Science.gov

Sample records for human p53 p63

  1. p53, p63 and p73 expression and angiogenesis in keratocystic odontogenic tumors

    PubMed Central

    Chandrangsu, Soranun

    2016-01-01

    Background Keratocystic odontogenic tumors (KCOTSs) are odontogenic tumors previously referred to as odontogenic keratocysts. Several studies have reported that KCOT behavior is more like that of a benign neoplasm than a cyst. KCOTs are locally destructive and exhibit a high recurrence rate. The objective of this study is to characterize the expression of p53, p63 and p73 in KCOTs together with the relationship between their expression and KCOT angiogenesis and recurrence. Material and Methods Standard indirect immunohistochemistry using monoclonal antibodies specific to human p53, p63, p73 and CD105 was performed in formalin-fixed paraffin-embedded tissue sections of 39 KCOT samples. Grading of p53, p63 and p73 immunohistochemical staining was divided into three groups, whereas microvessel density (MVD) was presented as the mean +/- standard deviation. Associations between p53, p63 and p73 expression and clinical-pathological parameters were analyzed by Fisher’s exact test, whereas associations among MVD levels, clinical and pathological parameters and p53, p63 and p73 expression were analyzed by the Mann-Whitney U test. Correlations among p53, p63, p73 and MVD levels were analyzed using Spearman’s correlation coefficients. For all analyses, p< 0.05 was considered to indicate statistical significance. Results p53, p63 and p73 expression was noted in 23, 32 and 26 of 39 KCOT cases, respectively. The mean MVD was 26.7 ± 15.8 per high-power field. In addition, correlations between the expression levels of p53, p63, p73 and MVD in KCOT were examined. Statistically significant positive relationships were noted for all proteins (p<0.001). Conclusions Three members of the p53 protein family are expressed in KCOTs, and their expression relates to angiogenesis in these tumors. Key words:p53, p63, p73, angiogenesis, keratocystic odontogenic tumors. PMID:27957261

  2. Binding kinetics of mutant p53R175H with wild type p53 and p63: A Surface Plasmon Resonance and Atomic Force Spectroscopy study.

    PubMed

    Moscetti, Ilaria; Bizzarri, Anna Rita; Cannistraro, Salvatore

    2017-09-01

    The oncogenic mutant p53R175H, one of the most frequently occurring in human cancers and usually associated with poor prognosis and chemo resistance, can exert a dominant negative effect over p53 family members, namely wild type p53, p63 and p73, inhibiting their oncosuppressive function. Novel anticancer strategies based on drugs able to prevent the formation of complexes between p53R175H and the p53 family members call for a deeper knowledge on the molecular mechanisms of their interaction. To this aim, p53R175H/p63 and p53R175H/p53 complexes were investigated in vitro by using Surface Plasmon Resonance and Atomic Force Spectroscopy, two emerging and complementary techniques able to provide interaction kinetic information, in near physiological conditions and without any labelling. Both approaches show that p53R175H forms a very specific and highly stable bimolecular complex with both p63 and p53; with these interactions being characterized by a very high affinity with equilibrium dissociation constant, KD, of about 10(-9)M. These kinetics results, discussed also in connection with those previously reported for the interaction of p53R175H with p73, could inspire the design of suitable anticancer drugs able to antagonize the interaction of p53R175H with the p53 family members, by restoring then their anti-tumour function. Copyright © 2017. Published by Elsevier B.V.

  3. Functional interplay between p63 and p53 controls RUNX1 function in the transition from proliferation to differentiation in human keratinocytes.

    PubMed

    Masse, I; Barbollat-Boutrand, L; Molina, M; Berthier-Vergnes, O; Joly-Tonetti, N; Martin, M T; Caron de Fromentel, C; Kanitakis, J; Lamartine, J

    2012-06-07

    The interfollicular epidermis is continuously renewed, thanks to a regulated balance between proliferation and differentiation. The ΔNp63 transcription factor has a key role in the control of this process. It has been shown that ΔNp63 directly regulates Runt-related transcription factor 1 (RUNX1) transcription factor expression in mouse keratinocytes. The present study showed for the first time that RUNX1 is expressed in normal human interfollicular epidermis and that its expression is tightly regulated during the transition from proliferation to differentiation. It demonstrated that ΔNp63 directly binds two different RUNX1 regulatory DNA sequences and modulates RUNX1 expression differentially in proliferative or differentiated human keratinocytes. It also showed that the regulation of RUNX1 expression by ΔNp63 is dependent on p53 and that this coregulation relies on differential binding and activation of RUNX1 regulatory sequences by ΔNp63 and p53. We also found that RUNX1 inhibits keratinocyte proliferation and activates directly the expression of KRT1, a critical actor in early keratinocyte differentiation. Finally, we described that RUNX1 expression, similar to ΔNp63 and p53, was strongly expressed and downregulated in basal cell carcinomas and squamous cell carcinomas respectively. Taken together, these data shed light on the importance of tight control of the functional interplay between ΔNp63 and p53 in regulating RUNX1 transcription factor expression for proper regulation of interfollicular epidermal homeostasis.

  4. Comparative study of p63 and p53 expression in tissue microarrays of malignant melanomas.

    PubMed

    Brinck, Ulrich; Ruschenburg, Ilka; Di Como, Charles J; Buschmann, Nadine; Betke, Herbert; Stachura, Jerzy; Cordon-Cardo, Carlos; Korabiowska, Monika

    2002-12-01

    p63 is a known homologue of p53. In contrast to p53, however, p63 mutations are rarely seen in tumours. There have been several reports that p63 plays a regulatory role in the normal differentiation of cells, whereas its role in tumour biology must still be elucidated. The main aim of this study was to compare p63 and p53 expression in tissue microarrays of malignant melanomas and to establish any prognostic significance. p63 expression was found in 2 out of 59 tumours, both pT4. The p63 index did not exceed 30%. p53 expression was found in 27 out of 59 melanomas, with maximal expression in up to 80% of tumour cells. There were no correlations observed between the two markers. Multivariate analysis confirmed the prognostically independent role of p53. This study also confirmed that tissue microarrays can be used effectively for evaluation of the expression of certain tumour markers.

  5. A Mutant-p53/Smad complex opposes p63 to empower TGFbeta-induced metastasis.

    PubMed

    Adorno, Maddalena; Cordenonsi, Michelangelo; Montagner, Marco; Dupont, Sirio; Wong, Christine; Hann, Byron; Solari, Aldo; Bobisse, Sara; Rondina, Maria Beatrice; Guzzardo, Vincenza; Parenti, Anna R; Rosato, Antonio; Bicciato, Silvio; Balmain, Allan; Piccolo, Stefano

    2009-04-03

    TGFbeta ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFbeta-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFbeta acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFbeta-dependent inhibition of p63 function.

  6. High thermostability and lack of cooperative DNA binding distinguish the p63 core domain from the homologous tumor suppressor p53.

    PubMed

    Klein, C; Georges, G; Künkele, K P; Huber, R; Engh, R A; Hansen, S

    2001-10-05

    The p53 protein is the major tumor suppressor in mammals. The discovery of the p53 homologs p63 and p73 defined a family of p53 members with distinct roles in tumor suppression, differentiation, and development. Here, we describe the biochemical characterization of the core DNA-binding domain of a human isoform of p63, p63-delta, and particularly novel features in comparison with p53. In contrast to p53, the free p63 core domain did not show specific binding to p53 DNA consensus sites. However, glutathione S-transferase-fused and thus dimerized p63 and p53 core domains had similar affinity and specificity for the p53 consensus sites p21, gadd45, cyclin G, and bax. Furthermore, the fold of p63 core was remarkably stable compared with p53 as judged by differential scanning calorimetry (T(m) = 61 degrees C versus 44 degrees C for p53) and equilibrium unfolding ([urea](50%) = 5.2 m versus 3.1 m for p53). A homology model of p63 core highlights differences at a segment near the H1 helix hypothetically involved in the formation of the dimerization interface in p53, which might reduce cooperativity of p63 core DNA binding compared with p53. The model also shows differences in the electrostatic and hydrophobic potentials of the domains relevant to folding stability.

  7. p63 in Mytilus galloprovincialis and p53 family members in the phylum Mollusca.

    PubMed

    Stifanić, Mauro; Micić, Milena; Ramsak, Andreja; Blasković, Sanja; Ruso, Ana; Zahn, Rudolf K; Batel, Renato

    2009-11-01

    Genes of the p53 family are known to be critical regulators of the cell cycle. They have already been established as possible biomarkers. Elaborate regulation mechanisms result in numerous cDNA and protein isoforms being expressed from each gene of the p53 family. Their similarity caused an often misleading nomenclature in non-vertebrate species. The aim of the present work is a clarification of the nomenclature of molluscan p53 family sequences, an essential prerequisite for reliable interpretation of gene expression and protein function studies. Here, we report five partial cDNA and one partial genomic p63 sequences, all originating from two Mytilus galloprovincialis individuals. DNA, deduced protein sequences, and the exon/intron architecture were analyzed and compared to p53, p63 and p73 sequences from other organisms. Along with our sequences, we analyzed all similar molluscan sequences found in the GenBank database. The analysis showed our cDNA sequences code for the TAp63gamma isoform of the p63 protein, and identified all other molluscan p53 family sequences as p63 genes or their expression isoforms. Our results also indicate p63 as the ancestral gene of the p53 family as well as the only gene of the family present in non-chordate metazoan species.

  8. Deficiency of the p53/p63 target Perp alters mammary gland homeostasis and promotes cancer

    PubMed Central

    2012-01-01

    Introduction Perp is a transcriptional target of both p53 during DNA damage-induced apoptosis and p63 during stratified epithelial development. Perp-/- mice exhibit postnatal lethality associated with dramatic blistering of the epidermis and oral mucosa, reflecting a critical role in desmosome-mediated intercellular adhesion in keratinocytes. However, the role of Perp in tissue homeostasis in other p63-dependent stratified epithelial tissues is poorly understood. Given that p63 is essential for proper mammary gland development and that cell adhesion is fundamental for ensuring the proper architecture and function of the mammary epithelium, here we investigate Perp function in the mammary gland. Methods Immunofluorescence and Western blot analysis were performed to characterize Perp expression and localization in the mouse mammary epithelium throughout development. The consequences of Perp deficiency for mammary epithelial development and homeostasis were examined by using in vivo mammary transplant assays. Perp protein levels in a variety of human breast cancer cell lines were compared with those in untransformed cells with Western blot analysis. The role of Perp in mouse mammary tumorigenesis was investigated by aging cohorts of K14-Cre/+;p53fl/fl mice that were wild-type or deficient for Perp. Mammary tumor latency was analyzed, and tumor-free survival was assessed using Kaplan-Meier analysis. Results We show that Perp protein is expressed in the mammary epithelium, where it colocalizes with desmosomes. Interestingly, although altering desmosomes through genetic inactivation of Perp does not dramatically impair mammary gland ductal development, Perp loss affects mammary epithelial homeostasis by causing the accumulation of inflammatory cells around mature mammary epithelium. Moreover, we show reduced Perp expression in many human breast cancer cell lines compared with untransformed cells. Importantly, Perp deficiency also promotes the development of mouse mammary

  9. Deficiency of the p53/p63 target Perp alters mammary gland homeostasis and promotes cancer.

    PubMed

    Dusek, Rachel L; Bascom, Jamie L; Vogel, Hannes; Baron, Sylvain; Borowsky, Alexander D; Bissell, Mina J; Attardi, Laura D

    2012-04-20

    Perp is a transcriptional target of both p53 during DNA damage-induced apoptosis and p63 during stratified epithelial development. Perp-/- mice exhibit postnatal lethality associated with dramatic blistering of the epidermis and oral mucosa, reflecting a critical role in desmosome-mediated intercellular adhesion in keratinocytes. However, the role of Perp in tissue homeostasis in other p63-dependent stratified epithelial tissues is poorly understood. Given that p63 is essential for proper mammary gland development and that cell adhesion is fundamental for ensuring the proper architecture and function of the mammary epithelium, here we investigate Perp function in the mammary gland. Immunofluorescence and Western blot analysis were performed to characterize Perp expression and localization in the mouse mammary epithelium throughout development. The consequences of Perp deficiency for mammary epithelial development and homeostasis were examined by using in vivo mammary transplant assays. Perp protein levels in a variety of human breast cancer cell lines were compared with those in untransformed cells with Western blot analysis. The role of Perp in mouse mammary tumorigenesis was investigated by aging cohorts of K14-Cre/+;p53fl/fl mice that were wild-type or deficient for Perp. Mammary tumor latency was analyzed, and tumor-free survival was assessed using Kaplan-Meier analysis. We show that Perp protein is expressed in the mammary epithelium, where it colocalizes with desmosomes. Interestingly, although altering desmosomes through genetic inactivation of Perp does not dramatically impair mammary gland ductal development, Perp loss affects mammary epithelial homeostasis by causing the accumulation of inflammatory cells around mature mammary epithelium. Moreover, we show reduced Perp expression in many human breast cancer cell lines compared with untransformed cells. Importantly, Perp deficiency also promotes the development of mouse mammary cancer. Together, these

  10. Novel therapeutic interventions for p53-altered tumors through manipulation of its family members, p63 and p73

    PubMed Central

    Venkatanarayan, Avinashnarayan; Raulji, Payal; Norton, William; Flores, Elsa R.

    2016-01-01

    ABSTRACT TP53 is highly mutated in human cancers, thus targeting this tumor suppressor pathway is highly desirable and will impact many cancer patients.1,2 Therapeutic strategies to reactivate the p53-pathway have been challenging,3,4 and no effective treatment exists.5 We utilized the p53-family members, p63 and p73, which are not frequently mutated in cancer, to treat p53-defective cancers. The N-terminal splice variants of p63 and p73 are denoted as the TA and ΔN isoforms. We recently demonstrated that deletion of either ΔNp63 or ΔNp73 in p53-deficient mouse tumors results in tumor regression mediated by metabolic programming. Using this strategy, we identified pramlintide, a synthetic analog of amylin, as an effective treatment for p53 deficient and mutant tumors. Here, we show the utility of using pramlintide, as a potential cancer preventive option for p53-deficient tumors in mouse models. Additionally, we found that in vivo inhibition of both ΔNp63 and ΔNp73 in combination accelerates tumor regression and increases survival of p53-deficient mice. We report that inhibition of both ΔNp63 and ΔNp73 in combination results in upregulation of 3 key metabolic regulators, IAPP, GLS2, and TIGAR resulting in an increase in apoptosis and tumor regression in ΔNp63/ΔNp73/p53 deficient thymic lymphomas. These data highlight the value of generating inhibitors that will simultaneously target ΔNp63 and ΔNp73 to treat cancer patients with alterations in p53. PMID:26652033

  11. p63 is an alternative p53 repressor in melanoma that confers chemoresistance and a poor prognosis

    PubMed Central

    Matin, Rubeta N.; Chikh, Anissa; Law Pak Chong, Stephanie; Mesher, David; Graf, Manuela; Sanza’, Paolo; Senatore, Valentina; Scatolini, Maria; Moretti, Francesca; Leigh, Irene M.; Proby, Charlotte M.; Costanzo, Antonio; Chiorino, Giovanna; Cerio, Rino; Harwood, Catherine A.

    2013-01-01

    The role of apoptosis in melanoma pathogenesis and chemoresistance is poorly characterized. Mutations in TP53 occur infrequently, yet the TP53 apoptotic pathway is often abrogated. This may result from alterations in TP53 family members, including the TP53 homologue TP63. Here we demonstrate that TP63 has an antiapoptotic role in melanoma and is responsible for mediating chemoresistance. Although p63 was not expressed in primary melanocytes, up-regulation of p63 mRNA and protein was observed in melanoma cell lines and clinical samples, providing the first evidence of significant p63 expression in this lineage. Upon genotoxic stress, endogenous p63 isoforms were stabilized in both nuclear and mitochondrial subcellular compartments. Our data provide evidence of a physiological interaction between p63 with p53 whereby translocation of p63 to the mitochondria occurred through a codependent process with p53, whereas accumulation of p53 in the nucleus was prevented by p63. Using RNA interference technology, both isoforms of p63 (TA and ΔNp63) were demonstrated to confer chemoresistance, revealing a novel oncogenic role for p63 in melanoma cells. Furthermore, expression of p63 in both primary and metastatic melanoma clinical samples significantly correlated with melanoma-specific deaths in these patients. Ultimately, these observations provide a possible explanation for abrogation of the p53-mediated apoptotic pathway in melanoma, implicating novel approaches aimed at sensitizing melanoma to therapeutic agents. PMID:23420876

  12. Alterations of p63 and p73 in human cancers.

    PubMed

    Inoue, Kazushi; Fry, Elizabeth A

    2014-01-01

    p53 and its related genes, p63 and p73 constitute the p53 gene family. While p53 is the most frequently mutated gene in human tumors, p63 and p73 are rarely mutated or deleted in cancers. Many studies have reported p63/p73 overexpression in human cancers while others showed that a loss of p63/p73 is associated with tumor progression and metastasis. Thus, whether p63 or p73 is a tumor suppressor gene or an oncogene has been a matter of debate. This controversy has been attributed to the existence of multiple splicing isoforms with distinct functions; the full-length TA isoform of p63 has structural and functional similarity to wild-type p53, whereas the ΔNp63 acts primarily in dominant-negative fashion against all family members of p53. Differential activities of TA and ΔN isoforms have been shown in vivo by creating isform-specific gene knockout mice. All p53, p63, p73 proteins bind to and activate target genes with p53-response elements; p63 also binds to distinct p63-response elements and regulate expression of specific target genes involved in skin, limb, and craniofacial development. Interestingly, several studies have shown that both p63 and p73 are involved in cellular response to cancer therapy and others have indicated that both of these molecules are required for p53-induced apoptosis, suggesting functional interplay among p53 family proteins. Consistent with these findings, aberrant splicing that result in ΔNp63 or ΔNp73 overexpression are frequently found in human cancers, and is associated with poor clinical outcomes of patients in the latter. Thus immunohistochemical staining of tumor specimen with ΔNp73-specific antibody might have diagnostic values in cancer clinics.

  13. A Subset of Tumor-Derived Mutant Forms of p53 Down-Regulate p63 and p73 through a Direct Interaction with the p53 Core Domain

    PubMed Central

    Gaiddon, C.; Lokshin, M.; Ahn, J.; Zhang, T.; Prives, C.

    2001-01-01

    The p53 protein is related by sequence homology and function to the products of two other genes, p63 and p73, that each encode several isoforms. We and others have discovered previously that certain tumor-derived mutants of p53 can associate and inhibit transcriptional activation by the α and β isoforms of p73. In this study we have extended these observations to show that in transfected cells a number of mutant p53 proteins could bind and down-regulate several isoforms not only of p73 (p73α, -β, -γ, and -δ) but also of p63 (p63α and -γ; ΔNp63α and -γ). Moreover, a correlation existed between the efficiency of p53 binding and the inhibition of p63 or p73 function. We also found that wild-type p63 and p73 interact efficiently with each other when coexpressed in mammalian cells. The interaction between p53 mutants and p63 or p73 was confirmed in a physiological setting by examining tumor cell lines that endogenously express these proteins. We also demonstrated that purified p53 and p73 proteins interact directly and that the p53 core domain, but not the tetramerization domain, mediates this interaction. Using a monoclonal antibody (PAb240) that recognizes an epitope within the core domain of a subset of p53 mutants, we found a correlation between the ability of p53 proteins to be immunoprecipitated by this antibody and their ability to interact with p73 or p63 in vitro and in transfected cells. Based on these results and those of others, we propose that interactions between the members of the p53 family are likely to be widespread and may account in some cases for the ability of tumor-derived p53 mutants to promote tumorigenesis. PMID:11238924

  14. Influenza A Viruses Control Expression of Proviral Human p53 Isoforms p53β and Δ133p53α

    PubMed Central

    Marcel, Virginie; Cartet, Gaëlle; Lane, David P.; Lina, Bruno; Rosa-Calatrava, Manuel

    2012-01-01

    Previous studies have described the role of p53 isoforms, including p53β and Δ133p53α, in the modulation of the activity of full-length p53, which regulates cell fate. In the context of influenza virus infection, an interplay between influenza viruses and p53 has been described, with p53 being involved in the antiviral response. However, the role of physiological p53 isoforms has never been explored in this context. Here, we demonstrate that p53 isoforms play a role in influenza A virus infection by using silencing and transient expression strategies in human lung epithelial cells. In addition, with the help of a panel of different influenza viruses from different subtypes, we also show that infection differentially regulates the expressions of p53β and Δ133p53α. Altogether, our results highlight the role of p53 isoforms in the viral cycle of influenza A viruses, with p53β and Δ133p53α acting as regulators of viral production in a p53-dependent manner. PMID:22647703

  15. Transforming growth factor-beta and mutant p53 conspire to induce metastasis by antagonizing p63: a (ternary) complex affair

    PubMed Central

    Marine, Jean-Christophe; Berx, Geert

    2009-01-01

    How and when a tumor acquires metastatic properties remain largely unknown. Recent work has uncovered an intricate new mechanism through which transforming growth factor-beta (TGFβ) acts in concert with oncogenic Ras to antagonize p63-metastasis protective function. p63 inhibition requires the combined action of Ras-activated mutant p53 and TGFβ-induced Smads. Mechanistically, it involves the formation of a p63-Smads-mutant p53 ternary complex. Remarkably, just two of the key downstream targets of p63 turn out to be sufficient as a prognostic tool for breast cancer metastasis. Moreover, the molecular mechanism of this inhibition points to novel therapeutic possibilities. PMID:19664188

  16. p53 responsive elements in human retrotransposons

    PubMed Central

    Harris, CR; DeWan, A; Zupnick, A; Normart, R; Gabriel, A; Prives, C; Levine, AJ; Hoh, J

    2011-01-01

    Long interspersed nuclear elements-1 (L1s) are highly repetitive DNA elements that are capable of altering the human genome through retrotransposition. To protect against L1 retroposition, the cell downregulates the expression of L1 proteins by various mechanisms, including high-density cytosine methylation of L1 promoters and DICER-dependent destruction of L1 mRNAs. In this report, a large number of p53 responsive elements, or p53 DNA binding sites, were detected in L1 elements within the human genome. At least some of these p53 responsive elements are functional and can act to increase the levels of L1 mRNA expression. The p53 protein can directly bind to a short 15-nucleotide sequence within the L1 promoter. This p53 responsive element within L1 is a recent addition to evolution, appearing ~20 million years ago. This suggests an interplay between L1 elements, which have a rich history of causing changes in the genome, and the p53 protein, the function of which is to protect against genomic changes. To understand these observations, a model is proposed in which the increased expression of L1 mRNAs by p53 actually increases, rather than decreases, the genomic stability through amplification of p53-dependent processes for genomic protection. PMID:19718052

  17. p53 responsive elements in human retrotransposons.

    PubMed

    Harris, C R; Dewan, A; Zupnick, A; Normart, R; Gabriel, A; Prives, C; Levine, A J; Hoh, J

    2009-11-05

    Long interspersed nuclear elements-1 (L1s) are highly repetitive DNA elements that are capable of altering the human genome through retrotransposition. To protect against L1 retroposition, the cell downregulates the expression of L1 proteins by various mechanisms, including high-density cytosine methylation of L1 promoters and DICER-dependent destruction of L1 mRNAs. In this report, a large number of p53 responsive elements, or p53 DNA binding sites, were detected in L1 elements within the human genome. At least some of these p53 responsive elements are functional and can act to increase the levels of L1 mRNA expression. The p53 protein can directly bind to a short 15-nucleotide sequence within the L1 promoter. This p53 responsive element within L1 is a recent addition to evolution, appearing approximately 20 million years ago. This suggests an interplay between L1 elements, which have a rich history of causing changes in the genome, and the p53 protein, the function of which is to protect against genomic changes. To understand these observations, a model is proposed in which the increased expression of L1 mRNAs by p53 actually increases, rather than decreases, the genomic stability through amplification of p53-dependent processes for genomic protection.

  18. Role of the p53 Tumor Suppressor Homolog, p63, in Breast Cancer

    DTIC Science & Technology

    2007-05-01

    evolutionarily conserved, we ex- amined the eight-way alignments of human, chimpan- zee, mouse, rat, dog, chicken , zebrafish, and fugu (Blanchette et al...dog, and (to a lesser extent) chicken as com- pared with randomly selected genomic sequences (Figure 3A). With respect to mouse, 65.6% of the sites...Vol 444 | 30 November 2006 LETTERS 627 Nature Publishing Group ©2006 replacing exons 2 and 3 with a neomycin resistance cassette in embryonic stem

  19. Mutant p53 uses p63 as a molecular chaperone to alter gene expression and induce a pro-invasive secretome.

    PubMed

    Neilsen, Paul M; Noll, Jacqueline E; Suetani, Rachel J; Schulz, Renee B; Al-Ejeh, Fares; Evdokiou, Andreas; Lane, David P; Callen, David F

    2011-12-01

    Mutations in the TP53 gene commonly result in the expression of a full-length protein that drives cancer cell invasion and metastasis. Herein, we have deciphered the global landscape of transcriptional regulation by mutant p53 through the application of a panel of isogenic H1299 derivatives with inducible expression of several common cancer-associated p53 mutants. We found that the ability of mutant p53 to alter the transcriptional profile of cancer cells is remarkably conserved across different p53 mutants. The mutant p53 transcriptional landscape was nested within a small subset of wild-type p53 responsive genes, suggesting that the oncogenic properties of mutant p53 are conferred by retaining its ability to regulate a defined set of p53 target genes. These mutant p53 target genes were shown to converge upon a p63 signalling axis. Both mutant p53 and wild-type p63 were co-recruited to the promoters of these target genes, thus providing a molecular basis for their selective regulation by mutant p53. We demonstrate that mutant p53 manipulates the gene expression pattern of cancer cells to facilitate invasion through the release of a pro-invasive secretome into the tumor microenvironment. Collectively, this study provides mechanistic insight into the complex nature of transcriptional regulation by mutant p53 and implicates a role for tumor-derived p53 mutations in the manipulation of the cancer cell secretome.

  20. p63 expression confers significantly better survival outcomes in high-risk diffuse large B-cell lymphoma and demonstrates p53-like and p53-independent tumor suppressor function

    PubMed Central

    Manyam, Ganiraju C.; Wang, Xiao-xiao; Xia, Yi; Visco, Carlo; Tzankov, Alexandar; Zhang, Li; Montes-Moreno, Santiago; Dybkaer, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Richards, Kristy L.; Hsi, Eric D.; Choi, William W.L.; van Krieken, J. Han; Huh, Jooryung; Ponzoni, Maurilio; Ferreri, Andrés J.M.; Zhao, Xiaoying; Møller, Michael B.; Parsons, Ben M.; Winter, Jane N.; Piris, Miguel A.; Medeiros, L. Jeffrey; Young, Ken H.

    2016-01-01

    The role of p53 family member, p63 in oncogenesis is the subject of controversy. Limited research has been done on the clinical implications of p63 expression in diffuse large B-cell lymphoma (DLBCL). In this study, we assessed p63 expression in de novo DLBCL samples (n=795) by immunohistochemistry with a pan-p63-monoclonal antibody and correlated it with other clinicopathologic factors and clinical outcomes. p63 expression was observed in 42.5% of DLBCL, did not correlate with p53 levels, but correlated with p21, MDM2, p16INK4A, Ki-67, Bcl-6, IRF4/MUM-1 and CD30 expression, REL gains, and BCL6 translocation. p63 was an independent favorable prognostic factor in DLBCL, which was most significant in patients with International Prognostic Index (IPI) >2, and in activated-B-cell–like DLBCL patients with wide-type TP53. The prognostic impact in germinal-center-B-cell–like DLBCL was not apparent, which was likely due to the association of p63 expression with high-risk IPI, and potential presence of ∆Np63 isoform in TP63 rearranged patients (a mere speculation). Gene expression profiling suggested that p63 has both overlapping and distinct functions compared with p53, and that p63 and mutated p53 antagonize each other. In summary, p63 has p53-like and p53-independent functions and favorable prognostic impact, however this protective effect can be abolished by TP53 mutations. PMID:26878872

  1. [Application of PLA Method for Detection of p53/p63/p73 Complexes in Situ in Tumour Cells and Tumour Tissue].

    PubMed

    Hrabal, V; Nekulová, M; Nenutil, R; Holčaková, J; Coates, P J; Vojtěšek, B

    2017-01-01

    PLA (proximity ligation assay) can be used for detection of protein-protein interactions in situ directly in cells and tissues. Due to its high sensitivity and specificity it is useful for detection, localization and quantification of protein complexes with single molecule resolution. One of the mechanisms of mutated p53 gain of function is formation of proten-protein complexes with other members of p53 family - p63 and p73. These interactions influences chemosensitivity and invasivity of cancer cells and this is why these complexes are potential targets of anti-cancer therapy. The aim of this work is to detect p53/p63/p73 interactions in situ in tumour cells and tumour tissue using PLA method. Unique in-house antibodies for specific detection of p63 and p73 isoforms were developed and characterized. Potein complexes were detected using PLA in established cell lines SVK14, HCC1806 and FaDu and in paraffin sections of colorectal carcinoma tissue. Cell lines were also processed to paraffin blocks. p53/T-antigen and ΔNp63/T-antigen protein complexes were detected in SVK14 cells using PLA. Interactions of ΔNp63 and TAp73 isoforms were found in HCC1806 cell line with endogenous expression of these proteins. In FaDu cell line mut-p53/TAp73 complex was localized but not mut-p53/ΔNp63 complex. p53 tetramer was detected directly in colorectal cancer tissue. During development of PLA method for detection of protein complexes between p53 family members we detected interactions of p53 and p63 with T-antigen and mut-p53 and ΔNp63 with TAp73 tumour suppressor in tumour cell lines and p53 tetramers in paraffin sections of colorectal cancer tissue. PLA will be further used for detection of p53/p63, p53/p73 and p63/p73 interactions in tumour tissues and it could be also used for screening of compounds that can block formation of p53/p63/p73 protein complexes.Key words: p53 protein family - protein interaction mapping - immunofluorescence This work was supported by MEYS - NPS I

  2. p63 the guardian of human reproduction

    PubMed Central

    Amelio, Ivano; Grespi, Francesca; Annicchiarico-Petruzzelli, Margherita; Melino, Gerry

    2012-01-01

    p63 is a transcriptional factor implicated in cancer and development. The presence in TP63 gene of alternative promoters allows expression of one isoform containing the N-terminal transactivation domain (TA isoform) and one N-terminal truncated isoform (ΔN isoform). Complete ablation of all p63 isoforms produced mice with fatal developmental abnormalities, including lack of epidermal barrier, limbs and other epidermal appendages. Specific TAp63-null mice, although they developed normally, failed to undergo in DNA damage-induced apoptosis during primordial follicle meiotic arrest, suggesting a p63 involvement in maternal reproduction. Recent findings have elucidated the role in DNA damage response of a novel Hominidae p63 isoform, GTAp63, specifically expressed in human spermatic precursors. Thus, these findings suggest a unique strategy of p63 gene, to evolve in order to preserve the species as a guardian of reproduction. Elucidation of the biological basis of p63 function in reproduction may provide novel approaches to the control of human fertility. PMID:23165243

  3. Glyoxalase II, a detoxifying enzyme of glycolysis byproduct methylglyoxal and a target of p63 and p73, is a pro-survival factor of the p53 family.

    PubMed

    Xu, Yang; Chen, Xinbin

    2006-09-08

    The p53 family proteins are transcription factors and have both common and distinct functions. p53 is a classic tumor suppressor, whereas p63 and p73 have fundamental functions in development. To gain an insight into the functional diversities among the p53 family, target genes specifically regulated by p63 and p73 were examined. Here, we found that the GLX2 gene, which encodes glyoxalase II enzyme, is up-regulated by p63 and p73. Accordingly, a specific responsive element was found in intron 1 of the GLX2 gene, which can be activated and bound by p63 and p73. We also found that, upon overexpression, the cytosolic, but not the mitochondrial, GLX2 inhibits the apoptotic response of a cell to methylglyoxal, a by-product of glycolysis. Likewise, we showed that cells deficient in GLX2 are hypersensitive to methylglyoxal-induced apoptosis. Interestingly, a deficiency in GLX2 also enhances the susceptibility of a cell to DNA damage-induced apoptosis in a p53-dependent manner. These observations reveal a novel link between the p53 family and the glyoxalase system. Given that methylglyoxal is frequently generated under both physiological and pathological conditions, we postulate that GLX2 serves as a pro-survival factor of the p53 family and plays a critical role in the normal development and in the pathogenesis of various human diseases, including cancer, diabetes, and neurodegenerative diseases.

  4. Differential expression of p53, p63 and p73 protein and mRNA for DMBA-induced hamster buccal-pouch squamous-cell carcinomas

    PubMed Central

    Chen, Yuk-Kwan; Huse, Shue-Sang; Lin, Li-Min

    2004-01-01

    Abnormalities in the p53 gene are regarded as the most consistent of the genetic abnormalities associated with oral squamous-cell carcinoma. Two related members of the p53 gene family, p73 and p63, have shown remarkable structural similarity to p53, suggesting possible functional and biological interactions. The purpose of this study was to investigate the differential expression of p73, p63 and p53 genes for DMBA-induced hamster buccal-pouch squamous-cell carcinoma. Immunohistochemical analysis for protein expression and reverse transcriptase-polymerase chain reaction (RT-PCR) for mRNA expression were performed for 40 samples of hamster buccal pouches, the total being separated into one experimental group (15-week DMBA-treated; 20 animals) and two control groups (untreated and mineral oil-treated; 10 animals each). Using immunohistochemical techniques, nuclear staining of p53 and p73 proteins was detected in a subset of hamster buccal-pouch tissue specimens treated with DMBA for a period of 15 weeks, whereas p63 proteins were noted for all of the 20 hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks as well as for all of the untreated and mineral oil-treated hamster buccal-pouch tissue specimens. Differential expression of p63, p73 and p53 protein for the experimental group was as follows: p63+/p73+/p53+ (n = 14; 70%); p63+/p73+/p53− (n = 2; 10%); p63+/p73−/p53− (n = 4; 20%) and p63+/p73−/p53− (untreated [n = 10] and mineral oil-treated mucosa [n = 10]; 100% each). Upon RT-PCR, ΔNp63mRNA was detected within all of the 20 hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks, whereas expression of TAp63 was not detected. Furthermore, p73 mRNA was identified for 16 of the hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks, whereas p53 mRNA was noted for 14 15-week DMBA-treated pouches. The proportional (percentage) expression of ΔNp63, p73 and p53 mRNA for the hamster buccal-pouch tissue specimens

  5. Oncogenic Intra-p53 Family Member Interactions in Human Cancers

    PubMed Central

    Ferraiuolo, Maria; Di Agostino, Silvia; Blandino, Giovanni; Strano, Sabrina

    2016-01-01

    The p53 gene family members p53, p73, and p63 display several isoforms derived from the presence of internal promoters and alternative splicing events. They are structural homologs but hold peculiar functional properties. p53, p73, and p63 are tumor suppressor genes that promote differentiation, senescence, and apoptosis. p53, unlike p73 and p63, is frequently mutated in cancer often displaying oncogenic “gain of function” activities correlated with the induction of proliferation, invasion, chemoresistance, and genomic instability in cancer cells. These oncogenic functions are promoted either by the aberrant transcriptional cooperation of mutant p53 (mutp53) with transcription cofactors (e.g., NF-Y, E2F1, Vitamin D Receptor, Ets-1, NF-kB and YAP) or by the interaction with the p53 family members, p73 and p63, determining their functional inactivation. The instauration of these aberrant transcriptional networks leads to increased cell growth, low activation of DNA damage response pathways (DNA damage response and DNA double-strand breaks response), enhanced invasion, and high chemoresistance to different conventional chemotherapeutic treatments. Several studies have clearly shown that different cancers harboring mutant p53 proteins exhibit a poor prognosis when compared to those carrying wild-type p53 (wt-p53) protein. The interference of mutantp53/p73 and/or mutantp53/p63 interactions, thereby restoring p53, p73, and p63 tumor suppression functions, could be among the potential therapeutic strategies for the treatment of mutant p53 human cancers. PMID:27066457

  6. Effect of poly(ADP-ribose)polymerase and DNA topoisomerase I inhibitors on the p53/p63-dependent survival of carcinoma cells.

    PubMed

    Montariello, Daniela; Troiano, Annaelena; Di Girolamo, Daniela; Beneke, Sascha; Calabrò, Viola; Quesada, Piera

    2015-04-01

    Depending on their genetic background (p53(wt) versus p53(null)), carcinoma cells are more or less sensitive to drug-induced cell cycle arrest and/or apoptosis. Among the members of the p53 family, p63 is characterized by two N-terminal isoforms, TAp63 and ΔNp63. TAp63 isoform has p53-like functions, while ΔNp63 acts as a dominant negative inhibitor of p53. We have previously published that TAp63 is involved in poly(ADP-ribose)polymerase-1 (PARP-1) signaling of DNA damage deriving from DNA topoisomerase I (TOP I) inhibition in carcinoma cells. In the present study, we treated MCF7 breast carcinoma cells (p53(+)/ΔNp63(-)) or SCC022 (p53(-)/ΔNp63(+)) squamous carcinoma cells with the TOP I inhibitor topotecan (TPT) and the PJ34 PARP inhibitor, to compare their effects in the two different cell contexts. In MCF7 cells, we found that PJ34 addition reverts TPT-dependent PARP-1 auto-modification and triggers caspase-dependent PARP-1 proteolysis. Moreover, TPT as single agent stimulates p53(ser15) phosphorylation, p53 PARylation and occupancy of the p21WAF promoter by p53 resulting in an increase of p21WAF expression. Interestingly, PJ34 in combination with TPT enhances p53 occupancy at the BAX promoter and is associated with increased BAX protein level. In SCC022 cells, instead, TPT+PJ34 combined treatment reduces the level of the anti-apoptotic ΔNp63α protein without inducing apoptosis. Remarkably, in such cells, either exogenous p53 or TAp63 can rescue the apoptotic program in response to the treatment. All together our results suggest that in cancer cells PARP inhibitor(s) can operate in the choice between growth arrest and apoptosis by modulating p53 family-dependent signal.

  7. Functional Diversification after Gene Duplication: Paralog Specific Regions of Structural Disorder and Phosphorylation in p53, p63, and p73

    PubMed Central

    Siltberg-Liberles, Jessica

    2016-01-01

    Conformational and functional flexibility promote protein evolvability. High evolvability allows related proteins to functionally diverge and perhaps to neostructuralize. p53 is a multifunctional protein frequently referred to as the Guardian of the Genome–a hub for e.g. incoming and outgoing signals in apoptosis and DNA repair. p53 has been found to be structurally disordered, an extreme form of conformational flexibility. Here, p53, and its paralogs p63 and p73, were studied for further insights into the evolutionary dynamics of structural disorder, secondary structure, and phosphorylation. This study is focused on the post gene duplication phase for the p53 family in vertebrates, but also visits the origin of the protein family and the early domain loss and gain events. Functional divergence, measured by rapid evolutionary dynamics of protein domains, structural properties, and phosphorylation propensity, is inferred across vertebrate p53 proteins, in p63 and p73 from fish, and between the three paralogs. In particular, structurally disordered regions are redistributed among paralogs, but within clades redistribution of structural disorder also appears to be an ongoing process. Despite its deemed importance as the Guardian of the Genome, p53 is indeed a protein with high evolvability as seen not only in rearranged structural disorder, but also in fluctuating domain sequence signatures among lineages. PMID:27003913

  8. Genetic basis for p53 overexpression in human breast cancer.

    PubMed Central

    Davidoff, A M; Humphrey, P A; Iglehart, J D; Marks, J R

    1991-01-01

    Overexpression of an activated form of the p53 protein may be involved in neoplastic transformation. We found widespread overexpression of p53 by immunohistochemical staining in 11 (22%) of 49 primary invasive human breast cancers. Northern blot analysis showed that this overexpression was not due to an increase in the steady-state level of p53 mRNA. The p53 gene was directly sequenced in 7 of these tumors with elevated levels of the protein and, in each case, a mutation that altered the coding sequence for p53 was found in a highly conserved region of the gene. Whereas 4 of these tumors contained only a mutant p53 allele, the other 3 tumors exhibited coding sequences from both a mutant and a wild-type allele. p53 mutations have previously been correlated with allelic loss of part of chromosome 17p that contains the p53 locus. Examination of all 49 breast tumors revealed a 61% frequency of deletion at or near the p53 locus. However, the presence of allelic deletion did not correlate with overexpression of the protein. Six tumors that were deleted but did not express high levels of the protein were sequenced and all retained a wild-type p53 allele. In this series of human breast cancers, overexpression of the p53 protein, not allelic loss on chromosome 17p, was always associated with mutation of the p53 gene. Images PMID:2052583

  9. Regulation of p53 during senescence in normal human keratinocytes

    PubMed Central

    Kim, Reuben H; Kang, Mo K; Kim, Terresa; Yang, Paul; Bae, Susan; Williams, Drake W; Phung, Samantha; Shin, Ki-Hyuk; Hong, Christine; Park, No-Hee

    2015-01-01

    p53, the guardian of the genome, is a tumor suppressor protein and critical for the genomic integrity of the cells. Many studies have shown that intracellular level of p53 is enhanced during replicative senescence in normal fibroblasts, and the enhanced level of p53 is viewed as the cause of senescence. Here, we report that, unlike in normal fibroblasts, the level of intracellular p53 reduces during replicative senescence and oncogene-induced senescence (OIS) in normal human keratinocytes (NHKs). We found that the intracellular p53 level was also decreased in age-dependent manner in normal human epithelial tissues. Senescent NHKs exhibited an enhanced level of p16INK4A, induced G2 cell cycle arrest, and lowered the p53 expression and transactivation activity. We found that low level of p53 in senescent NHKs was due to reduced transcription of p53. The methylation status at the p53 promoter was not altered during senescence, but senescent NHKs exhibited notably lower level of acetylated histone 3 (H3) at the p53 promoter in comparison with rapidly proliferating cells. Moreover, p53 knockdown in rapidly proliferating NHKs resulted in the disruption of fidelity in repaired DNA. Taken together, our study demonstrates that p53 level is diminished during replicative senescence and OIS and that such diminution is associated with H3 deacetylation at the p53 promoter. The reduced intracellular p53 level in keratinocytes of the elderly could be a contributing factor for more frequent development of epithelial cancer in the elderly because of the loss of genomic integrity of cells. PMID:26138448

  10. Influence of Human p53 on Plant Development

    PubMed Central

    2016-01-01

    Mammalian p53 is a super tumor suppressor and plays a key role in guarding genome from DNA damage. However, p53 has not been found in plants which do not bear cancer although they constantly expose to ionizing radiation of ultraviolet light. Here we introduced p53 into the model plant Arabidopsis and examined p53-conferred phenotype in plant. Most strikingly, p53 caused early senescence and fasciation. In plants, fasciation has been shown as a result of the elevated homologous DNA recombination. Consistently, a reporter with overlapping segments of the GUS gene (1445) showed that the frequency of homologous recombination was highly induced in p53-transgenic plants. In contrast to p53, SUPPRESSOR OF NPR1-1 INDUCIBLE 1 (SNI1), as a negative regulator of homologous recombination in plants, is not present in mammals. Comet assay and clonogenic survival assay demonstrated that SNI1 inhibited DNA damage repair caused by either ionizing radiation or hydroxyurea in human osteosarcoma U2OS cancer cells. RAD51D is a recombinase in homologous recombination and functions downstream of SNI1 in plants. Interestingly, p53 rendered the sni1 mutants madly branching of inflorescence, a phenotype of fasciation, whereas rad51d mutant fully suppressed the p53-induced phenotype, indicating that human p53 action in plant is mediated by the SNI1-RAD51D signaling pathway. The reciprocal species-swap tests of p53 and SNI1 in human and Arabidopsis manifest that these species-specific proteins play a common role in homologous recombination across kingdoms of animals and plants. PMID:27648563

  11. The p53 gene and protein in human brain tumors

    SciTech Connect

    Louis, D.N. )

    1994-01-01

    Because p53 gene alterations are commonplace in human tumors and because p53 protein is involved in a number of important cellular pathways, p53 has become a topic of intensive investigation, both by basic scientists and clinicians. p53 was initially identified by two independent laboratories in 1979 as a 53 kilodalton (kD) protein that complexes with the large T antigen of SV40 virus. Shortly thereafter, it was shown that the E1B oncoprotein of adenovirus also binds p53. The binding of two different oncogenic viral tumor proteins to the same cellular protein suggested that p53 might be integral to tumorigenesis. The human p53 cDNA and gene were subsequently cloned in the mid-1980s, and analysis of p53 gene alterations in human tumors followed a few year later. During these 10 years, researchers grappling with the vagaries of p53 first characterized the gene as an oncogene, then as a tumor suppressor gene, and most recently as both a tumor suppressor gene and a so-called [open quotes]dominant negative[close quotes] oncogene. The last few years have seen an explosion in work on this single gene and its protein product. A review of a computerized medical database revealed approximately 650 articles on p53 in 1992 alone. p53 has assumed importance in neuro-oncology because p53 mutations and protein alterations are frequent in the common diffuse, fibrillary astrocytic tumors of adults. p53 mutations in astrocytomas were first described in 1989 and were followed by more extensive analyses of gene mutations and protein alterations in adult astrocytomas. The gene has also been studied in less common brain tumors. Elucidating the role of p53 in brain tumorigenesis will not only enhance understanding of brain tumor biology but may also contribute to improved diagnosis and therapy. This discussion reviews key aspects of the p53 gene and protein, and describe their emerging roles in central nervous system neoplasia. 102 refs., 6 figs., 1 tab.

  12. Interaction of p53 with the human Rad51 protein.

    PubMed Central

    Buchhop, S; Gibson, M K; Wang, X W; Wagner, P; Stürzbecher, H W; Harris, C C

    1997-01-01

    p53 is thought to function in the maintenance of genomic stability by modulating transcription and interacting with cellular proteins to influence the cell cycle, DNA repair and apoptosis. p53 mutations occur in >50% of human cancers, and cells which lack wild type p53 accumulate karyotypic abnormalities such as amplifications, deletions, inversions and translocations. We propose that p53 hinders these promiscuous recombinational events by interacting with cellular recombination and repair machinery. We recently reported that p53 can directly bind in vivo to human Rad51 (hRad51) protein and in vitro to its bacterial homologue RecA. We used GST-fusion and his-tagged protein systems to further investigate the physical interaction between p53 and hRad51, homologue of the yeast Rad51 protein that is involved in recombination and DNA double strand repair. The hRad51 binds to wild-type p53 and to a lesser extent, point mutants 135Y, 249S and 273H. This binding is not mediated by a DNA or RNA intermediate. Mapping studies using a panel of p53 deletion mutants indicate that hRad51 could bind to two regions of p53; one between amino acids 94 and 160 and a second between 264 and 315. Addition of anti-p53 antibody PAb421 (epitope 372-381 amino acids) inhibited the interaction with hRad51. In contrast, p53 interacts with the region between aa 125 and 220 of hRad51, which is highly conserved among Rad51 related proteins from bacteria to human. In Escherichia coli ecA protein, this region is required for homo-oligomerization, suggesting that p53 might disrupt the interaction between RecA and Rad51 subunits, thus inhibiting biochemical functions of Rad51 like proteins. These data are consistent with the hypothesis that p53 interaction with hRAD51 may influence DNA recombination and repair and that additional modifications of p53 by mutation and protein binding may affect this interaction. PMID:9380510

  13. p53 regulates the mevalonate pathway in human glioblastoma multiforme

    PubMed Central

    Laezza, C; D'Alessandro, A; Di Croce, L; Picardi, P; Ciaglia, E; Pisanti, S; Malfitano, A M; Comegna, M; Faraonio, R; Gazzerro, P; Bifulco, M

    2015-01-01

    The mevalonate (MVA) pathway is an important metabolic pathway implicated in multiple aspects of tumorigenesis. In this study, we provided evidence that p53 induces the expression of a group of enzymes of the MVA pathway including 3′-hydroxy-3′-methylglutaryl-coenzyme A reductase, MVA kinase, farnesyl diphosphate synthase and farnesyl diphosphate farnesyl transferase 1, in the human glioblastoma multiforme cell line, U343 cells, and in normal human astrocytes, NHAs. Genetic and pharmacologic perturbation of p53 directly influences the expression of these genes. Furthermore, p53 is recruited to the gene promoters in designated p53-responsive elements, thereby increasing their transcription. Such effect was abolished by site-directed mutagenesis in the p53-responsive element of promoter of the genes. These findings highlight another aspect of p53 functions unrelated to tumor suppression and suggest p53 as a novel regulator of the MVA pathway providing insight into the role of this pathway in cancer progression. PMID:26469958

  14. Characterization of the human p53 gene promoter

    SciTech Connect

    Tuck, S.P.; Crawford, L.

    1989-05-01

    Transcriptional deregulation of the p53 gene may play an important part in the genesis of some tumors. The authors report here an accurate determination of the transcriptional start sites of the human p53 gene and show that the majority of p53 mRNA molecules do not contain a postulated stem-loop structure at their 5' ends. Recombinant plasmids of the human p53 promoter-leader region fused to the bacterial chloramphenicol acetyltransferase gene (cat) were constructed. After transfection into rodent or human cells, a 350-base-pair fragment spanning the promoter region conferred 4% of the CAT activity mediated by the simian virus 40 early promoter/enhancer. They monitored the efficiency with which 15 3' and 5' promoter deletion constructs initiated transcription. Their results show that an 85-base-pair fragment, previously thought to have resided in exon 1, is that is required for full promoter activity.

  15. The p53 status of cultured human premalignant oral keratinocytes.

    PubMed Central

    Burns, J. E.; Clark, L. J.; Yeudall, W. A.; Mitchell, R.; Mackenzie, K.; Chang, S. E.; Parkinson, E. K.

    1994-01-01

    Around 60% of oral squamous cell carcinomas (SCCs) have been shown to harbour p53 mutations, and other studies have demonstrated mutant p53 genes in normal and dysplastic squamous epithelium adjacent to these SCCs. In line with these earlier studies we show here that DOK, a keratinocyte cell line derived from a dysplasia, displays elevated levels of p53 protein and harbours a 12 bp in-frame deletion of the p53 gene spanning codons 188-191. In contrast, the coding region of the p53 gene was normal in a series of six benign recurrent laryngeal papillomas and a series of four premalignant oral erythroplakia biopsies and their cell cultures. All but one of these lesions were free of malignancy at the time of biopsy, in contrast to the premalignant lesions studied by previous investigators, but keratinocytes cultured from these lesions all displayed a partially transformed phenotype that was less pronounced than that of DOK. Since three out of four of the erythroplakia patients developed SCC within 1 year of biopsy, these lesions were by definition premalignant. The availability of strains of partially transformed keratinocytes from premalignant erythroplakias which possess normal p53 genes should enable us to test the role of mutant p53 in the progression of erythroplakia to SCC. The premalignant tissues and cultures were also tested for the presence of human papillomavirus (HPV), which is known to inactivate p53 function in some cases. Only the benign papillomas were shown to contain high levels of either HPV 6 or HPV 11 E6 DNA, but not both, and none of the samples contained detectable levels of HPV 16, HPV 18 or HPV 33 E6 DNA or L1 DNA of several other HPV types. There was therefore no evidence to suggest that p53 was being inactivated by a highly oncogenic HPV in these samples. Images Figure 1 Figure 2 Figure 3 PMID:7917902

  16. Mapping of UV photoproducts along the human P53 gene

    SciTech Connect

    Tornaletti, S.; Rozek, D.; Pfeifer, G.P.

    1994-12-31

    Methods to detect DNA adducts at the DNA sequence level in mammalian cells have been developed, and it is now possible to relate adduct frequency and repair efficiency with mutations at certain nucleotide positions in human cancer-relevant genes. Mutations in the p53 tumor suppressor gene have been found in a large proportion of human skin cancers. These mutations are predominantly C to T transitions and CC to TT double transition mutations, two types of base alterations specifically induced by UV light. In order to find possible correlations between adduct distribution and mutations at specific p53 sequences, we have mapped at single-base resolution the distribution of cyclobutane dimers (CBD) and (6-4) photoproducts along the p53 gene in UV-irradiated human skin fibroblasts by ligation-mediated polymerase chain reaction (LMPCR).

  17. p53-dependent and p53-independent anticancer activity of a new indole derivative in human osteosarcoma cells

    SciTech Connect

    Cappadone, C.; Stefanelli, C.; Malucelli, E.; Zini, M.; Onofrillo, C.; Locatelli, A.; Rambaldi, M.; Sargenti, A.; Merolle, L.; Farruggia, G.; Graziadio, A.; Montanaro, L.; Iotti, S.

    2015-11-13

    Osteosarcoma (OS) is the most common primary malignant tumor of bone, occurring most frequently in children and adolescents. The mechanism of formation and development of OS have been studied for a long time. Tumor suppressor pathway governed by p53 gene are known to be involved in the pathogenesis of osteosarcoma. Moreover, loss of wild-type p53 activity is thought to be a major predictor of failure to respond to chemotherapy in various human cancers. In previous studies, we described the activity of a new indole derivative, NSC743420, belonging to the tubulin inhibitors family, capable to induce apoptosis and arrest of the cell cycle in the G2/M phase of various cancer cell lines. However, this molecule has never been tested on OS cell line. Here we address the activity of NSC743420 by examine whether differences in the p53 status could influence its effects on cell proliferation and death of OS cells. In particular, we compared the effect of the tested molecule on p53-wild type and p53-silenced U2OS cells, and on SaOS2 cell line, which is null for p53. Our results demonstrated that NSC743420 reduces OS cell proliferation by p53-dependent and p53-independent mechanisms. In particular, the molecule induces proliferative arrest that culminate to apoptosis in SaOS2 p53-null cells, while it brings a cytostatic and differentiating effect in U2OS cells, characterized by the cell cycle arrest in G0/G1 phase and increased alkaline phosphatase activity. - Highlights: • The indole derivative NSC743420 induces antitumor effects on osteosarcoma cells. • p53 status could drive the activity of antitumor agents on osteosarcoma cells. • NSC743420 induces cytostatic and differentiating effects on U2OS cells. • NSC743420 causes apoptosis on p53-null SaOS2 cells.

  18. [Structural organization of the human p53 gene. I. Molecular cloning of the human p53 gene].

    PubMed

    Bukhman, V L; Ninkina, N N; Chumakov, P M; Khilenkova, M A; Samarina, O P

    1987-09-01

    Human p53 gene was cloned from the normal human placenta DNA and DNA from the strain of human kidney carcinoma transplanted into nude mice. Representative gene library from tumor strain of human kidney carcinoma and library of 15 kb EcoRI fragments of DNA from normal human placenta were constructed. Maniatis gene library was also used. Five clones were isolated from kidney carcinoma library; they covered 27 kb and included full-length p53 gene of 19.5 kb and flanking sequences. From normal placenta libraries three overlapped clones were obtained. Restriction map of cloned sequences was constructed and polarity of the p53 gene determined. The first intron of the gene is large (10.4 kb); polymorphic BglII site was observed in this intron, which allows to discriminate between allelic genes. One of these (BglII-) is ten times more abundant that the other (BglII+). Both allelic genes are able to synthesize the 2.8 kb p53 gene.

  19. Combination of p53-DC vaccine and rAd-p53 gene therapy induced CTLs cytotoxic against p53-deleted human prostate cancer cells in vitro.

    PubMed

    Saito, H; Kitagawa, K; Yoneda, T; Fukui, Y; Fujsawa, M; Bautista, D; Shirakawa, T

    2017-07-01

    Recently, the US FDA approved sipuleucel-T, which is composed of autologous DCs stimulated with a recombinant fusion protein of prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GM-CSF), as the first immunotherapeutic agent for metastatic castration resistant prostate cancer (mCRPC). However, sipuleucel-T demonstrated only modest efficacy in mCPRC patients. Researchers are now investigating the potential of p53 protein as a tumor-associated antigen (TAA) loaded in DC-based cancer vaccine. Approximately half of all tumors overexpress p53, and up to 20% of prostate cancer cells overexpresses p53. In this study, we evaluated the feasibility of combining p53-DC vaccine and rAd-p53 gene therapy, using the p53-overexpressing and non-expressing prostate cancer cells in vitro. We successfully generated the p53-DC vaccine by culturing autologous DCs infected with rAd-p53. This p53-DC vaccine can differentiate CTLs specifically cytotoxic to p53-overexpressing prostate cancer cells. In addition, rAd-p53 infection can induce overexpression of p53 and thus the cytotoxicity of CTLs differentiated by the p53-DC vaccine in p53 non-expressing prostate cancer cells. These findings suggest that this combination therapy using p53-DC vaccine and rAd-p53 gene therapy together may represent a new paradigm for the treatment of mCRPC.

  20. The 3p63d9-(3p53d10+3p63d84f) Transitions in Cobalt-like ions: As VII, Se VIII and Br IX

    NASA Astrophysics Data System (ADS)

    van Kleef, Th A. M.; Uylings, P.; Ryabtsev, A. N.; Joshi, Y. N.

    1986-01-01

    The spectra of As VII, Se VIII and Br IX have been studied in the 100 Å - 130 Å, 80 Å - 120 Å and 70 Å - 115 Å wavelength regions, respectively, using high dispersion spectrographs at various laboratories. A triggered vacuum spark was used as excitation source. It has been shown that substantial configuration interaction exists between the 3p5 3d10 and 3p63d84f configurations. In the 3p63d84f configuration, 34 levels have been established in As VII, 35 out of 37 levels reported earlier in Se VIII, have been confirmed, two have been revised and one additional level established, and 30 levels have been established in Br IX. Least-Squares-Fit calculations using a two-configuration model space support the analyses. Fifty-two (52), 5 and 40 newly classified lines are reported in As VII, Se VIII and Br IX, respectively.

  1. Ligand dependent restoration of human TLR3 signaling and death in p53 mutant cells

    PubMed Central

    Menendez, Daniel; Lowe, Julie M.; Snipe, Joyce; Resnick, Michael A.

    2016-01-01

    Diversity within the p53 transcriptional network can arise from a matrix of changes that include target response element sequences and p53 expression level variations. We previously found that wild type p53 (WT p53) can regulate expression of most innate immune-related Toll-like-receptor genes (TLRs) in human cells, thereby affecting immune responses. Since many tumor-associated p53 mutants exhibit change-of-spectrum transactivation from various p53 targets, we examined the ability of twenty-five p53 mutants to activate endogenous expression of the TLR gene family in p53 null human cancer cell lines following transfection with p53 mutant expression vectors. While many mutants retained the ability to drive TLR expression at WT levels, others exhibited null, limited, or change-of-spectrum transactivation of TLR genes. Using TLR3 signaling as a model, we show that some cancer-associated p53 mutants amplify cytokine, chemokine and apoptotic responses after stimulation by the cognate ligand poly(I:C). Furthermore, restoration of WT p53 activity for loss-of-function p53 mutants by the p53 reactivating drug RITA restored p53 regulation of TLR3 gene expression and enhanced DNA damage-induced apoptosis via TLR3 signaling. Overall, our findings have many implications for understanding the impact of WT and mutant p53 in immunological responses and cancer therapy. PMID:27533082

  2. Regulation of Human p53 Activity and Cell Localization by Alternative Splicing

    PubMed Central

    Ghosh, Anirban; Stewart, Deborah; Matlashewski, Greg

    2004-01-01

    The development of cancer is a multistep process involving mutations in proto-oncogenes, tumor suppressor genes, and other genes which control cell proliferation, telomere stability, angiogenesis, and other complex traits. Despite this complexity, the cellular pathways controlled by the p53 tumor suppressor protein are compromised in most, if not all, cancers. In normal cells, p53 controls cell proliferation, senescence, and/or mediates apoptosis in response to stress, cell damage, or ectopic oncogene expression, properties which make p53 the prototype tumor suppressor gene. Defining the mechanisms of regulation of p53 activity in normal and tumor cells has therefore been a major priority in cell biology and cancer research. The present study reveals a novel and potent mechanism of p53 regulation originating through alternative splicing of the human p53 gene resulting in the expression of a novel p53 mRNA. This novel p53 mRNA encodes an N-terminally deleted isoform of p53 termed p47. As demonstrated within, p47 was able to effectively suppress p53-mediated transcriptional activity and impair p53-mediated growth suppression. It was possible to select for p53-null cells expressing p47 alone or coexpressing p53 in the presence of p47 but not cells expressing p53 alone. This showed that p47 itself does not suppress cell viability but could control p53-mediated growth suppression. Interestingly, p47 was monoubiquitinated in an Mdm2-independent manner, and this was associated with its export out of the nucleus. In the presence of p47, there was a reduction in Mdm2-mediated polyubiquitination and degradation of p53, and this was also associated with increased monoubiquitination and nuclear export of p53. The expression of p47 through alternative splicing of the p53 gene thus has a major influence over p53 activity at least in part through controlling p53 ubiquitination and cell localization. PMID:15340061

  3. Cytoskeleton-anchoring of Conformational Mutant-like p53, but not shorter isoforms p53β and p47 (ΔN40p53) in Senescent Human fibroblasts.

    PubMed

    Nishio, Koji

    2017-05-23

    Cytoskeleton anchoring of conformational mutant-like p53 is prominent in human senescent cell. The present research investigated the structural basis of vimentin cytoskeleton-anchoring of human p53. GFP-fused wild type p53, mutant p53, those of the various truncated isoforms including p53β and p47, were expressed in the vimentin-expressing cells: mouse fibroblasts, COS-7 cells, young and senescent human fibroblasts, and HeLa cells (non-vimentin-expressing). A cancer-specific mutant p53V143A-GFP expressed in mouse fibroblasts, exclusively anchored on the vimentin cytoskeleton. Class I mutant p53R175C-GFP and class II mutant p53R175S-GFP localized in the nuclei of COS-7 cells. A class Ⅲmutant p53R175X-GFP (X: D, F, W or Y), cancer-specific mutant p53V143A-GFP and p53R249S-GFP, exclusively anchored on the vimentin cytoskeleton of COS-7 cells. The deletions of p53R249S and p53V143A at the C-terminus (ΔC63) exclusively promoted the nuclear import of the deleted mutant p53 in COS-7 and HeLa cells, whereas the deletions at the N-terminus (ΔN40) or C-terminus (ΔC33) were ineffective. Thus, the cancer-specific mutant p53R249S and p53V143A adopt distinct mutant conformation and thereby the C-terminal region (aa331-360) potently interacts with the vimentin cytoskeleton and HeLa cells' cytoskeleton. Wild type p53-GFP exclusively localized in the nuclei of growing young fibroblast, in contrast to the significant cytoplasmic retention in senescent human fibroblasts. The deletion of p53 at the N-terminus or at the C-terminus (ΔN40 or ΔC63) results in a significant nuclear import of the shorter isoforms, p53β and p47. Senescent fibroblasts promote p53 to adopt a hotspot mutant like-conformation which significantly overrides the nuclear import due to the potent cytoskeleton-anchoring. Interestingly, the shorter p53 isoforms can escape from the cytoskeleton-anchoring. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  4. PPAR{gamma} ligands induce growth inhibition and apoptosis through p63 and p73 in human ovarian cancer cells

    SciTech Connect

    Kim, Soyeon; Lee, Jae-Jung; Heo, Dae Seog

    2011-03-18

    Research highlights: {yields} PPAR{gamma} ligands increased the rate of apoptosis and inhibition of proliferation in ovarian cancer cells. {yields} PPAR{gamma} ligands induced p63 and p73 expression, but not p53. {yields} p63 and p73 leads to an increase in p21 expression and apoptosis in ovarian cancer cells with treatment PPAR{gamma} ligands. {yields} These findings suggest that PPAR{gamma} ligands suppressed growth of ovarian cancer cells through upregulation of p63 and p73. -- Abstract: Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) agonists, including thiazolidinediones (TZDs), can induce anti-proliferation, differentiation, and apoptosis in various cancer cell types. This study investigated the mechanism of the anticancer effect of TZDs on human ovarian cancer. Six human ovarian cancer cell lines (NIH:OVCAR3, SKOV3, SNU-251, SNU-8, SNU-840, and 2774) were treated with the TZD, which induced dose-dependent inhibition of cell growth. Additionally, these cell lines exhibited various expression levels of PPAR{gamma} protein as revealed by Western blotting. Flow cytometry showed that the cell cycle was arrested at the G1 phase, as demonstrated by the appearance of a sub-G1 peak. This observation was corroborated by the finding of increased levels of Bax, p21, PARP, and cleaved caspase 3 in TGZ-treated cells. Interestingly, when we determined the effect of p53-induced growth inhibition in these three human ovarian cancer cells, we found that they either lacked p53 or contained a mutant form of p53. Furthermore, TGZ induced the expression of endogenous or exogenous p63 and p73 proteins and p63- or p73-directed short hairpin (si) RNAs inhibited the ability of TGZ to regulate expression of p21 in these cells. Thus, our results suggest that PPAR{gamma} ligands can induce growth suppression of ovarian cancer cells and mediate p63 and p73 expression, leading to enhanced growth inhibition and apoptosis. The tumor suppressive effects of PPAR{gamma} ligands

  5. Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion

    PubMed Central

    Haque, Shabirul; Yan, Xiao Jie; Rosen, Lisa; McCormick, Steven; Chiorazzi, Nicholas; Mongini, Patricia K. A.

    2014-01-01

    Within T-cell-dependent germinal centers, p53 gene transcription is repressed by Bcl-6 and is thus less vulnerable to mutation. Malignant lymphomas within inflamed extranodal sites exhibit a relatively high incidence of p53 mutations. The latter might originate from normal B-cell clones manifesting activation-induced cytosine deaminase (AID) and up-regulated p53 following T-cell-independent (TI) stimulation. We here examine p53 gene transcription in such TI clones, with a focus on modulatory effects of prostaglandin E2 (PGE2), and evaluate progeny for p53 mutations. Resting IgM+IgD+CD27− B cells from human tonsils were labeled with CFSE and stimulated in vitro with complement-coated antigen surrogate, IL-4, and BAFF ± exogenous PGE2 (50 nM) or an analog specific for the EP2 PGE2 receptor. We use flow cytometry to measure p53 and AID protein within variably divided blasts, qRT-PCR of p53 mRNA from cultures with or without actinomycin D to monitor mRNA transcription/stability, and single-cell p53 RT-PCR/sequencing to assess progeny for p53 mutations. We report that EP2 signaling triggers increased p53 gene transcriptional activity in AID+ cycling blasts (P<0.01). Progeny exhibit p53 mutations at a frequency (8.5×10−4) greater than the baseline error rate (<0.8×10−4). We conclude that, devoid of the repressive influences of Bcl-6, dividing B lymphoblasts in inflamed tissues should display heightened p53 transcription and increased risk of p53 mutagenesis.—Haque, S., Yan, X. J., Rosen, L., McCormick, S., Chiorazzi, N., Mongini, P. K. A. Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion. PMID:24145719

  6. DDP-induced cytotoxicity is not influenced by p53 in nine human ovarian cancer cell lines with different p53 status.

    PubMed Central

    De Feudis, P.; Debernardis, D.; Beccaglia, P.; Valenti, M.; Graniela Siré, E.; Arzani, D.; Stanzione, S.; Parodi, S.; D'Incalci, M.; Russo, P.; Broggini, M.

    1997-01-01

    Nine human ovarian cancer cell lines that express wild-type (wt) or mutated (mut) p53 were used to evaluate the cytotoxicity induced by cisplatin (DDP). The concentrations inhibiting the growth by 50% (IC50) were calculated for each cell line, and no differences were found between cells expressing wt p53 and mut p53. Using, for each cell line, the DDP IC50, we found that these concentrations were able to induce an increase in p53 levels in all four wt-p53-expressing cell lines and in one out of five mut-p53-expressing cell lines. WAF1 and GADD45 mRNAs were also increased by DDP treatment, independently of the presence of a wt p53. Bax levels were only marginally affected by DDP, and this was observed in both wt-p53- and mut-p53-expressing cells. DDP-induced apoptosis was evident 72 h after treatment, and the percentage of cells undergoing apoptosis was slightly higher for wt-p53-expressing cells. However, at doses near the IC50, the percentage of apoptotic cells was less than 20% in all the cell lines investigated. We conclude that the presence of wt p53 is not a determinant for the cytotoxicity induced by DDP in human ovarian cancer cell lines. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:9275024

  7. Expression of the human tumor suppressor p53 induces cell death in Pichia pastoris.

    PubMed

    Abdelmoula-Souissi, Salma; Mabrouk, Imed; Gargouri, Ali; Mokdad-Gargouri, Raja

    2012-02-01

    The human tumor suppressor p53 is known as guardian of genome because of its involvement in many signals related to cell life or death. In this work, we report that human p53 induces cell death in the yeast Pichia pastoris. We showed a growth inhibition effect, which increased with the p53 protein expression level in recombinant Mut(s) (methanol utilization slow) strain of Pichia. However, no effect of p53 was observed in recombinant strain of Mut(+) (methanol utilization plus) phenotype. Interestingly, human p53 induces cell death in recombinant strains Mut(s) with characteristic markers of apoptosis such as DNA fragmentation, exposure of phosphatidylserine, and reactive oxygen species generation. Taken together, our results strongly suggest that human p53 is biologically active in this heterologous context. Thus, we propose that P. pastoris could be a useful tool to better understand the biological function of human p53.

