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Sample records for human p53 p63

  1. Role of p53 family members p73 and p63 in human hematological malignancies.

    PubMed

    Alexandrova, Evguenia M; Moll, Ute M

    2012-11-01

    p53, mutated in over half of human cancers and about 13% of all hematological malignancies, maintains genomic integrity and triggers cellular senescence and apoptosis of damaged cells. In contrast to p53, the homologs p73 and p63 play critical roles in development of the central nervous system and skin/limbs, respectively. Moreover, dependent on the context they can exert tumor suppressor activities that cooperate with p53. Unlike p53, p73 and p63 are rarely mutated in cancers. Instead, up-regulation of the anti-apoptotic dominant-negative ΔNp73 and ΔNp63 isoforms is the most frequent abnormality in solid cancers. In hematological malignancies the most frequent p73 defect is promoter methylation and loss of expression, associated with unfavorable clinical outcomes. This suggests an essential tumor suppressor role of p73 in blood cells, also supported by genetic mouse models. Many therapeutic approaches aiming to restore p73 activity are currently being investigated. In contrast, the most frequent p63 abnormality is protein overexpression, associated with higher disease grade and poorer prognosis. Surprisingly, although available data are still scarce, the emerging picture is up-regulation of transactivation-competent TAp63 isoforms, suggesting a tumor-promoting role in this context. PMID:22497596

  2. p63 is required beside p53 for PERP-mediated apoptosis in uveal melanoma

    PubMed Central

    Awais, Raheela; Spiller, David G; White, Michael R H; Paraoan, Luminita

    2016-01-01

    Background: PERP (p53 apoptosis effector related to PMP-22), a transcriptional target of p53, is downregulated and contributes to the impairment of apoptosis in uveal melanoma (UM). Intriguingly, PERP is not induced in UM despite functional p53. p63, located on chromosome 3, which is characteristically altered in high-risk UM, can transactivate PERP. Here, we determine the functional role of p63 expression in the initiation of p53/PERP-mediated apoptosis in UM. Methods: PERP expression was monitored by quantitative PCR (qPCR) and immunoblotting in UM cell lines treated with DNA-damaging agents. The functional role of p63 was assessed by transient expression of p63-turbo GFP (p63-tGFP) in the apoptosis- resistant, 3q-deficient OCM-1 cells. Expression and localisation of p63, PERP and p53, and induction of apoptosis were characterised by qPCR, immunoblotting and live cell confocal microscopy. Results: PERP expression was significantly downregulated in all UM cell lines. DNA-damaging treatments failed to induce apoptosis and activate PERP in OCM-1 cells, which displayed non-functional levels of p63. Expression of p63-tGFP induced apoptosis with marked increase in PERP expression and associated p53 accumulation. Conclusions: Lack of p63 contributes to reduced PERP levels and impaired p53-mediated apoptosis in UM. p63 expression is required for PERP-mediated apoptosis in UM. PMID:27584665

  3. 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

    2009-04-01

    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.

  4. P63 and P73: P53 mimics, menaces and more.

    PubMed

    Yang, A; McKeon, F

    2000-12-01

    Inactivation of the tumour suppressor p53 is the most common defect in cancer cells. The discovery of its two close relatives, p63 and p73, was therefore both provocative and confounding. Were these new genes tumour suppressors, p53 regulators, or evolutionary spin-offs? Both oncogenic and tumour-suppressor properties have now been attributed to the p53 homologues, perhaps reflecting the complex, often contradictory, protein products encoded by these genes. p63 and p73 are further implicated in many p53-independent pathways, including stem-cell regeneration, neurogenesis and sensory processes.

  5. SPATA18, a Spermatogenesis-Associated Gene, Is a Novel Transcriptional Target of p53 and p63

    PubMed Central

    Bornstein, Chamutal; Brosh, Ran; Molchadsky, Alina; Madar, Shalom; Kogan-Sakin, Ira; Goldstein, Ido; Chakravarti, Deepavali; Flores, Elsa R.; Goldfinger, Naomi; Sarig, Rachel; Rotter, Varda

    2011-01-01

    The transcription factor p53 functions not only to suppress tumorigenesis but also to maintain normal development and homeostasis. Although p53 was implicated in different aspects of fertility, including spermatogenesis and implantation, the mechanism underlying p53 involvement in spermatogenesis is poorly resolved. In this study we describe the identification of a spermatogenesis-associated gene, SPATA18, as a novel p53 transcriptional target and show that SPATA18 transcription is induced by p53 in a variety of cell types of both human and mouse origin. p53 binds a consensus DNA motif that resides within the first intron of SPATA18. We describe the spatiotemporal expression patterns of SPATA18 in mouse seminiferous tubules and suggest that SPATA18 transcription is regulated in vivo by p53. We also demonstrate the induction of SPATA18 by p63 and suggest that p63 can compensate for the loss of p53 activity in vivo. Our data not only enrich the known collection of p53 targets but may also provide insights on spermatogenesis defects that are associated with p53 deficiency. PMID:21300779

  6. 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.

  7. p53/p63/p73 in the Epidermis in Health and Disease

    PubMed Central

    Botchkarev, Vladimir A.; Flores, Elsa R.

    2014-01-01

    Although p53 has long been known as the “guardian of the genome” with a role in tumor suppression in many tissues, the discovery of two p53 ancestral genes, p63 and p73, more than a decade ago has triggered a considerable amount of research into the role of these genes in skin development and diseases. In this review, we primarily focus on mechanisms of action of p53 and p63, which are the best-studied p53 family members in the skin. The existence of multiple isoforms and their roles as transcriptional activators and repressors are key to their function in multiple biological processes including the control of skin morphogenesis, regeneration, tumorigenesis, and response to chemotherapy. Last, we provide directions for further research on this family of genes in skin biology and pathology. PMID:25085956

  8. Expression profiles of p53, p63, and p73 in benign salivary gland tumors.

    PubMed

    Weber, Anette; Langhanki, Larissa; Schütz, Alexander; Gerstner, Andreas; Bootz, Friedrich; Wittekind, Christian; Tannapfel, Andrea

    2002-11-01

    The tumor-suppressor protein p53 has recently been shown to belong to a family that includes two structurally related proteins, p63 and p73. In contrast to p53, p63 and p73 play an essential role in epithelial development, stem cell identity and cellular differentiation. Salivary gland tumors carry a wide spectrum of histopathological forms, which may share a common single-cell origin from the epithelial progenitor basal duct cells and have a different tendency of malignant progression. This study was performed to examine the expression of p53, p63, and p73 in benign salivary gland tumors. Expression and mutation of p53, p73, and p63 were examined by direct DNA sequencing, reverse transcription PCR using isoform-specific primers, and by immunohistochemistry in normal parotid tissue ( n=10), and various tumors of the salivary gland (42 pleomorphic adenomas, 12 myoepitheliomas, 8 basal cell adenomas, 5 oncocytomas, 5 canalicular adenomas, and 20 adenolymphomas). In normal parotid tissue the expression of p63 and p73 was restricted to few basal and myoepithelial cells. Ductal luminal and acinus cells were completely negative for the expression of all three family members. In contrast, in salivary gland tumors, strong nuclear staining for p63 and p73 was observed. Myoepithelial and basaloid cells and the basal epithelial layer of adenolyphomas and oncocytomas were positive for p63 and also, to a lesser extent, to p73. Mutations of p53 were detected in 4 of 42 (10%) pleomorphic adenomas, in 3 of 12 (25%) myoepitheliomas, and in 1 of 8 (13%) basal cell adenomas but not in other tumors. We failed to detect specific mutations of p63 and p73. Using isoform-specific PCR, we found that all isoforms of p63 were expressed in normal parotid tissue whereas the pleomorphic adenomas, myoepitehliomas, and basal cell adenomas dominantly expressed the transactivation-incompetent truncated isoforms. Our data indicate that p63 and p73 are upregulated in salivary gland tumors and may

  9. A dominant negative form of p63 inhibits apoptosis in a p53-independent manner

    SciTech Connect

    Lee, Hae-ock; Lee, Jung-Hwa; Choi, Eunhee; Seol, Ja Young; Yun, Yungdae; Lee, Hyunsook . E-mail: HL212@snu.ac.kr

    2006-05-26

    Stem cells are a source of differentiated cells in multiple tissues. If genetic alterations occur in stem cells, the problem persists and malignant cancers may arise. {delta}Np63{alpha}-a homologue of the tumor suppressor p53-is exclusively expressed in proliferating undifferentiated epithelial cells and cancer cells of epidermal origin. Here, we show that {delta}Np63{alpha} antagonizes DNA damage-induced apoptosis in a p53-independent manner. We found that upon cellular injury, {delta}Np63{alpha} must be downregulated before apoptotic program can be activated. The 5637 cell line has abundant levels of {delta}Np63{alpha} and mutant p53, and it is resistant to DNA damage-induced apoptosis. The knockdown of {delta}Np63{alpha} by RNA interference sensitized these cells to apoptosis upon genotoxic insult. This suggests that {delta}Np63{alpha} plays an anti-apoptotic role regardless of the p53 status. Considering the frequent mutations of p53 in tumor cells, our results provide important implications for the treatment of cancers in which p63 is amplified.

  10. 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

  11. 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

  12. 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

    Xu-Monette, Zijun Y; Zhang, Shanxiang; Li, Xin; 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

    2016-02-01

    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

  13. 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

  14. Immunohistochemical expression of p63, p53 and MIB-1 in urinary bladder carcinoma. A tissue microarray study of 158 cases.

    PubMed

    Compérat, Eva; Camparo, Philippe; Haus, Rachel; Chartier-Kastler, Emmanuel; Bart, Stephane; Delcourt, Annick; Houlgatte, Alain; François, Richard; Capron, Fréderique; Vieillefond, Annick

    2006-03-01

    P63 is a member of the p53 family, which plays a role in the differentiation of urothelium and is supposed to play a role in urothelial carcinogenesis. P53 and MIB-1 are recognised in many studies as predictive markers of progression, but few studies in the literature have examined p63. The aims of our study were to explore the expression of p63 in bladder carcinomas and to compare this expression to p53 and MIB-1, as well as to stage and grade. Tissue microarrays were performed on 158 urothelial carcinomas (56 pTa, 45 pT1 and 57>or=pT2). Immunohistochemical studies were performed with p63, p53 and MIB-1 antibodies. In our study we observed that p63 immunostaining is present in all cell layers in papillary urothelial neoplasm of low malignant potential (PUNLMP), but partially lost in non-invasive papillary urothelial carcinoma low grade (NILGC) and in pT1/>or=pT2 bladder cancers. P53 and MIB-1 displayed lower expression in PUNLMP/NILGC vs non-invasive papillary urothelial carcinoma high grade (NIHGC)/pT1, but there was no correlation between the expression of p63, p53 and MIB-1. Our study demonstrates that p63 expression distinguishes between PUNLMP/NILGC and NIHGC/pT1 (p=4.10(5)). A statistical difference disserving pTa and pT1/>or=pT2 with a statistical significance (p<10(-6)) could also be observed. P63 should be considered as an additional biomarker that might help pathologists to classify their patients.

  15. 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

  16. Transcriptional control of human p53-regulated genes.

    PubMed

    Riley, Todd; Sontag, Eduardo; Chen, Patricia; Levine, Arnold

    2008-05-01

    The p53 protein regulates the transcription of many different genes in response to a wide variety of stress signals. Following DNA damage, p53 regulates key processes, including DNA repair, cell-cycle arrest, senescence and apoptosis, in order to suppress cancer. This Analysis article provides an overview of the current knowledge of p53-regulated genes in these pathways and others, and the mechanisms of their regulation. In addition, we present the most comprehensive list so far of human p53-regulated genes and their experimentally validated, functional binding sites that confer p53 regulation. PMID:18431400

  17. 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

  18. Influence of Human p53 on Plant Development.

    PubMed

    Ma, Huimin; Song, Teng; Wang, Tianhua; Wang, Shui

    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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. Loss of the p53/p63 Target PERP is an Early Event in Oral Carcinogenesis and Correlates with Higher Rate of Local Relapse

    PubMed Central

    Kong, Christina S.; Cao, Hongbin; Kwok, Shirley; Nguyen, Catherine M.; Jordan, Richard C.; Beaudry, Veronica G.; Attardi, Laura D.; Le, Quynh-Thu

    2012-01-01

    BACKGROUND PERP is a p53/p63 regulated gene encoding a desmosomal protein that plays a critical role in cell-cell adhesion and tumor suppression. STUDY DESIGN We evaluated PERP expression in different grades of oral dysplasia (34 cases) and at different stages of invasive squamous cell carcinoma (SCC), and correlated the latter with clinical outcome. A tissue microarray (TMA) consisting of non-dysplastic mucosa, carcinoma in situ, SCC and nodal metastases from 33 patients with HPV-negative SCC was stained for PERP and E-cadherin. RESULTS Complete loss of PERP expression was associated with worse local control in patients with SCC. The 5-year local control rate was 91% for patients with partial PERP loss versus 31% for those with complete loss (p = 0.01). CONCLUSIONS This is the first study to show that loss of PERP expression correlates with the transition to SCC and with increased local relapse in patients with oral cavity SCC. PMID:23217540

  4. Transcriptional activation of the human epidermal growth factor receptor promoter by human p53.

    PubMed Central

    Ludes-Meyers, J H; Subler, M A; Shivakumar, C V; Munoz, R M; Jiang, P; Bigger, J E; Brown, D R; Deb, S P; Deb, S

    1996-01-01

    The human epidermal growth factor receptor (EGFR) promoter is activated by both wild-type and tumor-derived mutant p53. In this communication, we demonstrate that EGFR promoter sequence requirements for transactivation by wild-type and mutant p53 are different. Transient-expression assays with EGFR promoter deletions identified a wild-type human p53 response element, 5'-AGCTAGACGTCCGGGCAGCCCCCGGCG -3', from positions --265 to --239. Electrophoretic mobility shift analysis and DNase I footprinting assays indicated that wild-type p53 binds sequence specifically to the response element. Using circularly permuted DNA fragments containing the p53-binding site, we show that wild-type p53 binding induces DNA bending at this site. We further show that the EGFR promoter is also activated by tumor-derived p53 mutants p53-143A, p53-175H, p53-248W, p53-273H, and p53-281G. However, the transactivation by mutant p53 does not require the wild-type p53-binding site. The minimal EGFR promoter from positions --104 to --20 which does not contain the wild-type p53-binding site is transactivated by the p53 mutants but not by the wild-type protein, showing a difference in the mechanism of transactivation by wild-type and mutant p53. Transactivation of the EGFR promoter by p53 may represent a novel mechanism of cell growth regulation. PMID:8887630

  5. Expression pattern of the apoptosis-stimulating protein of p53 family in p53+ human breast cancer cell lines

    PubMed Central

    2013-01-01

    Background The apoptosis-stimulating protein of p53 (ASPP) family comprises three members, namely, ASPP1, ASPP2, and iASPP. They regulate the promotive effect of p53 on apoptosis. Breast cancer (BC) remains as one of the leading causes of cancer or cancer-related mortality among women. However, the relationship between the ASPP family members and p53, as well as the dissemination and expression pattern of ASPP family members in p53+ BC, has not been elucidated. Our objectives are to detect the expression of ASPP family members in p53+ BC cell lines and determine its significance in tumor cell apoptosis. Methods The mRNA expression of ASPP family members in five p53+ BC cell lines was detected through RT-PCR and assayed using Quality-one software. The p53 protein expression was detected by immunohistochemistry. Afterward, the apoptosis indices of the five BC cell lines were detected by flow cytometry. Results The iASPP mRNA was expressed in Bcap-37, MCF-7, and HBL-100. Compared with the human peripheral blood mononuclear cells, significant differences were found in the ASPP1 mRNA in Bcap-37, MDA-MB-231, MCF-7, and HBL-100 (p < 0.05), except that in ZR-75-30 (p > 0.05). The ASPP2 mRNA was expressed in MDA-MB-231, Bcap-37, and MCF-7, but not in HBL-100 and ZR-75-30. The p53 protein was expressed in five breast cancer cell lines. ZR-75-30 and MDA-MB-231 apoptosis indices were higher than those of other breast cancer cell line and peripheral blood mononuclear cells (p < 0.01). Conclusions The mRNA expression of ASPP family members varied in the five p53+ BC cell lines. The results also verified that the family members have an important function in apoptosis, which was promoted by p53 protein. ZR-75-30 BC showed high apoptosis index, without expression of any ASPP family members, indicating that the pathway of apoptosis in this cell line may be related to other cell transduction pathway. MDA-MB-231, Bcap37, and MCF-7 cell lines all expressed ASPP1/2. However, the

  6. Wild-type human p53 transactivates the human proliferating cell nuclear antigen promoter

    SciTech Connect

    Shivakumar, C.V.; Brown, D.R.; Deb, S.; Deb, S.P.

    1995-12-01

    The p53 tumor suppressor protein negatively regulates cell growth and somatic mutations in the p53 gene lead to uncontrolled cell growth and oncogenesis. This report describes research which demonstrates, using a number of different cell lines, that at low levels, wild-type p53 transactivates the human proliferating cell nuclear antigen (PCNA) promoter. When expressed at similar levels, tumor-derived p53 mutants did not transactivate the PCNA promoter. It also reports the identification of a wild-type human p53-binding site on the human PCNA promote. 84 refs., 5 figs, 3 tabs.

  7. Proteasome inhibitors induce p53-independent apoptosis in human cancer cells.

    PubMed

    Pandit, Bulbul; Gartel, Andrei L

    2011-01-01

    Proteasome inhibitors are used against human cancer, but their mechanisms of action are not entirely understood. For example, the role of the tumor suppressor p53 is controversial. We reevaluated the role of p53 in proteasome inhibitor-induced apoptosis by using isogenic human cancer cell lines with different p53 status. We found that well-known proteasome inhibitors such as MG132 and bortezomib, as well as the recently discovered proteasome inhibitor thiostrepton, induced p53-independent apoptosis in human cancer cell lines that correlated with p53-independent induction of proapoptotic Noxa but not Puma protein. In addition, these drugs inhibited growth of several cancer cell lines independently of p53 status. Notably, thiostrepton induced more potent apoptosis in HepG2 cells with p53 knockdown than in parental cells with wild-type p53. Our data confirm that proteasome inhibitors generally induce p53-independent apoptosis in human cancer cells.

  8. p53 acetylation enhances Taxol-induced apoptosis in human cancer cells.

    PubMed

    Kim, Jae Hyeong; Yoon, Eun-Kyung; Chung, Hye-Jin; Park, Seong-Yeol; Hong, Kyeong-Man; Lee, Chang-Hun; Lee, Yeon-Su; Choi, Kyungho; Yang, Young; Kim, Kyungtae; Kim, In-Hoo

    2013-01-01

    Microtubule inhibitors (MTIs) such as Taxol have been used for treating various malignant tumors. Although MTIs have been known to induce cell death through mitotic arrest, other mechanisms can operate in MTI-induced cell death. Especially, the role of p53 in this process has been controversial for a long time. Here we investigated the function of p53 in Taxol-induced apoptosis using p53 wild type and p53 null cancer cell lines. p53 was upregulated upon Taxol treatment in p53 wild type cells and deletion of p53 diminished Taxol-induced apoptosis. p53 target proteins including MDM2, p21, BAX, and β-isoform of PUMA were also upregulated by Taxol in p53 wild type cells. Conversely, when the wild type p53 was re-introduced into two different p53 null cancer cell lines, Taxol-induced apoptosis was enhanced. Among post-translational modifications that affect p53 stability and function, p53 acetylation, rather than phosphorylation, increased significantly in Taxol-treated cells. When acetylation was enhanced by anti-Sirt1 siRNA or an HDAC inhibitor, Taxol-induced apoptosis was enhanced, which was not observed in p53 null cells. When an acetylation-defective mutant of p53 was re-introduced to p53 null cells, apoptosis was partially reduced compared to the re-introduction of the wild type p53. Thus, p53 plays a pro-apoptotic role in Taxol-induced apoptosis and acetylation of p53 contributes to this pro-apoptotic function in response to Taxol in several human cancer cell lines, suggesting that enhancing acetylation of p53 could have potential implication for increasing the sensitivity of cancer cells to Taxol.

  9. Flexible lid to the p53-binding domain of human Mdm2: implications for p53 regulation.

    PubMed

    McCoy, Mark A; Gesell, Jennifer J; Senior, Mary M; Wyss, Daniel F

    2003-02-18

    The stabilization of p53 against Mdm2-mediated degradation is an important event in DNA damage response. Initial models of p53 stabilization focused on posttranslational modification of p53 that would disrupt the p53-Mdm2 interaction. The N-terminal regions of both p53 and Mdm2 are modified in vivo in response to cellular stress, suggesting that modifications to Mdm2 also may affect the p53-Mdm2 interaction. Our NMR studies of apo-Mdm2 have found that, in addition to Mdm2 residues 25-109 that form the well ordered p53-binding domain that was observed in the p52-Mdm2 complex, Mdm2 residues 16-24 form a lid that closes over the p53-binding site. The Mdm2 lid, which is strictly conserved in mammals, may help to stabilize apo-Mdm2. It also competes weakly with peptidic and nonpeptidic antagonists. Modifications to the Mdm2 lid may disrupt p53-Mdm2 binding leading to p53 stabilization. Mdm2 and Mdm4 possess nearly identical p53-binding domains but different lids suggesting that lid modifications may select for p53 binding.

  10. 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.

  11. p53 binds human telomeric G-quadruplex in vitro.

    PubMed

    Adámik, Matej; Kejnovská, Iva; Bažantová, Pavla; Petr, Marek; Renčiuk, Daniel; Vorlíčková, Michaela; Brázdová, Marie

    2016-01-01

    The tumor suppressor protein p53 is a key factor in genome stability and one of the most studied of DNA binding proteins. This is the first study on the interaction of wild-type p53 with guanine quadruplexes formed by the human telomere sequence. Using electromobility shift assay and ELISA, we show that p53 binding to telomeric G-quadruplexes increases with the number of telomeric repeats. Further, p53 strongly favors G-quadruplexes folded in potassium over those formed in sodium, thus indicating the telomeric G-quadruplex conformational selectivity of p53. The presence of the quadruplex-stabilizing ligand, N-methyl mesoporphyrin IX (NMM), increases p53 recognition of G-quadruplexes in potassium. Using deletion mutants and selective p53 core domain oxidation, both p53 DNA binding domains are shown to be crucial for telomeric G-quadruplex recognition.

  12. 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

  13. Reappraisal of p53 mutations in human malignant astrocytic neoplasms by p53 functional assay: comparison with conventional structural analyses.

    PubMed

    Tada, M; Iggo, R D; Waridel, F; Nozaki, M; Matsumoto, R; Sawamura, Y; Shinohe, Y; Ikeda, J; Abe, H

    1997-03-01

    We previously reported clonal expansion of p53 mutations in malignant astrocytic tumors detected with a yeast p53 functional assay that measures mutant p53 alleles quantitatively and loss of p53 transcriptional competence qualitatively (Tada et al., Int J Cancer 67:447-450, 1996). This method selectively detects inactivating mutations and is relatively insensitive to contamination of tumor samples with normal tissue. To determine whether the mutation frequency and spectrum detected in this way differ from those seen with conventional techniques, 54 malignant astrocytomas were tested with the yeast assay, and the abnormalities detected were characterized by DNA sequencing. Inactivating p53 mutations were found in 67% of anaplastic astrocytomas and 41% of glioblastomas. Overall, mutations were found in 48% of tumors, compared with only 29% in previous studies (P < 0.005), a difference that probably reflects the greater sensitivity of the yeast assay than of conventional techniques. The frequency of mutations in anaplastic astrocytomas (in our study plus published studies) was significantly higher than in glioblastomas (39% vs 29%; P < 0.05). This suggests that acquisition of p53 mutations is not rate limiting for progression to glioblastoma and that many glioblastomas develop by p53-independent pathways. Sequencing of mutant p53 cDNAs rescued from yeast showed that the mutation spectrum for functionally inactive mutants was nearly identical to the spectra from previous studies on structural mutants, indicating that transcriptional activity is the critical biological target of p53 mutation in malignant astrocytomas.

  14. Loss of p53 induces epidermal growth factor receptor promoter activity in normal human keratinocytes

    PubMed Central

    Bheda, A; Creek, KE; Pirisi, L

    2008-01-01

    Overexpression of the epidermal growth factor receptor (EGFR) in human papillomavirus type 16-immortalized human keratinocytes (HKc) is caused by the viral oncoprotein E6, which targets p53 for degradation. We have previously observed that expression of p53 RNAi in normal HKc is associated with an increase in EGFR mRNA and protein. We now report that p53 RNAi induces EGFR promoter activity up to approximately 10-fold in normal HKc, and this effect does not require intact p53 binding sites on the EGFR promoter. Exogenous wild-type p53 inhibits the EGFR promoter at low levels, and activates it at higher concentrations. Yin Yang 1 (YY1), which negatively regulates p53, induces EGFR promoter activity, and this effect is augmented by p53 RNAi. Intact p53 binding sites on the EGFR promoter are not required for activation by YY1. In addition, Sp1 and YY1 synergistically induce the EGFR promoter in normal HKc, indicating that Sp1 may recruit YY1 as a co-activator. Wild-type p53 suppressed Sp1- and YY1-mediated induction of the EGFR promoter. We conclude that acute loss of p53 in normal HKc induces EGFR expression bya mechanism that involves YY1 and Sp1 and does not require p53 binding to the EGFR promoter. PMID:18391986

  15. Human TAFII31 protein is a transcriptional coactivator of the p53 protein.

    PubMed Central

    Lu, H; Levine, A J

    1995-01-01

    The p53 protein activates transcription of a target gene by binding to a specific DNA response element and interacting with the transcriptional apparatus of RNA polymerase II. The amino-terminal domain of p53 interacts with a component of the TFIID basal transcription complex. The human TATA-binding-protein-associated factor TAFII31, a component of TFIID, has been identified as a critical protein required for p53-mediated transcriptional activation. TAFII31 and p53 proteins bind to each other via amino acid residues in the amino-terminal domain of p53 that are essential for transcription. Antibodies directed against TAFII31 protein inhibit p53-activated but not basal transcription in vitro. These results demonstrate that TAFII31 is a coactivator for the p53 protein. Images Fig. 3 Fig. 4 Fig. 6 PMID:7761466

  16. p53 Response to Ultrasound: Preliminary Observations in MCF7 Human Breast Cancer Cells

    NASA Astrophysics Data System (ADS)

    Burns, Janis M.; Campbell, Paul A.

