Transient gene expression assays showed that human PSA promoter activity was blocked by doxorubicin treatment. Mutation of the p53 response element GGGCATGTCT to GGGAGGATCT abolished the blocking effects of doxorubicin on PSA gene promoter activity. Results demonstrate that p53 regulates PSA gene expression through a putative p53 response element in the PSA promoter within human prostate cancer cells.

Who's Up First?

The tumor suppressor p53, a transcription factor activated in response to various stresses, promotes apoptosis and cell cycle arrest. PML, the protein product of the promyelocytic leukemia gene ( PML ), also acts as a tumor suppressor and is believed to act upstream of p53 to regulate its activity. De Stanchina et al. investigated the role of PML in cellular senescence and discovered that PML functioned as a direct p53 target. The authors used Western analysis to show that, in mouse embryo fibroblasts, the expression of p53 and PML was stimulated by oncogenic Ras. Stimulation of PML expression by Ras or in response to DNA damage depended on the presence of p53, and transfer of p53 into mouse and human cells lacking p53 increased the expression of PML mRNA and protein. Regions of the PML gene that contained putative p53 response elements (REs) enabled p53 to activate a heterologous gene reporter. Mutational analysis indicated that this sensitivity depended on REs. Moreover, electrophoretic mobility-shift analysis indicated that p53 bound to PML REs in vitro, and chromatin immunoprecipitation analysis indicated that p53 bound to the PML gene of cells expressing oncogenic Ras. Cells in which PML was suppressed (with short-hairpin RNA) showed specific defects in p53-mediated cell cycle arrest, senescence, and apoptosis (but not in some other p53 effects). Thus, PML appears to function as a direct target and an effector of p53. The authors discuss scenarios in which these data can be reconciled with previous research suggesting that PML acts upstream of p53. E. de Stanchina, E. Querido, M. Narita, R. V. Davuluri, P. P. Pandolfi, G. Ferbeyre, S. W. Lowe, PML is a direct p53 target that modulates p53 effector functions. Mol. Cell 13 , 523-535 (2004). [Online Journal]

P53 and DNA damage-inducible expression of the xeroderma pigmentosum group C gene.

The p53 tumor suppressor gene product is a transcription factor involved in cell-cycle regulation, apoptosis, and DNA repair. We and others have shown that p53 is required for efficient nucleotide excision repair (NER) of UV-induced DNA lesions. p53-deficient cells are defective in the repair of UV photoproducts in genomic DNA but proficient for transcription-coupled repair. Therefore, we examined whether p53 regulates the expression of genes required for global genomic repair. In this study, we demonstrate that the mRNA and protein products of the xeroderma pigmentosum group C (XPC) gene are UV-inducible in a time- and dose-dependent manner in human WI38 fibroblasts and HCT116 colorectal cancer cells wild type for p53. However, no significant induction of XPC was observed in p53-deficient counterparts to these cells. Furthermore, regulated expression of wild-type p53 in p53 null Li-Fraumeni syndrome human fibroblasts significantly augmented the expression of XPC protein. Analysis of the human XPC gene sequence revealed a putative p53 response element in the XPC promoter that was capable of mediating sequence-specific DNA binding to p53 in vitro. These results provide strong evidence that the NER gene XPC is a DNA damage-inducible and p53-regulated gene and likely plays a role in the p53-dependent NER pathway.