  8. Electrophoretic detection of protein p53 in human leukocytes

    SciTech Connect

    Paponov, V.D.; Kupsik, E.G.; Shcheglova, E.G.; Yarullin, N.N.

    1986-01-01

    The authors have found an acid-soluble protein with mol. wt. of about 53 kD in peripheral blood leukocytes of persons with Down's syndrome. It was present in different quantities in all 20 patients tested, but was virtually not discovered in 12 healthy blood donors. This paper determines the possible identity of this protein with protein p53 from mouse ascites carcinoma by comparing their electrophoretic mobilities, because the accuracy of electrophoretic determination of the molecular weight of proteins is not sufficient to identify them. The paper also describes experiments to detect a protein with electrophoretic mobility identical with that of a protein in the leukocytes of patients with Down's syndrome in leukocytes of patients with leukemia. To discover if protein p53 is involved in cell proliferation, the protein composition of leukocytes from healthy blood donors, cultured in the presence and absence of phytohemagglutinin (PHA), was compared. Increased incorporation of H 3-thymidine by leukocytes of patients with Down's syndrome is explained by the presence of a population of immature leukocytes actively synthesizing DNA in the peripheral blood of these patients, and this can also explain the presence of protein p53 in the leukocytes of these patients.

  9. Endogenous retrovirus drives hitherto unknown proapoptotic p63 isoforms in the male germ line of humans and great apes.

    PubMed

    Beyer, Ulrike; Moll-Rocek, Julian; Moll, Ute M; Dobbelstein, Matthias

    2011-03-01

    TAp63, but not its homolog p53, eliminates oocytes that suffered DNA damage. An equivalent gene for guarding the male germ line is currently not known. Here we identify hitherto unknown human p63 transcripts with unique 5'-ends derived from incorporated exons upstream of the currently mapped TP63 gene. These unique p63 transcripts are highly and specifically expressed in testis. Their most upstream region corresponds to a LTR of the human endogenous retrovirus 9 (ERV9). The insertion of this LTR upstream of the TP63 locus occurred only recently in evolution and is unique to humans and great apes (Hominidae). A corresponding p63 protein is the sole p63 species in healthy human testis, and is strongly expressed in spermatogenic precursors but not in mature spermatozoa. In response to DNA damage, this human male germ-cell-encoded TAp63 protein (designated GTAp63) is activated by caspase cleavage near its carboxyterminal domain and induces apoptosis. Human testicular cancer tissues and cell lines largely lost p63 expression. However, pharmacological inhibition of histone deacetylases completely restores p63 expression in testicular cancer cells (>3,000-fold increase). Our data support a model whereby testis-specific GTAp63 protects the genomic integrity of the male germ line and acts as a tumor suppressor. In Hominidae, this guardian function was greatly enhanced by integration of an endogenous retrovirus upstream of the TP63 locus that occurred 15 million years ago. By providing increased germ-line stability, this event may have contributed to the evolution of hominids and enabled their long reproductive periods.

  10. Endogenous retrovirus drives hitherto unknown proapoptotic p63 isoforms in the male germ line of humans and great apes

    PubMed Central

    Beyer, Ulrike; Moll-Rocek, Julian; Moll, Ute M.; Dobbelstein, Matthias

    2011-01-01

    TAp63, but not its homolog p53, eliminates oocytes that suffered DNA damage. An equivalent gene for guarding the male germ line is currently not known. Here we identify hitherto unknown human p63 transcripts with unique 5′-ends derived from incorporated exons upstream of the currently mapped TP63 gene. These unique p63 transcripts are highly and specifically expressed in testis. Their most upstream region corresponds to a LTR of the human endogenous retrovirus 9 (ERV9). The insertion of this LTR upstream of the TP63 locus occurred only recently in evolution and is unique to humans and great apes (Hominidae). A corresponding p63 protein is the sole p63 species in healthy human testis, and is strongly expressed in spermatogenic precursors but not in mature spermatozoa. In response to DNA damage, this human male germ-cell–encoded TAp63 protein (designated GTAp63) is activated by caspase cleavage near its carboxyterminal domain and induces apoptosis. Human testicular cancer tissues and cell lines largely lost p63 expression. However, pharmacological inhibition of histone deacetylases completely restores p63 expression in testicular cancer cells (>3,000-fold increase). Our data support a model whereby testis-specific GTAp63 protects the genomic integrity of the male germ line and acts as a tumor suppressor. In Hominidae, this guardian function was greatly enhanced by integration of an endogenous retrovirus upstream of the TP63 locus that occurred 15 million years ago. By providing increased germ-line stability, this event may have contributed to the evolution of hominids and enabled their long reproductive periods. PMID:21300884

  11. [Effect of recombinant human p53 adenovirus (Ad-p53) combined with EGFR inhibitor gefitinib on the sensitivity of breast cancer MDA-MB-468 cells].

    PubMed

    Wang, Xinzhao; Guan, Xiyun; Wang, Leilei; Xie, Li; Liu, Qi; Yu, Zhiyong

    2014-12-01

    To observe the impact of concurrent administration of recombinant human p53 adenovirus (Ad-p53) with EGFR inhibitor gefitinib on breast cancer MDA-MB-468 cells. MDA-MB-468 cells were treated with Ad-p53 and/or gefitinib. The effect of Ad-p53 and gefitinib on the growth of MDA-MB-468 cells was evaluated by MTT assay. Cell apoptosis was detected by flow cytometry. Western blot analysis was used to detect the alteration of p53,EGFR, phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and apoptosis-related proteins. Ad-p53 combined with gefitinib was used in vivo to explore their effect on tumor xenograft in nude mice. Immunohistochemistry was used to detect the p53 expression in vivo. The MTT assay showed a stronger inhibitory effect of gefitinib on MDA-MB-468 cells infected with Ad-p53 than on the control cells. Cell apoptosis assay revealed that the apoptosis rates of MDA-MB-468 cells in vehicle-treated group, Ad-p53 group, gefitinib group, and combination group were 8.5%, 17.4%, 20.5% and 32.6%, respectively. The apoptosis rate of MDA-MB-468 cells in the combination group was higher than that in other groups (P < 0.05, for all) . Western blot analysis revealed that the expression of p53 was significantly up-regulated in the presence of Ad-p53. The combination of Ad-p53 and gefitinib significantly down-regulated p-Akt (S473)(P < 0.01) and up-regulated caspase-9 and cleaved caspase-3 (P < 0.01 for both). Tumor inhibition rates (TIR) in the Ad-p53, gefitinib, and combination groups were 35.7%, 28.7% and 74.4%, respectively. Ad-p53 and gefitinib combination therapy significantly reduced the tumor burden in nude mice (P < 0.05 for all).Immunohistochemistry showed that after intratumoral administration of Ad-p53, wild-type p53 was increased (P < 0.01). p53 expressions in the vehicle-treated, Ad-p53, gefitinib and combination groups were 45.2%, 80.1%, 50.7% and 90.6%, respectively. Wild-type p53 may reverse the sensitivity of MDA-MB-468 cells to gefitinib through

  12. Temperature sensitivity of human wild-type and mutant p53 proteins expressed in vivo.

    PubMed Central

    Ponchel, F.; Milner, J.

    1998-01-01

    p53 is activated in response to DNA damage and functions in the maintenance of genetic integrity. Loss of p53 function because of mutation of the p53 gene is associated with over half all human cancers. Certain human p53 mutants are conformationally flexible in vitro and are temperature sensitive, with partial or complete recovery of wild-type (wt) properties at 32 degrees C. We have now tested the functional capacities of selected p53 mutants in vivo, by transfection into established human cell lines. Unexpectedly, we found that wt p53 can be temperature sensitive for transactivation of a co-transfected target gene in vivo. Flexible mutants retained varying degrees of functional capacity in transfected cells, and the recipient cell line appeared to be a significant determinant of both wt and mutant p53 function; importantly, two p53 null cell lines commonly used to study p53 function (Saos-2 and Hep3B) differed markedly in this latter respect. We also show that the p53 mutant V272M, which exhibits sequence-specific DNA binding in vitro, is nonetheless defective for transactivation and is unable to induce apoptosis in vivo. The valine 272 residue may thus be crucial for properties (other than sequence-specific DNA binding) that are important for p53 function(s) in vivo. Images Figure 4 PMID:9635828

  13. Reactivation of mutant p53 and induction of apoptosis in human tumor cells by maleimide analogs.

    PubMed

    Bykov, Vladimir J N; Issaeva, Natalia; Zache, Nicole; Shilov, Alexandre; Hultcrantz, Monica; Bergman, Jan; Selivanova, Galina; Wiman, Klas G

    2005-08-26

    Reactivation of mutant p53 is likely to provide important benefits for treatment of chemotherapy- and radiotherapy-resistant tumors. We demonstrate here that the maleimide-derived molecule MIRA-1 can reactivate DNA binding and preserve the active conformation of mutant p53 protein in vitro and restore transcriptional transactivation to mutant p53 in living cells. MIRA-1 induced mutant p53-dependent cell death in different human tumor cells carrying tetracycline-regulated mutant p53. The structural analog MIRA-3 showed antitumor activity in vivo against human mutant p53-carrying tumor xenografts in SCID mice. The MIRA scaffold is a novel lead for the development of anticancer drugs specifically targeting mutant p53.

  14. Characterization of a murine p53ser246 mutant equivalent to the human p53ser249 associated with hepatocellular carcinoma and aflatoxin exposure.

    PubMed

    Ghebranious, N; Knoll, B J; Wu, H; Lozano, G; Sell, S

    1995-06-01

    A mutation in the tumor suppressor p53 gene resulting in an Arg-->Ser substitution in position 249 is found frequently in human hepatocellular carcinomas associated with hepatitis B infection and with aflatoxin exposure. To determine the significance of this mutation in an in vivo experimental model using transgenic mice, we introduced a two-nucleotide change in the mouse p53 gene at amino-acid position 246, which is equivalent to position 249 in human p53, by the recombinant polymerase chain reaction mismatched primer method. This p53 mutation resulted in the same change, an Arg-->Ser substitution, as in the human p53 gene at position 249. We now report that the protein product of this mutant mouse p53ser246 had properties similar to those of the wild-type protein when tested by binding to (i) monoclonal antibodies PAb246 and PAb240, ii) simian virus 40 large T antigen, and (iii) heat-shock protein. However, it had mutant-type transforming properties when tested for colony formation with an osteosarcoma cell line. It was not active, as is wild-type p53, in transcription activation of the muscle creatine kinase promoter. These properties are the same as those found in the p53trp248 product of the p53 mutation associated with the Li-Fraumeni syndrome. Although less is known about the human p53ser249 product associated with hepatocellular carcinoma, the mutant murine p53ser246 protein shares the known properties of the human gene product.

  15. p53 and NF-κB coregulate proinflammatory gene responses in human macrophages.

    PubMed

    Lowe, Julie M; Menendez, Daniel; Bushel, Pierre R; Shatz, Maria; Kirk, Erin L; Troester, Melissa A; Garantziotis, Stavros; Fessler, Michael B; Resnick, Michael A

    2014-04-15

    Macrophages are sentinel immune cells that survey the tissue microenvironment, releasing cytokines in response to both exogenous insults and endogenous events such as tumorigenesis. Macrophages mediate tumor surveillance and therapy-induced tumor regression; however, tumor-associated macrophages (TAM) and their products may also promote tumor progression. Whereas NF-κB is prominent in macrophage-initiated inflammatory responses, little is known about the role of p53 in macrophage responses to environmental challenge, including chemotherapy or in TAMs. Here, we report that NF-κB and p53, which generally have opposing effects in cancer cells, coregulate induction of proinflammatory genes in primary human monocytes and macrophages. Using Nutlin-3 as a tool, we demonstrate that p53 and NF-κB rapidly and highly induce interleukin (IL)-6 by binding to its promoter. Transcriptome analysis revealed global p53/NF-κB co-regulation of immune response genes, including several chemokines, which effectively induced human neutrophil migration. In addition, we show that p53, activated by tumor cell paracrine factors, induces high basal levels of macrophage IL-6 in a TAM model system [tumor-conditioned macrophages (TCM)]. Compared with normal macrophages, TCMs exhibited higher p53 levels, enhanced p53 binding to the IL-6 promoter, and reduced IL-6 levels upon p53 inhibition. Taken together, we describe a mechanism by which human macrophages integrate signals through p53 and NF-κB to drive proinflammatory cytokine induction. Our results implicate a novel role for macrophage p53 in conditioning the tumor microenvironment and suggest a potential mechanism by which p53-activating chemotherapeutics, acting upon p53-sufficient macrophages and precursor monocytes, may indirectly impact tumors lacking functional p53. ©2014 AACR.

  16. Transgenic mouse with human mutant p53 expression in the prostate epithelium.

    PubMed

    Elgavish, Ada; Wood, Philip A; Pinkert, Carl A; Eltoum, Isam-Eldin; Cartee, Todd; Wilbanks, John; Mentor-Marcel, Roycelynn; Tian, Liqun; Scroggins, Samuel E

    2004-09-15

    Apoptosis is disrupted in prostate tumor cells, conferring a survival advantage. p53 is a nuclear protein believed to regulate cancer progression, in part by inducing apoptosis. To test this possibility in future studies, the objective of the present study was to generate a transgenic mouse model expressing mutant p53 in the prostate (PR). Transgene incorporation was tested using Southern analysis. Expression of mutant p53 protein was examined using immunofluorescence microscopy. Apoptosis in the PR was evaluated using the Tunnel method. A construct, consisting of the rat probasin promoter and a mutant human p53 fragment, was prepared and used to generate transgenic mice. rPB-mutant p53 transgene incorporation, as well as nuclear accumulation of mutant human p53 protein, was demonstrated. Prostatic intraepithelial neoplasia (PIN) III and IV were found in PR of 52-week old transgenic mice, whereas no pathological changes were found in the other organs examined. PR ability to undergo apoptosis following castration was reduced in rPB-mutant p53 mice as compared to non transgenic littermates. Transgenic rPB-mutant p53 mice accumulate mutant p53 protein in PR, resulting in neoplastic lesions and reduced apoptotic potential in the PR. Breeding rPB-mutant p53 mice with mice expressing an oncogene in their PR will be useful in examining interactions of multiple genes that result in progression of slow growing prostate tumors expressing oncogenes alone to metastatic cancer. Copyright 2004 Wiley-Liss, Inc.

  17. Alternate splicing of the p53 inhibitor HDMX offers a superior prognostic biomarker than p53 mutation in human cancer.

    PubMed

    Lenos, Kristiaan; Grawenda, Anna M; Lodder, Kirsten; Kuijjer, Marieke L; Teunisse, Amina F A S; Repapi, Emmanouela; Grochola, Lukasz F; Bartel, Frank; Hogendoorn, Pancras C W; Wuerl, Peter; Taubert, Helge; Cleton-Jansen, Anne-Marie; Bond, Gareth L; Jochemsen, Aart G

    2012-08-15

    Conventional high-grade osteosarcoma is the most common primary bone malignancy. Although altered expression of the p53 inhibitor HDMX (Mdmx/Mdm4) is associated with cancer risk, progression, and outcome in other tumor types, little is known about its role in osteosarcoma. High expression of the Hdmx splice variant HDMX-S relative to the full-length transcript (the HDMX-S/HDMX-FL ratio) correlates with reduced HDMX protein expression, faster progression, and poorer survival in several cancers. Here, we show that the HDMX-S/HDMX-FL ratio positively correlates with less HDMX protein expression, faster metastatic progression, and a trend to worse overall survival in osteosarcomas. We found that the HDMX-S/HDMX-FL ratio associated with common somatic genetic lesions connected with p53 inhibition, such as p53 mutation and HDM2 overexpression in osteosarcoma cell lines. Interestingly, this finding was not limited to osteosarcomas as we observed similar associations in breast cancer and a variety of other cancer cell lines, as well as in tumors from patients with soft tissue sarcoma. The HDMX-S/HDMX-FL ratio better defined patients with sarcoma with worse survival rates than p53 mutational status. We propose a novel role for alternative splicing of HDMX, whereby it serves as a mechanism by which HDMX protein levels are reduced in cancer cells that have already inhibited p53 activity. Alternative splicing of HDMX could, therefore, serve as a more effective biomarker for p53 pathway attenuation in cancers than p53 gene mutation.

  18. The p53 tumor suppressor protein protects against chemotherapeutic stress and apoptosis in human medulloblastoma cells

    PubMed Central

    Parasido, Erika; Tricoli, Lucas; Sivakumar, Angiela; Mikhaiel, John P.; Yenugonda, Venkata; Rodriguez, Olga C.; Karam, Sana D.; Rood, Brian R.; Avantaggiati, Maria Laura; Albanese, Chris

    2015-01-01

    Medulloblastoma (MB), a primitive neuroectodermal tumor, is the most common malignant childhood brain tumor and remains incurable in about a third of patients. Currently, survivors carry a significant burden of late treatment effects. The p53 tumor suppressor protein plays a crucial role in influencing cell survival in response to cellular stress and while the p53 pathway is considered a key determinant of anti-tumor responses in many tumors, its role in cell survival in MB is much less well defined. Herein, we report that the experimental drug VMY-1-103 acts through induction of a partial DNA damage-like response as well induction of non-survival autophagy. Surprisingly, the genetic or chemical silencing of p53 significantly enhanced the cytotoxic effects of both VMY and the DNA damaging drug, doxorubicin. The inhibition of p53 in the presence of VMY revealed increased late stage apoptosis, increased DNA fragmentation and increased expression of genes involved in apoptosis, including CAPN12 and TRPM8, p63, p73, BIK, EndoG, CIDEB, P27Kip1 and P21cip1. These data provide the groundwork for additional studies on VMY as a therapeutic drug and support further investigations into the intriguing possibility that targeting p53 function may be an effective means of enhancing clinical outcomes in MB. PMID:26540407

  19. p53 splice variants generated by atypical mRNA processing confer complexity of p53 transcripts in the human brain.

    PubMed

    Nikoshkov, Andrej; Hurd, Yasmin L

    2006-12-15

    Very limited is known about p53 expression in the normal mammalian brain and only few alternative splice variants have been reported thus far in human and rat peripheral tissues. Here, we detected eight new p53 transcripts in the human brain generated by alternative splicing, whereas two were present in the rat brain. Almost all alternative splice events occurred due to atypical splice mechanism employing direct repeats at splice sites. All discovered transcripts retain untranslated 5' area of the p53 gene and thus could be translated into peptides consisting of different functional domains.

  20. Mouse Models for the p53 R72P Polymorphism Mimic Human Phenotypes

    PubMed Central

    Zhu, Feng; Dollé, Martijn E.T.; Berton, Thomas R.; Kuiper, Raoul V.; Capps, Carrie; Espejo, Alexsandra; McArthur, Mark J.; Bedford, Mark T.; van Steeg, Harry; de Vries, Annemieke; Johnson, David G.

    2010-01-01

    The p53 tumor suppressor gene contains a common single nucleotide polymorphism (SNP) that results in either an arginine or proline at position 72 of the p53 protein. This polymorphism affects the apoptotic activity of p53 but the mechanistic basis and physiological relevance of this phenotypic difference remain unclear. Here we describe the development of mouse models for the p53 R72P SNP using two different approaches. In both sets of models the human or humanized p53 proteins are functional as evidenced by the transcriptional induction of p53 target genes in response to DNA damage and the suppression of early lymphomagenesis. Consistent with in vitro studies, mice expressing the 72R variant protein (p53R) have a greater apoptotic response to several stimuli compared to mice expressing the p53P variant. Molecular studies suggest that both transcriptional and non-transcriptional mechanisms may contribute to the differential abilities of the p53 variants to induce apoptosis. Despite a difference in the acute response to ultraviolet (UV) radiation, no difference in the tumorigenic response to chronic UV exposure was observed between the polymorphic mouse models. These findings suggest that under at least some conditions, the modulation of apoptosis by the R72P polymorphism does not impact the process of carcinogenesis. PMID:20587514

  1. Transduction of Recombinant M3-p53-R12 Protein Enhances Human Leukemia Cell Apoptosis

    PubMed Central

    Lu, Tsung Chi; Zhao, Guan- Hao; Chen, Yao Yun; Chien, Chia-Ying; Huang, Chi-Hung; Lin, Kwang Hui; Chen, Shen Liang

    2016-01-01

    Tumor suppressor protein p53 plays important roles in initiating cell cycle arrest and promoting tumor cell apoptosis. Previous studies have shown that p53 is either mutated or defective in approximately 50% of human cancers; therefore restoring normal p53 activity in cancer cells might be an effective anticancer therapeutic approach. Herein, we designed a chimeric p53 protein flanked with the MyoD N-terminal transcriptional activation domain (amino acids 1-62, called M3) and a poly-arginine (R12) cell penetrating signal in its N-and C-termini respectively. This chimeric protein, M3-p53-R12, can be expressed in E. coli and purified using immobilized metal ion chromatography followed by serial refolding dialysis. The purified M3-p53-R12 protein retains DNA-binding activity and gains of cell penetrating ability. Using MTT assay, we demonstrated that M3-p53-R12 inhibited the growth of K562, Jurkat as well as HL-60 leukemia cells carrying mutant p53 genes. Results from FACS analysis also demonstrated that transduction of M3-p53-R12 protein induced cell cycle arrest of these leukemia cells. Of special note, M3-p53-R12 has no apoptotic effect on normal mesenchymal stem cells (MSC) and leukocytes, highlighting its differential effects on normal and tumor cells. To sum up, our results reveal that purified recombinant M3-p53-R12 protein has functions of suppressing the leukemia cell lines' proliferation and launching cell apoptosis, suggesting the feasibility of using M3-p53-R12 protein as an anticancer drug. In the future we will test whether this chimeric protein can preferentially trigger the death of malignant cancer cells without affecting normal cells in animals carrying endogenous or xenographic tumors. PMID:27390612

  2. Biophysical characterizations of human mitochondrial transcription factor A and its binding to tumor suppressor p53

    PubMed Central

    Wong, Tuck Seng; Rajagopalan, Sridharan; Freund, Stefan M.; Rutherford, Trevor J.; Andreeva, Antonina; Townsley, Fiona M.; Petrovich, Miriana; Fersht, Alan R.

    2009-01-01

    Human mitochondrial transcription factor A (TFAM) is a multi-functional protein, involved in different aspects of maintaining mitochondrial genome integrity. In this report, we characterized TFAM and its interaction with tumor suppressor p53 using various biophysical methods. DNA-free TFAM is a thermally unstable protein that is in equilibrium between monomers and dimers. Self-association of TFAM is modulated by its basic C-terminal tail. The DNA-binding ability of TFAM is mainly contributed by its first HMG-box, while the second HMG-box has low-DNA-binding capability. We also obtained backbone resonance assignments from the NMR spectra of both HMG-boxes of TFAM. TFAM binds primarily to the N-terminal transactivation domain of p53, with a Kd of 1.95 ± 0.19 μM. The C-terminal regulatory domain of p53 provides a secondary binding site for TFAM. The TFAM–p53-binding interface involves both TAD1 and TAD2 sub-domains of p53. Helices α1 and α2 of the HMG-box constitute the main p53-binding region. Since both TFAM and p53 binds preferentially to distorted DNA, the TFAM–p53 interaction is implicated in DNA damage and repair. In addition, the DNA-binding mechanism of TFAM and biological relevance of the TFAM–p53 interaction are discussed. PMID:19755502

  3. Human pregnane X receptor compromises the function of p53 and promotes malignant transformation

    PubMed Central

    Robbins, D; Cherian, M; Wu, J; Chen, T

    2016-01-01

    The pregnane X receptor (PXR) is well established as a nuclear receptor that has a central role in xenobiotic metabolism and disposition. However, emerging evidence suggests that PXR is also a regulator of apoptosis, promoting a malignant phenotype both in vitro and in vivo. The tumor suppressor p53 can be activated in the presence of DNA damage and induce cell cycle arrest to allow for DNA repair or, ultimately, apoptosis to suppress tumor formation. We previously identified p53 as a novel PXR-associated protein by using a mass spectrometric approach. In the current study, we identified a novel inhibitory effect of PXR on p53, revealing an anti-apoptotic function of PXR in colon carcinogenesis. PXR expression reduced p53 transactivation and the expression of its downstream target genes involved in cell cycle arrest and apoptosis by decreasing p53 recruitment to the promoter regions of these genes. Consistent with the inhibitory effect of PXR on p53, elevated PXR levels decreased doxorubicin- or nutlin-3a-mediated toxicity and promoted malignant transformation in colon cancer cells. Our findings show for the first time that PXR expression modulates p53 target gene promoter binding and contributes to the downregulation of p53 function in human colon cancer cells. These results define the functional significance of PXR expression in modulating p53-mediated mechanisms of tumor suppression. PMID:27547448

  4. Mutations in p53 as potential molecular markers for human breast cancer

    SciTech Connect

    Runnebaum, I.B.; Nagarajan, M.; Bowman, M.; Soto, D.; Sukumar, S. )

    1991-12-01

    Based on the high incidence of loss of heterozygosity for loci on chromosome 17p in the vicinity of the p53 locus in human breast tumors. The authors investigated the frequency and effects of mutations in the p53 tumor suppressor gene in mammary neoplasia. They examined the p53 gene in 20 breast cancer cell lines and 59 primary breast tumors. Northern blot analysis, immunoprecipitation, and nucleotide sequencing analysis revealed aberrant mRNA expression, over-expression of protein, and point mutations in the p53 gene in 50% of the cell line tested. A multiplex PCR assay was developed to search for deletions in the p53 genomic locus. Multiplex PCR of genomic DNA showed that up to 36% of primary tumors contained aberrations in the p53 locus. Mutations in exons 5-9 of the p53 gene were found in 10 out of 59 (17%) of the primary tumors studied by single-stranded conformation polymorphism analysis. They conclude that, compared to amplification of HER2/NEU, MYC, or INT2 oncogene loci, p53 gene mutations and deletions are the most frequently observed genetic change in breast cancer related to a single gene. Correlated to disease status, p53 gene mutations could prove to be a valuable marker for diagnosis and/or prognosis of breast neoplasia.

  5. Arsenic promotes centrosome abnormalities and cell colony formation in p53 compromised human lung cells

    SciTech Connect

    Liao Weiting; Lin Pinpin; Cheng, T.-S.; Yu, H.-S.; Chang, Louis W.

    2007-12-01

    Epidemiological evidence indicated that residents, especially cigarette smokers, in arseniasis areas had significantly higher lung cancer risk than those living in non-arseniasis areas. Thus, an interaction between arsenic and cigarette smoking in lung carcinogenesis was suspected. p53 dysfunction or mutation in lung epithelial cells was frequently observed in cigarette smokers. Our present study was to explore the differential effects by arsenic on H1355 cells (human lung adenocarcinoma cell line with mutation in p53), BEAS-2B (immortalized lung epithelial cell with functional p53) and pifithrin-{alpha}-treated BEAS-2B cells (p53-inhibited cells). These cells were treated with different doses of sodium arsenite (0, 0.1, 1, 5 and 10 {mu}M) for 48 h. A greater reduction in cell viability was observed in the BEAS-2B cells vs. p53 compromised cells (H1355 or p53-inhibited BEAS-2B). Similar observation was also made on 7-day cell survival (growth) study. TUNEL analysis confirmed that there was indeed a significantly reduced arsenite-induced apoptosis found in p53-compromised cells. Centrosomal abnormality has been attributed to eventual chromosomal missegregation, aneuploidy and tumorigenesis. In our present study, reduced p21 and Gadd45a expressions and increased centrosomal abnormality (atopic and multiple centrosomes) were observed in both arsenite-treated H1355 and p53-inhibited BEAS-2B cells as compared with similarly treated BEAS-2B cells. Increased anchorage-independent growth (colony formation) of BEAS-2B cells co-treated with pifithrin-{alpha} and 5 {mu}M sodium arsenite was also observed in soft agar. Our present investigation demonstrated that arsenic would act specifically on p53 compromised cells (either with p53 dysfunction or inhibited) to induce centrosomal abnormality and colony formation. These findings provided strong evidence on the carcinogenic promotional role of arsenic, especially under the condition of p53 dysfunction.

  6. Uncovering the role of p53 splice variants in human malignancy: a clinical perspective

    PubMed Central

    Surget, Sylvanie; Khoury, Marie P; Bourdon, Jean-Christophe

    2014-01-01

    Thirty-five years of research on p53 gave rise to more than 68,000 articles and reviews, but did not allow the uncovering of all the mysteries that this major tumor suppressor holds. How p53 handles the different signals to decide the appropriate cell fate in response to a stress and its implication in tumorigenesis and cancer progression remains unclear. Nevertheless, the uncovering of p53 isoforms has opened new perspectives in the cancer research field. Indeed, the human TP53 gene encodes not only one but at least twelve p53 protein isoforms, which are produced in normal tissues through alternative initiation of translation, usage of alternative promoters, and alternative splicing. In recent years, it became obvious that the different p53 isoforms play an important role in regulating cell fate in response to different stresses in normal cells by differentially regulating gene expression. In cancer cells, abnormal expression of p53 isoforms contributes actively to cancer formation and progression, regardless of TP53 mutation status. They can also be associated with response to treatment, depending on the cell context. The determination of p53 isoform expression and p53 mutation status helps to define different subtypes within a particular cancer type, which would have different responses to treatment. Thus, the understanding of the regulation of p53 isoform expression and their biological activities in relation to the cellular context would constitute an important step toward the improvement of the diagnostic, prognostic, and predictive values of p53 in cancer treatment. This review aims to summarize the involvement of p53 isoforms in cancer and to highlight novel potential therapeutic targets. PMID:24379683

  7. The role of p53 inactivation in human cervical cell carcinoma development.

    PubMed Central

    Miwa, K.; Miyamoto, S.; Kato, H.; Imamura, T.; Nishida, M.; Yoshikawa, Y.; Nagata, Y.; Wake, N.

    1995-01-01

    We investigated the association between human papillomavirus (HPV) infection and p53 gene mutation in 47 primary uterine cervical cancers. HPV DNA sequences were present in 43 cancers (91.5%), and one of these cancers contained a p53 gene mutation. In addition, one of the remaining four HPV-negative cancers also contained a p53 gene mutation. As a result, p53 inactivation corresponded to the development of 44 of the primary uterine cervical cancers studied (93.6%). We obtained both primary and recurrent tumours from four cases. In two of these cases, the HPV genomes that were present in an episomal state in the primary tumours were observed to have disappeared in the recurrent tumours. One of these recurrent tumours also contained a p53 gene mutation, which suggested the possibility that p53 inactivation was required in order to maintain the aggressive behaviour in this cancer either by an HPV infection or by a p53 gene mutation. No MDM2 gene amplification was observed in the tumours that carried neither HPV DNAs nor p53 gene mutations. Images Figure 1 Figure 3 Figure 2 Figure 4 PMID:7841033

  8. p53 Mutations in human adrenocortical neoplasms: Immunohistochemical and molecular studies

    SciTech Connect

    Reincke, M.; Allolio, B.; Travis, W.H.; Linehan, H.M.; Karl, M.; Mastorakos, G.; Chrousos, G.P.

    1994-03-01

    p53 is a recessive tumor suppressor gene located on chromosome 17p. Mutations in the p53 gene play an important role in the tumorigenesis of diverse types of human neoplasms including breast and colon cancers. More than 90% of all mutations discovered in such tumors have been detected in 4 hot spot areas that lie between exons 5 and 8. In contrast to wild-type p53, mutant p53 accumulates intracellularly and can be easily detected by immunohistochemistry. The authors therefore investigated the frequency of p53 mutations in human adrenocortical neoplasms using molecular biology and immunohistochemistry techniques. Five patients with adrenocortical adenomas (5 female; ages 39-72 yr), 11 patients with adrenocortical carcinomas (8 female, 3 male; ages 15-50 yr), and two adrenocortical tumor cell lines were studied. After DNA extraction from frozen tumor tissue or paraffin-embedded material, exons 5 through 8 were amplified using the polymerase chain reaction and directly sequenced by the dideoxy termination method. Immunohistochemistry was performed on paraffin-embedded tumor specimens obtained during adrenalectomy using a monoclonal antibody reacting with both wild-type and mutant p53. Prevalence of mutations was adenomas, 0/5, carcinomas, 3/11, and adrenocortical cell lines, 2/2. Single point mutations were detected in 3 cases (exons 5, 6, and 7, respectively), and rearrangements of exon 7/8 and 8 were found in 2 cases. Immunohistochemistry detected strong nuclear and/or cytoplasmic p53 immunoreactivity in all adrenocortical carcinomas with point mutations of the p53 gene but not in adenomas and carcinomas with the wild-type sequence or with deletion/rearrangement of the p53 gene. They conclude that p53 plays a role in the tumorigenesis of adrenocortical carcinomas but is of less importance to benign adenomas. 27 refs., 3 figs., 2 tabs.

  9. Distinct p53 genomic binding patterns in normal and cancer-derived human cells

    SciTech Connect

    Botcheva K.; McCorkle S. R.; McCombie W. R.; Dunn J. J.; Anderson C. W.

    2011-12-15

    We report here genome-wide analysis of the tumor suppressor p53 binding sites in normal human cells. 743 high-confidence ChIP-seq peaks representing putative genomic binding sites were identified in normal IMR90 fibroblasts using a reference chromatin sample. More than 40% were located within 2 kb of a transcription start site (TSS), a distribution similar to that documented for individually studied, functional p53 binding sites and, to date, not observed by previous p53 genome-wide studies. Nearly half of the high-confidence binding sites in the IMR90 cells reside in CpG islands, in marked contrast to sites reported in cancer-derived cells. The distinct genomic features of the IMR90 binding sites do not reflect a distinct preference for specific sequences, since the de novo developed p53 motif based on our study is similar to those reported by genome-wide studies of cancer cells. More likely, the different chromatin landscape in normal, compared with cancer-derived cells, influences p53 binding via modulating availability of the sites. We compared the IMR90 ChIPseq peaks to the recently published IMR90 methylome1 and demonstrated that they are enriched at hypomethylated DNA. Our study represents the first genome-wide, de novo mapping of p53 binding sites in normal human cells and reveals that p53 binding sites reside in distinct genomic landscapes in normal and cancer-derived human cells.

  10. Effects of recombinant human adenovirus-p53 on the regression of hepatic fibrosis

    PubMed Central

    Liu, Yehong; Yang, Puye; Chen, Na; Lin, Shumei; Liu, Min

    2016-01-01

    Hepatic fibrosis is a scarring process that may progress to hepatic cirrhosis and even hepatic carcinoma if left untreated. Hepatic stellate cells (HSCs) play essential roles in the development of hepatic fibrosis. The tumor suppressor protein p53 is a transcription factor that is involved in cell proliferation, cell cycle regulation, apoptosis and DNA repair. Recombinant human adenovirus-p53 (Ad-p53) has been demonstrated to act as a promising antitumor gene therapy in various types of cancer. However, there is limited infomration regarding the therapeutic effect of Ad-p53 on the regression of hepatic fibrosis. In order to examine the underlying molecular mechanism responsible for the effects of Ad-p53 on HSCs, a rat model of hepatic fibrosis was established and HSC-T6 cells were cultured under different conditions. The expression of p53, transforming growth factor (TGF-β1) and α-smooth muscle actin (α-SMA), which is a marker of activated HSCs, was detected by immunohistochemical assays and RT-qPCR. In vitro, five different concentrations (1×106, 5×106, 1×107, 2×107 and 5×107 PFU/ml) of Ad-p53 were selected for use in the MTT assay to analyze the proliferation of HSCs at 0, 24, 48 and 72 h. Flow cytometric analysis was applied to determine the effect of three different concentrations of Ad-p53 (5×106, 1×107 and 2×107 PFU/ml) on the cell cycle and the apoptosis of HSC-T6 cells at 24 and 48 h. The results of immunohistochemical studies and RT-qPCR showed that Ad-p53 upregulated the expression of p53, and downregulated the expression of TGF-β1 and α-SMA. The MTT assay revealed that when treated with various doses of Ad-p53, the proliferation of HSCs was inhibited within a certain range of concentrations and time periods. Analysis of flow cytometric data showed that Ad-p53 arrested the cell cycle in G1 phase and significantly induced apoptosis. Taken together, these findings suggest that Ad-p53 promotes apoptosis and inhibits the proliferation of HSCs in

  11. ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly and p53

    PubMed Central

    Ghouzzi, Vincent El; Bianchi, Federico T; Molineris, Ivan; Mounce, Bryan C; Berto, Gaia E; Rak, Malgorzata; Lebon, Sophie; Aubry, Laetitia; Tocco, Chiara; Gai, Marta; Chiotto, Alessandra MA; Sgrò, Francesco; Pallavicini, Gianmarco; Simon-Loriere, Etienne; Passemard, Sandrine; Vignuzzi, Marco; Gressens, Pierre; Di Cunto, Ferdinando

    2016-01-01

    Epidemiological evidence from the current outbreak of Zika virus (ZIKV) and recent studies in animal models indicate a strong causal link between ZIKV and microcephaly. ZIKV infection induces cell-cycle arrest and apoptosis in proliferating neural progenitors. However, the mechanisms leading to these phenotypes are still largely obscure. In this report, we explored the possible similarities between transcriptional responses induced by ZIKV in human neural progenitors and those elicited by three different genetic mutations leading to severe forms of microcephaly in mice. We found that the strongest similarity between all these conditions is the activation of common P53 downstream genes. In agreement with these observations, we report that ZIKV infection increases total P53 levels and nuclear accumulation, as well as P53 Ser15 phosphorylation, correlated with genotoxic stress and apoptosis induction. Interestingly, increased P53 activation and apoptosis are induced not only in cells expressing high levels of viral antigens but also in cells showing low or undetectable levels of the same proteins. These results indicate that P53 activation is an early and specific event in ZIKV-infected cells, which could result from cell-autonomous and/or non-cell-autonomous mechanisms. Moreover, we highlight a small group of P53 effector proteins that could act as critical mediators, not only in ZIKV-induced microcephaly but also in many genetic microcephaly syndromes. PMID:27787521

  12. P53 alters the cytotoxicity and genotoxicity for oxidized graphene in human B-lymphoblastoid cells

    NASA Astrophysics Data System (ADS)

    Petibone, Dayton Matthew

    Widespread use of oxidized graphene nanomaterials in industry, medicine, and consumer products raises concern about potential adverse impacts on human health. The p53 tumor suppressor protein is crucial to maintaining cellular and genetic stability to prevent carcinogenesis. Here, we show that oxygen functionalized graphene (f-G) absorption and p53 functional status correlate with cytotoxicity and genotoxicity in human B-lymphoblastoid cells. Trends in f-G absorption by were dose-dependent. Cells with functional p53 exposed to f-G arrested in G0/G1 phase of the cell cycle, suppressed f-G induced reactive oxygen species (ROS), and had elevated apoptosis. While compared to p53 competent cells, the p53 deficient cells exposed to f-G accumulated in S-phase of the cell cycle, had elevated ROS levels, and evaded apoptosis. The f-G genotoxicity was evident as increased loss-of-heterozygosity mutants independent of p53 status, and structural chromosome damage in p53 deficient cells. These findings have broad implications for the safety and efficacy of oxidized graphene nanomaterials in industrial, consumer products and biomedical applications.

  13. Cadmium induces p53-dependent apoptosis in human prostate epithelial cells.

    PubMed

    Aimola, Pierpaolo; Carmignani, Marco; Volpe, Anna Rita; Di Benedetto, Altomare; Claudio, Luigi; Waalkes, Michael P; van Bokhoven, Adrie; Tokar, Erik J; Claudio, Pier Paolo

    2012-01-01

    Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl(2) and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl(2) concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis.

  14. Cadmium Induces p53-Dependent Apoptosis in Human Prostate Epithelial Cells

    PubMed Central

    Aimola, Pierpaolo; Carmignani, Marco; Volpe, Anna Rita; Di Benedetto, Altomare; Claudio, Luigi; Waalkes, Michael P.; van Bokhoven, Adrie; Tokar, Erik J.; Claudio, Pier Paolo

    2012-01-01

    Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl2 and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl2 concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis. PMID:22448262

  15. The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses

    NASA Technical Reports Server (NTRS)

    Kohli, M.; Jorgensen, T. J.

    1999-01-01

    The simian virus 40 large tumor antigen (SV40 Tag) has been ascribed many functions critical to viral propagation, including binding to the mammalian tumor suppressor p53. Recent studies have demonstrated that SV40-transformed murine cells have functional p53. The status of p53 in SV40-immortalized human cells, however, has not been characterized. We have found that in response to ionizing radiation, p53-dependent p21 transactivation activity is present, albeit reduced, in SV40-immortalized cells and that this activity can be further reduced with either dominant negative p53 expression or higher SV40 Tag expression. Furthermore, overexpression of p53 in SV40-immortalized ataxia-telangiectasia (A-T) cells restores p53-dependent p21 induction to typical A-T levels. All SV40-immortalized cell lines exhibited an absence of G1 arrest. Moreover, all SV40-immortalized cell lines exhibited increased apoptosis relative to primary cells in response to ionizing radiation, suggesting that SV40 immortalization results in a unique phenotype with regard to DNA damage responses. Copyright 1999 Academic Press.

  16. The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses

    NASA Technical Reports Server (NTRS)

    Kohli, M.; Jorgensen, T. J.

    1999-01-01

    The simian virus 40 large tumor antigen (SV40 Tag) has been ascribed many functions critical to viral propagation, including binding to the mammalian tumor suppressor p53. Recent studies have demonstrated that SV40-transformed murine cells have functional p53. The status of p53 in SV40-immortalized human cells, however, has not been characterized. We have found that in response to ionizing radiation, p53-dependent p21 transactivation activity is present, albeit reduced, in SV40-immortalized cells and that this activity can be further reduced with either dominant negative p53 expression or higher SV40 Tag expression. Furthermore, overexpression of p53 in SV40-immortalized ataxia-telangiectasia (A-T) cells restores p53-dependent p21 induction to typical A-T levels. All SV40-immortalized cell lines exhibited an absence of G1 arrest. Moreover, all SV40-immortalized cell lines exhibited increased apoptosis relative to primary cells in response to ionizing radiation, suggesting that SV40 immortalization results in a unique phenotype with regard to DNA damage responses. Copyright 1999 Academic Press.

  17. Concurrent expression of heme oxygenase-1 and p53 in human retinal pigment epithelial cell line

    SciTech Connect

    Lee, Sang Yull; Jo, Hong Jae; Kim, Kang Mi; Song, Ju Dong; Chung, Hun Taeg; Park, Young Chul

    2008-01-25

    Heme oxygenase-1 (HO-1) is a stress-responsive protein that is known to regulate cellular functions such as cell proliferation, inflammation, and apoptosis. Here, we investigated the effects of HO activity on the expression of p53 in the human retinal pigment epithelium (RPE) cell line ARPE-19. Cobalt protoporphyrin (CoPP) induced the expression of both HO-1 and p53 without significant toxicity to the cells. In addition, the blockage of HO activity with the iron chelator DFO or with HO-1 siRNA inhibited the CoPP-induced expression of p53. Similarly, zinc protoporphyrin (ZnPP), an inhibitor of HO, suppressed p53 expression in ARPE-19 cells, although ZnPP increased the level of HO-1 protein while inhibiting HO activity. Also, CoPP-induced p53 expression was not affected by the formation of reactive oxygen species (ROS). Based on these results, we conclude that HO activity is involved in the regulation of p53 expression in a ROS-independent mechanism, and also suggest that the expression of p53 in ARPE-19 cells is associated with heme metabolites such as biliverdin/bilirubin, carbon monoxide, and iron produced by the activity of HO.

  18. The state of the p53 and retinoblastoma genes in human cervical carcinoma cell lines

    SciTech Connect

    Scheffner, M.; Muenger, K.; Byrne, J.C.; Howley, P.M. )

    1991-07-01

    Human cervical carcinoma cell lines that were either positive or negative for human papillomavirus (HPV) DNA sequences were analyzed for evidence of mutation of the p53 and retinoblastoma genes. Each of five HPV-positive cervical cancer cell lines expressed normal pRB and low levels of wild-type p53 proteins, which are presumed to be altered in function as a consequence of association with HPV E7 and E6 oncoproteins, respectively. In contrast, mutations were identified in the p53 and RB genes expressed in the C-33A and HT-3 cervical cancer cell lines, which lack HPV DNA sequences. Mutations in the p53 genes mapped to codon 273 and codon 245 in the C33-A and HT-3 cell lines, respectively, located in the highly conserved regions of p53, where mutations appear in a variety of human cancers. Mutations in RB occurred at splice junctions, resulting in in-frame deletions, affecting exons 13 and 20 in the HT-3 and C-33A cell lines, respectively. These mutations resulted in aberrant proteins that were not phosphorylated and were unable to complex with the adenovirus E1A oncoprotein. These results support the hypothesis that the inactivation of the normal functions of the tumor-suppressor proteins pRB and p53 are important steps in human cervical carcinogenesis, either by mutation or from complex formation with the HPV E6 and E7 oncoproteins.

  19. Teroxirone inhibited growth of human non-small cell lung cancer cells by activating p53

    SciTech Connect

    Wang, Jing-Ping; Lin, Kai-Han; Liu, Chun-Yen; Yu, Ya-Chu; Wu, Pei-Tsun; Chiu, Chien-Chih; Su, Chun-Li; Chen, Kwun-Min; Fang, Kang

    2013-11-15

    In this work, we demonstrated that the growth of human non-small-cell-lung-cancer cells H460 and A549 cells can be inhibited by low concentrations of an epoxide derivative, teroxirone, in both in vitro and in vivo models. The cytotoxicity was mediated by apoptotic cell death through DNA damage. The onset of ultimate apoptosis is dependent on the status of p53. Teroxirone caused transient elevation of p53 that activates downstream p21 and procaspase-3 cleavage. The presence of caspase-3 inhibitor reverted apoptotic phenotype. Furthermore, we showed the cytotoxicity of teroxirone in H1299 cells with stable ectopic expression of p53, but not those of mutant p53. A siRNA-mediated knockdown of p53 expression attenuated drug sensitivity. The in vivo experiments demonstrated that teroxirone suppressed growth of xenograft tumors in nude mice. Being a potential therapeutic agent by restraining cell growth through apoptotic death at low concentrations, teroxirone provides a feasible perspective in reversing tumorigenic phenotype of human lung cancer cells. - Highlights: • Teroxirone repressed tumor cell growth in nude mice of human lung cancer cells. • The apoptotic cell death reverted by caspase-3 inhibitor is related to p53 status. • Teroxirone provides a good candidate for lung cancer treatment.

  20. Calpain-mediated Processing of p53-associated Parkin-like Cytoplasmic Protein (PARC) Affects Chemosensitivity of Human Ovarian Cancer Cells by Promoting p53 Subcellular Trafficking*

    PubMed Central

    Woo, Michael G.; Xue, Kai; Liu, Jiayin; McBride, Heidi; Tsang, Benjamin K.

    2012-01-01

    Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA), and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways, and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA are unknown. Here, we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation, and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibition of calpain but not of caspases or the 26 S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin-induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicate the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity. PMID:22117079

  1. P53 Regulation-Association Long Non-Coding RNA (LncRNA PRAL) Inhibits Cell Proliferation by Regulation of P53 in Human Lung Cancer.

    PubMed

    Su, Pengxiao; Wang, Fengqin; Qi, Bin; Wang, Ting; Zhang, Shaobo

    2017-04-11

    BACKGROUND Lung cancer is among the most common causes of cancer-related deaths worldwide, but its tumorigenic mechanisms are largely unknown. Long non-coding RNAs (LncRNAs) have been shown to have significant roles in multiple cancers. Herein, we aimed to elucidate the detailed effects of a newly-discovered LncRNA, termed PRAL, on cell proliferation in lung cancer. MATERIAL AND METHODS A total of 100 lung cancer patients were subjected to RT-PCR analysis to detect the expressions of PRAL. Western blot analysis was performed to examine P53 protein levels. PRAL plasmid and specific siRNA against P53 was transfected into lung cancer cell lines NCI-H929 and A549. Cell viability assay was conducted in the presence or absence of siP53. RESULTS The transcript level of PRAL in human lung cancer was remarkably decreased in vivo compared with their adjacent non-cancerous counterparts, and the protein levels of P53 were accordingly suppressed. Moreover, the expression of PRAL was also decreased in all of the 5 lung cancer cell lines. Transfection of PRAL plasmid inhibited cell proliferation in NCI-H929 and A549 cells and promoted the transcription of P53; however, knockdown of P53 caused no notable effects on PRAL transcription, but it retarded the inhibitory effects mediated by PRAL. CONCLUSIONS The transcript level of PRAL was decreased in lung cancer in vivo and in vitro. Overexpression of PRAL inhibited cell proliferation by upregulating the expression of P53. Our results indicate that PRAL might be a tumor suppressor in lung cancer and thus provides novel clues for the diagnosis and treatment for lung cancer in clinical practice.

  2. Peptide interactions stabilize and restructure human papillomavirus type 16 E6 to interact with p53.

    PubMed

    Ansari, Tina; Brimer, Nicole; Vande Pol, Scott B

    2012-10-01

    Human papillomavirus type 16 (HPV-16) E6 (16E6) binds the E3 ubiquitin ligase E6AP and p53, thereby targeting degradation of p53 (M. Scheffner, B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M. Howley, Cell 63:1129-1136, 1990). Here we show that minimal 16E6-binding LXXLL peptides reshape 16E6 to confer p53 interaction and stabilize 16E6 in vivo but that degradation of p53 by 16E6 requires E6AP expression. These experiments establish a general mechanism for how papillomavirus E6 binding to LXXLL peptides reshapes E6 to then act as an adapter molecule.

  3. Peptide Interactions Stabilize and Restructure Human Papillomavirus Type 16 E6 To Interact with p53

    PubMed Central

    Ansari, Tina; Brimer, Nicole

    2012-01-01

    Human papillomavirus type 16 (HPV-16) E6 (16E6) binds the E3 ubiquitin ligase E6AP and p53, thereby targeting degradation of p53 (M. Scheffner, B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M. Howley, Cell 63:1129–1136, 1990). Here we show that minimal 16E6-binding LXXLL peptides reshape 16E6 to confer p53 interaction and stabilize 16E6 in vivo but that degradation of p53 by 16E6 requires E6AP expression. These experiments establish a general mechanism for how papillomavirus E6 binding to LXXLL peptides reshapes E6 to then act as an adapter molecule. PMID:22896608

  4. Non-thermal Plasma Causes p53-Dependent Apoptosis in Human Colon Carcinoma Cells

    PubMed Central

    Tuhvatulin, A.I.; Sysolyatina, E.V.; Scheblyakov, D.V.; Logunov, D.Yu.; Vasiliev, M.M.; Yurova, M.A.; Danilova, M.A.; Petrov, O.F.; Naroditsky, B.S.; Morfill, G.E.; Grigoriev, A.I.; Fortov, V.E.; Gintsburg, A.L.; Ermolaeva, S.A.

    2012-01-01

    Non-thermal plasma (NTP) consists of a huge amount of biologically active particles, whereas its temperature is close to ambient. This combination allows one to use NTP as a perspective tool for solving different biomedical tasks, including antitumor therapy. The treatment of tumor cells with NTP caused dose-dependent effects, such as growth arrest and apoptosis. However, while the outcome of NTP treatment has been established, the molecular mechanisms of the interaction between NTP and eukaryotic cells have not been thoroughly studied thus far. In this work, the mechanisms and the type of death of human colon carcinoma HCT 116 cells upon application of non-thermal argon plasma were studied. The effect of NTP on the major stress-activated protein p53 was investigated. The results demonstrate that the viability of HCT116 cells upon plasma treatment is dependent on the functional p53 protein. NTP treatment caused an increase in the intracellular concentration of p53 and the induction of the p53-controlled regulon. The p53-dependent accumulation of active proapoptotic caspase-3 was shown in NTP-treated cells. The study was the first to demonstrate that treatment of human colon carcinoma cells with NTP results in p53-dependent apoptosis. The results obtained contribute to our understanding of the applicability of NTP in antitumor therapy. PMID:23150806

  5. Dipeptide analysis of p53 mutations and evolution of p53 family proteins.

    PubMed

    Huang, Qiang; Yu, Long; Levine, Arnold J; Nussinov, Ruth; Ma, Buyong

    2014-01-01

    p53 gain-of-function mutations are similar to driver mutations in cancer genes, with both promoting tumorigenesis. Most previous studies focused on residues lost by mutations, providing information related to a dominantly-negative effect. However, to understand gain-of-function mutations, it is also important to investigate what are the distributions of residues gained by mutations. We compile available p53/p63/p73 protein sequences and construct a non-redundant dataset. We analyze the amino acid and dipeptide composition of p53/p63/p73 proteins across evolution and compare them with the gain/loss of amino acids and dipeptides in human p53 following cancer-related somatic mutations. We find that the ratios of amino acids gained via somatic mutations during evolution to those lost through p53 cancer mutations correlate with the ratios found in single nucleotide polymorphisms in the human proteome. The dipeptide mutational gain/loss ratios are inversely correlated with those observed over p53 evolution but tend to follow the increasing p63/p73-like dipeptide propensities. We successfully simulated the p53 cancer mutation spectrum using the dipeptide composition across the p53 family accounting for the likelihood of mutations in p53 codons. The results revealed that the p53 mutation spectrum is dominated not only by p53 evolution but also by reversal of evolution to a certain degree. This article is part of a Special Issue entitled: Computational Proteomics, Systems Biology & Clinical Implications. Guest Editor: Yudong Cai.

  6. Human papillomavirus, p16 and p53 expression associated with survival of head and neck cancer

    PubMed Central

    2010-01-01

    Background P16 and p53 protein expression, and high-risk human papillomavirus (HPV-HR) types have been associated with survival in head and neck cancer (HNC). Evidence suggests that multiple molecular pathways need to be targeted to improve the poor prognosis of HNC. This study examined the individual and joint effects of tumor markers for differences in predicting HNC survival. P16 and p53 expression were detected from formalin-fixed, paraffin-embedded tissues by immunohistochemical staining. HPV DNA was detected by PCR and DNA sequencing in 237 histologically confirmed HNC patients. Results Overexpression of p16 (p16+) and p53 (p53+) occurred in 38% and 48% of HNC tumors, respectively. HPV-HR was detected in 28% of tumors. Worse prognosis was found in tumors that were p53+ (disease-specific mortality: adjusted hazard ratios, HR = 1.9, 95% CI: 1.04-3.4) or HPV- (overall survival: adj. HR = 2.1, 1.1-4.3) but no association in survival was found by p16 status. Compared to the molecular marker group with the best prognosis (p16+/p53-/HPV-HR: referent), the p16-/p53+/HPV- group had the lowest overall survival (84% vs. 60%, p < 0.01; HR = 4.1, 1.7-9.9) and disease-specific survival (86% vs. 66%, p < 0.01; HR = 4.0, 1.5-10.7). Compared to the referent, the HRs of the other six joint biomarker groups ranged from 1.6-3.4 for overall mortality and 0.9-3.9 for disease-specific mortality. Conclusion The p16/p53/HPV joint groups showed greater distinction in clinical outcomes compared to results based on the individual biomarkers alone. This finding suggests that assessing multiple molecular markers in HNC patients will better predict the diverse outcomes and potentially the type of treatment targeted to those markers. PMID:20181227

  7. p53/PUMA expression in human pulmonary fibroblasts mediates cell activation and migration in silicosis

    PubMed Central

    Wang, Wei; Liu, Haijun; Dai, Xiaoniu; Fang, Shencun; Wang, Xingang; Zhang, Yingming; Yao, Honghong; Zhang, Xilong; Chao, Jie

    2015-01-01

    Phagocytosis of SiO2 into the lung causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Clinical evidence has indicated that the activation of alveolar macrophages by SiO2 produces rapid and sustained inflammation characterized by the generation of monocyte chemotactic protein 1, which, in turn, induces fibrosis. However, the details of events downstream of monocyte chemotactic protein 1 activity in pulmonary fibroblasts remain unclear. Here, to elucidate the role of p53 in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Experiments using primary cultured adult human pulmonary fibroblasts led to the following results: 1) SiO2 treatment resulted in a rapid and sustained increase in p53 and PUMA protein levels; 2) the MAPK and PI3K pathways were involved in the SiO2-induced alteration of p53 and PUMA expression; and 3) RNA interference targeting p53 and PUMA prevented the SiO2-induced increases in fibroblast activation and migration. Our study elucidated a link between SiO2-induced p53/PUMA expression in fibroblasts and cell migration, thereby providing novel insight into the potential use of p53/PUMA in the development of novel therapeutic strategies for silicosis treatment. PMID:26576741

  8. Loss of p53 protein during radiation transformation of primary human mammary epithelial cells.

    PubMed Central

    Wazer, D E; Chu, Q; Liu, X L; Gao, Q; Safaii, H; Band, V

    1994-01-01

    The causative factors leading to breast cancer are largely unknown. Increased incidence of breast cancer following diagnostic or therapeutic radiation suggests that radiation may contribute to mammary oncogenesis. This report describes the in vitro neoplastic transformation of a normal human mammary epithelial cell strain, 76N, by fractionated gamma-irradiation at a clinically used dose (30 Gy). The transformed cells (76R-30) were immortal, had reduced growth factor requirements, and produced tumors in nude mice. Remarkably, the 76R-30 cells completely lacked the p53 tumor suppressor protein. Loss of p53 was due to deletion of the gene on one allele and a 26-bp deletion within the third intron on the second allele which resulted in abnormal splicing out of either the third or fourth exon from the mRNA. PCR with a mutation-specific primer showed that intron 3 mutation was present in irradiated cells before selection for immortal phenotype. 76R-30 cells did not exhibit G1 arrest in response to radiation, indicating a loss of p53-mediated function. Expression of the wild-type p53 gene in 76R-30 cells led to their growth inhibition. Thus, loss of p53 protein appears to have contributed to neoplastic transformation of these cells. This unique model should facilitate analyses of molecular mechanisms of radiation-induced breast cancer and allow identification of p53-regulated cellular genes in breast cells. Images PMID:7511207

  9. Phenylbutyrate Sensitizes Human Glioblastoma Cells Lacking Wild-Type P53 Function to Ionizing Radiation

    SciTech Connect

    Lopez, Carlos A. Feng, Felix Y.; Herman, Joseph M.; Nyati, Mukesh K.; Lawrence, Theodore S.; Ljungman, Mats

    2007-09-01

    Purpose: Histone deacetylase (HDAC) inhibitors induce growth arrest, differentiation, and apoptosis in cancer cells. Phenylbutyrate (PB) is a HDAC inhibitor used clinically for treatment of urea cycle disorders. Because of its low cytotoxicity, cerebrospinal fluid penetration, and high oral bioavailability, we investigated PB as a potential radiation sensitizer in human glioblastoma cell lines. Methods and Materials: Four glioblastoma cell lines were selected for this study. Phenylbutyrate was used at a concentration of 2 mM, which is achievable in humans. Western blots were used to assess levels of acetylated histone H3 in tumor cells after treatment with PB. Flow cytometry was used for cell cycle analysis. Clonogenic assays were performed to assess the effect of PB on radiation sensitivity. We used shRNA against p53 to study the role of p53 in radiosensitization. Results: Treatment with PB alone resulted in hyperacetylation of histones, confirmed by Western blot analysis. The PB alone resulted in cytostatic effects in three cell lines. There was no evidence of G{sub 1} arrest, increase in sub-G{sub 1} fraction or p21 protein induction. Clonogenic assays showed radiosensitization in two lines harboring p53 mutations, with enhancement ratios ({+-} SE) of 1.5 ({+-} 0.2) and 1.3 ({+-} 0.1), respectively. There was no radiopotentiating effect in two cell lines with wild-type p53, but knockdown of wild-type p53 resulted in radiosensitization by PB. Conclusions: Phenylbutyrate can produce p21-independent cytostasis, and enhances radiation sensitivity in p53 mutant human glioblastoma cells in vitro. This suggests the potential application of combined PB and radiotherapy in glioblastoma harboring mutant p53.

  10. CPEB regulation of human cellular senescence, energy metabolism, and p53 mRNA translation.

    PubMed

    Burns, David M; Richter, Joel D

    2008-12-15

    Cytoplasmic polyadenylation element-binding protein (CPEB) stimulates polyadenylation and translation in germ cells and neurons. Here, we show that CPEB-regulated translation is essential for the senescence of human diploid fibroblasts. Knockdown of CPEB causes skin and lung cells to bypass the M1 crisis stage of senescence; reintroduction of CPEB into the knockdown cells restores a senescence-like phenotype. Knockdown cells that have bypassed senescence undergo little telomere erosion. Surprisingly, knockdown of exogenous CPEB that induced a senescence-like phenotype results in the resumption of cell growth. CPEB knockdown cells have fewer mitochondria than wild-type cells and resemble transformed cells by having reduced respiration and reactive oxygen species (ROS), normal ATP levels, and enhanced rates of glycolysis. p53 mRNA contains cytoplasmic polyadenylation elements in its 3' untranslated region (UTR), which promote polyadenylation. In CPEB knockdown cells, p53 mRNA has an abnormally short poly(A) tail and a reduced translational efficiency, resulting in an approximately 50% decrease in p53 protein levels. An shRNA-directed reduction in p53 protein by about 50% also results in extended cellular life span, reduced respiration and ROS, and increased glycolysis. Together, these results suggest that CPEB controls senescence and bioenergetics in human cells at least in part by modulating p53 mRNA polyadenylation-induced translation.

  11. CPEB regulation of human cellular senescence, energy metabolism, and p53 mRNA translation

    PubMed Central

    Burns, David M.; Richter, Joel D.

    2008-01-01

    Cytoplasmic polyadenylation element-binding protein (CPEB) stimulates polyadenylation and translation in germ cells and neurons. Here, we show that CPEB-regulated translation is essential for the senescence of human diploid fibroblasts. Knockdown of CPEB causes skin and lung cells to bypass the M1 crisis stage of senescence; reintroduction of CPEB into the knockdown cells restores a senescence-like phenotype. Knockdown cells that have bypassed senescence undergo little telomere erosion. Surprisingly, knockdown of exogenous CPEB that induced a senescence-like phenotype results in the resumption of cell growth. CPEB knockdown cells have fewer mitochondria than wild-type cells and resemble transformed cells by having reduced respiration and reactive oxygen species (ROS), normal ATP levels, and enhanced rates of glycolysis. p53 mRNA contains cytoplasmic polyadenylation elements in its 3′ untranslated region (UTR), which promote polyadenylation. In CPEB knockdown cells, p53 mRNA has an abnormally short poly(A) tail and a reduced translational efficiency, resulting in an ∼50% decrease in p53 protein levels. An shRNA-directed reduction in p53 protein by about 50% also results in extended cellular life span, reduced respiration and ROS, and increased glycolysis. Together, these results suggest that CPEB controls senescence and bioenergetics in human cells at least in part by modulating p53 mRNA polyadenylation-induced translation. PMID:19141477

  12. Cellular localization of human p53 expressed in the yeast Saccharomyces cerevisiae: effect of NLSI deletion.

    PubMed

    Abdelmoula-Souissi, Salma; Delahodde, Agnès; Bolotin-Fukuhara, Monique; Gargouri, Ali; Mokdad-Gargouri, Raja

    2011-07-01

    The tumor suppressor p53 plays a central role in the regulation of cellular growth and apoptosis. In Saccharomyces cerevisiae, over-expression of the human wtp53 leads to growth inhibition and cell death on minimal medium. In the present work, we showed that deletion of the nuclear localization signal (NLSI) of p53 restores the yeast growth. In this heterologous context, the level of p53∆NLSI was low and the protein mainly located in the cytoplasm while the wtp53 was observed in both the cytoplasmic and nuclear compartments. Interestingly, the wtp53 protein was observed in the mitochondria, whereas the p53∆NLSI protein failed to localize in mitochondria. Moreover, mitochondrial morphology defect and release of cytochrome c in the cytosol were noticed only in the yeast strain expressing the wtp53. In conclusion, our results provide evidence that the human wtp53 is active in S. cerevisiae probably through dependent and independent transcriptional mechanisms leading to cell death. The deletion of the NLSI sequence decreases p53 nuclear translocation as well as its mitochondrial localization and consequently its effect on yeast growth.

  13. Quiescence does not affect p53 and stress response by irradiation in human lung fibroblasts

    SciTech Connect

    Dai, Jiawen; Itahana, Koji; Baskar, Rajamanickam

    2015-02-27

    Cells in many organs exist in both proliferating and quiescent states. Proliferating cells are more radio-sensitive, DNA damage pathways including p53 pathway are activated to undergo either G{sub 1}/S or G{sub 2}/M arrest to avoid entering S and M phase with DNA damage. On the other hand, quiescent cells are already arrested in G{sub 0}, therefore there may be fundamental difference of irradiation response between proliferating and quiescent cells, and this difference may affect their radiosensitivity. To understand these differences, proliferating and quiescent human normal lung fibroblasts were exposed to 0.10–1 Gy of γ-radiation. The response of key proteins involved in the cell cycle, cell death, and metabolism as well as histone H2AX phosphorylation were examined. Interestingly, p53 and p53 phosphorylation (Ser-15), as well as the cyclin-dependent kinase inhibitors p21 and p27, were induced similarly in both proliferating and quiescent cells after irradiation. Furthermore, the p53 protein half-life, and expression of cyclin A, cyclin E, proliferating cell nuclear antigen (PCNA), Bax, or cytochrome c expression as well as histone H2AX phosphorylation were comparable after irradiation in both phases of cells. The effect of radioprotection by a glycogen synthase kinase 3β inhibitor on p53 pathway was also similar between proliferating and quiescent cells. Our results showed that quiescence does not affect irradiation response of key proteins involved in stress and DNA damage at least in normal fibroblasts, providing a better understanding of the radiation response in quiescent cells, which is crucial for tissue repair and regeneration. - Highlights: • p53 response by irradiation was similar between proliferating and quiescent cells. • Quiescent cells showed similar profiles of cell cycle proteins after irradiation. • Radioprotection of GSK-3β inhibitor caused similar effects between these cells. • Quiescence did not affect p53 response despite its

  14. Reciprocal regulation of p63 by C/EBP delta in human keratinocytes

    PubMed Central

    Borrelli, Serena; Testoni, Barbara; Callari, Maurizio; Alotto, Daniela; Castagnoli, Carlotta; Romano, Rose-Anne; Sinha, Satrajit; Viganò, Alessandra M; Mantovani, Roberto

    2007-01-01

    Background Genetic experiments have clarified that p63 is a key transcription factor governing the establishment and maintenance of multilayered epithelia. Key to our understanding of p63 strategy is the identification of target genes. We perfomed an RNAi screening in keratinocytes for p63, followed by profiling analysis. Results C/EBPδ, member of a family with known roles in differentiation pathways, emerged as a gene repressed by p63. We validated C/EBPδ as a primary target of ΔNp63α by RT-PCR and ChIP location analysis in HaCaT and primary cells. C/EBPδ is differentially expressed in stratification of human skin and it is up-regulated upon differentiation of HaCaT and primary keratinocytes. It is bound to and activates the ΔNp63 promoter. Overexpression of C/EBPδ leads to alteration in the normal profile of p63 isoforms, with the emergence of ΔNp63β and γ, and of the TA isoforms, with different kinetics. In addition, there are changes in the expression of most p63 targets. Inactivation of C/EBPδ leads to gene expression modifications, in part due to the concomitant repression of ΔNp63α. Finally, C/EBPδ is found on the p63 targets in vivo by ChIP analysis, indicating that coregulation is direct. Conclusion Our data highlight a coherent cross-talk between these two transcription factors in keratinocytes and a large sharing of common transcriptional targets. PMID:17903252

  15. Human Glioblastoma Multiforme: p53 Reactivation by a Novel MDM2 Inhibitor

    PubMed Central

    Costa, Barbara; Bendinelli, Sara; Gabelloni, Pamela; Da Pozzo, Eleonora; Daniele, Simona; Scatena, Fabrizio; Vanacore, Renato; Campiglia, Pietro; Bertamino, Alessia; Gomez-Monterrey, Isabel; Sorriento, Daniela; Del Giudice, Carmine; Iaccarino, Guido; Novellino, Ettore; Martini, Claudia

    2013-01-01

    Cancer development and chemo-resistance are often due to impaired functioning of the p53 tumor suppressor through genetic mutation or sequestration by other proteins. In glioblastoma multiforme (GBM), p53 availability is frequently reduced because it binds to the Murine Double Minute-2 (MDM2) oncoprotein, which accumulates at high concentrations in tumor cells. The use of MDM2 inhibitors that interfere with the binding of p53 and MDM2 has become a valid approach to inhibit cell growth in a number of cancers; however little is known about the efficacy of these inhibitors in GBM. We report that a new small-molecule inhibitor of MDM2 with a spirooxoindolepyrrolidine core structure, named ISA27, effectively reactivated p53 function and inhibited human GBM cell growth in vitro by inducing cell cycle arrest and apoptosis. In immunoincompetent BALB/c nude mice bearing a human GBM xenograft, the administration of ISA27 in vivo activated p53, inhibited cell proliferation and induced apoptosis in tumor tissue. Significantly, ISA27 was non-toxic in an in vitro normal human cell model and an in vivo mouse model. ISA27 administration in combination with temozolomide (TMZ) produced a synergistic inhibitory effect on GBM cell viability in vitro, suggesting the possibility of lowering the dose of TMZ used in the treatment of GBM. In conclusion, our data show that ISA27 releases the powerful antitumor capacities of p53 in GBM cells. The use of this MDM2 inhibitor could become a novel therapy for the treatment of GBM patients. PMID:23977270

  16. MK-8776, a novel chk1 kinase inhibitor, radiosensitizes p53-defective human tumor cells

    PubMed Central

    Bridges, Kathleen A.; Chen, Xingxing; Liu, Huifeng; Rock, Crosby; Buchholz, Thomas A.; Shumway, Stuart D.; Skinner, Heath D.; Meyn, Raymond E.