    2011-09-01

    Mutated p53 can be found in approximately half of all human cancers. Strategies which seek to restore, or at least exercise a level of external control over, p53 functionality are thus potentially useful as adjuncts to therapy. Here, we report our preliminary measurements in this area, and demonstrate that short-burst pulsed ultrasound can indeed affect p53 activity. Specifically, we have observed that expression of the p53 protein can be regulated in the period immediately following low intensity short pulse (millisecond) ultrasound exposure, and that altered activity levels return to basal levels over a 24 hour period post-insonation.

  17. Genome-scale functional analysis of the human genes modulating p53 activity by regulating MDM2 expression in a p53-independent manner.

    PubMed

    Kim, Dong Min; Choi, Seung-Hyun; Yeom, Young Il; Min, Sang-Hyun; Kim, Il-Chul

    2016-09-16

    MDM2, a critical negative regulator of p53, is often overexpressed in leukemia, but few p53 mutations are found, suggesting that p53-independent MDM2 expression occurs due to alterations in MDM2 upstream regulators. In this study, a high MDM2 transcription level was observed (41.17%) regardless of p53 expression in patient with acute myeloid leukemia (AML). Therefore, we performed genome-scale functional screening of the human genes modulating MDM2 expression in a p53-independent manner. We searched co-expression profiles of genes showing a positive or negative pattern with MDM2 expression in a DNA microarray database, selected1089 links, and composed a screening library of 368 genes. Using MDM2 P1 and P2 promoter-reporter systems, we screened clones regulating MDM2 transcriptions in a p53-independent manner by overexpression. Nine clones from the screening library showed enhanced MDM2 promoter activity and MDM2 expression in p53-deficient HCT116 cells. Among them, six clones, including NTRK2, GNA15, SFRS2, EIF5A, ELAVL1, and YWHAB mediated MAPK signaling for expressing MDM2. These results indicate that p53-independent upregulation of MDM2 by increasing selected clones may lead to oncogenesis in AML and that MDM2-modulating genes are novel potential targets for AML treatment. PMID:27524244

  18. Genome-scale functional analysis of the human genes modulating p53 activity by regulating MDM2 expression in a p53-independent manner.

    PubMed

    Kim, Dong Min; Choi, Seung-Hyun; Yeom, Young Il; Min, Sang-Hyun; Kim, Il-Chul

    2016-09-16

    MDM2, a critical negative regulator of p53, is often overexpressed in leukemia, but few p53 mutations are found, suggesting that p53-independent MDM2 expression occurs due to alterations in MDM2 upstream regulators. In this study, a high MDM2 transcription level was observed (41.17%) regardless of p53 expression in patient with acute myeloid leukemia (AML). Therefore, we performed genome-scale functional screening of the human genes modulating MDM2 expression in a p53-independent manner. We searched co-expression profiles of genes showing a positive or negative pattern with MDM2 expression in a DNA microarray database, selected1089 links, and composed a screening library of 368 genes. Using MDM2 P1 and P2 promoter-reporter systems, we screened clones regulating MDM2 transcriptions in a p53-independent manner by overexpression. Nine clones from the screening library showed enhanced MDM2 promoter activity and MDM2 expression in p53-deficient HCT116 cells. Among them, six clones, including NTRK2, GNA15, SFRS2, EIF5A, ELAVL1, and YWHAB mediated MAPK signaling for expressing MDM2. These results indicate that p53-independent upregulation of MDM2 by increasing selected clones may lead to oncogenesis in AML and that MDM2-modulating genes are novel potential targets for AML treatment.

  19. Transcriptional repression in normal human keratinocytes by wild-type and mutant p53.

    PubMed

    Alvarez-Salas, L M; Velazquez, A; Lopez-Bayghen, E; Woodworth, C D; Garrido, E; Gariglio, P; DiPaolo, J A

    1995-05-01

    Wild-type p53 is a nuclear phosphoprotein that inhibits cell proliferation and represses transcriptionally most TATA box-containing promoters in transformed or tumor-derived cell lines. This study demonstrates that p53 alters transcription of the long control region (LCR) of human papillomavirus type 18 (HPV-18). Wild-type and mutant p53 143Val to Ala repressed the HPV-18 LCR promoter in normal human keratinocytes, the natural host cell for HPV infections. Repression by wild-type p53 was also observed in C-33A cells and in an HPV-16-immortalized cell line with an inducible wild-type p53. However, when C-33A cells were cotransfected with the HPV-18 LCR and mutant 143Val to Ala, repression did not occur. Mutant p53 135Cys to Ser did not induce repression in either normal human keratinocytes or in the C-33A line; although like 143Val to Ala, it is thought to affect the DNA binding activity of the wild-type protein. The ability of mutant p53 143Val to Ala to inactivate the HPV early promoter in normal cells (by approximately 60% reduction) suggests that this mutant may be able to associate with wild-type p53 and interact with TATA box-binding proteins. Therefore, these results demonstrate that the transcriptional activities of p53 mutants may be dependent upon the cell type assayed and the form of its endogenous p53. Furthermore, normal human keratinocytes represent an alternative model for determining the activities of p53 mutants.

  20. 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

  1. 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.

  2. 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

  3. Strategies for manipulating the p53 pathway in the treatment of human cancer.

    PubMed Central

    Hupp, T R; Lane, D P; Ball, K L

    2000-01-01

    Human cancer progression is driven in part by the mutation of oncogenes and tumour-suppressor genes which, under selective environmental pressures, give rise to evolving populations of biochemically altered cells with enhanced tumorigenic and metastatic potential. Given that human cancers are biologically and pathologically quite distinct, it has been quite surprising that a common event, perturbation of the p53 pathway, occurs in most if not all types of human cancers. The central role of p53 as a tumour-suppressor protein has fuelled interest in defining its mechanism of function and regulation, determining how its inactivation facilitates cancer progression, and exploring the possibility of restoring p53 function for therapeutic benefit. This review will highlight the key biochemical properties of p53 protein that affect its tumour-suppressor function and the experimental strategies that have been developed for the re-activation of the p53 pathway in cancers. PMID:11062053

  4. Serum withdrawal up-regulates human SIRT1 gene expression in a p53-dependent manner.

    PubMed

    Shang, Linshan; Zhou, Haibin; Xia, Yu; Wang, Hui; Gao, Guimin; Chen, Bingxi; Liu, Qiji; Shao, Changshun; Gong, Yaoqin

    2009-10-01

    SIRT1, a nicotinamide adenine dinucleotide (NAD(+))-dependent histone/protein deacetylase, has been extensively studied recently for its critical role in the regulation of physiology, calorie restriction and aging. Studies on laboratory mice showed that expression of SIRT1 can be induced by starvation in a p53-dependent manner and requires the p53-binding sites present in the Sirt1 promoter. However, it remains to be determined whether these findings based on rodents apply to human beings. In this paper, we characterized a putative p53-binding element in the human SIRT1 promoter that might be required for the up-regulation of SIRT1 in response to nutritional stress. The p53-binding site in the promoter of human SIRT1 is more deviant from the consensus sequence than the corresponding sequence in the mouse Sirt1. There is a C to A change at the second half site in human SIRT1, thus disrupting the core-binding element CWWG in the canonical RRRCWWGYYY. To test whether such sequence change would affect its binding with p53 and the SIRT1 expression under stress, we studied various human cell lines with different p53 status and cells with ectopic expression of functionally distinct p53. We found that serum withdrawal also up-regulates human SIRT1 gene expression in a p53-dependent manner and that the p53-binding element in SIRT1 is required for the up-regulation. Thus, the mechanism responsible for the regulation of SIRT1 expression by p53 is conserved between mice and human beings.

  5. Human papillomavirus type 16 E6 and E7 oncogenes abrogate radiation-induced DNA damage responses in vivo through p53-dependent and p53-independent pathways.

    PubMed

    Song, S; Gulliver, G A; Lambert, P F

    1998-03-01

    E6 and E7 oncoproteins from high risk human papillomaviruses (HPVs) transform cells in tissue culture and induce tumors in vivo. Both E6, which inhibits p53 functions, and E7, which inhibits pRb, can also abrogate growth arrest induced by DNA-damaging agents in cultured cells. In this study, we have used transgenic mice that express HPV-16 E6 or E7 in the epidermis to determine how these two proteins modulate DNA damage responses in vivo. Our results demonstrate that both E6 and E7 abrogate the inhibition of DNA synthesis in the epidermis after treatment with ionizing radiation. Increases in the levels of p53 and p21 proteins after irradiation were suppressed by E6 but not by E7. Through the study of p53-null mice, we found that radiation-induced growth arrest in the epidermis is mediated through both p53-dependent and p53-independent pathways. The abrogation of radiation responses in both E6 and E7 transgenic mice was more complete than was seen in the p53-null epidermis. We conclude that E6 and E7 each have the capacity to modulate p53-dependent as well as p53-independent cellular responses to radiation. Additionally, we found that the conserved region (CR) 1 and CR2 domains in E7 protein, which are involved in the inactivation of pRb function and required for E7's transforming function, were also required for E7 to modulate DNA damage responses in vivo. Thus pRb and/or pRb-like proteins likely mediate both p53-dependent and p53-independent responses to radiation.

  6. Inhibition of p53 DNA binding by human papillomavirus E6 proteins.

    PubMed Central

    Lechner, M S; Laimins, L A

    1994-01-01

    Transformation by the human papillomavirus (HPV) early gene products, E6 and E7, involves their interaction with cellular proteins p53 and Rb. Using glutathione S-transferase (GST) fusion proteins, we found that HPV E6 bound human p53 and that the relative efficiency of binding varied such that the GST-HPV type 16 E6 (16E6) protein bound p53 with highest affinity, followed by GST-31E6, GST-18E6, and GST-11E6. The GST-E6 fusion proteins were sufficient for binding p53 purified from a baculovirus expression system as well as in vitro translation sources, while no association was observed with GST-18E7 or a GST-16E6 mutant bearing a five-amino-acid deletion in E6. When the site-specific DNA binding activity of p53 was examined in the presence of GST-E6 proteins, an inhibition of DNA binding was observed. The degree of inhibition correlated with the relative affinity of different E6 proteins for p53; thus, GST-16E6 was the most potent inhibitor of p53 DNA binding activity, and GST-11E6 was the least effective. Prevention of p53 DNA binding is likely to play a role in the abrogation of the transcriptional activity of p53 by HPV E6 and provides a further mechanism for E6 disruption of p53 growth suppressor function in addition to its role in directing specific degradation of p53 through the ubiquitin-mediated pathway. The variation in inhibition of DNA binding seen with the various E6 proteins may thus contribute to the differences in oncogenic potential seen among the HPV types. Images PMID:8207801

  7. Adenoviral-E2F-1 radiosensitizes p53{sup wild-type} and p53{sup null} human prostate cancer cells

    SciTech Connect

    Nguyen, Khanh H.; Hachem, Paul; Khor, L.-Y.; Salem, Naji; Hunt, Kelly K.; Calkins, Peter R.; Pollack, Alan . E-mail: Alan.Pollack@fccc.edu

    2005-09-01

    Purpose: E2F-1 is a transcription factor that enhances the radiosensitivity of various cell lines by inducing apoptosis. However, there are conflicting data concerning whether this enhancement is mediated via p53 dependent pathways. Additionally, the role of E2F-1 in the response of human prostate cancer to radiation has not been well characterized. In this study, we investigated the effect of Adenoviral-E2F-1 (Ad-E2F-1) on the radiosensitivity of p53{sup wild-type} (LNCaP) and p53{sup null} (PC3) prostate cancer cell lines. Methods and Materials: LNCaP and PC3 cells were transduced with Ad-E2F-1, Adenoviral-Luciferase (Ad-Luc) control vector, or Adenoviral-p53 (Ad-p53). Expression of E2F-1 and p53 was examined by Western blot analysis. Annexin V and caspase 3 + 7 assays were performed to estimate the levels of apoptosis. Clonogenic survival assays were used to determine overall cell death. Statistical significance was determined by analysis of variance, using the Bonferroni method to correct for multiple comparisons. Results: Western blot analysis confirmed the efficacy of transductions with Ad-E2F-1 and Ad-p53. Ad-E2F-1 transduction significantly enhanced apoptosis and decreased clonogenic survival in both cell lines. These effects were compounded by the addition of RT. Although E2F-1-mediated radiosensitization was independent of p53 status, this effect was more pronounced in p53{sup wild-type} LNCaP cells. When PC3 cells were treated with Ad-p53 in combination with RT and Ad-E2F-1, there was at least an additive reduction in clonogenic survival. Conclusions: Our results suggest that Ad-E2F-1 significantly enhances the response of p53{sup wild-type} and p53{sup null} prostate cancer cells to radiation therapy, although radiosensitization is more pronounced in the presence of p53. Ad-E2F-1 may be a useful adjunct to radiation therapy in the treatment of prostate cancer.

  8. Analytical Validation of AmpliChip p53 Research Test for Archival Human Ovarian FFPE Sections.

    PubMed

    Marton, Matthew J; McNamara, Andrew R; Nikoloff, D Michele; Nakao, Aki; Cheng, Jonathan

    2015-01-01

    The p53 tumor suppressor gene (TP53) is reported to be mutated in nearly half of all tumors and plays a central role in genome integrity. Detection of mutations in p53 can be accomplished by many assays, including the AmpliChip p53 Research Test. The AmpliChip p53 Research Test has been successfully used to determine p53 status in hematologic malignancies and fresh frozen solid tissues but there are few reports of using the assay with formalin fixed, paraffin-embedded (FFPE) tissue. The objective of this study was to describe analytical performance characterization of the AmpliChip p53 Research Test to detect p53 mutations in genomic DNA isolated from archival FFPE human ovarian tumor tissues. Method correlation with sequencing showed 96% mutation-wise agreement and 99% chip-wise agreement. We furthermore observed 100% agreement (113/113) of the most prevalent TP53 mutations. Workflow reproducibility was 96.8% across 8 samples, with 2 operators, 2 reagent lots and 2 instruments. Section-to-section reproducibility was 100% for each sample across a 60 μm region of the FFPE block from ovarian tumors. These data indicate that the AmpliChip p53 Research Test is an accurate and reproducible method for detecting mutations in TP53 from archival FFPE human ovarian specimens.

  9. Analytical Validation of AmpliChip p53 Research Test for Archival Human Ovarian FFPE Sections.

    PubMed

    Marton, Matthew J; McNamara, Andrew R; Nikoloff, D Michele; Nakao, Aki; Cheng, Jonathan

    2015-01-01

    The p53 tumor suppressor gene (TP53) is reported to be mutated in nearly half of all tumors and plays a central role in genome integrity. Detection of mutations in p53 can be accomplished by many assays, including the AmpliChip p53 Research Test. The AmpliChip p53 Research Test has been successfully used to determine p53 status in hematologic malignancies and fresh frozen solid tissues but there are few reports of using the assay with formalin fixed, paraffin-embedded (FFPE) tissue. The objective of this study was to describe analytical performance characterization of the AmpliChip p53 Research Test to detect p53 mutations in genomic DNA isolated from archival FFPE human ovarian tumor tissues. Method correlation with sequencing showed 96% mutation-wise agreement and 99% chip-wise agreement. We furthermore observed 100% agreement (113/113) of the most prevalent TP53 mutations. Workflow reproducibility was 96.8% across 8 samples, with 2 operators, 2 reagent lots and 2 instruments. Section-to-section reproducibility was 100% for each sample across a 60 μm region of the FFPE block from ovarian tumors. These data indicate that the AmpliChip p53 Research Test is an accurate and reproducible method for detecting mutations in TP53 from archival FFPE human ovarian specimens. PMID:26125596

  10. 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.

  11. 5-Lipoxygenase is a direct p53 target gene in humans.

    PubMed

    Gilbert, Bianca; Ahmad, Khalil; Roos, Jessica; Lehmann, Christoph; Chiba, Tomohiro; Ulrich-Rückert, Sandra; Smeenk, Leonie; van Heeringen, Simon; Maier, Thorsten J; Groner, Bernd; Steinhilber, Dieter

    2015-08-01

    The p53 tumor suppressor plays a critical role in cancer, and more than 50% of human tumors contain mutations or deletions of the TP53 gene. p53 can transactivate or repress target genes in response to diverse stress signals, such as transient growth arrest, DNA repair, cellular differentiation, senescence and apoptosis. Through an unbiased genome-wide ChIP-seq analysis, we have found that 5-lipoxygenase (ALOX5, 5-LO) which is a key enzyme of leukotriene (LT) biosynthesis, is a direct target gene of p53 and its expression is induced by genotoxic stress via actinomycin D (Act.D) or etoposide (Eto) treatment. 5-LO and LTs play a role in immunological diseases as well as in tumorigenesis and tumor growth. p53 binds to a specific binding site consisting of a complete p53 consensus-binding motif in ALOX5 intron G which is located about 64kbp downstream of the transcriptional start site. We confirmed the strong binding of p53 to the 5-LO target site in ChIP-qPCR experiments. Expression analyses by qRT-PCR and immunoblot further revealed that genotoxic stress induces the ALOX5 mRNA and protein expression in a p53-dependent manner. Knockdown of p53 in U2OS cells leads to a downregulation of 5-LO mRNA and protein expression. In addition, immunofluorescence and immunoprecipitation assays indicate the direct binding of 5-LO to p53 protein. Furthermore, we found that 5-LO can inhibit the transcriptional activity of p53 suggesting that 5-LO acts in a negative feedback loop to limit induction of p53 target genes.

  12. Dual regulation of energy metabolism by p53 in human cervix and breast cancer cells.

    PubMed

    Hernández-Reséndiz, Ileana; Román-Rosales, Alejandra; García-Villa, Enríque; López-Macay, Ambar; Pineda, Erika; Saavedra, Emma; Gallardo-Pérez, Juan Carlos; Alvarez-Ríos, Elizabeth; Gariglio, Patricio; Moreno-Sánchez, Rafael; Rodríguez-Enríquez, Sara

    2015-12-01

    The role of p53 as modulator of OxPhos and glycolysis was analyzed in HeLa-L (cells containing negligible p53 protein levels) and HeLa-H (p53-overexpressing) human cervix cancer cells under normoxia and hypoxia. In normoxia, functional p53, mitochondrial enzyme contents, mitochondrial electrical potential (ΔΨm) and OxPhos flux increased in HeLa-H vs. HeLa-L cells; whereas their glycolytic enzyme contents and glycolysis flux were unchanged. OxPhos provided more than 70% of the cellular ATP and proliferation was abolished by anti-mitochondrial drugs in HeLa-H cells. In hypoxia, both cell proliferations were suppressed, but HeLa-H cells exhibited a significant decrease in OxPhos protein contents, ΔΨm and OxPhos flux. Although glycolytic function was also diminished vs. HeLa-L cells in hypoxia, glycolysis provided more than 60% of cellular ATP in HeLa-H cells. The energy metabolism phenotype of HeLa-H cells was reverted to that of HeLa-L cells by incubating with pifithrin-α, a p53-inhibitor. In normoxia, the energy metabolism phenotype of breast cancer MCF-7 cells was similar to that of HeLa-H cells, whereas p53shRNAMCF-7 cells resembled the HeLa-L cell phenotype. In hypoxia, autophagy proteins and lysosomes contents increased 2-5 times in HeLa-H cells suggesting mitophagy activation. These results indicated that under normoxia p53 up-regulated OxPhos without affecting glycolysis, whereas under hypoxia, p53 down-regulated both OxPhos (severely) and glycolysis (weakly). These p53 effects appeared mediated by the formation of p53-HIF-1α complexes. Therefore, p53 exerts a dual and contrasting regulatory role on cancer energy metabolism, depending on the O₂level.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. p63 regulates proliferation and differentiation of developmentally mature keratinocytes

    PubMed Central

    Truong, Amy B.; Kretz, Markus; Ridky, Todd W.; Kimmel, Robin; Khavari, Paul A.

    2006-01-01

    p63 is a multi-isoform p53 family member required for epidermal development. Contrasting roles for p63 in either the initial commitment to the stratified epithelial cell fate or in stem cell-based self-renewal have been proposed. To investigate p63 function in a post-developmental context, we used siRNAs directed against p63 to down-regulate p63 expression in regenerating human epidermis. Loss of p63 resulted in severe tissue hypoplasia and inhibited both stratification and differentiation in a cell-autonomous manner. Although p63-deficient cells exhibited hypoproliferation, differentiation defects were not due to tissue hypoplasia. Simultaneous p63 and p53 knockdown rescued the cell proliferation defect of p63 knockdown alone but failed to restore differentiation, suggesting that defects in epidermal proliferation and differentiation are mediated via p53-dependent and -independent mechanisms, respectively. Furthermore, ΔNp63 isoforms are the main mediators of p63 effects, although TAp63 isoforms may contribute to late differentiation. These data indicate that p63 is required for both the proliferative and differentiation potential of developmentally mature keratinocytes. PMID:17114587

  19. Attenuating the p53 Pathway in Human Cancers: Many Means to the Same End.

    PubMed

    Wasylishen, Amanda R; Lozano, Guillermina

    2016-08-01

    The p53 pathway is perturbed in the majority of human cancers. Although this most frequently occurs through the direct mutation or deletion of p53 itself, there are a number of other alterations that can attenuate the pathway and contribute to tumorigenesis. For example, amplification of important negative regulators, MDM2 and MDM4, occurs in a number of cancers. In this work, we will review both the normal regulation of the p53 pathway and the different mechanisms of pathway inhibition in cancer, discuss these alterations in the context of the global genomic analyses that have been conducted across tumor types, and highlight the translational implications for cancer diagnosis and treatment.

  20. Mutations in the p53 gene occur in diverse human tumour types.

    PubMed

    Nigro, J M; Baker, S J; Preisinger, A C; Jessup, J M; Hostetter, R; Cleary, K; Bigner, S H; Davidson, N; Baylin, S; Devilee, P

    1989-12-01

    The p53 gene has been a constant source of fascination since its discovery nearly a decade ago. Originally considered to be an oncogene, several convergent lines of research have indicated that the wild-type gene product actually functions as a tumour suppressor gene. For example, expression of the neoplastic phenotype is inhibited, rather than promoted, when rat cells are transfected with the murine wild-type p53 gene together with mutant p53 genes and/or other oncogenes. Moreover, in human tumours, the short arm of chromosome 17 is often deleted. In colorectal cancers, the smallest common region of deletion is centred at 17p13.1; this region harbours the p53 gene, and in two tumours examined in detail, the remaining (non-deleted) p53 alleles were found to contain mutations. This result was provocative because allelic deletion coupled with mutation of the remaining allele is a theoretical hallmark of tumour-suppressor genes. In the present report, we have attempted to determine the generality of this observation; that is, whether tumours with allelic deletions of chromosome 17p contain mutant p53 genes in the allele that is retained. Our results suggest that (1) most tumours with such allelic deletions contain p53 point mutations resulting in amino-acid substitutions, (2) such mutations are not confined to tumours with allelic deletion, but also occur in at least some tumours that have retained both parental 17p alleles, and (3) p53 gene mutations are clustered in four 'hot-spots' which exactly coincide with the four most highly conserved regions of the gene. These results suggest that p53 mutations play a role in the development of many common human malignancies.

  1. High-level expression of human tumour suppressor P53 in the methylotrophic yeast: Pichia pastoris.

    PubMed

    Abdelmoula-Souissi, Salma; Rekik, Leila; Gargouri, Ali; Mokdad-Gargouri, Raja

    2007-08-01

    The human tumour suppressor P53 is a key protein involved in tumour suppression. P53 acts as a "guardian of genome" by regulating many target genes involved in cell cycle regulation, DNA repair and apoptosis. We report the P53 expression by the methylotrophic yeast Pichia pastoris using the methanol inducible AOX1 promoter. We have produced the rP53 in intracellular form as well as secreted using the Saccharomyces cerevisiae alpha-mating factor prepro-leader sequence in two genetic contexts of Pichia, Mut(s) and Mut(+). The intracellular P53 was successfully produced by Mut(s) (KM71) as well as Mut(+) (X33) strains, however, the secreted form was mainly observed in the Mut(s) strain, despite a higher number of p53 copies integrated in the Mut(+) strain. Interestingly, in Mut(s) phenotype, the medium pH influences markedly the rP53 production since it was higher at pH 7 than 6. PMID:17482479

  2. 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.

  3. p53 mutations and human papillomavirus DNA in oral squamous cell carcinoma: correlation with apoptosis.

    PubMed Central

    Koh, J. Y.; Cho, N. P.; Kong, G.; Lee, J. D.; Yoon, K.

    1998-01-01

    Forty-two oral squamous cell carcinomas (SCCs) were analysed for p53 mutations and human papillomavirus (HPV) infection to examine the prevalency of these factors and correlation with apoptotic index (AI; number of apoptotic cells per 100 tumour cells) of the tumour tissue. In polymerase chain reaction (PCR)-Southern blot analysis, HPV DNAs were detected from 22 out of 42 SCCs (52%) with predominance of HPV-16 (68%). p53 mutations in exons 5-8, screened by nested PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, were observed in 16 of 42 tumours (38%). The state of the p53 gene did not show any correlation with HPV infection. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labelling (TUNEL) method was used for detection of apoptotic cells. The mean AI was 2.35, ranging from 0.31 to 6.63. SCCs associated with p53 mutation had significantly lower AI than those without p53 mutation (P < 0.01), whereas no difference in AI was found between SCCs with and without HPV infection. The results of this study confirmed that HPV infection and/or p53 mutations are implicated, but are not mutually exclusive events, in carcinogenesis of oral SCC and also showed that decrease in apoptosis is more closely related to p53 mutation than HPV infection. Images Figure 1 Figure 2 Figure 3 PMID:9703282

  4. 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.