    2016-01-01

    Radiotherapy is commonly used to treat a variety of solid tumors but improvements in the therapeutic ratio are sorely needed. The aim of this study was to assess the Chk1 kinase inhibitor, MK-8776, for its ability to radiosensitize human tumor cells. Cells derived from NSCLC and HNSCC cancers were tested for radiosensitization by MK-8776. The ability of MK-8776 to abrogate the radiation-induced G2 block was determined using flow cytometry. Effects on repair of radiation-induced DNA double strand breaks (DSBs) were determined on the basis of rad51, γ-H2AX and 53BP1 foci. Clonogenic survival analyses indicated that MK-8776 radiosensitized p53-defective tumor cells but not lines with wild-type p53. Abrogation of the G2 block was evident in both p53-defective cells and p53 wild-type lines indicating no correlation with radiosensitization. However, only p53-defective cells entered mitosis harboring unrepaired DSBs. MK-8776 appeared to inhibit repair of radiation-induced DSBs at early times after irradiation. A comparison of MK-8776 to the wee1 inhibitor, MK-1775, suggested both similarities and differences in their activities. In conclusion, MK-8776 radiosensitizes tumor cells by mechanisms that include abrogation of the G2 block and inhibition of DSB repair. Our findings support the clinical evaluation of MK-8776 in combination with radiation. PMID:27690219

  17. Abnormal mitosis triggers p53-dependent cell cycle arrest in human tetraploid cells.

    PubMed

    Kuffer, Christian; Kuznetsova, Anastasia Yurievna; Storchová, Zuzana

    2013-08-01

    Erroneously arising tetraploid mammalian cells are chromosomally instable and may facilitate cell transformation. An increasing body of evidence shows that the propagation of mammalian tetraploid cells is limited by a p53-dependent arrest. The trigger of this arrest has not been identified so far. Here we show by live cell imaging of tetraploid cells generated by an induced cytokinesis failure that most tetraploids arrest and die in a p53-dependent manner after the first tetraploid mitosis. Furthermore, we found that the main trigger is a mitotic defect, in particular, chromosome missegregation during bipolar mitosis or spindle multipolarity. Both a transient multipolar spindle followed by efficient clustering in anaphase as well as a multipolar spindle followed by multipolar mitosis inhibited subsequent proliferation to a similar degree. We found that the tetraploid cells did not accumulate double-strand breaks that could cause the cell cycle arrest after tetraploid mitosis. In contrast, tetraploid cells showed increased levels of oxidative DNA damage coinciding with the p53 activation. To further elucidate the pathways involved in the proliferation control of tetraploid cells, we knocked down specific kinases that had been previously linked to the cell cycle arrest and p53 phosphorylation. Our results suggest that the checkpoint kinase ATM phosphorylates p53 in tetraploid cells after abnormal mitosis and thus contributes to proliferation control of human aberrantly arising tetraploids.

  18. Relative biological effectiveness of light ions in human tumoural cell lines: role of protein p53

    NASA Technical Reports Server (NTRS)

    Baggio, L.; Cavinato, M.; Cherubini, R.; Conzato, M.; Cucinotta, F.; Favaretto, S.; Gerardi, S.; Lora, S.; Stoppa, P.; Williams, J. R.

    2002-01-01

    Protons and alpha particles of high linear energy transfer (LET) have shown an increased relative biological effectiveness (RBE) with respect to X/gamma rays for several cellular and molecular endpoints in different in vitro cell systems. To contribute to understanding the biochemical mechanisms involved in the increased effectiveness of high LET radiation, an extensive study has been designed. The present work reports the preliminary result of this study on two human tumoural cell lines, DLD1 and HCT116, (with different p53 status), which indicate that for these cell lines, p53 does not appear to take a part in the response to radiation induced DNA damage, suggesting an alternative p53-independent pathway and a cell biochemical mechanism dependent on the cell type.

  19. Relative biological effectiveness of light ions in human tumoural cell lines: role of protein p53

    NASA Technical Reports Server (NTRS)

    Baggio, L.; Cavinato, M.; Cherubini, R.; Conzato, M.; Cucinotta, F.; Favaretto, S.; Gerardi, S.; Lora, S.; Stoppa, P.; Williams, J. R.

    2002-01-01

    Protons and alpha particles of high linear energy transfer (LET) have shown an increased relative biological effectiveness (RBE) with respect to X/gamma rays for several cellular and molecular endpoints in different in vitro cell systems. To contribute to understanding the biochemical mechanisms involved in the increased effectiveness of high LET radiation, an extensive study has been designed. The present work reports the preliminary result of this study on two human tumoural cell lines, DLD1 and HCT116, (with different p53 status), which indicate that for these cell lines, p53 does not appear to take a part in the response to radiation induced DNA damage, suggesting an alternative p53-independent pathway and a cell biochemical mechanism dependent on the cell type.

  20. Zinc Deficiency Induces Apoptosis via Mitochondrial p53- and Caspase-Dependent Pathways in Human Neuronal Precursor Cells

    ERIC Educational Resources Information Center

    Seth, Rohit; Corniola, Rikki S.; Gower-Winter, Shannon D.; Morgan, Thomas J., Jr.; Bishop, Brian; Levenson, Cathy W.

    2015-01-01

    Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent…

  1. Undecylprodigiosin selectively induces apoptosis in human breast carcinoma cells independent of p53

    SciTech Connect

    Ho, T.-F.; Ma, C.-J.; Lu, C.-H.; Tsai, Yo-Ting; Wei, Y.-H.; Chang, J.-S.; Lai, J.-K.; Cheuh, Pin-Ju; Yeh, C.-T.; Tang, P.-C.; Jingua, T.C.; Ko, J.-L.; Liu, F.-S.; Yen, H.E.

    2007-12-15

    Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP's cytotoxic effect is selective for malignant cells. UP's cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspase 9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-X{sub L}, Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP's cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status.

  2. Frequent mutation of the p53 gene in human esophageal cancer

    SciTech Connect

    Hollstein, M.C.; Montesano, R. ); Metcalf, R.A.; Welsh, J.A.; Harris, C.C. )

    1990-12-01

    Sequence alterations in the p53 gene have been detected in human tumors of the brain, breast, lung, and colon, and it has been proposed that p53 mutations spanning a major portion of the coding region inactivate the tumor suppressor function of this gene. To our knowledge, neither transforming mutations in oncogenes nor mutations in tumor suppressor genes have been reported in human esophageal tumors. The authors examined four human esophageal carcinoma cell lines and 14 human esophageal squamous cell carcinomas by polymerase chain reaction amplification and direct sequencing for the presence of p53 mutations in exons 5,6,7,8, and 9. Two cell lines and five of the tumor speicmens contained a mutated allele (one frameshift and six missense mutations). All missense mutations detected occurred at G{center dot}C base pairs in codons at or adjacent to mutations previously reported in other cancers. The identification of aberrant p53 genes alleles in one-third of the tumors they tested suggests that mutations at this locus are common genetic events in the pathogenesis of squamous cell carcinomas of the esophagus.

  3. Alterations of p53 in tumorigenic human bronchial epithelial cells correlate with metastatic potential

    NASA Technical Reports Server (NTRS)

    Piao, C. Q.; Willey, J. C.; Hei, T. K.; Hall, E. J. (Principal Investigator)

    1999-01-01

    The cellular and molecular mechanisms of radiation-induced lung cancer are not known. In the present study, alterations of p53 in tumorigenic human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells induced by a single low dose of either alpha-particles or 1 GeV/nucleon (56)Fe were analyzed by PCR-single-stranded conformation polymorphism (SSCP) coupled with sequencing analysis and immunoprecipitation assay. A total of nine primary and four secondary tumor cell lines, three of which were metastatic, together with the parental BEP2D and primary human bronchial epithelial (NHBE) cells were studied. The immunoprecipitation assay showed overexpression of mutant p53 proteins in all the tumor lines but not in NHBE and BEP2D cells. PCR-SSCP and sequencing analysis found band shifts and gene mutations in all four of the secondary tumors. A G-->T transversion in codon 139 in exon 5 that replaced Lys with Asn was detected in two tumor lines. One mutation each, involving a G-->T transversion in codon 215 in exon 6 (Ser-->lle) and a G-->A transition in codon 373 in exon 8 (Arg-->His), was identified in the remaining two secondary tumors. These results suggest that p53 alterations correlate with tumorigenesis in the BEP2D cell model and that mutations in the p53 gene may be indicative of metastatic potential.

  4. Alterations of p53 in tumorigenic human bronchial epithelial cells correlate with metastatic potential

    NASA Technical Reports Server (NTRS)

    Piao, C. Q.; Willey, J. C.; Hei, T. K.; Hall, E. J. (Principal Investigator)

    1999-01-01

    The cellular and molecular mechanisms of radiation-induced lung cancer are not known. In the present study, alterations of p53 in tumorigenic human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells induced by a single low dose of either alpha-particles or 1 GeV/nucleon (56)Fe were analyzed by PCR-single-stranded conformation polymorphism (SSCP) coupled with sequencing analysis and immunoprecipitation assay. A total of nine primary and four secondary tumor cell lines, three of which were metastatic, together with the parental BEP2D and primary human bronchial epithelial (NHBE) cells were studied. The immunoprecipitation assay showed overexpression of mutant p53 proteins in all the tumor lines but not in NHBE and BEP2D cells. PCR-SSCP and sequencing analysis found band shifts and gene mutations in all four of the secondary tumors. A G-->T transversion in codon 139 in exon 5 that replaced Lys with Asn was detected in two tumor lines. One mutation each, involving a G-->T transversion in codon 215 in exon 6 (Ser-->lle) and a G-->A transition in codon 373 in exon 8 (Arg-->His), was identified in the remaining two secondary tumors. These results suggest that p53 alterations correlate with tumorigenesis in the BEP2D cell model and that mutations in the p53 gene may be indicative of metastatic potential.

  5. Human papillomavirus and p53 mutations in head and neck squamous cell carcinoma among Japanese population.

    PubMed

    Maruyama, Hiromi; Yasui, Toshimichi; Ishikawa-Fujiwara, Tomoko; Morii, Eiichi; Yamamoto, Yoshifumi; Yoshii, Tadashi; Takenaka, Yukinori; Nakahara, Susumu; Todo, Takeshi; Hongyo, Tadashi; Inohara, Hidenori

    2014-04-01

    We aimed to reveal the prevalence and pattern of human papillomavirus (HPV) infection and p53 mutations among Japanese head and neck squamous cell carcinoma (HNSCC) patients in relation to clinicopathological parameters. Human papillomavirus DNA and p53 mutations were examined in 493 HNSCCs and its subset of 283 HNSCCs. Oropharyngeal carcinoma was more frequently HPV-positive than non-oropharyngeal carcinoma (34.4% vs 3.6%, P < 0.001), and HPV16 accounted for 91.1% of HPV-positive tumors. In oropharyngeal carcinoma, which showed an increasing trend of HPV prevalence over time (P < 0.001), HPV infection was inversely correlated with tobacco smoking, alcohol drinking, p53 mutations, and a disruptive mutation (P = 0.003, <0.001, <0.001, and <0.001, respectively). The prevalence of p53 mutations differed significantly between virus-unrelated HNSCC and virus-related HNSCC consisting of nasopharyngeal and HPV-positive oropharyngeal carcinomas (48.3% vs 7.1%, P < 0.001). Although p53 mutations were associated with tobacco smoking and alcohol drinking, this association disappeared in virus-unrelated HNSCC. A disruptive mutation was never found in virus-related HNSCC, whereas it was independently associated with primary site, such as the oropharynx and hypopharynx (P = 0.01 and 0.03, respectively), in virus-unrelated HNSCC. Moreover, in virus-unrelated HNSCC, G:C to T:A transversions were more frequent in ever-smokers than in never-smokers (P = 0.04), whereas G:C to A:T transitions at CpG sites were less frequent in ever-smokers than in never-smokers (P = 0.04). In conclusion, HNSCC is etiologically classified into virus-related and virus-unrelated subgroups. In virus-related HNSCC, p53 mutations are uncommon with the absence of a disruptive mutation, whereas in virus-unrelated HNSCC, p53 mutations are common, and disruptive mutagenesis of p53 is related with oropharyngeal and hypopharyngeal carcinoma.

  6. Heterozygous p53V172F mutation in cisplatin-resistant human tumor cells promotes MDM4 recruitment and decreases stability and transactivity of p53

    PubMed Central

    Xie, Xiaolei; Lozano, Guillermina; Siddik, Zahid H.

    2017-01-01

    Cisplatin is an important antitumor agent, but its clinical utility is often limited by multifactorial mechanism of resistance. Loss of tumor suppressor p53 function is a major mechanism, affected by either mutation in the DNA binding domain or dysregulation by overexpression of p53 inhibitors MDM2 and MDM4 that destabilize p53 by increasing its proteosomal degradation. In the present study, cisplatin-resistant 2780CP/Cl-16 ovarian tumor cells expressed a heterozygous, temperature-sensitive p53V172F mutation, which reduced p53 half-life by 2- to 3-fold compared to homozygous wild-type p53 in parental A2780 cells. Although reduced p53 stability in 2780CP/Cl-16 cells was associated with moderate cellular overexpression of MDM2 or MDM4 (<1.5-fold), their binding to p53 was substantially enhanced (5- to 8-fold). The analogous cisplatin-resistant 2780CP/Cl-24 cells, which express loss of p53 heterozygosity, retained the p53V172F mutation and high p53-MDM4 binding, but demonstrated lower p53-bound MDM2 that was associated with reduced p53 ubiquitination and enhanced p53 stability. The inference that p53 was unstable as a hetromeric p53wt/p53V172F complex was confirmed in 2780CP/Cl-24 cells transfected with wild-type (wt) p53 or multimer-inhibiting p53L344P mutant, and further supported by normalization of p53 stability in both resistant cell lines grown at the permissive temperature of 32.5°C. Surprisingly, in 2780CP/Cl-16 and 2780CP/Cl-24 models, cisplatin-induced transactivity of p53 was attenuated at 37°C, and this correlated with cisplatin resistance. However, downregulation of MDM2 or MDM4 by siRNA in either resistant cell line induced p53 and restored p21 transactivation at 37°C, as did cisplatin-induced DNA damage at 32.5°C that coincided with reduced p53-MDM4 binding and cisplatin resistance. These results demonstrate that cisplatin-mediated p53V172F mutation regulates p53 stability at the normothermic temperature, but it is the increased recruitment of MDM4

  7. Mitochondrially targeted wild-type p53 induces apoptosis in a solid human tumor xenograft model

    PubMed Central

    Palacios, Gustavo; Crawford, Howard C.; Vaseva, Angelina; Moll, Ute M.

    2013-01-01

    Classic but also novel roles of p53 are becoming increasingly well characterized. We previously showed that ex vivo retroviral transfer of mitochondrially targeted wild type p53 (mitop53) in the Eμ-myc mouse lymphoma model efficiently induces tumor cell killing in vivo. In an effort to further explore the therapeutic potential of mitop53 for its pro-apoptotic effect in solid tumors, we generated replication-deficient recombinant human Adenovirus type 5 vectors. We show here that adenoviral delivery of mitop53 by intratumoral injection into HCT116 human colon carcinoma xenograft tumors in nude mice is surprisingly effective, resulting in tumor cell death of comparable potency to conventional p53. These apoptotic effects in vivo were confirmed by Ad5-mitop53 mediated cell death of HCT116 cells in culture. Together, these data provide encouragement to further explore the potential for novel mitop53 proteins in cancer therapy to execute the shortest known circuitry of p53 death signaling. PMID:18719383

  8. Aciculatin Induces p53-Dependent Apoptosis via MDM2 Depletion in Human Cancer Cells In Vitro and In Vivo

    PubMed Central

    Lai, Chin-Yu; Tsai, An-Chi; Chen, Mei-Chuan; Chang, Li-Hsun; Sun, Hui-Lung; Chang, Ya-Ling; Chen, Chien-Chih

    2012-01-01

    Aciculatin, a natural compound extracted from the medicinal herb Chrysopogon aciculatus, shows potent anti-cancer potency. This study is the first to prove that aciculatin induces cell death in human cancer cells and HCT116 mouse xenografts due to G1 arrest and subsequent apoptosis. The primary reason for cell cycle arrest and cell death was p53 accumulation followed by increased p21 level, dephosphorylation of Rb protein, PUMA expression, and induction of apoptotic signals such as cleavage of caspase-9, caspase-3, and PARP. We demonstrated that p53 allele-null (−/−) (p53-KO) HCT116 cells were more resistant to aciculatin than cells with wild-type p53 (+/+). The same result was achieved by knocking down p53 with siRNA in p53 wild-type cells, indicating that p53 plays a crucial role in aciculatin-induced apoptosis. Although DNA damage is the most common event leading to p53 activation, we found only weak evidence of DNA damage after aciculatin treatment. Interestingly, the aciculatin-induced downregulation of MDM2, an important negative regulator of p53, contributed to p53 accumulation. The anti-cancer activity and importance of p53 after aciculatin treatment were also confirmed in the HCT116 xenograft models. Collectively, these results indicate that aciculatin treatment induces cell cycle arrest and apoptosis via inhibition of MDM2 expression, thereby inducing p53 accumulation without significant DNA damage and genome toxicity. PMID:22912688

  9. 2.6 Å X-ray Crystal Structure of Human p53R2, a p53 Inducible Ribonucleotide Reductase

    PubMed Central

    Smith, Peter; Zhou, Bingsen; Ho, Nam; Yuan, Yate-Ching; Su, Leila; Tsai, Shiou-Chuan; Yen, Yun

    2009-01-01

    Human p53R2 (hp53R2) is a 351 residue p53-inducible ribonucleotide reductase (RNR) small subunit. It shares >80% sequence identity with hRRM2, the small RNR subunit responsible for normal maintenance of the deoxyribonucleotide (dNTP) pool used for DNA replication, which is active during the S-phase in a cell-cycle dependent fashion. But rather than cyclic dNTP synthesis, hp53R2 has been shown to supply dNTPs for DNA repair to cells in G0-G1 in a p53-dependent fashion. The first x-ray crystal structure of hp53R2 is solved to 2.6 Å, in which monomers A and B exhibit mono- and bi-nuclear iron occupancy, respectively. The pronounced structural differences at three regions between hp53R2 and hRRM2 highlight the possible regulatory role in iron assimilation, and help explain previously observed physical and biochemical differences in the mobility and accessibility of the radical-iron center, as well as radical transfer pathways between the two enzymes. The sequence-structure-function correlations that differentiate hp53R2 and hRRM2 are revealed for the first time. Insight gained from this structural work will be used toward the identification of biological function, regulation mechanism and inhibitors selection in RNR small subunits. PMID:19728742

  10. Definition of a DNA repair domain in the genomic region containing the human p53 gene.

    PubMed

    Tolbert, D M; Kantor, G J

    1996-07-15

    The human p53 gene is repaired in UV (254 nm)-irradiated xeroderma pigmentosum group C (XP-C) cells as part of a large genomic region that is about twice the size of the gene. Surrounding genomic regions are not repaired. Through DNA cloning and measurements of DNA repair, we mapped the location of the repair domain, including the terminal regions, relative to the topological features of the gene. The domain includes only the DNA strand that is transcribed and extends in both 3' and 5' directions beyond the promoter and transcription termination sites. No transcriptional activity other than that associated with the p53 gene was detected. The results suggest that nontranscribed regions adjacent to the p53 transcribed regions are efficiently repaired in XP-C cells. This means that factors associated with transcription other than RNA polymerase II and the associated transcription repair coupling factor must also play a role in the selective repair process in XP-C cells. We also found that a DNA fragment that contains the p53 promoters is nearly twice as sensitive to cyclobutane pyrimidine dimer induction by UV irradiation than are the surrounding fragments, which have the expected sensitivity.

  11. {sub p}53-Dependent Adaptive Responses in Human Cells Exposed to Space Radiations

    SciTech Connect

    Takahashi, Akihisa; Su Xiaoming; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki; Iwasaki, Toshiyasu; Ohnishi, Takeo

    2010-11-15

    Purpose: It has been reported that priming irradiation or conditioning irradiation with a low dose of X-rays in the range of 0.02-0.1 Gy induces a p53-dependent adaptive response in mammalian cells. The aim of the present study was to clarify the effect of space radiations on the adaptive response. Methods and Materials: Two human lymphoblastoid cell lines were used; one cell line bears a wild-type p53 (wtp53) gene, and another cell line bears a mutated p53 (mp53) gene. The cells were frozen during transportation on the space shuttle and while in orbit in the International Space Station freezer for 133 days between November 15, 2008 and March 29, 2009. After the frozen samples were returned to Earth, the cells were cultured for 6 h and then exposed to a challenging X-ray-irradiation (2 Gy). Cellular sensitivity, apoptosis, and chromosome aberrations were scored using dye-exclusion assays, Hoechst33342 staining assays, and chromosomal banding techniques, respectively. Results: In cells exposed to space radiations, adaptive responses such as the induction of radioresistance and the depression of radiation-induced apoptosis and chromosome aberrations were observed in wtp53 cells but not in mp53 cells. Conclusion: These results have confirmed the hypothesis that p53-dependent adaptive responses are apparently induced by space radiations within a specific range of low doses. The cells exhibited this effect owing to space radiations exposure, even though the doses in space were very low.

  12. Silver-based nanoparticles induce apoptosis in human colon cancer cells mediated through p53.

    PubMed

    Satapathy, Shakti Ranjan; Mohapatra, Purusottam; Preet, Ranjan; Das, Dipon; Sarkar, Biplab; Choudhuri, Tathagata; Wyatt, Michael D; Kundu, Chanakya Nath

    2013-08-01

    The authors have systematically investigated the anticancer potentiality of silver-based nanoparticles (AgNPs) and the mechanism underlying their biological activity in human colon cancer cells. Starch-capped AgNPs were synthesized, characterized and their biological activity evaluated through multiple biochemical assays. AgNPs decreased the growth and viability of HCT116 colon cancer cells. AgNP exposure increased apoptosis, as demonstrated by an increase in 4´,6-diamidino-2-phenylindole-stained apoptotic nuclei, BAX/BCL-XL ratio, cleaved poly(ADP-ribose) polymerase, p53, p21 and caspases 3, 8 and 9, and by a decrease in the levels of AKT and NF-κB. The cell population in the G1 phase decreased, and the S-phase population increased after AgNP treatment. AgNPs caused DNA damage and reduced the interaction between p53 and NF-κB. Interestingly, no significant alteration was noted in the levels of p21, BAX/BCL-XL and NF-κB after AgNP treatment in a p53-knockout HCT116 cell line. AgNPs are bona fide anticancer agents that act in a p53-dependent manner. Original submitted 16 March 2012; Revised submitted 25 August 2012; Published online 21 March 2013.

  13. Viral Single-Strand DNA Induces p53-Dependent Apoptosis in Human Embryonic Stem Cells

    PubMed Central

    Hirsch, Matthew L.; Fagan, B. Matthew; Dumitru, Raluca; Bower, Jacquelyn J.; Yadav, Swati; Porteus, Matthew H.; Pevny, Larysa H.; Samulski, R. Jude

    2011-01-01

    Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication. PMID:22114676

  14. Involvement of human ribosomal proteins in nucleolar structure and p53-dependent nucleolar stress

    PubMed Central

    Nicolas, Emilien; Parisot, Pascaline; Pinto-Monteiro, Celina; de Walque, Roxane; De Vleeschouwer, Christophe; Lafontaine, Denis L. J.

    2016-01-01

    The nucleolus is a potent disease biomarker and a target in cancer therapy. Ribosome biogenesis is initiated in the nucleolus where most ribosomal (r-) proteins assemble onto precursor rRNAs. Here we systematically investigate how depletion of each of the 80 human r-proteins affects nucleolar structure, pre-rRNA processing, mature rRNA accumulation and p53 steady-state level. We developed an image-processing programme for qualitative and quantitative discrimination of normal from altered nucleolar morphology. Remarkably, we find that uL5 (formerly RPL11) and uL18 (RPL5) are the strongest contributors to nucleolar integrity. Together with the 5S rRNA, they form the late-assembling central protuberance on mature 60S subunits, and act as an Hdm2 trap and p53 stabilizer. Other major contributors to p53 homeostasis are also strictly late-assembling large subunit r-proteins essential to nucleolar structure. The identification of the r-proteins that specifically contribute to maintaining nucleolar structure and p53 steady-state level provides insights into fundamental aspects of cell and cancer biology. PMID:27265389

  15. Human papillomavirus oncogenic E6 protein regulates human β-defensin 3 (hBD3) expression via the tumor suppressor protein p53

    PubMed Central

    Yue, Hong; Wang, Liming; Jin, Jessica; Ghosh, Santosh K.; Kawsar, Hameem I.; Zender, Chad; Androphy, Elliot J.; Weinberg, Aaron; McCormick, Thomas S.; Jin, Ge

    2016-01-01

    Human β-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer. PMID:27034006

  16. Mouse p63 variants and chondrogenesis

    PubMed Central

    Gu, Junxia; Lu, Yaojuan; Qiao, Longwei; Ran, Deyuan; Li, Na; Cao, Hong; Gao, Yan; Zheng, Qiping

    2013-01-01

    As a critical member of the p53 family of transcription factors, p63 has been implicated a role in development than in tumor formation, because p63 is seldom mutated in human cancers, while p63 null mice exhibit severe developmental abnormalities without increasing cancer susceptibility. Notably, besides the major epithelial and cardiac defect, p63 deficient mice show severe limb and craniofacial abnormalities. In addition, humans with p63 mutations also show severe limb and digit defects, suggesting a putative role of p63 in skeletal development. There are eight p63 variants which encode for the TAp63 and ΔNp63 isoforms by alternative promoters. How these isoforms function during skeletal development is currently largely unknown. Our recent transgenic studies suggest a role of TAP63α, but not ΔNP63α, during embryonic long bone development. However, the moderate skeletal phenotypes in the TAP63α transgenic mice suggest requirement of additional p63 isoform(s) for the limb defects in p63 null mice. Here, we report analysis of mouse p63 variants in MCT and ATDC5 cells, two cell models undergo hypertrophic differentiation and mimic the process of endochondral bone formation upon growth arrest or induction. We detected increased level of p63 variants in hypertrophic MCT cells by regular RT-PCR analysis. Further analysis by qRT-PCR, we detected significantly upregulated level of γ variant (p<0.05), but not α or β variant (p>0.05), in hypertrophic MCT cells than in proliferative MCT cells. Moreover, we detected upregulated TAP63γ in ATDC5 cells undergoing hypertrophic differentiation. Our results suggest that TAp63γ plays a positive role during endochondral bone formation. PMID:24294373

  17. Knockdown of CDK2AP1 in Primary Human Fibroblasts Induces p53 Dependent Senescence

    PubMed Central

    Alsayegh, Khaled N.; Gadepalli, Venkat S.; Iyer, Shilpa; Rao, Raj R.

    2015-01-01

    Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a) no increase in senescence associated beta-galactosidase activity, (b) decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c) decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1 knockdown. Altogether

  18. Genome-wide analysis of the human p53 transcriptional network unveils a lncRNA tumour suppressor signature.

    PubMed

    Sánchez, Yolanda; Segura, Victor; Marín-Béjar, Oskar; Athie, Alejandro; Marchese, Francesco P; González, Jovanna; Bujanda, Luis; Guo, Shuling; Matheu, Ander; Huarte, Maite

    2014-12-19

    Despite the inarguable relevance of p53 in cancer, genome-wide studies relating endogenous p53 activity to the expression of lncRNAs in human cells are still missing. Here, by integrating RNA-seq with p53 ChIP-seq analyses of a human cancer cell line under DNA damage, we define a high-confidence set of 18 lncRNAs that are p53 transcriptional targets. We demonstrate that two of the p53-regulated lncRNAs are required for the efficient binding of p53 to some of its target genes, modulating the p53 transcriptional network and contributing to apoptosis induction by DNA damage. We also show that the expression of p53-lncRNAs is lowered in colorectal cancer samples, constituting a tumour suppressor signature with high diagnostic power. Thus, p53-regulated lncRNAs establish a positive regulatory feedback loop that enhances p53 tumour suppressor activity. Furthermore, the signature defined by p53-regulated lncRNAs supports their potential use in the clinic as biomarkers and therapeutic targets.

  19. Mutations of the p53 gene in human functional adrenal neoplasms

    SciTech Connect

    Shiu-Ru Lin; Yau-Jiunn Lee; Juei-Hsiung Tsai

    1994-02-01

    To clarify gene alterations in functional human adrenal tumors, the authors performed molecular analysis for p53 abnormalities in 23 cases with adrenal neoplasms. The immunohistochemical study with anti-p53 monoclonal antibody pAb1801 demonstrated that 10 of 23 (43.5%) cases overexpressed p53 protein in the tumor cells. Using a polymerase chain reaction-single strand conformation polymorphism study, 5 of 6 (83.3%) pheochromocytoma tissues (1 malignant and 5 benign) and 11 of 15 (73.3%) adrenocortical adenomas (2 with Cushing`s syndrome and 13 with primary aldosteronism, all benign) showed an apparent electrophoretic mobility shift between the tumor and its paired adjacent normal adrenal tissue. Such differences were detected in exon 4 (12 cases), exon 5 (2 cases), and exon 7 (3 cases). The types of these mutations in exon 4 were a substitution from threonine (ACC) to isoleucine (ATC) at codon 102 in 5 cases, from glutamine (CAG) to histidine (CAC) at codon 104 in 1 case, from glycine (GGG) to alanine (CGG) at codon 117 in 1 case, from glutamate (GAG) to glutamine (CAG) at codon 68 in 1 case, and single base changes resulting in a premature stop codon at codon 100 in 2 cases. A 2-basepair deletion at codon 175 in exon 5 resulting in a frame shift was identified in 1 case. A single point mutation was identified, resulting in the substitution of glutamine (CAG) for arginine (CGG) at codon 248 of exon 7 in 1 case. A single basepair deletion at codon 249 resulted in a frame shift in 2 cases. There was 1 case with malignant pheochromocytoma that combined a single point mutation in exon 4 and a single base deletion in exon 7. Only 2 of 23 cases showed a loss of a normal allele encoding in the p53 gene. Northern blot analysis with 1.8-kilobase p53 cDNA revealed that p53 mRNA was overexpressed in 6 cases. The results indicate that high frequencies of p53 gene mutation, especially in exon 4, exist in functional adrenal tumors. 39 refs., 6 figs., 4 tabs.

  20. p53 mutations in human lymphoid malignancies: Association with Burkitt lymphoma and chronic lymphocytic leukemia

    SciTech Connect

    Gaidano, G.; Ballerini, P.; Gong, J.Z.; Inghirami, G.; Knowles, D.M.; Dalla-Favera, R. ); Neri, A, Centro Malattie del Sangue G. Marcora, Milan ); Newcomb, E.W. ); Magrath, I.T. )

    1991-06-15

    The authors have investigated the frequency of p53 mutations in B- and T-cell human lymphoid malignancies, including acute lymphoblastic leukemia, the major subtypes of non-Hodgkin lymphoma, and chronic lymphocytic leukemia. p53 exons 5-9 were studied by using genomic DNA from 197 primary tumors and 27 cell lines by single-strand conformation polymorphism analysis and by direst sequencing of PCR-amplified fragments. Mutations were found associated with (i) Burkitt lymphoma (9/27 biopsoes; 17/27 cell lines) and its leukemic counterpart L{sub 3}-type B-cell acute lymphoblastic leukemia (5/9), both of which also carry activated c-myc oncogenes, and (ii) B-cell chronic lymphocytic leukemia (6/40) and, in particular, its stage of progression known as Richter's transformation (3/7). Mutations were not found at any significant frequency in other types of non-Hodgkin lymphoma or acute lymphoblastic leukemia. In many cases, only the mutated allele was detectable, implying loss of the normal allele. These results suggest that (1) significant differences in the frequency of p53 mutations are present among subtypes of neoplasms derived from the same tissue; (2) p53 may play a role in tumor progression in B-cell chronic lymphocytic leukemia; (3) the presence of both p53 loss/inactivation and c-myc oncogene activation may be important in the pathogenesis of Burkitt lymphoma and its leukemia form L{sub 3}-type B-cell acute lymphoblastic leukemia.

  1. HJURP regulates cellular senescence in human fibroblasts and endothelial cells via a p53-dependent pathway.

    PubMed

    Heo, Jong-Ik; Cho, Jung Hee; Kim, Jae-Ryong

    2013-08-01

    Holliday junction recognition protein (HJURP), a centromere protein-A (CENP-A) histone chaperone, mediates centromere-specific assembly of CENP-A nucleosome, contributing to high-fidelity chromosome segregation during cell division. However, the role of HJURP in cellular senescence of human primary cells remains unclear. We found that the expression levels of HJURP decreased in human dermal fibroblasts and umbilical vein endothelial cells in replicative or premature senescence. Ectopic expression of HJURP in senescent cells partially overcame cell senescence. Conversely, downregulation of HJURP in young cells led to premature senescence. p53 knockdown, but not p16 knockdown, abolished senescence phenotypes caused by HJURP reduction. These data suggest that HJURP plays an important role in the regulation of cellular senescence through a p53-dependent pathway and might contribute to tissue or organismal aging and protection of cellular transformation.

  2. Mutability of p53 hotspot codons to benzo(a)pyrene diol epoxide (BPDE) and the frequency of p53 mutations in nontumorous human lung.

    PubMed

    Hussain, S P; Amstad, P; Raja, K; Sawyer, M; Hofseth, L; Shields, P G; Hewer, A; Phillips, D H; Ryberg, D; Haugen, A; Harris, C C

    2001-09-01

    p53 mutations are common in lung cancer. In smoking-associated lung cancer,the occurrence of G:C to T:A transversions at hotspot codons, e.g., 157, 248, 249,and 273, has been linked to the presence of carcinogenic chemicalsin tobacco smoke including polycyclic aromatic hydrocarbons suchas benzo(a)pyrene (BP). In the present study, we have used a highly sensitive mutation assay to determine the p53 mutation load in nontumorous human lung and to study the mutability of p53 codons 157, 248, 249, and 250 to benzo(a)pyrene-diol-epoxide (BPDE), an active metabolite of BP in human bronchial epithelial BEAS-2B cells. We determined the p53 mutational load at codons 157, 248, 249, and 250 in nontumorous peripheral lung tissue either from lung cancer cases among smokers or noncancer controls among smokers and nonsmokers. A 5-25-fold higher frequency of GTC(val) to TTC(phe) transversions at codon 157 was found in nontumorous samples (57%) from cancer cases (n = 14) when compared with noncancer controls (n = 8; P < 0.01). Fifty percent (7/14) of the nontumorous samples from lung cancer cases showed a high frequency of codon 249 AGG(arg) to AGT(ser) mutations (P < 0.02). Four of these seven samples with AGT(ser) mutations also showed a high frequency of codon 249 AGG(arg) to ATG(met) mutations, whereas only one sample showed a codon 250 CCC to ACC transversion. Tumor tissue from these lung cancer cases (38%) contained p53 mutations but were different from the above mutations found in the nontumorous pair. Noncancer control samples from smokers or nonsmokers did not contain any detectable mutations at codons 248, 249, or 250. BEAS-2B bronchial epithelial cells exposed to doses of 0.125, 0.5, and 1.0 microM BPDE, showed G:C to T:A transversions at codon 157 at a frequency of 3.5 x 10(-7), 4.4 x 10(-7), and 8.9 x 10(-7), respectively. No mutations at codon 157 were found in the DMSO-treated controls. These doses of BPDE induced higher frequencies, ranging from 4-12-fold, of G:C to

  3. Depression of p53-independent Akt survival signals in human oral cancer cells bearing mutated p53 gene after exposure to high-LET radiation

    SciTech Connect

    Nakagawa, Yosuke; Takahashi, Akihisa; Kajihara, Atsuhisa; Yamakawa, Nobuhiro; Imai, Yuichiro; Ota, Ichiro; Okamoto, Noritomo; Mori, Eiichiro; Noda, Taichi; Furusawa, Yoshiya; Kirita, Tadaaki; Ohnishi, Takeo

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer High-LET radiation induces efficiently apoptosis regardless of p53 gene status. Black-Right-Pointing-Pointer We examined whether high-LET radiation depresses the Akt-survival signals. Black-Right-Pointing-Pointer High-LET radiation depresses of survival signals even in the mp53 cancer cells. Black-Right-Pointing-Pointer High-LET radiation activates Caspase-9 through depression of survival signals. Black-Right-Pointing-Pointer High-LET radiation suppresses cell growth through depression of survival signals. -- Abstract: Although mutations and deletions in the p53 tumor suppressor gene lead to resistance to low linear energy transfer (LET) radiation, high-LET radiation efficiently induces cell lethality and apoptosis regardless of the p53 gene status in cancer cells. Recently, it has been suggested that the induction of p53-independent apoptosis takes place through the activation of Caspase-9 which results in the cleavage of Caspase-3 and poly (ADP-ribose) polymerase (PARP). This study was designed to examine if high-LET radiation depresses serine/threonine protein kinase B (PKB, also known as Akt) and Akt-related proteins. Human gingival cancer cells (Ca9-22 cells) harboring a mutated p53 (mp53) gene were irradiated with 2 Gy of X-rays or Fe-ion beams. The cellular contents of Akt-related proteins participating in cell survival signaling were analyzed with Western Blotting 1, 2, 3 and 6 h after irradiation. Cell cycle distributions after irradiation were assayed with flow cytometric analysis. Akt-related protein levels decreased when cells were irradiated with high-LET radiation. High-LET radiation increased G{sub 2}/M phase arrests and suppressed the progression of the cell cycle much more efficiently when compared to low-LET radiation. These results suggest that high-LET radiation enhances apoptosis through the activation of Caspase-3 and Caspase-9, and suppresses cell growth by suppressing Akt-related signaling, even in mp

  4. Expression of p53 protein related to the presence of human papillomavirus infection in precancer lesions of the larynx.

    PubMed Central

    Fouret, P.; Dabit, D.; Sibony, M.; Alili, D.; Commo, F.; Saint-Guily, J. L.; Callard, P.

    1995-01-01

    The aim of this study was to gain some insight into the relationship of human papillomavirus (HPV) infection to p53 expression and to some pathological parameters in precancerous lesions of the larynx. Formalin-fixed paraffin-embedded tissue sections containing human laryngeal precancerous lesions were screened for p53 protein by immunohistochemistry with the monoclonal antibody DO7 and for the presence of HPV infection by polymerase chain reaction with consensus primers directed against the E6 gene. The presence of p53 protein was detected in 31 of 57 specimens (54.4%) including 7 of 9 cases with mild dysplasia (78%), in 4 of 9 cases with moderate dysplasia (44%), and in 15 of 23 cases with severe dysplasia (65%). Of 16 samples with keratotic benign squamous metaplasia, 5 were also p53 positive (31%). Of 6 samples that were HPV positive, all were of type 16. Interestingly, 3 of the 6 HPV-positive samples were p53 negative. There was 1 HPV-positive case with strong p53 staining and 2 HPV-positive cases with minimal p53 staining. The 2 HPV-positive cases with minimal p53 staining had mild dysplasia. The HPV-positive case with strong p53 staining displayed severe dysplasia. Of 23 cases that were both HPV and p53 negative, 11 presented with keratosis and no dysplasia, 5 with moderate dysplasia, and 7 with severe dysplasia. Our data indicate that nuclear accumulation of p53 protein, presumably resulting from p53 gene mutation, may occur in HPV-infected epithelial tissues. On the other hand, there are many precancer lesions, some exhibiting moderate or severe dysplasia, that are both HPV negative and p53 unreactive, suggesting that alterations of genes other than the E6 oncogene and the p53 tumor suppressor gene play a role in early laryngeal carcinogenesis. Images Figure 1 PMID:7887442

  5. Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation.

    PubMed

    Hollstein, M; Shomer, B; Greenblatt, M; Soussi, T; Hovig, E; Montesano, R; Harris, C C

    1996-01-01

    In 1994 we described a list of approximately 2500 point mutations in the p53 gene of human tumors and cell lines which we had compiled from the published literature and made available electronically through the file server at the EMBL Data Library. This database, updated twice a year, now contains records on 4496 published mutations (July 1995 release) and can be obtained from the EMBL Outstation-the European Bioinformatics Institute (EBI) through the network or on CD-ROM. This report describes the criteria for inclusion of data in this database, a description of the current format and a brief discussion of the current relevance of p53 mutation analysis to clinical and biological questions.

  6. Involvement of Nuclear Export in Human Papillomavirus Type 18 E6-Mediated Ubiquitination and Degradation of p53

    PubMed Central

    Stewart, Deborah; Ghosh, Anirban; Matlashewski, Greg

    2005-01-01

    The E6 protein from high-risk human papillomaviruses (HPVs) targets the p53 tumor suppressor for degradation by the proteasome pathway. This ability contributes to the oncogenic potential of these viruses. However, several aspects concerning the mechanism of E6-mediated p53 degradation at the cellular level remain to be clarified. This study therefore examined the role of cell localization and ubiquitination in the E6-mediated degradation of p53. As demonstrated within, following coexpression both p53 and high-risk HPV type 18 (HPV-18) E6 (18E6) shuttle from the nucleus to the cytoplasm. Mutation of the C-terminal nuclear export signal (NES) of p53 or treatment with leptomycin B inhibited the 18E6-mediated nuclear export of p53. Impairment of nuclear export resulted in only a partial reduction in 18E6-mediated degradation, suggesting that both nuclear and cytoplasmic proteasomes can target p53 for degradation. This was also consistent with the observation that 18E6 mediated the accumulation of polyubiquitinated p53 in the nucleus. In comparison, a p53 isoform that localizes predominantly to the cytoplasm was not targeted for degradation by 18E6 in vivo but could be degraded in vitro, arguing that nuclear p53 is the target for E6-mediated degradation. This study supports a model in which (i) E6 mediates the accumulation of polyubiquitinated p53 in the nucleus, (ii) E6 is coexported with p53 from the nucleus to the cytoplasm via a CRM1 nuclear export mechanism involving the C-terminal NES of p53, and (iii) E6-mediated p53 degradation can be mediated by both nuclear and cytoplasmic proteasomes. PMID:15994771

  7. Tracing the Evolution of the p53 Tetramerization Domain

    PubMed Central

    Joerger, Andreas C.; Wilcken, Rainer; Andreeva, Antonina

    2014-01-01

    Summary The tetrameric transcription factors p53, p63, and p73 evolved from a common ancestor and play key roles in tumor suppression and development. Surprisingly, p63 and p73 require a second helix in their tetramerization domain for the formation of stable tetramers that is absent in human p53, raising questions about the evolutionary processes leading to diversification. Here we determined the crystal structure of the zebrafish p53 tetramerization domain, which contains a second helix, reminiscent of p63 and p73, combined with p53-like features. Through comprehensive phylogenetic analyses, we systematically traced the evolution of vertebrate p53 family oligomerization domains back to the beginning of multicellular life. We provide evidence that their last common ancestor also had an extended p63/p73-like domain and pinpoint evolutionary events that shaped this domain during vertebrate radiation. Domain compaction and transformation of a structured into a flexible, intrinsically disordered region may have contributed to the expansion of the human p53 interactome. PMID:25185827

  8. p53 regulates cell cycle and microRNAs to promote differentiation of human embryonic stem cells.

    PubMed

    Jain, Abhinav K; Allton, Kendra; Iacovino, Michelina; Mahen, Elisabeth; Milczarek, Robert J; Zwaka, Thomas P; Kyba, Michael; Barton, Michelle Craig

    2012-01-01

    Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells, p53 in hESCs is maintained at low levels in the nucleus, albeit in a deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G(1) phase of cell cycle without activation of cell death pathways. In parallel, p53 activates expression of miR-34a and miR-145, which in turn repress stem cell factors OCT4, KLF4, LIN28A, and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation, whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs, independently of retinoic acid. Ectopic expression of p53R175H, a mutated form of p53 that does not bind DNA or regulate transcription, failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state.

  9. The human long non-coding RNA-RoR is a p53 repressor in response to DNA damage

    PubMed Central

    Zhang, Ali; Zhou, Nanjiang; Huang, Jianguo; Liu, Qian; Fukuda, Koji; Ma, Ding; Lu, Zhaohui; Bai, Cunxue; Watabe, Kounosuke; Mo, Yin-Yuan

    2013-01-01

    It is well known that upon stress, the level of the tumor suppressor p53 is remarkably elevated. However, despite extensive studies, the underlying mechanism involving important inter-players for stress-induced p53 regulation is still not fully understood. We present evidence that the human lincRNA-RoR (RoR) is a strong negative regulator of p53. Unlike MDM2 that causes p53 degradation through the ubiquitin-proteasome pathway, RoR suppresses p53 translation through direct interaction with the heterogeneous nuclear ribonucleoprotein I (hnRNP I). Importantly, a 28-base RoR sequence carrying hnRNP I binding motifs is essential and sufficient for p53 repression. We further show that RoR inhibits p53-mediated cell cycle arrest and apoptosis. Finally, we demonstrate a RoR-p53 autoregulatory feedback loop where p53 transcriptionally induces RoR expression. Together, these results suggest that the RoR-hnRNP I-p53 axis may constitute an additional surveillance network for the cell to better respond to various stresses. PMID:23208419

  10. A Synthetic Interaction Screen Identifies Factors Selectively Required for Proliferation and TERT Transcription in p53-Deficient Human Cancer Cells

    PubMed Central

    Park, Sung Mi; Zhu, Lihua J.; Debily, Marie-anne; Kittler, Ellen L. W.; Zapp, Maria L.; Lapointe, David; Gobeil, Stephane; Virbasius, Ching-Man; Green, Michael R.

    2012-01-01

    Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi)–based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53−) human cancer cells. We find that compared to p53-competent (p53+) human cancer cell lines, diverse p53human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53− cells, RNAi–mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53− but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53− cancer cells. PMID:23284306

  11. Mutant p53 stimulates cell invasion through an interaction with Rad21 in human ovarian cancer cells.

    PubMed

    Ahn, Ji-Hye; Kim, Tae Jin; Lee, Jae Ho; Choi, Jung-Hye

    2017-08-22

    Missense mutations of TP53 are extremely common, and mutant p53 accumulation and gain-of-function play crucial roles in human ovarian cancer. Here, we investigated the role of mutant p53 in cell migration and invasion as well as its underlying molecular mechanisms in human ovarian cancer cells. Overexpression of mutant p53 significantly increased migration and invasion in p53-null SKOV3 cells. In contrast, knockdown of mutant p53 significantly compromised mutant p53-induced cell migration and invasion. Microarray analysis revealed that several migration/invasion-related genes, including S1PR1 (Sphingosine-1-phosphate receptor 1) and THBS1 (Thrombospodin 1), were significantly upregulated in SKOV3 cells that overexpressed mutant p53-R248 (SKOV3(R248)). We found that Rad21 is involved in the transcriptional regulation of the migration/invasion-related genes induced by mutant p53-R248. Knockdown of Rad21 significantly attenuated the mutant p53-R248-induced invasion and the expressions of S1PR1 and THBS1. Moreover, co-immunoprecipitation and chromatin immunoprecipitation assays revealed that mutant p53 interacts with Rad21 and binds to the Rad21-binding elements in the S1PR1 and THBS1 genes. Finally, downregulation of S1PR1 significantly attenuated the invasion driven by mutant p53-R248. These novel findings reveal that mutant p53-R248 maintains gain-of-function activity to stimulate cell invasion and induces the related gene expressions through an interaction with Rad21 in human ovarian cancer cells.

  12. p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells

    SciTech Connect

    Huang, Shi-Wei; Wu, Chun-Ying; Wang, Yen-Ting; Kao, Jun-Kai; Lin, Chi-Chen; Chang, Chia-Che; Mu, Szu-Wei; Chen, Yu-Yu; Chiu, Husan-Wen; Chang, Chuan-Hsun; Liang, Shu-Mei; Chen, Yi-Ju; Huang, Jau-Ling; Shieh, Jeng-Jer

    2013-02-15

    Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status. - Highlights: ► Compound C caused more apoptosis in p53 wildtype than p53-mutant skin cancer cells. ► Compound C can upregulate p53 expression and induce p53 activation. ► Compound C induced p53 effects were dependent on ROS induced DNA damage pathway. ► p53-knockdown attenuated compound C-induced apoptosis but not autophagy. ► Compound C-induced apoptosis in skin cancer cells was dependent on p53 status.

  13. Hepatic p63 regulates steatosis via IKKβ/ER stress

    PubMed Central

    Porteiro, Begoña; Fondevila, Marcos F.; Delgado, Teresa C.; Iglesias, Cristina; Imbernon, Monica; Iruzubieta, Paula; Crespo, Javier; Zabala-Letona, Amaia; Fernø, Johan; González-Terán, Bárbara; Matesanz, Nuria; Hernández-Cosido, Lourdes; Marcos, Miguel; Tovar, Sulay; Vidal, Anxo; Sánchez-Ceinos, Julia; Malagon, Maria M.; Pombo, Celia; Zalvide, Juan; Carracedo, Arkaitz; Buque, Xabier; Dieguez, Carlos; Sabio, Guadalupe; López, Miguel; Aspichueta, Patricia; Martínez-Chantar, María L.; Nogueiras, Ruben

    2017-01-01

    p53 family members control several metabolic and cellular functions. The p53 ortholog p63 modulates cellular adaptations to stress and has a major role in cell maintenance and proliferation. Here we show that p63 regulates hepatic lipid metabolism. Mice with liver-specific p53 deletion develop steatosis and show increased levels of p63. Down-regulation of p63 attenuates liver steatosis in p53 knockout mice and in diet-induced obese mice, whereas the activation of p63 induces lipid accumulation. Hepatic overexpression of N-terminal transactivation domain TAp63 induces liver steatosis through IKKβ activation and the induction of ER stress, the inhibition of which rescues the liver functions. Expression of TAp63, IKKβ and XBP1s is also increased in livers of obese patients with NAFLD. In cultured human hepatocytes, TAp63 inhibition protects against oleic acid-induced lipid accumulation, whereas TAp63 overexpression promotes lipid storage, an effect reversible by IKKβ silencing. Our findings indicate an unexpected role of the p63/IKKβ/ER stress pathway in lipid metabolism and liver disease. PMID:28480888

  14. Enhancement of P53-Mutant Human Colorectal Cancer Cells Radiosensitivity by Flavonoid Fisetin

    SciTech Connect

    Chen Wenshu; Lee Yijang; Yu Yichu; Hsaio Chinghui

    2010-08-01

    Purpose: The aim of this study was to investigate whether fisetin is a potential radiosensitizer for human colorectal cancer cells, which are relatively resistant to radiotherapy. Methods and Materials: Cell survival was examined by clonogenic survival assay, and DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The effects of treatments on cell cycle distribution and apoptosis were examined by flow cytometry. Western blot analysis was performed to ascertain the protein levels of {gamma}-H2AX, phospho-Chk2, active caspase-3, PARP cleavage, phospho-p38, phospho-AKT, and phospho-ERK1/2. Results: Fisetin pretreatment enhanced the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells but not human keratocyte HaCaT cells; it also prolonged radiation-induced G{sub 2}/M arrest, enhanced radiation-induced cell growth arrest in HT-29 cells, and suppressed radiation-induced phospho-H2AX (Ser-139) and phospho-Chk2 (Thr-68) in p53-mutant HT-29 cells. Pretreatment with fisetin enhanced radiation-induced caspase-dependent apoptosis in HT-29 cells. Fisetin pretreatment augmented radiation-induced phosphorylation of p38 mitogen-activated protein kinase, which is involved in caspase-mediated apoptosis, and SB202190 significantly reduced apoptosis and radiosensitivity in fisetin-pretreated HT-29 cells. By contrast, both phospho-AKT and phospho-ERK1/2, which are involved in cell proliferation and antiapoptotic pathways, were suppressed after irradiation combined with fisetin pretreatment. Conclusions: To our knowledge, this study is the first to provide evidence that fisetin exerts a radiosensitizing effect in p53-mutant HT-29 cells. Fisetin could potentially be developed as a novel radiosensitizer against radioresistant human cancer cells.

  15. p53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200.

    PubMed

    Morselli, Eugenia; Shen, Shensi; Ruckenstuhl, Christoph; Bauer, Maria Anna; Mariño, Guillermo; Galluzzi, Lorenzo; Criollo, Alfredo; Michaud, Mickael; Maiuri, Maria Chiara; Chano, Tokuhiro; Madeo, Frank; Kroemer, Guido

    2011-08-15

    The tumor suppressor protein p53 tonically suppresses autophagy when it is present in the cytoplasm. This effect is phylogenetically conserved from mammals to nematodes, and human p53 can inhibit autophagy in yeast, as we show here. Bioinformatic investigations of the p53 interactome in relationship to the autophagy-relevant protein network underscored the possible relevance of a direct molecular interaction between p53 and the mammalian ortholog of the essential yeast autophagy protein Atg17, namely RB1-inducible coiled-coil protein 1 (RB1CC1), also called FAK family kinase-interacting protein of 200 KDa (FIP200). Mutational analyses revealed that a single point mutation in p53 (K382R) abolished its capacity to inhibit autophagy upon transfection into p53-deficient human colon cancer or yeast cells. In conditions in which wild-type p53 co-immunoprecipitated with RB1CC1/FIP200, p53 (K382R) failed to do so, underscoring the importance of the physical interaction between these proteins for the control of autophagy. In conclusion, p53 regulates autophagy through a direct molecular interaction with RB1CC1/FIP200, a protein that is essential for the very apical step of autophagy initiation.

  16. Relationship of p53 Mutations to Epidermal Cell Proliferation and Apoptosis in Human UV-Induced Skin Carcinogenesis1

    PubMed Central

    Einspahr, Janine G; Alberts, David S; Warneke, James A; Bozzo, Paul; Basye, Jenny; Grogan, Thomas M; Nelson, Mark A; Bowden, G Tim

    1999-01-01

    Abstract Human skin is continually subjected to UV-irradiation with the p53 gene playing a pivotal role in repair of UV-induced DNA damage and apoptosis. Consequently, p53 alterations are early events in human UV-induced skin carcinogenesis. We studied 13 squamous cell carcinomas (SCC), 16 actinic keratoses (AK), 13 samples adjacent to an AK (chronically sun-damaged), and 14 normal-appearing skin samples for p53 mutation, p53 immunostaining (IHC), apoptosis (in situ TUNEL and morphology), and proliferation (PCNA). The frequency of p53 mutation increased from 14% in normal skin, to 38.5% in sun-damaged skin, 63% in AK, and 54% in SCC. p53 IHC increased similarly. Apoptosis (TUNEL) increased from 0.06 ± 0.02%, to 0.1 ± 0.2, 0.3 ± 0.3, and 0.4 ± 0.3 in normal skin, sun-damaged skin, AK, and SCC, respectively. Apoptosis was strongly correlated with proliferation (i.e., TUNEL and PCNA, r = 0.7, P < 0.0001), and proliferation was significantly increased in the progression from normal skin to SCC. Bax was significantly increased in SCC compared to AK. These data imply that apoptosis in samples with a high frequency of p53 mutation may not necessarily be p53-dependent. We suggest that there is a mechanism for apoptosis in response to increased cellular proliferation that is p53-independent. PMID:10933063

  17. Human p53 oncogene contains one promoter upstream of exon 1 and a second, stronger promoter within intron 1

    SciTech Connect

    Reisman, D.; Greenberg, M.; Rotter, V. )

    1988-07-01

    To gain insight into how transcription of the human p53 oncogene is controlled, the authors characterized the regulatory regions of the gene. A 3.8-kilobase-pair (kbp) EcoRI restriction fragment encompassing the 5{prime} end of the human p53 gene, as well as subfragments generated by restriction digests, was cloned upstream of the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and CAT activity was assayed in extracts of transfected cells. Two types of CAT vectors were used: Epstein-Barr virus oriP-derived constructs that were stably introduced into the human cell lines K562, Raji, and HL-60, and pSVO-CAT-derived constructs that were transiently introduced into the monkey cell line COS. By this approach they have identified two promoters for the human p53 gene. One promoter, p53P1, is located 100-250 bp upstream of the 218-bp noncoding first exon; a second, stronger promoter, p53P2, maps within the first intron. CAT activity and expression of CAT RNA indicate that p53P2 functions up to 50-fold more efficiently than p53P1. They conclude that the expression of the human p53 gene may be controlled by two promoters and that differential regulation of these promoters may play an important role in the altered expression of the gene in both normal and transformed cells.

  18. Preimplantation factor is an anti-apoptotic effector in human trophoblasts involving p53 signaling pathway

    PubMed Central

    Moindjie, Hadia; Santos, Esther Dos; Gouesse, Rita-Josiane; Swierkowski-Blanchard, Nelly; Serazin, Valérie; Barnea, Eytan R; Vialard, François; Dieudonné, Marie-Noëlle

    2016-01-01

    From the earliest stages of gestation, embryonic–maternal interaction has a key role in a successful pregnancy. Various factors present during gestation may significantly influence this type of juxta/paracrine interaction. PreImplantation Factor (PIF) is a recently identified factor with activity at the fetomaternal interface. PIF is secreted by viable embryos and directly controls placental development by increasing the invasive capacity of human extravillous trophoblasts (EVTs). To further specify PIF's role in the human placenta, we analyzed the genome-wide expression profile of the EVT in the presence of a synthetic PIF analog (sPIF). We found that sPIF exposure altered several pathways related to p53 signaling, survival and the immune response. Functional assays revealed that sPIF acts through the p53 pathway to reduce both early and late trophoblast apoptosis. More precisely, sPIF (i) decreases the phosphorylation of p53 at Ser-15, (ii) enhances the B-cell lymphoma-2 (BCL2) expression and (iii) reduces the BCL2-associated X protein (BAX) and BCL2 homologous antagonist killer (BAK) mRNA expression levels. Furthermore, invalidation experiments of TP53 allowed us to demonstrate that PIF's effects on placental apoptosis seemed to be essentially mediated by this gene. We have clearly shown that p53 and sPIF pathways could interact in human trophoblast and thus promotes cell survival. Furthermore, sPIF was found to regulate a gene network related to immune tolerance in the EVT, which emphasizes the beneficial effect of this peptide on the human placenta. Finally, the PIF protein levels in placentas from pregnancies affected by preeclampsia or intra-uterine growth restriction were significantly lower than in gestational age-matched control placentas. Taken as a whole, our results suggest that sPIF protects the EVT's functional status through a variety of mechanisms. Clinical application of sPIF in the treatment of disorders of early pregnancy can be envisioned

  19. Rosiglitazone enhances the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells

    SciTech Connect

    Chiu, Shu-Jun; Hsaio, Ching-Hui; Tseng, Ho-Hsing; Su, Yu-Han; Shih, Wen-Ling; Lee, Jeng-Woei; Chuah, Jennifer Qiu-Yu

    2010-04-09

    Combined-modality treatment has improved the outcome in cases of various solid tumors, and radiosensitizers are used to enhance the radiotherapeutic efficiency. Rosiglitazone, a synthetic ligand of peroxisome proliferator-activated receptors {gamma} used in the treatment of type-2 diabetes, has been shown to reduce tumor growth and metastasis in human cancer cells, and may have the potential to be used as a radiosensitizer in radiotherapy for human colorectal cancer cells. In this study, rosiglitazone treatment significantly reduced the cell viability of p53-wild type HCT116 cells but not p53-mutant HT-29 cells. Interestingly, rosiglitazone pretreatment enhanced radiosensitivity in p53-mutant HT-29 cells but not HCT116 cells, and prolonged radiation-induced G{sub 2}/M arrest and enhanced radiation-induced cell growth inhibition in HT-29 cells. Pretreatment with rosiglitazone also suppressed radiation-induced H2AX phosphorylation in response to DNA damage and AKT activation for cell survival; on the contrary, rosiglitazone pretreatment enhanced radiation-induced caspase-8, -9, and -3 activation and PARP cleavage in HT-29 cells. In addition, pretreatment with a pan-caspase inhibitor, zVAD-fmk, attenuated the levels of caspase-3 activation and PARP cleavage in radiation-exposed cancer cells in combination with rosiglitazone pretreatment. Our results provide proof for the first time that rosiglitazone suppresses radiation-induced survival signals and DNA damage response, and enhances the radiation-induced apoptosis signaling cascade. These findings can assist in the development of rosiglitazone as a novel radiosensitizer.

  20. Cytogenetic damage, oncogenic transformation and p53 induction in human epithelial cells in response to irradiation

    NASA Astrophysics Data System (ADS)

    Armitage, Mark

    Ionizing radiation can have several different effects on cells, some are almost instantaneous such as the generation of DNA damage, other cellular responses take a matter of minutes or hours - DNA repair protein induction/activation, and others may take months or even years to be manifested - carcinogenesis. Human epithelial cell lines derived from both normal, non-neoplastic tissues and from a malignant source were cultured in order to examine several effects of ionizing radiation on such cell types. Cells not from a malignant source were previously immortalized by viral infection or by transfection with viral sequences. Simian virus 40 immortalised uroepithelial cells (SV-HUC) were found to be approximately a factor of two fold more radioresistant than cells of malignant origin (T24) in terms of unrepaired clastogenic damage i.e. assessment of micronuclei levels following irradiation. SV-HUC lines unlike T24 cells are non-tumourigenic when inoculated into nude athymic mice. SV-HUC lines proved very resistant to full oncogenic transformation using radiation and chemical carcinogens. However, morphological alterations and decreased anchorage dependant growth was observed in post carcinogen treated cells after appropriate cell culture conditions were utilized. The progression from this phenotype to a fully tumourigenic one was not recorded in this study. The ability of ionizing radiation to induce increased levels of the nuclear phosphoprotein p53 was also assessed using several different cell lines. SV- HUC and T24 cell lines failed to exhibit any increased p53 stabilization following irradiation. One cell line, a human papilloma virus transformed line (HPV) did show an approximate two fold increase of the wild type p53 protein after treatment with radiation. Only the cell line HPV showed any cell cycle delay, resulting in accumulation of cells in the G2/M compartment in post irradiation cell cycle analysis. The status of p53 was also assessed i.e. wild type or

  1. The Enigma of p53.

    PubMed

    Lozano, Guillermina

    2016-12-08

    This perspective will focus on the physiological impact of wild-type and mutant p53 activities. In particular, the tissue-specific nature of activation of p53 targets and their subsequent effects on cell behavior will be discussed. Because mutations in p53 are common in human cancers, the regulation and physiological consequences of mutant p53 proteins will also be discussed.

  2. Allicin induces p53-mediated autophagy in Hep G2 human liver cancer cells.

    PubMed

    Chu, Yung-Lin; Ho, Chi-Tang; Chung, Jing-Gung; Rajasekaran, Raghu; Sheen, Lee-Yan

    2012-08-29

    Garlic has been used throughout history for both culinary and medicinal purpose. Allicin is a major component of crushed garlic. Although it is sensitive to heat and light and easily metabolized into various compounds such as diallyl disulfide, diallyl trisulfide, and diallyl sulfide, allicin is still a major bioactive compound of crushed garlic. The mortality of hepatocellular carcinoma is quite high and ranks among the top 10 cancer-related deaths in Taiwan. Although numerous studies have shown the cancer-preventive properties of garlic and its components, there is no study on the effect of allicin on the growth of human liver cancer cells. In this study, we focused on allicin-induced autophagic cell death in human liver cancer Hep G2 cells. Our results indicated that allicin induced p53-mediated autophagy and inhibited the viability of human hepatocellular carcinoma cell lines. Using Western blotting, we observed that allicin decreased the level of cytoplasmic p53, the PI3K/mTOR signaling pathway, and the level of Bcl-2 and increased the expression of AMPK/TSC2 and Beclin-1 signaling pathways in Hep G2 cells. In addition, the colocalization of LC3-II with MitoTracker-Red (labeling mitochondria), resulting in allicin-induced degradation of mitochondria, could be observed by confocal laser microscopy. In conclusion, allicin of garlic shows great potential as a novel chemopreventive agent for the prevention of liver cancer.

  3. p53 stimulates transcription from the human transforming growth factor alpha promoter: a potential growth-stimulatory role for p53.

    PubMed Central

    Shin, T H; Paterson, A J; Kudlow, J E

    1995-01-01

    Physical and chemical agents can damage the genome. Part of the protective response to this damage is the increased expression of p53. p53, a transcription factor, controls the expression of genes, leading to cell cycle arrest and apoptosis. Another protective mechanism is the proliferative response required to replace the damaged cells. This proliferation is likely to be signaled by growth factors. In this communication, we show that the transforming growth factor alpha (TGF-alpha) gene is a direct target for p53-mediated transcriptional activation. In a stable cell line containing an inducible p53 construct, p53 induction leads to a threefold accumulation of the native TGF-alpha mRNA. IN cotransfection assays using a TGF-alpha promoter reporter construct, we show that expression of wild-type but not mutant p53 increases transcriptional activity of the TGF-alpha promoter by approximately 2.5-fold. In vitro, wild-type p53 binds to a consensus binding site found in the proximal portion of the promoter, and this sequence is necessary for the p53 transcriptional response. Furthermore, this element confers p53 induction to the otherwise nonresponsive adenovirus major late promoter. In addition to these results, we found that the TGF-alpha promoter contains a nonconsensus but functional TATA box-binding protein-binding site approximately 30 bp upstream of the transcription start site. Although p53 can repress transcription from promoters containing a TATA box, the nonconsensus TGF-alpha TATA motif is resistant to this effect. On the basis of these results, we propose that p53 may play a dual role, which includes both the elimination of irreparably genetically damage cells and the proliferative response necessary for their replacement, in the response to physical-chemical damage. PMID:7651386

  4. An immunohistochemical study of primary signet-ring cell carcinoma of the stomach and colorectum: III. expressions of EMA, CEA, CA19-9, CDX-2, p53, Ki-67 antigen, TTF-1, vimentin, and p63 in normal mucosa and in 42 cases

    PubMed Central

    Terada, Tadashi

    2013-01-01

    There have no comprehensive immunohistochemical studies of primary signet ring cell carcinoma (SRCC) in the stomach and colorectum. The author examined the expression of nine common antigens (EMA, CEA, CA19-9, CDX-2, p53, Ki-67 antigen, TTF-1, vimentin, and p63) in the non-tumorous normal epithelium of the stomach and colorectum and in 42 cases of primary SRCC of the stomach (30 cases) and colorectum (12 cases). The normal epithelium of the stomach and colon consistently (100%) expressed EMA, CEA, CA19-9, CDX-2, and Ki-67 (labeling <15%). Normal epithelium of these locations never expressed p53, TTF-1, vimentin, and p63. In the primary gastric SRCC, the expression percentage of EMA was 57% (17/30), CEA 100% (30/30), CA19-9 100% (30/30), CDX-2 43% (13/30), p53 83% (25/30), Ki-67 100% (30/30) (labeling index= 36 ± 23 %), TTF-1 0% (0/30), vimentin 0% (0/30), and p63 0% (0/30). In primary colorectal SRCC, the expression percentage of EMA was 25% (3/12), CEA 100% (12/12), CA19-9 100% (12/12), CDX-2 93% (28/30), p53 75% (9/12), Ki-67 100% (30/30) (labeling index= 47% ± 26 %), TTF-1 0% (0/12), vimentin 0% (0/12), and p63 0% (0/12). A comparative statistical analysis showed significant difference in EMA (gastric SRCC 57% vs colorectal SRCC 25%) and CDX-2 (43% vs 93%). There were no significant differences in the other seven antigens’ expression between primary gastric SRCC and primary colorectal SRCC. These findings provide much knowledge of primary SRCC of the stomach and colorectum. The data indicated primary gastric SRCC frequently express EMA but not CDX-2 whereas primary colorectal SRCC frequently express CDX-2 but not EMA. These findings also suggest that EMA and CDX-2 are down-regulated during the gastric SRCC carcinogenesis. This down regulations may be associated with the malignant transformation of gastric SRCC. The data of colorectal SRCC suggest EMA is markedly down-regulated and also suggest that this EMA down-regulation may be associated with the

  5. An immunohistochemical study of primary signet-ring cell carcinoma of the stomach and colorectum: III. Expressions of EMA, CEA, CA19-9, CDX-2, p53, Ki-67 antigen, TTF-1, vimentin, and p63 in normal mucosa and in 42 cases.