  5. Zinc Induces Apoptosis of Human Melanoma Cells, Increasing Reactive Oxygen Species, p53 and FAS Ligand.

    PubMed

    Provinciali, Mauro; Pierpaoli, Elisa; Bartozzi, Beatrice; Bernardini, Giovanni

    2015-10-01

    The aim of this study was to examine the in vitro effect of zinc on the apoptosis of human melanoma cells, by studying the zinc-dependent modulation of intracellular levels of reactive oxygen species (ROS) and of p53 and FAS ligand proteins. We showed that zinc concentrations ranging from 33.7 μM to 75 μM Zn(2+) induced apoptosis in the human melanoma cell line WM 266-4. This apoptosis was associated with an increased production of intracellular ROS, and of p53 and FAS ligand protein. Treatment of tumor cells with the antioxidant N-acetylcysteine was able to prevent Zn(2+)-induced apoptosis, as well as the increase of p53 and FAS ligand protein induced by zinc. Zinc induces apoptosis in melanoma cells by increasing ROS and this effect may be mediated by the ROS-dependent induction of p53 and FAS/FAS ligand.

  6. 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.

  7. 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.

  8. p53 codon 72 polymorphism and human papillomavirus associated skin cancer

    PubMed Central

    O'Connor, D; Kay, E; Leader, M; Atkins, G; Murphy, G; Mabruk, M

    2001-01-01

    Background/Aims—Non-melanoma skin cancers frequently harbour multiple human papillomavirus (HPV) types. A recent report suggests that a polymorphism of the p53 tumour suppressor gene that results in the substitution of a proline residue with an arginine residue at position 72 of the p53 protein might act as a risk factor in HPV associated malignancies. This study aimed to determine the following: (1) the relation between HPV infection and the development of cutaneous squamous cell carcinoma (SCC), and (2) whether there is a correlation between p53 codon 72 polymorphism and the development of SCC. Methods—Blood samples were taken from 55 patients with skin cancer (both renal transplant recipients and immunocompetent patients with skin cancer) and 115 ethnically matched volunteers. A polymerase chain reaction based assay was used to determine p53 codon 72 genotypes. In addition, 49 benign and malignant lesions from 34 of the patients with skin cancer and 20 normal human skin samples from 20 of the control volunteers were examined for HPV. Results—The proportions of p53 codon 72 genotypes found were 78% arginine homozygous, 2% proline homozygous, and 20% heterozygous among patients with skin cancer and 79% arginine homozygous, 3.5% proline homozygous, and 17.5% heterozygous among the control population. Statistical analysis showed no significant differences in the distribution of the two p53 isoforms between the patients with skin cancer and the control population. The predominant viral types detected in both the patients and the control group were EV associated HPVs, although the incidence was lower in normal skin samples than in malignant lesions or viral warts. Conclusions—These results suggest that in a Celtic population there is no correlation between the presence of HPV, the p53 codon 72 arginine polymorphism, and the development of skin cancer. Key Words: p53 codon 72 polymorphism • human papillomavirus • skin cancer PMID:11429426

  9. Chloroquine activates the p53 pathway and induces apoptosis in human glioma cells

    PubMed Central

    Kim, Ella L.; Wüstenberg, Robin; Rübsam, Anne; Schmitz-Salue, Christoph; Warnecke, Gabriele; Bücker, Eva-Maria; Pettkus, Nadine; Speidel, Daniel; Rohde, Veit; Schulz-Schaeffer, Walter; Deppert, Wolfgang; Giese, Alf

    2010-01-01

    Glioblastoma is the most common malignant brain tumor in adults. The currently available treatments offer only a palliative survival advantage and the need for effective treatments remains an urgent priority. Activation of the p53 growth suppression/apoptotic pathway is one of the promising strategies in targeting glioma cells. We show that the quinoline derivative chloroquine activates the p53 pathway and suppresses growth of glioma cells in vitro and in vivo in an orthotopic (U87MG) human glioblastoma mouse model. Induction of apoptosis is one of the mechanisms underlying the effects of chloroquine on suppressing glioma cell growth and viability. siRNA-mediated downregulation of p53 in wild-type but not mutant p53 glioblastoma cells substantially impaired chloroquine-induced apoptosis. In addition to its p53-activating effects, chloroquine may also inhibit glioma cell growth via p53-independent mechanisms. Our results clarify the mechanistic basis underlying the antineoplastic effect of chloroquine and reveal its therapeutic potential as an adjunct to glioma chemotherapy. PMID:20308316

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

    PubMed

    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

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

    PubMed

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

    2015-11-18

    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.

  12. Lack of p53 augments thymoquinone-induced apoptosis and caspase activation in human osteosarcoma cells.

    PubMed

    Roepke, Martin; Diestel, Antje; Bajbouj, Khuloud; Walluscheck, Diana; Schonfeld, Peter; Roessner, Albert; Schneider-Stock, Regine; Gali-Muhtasib, Hala

    2007-02-01

    We have recently shown that thymoquinone (TQ) is an antineoplastic drug that induces p53-dependent apoptosis in human colon cancer cells. This study evaluated the antiproliferative and pro-apoptotic effects of TQ in two human osteosarcoma cell lines with different p53 mutation status. TQ decreased cell survival dose-dependently and, more significantly, in p53-null MG63 cells (IC(50) = 17 muM) than in p53-mutant MNNG/HOS cells (IC(50) = 38 muM). Cell viability was reduced more selectively in MG63 tumor cells than in normal human osteoblasts. Flow cytometric analysis showed that TQ induced a much greater increase in the PreG(1) (apoptotic) cell population, but no cell cycle arrest in MG63. G(2)/M arrest in MNNG/HOS cells was associated with p21(WAF1) upregulation. Using three DNA damage assays, TQ was confirmed to result in a significantly greater extent of apoptosis in p53 null MG63 cells. Although the Bax/Bcl-2 ratios were not differentially modulated in both cell lines, the mitochondrial pathway appeared to be involved in TQ-induced apoptosis in MG63 by showing the cleavage of caspases-9 and -3. Oxidative stress and mitochondrial O(2)(*-) generation in isolated rat mitochondria were enhanced by TQ as measured by the dose-dependent reduction in aconitase enzyme activity and Amplex Red oxidation respectively. TQ-induced oxidative damage, reflected by an increase in gamma-H2AX foci and increased protein expression levels of gamma-H2AX and the DNA repair enzyme, NBS1, was more pronounced in MNNG/HOS than in MG63. We suggest that the resistance of MNNG/HOS cells to drug-induced apoptosis is caused by the up-regulation of p21(WAF1) by the mutant p53 (transcriptional activity was shown by p53 siRNA treatment) which induces cell cycle arrest and allows to repair DNA damage. Collectively, these findings show that TQ induces p53-independent apoptosis in human osteosarcoma cells. As the loss of p53 function is frequently observed in osteosarcoma patients, our data suggest

  13. Modulation of Melanogenesis by Heme Oxygenase-1 via p53 in Normal Human Melanocytes.

    PubMed

    Lim, Hee-Sun; Jin, Suna; Yun, Sook Jung

    2016-01-01

    As a key regulator of melanogenesis, p53 controls microphthalmia-associated transcription factor (MITF) and tyrosinase expression. The anti-oxidant enzyme heme oxygenase-1 (HO-1) is induced by various forms of cellular stress and diverse oxidative stimuli. However, few studies have examined the role of HO-1 in melanogenesis. Therefore, the aim of this study was to determine the role of HO-1 in melanogenesis and the mechanism underlying this relationship. Cultures of normal human melanocytes were treated with the HO-1 inducer cobalt protoporphyrin (CoPP) or the HO-1 inhibitor zinc protoporphyrin (ZnPP). We then measured the melanin content of the cells. Additional analyses consisted of Western blotting and RT-PCR. The results showed that the cellular melanin content was increased by CoPP and decreased by ZnPP. The Western blot and RT-PCR analyses showed that CoPP increased p53, MITF and tyrosinase levels, and ZnPP reduced all of them. The knockdown of p53 by siRNA transfection was followed by large decreases in the expression levels of p53, MITF and tyrosinase at 3 h of transfection. The presence of CoPP or ZnPP had no significant increased or decreased effects on MITF and tyrosinase levels from 15 h in the siRNA transfectants. Our results suggest that HO-1 modulates melanogenesis in human melanocytes via a p53-dependent pathway. PMID:26865999

  14. 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.

  15. 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…

  16. 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

  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. 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

  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. Attenuating the p53 Pathway in Human Cancers: Many Means to the Same End.

    PubMed

    Wasylishen, Amanda R; Lozano, Guillermina

    2016-01-01

    The p53 pathway is perturbed in the majority of human cancers. Although this most frequently occurs through the direct mutation or deletion of p53 itself, there are a number of other alterations that can attenuate the pathway and contribute to tumorigenesis. For example, amplification of important negative regulators, MDM2 and MDM4, occurs in a number of cancers. In this work, we will review both the normal regulation of the p53 pathway and the different mechanisms of pathway inhibition in cancer, discuss these alterations in the context of the global genomic analyses that have been conducted across tumor types, and highlight the translational implications for cancer diagnosis and treatment. PMID:27329033

  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. 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

  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. 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

  5. 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

  6. Applications of the human p53 knock-in (Hupki) mouse model for human carcinogen testing

    PubMed Central

    Besaratinia, Ahmad; Pfeifer, Gerd P.

    2010-01-01

    Tumor-driving mutations in the TP53 gene occur frequently in human cancers. These inactivating mutations arise predominantly from a single-point mutation in the DNA-binding domain of this tumor suppressor gene (i.e., exons 4–9). The human p53 knock-in (Hupki) mouse model was constructed using gene-targeting technology to create a mouse strain that harbors human wild-type TP53 DNA sequences in both copies of the mouse TP53 gene. Replacement of exons 4–9 of the endogenous mouse TP53 alleles in the Hupki mouse with the homologous normal human TP53 gene sequences has offered a humanized replica of the TP53 gene in a murine genetic environment. The Hupki mouse model system has proven to be an invaluable research tool for studying the underlying mechanisms of human TP53 mutagenesis. The utility of the Hupki mouse model system for exploring carcinogen-induced TP53 mutagenesis has been demonstrated in both in vivo animal experiments and in vitro cell culture experiments. Here, we highlight applications of the Hupki mouse model system for investigating mutagenesis induced by a variety of environmental carcinogens, including sunlight ultraviolet radiation, benzo[a]pyrene (a tobacco smoke-derived carcinogen), 3-nitrobenzanthrone (an urban air pollutant), aristolochic acid (a component of Chinese herbal medicine), and aflatoxin B1 (a food contaminant). We summarize the salient findings of the respective studies and discuss their relevance to human cancer etiology.—Besaratinia, A., Pfeifer, G. P. Applications of the human p53 knock-in (Hupki) mouse model for human carcinogen testing. PMID:20371617

  7. Hyperthermia Selectively Targets Human Papillomavirus in Cervical Tumors via p53-Dependent Apoptosis.

    PubMed

    Oei, Arlene L; van Leeuwen, Caspar M; ten Cate, Rosemarie; Rodermond, Hans M; Buist, Marrije R; Stalpers, Lukas J A; Crezee, Johannes; Kok, H Petra; Medema, Jan Paul; Franken, Nicolaas A P

    2015-12-01

    Human papillomavirus (HPV) is associated with cervical cancer, the third most common cancer in women. The high-risk HPV types 16 and 18 are found in over 70% of cervical cancers and produce the oncoprotein, early protein 6 (E6), which binds to p53 and mediates its ubiquitination and degradation. Targeting E6 has been shown to be a promising treatment option to eliminate HPV-positive tumor cells. In addition, combined hyperthermia with radiation is a very effective treatment strategy for cervical cancer. In this study, we examined the effect of hyperthermia on HPV-positive cells using cervical cancer cell lines infected with HPV 16 and 18, in vivo tumor models, and ex vivo-treated patient biopsies. Strikingly, we demonstrate that a clinically relevant hyperthermia temperature of 42 °C for 1 hour resulted in E6 degradation, thereby preventing the formation of the E6-p53 complex and enabling p53-dependent apoptosis and G2-phase arrest. Moreover, hyperthermia combined with p53 depletion restored both the cell-cycle distribution and apoptosis to control levels. Collectively, our findings provide new insights into the treatment of HPV-positive cervical cancer and suggest that hyperthermia therapy could improve patient outcomes.

  8. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

    PubMed

    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.

  9. {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.

  10. Correlation between radiation dose and p53 protein expression levels in human lymphocytes.

    PubMed

    Cavalcanti, Mariana B; Fernandes, Thiago S; Silva, Edvane B; Amaral, Ademir

    2015-09-01

    The aim of this research was to evaluate the relationship between p53 protein levels and absorbed doses from in vitro irradiated human lymphocytes. For this, samples of blood from 23 donors were irradiated with 0.5; 1; 2; and 4 Gy from a Cobalt-60 source, and the percentages of lymphocytes expressing p53 were scored using Flow Cytometry. The subjects were divided into 3 groups, in accordance with the p53 levels expressed per radiation dose: low (Group I), high (Group II), and excessive levels (Group III). For all groups, the analyses showed that the p53 expression levels increase with the absorbed dose. Particularly for groups I and II, the correlation between this protein expression and the dose follows the linear-quadratic model, such as for radioinduced chromosomal aberrations. In conclusion, our findings indicate possible applications of this approach in evaluating individual radiosensitivity prior to radiotherapeutical procedures as well as in medical surveillance of occupationally exposed workers. Furthermore, due to the rapidity of flow-cytometric analyses, the methodology here employed would play an important role in emergency responses to a large-scale radiation incident where many people may have been exposed. PMID:26312422

  11. 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

  12. 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

  13. The effect of p53-RNAi and p53 knockout on human 8-oxoguanine DNA glycosylase (hOgg1) activity.

    PubMed

    Chatterjee, Aditi; Mambo, Elizabeth; Osada, Motonobu; Upadhyay, Sunil; Sidransky, David

    2006-01-01

    Recent evidence indicates that in vitro p53 augments base excision repair (BER) activities in mammalian cells. To understand the role of p53 in BER, we analyzed the repair activity of hOgg1 in isogenic cell lines HCT116p53+/+ and HCT116p53-/-. We found that hOgg1 activity was significantly decreased in HCT116p53-/- cells as compared with HCT116p53+/+ cells, indicating a functional role for p53 in the regulation of hOGG1. Using gel-shift assays, we showed that p53 binds to its putative cis-elements within the hOGG1 promoter. In addition we demonstrated that supplementing p53 in HCT116p53-/- cells enhanced the transcription of hOGG1. To further strengthen our findings, we used p53-RNAi to study the effects of decreased p53 levels on hOgg1 activity. We observed that p53-RNAi resulted in decreased hOGG1 expression both at the mRNA and protein levels. This decrease in hOGG1 expression was associated with reduced cell viability upon oxidative damage and reduced hOgg1 activity as evidenced by the 8-oxoG incision assay. Taken together, our results indicate that loss of p53 function can lead to decreased hOgg1 repair activity.

  14. 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.

  15. Capsaicin mediates cell cycle arrest and apoptosis in human colon cancer cells via stabilizing and activating p53.

    PubMed

    Jin, Junzhe; Lin, Guofu; Huang, Hong; Xu, Dong; Yu, Hao; Ma, Xu; Zhu, Lisi; Ma, Dongyan; Jiang, Honglei

    2014-01-01

    Capsaicin is the major pungent ingredient in red peppers which is world widely consumed. Except its potent pain relieving efficacy as reported, capsaicin also exerted its antitumor activity in several tumor models. Here, we reported that capsaicin had a profound anti-proliferative effect on human colon cancer cells via inducing cell cycle G0/G1 phase arrest and apoptosis, which was associated with an increase of p21, Bax and cleaved PARP. The underlying mechanism of capsaicin's antitumor potency was mainly attributed to the stabilization and activation of p53. Capsaicin substantially prolonged the half-life of p53 and significantly elevated the transcriptional activity of p53. Through suppressing the interaction between p53 and MDM2, MDM2-mediated p53 ubiquitination was remarkably decreased after capsaicin treatment, which resulted in the stabilization and accumulation of p53. The results of p53-shRNA experiment further demonstrated that p53 knockdown severely impaired the sensitivity of tested cells to capsaicin, G0/G1 phase arrest and the apoptosis induced by capsaicin in p53-knockdown cells was also dramatically decreased, implicating the important role of p53 played in capsaicin's antitumor activity. In summary, our data suggested that capsaicin, or a related analogue, may have a role in the management of human colon cancer.

  16. 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

  17. Human p53 is Inhibited by Glutathionylation of Cysteines Present in the Proximal DNA-Binding Domain During Oxidative Stress†

    PubMed Central

    Velu, Chinavenmeni S.; Niture, Suryakant K.; Doneanu, Catalin E.; Pattabiraman, Nagarajan; Srivenugopal, Kalkunte S.

    2008-01-01

    The cellular mechanisms that modulate the redox state of p53 tumor suppressor remain unclear, although its DNA-binding function is known to be strongly inhibited by oxidative and nitrosative stresses. We show that human p53 is subjected to a new and reversible posttranslational modification, namely, S-glutathionylation in stressed states including DNA damage. First, a rapid and direct incorporation of biotinylated GSH or GSSG into the purified recombinant p53 protein was observed. The modified p53 had significantly decreased ability to bind its consensus DNA sequence. Reciprocal immunoprecipitations and a GST-overlay assay showed that p53 in tumor cells was marginally glutathionylated, however, the modification increased greatly after oxidant and DNA-damaging treatments. GSH-modification coexisted with the serine phophorylations in activated p53, and the thiol-conjugated protein was present in nuclei. When tumor cells treated with camptothecin or cisplatin were subsequently exposed to glutathione-enhancing agents, p53 underwent dethiolation accompanied by detectable increases in p21waf1 expression, relative to the DNA damaging drugs alone. Mass spectrometry of GSH-modified p53 protein identified the cysteines 124, 141 and 182, all present in the proximal DNA-binding domain, as the sites of glutathionylation. Biotinylated maleimide also reacted rapidly with Cys141, implying this to be the most reactive cysteine on p53 surface. The glutathionylatable cysteines were found to exist in a negatively-charged microenvironment in cellular p53. Molecular modeling studies located Cys124 and 141 to the dimer interface of p53 and showed glutathionylation of either residue would inhibit p53-DNA association, and also interfere with protein dimerization. These results show for the first time that shielding of reactive cysteines contributes to a negative regulation for human p53, and imply that such an inactivation of the transcription factor may represent an acute defensive

  18. Inhibitor or promoter? The performance of polysaccharides from Ganoderma lucidum on human tumor cells with different p53 statuses.

    PubMed

    Zhang, Jue; Chen, Jun-ming; Wang, Xiao-xia; Xia, Yong-mei; Cui, Steve W; Li, Jian; Ding, Zhong-yang

    2016-04-01

    Polysaccharides from Ganoderma lucidum (GLPs) have been taken as effective supplements by both healthy people and cancer patients for many years. However, this short survey indicates that instead of inhibiting cancer cell growth, both submerge-cultured intracellular GLP and fruiting body GLP can stimulate the growth of human carcinoma cell lines lacking functional p53, such as HCT-116 p53(-/-), Saos-2, H1299, HL-60, MDA-MB-157. Conversely, the two GLPs inhibit all other assayed cells with functional p53. These results could be an alert since mutational inactivation of the tumor suppressor protein p53 is the most frequent genetic alteration found in human tumors. PMID:26999513

  19. Applications of the human p53 knock-in (Hupki) mouse model for human carcinogen testing.

    PubMed

    Besaratinia, Ahmad; Pfeifer, Gerd P

    2010-08-01

    Tumor-driving mutations in the TP53 gene occur frequently in human cancers. These inactivating mutations arise predominantly from a single-point mutation in the DNA-binding domain of this tumor suppressor gene (i.e., exons 4-9). The human p53 knock-in (Hupki) mouse model was constructed using gene-targeting technology to create a mouse strain that harbors human wild-type TP53 DNA sequences in both copies of the mouse TP53 gene. Replacement of exons 4-9 of the endogenous mouse TP53 alleles in the Hupki mouse with the homologous normal human TP53 gene sequences has offered a humanized replica of the TP53 gene in a murine genetic environment. The Hupki mouse model system has proven to be an invaluable research tool for studying the underlying mechanisms of human TP53 mutagenesis. The utility of the Hupki mouse model system for exploring carcinogen-induced TP53 mutagenesis has been demonstrated in both in vivo animal experiments and in vitro cell culture experiments. Here, we highlight applications of the Hupki mouse model system for investigating mutagenesis induced by a variety of environmental carcinogens, including sunlight ultraviolet radiation, benzo[a]pyrene (a tobacco smoke-derived carcinogen), 3-nitrobenzanthrone (an urban air pollutant), aristolochic acid (a component of Chinese herbal medicine), and aflatoxin B1 (a food contaminant). We summarize the salient findings of the respective studies and discuss their relevance to human cancer etiology.

  20. Human Cytomegalovirus nuclear egress and secondary envelopment are negatively affected in the absence of cellular p53.

    PubMed

    Kuan, Man I; O'Dowd, John M; Chughtai, Kamila; Hayman, Ian; Brown, Celeste J; Fortunato, Elizabeth A

    2016-10-01

    Human Cytomegalovirus (HCMV) infection is compromised in cells lacking p53, a transcription factor that mediates cellular stress responses. In this study we have investigated compromised functional virion production in cells with p53 knocked out (p53KOs). Infectious center assays found most p53KOs released functional virions. Analysis of electron micrographs revealed modestly decreased capsid production in infected p53KOs compared to wt. Substantially fewer p53KOs displayed HCMV-induced infoldings of the inner nuclear membrane (IINMs). In p53KOs, fewer capsids were found in IINMs and in the cytoplasm. The deficit in virus-induced membrane remodeling within the nucleus of p53KOs was mirrored in the cytoplasm, with a disproportionately smaller number of capsids re-enveloped. Reintroduction of p53 substantially recovered these deficits. Overall, the absence of p53 contributed to inhibition of the formation and function of IINMs and re-envelopment of the reduced number of capsids able to reach the cytoplasm.

  1. p53 suppresses type II endometrial carcinomas in mice and governs endometrial tumour aggressiveness in humans

    PubMed Central

    Wild, Peter J; Ikenberg, Kristian; Fuchs, Thomas J; Rechsteiner, Markus; Georgiev, Strahil; Fankhauser, Niklaus; Noske, Aurelia; Roessle, Matthias; Caduff, Rosmarie; Dellas, Athanassios; Fink, Daniel; Moch, Holger; Krek, Wilhelm; Frew, Ian J

    2012-01-01

    Type II endometrial carcinomas are a highly aggressive group of tumour subtypes that are frequently associated with inactivation of the TP53 tumour suppressor gene. We show that mice with endometrium-specific deletion of Trp53 initially exhibited histological changes that are identical to known precursor lesions of type II endometrial carcinomas in humans and later developed carcinomas representing all type II subtypes. The mTORC1 signalling pathway was frequently activated in these precursor lesions and tumours, suggesting a genetic cooperation between this pathway and Trp53 deficiency in tumour initiation. Consistent with this idea, analyses of 521 human endometrial carcinomas identified frequent mTORC1 pathway activation in type I as well as type II endometrial carcinoma subtypes. mTORC1 pathway activation and p53 expression or mutation status each independently predicted poor patient survival. We suggest that molecular alterations in p53 and the mTORC1 pathway play different roles in the initiation of the different endometrial cancer subtypes, but that combined p53 inactivation and mTORC1 pathway activation are unifying pathogenic features among histologically diverse subtypes of late stage aggressive endometrial tumours. PMID:22678923

  2. Liriodenine induces the apoptosis of human laryngocarcinoma cells via the upregulation of p53 expression

    PubMed Central

    LI, LIANG; XU, YING; WANG, BINQUAN

    2015-01-01

    Laryngocarcinoma is one of the most aggressive cancers that affects the head and neck region. The survival rate of patients with laryngocarcinoma is low due to late metastases and the resistance of the disease to chemotherapy and radiotherapy. Liriodenine, an alkaloid extracted from a number of plant species, has demonstrated antitumor effects on multiple types of cancer. However, the effects of liriodenine upon laryngocarcinoma, and the underlying mechanisms, are yet to be elucidated. The present study therefore investigated the potential antitumor effects of liriodenine on HEp-2 human laryngocarcinoma cells in vitro and HEp-2-implanted nude mice in vivo. Liriodenine induced significant apoptosis and inhibition of cell migration in the HEp-2 cells. Furthermore, the rate of tumor growth in the HEp-2-implanted nude mice was inhibited by the administration of liriodenine. The potential mechanism underlying the antitumor effects of liriodenine may result from an upregulative effect upon p53 expression, which ultimately induces cellular apoptosis. By contrast, the downregulation of p53 significantly reduced the antitumor effects of liriodenine. Together, these results suggest that liriodenine exhibits potent antitumor activities in laryngocarcinoma HEp-2 cells, in vitro and in vivo, via the upregulation of p53 expression. Liriodenine may therefore be a potential therapy for the treatment of laryngocarcinoma. PMID:25663867

  3. Matrine-induced autophagy regulated by p53 through AMP-activated protein kinase in human hepatoma cells.

    PubMed

    Xie, Shan-Bu; He, Xing-Xing; Yao, Shu-Kun

    2015-08-01

    Matrine, one of the main extract components of Sophora flavescens, has been shown to exhibit inhibitory effects on some tumors through autophagy. However, the mechanism underlying the effect of matrine remains unclear. The cultured human hepatocellular carcinoma cell line HepG2 and SMMC‑7721 were treated with matrine. Signal transduction and gene expression profile were determined. Matrine stimulated autophagy in SMMC‑7721 cells in a mammalian target of rapamycin (mTOR)-dependent manner, but in an mTOR-independent manner in HepG2 cells. Next, in HepG2 cells, autophagy induced by matrine was regulated by p53 inactivation through AMP-activated protein kinase (AMPK) signaling transduction, then AMPK suppression switched autophagy to apoptosis. Furthermore, the interferon (IFN)-inducible genes, including interferon α-inducible protein 27 (IFI27) and interferon induced transmembrane protein 1 (IFITM1), which are downstream effector of p53, might be modulated by matrine-induced autophagy. In addition, we found that the p53 protein isoforms, p53β, p53γ, ∆133p53, and ∆133p53γ, due to alternative splicing of intron 9, might be regulated by the p53-mediated autophagy. These results show that matrine induces autophagy in human hepatoma cells through a novel mechanism, which is p53/AMPK signaling pathway involvement in matrine-promoted autophagy.

  4. Acute Endurance Exercise Induces Nuclear p53 Abundance in Human Skeletal Muscle

    PubMed Central

    Tachtsis, Bill; Smiles, William J.; Lane, Steven C.; Hawley, John A.; Camera, Donny M.

    2016-01-01

    Purpose: The tumor suppressor protein p53 may have regulatory roles in exercise response-adaptation processes such as mitochondrial biogenesis and autophagy, although its cellular location largely governs its biological role. We investigated the subcellular localization of p53 and selected signaling targets in human skeletal muscle following a single bout of endurance exercise. Methods: Sixteen, untrained individuals were pair-matched for aerobic capacity (VO2peak) and allocated to either an exercise (EX, n = 8) or control (CON, n = 8) group. After a resting muscle biopsy, EX performed 60 min continuous cycling at ~70% of VO2peak during which time CON subjects rested. A further biopsy was obtained from both groups 3 h post-exercise (EX) or 4 h after the first biopsy (CON). Results: Nuclear p53 increased after 3 h recovery with EX only (~48%, p < 0.05) but was unchanged in the mitochondrial or cytoplasmic fractions in either group. Autophagy protein 5 (Atg-5) decreased in the mitochondrial protein fraction 3 h post-EX (~69%, P < 0.05) but remained unchanged in CON. There was an increase in cytoplasmic levels of the mitophagy marker PINK1 following 3 h of rest in CON only (~23%, P < 0.05). There were no changes in mitochondrial, nuclear, or cytoplasmic levels of PGC-1α post-exercise in either group. Conclusions: The selective increase in nuclear p53 abundance following endurance exercise suggests a potential pro-autophagy response to remove damaged proteins and organelles prior to initiating mitochondrial biogenesis and remodeling responses in untrained individuals. PMID:27199762

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

    PubMed Central

    Reisman, D; Greenberg, M; Rotter, V

    1988-01-01

    To gain insight into how transcription of the human p53 oncogene is controlled, we characterized the regulatory regions of the gene. A 3.8-kilobase-pair (kbp) EcoRI restriction fragment encompassing the 5' 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 pSV0-CAT-derived constructs that were transiently introduced into the monkey cell line COS. By this approach we 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. We 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. Images PMID:2839831

  6. The specificity of p53 mutation spectra in sunlight induced human cancers.

    PubMed

    Daya-Grosjean, L; Dumaz, N; Sarasin, A

    1995-05-01

    Ultraviolet (UV) irradiation emitted by the sun has been clearly implicated as a major carcinogen in the formation of skin cancers in man. Indeed, the high levels of cutaneous tumors in xeroderma pigmentosum patients (XP) who are deficient in repair of UV-induced lesions have confirmed that DNA damage produced by sunlight is directly involved in the cancer development. The tumor suppressor gene, p53, very frequently found modified in human cancers, has proved to be a perfect target gene for correlating mutation spectra with different cancer causing agents as there are nearly 300 potential mutation sites available for analysis. In a comparative analysis of p53 mutations found in internal cancers with those in skin tumours we show here that clear differences exist between the types of spectra obtained. The specificity of UV induced mutations in skin cancers is confirmed when single and tandem mutations are compared. Most of the p53 point mutations found are GC to AT transitions both in skin and internal tumors where in the latter they are located mainly at CpG sequences probably due to the deamination of the unstable 5-MeC. Moreover, mutations are targeted at py-py sequences in over 90% of skin tumors whereas in internal cancers the distribution is proportional to the frequency of bipyrimidine sequences in the p53 gene. Most significantly, all mutations found in XP skin tumors are targeted at py-py sites and more than 50% are tandem CC to TT transitions considered as veritable signatures of UV-induced lesions. Tandem mutations are also relatively common (14%) in skin tumors from normal individuals compared to their very rare occurrence in internal malignancies (0.8%). Finally, nearly all mutations observed in XP skin tumors are due to unrepaired lesions remaining on the coding strand whereas no strand bias is seen in mutation location of internal or skin tumors from normal individuals. In fact the mutation spectrum analysed in XP skin cancers has permitted the first

  7. 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.

  8. Cross-triggered and cascaded recycling amplification for ultrasensitive electrochemical sensing of the mutant human p53 gene.

    PubMed

    Yang, Cuiyun; Dou, Baoting; Yang, Jianmei; Yuan, Ruo; Xiang, Yun

    2016-07-01

    Based on an endonuclease-assisted, cross-triggered and cascaded recycling amplification strategy, the construction of a simple electrochemical sensing platform for the ultrasensitive detection of the mutant p53 gene in human serum is described. Using this new signal amplification approach, the sub-femtomolar level of the mutant p53 gene can be selectively detected.

  9. Transcriptional regulation of basic fibroblast growth factor gene by p53 in human glioblastoma and hepatocellular carcinoma cells.

    PubMed Central

    Ueba, T; Nosaka, T; Takahashi, J A; Shibata, F; Florkiewicz, R Z; Vogelstein, B; Oda, Y; Kikuchi, H; Hatanaka, M

    1994-01-01

    Mutations of the p53 gene are found in various human cancers. The frequency of its mutation is reported to increase during tumor progression in most tumors. In human gliomas, mutations of the p53 gene are found in about one-third of the malignant forms and in few of the benign ones, indicating their possible involvement in tumor progression. On the other hand, we have recently shown that basic fibroblast growth factor (basic FGF) plays a crucial role in tumor progression as an autocrine growth factor in tissues of human gliomas. Therefore, we hypothesized that p53 might regulate the promoter activity of the basic FGF gene, which has several GC boxes and no typical TATA box. In this study, cotransfection assays using human glioblastoma and hepatocellular carcinoma cells and establishment of stable cell lines expressing mutant-type p53 were performed. The basic FGF gene promoter was demonstrated to be regulated by p53 at the transcriptional level and its basal core promoter was found to be responsive to p53. Expression of endogenous basic FGF was also demonstrated to be activated by mutant type p53. Wild-type p53 repressed gene expression of the basic FGF and its mutant activated it in vitro, implying one of the possible pathways in tumor progression. Images PMID:8090761

  10. 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.

  11. Resistance of mitochondrial p53 to dominant inhibition

    PubMed Central

    Heyne, Kristina; Schmitt, Katrin; Mueller, Daniel; Armbruester, Vivienne; Mestres, Pedro; Roemer, Klaus

    2008-01-01

    Background Mutation of a tumor suppressor allele leaves the second as backup. Not necessarily so with p53. This homo-tetrameric transcription factor can become contaminated with mutant p53 through hetero-tetramerization. In addition, it can be out-competed by the binding to p53 DNA recognition motifs of transactivation-incompetent isoforms (ΔN and ΔTA-isoforms) of the p53/p63/p73 family of proteins. Countermeasures against such dominant-negative or dominant-inhibitory action might include the evolutionary gain of novel, transactivation-independent tumor suppressor functions by the wild-type monomer. Results Here we have studied, mostly in human HCT116 colon adenocarcinoma cells with an intact p53 pathway, the effects of dominant-inhibitory p53 mutants and of Δex2/3p73, a tumor-associated ΔTA-competitor of wild-type p53, on the nuclear transactivation-dependent and extra-nuclear transactivation-independent functions of wild-type p53. We report that mutant p53 and Δex2/3p73, expressed from a single gene copy per cell, interfere with the stress-induced expression of p53-responsive genes but leave the extra-nuclear apoptosis by mitochondrial p53 largely unaffected, although both wild-type and mutant p53 associate with the mitochondria. In accord with these observations, we present evidence that in contrast to nuclear p53 the vast majority of mitochondrial p53, be it wild-type or mutant, is consisting of monomeric protein. Conclusion The extra-nuclear p53-dependent apoptosis may constitute a fail-safe mechanism against dominant inhibition. PMID:18547443

  12. Comparative study of p53 gene and protein alterations in human astrocytic tumors.

    PubMed

    Louis, D N; von Deimling, A; Chung, R Y; Rubio, M P; Whaley, J M; Eibl, R H; Ohgaki, H; Wiestler, O D; Thor, A D; Seizinger, B R

    1993-01-01

    The p53 gene is a tumor suppressor gene involved in many common malignancies, including astrocytomas. Genetic analysis of the p53 gene and immunohistochemistry of the p53 protein have each been used to screen astrocytomas. To compare these methods, we performed immunohistochemistry with the monoclonal antibody PAb 1801 and single-strand conformational polymorphism (SSCP) with sequence analysis on 34 astrocytic tumors (WHO grades II, III and IV). Seven cases had detectable p53 protein and gene mutations, while twelve cases had neither detectable protein nor gene mutations. Four tumors had frameshift mutations in the p53 gene that were not revealed by immunohistochemistry. One tumor had a genetic polymorphism and no detectable p53 protein. Ten tumors had p53 protein accumulation but no mutations by SSCP; these cases may represent p53 mutations outside of the conserved exons or elevated levels of wild-type p53 protein. Thus, some p53 mutations are missed with PAb 1801 immunohistochemistry alone. p53 immunohistochemistry, however, may reveal p53 accumulation independent of mutations in the conserved portions of the gene. Finally, we suggest that glioblastomas with p53 mutations in the conserved region of the gene may be a subset that are more common in women and in younger patients.

  13. Enhancing the efficiency of direct reprogramming of human primary fibroblasts into dopaminergic neuron-like cells through p53 suppression.

    PubMed

    Liu, XinJian; Huang, Qian; Li, Fang; Li, Chuan-Yuan

    2014-09-01

    Dopaminergic (DA) neuron-like cells obtained through direct reprogramming of primary human fibroblasts offer exciting opportunities for treatment of Parkinson's disease. A significant obstacle is the low efficiency of conversion during the reprogramming process. Here, we demonstrate that the suppression of p53 significantly enhances the efficiency of transcription factor-mediated conversion of human fibroblasts into functional dopaminergic neurons. In particular, blocking p53 activity using a dominant-negative p53 (p53-DN) in IMR90 cells increases the conversion efficiency by 5-20 fold. The induced DA neuron-like cells exhibit dopamine neuron-specific gene expression, significant dopamine uptake and production capacities, and enables symptomatic relief in a rat Parkinson's disease model. Taken together, our findings suggest that p53 is a critical barrier in direct reprogramming of fibroblast into dopaminergic neurons. PMID:25129808

  14. 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.

  15. 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.

  16. Binding sequence-dependent regulation of the human proliferating cell nuclear antigen promoter by p53

    SciTech Connect

    Shan Bin . E-mail: gmorris2@tulane.edu

    2005-04-15

    Exposure of a lung epithelial cell line to ionizing radiation (IR) arrests cell cycle progression through 48 h post-exposure. Coincidentally, IR differentially activates expression of the cell cycle inhibitor, p21/WAF1, and the DNA replication protein, proliferating cell nuclear antigen (PCNA). p21/WAF1 mRNA levels remain elevated through 48 h post-exposure to IR, while PCNA mRNA levels increase transiently at early times. Since p21/WAF1 inhibits DNA replication by directly binding PCNA, the relative levels of the two proteins can determine cell cycle progression. The PCNA p53-binding site displayed reduced p53 binding affinity in vitro relative to the distal p21/WAF1 p53-binding site. Substitution of the p21/WAF1 site for the resident p53-binding site in the PCNA promoter altered the responses to increasing amounts of p53 or IR in transient expression assays. The p21/WAF1 p53-binding site sustained activation of the chimeric PCNA promoter under conditions (high p53 levels or high dose IR) that the PCNA p53-binding site did not. Binding site-specific regulation by wild-type p53 was not observed with mutant p53 harboring a serine to alanine change at amino acid 46. Limited activation of the PCNA promoter by p53 and its modified forms would restrict the amount of PCNA made available for DNA repair.

  17. Mutant p53 interactome identifies nardilysin as a p53R273H-specific binding partner that promotes invasion.

    PubMed

    Coffill, Cynthia R; Muller, Patricia A J; Oh, Hue Kian; Neo, Suat Peng; Hogue, Kelly A; Cheok, Chit Fang; Vousden, Karen H; Lane, David P; Blackstock, Walter P; Gunaratne, Jayantha

    2012-07-01

    The invasiveness of tumour cells depends on changes in cell shape, polarity and migration. Mutant p53 induces enhanced tumour metastasis in mice, and human cells overexpressing p53R273H have aberrant polarity and increased invasiveness, demonstrating the 'gain of function' of mutant p53 in carcinogenesis. We hypothesize that p53R273H interacts with mutant p53-specific binding partners that control polarity, migration or invasion. Here we analyze the p53R273H interactome using stable isotope labelling by amino acids in cell culture and quantitative mass spectrometry, and identify at least 15 new potential mutant p53-specific binding partners. The interaction of p53R273H with one of them--nardilysin (NRD1)--promotes an invasive response to heparin binding-epidermal growth factor-like growth factor that is p53R273H-dependant but does not require Rab coupling protein or p63. Advanced proteomics has thus allowed the detection of a new mechanism of p53-driven invasion.

  18. Cell cycle and p53 gate the direct conversion of human fibroblasts to dopaminergic neurons.

    PubMed

    Jiang, Houbo; Xu, Zhimin; Zhong, Ping; Ren, Yong; Liang, Gaoyang; Schilling, Haley A; Hu, Zihua; Zhang, Yi; Wang, Xiaomin; Chen, Shengdi; Yan, Zhen; Feng, Jian

    2015-01-01

    The direct conversion of fibroblasts to induced dopaminergic (iDA) neurons and other cell types demonstrates the plasticity of cell fate. The low efficiency of these relatively fast conversions suggests that kinetic barriers exist to safeguard cell-type identity. Here we show that suppression of p53, in conjunction with cell cycle arrest at G1 and appropriate extracellular environment, markedly increase the efficiency in the transdifferentiation of human fibroblasts to iDA neurons by Ascl1, Nurr1, Lmx1a and miR124. The conversion is dependent on Tet1, as G1 arrest, p53 knockdown or expression of the reprogramming factors induces Tet1 synergistically. Tet1 knockdown abolishes the transdifferentiation while its overexpression enhances the conversion. The iDA neurons express markers for midbrain DA neurons and have active dopaminergic transmission. Our results suggest that overcoming these kinetic barriers may enable highly efficient epigenetic reprogramming in general and will generate patient-specific midbrain DA neurons for Parkinson's disease research and therapy. PMID:26639555

  19. Aspirin inhibits human coronary artery endothelial cell proliferation by upregulation of p53.

    PubMed

    Ranganathan, Subramanian; Joseph, Jacob; Mehta, Jawahar L

    2003-01-31

    Aspirin (acetylsalicylic acid, ASA) is effective in the primary and secondary prevention of vascular events. This effect is mediated in large part by platelet inhibition; however, non-platelet-mediated effects may also be relevant in the overall efficacy of ASA. We determined the effect of ASA on the synthesis of DNA and total proteins in cultured human coronary endothelial cells (HCAECs). Fourth generation HCAECs were cultured and treated with ASA and rate of synthesis of DNA and total proteins was determined by incorporation of [3H]thymidine and [3H]proline, respectively. ASA inhibited DNA synthesis by 50% at a concentration of 1mM and protein synthesis by 50% at a concentration of 2mM. The inhibitory effect of ASA was observed as early as 2h after treatment of HCAECs. The inhibition of DNA and protein synthesis could be reversed within 24h after removal of the drug from the culture medium. Indomethacin also inhibited DNA and protein synthesis. Western blot analysis revealed that the expression of p53 protein was increased after treatment of the cells with ASA. These observations indicate that ASA decreases endothelial cell proliferation through cell cycle arrest mediated by enhanced p53 expression. Arrest of endothelial proliferation and activation may be an important mechanism of the beneficial effect of ASA in acute coronary syndromes.

  20. Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53

    SciTech Connect

    Mercer, W.E.; Shields, M.T.; Amin, M.; Sauve, G.J. ); Appella, E.; Romano, J.W.; Ullrich, S.J. )

    1990-08-01

    To investigate the effect that human wild-type p53 (wt-p53) expression has on cell proliferation the authors constructed a recombinant plasmid, pM47, in which wt-p53 cDNA is under transcriptional control of the hormone-inducible mouse mammary tumor virus promoter linked to the dominant biochemical selection marker gene Eco gpt. The pM47 plasmid was introduced into T98G cells derived from a human glioblastomas multiforme tumor, and a stable clonal cell line, GM47.23, was derived that conditionally expressed wt-p53 following exposure to dexamethasone. The authors show that induction of wt-p53 expression in exponentially growing cells inhibits cell cycle progression and that the inhibitory effect is reversible upon removal of the inducer or infection with simian virus 40. Moreover, when growth-arrested cells are stimulated to proliferate, induction of wt-p53 expression inhibits G{sub 0}/G{sub 1} progression into S phase and the cells accumulate with a DNA content equivalent to cells arrested in the G{sub 0}/G{sub 1} phase of the cell cycle. Taken together, these studies suggest that wt-p53 may play a negative role in growth regulation.

  1. Histone modifications and p53 binding poise the p21 promoter for activation in human embryonic stem cells

    PubMed Central

    Itahana, Yoko; Zhang, Jinqiu; Göke, Jonathan; Vardy, Leah A.; Han, Rachel; Iwamoto, Kozue; Cukuroglu, Engin; Robson, Paul; Pouladi, Mahmoud A.; Colman, Alan; Itahana, Koji

    2016-01-01

    The high proliferation rate of embryonic stem cells (ESCs) is thought to arise partly from very low expression of p21. However, how p21 is suppressed in ESCs has been unclear. We found that p53 binds to the p21 promoter in human ESCs (hESCs) as efficiently as in differentiated human mesenchymal stem cells, however it does not promote p21 transcription in hESCs. We observed an enrichment for both the repressive histone H3K27me3 and activating histone H3K4me3 chromatin marks at the p21 locus in hESCs, suggesting it is a suppressed, bivalent domain which overrides activation by p53. Reducing H3K27me3 methylation in hESCs rescued p21 expression, and ectopic expression of p21 in hESCs triggered their differentiation. Further, we uncovered a subset of bivalent promoters bound by p53 in hESCs that are similarly induced upon differentiation in a p53-dependent manner, whereas p53 promotes the transcription of other target genes which do not show an enrichment of H3K27me3 in ESCs. Our studies reveal a unique epigenetic strategy used by ESCs to poise undesired p53 target genes, thus balancing the maintenance of pluripotency in the undifferentiated state with a robust response to differentiation signals, while utilizing p53 activity to maintain genomic stability and homeostasis in ESCs. PMID:27346849

  2. Histone modifications and p53 binding poise the p21 promoter for activation in human embryonic stem cells.

    PubMed

    Itahana, Yoko; Zhang, Jinqiu; Göke, Jonathan; Vardy, Leah A; Han, Rachel; Iwamoto, Kozue; Cukuroglu, Engin; Robson, Paul; Pouladi, Mahmoud A; Colman, Alan; Itahana, Koji

    2016-06-27

    The high proliferation rate of embryonic stem cells (ESCs) is thought to arise partly from very low expression of p21. However, how p21 is suppressed in ESCs has been unclear. We found that p53 binds to the p21 promoter in human ESCs (hESCs) as efficiently as in differentiated human mesenchymal stem cells, however it does not promote p21 transcription in hESCs. We observed an enrichment for both the repressive histone H3K27me3 and activating histone H3K4me3 chromatin marks at the p21 locus in hESCs, suggesting it is a suppressed, bivalent domain which overrides activation by p53. Reducing H3K27me3 methylation in hESCs rescued p21 expression, and ectopic expression of p21 in hESCs triggered their differentiation. Further, we uncovered a subset of bivalent promoters bound by p53 in hESCs that are similarly induced upon differentiation in a p53-dependent manner, whereas p53 promotes the transcription of other target genes which do not show an enrichment of H3K27me3 in ESCs. Our studies reveal a unique epigenetic strategy used by ESCs to poise undesired p53 target genes, thus balancing the maintenance of pluripotency in the undifferentiated state with a robust response to differentiation signals, while utilizing p53 activity to maintain genomic stability and homeostasis in ESCs.

  3. 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.

  4. Histone modifications and p53 binding poise the p21 promoter for activation in human embryonic stem cells.

    PubMed

    Itahana, Yoko; Zhang, Jinqiu; Göke, Jonathan; Vardy, Leah A; Han, Rachel; Iwamoto, Kozue; Cukuroglu, Engin; Robson, Paul; Pouladi, Mahmoud A; Colman, Alan; Itahana, Koji

    2016-01-01

    The high proliferation rate of embryonic stem cells (ESCs) is thought to arise partly from very low expression of p21. However, how p21 is suppressed in ESCs has been unclear. We found that p53 binds to the p21 promoter in human ESCs (hESCs) as efficiently as in differentiated human mesenchymal stem cells, however it does not promote p21 transcription in hESCs. We observed an enrichment for both the repressive histone H3K27me3 and activating histone H3K4me3 chromatin marks at the p21 locus in hESCs, suggesting it is a suppressed, bivalent domain which overrides activation by p53. Reducing H3K27me3 methylation in hESCs rescued p21 expression, and ectopic expression of p21 in hESCs triggered their differentiation. Further, we uncovered a subset of bivalent promoters bound by p53 in hESCs that are similarly induced upon differentiation in a p53-dependent manner, whereas p53 promotes the transcription of other target genes which do not show an enrichment of H3K27me3 in ESCs. Our studies reveal a unique epigenetic strategy used by ESCs to poise undesired p53 target genes, thus balancing the maintenance of pluripotency in the undifferentiated state with a robust response to differentiation signals, while utilizing p53 activity to maintain genomic stability and homeostasis in ESCs. PMID:27346849

  5. Generation and immunosuppressive functions of p53-induced human adaptive regulatory T cells

    PubMed Central

    Mandapathil, Magis; Visus, Carmen; Finn, Olivera J; Lang, Stephan; Whiteside, Theresa L

    2013-01-01

    Inducible regulatory T cells (iTregs, also called Tr1 cells) are generated in the periphery (circulation or tissue) of cancer patients upon the encounter of naïve CD4+ T cells with tumor-associated antigens. As p53 is often inactivated by genetic or epigenetic events during oncogenesis, p53-induced Tr1 cells might play a key role in establishing immunosuppressive networks in cancer patients. Tr1 cells were generated by co-culturing circulating CD4+CD25− T cells with autologous immature dendritic cells pulsed with a wild-type (WT) p53-derived peptide or an unrelated peptide derived from mucin 1 (MUC1). The Tr1 phenotype and the specificity for p53 of these cells were confirmed by multicolor flow cytometry. Moreover, the Tr1 cell-mediated suppression of T-cell proliferation was evaluated by CFSE-based flow cytometry, while their ability to alter the T-cell cytokine profile by ELISA and Luminex assays. The capacity of p53-induced Tr1 cells to suppress the generation and function of cytotoxic T lymphcoytes (CTLs) was assessed by flow cytometry and ELISPOT. Of note, low doses of the p53-derived peptide (p53low) induced greater numbers of Tr1 cells than the same peptide employed at high doses (p53high). Moreover, Tr1/p53low cells not secreted higher levels of interleukin-10 and transforming growth factor β1, but also mediated more robust suppressive effects on CTL proliferation than Tr1/p53high cells. Tr1/p53low cells, Tr1/p53high cells, as well as Tr1 cells generated with low doses of an unrelated MUC1-derived peptide were equally effective in suppressing the expansion and antitumor activity of p53-reactive CTLs. p53low induced the expansion of highly suppressive p53-reactive Tr1 cells. However, the capacity of these Tr1 cells to suppress the generation and function of p53-reactive CTLs was independent of their antigen-specificity. PMID:24073385

  6. New insights into the anticancer activity of carnosol: p53 reactivation in the U87MG human glioblastoma cell line.

    PubMed

    Giacomelli, Chiara; Natali, Letizia; Trincavelli, Maria Letizia; Daniele, Simona; Bertoli, Alessandra; Flamini, Guido; Braca, Alessandra; Martini, Claudia

    2016-05-01

    Glioblastoma multiforme (GBM) is an aggressive brain tumour with high resistance to radio- and chemotherapy. As such, increasing attention has focused on developing new therapeutic strategies to improve treatment responses. Recently, attention has been shifted to natural compounds that are able to halt tumour development. Among them, carnosol (CAR), a phenolic diterpene present in rosemary, has become a promising molecule that is able to prevent certain types of solid cancer. However, no data are available on the effects of CAR in GBM. Here, CAR activity decreased the proliferation of different human glioblastoma cell lines, particularly cells that express wild type p53. The p53 pathway is involved in the control of apoptosis and is often impaired in GBM. Notably, CAR, through the dissociation of p53 from its endogenous inhibitor MDM2, was able to increase the intracellular p53 levels in GBM cells. Accordingly, functional reactivation of p53 was demonstrated by the stimulation of p53 target genes' transcription, the induction of apoptosis and cell cycle blockade. Most importantly, CAR produced synergistic effects with temozolomide (TMZ) and reduced the restoration of the tumour cells' proliferation after drug removal. Thus, for the first time, these data highlighted the potential use of the diterpene in the sensitization of GBM cells to chemotherapy through a direct re-activation of p53 pathway. Furthermore, progress has been made in delineating the biochemical mechanisms underlying the pro-apoptotic effects of this molecule. PMID:26939786

  7. 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. PMID:27586146

  8. Expression of Cell Competition Markers at the Interface between p53 Signature and Normal Epithelium in the Human Fallopian Tube

    PubMed Central

    Kito, Masahiko; Maeda, Daichi; Kudo-Asabe, Yukitsugu; Sato, Naoki; Shih, Ie-Ming; Wang, Tian-Li; Tanaka, Masamitsu; Terada, Yukihiro; Goto, Akiteru

    2016-01-01

    There is a growing body of evidence regarding cell competition between normal and mutant mammalian cells, which suggest that it may play a defensive role in the early phase of carcinogenesis. In vitro study in the past has shown that overexpression of vimentin in normal epithelial cells at the contact surface with transformed cells is essential for the cell competition involved in epithelial defense against cancer. In this study, we attempted to examine cell competition in human tissue in vivo by investigating surgically resected human fallopian tubes that contain p53 signatures and serous tubal intraepithelial lesions (STILs), a linear expansion of p53-immunopositive/TP53 mutant tubal epithelial cells that are considered as precursors of pelvic high grade serous carcinoma. Immunofluorescence double staining for p53 and the cell competition marker vimentin was performed in 21 sections of human fallopian tube tissue containing 17 p53 signatures and 4 STILs. The intensities of vimentin expression at the interface between p53-positive cells at the end of the p53 signature/STIL and adjacent p53-negative normal tubal epithelial cells were compared with the background tubal epithelium. As a result, the average vimentin intensity at the interfaces relative to the background intensity was 1.076 (95% CI, 0.9412 – 1.211 for p53 signature and 0.9790 (95% CI, 0.7206 – 1.237) for STIL. Thus, it can be concluded that overexpression of the cell competition marker vimentin are not observed in human tissue with TP53 alterations. PMID:27258067

  9. Sunlight and sunburn in human skin cancer: p53, apoptosis, and tumor promotion.

    PubMed

    Brash, D E; Ziegler, A; Jonason, A S; Simon, J A; Kunala, S; Leffell, D J

    1996-04-01

    Sunlight is a carcinogen to which everyone is exposed. Epidemiology indicates that most carcinogenic sunlight exposure takes place several decades before the tumor arises. Some of the early events have been identified by searching for genes having ultraviolet (UV)-specific mutations. Over 90% of squamous cell carcinomas and more than 50% of basal cell carcinomas from New England patients contain UV-like mutations in the p53 tumor suppressor gene. From the mutation pattern, it can be concluded that the carcinogenic DNA lesions were pyrimidine-cytosine photoproducts caused by the UVB portion of sunlight. Particular codons of the p53 gene are most susceptible, apparently because of slower DNA repair at specific sites. Sunlight is sufficiently mutagenic often to mutate both p53 alleles. These mutations are also found in the precancer for squamous cell carcinoma, actinic keratosis, implying an early role. The function of p53 in normal skin is indicated by the observation that inactivating p53 in mouse skin reduces the appearance of sunburn cells, apoptotic keratinocytes generated by UV overexposure. Skin thus appears to possess a p53-dependent "cellular proofreading" response to DNA damage in which precancerous cells self-destruct. If this response is reduced in a single cell by a prior p53 mutation, sunburn can thereafter select for clonal expansion of the p53-mutated cell into an actinic keratosis. Sunlight appears to act twice: as tumor initiator and as tumor promoter.