    PubMed

    Terada, Tadashi

    2013-01-01

    There have no comprehensive immunohistochemical studies of primary signet ring cell carcinoma (SRCC) in the stomach and colorectum. The author examined the expression of nine common antigens (EMA, CEA, CA19-9, CDX-2, p53, Ki-67 antigen, TTF-1, vimentin, and p63) in the non-tumorous normal epithelium of the stomach and colorectum and in 42 cases of primary SRCC of the stomach (30 cases) and colorectum (12 cases). The normal epithelium of the stomach and colon consistently (100%) expressed EMA, CEA, CA19-9, CDX-2, and Ki-67 (labeling <15%). Normal epithelium of these locations never expressed p53, TTF-1, vimentin, and p63. In the primary gastric SRCC, the expression percentage of EMA was 57% (17/30), CEA 100% (30/30), CA19-9 100% (30/30), CDX-2 43% (13/30), p53 83% (25/30), Ki-67 100% (30/30) (labeling index= 36 ± 23 %), TTF-1 0% (0/30), vimentin 0% (0/30), and p63 0% (0/30). In primary colorectal SRCC, the expression percentage of EMA was 25% (3/12), CEA 100% (12/12), CA19-9 100% (12/12), CDX-2 93% (28/30), p53 75% (9/12), Ki-67 100% (30/30) (labeling index= 47% ± 26 %), TTF-1 0% (0/12), vimentin 0% (0/12), and p63 0% (0/12). A comparative statistical analysis showed significant difference in EMA (gastric SRCC 57% vs colorectal SRCC 25%) and CDX-2 (43% vs 93%). There were no significant differences in the other seven antigens' expression between primary gastric SRCC and primary colorectal SRCC. These findings provide much knowledge of primary SRCC of the stomach and colorectum. The data indicated primary gastric SRCC frequently express EMA but not CDX-2 whereas primary colorectal SRCC frequently express CDX-2 but not EMA. These findings also suggest that EMA and CDX-2 are down-regulated during the gastric SRCC carcinogenesis. This down regulations may be associated with the malignant transformation of gastric SRCC. The data of colorectal SRCC suggest EMA is markedly down-regulated and also suggest that this EMA down-regulation may be associated with the

  6. Tumor suppressor protein p53 exerts negative transcriptional regulation on human sodium iodide symporter gene expression in breast cancer.

    PubMed

    Kelkar, Madhura G; Thakur, Bhushan; Derle, Abhishek; Chatterjee, Sushmita; Ray, Pritha; De, Abhijit

    2017-08-01

    Aberrant expression of human sodium iodide symporter (NIS) in breast cancer (BC) is well documented but the transcription factors (TF) regulating its aberrant expression is poorly known. We identify the presence of three p53 binding sites on the human NIS promoter sequence by conducting genome-wide TF analysis, and further investigate their regulatory role. The differences in transcription and translation were measured by real-time PCR, luciferase reporter assay, site-directed mutagenesis, in vivo optical imaging, and chromatin immunoprecipitation. The relation of NIS and p53 in clinical samples was judged by TCGA data analysis and immunohistochemistry. Overexpression of wild-type p53 as a transgene or pharmacological activation by doxorubicin drug treatment shows significant suppression of NIS transcription in multiple BC cell types which also results in lowered NIS protein content and cellular iodide intake. NIS repression by activated p53 is further confirmed by non-invasive bioluminescence imaging in live cell and orthotropic tumor model. Abrogation of p53-binding sites by directional mutagenesis confirms reversal of transcriptional activity in wild-type p53-positive BC cells. We also observe direct binding of p53 to these sites on the human NIS promoter. Importantly, TCGA data analysis of NIS and p53 co-expression registers an inverse relationship between the two candidates. Our data for the first time highlight the role of p53 as a negative regulator of functional NIS expression in BC, where the latter is a potential targeted radioiodine therapy candidate. Thus, the study provides an important insight into prospective clinical application of this approach that may significantly impact the patient with mutant versus wild-type p53 profile.

  7. Lack of dependence on p53 for DNA double strand break repair of episomal vectors in human lymphoblasts

    NASA Technical Reports Server (NTRS)

    Kohli, M.; Jorgensen, T. J.

    1999-01-01

    The p53 tumor suppressor gene has been shown to be involved in a variety of repair processes, and recent findings have suggested that p53 may be involved in DNA double strand break repair in irradiated cells. The role of p53 in DNA double strand break repair, however, has not been fully investigated. In this study, we have constructed a novel Epstein-Barr virus (EBV)-based shuttle vector, designated as pZEBNA, to explore the influence of p53 on DNA strand break repair in human lymphoblasts, since EBV-based vectors do not inactivate the p53 pathway. We have compared plasmid survival of irradiated, restriction enzyme linearized, and calf intestinal alkaline phosphatase (CIP)-treated pZEBNA with a Simian virus 40 (SV40)-based shuttle vector, pZ189, in TK6 (wild-type p53) and WTK1 (mutant p53) lymphoblasts and determined that p53 does not modulate DNA double strand break repair in these cell lines. Copyright 1999 Academic Press.

  8. Specific loss of apoptotic but not cell-cycle arrest function in a human tumor derived p53 mutant.

    PubMed Central

    Rowan, S; Ludwig, R L; Haupt, Y; Bates, S; Lu, X; Oren, M; Vousden, K H

    1996-01-01

    The p53 tumor-suppressor gene product is frequently inactivated in malignancies by point mutation. Although most tumor-derived p53 mutants show loss of sequence specific transcriptional activation, some mutants have been identified which retain this activity. One such mutant, p53175P, is defective for the suppression of transformation in rodent cells, despite retaining the ability to suppress the growth of p53-null human cells. We now demonstrate that p53175P can induce a cell-cycle arrest in appropriate cell types but shows loss of apoptotic function. Our results therefore support a direct role of p53 transcriptional activation in mediating a cell-cycle arrest and demonstrate that such activity is not sufficient for the full apoptotic response. These data suggest that either p53 can induce apoptosis through a transcriptionally independent mechanism, a function lost by p53175P, or that this mutant has specifically lost the ability to activate genes which contribute to cell death, despite activation of genes responsible for the G1 arrest. This dissociation of the cell-cycle arrest and apoptotic activities of p53 indicates that inactivation of p53 apoptotic function without concomitant loss of growth inhibition can suffice to relieve p53-dependent tumor-suppression in vivo and thereby contribute to tumor development. Images PMID:8631304

  9. Lack of dependence on p53 for DNA double strand break repair of episomal vectors in human lymphoblasts

    NASA Technical Reports Server (NTRS)

    Kohli, M.; Jorgensen, T. J.

    1999-01-01

    The p53 tumor suppressor gene has been shown to be involved in a variety of repair processes, and recent findings have suggested that p53 may be involved in DNA double strand break repair in irradiated cells. The role of p53 in DNA double strand break repair, however, has not been fully investigated. In this study, we have constructed a novel Epstein-Barr virus (EBV)-based shuttle vector, designated as pZEBNA, to explore the influence of p53 on DNA strand break repair in human lymphoblasts, since EBV-based vectors do not inactivate the p53 pathway. We have compared plasmid survival of irradiated, restriction enzyme linearized, and calf intestinal alkaline phosphatase (CIP)-treated pZEBNA with a Simian virus 40 (SV40)-based shuttle vector, pZ189, in TK6 (wild-type p53) and WTK1 (mutant p53) lymphoblasts and determined that p53 does not modulate DNA double strand break repair in these cell lines. Copyright 1999 Academic Press.

  10. The Origins and Evolution of the p53 Family of Genes

    PubMed Central

    Belyi, Vladimir A.; Ak, Prashanth; Markert, Elke; Wang, Haijian; Hu, Wenwei; Puzio-Kuter, Anna; Levine, Arnold J.

    2010-01-01

    A common ancestor to the three p53 family members of human genes p53, p63, and p73 is first detected in the evolution of modern‐day sea anemones, in which both structurally and functionally it acts to protect the germ line from genomic instabilities in response to stresses. This p63/p73 common ancestor gene is found in almost all invertebrates and first duplicates to produce a p53 gene and a p63/p73 ancestor in cartilaginous fish. Bony fish contain all three genes, p53, p63, and p73, and the functions of these three transcription factors diversify in the higher vertebrates. Thus, this gene family has preserved its structural features and functional activities for over one billion years of evolution. PMID:20516129

  11. Unequal prognostic potentials of p53 gain-of-function mutations in human cancers associate with drug-metabolizing activity.

    PubMed

    Xu, J; Wang, J; Hu, Y; Qian, J; Xu, B; Chen, H; Zou, W; Fang, J-Y

    2014-03-06

    Mutation of p53 is the most common genetic change in human cancer, causing complex effects including not only loss of wild-type function but also gain of novel oncogenic functions (GOF). It is increasingly likely that p53-hotspot mutations may confer different types and magnitudes of GOF, but the evidences are mainly supported by cellular and transgenic animal models. Here we combine large-scale cancer genomic data to characterize the prognostic significance of different p53 mutations in human cancers. Unexpectedly, only mutations on the Arg248 and Arg282 positions displayed significant association with shorter patient survival, but such association was not evident for other hotspot GOF mutations. Gene set enrichment analysis on these mutations revealed higher activity of drug-metabolizing enzymes, including the CYP3A4 cytochrome P450. Ectopic expression of p53 mutant R282W in H1299 and SaOS2 cells significantly upregulated CYP3A4 mRNA and protein levels, and cancer cell lines bearing mortality-associated p53 mutations display higher CYP3A4 expression and resistance to several CYP3A4-metabolized chemotherapeutic drugs. Our results suggest that p53 mutations have unequal GOF activities in human cancers, and future evaluation of p53 as a cancer biomarker should consider which mutation is present in the tumor, rather than having comparison between wild-type and mutant genotypes.

  12. p53 elevation in human cells halt SV40 infection by inhibiting T-ag expression

    PubMed Central

    Drayman, Nir; Ben-nun-Shaul, Orly; Butin-Israeli, Veronika; Srivastava, Rohit; Rubinstein, Ariel M.; Mock, Caroline S.; Elyada, Ela; Ben-Neriah, Yinon; Lahav, Galit; Oppenheim, Ariella

    2016-01-01

    SV40 large T-antigen (T-ag) has been known for decades to inactivate the tumor suppressor p53 by sequestration and additional mechanisms. Our present study revealed that the struggle between p53 and T-ag begins very early in the infection cycle. We found that p53 is activated early after SV40 infection and defends the host against the infection. Using live cell imaging and single cell analyses we found that p53 dynamics are variable among individual cells, with only a subset of cells activating p53 immediately after SV40 infection. This cell-to-cell variabilty had clear consequences on the outcome of the infection. None of the cells with elevated p53 at the beginning of the infection proceeded to express T-ag, suggesting a p53-dependent decision between abortive and productive infection. In addition, we show that artificial elevation of p53 levels prior to the infection reduces infection efficiency, supporting a role for p53 in defending against SV40. We further found that the p53-mediated host defense mechanism against SV40 is not facilitated by apoptosis nor via interferon-stimulated genes. Instead p53 binds to the viral DNA at the T-ag promoter region, prevents its transcriptional activation by Sp1, and halts the progress of the infection. These findings shed new light on the long studied struggle between SV40 T-ag and p53, as developed during virus-host coevolution. Our studies indicate that the fate of SV40 infection is determined as soon as the viral DNA enters the nucleus, before the onset of viral gene expression. PMID:27462916

  13. The p63 Protein Isoform ΔNp63α Inhibits Epithelial-Mesenchymal Transition in Human Bladder Cancer Cells

    PubMed Central

    Tran, Mai N.; Choi, Woonyoung; Wszolek, Matthew F.; Navai, Neema; Lee, I-Ling C.; Nitti, Giovanni; Wen, Sijin; Flores, Elsa R.; Siefker-Radtke, Arlene; Czerniak, Bogdan; Dinney, Colin; Barton, Michelle; McConkey, David J.

    2013-01-01

    Epithelial-mesenchymal transition (EMT) is a physiological process that plays important roles in tumor metastasis, “stemness,” and drug resistance. EMT is typically characterized by the loss of the epithelial marker E-cadherin and increased expression of EMT-associated transcriptional repressors, including ZEB1 and ZEB2. The miR-200 family and miR-205 prevent EMT through suppression of ZEB1/2. p53 has been implicated in the regulation of miR-200c, but the mechanisms controlling miR-205 expression remain elusive. Here we report that the p53 family member and p63 isoform, ΔNp63α, promotes miR-205 transcription and controls EMT in human bladder cancer cells. ΔNp63α, E-cadherin and miR-205 were coexpressed in a panel of bladder cancer cell lines (n = 28) and a cohort of primary bladder tumors (n = 98). Stable knockdown of ΔNp63α in the “epithelial” bladder cancer cell line UM-UC6 decreased the expression of miR-205 and induced the expression of ZEB1/2, effects that were reversed by expression of exogenous miR-205. Conversely, overexpression of ΔNp63α in the “mesenchymal” bladder cancer cell line UM-UC3 induced miR-205 and suppressed ZEB1/2. ΔNp63α knockdown reduced the expression of the primary and mature forms of miR-205 and the miR-205 “host” gene (miR-205HG) and decreased binding of RNA Pol II to the miR-205HG promoter, inhibiting miR-205HG transcription. Finally, high miR-205 expression was associated with adverse clinical outcomes in bladder cancer patients. Together, our data demonstrate that ΔNp63α-mediated expression of miR-205 contributes to the regulation of EMT in bladder cancer cells and identify miR-205 as a molecular marker of the lethal subset of human bladder cancers. PMID:23239884

  14. SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes.

    PubMed

    Herbert, Katharine J; Cook, Anthony L; Snow, Elizabeth T

    2014-06-15

    Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment.

  15. Human T-cell leukemia virus I tax protein sensitizes p53-mutant cells to DNA damage.

    PubMed

    Mihaylova, Valia T; Green, Allison M; Khurgel, Moshe; Semmes, Oliver J; Kupfer, Gary M

    2008-06-15

    Mutations in p53 are a common cause of resistance of cancers to standard chemotherapy and, thus, treatment failure. Reports have shown that Tax, a human T-cell leukemia virus type I encoded protein that has been associated with genomic instability and perturbation of transcription and cell cycle, sensitizes HeLa cells to UV treatment. The extent to which Tax can sensitize cells and the mechanism by which it exerts its effect are unknown. In this study, we show that Tax sensitizes p53-mutant cells to a broad range of DNA-damaging agents, including mitomycin C, a bifunctional alkylator, etoposide, a topoisomerase II drug, and UV light, but not ionizing radiation, a double-strand break agent, or vinblastine, a tubulin poison. Tax caused hypersensitivity in all p53-deleted cell lines and several, but not all, mutant-expressed p53-containing cell lines, while unexpectedly being protective in p53 wild-type (wt) cells. The effect observed in p53-deleted lines could be reversed for this by transfection of wt p53. We also show that Tax activates a p53-independent proapoptotic program through decreased expression of the retinoblastoma protein and subsequent increased E2F1 expression. The expression of several proapoptotic proteins was also induced by Tax, including Puma and Noxa, culminating in a substantial increase in Bax dimerization. Our results show that Tax can sensitize p53-mutant cells to DNA damage while protecting p53 wt cells, a side benefit that might result in reduced toxicity in normal cells. Such studies hold the promise of a novel adjunctive therapy that could make cancer chemotherapy more effective.

  16. Chemotherapy-induced Dkk-1 expression by primary human mesenchymal stem cells is p53 dependent.

    PubMed

    Hare, Ian; Evans, Rebecca; Fortney, James; Moses, Blake; Piktel, Debbie; Slone, William; Gibson, Laura F

    2016-10-01

    Mesenchymal stem cells (MSCs) are abundant throughout the body and regulate signaling within tumor microenvironments. Wnt signaling is an extrinsically regulated pathway that has been shown to regulate tumorigenesis in many types of cancer. After evaluating a panel of Wnt activating and inhibiting molecules, we show that primary human MSCs increase the expression of Dkk-1, an inhibitor of Wnt signaling, into the extracellular environment following chemotherapy exposure in a p53-dependent manner. Dkk-1 has been shown to promote tumor growth in several models of malignancy, suggesting that MSC-derived Dkk-1 could counteract the intent of cytotoxic chemotherapy, and that pharmacologic inhibition of Dkk-1 in patients receiving chemotherapy treatment for certain malignancies may be warranted.

  17. Wild-type p53-mediated down-modulation of interleukin 15 and interleukin 15 receptors in human rhabdomyosarcoma cells.

    PubMed Central

    De Giovanni, C.; Nanni, P.; Sacchi, A.; Soddu, S.; Manni, I.; D'Orazi, G.; Bulfone-Paus, S.; Pohl, T.; Landuzzi, L.; Nicoletti, G.; Frabetti, F.; Rossi, I.; Lollini, P. L.

    1998-01-01

    We recently reported that rhabdomyosarcoma cell lines express and secrete interleukin 15 (IL-15), a tightly regulated cytokine with IL-2-like activity. To test whether the p53-impaired function that is frequently found in this tumour type could play a role in the IL-15 production, wild-type p53 gene was transduced in the human rhabdomyosarcoma cell line RD (which harbours a mutated p53 gene), and its effect on proliferation and expression of IL-15 was studied. Arrest of proliferation was induced by wild-type p53; increased proportions of G1-arrested cells and of apoptotic cells were observed. A marked down-modulation of IL-15 expression, at both the mRNA and protein level, was found in p53-transduced cells. Because a direct effect of IL-15 on normal muscle cells has been reported, the presence of IL-15 membrane receptors was studied by cytofluorometric analysis. Rhabdomyosarcoma cells showed IL-15 membrane receptors, which are down-modulated by wild-type p53 transfected gene. In conclusion, wild-type p53 transduction in human rhabdomyosarcoma cells induces the down-modulation of both IL-15 production and IL-15 receptor expression. Images Figure 3 PMID:9862562

  18. Tetraploidization or autophagy: The ultimate fate of senescent human endometrial stem cells under ATM or p53 inhibition.

    PubMed

    Borodkina, Aleksandra V; Shatrova, Alla N; Deryabin, Pavel I; Grukova, Anastasiya A; Nikolsky, Nikolay N; Burova, Elena B

    2016-01-01

    Previously we demonstrated that endometrium-derived human mesenchymal stem cells (hMESCs) via activation of the ATM/p53/p21/Rb pathway enter the premature senescence in response to oxidative stress. Down regulation effects of the key components of this signaling pathway, particularly ATM and p53, on a fate of stressed hMESCs have not yet been investigated. In the present study by using the specific inhibitors Ku55933 and Pifithrin-α, we confirmed implication of both ATM and p53 in H(2)O(2)-induced senescence of hMESCs. ATM or p53 down regulation was shown to modulate differently the cellular fate of H(2)O(2)-treated hMESCs. ATM inhibition allowed H(2)O(2)-stimulated hMESCs to escape the permanent cell cycle arrest due to loss of the functional ATM/p53/p21/Rb pathway, and induced bypass of mitosis and re-entry into S phase, resulting in tetraploid cells. On the contrary, suppression of the p53 transcriptional activity caused a pronounced cell death of H(2)O(2)-treated hMESCs via autophagy induction. The obtained data clearly demonstrate that down regulation of ATM or p53 shifts senescence of human endometrial stem cells toward tetraploidization or autophagy.

  19. Antitumor effects of recombinant human adenovirus-p53 against human cutaneous squamous cell carcinoma in mice

    PubMed Central

    Li, Yuanchao; He, Wei; Wang, Rupeng; Yang, Libin; Zhou, Chunli; Zhang, Bin

    2016-01-01

    The present study was conducted to identify the anti-tumor effects of rAd/p53, which is a recombinant human serotype 5 adenovirus, in cutaneous squamous cell carcinoma (cSCC). Mouse models of human cSCC were constructed by injecting human cutaneous squamous cell carcinoma cells into both flanks of nude mice. Subsequently, the 75 nude mice with cSCC xenograft tumors were randomly divided into recombinant human serotype 5 adenovirus (rAd)/p53, rAd/p53 + 5-fluorouracil (5-Fu) and 5-Fu groups. One side of the tumors was administered the therapeutic agents as the therapeutic group, whereas the remaining side was treated with medical saline as the control. At 24, 48, 72, 120 and 168 h post-intratumoral injection, alterations in tumor volume, tumor necrosis and the expression of several tumor-associated genes, including Smad4, Brca1 and matrix metalloproteinase (MMP-2), were analyzed. Compared with its control group, the rAd/P53 group exhibited a significantly increased tumor necrosis ratio. In addition, Smad4 and Brca1 expression levels increased significantly at various time points (P<0.05), and MMP-2 expression decreased significantly (P<0.05). In the rAd/p53 + 5-Fu group, the tumor necrosis ratio, and Smad4 and Brca1 expression levels also significantly increased at various time points (P<0.05). MMP-2 gene transcription gradually decreased, high expression of Smad4 was prolonged, and high expression of Brca1 was observed in the early period following treatment compared with the rAd/P53 group. In addition, p53 expression exhibited a positive correlation with the tumor necrosis ratio and Smad4 expression, and showed a negative correlation with MMP-2 gene transcription (P<0.05). These findings indicate that rAd/p53 has a potent anti-tumor effect in cSCC via the promotion of tumor necrosis and regulating the expression of various tumor-associated genes. PMID:28105142

  20. DN-R175H p53 mutation is more effective than p53 interference in inducing epithelial disorganization and activation of proliferation signals in human carcinoma cells: role of E-cadherin.

    PubMed

    Rieber, Manuel; Strasberg Rieber, Mary

    2009-10-01

    One of the hallmarks of carcinomas is epithelial disorganization, linked to overexpression of matrix metalloproteases (MMP) like MMP-9, loss of intercellular E-cadherin and activation of epidermal growth receptor (EGFR/erbB1). Since the p53 tumor suppressor pathway is inactivated in most human cancers due to gene mutations or defective wt p53 signaling, we now investigated in human wt p53 breast carcinoma MCF-7 cells, whether single treatment with the p53 transactivation pharmacological inhibitor pifithrin-alpha, transient p53 siRNA interference or stable insertion of a dominant-negative (DN) R175H p53 mutant increase: (i) EGFR/erbB1 activation, (ii) MMP-9 expression and (iii) loss of surface E-cadherin. Transient abrogation of wt p53 function increased phosphorylation of EGFR/erbB1 and MMP-9 expression. However, all these effects were much more pronounced in cells stably transduced with the dominant negative-Arg-175His mutant p53 (DN-R175H mutant p53), which also showed loss of epithelial cytoarchitecture and extensive E-cadherin downregulation. Collectively, these results support the notion that the DN-R175H mutant p53 exerts a gain of oncogenic function by promoting disruption of E-cadherin intercellular contacts and activation of proliferation signals. Our data suggests that epithelial shape and growth control are unequally affected depending on how wt p53 function is impaired and whether partial or full tumor suppressor activity is lost.

  1. rAd-p53 enhances the sensitivity of human gastric cancer cells to chemotherapy

    PubMed Central

    Chen, Guang-Xia; Zheng, Li-Hong; Liu, Shi-Yu; He, Xiao-Hua

    2011-01-01

    AIM: To investigate potential antitumor effects of rAd-p53 by determining if it enhanced sensitivity of gastric cancer cells to chemotherapy. METHODS: Three gastric cancer cell lines with distinct levels of differentiation were treated with various doses of rAd-p53 alone, oxaliplatin (OXA) alone, or a combination of both. Cell growth was assessed with an 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide assay and the expression levels of p53, Bax and Bcl-2 were determined by immunohistochemistry. The presence of apoptosis and the expression of caspase-3 were determined using flow cytometry. RESULTS: Treatment with rAd-p53 or OXA alone inhibited gastric cancer cell growth in a time- and dose-dependent manner; moreover, significant synergistic effects were observed when these treatments were combined. Immunohistochemical analysis demonstrated that treatment with rAd-p53 alone, OXA alone or combined treatment led to decreased Bcl-2 expression and increased Bax expression in gastric cancer cells. Furthermore, flow cytometry showed that rAd-p53 alone, OXA alone or combination treatment induced apoptosis of gastric cancer cells, which was accompanied by increased expression of caspase-3. CONCLUSION: rAd-p53 enhances the sensitivity of gastric cancer cells to chemotherapy by promoting apoptosis. Thus, our results suggest that p53 gene therapy combined with chemotherapy represents a novel avenue for gastric cancer treatment. PMID:22090785

  2. Persistent p53 Mutations in Single Cells from Normal Human Skin

    PubMed Central

    Ling, Gao; Persson, Åsa; Berne, Berit; Uhlén, Mathias; Lundeberg, Joakim; Ponten, Fredrik

    2001-01-01

    Epidermal clones of p53-mutated keratinocytes are abundant in chronically sun-exposed skin and may play an important role in early development of skin cancer. Advanced laser capture microdissection enables genetic analysis of targeted cells from tissue sections without contamination from neighboring cells. In this study p53 gene mutations were characterized in single cells from normal, chronically sun-exposed skin. Biopsies were obtained from skin subjected to daily summer sun and skin totally protected from the sun by blue denim fabric. Using laser capture microdissection, 172 single-cell samples were retrieved from four biopsies and analyzed using single-cell polymerase chain reaction and direct DNA sequencing. A total of 14 different mutations were identified in 26 of 99 keratinocytes from which the p53 gene could be amplified. Mutations displayed a typical UV signature and were detected in both scattered keratinocytes and in a small cluster of p53-immunoreactive keratinocytes. This minute epidermal p53 clone had a diameter of 10 to 15 basal cells. Two missense mutations were found in all layers of epidermis within the p53 clone. The presented data show that p53 mutations are common in normal skin and that a clone of keratinocytes with a mutated p53 gene prevailed despite 2 months of total protection from ultraviolet light. PMID:11583952

  3. Apoptosis induced by selenomethionine and methioninase is superoxide-mediated and p53-dependent in human prostate cancer cells

    PubMed Central

    Zhao, Rui; Domann, Frederick E.

    2006-01-01

    Selenomethionine (SeMet) is the chemical form or major component of selenium used for cancer chemoprevention in several clinical trials. However, evidence from experimental studies indicates that SeMet has weaker anticancer effects than most other forms of selenium. Recent studies showed that the anticancer activity of SeMet can be enhanced by methioninase (METase), indicating that SeMet metabolites are responsible for its anticancer activity. In the present study, we demonstrated that wild-type p53-expressing LNCaP human prostate cancer cells were more sensitive to co-treatment with SeMet and METase than p53-null PC3 human prostate cancer cells. SeMet and METase co-treatment significantly increased levels of superoxide and apoptosis in LNCaP cells. Co-treatment with SeMet and METase resulted in increased levels of phosphorylated p53 (serine15), total p53, Bax, and p21Waf1 proteins. LNCaP cells treated with SeMet and METase also showed p53 translocation to mitochondria, decreased mitochondrial membrane potential, cytochrome c release into the cytosol, and activation of caspase 9. The effects of SeMet and METase were suppressed by pre-treatment with a synthetic superoxide dismutase mimic or by knockdown of p53 via RNA interference. Reexpression of wild-type p53 in PC3 cells resulted in increases in superoxide production, apoptosis, and caspase 9 activity, and a decrease in mitochondrial membrane potential following co-treatment with SeMet and METase. Our study demonstrates that apoptosis induced by SeMet plus METase is superoxide-mediated and p53-dependent via mitochondrial pathway(s). These results suggest that superoxide and p53 may play a role in cancer chemoprevention by selenium. PMID:17172431

  4. p53 Degradation Activity, Expression, and Subcellular Localization of E6 Proteins from 29 Human Papillomavirus Genotypes

    PubMed Central

    Mesplède, Thibault; Gagnon, David; Bergeron-Labrecque, Fanny; Azar, Ibrahim; Sénéchal, Hélène; Coutlée, François

    2012-01-01

    Human papillomaviruses (HPVs) are the etiological agents of cervical cancer and other human malignancies. HPVs are classified into high- and low-risk genotypes according to their association with cancer. Host cell transformation by high-risk HPVs relies in part on the ability of the viral E6 protein to induce the degradation of p53. We report the development of a cellular assay that accurately quantifies the p53 degradation activity of E6 in vivo, based on the fusion of p53 to Renilla luciferase (RLuc-p53). This assay was used to measure the p53 degradation activities of E6 proteins from 29 prevalent HPV types and variants of HPV type 16 (HPV16) and HPV33 by determining the amount of E6 expression vector required to reduce by half the levels of RLuc-p53 (50% effective concentration [EC50]). These studies revealed an unexpected variability in the p53 degradation activities of different E6 proteins, even among active types whose EC50s span more than 2 log units. Differences in activity were greater between types than between variants and did not correlate with differences in the intracellular localization of E6, with most being predominantly nuclear. Protein and mRNA expression of the 29 E6 proteins was also examined. For 16 high-risk types, spliced transcripts that encode shorter E6*I proteins of variable sizes and abundances were detected. Mutation of the splice donor site in five different E6 proteins increased their p53 degradation activity, suggesting that mRNA splicing can limit the activity of some high-risk E6 types. The quantification of p53 degradation in vivo represents a novel tool to systematically compare the oncogenic potentials of E6 proteins from different HPV types and variants. PMID:22013048

  5. SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

    SciTech Connect

    Herbert, Katharine J.; Cook, Anthony L. Snow, Elizabeth T.

    2014-06-15

    Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment. - Highlights: • Impaired microRNA biogenesis promotes apoptotic resistance in HaCaT keratinocytes. • TP53 mutations suppress miR-34a-mediated regulation of SIRT1 expression. • SIRT1 inhibition increases p53 acetylation in HaCaTs, restoring apoptosis.

  6. Species-specific mutual regulation of p53 and miR-138 between human, rat and mouse

    PubMed Central

    Li, Jie; Xia, Wei; Su, Xueting; Qin, Xingliang; Chen, Ying; Li, Shaohua; Dong, Jie; Ding, Hongmei; Li, Hui; Huang, Aixue; Ge, Xingfeng; Hou, Lvbin; Wang, Chaonan; Sun, Leqiao; Bai, Chenjun; Shen, Xuelian; Fang, Tao; Liu, Yuanlin; Zhang, Yi; Zhang, Hongru; Zhang, Hongwen; Shao, Ningsheng

    2016-01-01

    In recent years, p53 was identified to regulate the expression of many miRNAs and was also regulated by miRNAs. In this paper, we found that miR-138 showed a pronounced increase after p53 activation in human non-small cell lung cancer (NSCLC) cells, which is mediated by p53 binding sites in the promoter region of its host gene, but this did not happen with rat and mouse cells. More interestingly, we found that p53 could be also regulated by miR-138 in mouse and rat cells, but not in the human NSCLC cells. Our results suggest the existence of species-specific differences of the regulations of miRNA against its targets and the regulations of miRNA itself by other proteins. PMID:27183959

  7. Imiquimod activates p53-dependent apoptosis in a human basal cell carcinoma cell line.

    PubMed

    Huang, Shi-Wei; Chang, Shu-Hao; Mu, Szu-Wei; Jiang, Hsin-Yi; Wang, Sin-Ting; Kao, Jun-Kai; Huang, Jau-Ling; Wu, Chun-Ying; Chen, Yi-Ju; Shieh, Jeng-Jer

    2016-03-01

    The tumor suppressor p53 controls DNA repair, cell cycle, apoptosis, autophagy and numerous other cellular processes. Imiquimod (IMQ), a synthetic toll-like receptor (TLR) 7 ligand for the treatment of superficial basal cell carcinoma (BCC), eliminates cancer cells by activating cell-mediated immunity and directly inducing apoptosis and autophagy in cancer cells. To evaluate the role of p53 in IMQ-induced cell death in skin cancer cells. The expression, phosphorylation and subcellular localization of p53 were detected by real-time PCR, luciferase reporter assay, cycloheximide chase analysis, immunoblotting and immunocytochemistry. Using BCC/KMC1 cell line as a model, the upstream signaling of p53 activation was dissected by over-expression of TLR7/8, the addition of ROS scavenger, ATM/ATR inhibitors and pan-caspase inhibitor. The role of p53 in IMQ-induced apoptosis and autophagy was assessed by genetically silencing p53 and evaluated by a DNA content assay, immunoblotting, LC3 puncta detection and acridine orange staining. IMQ induced p53 mRNA expression and protein accumulation, increased Ser15 phosphorylation, promoted nuclear translocation and up-regulated its target genes in skin cancer cells in a TLR7/8-independent manner. In BCC/KMC1 cells, the induction of p53 by IMQ was achieved through increased ROS production to stimulate the ATM/ATR-Chk1/Chk2 axis but was not mediated by inducing DNA damage. The pharmacological inhibition of ATM/ATR significantly suppressed IMQ-induced p53 activation and apoptosis. Silencing of p53 significantly decreased the IMQ-induced caspase cascade activation and apoptosis but enhanced autophagy. Mutant p53 skin cancer cell lines were more resistant to IMQ-induced apoptosis than wildtype p53 skin cancer cell lines. IMQ induced ROS production to stimulate ATM/ATR pathways and contributed to p53-dependent apoptosis in a skin basal cell carcinoma cell line BCC/KMC1. Copyright © 2015 Japanese Society for Investigative Dermatology

  8. Transcriptional regulation of the tumor suppressor FHL2 by p53 in human kidney and liver cells.

    PubMed

    Xu, Jiaying; Zhou, Junwei; Li, Man-Shan; Ng, Chor-Fung; Ng, Yuen-Keng; Lai, Paul Bo-San; Tsui, Stephen Kwok-Wing

    2014-01-01

    Four and a Half LIM protein 2 (FHL2) is a LIM domain only protein that is able to form various protein complexes and regulate gene transcription. Recent findings showed that FHL2 is a potential tumor suppressor gene that was down-regulated in hepatocellular carcinoma (HCC). Moreover, FHL2 can bind to and activate the TP53 promoter in hepatic cells. In this study, the activity of the two promoters of FHL2, 1a and 1b, were determined in the human embryonic kidney cell line HEK293 and the activation of these two promoters by p53 was investigated. Our results showed that the 1b promoter has a higher activity than the 1a promoter in HEK 293 cells but the 1a promoter is more responsive to the activation by p53 when compared with the 1b promoter. The regulation of FHL2 by p53 was further confirmed in liver cells by the overexpression of p53 in Hep3B cells and the knockdown of p53 in HepG2 cells. Combining promoter activity results of truncated mutants and predictions by bioinformatics tools, a putative p53 binding site was found in the exon 1a of FHL2 from +213 to +232. The binding between the p53 protein and the putative p53 binding site was then validated by the ChIP assay. Furthermore, the expression of FHL2 and TP53 were down-regulated in majority of HCC tumour samples (n = 41) and significantly correlated (P = 0.026). Finally, we found that the somatic mutation 747 (G→T), a hot spot mutation of the TP53 gene, is potentially associated with a higher expression of FHL2 in HCC tumour samples. Taken together, this is the first in-depth study about the transcriptional regulation of FHL2 by p53.

  9. Novel small molecule induces p53-dependent apoptosis in human colon cancer cells

    SciTech Connect

    Park, Sang Eun; Min, Yong Ki; Ha, Jae Du; Kim, Bum Tae; Lee, Woo Ghil . E-mail: bigguy@krict.re.kr

    2007-07-06

    Using high-throughput screening with small-molecule libraries, we identified a compound, KCG165 [(2-(3-(2-(pyrrolidin-1-yl)ethoxy)-1,10b-dihydro-[1,2,4]triazolo[1,5-c] quinazolin-5(6H)-one)], which strongly activated p53-mediated transcriptional activity. KCG165-induced phosphorylations of p53 at Ser{sup 6}, Ser{sup 15}, and Ser{sup 20}, which are all key residues involved in the activation and stabilization of p53. Consistent with these findings, KCG165 increased level of p53 protein and led to the accumulation of transcriptionally active p53 in the nucleus with the increased occupancy of p53 in the endogenous promoter region of its downstream target gene, p21{sup WAF1/CIP}. Notably, KCG165-induced p53-dependent apoptosis in cancer cells. Furthermore, we suggested topoisomerase II as the molecular target of KCG165. Together, these results indicate that KCG165 may have potential applications as an antitumor agent.

  10. A regulatory feedback loop involving p63 and IRF6 links the pathogenesis of 2 genetically different human ectodermal dysplasias.

    PubMed

    Moretti, Francesca; Marinari, Barbara; Lo Iacono, Nadia; Botti, Elisabetta; Giunta, Alessandro; Spallone, Giulia; Garaffo, Giulia; Vernersson-Lindahl, Emma; Merlo, Giorgio; Mills, Alea A; Ballarò, Costanza; Alemà, Stefano; Chimenti, Sergio; Guerrini, Luisa; Costanzo, Antonio

    2010-05-01

    The human congenital syndromes ectrodactyly ectodermal dysplasia-cleft lip/palate syndrome, ankyloblepharon ectodermal dysplasia clefting, and split-hand/foot malformation are all characterized by ectodermal dysplasia, limb malformations, and cleft lip/palate. These phenotypic features are a result of an imbalance between the proliferation and differentiation of precursor cells during development of ectoderm-derived structures. Mutations in the p63 and interferon regulatory factor 6 (IRF6) genes have been found in human patients with these syndromes, consistent with phenotypes. Here, we used human and mouse primary keratinocytes and mouse models to investigate the role of p63 and IRF6 in proliferation and differentiation. We report that the DeltaNp63 isoform of p63 activated transcription of IRF6, and this, in turn, induced proteasome-mediated DeltaNp63 degradation. This feedback regulatory loop allowed keratinocytes to exit the cell cycle, thereby limiting their ability to proliferate. Importantly, mutations in either p63 or IRF6 resulted in disruption of this regulatory loop: p63 mutations causing ectodermal dysplasias were unable to activate IRF6 transcription, and mice with mutated or null p63 showed reduced Irf6 expression in their palate and ectoderm. These results identify what we believe to be a novel mechanism that regulates the proliferation-differentiation balance of keratinocytes essential for palate fusion and skin differentiation and links the pathogenesis of 2 genetically different groups of ectodermal dysplasia syndromes into a common molecular pathway.

  11. Evidence for activation of mutated p53 by apigenin in human pancreatic cancer

    PubMed Central

    King, Jonathan C; Lu, Qing-Yi; Li, Gang; Moro, Aune; Takahashi, Hiroki; Chen, Monica; Go, Vay Liang W; Reber, Howard A; Eibl, Guido; Hines, O. Joe

    2012-01-01

    Pancreatic cancer is an exceedingly lethal disease with a five-year survival that ranks among the lowest of gastrointestinal malignancies. Part of its lethality is attributable to a generally poor response to existing chemotherapeutic regimens. New therapeutic approaches are urgently needed. We aimed to elucidate the anti-neoplastic mechanisms of apigenin-an abundant, naturally-occurring plant flavonoid-with a particular focus on p53 function. Pancreatic cancer cells (BxPC-3, MiaPaCa-2) experienced dose and time-dependent growth inhibition and increased apoptosis with apigenin treatment. p53 post-translational modification, nuclear translocation, DNA binding, and upregulation of p21 and PUMA were all enhanced by apigenin treatment despite mutated p53 in both cell lines. Transcription-dependent p53 activity was reversed by pifithrin-α, a specific DNA binding inhibitor of p53, but not growth inhibition or apoptosis suggesting transcription-independent p53 activity. This was supported by immunoprecipitation assays which demonstrated disassociation of p53/BclXL and PUMA/BclXL and formation of complexes with Bak followed by Cytochrome c release. Treated animals grew smaller tumors with increased cellular apoptosis than those fed control diet. These results suggest that despite deactivating mutation, p53 retains some of its function which is augmented following treatment with apigenin. Cell cycle arrest and apoptosis induction may be mediated by transcription-independent p53 function via interactions with BclXL and PUMA. Further study of flavonoids as chemotherapeutics is warranted PMID:22227579

  12. Efficacy of recombinant adenoviral human p53 gene in the treatment of lung cancer-mediated pleural effusion

    PubMed Central

    LI, KUN-LIN; KANG, JUN; ZHANG, PENG; LI, LI; WANG, YU-BO; CHEN, HENG-YI; HE, YONG

    2015-01-01

    Pleural effusion induced by lung cancer exerts a negative impact on quality of life and prognosis. The aim of the present study was to evaluate the value of the recombinant adenoviral human p53 gene (rAd-p53) in the local treatment of lung cancer and its synergistic effect with chemotherapy. The present study retrospectively recruited 210 patients with lung cancer-mediated pleural effusion who had adopted a treatment strategy of platinum chemotherapy. Pleurodesis was performed via the injection of cisplatin or rAd-p53. Long-term follow-up was conducted to investigate the therapeutic effects of cisplatin and rAd-p53 administration on pleural effusion and other relevant clinical indicators. The short-term effect of pleurodesis was as follows: The efficacy rate of rAd-p53 therapy was significantly higher compared with cisplatin therapy (71.26 vs. 54.47%), and the efficacy of treatment with ≥2×1012 viral particles of rAd-p53 for pleurodesis was significantly greater than treatment with 40 mg cisplatin (P<0.05). Furthermore, efficacy analysis performed 6 and 12 months after pleurodesis indicated that the efficacy rate of rAd-p53 was significantly greater than that of cisplatin (P<0.05). A comparison of median progression-free survival (PFS) time identified a significant difference (P<0.05) between rAd-p53 and cisplatin therapy (3.3 vs. 2.7 months); however, a comparison of median overall survival time identified no significant difference (P>0.05) between rAd-p53 and cisplatin therapy (9.6 vs. 8.7 months). In addition, Cox regression analysis indicated that PFS was not affected by clinical indicators such as age, gender, prognostic staging and smoking status; however, PFS was affected by pathological subtype (adenocarcinoma or squamous carcinoma) in the rAd-p53 group. rAd-p53 administration for pleurodesis exerts long-term therapeutic effects on the local treatment of lung cancer. Thus, a combination of rAd-p53 and chemotherapy may exert a synergistic effect and

  13. Involvement of RB-1, p53, p16INK4 and telomerase in immortalisation of human cells.

    PubMed

    Whitaker, N J; Bryan, T M; Bonnefin, P; Chang, A C; Musgrove, E A; Braithwaite, A W; Reddel, R R

    1995-09-07

    Involvement of the retinoblastoma susceptibility (RB-1), p16INK4, p53 and telomerase genes in immortalisation was examined by determining their status in 15 human cell lines representing four immortalisation complementation groups. No abnormalities of RB-1, p53 and p16INK4 were detected in cell lines containing DNA tumour virus proteins known to bind to the protein products of the RB-1 and p53 genes. In contrast, in all other cell lines from each of the four groups either RB-1 was mutant or p16INK4 protein was undetectable and there were cell lines containing p53 mutations in three of the groups. Telomerase activity was detected in 12/15 lines, including some of the virally immortalised lines and in some lines from each group. Since none of these changes correlated with complementation group, other genetic changes must be required for immortalisation.

  14. Endogenous Human MDM2-C Is Highly Expressed in Human Cancers and Functions as a p53-Independent Growth Activator

    PubMed Central

    Okoro, Danielle R.; Arva, Nicoleta; Gao, Chong; Polotskaia, Alla; Puente, Cindy; Rosso, Melissa; Bargonetti, Jill

    2013-01-01

    Human cancers over-expressing mdm2, through a T to G variation at a single nucleotide polymorphism at position 309 (mdm2 SNP309), have functionally inactivated p53 that is not effectively degraded. They also have high expression of the alternatively spliced transcript, mdm2-C. Alternatively spliced mdm2 transcripts are expressed in many forms of human cancer and when they are exogenously expressed they transform human cells. However no study to date has detected endogenous MDM2 protein isoforms. Studies with exogenous expression of splice variants have been carried out with mdm2-A and mdm2-B, but the mdm2-C isoform has remained virtually unexplored. We addressed the cellular influence of exogenously expressed MDM2-C, and asked if endogenous MDM2-C protein was present in human cancers. To detect endogenous MDM2-C protein, we created a human MDM2-C antibody to the splice junction epitope of exons four and ten (MDM2 C410) and validated the antibody with in vitro translated full length MDM2 compared to MDM2-C. Interestingly, we discovered that MDM2-C co-migrates with MDM2-FL at approximately 98 kDa. Using the validated C410 antibody, we detected high expression of endogenous MDM2-C in human cancer cell lines and human cancer tissues. In the estrogen receptor positive (ER+) mdm2 G/G SNP309 breast cancer cell line, T47D, we observed an increase in endogenous MDM2-C protein with estrogen treatment. MDM2-C localized to the nucleus and the cytoplasm. We examined the biological activity of MDM2-C by exogenously expressing the protein and observed that MDM2-C did not efficiently target p53 for degradation or reduce p53 transcriptional activity. Exogenous expression of MDM2-C in p53-null human cancer cells increased colony formation, indicating p53-independent tumorigenic properties. Our data indicate a role for MDM2-C that does not require the inhibition of p53 for increasing cancer cell proliferation and survival. PMID:24147044

  15. Endogenous human MDM2-C is highly expressed in human cancers and functions as a p53-independent growth activator.

    PubMed

    Okoro, Danielle R; Arva, Nicoleta; Gao, Chong; Polotskaia, Alla; Puente, Cindy; Rosso, Melissa; Bargonetti, Jill

    2013-01-01

    Human cancers over-expressing mdm2, through a T to G variation at a single nucleotide polymorphism at position 309 (mdm2 SNP309), have functionally inactivated p53 that is not effectively degraded. They also have high expression of the alternatively spliced transcript, mdm2-C. Alternatively spliced mdm2 transcripts are expressed in many forms of human cancer and when they are exogenously expressed they transform human cells. However no study to date has detected endogenous MDM2 protein isoforms. Studies with exogenous expression of splice variants have been carried out with mdm2-A and mdm2-B, but the mdm2-C isoform has remained virtually unexplored. We addressed the cellular influence of exogenously expressed MDM2-C, and asked if endogenous MDM2-C protein was present in human cancers. To detect endogenous MDM2-C protein, we created a human MDM2-C antibody to the splice junction epitope of exons four and ten (MDM2 C410) and validated the antibody with in vitro translated full length MDM2 compared to MDM2-C. Interestingly, we discovered that MDM2-C co-migrates with MDM2-FL at approximately 98 kDa. Using the validated C410 antibody, we detected high expression of endogenous MDM2-C in human cancer cell lines and human cancer tissues. In the estrogen receptor positive (ER+) mdm2 G/G SNP309 breast cancer cell line, T47D, we observed an increase in endogenous MDM2-C protein with estrogen treatment. MDM2-C localized to the nucleus and the cytoplasm. We examined the biological activity of MDM2-C by exogenously expressing the protein and observed that MDM2-C did not efficiently target p53 for degradation or reduce p53 transcriptional activity. Exogenous expression of MDM2-C in p53-null human cancer cells increased colony formation, indicating p53-independent tumorigenic properties. Our data indicate a role for MDM2-C that does not require the inhibition of p53 for increasing cancer cell proliferation and survival.

  16. Distinctive patterns of p53 protein expression and microsatellite instability in human colorectal cancer.

    PubMed

    Nyiraneza, Christine; Jouret-Mourin, Anne; Kartheuser, Alex; Camby, Philippe; Plomteux, Olivier; Detry, Roger; Dahan, Karin; Sempoux, Christine

    2011-12-01

    Although evidence suggests an inverse relationship between microsatellite instability and p53 alterations in colorectal cancer, no study has thoroughly examined the use of p53 immunohistochemistry in phenotyping colorectal cancers. We investigated the value of p53 immunohistochemistry in microsatellite instability-positive colorectal cancers prescreening and attempted to clarify the relationship between DNA mismatch repair system and p53 pathway. In a series of 104 consecutive colorectal cancers, we performed p53 immunohistochemistry, TP53 mutational analysis, DNA mismatch repair system efficiency evaluation (DNA mismatch repair system immunohistochemistry, microsatellite instability status, MLH1/MSH2 germ line, and BRAF, murine double minute 2, and p21 immunohistochemistry. Microsatellite instability high was observed in 25 of 104 colorectal cancers, with DNA mismatch repair system protein loss (24/25) and germ line (8/25) or BRAF mutations (8/25). p53 immunohistochemistry revealed 3 distinct patterns of expression: complete negative immunostaining associated with truncating TP53 mutations (P < .0001), diffuse overexpression associated with missense TP53 mutations (P < .0001), and restricted overexpression characterized by a limited number of homogenously scattered strongly positive tumor cells in 36.5% of colorectal cancers. This latest pattern was associated with wild-type TP53 and microsatellite instability high colorectal cancers (P < .0001) including all Lynch tumors (8/8), but its presence among 22% of DNA mismatch repair system-competent colorectal cancers decreased its positive predictive value (55.2% [95% confidence interval, 45%-65%]). It was also correlated with murine double minute 2 overexpression (P < .0001) and inversely with p21 loss (P = .0002), independently of microsatellite instability status. In conclusion, a restricted pattern of p53 overexpression is preferentially associated with microsatellite instability high phenotype and could

  17. Clinical utility of recombinant adenoviral human p53 gene therapy: current perspectives

    PubMed Central

    Chen, Guang-xia; Zhang, Shu; He, Xiao-hua; Liu, Shi-yu; Ma, Chao; Zou, Xiao-Ping

    2014-01-01

    Gene therapy has promised to be a highly effective antitumor treatment by introducing a tumor suppressor gene or the abrogation of an oncogene. Among the potential therapeutic transgenes, the tumor suppressor gene p53 serves as an attractive target. Restoration of wild-type p53 function in tumors can be achieved by introduction of an intact complementary deoxyribonucleic acid copy of the p53 gene using a suitable viral vector, in most cases an adenoviral vector (Adp53). Preclinical in vitro and in vivo studies have shown that Adp53 triggers a dramatic tumor regression response in various cancers. These viruses are engineered to lack certain early proteins and are thus replication defective, including Gendicine, SCH-58500, and Advexin. Several types of tumor-specific p53-expressing conditionally replicating adenovirus vectors (known as replication-competent CRAdp53 vectors) have been developed, such as ONYX 015, AdDelta24-p53, SG600-p53, OBP-702, and H101. Various clinical trials have been conducted to investigate the safety and efficiency of these adenoviral vectors. In this review we will talk about the biological mechanisms, clinical utility, and therapeutic potentials of the replication-deficient Adp53-based and replication-competent CRAdp53-based gene therapy. PMID:25364261

  18. Apoptosis in human hepatocellular carcinoma and in liver cell dysplasia is correlated with p53 protein immunoreactivity.

    PubMed Central

    Zhao, M; Zimmermann, A

    1997-01-01

    AIMS: To investigate the prevalence of apoptosis in human hepatocellular carcinomas (HCC) of different types and grades and in liver cell dysplasia, and to test whether the apoptotic rate is correlated with the p53 protein status. METHODS: 37 HCC and 66 six liver samples with liver cell dysplasia were analysed for apoptosis using in situ DNA end labelling (ISEL), and for p53 protein expression by immunohistochemistry. In HCCs, proliferative activity was quantitatively assessed using proliferating cell nuclear antigen labelling. RESULTS: The apoptotic index in HCC as based on ISEL ranged from 0.1 to 13.5 per 1000 cells analysed and was not related to type or grade. No nuclear staining was observed in multinuclear tumour cells. There was a significant correlation between the apoptotic rate and both the proliferative activity and p53 protein reactivity. In liver samples containing p53 protein positive liver cell dysplasia cells, there was a significantly higher apoptotic rate of these cells. CONCLUSIONS: Apoptosis is detectable in HCC, and is not related to type and grade. There is a highly significant positive correlation between the apoptotic rate in HCC and both the proliferative activity and p53 protein expression. A similar phenomenon occurs for putative cancer precursors. The findings support the role of p53 in regulating apoptosis in preneoplastic and neoplastic liver lesions. Images PMID:9215122

  19. p53 associates with and targets ΔNp63 into a protein degradation pathway

    PubMed Central

    Ratovitski, Edward A.; Patturajan, Meera; Hibi, Kenji; Trink, Barry; Yamaguchi, Kengo; Sidransky, David

    2001-01-01

    A human p53 homologue, p63 (p40/p51/p73L/CUSP) that maps to the chromosomal region 3q27–29 was found to produce a variety of transcripts that encode DNA-binding proteins with and without a trans-activation domain (TA- or ΔN-, respectively). The p63 gene locus was found to be amplified in squamous cell carcinoma, and overexpression of ΔNp63 (p40) led to increased growth of transformed cells in vitro and in vivo. Moreover, p63-null mice displayed abnormal epithelial development and germ-line human mutations were found to cause ectodermal dysplasia. We now demonstrate that certain p63 isotypes form complexes with p53. p53 mutations R175H or R248W abolish the association of p53 with p63, whereas V143A or R273H has no effect. Deletion studies suggest that the DNA-binding domains of both p53 and p63 mediate the association. Overexpression of wild type but not mutant (R175H) p53 results in the caspase-dependent degradation of certain ΔNp63 proteins (p40 and ΔNp63α). The association between p53 and ΔNp63 supports a previously unrecognized role for p53 in regulation of ΔNp63 stability. The ability of p53 to mediate ΔNp63 degradation may balance the capacity of ΔNp63 to accelerate tumorigenesis or to induce epithelial proliferation. PMID:11172034

  20. Allicin induces anti-human liver cancer cells through the p53 gene modulating apoptosis and autophagy.

    PubMed

    Chu, Yung-Lin; Ho, Chi-Tang; Chung, Jing-Gung; Raghu, Rajasekaran; Lo, Yi-Chen; Sheen, Lee-Yan

    2013-10-16

    Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer globally and ranks first among the cancer-related mortalities in Taiwan. This study aims to understand the modes of cell death mechanism induced by allicin, a major phytochemical of crushed garlic, in human hepatoma cells. Our earlier study indicated that allicin induced autophagic cell death in human HCC Hep G2 (p53(wild type)) cells, whereas in the present study, allicin induced apoptotic cell death through caspase-dependent and caspase-independent pathways by reactive oxygen species (ROS) overproduction in human HCC Hep 3B (p53(mutation)) cells. To gain insight into the cell death mechanism in p53 knocked down Hep G2, we silenced the p53 gene using siRNA-mediated silencing. Allicin treatment induced apoptotic cell death in p53 knocked down Hep G2 cells similar to that of Hep 3B cells. These results suggest that allicin induced cell death in human hepatoma cells through either autophagy or apoptosis and might be a potential novel complementary gene therapeutic agent for the treatment of apoptosis-resistant cancer cells.

  1. High prevalence of mutations of the p53 gene in poorly differentiated human thyroid carcinomas.

    PubMed Central

    Fagin, J A; Matsuo, K; Karmakar, A; Chen, D L; Tang, S H; Koeffler, H P

    1993-01-01

    The development and progression of thyroid tumors is signaled by phenotype-specific mutations of genes involved in growth control. Molecular events associated with undifferentiated thyroid cancer are not known. We examined normal, benign, and malignant thyroid tissue for structural abnormalities of the p53 tumor suppressor gene. Mutations were detected by single-strand conformation polymorphisms of PCR-amplified DNA, using primers bracketing the known hot spots on either exons 5, 6, 7, or 8. The prevalence of mutations was as follows: normal thyroid 0/6; follicular adenomas 0/31; papillary carcinomas 0/37; medullary carcinomas 0/2; follicular carcinomas 1/11; anaplastic carcinomas 5/6; thyroid carcinoma cell lines 3/4. Positive cases were confirmed by direct sequencing of the PCR products. All five anaplastic carcinoma tissues and the anaplastic carcinoma cell line ARO had G:C to A:T transitions leading to an Arg to His substitution at codon 273. In both tumors and cell lines, examples of heterozygous and homozygous p53 mutations were identified. The only thyroid carcinoma cell line in which p53 mutations were not detected in exons 5-8 had markedly decreased p53 mRNA levels, suggesting the presence of a structural abnormality of either p53 itself or of some factor controlling its expression. The presence of p53 mutations almost exclusively in poorly differentiated thyroid tumors and thyroid cancer cell lines suggests that inactivation of p53 may confer these neoplasms with aggressive properties, and further loss of differentiated function. Images PMID:8423216

  2. Human transcription factors in yeast: the fruitful examples of P53 and NF-кB.

    PubMed

    Sharma, Vasundhara; Monti, Paola; Fronza, Gilberto; Inga, Alberto

    2016-11-01

    The observation that human transcription factors (TFs) can function when expressed in yeast cells has stimulated the development of various functional assays to investigate (i) the role of binding site sequences (herein referred to as response elements, REs) in transactivation specificity, (ii) the impact of polymorphic nucleotide variants on transactivation potential, (iii) the functional consequences of mutations in TFs and (iv) the impact of cofactors or small molecules. These approaches have found applications in basic as well as applied research, including the identification and the characterisation of mutant TF alleles from clinical samples. The ease of genome editing of yeast cells and the availability of regulated systems for ectopic protein expression enabled the development of quantitative reporter systems, integrated at a chosen chromosomal locus in isogenic yeast strains that differ only at the level of a specific RE targeted by a TF or for the expression of distinct TF alleles. In many cases, these assays were proven predictive of results in higher eukaryotes. The potential to work in small volume formats and the availability of yeast strains with modified chemical uptake have enhanced the scalability of these approaches. Next to well-established one-, two-, three-hybrid assays, the functional assays with non-chimeric human TFs enrich the palette of opportunities for functional characterisation. We review ∼25 years of research on human sequence-specific TFs expressed in yeast, with an emphasis on the P53 and NF-кB family of proteins, highlighting outcomes, advantages, challenges and limitations of these heterologous assays.

  3. Pleurotus ostreatus inhibits proliferation of human breast and colon cancer cells through p53-dependent as well as p53-independent pathway

    PubMed Central

    JEDINAK, ANDREJ; SLIVA, DANIEL

    2009-01-01

    In spite of the global consumption of mushrooms, only two epidemiological studies demonstrated an inverse correlation between mushroom intake and the risk of cancer. Therefore, in the present study we evaluated whether extracts from edible mushrooms Agaricus bisporus (portabella), Flammulina velutipes (enoki), Lentinula edodes (shiitake) and Pleurotus ostreatus (oyster) affect the growth of breast and colon cancer cells. Here, we identified as the most potent, P. ostreatus (oyster mushroom) which suppressed proliferation of breast cancer (MCF-7, MDA-MB-231) and colon cancer (HT-29, HCT-116) cells, without affecting proliferation of epithelial mammary MCF-10A and normal colon FHC cells. Flow cytometry revealed that the inhibition of cell proliferation by P. ostreatus was associated with the cell cycle arrest at G0/G1 phase in MCF-7 and HT-29 cells. Moreover, P. ostreatus induced the expression of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21(CIP1/WAF1), whereas inhibited the phosphorylation of retinoblastoma Rb protein in MCF-7 cells. In addition, P. ostreatus also up-regulated expression of p21 and inhibited Rb phosphorylation in HT-29 cells, suggesting that that P. ostreatus suppresses the proliferation of breast and colon cancer cells via p53-dependent as well as p53-independent pathway. In conclusion, our results indicated that the edible oyster mushroom has potential therapeutic/preventive effects on breast and colon cancer. PMID:19020765

  4. Sirt 1 activator inhibits the AGE-induced apoptosis and p53 acetylation in human vascular endothelial cells.

    PubMed

    Li, Peng; Zhang, Lina; Zhou, Changyong; Lin, Nan; Liu, Aiguo

    2015-01-01

    Advanced glycation end products (AGEs) by nonenzymatic glycation reactions are extremely accumulated in the diabetic vascular cells, neurons, and glia, and are confirmed to play important role in the pathogenesis of diabetes mellitus -induced cardiovascular complications. Sirt 1, known as mammalian sirtuin, has been recognized to regulate insulin secretion and protect cells against oxidative stress, which is promoted by the accumulated AGEs in cardiovascular cells. In the present study, we treated human endothelial Eahy926 cells with AGEs, and determined the apoptosis induction, caspase activation, the Sirt 1 activity, the expression and acetylation of p53. Then we manipulated Sirt 1 activity with a Sirt 1 activator, Resveratrol (RSV), and a Sirt 1 inhibitor, sirtinol, in the AGE-BSA-treated Eahy926 cells, and then re-evaluated the apoptosis induction, caspase activation, the expression and acetylation of p53. Results demonstrated that AGEs induced apoptosis in the human endothelial Eahy926 cells, by promoting the cytochrome c release, activation of caspase 9/3. Also, the AGE-BSA treatment promoted the total p53 level and acetylated (Ac) p53, but reduced the Sirt 1 level and activity. On the other hand, the Sirt 1 inhibitor/activator not only deteriorated/ameliorated the promotion to p53 level and Ac p53, but also aggravated/inhibited the AGE-induced apoptosis and the promotion to apoptosis-associated signaling molecules. In conclusion, the present study confirmed the apoptosis promotion by AGEs in endothelial Eahy926 cells, by regulating the Sirt 1 activity and p53 signaling, it also implies the protective role of Sirt 1 activator against the AGE-induced apoptosis.

  5. p63 regulates glutaminase 2 expression

    PubMed Central

    Giacobbe, Arianna; Bongiorno-Borbone, Lucilla; Bernassola, Francesca; Terrinoni, Alessandro; Markert, Elke Katrin; Levine, Arnold J.; Feng, Zhaohui; Agostini, Massimilano; Zolla, Lello; Agrò, Alessandro Finazzi; Notterman, Daniel A.; Melino, Gerry; Peschiaroli, Angelo

    2013-01-01

    The transcription factor p63 is critical for many biological processes, including development and maintenance of epidermal tissues and tumorigenesis. Here, we report that the TAp63 isoforms regulate cell metabolism through the induction of the mitochondrial glutaminase 2 (GLS2) gene both in primary cells and tumor cell lines. By ChIP analysis and luciferase assay, we confirmed that TAp63 binds directly to the p53/p63 consensus DNA binding sequence within the GLS2 promoter region. Given the critical role of p63 in epidermal differentiation, we have investigated the regulation of GLS2 expression during this process. GLS2 and TAp63 expression increases during the in vitro differentiation of primary human keratinocytes, and depletion of GLS2 inhibits skin differentiation both at molecular and cellular levels. We found that GLS2 and TAp63 expression are concomitantly induced in cancer cells exposed to oxidative stresses. siRNA-mediated depletion of GLS2 sensitizes cells to ROS-induced apoptosis, suggesting that the TAp63/GLS2 axis can be functionally important as a cellular antioxidant pathway in the absence of p53. Accordingly, we found that GLS2 is upregulated in colon adenocarcinoma. Altogether, our findings demonstrate that GLS2 is a bona fide TAp63 target gene, and that the TAp63-dependent regulation of GLS2 is important for both physiological and pathological processes. PMID:23574722

  6. Loss of p53 and altered miR15-a/16-1→MCL-1 pathway in CLL: insights from TCL1-Tg:p53−/− mouse model and primary human leukemia cells

    PubMed Central

    Liu, Jinyun; Chen, Gang; Feng, Li; Zhang, Wan; Pelicano, Helene; Wang, Feng; Ogasawara, Marcia A; Lu, Weiqin; Amin, Hesham M.; Croce, Carlo M.; Keating, Michael J.; Huang, Peng

    2013-01-01

    Chronic lymphocytic leukemia (CLL) patients with deletion of chromosome 17p, where the tumor suppressor p53 gene is located, often develop more aggressive disease with poor clinical outcomes. To investigate the underlying mechanisms responsible for the highly malignant phenotype of 17p- CLL and to facilitate the in vivo evaluation of potential drugs against CLL with p53 deletion, we have created a mouse model with TCL1-Tg:p53−/− genotype. The TCL1-Tg:p53−/− mice develop B-cell leukemia at very early age and follow an aggressive path of disease development that resembles human CLL with 17p deletion, with an early appearance of CD5+/IgM+ B cells in the peritoneal cavity, spleen and bone marrow. These TCL1-Tg:p53−/− leukemia cells exhibit higher survival capacity and are more resistant to drug treatment than the leukemia cells from the TCL1-Tg:p53wt (TCL1-Tg) mice. Analysis of microRNA expression reveals that the p53 deletion resulted in a significant decrease of miR-15a and miR-16-1, leading to a substantial elevated expression of Mcl-1. Primary leukemia cells from CLL patients with 17p deletion also show a decrease in miR-15a/miR-16-1 and an increase in Mcl-1 expression. Our study has created a novel CLL mouse model, and suggests that the p53/miR15a/16-Mcl-1 axis may contribute to the aggressive phenotype and drug resistance in CLL cells with loss of p53. PMID:23608884

  7. A universal gene carrier platform for treatment of human prostatic carcinoma by p53 transfection.

    PubMed

    Han, Lu; Zhao, Jing; Liu, Juan; Duan, Xiang-Lin; Li, Lu-Hai; Wei, Xian-Fu; Wei, Yen; Liang, Xing-Jie

    2014-03-01

    Our previous work showed that a charge-reversal layer-by-layer nanosystem, PEI/PAH-Cit/AuNP-CS, effectively facilitates cellular uptake of siRNA and enhances the silencing efficacy of MDR1 siRNA. Here, the plasmid loading capacity of this vehicle was examined using EGFP-N1, and the plasmid release profile was determined in response to pH changes. The cytotoxicity of the EGFP-N1/PEI/PAH-Cit/AuNP-CS complex against HeLa and 293T cells was almost negligible. PEI/PAH-Cit/AuNP-CS efficaciously delivered the plasmids EGFP-N1 (encoding green fluorescent protein) and pGL3.0 (encoding luciferase) into 293T and HeLa cells, thus verifying the universality of PEI/PAH-Cit/AuNP-CS as a gene carrier. The results of an inverted fluorescence microscopy, flow cytometry (FCM) and western blotting methods demonstrated that PC-3 prostate cancer cells treated with EGFP-p53/PEI/PAH-Cit/AuNP-CS expressed higher levels of GFP than cells treated with EGFP-p53/PEI. Furthermore, PC-3 cells treated with EGFP-p53/PEI/PAH-Cit/AuNP-CS showed reduced cellular viability and increased nuclear fragmentation, consistent with elevated p53 expression. Propidium iodide (PI) flow cytometric assays were conducted to demonstrate that EGFP-p53/PEI/PAH-Cit/AuNP-CS elevated the level of apoptosis in PC-3 cells. Western blotting and caspase activation studies revealed that EGFP-p53/PEI/PAH-Cit/AuNP-CS complexes may induce PC-3 apoptosis via the mitochondria-mediated signaling pathway by up-regulation of Bax, down-regulation of Bcl-2, and activation of caspase-3. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. The evaluation of human papillomavirus and p53 gene mutation in benign and malignant conjunctiva and eyelid lesions.

    PubMed

    Joanna, Reszec; Renata, Zalewska; Witold, Pepiński; Małgorzata, Skawronska; Bernaczyk, Piotr; Chyczewski, Lech

    2010-12-01

    Papillomas and squamous cell carcinomas are the most common conjunctival and eyelid lesions. The etiology is still unclear and recently human papillomavirus infection and p53 gene mutation have been taken into consideration. The aim of our study was the evaluation of HPV DNApresence and p53 gene mutation in 45 benign and 38 malignant squamous lesions of the conjunctiva and eyelid. For HPV detection PCR-RFLP and immunohistochemical reaction were used; for p53 gene mutation PCR-SSCP was used. Only 8.8% papillomas, 9.1% squamous cell cancers and 3.7% basal cell cancers (using PCR-RFLP method) and 26.6% papillomas, 7.4% squamous cell cancers and 9.1% basal cell cancers (using immunohisto-chemical reaction) were HPV positive. p53 gene mutation was evaluated in 24.4% papillomas, 54.5% squamous cell cancers and 22.2% basal cell cancers; most commonly in 6 and 7 exon. Human papillomavirus infection, opposite to p53 gene mutation, is not a significant etiological factor of the benign and malignant conjunctival and eyelid lesions development.

  9. A novel charged trinuclear platinum complex effective against cisplatin-resistant tumours: hypersensitivity of p53-mutant human tumour xenografts

    PubMed Central

    Pratesi, G; Perego, P; Polizzi, D; Righetti, S C; Supino, R; Caserini, C; Manzotti, C; Giuliani, F C; Pezzoni, G; Tognella, S; Spinelli, S; Farrell, N; Zunino, F

    1999-01-01

    Multinuclear platinum compounds were rationally designed to bind to DNA in a different manner from that of cisplatin and its mononuclear analogues. A triplatinum compound of the series (BBR 3464) was selected for preclinical development, since, in spite of its charged nature, it was very potent as cytotoxic agent and effective against cisplatin-resistant tumour cells. Anti-tumour efficacy studies were performed in a panel of human tumour xenografts refractory or poorly responsive to cisplatin. The novel platinum compound exhibited efficacy in all tested tumours and an impressive efficacy (including complete tumour regressions) was displayed in two lung carcinoma models, CaLu-3 and POCS. Surprisingly, BBR 3464 showed a superior activity against p53-mutant tumours as compared to those carrying the wild-type gene. The involvement of p53 in tumour response was investigated in an osteosarcoma cell line, SAOS, which is null for p53 and is highly sensitive to BBR 3464, and in the same cells following introduction of the wild-type p53 gene. Thus the pattern of cellular response was investigated in a panel of human tumour cells with a different p53 gene status. The results showed that the transfer of functional p53 resulted in a marked (tenfold) reduction of cellular chemosensitivity to the multinuclear platinum complex but in a moderate sensitization to cisplatin. In addition, in contrast to cisplatin, the triplatinum complex was very effective as an inducer of apoptosis in a lung carcinoma cell line carrying mutant p53. The peculiar pattern of anti-tumour activity of the triplatinum complex and its ability to induce p53-independent cell death may have relevant pharmacological implications, since p53, a critical protein involved in DNA repair and induction of apoptosis by conventional DNA-damaging agents, is defective in several human tumours. We suggest that the peculiar DNA binding properties of the triplatinum complex may contribute to the striking profile of anti

  10. p53 mutations associated with increased sensitivity to ionizing radiation in human head and neck cancer cell lines.

    PubMed

    Servomaa, K; Kiuru, A; Grénman, R; Pekkola-Heino, K; Pulkkinen, J O; Rytömaa, T

    1996-05-01

    The p53 tumour suppressor gene is activated following cellular exposure to DNA-damaging agents. The functions of wild-type p53 protein include transient blocking of cell cycle progression, direct or indirect stimulation of DNA repair machinery and triggering of apoptosis if DNA repair fails. Therefore, the status of p53 protein may be critically associated with tumour cell radiosensitivity. In the present study we examine the intrinsic radiosensitivity of 20 human carcinoma cell lines derived from 15 patients with different types of head and neck tumour. Radiosensitivities were measured in a 96-well plate clonogenic assay in terms of the mean inactivation dose, surviving fraction at 2 Gy, and constants alpha and beta in the linear quadratic survival curve. The p53 allele status was determined by amplifying exons 4-10 by the polymerase chain reaction (PCR), screening for mutations using single-strand conformation polymorphism (SSCP) analysis and determining the exact type and location of a mutation by direct sequencing. The results showed that prevalence of p53 mutations in squamous cell carcinoma (SCC) cell lines is high (80%), and that deletion of one or both wild-type alleles is common (75%). Intrinsic radiosensitivity of the cell lines varied greatly in terms of mean inactivation dose, from 1.4 +/- 0.1 to 2.6 +/- 0.2 Gy. Radiosensitivity correlated well with the p53 allele status so that cell lines carrying a wild-type p53 allele were significantly (P < 0.01) more radioresistant (mean inactivation dose 2.23 +/- 0.15 Gy) than cell lines which lacked a wild-type gene (1.82 +/- 0.24 Gy). Evaluation of our own results and those published in the literature lead us to conclude that absence of the wild-type p53 allele in human head and neck cancer cell lines is associated with increased radiosensitivity. However, the sensitivity is also strongly dependent on the exact type and location of the p53 mutation.

  11. Effect of Mir-122 on Human Cholangiocarcinoma Proliferation, Invasion, and Apoptosis Through P53 Expression

    PubMed Central

    Wu, Cuiping; Zhang, Jinmei; Cao, Xiangang; Yang, Qian; Xia, Dequan

    2016-01-01

    Background Bile duct carcinoma is a common digestive tract tumor with high morbidity and mortality. As a kind of important non-coding RNA, microRNA (miR) plays an important role in post-transcriptional regulation. MiR-122 is the most abundant miR in the liver. Multiple studies have shown that miR-122 level is reduced in a variety of liver tumors and can be used as a specific marker for liver injury. P53 is a classic tumor suppressor gene that can induce tumor cell apoptosis through various pathways. Whether miR-122 affects p53 in bile duct carcinoma still needs investigation. Material/Methods miR inhibitor or mimics was transfected to bile duct carcinoma cells to evaluate its function on proliferation, invasion, apoptosis, and p53 expression. Results MiR-122 overexpression reduced cell invasion and migration ability, and inhibited cell apoptosis and p53 expression. Inhibiting miR-122 caused the opposite results. Conclusions Upregulating miR-122 can suppress bile duct carcinoma cell proliferation and induce apoptosis. MiR-122 could be used as a target for bile duct carcinoma treatment, which provides a new strategy for cholangiocarcinoma patients. PMID:27472451

  12. Regulation of The Tumor Suppressor Activity of P53 In Human Breast Cancer

    DTIC Science & Technology

    1999-09-01

    Lenglet, S., Moreau , V., Iggo, R., and Frehourg. T. Michael Datto and Xiao-Fan Wang (Duke University) for the p21 pro- (1998) Oncgene 16, 1369-1372...School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA Depending upon particular cellular conditions, the tumor suppressor protein p53

  13. Misfolding, Aggregation, and Disordered Segments in c-Abl and p53 in Human Cancer

    PubMed Central

    de Oliveira, Guilherme A. P.; Rangel, Luciana P.; Costa, Danielly C.; Silva, Jerson L.

    2015-01-01

    The current understanding of the molecular mechanisms that lead to cancer is not sufficient to explain the loss or gain of function in proteins related to tumorigenic processes. Among them, more than 100 oncogenes, 20–30 tumor-suppressor genes, and hundreds of genes participating in DNA repair and replication have been found to play a role in the origins of cancer over the last 25 years. The phosphorylation of serine, threonine, or tyrosine residues is a critical step in cellular growth and development and is achieved through the tight regulation of protein kinases. Phosphorylation plays a major role in eukaryotic signaling as kinase domains are found in 2% of our genes. The deregulation of kinase control mechanisms has disastrous consequences, often leading to gains of function, cell transformation, and cancer. The c-Abl kinase protein is one of the most studied targets in the fight against cancer and is a hotspot for drug development because it participates in several solid tumors and is the hallmark of chronic myelogenous leukemia. Tumor suppressors have the opposite effects. Their fundamental role in the maintenance of genomic integrity has awarded them a role as the guardians of DNA. Among the tumor suppressors, p53 is the most studied. The p53 protein has been shown to be a transcription factor that recognizes and binds to specific DNA response elements and activates gene transcription. Stress triggered by ionizing radiation or other mutagenic events leads to p53 phosphorylation and cell-cycle arrest, senescence, or programed cell death. The p53 gene is the most frequently mutated gene in cancer. Mutations in the DNA-binding domain are classified as class I or class II depending on whether substitutions occur in the DNA contact sites or in the protein core, respectively. Tumor-associated p53 mutations often lead to the loss of protein function, but recent investigations have also indicated gain-of-function mutations. The prion-like aggregation of mutant p

  14. Human papillomavirus E6 proteins mediate resistance to interferon-induced growth arrest through inhibition of p53 acetylation.

    PubMed

    Hebner, Christy; Beglin, Melanie; Laimins, Laimonis A

    2007-12-01

    The high-risk human papillomavirus (HPV) E6 and E7 proteins act cooperatively to mediate multiple activities in viral pathogenesis. For instance, E7 acts to increase p53 levels while E6 accelerates its rate of turnover through the binding of the cellular ubiquitin ligase E6AP. Interferons are important antiviral agents that modulate both the initial and persistent phases of viral infection. The expression of HPV type 16 E7 was found to sensitize keratinocytes to the growth-inhibitory effects of interferon, while coexpression of E6 abrogates this inhibition. Treatment of E7-expressing cells with interferon ultimately resulted in cellular senescence through a process that is dependent upon acetylation of p53 by p300/CBP at lysine 382. Cells expressing mutant forms of E6 that are unable to bind p300/CBP or bind p53 failed to block acetylation of p53 at lysine 382 and were sensitive to growth arrest by interferon. In contrast, mutant forms of E6 that are unable to bind E6AP remain resistant to the effects of interferon, demonstrating that the absolute levels of p53 are not the major determinants of this activity. Finally, p53 acetylation at lysine 382 was found not to be an essential determinant of other types of senescence such as that induced by overexpression of Ras in human fibroblasts. This study identifies an important physiological role for E6 binding to p300/CBP in blocking growth arrest of human keratinocytes in the presence of interferon and so contributes to the persistence of HPV-infected cells.

  15. Central role of mitochondria and p53 in PUVA-induced apoptosis in human keratinocytes cell line NCTC-2544

    SciTech Connect

    Viola, Giampietro Fortunato, Elena; Cecconet, Laura; Del Giudice, Laura; Dall'Acqua, Francesco; Basso, Giuseppe

    2008-02-15

    Despite strong evidence concerning the high efficiency of PUVA therapy (psoralen plus UVA light), its mechanism of action has not yet been fully elucidated. In this study, we have evaluated in a cell line of human keratinocytes (NCTC-2544) the effects of two linear psoralen derivatives, 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP), that are widely used in PUVA therapy and two angular derivatives, Angelicin (ANG) and 4,6,4'-trymetyl angelicin (TMA). All derivatives photoinduce cellular death, TMA being the most active compound. The cell cycle analysis showed that the four derivatives induce, 24 h after irradiation, a cell cycle arrest in G1 phase later followed by massive apoptosis. The G1 arrest is correlated to an increase in the expression of p21{sup Waf1/Cip1}, a protein associated with the cell cycle block and apoptosis. Furthermore, treatment of NCTC-2544 resulted in p53 activation by 5-MOP, 8-MOP, and ANG but not TMA and its phosphorylation at serine-15. The levels of p21{sup Waf1/Cip1} paralleled p53 protein staining pattern suggesting that p53 activation correlated with p21{sup Waf1/Cip1} induction. Simultaneous to p53 activation, psoralens induced mitochondrial depolarization, cytochrome c release, mitochondrial production of reactive oxygen species, as well as caspase-3 and -9 activation. Thus these results strongly indicate the necessity of p53 activation and the induction of the apoptotic machinery downstream of mitochondria.

  16. Delayed expression of apoptosis in X-irradiated human leukemic MOLT-4 cells transfected with mutant p53.

    PubMed

    Nakano, Hisako; Yonekawa, Hiromichi; Shinohara, Kunio

    2003-06-01

    The effects of X-rays on cell survival, apoptosis, and long-term response in the development of cell death as measured by the dye exclusion test were studied in human leukemic MOLT-4 cells (p53 wild-type) stably transfected with a mutant p53 cDNA expression vector. Cell survival, as determined from colony-forming ability, was increased in an expression level dependent manner, but the increase was partial even with the highest-expressing clone (B3). This contrasts with the prior observation that cell death and apoptosis in B3 are completely inhibited at 24 h after irradiation with 1.8 Gy of X-rays. The examination of B3 cells incubated for longer than 24 h after X-irradiation showed a delay in the induction of cell death and apoptosis. Western blot analysis revealed that the time required to reach the highest level of wild-type p53 protein in B3 was longer than the time in MOLT-4 and that the p53 may be stabilized by the phosphorylation at Ser-15. These results suggest that the introduction of mutant p53 into MOLT-4 merely delays the development of apoptosis, during which the cells could repair the damage induced by X-rays, and results in the partial increase in cell survival.

  17. Requirement of E6AP and the features of human papillomavirus E6 necessary to support degradation of p53.

    PubMed

    Cooper, Brooke; Schneider, Steven; Bohl, Joanna; Jiang, Yong hui; Beaudet, Arthur; Vande Pol, Scott

    2003-02-01

    E6 oncoproteins from human papillomavirus type 16 (16E6) and Bovine Papillomavirus type 1 (BE6) bind to leucine rich peptides (called charged leucine, LXXLL, or signature peptides) found on target cellular proteins. BE6 and 16E6 both bind the product of the UBE3A gene called E6AP on a charged leucine peptide, LQELL. E6AP is an E3 ubiquitin ligase that together with 16E6 interacts with p53 to target p53 degradation. Although both BE6 and 16E6 bind the LQELL peptide of E6AP, only 16E6 acts as an adapter to then bring p53 to E6AP. In order to determine how E6 proteins function as adapters, 16E6, p53, and E6AP were expressed in yeast, and were shown to form a tri-molecular complex. 16E6 mutants were selected that retained interactions with E6AP yet were defective for interaction with p53. Such 16E6 mutations were typically within the amino-terminus of 16E6. Through the use of E6AP null cells, transfected E6AP was shown to be necessary and sufficient for the degradation of p53 in the presence of 16E6. However, the interaction of 16E6 with E6AP was complex. While BE6 interacts only with the LQELL motif of E6AP, an intact LQELL motif is not necessary either for interaction of 16E6 with E6AP or for p53 degradation. In addition, 16E6 mutants that fail to bind the LQELL motif of E6AP can support p53 degradation. These results indicate that 16E6 may have multiple modes of interaction with E6AP and that assembly of p53 containing complexes for targeted degradation by E6AP may occur in more than one way. These results have implications for potential targeting of the interaction of 16E6 and E6AP in the therapy of HPV-induced cancer.

  18. Influence of zinc deficiency on AKT-MDM2-P53 signaling axes in normal and malignant human prostate cells

    USDA-ARS?s Scientific Manuscript database

    With prostate being the highest zinc-accumulating tissue before the onset of cancer, the effects of physiologic levels of zinc on Akt-Mdm2-p53 and Akt-p21 signaling axes in human normal prostate epithelial cells (PrEC) and malignant prostate LNCaP cells were examined. Cells were cultured for 6 d in...

  19. Zinc Induced G2/M Blockage is p53 and p21 Dependent in Normal Human Bronchial Epithelial Cells

    USDA-ARS?s Scientific Manuscript database

    The involvement of the p53 and p21 signal pathway in the G2/M cell cycle progression of zinc supplemented normal human bronchial epithelial (NHBE) cells was examined using the siRNA approach. Cells were cultured for one passage in different concentrations of zinc: <0.4 microM (ZD) as zinc-deficient;...

  20. Genus Beta Human Papillomavirus E6 Proteins Vary in Their Effects on the Transactivation of p53 Target Genes

    PubMed Central

    White, Elizabeth A.; Walther, Johanna; Javanbakht, Hassan

    2014-01-01

    ABSTRACT The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. IMPORTANCE This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help

  1. Genus beta human papillomavirus E6 proteins vary in their effects on the transactivation of p53 target genes.

    PubMed

    White, Elizabeth A; Walther, Johanna; Javanbakht, Hassan; Howley, Peter M

    2014-08-01

    The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help to explain the

  2. Cyclobutane pyrimidine dimer formation and p53 production in human skin after repeated UV irradiation.

    PubMed

    Yamaguchi, Yuji; Coelho, Sergio G; Zmudzka, Barbara Z; Takahashi, Kaoruko; Beer, Janusz Z; Hearing, Vincent J; Miller, Sharon A

    2008-11-01

    Substantial differences in DNA damage caused by a single UV irradiation were found in our previous study on skin with different levels of constitutive pigmentation. In this study, we assessed whether facultative pigmentation induced by repeated UV irradiation is photoprotective. Three sites on the backs of 21 healthy subjects with type II-III skin were irradiated at 100-600 J/m(2) every 2-7 days over a 4- to 5-week period. The three sites received different cumulative doses of UV (1900, 2900 or 4200 J/m(2)) and were biopsied 1 day after the last irradiation. Biomarkers examined included pigment content assessed by Fontana-Masson staining, melanocyte function by expression of melanocyte-specific markers, DNA damage as cyclobutane pyrimidine dimers (CPD), nuclear accumulation of p53, apoptosis determined by TUNEL assay, and levels of p21 and Ser46-phosphorylated p53. Increases in melanocyte function and density, and in levels of apoptosis were similar among the 3 study sites irradiated with different cumulative UV doses. Levels of CPD decreased while the number of p53-positive cells increased as the cumulative dose of UV increased. These results suggest that pigmentation induced in skin by repeated UV irradiation protects against subsequent UV-induced DNA damage but not as effectively as constitutive pigmentation.

  3. Curcumin enhances temsirolimus-induced apoptosis in human renal carcinoma cells through upregulation of YAP/p53

    PubMed Central

    Xu, Shan; Yang, Zheng; Fan, Yizeng; Guan, Bing; Jia, Jing; Gao, Yang; Wang, Ke; Wu, Kaijie; Wang, Xinyang; Zheng, Pengsheng; He, Dalin; Guo, Peng

    2016-01-01

    Curcumin has frequently been used as a therapeutic agent in the treatment of various types of disease and is known to enhance the drug sensitivity of cells. In the present study, the combined effect of curcumin and temsirolimus treatment on apoptosis in human renal cell carcinoma (RCC) cells was investigated. Temsirolimus is an inhibitor of the mechanistic target of rapamycin signaling pathway and used in the first-line treatment of metastatic RCC. It was demonstrated that curcumin combined with temsirolimus markedly induced apoptosis in RCC cells, however this effect was not observed following curcumin or temsirolimus treatment alone. Co-treatment with temsirolimus and curcumin led to the activation of cleaved poly ADP-ribose polymerase and caspase 3, upregulation of p53 expression and nuclear translocation, and downregulation of B-cell lymphoma 2 protein expression. Furthermore, curcumin treatment was demonstrated to increase Yes-associated protein (YAP) expression in a time-dependent manner, which was concurrent with the curcumin-induced expression pattern of p53 after 2 h. In addition, knockdown of YAP by small interfering RNA caused the attenuation of curcumin-induced increased p53 expression in RCC cells. In conclusion, the present results indicate that combined curcumin and temsirolimus treatment has a synergistic effect on apoptosis in human RCC cells, through the activation of p53. Mechanistically, YAP is essential in the induction of p53 expression by curcumin. Furthermore, the results suggest that pre-treatment or co-treatment of cells with low concentration curcumin enhances the response to targeted drugs, and this presents a potentially novel and efficient strategy to overcome drug resistance in human RCC. PMID:28105206

  4. Downregulation of Polo-like kinase 1 induces cellular senescence in human primary cells through a p53-dependent pathway.

    PubMed

    Kim, Hee-Jin; Cho, Jung Hee; Kim, Jae-Ryong

    2013-10-01

    Polo-like kinase 1 (PLK1) plays a key role in various stages of mitosis from entry into M phase to exit from mitosis. However, its role in cellular senescence remains to be determined. Therefore, the effects of PLK1 on cellular senescence in human primary cells were investigated. We found that expression of PLK1 decreased in human dermal fibroblasts and human umbilical vein endothelial cells under replicative senescence and premature senescence induced by adriamycin. PLK1 knockdown with PLK1 small interfering RNAs in young cells induced premature senescence. In contrast, upregulation of PLK1 in old cells partially reversed senescence phenotypes. Cellular senescence by PLK1 inhibition was observed in p16 knockdown cells but not in p53 knockdown cells. Our data suggest that PLK1 repression might result in cellular senescence in human primary cells via a p53-dependent pathway.

  5. Metabolic pathways regulated by p63.

    PubMed

    Candi, Eleonora; Smirnov, Artem; Panatta, Emanuele; Lena, Anna Maria; Novelli, Flavia; Mancini, Mara; Viticchiè, Giuditta; Piro, Maria Cristina; Di Daniele, Nicola; Annicchiarico-Petruzzelli, Margherita; Melino, Gerry

    2017-01-15

    The transcription factor p63 belongs to the p53-family and is a master regulator of proliferative potential, lineage specification, and differentiation in epithelia during development and tissue homeostasis. In cancer, p63 contribution is isoform-specific, with both oncogenic and tumour suppressive roles attributed, for ΔNp63 and TAp63, respectively. Recently, p53 and TAp73, in line with other tumour suppressor genes, have emerged as important regulators of energy metabolism and metabolic reprogramming in cancer. To date, p63 contributions in controlling energy metabolism have been partially investigated; given the extensive interaction of the p53 family members, these studies have potential implications in tumour cells for metabolic reprogramming. Here, we review the role of p63 isoforms, TAp63 and ΔNp63, in controlling cell metabolism, focusing on their specific metabolic target genes and their physiological/functional context of action. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Synergistic anticancer effect of rAd/P53 combined with 5-fluorouracil or iodized oil in the early therapeutic response of human colon cancer in vivo.

    PubMed

    Xie, Qi; Liang, Bi-Ling; Wu, Yan-Heng; Zhang, Jing; Chen, Ming-Wang; Liu, Hong-Yan; Gu, Xiong-Fei; Xu, Jin

    2012-05-15

    Exogenous wild-type p53 (wt-p53) tumor suppression increases the sensitivity of tumor cells to radiotherapy and chemotherapy. An iodized oil emulsion was used as a p53 vector for intra-arterial gene delivery to treat hepatic tumors. Whether the chemotherapeutic agent or the iodized oil affects exogenous wt-p53 activity remains poorly understood. In the present study, the early therapeutic response of rAd/p53, combined with 5-fluorouracil (5-FU) or with iodized oil, was observed in a human colon cancer model. Allograft models in 82 nude mice with human colon carcinoma SW480 were divided randomly into four groups and administered with physiologic saline, rAd/p53, rAd/p53+5-FU, and rAd/p53+iodized oil by intratumoral injection. At 24, 48, 72, 120, and 168 h after treatment, p53 expression, the Ki-67 index (KI), and the degree of tumor necrosis were assessed. The p53 expression and tumor necrosis in the therapeutic groups were higher than those in the control group. p53 expression reached its peak at 120 h in the rAd/p53 group, at 72 h in the rAd/p53+5-FU group, and at 48 h in the rAd/p53+iodized oil group. The p53 expression in the rAd/P53+5-FU group and the iodized oil group was significantly higher than those in the rAd/P53 group at 24 and 48 h. The results revealed that tumor necrosis is positively correlated with p53 expression. The KI of the rAd/p53+5-FU group increased significantly at 24 h. 5-FU and iodized oil increase the anticancer effect of rAd/p53, and 5-FU combined with rAd/p53 has a synergistic anticancer effect. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Space experiment "Rad Gene"-report 1; p53-Dependent gene expression in human cultured cells exposed to space environment

    NASA Astrophysics Data System (ADS)

    Takahashi, Akihisa; Ohnishi, Takeo; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki

    The space environment contains two major biologically significant influences: space radiations and microgravity. A p53 tumor suppressor protein plays a role as a guardian of the genome through the activity of p53-centered signal transduction pathways. The aim of this study was to clarify the biological effects of space radiations, microgravity and a space environment on the gene and protein expression of p53-dependent regulated genes. Space experiments were performed with two human cultured lymphoblastoid cell lines: one cells line (TSCE5) bears a wild-type p53 gene status, and another cells line (WTK1) bears a mutated p53 gene status. Un-der one gravity or microgravity condition, the cells were grown in the cell biology experimental facility (CBEF) of the International Space Station (ISS) for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. Ground control samples also were cultured for 8 days in the CBEF on the ground during the same periods as space flight. Gene and protein expression was analyzed by using DNA chip (a 44k whole human genome microarray, Agilent Technologies Inc.) and protein chip (PanoramaTM Ab MicroArray, Sigma-Aldrich Co.), respectively. In addition, we analyzed the gene expression in cultured cells after space flight during 133 days with frozen condition. We report the results and discussion from the viewpoint of the functions of the up-regulated and down-regulated genes after an exposure to space radiations and/or microgravity. The initial goal of this space experiment was completely achieved. It is expected that data from this type of work will be helpful in designing physical protection from the deleterious effects of space radiations during long term stays in space.

  8. Special AT-rich Binding Protein-2 (SATB2) Differentially Affects Disease-causing p63 Mutant Proteins*

    PubMed Central

    Chung, Jacky; Grant, R. Ian; Kaplan, David R.; Irwin, Meredith S.

    2011-01-01

    p63, a p53 family member, is critical for proper skin and limb development and directly regulates gene expression in the ectoderm. Mice lacking p63 exhibit skin and craniofacial defects including cleft palate. In humans p63 mutations are associated with several distinct developmental syndromes. p63 sterile-α-motif domain, AEC (ankyloblepharon-ectodermal dysplasia-clefting)-associated mutations are associated with a high prevalence of orofacial clefting disorders, which are less common in EEC (ectrodactyly-ectodermal dysplasia-clefting) patients with DNA binding domain p63 mutations. However, the mechanisms by which these mutations differentially influence p63 function remain unclear, and interactions with other proteins implicated in craniofacial development have not been identified. Here, we show that AEC p63 mutations affect the ability of the p63 protein to interact with special AT-rich binding protein-2 (SATB2), which has recently also been implicated in the development of cleft palate. p63 and SATB2 are co-expressed early in development in the ectoderm of the first and second branchial arches, two essential sites where signaling is required for craniofacial patterning. SATB2 attenuates p63-mediated gene expression of perp (p53 apoptosis effector related to PMP-22), a critical downstream target gene during development, and specifically decreases p63 perp promoter binding. Interestingly, AEC but not EEC p63 mutations affect the ability of p63 to interact with SATB2 and the inhibitory effects of SATB2 on p63 transactivation of perp are most pronounced for AEC-associated p63 mutations. Our findings reveal a novel gain-of-function property of AEC-causing p63 mutations and identify SATB2 as the first p63 binding partner that differentially influences AEC and EEC p63 mutant proteins. PMID:21965674

  9. Effect of Boswellia Thurifera Gum Methanol Extract on Cytotoxicity and P53 Gene Expression in Human Breast Cancer Cell Line

    PubMed Central

    Yazdanpanahi, Nasrin; Behbahani, Mandana; Yektaeian, Afsaneh

    2014-01-01

    Boswellia has been widely used in traditional medicine for the treatment of different diseases such as cancer in Iran. The aim of this study was to evaluate the effect of the gum methanol extract of Boswellia thurifera on the viability and P53 gene expression of cultured breast cancer cells. The gum methanol extract was obtained in various concentrations using the maceration method. Normal (HEK-293) and cancer (MDA-MB-231) human cells were cultured and treated with various concentrations of the extract. Then MTT assay was used for the study of cytotoxic effect of the extract and real time PCR method was also applied for the investigation of P53 gene expression in cancer cells. The IC50 of the extract against cancer cells was 80 µg/mL and had less cytotoxic effect in normal cells. The effect of the extract was dose dependent. Induction of P53 expression by extract was also significantly more in treated cancer cells than untreated cells. This inductive effect in cells was higher after 12 h treatment than it was after 6 h. The results of the current study show that gum methanol extract of Boswellia thurifera has probably anti-cancer effects and could induce P53 gene transcription and toxicity in the cultured breast cancer cell line. The increase of P53 gene specific mRNA may be a mechanism of gum methanol extract induced cytotoxicity. However, for a definitive conclusion, further studies on other cell lines as well as animal models and subsequent clinical studies are warranted. PMID:25237368

  10. Mouse p53-Deficient Cancer Models as Platforms for Obtaining Genomic Predictors of Human Cancer Clinical Outcomes

    PubMed Central

    Dueñas, Marta; Santos, Mirentxu; Aranda, Juan F.; Bielza, Concha; Martínez-Cruz, Ana B.; Lorz, Corina; Taron, Miquel; Ciruelos, Eva M.; Rodríguez-Peralto, José L.; Martín, Miguel; Larrañaga, Pedro; Dahabreh, Jubrail; Stathopoulos, George P.; Rosell, Rafael; Paramio, Jesús M.; García-Escudero, Ramón

    2012-01-01

    Mutations in the TP53 gene are very common in human cancers, and are associated with poor clinical outcome. Transgenic mouse models lacking the Trp53 gene or that express mutant Trp53 transgenes produce tumours with malignant features in many organs. We previously showed the transcriptome of a p53-deficient mouse skin carcinoma model to be similar to those of human cancers with TP53 mutations and associated with poor clinical outcomes. This report shows that much of the 682-gene signature of this murine skin carcinoma transcriptome is also present in breast and lung cancer mouse models in which p53 is inhibited. Further, we report validated gene-expression-based tests for predicting the clinical outcome of human breast and lung adenocarcinoma. It was found that human patients with cancer could be stratified based on the similarity of their transcriptome with the mouse skin carcinoma 682-gene signature. The results also provide new targets for the treatment of p53-defective tumours. PMID:22880004

  11. Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53.

    PubMed

    Kelly, Gemma L; Grabow, Stephanie; Glaser, Stefan P; Fitzsimmons, Leah; Aubrey, Brandon J; Okamoto, Toru; Valente, Liz J; Robati, Mikara; Tai, Lin; Fairlie, W Douglas; Lee, Erinna F; Lindstrom, Mikael S; Wiman, Klas G; Huang, David C S; Bouillet, Philippe; Rowe, Martin; Rickinson, Alan B; Herold, Marco J; Strasser, Andreas

    2014-01-01

    The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.

  12. Intratumoral heterogeneity in a p53 null mouse model of human breast cancer

    PubMed Central

    Zhang, Mei; Tsimelzon, Anna; Chang, Chi-Hsuan; Fan, Cheng; Wolff, Andrew; Perou, Charles M.; Hilsenbeck, Susan G.; Rosen, Jeffrey M.

    2015-01-01

    Intratumoral heterogeneity correlates with clinical outcome and reflects the cellular complexity and dynamics within a tumor. Such heterogeneity is thought to contribute to radio- and chemoresistance since many treatments may only target certain tumor cell subpopulations. A better understanding of the functional interactions between various subpopulations of cells, therefore, may help in the development of effective cancer treatments. We identified a unique subpopulation of tumor cells expressing mesenchymal-like markers in a p53 null mouse model of basal-like breast cancer using fluorescence-activated cell sorting and microarray analysis. Both in vitro and in vivo experiments revealed the existence of crosstalk between these “mesenchymal-like” cells and tumor-initiating cells. Knockdown of genes encoding ligands upregulated in the mesenchymal cells and their corresponding receptors in the tumor-initiating cells resulted in reduced tumorigenicity and increased tumor latency. These studies illustrate the non-cell autonomous properties and importance of cooperativity between tumor subpopulations. PMID:25735774

  13. Differential response between the p53 ubiquitin-protein ligases Pirh2 and MdM2 following DNA damage in human cancer cells

    SciTech Connect

    Duan Wenrui; Gao, Li; Wu Xin; Zhang Yang; Otterson, Gregory A.; Villalona-Calero, Miguel A. . E-mail: Miguel.villalona@osumc.edu

    2006-10-15

    Pirh2, a recently identified ubiquitin-protein ligase, has been reported to promote p53 degradation. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of MDM2. Like MDM2, Pirh2 is thought to participate in an autoregulatory feedback loop that controls p53 function. We have previously reported that Pirh2 was overexpressed in human and murine lung cancers as compared to uninvolved lung tissue. Pirh2 increase could potentially cause degradation of wildtype p53 and reduce its tumor suppression function in the lung tumor cells. Since Pirh2 has been reported to be transactivated by p53, however, the mechanisms by which a high level of Pirh2 expression is maintained in tumor cells despite low level of wildtype p53 protein are unclear. In order to evaluate p53 involvement in the transactivation of Pirh2, we evaluated Pirh2, MDM2, p53 and p21 expression with Western blot analysis and real time PCR after {gamma} irradiation or cisplatin DNA damage treatment using human cancer cell lines containing wildtype (A549, MCF-7), mutant (H719) and null (H1299) p53. Surprisingly, Pirh2 expression was not affected by the presence of wildtype p53 in the cancer cells. In contrast, MDM2 was upregulated by wildtype p53 in A549 and MCF-7 cells and was absent from the H1299 and the H719 cells. We conclude that Pirh2 operates in a distinct manner from MDM2 in response to DNA damage in cancer cells. Pirh2 elevation in p53 null cells indicates the existence of additional molecular mechanisms for Pirh2 upregulation and suggests that p53 is not the sole target of Pirh2 ubiquitin ligase activity.

  14. Efficacy of recombinant adenoviral human p53 gene in treatment of malignant pleural or peritoneal effusions.

    PubMed

    Zhang, Xin; Hu, Yi; Wang, Jinliang; Zhang, Sujie; Tao, Haitao; Jing, Sun; Wu, Baishou

    2013-03-01

    Once the malignant pleural or peritoneal effusion is developed it is difficult to control. This report presents a new method for controlling the malignant effusions. Forty-eight patients, 29 males and 19 females with an average age of 61.2 years old, who were satisfied with the study inclusion criteria, were recruited in this study. Twenty-seven and 21 patients had a malignant pleural and peritoneal effusion, respectively. After draining most of fluids, these patients received intra-cavity infusion of rAd-p53 once per week for 4 weeks, at dose of 2×10¹² viral particles (VP) diluted into 200 mL of saline solution for pleural effusions, and 4×10¹² VP diluted into 500 mL of saline solution for peritoneal effusions. Participants were followed up for a median time of 13.6 month. A total of 11 cases, 7 with pleural effusions and 4 with peritoneal effusions achieved a complete response (CR), and 20 cases (12 pleural effusions and 8 peritoneal effusions) had a partial response (PR). The overall response rate is 64.6%. Patients' quality of life, assessed by using Karnofsky performance scale (KPS) scores, was improved by an average of 26.4. The one-year of overall survival rate was 54.2% with a median survival time of 12.5 months. There were no serious side effects observed except for self-limited fever found in 79.8% of the cases. Intra-cavity infusion of rAd-p53 is an effective and safe treatment for the patients with malignant pleural or peritoneal effusions, especially for those patients who can't tolerate the standard treatments.

  15. Cytoplasmic sequestration of the tumor suppressor p53 by a heat shock protein 70 family member, mortalin, in human colorectal adenocarcinoma cell lines

    SciTech Connect

    Gestl, Erin E.; Anne Boettger, S.

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer Eight human colorectal cell lines were evaluated for p53 and mortalin localization. Black-Right-Pointing-Pointer Six cell lines displayed cytoplasmic sequestration of the tumor suppressor p53. Black-Right-Pointing-Pointer Direct interaction between mortalin and p53 was shown in five cell lines. Black-Right-Pointing-Pointer Cell lines positive for p53 sequestration yielded elevated p53 expression levels. Black-Right-Pointing-Pointer This study yields the first evidence of cytoplasmic sequestration p53 by mortalin. -- Abstract: While it is known that cytoplasmic retention of p53 occurs in many solid tumors, the mechanisms responsible for this retention have not been positively identified. Since heatshock proteins like mortalin have been associated with p53 inactivation in other tumors, the current study sought to characterize this potential interaction in never before examined colorectal adenocarcinoma cell lines. Six cell lines, one with 3 different fractions, were examined to determine expression of p53 and mortalin and characterize their cellular localization. Most of these cell lines displayed punctate p53 and mortalin localization in the cell cytoplasm with the exception of HCT-8 and HCT116 379.2 cells, where p53 was not detected. Nuclear p53 was only observed in HCT-116 40-16, LS123, and HT-29 cell lines. Mortalin was only localized in the cytoplasm in all cell lines. Co-immunoprecipitation and immunohistochemistry revealed that p53 and mortalin were bound and co-localized in the cytoplasmic fraction of four cell lines, HCT-116 (40-16 and 386; parental and heterozygous fractions respectively of the same cell line), HT-29, LS123 and LoVo, implying that p53 nuclear function is limited in those cell lines by being restricted to the cytoplasm. Mortalin gene expression levels were higher than gene expression levels of p53 in all cell lines. Cell lines with cytoplasmic sequestration of p53, however, also displayed elevated p53

  16. Tacrolimus ointment neither blocks ultraviolet B nor affects expression of thymine dimers and p53 in human skin.

    PubMed

    Gambichler, Thilo; Schlaffke, Axel; Tomi, Nordwig S; Othlinghaus, Nick; Altmeyer, Peter; Kreuter, Alexander

    2008-05-01

    There is a lack of data with regard to the interaction between ultraviolet (UV) radiation and topical calcineurin inhibitors. We aimed to investigate (1) the UV transmission through tacrolimus ointment and (2) the impact of topical exposure to tacrolimus on the protein expression of thymine dimers (TD) and p53 in human skin. Spectrophotometric measurements (290-400 nm) of tacrolimus ointment and the vehicle were performed. Eight subjects were treated with tacrolimus ointment and the vehicle thrice daily over a 3-day period on the back. Pre-treated sites and one control site were exposed to two minimal erythema doses UVB. Skin biopsies were taken 1h and 24h after irradiation. Immunohistochemical procedures were used for the detection of TD and p53. Mean UV transmission was over 94% and did not significantly differ between tacrolimus ointment and the vehicle. Immunohistological examinations of TD and p53 expression did not demonstrate significant differences between irradiated sites, irradiated plus vehicle treated sites, and irradiated plus tacrolimus treated sites both 1h and 24h post-irradiation. The present data suggest that tacrolimus ointment hardly has UV blocking capacities and does not significantly interfere with development and/or removal of local DNA damage in human skin.

  17. Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro by inhibiting the p53-dependent mitochondrial apoptotic pathway

    PubMed Central

    Lee, Yoon-Jin; Kim, Soo A; Lee, Sang-Han

    2016-01-01

    Aim: Intra-articular injection of local anesthetics (LAs) is a common procedure for therapeutic purposes. However, LAs have been found toxic to articular cartilage, and hyaluronan may attenuate this toxicity. In this study we investigated whether hyaluronan attenuated lidocaine-induced chondrotoxicity, and if so, to elucidate the underlying mechanisms. Methods: Human chondrocyte cell line SW1353 and newly isolated murine chondrocytes were incubated in culture medium containing hyaluronan and/or lidocaine for 72 h. Cell viability was evaluated using MTT assay. Cell apoptosis was detected with DAPI staining, caspase 3/7 activity assay and flow cytometry. Cell cycle distributions, ROS levels and mitochondrial membrane potential (ΔΨm) were determined using flow cytometry. The expression of p53 and p53-regulated gene products was measured with Western blotting. Results: Lidocaine (0.005%−0.03%) dose-dependently decreased the viability of SW1353 cells. This local anesthetic (0.015%, 0.025%) induced apoptosis, G2/M phase arrest and loss of ΔΨm, and markedly increased ROS production in SW1353 cells. Hyaluronan (50−800 μg/mL) alone did not affect the cell viability, but co-treatment with hyaluronan (200 μg/mL) significantly attenuated lidocaine-induced apoptosis and other abnormalities in SW1353 cells. Furthermore, co-treatment with lidocaine and hyaluronan significantly decreased the levels of p53 and its transcription targets Bax and p21 in SW1353 cells, although treatment with lidocaine alone did not significantly change these proteins. Similar results were obtained in ex vivo cultured murine chondrocytes. Conclusion: Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro through inhibiting the p53-dependent mitochondrial apoptotic pathway. PMID:27041463

  18. p53 activation by Ni(II) is a HIF-1α independent response causing caspases 9/3-mediated apoptosis in human lung cells

    SciTech Connect

    Wong, Victor C.; Morse, Jessica L.; Zhitkovich, Anatoly

    2013-06-15

    Hypoxia mimic nickel(II) is a human respiratory carcinogen with a suspected epigenetic mode of action. We examined whether Ni(II) elicits a toxicologically significant activation of the tumor suppressor p53, which is typically associated with genotoxic responses. We found that treatments of H460 human lung epithelial cells with NiCl{sub 2} caused activating phosphorylation at p53-Ser15, accumulation of p53 protein and depletion of its inhibitor MDM4 (HDMX). Confirming the activation of p53, its knockdown suppressed the ability of Ni(II) to upregulate MDM2 and p21 (CDKN1A). Unlike DNA damage, induction of GADD45A by Ni(II) was p53-independent. Ni(II) also increased p53-Ser15 phosphorylation and p21 expression in normal human lung fibroblasts. Although Ni(II)-induced stabilization of HIF-1α occurred earlier, it had no effect on p53 accumulation and Ser15 phosphorylation. Ni(II)-treated H460 cells showed no evidence of necrosis and their apoptosis and clonogenic death were suppressed by p53 knockdown. The apoptotic role of p53 involved a transcription-dependent program triggering the initiator caspase 9 and its downstream executioner caspase 3. Two most prominently upregulated proapoptotic genes by Ni(II) were PUMA and NOXA but only PUMA induction required p53. Knockdown of p53 also led to derepression of antiapoptotic MCL1 in Ni(II)-treated cells. Overall, our results indicate that p53 plays a major role in apoptotic death of human lung cells by Ni(II). Chronic exposure to Ni(II) may promote selection of resistant cells with inactivated p53, providing an explanation for the origin of p53 mutations by this epigenetic carcinogen. - Highlights: • Ni(II) is a strong activator of the transcription factor p53. • Apoptosis is a principal form of death by Ni(II) in human lung epithelial cells. • Ni(II)-activated p53 triggers caspases 9/3-mediated apoptotic program. • NOXA and PUMA are two main proapoptotic genes induced by Ni(II). • HIF-1α and p53 are independent

  19. Age-Related Susceptibility to Apoptosis in Human Retinal Pigment Epithelial Cells Is Triggered by Disruption of p53–Mdm2 Association

    PubMed Central

    Bhattacharya, Sujoy; Chaum, Edward; Johnson, Dianna A.; Johnson, Leonard R.

    2012-01-01

    Purpose. Relatively little is known about the contribution of p53/Mdm2 pathway in apoptosis of retinal pigment epithelial (RPE) cells or its possible link to dysfunction of aging RPE or to related blinding disorders such as age-related macular degeneration (AMD). Methods. Age-associated changes in p53 activation were evaluated in primary RPE cultures from human donor eyes of various ages. Apoptosis was evaluated by activation of caspases and DNA fragmentation. Gene-specific small interfering RNA was used to knock down expression of p53. Results. We observed that the basal rate of p53-dependent apoptosis increased in an age-dependent manner in human RPE. The age-dependent increase in apoptosis was linked to alterations in several aspects of the p53 pathway. p53 phosphorylation Ser15 was increased through the stimulation of ATM-Ser1981. p53 acetylation Lys379 was increased through the inhibition of SIRT1/2. These two posttranslational modifications of p53 blocked the sequestration of p53 by Mdm2, thus resulting in an increase in free p53 and of p53 stimulation of apoptosis through increased expression of PUMA (p53 upregulated modulator of apoptosis) and activation of caspase-3. Aged RPE also had reduced expression of antiapoptotic Bcl-2, which contributed to the increase in apoptosis. Of particular interest in these studies was that pharmacologic treatments to block p53 phosphorylation, acetylation, or expression were able to protect RPE cells from apoptosis. Conclusions. Our studies suggest that aging in the RPE leads to alterations of specific checkpoints in the apoptotic pathway, which may represent important molecular targets for the treatment of RPE-related aging disorders such as AMD. PMID:23139272

  20. Gene expression profiling of mouse p53-deficient epidermal carcinoma defines molecular determinants of human cancer malignancy

    PubMed Central

    2010-01-01

    Background The epidermal specific ablation of Trp53 gene leads to the spontaneous development of aggressive tumors in mice through a process that is accelerated by the simultaneous ablation of Rb gene. Since alterations of p53-dependent pathway are common hallmarks of aggressive, poor prognostic human cancers, these mouse models can recapitulate the molecular features of some of these human malignancies. Results To evaluate this possibility, gene expression microarray analysis was performed in mouse samples. The mouse tumors display increased expression of cell cycle and chromosomal instability associated genes. Remarkably, they are also enriched in human embryonic stem cell gene signatures, a characteristic feature of human aggressive tumors. Using cross-species comparison and meta-analytical approaches, we also observed that spontaneous mouse tumors display robust similarities with gene expression profiles of human tumors bearing mutated TP53, or displaying poor prognostic outcome, from multiple body tissues. We have obtained a 20-gene signature whose genes are overexpressed in mouse tumors and can identify human tumors with poor outcome from breast cancer, astrocytoma and multiple myeloma. This signature was consistently overexpressed in additional mouse tumors using microarray analysis. Two of the genes of this signature, AURKA and UBE2C, were validated in human breast and cervical cancer as potential biomarkers of malignancy. Conclusions Our analyses demonstrate that these mouse models are promising preclinical tools aimed to search for malignancy biomarkers and to test targeted therapies of prospective use in human aggressive tumors and/or with p53 mutation or inactivation. PMID:20630075

  1. Disposable Amperometric Immunosensor for the Determination of Human P53 Protein in Cell Lysates Using Magnetic Micro-Carriers

    PubMed Central

    Pedrero, María; Manuel de Villena, F. Javier; Muñoz-San Martín, Cristina; Campuzano, Susana; Garranzo-Asensio, María; Barderas, Rodrigo; Pingarrón, José M.

    2016-01-01

    An amperometric magnetoimmunosensor for the determination of human p53 protein is described in this work using a sandwich configuration involving the covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a mixture of the target protein and horseradish peroxidase-labeled antibody (HRP-anti-p53). The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are measured at −0.20 V (vs. an Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as a redox mediator and H2O2 as the enzyme substrate. The magnetoimmunosensing platform was successfully applied for the detection of p53 protein in different cell lysates without any matrix effect after a simple sample dilution. The results correlated accurately with those provided by a commercial ELISA kit, thus confirming the immunosensor as an attractive alternative for rapid and simple determination of this protein using portable and affordable instrumentation. PMID:27879639

  2. Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells.

    PubMed

    Min, Kyoung-Jin; Nam, Ju-Ock; Kwon, Taeg Kyu

    2017-08-02

    Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki) cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose) polymerase (PARP), which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk) inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5) expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.

  3. Human T-cell leukemia virus type-1-encoded protein HBZ represses p53 function by inhibiting the acetyltransferase activity of p300/CBP and HBO1.

    PubMed

    Wright, Diana G; Marchal, Claire; Hoang, Kimson; Ankney, John A; Nguyen, Stephanie T; Rushing, Amanda W; Polakowski, Nicholas; Miotto, Benoit; Lemasson, Isabelle

    2016-01-12

    Adult T-cell leukemia (ATL) is an often fatal malignancy caused by infection with the complex retrovirus, human T-cell Leukemia Virus, type 1 (HTLV-1). In ATL patient samples, the tumor suppressor, p53, is infrequently mutated; however, it has been shown to be inactivated by the viral protein, Tax. Here, we show that another HTLV-1 protein, HBZ, represses p53 activity. In HCT116 p53+/+ cells treated with the DNA-damaging agent, etoposide, HBZ reduced p53-mediated activation of p21/CDKN1A and GADD45A expression, which was associated with a delay in G2 phase-arrest. These effects were attributed to direct inhibition of the histone acetyltransferase (HAT) activity of p300/CBP by HBZ, causing a reduction in p53 acetylation, which has be linked to decreased p53 activity. In addition, HBZ bound to, and inhibited the HAT activity of HBO1. Although HBO1 did not acetylate p53, it acted as a coactivator for p53 at the p21/CDKN1A promoter. Therefore, through interactions with two separate HAT proteins, HBZ impairs the ability of p53 to activate transcription. This mechanism may explain how p53 activity is restricted in ATL cells that do not express Tax due to modifications of the HTLV-1 provirus, which accounts for a majority of patient samples.

  4. Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins.

    PubMed Central

    Huibregtse, J M; Scheffner, M; Howley, P M

    1993-01-01

    E6-AP is a 100-kDa cellular protein that mediates the interaction of the human papillomavirus type 16 and 18 E6 proteins with p53. The association of p53 with E6 and E6-AP promotes the specific ubiquitination and subsequent proteolytic degradation of p53 in vitro. We recently isolated a cDNA encoding E6-AP and have now mapped functional domains of E6-AP involved in binding E6, association with p53, and ubiquitination of p53. The E6 binding domain consists of an 18-amino-acid region within the central portion of the molecule. Deletion of these 18 amino acids from E6-AP results in loss of both E6 and p53 binding activities. The region that directs p53 binding spans the E6 binding domain and consists of approximately 500 amino acids. E6-AP sequences in addition to those required for formation of a stable ternary complex with E6 and p53 are necessary to stimulate the ubiquitination of p53. These sequences lie within the C-terminal 84 amino acids of E6-AP. The entire region required for E6-dependent ubiquitination of p53 is also required for the ubiquitination of an artificial E6 fusion protein. Images PMID:8393140

  5. Honokiol induces cell cycle arrest and apoptosis via p53 activation in H4 human neuroglioma cells.

    PubMed

    Guo, Yun-Bao; Bao, Xin-Jie; Xu, Song-Bai; Zhang, Xing-Dong; Liu, Hai-Yan

    2015-01-01

    To investigate the signal pathway of honokiol-induced apoptosis in H4 human neuroglioma cells and to evaluate whether p53 signaling and cell cycle arrest were involved in honokiol-treated H4 human neuroglioma cells. The cell viability was detected by the CCK8 assay. The cell apoptosis was assessed by annexin V-PI double-labeling staining and hoechst 33342 staining. The protein expression of cell cycle regulators and tumor suppressors were analyzed by western blotting. Treatment of H4 human neuroglioma cells with honokiol induced cell death in a dose-and time-dependent manner by using CCK8 assay. Consistent with the CCK8 assay, the flow cytometry results showed that the proportion of the apoptosis cells increased after honokiol when compared with untreated group. Moreover, H4 human neuroglioma cells exposed to honokiol, resulted in an accumulation of cells in S and G2/M phase. Apoptotic bodies were clearly observed in human neuroglioma cells when treated with honokiol and then stained with Hoechst 33342. The expression of Cyclin B1, CDC2 and cdc25C were downregulated, however, the expression of p-CDC2 and p-cdc25c was significantly upregulated when the neuroglioma cells were exposed to honokiol. Moreover, p53, p21 and Bax/Bcl-2 were significantly upregulated by honokiol treatment. These results confirmed that honokiol could induce apoptosis in human neuroglioma cells, the underlying molecular mechanisms, at least partially, through activation p53 signaling and induction of cell cycle arrest.

  6. Discovery of novel tumor suppressor p53 response elements using information theory

    PubMed Central

    Lyakhov, Ilya G.; Krishnamachari, Annangarachari; Schneider, Thomas D.

    2008-01-01

    An accurate method for locating genes under tumor suppressor p53 control that is based on a well-established mathematical theory and built using naturally occurring, experimentally proven p53 sites is essential in understanding the complete p53 network. We used a molecular information theory approach to create a flexible model for p53 binding. By searching around transcription start sites in human chromosomes 1 and 2, we predicted 16 novel p53 binding sites and experimentally demonstrated that 15 of the 16 (94%) sites were bound by p53. Some were also bound by the related proteins p63 and p73. Thirteen of the adjacent genes were controlled by at least one of the proteins. Eleven of the 16 sites (69%) had not been identified previously. This molecular information theory approach can be extended to any genetic system to predict new sites for DNA-binding proteins. PMID:18495754

  7. The carcinogenic air pollutant 3-nitrobenzanthrone induces GC to TA transversion mutations in human p53 sequences.

    PubMed

    vom Brocke, Jochen; Krais, Annette; Whibley, Catherine; Hollstein, Monica C; Schmeiser, Heinz H

    2009-01-01

    3-Nitrobenzanthrone (3-NBA) is a potent mutagen and a suspected human carcinogen present in particulate matter of diesel exhaust and ambient air pollution. Employing an assay with human p53 knock-in (Hupki) murine embryonic fibroblasts (HUFs), we examined p53 mutations induced by 3-NBA and its active metabolite, N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA). Twenty-nine immortalized cultures (cell lines) from 89 HUF primary cultures exposed at passage 1 for 5 days to 2 microM 3-NBA harboured 22 different mutations in the human DNA-binding domain sequence of the Hupki p53 tumour suppressor gene. The most frequently observed mutation was GC to TA transversion (46%), corroborating previous mutation studies with 3-NBA, and consistent with the presence of persistent 3-NBA-guanosine adducts found in DNA of exposed rodents. Six of the transversions found solely in 3-NBA-treated HUFs have not been detected thus far in untreated HUFs, but have been found repeatedly in human lung tumours. (32)P-post-labelling adduct analysis of DNA from HUF cells treated with 2 microM 3-NBA for 5 days showed a pattern similar to that found in vivo, indicating the metabolic competence of HUF cells to metabolize 3-NBA to electrophilic intermediates. Total DNA binding was 160 +/- 56 per 10(7) normal nucleotides with N(2)-guanosine being the major adduct. In contrast, identical treatment with N-OH-3-ABA resulted in a 100-fold lower level of specific DNA adducts and no carcinogen-specific mutation pattern in the Hupki assay. This indicates that the level of DNA adduct formation by the mutagen is critical to obtain specific mutation spectra in the assay. Our results are consistent with previous experiments in Muta Mouse and are compatible with the possibility that diesel exhaust exposure contributes to mutation load in humans and to lung cancer risk.

  8. Human T-Cell Lymphotropic/Leukemia Virus Type 1 Tax Abrogates p53-Induced Cell Cycle Arrest and Apoptosis through Its CREB/ATF Functional Domain

    PubMed Central

    Mulloy, J. C.; Kislyakova, T.; Cereseto, A.; Casareto, L.; LoMonico, A.; Fullen, J.; Lorenzi, M. V.; Cara, A.; Nicot, C.; Giam, C.-Z.; Franchini, G.

    1998-01-01

    Human T-cell lymphotropic/leukemia virus type 1 (HTLV-1) transforms human T cells in vitro, and Tax, a potent transactivator of viral and cellular genes, plays a key role in cell immortalization. Tax activity is mediated by interaction with cellular transcription factors including members of the CREB/ATF family, the NF-κB/c-Rel family, serum response factor, and the coactivators CREB binding protein-p300. Although p53 is usually not mutated in HTLV-1-infected T cells, its half-life is increased and its function is impaired. Here we report that transient coexpression of p53 and Tax results in the suppression of p53 transcriptional activity. Expression of Tax abrogates p53-induced G1 arrest in the Calu-6 cell line and prevents the apoptosis induced by overexpressing p53 in the HeLa/Tat cell line. The Tax mutants M22 and G148V, which selectively activate the CREB/ATF pathway, exert these same biological effects on p53 function. In contrast, the NF-κB-active Tax mutant M47 has no effect on p53 activity in any of these systems. Consistent with the negative effect of Tax on p53, no activity on a p53-responsive promoter was observed upon transfection of HTLV-1-infected T-cell lines. The p53 protein is expressed at high levels in the nucleus, and nuclear extracts of HTLV-1-infected T cells bind constitutively to a DNA oligonucleotide containing the p53 response element, indicating that Tax does not interfere with p53 binding to DNA. Tax is able to suppress the transactivation function of p53 in three different cell lines, and this suppression required Tax-mediated activation of the CREB/ATF, but not the NF-κB/c-Rel, pathway. Tax and the active Tax mutants were able to abrogate the G1 arrest and apoptosis induced by p53, and this effect does not correlate with an altered localization of nuclear p53 or with the disruption of p53-DNA complexes. The suppression of p53 activity by Tax could be important in T-cell immortalization induced by HTLV-1. PMID:9765430

  9. Differences in the Ability of Human T-Cell Lymphotropic Virus Type 1 (HTLV-1) and HTLV-2 Tax To Inhibit p53 Function

    PubMed Central

    Mahieux, Renaud; Pise-Masison, Cynthia A.; Lambert, Paul F.; Nicot, Christophe; De Marchis, Laura; Gessain, Antoine; Green, Patrick; Hall, William; Brady, John N.

    2000-01-01

    We have analyzed the functional activity of the p53 tumor suppressor in human T-cell lymphotropic virus type 2 (HTLV-2)-transformed cells. Abundant levels of the p53 protein were detected in both HTLV-2A and -2B virus-infected cell lines. The p53 was functionally inactive, however, both in transient-transfection assays using a p53 reporter plasmid and in induction of p53-responsive genes in response to gamma irradiation. We further investigated HTLV-2A Tax and HTLV-2B Tax effects on p53 activity. Interestingly, although Tax-2A and -2B inactivate p53, the Tax-2A protein appears to inhibit p53 function less efficiently than either Tax-1 or Tax-2B. In transient-cotransfection assays, Tax-1 and Tax-2B inactivated p53 by 80%, while Tax2A reduced p53 activity by 20%. In addition, Tax-2A does not increase the steady-state level of cellular p53 as well as Tax-1 or -2B does in the same assays. Cotransfection assays demonstrated that Tax-2A could efficiently transactivate CREB-responsive promoters to the same level as Tax-1 and Tax-2B, indicating that the protein was functional. This report provides evidence of the first functional difference between the HTLV-2A and -2B subtypes. This comparison of the action of HTLV-1 and HTLV-2 Tax proteins on p53 function will provide important insights into the mechanism of HTLV transformation. PMID:10888626

  10. Differences in the ability of human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 tax to inhibit p53 function.

    PubMed

    Mahieux, R; Pise-Masison, C A; Lambert, P F; Nicot, C; De Marchis, L; Gessain, A; Green, P; Hall, W; Brady, J N

    2000-08-01

    We have analyzed the functional activity of the p53 tumor suppressor in human T-cell lymphotropic virus type 2 (HTLV-2)-transformed cells. Abundant levels of the p53 protein were detected in both HTLV-2A and -2B virus-infected cell lines. The p53 was functionally inactive, however, both in transient-transfection assays using a p53 reporter plasmid and in induction of p53-responsive genes in response to gamma irradiation. We further investigated HTLV-2A Tax and HTLV-2B Tax effects on p53 activity. Interestingly, although Tax-2A and -2B inactivate p53, the Tax-2A protein appears to inhibit p53 function less efficiently than either Tax-1 or Tax-2B. In transient-cotransfection assays, Tax-1 and Tax-2B inactivated p53 by 80%, while Tax2A reduced p53 activity by 20%. In addition, Tax-2A does not increase the steady-state level of cellular p53 as well as Tax-1 or -2B does in the same assays. Cotransfection assays demonstrated that Tax-2A could efficiently transactivate CREB-responsive promoters to the same level as Tax-1 and Tax-2B, indicating that the protein was functional. This report provides evidence of the first functional difference between the HTLV-2A and -2B subtypes. This comparison of the action of HTLV-1 and HTLV-2 Tax proteins on p53 function will provide important insights into the mechanism of HTLV transformation.

  11. p53 amplifies Toll-like receptor 5 response in human primary and cancer cells through interaction with multiple signal transduction pathways

    PubMed Central

    Shatz, Maria; Shats, Igor; Menendez, Daniel; Resnick, Michael A.

    2015-01-01

    The p53 tumor suppressor regulates transcription of genes associated with diverse cellular functions including apoptosis, growth arrest, DNA repair and differentiation. Recently, we established that p53 can modulate expression of Toll-like receptor (TLR) innate immunity genes but the degree of cross-talk between p53 and TLR pathways remained unclear. Here, using gene expression profiling we characterize the global effect of p53 on the TLR5-mediated transcription in MCF7 cells. We found that combined activation of p53 and TLR5 pathways synergistically increases expression of over 200 genes, mostly associated with immunity and inflammation. The synergy was observed in several human cancer cells and primary lymphocytes. The p53-dependent amplification of transcriptional response to TLR5 activation required expression of NFκB subunit p65 and was mediated by several molecular mechanisms including increased phosphorylation of p38 MAP kinase, PI3K and STAT3 signaling. Additionally, p53 induction increased cytokine expression in response to TNFα, another activator of NFκB and MAP kinase pathways, suggesting a broad interaction between p53 and these signaling pathways. The expression of many synergistically induced genes is elevated in breast cancer patients responsive to chemotherapy. We suggest that p53's capacity to enhance immune response could be exploited to increase antitumor immunity and to improve cancer treatment. PMID:26220208

  12. The role of tumor protein 53 mutations in common human cancers and targeting the murine double minute 2-p53 interaction for cancer therapy.

    PubMed

    Hamzehloie, Tayebeh; Mojarrad, Majid; Hasanzadeh Nazarabadi, Mohammad; Shekouhi, Sahar

    2012-03-01

    The gene TP53 (also known as protein 53 or tumor protein 53), encoding transcription factor P53, is mutated or deleted in half of human cancers, demonstrating the crucial role of P53 in tumor suppression. There are reports of nearly 250 independent germ line TP53 mutations in over 100 publications. The P53 protein has the structure of a transcription factor and, is made up of several domains. The main function of P53 is to organize cell defense against cancerous transformation. P53 is a potent transcription factor that is activated in response to diverse stresses, leading to the induction of cell cycle arrest, apoptosis or senescence. The P53 tumor suppressor is negatively regulated in cells by the murine double minute 2 (MDM2) protein. Murine double minute 2 favors its nuclear export, and stimulates its degradation. Inhibitors of the P53-MDM2 interaction might be attractive new anticancer agents that could be used to activate wild-type P53 in tumors. Down regulation of MDM2 using an small interfering RNA (siRNA) approach has recently provided evidence for a new role of MDM2 in the P53 response, by modulating the inhibition of the cyclindependent kinase 2 (cdk2) by P21/WAF1 (also known as cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1).

  13. Depletion of Securin Induces Senescence After Irradiation and Enhances Radiosensitivity in Human Cancer Cells Regardless of Functional p53 Expression

    SciTech Connect

    Chen Wenshu; Yu Yichu; Lee Yijang; Chen, J.-H.; Hsu, H.-Y.; Chiu, S.-J.

    2010-06-01

    Purpose: Radiotherapy is one of the best choices for cancer treatment. However, various tumor cells exhibit resistance to irradiation-induced apoptosis. The development of new strategies to trigger cancer cell death besides apoptosis is necessary. This study investigated the role of securin in radiation-induced apoptosis and senescence in human cancer cells. Methods and Materials: Cell survival was determined using clonogenic assays. Western blot analysis was used to analyze levels of securin, caspase-3, PARP, p53, p21, Rb, gamma-H2AX, and phospho-Chk2. Senescent cells were analyzed using a beta-galactosidase staining assay. A securin-expressed vector (pcDNA-securin) was stably transfected into securin-null HCT116 cells. Securin gene knockdown was performed by small interfering RNA and small hairpin RNA in HCT116 and MDA-MB-231 cells, respectively. Results: Radiation was found to induce apoptosis in securin wild type HCT116 cells but induced senescence in securin-null cells. Restoration of securin reduced senescence and increased cell survival in securin-null HCT116 cells after irradiation. Radiation-induced gamma-H2AX and Chk2 phosphorylation were induced transiently in securin-wild-type cells but exhibited sustained activation in securin-null cells. Securin gene knockdown switches irradiation-induced apoptosis to senescence in both HCT116 p53-null and MDA-MB-231 cells. Conclusions: Our results demonstrated that the level of securin expression plays a determining role in the radiosensitivity and fate of cells. Depletion of securin impairs DNA repair after irradiation, increasing DNA damage and promoting senescence in the residual surviving cells regardless of functional p53 expression. The knockdown of securin may contribute to a novel radiotherapy protocol for the treatment of human cancer cells that are resistant to irradiation.

  14. p53 is preferentially recruited to the promoters of growth arrest genes p21 and GADD45 during replicative senescence of normal human fibroblasts.

    PubMed

    Jackson, James G; Pereira-Smith, Olivia M

    2006-09-01

    Replicative senescence is the terminal growth arrest that most normal human cells enter into after a fixed number of divisions in vitro, limiting the proliferative potential of a cell and preventing genomic instability caused by critically short telomeres. Thus, senescence presents a tumor-suppressive mechanism and a barrier to tumor formation. However, senescent cells are inherently resistant to apoptosis and, as they accumulate in aging tissues, may contribute to organ dysfunction and promote tumor progression as part of the stromal environment. Replicative life span in normal human cells can be extended by inactivation of the tumor suppressor gene p53 or its direct target, the cyclin-dependent kinase inhibitor p21, suggesting a direct role for this pathway in senescence. However, p53 recruitment to promoters of target genes during replicative senescence has not been shown in live cells. In this study, we used chromatin immunoprecipitation to determine that p53 preferentially occupied the promoters of growth arrest genes p21 and GADD45 in senescent normal human diploid fibroblasts but not the promoters of other target genes that recruited p53 following doxorubicin-induced DNA damage, such as apoptosis regulators TNFRSF10b, TNFRSF6, and PUMA. This differential recruitment of p53 in senescent versus doxorubicin-treated fibroblasts was accompanied by differences in post-translational modification of p53. These data provide mechanisms for both the growth arrest mediated by p53 and the resistant nature of senescent cells to apoptosis despite p53 activity.

  15. Human thyroid cancer cells as a source of iso-genic, iso-phenotypic cell lines with or without functional p53

    PubMed Central

    Wyllie, F S; Haughton, M F; Rowson, J M; Wynford-Thomas, D

    1999-01-01

    Differentiated thyroid carcinomas (in contrast to the rarer anaplastic form) are unusual among human cancers in displaying a remarkably low frequency of p53 mutation and appear to retain wild-type (wt) p53 function as assessed by the response of derived cell lines to DNA damage. Using one such cell line, K1, we have tested the effect of experimental abrogation of p53 function by generating matched sub-clones stably expressing either a neo control gene, a dominant-negative mutant p53 (143ala) or human papilloma virus protein HPV16 E6. Loss of p53 function in the latter two groups was confirmed by abolition of p53-dependent ‘stress’ responses including induction of the cyclin/CDK inhibitor p21WAF1 and G1/S arrest following DNA-damage. In contrast, no change was detected in the phenotype of ‘unstressed’ clones, with respect to any of the following parameters: proliferation rate in monolayer, serum-dependence for proliferation or survival, tumorigenicity, cellular morphology, or tissue-specific differentiation markers. The K1 line therefore represents a ‘neutral’ background with respect to p53 function, permitting the derivation of functionally p53 + or − clones which are not only iso-genic but also iso-phenotypic. Such a panel should be an ideal tool with which to test the p53-dependence of cellular stress responses, particularly the sensitivity to potential therapeutic agents, free from the confounding additional phenotypic differences which usually accompany loss of p53 function. The results also further support the hypothesis that p53 mutation alone is not sufficient to drive progression of thyroid cancer to the aggressive anaplastic form. © 1999 Cancer Research Campaign PMID:10098744

  16. Kaempferol induces ATM/p53-mediated death receptor and mitochondrial apoptosis in human umbilical vein endothelial cells.

    PubMed

    Lee, Chiu-Fang; Yang, Jai-Sing; Tsai, Fuu-Jen; Chiang, Ni-Na; Lu, Chi-Cheng; Huang, Yu-Syuan; Chen, Chun; Chen, Fu-An

    2016-05-01

    Kaempferol is a member of the flavonoid compounds found in vegetables and fruits. It is shown to exhibit biological impact and anticancer activity, but no report exists on the angiogenic effect of kaempferol and induction of cell apoptosis in vitro. In this study, we investigated the role of kaempferol on anti-angiogenic property and the apoptotic mechanism of human umbilical vein endothelial cells (HUVECs). Our results demonstrated that kaempferol decreased HUVEC viability in a time- and concentration-dependent manner. Kaempferol also induced morphological changes and sub-G1 phase cell population (apoptotic cells). Kaempferol triggered apoptosis of HUVECs as detecting by DNA fragmentation, comet assay and immunofluorescent staining for activated caspase-3. The caspase signals, including caspase-8, -9 and -3, were time-dependently activated in HUVECs after kaempferol exposure. Furthermore, pre-treatment with a specific inhibitor of caspase-8 (Z-IETD-FMK) significantly reduced the activity of caspase-8, -9 and -3, indicating that extrinsic pathway is a major signaling pathway in kaempferol-treated HUVECs. Importantly, kaempferol promoted reactive oxygen species (ROS) evaluated using flow cytometric assay in HUVECs. We further investigated the upstream extrinsic pathway and showed that kaempferol stimulated death receptor signals [Fas/CD95, death receptor 4 (DR4) and DR5] through increasing the levels of phosphorylated p53 and phosphorylated ATM pathways in HUVECs, which can be individually confirmed by N-acetylcysteine (NAC), ATM specific inhibitor (caffeine) and p53 siRNA. Based on these results, kaempferol-induced HUVEC apoptosis was involved in an ROS-mediated p53/ATM/death receptor signaling. Kaempferol might possess therapeutic effects on cancer treatment in anti-vascular targeting.