  10. 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

  11. 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.

  12. Characterisation of human fibroblasts as keratinocyte feeder layer using p63 isoforms status.

    PubMed

    Auxenfans, Céline; Thépot, Amélie; Justin, Virginie; Hautefeuille, Agnès; Shahabeddin, Lili; Damour, Odile; Hainaut, Pierre

    2009-01-01

    Large-scale culture of primary keratinocytes allows the production of large epidermal sheet surfaces for the treatment of extensive skin burns. This method is dependent upon the capacity to establish cultures of proliferating keratinocytes in conditions compatible with their clonal expansion while maintaining their capacity to differentiate into the typical squamous pattern of human epidermis. Feeder layers are critical in this process because the fibroblasts that compose this layer serve as a source of adhesion, growth and differentiation factors. In this report, we have characterise the expression patterns of p63 isoforms in primary keratinocytes cultured on two different feeder layer systems, murine 3T3 and human fibroblasts. We show that with the latter, keratinocytes express a higher ratio of Delta N to TAp63 isoform, in relation with higher clonogenic potential. These results indicate that human fibroblasts represent an adequate feeder layer system to support the culture of primary human keratinocytes. PMID:20042803

  13. 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.

  14. 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

  15. Functional inactivation of endogenous MDM2 and CHIP by HSP90 causes aberrant stabilization of mutant p53 in human cancer cells.

    PubMed

    Li, Dun; Marchenko, Natalia D; Schulz, Ramona; Fischer, Victoria; Velasco-Hernandez, Talia; Talos, Flaminia; Moll, Ute M

    2011-05-01

    The tight control of wild-type p53 by mainly MDM2 in normal cells is permanently lost in tumors harboring mutant p53, which exhibit dramatic constitutive p53 hyperstabilization that far exceeds that of wild-type p53 tumors. Importantly, mutant p53 hyperstabilization is critical for oncogenic gain of function of mutant p53 in vivo. Current insight into the mechanism of this dysregulation is fragmentary and largely derived from ectopically constructed cell systems. Importantly, mutant p53 knock-in mice established that normal mutant p53 tissues have sufficient enzymatic reserves in MDM2 and other E3 ligases to maintain full control of mutant p53. We find that in human cancer cells, endogenous mutant p53, despite its ability to interact with MDM2, suffers from a profound lack of ubiquitination as the root of its degradation defect. In contrast to wild-type p53, the many mutant p53 proteins which are conformationally aberrant are engaged in complexes with the HSP90 chaperone machinery to prevent its aggregation. In contrast to wild-type p53 cancer cells, we show that in mutant p53 cancer cells, this HSP90 interaction blocks the endogenous MDM2 and CHIP (carboxy-terminus of Hsp70-interacting protein) E3 ligase activity. Interference with HSP90 either by RNA interference against HSF1, the transcriptional regulator of the HSP90 pathway, or by direct knockdown of Hsp90 protein or by pharmacologic inhibition of Hsp90 activity with 17AAG (17-allylamino-17-demethoxygeldanamycin) destroys the complex, liberates mutant p53, and reactivates endogenous MDM2 and CHIP to degrade mutant p53. Of note, 17AAG induces a stronger viability loss in mutant p53 than in wild-type p53 cancer cells. Our data support the rationale that suppression of mutant p53 levels in vivo in established cancers might achieve clinically significant effects.

  16. 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

  17. Inactivation of p53 in Human Keratinocytes Leads to Squamous Differentiation and Shedding via Replication Stress and Mitotic Slippage.

    PubMed

    Freije, Ana; Molinuevo, Rut; Ceballos, Laura; Cagigas, Marta; Alonso-Lecue, Pilar; Rodriguez, René; Menendez, Pablo; Aberdam, Daniel; De Diego, Ernesto; Gandarillas, Alberto

    2014-11-20

    Tumor suppressor p53 is a major cellular guardian of genome integrity, and its inactivation is the most frequent genetic alteration in cancer, rising up to 80% in squamous cell carcinoma (SCC). By adapting the small hairpin RNA (shRNA) technology, we inactivated endogenous p53 in primary epithelial cells from the epidermis of human skin. We show that either loss of endogenous p53 or overexpression of a temperature-sensitive dominant-negative conformation triggers a self-protective differentiation response, resulting in cell stratification and expulsion. These effects follow DNA damage and exit from mitosis without cell division. p53 preserves the proliferative potential of the stem cell compartment and limits the power of proto-oncogene MYC to drive cell cycle stress and differentiation. The results provide insight into the role of p53 in self-renewal homeostasis and help explain why p53 mutations do not initiate skin cancer but increase the likelihood that cancer cells will appear.

  18. Regulation of apoptosis by p53 in UV-irradiated human epidermis, psoriatic plaques and senescent keratinocytes.

    PubMed

    Qin, Jian-Zhong; Chaturvedi, Vijaya; Denning, Mitchell F; Bacon, Patricia; Panella, Jeffry; Choubey, Divaker; Nickoloff, Brian J

    2002-05-01

    The carcinogenic effects of sunlight in human epidermis may be thwarted by either: transient growth arrest and repair of DNA photodamage in keratinocytes (KCs); elimination of KCs with damaged DNA via apoptosis; or by stimulating a senescence switch whereby KCs become irreversibly growth arrested. Using normal human skin organ cultures and living epidermal equivalents, we demonstrate that in the proliferative basal layer, removal of KCs via apoptosis had a rapid onset (beginning within 2 h) following UV-light exposure generating progressively greater numbers of KCs with thymine dimers as the dose of UV-light was increased; involved induction of Apaf-1, activation of caspase-3, and was dependent on p53 activation as addition of a p53 chemical inhibitor blocked the apoptotic response. Suprabasal layer KCs underwent apoptosis at much later time points (>8 h). KCs in the basal layer repaired DNA damage more rapidly than KCs in suprabasal layers. Steady state levels of p53 increased in irradiated cells, and the increase was accompanied by phosphorylation of serine 9 and serine 15, but not serine 6 residues. By contrast, cultured KCs undergoing spontaneous replicative senescence were resistant to UV-induced apoptosis. Senescent KCs constitutively contained low levels of p53, which were neither increased nor phosphorylated or acetylated after UV-exposure and possessed minimal DNA binding activity, indicative of functional inactivation. Furthermore, treatment of senescent KCs with DNA damaging agent adriamycin did not result in activation of latent p53 or apoptosis. When KCs within psoriatic plaques were examined, they resembled senescent KCs in that they expressed p53, which was not phosphorylated or acetylated. Thus, UV-light induces DNA damage in human epidermal KCs triggering p53 activation, and subsequent apoptosis involving distinct cell layers and kinetics. However, the lack of p53 activation as seen in senescent KCs and psoriatic plaques, is associated with a

  19. DIETARY PHYTOCHEMICALS INDUCE p53- AND CASPASE-INDEPENDENT CELL DEATH IN HUMAN NEUROBLASTOMA CELLS

    PubMed Central

    Sukumari-Ramesh, Sangeetha; Bentley, J. Nicole; Laird, Melissa D.; Singh, Nagendra; Vender, John R.; Dhandapani, Krishnan M

    2013-01-01

    Neuroblastoma (NB) is the most prevalent pediatric solid tumor and a leading cause of cancer-related death in children. In the present study, a novel cytotoxic role for the dietary compounds, curcumin, andrographolide, wedelolactone, dibenzoylmethane, and tanshinone IIA was identified in human S-type NB cells, SK-N-AS and SK-N-BE(2). Mechanistically, cell death appeared apoptotic by flow cytometry; however, these effects proceeded independently from both caspase-3 and p53 activation, as assessed by both genetic (shRNA) and pharmacological approaches. Notably, cell death induced by both curcumin and andrographolide was associated with decreased NFκB activity and a reduction in Bcl-2 and Bcl-xL expression. Finally, curcumin and andrographolide increased cytotoxicity following co-treatment with either cisplatin or doxorubicin, two chemotherapeutic agents widely used in the clinical management of NB. Coupled with the documented safety in humans, dietary compounds may represent a potential adjunct therapy for NB. PMID:21704149

  20. How contemporary human reproductive behaviors influence the role of fertility-related genes: the example of the p53 gene.

    PubMed

    Corbo, Rosa Maria; Gambina, Giuseppe; Scacchi, Renato

    2012-01-01

    Studies on human fertility genes have identified numerous risk/protective alleles involved in the occurrence of reproductive system diseases causing infertility or subfertility. Investigations we carried out in populations at natural fertility seem to suggest that the clinical relevance that some fertility genes are now acquiring depends on their interaction with contemporary reproductive behaviors (birth control, delayed childbearing, and spacing birth order, among others). In recent years, a new physiological role in human fertility regulation has emerged for the tumor- suppressor p53 gene (P53), and the P53 Arg72Pro polymorphism has been associated with recurrent implantation failure in humans. To lend support to our previous observations, we examined the impact of Arg72Pro polymorphism on fertility in two samples of Italian women not selected for impaired fertility but collected from populations with different (premodern and modern) reproductive behaviors. Among the women at near-natural fertility (n = 98), the P53 genotypes were not associated with different reproductive efficiency, whereas among those with modern reproductive behaviors (n = 68), the P53 genotypes were associated with different mean numbers of children [Pro/Pro = 0.75P53 Pro allele frequencies (p = 0.028) was observed. These results are consistent with those of clinical studies reporting an association between the P53 Pro allele and recurrent implantation failure. By combining these findings with previous ones, we suggest here that some common variants of fertility genes may have become "detrimental" following exposure to modern reproductive patterns and might therefore be associated with reduced reproductive success. Set within an evolutionary framework, this change could lead to the selection of a set of gene variants fitter to current reproductive behaviors as the shift to

  1. Selective induction of apoptosis through the FADD/caspase-8 pathway by a p53 c-terminal peptide in human pre-malignant and malignant cells.

    PubMed

    Li, Yin; Mao, Yuehua; Rosal, Ramon V; Dinnen, Richard D; Williams, Ann C; Brandt-Rauf, Paul W; Fine, Robert L

    2005-05-20

    A p53 C-terminal peptide (aa 361-382, p53p), fused at its C-terminus to the minimal carrier peptide of antennapedia (17 aa, Ant; p53p-Ant), induced rapid apoptosis in human cancer cells, via activation of the Fas pathway. We examined p53p-Ant mechanism of action, toxicity in various human normal, non-malignant, pre-malignant and malignant cancer cells and investigated its biophysical characteristics. p53p-Ant selectively induced cell death in only pre-malignant or malignant cells in a p53-dependent manner and was not toxic to normal and non-malignant cells. p53p-Ant was more toxic to the mutant p53 than wild-type p53 phenotype in H1299 lung cancer cells stably expressing human temperature-sensitive p53 mutant 143Ala. Surface plasmon resonance (BIACORE) analysis demonstrated that this peptide had higher binding affinity to mutant p53 as compared to wild-type p53. p53p-Ant induced-cell death had the classical morphological characteristics of apoptosis and had no features of necrosis. The mechanism of cell death by p53p-Ant was through the FADD/caspase-8-dependent pathway without the involvement of the TRAIL pathway, Bcl-2 family and cell cycle changes. Blocking Fas with antibody did not alter the peptide's effect, suggesting that Fas itself did not interact with the peptide. Transfection with a dominant-negative FADD with a deleted N-terminus inhibited p53p-Ant-induced apoptosis. Its mechanism of action is related to the FADD-induced pathway without restoration of other p53 functions. p53p-Ant is a novel anticancer agent with unique selectivity for human cancer cells and could be useful as a prototype for the development of new anti-cancer agents. PMID:15645452

  2. Heterogeneity in intratumor distribution of p53 mutations in human prostate cancer.

    PubMed Central

    Mirchandani, D.; Zheng, J.; Miller, G. J.; Ghosh, A. K.; Shibata, D. K.; Cote, R. J.; Roy-Burman, P.

    1995-01-01

    Prostatic carcinoma from 65 patients have been examined for the occurrence of point mutations in the p53 tumor suppressor gene locus within the region of exons 5 to 8. Overall, only a small fraction of tumors (12.3%) was found to contain p53 mutations. No significant correlation was detected between the presence of the mutant gene and either tumor volume or histopathological grade. However, metastatic prostatic tumors are found to display a higher percentage (21.4%) of p53 mutations compared with primary adenocarcinomas (9.8%). Analysis of the topographical distribution of the p53 mutant genotype revealed two remarkable findings. First, multifocal tumors within a prostate appear to differ in harboring the mutant gene, and second, evidence is obtained for intratumor heterogeneity in the distribution of the mutant p53 allele. Together these findings appear to explain, at least in part, why there has been a wide discrepancy in the reported detection frequency of p53 mutations in prostate cancer specimens. It appears that the outcome of mutation analysis would depend not only on which tumors but also which regions of the tumors are included in the study. Furthermore, the observed heterogeneous topographical distribution of the mutation, if confirmed to be unique to prostate cancer, may have important implications in the understanding of the biology of prostate carcinogenesis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7604888

  3. Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses.

    PubMed

    Porter, Paul C; Clark, Denise R; McDaniel, Lisa D; McGregor, W Glenn; States, J Christopher

    2006-01-01

    Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.

  4. Activation of p53 contributes to pseudolaric acid B-induced senescence in human lung cancer cells in vitro

    PubMed Central

    Yao, Guo-dong; Yang, Jing; Li, Qiang; Zhang, Ye; Qi, Min; Fan, Si-miao; Hayashi, Toshihiko; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

    2016-01-01

    Aim: Pseudolaric acid B (PAB), a diterpene acid isolated from the root bark of Pseudolarix kaempferi Gordon, has shown to exert anti-tumor effects via inducing cell cycle arrest followed by apoptosis in several cancer cell lines. Here we reported that PAB induced a mitotic catastrophe in human lung cancer A549 cells, which resulted in senescence without apoptosis or necrosis. Methods: Three human lung cancer cell lines (A549, H460 and H1299 cells) were examined. Cell growth inhibition was assessed with MTT assay. Cell cycle distribution was determined using a flow cytometer. Cell nuclear morphology was observed under a fluorescence microscope. Senescent cells were detected using SA-β-Gal staining. Apoptotic and senescent protein expression was examined using Western blot analysis. The expression of p53 and p21 in the cells was downregulated by siRNAs. Results: Treatment with PAB (5–80 μmol/L) inhibited the growth of A549 cells in dose- and time-dependent manners. Prolonged treatment with PAB (20 μmol/L) caused G2/M arrest at day 1 followed by mitotic catastrophe from day 2, which eventually resulted in cell senescence between days 3 and 4 without cell death (apoptosis or necrosis). Knockdown of p53 expression with siRNA significantly suppressed PAB-induced senescence in A549 cells (p53 wild). Furthermore, PAB-induced senescence was also observed in human lung cancer H460 cells (p53 wild), but not in human lung cancer H1299 cells (p53 null). Conclusion: The anti-tumor action of PAB against human lung cancer A549 cells in vitro involves the induction of senescence through activation of the p53 pathway. PMID:27041461

  5. p53-induced microRNA-1246 inhibits the cell growth of human hepatocellular carcinoma cells by targeting NFIB.

    PubMed

    Zhang, Quan; Cao, Li-Ye; Cheng, Shu-Jie; Zhang, Ai-Min; Jin, Xiao-Shi; Li, Yong

    2015-03-01

    In recent years, miR-1246 has been identified as a transcriptional target of p53 in Down syndrome and may provide a new p53-miR-1246-DYRK1A-NFAT pathway in cancer. The present study aimed to explore the role of miR-1246 in the tumorigenesis of human hepatocellular carcinoma (HCC). We found that wild-type p53 regulated the expression of miR-1246 in HCC cell lines, and alteration of miR-1246 modulated cell proliferation, colony formation ability and apoptosis. The nuclear factor I/B (NFIB), an oncogene, was identified as a direct target gene of miR-1246 using a fluorescent reporter assay. Overexpression of NFIB abolished the regulation of cell apoptosis caused by miR-1246 in HepG2 cells. This finding suggests that miR-1246 is regulated by p53 and suppresses the growth of human HCC by targeting NFIB. Here, we propose a new p53-miR-1246-NFIB pathway in HCC. PMID:25591821

  6. 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

  7. 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

  8. Expression of heat shock protein 70 and p53 in human lung cancer.

    PubMed

    Costa, M; Rosas, S; Chindano, A; Lima, P; Madi, K; Carvalho, M

    1997-01-01

    Bronchial biopsies of 21 patients with lung cancer were analyzed by Western blot for stress protein HSP70 and p53 proteins. Squamous carcinoma was the most common type found. The p53 protein was detectable in 14 cases. The HSP70 was detectable in 17 and overexpressed in 9 patients. Eleven patients showed positivity for both protein expressions, however no statistical significance was found (Kappa's test, p>0.05). Specific associations were not observed for HSP70 overexpression and p53 detection that could be related to clinical finds or tabagism. Our results indicate that the stress protein HSP70 is detectable and may be involved in the tumor development. PMID:21590207

  9. The Mdm2 and p53 genes are conserved in the Arachnids.

    PubMed

    Lane, David P; Cheok, Chit Fang; Brown, Christopher J; Madhumalar, Arumugam; Ghadessy, Farid J; Verma, Chandra

    2010-02-15

    The p53 protein and its negative regulator the ubiquitin E3 ligase Mdm2 have been shown to be conserved from the T. adhaerens to man. In common with D. melanogaster and C. elegans, there is a single copy of the p53 gene in T. adhaerens, while in the vertebrates three p53-like genes can be found: p53, p63 and p73. The Mdm2 gene is not present within the fully sequenced and highly annotated genomes of C. elegans and D. melanogaster. However, it is present in Placazoanand the presence of multiple distinct p53 genes in the Sea anemone N. vectensis led us to examine the genomes of other phyla for p53 and Mdm2-like genes. We report here the discovery of an Mdm2-like gene and two distinct p53-like genes in the Arachnid Ioxodes scapularis (Northern Deer Tick). The two predicted Deer Tick p53 proteins are much more highly related to the human p53 protein in sequence than are the fruit fly and nematode proteins. One of the Deer Tick genes encodes a p53 protein that is initiated within the DNA binding domain of p53 and shows remarkable homology to the newly described N-terminally truncated delta isoforms of human and zebrafish p53. PMID:20160485

  10. Punicalagin, a polyphenol in pomegranate juice, downregulates p53 and attenuates hypoxia-induced apoptosis in cultured human placental syncytiotrophoblasts.

    PubMed

    Chen, Baosheng; Longtine, Mark S; Nelson, D Michael

    2013-11-15

    Oxidative stress is associated with placental dysfunction and suboptimal pregnancy outcomes. Therapeutic interventions to limit placental injury from oxidative stress are lacking. Punicalagin is an ellagitannin and a potent antioxidant in pomegranate juice. We showed that both pomegranate juice and punicalagin decrease oxidative stress and apoptosis in cultured syncytiotrophoblasts. p53 is involved in the oxidative stress-induced apoptosis in trophoblasts. We now test the hypothesis that punicalagin limits trophoblast injury in vitro by regulating the levels of p53. We examined the expression of p53, mouse double minute 2 homolog, p21, hypoxia-inducible factor (HIF) α, and selected members of the B cell lymphoma 2 (BCL2) family of proteins in cultured syncytiotrophoblasts exposed to ≤1% oxygen in the absence or presence of punicalagin. We found that punicalagin attenuated hypoxia-induced apoptosis in syncytiotrophoblasts, as quantified by levels of cleaved poly-ADP ribose polymerase. This protective effect was in part mediated by reduced p53 activity shown by decreased expression of p21, lower HIF1α expression, and limited activity of caspases 9 and 3. There was no change in expression of proteins in the BCL2 family, which are also important in apoptosis. The data support a role for downregulation of p53 in the protection of human trophoblasts by punicalagin.

  11. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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;...

  14. 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

  15. 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

  16. New Plays in the p53 Theater

    PubMed Central

    Aylon, Yael; Oren, Moshe

    2010-01-01

    Summary The p53 tumor suppressor and its paralogs p63 and p73 are at the crux of a network modulating cellular responses against potentially tumorigenic events. p53 acts primarily as a transcription factor, regulating the expression of both coding and non-coding RNAs, as well as the activity of RNA processing complexes. In line with their anti-tumorigenic function, p53 and p63 have recently been implicated in restricting tumor cell invasion. In parallel, a growing number of non-canonical target genes have been added to the p53 repertoire. These include genes encoding for proteins that impinge on a broad spectrum of cellular functions, from cell metabolism to stem cell renewal. The p53 story is still far from being fully told. PMID:21317061

  17. Upregulation of Acetylcholinesterase Mediated by p53 Contributes to Cisplatin-Induced Apoptosis in Human Breast Cancer Cell

    PubMed Central

    Ye, Xiaolei; Zhang, Changsong; Chen, Yichen; Zhou, Tianbao

    2015-01-01

    Background: The expression of acetylcholinesterase (AChE) could be induced during apoptosis in various cell types. And reduced AChE expression either by siRNA could prevent apoptosis. However, the detailed mechanisms underlying the AChE regulation are largely unknown in human breast cancer cell. Material and methods: MCF-7 cells were cultured and treated by cisplatin in the absence or presence of p53 siRNA. Results: In this study, the regulation of AChE expression during apoptosis induced by cisplatin, a current used anticancer drug, was investigated in human breast cancer cell line MCF-7. Exposure of MCF-7 cells to cisplatin resulted in apoptosis in a time- and concentration-dependent manner. Meanwhile, the upregulated AChE and p53 were also observed during apoptosis. Silencing interfering RNA directed against p53 blocked the expression of AChE. Conclusion: Taken together, these results suggested that AChE expression could be upregulated by the activation of p53 during apoptosis induced by cisplatin in MCF-7 cells. PMID:25553088

  18. p53 directly regulates the transcription of the human frataxin gene and its lack of regulation in tumor cells decreases the utilization of mitochondrial iron.