  17. Addition of TAT protein transduction domain and GrpE to human p53 provides soluble fusion proteins that can be transduced into dendritic cells and elicit p53-specific T-cell responses in HLA-A*0201 transgenic mice

    PubMed Central

    Justesen, S; Buus, S; Claesson, M H; Pedersen, A E

    2007-01-01

    The protein p53 has been shown to be an efficient tumour antigen in both murine and human cancer vaccine studies and cancer vaccines targeting p53 based on major histocompatibility complex (MHC) class I binding p53-derived peptides that induce cytotoxic T lymphocytes (CTLs) without p53-specific CD4+ T-cell help have been tested by several research groups including ours. To obtain such CD4+ T-cell help and cover a broader repertoire of MHC haplotypes we have previously attempted to produce recombinant human p53 for vaccination purposes. However, attempts to refold a hexahis-tagged p53 protein in our laboratory were unsuccessful. Here, we show that fusion of an 11-amino-acid region of the human immunodeficiency virus TAT protein transduction domain (PTD) to human p53 increases the solubility of the otherwise insoluble p53 protein and this rTAT-p53 protein can be transduced into human monocyte-derived dendritic cells (DCs). The induction of a p53-specific HLA-A*0201 immune response was tested in HLA-A*0201/Kb transgenic mice after immunization with rTAT-p53-transduced bone-marrow-derived DCs. In these mice, p53-specific CD4+ and CD8+ T-cell proliferation was observed and immunization resulted in the induction of HLA-A*0201-restricted CTLs specific for two human p53-derived HLA-A*0201-binding peptides, p5365−73 and p53149−157. Addition of GrpE to generate rTAT-GrpE-p53 led to a further increase in protein solubility and to a small increase in DC maturation but did not increase the observed p53-specific T-cell responses. The use of rTAT-p53 in ongoing clinical protocols should be applicable and offers advantages to current strategies omitting the use of HLA-typed patients. PMID:17610503

  18. Perfluorooctanoic acid induces apoptosis through the p53-dependent mitochondrial pathway in human hepatic cells: a proteomic study.

    PubMed

    Huang, Qingyu; Zhang, Jie; Martin, Francis L; Peng, Siyuan; Tian, Meiping; Mu, Xiaoli; Shen, Heqing

    2013-11-25

    Perfluorooctanoic acid (PFOA) is one of the most commonly used perfluorinated compounds, and exposure to it has been associated with a number of adverse health effects. However, the molecular mechanisms involved in PFOA toxicity are still not well characterized. In the present study, flow cytometry analysis revealed that PFOA induced oxidative stress, cell cycle arrest and apoptosis in human non-tumor hepatic cells (L-02). Furthermore, we investigated the alterations in protein profile within L-02 cells exposed to PFOA, aiming to explore the mechanisms underlying PFOA hepatotoxicity on the proteome level. Of the 28 proteins showing significant differential expression in response to PFOA, 24 were down-regulated and 4 were up-regulated. This proteomic study proposed that the inhibition of some proteins, including GRP78, HSP27, CTSD and hnRNPC may be involved in the activation of p53, which consequently triggered the apoptotic process in L-02 cells. Induction of apoptosis via the p53-dependent mitochondrial pathway is further suggested as one of the key toxicological events occurring in L-02 cells under PFOA stress. We hope these data will shed new light on the molecular mechanisms responsible for PFOA-mediated toxicity in human liver cells, and from such studies useful biomarkers indicative of PFOA exposure could be developed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. p53 missense but not truncation mutations are associated with low levels of p21CIP1/WAF1 mRNA expression in primary human sarcomas

    PubMed Central

    Mousses, S; Gokgoz, N; Wunder, J S; Ozcelik, H; Bull, S; Bell, R S; Andrulis, I L

    2001-01-01

    Many growth-suppressing signals converge to control the levels of the CDK inhibitor p21CIP1/WAF1. Some human cancers exhibit low levels of expression of p21CIP1/WAF1and mutations in p53 have been implicated in this down-regulation. To evaluate whether the presence of p53 mutations was related to the in vivo expression of p21CIP1/WAF1 mRNA in sarcomas we measured the p21CIP1/WAF1 mRNA levels for a group of 71 primary bone and soft tissue tumours with known p53 status. As expected, most tumours with p53 mutations expressed low levels of p21CIP1/WAF1 mRNA. However, we identified a group of tumours with p53 gene mutations that exhibited normal or higher levels of p21CIP1/WAF1 mRNA. The p53 mutations in the latter group were not the common missense mutations in exons 4–9, but were predominantly nonsense mutations predicted to result in truncation of the p53 protein. The results of this study suggest that different types of p53 mutations can have different effects on the expression of downstream genes such as p21CIP1/WAF1 in human sarcomas. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11401317

  20. Effect of an hdm-2 antagonist peptide inhibitor on cell cycle progression in p53-deficient H1299 human lung carcinoma cells.

    PubMed

    VanderBorght, A; Valckx, A; Van Dun, J; Grand-Perret, T; De Schepper, S; Vialard, J; Janicot, M; Arts, J

    2006-10-26

    The hdm-2 oncogene is overexpressed in several types of malignancies including osteosarcomas, soft tissue sarcomas and gliomas and hdm-2 has been associated with accelerated tumor formation in both hereditary and sporadic cancers. Among the other key binding partners, hdm-2 forms a complex with the tumor suppressor p53, resulting in a rapid proteasome-mediated degradation of the p53 protein. This positions the hdm-2-p53 complex as an attractive target for the development of anticancer therapy and recently the first small molecule hdm-2 antagonist has been reported. Development of hdm-2 antagonists is currently focused on malignancies containing a wild-type p53 genotype, which is the case in approximately half of human cancer indications. However, hdm-2 has also been implicated in oncogenesis in the absence of p53. We therefore studied the effect of hdm-2 antagonists in p53-deficient human H1299 lung carcinoma cells. The hdm-2 antagonistic peptide caused G1 cell cycle arrest, inhibited colony growth and induced expression of G1 checkpoint regulatory proteins, such as p21(waf1,cip1). These data demonstrate that hdm-2 regulates the G1 cell cycle checkpoint in a p53-independent manner, suggesting that hdm-2 antagonists represent a novel class of anticancer therapeutics with broad applicability towards tumors with different p53 genetic backgrounds.

  1. MicroRNA-1301 suppresses tumor cell migration and invasion by targeting the p53/UBE4B pathway in multiple human cancer cells.

    PubMed

    Wang, Benfan; Wu, Hong; Chai, Chengsen; Lewis, John; Pichiorri, Flavia; Eisenstat, David D; Pomeroy, Scott L; Leng, Roger P

    2017-08-10

    The p53 protein plays a critical role in preventing tumor development. Although numerous factors have been shown to directly or indirectly regulate p53, the mechanism of how microRNAs (miRNAs) modulate p53 remains unclear. Here, we identified miR-1301, a microRNA that regulates the activity and function of p53, by directly targeting the ubiquitination factor E4B (UBE4B), an E3 and E4 ubiquitin ligase. Notably, ectopic expression of miR-1301 inhibits dissemination and metastasis of tumor cells in a p53-dependent manner. Depletion of miR-1301 downregulates p53 function. Our results reveal that there is an inverse correlation between miR-1301 and UBE4B expression and p53 status in prostate cancer. Furthermore, low miR-1301 expression is often associated with incomplete methylation of its gene in human prostate tumors. Together, our results provide the first report indicating that miR-1301 functions as a tumor suppressor that inhibits tumor cell migration and invasion in multiple human cancer cells by regulating the UBE4B-p53 pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. UVB-mediated activation of p38 mitogen-activated protein kinase enhances resistance of normal human keratinocytes to apoptosis by stabilizing cytoplasmic p53.

    PubMed

    Chouinard, Nadine; Valerie, Kristoffer; Rouabhia, Mahmoud; Huot, Jacques

    2002-07-01

    Human keratinocytes respond to UV rays by developing a fast adaptive response that contributes to maintaining their functions and survival. We investigated the role of the mitogen-activated protein kinase pathways in transducing the UV signals in normal human keratinocytes. We found that UVA, UVB or UVC induced a marked and persistent activation of p38, whereas c-Jun N-terminal kinase or extracellular signal-regulated kinase were less or not activated respectively. Inhibition of p38 activity by expression of a dominant-negative mutant of p38 or with SB203580 impaired cell viability and led to an increase in UVB-induced apoptosis. This sensitization to apoptosis was independent of caspase activities. Inhibition of p38 did not sensitize transformed HaCaT keratinocytes to UVB-induced apoptosis. In normal keratinocytes, expression of a dominant-negative mutant of p53 increased UVB-induced cell death, pointing to a role for p53. In these cells, UVB triggered a p38-dependent phosphorylation of p53 on Ser-15. This phosphorylation was associated with an SB203580-sensitive accumulation of p53, even in the presence of a serine phosphatase inhibitor. Accumulated p53 was localized mainly in the cytoplasm, independently of CRM1 nuclear export. In HaCaT cells, p53 was localized exclusively in the nucleus and its distribution and level were not affected by UVB or p38 inhibition. However, UVB induced an SB203580-insensitive phosphorylation on Ser-15 of mutated p53. Overall, our results suggest that, in normal human keratinocytes, protection against UVB depends on p38-mediated phosphorylation and stabilization of p53 and is tightly associated with the cytoplasmic sequestration of wild-type p53. We conclude that the p38/p53 pathway plays a key role in the adaptive response of normal human keratinocytes against UV stress.

  3. Control of keratinocyte proliferation and differentiation by p63.

    PubMed

    Truong, Amy B; Khavari, Paul A

    2007-02-01

    The p53 family member p63 has been implicated in both the development and maintenance of stratified epithelial tissues, including the epidermis. Increasing data support p63 function in the regenerative capacity of basal keratinocytes by maintaining cell proliferation. Recent studies further suggest this regulation relies on inhibition of p53 activity. In addition, p63 appears to exert separate control over epidermal differentiation, which may involve control of such key signaling molecules as IKKalpha and Notch. While studies over the past decade have greatly expanded our knowledge of p63 function, much remains to be understood regarding how p63 regulates epidermal homeostasis. Future efforts to identify and validate direct p63 target genes as well as to understand the expression and function of individual p63 isoforms will be important to further define how p63 functions in the control of keratinocyte proliferation and differentiation.

  4. Modulation of the DNA damage response in UV-exposed human lymphoblastoid cells through genetic-versus functional-inactivation of the p53 tumor suppressor.

    PubMed

    Léger, Caroline; Drobetsky, Elliot A

    2002-10-01

    The global cellular response to UV-induced DNA damage has been analyzed in the p53-proficient human lymphoblastoid strain TK6 versus two isogenic derivatives wherein p53 activity was abrogated by diverse experimental approaches: (i) NH32, carrying a homozygous genetic knockout of p53; and (ii) TK6-5E, expressing the human papillomavirus E6 oncoprotein which binds and functionally inactivates p53 protein. Although widely employed as such, the extent to which intracellular E6 expression faithfully models the p53 deficient state still remains uncertain. Following irradiation with UV (either monochromatic 254 nm UV or broad-spectrum simulated sunlight), relative to wild-type TK6, p53-null NH32 exhibited virtually identical clonogenic survival and kinetics of G1-S progression but was nonetheless profoundly resistant to apoptosis. In addition, there were significant qualitative and quantitative differences between NH32 and TK6 with respect to UV mutagenesis at the endogenous hypoxanthine phosphoribosyltransferase (hprt) locus. However, important disparities were observed between genetically p53-deficient NH32 and E6-expressing TK6-5E regarding the manner in which they responded to UV-induced genotoxic stress in relation to wild-type TK6. Indeed, although NH32 and TK6-5E behaved similarly with respect to UV mutagenesis at the hprt locus, there were significant differences between these strains in clonogenic survival, apoptosis, and G1-S progression. Using a well-defined isogenic system, our data clearly reveal the influence of p53 inactivation on the global response of human cells to UV-induced DNA damage, and highlight an important caveat in the field of p53 biology by directly demonstrating that this influence varies substantially depending upon whether p53 function is abrogated genetically, or through E6 oncoprotein expression.

  5. Antiproliferation and apoptosis induced by tamoxifen in human bile duct carcinoma QBC939 cells via upregulated p53 expression

    SciTech Connect

    Han, Peng; Kang, Jin-He; Li, Hua-Liang; Hu, Su-Xian; Lian, Hui-Hui; Qiu, Ping-Ping; Zhang, Jian; Li, Wen-Gang; Chen, Qing-Xi

    2009-07-24

    Tamoxifen (TAM) is a nonsteroidal antiestrogen that has been used in the treatment of breast cancer for over 30 years. Recently, it was shown that TAM also has efficacy on gastrointestinal neoplasms such as hepatocarcinoma and pancreatic carcinoma, and that the chemopreventive activities of TAM might be due to its abilities to inhibit cell growth and induce apoptosis. In the present study, we investigated the effects of tamoxifen on growth and apoptosis in the human bile duct carcinoma (BDC) cell line QBC939 using MTT assay, inverted microscopy, fluorescence microscopy, transmission electron microscopy, classic DNA fragmentation agarose gel electrophoresis assay, PI single- and FITC/PI double-staining flow cytometry, and Western blotting. Our data revealed that TAM could significantly inhibit growth and induce apoptosis in QBC939 cells. Increased expression of p53 was observed in TAM-treated cells, indicating that p53 might play an important role in TAM-induced apoptosis in QBC939 cells. These results provide significant insight into the anticarcinogenic action of TAM on BDC.

  6. Biological functions of p53 isoforms through evolution: lessons from animal and cellular models.

    PubMed

    Marcel, V; Dichtel-Danjoy, M-L; Sagne, C; Hafsi, H; Ma, D; Ortiz-Cuaran, S; Olivier, M; Hall, J; Mollereau, B; Hainaut, P; Bourdon, J-C

    2011-12-01

    The TP53 tumour-suppressor gene is expressed as several protein isoforms generated by different mechanisms, including use of alternative promoters, splicing sites and translational initiation sites, that are conserved through evolution and within the TP53 homologues, TP63 and TP73. Although first described in the eighties, the importance of p53 isoforms in regulating the suppressive functions of p53 has only become evident in the last 10 years, by analogy with observations that p63 and p73 isoforms appeared indispensable to fully understand the biological functions of TP63 and TP73. This review summarizes recent advances in the field of 'p53 isoforms', including new data on p63 and p73 isoforms. Details of the alternative mechanisms that produce p53 isoforms and cis- and trans-regulators identified are provided. The main focus is on their biological functions (apoptosis, cell cycle, aging and so on) in cellular and animal models, including mouse, zebrafish and Drosophila. Finally, the deregulation of p53 isoform expression in human cancers is reviewed. Based on these latest results, several developments are expected in the future: the identification of drugs modulating p53 isoform expression; the generation of animal models and the evaluation of the use of p53 isoform as biomarkers in human cancers.

  7. Mutant p53 can induce tumorigenic conversion of human bronchial epithelial cells and reduce their responsiveness to a negative growth factor, transforming growth factor beta 1.

    PubMed Central

    Gerwin, B I; Spillare, E; Forrester, K; Lehman, T A; Kispert, J; Welsh, J A; Pfeifer, A M; Lechner, J F; Baker, S J; Vogelstein, B

    1992-01-01

    Loss of normal functions and gain of oncogenic functions when the p53 tumor suppressor gene is mutated are considered critical events in the development of the majority of human cancers. Human bronchial epithelial cells (BEAS-2B) provide an in vitro model system to study growth, differentiation, and neoplastic transformation of progenitor cells of lung carcinoma. When wild-type (WT) or mutant (MT; codon 143Val-Ala) human p53 cDNA was transfected into nontumorigenic BEAS-2B cells, we observed that (i) transfected WT p53 suppresses and MT p53 enhances the colony-forming efficiency of these cells, (ii) MT p53 increases resistance to transforming growth factor beta 1, and (iii) clones of MT p53 transfected BEAS-2B cells are tumorigenic when inoculated into athymic nude mice. These results are consistent with the hypothesis that certain mutations in p53 may function in multistage lung carcinogenesis by reducing the responsiveness of bronchial epithelial cells to negative growth factors. Images PMID:1557382

  8. R280T mutation of p53 gene promotes proliferation of human glioma cells through GSK-3β/PTEN pathway.

    PubMed

    Lin, Chenli; Liang, Yinji; Zhu, Huili; Zhang, Jijun; Zhong, Xueyun

    2012-10-31

    p53 mutation is associated with "gain-of-function" capabilities of human cancers. We aim to identify p53 mutations in human glioma cells and to explore the potential mechanism for mutant p53-promoted cellular growth. Whole genomic DNA was isolated from SWO-38, a human glioma cell line and amplified for the region of exons 5, 6, and 8 in p53 gene using polymerase chain reaction (PCR). By means of direct sequencing of PCR products and alignment analysis using BLAST database, a mutation of G to C transition at codon 280 of p53 exon 8 (AGA→ACA), i.e. R280T was detected in SWO-38 cells. Knockdown of R280T mutant p53 by RNA interference inhibited the GSK-3β/PTEN associated cell proliferation, and PI3K/Akt but not Wnt/β-catenin signaling pathway was involved in this process. Furthermore, depletion or overexpression of PTEN alone did not affect cell proliferation and cell cycle, implicating the impairment of PTEN function in SWO-38 cells. However, knockdown of both PTEN and p53 mutation could significantly rescue the p53 depletion-mediated growth inhibition, suggesting that the R280T mutation in glioma may promote the proliferation through an underlying mechanism related to PTEN. Our observations indicate that the R280T mutation of p53 regulates the proliferation of human glioma cells related to the GSK-3β/PTEN pathway. These findings provide valuable insights for better understanding the molecular mechanism of uncontrolled growth of glioma cells.

  9. Hypoxia induces p53 accumulation in the S-phase and accumulation of hypophosphorylated retinoblastoma protein in all cell cycle phases of human melanoma cells.

    PubMed Central

    Danielsen, T.; Hvidsten, M.; Stokke, T.; Solberg, K.; Rofstad, E. K.

    1998-01-01

    Hypoxia has been shown to induce accumulation of p53 and of hypophosphorylated retinoblastoma protein (pRb) in tumour cells. In this study, the cell cycle dependence of p53 accumulation and pRb hypophosphorylation in four human melanoma cell lines that are wild type for p53 was investigated using two-parameter flow cytometry measurements of p53 or pRb protein content and DNA content. The hypoxia-induced increase in p53 protein was higher in S-phase than in G1 and G2 phases in all cell lines. The accumulation of p53 in S-phase during hypoxia was not related to hypoxia-induced apoptosis or substantial cell cycle specific cell inactivation during the first 24 h of reoxygenation. pRb was hypophosphorylated in all cell cycle phases by hypoxia treatment. The results did not support a direct link between p53 and pRb during hypoxia because p53 was induced in a cell cycle-specific manner, whereas no cell cycle-dependent differences in pRb hypophosphorylation were detected. Only a fraction of the cell populations (0.60+/-0.10) showed hypophosphorylated pRb. Thus, pRb is probably not the only mediator of the hypoxia-induced cell cycle block seen in all cells and all cell cycle phases. Moreover, the cell cycle-dependent induction of p53 by hypoxia suggests that the primary function of p53 accumulation during hypoxia is other than to arrest the cells. Images Figure 4 Figure 7 PMID:9862563

  10. Human p53 interacts with the elongating RNAPII complex and is required for the release of actinomycin D induced transcription blockage

    PubMed Central

    Borsos, Barbara N.; Huliák, Ildikó; Majoros, Hajnalka; Ujfaludi, Zsuzsanna; Gyenis, Ákos; Pukler, Peter; Boros, Imre M.; Pankotai, Tibor

    2017-01-01

    The p53 tumour suppressor regulates the transcription initiation of selected genes by binding to specific DNA sequences at their promoters. Here we report a novel role of p53 in transcription elongation in human cells. Our data demonstrate that upon transcription elongation blockage, p53 is associated with genes that have not been reported as its direct targets. p53 could be co-immunoprecipitated with active forms of DNA-directed RNA polymerase II subunit 1 (RPB1), highlighting its association with the elongating RNA polymerase II. During a normal transcription cycle, p53 and RPB1 are localised at distinct regions of selected non-canonical p53 target genes and this pattern of localisation was changed upon blockage of transcription elongation. Additionally, transcription elongation blockage induced the proteasomal degradation of RPB1. Our results reveal a novel role of p53 in human cells during transcription elongation blockage that may facilitate the removal of RNA polymerase II from DNA. PMID:28102346

  11. Cellular transcriptional profiling in human lung epithelial cells infected by different subtypes of influenza A viruses reveals an overall down-regulation of the host p53 pathway

    PubMed Central

    2011-01-01

    Background Influenza viruses can modulate and hijack several cellular signalling pathways to efficiently support their replication. We recently investigated and compared the cellular gene expression profiles of human lung A549 cells infected by five different subtypes of human and avian influenza viruses (Josset et al. Plos One 2010). Using these transcriptomic data, we have focused our analysis on the modulation of the p53 pathway in response to influenza infection. Results Our results were supported by both RT-qPCR and western blot analyses and reveal multiple alterations of the p53 pathway during infection. A down-regulation of mRNA expression was observed for the main regulators of p53 protein stability during infection by the complete set of viruses tested, and a significant decrease in p53 mRNA expression was also observed in H5N1 infected cells. In addition, several p53 target genes were also down-regulated by these influenza viruses and the expression of their product reduced. Conclusions Our data reveal that influenza viruses cause an overall down-regulation of the host p53 pathway and highlight this pathway and p53 protein itself as important viral targets in the altering of apoptotic processes and in cell-cycle regulation. PMID:21651802

  12. Preventive effects of epigallocatechin-3-O-gallate against replicative senescence associated with p53 acetylation in human dermal fibroblasts.

    PubMed

    Han, Dong-Wook; Lee, Mi Hee; Kim, Bongju; Lee, Jun Jae; Hyon, Suong-Hyu; Park, Jong-Chul

    2012-01-01

    Considering the various pharmacological activities of epigallocatechin-3-O-gallate (EGCG) including anticancer, and anti-inflammatory, antidiabetic, and so forth, relatively less attention has been paid to the antiaging effect of EGCG on primary cells. In this study, the preventive effects of EGCG against serial passage-induced senescence were investigated in primary cells including rat vascular smooth muscle cells (RVSMCs), human dermal fibroblasts (HDFs), and human articular chondrocytes (HACs). The involvement of Sirt1 and acetylated p53 was examined as an underlying mechanism for the senescence preventive activity of EGCG in HDFs. All cells were employed with the initial passage number (PN) between 3 and 7. For inducing senescence, the cells were serially passaged at the predetermined times and intervals in the absence or presence of EGCG (50 or 100 μM). Serial passage-induced senescence in RVSMCs and HACs was able to be significantly prevented at 50 μM EGCG, while in HDFs, 100 μM EGCG could significantly prevent senescence and recover their cell cycle progression close to the normal level. Furthermore, EGCG was found to prevent serial passage- and H(2)O(2)-induced senescence in HDFs by suppressing p53 acetylation, but the Sirt1 activity was unaffected. In addition, proliferating HDFs showed similar cellular uptake of FITC-conjugated EGCG into the cytoplasm with their senescent counterparts but different nuclear translocation of it from them, which would partly account for the differential responses to EGCG in proliferating versus senescent cells. Taking these results into consideration, it is suggested that EGCG may be exploited to craft strategies for the development of an antiaging or age-delaying agent.

  13. Deficiency in p53 is required for doxorubicin induced transcriptional activation of NF-κB target genes in human breast cancer

    PubMed Central

    Dalmases, Alba; González, Irene; Menendez, Silvia; Arpí, Oriol; Corominas, Josep Maria; Servitja, Sonia; Tusquets, Ignasi; Chamizo, Cristina; Rincón, Raúl; Espinosa, Lluis; Bigas, Anna; Eroles, Pilar; Furriol, Jessica; Lluch, Anna; Rovira, Ana; Albanell, Joan; Rojo, Federico

    2014-01-01

    NF-κB has been linked to doxorubicin resistance in breast cancer patients. NF-κB nuclear translocation and DNA binding in doxorubicin treated-breast cancer cells have been extensively examined; however its functional relevance at transcriptional level on NF-κB -dependent genes and the biological consequences are unclear. We studied NF-κB -dependent gene expression induced by doxorubicin in breast cancer cells and fresh human cancer specimens with different genetic backgrounds focusing on their p53 status. NF-κB -dependent signature of doxorubicin was identified by gene expression microarrays in breast cancer cells treated with doxorubicin and the IKKβ-inhibitor MLN120B, and confirmed ex vivo in human cancer samples. The association with p53 was functionally validated. Finally, NF-κB activation and p53 status was determined in a cohort of breast cancer patients treated with adjuvant doxorubicin-based chemotherapy. Doxorubicin treatment in the p53-mutated MDA-MB-231 cells resulted in NF NF-κB driven-gene transcription signature. Modulation of genes related with invasion, metastasis and chemoresistance (ICAM-1, CXCL1, TNFAIP3, IL8) were confirmed in additional doxorubicin-treated cell lines and fresh primary human breast tumors. In both systems, p53-defcient background correlated with the activation of the NF-κB -dependent signature. Furthermore, restoration of p53WT in the mutant p53 MDA-MB-231 cells impaired NF-κB driven transcription induced by doxorubicin. Moreover, a p53 deficient background and nuclear NF-κB /p65 in breast cancer patients correlated with reduced disease free-survival. This study supports that p53 deficiency is necessary for a doxorubicin driven NF-κB -response that limits doxorubicin cytotoxicity in breast cancer and is linked to an aggressive clinical behavior. PMID:24344116

  14. Deficiency in p53 is required for doxorubicin induced transcriptional activation of NF-кB target genes in human breast cancer.

    PubMed

    Dalmases, Alba; González, Irene; Menendez, Silvia; Arpí, Oriol; Corominas, Josep Maria; Servitja, Sonia; Tusquets, Ignasi; Chamizo, Cristina; Rincón, Raúl; Espinosa, Lluis; Bigas, Anna; Eroles, Pilar; Furriol, Jessica; Lluch, Anna; Rovira, Ana; Albanell, Joan; Rojo, Federico

    2014-01-15

    NF-кB has been linked to doxorubicin resistance in breast cancer patients. NF-кB nuclear translocation and DNA binding in doxorubicin treated-breast cancer cells have been extensively examined; however its functional relevance at transcriptional level on NF-кB-dependent genes and the biological consequences are unclear. We studied NF-кB-dependent gene expression induced by doxorubicin in breast cancer cells and fresh human cancer specimens with different genetic backgrounds focusing on their p53 status. NF-кB-dependent signature of doxorubicin was identified by gene expression microarrays in breast cancer cells treated with doxorubicin and the IKKβ-inhibitor MLN120B, and confirmed ex vivo in human cancer samples. The association with p53 was functionally validated. Finally, NF-кB activation and p53 status was determined in a cohort of breast cancer patients treated with adjuvant doxorubicin-based chemotherapy. Doxorubicin treatment in the p53-mutated MDA-MB-231 cells resulted in NF-кB driven-gene transcription signature. Modulation of genes related with invasion, metastasis and chemoresistance (ICAM-1, CXCL1, TNFAIP3, IL8) were confirmed in additional doxorubicin-treated cell lines and fresh primary human breast tumors. In both systems, p53-deficient background correlated with the activation of the NF-кB-dependent signature. Furthermore, restoration of p53WT in the mutant p53 MDA-MB-231 cells impaired NF-кB driven transcription induced by doxorubicin. Moreover, a p53 deficient background and nuclear NF-кB/p65 in breast cancer patients correlated with reduced disease free-survival. This study supports that p53 deficiency is necessary for a doxorubicin driven NF-кB-response that limits doxorubicin cytotoxicity in breast cancer and is linked to an aggressive clinical behavior.

  15. Expression of VEGF-A, Otx Homeobox and p53 Family Genes in Proliferative Vitreoretinopathy

    PubMed Central

    Azzolini, Claudio; Pagani, Ilaria Stefania; Pirrone, Cristina; Al Oum, Muna; Pigni, Diana; Chiaravalli, Anna Maria; Vinciguerra, Riccardo; Simonelli, Francesca; Porta, Giovanni

    2013-01-01

    Introduction. Proliferative vitreoretinopathy (PVR) is a severe inflammatory complication of retinal detachment. Pathological epiretinal membranes grow on the retina surface leading to contraction, and surgery fails in 5% to 10% of the cases. We evaluated the expression of VEGF-A, Otx1, Otx2, Otx3, and p53 family members from PVR specimens to correlate their role in inducing or preventing the pathology. Methods. Twelve retinal samples were taken from patients affected by PVR during therapeutic retinectomies in vitreoretinal surgery. Gene expression was evaluated using quantitative real-time reverse transcriptase PCR analysis and immunohistochemistry, using four healthy human retinae as control. Result. Controls showed basal expression of all genes. PVR samples showed little or no expression of Otx1 and variable expression of VEGF-A, Otx2, Otx3, p53, and p63 genes. Significant correlation was found among VEGF-A, Otx2, p53, and p63 and between Otx1 and Otx3. Conclusions. Otx homeobox, p53 family, and VEGF-A genes are expressed in PVR human retina. We individuated two possible pathways (VEGF-A, Otx2, p53, p63 and Otx1 and Otx3) involved in PVR progression that could influence in different manners the course of the pathology. Individuating the genetic pathways of PVR represents a novel approach to PVR therapies. PMID:24227910

  16. The p63 Gene Is Regulated by Grainyhead-like 2 (GRHL2) through Reciprocal Feedback and Determines the Epithelial Phenotype in Human Keratinocytes*

    PubMed Central

    Mehrazarin, Shebli; Chen, Wei; Oh, Ju-Eun; Liu, Zi X.; Kang, Kyung L.; Yi, Jin K.; Kim, Reuben H.; Shin, Ki-Hyuk; Park, No-Hee; Kang, Mo K.

    2015-01-01

    In this study, we investigated the effects of p63 modulation in epithelial plasticity in human keratinocytes. The p63 isoforms ΔNp63α, ΔNp63β, and ΔNp63γ were ectopically expressed in normal human epidermal keratinocytes (NHEKs). The epithelial or mesenchymal state was determined by morphological changes and altered expression of various markers, e.g. fibronectin, E-Cadherin, and keratin 14. Overexpression of ΔNp63α and ΔNp63β but not ΔNp63γ isoforms led to morphological changes consistent with epithelial-mesenchymal transition (EMT). However, only ΔNp63α overexpression was able to maintain the morphological changes and molecular phenotype consistent with EMT. Interestingly, knockdown of all p63 isoforms by transfection of p63 siRNA also led to the EMT phenotype, further confirming the role of p63 in regulating the epithelial phenotype in NHEKs. EMT in NHKs accompanied loss of Grainyhead-Like 2 (GHRL2) and miR-200 family gene expression, both of which play crucial roles in determining the epithelial phenotype. Modulation of GRHL2 in NHKs also led to congruent changes in p63 expression. ChIP revealed direct GRHL2 binding to the p63 promoter. GRHL2 knockdown in NHK led to impaired binding of GRHL2 and changes in the histone marks consistent with p63 gene silencing. These data indicate the presence of a reciprocal feedback regulation between p63 and GRHL2 in NHEKs to regulate epithelial plasticity. PMID:26085095

  17. Different Mutant/Wild-Type p53 Combinations Cause a Spectrum of Increased Invasive Potential in Nonmalignant Immortalized Human Mammary Epithelial Cells1

    PubMed Central

    Junk, Damian J; Vrba, Lukas; Watts, George S; Oshiro, Marc M; Martinez, Jesse D; Futscher, Bernard W

    2008-01-01

    Aberrations of p53 occur in most, if not all, human cancers. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Immortalized human mammary epithelial cells resemble the earliest forms of aberrant breast tissue growth but do not express many malignancy-associated phenotypes. We created a model of human mammary epithelial tumorigenesis by infecting hTERT-HME1 immortalized human mammary epithelial cells expressing wild-type p53 with four different mutant p53 constructs to determine the role of p53 mutation on the evolution of tumor phenotypes. We demonstrate that different mutant/wild-type p53 heterozygous models generate loss of function, dominant negative activity, and a spectrum of gain of function activities that induce varying degrees of invasive potential. We suggest that this model can be used to elucidate changes that occur in early stages of human mammary epithelial tumorigenesis. These changes may constitute novel biomarkers or reveal novel treatment modalities that could inhibit progression from primary to metastatic breast disease. PMID:18472962

  18. APR-246/PRIMA-1(MET) rescues epidermal differentiation in skin keratinocytes derived from EEC syndrome patients with p63 mutations.

    PubMed

    Shen, Jinfeng; van den Bogaard, Ellen H; Kouwenhoven, Evelyn N; Bykov, Vladimir J N; Rinne, Tuula; Zhang, Qiang; Tjabringa, Geuranne S; Gilissen, Christian; van Heeringen, Simon J; Schalkwijk, Joost; van Bokhoven, Hans; Wiman, Klas G; Zhou, Huiqing

    2013-02-05

    p53 and p63 share extensive sequence and structure homology. p53 is frequently mutated in cancer, whereas mutations in p63 cause developmental disorders manifested in ectodermal dysplasia, limb defects, and orofacial clefting. We have established primary adult skin keratinocytes from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients with p63 mutations as an in vitro human model to study the disease mechanism in the skin of EEC patients. We show that these patient keratinocytes cultured either in submerged 2D cultures or in 3D skin equivalents have impaired epidermal differentiation and stratification. Treatment of these patient keratinocytes with the mutant p53-targeting compound APR-246/PRIMA-1(MET) (p53 reactivation and induction of massive apoptosis) that has been successfully tested in a phase I/II clinical trial in cancer patients partially but consistently rescued morphological features and gene expression during epidermal stratification in both 2D and 3D models. This rescue coincides with restoration of p63 target-gene expression. Our data show that EEC patient keratinocytes with p63 mutations can be used for characterization of the abnormal molecular circuitry in patient skin and may open possibilities for the design of novel pharmacological treatment strategies for patients with mutant p63-associated developmental abnormalities.

  19. Synergistic anticancer effect of exogenous wild-type p53 gene combined with 5-FU in human colon cancer resistant to 5-FU in vivo

    PubMed Central

    Xie, Qi; Wu, Min-Yi; Zhang, Ding-Xuan; Yang, Yi-Ming; Wang, Bao-Shuai; Zhang, Jing; Xu, Jin; Zhong, Wei-De; Hu, Jia-Ni

    2016-01-01

    AIM To investigate the anticancer effect of a recombinant adenovirus-mediated p53 (rAd-p53) combined with 5-fluorouracil (5-FU) in human colon cancer resistant to 5-FU in vivo and the mechanism of rAd-p53 in reversal of 5-FU resistance. METHODS Nude mice bearing human colon cancer SW480/5-FU (5-FU resistant) were randomly assigned to four groups (n = 25 each): control group, 5-FU group, rAd-p53 group, and rAd-p53 + 5-FU group. At 24 h, 48 h, 72 h, 120 h and 168 h after treatment, 5 mice were randomly selected from each group and sacrificed using an overdose of anesthetics. The tumors were removed and the protein expressions of p53, protein kinase C (PKC), permeability-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) (Western blot) and apoptosis (TUNEL) were determined. RESULTS The area ratios of tumor cell apoptosis were larger in the rAd/p53 + 5-FU group than that in the control, 5-FU and rAd/p53 groups (P < 0.05), and were larger in the rAd/p53 group than that of the control group (P < 0.05) and the 5-FU group at more than 48 h (P < 0.05). The p53 expression was higher in the rAd/p53 and the rAd/p53 + 5-FU groups than that of the control and 5-FU groups (P < 0.05), and were higher in the rAd/p53 + 5-FU group than that of the rAd/p53 group (P < 0.05). Overexpression of PKC, P-gp and MRP1 was observed in the 5-FU and control groups. In the rAd/p53 + 5-FU group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups (P < 0.05), and the expression of PKC was lower than that of the control, 5-FU and rAd/p53 groups at more than 48 h (P < 0.05). In the rAd/p53 group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups at more than 48 h (P < 0.05), and the expression of PKC was lower than that of the control and 5-FU groups at more than 120 h (P < 0.05). CONCLUSION 5-FU combined with rAd-p53 has a synergistic anticancer effect in SW480/5-FU (5-FU resistance), which contributes to reversal of 5-FU

  20. p53 mutations promote proteasomal activity.

    PubMed

    Oren, Moshe; Kotler, Eran

    2016-07-27

    p53 mutations occur very frequently in human cancer. Besides abrogating the tumour suppressive functions of wild-type p53, many of those mutations also acquire oncogenic gain-of-function activities. Augmentation of proteasome activity is now reported as a common gain-of-function mechanism shared by different p53 mutants, which promotes cancer resistance to proteasome inhibitors.

  1. Mutant p53 R248Q but not R248W enhances in vitro invasiveness of human lung cancer NCI-H1299 cells.

    PubMed

    Yoshikawa, Kazuhito; Hamada, Jun-ichi; Tada, Mitsuhiro; Kameyama, Takeshi; Nakagawa, Koji; Suzuki, Yukiko; Ikawa, Mayumi; Hassan, Nur Mohammad Monsur; Kitagawa, Yoshimasa; Moriuchi, Tetsuya

    2010-12-01

    More than half of all human cancers are associated with mutations of the TP53 gene. In regard to the functional interaction with the remaining wild-type (WT) p53 allele, p53 mutations are classified into two types, recessive and dominant-negative (DN) mutations. The latter mutant protein has a DN activity over the remaining WT allele. We previously showed that the DN p53 mutant was useful as a predictor of poor outcome or a risk factor for metastatic recurrence in patients with some types of cancers, regardless of the presence or absence of loss of heterozygosity (LOH) of WT p53, suggesting that the DN p53 had 'gain-of-function (GOF)' activity besides the transdominance function. In this study, we investigated GOF activity of two DN p53 mutants which had a point mutation at codon 248 (R248Q and R248W), one of the hot spots, by transfecting them respectively into H1299 cells which originally expressed no p53 protein. Growth activity of the transfectants with the two mutants was not different from that of parent or Mock transfectants. Meanwhile, in vitro invasions of Matrigel and type I collagen gel by R248Q-transfectants were significantly higher than those by R248W-transfectants or the control cells. However, there were no differences in cell motile activities, expressions of extracellular matrix-degradative enzymes such as matrix metalloproteinases, urokinase-type plasminogen activator and heparanase, and their inhibitors, between R248Q- and R248W-transfectants. These findings indicate that the p53 mutants have a different quality in GOF activities even if the mutations occurred at the same codon. And detailed information of the status of p53, including transdominancy and GOF activity, is expected to be useful for diagnosis and therapeutic strategy fitting the individual patients.

  2. The biological impact of the human master regulator p53 can be altered by mutations that change the spectrum and expression of its target genes.

    PubMed

    Menendez, Daniel; Inga, Alberto; Resnick, Michael A

    2006-03-01

    Human tumor suppressor p53 is a sequence-specific master regulatory transcription factor that targets response elements (REs) in many genes. p53 missense mutations in the DNA-binding domain are often cancer associated. As shown with systems based on the yeast Saccharomyces cerevisiae, p53 mutants can alter the spectra and intensities of transactivation from individual REs. We address directly in human cells the relationship between changes in the p53 master regulatory network and biological outcomes. Expression of integrated, tightly regulated DNA-binding domain p53 mutants resulted in many patterns of apoptosis and survival following UV or ionizing radiation, or spontaneously. These patterns reflected changes in the spectra and activities of target genes, as demonstrated for P21, MDM2, BAX, and MSH2. Thus, as originally proposed for "master genes of diversity," p53 mutations in human cells can differentially influence target gene transactivation, resulting in a variety of biological consequences which, in turn, might be expected to influence tumor development and therapeutic efficacy.

  3. Anti-cancer effect of adenovirus p53 on human cervical cancer cell growth in vitro and in vivo.

    PubMed

    Ahn, W S; Bae, S M; Lee, J M; Namkoong, S E; Yoo, J Y; Seo, Y-S; Nam, S L; Cho, Y-L; Nam, K H; Kim, C K; Kim, Y-W

    2004-01-01

    To evaluate anti-tumor effects of recombinant adenovirus p53, time-course p53, E6 expression, and cell growth inhibition were investigated in vitro and in vivo using cervical cancer cell lines such as CaSki, SiHa, HeLa, HeLaS3, C33A, and HT3. The cell growth inhibition was studied via cell count assay, MTT assay and neutral red assay. After transfecting AdCMVp53 into SiHa cells-xenografted nude mice, the transduction efficiency and anti-tumor effect were investigated for a month. The results showed that adenoviral p53 expression induced significant growth suppression on the cancer cells, in which E6 transcript was strongly repressed, and that the expression of p53 and E6 were remarkably dependent on each cell type. The transduction efficiency was highly maintained in vivo as well as in vitro, and the size of tumor was remarkably decreased in comparison with AdCMVLacZ control. The results suggest that the adenovirus-mediated p53 gene transfection was done very effectively in vitro and in vivo experiment, and the cell growth was suppressed via p53-dependent apoptotic cell death, and that the anti-tumor effect could be related to E6 and p53 expression pattern.

  4. Noncanonical DNA Motifs as Transactivation Targets by Wild Type and Mutant p53

    PubMed Central

    Jordan, Jennifer J.; Menendez, Daniel; Inga, Alberto; Nourredine, Maher; Bell, Douglas; Resnick, Michael A.

    2008-01-01

    Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0–13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0–13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi–in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical ½-(a single decamer) and ¾-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of ½- and ¾-site REs greatly expands the p53 master regulatory network. PMID:18714371

  5. p53 mutation and cyclin D1 amplification correlate with cisplatin sensitivity in xenografted human squamous cell carcinomas from head and neck.

    PubMed

    Henriksson, Eva; Baldetorp, Bo; Borg, Ake; Kjellen, Elisabeth; Akervall, Jan; Wennerberg, Johan; Wahlberg, Peter

    2006-01-01

    To investigate the response of tumour growth to cisplatin treatment, in relation to p53 mutation and cyclin D1 dysregulation on DNA and protein level, biopsies from seven xenografted human squamous cell carcinomas from the head and neck were analysed with immunohistochemistry for p53 expression and cyclin D1 expression. Polymerase chain reaction-singlestranded conformation polymorphism was used to determine p53 mutations. Fluorescence in situ hybridization was performed to analyse cyclin D1 amplification. The mice were injected i.p. with NaCl (controls) or cisplatin. After injection the tumour volume were measured. The inhibition of tumour growth by cisplatin was defined as the area under the growth curves, and compared with the growth curves of the tumours in the control group. Xenografts with p53 mutation showed significantly higher resistance to cisplatin (p < 0.001) and also tumours with cyclin D1 amplification showed significantly higher resistance (p < 0.001).

  6. Human papillomavirus infection in Bowen disease: negative p53 expression, not p16(INK4a) overexpression, is correlated with human papillomavirus-associated Bowen disease.

    PubMed

    Murao, Kazutoshi; Yoshioka, Rika; Kubo, Yoshiaki

    2014-10-01

    Genital Bowen disease (BD) has been linked to the high-risk types of human papillomavirus (HPV) infection. Recently, it has been recognized that HPV also can be associated with extragenital BD. HPV oncoproteins E6 and E7 interfere with the function of p53 and pRb, respectively, leading carcinogenesis. p16(INK4a) overexpression induced by inactivation of pRb is recognized as a surrogate marker for HPV-associated cervical cancer. In this study, we examined the presence of HPV DNA in 142 BD lesions by polymerase chain reaction (PCR), and determined the type of HPV by PCR restriction fragment length polymorphism or direct DNA sequencing. HPV DNA was detected in 66.7% of genital BD and 8.3% of extragenital BD. The types of HPV detected were HPV types 6, 16, 33, 52, 56, 58 and 59. We also investigated the expression of p16(INK4a) , pRb and p53 by immunohistochemistry. Positive expression was detected in 88.6% for p16(INK4a) , 25.2% for pRb, and 63.8% for p53. There was no significant difference in p16(INK4a) and pRb expression between HPV-positive and -negative BD. However, a strong correlation of HPV positivity with p53 negativity was found. A total of 66.7% of HPV-positive BD showed no p53 expression, whereas the corresponding rate was 32.8% of HPV-negative BD. This study demonstrated that HPV can participate in the development of BD, not only in the genital lesion, but also in extragenital lesion. p16(INK) (4a) overexpression is not a marker for HPV infection in BD. Instead, negative p53 expression is correlated with HPV-associated BD.

  7. Human papillomavirus oncoprotein E7 targets the promyelocytic leukemia protein and circumvents cellular senescence via the Rb and p53 tumor suppressor pathways.

    PubMed

    Bischof, Oliver; Nacerddine, Karim; Dejean, Anne

    2005-02-01

    Cellular senescence can be triggered by a variety of signals, including loss of telomeric integrity or intense oncogenic signaling, and is considered a potent, natural tumor suppressor mechanism. Previously, it was shown that the promyelocytic leukemia protein (PML) induces cellular senescence when overexpressed in primary human fibroblasts. The mechanism by which the PML IV isoform elicits this irreversible growth arrest is believed to involve activation of the tumor suppressor pathways p21/p53 and p16/Rb; however, a requirement for either pathway has not been demonstrated unequivocally. To investigate the individual contributions of p53 and Rb to PML-induced senescence, we used oncoproteins E6 and E7 from human papillomaviruses (HPVs), which predominantly target p53 and Rb. We show that E7, but not E6, circumvents PML-induced senescence. Using different E7 mutant proteins, dominant negative cyclin-dependent kinase 4, and p16 RNA interference, we demonstrate that Rb-related and Rb-independent mechanisms of E7 are necessary for subversion of PML-induced senescence and we identify PML as a novel target for E7. Interaction between E7 and a functional prosenescence complex composed of PML, p53, and CBP perturbs transcriptional activation of p53, thus highlighting a significant effect also on the p53 tumor suppressor pathway. Given the importance of HPV in the pathogenesis of cervical cancer, our results warrant a more detailed analyses of PML in HPV infections.

  8. Liposome-mediated transfection of wild-type P53 DNA into human prostate cancer cells is improved by low-frequency ultrasound combined with microbubbles

    PubMed Central

    BAI, WEN-KUN; ZHANG, WEI; HU, BING; YING, TAO

    2016-01-01

    Prostate cancer is a common type of cancer in elderly men. The aim of the present study was to evaluate the effects of ultrasound exposure in combination with SonoVue microbubbles on liposome-mediated transfection of wild-type P53 genes into human prostate cancer cells. PC-3 human prostate cancer cells were exposed to ultrasound; duty cycle was controlled at 20% (2 sec on, 8 sec off) for 5 min with and without SonoVue microbubble echo-contrast agent using a digital sonifier (frequency, 21 kHz; intensity, 46 mW/cm2). The cells were divided into eight groups, as follows: Group A (SonoVue + wild-type P53), group B (ultrasound + wild-type P53), group C (SonoVue + ultrasound + wild-type P53), group D (liposome + wild-type P53), group E (liposome + SonoVue + wild-type P53), group F (liposome + wild-type P53 + ultrasound), group G (liposome + wild-type P53 + ultrasound + SonoVue) and the control group (wild-type P53). Following treatment, a hemocytometer was used to measure cell lysis, reverse transcription-quantitative polymerase chain reaction and western blotting were performed to detect P53 gene transfection efficiency, Cell Counting Kit-8 was employed to reveal cell proliferation and Annexin V/propidium iodide staining was used to determine cell apoptosis. Cell lysis was minimal in each group. Wild-type P53 gene and protein expression were significantly increased in the PC-3 cells in group G compared with the control and all other groups (P<0.01). Cell proliferation was significantly suppressed in group G compared with the control group and all other groups (P<0.01). Cell apoptosis levels in group G were significantly improved compared with the control group and all other groups (P<0.01). Thus, the results of the present study indicate that the use of low-frequency and low-energy ultrasound in combination with SonoVue microbubbles may be a potent physical method for increasing liposome gene delivery efficiency. PMID:27313702

  9. Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the p53 or BRCA1 Genes

    DTIC Science & Technology

    1999-01-01

    development of breast cancers. To study the effects of inactivating mutations in these tumor suppressor genes early in the breast-cancer pathway, we have...the effects of inactivating mutations in these tumor suppressor genes early in the breast-cancer pathway. The consequences of transduction of these...proposed three approaches for constructing p53-deficient cells; i.e., by mutating the p53 gene directly, by abrogating the protein’s normal cellular

  10. Caspase-dependent Proteolysis of Human Ribonucleotide Reductase Small Subunits R2 and p53R2 during Apoptosis*

    PubMed Central

    Tebbi, Ali; Guittet, Olivier; Tuphile, Karine; Cabrié, Aimeric; Lepoivre, Michel

    2015-01-01

    Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. In mammals, the R1 catalytic subunit forms an active complex with either one of the two small subunits R2 and p53R2. Expression of R2 is S phase-specific and required for DNA replication. The p53R2 protein is expressed throughout the cell cycle and in quiescent cells where it provides dNTPs for mitochondrial DNA synthesis. Participation of R2 and p53R2 in DNA repair has also been suggested. In this study, we investigated the fate of the RnR subunits during apoptosis. The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase and in HeLa 229 cells incubated with TNF-α and cycloheximide. The cleavage site was mapped between Asp342 and Asn343. Caspase attack released a C-terminal p53R2 peptide of nine residues containing the conserved heptapeptide essential for R1 binding. As a consequence, the cleaved p53R2 protein was inactive. In vitro, purified caspase-3 and -8 could release the C-terminal tail of p53R2. Knocking down these caspases, but not caspase-2, -7, and -10, also inhibited p53R2 cleavage in cells committed to die via the extrinsic death receptor pathway. The R2 subunit was subjected to caspase- and proteasome-dependent proteolysis, which was prevented by siRNA targeting caspase-8. Knocking down caspase-3 was ineffective. Protein R1 was not subjected to degradation. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis. These data identify RnR activity as a prosurvival function inactivated by proteolysis during apoptosis. PMID:25878246

  11. Heat stress induces apoptosis through transcription-independent p53-mediated mitochondrial pathways in human umbilical vein endothelial cell.

    PubMed

    Gu, Z T; Wang, H; Li, L; Liu, Y S; Deng, X B; Huo, S F; Yuan, F F; Liu, Z F; Tong, H S; Su, L

    2014-03-26

    Cells apoptosis induced by intense heat stress is the prominent feature of heat-related illness. However, little is known about the biological effects of heat stress on cells apoptosis. Herein, we presented evidence that intense heat stress could induce early apoptosis of HUVEC cells through activating mitochondrial pathway with changes in mitochondrial membrane potential(ΔΨm), release of cytochrome c, and activation of caspase-9 and -3. We further revealed that p53 played a crucial role in heat stress-induced early apoptosis, with p53 protein rapidly translocated into mitochondria. Using pifithrin-α(PFT), a p53's mitochondrial translocation inhibitor, we found that pretreated with PFT, heat stress induced mitochondrial p53 translocation was significantly suppressed, accompanied by a significant alleviation in the loss of ΔΨm, cytochrome c release and caspase-9 activation. Furthermore, we also found that generation of reactive oxygen species (ROS) was a critical mediator in heat stress-induced apoptosis. In addition, the antioxidant MnTMPyP significantly decreased the heat stress-induced p53's mitochondrial translocation, followed by the loss of ΔΨm, cytochrome c release, caspase-9 activation and heat stress-mediated apoptosis. Conclusively, these findings indicate the contribution of the transcription-independent mitochondrial p53 pathway to early apoptosis in HUVEC cells induced by oxidative stress in response to intense heat stress.

  12. p53: out of Africa.

    PubMed

    Lane, David

    2016-04-15

    Somatic mutations in the tumor suppressor gene p53 occur in more than half of all human cancers. Rare germline mutations result in the Li-Fraumeni cancer family syndrome. In this issue ofGenes&Development, Jennis and colleagues (pp. 918-930) use an elegant mouse model to examine the affect of a polymorphism, P47S (rs1800371), in the N terminus of p53 that is found in Africans as well as more than a million African Americans. Remarkably, the single nucleotide change causes the mice to be substantially tumor-prone compared with littermates, suggesting that this allele causes an increased risk of developing cancer. The defect in p53 function is traced to a restriction in downstream gene regulation that reduces cell death in response to stress.

  13. Potential therapeutic role of Tridham in human hepatocellular carcinoma cell line through induction of p53 independent apoptosis

    PubMed Central

    2013-01-01

    Background Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths reported worldwide. The incidence is higher in Asia and Africa, where there is greater endemic prevalence of hepatitis B and C. The devastating outcome of cancer can be minimized only by the use of potent therapeutic agents. Tridham (TD) has been acknowledged since olden days for its wide spectrum of biological properties and was used by traditional practitioners of Siddha and other indigenous systems of medicine. The present study aims at investigating the mechanistic action of TD by assessing the antiproliferative and pro-apoptotic effects on human hepatocellular carcinoma cell line (Huh7). Methods Cell viability and apoptosis assay using MTT analysis and trypan blue staining, DAPI staining, DNA fragmentation, cell cycle analysis, mitochondrial membrane potential, real-time reverse transcription-polymerase chain reaction, western blotting and immunofluorescence staining were determined in Huh7 cells. Results Viability studies of TD treated Huh7 cells showed an inhibition in cell growth in time and dose dependent manner. Chromatin condensation, DNA fragmentation and apoptotic bodies, which are structural changes characteristic of apoptosis, were found following TD treatment of Huh7 cells. DAPI staining and agarose gel electrophoresis confirmed the induction of apoptosis by TD. Cell cycle analysis of Huh7 cells treated with TD exhibited a marked accumulation of cells in the sub-G1 phase of the cell cycle in a dose dependent manner. Immunofluorescent staining for Ki-67 showed a higher level of expression in untreated cells as compared to TD treated cells. We observed a significant loss in the mitochondrial membrane potential and the release of cytochrome c into the cytosol in TD treated cells. Down regulation of Bcl-2, up regulation of Bax and Bad as well as activation of caspases-3 and 9 were also observed. The p53 gene expression was found to be unaltered in TD treated cells

  14. CQ synergistically sensitizes human colorectal cancer cells to SN-38/CPT-11 through lysosomal and mitochondrial apoptotic pathway via p53-ROS cross-talk.

    PubMed

    Chen, Pinjia; Luo, Xiaoyong; Nie, Peipei; Wu, Baoyan; Xu, Wei; Shi, Xinpeng; Chang, Haocai; Li, Bing; Yu, Xiurong; Zou, Zhengzhi

    2017-03-01

    Autophagy plays a key role in supporting cell survival against chemotherapy-induced apoptosis. In this study, we found the chemotherapy agent SN-38 induced autophagy in colorectal cancer (CRC) cells. However, inhibition of autophagy using a small molecular inhibitor 3-methyladenine (3-MA) and ATG5 siRNA did not increase SN-38-induced cytotoxicity in CRC cells. Notably, another autophagy inhibitor chloroquine (CQ) synergistically enhanced the anti-tumor activity of SN-38 in CRC cells with wild type (WT) p53. Subsequently, we identified a potential mechanism of this cooperative interaction by showing that CQ and SN-38 acted together to trigger reactive oxygen species (ROS) burst, upregulate p53 expression, elicit the loss of lysosomal membrane potential (LMP) and mitochondrial membrane potential (∆ψm). In addition, ROS induced by CQ plus SN-38 upregulated p53 levels by activating p38, conversely, p53 stimulated ROS. These results suggested that ROS and p53 reciprocally promoted each other's production and cooperated to induce CRC cell death. Moreover, we showed induction of ROS and p53 by the two agents provoked the loss of LMP and ∆ψm. Altogether, all results suggested that CQ synergistically sensitized human CRC cells with WT p53 to SN-38 through lysosomal and mitochondrial apoptotic pathway via p53-ROS cross-talk. Lastly, we showed that CQ could enhance CRC cells response to CPT-11 (a prodrug of SN-38) in xenograft models. Thus the combined treatment might represent an attractive therapeutic strategy for the treatment of CRC.

  15. Involvement of p53 mutation and mismatch repair proteins dysregulation in NNK-induced malignant transformation of human bronchial epithelial cells.

    PubMed

    Shen, Ying; Zhang, Shuilian; Huang, Xiaobin; Chen, Kailin; Shen, Jing; Wang, Zhengyang

    2014-01-01

    Genome integrity is essential for normal cellular functions and cell survival. Its instability can cause genetic aberrations and is considered as a hallmark of most cancers. To investigate the carcinogenesis process induced by tobacco-specific carcinogen NNK, we studied the dynamic changes of two important protectors of genome integrity, p53 and MMR system, in malignant transformation of human bronchial epithelial cells after NNK exposure. Our results showed that the expression of MLH1, one of the important MMR proteins, was decreased early and maintained the downregulation during the transformation in a histone modification involved and DNA methylation-independent manner. Another MMR protein PMS2 also displayed a declined expression while being in a later stage of transformation. Moreover, we conducted p53 mutation analysis and revealed a mutation at codon 273 which led to the replacement of arginine by histidine. With the mutation, DNA damage-induced activation of p53 was significantly impaired. We further reintroduced the wild-type p53 into the transformed cells, and the malignant proliferation can be abrogated by inducing cell cycle arrest and apoptosis. These findings indicate that p53 and MMR system play an important role in the initiation and progression of NNK-induced transformation, and p53 could be a potential therapeutic target for tobacco-related cancers.

  16. Selenite-induced p53 Ser-15 phosphorylation and caspase-mediated apoptosis in LNCaP human prostate cancer cells.

    PubMed

    Jiang, Cheng; Hu, Hongbo; Malewicz, Barbara; Wang, Zaisen; Lü, Junxuan

    2004-07-01

    The issue of p53 requirement for the caspase-mediated apoptosis induced by selenium in a cancer chemoprevention or chemotherapy context has not been critically addressed. We and others have shown that selenite induces apoptotic DNA laddering in the p53-mutant DU145 prostate cancer cells and the p53-null HL60 leukemia cells without the cleavage of poly(ADP-ribose) polymerase (PARP; i.e., caspase-independent apoptosis), whereas selenium compounds leading to the formation of methylselenol induce caspase-mediated apoptosis in these cells. Because selenite induces DNA single strand breaks, and because certain types of DNA damage activate p53, we investigated whether the human LNCaP prostate cancer cells, which contain a wild-type p53, execute selenite-induced apoptosis through caspase pathways. The results showed that exposure of LNCaP cells for 24 hours to lower micromolar concentrations of selenite led to DNA laddering, and to the cleavage of PARP and several pro-caspases. In contrast to this apoptosis sensitivity, LNCaP cells were rather resistant to similar concentrations of the methylselenol precursor methylseleninic acid. Selenite treatment led to a significant increase in p53 phosphorylation on Ser-15 (Ser15P). Time course experiments showed that p53 Ser15P occurred several hours before caspase activation and PARP cleavage. The general caspase inhibitor zVADfmk completely blocked PARP cleavage, and significantly decreased DNA laddering, but did not affect p53 Ser15P. An inhibitor for caspase-8 was equally as protective as that for caspase-9 against the selenite-induced apoptosis. Attenuating p53 by a chemical inhibitor pifithrin-alpha decreased the selenite-induced p53 Ser15P and led to concordant reductions of PARP cleavage and apoptosis. In summary, selenite-induced p53 Ser15P appeared to be important for activating the caspase-mediated apoptosis involving both the caspase-8 and the caspase-9 pathways in the LNCaP cells.

  17. The p53-Mdm2 interaction and the E3 ligase activity of Mdm2/Mdm4 are conserved from lampreys to humans.

    PubMed

    Coffill, Cynthia R; Lee, Alison P; Siau, Jia Wei; Chee, Sharon M; Joseph, Thomas L; Tan, Yaw Sing; Madhumalar, Arumugam; Tay, Boon-Hui; Brenner, Sydney; Verma, Chandra S; Ghadessy, Farid J; Venkatesh, Byrappa; Lane, David P

    2016-02-01

    The extant jawless vertebrates, represented by lampreys and hagfish, are the oldest group of vertebrates and provide an interesting genomic evolutionary pivot point between invertebrates and jawed vertebrates. Through genome analysis of one of these jawless vertebrates, the Japanese lamprey (Lethenteron japonicum), we identified all three members of the important p53 transcription factor family--Tp53, Tp63, and Tp73--as well as the Mdm2 and Mdm4 genes. These genes and their products are significant cellular regulators in human cancer, and further examination of their roles in this most distant vertebrate relative sheds light on their origin and coevolution. Their important role in response to DNA damage has been highlighted by the discovery of multiple copies of the Tp53 gene in elephants. Expression of lamprey p53, Mdm2, and Mdm4 proteins in mammalian cells reveals that the p53-Mdm2 interaction and the Mdm2/Mdm4 E3 ligase activity existed in the common ancestor of vertebrates and have been conserved for >500 million years of vertebrate evolution. Lamprey Mdm2 degrades human p53 with great efficiency, but this interaction is not blocked by currently available small molecule inhibitors of the human HDM2 protein, suggesting utility of lamprey Mdm2 in the study of the human p53 signaling pathway.

  18. The p53–Mdm2 interaction and the E3 ligase activity of Mdm2/Mdm4 are conserved from lampreys to humans

    PubMed Central

    Coffill, Cynthia R.; Lee, Alison P.; Siau, Jia Wei; Chee, Sharon M.; Joseph, Thomas L.; Tan, Yaw Sing; Madhumalar, Arumugam; Tay, Boon-Hui; Brenner, Sydney; Verma, Chandra S.; Ghadessy, Farid J.; Venkatesh, Byrappa; Lane, David P.

    2016-01-01

    The extant jawless vertebrates, represented by lampreys and hagfish, are the oldest group of vertebrates and provide an interesting genomic evolutionary pivot point between invertebrates and jawed vertebrates. Through genome analysis of one of these jawless vertebrates, the Japanese lamprey (Lethenteron japonicum), we identified all three members of the important p53 transcription factor family—Tp53, Tp63, and Tp73—as well as the Mdm2 and Mdm4 genes. These genes and their products are significant cellular regulators in human cancer, and further examination of their roles in this most distant vertebrate relative sheds light on their origin and coevolution. Their important role in response to DNA damage has been highlighted by the discovery of multiple copies of the Tp53 gene in elephants. Expression of lamprey p53, Mdm2, and Mdm4 proteins in mammalian cells reveals that the p53–Mdm2 interaction and the Mdm2/Mdm4 E3 ligase activity existed in the common ancestor of vertebrates and have been conserved for >500 million years of vertebrate evolution. Lamprey Mdm2 degrades human p53 with great efficiency, but this interaction is not blocked by currently available small molecule inhibitors of the human HDM2 protein, suggesting utility of lamprey Mdm2 in the study of the human p53 signaling pathway. PMID:26798135

  19. Modeling Human Epithelial Ovarian Cancer in Mice by Alteration of Expression of the BRCA1 and/or P53 Genes

    DTIC Science & Technology

    2005-02-01

    later by injection with 5 U of human chorionic gonadotropin (hormones purchased from Sigma, St. Louis, MO). 1.5 days following the last hormone...AD Award Number: W81XWH-04-1-0063 TITLE: Modeling Human Epithelial Ovarian Cancer in Mice by Alteration of Expression of the BRCA1 and/or P53 Genes...FUNDING NUMBERS Modeling Human Epithelial Ovarian Cancer in Mice by W81XWH-04-1-0063 Alteration of Expression of the BRCAI and/or P53 Genes 6. AUTHOR(S

  20. Frequent mutations of p53 gene in oesophageal squamous cell carcinomas with and without human papillomavirus (HPV) involvement suggest the dominant role of environmental carcinogens in oesophageal carcinogenesis.

    PubMed Central

    Chang, F.; Syrjänen, S.; Tervahauta, A.; Kurvinen, K.; Wang, L.; Syrjänen, K.

    1994-01-01

    Epidemiological evidence suggests that alcohol intake, use of tobacco, ingestion of mycotoxins and nitrosamines and nutritional deficiencies are high-risk factors for the development of oesophageal cancer. Similarly, viral infections have been postulated to play a role in some tumours. However, the molecular events underlying the development of oesophageal carcinoma are poorly understood as yet. Loss of p53 tumour-suppressor gene function has been found in different human malignancies, and it can occur in a variety of ways, including gene mutation and interaction with the E6 protein of oncogenic human papillomaviruses (HPVs). Because the oesophageal mucosa is potentially exposed to mutagens and HPVs, we studied DNA samples derived from nine HPV-positive squamous cell carcinomas and 12 HPV-negative tumours. Exons 5-9 of the p53 gene containing phylogenetically conserved domains were examined using the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique. HPV detection was done using DNA in situ hybridisation with biotin-labelled HPV DNA probes. Mutations were detected in eight (38%) out of the 21 cases. Three mutations were found in exons 5/6, three in exon 7 and two in exon 8/9. Six (50%) of the 12 HPV-negative carcinomas showed p53 mutations. Two (22.2%) of the nine HPV-positive carcinomas were found to contain p53 mutations as well; one contained HPV 16 DNA sequences and showed p53 mutation in exon 8/9, and the other was HPV 6/11 positive with the mutation in exon 5/6. Although mutations were more common in HPV-negative tumours (50.0% vs 22.2%), the difference in p53 mutations in HPV-positive and -negative tumours did not reach statistical significance (P = 0.1946). These data indicate that inactivation of the p53 gene is a frequent event in oesophageal squamous cell carcinomas and such an inactivation might be an important molecular pathway for the development of oesophageal cancer. The findings of p53 mutations in HPV

  1. Eradication of Helicobacter pylori Infection Restores ki67, p53, and Cyclin D1 Immunoreactivity in the Human Gastric Epithelium

    PubMed Central

    Triantafyllou, Konstantinos; Papadopoulos, Vasilios; Emanouil, Theodoros; Gkolfakis, Paraskevas; Damaskou, Vasileia; Tziatzios, Georgios; Panayiotides, Ioannis G.; Vafiadis, Irene; Ladas, Spiros D.

    2016-01-01

    INTRODUCTION We evaluated the effect of Helicobacter pylori (HP) eradication on p53, cyclin D1 expression, and cell proliferation in gastric mucosa. MATERIALS AND METHODS We assessed p53, cyclin D1, and ki67 immunoexpression in gastric mucosa from 31 HP chronic gastritis patients and 12 controls. Reassessment was performed 6 months after successful HP eradication. RESULTS Successful eradication resulted in significant decrease of p53 (1.53 ± 0.16 vs 0.83 ± 0.19, P = 0.01) and ki67 (9.84 ± 0.96 vs 4.77 ± 0.27, P < 0.001) staining in the antrum. Similarly, p53 immunoreactivity significantly decreased in the corpus (1.27 ± 0.20 vs 0.46 ± 0.15, P = 0.02), while there was a trend for decreased corpus cyclin D1 and ki67 expression (0.17 ± 0.07 vs 0.0, P = 0.08 and 8.71 ± 1.24 vs 5.85 ± 0.54, P = 0.09, respectively). Importantly, after successful HP eradication, the immunoreactivity of the studied parameters was similar to that of controls. CONCLUSION Successful HP infection eradication restores p53, cyclin D1, and ki67 immunoreactivity in the gastric mucosa to the level of controls. PMID:27891056

  2. Shikonin Induces Apoptotic Cell Death via Regulation of p53 and Nrf2 in AGS Human Stomach Carcinoma Cells

    PubMed Central

    Ko, Hyeonseok; Kim, Sun-Joong; Shim, So Hee; Chang, HyoIhl; Ha, Chang Hoon

    2016-01-01

    Shikonin, which derives from Lithospermum erythrorhizon, has been traditionally used against a variety of diseases, including cancer, in Eastern Asia. Here we determined that shikonin inhibits proliferation of gastric cancer cells by inducing apoptosis. Shikonin’s biological activity was validated by observing cell viability, caspase 3 activity, reactive oxygen species (ROS) generation, and apoptotic marker expressions in AGS stomach cancer cells. The concentration range of shikonin was 35–250 nM with the incubation time of 6 h. Protein levels of Nrf2 and p53 were evaluated by western blotting and confirmed by real-time PCR. Our results revealed that shikonin induced the generation of ROS as well as caspase 3-dependent apoptosis. c-Jun-N-terminal kinases (JNK) activity was significantly elevated in shikonin-treated cells, thereby linking JNK to apoptosis. Furthermore, our results revealed that shikonin induced p53 expression but repressed Nrf2 expression. Moreover, our results suggested that there may be a co-regulation between p53 and Nrf2, in which transfection with siNrf2 induced the p53 expression. We demonstrated for the first time that shikonin activated cell apoptosis in AGS cells via caspase 3- and JNK-dependent pathways, as well as through the p53-Nrf2 mediated signal pathway. Our study validates in partly the contribution of shikonin as a new therapeutic approaches/ agent for cancer chemotherapy. PMID:27257011

  3. Arecoline, a major alkaloid of areca nut, inhibits p53, represses DNA repair, and triggers DNA damage response in human epithelial cells.

    PubMed

    Tsai, Yi-Shan; Lee, Ka-Wo; Huang, Jau-Ling; Liu, Yu-Sen; Juo, Suh-Hang Hank; Kuo, Wen-Rei; Chang, Jan-Gowth; Lin, Chang-Shen; Jong, Yuh-Jyh

    2008-07-30

    The International Agency for Research on Cancer declared that areca nut was carcinogenic to human. Areca nut is the main component of betel quid (BQ), which is commonly consumed in Asia. Epidemiological studies have shown that BQ chewing is a predominant risk factor for oral and pharyngeal cancers. It has been known that areca nut is genotoxic to human epithelial cells. However, the molecular and cellular mechanisms underlying areca nut-associated genotoxicity are not fully understood. Here we showed that arecoline, a major alkaloid of areca nut, might contribute to oral carcinogenesis through inhibiting p53 and DNA repair. We found, on the biological aspect, that arecoline could induce gamma-H2AX phosphorylation, a sensitive DNA damage marker, in KB, HEp-2, and 293 cells, suggesting that DNA damages were elicited by arecoline. This phenomenon was supported by the observations of arecoline-induced hyperphosphorylation of ATM, Nbs1, Chk1/2, p53, and Cdc25C, as well as G2/M cell cycle arrest, indicating that a cellular DNA damage response was activated. To explore the possible mechanism accounting for arecoline-elicited DNA damages, we found that arecoline could inhibit p53 by its expression and transactivation function. As a result, the expression of p53-regulated p21(WAF1) and the p53-activated DNA repair were repressed by arecoline. Finally, we showed that p53 mRNA transcripts were frequently down-regulated in BQ-associated oral cancer, suggesting that arecoline-mediated p53 inhibition might play a role in BQ-associated tumorigenesis.

  4. IARC Database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualisation tools.

    PubMed Central

    Hainaut, P; Hernandez, T; Robinson, A; Rodriguez-Tome, P; Flores, T; Hollstein, M; Harris, C C; Montesano, R

    1998-01-01

    Since 1989, about 570 different p53 mutations have been identified in more than 8000 human cancers. A database of these mutations was initiated by M. Hollstein and C. C. Harris in 1990. This database originally consisted of a list of somatic point mutations in the p 53 gene of human tumors and cell lines, compiled from the published literature and made available in a standard electronic form. The database is maintained at the International Agency for Research on Cancer (IARC) and updated versions are released twice a year (January and July). The current version (July 1997) contains records on 6800 published mutations and will surpass the 8000 mark in the January 1998 release. The database now contains information on somatic and germline mutations in a new format to facilitate data retrieval. In addition, new tools are constructed to improve data analysis, such as a Mutation Viewer Java applet developed at the European Bioinformatics Institute (EBI) to visualise the location and impact of mutations on p53 protein structure. The database is available in different electronic formats at IARC (http://www.iarc. fr/p53/homepage.htm ) or from the EBI server (http://www.ebi.ac.uk ). The IARC p53 website also provides reports on database analysis and links with other p53 sites as well as with related databases. In this report, we describe the criteria for inclusion of data, the revised format and the new visualisation tools. We also briefly discuss the relevance of p 53 mutations to clinical and biological questions. PMID:9399837

  5. Enhanced Gadd45 expression and delayed G2/M progression are p53 dependent in zinc-supplemented human bronchial epithelial cells

    USDA-ARS?s Scientific Manuscript database

    Zinc is an essential nutrient for humans; however, this study demonstrated for the first time that an elevated zinc status, created by culturing cells at optimal plasma zinc concentration attainable by oral zinc supplementation, is cytotoxic for normal human bronchial epithelial (NHBE) cells. p53 p...

  6. Prostaglandin E2 inhibits p53 in human breast adipose stromal cells: a novel mechanism for the regulation of aromatase in obesity and breast cancer.

    PubMed

    Wang, Xuyi; Docanto, Maria M; Sasano, Hironobu; Lo, Camden; Simpson, Evan R; Brown, Kristy A

    2015-02-15

    Obesity is a risk factor for postmenopausal breast cancer and the majority of these cancers are estrogen dependent. Aromatase converts androgens into estrogens and its increased expression in breast adipose stromal cells (ASC) is a major driver of estrogen receptor-positive breast cancer. In particular, obesity-associated and tumor-derived factors, such as prostaglandin E2 (PGE2), have been shown to drive the expression of aromatase by stimulating the activity of the proximal promoter II (PII). The tumor-suppressor p53 is a key regulator of cell-cycle arrest and apoptosis and is frequently mutated in breast cancer. Mutations in p53 are rare in tumor-associated ASCs. Therefore, it was hypothesized that p53 is regulated by PGE2 and involved in the PGE2-mediated regulation of aromatase. Results demonstrate that PGE2 causes a significant decrease in p53 transcript and nuclear protein expression, as well as phosphorylation at Ser15 in primary human breast ASCs. Stabilization of p53 with RITA leads to a significant decrease in the PGE2-stimulated aromatase mRNA expression and activity, and PII activity. Interaction of p53 with PII was demonstrated and this interaction is decreased in the presence of PGE2. Moreover, mutation of the identified p53 response element leads to an increase in the basal activity of the promoter. Immunofluorescence on clinical samples demonstrates that p53 is decreased in tumor-associated ASCs compared with ASCs from normal breast tissue, and that there is a positive association between perinuclear (inactive) p53 and aromatase expression in these cells. Furthermore, aromatase expression is increased in breast ASCs from Li-Fraumeni patients (germline TP53 mutations) compared with non-Li-Fraumeni breast tissue. Overall, our results demonstrate that p53 is a negative regulator of aromatase in the breast and its inhibition by PGE2 provides a novel mechanism for aromatase regulation in obesity and breast cancer. ©2015 American Association for Cancer

  7. Estrogen receptor alpha (ERα/ESR1) mediates the p53-independent overexpression of MDM4/MDMX and MDM2 in human breast cancer

    PubMed Central

    Swetzig, Wendy M.; Wang, Jianmin; Das, Gokul M.

    2016-01-01

    MDM2 and MDM4 are heterodimeric, non-redundant oncoproteins that potently inhibit the p53 tumor suppressor protein. MDM2 and MDM4 also enhance the tumorigenicity of breast cancer cells in in vitro and in vivo models and are overexpressed in primary human breast cancers. Prior studies have characterized Estrogen Receptor Alpha (ERα/ESR1) as a regulator of MDM2 expression and an MDM2- and p53-interacting protein. However, similar crosstalk between ERα and MDM4 has not been investigated. Moreover, signaling pathways that mediate the overexpression of MDM4 in human breast cancer remain to be elucidated. Using the Cancer Genome Atlas (TCGA) breast invasive carcinoma patient cohort, we have analyzed correlations between ERα status and MDM4 and MDM2 expression in primary, treatment-naïve, invasive breast carcinoma samples. We report that the expression of MDM4 and MDM2 is elevated in primary human breast cancers of luminal A/B subtypes and associates with ERα-positive disease, independently of p53 mutation status. Furthermore, in cell culture models, ERα positively regulates MDM4 and MDM2 expression via p53-independent mechanisms, and these effects can be blocked by the clinically-relevant endocrine therapies fulvestrant and tamoxifen. Additionally, ERα also positively regulates p53 expression. Lastly, we report that endogenous MDM4 negatively regulates ERα expression and forms a protein complex with ERα in breast cancer cell lines and primary human breast tumor tissue. This suggests direct signaling crosstalk and negative feedback loops between ERα and MDM4 expression in breast cancer cells. Collectively, these novel findings implicate ERα as a central component of the p53-MDM2-MDM4 signaling axis in human breast cancer. PMID:26909605

  8. Predominant role of DNA polymerase eta and p53-dependent translesion synthesis in the survival of ultraviolet-irradiated human cells

    PubMed Central

    Lerner, Leticia K.; Francisco, Guilherme; Soltys, Daniela T.; Rocha, Clarissa R.R.; Quinet, Annabel; Vessoni, Alexandre T.; Castro, Ligia P.; David, Taynah I.P.; Bustos, Silvina O.; Strauss, Bryan E.; Gottifredi, Vanesa; Stary, Anne; Sarasin, Alain; Chammas, Roger

    2017-01-01

    Abstract Genome lesions trigger biological responses that help cells manage damaged DNA, improving cell survival. Pol eta is a translesion synthesis (TLS) polymerase that bypasses lesions that block replicative polymerases, avoiding continued stalling of replication forks, which could lead to cell death. p53 also plays an important role in preventing cell death after ultraviolet (UV) light exposure. Intriguingly, we show that p53 does so by favoring translesion DNA synthesis by pol eta. In fact, the p53-dependent induction of pol eta in normal and DNA repair-deficient XP-C human cells after UV exposure has a protective effect on cell survival after challenging UV exposures, which was absent in p53- and Pol H-silenced cells. Viability increase was associated with improved elongation of nascent DNA, indicating the protective effect was due to more efficient lesion bypass by pol eta. This protection was observed in cells proficient or deficient in nucleotide excision repair, suggesting that, from a cell survival perspective, proper bypass of DNA damage can be as relevant as removal. These results indicate p53 controls the induction of pol eta in DNA damaged human cells, resulting in improved TLS and enhancing cell tolerance to DNA damage, which parallels SOS responses in bacteria. PMID:28180309

  9. Predominant role of DNA polymerase eta and p53-dependent translesion synthesis in the survival of ultraviolet-irradiated human cells.

    PubMed

    Lerner, Leticia K; Francisco, Guilherme; Soltys, Daniela T; Rocha, Clarissa R R; Quinet, Annabel; Vessoni, Alexandre T; Castro, Ligia P; David, Taynah I P; Bustos, Silvina O; Strauss, Bryan E; Gottifredi, Vanesa; Stary, Anne; Sarasin, Alain; Chammas, Roger; Menck, Carlos F M

    2017-02-17

    Genome lesions trigger biological responses that help cells manage damaged DNA, improving cell survival. Pol eta is a translesion synthesis (TLS) polymerase that bypasses lesions that block replicative polymerases, avoiding continued stalling of replication forks, which could lead to cell death. p53 also plays an important role in preventing cell death after ultraviolet (UV) light exposure. Intriguingly, we show that p53 does so by favoring translesion DNA synthesis by pol eta. In fact, the p53-dependent induction of pol eta in normal and DNA repair-deficient XP-C human cells after UV exposure has a protective effect on cell survival after challenging UV exposures, which was absent in p53- and Pol H-silenced cells. Viability increase was associated with improved elongation of nascent DNA, indicating the protective effect was due to more efficient lesion bypass by pol eta. This protection was observed in cells proficient or deficient in nucleotide excision repair, suggesting that, from a cell survival perspective, proper bypass of DNA damage can be as relevant as removal. These results indicate p53 controls the induction of pol eta in DNA damaged human cells, resulting in improved TLS and enhancing cell tolerance to DNA damage, which parallels SOS responses in bacteria. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Degradation of NF-κB, p53 and other regulatory redox-sensitive proteins by thiol-conjugating and -nitrosylating drugs in human tumor cells

    PubMed Central

    Srivenugopal, Kalkunte S.

    2013-01-01

    The ionized cysteines present on the surfaces of many redox-sensitive proteins play functionally essential roles and are readily targeted by the reactive oxygen and reactive nitrogen species. Using disulfiram (DSF) and nitroaspirin (NCX4016) as the model compounds that mediate thiol-conjugating and nitrosylating reactions, respectively, we investigated the fate of p53, nuclear factor-kappaB (NF-κB) and other redox-responsive proteins following the exposure of human cancer cell lines to the drugs. Both drugs induced glutathionylation of bulk proteins in tumor cells and cell-free extracts. A prominent finding of this study was a time- and dose-dependent degradation of the redox-regulated proteins after brief treatments of tumor cells with DSF or NCX4016. DSF and copper-chelated DSF at concentrations of 50–200 µM induced the disappearance of wild-type p53, mutant p53, NF-κB subunit p50 and the ubiquitin-activating enzyme E1 (UBE1) in tumor cell lines. DSF also induced the glutathionylation of p53. The recombinant p53 protein modified by DSF was preferentially degraded by rabbit reticulocyte lysates. The proteasome inhibitor PS341 curtailed the DSF-induced degradation of p53 in HCT116 cells. Further, the NCX4016 induced a dose-dependent disappearance of the UBE1 and NF-κB p50 proteins in cell lines, besides a time-dependent degradation of aldehyde dehydrogenase in mouse liver after a single injection of 150mg/kg. The loss of p53 and NF-kB proteins correlated with decreases in their specific binding to DNA. Our results demonstrate the hitherto unrecognized ability of the non-toxic thiolating and nitrosylating agents to degrade regulatory proteins and highlight the exploitable therapeutic benefits. PMID:23354308

  11. Evaluation of efficacy and safety for recombinant human adenovirus-p53 in the control of the malignant pleural effusions via thoracic perfusion

    PubMed Central

    Biaoxue, Rong; Hui, Pan; Wenlong, Gao; Shuanying, Yang

    2016-01-01

    A certain number of studies have showed that p53 gene transfer has an anti-tumor activity in vitro and in vivo. This study was to evaluate the efficacy and safety of thoracic perfusion of recombinant human adenovirus p53 (rAd-p53, Gendicine) for controlling malignant pleural effusion (MPE). We searched for the relevant studies from the database of MEDLINE, Web of Science, EMBASE, Cochrance Library and CNKI to collect the trials concerning the efficacy and safety of rAd-p53 to treat MPE. Fourteen randomised controlled trials (RCTs) with 879 patients were involved in this analysis. The rAd-p53 combined with chemotherapeutic agents significantly improved the overall response rate (ORR) (P < 0.001; odds ratio = 3.73) and disease control rate (DCR) (P < 0.001; odds ratio = 2.32) of patients with MPE as well as the quality of life (QOL) of patients (P < 0.001; odds ratio = 4.27), compared with that of chemotherapeutic agents alone. In addition, the participation of rAd-p53 did not have an obvious impact on the most of incidence of adverse reactions (AEs) (P < 0.05) except the fever (P < 0.001). However, the fever was self-limited and could be tolerated well. The application of rAd-p53 through thoracic perfusion for treating MPE had a better efficacy and safety, which could be a potential choice for controlling MPE. PMID:27976709

  12. Regulation of p53 tetramerization and nuclear export by ARC

    PubMed Central

    Foo, Roger S.-Y.; Nam, Young-Jae; Ostreicher, Marc Jason; Metzl, Mark D.; Whelan, Russell S.; Peng, Chang-Fu; Ashton, Anthony W.; Fu, Weimin; Mani, Kartik; Chin, Suet-Feung; Provenzano, Elena; Ellis, Ian; Figg, Nichola; Pinder, Sarah; Bennett, Martin R.; Caldas, Carlos; Kitsis, Richard N.

    2007-01-01

    Inactivation of the transcription factor p53 is central to carcinogenesis. Yet only approximately one-half of cancers have p53 loss-of-function mutations. Here, we demonstrate a mechanism for p53 inactivation by apoptosis repressor with caspase recruitment domain (ARC), a protein induced in multiple cancer cells. The direct binding in the nucleus of ARC to the p53 tetramerization domain inhibits p53 tetramerization. This exposes a nuclear export signal in p53, triggering Crm1-dependent relocation of p53 to the cytoplasm. Knockdown of endogenous ARC in breast cancer cells results in spontaneous tetramerization of endogenous p53, accumulation of p53 in the nucleus, and activation of endogenous p53 target genes. In primary human breast cancers with nuclear ARC, p53 is almost always WT. Conversely, nearly all breast cancers with mutant p53 lack nuclear ARC. We conclude that nuclear ARC is induced in cancer cells and negatively regulates p53. PMID:18087040

  13. p53-independent structure-activity relationships of 3-ring mesogenic compounds' activity as cytotoxic effects against human non-small cell lung cancer lines.

    PubMed

    Fukushi, Saori; Yoshino, Hironori; Yoshizawa, Atsushi; Kashiwakura, Ikuo

    2016-07-25

    We recently demonstrated the cytotoxicity of liquid crystal precursors (hereafter referred to as "mesogenic compounds") in the human non-small cell lung cancer (NSCLC) cell line A549 which carry wild-type p53. p53 mutations are observed in 50 % of NSCLC and contribute to their resistance to chemotherapy. To develop more effective and cancer-specific agents, in this study, we investigated the structure-activity relationships of mesogenic compounds with cytotoxic effects against multiple NSCLC cells. The pharmacological effects of mesogenic compounds were examined in human NSCLC cells (A549, LU99, EBC-1, and H1299) and normal WI-38 human fibroblast. Analyses of the cell cycle, cell-death induction, and capsases expression were performed. The 3-ring compounds possessing terminal alkyl and hydroxyl groups (compounds C1-C5) showed cytotoxicity in NSCLC cells regardless of the p53 status. The compounds C1 and C3, which possess a pyrimidine at the center of the core, induced G2/M arrest, while the compounds without a pyrimidine (C2, C4, and C5) caused G1 arrest; all compounds produced caspase-mediated cell death. These events occurred in a p53-independent manner. Furthermore, it was suggested that compounds induced cell death through p53-independent DNA damage-signaling pathway. Compounds C2, C4, and C5 did not show strong cytotoxicity in WI-38 cells, whereas C1 and C3 did. However, the cytotoxicity of compound C1 against WI-38 cells was improved by modulating the terminal alkyl chain lengths of the compound. We showed the p53-indepdent structure-activity relationships of mesogenic compounds related to the cytotoxic effects. These structure-activity relationships will be helpful in the development of more effective and cancer-specific agents.

  14. High levels of stable p53 protein and the expression of c-myc in cultured human epithelial tissue after cobalt-60 irradiation

    SciTech Connect

    Mothersill, C.; Seymour, C.B. ); Harney, J.; Hennessy, T.P. )

    1994-03-01

    When explants of human uroepithelium or esophageal epithelium are exposed to acute doses of radiation (cobalt-60), the cells which grow out to form the primary cultures show a number of abnormal features. These include the development of characteristic nonsenescent foci. These foci have previously been shown to be c-myc positive and to have an abnormal, tumor-like ultrastructure. Expression of c-myc and the level of stable p53 proteins have now been examined in these cultures 2 weeks after irradiation. Both proteins occurred in dividing cells at the growing edge of the explant and in the foci. The expression of c-myc appeared to be correlated with growth. As expected, variation between individual cultures of normal human cells was noted in the expression of stable p53 protein. Most control uroepithelial cell cultures were negative, but a small cohort showed a wide range of values. The control cultures from the esophageal tissues had high expression of p53, and this decreased marginally after irradiation. Cells positive for p53 were always in cycle and were usually positive for c-myc as well. It would appear from these results that the expression of c-myc and the stable form of the p53 protein occur in irradiated primary cultures of normal human cells both in foci which also express a number of abnormalities and in [open quotes]edge[close quotes] cells which are dividing. Cultures of unirradiated cells from esophagus and a small number of uroepithelial samples had high levels of p53. Possible reasons for this are discussed. 33 refs., 2 figs., 3 tabs.

  15. Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3

    PubMed Central

    Chaudhary, Ritu; Gryder, Berkley; Woods, Wendy S; Subramanian, Murugan; Jones, Matthew F; Li, Xiao Ling; Jenkins, Lisa M; Shabalina, Svetlana A; Mo, Min; Dasso, Mary; Yang, Yuan; Wakefield, Lalage M; Zhu, Yuelin; Frier, Susan M; Moriarity, Branden S; Prasanth, Kannanganattu V; Perez-Pinera, Pablo; Lal, Ashish

    2017-01-01

    Thousands of long noncoding RNAs (lncRNAs) have been discovered, yet the function of the vast majority remains unclear. Here, we show that a p53-regulated lncRNA which we named PINCR (p53-induced noncoding RNA), is induced ~100-fold after DNA damage and exerts a prosurvival function in human colorectal cancer cells (CRC) in vitro and tumor growth in vivo. Targeted deletion of PINCR in CRC cells significantly impaired G1 arrest and induced hypersensitivity to chemotherapeutic drugs. PINCR regulates the induction of a subset of p53 targets involved in G1 arrest and apoptosis, including BTG2, RRM2B and GPX1. Using a novel RNA pulldown approach that utilized endogenous S1-tagged PINCR, we show that PINCR associates with the enhancer region of these genes by binding to RNA-binding protein Matrin 3 that, in turn, associates with p53. Our findings uncover a critical prosurvival function of a p53/PINCR/Matrin 3 axis in response to DNA damage in CRC cells. DOI: http://dx.doi.org/10.7554/eLife.23244.001 PMID:28580901

  16. [Effect of lycium bararum polysaccharides on angiotensin II-induced senescence of human umbilical vein endothelial cells and expressions of P53 and P16].

    PubMed

    Liu, Ling; Wang, Xue-ni; Liu, Ze; Wang, Lu-ni; Wu, Jun; Wang, Wei; Feng, Ju-xiang

    2011-06-01

    To investigate the role of lycium bararum polysaccharides (LBP) on angiotensin II (AngII)-induced senescence of human umbilical vein endothelial cells (HUVECs) and expressions of P53 and P16 and explore the mechanism of LBP against aging. HUVECs cultured in vitro were stimulated with 1×10(-6) mmol/L AngII to induce cell senescence, which was identified using β-gal staining. Flow cytometry was used for analyzing the cell cycle changes, and the cell viability was assessed using CCK-8 method. Western blotting was employed to detect the expression of P53 and P16 in the exposed cells. Compared with the control cells, the cells positive for β-gal staining was significantly increased in AngII group, and showed cell cycle arrest at G(0)/G(1) phase with decreased S-phase cell percentage and cell viability. The expression levels of P53 and P16 were significantly increased in the cells with AngII exposure (P<0.05). LBP treatment of AngII-exposed cells resulted in decreased β-gal-positive cells with a reduction in G(0)/G(1) phase cells and an increase in S phase cells. LBP treatment also increased the cell viability and significantly decreased the expression levels of P53 and P16 (P<0.05). LBP can delay AngII-induced aging of HUVECs possibly by down-regulating the expression of P53 and P15.

  17. FOXM1 allows human keratinocytes to bypass the oncogene-induced differentiation checkpoint in response to gain of MYC or loss of p53

    PubMed Central

    Molinuevo, R; Freije, A; de Pedro, I; Stoll, S W; Elder, J T; Gandarillas, A

    2017-01-01

    Tumour suppressor p53 or proto-oncogene MYC is frequently altered in squamous carcinomas, but this is insufficient to drive carcinogenesis. We have shown that overactivation of MYC or loss of p53 via DNA damage triggers an anti-oncogenic differentiation-mitosis checkpoint in human epidermal keratinocytes, resulting in impaired cell division and squamous differentiation. Forkhead box M1 (FOXM1) is a transcription factor recently proposed to govern the expression of a set of mitotic genes. Deregulation of FOXM1 occurs in a wide variety of epithelial malignancies. We have ectopically expressed FOXM1 in keratinocytes of the skin after overexpression of MYC or inactivation of endogenous p53. Ectopic FOXM1 rescues the proliferative capacity of MYC- or p53-mutant cells in spite of higher genetic damage and a larger cell size typical of differentiation. As a consequence, differentiation induced by loss of p53 or MYC is converted into increased proliferation and keratinocytes displaying genomic instability are maintained within the proliferative compartment. The results demonstrate that keratinocyte oncogene-induced differentiation is caused by mitosis control and provide new insight into the mechanisms driving malignant progression in squamous cancer. PMID:27452522

  18. Sprint-interval but not continuous exercise increases PGC-1α protein content and p53 phosphorylation in nuclear fractions of human skeletal muscle

    PubMed Central

    Granata, Cesare; Oliveira, Rodrigo S. F.; Little, Jonathan P.; Renner, Kathrin; Bishop, David J.

    2017-01-01

    Sprint interval training has been reported to induce similar or greater mitochondrial adaptations to continuous training. However, there is limited knowledge about the effects of different exercise types on the early molecular events regulating mitochondrial biogenesis. Therefore, we compared the effects of continuous and sprint interval exercise on key regulatory proteins linked to mitochondrial biogenesis in subcellular fractions of human skeletal muscle. Nineteen men, performed either 24 min of moderate-intensity continuous cycling at 63% of WPeak (CE), or 4 × 30-s “all-out” cycling sprints (SIE). Muscle samples (vastus lateralis) were collected pre-, immediately (+0 h) and 3 (+3 h) hours post-exercise. Nuclear p53 and PHF20 protein content increased at +0 h, with no difference between groups. Nuclear p53 phosphorylation and PGC-1α protein content increased at +0 h after SIE, but not CE. We demonstrate an exercise-induced increase in nuclear p53 protein content, an event that may relate to greater p53 stability - as also suggested by increased PHF20 protein content. Increased nuclear p53 phosphorylation and PGC-1α protein content immediately following SIE but not CE suggests these may represent important early molecular events in the exercise-induced response to exercise, and that SIE is a time-efficient and possibly superior option than CE to promote these adaptations. PMID:28281651

  19. Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3.

    PubMed

    Chaudhary, Ritu; Gryder, Berkley; Woods, Wendy S; Subramanian, Murugan; Jones, Matthew F; Li, Xiao Ling; Jenkins, Lisa M; Shabalina, Svetlana A; Mo, Min; Dasso, Mary; Yang, Yuan; Wakefield, Lalage M; Zhu, Yuelin; Frier, Susan M; Moriarity, Branden S; Prasanth, Kannanganattu V; Perez-Pinera, Pablo; Lal, Ashish

    2017-06-05

    Thousands of long noncoding RNAs (lncRNAs) have been discovered, yet the function of the vast majority remains unclear. Here, we show that a p53-regulated lncRNA which we named PINCR (p53-induced noncoding RNA), is induced ~100-fold after DNA damage and exerts a prosurvival function in human colorectal cancer cells (CRC) in vitro and tumor growth in vivo. Targeted deletion of PINCR in CRC cells significantly impaired G1 arrest and induced hypersensitivity to chemotherapeutic drugs. PINCR regulates the induction of a subset of p53 targets involved in G1 arrest and apoptosis, including BTG2, RRM2B and GPX1. Using a novel RNA pulldown approach that utilized endogenous S1-tagged PINCR, we show that PINCR associates with the enhancer region of these genes by binding to RNA-binding protein Matrin 3 that, in turn, associates with p53. Our findings uncover a critical prosurvival function of a p53/PINCR/Matrin 3 axis in response to DNA damage in CRC cells.

  20. Sprint-interval but not continuous exercise increases PGC-1α protein content and p53 phosphorylation in nuclear fractions of human skeletal muscle.

    PubMed

    Granata, Cesare; Oliveira, Rodrigo S F; Little, Jonathan P; Renner, Kathrin; Bishop, David J

    2017-03-10

    Sprint interval training has been reported to induce similar or greater mitochondrial adaptations to continuous training. However, there is limited knowledge about the effects of different exercise types on the early molecular events regulating mitochondrial biogenesis. Therefore, we compared the effects of continuous and sprint interval exercise on key regulatory proteins linked to mitochondrial biogenesis in subcellular fractions of human skeletal muscle. Nineteen men, performed either 24 min of moderate-intensity continuous cycling at 63% of WPeak (CE), or 4 × 30-s "all-out" cycling sprints (SIE). Muscle samples (vastus lateralis) were collected pre-, immediately (+0 h) and 3 (+3 h) hours post-exercise. Nuclear p53 and PHF20 protein content increased at +0 h, with no difference between groups. Nuclear p53 phosphorylation and PGC-1α protein content increased at +0 h after SIE, but not CE. We demonstrate an exercise-induced increase in nuclear p53 protein content, an event that may relate to greater p53 stability - as also suggested by increased PHF20 protein content. Increased nuclear p53 phosphorylation and PGC-1α protein content immediately following SIE but not CE suggests these may represent important early molecular events in the exercise-induced response to exercise, and that SIE is a time-efficient and possibly superior option than CE to promote these adaptations.

  1. Identification of epigallocatechin-3-gallate in green tea polyphenols as a potent inducer of p53-dependent apoptosis in the human lung cancer cell line A549.

    PubMed

    Yamauchi, Rieko; Sasaki, Kaori; Yoshida, Kenichi

    2009-08-01

    The effects of green tea polyphenols on cultured cancer cells have been well characterized, especially the effects of epigallocatechin-3-gallate (EGCg), since EGCg suppresses oncogenic signaling pathways and induces cell cycle arrest or apoptosis by regulating cell cycle-associated proteins. In the present study, we attempted to identify signaling pathways or target molecules regulated by each of or a mixture of green tea polyphenols, including epicatechin (EC), epicatechin-3-gallate (ECg), epigallocatechin (EGC), and EGCg, in the human lung cancer cell line A549. ECg, EGC, and a catechin mixture, in addition to EGCg, significantly decreased cell viability. In contrast, caspase 3/7 activity, an apoptosis indicator, was specifically induced by EGCg. By conducting a series of luciferase-based reporter assays, we revealed that the catechin mixture only up-regulates the p53 reporter. EGCg was a more potent inducer of p53-dependent transcription, and this induction was further supported by the induced level of p53 protein. RNA interference (RNAi)-mediated p53 knockdown completely abolished EGCg-induced apoptosis. Finally, a proteome and western blot analysis using approximately 70 different antibodies failed to detect up-regulated proteins in catechin mixture-treated A549 cells. Taken together, these results indicate that EGCg, among several green tea polyphenols, is a potent apoptosis inducer that functions exclusively through a p53-dependent pathway in A549 cells.

  2. Arsenite induced poly(ADP-ribosyl)ation of tumor suppressor P53 in human skin keratinocytes as a possible mechanism for carcinogenesis associated with arsenic exposure

    SciTech Connect

    Komissarova, Elena V.; Rossman, Toby G.

    2010-03-15

    Arsenite is an environmental pollutant. Exposure to inorganic arsenic in drinking water is associated with elevated cancer risk, especially in skin. Arsenite alone does not cause skin cancer in animals, but arsenite can enhance the carcinogenicity of solar UV. Arsenite is not a significant mutagen at non-toxic concentrations, but it enhances the mutagenicity of other carcinogens. The tumor suppressor protein P53 and nuclear enzyme PARP-1 are both key players in DNA damage response. This laboratory demonstrated earlier that in cells treated with arsenite, the P53-dependent increase in p21{sup WAF1/CIP1} expression, normally a block to cell cycle progression after DNA damage, is deficient. Here we show that although long-term exposure of human keratinocytes (HaCaT) to a nontoxic concentration (0.1 muM) of arsenite decreases the level of global protein poly(ADP-ribosyl)ation, it increases poly(ADP-ribosyl)ation of P53 protein and PARP-1 protein abundance. We also demonstrate that exposure to 0.1 muM arsenite depresses the constitutive expression of p21 mRNA and P21 protein in HaCaT cells. Poly(ADP-ribosyl)ation of P53 is reported to block its activation, DNA binding and its functioning as a transcription factor. Our results suggest that arsenite's interference with activation of P53 via poly(ADP-ribosyl)ation may play a role in the comutagenic and cocarcinogenic effects of arsenite.

  3. Alpha-melanocyte-stimulating hormone suppresses oxidative stress through a p53-mediated signaling pathway in human melanocytes.

    PubMed

    Kadekaro, Ana Luisa; Chen, Juping; Yang, Jennifer; Chen, Shuna; Jameson, Joshua; Swope, Viki B; Cheng, Tan; Kadakia, Madhavi; Abdel-Malek, Zalfa

    2012-06-01

    Epidermal melanocytes are skin cells specialized in melanin production. Activation of the melanocortin 1 receptor (MC1R) on melanocytes by α-melanocyte-stimulating hormone (α-MSH) induces synthesis of the brown/black pigment eumelanin that confers photoprotection from solar UV radiation (UVR). Contrary to keratinocytes, melanocytes are slow proliferating cells that persist in the skin for decades, in an environment with high levels of UVR-induced reactive oxygen species (ROS). We previously reported that in addition to its role in pigmentation, α-MSH also reduces oxidative stress and enhances the repair of DNA photoproducts in melanocytes, independent of melanin synthesis. Given the significance of ROS in carcinogenesis, here we investigated the mechanisms by which α-MSH exerts antioxidant effects in melanocytes. We show that activation of the MC1R by α-MSH contributes to phosphorylation of p53 on serine 15, a known requirement for stabilization and activation of p53, a major sensor of DNA damage. This effect is mediated by the cAMP/PKA pathway and by the activation of phosphoinositide 3-kinase (PI3K) ATR and DNA protein kinase (DNA-PK). α-MSH increases the levels of 8-oxoguanine DNA glycosylase (OGG1) and apurinic apyrimidinic endonuclease 1 (APE-1/Ref-1), enzymes essential for base excision repair. Nutlin-3, an HDM2 inhibitor, mimicked the effects of α-MSH resulting in reduced phosphorylation of H2AX (γ-H2AX), a marker of DNA damage. Conversely, the p53 inhibitor pifithrin-α or silencing of p53 abolished the effects of α-MSH and augmented oxidative stress. These results show that p53 is an important target of the downstream MC1R signaling that reduces oxidative stress and possibly malignant transformation of melanocytes.

  4. Gamma irradiation results in phosphorylation of p53 at serine-392 in human T-lymphocyte leukaemia cell line MOLT-4.

    PubMed

    Szkanderová, S; Vávrová, J; Rézacová, M; Vokurková, D; Pavlová, S; Smardová, J; Stulík, J

    2003-01-01

    Exposure of human leukaemia MOLT-4 cells to ionizing irradiation led to apoptosis, which was detected by flow cytometric analysis and degradation of the nuclear lamina. The multiple signalling pathways triggered by either membrane or DNA damage play a critical role in radiation-induced apoptosis. The response to DNA damage is typically associated with the p53 protein accumulation. In this study, we proved that the transcriptionally active p53 variant occurs in the MOLT-4 cells and its abundance alteration is triggered in the gamma-irradiated cell population concomitantly with phosphorylation at both the serine-392 and serine-15 residues. The p21 upregulation followed the p53 phosphorylation process in irradiated MOLT-4 cells.

  5. Protein degradation rate is the dominant mechanism accounting for the differences in protein abundance of basal p53 in a human breast and colorectal cancer cell line.

    PubMed

    Lakatos, Eszter; Salehi-Reyhani, Ali; Barclay, Michael; Stumpf, Michael P H; Klug, David R

    2017-01-01

    We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.

  6. Mutant p53 upregulates alpha-1 antitrypsin expression and promotes invasion in lung cancer.

    PubMed

    Shakya, R; Tarulli, G A; Sheng, L; Lokman, N A; Ricciardelli, C; Pishas, K I; Selinger, C I; Kohonen-Corish, M R J; Cooper, W A; Turner, A G; Neilsen, P M; Callen, D F

    2017-04-03

    Missense mutations in the TP53 tumor-suppressor gene inactivate its antitumorigenic properties and endow the incipient cells with newly acquired oncogenic properties that drive invasion and metastasis. Although the oncogenic effect of mutant p53 transcriptome has been widely acknowledged, the global influence of mutant p53 on cancer cell proteome remains to be fully elucidated. Here, we show that mutant p53 drives the release of invasive extracellular factors (the ‘secretome’) that facilitates the invasion of lung cancer cell lines. Proteomic characterization of the secretome from mutant p53-inducible H1299 human non-small cell lung cancer cell line discovered that the mutant p53 drives its oncogenic pathways through modulating the gene expression of numerous targets that are subsequently secreted from the cells. Of these genes, alpha-1 antitrypsin (A1AT) was identified as a critical effector of mutant p53 that drives invasion in vitro and in vivo, together with induction of epithelial–mesenchymal transition markers expression. Mutant p53 upregulated A1AT transcriptionally through the involvement with its family member p63. Conditioned medium containing secreted A1AT enhanced cell invasion, while an A1AT-blocking antibody attenuated the mutant p53-driven migration and invasion. Importantly, high A1AT expression correlated with increased tumor stage, elevated p53 staining and shorter overall survival in lung adenocarcinoma patients. Collectively, these findings suggest that A1AT is an indispensable target of mutant p53 with prognostic and therapeutic potential in mutant p53-expressing tumors. Oncogene advance online publication, 3 April 2017; doi:10.1038/onc.2017.66.

  7. p53-dependent G2 arrest associated with a decrease in cyclins A2 and B1 levels in a human carcinoma cell line

    PubMed Central

    Badie, C; Bourhis, J; Sobczak-Thépot, J; Haddada, H; Chiron, M; Janicot, M; Janot, F; Tursz, T; Vassal, G

    2000-01-01

    In vivo transfer of wild-type (wt) p53 gene via a recombinant adenovirus has been proposed to induce apoptosis and increase radiosensitivity in several human carcinoma models. In the context of combining p53 gene transfer and irradiation, we investigated the consequences of adenoviral-mediated wtp53 gene transfer on the cell cycle and radiosensitivity of a human head and neck squamous cell carcinoma line (SCC97) with a p53 mutated phenotype. We showed that ectopic expression of wtp53 in SCC97 cells resulted in a prolonged G1 arrest, associated with an increased expression of the cyclin-dependent kinase inhibitor WAF1/p21 target gene. A transient arrest in G2 but not in G1 was observed after irradiation. This G2 arrest was permanent when exponentially growing cells were transduced by Ad5CMV- p53 (RPR/INGN201) immediately after irradiation with 5 or 10 Gy. Moreover, levels of cyclins A2 and B1, which are known to regulate the G2/M transition, dramatically decreased as cells arrived in G2, whereas maximal levels of expression were observed in the absence of wtp53. In conclusion, adenoviral mediated transfer of wtp53 in irradiated SCC97 cells, which are mutated for p53, appeared to increase WAF1/p21 expression and decrease levels of the mitotic cyclins A2 and B1. These observations suggest that the G2 arrest resulted from a p53-dependent premature inactivation of the mitosis promoting factor. © 2000 Cancer Research Campaign PMID:10682678

  8. From Sea Anemone to Homo sapiens: The Evolution of the p53 Family of Genes

    SciTech Connect

    Levine, Arnold

    2009-09-14

    The human genome contains three transcription factors termed p53, p63 and p73 which are related orthologues. The function of the p53 protein is to respond to a wide variety of stresses which can disrupt the fidelity of DNA replication and cell division in somatic cells of the body. These stress signals, such as DNA damage, increase the mutation rate during DNA duplication and so an active p53 protein responds by eliminating clones of cells with mutations employing apoptosis, senescence or cell cycle arrest. In this way the p53 protein acts as a tumor suppressor preventing the mutations that can lead to cancers. The p63 and p73 proteins act in a similar fashion to protect the germ line cells in females (eggs). In addition the p63 protein plays a central role in the formation of epithelial cell layers and p73 plays a critical role in the formation of several structures in the central nervous system. Based upon their amino acid sequences and structural considerations the oldest organisms that contain an ancestor of the p53/p63/p73 gene are the sea anemone or hydra. The present day representatives of these animals contain a p63/p73 like ancestor gene and the protein functions in germ cells of this animal to enforce the fidelity of DNA replication after exposure to ultraviolet light. Thus the structure and functions of this gene family have been preserved for over one billion years of evolution. Other invertebrates such as the worm, the fly and the clam contain a very similar ancestor gene with a similar set of functions. The withdrawal of a food source from a worm results in the p63/p73 mediated apoptosis of the eggs so that new organisms will not be hatched into a poor environment. A similar response is thought to occur in humans. Thus this ancestor gene ensures the fidelity of the next generation of organisms. The first time a clearly distinct new p53 gene arises is in the cartilaginous fish and in the bony fish a separation of the p

  9. From Sea Anemone to Homo Sapiens: The Evolution of the p53 Family of Genes

    SciTech Connect

    Levine, Arnold

    2009-09-14

    The human genome contains three transcription factors termed p53, p63 and p73 which are related orthologues. The function of the p53 protein is to respond to a wide variety of stresses which can disrupt the fidelity of DNA replication and cell division in somatic cells of the body. These stress signals, such as DNA damage, increase the mutation rate during DNA duplication and so an active p53 protein responds by eliminating clones of cells with mutations employing apoptosis, senescence or cell cycle arrest. In this way the p53 protein acts as a tumor suppressor preventing the mutations that can lead to cancers. The p63 and p73 proteins act in a similar fashion to protect the germ line cells in females (eggs). In addition the p63 protein plays a central role in the formation of epithelial cell layers and p73 plays a critical role in the formation of several structures in the central nervous system. Based upon their amino acid sequences and structural considerations the oldest organisms that contain an ancestor of the p53/p63/p73 gene are the sea anemone or hydra. The present day representatives of these animals contain a p63/p73 like ancestor gene and the protein functions in germ cells of this animal to enforce the fidelity of DNA replication after exposure to ultraviolet light. Thus the structure and functions of this gene family have been preserved for over one billion years of evolution. Other invertebrates such as the worm, the fly and the clam contain a very similar ancestor gene with a similar set of functions. The withdrawal of a food source from a worm results in the p63/p73 mediated apoptosis of the eggs so that new organisms will not be hatched into a poor environment. A similar response is thought to occur in humans. Thus this ancestor gene ensures the fidelity of the next generation of organisms. The first time a clearly distinct new p53 gene arises is in the cartilaginous fish and in the bony fish a separation of the p

  10. Regulation of autophagy by cytoplasmic p53.

    PubMed

    Tasdemir, Ezgi; Maiuri, M Chiara; Galluzzi, Lorenzo; Vitale, Ilio; Djavaheri-Mergny, Mojgan; D'Amelio, Marcello; Criollo, Alfredo; Morselli, Eugenia; Zhu, Changlian; Harper, Francis; Nannmark, Ulf; Samara, Chrysanthi; Pinton, Paolo; Vicencio, José Miguel; Carnuccio, Rosa; Moll, Ute M; Madeo, Frank; Paterlini-Brechot, Patrizia; Rizzuto, Rosario; Szabadkai, Gyorgy; Pierron, Gérard; Blomgren, Klas; Tavernarakis, Nektarios; Codogno, Patrice; Cecconi, Francesco; Kroemer, Guido

    2008-06-01

    Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

  11. Epigenetic inactivation of the p53-induced long noncoding RNA TP53 target 1 in human cancer

    PubMed Central

    Diaz-Lagares, Angel; Crujeiras, Ana B.; Lopez-Serra, Paula; Soler, Marta; Setien, Fernando; Goyal, Ashish; Sandoval, Juan; Hashimoto, Yutaka; Martinez-Cardús, Anna; Gomez, Antonio; Heyn, Holger; Moutinho, Catia; Espada, Jesús; Vidal, August; Paúles, Maria; Galán, Maica; Sala, Núria; Akiyama, Yoshimitsu; Martínez-Iniesta, María; Farré, Lourdes; Villanueva, Alberto; Gross, Matthias; Diederichs, Sven; Guil, Sonia; Esteller, Manel

    2016-01-01

    Long noncoding RNAs (lncRNAs) are important regulators of cellular homeostasis. However, their contribution to the cancer phenotype still needs to be established. Herein, we have identified a p53-induced lncRNA, TP53TG1, that undergoes cancer-specific promoter hypermethylation-associated silencing. In vitro and in vivo assays identify a tumor-suppressor activity for TP53TG1 and a role in the p53 response to DNA damage. Importantly, we show that TP53TG1 binds to the multifaceted DNA/RNA binding protein YBX1 to prevent its nuclear localization and thus the YBX1-mediated activation of oncogenes. TP53TG1 epigenetic inactivation in cancer cells releases the transcriptional repression of YBX1-targeted growth-promoting genes and creates a chemoresistant tumor. TP53TG1 hypermethylation in primary tumors is shown to be associated with poor outcome. The epigenetic loss of TP53TG1 therefore represents an altered event in an lncRNA that is linked to classical tumoral pathways, such as p53 signaling, but is also connected to regulatory networks of the cancer cell. PMID:27821766

  12. Epigenetic inactivation of the p53-induced long noncoding RNA TP53 target 1 in human cancer.

    PubMed

    Diaz-Lagares, Angel; Crujeiras, Ana B; Lopez-Serra, Paula; Soler, Marta; Setien, Fernando; Goyal, Ashish; Sandoval, Juan; Hashimoto, Yutaka; Martinez-Cardús, Anna; Gomez, Antonio; Heyn, Holger; Moutinho, Catia; Espada, Jesús; Vidal, August; Paúles, Maria; Galán, Maica; Sala, Núria; Akiyama, Yoshimitsu; Martínez-Iniesta, María; Farré, Lourdes; Villanueva, Alberto; Gross, Matthias; Diederichs, Sven; Guil, Sonia; Esteller, Manel

    2016-11-22

    Long noncoding RNAs (lncRNAs) are important regulators of cellular homeostasis. However, their contribution to the cancer phenotype still needs to be established. Herein, we have identified a p53-induced lncRNA, TP53TG1, that undergoes cancer-specific promoter hypermethylation-associated silencing. In vitro and in vivo assays identify a tumor-suppressor activity for TP53TG1 and a role in the p53 response to DNA damage. Importantly, we show that TP53TG1 binds to the multifaceted DNA/RNA binding protein YBX1 to prevent its nuclear localization and thus the YBX1-mediated activation of oncogenes. TP53TG1 epigenetic inactivation in cancer cells releases the transcriptional repression of YBX1-targeted growth-promoting genes and creates a chemoresistant tumor. TP53TG1 hypermethylation in primary tumors is shown to be associated with poor outcome. The epigenetic loss of TP53TG1 therefore represents an altered event in an lncRNA that is linked to classical tumoral pathways, such as p53 signaling, but is also connected to regulatory networks of the cancer cell.

  13. Genomic Instability Associated with p53 Knockdown in the Generation of Huntington’s Disease Human Induced Pluripotent Stem Cells

    PubMed Central

    Tidball, Andrew M.; Neely, M. Diana; Chamberlin, Reed; Aboud, Asad A.; Kumar, Kevin K.; Han, Bingying; Bryan, Miles R.; Aschner, Michael; Ess, Kevin C.; Bowman, Aaron B.

    2016-01-01

    Alterations in DNA damage response and repair have been observed in Huntington’s disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis, while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX, indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus, increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown. PMID:26982737

  14. Noscapine induced apoptosis via downregulation of survivin in human neuroblastoma cells having wild type or null p53.

    PubMed

    Li, Shiwang; He, Jing; Li, Shuai; Cao, Guoqing; Tang, Shaotao; Tong, Qiangsong; Joshi, Harish C

    2012-01-01

    Neuroblastoma is the most common extracranial solid tumor of childhood. It accounts for 15% of pediatric cancer deaths. Chemotherapy is the mainstay of treatment in children with advanced neuroblastoma. Noscapine, a nontoxic natural compound, can trigger apoptosis in many cancer types. We now show that p53 is dispensable for Noscapine-induced cell death in neuroblastoma cell lines, proapoptotic response to this promising chemopreventive agent is mediated by suppression of survivin protein expression. The Noscapine treatment increased levels of total and Ser(15)-phosphorylated p53 protein in SK-SY5Y cells, but the proapoptotic response to this agent was maintained even after knockdown of the p53 protein level. Exposure of SK-SY5Y and LA1-5S cells to Noscapine resulted in a marked decrease in protein and mRNA level of survivin as early as 12 hours after treatment. Ectopic expression of survivin conferred statistically significant protection against Noscapine-mediated cytoplasmic histone-associated apoptotic DNA fragmentation. Also, the Noscapine-induced apoptosis was modestly but statistically significantly augmented by RNA interference of survivin in both cell lines. Furthermore, Noscapine-induced apoptotic cell death was associated with activation of caspase-3 and cleavage of PARP. In conclusion, the present study provides novel insight into the molecular circuitry of Noscapine-induced apoptosis to indicate suppression of survivin expression as a critical mediator of this process.

  15. p53 isoforms - a conspiracy to kidnap p53 tumor suppressor activity?

    PubMed

    Marcel, V; Hainaut, P

    2009-02-01

    For 25 years, the p53 tumor suppressor protein was considered the only protein expressed by the (TP53) gene. However, in several studies the existence of p53 alternative transcripts in mouse and human cells has been documented, while their expression patterns and functions remained a mystery. Since 2002, several groups have identified and described the existence of up to 10 p53 isoforms and have demonstrated their roles in modulation of p53 suppressive activity. It is now clear that the patterns of p53 expression are much more complex than previously recognized and that these isoforms have the potential to act either synergistically or antagonistically, depending on their structure and mechanism of production. This review focuses on the different ways to produce p53 isoforms, on their specific properties, on their effect on p53 suppressive activity as well as on their implication in a new potential mechanism involved in p53 deregulation in cancer.

  16. Study on X-ray-induced apoptosis and chromosomal damage in G2 human lymphocytes in the presence of pifithrin-α, an inhibitor of p53.

    PubMed

    Ortenzi, Vincenza; Meschini, Roberta; Berni, Andrea; Mancinelli, Pierluigi; Palitti, Fabrizio

    2011-11-27

    The aim of this study is to investigate the role of the cell-cycle phase in cells exposed to radiation and chemicals in relation to the cellular response. The analysis was focused on the G2 cell-cycle phase, exploring the impact of p53 inhibition in human lymphocytes irradiated with X-rays in the presence or absence of pifithrin-α (PFT-α), a p53-specific inhibitor. Lymphocytes, 44h after stimulation to proliferate, were X-irradiated with 0.5Gy both in the presence or the absence of PFT-α and post-treated with a pulse of 5-bromodeoxyuridine (BrdUrd) to distinguish cells in the S- or G2-phase at the moment of irradiation. At early sampling times after X-ray exposure the following parameters were analysed: cellular proliferation, apoptosis, chromosomal aberrations and p53 expression. The results show an enhancement of apoptotic cells in G2 at early sampling times after irradiation and no differences in terms of chromosomal aberration induction both in cells treated with X-rays alone and in cells treated with X-rays plus PFT-α. Expression of p53 was not detectable at all recovery times. The results suggest a p53-independent apoptotic pathway acting at early times after X-ray exposure in G2 lymphocytes. Furthermore, the same yield of X-ray-induced chromatid breaks was observed both in the presence or absence of PFT-α implying that in G2 X-irradiated lymphocytes this inhibitor of the p53 protein does not affect DNA repair.

  17. Destabilization of CARP mRNAs by aloe-emodin contributes to caspase-8-mediated p53-independent apoptosis of human carcinoma cells.

    PubMed

    Lin, Meng-Liang; Lu, Yao-Cheng; Su, Hong-Lin; Lin, Hsin-Ting; Lee, Chuan-Chun; Kang, Shang-En; Lai, Tan-Chen; Chung, Jing-Gung; Chen, Shih-Shun

    2011-04-01

    Using short hairpin RNA against p53, transient ectopic expression of wild-type p53 or mutant p53 (R248W or R175H), and a p53- and p21-dependent luciferase reporter assay, we demonstrated that growth arrest and apoptosis of FaDu (human pharyngeal squamous cell carcinoma), Hep3B (hepatoma), and MG-63 (osteosarcoma) cells induced by aloe-emodin (AE) are p53-independent. Co-immunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE caused S-phase cell cycle arrest by inducing the formation of cyclin A-Cdk2-p21 complexes through extracellular signal-regulated kinase (ERK) activation. Ectopic expression of Bcl-X(L) and siRNA-mediated Bax attenuation significantly inhibited apoptosis induced by AE. Cyclosporin A or the caspase-8 inhibitor Z-IETD-FMK blocked AE-induced loss of mitochondrial membrane potential and prevented increases in reactive oxygen species and Ca(++). Z-IETD-FMK inhibited AE-induced apoptosis, Bax expression, Bid cleavage, translocation of tBid to mitochondria, ERK phosphorylation, caspase-9 activation, and the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G from mitochondria. The stability of the mRNAs encoding caspase-8 and -10-associated RING proteins (CARPs) 1 and 2 was affected by AE, whereas CARP1 or 2 overexpression inhibited caspase-8 activation and apoptosis induced by AE. Collectively, our data indicate AE induces caspase-8-mediated activation of mitochondrial death pathways by decreasing the stability of CARP mRNAs in a p53-independent manner.

  18. Overexpression of the wip1 gene abrogates the p38 MAPK/p53/Wip1 pathway and silences p16 expression in human breast cancers.

    PubMed

    Yu, Eunsil; Ahn, Yeon Sun; Jang, Se Jin; Kim, Mi-Jung; Yoon, Ho Sung; Gong, Gyungyub; Choi, Jene

    2007-03-01

    Wild-type p53-induced phosphatase (Wip1 or PPM1D) is a serine/threonine protein phosphatase expressed under various stress conditions, which selectively inactivates p38 MAPK. The finding that this gene is amplified in association with frequent gain of 17q21-24 in breast cancers supports its role as a driver oncogene. However, the pathogenetic mechanism of the wip1 gene expression in breast carcinogenesis remains to be elucidated. In this study, we examine Wip1 mRNA and protein expression in 20 breast cancer tissues and six cell lines. We additionally investigate the relationship among Wip1, active p38 MAPK, p53, and p16 proteins. In our experiments, Wip1 mRNA was significantly upregulated in 7 of 20 (35%) invasive breast cancer samples. Overexpression of Wip1 was inversely correlated with that of active (phosphor-) p38 MAPK (P = 0.007). Furthermore, Wip1-overexpressing tumors exhibited no or low levels of p16, which normally accumulates upon p38 MAPK activation (P = 0.057). Loss of p16 expression was not associated with hypermethylation of its promoter or loss of heterozygosity on 9p21. Among the 135 primary breast carcinomas further examined, a significant association was found between the Wip1 overexpression and negative staining for p53 (P value = 0.057), indicating that the tumors are wild-type for p53. This is first report showing that Wip1 overexpression abrogates the homeostatic balance maintained through the p38-p53-Wip1 pathway, and contributes to malignant progression by inactivating wild-type p53 and p38 MAPK as well as decreasing p16 protein levels in human breast tissues.

  19. Dihydroptychantol A, a macrocyclic bisbibenzyl derivative, induces autophagy and following apoptosis associated with p53 pathway in human osteosarcoma U2OS cells

    SciTech Connect

    Li Xia; Wu, William K.K.; Sun Bin; Cui Min; Liu Shanshan; Gao Jian; Lou Hongxiang

    2011-03-01

    Dihydroptychantol A (DHA), a novel macrocyclic bisbibenzyl compound extracted from liverwort Asterella angusta, has antifungal and multi-drug resistance reversal properties. Here, the chemically synthesized DHA was employed to test its anti-cancer activities in human osteosarcoma U2OS cells. Our results demonstrated that DHA induced autophagy followed by apoptotic cell death accompanied with G{sub 2}/M-phase cell cycle arrest in U2OS cells. DHA-induced autophagy was morphologically characterized by the formation of double membrane-bound autophagic vacuoles recognizable at the ultrastructural level. DHA also increased the levels of LC3-II, a marker of autophagy. Surprisingly, DHA-mediated apoptotic cell death was potentiated by the autophagy inhibitor 3-methyladenine, suggesting that autophagy may play a protective role that impedes the eventual cell death. Furthermore, p53 was shown to be involved in DHA-meditated autophagy and apoptosis. In this connection, DHA increased nuclear expression of p53, induced p53 phosphorylation, and upregulated p53 target gene p21{sup Waf1/Cip1}. In contrast, cytoplasmic p53 was reduced by DHA, which contributed to the stimulation of autophagy. In relation to the cell cycle, DHA decreased the expression of cyclin B{sub 1}, a cyclin required for progression through the G{sub 2}/M phase. Taken together, DHA induces G{sub 2}/M-phase cell cycle arrest and apoptosis in U2OS cells. DHA-induced apoptosis was preceded by the induction of protective autophagy. DHA-mediated autophagy and apoptosis are associated with the cytoplasmic and nuclear functions of p53.

  20. p53 mutation heterogeneity in cancer

    SciTech Connect

    Soussi, T. . E-mail: thierry.soussi@free.fr; Lozano, G.

    2005-06-10

    The p53 gene is inactivated in about 50% of human cancers and the p53 protein is an essential component of the cell response induced by genotoxic stresses such as those generated by radiotherapy or chemotherapy. It is therefore highly likely that these alterations are an important component in tumor resistance to therapy. The particular characteristics of these alterations, 80% of which are missense mutations leading to functionally heterogeneous proteins, make p53 a unique gene in the class of tumor suppressor genes. A considerable number of mutant p53 proteins probably have an oncogenic activity per se and therefore actively participate in cell transformation. The fact that the apoptotic and antiproliferative functions of p53 can be dissociated in certain mutants also suggests another level of complexity in the relationships between p53 inactivation and neoplasia.

  1. p53: good cop/bad cop.

    PubMed

    Sharpless, Norman E; DePinho, Ronald A

    2002-07-12

    Activation of the p53 transcription factor in response to a variety of cellular stresses, including DNA damage and oncogene activation, initiates a program of gene expression that blocks the proliferative expansion of damaged cells. While the beneficial impact of the anticancer function of p53 is well established, several recent papers suggest that p53 activation may in some circumstances act in a manner detrimental to the long-term homeostasis of the organism. Here, we discuss the significant participation of p53 in three non-mutually exclusive theories of human aging involving DNA damage, telomere shortening, and oxidative stress. These "good cop/bad cop" functions of p53 appear to place it at the nexus of two opposing forces, cancer and aging. By extension, this relationship implies that therapies aimed to reduce cancer and postpone aging, and thereby increase longevity, will necessarily work either upstream or downstream, but not on the level of, p53.

  2. Suppression of tumorigenicity of breast cancer cells by transfer of human chromosome 17 does not require transferred BRCA1 and p53 genes.

    PubMed

    Theile, M; Hartmann, S; Scherthan, H; Arnold, W; Deppert, W; Frege, R; Glaab, F; Haensch, W; Scherneck, S

    1995-02-02

    A number of candidate tumor suppressor genes located on the human chromosome 17 are thought to have a role to play in the development of breast cancer. In addition to the p53 gene on 17p13.1 and the BRCA1 gene mapped to 17q12-21, other chromosomal regions for tumor suppressor genes have been suggested to exist on 17p13.3 and both the central and the distal parts of 17q, although definitive functional proof of their involvement in breast cancer tumorigenesis is still lacking. In this report we show that microcell transfer of a human chromosome 17 into wild-type p53 breast cancer cells CAL51 results in loss of tumorigenicity and anchorage-independent growth, changes in cell morphology and a reduction of cell growth rates of the neo-selected microcell hybrids. In the hybrid cells, which express the p53 wild-type protein, only the p- and the distal parts of the q arm of donor chromosome 17 are transferred. Thus, our results provide functional evidence for the presence of one or more tumor suppressor gene(s) on chromosome 17, which are distinct from the p53 and the BRCA1 genes.

  3. Establishment of human oral-cancer cell lines (KOSC-2 and -3) carrying p53 and c-myc abnormalities by geneticin treatment.

    PubMed

    Inagaki, T; Matsuwari, S; Takahashi, R; Shimada, K; Fujie, K; Maeda, S

    1994-01-15

    Two cultured cell lines derived from human squamous-cell carcinomas were established through xenografted tumors in nude mice by "Geneticin" treatment, which allows to eliminate contaminated mouse fibroblasts and obtain enriched tumor cells at the early stage of cultivation. Line KOSC-2 and KOSC-3 were each derived from a squamous-cell carcinoma of the oral floor and of the lower gingiva, respectively. Both lines grew in a cobblestone pattern, demonstrating their epithelial heritage. Giemsa-banding patterns by chromosome analysis confirmed that both lines are of human origin. Molecular analysis of cancer-related genes, including the Ha-ras, c-myc and p53 genes, was performed. KOSC-3 cells showed co-over-expression of p53 and c-myc mRNA, in addition to p53 point mutation at codon 248 with transition from CGG to TGG. However, loss of heterozygosity (LOH) on chromosome 17 was detected in both lines by Southern blotting. These cell lines provide a model for elucidating the mechanism involving p53 inactivation and c-myc-gene over-expression.

  4. Biphasic Effects of Nitric Oxide Radicals on Radiation-Induced Lethality and Chromosome Aberrations in Human Lung Cancer Cells Carrying Different p53 Gene Status

    SciTech Connect

    Su Xiaoming; Takahashi, Akihisa; Guo Guozhen; Mori, Eiichiro; Okamoto, Noritomo; Ohnishi, Ken; Iwasaki, Toshiyasu; Ohnishi, Takeo

    2010-06-01

    Purpose: The aim of this study was to clarify the effects of nitric oxide (NO) on radiation-induced cell killing and chromosome aberrations in two human lung cancer cell lines with a different p53 gene status. Methods and Materials: We used wild-type (wt) p53 and mutated (m) p53 cell lines that were derived from the human lung cancer H1299 cell line, which is p53 null. The wtp53 and mp53 cell lines were generated by transfection of the appropriate p53 constructs into the parental cells. Cells were pretreated with different concentrations of isosorbide dinitrate (ISDN) (an NO donor) and/or 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) (an NO scavenger) and then exposed to X-rays. Cell survival, apoptosis, and chromosome aberrations were scored by use of a colony-forming assay, Hoechst 33342 staining assay and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP [deoxyuridine triphosphate] nick end labeling) assay, and chromosomal banding techniques, respectively. Results: In wtp53 cells the induction of radioresistance and the inhibition of apoptosis and chromosome aberrations were observed in the presence of ISDN at low 2- to 10-{mu}mol/L concentrations before X-irradiation. The addition of c-PTIO and ISDN into the culture medium 6 h before irradiation almost completely suppressed these effects. However, at high concentrations of ISDN (100-500 {mu}mol/L), clear evidence of radiosensitization, enhancement of apoptosis, and chromosome aberrations was detected. However, these phenomena were not observed in mp53 cells at either concentration range with ISDN. Conclusions: These results indicate that low and high concentrations of NO radicals can choreograph inverse radiosensitivity, apoptosis, and chromosome aberrations in human lung cancer cells and that NO radicals can affect the fate of wtp53 cells.

  5. Mimulone-induced autophagy through p53-mediated AMPK/mTOR pathway increases caspase-mediated apoptotic cell death in A549 human lung cancer cells.

    PubMed

    An, Hyun-Kyu; Kim, Kyoung-Sook; Lee, Ji-Won; Park, Mi-Hyun; Moon, Hyung-In; Park, Shin-Ji; Baik, Ji-Sue; Kim, Cheorl-Ho; Lee, Young-Choon

    2014-01-01

    Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy.

  6. Mimulone-Induced Autophagy through p53-Mediated AMPK/mTOR Pathway Increases Caspase-Mediated Apoptotic Cell Death in A549 Human Lung Cancer Cells

    PubMed Central

    Lee, Ji-Won; Park, Mi-Hyun; Moon, Hyung-In; Park, Shin-Ji; Baik, Ji-Sue; Kim, Cheorl-Ho; Lee, Young-Choon

    2014-01-01

    Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy. PMID:25490748

  7. P63 regulates tubular formation via epithelial-to-mesenchymal transition

    PubMed Central

    Zhang, Y; Yan, W; Chen, X

    2016-01-01

    P63, a p53 family member, is expressed as TA and ΔN isoforms. Interestingly, both TAp63 and ΔNp63 are transcription factors, and regulate both common and distinct sets of target genes. p63 is required for survival of some epithelial cell lineages, and lack of p63 leads to loss of epidermis and other epithelia in humans and mice. Here, we explored the role of p63 isoforms in cell proliferation, migration and tubulogenesis by using Madin–Darby Canine Kidney (MDCK) tubular epithelial cells in two- or three-dimensional (2-D or 3-D) culture. We found that like downregulation of p53, downregulation of p63 and TAp63 decreases expression of growth-suppressing genes, including p21, PUMA and MIC-1, and consequently promotes cell proliferation and migration in 2-D culture. However, in 3-D culture, downregulation of p63, especially TAp63, but not p53, decapacitates MDCK cells to form a cyst structure through enhanced epithelial-to-mesenchymal transition (EMT). In contrast, downregulation of ΔNp63 inhibits MDCK cell proliferation and migration in 2-D culture, and delays but does not block MDCK cell cyst formation and tubulogenesis in 3-D culture. Consistent with this, downregulation of ΔNp63 markedly upregulates growth-suppressing genes, including p21, PUMA and MIC-1. Taken together, these data suggest that TAp63 is the major isoform required for tubulogenesis by maintaining an appropriate level of EMT, whereas ΔNp63 fine-tunes the rate of cyst formation and tubulogenesis by maintaining an appropriate expression level of genes involved in cell cycle arrest and apoptosis. PMID:23542170

  8. p53 dependent apoptosis and cell cycle delay induced by heteroleptic complexes in human cervical cancer cells.

    PubMed

    Sharma, Gunjan; Rana, Nishant Kumar; Singh, Priya; Dubey, Pradeep; Pandey, Daya Shankar; Koch, Biplob

    2017-04-01

    We previously reported synthesis of novel arene ruthenium (Ru) complexes and evaluated their antitumor activity in murine lymphoma (DL) cells. In this present study we further investigated the mechanism of action of two ruthenium complexes [complex 1 (η6-arene)RuCl(2-pcdpm)] and complex 2 (η6-arene)RuCl(4-mtdpm)] in cervical cancer cell line (HeLa). Our studies demonstrate that anticancer property of these two complexes was due to induction of apoptosis through p53 mediated pathway as well as arrest of cells in G2/M phase of cell cycle. It is worth to note that the complexes did not cause any substantial cytotoxic effect on normal cells. Further in comprehensive studies, apoptosis inducing property of both complexes were established in accordance with array of morphological changes ranging from membrane blebbing to formation of apoptotic bodies and followed by DNA fragmentation assay. Furthermore, Flow cytometry by Annexin V/PI staining delineate that complex 1 and 2 have strident impact to induce apoptosis in HeLa cells. The complex 1 and 2 treated cells show increased level of intracellular ROS generation which was preceded by p53 activation. Apoptosis induced by 1 and 2 was preceded by mitochondrial aggregations which were monitored by mitotracker. In addition flow cytometry analysis showed that both complexes also effectively arrest cells at G2/M phase of cell cycle. Western blot, RT-PCR as well as Real Time analysis were used to further confirm that the complexes induced apoptosis in p53 dependent pathway. Thus, our promising results can contribute to the rational design of novel potential anticancer agents. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  9. Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the p53 or BRCAl Genes

    DTIC Science & Technology

    1997-10-01

    We originally proposed the Differential Display method (Liang and Pardee 1992) to identify genes that are modulated by p53 and BRCA1 deficiency. As...retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette. Proc Natl Acad Sci U S A 93:5185-5190 (1996). Liang P, Pardee A...Shoemaker A, Dove W. ApcMin: a mouse model for intestinal and mammary tumorigenesis. Eur J Cancer 3 1A: 1061-4 (1995). Moser A, Mattes E, Dove W, Lindstrom M

  10. IFI16 Induction by Glucose Restriction in Human Fibroblasts Contributes to Autophagy through Activation of the ATM/AMPK/p53 Pathway

    PubMed Central

    Duan, Xin; Ponomareva, Larissa; Veeranki, Sudhakar; Choubey, Divaker

    2011-01-01

    Background Glucose restriction in cells increases the AMP/ATP ratio (energetic stress), which activates the AMPK/p53 pathway. Depending upon the energetic stress levels, cells undergo either autophagy or cell death. Given that the activated p53 induces the expression of IFI16 protein, we investigated the potential role of the IFI16 protein in glucose restriction-induced responses. Methodology/Principal Findings We found that glucose restriction or treatment of human diploid fibroblasts (HDFs) with the activators of the AMPK/p53 pathway induced the expression of IFI16 protein. The induced levels of IFI16 protein were associated with the induction of autophagy and reduced cell survival. Moreover, the increase in the IFI16 protein levels was dependent upon the expression of the functional ATM protein kinase. Importantly, the knockdown of the IFI16 expression in HDFs inhibited the activation of the ATM/AMPK/p53 pathway in response to glucose restriction and also increased the survival of HDFs. Conclusions/Significance Our observations demonstrate a role for the IFI16 protein in the energetic stress-induced regulation of autophagy and cell survival. Additionally, our findings also indicate that the loss of IFI16 expression, as found in certain cancers, may provide a survival advantage to cancer cells in microenvironments with low glucose levels. PMID:21573174

  11. The retinoblastoma protein/p16 INK4A pathway but not p53 is disrupted by human papillomavirus in penile squamous cell carcinoma.