    PubMed

    Shimizu, Rina; Lan, Nguyen Ngoc; Tai, Tran Tien; Adachi, Yuka; Kawazoe, Asako; Mu, Anfeng; Taketani, Shigeru

    2014-11-01

    Mitochondrial frataxin functions in iron homeostasis, biogenesis of iron-sulfur clusters, protection from oxidative stress and apoptosis, and as a tumor suppressor protein. We examined regulation of the expression of the human frataxin by p53. Pifithrin-α, an inhibitor of p53 function, and knockdown of p53 decreased the level of frataxin mRNA in human kidney HEK 293T cells. The transcriptional activity of the human frataxin gene is enhanced by the proximal promoter containing the p53-responsive element (p53RE) on the gene. Chromatin immunoprecipitation assay and electrophoretic mobility shift assay confirmed the binding of p53 to the human frataxin p53RE. The expression of wild-type p53 in human cancer HeLa cells increased the reporter activity carrying p53RE at the region of -209 to -200bp of the frataxin promoter. Finally, when the HeLa cells overexpressing frataxin were treated with 5-aminolevulinic acid (ALA), there was less accumulation of protoporphyrin than HeLa control cells, and it was sharply decreased by the addition of iron citrate, suggesting that the utilization of mitochondrial iron for heme biosynthesis can be dependent on the level of frataxin. Alternatively, the low expression of frataxin not regulated by p53 in tumor cells lowers the utilization of iron in mitochondria, causing the tumor-specific ALA-induced accumulation of protoporphyrin.

  19. Radon-induced alterations in p53-mediated energy metabolism of malignantly transformed human bronchial epithelial cells.

    PubMed

    Liu, Xing; Wang, Xu; Tong, Jian

    2016-01-01

    Radon and its progeny were confirmed to be a category I carcinogenic agent. However, the molecular basis underlying carcinogenesis induced by radon has not been fully elucidated. Expression of p53, a key regulator in glycolysis, is known to be decreased in carcinogenesis. The aim of this investigation was to determine changes in energy metabolism mediated by p53-related metabolic pathway using radon-induced transformation of human bronchial epithelial (HBE) cells. HBE cells were exposed to radon for 20 min at a concentration of 20,000 Bq/m(3) and cultured for 3 d, and exposed again at the same concentration and duration. This was repeated 10 times with culture for 35 passages until malignant transformation occurred. During the culturing process, the levels of lactate and lactate dehydrogenase (LDH) and ratio of NAD(+)/NADH gradually increased between passages. Between passages 30 and 35, p53 target gene synthesis of cytochrome c oxidase 2 (SCO2), TP53-induced glycolysis, and apoptosis regulator (TIGAR) expression were significantly decreased. Data demonstrated that p53-associated metabolic pathways may be altered in radon-mediated malignant transformation. PMID:27267826

  20. Structure of human MDM2 complexed with RPL11 reveals the molecular basis of p53 activation

    PubMed Central

    Zheng, Jiangge; Lang, Yue; Zhang, Qi; Cui, Di; Sun, Haili; Jiang, Lun; Chen, Zhenhang; Zhang, Rui; Gao, Yina; Tian, Wenli; Wu, Wei; Tang, Jun; Chen, Zhongzhou

    2015-01-01

    The central region of MDM2 is critical for p53 activation and tumor suppression. Upon ribosomal stress, this region is bound by ribosomal proteins, particularly ribosomal protein L11 (RPL11), leading to MDM2 inactivation and subsequent p53 activation. Here, we solved the complex structure of human MDM2–RPL11 at 2.4 Å. MDM2 extensively interacts with RPL11 through an acidic domain and two zinc fingers. Formation of the MDM2–RPL11 complex induces substantial conformational changes in both proteins. RPL11, unable to bind MDM2 mutants, fails to induce the activation of p53 in cells. MDM2 mimics 28S rRNA binding to RPL11. The C4 zinc finger determines RPL11 binding to MDM2 but not its homolog, MDMX. Our results highlight the essential role of the RPL11–MDM2 interaction in p53 activation and tumor suppression and provide a structural basis for potential new anti-tumor drug development. PMID:26220995

  1. 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.

  2. An expression signature for p53 status in human breast cancer predicts mutation status, transcriptional effects, and patient survival.

    PubMed

    Miller, Lance D; Smeds, Johanna; George, Joshy; Vega, Vinsensius B; Vergara, Liza; Ploner, Alexander; Pawitan, Yudi; Hall, Per; Klaar, Sigrid; Liu, Edison T; Bergh, Jonas

    2005-09-20

    Perturbations of the p53 pathway are associated with more aggressive and therapeutically refractory tumors. However, molecular assessment of p53 status, by using sequence analysis and immunohistochemistry, are incomplete assessors of p53 functional effects. We posited that the transcriptional fingerprint is a more definitive downstream indicator of p53 function. Herein, we analyzed transcript profiles of 251 p53-sequenced primary breast tumors and identified a clinically embedded 32-gene expression signature that distinguishes p53-mutant and wild-type tumors of different histologies and outperforms sequence-based assessments of p53 in predicting prognosis and therapeutic response. Moreover, the p53 signature identified a subset of aggressive tumors absent of sequence mutations in p53 yet exhibiting expression characteristics consistent with p53 deficiency because of attenuated p53 transcript levels. Our results show the primary importance of p53 functional status in predicting clinical breast cancer behavior. PMID:16141321

  3. 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.

  4. Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

    PubMed Central

    2010-01-01

    Background α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action. Methods MTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells. Results α-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G2/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G2/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells. Conclusions This study for the first time identifies effects of α-santalol in G2/M phase arrest and describes detailed mechanisms of G2/M phase arrest by this agent, which might be

  5. 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

  6. Effect of boswellia thurifera gum methanol extract on cytotoxicity and p53 gene expression in human breast cancer cell line.

    PubMed

    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

  7. 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

  8. 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

  9. Anticancer Activity of γ-Bisabolene in Human Neuroblastoma Cells via Induction of p53-Mediated Mitochondrial Apoptosis.

    PubMed

    Jou, Yu-Jen; Hua, Chun-Hung; Lin, Chen-Sheng; Wang, Ching-Ying; Wan, Lei; Lin, Ying-Ju; Huang, Su-Hua; Lin, Cheng-Wen

    2016-01-01

    γ-Bisabolene has demonstrated antiproliferative activities against several human cancer cell lines. This study first discloses the antiproliferative and apoptosis induction activities of γ-bisabolene to human neuroblastoma TE671 cells. A CC50 value of γ-bisabolene was 8.2 μM to TE671 cells. Cell cycle analysis with PI staining showed γ-bisabolene elevating the sub-G1 fractions in a time-dependent manner. In addition, annexin V-FITC/PI staining showed γ-bisabolene significantly triggering early (annexin-V positive/PI negative) and late (annexin-V positive/PI positive) apoptosis in dose-dependent manners. γ-Bisabolene induced caspase 3/8/9 activation, intracellular ROS increase, and mitochondrial membrane potential decrease in apoptosis of human neuro-blastoma cells. Moreover, γ-bisabolene increased p53 phosphorylation and up-regulated p53-mediated apoptotic genes Bim and PUMA, as well as decreased the mRNA and protein levels of CK2α. Notably, the results indicated the involvement of CK2α-p53 pathways in mitochondria-mediated apoptosis of human neuroblastoma cells treated with γ-bisabolene. This study elucidated the apoptosis induction pathways of γ-bisabolene-treated neuroblastoma cells, in which could be useful for developing anti-neuroblastoma drugs. PMID:27164076

  10. 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

  11. Paeoniflorin attenuates ultraviolet B-induced apoptosis in human keratinocytes by inhibiting the ROS-p38-p53 pathway.

    PubMed

    Kong, Lingwen; Wang, Shangshang; Wu, Xiao; Zuo, Fuguo; Qin, Haihong; Wu, Jinfeng

    2016-04-01

    Ultraviolet (UV) light is one of the most harmful environmental factors that contribute to skin damage. Exposure to UV induces extensive generation of reactive oxygen species (ROS), and results in photoaging and skin cancer development. One approach to protecting human skin against UV radiation is the use of antioxidants. In recent years, naturally occurring herbal compounds have gained considerable attention as protective agents for UV exposure. Paeoniflorin (PF) is a novel natural antioxidant, which is isolated from peony root (Radix Paeoniae Alba). The present study evaluated the protective effects of PF on UV‑induced skin damage in vitro, and demonstrated that the effects were mediated via the ROS‑p38‑p53 pathway. The results of the present study demonstrated that treatment with PF (25, 50, and 100 µM) significantly increased the percentage of viable keratinocytes after UV‑B exposure. In addition, cell death analysis indicated that PF treatment markedly reduced UV‑B‑radiation‑induced apoptosis in keratinocytes, which was accompanied by increased procaspase 3 expression and decreased cleaved caspase 3 expression. Treatment with PF markedly reduced the production of ROS, and inhibited the activation of p38 and p53 in human keratinocytes, thus suggesting that the ROS‑p38‑p53 pathway has a role in UV‑B‑induced skin damage. In conclusion, the present study reported that PF was able to attenuate UV‑B‑induced cell damage in human keratinocytes. Notably, these effects were shown to be mediated, at least in part, via inhibition of the ROS-p38-p53 pathway. PMID:26936104

  12. Sodium Arsenite ± Hyperthermia Sensitizes p53-Expressing Human Ovarian Cancer Cells to Cisplatin by Modulating Platinum-DNA Damage Responses

    PubMed Central

    Muenyi, Clarisse S.; Pinhas, Allan R.; Fan, Teresa W.; Brock, Guy N.; Helm, C. William; States, J. Christopher

    2012-01-01

    Epithelial ovarian cancer (EOC) is the leading cause of gynecological cancer death in the United States. Cisplatin is a DNA damaging agent initially effective against EOC but limited by resistance. P53 plays a critical role in cellular response to DNA damage and has been implicated in EOC response to platinum chemotherapy. In this study, we examined the role of p53 status in EOC response to a novel combination of cisplatin, sodium arsenite, and hyperthermia. Human EOC cells were treated with cisplatin ± 20μM sodium arsenite at 37°C or 39°C for 1 h. Sodium arsenite ± hyperthermia sensitized wild-type p53-expressing (A2780, A2780/CP70, OVCA 420, OVCA 429, and OVCA 433) EOC cells to cisplatin. Hyperthermia sensitized p53-null SKOV-3 and p53-mutant (OVCA 432 and OVCAR-3) cells to cisplatin. P53 small interfering RNA (siRNA) transfection abrogated sodium arsenite sensitization effect. XPC, a critical DNA damage recognition protein in global genome repair pathway, was induced by cisplatin only in wild-type p53-expressing cells. Cotreatment with sodium arsenite ± hyperthermia attenuated cisplatin-induced XPC in wild-type p53-expressing cells. XPC siRNA transfection sensitized wild-type p53-expressing cells to cisplatin, suggesting that sodium arsenite ± hyperthermia attenuation of XPC is a mechanism by which wild-type p53-expressing cells are sensitized to cisplatin. Hyperthermia ± sodium arsenite enhanced cellular and DNA accumulation of platinum in wild-type p53-expressing cells. Only hyperthermia enhanced platinum accumulation in p53-null cells. In conclusion, sodium arsenite ± hyperthermia sensitizes wild-type p53-expressing EOC cells to cisplatin by suppressing DNA repair protein XPC and increasing cellular and DNA platinum accumulation. PMID:22331493

  13. 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

  14. Elevated expression of mechanosensory polycystins in human carotid atherosclerotic plaques: association with p53 activation and disease severity

    PubMed Central

    Varela, Aimilia; Piperi, Christina; Sigala, Fragiska; Agrogiannis, George; Davos, Constantinos H.; Andri, Maria-Anastasia; Manopoulos, Christos; Tsangaris, Sokrates; Basdra, Efthimia K.; Papavassiliou, Athanasios G.

    2015-01-01

    Atherosclerotic plaque formation is associated with irregular distribution of wall shear stress (WSS) that modulates endothelial function and integrity. Polycystins (PC)-1/-2 constitute a flow-sensing protein complex in endothelial cells, able to respond to WSS and induce cell-proliferation changes leading to atherosclerosis. An endothelial cell-culture system of measurable WSS was established to detect alterations in PCs expression under conditions of low- and high-oscillatory shear stress in vitro. PCs expression and p53 activation as a regulator of cell proliferation were further evaluated in vivo and in 69 advanced human carotid atherosclerotic plaques (AAPs). Increased PC-1/PC-2 expression was observed at 30–60 min of low shear stress (LSS) in endothelial cells. Elevated PC-1 expression at LSS was followed by p53 potentiation. PCs immunoreactivity localizes in areas with macrophage infiltration and neovascularization. PC-1 mRNA and protein levels were significantly higher than PC-2 in stable fibroatherotic (V) and unstable/complicated (VI) AAPs. Elevated PC-1 immunostaining was detected in AAPs from patients with diabetes mellitus, dyslipidemia, hypertension and carotid stenosis, at both arteries (50%) or in one artery (90%). PCs seem to participate in plaque formation and progression. Since PC-1 upregulation coincides with p38 and p53 activation, a potential interplay of these molecules in atherosclerosis induction is posed. PMID:26286632

  15. Human herpes virus 8 (HHV-8) in Kaposi's sarcoma: lack of association with Bcl-2 and p53 protein expression.

    PubMed Central

    Kennedy, M M; O'Leary, J J; Oates, J L; Lucas, S B; Howells, D D; Picton, S; McGee, J O

    1998-01-01

    AIMS: Human herpes virus 8 (HHV-8) is the infectious agent implicated in the pathogenesis of Kaposi's sarcoma, although its mode of action is unclear. Recent work indicates that the HHV-8 genome encodes a viral Bcl-2 homologue (v-Bcl-2). The aim of this study was to explore Bcl-2 expression in Kaposi's sarcoma using a unique set of HHV-8 positive and negative cases, and to determine whether there is a relation with p53 expression. METHODS: Up to 18 specimens from 17 patients were selected. HHV-8 status was determined using nested polymerase chain reaction (PCR) to the open reading frame (ORF) 26, with further confirmation by TaqMan PCR. In addition, Bcl-2 and p53 immunohistochemistry were performed using standard protocols. RESULTS: The results suggest that Bcl-2 and p53 expression is independent of HHV-8 status. In addition, there does not appear to be a direct correlation with disease stage. CONCLUSIONS: HHV8 histopathogenesis is likely to be a multifactorial complex process, which may be mediated in part by viral genes and apoptosis regulating homologues. PMID:9850339

  16. Quantitative analysis of p53 expression in human normal and cancer tissue microarray with global normalization method

    PubMed Central

    Idikio, Halliday A

    2011-01-01

    Tissue microarray based immunohistochemical staining and proteomics are important tools to create and validate clinically relevant cancer biomarkers. Immunohistochemical stains using formalin-fixed tissue microarray sections for protein expression are scored manually and semi-quantitatively. Digital image analysis methods remove some of the drawbacks of manual scoring but may need other methods such as normalization to provide across the board utility. In the present study, quantitative proteomics-based global normalization method was used to evaluate its utility in the analysis of p53 protein expression in mixed human normal and cancer tissue microarray. Global normalization used the mean or median of β-actin to calculate ratios of individual core stain intensities, then log transformed the ratios, calculate a mean or median and subtracted the value from the log of ratios. In the absence of global normalization of p53 protein expression, 44% (42 of 95) of tissue cores were positive using the median of intensity values and 40% (38 of 95) using the mean of intensities as cut-off points. With global normalization, p53 positive cores changed to 20% (19 of 95) when using median of intensities and 15.8%(15 of 95) when the mean of intensities were used. In conclusion, the global normalization method helped to define positive p53 staining in the tissue microarray set used. The method used helped to define clear cut-off points and confirmed all negatively stained tissue cores. Such normalization methods should help to better define clinically useful biomarkers. PMID:21738821

  17. Vascular endothelial growth factor and nitric oxide synthase expression in human lung cancer and the relation to p53.

    PubMed Central

    Ambs, S.; Bennett, W. P.; Merriam, W. G.; Ogunfusika, M. O.; Oser, S. M.; Khan, M. A.; Jones, R. T.; Harris, C. C.

    1998-01-01

    Vascular endothelial growth factor (VEGF) expression and mutations of cancer-related genes increase with cancer progression. This correlation suggests the hypothesis that oncogenes and tumour suppressors regulate VEGF, and a significant correlation between p53 alteration and increased VEGF expression in human lung cancer was reported recently. To further examine this hypothesis, we analysed VEGF protein expression and mutations in p53 and K-ras in 27 non-small-cell lung cancers (NSCLC): 16 squamous cell, six adenocarcinomas, one large cell, two carcinoids and two undifferentiated tumours. VEGF was expressed in 50% of the squamous cell carcinomas (SCC) and carcinoids but none of the others. p53 mutations occurred in 14 tumours (52%), and K-ras mutations were found in two adenocarcinomas and one SCC; there was no correlation between the mutations and VEGF expression. As nitric oxide also regulates angiogenesis, we examined NOS expression in NSCLC. The Ca2+-dependent NOS activity, which indicates NOS1 and NOS3 expression, was significantly reduced in lung carcinomas compared with adjacent non-tumour tissue (P < 0.004). Although the Ca2+-independent NOS activity, which indicates NOS2 expression, was low or undetectable in non-tumour tissues and most carcinomas, significant activity occurred in three SCC. In summary, our data do not show a direct regulation of VEGF by p53 in NSCLC. Finally, we did not find the up-regulation of NOS isoforms during NSCLC progression that has been suggested for gynaecological and breast cancers. Images Figure 1 Figure 4 Figure 5 PMID:9683299

  18. Mitochondrial dysfunction and transactivation of p53-dependent apoptotic genes in BaP-treated human fetal lung fibroblasts.

    PubMed

    Yang, Guangtao; Jiang, Ying; Rao, Kaimin; Chen, Xi; Wang, Qian; Liu, Ailin; Xiong, Wei; Yuan, Jing

    2011-12-01

    Benzo(a)pyrene (BaP) has been shown to be an inducer of apoptosis. However, mechanisms involved in BaP-induced mitochondrial dysfunction are not well-known. In this study, human fetal lung fibroblasts cells were treated with BaP (8, 16, 32, 64 and 128 μM) for 4 and 12 h. Cell viability, intracellular level of reactive oxygen species (ROS), total antioxidant capacity (T-AOC), mitochondrial membrane potential (ΔΨ(m)) and cytochrome c release were determined. Changes in transcriptional levels of p53-dependent apoptotic genes (p53, APAF1, CASPASE3, CASPASE9, NOXA and PUMA) were measured. At time point of 4 h, BaP induced the intracellular ROS generation in 64 (p < .05) and 128 μM BaP groups (p < .01) but decreased the T-AOC activities in 32, 64 (p < .05 for both) and 128 μM BaP groups (p < .01). At time point of 12 h, ΔΨ(m) significantly decreased in ≥32 μM BaP groups (p < .05 for all). Amount of mitochondrial cytochrome c significantly increased in 128 μM BaP group (p < .01). Transcriptional levels of CASPASE3, CASPASE9, APAF1 and PUMA were up-regulated in all BaP groups (p < .05 for all) and in ≥32 μM groups for NOXA (p < .05). But only in 16 μM BaP group a relatively little expression of p53 mRNA was observed (p < .05). The results indicate that in the earlier period BaP promoted the generation of excessive ROS and subsequently the mitochondrial depolarization, whereas transactivations of the p53-dependent apoptotic genes were significantly induced at the later period.

  19. Human Papillomavirus E6 Knockdown Restores Adenovirus Mediated-estrogen Response Element Linked p53 Gene Transfer in HeLa Cells.

    PubMed

    Kajitani, Koji; Honda, Ken-Ichi; Terada, Hiroyuki; Yasui, Tomoyo; Sumi, Toshiyuki; Koyama, Masayasu; Ishiko, Osamu

    2015-01-01

    The p53 gene is inactivated by the human papillomavirus (HPV) E6 protein in the majority of cervical cancers. Treatment of HeLa S3 cells with siRNA for HPV E6 permitted adenovirus-mediated transduction of a p53 gene linked to an upstream estrogen response element (ERE). Our previous study in non-siRNA treated HHUA cells, which are derived from an endometrial cancer and express estrogen receptor β, showed enhancing effects of an upstream ERE on adenovirus-mediated p53 gene transduction. In HeLa S3 cells treated with siRNA for HPV E6, adenovirus-mediated transduction was enhanced by an upstream ERE linked to a p53 gene carrying a proline variant at codon 72, but not for a p53 gene with arginine variant at codon 72. Expression levels of p53 mRNA and Coxsackie/adenovirus receptor (CAR) mRNA after adenovirus-mediated transfer of an ERE-linked p53 gene (proline variant at codon 72) were higher compared with those after non-ERE-linked p53 gene transfer in siRNA-treated HeLa S3 cells. Western blot analysis showed lower β-tubulin levels and comparatively higher p53/β-tubulin or CAR /β-tubulin ratios in siRNA-treated HeLa S3 cells after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with those in non-siRNA-treated cells. Apoptosis, as measured by annexin V binding, was higher after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with that after non-ERE-linked p53 gene transfer in siRNA-treated cells.

  20. 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

  1. p53 is involved in clearance of ionizing radiation-induced RAD51 foci in a human colon cancer cell line

    SciTech Connect

    Orre, Lukas M. . E-mail: Lukas.Orre@ki.se; Stenerloew, Bo; Dhar, Sumeer; Larsson, Rolf; Lewensohn, Rolf; Lehtioe, Janne

    2006-04-21

    We have investigated p53-related differences in cellular response to DNA damaging agents, focusing on p53s effects on RAD51 protein level and sub-cellular localization post exposure to ionizing radiation. In a human colon cancer cell line, HCT116 and its isogenic p53-/- subcell line we show here p53-independent RAD51 foci formation but interestingly the resolution of RAD51 foci showed clear p53 dependence. In p53 wt cells, but not in p53-/- cells, RAD51 protein level decreased 48 h post irradiation and fluorescence immunostaining showed resolution of RAD51 foci and relocalization of RAD51 to nucleoli at time points corresponding to the decrease in RAD51 protein level. Both cell lines rejoined DNA double strand breaks efficiently with similar kinetics and p53 status did not influence sensitivity to DNA damaging agents. We suggest that p53 has a role in RAD51 clearance post DSB repair and that nucleoli might be sites of RAD51 protein degradation.

  2. Comparison of p53 and the PDZ domain containing protein MAGI-3 regulation by the E6 protein from high-risk human papillomaviruses

    PubMed Central

    Ainsworth, Julia; Thomas, Miranda; Banks, Lawrence; Coutlee, Francois; Matlashewski, Greg

    2008-01-01

    Central to cellular transformation caused by human papillomaviruses (HPVs) is the ability of E6 proteins to target cellular p53 and proteins containing PDZ domains, including MAGI-3, for degradation. The aim of this study was to compare E6-mediated degradation of p53 and MAGI-3 under parallel experimental conditions and further with respect to the involvement of proteasomes and ubiquitination. We also compared the degradation of p53 and MAGI-3 by E6 from several HPV types including different variants from HPV-33. All of the E6 genes from different HPV types displayed similar abilities to mediate the degradation of both p53 and MAGI-3 although there may be subtle differences observed with the different 33E6 variants. There were however differences in E6 mediated degradation of p53 and MAGI-3. Proteasome inhibition assays partially protected p53 from E6 mediated degradation, but did not protect MAGI-3. In addition, under conditions where p53 was ubiquitinated by E6 and MDM2 in vivo, ubiquitination of MAGI-3 was not detected. These results imply that although both p53 and MAGI-3 represent effective targets for oncogenic E6, the mechanisms by which E6 mediates p53 and MAGI-3 degradation are distinct with respect to the involvement of ubiquitination prior to proteasomal degradation. PMID:18518978

  3. A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins.

    PubMed

    Coppari, Emilia; Yamada, Tohru; Bizzarri, Anna Rita; Beattie, Craig W; Cannistraro, Salvatore

    2014-01-01

    p28 is an anionic, amphipathic, cell-penetrating peptide derived from the cupredoxin azurin that binds to the DNA-binding domain (DBD) of the tumor suppressor protein, p53, and induces a post-translational increase in the level of wild type and mutated p53 in a wide variety of human cancer cells. As p63 and p73, additional members of the p53 superfamily of proteins, also appear to be involved in the cellular response to cancer therapy and are reportedly required for p53-induced apoptosis, we asked whether p28 also binds to p63 and p73. Atomic force spectroscopy demonstrates that p28 forms a stable, high-affinity complex with full-length p63, the DBD of p63, and full-length p73. Exposure to p28 decreased the level of TAp63α and ΔNp63α, the truncated form of p63, in p53 wild type and mutated human breast cancer cells, respectively. p28 increased the level of TAp73α, but not ΔNp73α, in the same breast cancer cell lines. In contrast, p28 increased the level of the TA and ΔN isoforms of p63 in p53 wild type, but not in p53 mutated melanoma cells, while decreasing TA p73α in p53 wild type and mutated human melanoma cells. All changes were mirrored by an associated change in the expression of the HECT E3 ligases Itch/AIP4, AIP5, and the RING E3 ligase Pirh2, but not in the receptor for activated C kinase or the RING E3 ligases Mdm2 and Cop1. Collectively, the data suggest that molecules such as p28 bind with high affinity to the DBD of p63 and p73 and alter their expression independent of the Mdm2 and Cop1 pathways.

  4. p53-dependent but ATM-independent inhibition of DNA synthesis and G2 arrest in cadmium-treated human fibroblasts

    SciTech Connect

    Cao Feng |; Zhou Tong; Simpson, Dennis; Zhou Yingchun; Boyer, Jayne; Chen Bo |; Jin Taiyi; Cordeiro-Stone, Marila; Kaufmann, William . E-mail: wkarlk@med.unc.edu

    2007-01-15

    This study focused on the activation of cell cycle checkpoint responses in diploid human fibroblasts that were treated with cadmium chloride and the potential roles of ATM and p53 signaling pathways in cadmium-induced responses. The alkaline comet assay indicated that cadmium caused a dose-dependent increase in DNA damage. Cells that were rendered p53-defective by expression of a dominant-negative p53 allele or knockdown of p53 mRNA were more resistant to cadmium-induced inactivation of colony formation than normal and ataxia telangiectasia (AT) cells. Synchronized fibroblasts in S were more sensitive to cadmium toxicity than cells in G1, suggesting that cadmium may target some element of DNA replication. Cadmium produced a dose- and time-dependent inhibition of DNA synthesis. An immediate inhibition was associated with severe delay in progression through S phase and a delayed inhibition seen 24 h after treatment was associated with accumulation of cells in G2. AT and normal cells displayed similar patterns of inhibition of DNA synthesis and G2 delay after treatment with cadmium, while p53-defective cells displayed significantly less of the delayed inhibition of DNA synthesis and accumulation in G2 post-treatment. Total p53 protein and ser15-phosphorylated p53 were induced by cadmium in normal and AT cells. The p53 transactivation target Gadd45{alpha} was induced in both p53-effective and p53-defective cells after 4 h cadmium treatment, and this was associated with an acute inhibition of mitosis. Cadmium produced a very unusual pattern of toxicity in human fibroblasts, inhibiting DNA replication and inducing p53-dependent growth arrest but without induction of p21{sup Cip1/Waf1} or activation of Chk1.

  5. 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.

  6. Biochemical and functional evidence of p53 homology is inconsistent with molecular phylogenetics for distant sequences.

    PubMed

    Fernandes, Andrew D; Atchley, William R

    2008-07-01

    The tumor suppressor p53 is mutated in approximately 50% of all human cancer cases worldwide. It is commonly assumed that the phylogenetic history of this important tumor suppressor has been thoroughly studied; however, few detailed studies of the entire extended p53 protein family have been reported, and none comprehensively and simultaneously consider functional, molecular, and phylogenetic data. Herein we examine a diverse collection of reported p53-like protein sequences, including representatives from the arthropods, nematodes, and protists, with the goal of answering several important questions. First, what evidence supports these highly divergent proteins being true homologues to the p53 family? Second, is the inferred overall family phylogeny concordant with known structures and functions? Third, does the extended p53 family possess recognizable conserved sites outside of the within-chordate, highly-conserved DNA-binding domain? Our study shows that the biochemical and functional evidence of p53 homology for nematodes, arthropods, and protists is inconsistent with their implied phylogenetic relationship within the overall family. Although these divergent sequences are always reported as functionally similar to human p53, our results confirm and extend the hypothesis that p63 is a far more appropriate protein for comparison. Within these divergent sequences, we find minimal conservation within the DNA-binding domain, and no conservation elsewhere. Taken together, our findings suggest that these sequences are not bona fide homologues of the extended p53 family and provide baseline criteria for the future identification and characterization of distant p53-family homologues.

  7. Estradiol agonists inhibit human LoVo colorectal-cancer cell proliferation and migration through p53

    PubMed Central

    Hsu, Hsi-Hsien; Kuo, Wei-Wen; Ju, Da-Tong; Yeh, Yu-Lan; Tu, Chuan-Chou; Tsai, Ying-Lan; Shen, Chia-Yao; Chang, Sheng-Huang; Chung, Li-Chin; Huang, Chih-Yang

    2014-01-01

    AIM: To investigate the effects of 17β-estradiol via estrogen receptors (ER) or direct administration of ER agonists on human colorectal cancer. METHODS: LoVo cells were established from the Bioresource Collection and Research Center and cultured in phenol red-free DMEM (Sigma, United States). To investigate the effects of E2 and/or ER selective agonists on cellular proliferation, LoVo colorectal cells were treated with E2 or ER-selective agonists for 24 h and 48 h and subjected to the MTT (Sigma) assay to find the concentration. And investigate the effects of E2 and/or ER selective agonists on cell used western immunoblotting to find out the diversification of signaling pathways. In order to observe motility and migration the wound healing assay and a transwell chamber (Neuro Probe) plate were tased. For a quantitative measure, we counted the number of migrating cells to the wound area post-wounding for 24 h. We further examined the cellular migration-regulating factors urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and matrix metalloproteinase (MMP)-9 in human LoVo cells so gelatin zymography that we used and gelatinolytic activity was visualized by Coomassie blue staining. And these results are presented as means ± SE, and statistical comparisons were made using Student’s t-test. RESULTS: The structure was first compared with E2 and ER agonists. We then treated the LoVo cells with E2 and ER agonists (10-8 mol/L) for 24 h and 48 h and subsequently measured the cell viability using MTT assay. Our results showed that treatment with 17β-estradiol and/or ER agonists in human LoVo colorectal cancer cells activated p53 and then up-regulated p21 and p27 protein levels, subsequently inhibiting the downstream target gene, cyclin D1, which regulates cell proliferation. Taken together, our findings demonstrate the anti-tumorigenesis effects of 17β-estradiol and/or ER agonists and suggest that these compounds may prove to be a

  8. 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.

  9. Expression of p53-regulated genes in human cultured lymphoblastoid TSCE5 and WTK1 cell lines after spaceflight in a frozen state

    NASA Astrophysics Data System (ADS)

    Takahashi, A.; Suzuki, H.; Omori, K.; Seki, M.; Hashizume, T.; Shimazu, T.; Ishioka, N.; Ohnishi, T.

    2011-03-01

    The 53 kDa tumor suppressor protein p53 is generally thought to contribute to the genetic stability of cells and to protect cells from DNA damage through the activity of p53-centered signal transduction pathways. To clarify the effect of space radiation on the expression of p53-dependent regulated genes, gene expression profiles were compared between two human cultured lymphoblastoid cell lines: one line (TSCE5) has a wild-type p53 gene status, and the other line (WTK1) has a mutated p53 gene status. Frozen human lymphoblastoid cells were stored in a freezer in the International Space Station (ISS) for 133 days. Gene expression was analyzed using DNA chips after culturing the space samples for 6 h on the ground after their return from space. Ground control samples were also cultured for 6 h after being stored in a frozen state on the ground for the same time period that the frozen cells were in space. p53-Dependent gene expression was calculated from the ratio of the gene expression values in wild-type p53 cells and in mutated p53 cells. The expression of 50 p53-dependent genes was up-regulated, and the expression of 94 p53-dependent genes was down-regulated after spaceflight. These expression data identified genes which could be useful in advancing studies in basic space radiation biology. The biological meaning of these results is discussed from the aspect of gene functions in the up- and down-regulated genes after exposure to low doses of space radiation.

  10. Sensitivity to CPT-11 of xenografted human colorectal cancers as a function of microsatellite instability and p53 status

    PubMed Central

    Bras-Gonçalves, R A; Rosty, C; Laurent-Puig, P; Soulié, P; Dutrillaux, B; Poupon, M-F

    2000-01-01

    Biological parameters influencing the response of human colorectal cancers (CRCs) to CPT-11, a topoisomerase 1 (top1) inhibitor, were investigated using a panel of nine CRCs xenografted into nude mice. CRC xenografts differed in their p53 status (wt or mut) and in their microsatellite instability phenotype (MSI+when altered). Five CRC xenografts were established from clinical samples. All five had a functional p53, two were MSI+and three were MSI–. Tumour-bearing nude mice were treated intraperitonealy (i.p.) with CPT-11. At 10 mg kg–1of CPT-11, four injections at 4-day intervals, four of the five xenografts responded to CPT-11 (growth delay of up to 10 days); the non-responder tumour was MSI−. At 40 mg kg−1of CPT-11, six injections at 4-day intervals, the five CRCs displayed variable but marked responses with complete regressions. In order to assess the role of p53 status in CPT-11 response, four CRC lines were used. HT29 cell line was MSI−/ Ala273-mutp53, its subclone HT29A3 being transfected by wtp53. LoVo cell line was MSI+/ wtp53, its subclone X17LoVo dominantly expressed Ala273-mutp53 after transfection. LoVo tumours (MSI+/ mutp53) were more sensitive than X17LoVo (MSI+/ mutp53. HT 29 tumours (MSI−Imutp53), were refractory to CPT-11 while HT29A3 tumours (MSI−/ wtp53) were sensitive, showing that wtp53 improves the drug-response in these MSI−tumours. Levels of mRNA expression of top1, fasR, TP53 and mdr1 were semi-quantified by reverse transcription polymerase chain reaction. None of these parameters correlated with CPT-11 response. Taken together, these observations indicate that MSI and p53 alterations could be associated with different CPT-11 sensitivities; MSI phenotype moderately influences the CPT-11 sensitivity, MSI+being more sensitive than MSI−CRC freshly obtained from patients, mutp53 status being associated with a poor response to CPT-11. © 2000 Cancer Research Campaign PMID:10732766

  11. Volatile Oil of Acori Graminei Rhizoma-Induced Apoptosis and Autophagy are dependent on p53 Status in Human Glioma Cells

    PubMed Central

    Chen, Lu; Jiang, Zhuyun; Ma, Hui; Ning, Ling; Chen, Hongdan; Li, Li; Qi, Hongyi

    2016-01-01

    Acori Graminei Rhizoma is well known for the beneficial effects on CNS disorders in traditional medicine. Though it is frequently prescribed in formulations for brain tumors, the anti-glioma effect has not been examined. We used volatile oil of Acori Graminei Rhizoma (VOA) and human glioblastoma multiforme (GBM) cells in this study. We found that VOA exhibited greater growth suppression in p53 wild-type cells than p53 mutant cells and very low effect on fibroblasts and human glial HEB cells. Apoptosis was triggered by VOA with a caspase-dependent way in p53 wild-type A172 cells, while a caspase-independent way in p53 mutant U251 cells. Meanwhile, both A172 and U251 cells treated by VOA displayed autophagic features. Furthermore, p53 decrease was observed along with VOA-induced apoptosis and autophagy in A172 cells. VOA-induced autophagy was mediated through a p53/AMPK/mTOR signaling pathway in A172 cells, while an mTOR-independent signaling pathway in U251 cells. Finally, blockage of autophagy potentiated the proapoptotic effect in both A172 and U251 cells, indicating a protective role of autophagy in VOA-induced cell death. Together, VOA exhibited anti-tumor activity in human GBM cells and induced apoptotic cell death and protective autophagy, which is cell type specific and dependent on p53 status. PMID:26892186

  12. Analysis of p53 codon 72 polymorphism and its association with human papillomavirus 16 and 18 E6 in Chinese cervical lesions.

    PubMed

    Min-min, H; Ming-rong, X; Ze-yi, C; Kai-xuan, Y; Zhi-lin, S

    2006-01-01

    The aim of this study was to analysis the relationship between p53 codon 72 polymorphism with human papillomavirus (HPV) 16 and 18 E6 in Chinese cervical cancer. A total of 81 cervical squamous cancer (specimens of G1, G2, and G3 are 13, 24, and 44, respectively; and of stage IB, IIA, IIB, and IIIA are 15, 37, 24, and 5, respectively), 18 cervical adenocarcinoma, 88 cervical intraepithelial neoplasm (CIN) (specimens of CIN II and III are 30 and 58), and 60 normal cervical specimens were included in this study. Polymerase chain reaction was used to examine p53 genotypes and HPV 16 and 18 E6. The frequencies of p53 Arg homozygosity in cervical squamous cancer, cervical adenocarcinoma, and CIN (II-III) were 58.02%, 55.55%, and 59.09%, respectively, that was much higher than that of p53 Arg/Pro heterozygosity (30.86%, 27.78%, and 21.59%) and of p53 Pro homozygosity (11.12%, 16.67%, and 19.32%) in each groups and higher than the frequency of p53 Arg homozygosity in normal samples (23.33%). There is no statistic difference in the normal samples for the frequency of p53 Arg homozygosity, p53 Arg/Pro heterozygosity, and p53 Pro homozygosity (23.33%, 40.00%, and 36.67%, respectively). The frequency of p53 Arg homozygosity in high risk (HR)-HPV E6-positive cervical squamous cancer samples (64.06%) is much higher than that in (HR)-HPV E6-negative cervical squamous cancer samples (35.29%) and in HR-HPV E6-positive normal samples (33.33%). No difference of p53 codon 72 polymorphism was found according to FIGO staging and grades. In conclusion, based on the findings of this study, it is suggested that p53 Arg homozygosity could act as a potential risk factor for the tumorigenesis of the cervix. p53 codon 72 polymorphism has no relation with the FIGO staging and grades of cervical cancer. p53 Arg homozygosity and HR-HPV E6 positive simultaneously can predict the fate of cervical lesions. PMID:17177838

  13. 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.

  14. Human Cytomegalovirus pUL29/28 and pUL38 Repression of p53-Regulated p21CIP1 and Caspase 1 Promoters during Infection

    PubMed Central

    Savaryn, John P.; Reitsma, Justin M.; Bigley, Tarin M.; Halligan, Brian D.; Qian, Zhikang; Yu, Dong

    2013-01-01

    During infection by human cytomegalovirus (HCMV), the tumor suppressor protein p53, which promotes efficient viral gene expression, is stabilized. However, the expression of numerous p53-responsive cellular genes is not upregulated. The molecular mechanism used to manipulate the transcriptional activity of p53 during infection remains unclear. The HCMV proteins IE1, IE2, pUL44, and pUL84 likely contribute to the regulation of p53. In this study, we used a discovery-based approach to identify the protein targets of the HCMV protein pUL29/28 during infection. Previous studies have demonstrated that pUL29/28 regulates viral gene expression by interacting with the chromatin remodeling complex NuRD. Here, we observed that pUL29/28 also associates with p53, an additional deacetylase complex, and several HCMV proteins, including pUL38. We confirmed the interaction between p53 and pUL29/28 in both the presence and absence of infection. HCMV pUL29/28 with pUL38 altered the activity of the 53-regulatable p21CIP1 promoter. During infection, pUL29/28 and pUL38 contributed to the inhibition of p21CIP1 as well as caspase 1 expression. The expression of several other p53-regulating genes was not altered. Infection using a UL29-deficient virus resulted in increased p53 binding and histone H3 acetylation at the responsive promoters. Furthermore, expression of pUL29/28 and its interacting partner pUL38 contributed to an increase in the steady-state protein levels of p53. This study identified two additional HCMV proteins, pUL29/28 and pUL38, which participate in the complex regulation of p53 transcriptional activity during infection. PMID:23236067

  15. Active oxygen species mediate the solar ultraviolet radiation-dependent increase in the tumour suppressor protein p53 in human skin fibroblasts.

    PubMed

    Vile, G F

    1997-07-21

    Active oxygen species mediate many of the biological consequences of exposing cultured human skin cells to solar ultraviolet (UV) radiation (290-380 nm). A critical step in the escape from the carcinogenic potential of UV radiation is mediated by the protein p53. P53 activates growth arrest, allowing for DNA repair, and apoptosis, which removes damaged cells. Here I show that p53 in cultured human skin fibroblasts is elevated after treatment with hydrogen peroxide, an oxidant produced in cells during exposure to solar UV radiation. Simulated solar UV radiation increased p53, and agents that scavenge active oxygen species, N-acetylcysteine, ascorbate and alpha-tocopherol, inhibited the increase. The generation of DNA single strand breaks has been proposed to be an important step in the pathway leading to the increase in p53 initiated by a variety of cytotoxic agents. In this study I show that compounds that allow the accumulation of DNA single strand breaks, ara c and hydroxyurea, enhanced the UVC radiation (254 nm)-dependent increase in p53, but had no effect on the solar UV radiation-dependent increase. Thus, while DNA single strand breaks are involved in the UVC radiation-dependent increase in p53, the increase caused by solar UV radiation occurs by an alternative mechanism involving active oxygen species.

  16. 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

  17. Increased expression of p53 enhances transcription-coupled repair and global genomic repair of a UVC-damaged reporter gene in human cells.

    PubMed

    Dregoesc, Diana; Rybak, Adrian P; Rainbow, Andrew J

    2007-05-01

    Ultraviolet (UV) light-induced DNA damage is repaired by nucleotide excision repair, which is divided into two sub-pathways: global genome repair (GGR) and transcription-coupled repair (TCR). While it is well established that the GGR pathway is dependent on the p53 tumour suppressor protein in human cells, both p53-dependent and p53-independent pathways have been reported for TCR. In the present work, we investigated the role of p53 in both GGR and TCR of a UVC-damaged reporter gene in human fibroblasts. We employed a non-replicating recombinant human adenovirus, AdCA17lacZ, that can efficiently infect human fibroblasts and express the beta-galactosidase (beta-gal) reporter gene under the control of the human cytomegalovirus promoter. We examined host cell reactivation (HCR) of beta-gal expression for the UVC-treated reporter construct in normal fibroblasts and in xeroderma pigmentosum (XP) and Cockayne syndrome (CS) fibroblasts deficient in GGR, TCR, or both. HCR was examined in fibroblasts that had been pre-infected with Ad5p53wt, which expresses wild-type p53, or a control adenovirus, AdCA18luc, which expresses the luciferase gene. We show that increased expression of p53 results in enhanced HCR of the UVC-damaged reporter gene in both untreated and UVC-treated cells for normal, CS-B (TCR-deficient), and XP-C (GGR-deficient), but not XP-A (TCR- and GGR-deficient) fibroblasts. These results indicate an involvement of p53 in both TCR and GGR of the UV-damaged reporter gene in human cells. PMID:17196445

  18. Ganoderma lucidum Polysaccharides Induce Macrophage-Like Differentiation in Human Leukemia THP-1 Cells via Caspase and p53 Activation.

    PubMed

    Hsu, Jia-Wei; Huang, Hsuan-Cheng; Chen, Shui-Tein; Wong, Chi-Huey; Juan, Hsueh-Fen

    2011-01-01

    Differentiation therapy by induction of tumor cells is an important method in the treatment of hematological cancers such as leukemia. Tumor cell differentiation ends cancer cells' immortality, thus stopping cell growth and proliferation. In our previous study, we found that fucose-containing polysaccharide fraction F3 extracted from Ganoderma lucidum can bring about cytokine secretion and cell death in human leukemia THP-1 cells. This prompted us to further investigate on how F3 induces the differentiation in human leukemia cells. We integrated time-course microarray analysis and network modeling to study the F3-induced effects on THP-1 cells. In addition, we determined the differentiation effect using Liu's staining, nitroblue tetrazolium (NBT) reduction assay, flow cytometer, western blotting and Q-PCR. We also examined the modulation and regulation by F3 during the differentiation process. Dynamic gene expression profiles showed that cell differentiation was induced in F3-treated THP-1 cells. Furthermore, F3-treated THP-1 cells exhibited enhanced macrophage differentiation, as demonstrated by changes in cell adherence, cell cycle arrest, NBT reduction and expression of differentiation markers including CD11b, CD14, CD68, matrix metalloproteinase-9 and myeloperoxidase. In addition, caspase cleavage and p53 activation were found to be significantly enhanced in F3-treated THP-1 cells. We unraveled the role of caspases and p53 in F3-induced THP-1 cells differentiation into macrophages. Our results provide a molecular explanation for the differentiation effect of F3 on human leukemia THP-1 cells and offer a prospect for a potential leukemia differentiation therapy.

  19. High-risk human papillomavirus infections and overexpression of p53 protein as prognostic indicators in transitional cell carcinoma of the urinary bladder.

    PubMed

    Furihata, M; Inoue, K; Ohtsuki, Y; Hashimoto, H; Terao, N; Fujita, Y

    1993-10-15

    Ninety Japanese patients with transitional cell carcinoma of the urinary bladder were investigated for tumor incorporation of DNA for high-risk human papillomavirus (HPV) types 16, 18, and 33 by in situ hybridization with biotinylated DNA probes. In addition, immunohistochemical analysis of p53 protein expression was performed with an antibody to p53 protein. Twenty-eight tumors were positive for HPV DNA, and multiple HPV infection was detected in 17 cases. Positive nuclear staining of cancer cells by the antibody to p53 protein was detected in 32 cases. DNA for HPV 16, 18, and/or 33 and the overexpression of p53 protein were simultaneously observed in 6 tumors by using a mirror section method. The overexpression of p53 protein was frequently detected in invasive and nonpapillary tumors (P < 0.05) and in high grade tumors (P < 0.05). In contrast, HPV infection was more common in noninvasive and papillary tumors (P < 0.01). The patients with tumors positive for HPV DNA and/or p53 antibody had a significantly worse survival rate (P < 0.05). These results suggest that HPV infection or overexpression of p53 protein may be related to tumor behavior and may indicate a relatively poor prognosis in patients with transitional cell carcinoma.

  20. 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

  1. 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

  2. Loss of transcription factor KLF5 in the context of p53 ablation drives invasive progression of human squamous cell cancer.

    PubMed

    Yang, Yizeng; Nakagawa, Hiroshi; Tetreault, Marie-Pier; Billig, Janelle; Victor, Noel; Goyal, Abha; Sepulveda, Antonia R; Katz, Jonathan P

    2011-10-15

    Squamous cell cancers account for more than half of all human cancers, and esophageal cancer is the sixth leading cause of cancer death worldwide. The majority of esophageal squamous cell carcinomas have identifiable p53 mutations, yet the same p53 mutations are found at comparable frequencies in precancerous dysplasia, indicating that transformation requires additional somatic changes yet to be defined. Here, we show that the zinc finger transcription factor Krüppel-like factor 5 (KLF5) transactivates NOTCH1 in the context of p53 mutation or loss. KLF5 loss limited NOTCH1 activity and was sufficient on its own to transform primary human keratinocytes harboring mutant p53, leading to the formation of invasive tumors. Restoration of NOTCH1 blocked transformation of KLF5-deficient and p53-mutant keratinocytes. Although human dysplastic epithelia accumulated KLF5, KLF5 expression was lost concurrently with NOTCH1 in squamous cell cancers. Taken together, these results define KLF5 loss as a critical event in squamous cell transformation and invasion. Our findings suggest that KLF5 may be a useful diagnostic and therapeutic target in esophageal squamous carcinomas and possibly more generally in other cancers associated with p53 loss of function.

  3. p53 controls global nucleotide excision repair of low levels of structurally diverse benzo(g)chrysene-DNA adducts in human fibroblasts.

    PubMed

    Lloyd, Daniel R; Hanawalt, Philip C

    2002-09-15

    Benzo(g)chrysene is a widespread environmental contaminant and potent carcinogen. We have measured the formation and nucleotide excision repair of covalent DNA adducts formed by the DNA-reactive metabolite of this compound in human fibroblasts, in which expression of the p53 tumor suppressor gene could be controlled by a tetracycline-inducible promoter. Cells were exposed for 1 h to 0.01, 0.1, or 1.2 microM (+/-)-anti-benzo(g)chrysene diol-epoxide, and DNA adducts were assessed at various post-treatment times by subjecting isolated DNA to (32)P-postlabeling analysis. Four major DNA adducts were detected, corresponding to the reaction of either the (+)- or (-)-anti-benzo(g)chrysene diol-epoxide stereoisomer with adenine or guanine. Treatment with 1.2 microM resulted in a level of 1100 total adducts/10(8) nucleotides for both p53-proficient and -deficient cells; removal of adducts was not observed in either case. In cells treated with 0.1 microM, the maximum level of total adducts at 24 h was 150/10(8) nucleotides in p53-proficient cells and 210 adducts/10(8) nucleotides in p53-deficient cells. A concentration of 0.01 microM resulted in a maximum of 20 adducts/10(8) nucleotides in p53-proficient cells at 4 h, but 40 adducts/10(8) nucleotides persisted in p53-deficient cells at 24 h. Whereas there were clear differences in the time course of adduct levels in p53-proficient compared with p53-deficient cells treated with 0.1 microM or 0.01 microM, these levels did not decrease extensively over 3 days. This is likely because of the stabilization of the diol-epoxide in cells, and consequent exposure and formation of adducts for many hours after the initial treatment. Furthermore, despite minor quantitative differences, all 4 of the adducts behaved similarly with respect to the effect of p53 expression on their removal. p53 appears to minimize the appearance of benzo(g)chrysene adducts in human cells by up-regulating global nucleotide excision repair and reducing the

  4. P-Glycoprotein/MDR1 regulates pokemon gene transcription through p53 expression in human breast cancer cells.

    PubMed

    He, Shengnan; Liu, Feng; Xie, Zhenhua; Zu, Xuyu; Xu, Wei; Jiang, Yuyang

    2010-01-01

    P-glycoprotein (Pgp), encoded by the multidrug resistance 1 (MDR1) gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of p53 as seen by use of p53 siRNA transfected MCF-7 cells or p53 mutated MDA-MB-231 cells. Moreover, p53 was detected to bind with Pgp in vivo using immunoprecipitation assay. Taken together, we conclude that Pgp can regulate the expression of pokemon through the presence of p53, suggesting that Pgp is a potent regulator and may offer an effective novel target for cancer therapy. PMID:20957096

  5. P-Glycoprotein/MDR1 regulates pokemon gene transcription through p53 expression in human breast cancer cells.

    PubMed

    He, Shengnan; Liu, Feng; Xie, Zhenhua; Zu, Xuyu; Xu, Wei; Jiang, Yuyang

    2010-01-01

    P-glycoprotein (Pgp), encoded by the multidrug resistance 1 (MDR1) gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of p53 as seen by use of p53 siRNA transfected MCF-7 cells or p53 mutated MDA-MB-231 cells. Moreover, p53 was detected to bind with Pgp in vivo using immunoprecipitation assay. Taken together, we conclude that Pgp can regulate the expression of pokemon through the presence of p53, suggesting that Pgp is a potent regulator and may offer an effective novel target for cancer therapy.