    PubMed

    Stankiewicz, Elzbieta; Prowse, David M; Ktori, Elena; Cuzick, Jack; Ambroisine, Laurence; Zhang, Xiaoxi; Kudahetti, Sakunthala; Watkin, Nicholas; Corbishley, Catherine; Berney, Daniel M

    2011-02-01

    The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood. Human papillomavirus (HPV) may be involved in carcinogenesis, but few studies have compared cell-cycle protein expression in HPV positive and negative cancers. The aim was to determine the extent of HPV infection in different histological subtypes of PSCC and its impact on the expression of key cell-cycle proteins: p53, p21, p16(INK4A) and retinoblastoma (RB) protein. One hundred and forty-eight PSCC samples were examined immunohistochemically for RB, p16(INK4A) , p53 and p21 protein expression. One hundred and two cases were typed for HPV by PCR. HPV DNA was detected in 56% of tumours, with HPV16 present in 81%. Basaloid tumours were related strongly to HPV infection (10 of 13), while verrucous were not (three of 13). Fifty-nine per cent (38 of 64) of usual type SCCs had HPV infection. RB protein correlated negatively (P<0.0001) and p16(INK4A) (P<0.0001) and p21 (P=0.0002) correlated positively with HPV infection. p53 did not correlate with HPV infection. HPV infection is present in more than half of penile cancers and it is responsible for RB pathway disruption. However, no link between HPV and p53 immunodetection was found. Only basaloid and half of usual-type PSSCs correlate with HPV infection, confirming possible separate aetiologies for those tumours. © 2011 Blackwell Publishing Limited.

  12. Cellular Stress and p53-Associated Apoptosis by Juniperus communis L. Berry Extract Treatment in the Human SH-SY5Y Neuroblastoma Cells

    PubMed Central

    Lantto, Tiina A.; Laakso, Into; Dorman, H. J. Damien; Mauriala, Timo; Hiltunen, Raimo; Kõks, Sulev; Raasmaja, Atso

    2016-01-01

    Plant phenolics have shown to activate apoptotic cell death in different tumourigenic cell lines. In this study, we evaluated the effects of juniper berry extract (Juniperus communis L.) on p53 protein, gene expression and DNA fragmentation in human neuroblastoma SH-SY5Y cells. In addition, we analyzed the phenolic composition of the extract. We found that juniper berry extract activated cellular relocalization of p53 and DNA fragmentation-dependent cell death. Differentially expressed genes between treated and non-treated cells were evaluated with the cDNA-RDA (representational difference analysis) method at the early time point of apoptotic process when p53 started to be activated and no caspase activity was detected. Twenty one overexpressed genes related to cellular stress, protein synthesis, cell survival and death were detected. Interestingly, they included endoplasmic reticulum (ER) stress inducer and sensor HSPA5 and other ER stress-related genes CALM2 and YKT6 indicating that ER stress response was involved in juniper berry extract mediated cell death. In composition analysis, we identified and quantified low concentrations of fifteen phenolic compounds. The main groups of them were flavones, flavonols, phenolic acids, flavanol and biflavonoid including glycosides of quercetin, apigenin, isoscutellarein and hypolaetin. It is suggested that juniper berry extract induced the p53-associated apoptosis through the potentiation and synergism by several phenolic compounds. PMID:27420050

  13. Biomarkers p16, Human Papillomavirus and p53 Predict Recurrence and Survival in Early Stage Squamous Cell Carcinoma of the Vulva.

    PubMed

    Hay, Casey M; Lachance, Jason A; Lucas, F L; Smith, Kahsi A; Jones, Michael A

    2016-07-01

    Vulvar squamous cell carcinoma (VSCC) develops through 2 distinct molecular pathways, one involving high-risk human papillomavirus (HPV) infection and the other through early p53 suppressor gene mutation. We sought to evaluate the influence of p53 mutation, HPV status, and p16 expression on local recurrence and disease-specific mortality in early stage VSCC. We performed a retrospective chart review of all patients with stage I VSCC at the Maine Medical Center from 1998 to 2007 (n = 92). Tumor size, depth of invasion, lymphatic/vascular space invasion, and growth pattern were recorded. Paraffin-embedded tissue blocks were stained by immunohistochemistry for p16 and p53; high-risk HPV was detected by polymerase chain reaction assay. Margin distance was determined by a gynecologic pathologist. Survival analyses were conducted to examine predictors of VSCC recurrence and disease-specific mortality. Age, depth of invasion, lymphatic/vascular space invasion, growth pattern, and margin status were not significant predictors of recurrence or disease-specific mortality. Tumor size of greater than 4.0 cm indicated a 4-fold increase in disease-specific mortality but did not significantly increase recurrence. p16-Positive patients were less likely to recur and had no VSCC-related deaths. Human papillomavirus-positive patients were less likely to recur and had no VSCC-related deaths. p53-positive patients were 3 times more likely to recur and nearly 7 times more likely to die from vulvar cancer. Our findings suggest that HPV and the surrogate biomarker p16 indicate a less aggressive type of vulvar cancer. p53 positivity was associated with poor prognosis and significantly increased both recurrence and disease-specific mortality.

  14. Anticancer effect of xanthohumol induces growth inhibition and apoptosis of human liver cancer through NF-κB/p53-apoptosis signaling pathway

    PubMed Central

    ZHAO, XIANGQIAN; JIANG, KAI; LIANG, BIN; HUANG, XIAOQIANG

    2016-01-01

    Xanthohumol may prevent and cure diabetes and atherosis, have oxidation resistance and antiviral function as well as anticancer effect preventing cancer cell metastasis. We investigate whether the anticancer effect of xanthohumol induces growth inhibition and apoptosis of human liver cancer through NF-κB/p53-apoptosis signaling pathway. Human liver cancer HepG2 cell were treated with 10, 20, 30 and 40 µM xanthohumol for 48 h. The present study showed that the anticancer effect of xanthohumol was effective in inhibiting proliferation and inducing apoptosis of human liver cancer HepG2 cells. Furthermore, the caspase-3 activity of human liver cancer HepG2 cells was increased by xanthohumol. In addition, 48-h treatment with xanthohumol suppressed NF-κB expression and promoted p53, cleaved PARP, AIF and cytochrome c expression and downregulated XIAP and Bcl-2/Bax expression in human liver cancer HepG2 cells. Therefore, the anticancer effect of xanthohumol induces growth inhibition and apoptosis of human liver cancer through the NF-κB/p53-apoptosis signaling pathway. PMID:26718026

  15. Anticancer effect of xanthohumol induces growth inhibition and apoptosis of human liver cancer through NF-κB/p53-apoptosis signaling pathway.

    PubMed

    Zhao, Xiangqian; Jiang, Kai; Liang, Bin; Huang, Xiaoqiang

    2016-02-01

    Xanthohumol may prevent and cure diabetes and atherosis, have oxidation resistance and antiviral function as well as anticancer effect preventing cancer cell metastasis. We investigate whether the anticancer effect of xanthohumol induces growth inhibition and apoptosis of human liver cancer through NF-κB/p53-apoptosis signaling pathway. Human liver cancer HepG2 cell were treated with 10, 20, 30 and 40 µM xanthohumol for 48 h. The present study showed that the anticancer effect of xanthohumol was effective in inhibiting proliferation and inducing apoptosis of human liver cancer HepG2 cells. Furthermore, the caspase-3 activity of human liver cancer HepG2 cells was increased by xanthohumol. In addition, 48-h treatment with xanthohumol suppressed NF-κB expression and promoted p53, cleaved PARP, AIF and cytochrome c expression and downregulated XIAP and Bcl-2/Bax expression in human liver cancer HepG2 cells. Therefore, the anticancer effect of xanthohumol induces growth inhibition and apoptosis of human liver cancer through the NF-κB/p53-apoptosis signaling pathway.

  16. Role of p16, CK17, p63, and human papillomavirus in diagnosis of cervical intraepithelial neoplasia and distinction from its mimics.

    PubMed

    Selvi, Kalaivani; Badhe, Bhawana Ashok; Papa, Dasari; Ganesh, Rajesh Nachiappa

    2014-05-01

    Diagnosis of cervical intraepithelial neoplasia (CIN), the precursor forms of cervical cancer, can be tricky and it has led to discrepancy between pathologists in distinguishing them from its mimics such as atypical immature metaplasia (AIM), immature squamous metaplasia (ISM), reactive atypia (RA), atrophy, and basal cell hyperplasia (BCH). To overcome this problem this study aims at using immunohistochemical (IHC) markers p16, p63, CK17, and human papillomavirus (HPV) to differentiate CIN from its mimics. This study analyzed 350 cervical samples with histomorphological diagnosis of CIN and its mimics and the utility of IHC markers p16, p63, CK17, and HPV in distinction was analyzed. p16 showed 67.76% sensitivity and 99.4% specificity whereas HPV showed 57.9% sensitivity and 91.6% specificity in detecting CIN. CK17 and p63 did not show any significance in distinguishing CIN from its mimics. After IHC of AIM cases, 66.7% were reclassified as CIN III, 27.8% as ISM with reactive atypia (ISMRA), and 5.5% case as immature condyloma. In total, 3.7% of diagnosis was upgraded to CIN and 0.6% of pre-IHC diagnosis was downgraded from CIN to reactive lesions. IHC panel comprising p16, p63, CK17, and HPV are useful adjuncts in distinguishing CIN from its mimics particularly when histomorphology has overlapping morphological features.

  17. Regulation of autophagy by cytoplasmic p53

    PubMed Central

    Tasdemir, Ezgi; Maiuri, M. Chiara; Galluzzi, Lorenzo; Vitale, Ilio; Djavaheri-Mergny, Mojgan; D'Amelio, Marcello; Criollo, Alfredo; Morselli, Eugenia; Zhu, Changlian; Harper, Francis; Nannmark, Ulf; Samara, Chrysanthi; Pinton, Paolo; Vicencio, José Miguel; Carnuccio, Rosa; Moll, Ute M.; Madeo, Frank; Paterlini-Brechot, Patrizia; Rizzuto, Rosario; Szabadkai, Gyorgy; Pierron, Gérard; Blomgren, Klas; Tavernarakis, Nektarios; Codogno, Patrice; Cecconi, Francesco; Kroemer, Guido

    2009-01-01

    Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that knockout, knockdown or pharmacological inhibition of p53 can induce autophagy in human, mouse and nematode cells. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53-/- cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53. PMID:18454141

  18. [Investigation of detoxification polymorphisms genes, methylenetetrahydrofolate-reductase (MTHFR) and P53 in the radiosensitive human cells].

    PubMed

    Shagirova, Zh M; Ushenkova, L N; Mikhaĭlov, V F; Kurbatova, L A; Kuz'mina, N S; Semiachkina, A N; Vasil'eva, I M; Mazurik, V K; Zasukhina, G D

    2010-01-01

    The genes of detoxication, MTHFR and p53 were studied in Down' and Ehlers-Danlos syndrome cells. The frequency GSTM1(0/0) genotype in Down syndrome patients was in 1.5 times higher than in control cells (p < 0.069). Opposite the frequency GSTM1(0/0) genotype in Ehlers-Danlos syndrome was 23.3% 2 times lower than in control cells (p < 0.034). This indication was in 2 times lower in women cells than in men cells and in 3 times lower than in control cells (p < 0.026). The mutations of p53 gene (7th exon) were detected in 4 from 11 Down patients (36.7%; in 2 cases af women and men), in Ehlers-Danlos patients--in 5 cases and only in men (29.4% among all the observed patients). The observations 24 healthy donors weren't revealed any mutations (p < 0.013-0.001). The hypothesis about the connection between gene polymorphisms which take a part in genome stability and radiosensitivity in Down and Ehlers-Danlos patients was developed.

  19. Plumbagin induces apoptosis via the p53 pathway and generation of reactive oxygen species in human osteosarcoma cells.

    PubMed

    Tian, Linqiang; Yin, Delong; Ren, Ye; Gong, Chen; Chen, Anmin; Guo, Feng-Jin

    2012-01-01

    Osteosarcoma, which is the most common primary bone tumor, occurs most frequently in adolescents. A number of studies have indicated that plumbagin (PL) (5-hydroxy-2-methyl-1, 4-naphthoquinone), a compound found in the plants of the Plumbaginaceae and Droseraceae families, possesses anticancer activity. However, its anticancer effects and mechanisms against osteosarcoma have not been explored. To determine the anticancer effect of PL on osteosarcoma cell lines MG-63 and U2OS, cell viability, apoptosis, cell cycle distribution, caspase-3 and caspase-9 activity and intracellular reactive oxygen species (ROS) generation were measured, and Western blot analyses were performed. PL significantly inhibited the growth of osteosarcoma cells, particularly U2OS cells. PL up-regulated the expression of p53 in U2OS cells and p21 in the two osteosarcoma cell lines causing cell cycle arrest by decreasing the expression of murine double minute 2 (MDM2)/cyclin B1 and cyclin D1. Furthermore, PL altered the ratio of Bax/Bcl-2, and may have triggered the mitochondrial apoptotic pathway, resulting in caspase-3 and caspase-9 activation. We also found that PL induced the generation of ROS in osteosarcoma cell lines. To conclude, PL exerted anticancer activity on osteosarcoma cells by inducing pro-apoptotic signaling and modulating the intracellular ROS that causes induction of apoptosis. These effects may relate to the p53 status.

  20. Solution structure and binding specificity of the p63 DNA binding domain

    PubMed Central

    Enthart, Andreas; Klein, Christian; Dehner, Alexander; Coles, Murray; Gemmecker, Gerd; Kessler, Horst; Hagn, Franz

    2016-01-01

    p63 is a close homologue of p53 and, together with p73, is grouped into the p53 family of transcription factors. p63 is known to be involved in the induction of controlled apoptosis important for differentiation processes, germ line integrity and development. Despite its high homology to p53, especially within the DNA binding domain (DBD), p63-DBD does not show cooperative DNA binding properties and is significantly more stable against thermal and chemical denaturation. Here, we determined the solution structure of p63-DBD and show that it is markedly less dynamic than p53-DBD. In addition, we also investigate the effect of a double salt bridge present in p53-DBD, but not in p63-DBD on the cooperative binding behavior and specificity to various DNA sites. Restoration of the salt bridges in p63-DBD by mutagenesis leads to enhanced binding affinity to p53-specific, but not p63-specific response elements. Furthermore, we show that p63-DBD is capable of binding to anti-apoptotic BclxL via its DNA binding interface, a feature that has only been shown for p53 so far. These data suggest that all p53 family members - despite alterations in the specificity and binding affinity - are capable of activating pro-apoptotic pathways in a tissue specific manner. PMID:27225672

  1. Solution structure and binding specificity of the p63 DNA binding domain.

    PubMed

    Enthart, Andreas; Klein, Christian; Dehner, Alexander; Coles, Murray; Gemmecker, Gerd; Kessler, Horst; Hagn, Franz

    2016-05-26

    p63 is a close homologue of p53 and, together with p73, is grouped into the p53 family of transcription factors. p63 is known to be involved in the induction of controlled apoptosis important for differentiation processes, germ line integrity and development. Despite its high homology to p53, especially within the DNA binding domain (DBD), p63-DBD does not show cooperative DNA binding properties and is significantly more stable against thermal and chemical denaturation. Here, we determined the solution structure of p63-DBD and show that it is markedly less dynamic than p53-DBD. In addition, we also investigate the effect of a double salt bridge present in p53-DBD, but not in p63-DBD on the cooperative binding behavior and specificity to various DNA sites. Restoration of the salt bridges in p63-DBD by mutagenesis leads to enhanced binding affinity to p53-specific, but not p63-specific response elements. Furthermore, we show that p63-DBD is capable of binding to anti-apoptotic BclxL via its DNA binding interface, a feature that has only been shown for p53 so far. These data suggest that all p53 family members - despite alterations in the specificity and binding affinity - are capable of activating pro-apoptotic pathways in a tissue specific manner.

  2. Molecular dynamics of the full-length p53 monomer

    PubMed Central

    Chillemi, Giovanni; Davidovich, Pavel; D’Abramo, Marco; Mametnabiev, Tazhir; Garabadzhiu, Alexander Vasilievich; Desideri, Alessandro; Melino, Gerry

    2013-01-01

    The p53 protein is frequently mutated in a very large proportion of human tumors, where it seems to acquire gain-of-function activity that facilitates tumor onset and progression. A possible mechanism is the ability of mutant p53 proteins to physically interact with other proteins, including members of the same family, namely p63 and p73, inactivating their function. Assuming that this interaction might occurs at the level of the monomer, to investigate the molecular basis for this interaction, here, we sample the structural flexibility of the wild-type p53 monomeric protein. The results show a strong stability up to 850 ns in the DNA binding domain, with major flexibility in the N-terminal transactivations domains (TAD1 and TAD2) as well as in the C-terminal region (tetramerization domain). Several stable hydrogen bonds have been detected between N-terminal or C-terminal and DNA binding domain, and also between N-terminal and C-terminal. Essential dynamics analysis highlights strongly correlated movements involving TAD1 and the proline-rich region in the N-terminal domain, the tetramerization region in the C-terminal domain; Lys120 in the DNA binding region. The herein presented model is a starting point for further investigation of the whole protein tetramer as well as of its mutants. PMID:23974096

  3. Wild-type and mutated presenilins 2 trigger p53-dependent apoptosis and down-regulate presenilin 1 expression in HEK293 human cells and in murine neurons

    PubMed Central

    Alves da Costa, Cristine; Paitel, Erwan; Mattson, Mark P.; Amson, Robert; Telerman, Adam; Ancolio, Karine; Checler, Frédéric

    2002-01-01

    Presenilins 1 and 2 are two homologous proteins that, when mutated, account for most early onset Alzheimer's disease. Several lines of evidence suggest that, among various functions, presenilins could modulate cell apoptotic responses. Here we establish that the overexpression of presenilin 2 (PS2) and its mutated form Asn-141-Ile-PS2 alters the viability of human embryonic kidney (HEK)293 cells as established by combined trypan blue exclusion, sodium 3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate assay, and propidium iodide incorporation FACS analyses. The two parent proteins increase the acetyl-DEVD-al-sensitive caspase-3-like activity in both HEK293 cells and Telencephalon specific murine neurons, modulate Bax and bcl-2 expressions, and enhance cytochrome C translocation into the cytosol. We show that overexpression of both wild-type and mutated PS2 increases p53-like immunoreactivity and transcriptional activity. We also establish that wild-type- and mutated PS2-induced caspase activation is reduced by p53 antisense approach and by pifithrin-α, a chemical inhibitor of p53. Furthermore, mouse fibroblasts in which the PS2 gene has been knocked out exhibited strongly reduced p53-transcriptional activity. Finally, we establish that the overexpression of both wild-type and mutated PS2 is accompanied by a drastic reduction of endogenous presenilin 1 (PS1) expression. Interestingly, pifithrin-α diminished endogenous PS2 immunoreactivity, whereas the inhibitor increases PS1 expression. Altogether, our data demonstrate that wild-type and familial Alzheimer's disease-linked PS2 trigger apoptosis and down-regulate PS1 expression through p53-dependent mechanisms. PMID:11904448

  4. An anthraquinone derivative from Luffa acutangula induces apoptosis in human lung cancer cell line NCI-H460 through p53-dependent pathway.

    PubMed

    Vanajothi, Ramar; Srinivasan, Pappu

    2016-01-01

    The current study was designed to evaluate the in vitro antiproliferative activity of 1,8-dihydroxy-4-methylanthracene-9,10-dione (DHMA) isolated from the Luffa acutangula against human non-small cell lung cancer cell line (NCI-H460). Induction of apoptosis and reactive oxygen species (ROS) generation was determined through fluorescence microscopic technique. Quantitative real-time PCR and western blotting analysis was carried out to detect the expression of pro-apoptotic (p53, p21, caspase-3, Bax, GADD45A, and ATM) and anti-apoptotic (NF-κB) proteins in NCI-H460 cell line. In silico studies also performed to predict the binding mechanism of DHMA with MDM2-p53 protein. The DHMA inhibited the cell viability of NCI-H460 cells in a dose-dependent manner with an IC(50) of about 50 µg/ml. It significantly reduced cell viability correlated with induction of apoptosis, which was associated with ROS generation. The apoptotic cell death was further confirmed through dual staining and DNA fragmentation assay. DHMA significantly increased the expression of anti-apoptotic protein such as p53, p21, Bax, and caspase-3 but downregulated the expression of NF-κB in NCI-H460 cell line. In silico studies demonstrate that DHMA formed hydrogen bond interaction with key residues Trp26, Phe55 and Lys24 by which it disrupt the binding of p53 with MDM2 receptor. These findings suggested that DHMA induces apoptosis in NCI-H460 via a p53-dependent pathway. This the first study on cytotoxic and apoptosis inducing activity of DHMA from L. acutangula against NCI-H460 cell line. Therefore, DHMA has therapeutic potential for lung cancer treatment.

  5. Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers.

    PubMed

    Denissenko, M F; Pao, A; Pfeifer, G P; Tang, M

    1998-03-12

    Using UvrABC incision in combination with ligation-mediated PCR (LMPCR) we have previously shown that benzo(a)pyrene diol epoxide (BPDE) adduct formation along the nontranscribed strand of the human p53 gene is highly selective; the preferential binding sites coincide with the major mutation hotspots found in human lung cancers. Both sequence-dependent adduct formation and repair may contribute to these mutation hotspots in tumor tissues. To test this possibility, we have extended our previous studies by mapping the BPDE adduct distribution in the transcribed strand of the p53 gene and quantifying the rates of repair for individual damaged bases in exons 5, 7, and 8 for both DNA strands of this gene in normal human fibroblasts. We found that: (i) on both strands, BPDE adducts preferentially form at CpG sequences, and (ii) repair of BPDE adducts in the transcribed DNA strand is consistently faster than repair of adducts in the nontranscribed strand, while repair at the major damage hotspots (guanines at codons 157, 248 and 273) in the nontranscribed strand is two to four times slower than repair at other damage sites. These results strongly suggest that both preferential adduct formation and slow repair lead to hotspots for mutations at codons 157, 248 and 273, and that the strand bias of bulky adduct repair is primarily responsible for the strand bias of G to T transversion mutations observed in the p53 gene in human cancers.

  6. Mutant p53: Multiple Mechanisms Define Biologic Activity in Cancer.

    PubMed

    Kim, Michael Paul; Zhang, Yun; Lozano, Guillermina

    2015-01-01

    The functional importance of p53 as a tumor suppressor gene is evident through its pervasiveness in cancer biology. The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of alterations involve p53 missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may concomitantly gain novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in various model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 in various organ systems are reviewed and their limitations discussed.

  7. Mutant p53: Multiple Mechanisms Define Biologic Activity in Cancer

    PubMed Central

    Kim, Michael Paul; Zhang, Yun; Lozano, Guillermina

    2015-01-01

    The functional importance of p53 as a tumor suppressor gene is evident through its pervasiveness in cancer biology. The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of alterations involve p53 missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may concomitantly gain novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in various model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 in various organ systems are reviewed and their limitations discussed. PMID:26618142

  8. Studying p53 family proteins in yeast: Induction of autophagic cell death and modulation by interactors and small molecules

    SciTech Connect

    Leão, Mariana; Gomes, Sara; Bessa, Cláudia; Soares, Joana; Raimundo, Liliana; Monti, Paola; Fronza, Gilberto; Pereira, Clara; Saraiva, Lucília

    2015-01-01

    In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions. - Highlights: • p53, p63 and p73 are individually studied in the yeast S. cerevisiae. • p53 family members induce ROS production, cell cycle arrest and autophagy in yeast. • p53 family members increase actin depolarization and expression levels in yeast. • MDM2 and MDMX inhibit the activity of p53 family members in yeast. • Yeast can be a useful tool to study the biology and drugability of p53, p63 and p73.

  9. The p53-dependent radioadaptive response

    NASA Astrophysics Data System (ADS)

    Ohnishi, Takeo

    We already reported that conditioning exposures at low doses, or at low dose-rates, lowered radiation-induced p53-dependent apoptosis in cultured cells in vitro and in the spleens of mice in vivo. In this study, the aim was to characterize the p53-dependent radioadaptive response at the molecular level. We used wild-type (wt) p53 and mutated (m) p53 containing cells derived from the human lung cancer H1299 cell line, which is p53-null. Cellular radiation sensitivities were determined with a colony-forming assay. The accumulation of p53, Hdm2, and iNOS was analyzed with Western blotting. The quantification of chromosomal aberrations was estimated by scoring dicentrics per cell. In wtp53 cells, it was demonstrated that the lack of p53 accumulation was coupled with the activation of Hdm2 after low dose irradiation (0.02 Gy). Although NO radicals were only minimally induced in wtp53 cells irradiated with a challenging irradiation (6 Gy) alone, NO radicals were seen to increase about 2-4 fold after challenging irradiation following a priming irradiation (0.02 Gy). Under similar irradiation conditions with a priming and challenging irradiation in wtp53 cells, induction of radioresistance and a depression of chromosomal aberrations were observed only in the absence of Pifithrin-α (a p53 inhibitor), RITA or Nutlin-3 (p53-Hdm2 interaction inhibitors), aminoguanidine (an iNOS inhibitor) and c-PTIO (an NO radical scavenger). On the other hand, in p53 dysfunctional cells, a radioadaptive response was not observed in the presence or absence of those inhibitors. Moreover, radioresistance developed when wtp53 cells were treated with ISDN (an NO generating agent) alone. These findings suggest that NO radicals are an initiator of the radioadaptive response acting through the activation of Hdm2 and the depression of p53 accumulations.

  10. Silencing of RUNX2 enhances gemcitabine sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the stimulation of TAp63-mediated cell death

    PubMed Central

    Sugimoto, H; Nakamura, M; Yoda, H; Hiraoka, K; Shinohara, K; Sang, M; Fujiwara, K; Shimozato, O; Nagase, H; Ozaki, T

    2015-01-01

    Runt-related transcription factor 2 (RUNX2) has been considered to be one of master regulators for osteoblast differentiation and bone formation. Recently, we have described that RUNX2 attenuates p53/TAp73-dependent cell death of human osteosarcoma U2OS cells bearing wild-type p53 in response to adriamycin. In this study, we have asked whether RUNX2 silencing could enhance gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells. Under our experimental conditions, GEM treatment increased the expression level of p53 family TAp63, whereas RUNX2 was reduced following GEM exposure, indicating that there exists an inverse relationship between the expression level of TAp63 and RUNX2 following GEM exposure. To assess whether TAp63 could be involved in the regulation of GEM sensitivity of AsPC-1 cells, small interfering RNA-mediated knockdown of TAp63 was performed. As expected, silencing of TAp63 significantly prohibited GEM-dependent cell death as compared with GEM-treated non-silencing cells. As TAp63 was negatively regulated by RUNX2, we sought to examine whether RUNX2 knockdown could enhance the sensitivity to GEM. Expression analysis demonstrated that depletion of RUNX2 apparently stimulates the expression of TAp63, as well as proteolytic cleavage of poly ADP ribose polymerase (PARP) after GEM exposure, and further augmented GEM-mediated induction of p53/TAp63-target genes, such as p21WAF1, PUMA and NOXA, relative to GEM-treated control-transfected cells, implying that RUNX2 has a critical role in the regulation of GEM resistance through the downregulation of TAp63. Notably, ablation of TAp63 gave a decrease in number of γH2AX-positive cells in response to GEM relative to control-transfected cells following GEM exposure. Consistently, GEM-dependent phosphorylation of ataxia telangiectasia-mutated protein was remarkably impaired in TAp63 knockdown cells. Collectively, our present findings strongly suggest that RUNX2-mediated repression of

  11. Moraxella catarrhalis decreases antiviral innate immune responses by down-regulation of TLR3 via inhibition of p53 in human bronchial epithelial cells.

    PubMed

    Heinrich, Annina; Haarmann, Helge; Zahradnik, Sabrina; Frenzel, Katrin; Schreiber, Frauke; Klassert, Tilman E; Heyl, Kerstin A; Endres, Anne-Sophie; Schmidtke, Michaela; Hofmann, Jörg; Slevogt, Hortense

    2016-06-01

    Chronic obstructive pulmonary disease (COPD) is complicated by infectious exacerbations with acute worsening of respiratory symptoms. Coinfections of bacterial and viral pathogens are associated with more severe exacerbations. Moraxella catarrhalis is one of the most frequent lower respiratory tract pathogens detected in COPD. We therefore studied the impact of M. catarrhalis on the antiviral innate immune response that is mediated via TLR3 and p53. Molecular interactions between M. catarrhalis and normal human bronchial epithelial (NHBE) cells as well as Beas-2B cells were studied using flow cytometry, quantitative PCR analysis, chromatin immunoprecipitation, RNA interference, and ELISA. M. catarrhalis induces a significant down-regulation of TLR3 in human bronchial epithelial cells. In M. catarrhalis-infected cells, expression of p53 was decreased. We detected a reduced binding of p53 to the tlr3 promoter, resulting in reduced TLR3 gene transcription. M. catarrhalis diminished the TLR3-dependent secretion of IFN-β, IFN-λ, and chemokine (C-X-C motif) ligand 8. In addition in M. catarrhalis infected cells, expression of rhinovirus type 1A RNA was increased compared with uninfected cells. M. catarrhalis reduces antiviral defense functions of bronchial epithelial cells, which may increase susceptibility to viral infections.-Heinrich, A., Haarmann, H., Zahradnik, S., Frenzel, K., Schreiber, F., Klassert, T. E., Heyl, K. A., Endres, A.-S., Schmidtke, M., Hofmann, J., Slevogt, H. Moraxella catarrhalis decreases antiviral innate immune responses by down-regulation of TLR3 via inhibition of p53 in human bronchial epithelial cells.

  12. SCH529074, a small molecule activator of mutant p53, which binds p53 DNA binding domain (DBD), restores growth-suppressive function to mutant p53 and interrupts HDM2-mediated ubiquitination of wild type p53.

    PubMed

    Demma, Mark; Maxwell, Eugene; Ramos, Robert; Liang, Lianzhu; Li, Cheng; Hesk, David; Rossman, Randall; Mallams, Alan; Doll, Ronald; Liu, Ming; Seidel-Dugan, Cynthia; Bishop, W Robert; Dasmahapatra, Bimalendu

    2010-04-02

    Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 microm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

  13. p63 Expression Defines a Lethal Subset of Muscle-Invasive Bladder Cancers

    PubMed Central

    Choi, Woonyoung; Shah, Jay B.; Tran, Mai; Svatek, Robert; Marquis, Lauren; Lee, I-Ling; Yu, Dasom; Adam, Liana; Wen, Sijin; Shen, Yu; Dinney, Colin; McConkey, David J.; Siefker-Radtke, Arlene

    2012-01-01

    Background p63 is a member of the p53 family that has been implicated in maintenance of epithelial stem cell compartments. Previous studies demonstrated that p63 is downregulated in muscle-invasive bladder cancers, but the relationship between p63 expression and survival is not clear. Methodology/Principal Findings We used real-time PCR to characterize p63 expression and several genes implicated in epithelial-to-mesenchymal transition (EMT) in human bladder cancer cell lines (n = 15) and primary tumors (n = 101). We correlated tumor marker expression with stage, disease-specific (DSS), and overall survival (OS). Expression of E-cadherin and p63 correlated directly with one another and inversely with expression of the mesenchymal markers Zeb-1, Zeb-2, and vimentin. Non-muscle-invasive (Ta and T1) bladder cancers uniformly expressed high levels of E-cadherin and p63 and low levels of the mesenchymal markers. Interestingly, a subset of muscle-invasive (T2–T4) tumors maintained high levels of E-cadherin and p63 expression. As expected, there was a strongly significant correlation between EMT marker expression and muscle invasion (p<0.0001). However, OS was shorter in patients with muscle-invasive tumors that retained p63 (p = 0.007). Conclusions/Significance Our data confirm that molecular markers of EMT are elevated in muscle-invasive bladder cancers, but interestingly, retention of the “epithelial” marker p63 in muscle-invasive tumors is associated with a worse outcome. PMID:22253920

  14. The Telomerase Activity of Selenium-Induced Human Umbilical Cord Mesenchymal Stem Cells Is Associated with Different Levels of c-Myc and p53 Expression.

    PubMed

    Hosseinzadeh Anvar, Leila; Hosseini-Asl, Saeid; Mohammadzadeh-Vardin, Mohammad; Sagha, Mohsen

    2017-01-01

    Selenium-as a trace element-is nutritionally essential for humans. It prevents cancerous growth by inhibiting the telomerase activity but the mechanism involved in regulation of telomerase activity in normal telomerase-positive cells remains to be elucidated. Here, we find out whether the effect of sodium selenite and selenomethionine on telomerase activity in human umbilical cord-derived mesenchymal stem cells (hUCMSCs) is associated with different levels of c-Myc and p53 expression. The use of different staining methods including ethidium bromide/acridine orange and DAPI in addition to telomeric repeat amplification protocol assay and real-time PCR indicated that different forms of selenium have opposite impacts on c-Myc and p53 expressions in both hUCMSCs and AGS, a gastric adenocarcinoma cell line, as a positive control. Our findings suggest that the signaling pathways involved in the regulation of telomerase activity in malignant and normal telomerase-positive cell types are somewhat different, at least on the c-Myc and P53 expression levels.

  15. Role of NF-κB-p53 crosstalk in ultraviolet A-induced cell death and G1 arrest in human dermal fibroblasts.

    PubMed

    Lee, Yun Kyung; Cha, Hwa Jun; Hong, Misun; Yoon, Yeongmin; Lee, Hyunjin; An, Sungkwan

    2012-01-01

    Photoaging is the premature aging of the skin caused by repeated exposure to sunlight and is characterized by a depletion of the dermal extracellular matrix. This depletion is due to the loss of fibroblast cells and their multiple functions. UVA was revealed as a major inducer of photoaging in various clinical studies. As UVA photons have long wavelength spectra, UVA penetrates deeper into the dermis than UVB and UVC, leading to the induction of cell death, the destruction of the dermal extracellular matrix through the induction of matrix metalloproteinase expression, and the repression of collagen expression. However, the exact effects of UVA on the skin remain a matter of debate. Here, we assess cell cycle stage to demonstrate that NF-κB-p53 crosstalk induces apoptosis and growth arrest in UVA-irradiated human dermal fibroblasts. In addition, UVA irradiation led to an increase of NF-κB-HDAC1 complexes, which in turn repressed cyclin D1 expression in UVA-irradiated human dermal fibroblasts. We provide direct evidence that UVA irradiation induces changes in the p53-dependent NF-κB complex that lead to growth arrest and apoptosis through the repression of cyclin D1. These studies uncovered that NF-κB-p53 crosstalk is a key regulator of UVA-dependent growth arrest and apoptosis.

  16. The Hsp90 inhibitor SNX-7081 is synergistic with fludarabine nucleoside via DNA damage and repair mechanisms in human, p53-negative chronic lymphocytic leukemia

    PubMed Central

    Alomari, Munther; Mirzaei, Mehdi; Best, O. Giles; Pascovici, Dana; Mactier, Swetlana; Mulligan, Stephen P.; Haynes, Paul A.; Christopherson, Richard I.

    2015-01-01

    Clinical trials of heat shock protein 90 (Hsp90) inhibitors have been limited by high toxicity. We previously showed that the Hsp90 inhibitor, SNX-7081, synergizes with and restores sensitivity to fludarabine nucleoside (2-FaraA) in human chronic lymphocytic leukemia (CLL) cells with lesions in the p53 pathway (Best OG, et al., Leukemia Lymphoma 53:1367-75, 2012). Here, we used label-free quantitative shotgun proteomics and comprehensive bioinformatic analysis to determine the mechanism of this synergy. We propose that 2-FaraA-induced DNA damage is compounded by SNX-7081-mediated inhibition of DNA repair, resulting in enhanced induction of apoptosis. DNA damage responses are impaired in part due to reductions in checkpoint regulators BRCA1 and cyclin D1, and cell death is triggered following reductions of MYC and nucleolin and an accumulation of apoptosis-inducing NFkB2 p100 subunit. Loss of nucleolin can activate Fas-mediated apoptosis, leading to the increase of pro-apoptotic proteins (BID, fas-associated factor-2) and subsequent apoptosis of p53-negative, 2-FaraA refractory CLL cells. A significant induction of DNA damage, indicated by increases in DNA damage marker ϕH2AX, was observed following the dual drug treatment of additional cell lines, indicating that a similar mechanism may operate in other p53-mutated human B-lymphoid cancers. These results provide valuable insight into the synergistic mechanism between SNX-7081 and 2-FaraA that may provide an alternative treatment for CLL patients with p53 mutations, for whom therapeutic options are currently limited. Moreover, this drug combination reduces the effective dose of the Hsp90 inhibitor and may therefore alleviate any toxicity encountered. PMID:26556860

  17. Chromium (VI) induces both bulky DNA adducts and oxidative DNA damage at adenines and guanines in the p53 gene of human lung cells

    PubMed Central

    Arakawa, Hirohumi; Weng, Mao-wen; Tang, Moon-shong

    2012-01-01

    Chromium (VI) [Cr(VI)], a ubiquitous environmental carcinogen, is generally believed to induce mainly mutagenic binary and ternary Cr(III)–deoxyguanosine (dG)-DNA adducts in human cells. However, both adenine (A) and guanine (G) mutations are found in the p53 gene in Cr exposure-related lung cancer. Using UvrABC nuclease and formamidopyrimidine glycosylase (Fpg), and ligation-mediated PCR methods, we mapped the distribution of bulky DNA adducts (BDA) and oxidative DNA damage (ODD) in the p53 gene in Cr(VI)-treated human lung cells. We found that both BDA and ODD formed at 2ʹ-deoxyadenosine (dA) and dG bases. To understand the causes for these Cr-induced DNA damages, we mapped the distribution of BDA adducts and ODD in the p53 gene DNA fragments induced by Cr(III), Cr(VI) and Cr(V), the three major cellular Cr forms. We found that (i) dA at –CA- is a major Cr(VI) binding site followed by -GG- and –G-. Cr(VI) does not bind to –GGG-, (ii) Cr(VI)–DNA binding specificity is distinctly different from the Cr(III)–DNA binding in which –GGG- and –GG- are preferential sites, (iii) Cr(V) binding sites include all of Cr(VI) and Cr(III)–DNA binding sites and (iv) Cr(VI) and Cr(V) induce Fpg-sensitive sites at –G-. Together, these results suggest that Cr(VI) induction of BDA and ODD at dA and dG residues is through Cr(V) intermediate. We propose that these Cr(VI)-induced BDA and ODD contribute to mutagenesis of the p53 gene that leads to lung carcinogenesis. PMID:22791815

  18. Phytochemical regulation of the tumor suppressive microRNA, miR-34a, by p53-dependent and independent responses in human breast cancer cells.

    PubMed

    Hargraves, Kris G; He, Lin; Firestone, Gary L

    2016-05-01

    The tumor suppressive microRNA miR-34a is transcriptionally regulated by p53 and shown to inhibit breast cancer cell proliferation as well as being a marker of increased disease free survival. Indole-3-carbinol (I3C) derived from cruciferous vegetables, artemisinin, extracted from the sweet wormwood plant, and artesunate, a semi-synthetic derivative of artemisinin, are phytochemicals with anti-tumorigenic properties however, little is known about the role of microRNAs in their mechanism of action. Human breast cancer cells expressing wild-type (MCF-7) or mutant p53 (T47D) were treated with a concentration range and time course of each phytochemical under conditions of cell cycle arrest as detected by flow cytometry to examine the potential connection between miR-34a expression and their anti-proliferative responses. Real-time PCR and western blot analysis of extracted RNA and total protein revealed artemsinin and artesunate increased miR-34a expression in a dose-dependent manner correlating with down-regulation of the miR-34a target gene, CDK4. I3C stimulation of miR-34a expression required functional p53, whereas, both artemisinin and artesunate up-regulated miR-34a expression regardless of p53 mutational status or in the presence of dominant negative p53. Phytochemical treatments inhibited the luciferase activity of a construct containing the wild-type 3'UTR of CDK4, but not those with a mutated miR-34a binding site, whereas, transfection of miR-34a inhibitors ablated the phytochemical mediated down-regulation of CDK4 and induction of cell cycle arrest. Our results suggest that miR-34a is an essential component of the anti-proliferative activities of I3C, artemisinin, and artesunate and demonstrate that both wild-type p53 dependent and independent pathways are responsible for miR-34a induction. © 2015 Wiley Periodicals, Inc.

  19. p53 Superfamily proteins in marine bivalve cancer and stress biology.

    PubMed

    Walker, Charles W; Van Beneden, Rebecca J; Muttray, Annette F; Böttger, S Anne; Kelley, Melissa L; Tucker, Abraham E; Thomas, W Kelley

    2011-01-01

    The human p53 tumour suppressor protein is inactivated in many cancers and is also a major player in apoptotic responses to cellular stress. The p53 protein and the two other members of this protein family (p63, p73) are encoded by distinct genes and their functions have been extensively documented for humans and some other vertebrates. The structure and relative expression levels for members of the p53 superfamily have also been reported for most major invertebrate taxa. The functions of homologous proteins have been investigated for only a few invertebrates (specifically, p53 in flies, nematodes and recently a sea anemone). These studies of classical model organisms all suggest that the gene family originally evolved to mediate apoptosis of damaged germ cells or to protect germ cells from genotoxic stress. Here, we have correlated data from a number of molluscan and other invertebrate sequencing projects to provide a framework for understanding p53 signalling pathways in marine bivalve cancer and stress biology. These data suggest that (a) the two identified p53 and p63/73-like proteins in soft shell clam (Mya arenaria), blue mussel (Mytilus edulis) and Northern European squid (Loligo forbesi) have identical core sequences and may be splice variants of a single gene, while some molluscs and most other invertebrates have two or more distinct genes expressing different p53 family members; (b) transcriptional activation domains (TADs) in bivalve p53 and p63/73-like protein sequences are 67-69% conserved with human p53, while those in ecdysozoan, cnidarian, placozoan and choanozoan eukaryotes are ≤33% conserved; (c) the Mdm2 binding site in the transcriptional activation domain is 100% conserved in all sequenced bivalve p53 proteins (e.g. Mya, Mytilus, Crassostrea and Spisula) but is not present in other non-deuterostome invertebrates; (d) an Mdm2 homologue has been cloned for Mytilus trossulus; (e) homologues for both human p53 upstream regulatory and

  20. What's new in p53

    PubMed Central

    Maritsi, D; Stagikas, D; Charalabopoulos, K; Batistatou, A

    2006-01-01

    p53 is the main intrinsic factor inducing apoptosis by recognizing the external stimuli and activating the p53 responsive genes to an irreversible series of events. P53 activates the transcription of specific proapoptotic genes called p53 target genes. A growing number of p53 responsive genes have been identified and numerous studies have demonstrated that p53 proapoptotic factors such as Noxa, Puma and Perp play cell type specific roles in p53's mediated response to certain stimuli. Perp (p53 apoptosis effector related to PMP-22) is a direct proapoptotic target gene encoding a tetraspan protein. Perp is highly expressed in cells undergoing apoptosis compared to cells under G1 arrest and its overexpression is sufficient to cause cell death in fibroblasts. Noxa is another member of the preapoptotic p53 genes family. When expressed Noxa acts in a BH3 motif-dependent localization to mitochondria, causing structural changes, activation of caspase 9 and release of cytochrome c from mitochondria to cytosol. Puma (p53 mutant of apoptosis) is another critical mediator of p53-dependent apoptosis. P53 binds to Puma-promoter gene sites, leading to puma production. The mtCLIC, a member of intracellular chloride channels, is a cytoplasmic and mitochondrial protein positively regulated by p53. Caspase 10 is induced in p53-dependent manner leading to cellular apoptosis. Other newly announced factors are also involved in p53-regulated apoptosis such as brain-specific angiogenesis inhibitor - 1 (BSAI1), MSOD and GPX genes. A global discussion on this topic is attempted in the present review article. PMID:20351806

  1. What's new in p53?

    PubMed

    Maritsi, D; Stagikas, D; Charalabopoulos, K; Batistatou, A

    2006-07-01

    p53 is the main intrinsic factor inducing apoptosis by recognizing the external stimuli and activating the p53 responsive genes to an irreversible series of events. P53 activates the transcription of specific proapoptotic genes called p53 target genes. A growing number of p53 responsive genes have been identified and numerous studies have demonstrated that p53 proapoptotic factors such as Noxa, Puma and Perp play cell type specific roles in p53's mediated response to certain stimuli. Perp (p53 apoptosis effector related to PMP-22) is a direct proapoptotic target gene encoding a tetraspan protein. Perp is highly expressed in cells undergoing apoptosis compared to cells under G1 arrest and its overexpression is sufficient to cause cell death in fibroblasts. Noxa is another member of the preapoptotic p53 genes family. When expressed Noxa acts in a BH3 motif-dependent localization to mitochondria, causing structural changes, activation of caspase 9 and release of cytochrome c from mitochondria to cytosol. Puma (p53 mutant of apoptosis) is another critical mediator of p53-dependent apoptosis. P53 binds to Puma-promoter gene sites, leading to puma production. The mtCLIC, a member of intracellular chloride channels, is a cytoplasmic and mitochondrial protein positively regulated by p53. Caspase 10 is induced in p53-dependent manner leading to cellular apoptosis. Other newly announced factors are also involved in p53-regulated apoptosis such as brain-specific angiogenesis inhibitor-1 (BSAI1), MSOD and GPX genes. A global discussion on this topic is attempted in the present review article.

  2. Apoptotic effects of Physalis minima L. chloroform extract in human breast carcinoma T-47D cells mediated by c-myc-, p53-, and caspase-3-dependent pathways.

    PubMed

    Ooi, Kheng Leong; Tengku Muhammad, Tengku Sifzizul; Lim, Chui Hun; Sulaiman, Shaida Fariza

    2010-03-01

    The chloroform extract of Physalis minima produced a significant growth inhibition against human T-47D breast carcinoma cells as compared with other extracts with an EC(50) value of 3.8 microg/mL. An analysis of cell death mechanisms indicated that the extract elicited an apoptotic cell death. mRNA expression analysis revealed the coregulation of apoptotic genes, that is, c-myc , p53, and caspase-3. The c-myc was significantly induced by the chloroform extract at the earlier phase of treatment, followed by p53 and caspase-3. Biochemical assay and ultrastructural observation displayed typical apoptotic features in the treated cells, including DNA fragmentation, blebbing and convolution of cell membrane, clumping and margination of chromatin, and production of membrane-bound apoptotic bodies. The presence of different stages of apoptotic cell death and phosphatidylserine externalization were further reconfirmed by annexin V and propidium iodide staining. Thus, the results from this study strongly suggest that the chloroform extract of P. minima induced apoptotic cell death via p53-, caspase-3-, and c-myc-dependent pathways.

  3. Nobiletin induces apoptosis and potentiates the effects of the anticancer drug 5-fluorouracil in p53-mutated SNU-16 human gastric cancer cells.

    PubMed

    Moon, Jeong Yong; Cho, Moonjae; Ahn, Kwang Seok; Cho, Somi Kim

    2013-01-01

    Nobiletin is a typical polymethoxyl flavone from citrus fruits that has anticancer properties, but the molecular mechanism of its inhibitory effects on the growth of p53-mutated SNU-16 human gastric cancer cells has not been explored. In this study, nobiletin was found to be effective at inhibiting the proliferation of SNU-16 cells than other flavonoids. Nobiletin induced the death of SNU-16 cells through apoptosis, as evidenced by the increased cell population in the sub-G1 phase, the appearance of fragmented nuclei, an increase in the Bax/Bcl-2 ratio, the proteolytic activation of caspase-9, an increase in caspase-3 activity, and the degradation of poly(ADP-ribose) polymerase (PARP) protein. We found that the combination of nobiletin plus the anticancer drug 5-fluorouracil (5-FU) reduced the viability of SNU-16 cells in a concentration-dependent manner and exhibited a synergistic anticancer effect (combination index = 0.38) when 5-FU was used at relatively low concentrations. The expression of p53 protein increased after treatment with 5-FU, but not nobiletin, whereas the expression of p21 (WAF1/CIP1) protein increased after treatment with nobiletin, but not 5-FU. The cellular responses to nobiletin and 5-FU occurred through different pathways. The results of this study suggest the potential application of nobiletin to the enhancement of 5-FU efficiency in p53 mutant tumors.

  4. Cell cycle arrest and apoptosis induced by aspidin PB through the p53/p21 and mitochondria-dependent pathways in human osteosarcoma cells.

    PubMed

    Wan, Daqian; Jiang, Chaoyin; Hua, Xin; Wang, Ting; Chai, Yimin

    2015-10-01

    Aspidin PB is a natural product extracted from Dryopteris fragrans (L.) Schott, which has been characterized for its various biological activities. We reported that aspidin PB induced cell cycle arrest and apoptosis through the p53/p21 and mitochondria-dependent pathways in human osteosarcoma cells. Aspidin PB inhibited the proliferation of Saos-2, U2OS, and HOS cells in a dose-dependent and time-dependent manner. Aspidin PB induced changes in the cell cycle regulators (cyclin A, pRb, CDK2, p53, and p21), which caused cell cycle arrest in the S phase. We also explored the role of siRNA targeted to p53; it led to a dose-dependent attenuation of aspidin PB-induced apoptosis signaling. Moreover, after treatment with aspidin PB, the p21-silenced cells decreased significantly at the S phase. Aspidin PB increased the percentage of cells with mitochondrial membrane potential disruption. Western blot analysis showed that aspidin PB inhibited Bcl-2 expression and induced Bax expression to disintegrate the outer mitochondrial membrane and caused cytochrome C release. Mitochondrial cytochrome C release was associated with the activation of caspase-9 and caspase-3 cascades. Furthermore, the double-stranded DNA breaks and reactive oxygen species signaling were both involved in aspidin PB-induced DNA damage. In addition, aspidin PB inhibited tumor growth significantly in U2OS xenografts. Above all, we conclude that aspidin PB represents a valuable natural source and may potentially be applicable in osteosarcoma therapy.

  5. Detection of human papillomavirus (HPV) DNA prevalence and p53 codon 72 (Arg72Pro) polymorphism in prostate cancer in a Greek group of patients.

    PubMed

    Michopoulou, Vasiliki; Derdas, Stavros P; Symvoulakis, Emmanouil; Mourmouras, Nikolaos; Nomikos, Alexandros; Delakas, Dimitris; Sourvinos, George; Spandidos, Demetrios A

    2014-12-01

    Prostate cancer is the most common neoplasm found in males and the second most frequent cause of cancer-related mortality in males in Greece. Among other pathogens, the detection frequency of human papillomavirus (HPV) has been found to be significantly increased in tumor tissues among patients with sexually transmitted diseases (STDs), depending on the geographical distribution of each population studied. The present study focused on the detection of HPV and the distribution of Arg72Pro p53 polymorphism in a cohort of healthy individuals, as well as prostate cancer patients. We investigated the presence of HPV in 50 paraffin-embedded prostate cancer tissues, as well as in 30 physiological tissue samples from healthy individuals by real-time PCR. Furthermore, the same group of patients was also screened for the presence of the Arg72Pro polymorphism of the p53 gene, a p53 polymorphism related to HPV. Out of the 30 control samples, only 1 was found positive for HPV (3.33 %). On the contrary, HPV DNA was detected in 8 out of the total 50 samples (16 %) in the prostate cancer samples. The distribution of the three genotypes, Arg/Arg, Arg/Pro, and Pro/Pro, was 69.6, 21.7, and 8.7 % in the cancer patients and 75.0, 17.86, and 7.14 % in healthy controls, respectively. No statistically significant association was observed between the HPV presence and the age, stage, p53 polymorphism status at codon 72, or PSA. The increased prevalence of HPV detected in the prostate cancer tissues is in agreement with that reported in previous studies, further supporting the association of HPV infection and prostate cancer.

  6. Emulsified isoflurane treatment inhibits the cell cycle and respiration of human bronchial epithelial 16HBE cells in a p53-independent manner.

    PubMed

    Yang, Hui; Deng, Jia; Jiang, Yingying; Chen, Jiao; Zeng, Xianzheng; He, Zhiyang; Jiang, Xiaojuan; Li, Zhuoning; Jiang, Chunling

    2016-07-01

    Emulsified isoflurane (EIso), as a result of its rapid anesthetic induction, recovery and convenience, is widely used as a novel intravenous general anesthetic. Treatment with EIso can reduce injuries caused by ischemia/reperfusion (I/R) to organs, including the heart, lung and liver, without knowing understanding the molecular mechanism. The present study hypothesized that treatment with EIso can affect the physiological processes of human lung bronchial epithelial cells (16HBE) prior to I/R. To test this hypothesis, the present study first constructed stable p53 knockdown and synthesis of cytochrome c oxidase (SCO)2 knockdown 16HBE cells. The above cells were subsequently treated with EIso at a concentration of 0.1 and 0.2% for 24 h. The relevant concentration of fat emulsion was used as a negative control. The expression levels of p53, p21, SCO1, SCO2 and Tp53‑induced glycolysis and apoptosis regulator (TIGAR) were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting. Subsequently, the cell proliferation, respiration and glycolysis were investigated. The results revealed that EIso treatment significantly decreased the transcription of TIGAR, SCO1 and SCO2, and increased the transcription of p21, which are all p53 target genes, in a p53-independent manner. The cell cycle was inhibited by arresting cells at the G0/G1 phase. Respiration was reduced, which caused a decrease in oxygen consumption and the accumulation of lactate and reactive oxygen species. Taken together, EIso treatment inhibited the proliferation and respiration, and promoted glycolysis in 16HBE cells. This regulatory pathway may represent a protective mechanism of EIso treatment by inhibiting cell growth and decreasing the oxygen consumption from I/R.

  7. Brahma-related gene 1 induces apoptosis in a p53-dependent manner in human rheumatoid fibroblast-like synoviocyte MH7A

    PubMed Central

    Hou, Hongli; Xing, Weipeng; Li, Wuyin

    2016-01-01

    Abstract Blocked apoptosis and aggressive inflammatory responses occur in fibroblast-like synoviocyte (FLS) of rheumatoid arthritis (RA) patients. Although Brahma-related gene 1 (BRG1) is considered as a tumor suppressor, few research covers its role in RA. This study aims to reveal effects and potential mechanisms of BRG1 in human FLS cell line MH7A. BRG1 expression in MH7A cells was altered by transfection of overexpression vectors or short hairpin RNAs (shRNAs). Cell viability and apoptosis were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry after transfection. Factors involved in inflammation and apoptosis were quantified by qPCR and Western blot. The interaction between BRG1 and p53 was assessed by immunoprecipitation (IP). Results showed that BRG1 overexpression significantly suppressed MH7A cell viability and induced apoptosis (P < 0.01), and its knockdown had opposite effects. BRG1 reduced mRNA levels of matrix metallopeptidase 3, TIMP metallopeptidase inhibitor 2, cyclooxygenase 2, and interleukin 6, implying its suppressive effects on inflammation. BRG1 interacted with and promoted p53 (P < 0.05). B-cell chronic lymphocytic leukemia/lymphoma 2 was suppressed (P < 0.05), while cytochrome c, caspase 3 (CASP3) and CASP9 were activated (P < 0.01) by BRG1. However, the regulation on these factors was abrogated by p53 knockdown (P < 0.01). These findings suggest that BRG1 may induce apoptosis and suppress inflammation in MH7A cells. Potential functional mechanisms involve the regulation of apoptotic factors by BRG1, which may depend on the recruitment and promotion of p53. This study provides the essential proof for applying BRG1 to the molecular therapy of RA. PMID:28002318

  8. Quercetin induces p53-independent apoptosis in human prostate cancer cells by modulating Bcl-2-related proteins: a possible mediation by IGFBP-3.

    PubMed

    Vijayababu, Marati R; Kanagaraj, P; Arunkumar, A; Ilangovan, R; Dharmarajan, A; Arunakaran, J

    2006-01-01

    Quercetin, a flavonoid found in onion, grapes, green vegetables, etc., has been shown to possess potent antiproliferative effects against various malignant cells. We report insulin-like growth factor-binding protein-3 (IGFBP-3) as an effector of quercetin-induced apoptosis in human prostate cancer cell lines in a p53-independent manner. We evaluated the production of IGFBP-3 in quercetin-treated cells. Apoptosis was studied in quercetin-treated cells to study the IGFBP-3-mediated role with flow cytometry and DNA fragmentation. Protein expressions of Bcl-2, Bcl-x(L), and Bax were studied by Western blot. Increased production of IGFBP-3 was associated with the increased ratio of proapoptotic to antiapoptotic members of the Bcl-2 family. In quercetin-treated PC-3 cells, an increase in Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein were observed. As PC-3 is a p53-negative cell line, these modulations of proapoptotic proteins and induction of apoptosis were independent of p53. The level of IGFBP-3 on the response of PC-3 cells to quercetin was examined. There was a twofold increase in IGFBP-3 level in conditioned media of 100 microM quercetin-treated cells. Quercetin also brought a peak at sub-G1 in PC-3 cells. Thus, increased level of IGFBP-3 was associated with increased proapoptotic proteins and apoptosis in response to quercetin, suggesting it may be a p53-independent effector of apoptosis in prostate cancer cells via its modulation of the Bax/Bcl-2 protein ratio.

  9. Peroxisome proliferator-activated receptor-β/δ inhibits human neuroblastoma cell tumorigenesis by inducing p53- and SOX2-mediated cell differentiation.

    PubMed

    Yao, Pei-Li; Chen, Liping; Dobrzański, Tomasz P; Zhu, Bokai; Kang, Boo-Hyon; Müller, Rolf; Gonzalez, Frank J; Peters, Jeffrey M

    2016-12-20

    Neuroblastoma is a common childhood cancer typically treated by inducing differentiation with retinoic acid (RA). Peroxisome proliferator-activated receptor-β/δ, (PPARβ/δ) is known to promote terminal differentiation of many cell types. In the present study, PPARβ/δ was over-expressed in three human neuroblastoma cell lines, NGP, SK-N-BE(2), and IMR-32, that exhibit high, medium, and low sensitivity, respectively, to retinoic acid-induced differentiation to determine if PPARβ/δ and retinoic acid receptors (RARs) could be jointly targeted to increase the efficacy of treatment. All-trans-RA (atRA) decreased expression of SRY (sex determining region Y)-box 2 (SOX2), a stem cell regulator and marker of de-differentiation, in NGP and SK-N-BE(2) cells with inactive or mutant tumor suppressor p53, respectively. However, atRA did not suppress SOX2 expression in IMR-32 cells carrying wild-type p53. Over-expression and/or ligand activation of PPARβ/δ reduced the average volume and weight of ectopic tumor xenografts from NGP, SK-N-BE(2), or IMR-32 cells compared to controls. Compared with that found with atRA, PPARβ/δ suppressed SOX2 expression in NGP and SK-N-BE(2) cells and ectopic xenografts, and was also effective in suppressing SOX2 expression in IMR-32 cells that exhibit higher p53 expression compared to the former cell lines. Combined, these observations demonstrate that activating or over-expressing PPARβ/δ induces cell differentiation through p53- and SOX2-dependent signaling pathways in neuroblastoma cells and tumors. This suggests that combinatorial activation of both RARα and PPARβ/δ may be suitable as an alternative therapeutic approach for RA-resistant neuroblastoma patients.

  10. p63 gene structure in the phylum mollusca.

    PubMed

    Baričević, Ana; Štifanić, Mauro; Hamer, Bojan; Batel, Renato

    2015-08-01

    Roles of p53 family ancestor (p63) in the organisms' response to stressful environmental conditions (mainly pollution) have been studied among molluscs, especially in the genus Mytilus, within the last 15 years. Nevertheless, information about gene structure of this regulatory gene in molluscs is scarce. Here we report the first complete genomic structure of the p53 family orthologue in the mollusc Mediterranean mussel Mytilus galloprovincialis and confirm its similarity to vertebrate p63 gene. Our searches within the available molluscan genomes (Aplysia californica, Lottia gigantea, Crassostrea gigas and Biomphalaria glabrata), found only one p53 family member present in a single copy per haploid genome. Comparative analysis of those orthologues, additionally confirmed the conserved p63 gene structure. Conserved p63 gene structure can be a helpful tool to complement or/and revise gene annotations of any future p63 genomic sequence records in molluscs, but also in other animal phyla. Knowledge of the correct gene structure will enable better prediction of possible protein isoforms and their functions. Our analyses also pointed out possible mis-annotations of the p63 gene in sequenced molluscan genomes and stressed the value of manual inspection (based on alignments of cDNA and protein onto the genome sequence) for a reliable and complete gene annotation. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. The function of Drosophila p53 isoforms in apoptosis

    PubMed Central

    Zhang, B; Rotelli, M; Dixon, M; Calvi, B R

    2015-01-01

    The p53 protein is a major mediator of the cellular response to genotoxic stress and is a crucial suppressor of tumor formation. In a variety of organisms, p53 and its paralogs, p63 and p73, each encode multiple protein isoforms through alternative splicing, promoters, and translation start sites. The function of these isoforms in development and disease are still being defined. Here, we evaluate the apoptotic potential of multiple isoforms of the single p53 gene in the genetic model Drosophila melanogaster. Most previous studies have focused on the p53A isoform, but it has been recently shown that a larger p53B isoform can induce apoptosis when overexpressed. It has remained unclear, however, whether one or both isoforms are required for the apoptotic response to genotoxic stress. We show that p53B is a much more potent inducer of apoptosis than p53A when overexpressed. Overexpression of two newly identified short isoforms perturbed development and inhibited the apoptotic response to ionizing radiation. Analysis of physiological protein expression indicated that p53A is the most abundant isoform, and that both p53A and p53B can form a complex and co-localize to sub-nuclear compartments. In contrast to the overexpression results, new isoform-specific loss-of-function mutants indicated that it is the shorter p53A isoform, not full-length p53B, that is the primary mediator of pro-apoptotic gene transcription and apoptosis after ionizing radiation. Together, our data show that it is the shorter p53A isoform that mediates the apoptotic response to DNA damage, and further suggest that p53B and shorter isoforms have specialized functions. PMID:25882045

  12. A novel alkaloid, evodiamine causes nuclear localization of cytochrome-c and induces apoptosis independent of p53 in human lung cancer cells

    SciTech Connect

    Mohan, Vijay; Agarwal, Rajesh; Singh, Rana P.

    2016-09-02

    Lung cancer is the most frequently diagnosed malignancy that contributes to high proportion of deaths globally among patients who die due to cancer. Chemotherapy remains the common mode of treatment for lung cancer patients though with limited success. We assessed the biological effects and associated molecular changes of evodiamine, a plant alkaloid, on human lung cancer A549 and H1299 cells along with other epithelial cancer and normal lung SAEC cells. Our data showed that 20–40 μM evodiamine treatment for 24–48 h strongly (up to 73%, P < 0.001) reduced the growth and survival of these cancer cells. However, it also moderately inhibited growth and survival of SAEC cells. A strong inhibition (P < 0.001) was observed on clonogenicity of A549 cells. Further, evodiamine increased (4-fold) mitochondrial membrane depolarization with 6-fold increase in apoptosis and a slight increase in Bax/Bcl-2 ratio. It increased the cytochrome-c release from mitochondria into the cytosol as well as nucleus. Cytosolic cytochrome-c activated cascade of caspase-9 and caspase-3 intrinsic pathway, however, DR5 and caspase-8 extrinsic pathway was also activated which could be due to nuclear cytochrome-c. Pan-caspase inhibitor (z-VAD.fmk) partially reversed evodiamine induced apoptosis. An increase in p53 as well as its serine 15 phosphorylation was also observed. Pifithrin-α, a p53 inhibitor, slightly inhibited growth of A549 cells and under p53 inhibitory condition evodiamine-induced apoptosis could not be reversed. Together these findings suggest that evodiamine is a strong inducer of apoptosis in lung epithelial cancer cells independent of their p53 status and that could involve both intrinsic as well as extrinsic pathway of apoptosis. Thus evodiamine could be a potential anticancer agent against lung cancer. - Highlights: • Evodiamine, a novel plant alkaloid, relatively selectively inhibited growth and survival of human lung cancer cells. • Increased cancer cell

  13. Mutant p53 accumulation in human breast cancer is not an intrinsic property or dependent on structural or functional disruption but is regulated by exogenous stress and receptor status.

    PubMed

    Bouchalova, Pavla; Nenutil, Rudolf; Muller, Petr; Hrstka, Roman; Appleyard, M Virginia; Murray, Karen; Jordan, Lee B; Purdie, Colin A; Quinlan, Philip; Thompson, Alastair M; Vojtesek, Borivoj; Coates, Philip J

    2014-07-01

    Many human cancers contain missense TP53 mutations that result in p53 protein accumulation. Although generally considered as a single class of mutations that abrogate wild-type function, individual TP53 mutations may have specific properties and prognostic effects. Tumours that contain missense TP53 mutations show variable p53 stabilization patterns, which may reflect the specific mutation and/or aspects of tumour biology. We used immunohistochemistry on cell lines and human breast cancers with known TP53 missense mutations and assessed the effects of each mutation with four structure-function prediction methods. Cell lines with missense TP53 mutations show variable percentages of cells with p53 stabilization under normal growth conditions, ranging from approximately 50% to almost 100%. Stabilization is not related to structural or functional disruption, but agents that stabilize wild-type p53 increase the percentages of cells showing missense mutant p53 accumulation in cell lines with heterogeneous stabilization. The same heterogeneity of p53 stabilization occurs in primary breast cancers, independent of the effect of the mutation on structural properties or functional disruption. Heterogeneous accumulation is more common in steroid receptor-positive or HER2-positive breast cancers and cell lines than in triple-negative samples. Immunohistochemcal staining patterns associate with Mdm2 levels, proliferation, grade and overall survival, whilst the type of mutation reflects downstream target activity. Inhibiting Mdm2 activity increases the extent of p53 stabilization in some, but not all, breast cancer cell lines. The data indicate that missense mutant p53 stabilization is a complex and variable process in human breast cancers that associates with disease characteristics but is unrelated to structural or functional properties. That agents which stabilize wild-type p53 also stabilize mutant p53 has implications for patients with heterogeneous mutant p53 accumulation

  14. p53 in the game of transposons.

    PubMed

    Wylie, Annika; Jones, Amanda E; Abrams, John M

    2016-11-01

    Throughout the animal kingdom, p53 genes function to restrain mobile elements and recent observations indicate that transposons become derepressed in human cancers. Together, these emerging lines of evidence suggest that cancers driven by p53 mutations could represent "transpospoathies," i.e. disease states linked to eruptions of mobile elements. The transposopathy hypothesis predicts that p53 acts through conserved mechanisms to contain transposon movement, and in this way, prevents tumor formation. How transposon eruptions provoke neoplasias is not well understood but, from a broader perspective, this hypothesis also provides an attractive framework to explore unrestrained mobile elements as inciters of late-onset idiopathic disease. Also see the video abstract here.

  15. Targeting Oncogenic Mutant p53 for Cancer Therapy.

    PubMed

    Parrales, Alejandro; Iwakuma, Tomoo

    2015-01-01

    Among genetic alterations in human cancers, mutations in the tumor suppressor p53 gene are the most common, occurring in over 50% of human cancers. The majority of p53 mutations are missense mutations and result in the accumulation of dysfunctional p53 protein in tumors. These mutants frequently have oncogenic gain-of-function activities and exacerbate malignant properties of cancer cells, such as metastasis and drug resistance. Increasing evidence reveals that stabilization of mutant p53 in tumors is crucial for its oncogenic activities, while depletion of mutant p53 attenuates malignant properties of cancer cells. Thus, mutant p53 is an attractive druggable target for cancer therapy. Different approaches have been taken to develop small-molecule compounds that specifically target mutant p53. These include compounds that restore wild-type conformation and transcriptional activity of mutant p53, induce depletion of mutant p53, inhibit downstream pathways of oncogenic mutant p53, and induce synthetic lethality to mutant p53. In this review article, we comprehensively discuss the current strategies targeting oncogenic mutant p53 in cancers, with special focus on compounds that restore wild-type p53 transcriptional activity of mutant p53 and those reducing m