  6. Fangchinoline induces autophagic cell death via p53/sestrin2/AMPK signalling in human hepatocellular carcinoma cells

    PubMed Central

    Wang, Ning; Pan, Weidong; Zhu, Meifen; Zhang, Maosheng; Hao, Xiaojian; Liang, Guangyi; Feng, Yibin

    2011-01-01

    BACKGROUND AND PURPOSE Fangchinoline is a novel anti-tumour agent with little known of its cellular and molecular mechanisms of action. Here we have investigated the mode of cell death induced by fangchinoline and its underlying mechanism in two human hepatocellular carcinoma cell lines, HepG2 and PLC/PRF/5. EXPERIMENTAL APPROACH Apoptosis and autophagy were monitored in fangchinoline-treated HepG2 and PLC/PRF/5 cells by histological methods. The signal transduction pathways involved in activation of autophagy were examined, using immunoblotting, real-time PCR and siRNA techniques. KEY RESULTS Fangchinoline did not induce apoptosis in HepG2 and PLC/PRF/5 cells but triggered, dose-dependently, autophagy, an alternative mode of cell death which may contribute to fangchinoline's anti-tumour action. Nuclear translocation of p53 was involved in induction of autophagy by fangchinoline, followed by selective transactivation of the autophagy-related gene sestrin2 and initiation of the autophagic process. Signalling by the AMP-activated protein kinase was also involved as a downstream target of sestrin2 and induced mTOR-independent autophagic cell death in both cell lines. siRNA for Atg 5 or pharmacological block of p53 abolished fangchinoline-induced autophagy and inhibition of autophagy switched cell death to apoptosis in these cells, suggesting that cell death is irreversible once autophagy is induced by fangchinoline. CONCLUSIONS AND IMPLICATIONS Fangchinoline is a highly specific agent inducing autophagic cell death in hepatocellular carcinoma cells with a novel mechanism, which elucidates the potential of fangchinoline to potentiate programmed cell death in cancer cells. PMID:21418191

  7. Critical roles of DMP1 in human epidermal growth factor receptor 2/neu-Arf-p53 signaling and breast cancer development.

    PubMed

    Taneja, Pankaj; Maglic, Dejan; Kai, Fumitake; Sugiyama, Takayuki; Kendig, Robert D; Frazier, Donna P; Willingham, Mark C; Inoue, Kazushi

    2010-11-15

    Human epidermal growth factor receptor 2 (HER2) overexpression stimulates cell growth in p53-mutated cells while it inhibits cell proliferation in those with wild-type p53, but the molecular mechanism is unknown. The Dmp1 promoter was activated by HER2/neu through the phosphatidylinositol-3'-kinase-Akt-NF-κB pathway, which in turn stimulated Arf transcription. Binding of p65 and p52 subunits of NF-κB was shown to the Dmp1 promoter and that of Dmp1 to the Arf promoter on HER2/neu overexpression. Both Dmp1 and p53 were induced in premalignant lesions from mouse mammary tumor virus-neu mice, and mammary tumorigenesis was significantly accelerated in both Dmp1+/- and Dmp1-/- mice. Selective deletion of Dmp1 and/or overexpression of Tbx2/Pokemon was found in >50% of wild-type HER2/neu carcinomas, although the involvement of Arf, Mdm2, or p53 was rare. Tumors from Dmp1+/-, Dmp1-/-, and wild-type neu mice with hemizygous Dmp1 deletion showed significant downregulation of Arf and p21Cip1/WAF1, showing p53 inactivity and more aggressive phenotypes than tumors without Dmp1 deletion. Notably, endogenous hDMP1 mRNA decreased when HER2 was depleted in human breast cancer cells. Our study shows the pivotal roles of Dmp1 in HER2/neu-p53 signaling and breast carcinogenesis. PMID:21062982

  8. Critical roles of DMP1 in human epidermal growth factor receptor 2/neu-Arf-p53 signaling and breast cancer development.

    PubMed

    Taneja, Pankaj; Maglic, Dejan; Kai, Fumitake; Sugiyama, Takayuki; Kendig, Robert D; Frazier, Donna P; Willingham, Mark C; Inoue, Kazushi

    2010-11-15

    Human epidermal growth factor receptor 2 (HER2) overexpression stimulates cell growth in p53-mutated cells while it inhibits cell proliferation in those with wild-type p53, but the molecular mechanism is unknown. The Dmp1 promoter was activated by HER2/neu through the phosphatidylinositol-3'-kinase-Akt-NF-κB pathway, which in turn stimulated Arf transcription. Binding of p65 and p52 subunits of NF-κB was shown to the Dmp1 promoter and that of Dmp1 to the Arf promoter on HER2/neu overexpression. Both Dmp1 and p53 were induced in premalignant lesions from mouse mammary tumor virus-neu mice, and mammary tumorigenesis was significantly accelerated in both Dmp1+/- and Dmp1-/- mice. Selective deletion of Dmp1 and/or overexpression of Tbx2/Pokemon was found in >50% of wild-type HER2/neu carcinomas, although the involvement of Arf, Mdm2, or p53 was rare. Tumors from Dmp1+/-, Dmp1-/-, and wild-type neu mice with hemizygous Dmp1 deletion showed significant downregulation of Arf and p21Cip1/WAF1, showing p53 inactivity and more aggressive phenotypes than tumors without Dmp1 deletion. Notably, endogenous hDMP1 mRNA decreased when HER2 was depleted in human breast cancer cells. Our study shows the pivotal roles of Dmp1 in HER2/neu-p53 signaling and breast carcinogenesis.

  9. Association of Human Papilloma Virus 16 Infection and p53 Polymorphism among Tobacco using Oral Leukoplakia Patients: A Clinicopathologic and Genotypic Study

    PubMed Central

    Sikka, Seema; Sikka, Pranav

    2014-01-01

    Background: Human papillomavirus (HPV) and p53 alterations are speculated to play a role in carcinogenesis. This study was carried out to find out the association of HPV and p53 with precancerous lesions of the oral cavity such as leukoplakia: The objective of this study was to find the association among human papilloma virus (HPV) 16 infections and p53 polymorphism in tobacco using the oral leukoplakia patients. Methods: A total of 91 oral leukoplakia patients and 100 controls were randomly selected from the out-patient department of a tertiary care dental hospital of North-east India. Blood samples were drawn incisional biopsy was performed from the lesion proper and the tissue was processed for histopathological grading. Cytological smears were taken from the lesional site of leukoplakia patients and buccal mucosa of controls. The rate of HPV infection and p53 polymorphism was detected with the help of polymerase chain reaction, gel electrophoresis and deoxyribonucleic acid sequencing. Results: The rate of HPV 16 infection was found significantly high in the oral leukoplakia patients. No particular p53 genotype at exon 4 of codon 72 was found to be associated with oral leukoplakia, but “C” allele (proline) at exon 4 of codon 72 was significantly raised in these patients. Conclusions: Oral leukoplakia, a well-known pre-cancerous lesion, has been shown to be associated with tobacco, but certain other factors like HPV infection and p53 polymorphism may play an important role in its development. PMID:24829730

  10. Kinase requirements in human cells: V. Synthetic lethal interactions between p53 and the protein kinases SGK2 and PAK3.

    PubMed

    Baldwin, Amy; Grueneberg, Dorre A; Hellner, Karin; Sawyer, Jacqueline; Grace, Miranda; Li, Wenliang; Harlow, Ed; Munger, Karl

    2010-07-13

    Cervical carcinomas are initiated through a series of well-defined stages that rely on the expression of human papillomavirus (HPV) oncogenes. A panel of 100 small hairpin RNAs that target essential kinases in many tumor types was used to study the stepwise appearance of kinase requirements during cervical tumor development. Twenty-six kinases were commonly required in three cell lines derived from frank carcinomas, and each kinase requirement was traced to the specific stage in which the requirement emerged. Six kinases became required following HPV-induced immortalization, and the requirement for two kinases, SGK2 and PAK3, was mapped to the inactivation of p53 in primary human epithelial cells. Loss of the p53 tumor suppressor in other primary epithelial cells also induced dependence on SGK2 and PAK3. Hence, SGK2 and PAK3 provide important cellular functions following p53 inactivation, fulfilling the classical definition of synthetic lethality; loss of p53, SGK2, or PAK3 alone has little effect on cell viability, whereas loss of p53 together with either SGK2 or PAK3 loss leads to cell death. Whereas tumor suppressor gene mutations are not directly druggable, other proteins or pathways that become obligatory to cell viability following tumor suppressor loss provide theoretical targets for tumor suppressor-specific drug discovery efforts. The kinases SGK2 and PAK3 may thus represent such targets for p53-specific drug development.

  11. Subtype-selective induction of wild-type p53 and apoptosis, but not cell cycle arrest, by human somatostatin receptor 3.

    PubMed

    Sharma, K; Patel, Y C; Srikant, C B

    1996-12-01

    Somatostatin (SST) exerts direct antiproliferative effects in tumor cells, triggering either growth arrest or apoptosis. The cellular actions of SST are transduced through a family of five distinct somatostatin receptor subtypes (SSTR1-5). Whereas growth inhibition has been reported to follow stimulation of protein tyrosine phosphatase via SSTR2 or inhibition of Ca2+ channels via SSTR5 in heterologous expression systems, the subtype selectivity for signaling apoptosis has not been investigated. The tumor suppressor protein p53 and the protooncogene product c-Myc regulate cell cycle progression (growth factors present) or apoptosis (growth factors absent). The p53-induced G1 arrest requires induction of p21, an inhibitor of cyclin-dependent kinases, whereas apoptosis requires induction of Bax. c-Myc is capable of abrogating p53-induced G1 arrest by interfering with the inhibitory action of p21 on cyclin-dependent kinases. We have, therefore, investigated the regulation of p53, p21, c-Myc, and Bax and cellular apoptosis in relation to cell cycle progression in CHO-K1 cells stably expressing individual human SSTR1-5. We demonstrate that apoptosis is signaled uniquely through human SSTR3 and is associated with dephosphorylation-dependent conformational change in wild-type (wt) p53 as well as induction of Bax. The induction of wt p53 occurs rapidly and precedes the onset of apoptosis. We show that the increase in wt p53 is not associated with the induction of p21 or c-Myc when octreotide-induced apoptosis becomes evident, suggesting that such apoptosis does not require G1 arrest and is not c-Myc dependent. These findings provide the first evidence for hormonal induction of wt p53-associated apoptosis via G protein-coupled receptor in a subtype-selective manner.

  12. 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

  13. Paclitaxel/Taxol sensitivity in human renal cell carcinoma is not determined by the p53 status.

    PubMed

    Reinecke, Petra; Kalinski, Thomas; Mahotka, Csaba; Schmitz, Michael; Déjosez, Marion; Gabbert, Helmut Erich; Gerharz, Claus Dieter

    2005-05-26

    In this study, we analyzed the role of the p53 status for paclitaxel/Taxol sensitivity in renal cell carcinomas (RCCs) of the clear cell type. Using immunohistochemistry, nuclear p53 accumulation could not be correlated to the paclitaxel/Taxol sensitivity. DNA sequencing detected a p53 gene mutation in two out of eight RCC cell lines, i.e. in exon 8 (cell line clearCa-6), and in exon 9 (cell line clearCa-5). No correlation, however, was found between the p53 status of our RCC cell lines and their paclitaxel/Taxol sensitivity as indicated by the IC50 values. However, paclitaxel-induced growth inhibition in paclitaxel-sensitive RCC cell lines was accompanied by an increase in apoptosis, irrespective of their p53 status. Although CD95 up-regulation was observed in renal cell carcinoma with wild-type p53 upon paclitaxel treatment, paclitaxel-induced apoptosis itself is triggered independently from the CD95 system. In conclusion, the p53 status cannot predict paclitaxel/Taxol sensitivity in RCC cell lines of the clear cell type.

  14. Simulated sunlight and benzo[a]pyrene diol epoxide induced mutagenesis in the human p53 gene evaluated by the yeast functional assay: lack of correspondence to tumor mutation spectra.

    PubMed

    Yoon, Jung-Hoon; Lee, Chong-Soon; Pfeifer, Gerd P

    2003-01-01

    Many mutations in the p53 gene destroy the transcriptional transactivation function of the p53 protein. This function of p53 can be determined in a yeast assay using a p53 responsive reporter gene. The yeast assay could hold promise for the identification of mutagens implicated in human cancer if the p53 mutational spectra obtained with this assay would match human tumor mutation data. Ultraviolet (UV) light from the sun and polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, are strongly implicated in the spectrum of p53 mutations found in human non-melanoma skin cancers and smoking-associated lung cancers, respectively. We have used these two model mutagens to assess the feasibility of using the p53 yeast assay in cancer epidemiology. After treatment of CpG methylated p53 DNA with a solar UV simulator or with benzo[a]pyrene diol epoxide (BPDE), the modified p53 sequences were assayed in yeast for mutational outcome. As expected, BPDE produced predominantly G to T transversions and simulated sunlight produced mostly C to T transitions at dipyrimidine sites in the p53 coding sequence. However, the preferentially mutated p53 sequences (hotspots) in the yeast assay were completely different from those in the mutational spectra found in human lung and skin cancers. The data indicate that this assay is not a reliable measurement of p53 mutagenesis in human tissues and that, perhaps, transcriptional activation is not the primary function of p53 in tumor suppression. PMID:12538356

  15. Analysis of p53 gene mutations in human gliomas by polymerase chain reaction-based single-strand conformation polymorphism and DNA sequencing.

    PubMed

    Sarkar, F H; Kupsky, W J; Li, Y W; Sreepathi, P

    1994-03-01

    Mutations in the p53 gene have been recognized in brain tumors, and clonal expansion of p53 mutant cells has been shown to be associated with glioma progression. However, studies on the p53 gene have been limited by the need for frozen tissues. We have developed a method utilizing polymerase chain reaction (PCR) for the direct analysis of p53 mutation by single-strand conformation polymorphism (SSCP) and by direct DNA sequencing of the p53 gene using a single 10-microns paraffin-embedded tissue section. We applied this method to screen for p53 gene mutations in exons 5-8 in human gliomas utilizing paraffin-embedded tissues. Twenty paraffin blocks containing tumor were selected from surgical specimens from 17 different adult patients. Tumors included six anaplastic astrocytomas (AAs), nine glioblastomas (GBs), and two mixed malignant gliomas (MMGs). The tissue section on the stained glass slide was used to guide microdissection of an unstained adjacent tissue section to ensure > 90% of the tumor cell population for p53 mutational analysis. Simultaneously, microdissection of the tissue was also carried out to obtain normal tissue from adjacent areas as a control. Mutations in the p53 gene were identified in 3 of 17 (18%) patients by PCR-SSCP analysis and subsequently confirmed by PCR-based DNA sequencing. Mutations in exon 5 resulting in amino acid substitution were found in one thalamic AA (codon 158, CGC > CTT: Arg > Leu) and one cerebral hemispheric GB (codon 151, CCG > CTG: Pro > Leu).(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Quantitative phosphoproteomic analysis reveals γ-bisabolene inducing p53-mediated apoptosis of human oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2 activation.

    PubMed

    Jou, Yu-Jen; Chen, Chao-Jung; Liu, Yu-Ching; Way, Tzong-Der; Lai, Chih-Ho; Hua, Chun-Hung; Wang, Ching-Ying; Huang, Su-Hua; Kao, Jung-Yie; Lin, Cheng-Wen

    2015-10-01

    γ-Bisabolene, one of main components in cardamom, showed potent in vitro and in vivo anti-proliferative activities against human oral squamous cell carcinoma (OSCC). γ-Bisabolene activated caspases-3/9 and decreased mitochondrial memebrane potential, leading to apoptosis of OSCC cell lines (Ca9-22 and SAS), but not normal oral fibroblast cells. Phosphoproteome profiling of OSCC cells treated with γ-bisabolene was identified using TiO2-PDMS plate and LC-MS/MS, then confirmed using Western blotting and real-time RT-PCR assays. Phosphoproteome profiling revealed that γ-bisabolene increased the phosphorylation of ERK1/2, protein phosphatases 1 (PP1), and p53, as well as decreased the phosphorylation of histone deacetylase 2 (HDAC2) in the process of apoptosis induction. Protein-protein interaction network analysis proposed the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in γ-bisabolene-induced apoptosis. Subsequent assays indicated γ-bisabolene eliciting p53 acetylation that enhanced the expression of p53-regulated apoptotic genes. PP1 inhibitor-2 restored the status of HDAC2 phosphorylation, reducing p53 acetylation and PUMA mRNA expression in γ-bisabolene-treated Ca9-22 and SAS cells. Meanwhile, MEK and ERK inhibitors significantly decreased γ-bisabolene-induced PUMA expression in both cancer cell lines. Notably, the results ascertained the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in mitochondria-mediated apoptosis of γ-bisabolene-treated cells. This study demonstrated γ-bisabolene displaying potent anti-proliferative and apoptosis-inducing activities against OSCC in vitro and in vivo, elucidating molecular mechanisms of γ-bisabolene-induced apoptosis. The novel insight could be useful for developing anti-cancer drugs. PMID:26194454

  17. [RELATIONSHIP BETWEEN p53/p21/Rb AND MAPK SIGNALING PATHWAYS IN HUMAN ENDOMETRIUM-DERIVED STEM CELLS UNDER OXIDATIVE STRESS].

    PubMed

    Deryabin, P I; Borodkina, A V; Nikolsky, N N; Burova, E B

    2015-01-01

    Human endometrium-derived mesenchymal stem cells (hMESC) under the sublethal oxidative stress induced by H2O2 activate both p53/p21/Rb and p38MAPK/MAPKAPK-2 pathways that are responsible for the induction of hMESC premature senescence (Borodkina et al., 2014). However the mutual relations between p53/p21/Rb and MAPK signaling pathways, including ERK, p38 and JNK remain unexplored as yet. Here, we used the specific inhibitors--pifithrin-α (PFT), U0126, SB203580 and SP600125 to "switch off" one of the proteins in these cascades and to evaluate the functional status alterations of the rest proteins. Suppression each of the MAPK significantly increased the p53 phosphorylation levels, as well as p21 protein expression followed by Rb hypophosphorylation. On the other hand, PFT-induced p53 inhibition enhanced mostly the ERK1/2 activation compared with p38 and JNK. These results suppose the existence of the reciprocal negative regulation between p53- and MAPK-dependent signaling pathways. Analyzing the possible interactions among the members of the MAPK family, we showed that p38 and JNK can function as the ERK antagonists: JNK is capable to activate ERK, while p38 may block the ERK activation. Together, these results demonstrate complex links between different signaling cascades in stressed hMESC, implicating ERK, p38 and JNK in regulation of the premature senescence via p53/p21/Rb pathway.

  18. To analyze the concomitant expression of human papillomavirus-16 in the pathogenetic model of p53-dependant pathway in oral squamous cell carcinoma

    PubMed Central

    Rathore, Ajit Singh; Gulati, Nikita; Shetty, Devi Charan; Jain, Anshi

    2016-01-01

    Background: Oral squamous cell carcinoma (OSCC) accounts for 90% of all primary oral malignancies. Association between human papillomavirus (HPV) as a risk factor of cervical cancer is well known; there is a need to widen the knowledge for its role in oral cancer development. The viral E6 protein of HPV binds to p53 making it nonfunctional. Aims and Objective: To study mutated/wild type p53 expression using immunohistochemistry and detect HPV-16 presence using polymerase chain reaction (PCR), in OSCC and correlating their expression. Materials and Methods: Immunohistochemical staining for p53 molecule in 24 sections of OSCC followed by DNA extraction of the cases using qiagen extraction kit and subsequent HPV-16 detection using PCR technique. Statistical Analysis: The data were analysed using SPSS software version 19. Results: Out of 24 cases of OSCC, twenty cases were positive for P 53 expression and four cases were negative for P 53 expression. Out of the four negative cases, one case was detected positive for HPV-16. Conclusion: HPV infection along with p53 expression helps in understanding its exact pathogenesis which further helps in expanding our spectrum of therapeutic modalities PMID:27721595

  19. 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

  20. 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.

  1. Interferon-β Induces Cellular Senescence in Cutaneous Human Papilloma Virus-Transformed Human Keratinocytes by Affecting p53 Transactivating Activity

    PubMed Central

    Chiantore, Maria V.; Vannucchi, Serena; Accardi, Rosita; Tommasino, Massimo; Percario, Zulema A.; Vaccari, Gabriele; Affabris, Elisabetta; Fiorucci, Gianna; Romeo, Giovanna

    2012-01-01

    Interferon (IFN)-β inhibits cell proliferation and affects cell cycle in keratinocytes transformed by both mucosal high risk Human Papilloma Virus (HPV) and cutaneous HPV E6 and E7 proteins. In particular, upon longer IFN-β treatments, cutaneous HPV38 expressing cells undergo senescence. IFN-β appears to induce senescence by upregulating the expression of the tumor suppressor PML, a well known IFN-induced gene. Indeed, experiments in gene silencing via specific siRNAs have shown that PML is essential in the execution of the senescence programme and that both p53 and p21 pathways are involved. IFN-β treatment leads to a modulation of p53 phosphorylation and acetylation status and a reduction in the expression of the p53 dominant negative ΔNp73. These effects allow the recovery of p53 transactivating activity of target genes involved in the control of cell proliferation. Taken together, these studies suggest that signaling through the IFN pathway might play an important role in cellular senescence. This additional understanding of IFN antitumor action and mechanisms influencing tumor responsiveness or resistance appears useful in aiding further promising development of biomolecular strategies in the IFN therapy of cancer. PMID:22615843

  2. Disruption of the p53-mediated G{sub 1}/S cell cycle checkpoint results in elevated rates of spontaneous genetic recombination in human fibroblasts

    SciTech Connect

    Strasfeld, L.; Brainerd, E.; Meyn, M.S.

    1994-09-01

    A key feature of the cancer-prone inherited disease ataxia-telangiectasia (A-T) is genetic instability. We recently demonstrated that one aspect of genetic instability in A-T is a marked elevation in the spontaneous rates of intrachromosomal mitotic recombination. We have proposed a model for A-T that attributes these high recombination rates to a lack of DNA damage-sensitive cell cycle checkpoints. One prediction of this model is that disrupting p53 function in normal cells should increase their spontaneous rates of recombination by interfering with their p53-dependent G{sub 1}/S cell cycle checkpoint. To test this prediction, we transfected control and A-T fibroblast lines that each harbor a single integrated copy of lacZ-based recombination vector (pLrec) with derivatives of a eukaryotic expression vector (pRep5) that contain either a dominant-negative p53 mutant (143{sup val{yields}ala}) or a human papilloma virus E6 gene (HPV18 E6). Expression of either of these genes results in loss of p53 function and abolition of the G{sub 1}/S cell cycle checkpoint. Four independent p53{sup 143ala} transformants of the control line showed 25-80 fold elevations in spontaneous recombination rates when compared to their parent cell line. Elevations in spontaneous recombination rates were also detected following transfection with the HPV18 E6 gene. In contrast, four independent p53{sup 143ala} transformants of the A-T cell line showed no significant changes in their already high spontaneous recombination rates. We are now extending these observations to additional normal human fibroblast lines and carrying out molecular analyses of the products of these recombinational events. Our results support our hypothesis that the lack of a p53-dependent G{sub 1}/S cell cycle checkpoint contributes to the hyperrecombination seen in A-T.

  3. 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

  4. The G12 family proteins upregulate matrix metalloproteinase-2 via p53 leading to human breast cell invasion.

    PubMed

    Kim, Eun-Sook; Jeong, Jae-Boon; Kim, Seonhoe; Lee, Kyung-Min; Ko, Eunyoung; Noh, Dong-Young; Hwang, Ki-Tae; Ha, Ji Hee; Lee, Chang Ho; Kim, Sang Geon; Moon, Aree

    2010-11-01

    Although mounting evidence suggests a role for G(12) proteins, G(α12) and G(α13), in tumor progression, a direct role of G(12) proteins has not been determined. This study aims to elucidate the molecular mechanism for a tumorigenic and invasive potential of G(α12) and G(α13) in MCF10A human breast epithelial cells. Here, we report, for the first time, that G(α12) and G(α13) induce upregulation of matrix metalloproteinase (MMP)-2 leading to the invasive and migratory phenotypes in MCF10A cells. We further show that p53 is an important transcription factor for induction of MMP-2 transcriptional activation by G(α12/13). G(α12/13)-induced MMP-2 upregulation, invasion, and migration are dependent on the activation of Ras, Rac1, MKK3/6, p38, and Akt. Using human breast tissue samples, we demonstrate that the expression levels of G(α12) and MMP-2 are strongly correlated with the pathogenically diagnosed cancer (P < 0.0001). Moreover, the expression of G(α12) shows a strong correlation with that of MMP-2 in human breast cancer tissues, implicating the in vivo tumorigenic potential of G(α12). Taken together, this study elucidated the role of G(12) proteins in regulating processes for MMP-2 expression and malignant phenotypic conversion of MCF10A human breast epithelial cells, providing a molecular basis for the promoting role of G(α12) and G(α13) in breast cell invasion.

  5. 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

  6. 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

  7. Transcriptome analysis of human OXR1 depleted cells reveals its role in regulating the p53 signaling pathway

    PubMed Central

    Yang, Mingyi; Lin, Xiaolin; Rowe, Alexander; Rognes, Torbjørn; Eide, Lars; Bjørås, Magnar

    2015-01-01

    The oxidation resistance gene 1 (OXR1) is crucial for protecting against oxidative stress; however, its molecular function is unknown. We employed RNA sequencing to examine the role of human OXR1 for genome wide transcription regulation. In total, in non-treated and hydrogen peroxide exposed HeLa cells, OXR1 depletion resulted in down-regulation of 554 genes and up-regulation of 253 genes. These differentially expressed genes include transcription factors (i.e. HIF1A, SP6, E2F8 and TCF3), antioxidant genes (PRDX4, PTGS1 and CYGB) and numerous genes of the p53 signaling pathway involved in cell-cycle arrest (i.e. cyclin D, CDK6 and RPRM) and apoptosis (i.e. CytC and CASP9). We demonstrated that OXR1 depleted cells undergo cell cycle arrest in G2/M phase during oxidative stress and increase protein expression of the apoptosis initiator protease CASP9. In summary, OXR1 may act as a sensor of cellular oxidative stress to regulate the transcriptional networks required to detoxify reactive oxygen species and modulate cell cycle and apoptosis. PMID:26616534

  8. Sonoporation Delivery of Monoclonal Antibodies against Human Papillomavirus 16 E6 Restores p53 Expression in Transformed Cervical Keratinocytes

    PubMed Central

    Togtema, Melissa; Pichardo, Samuel; Jackson, Robert; Lambert, Paul F.; Curiel, Laura; Zehbe, Ingeborg

    2012-01-01

    High-risk types of human papillomavirus (HPV), such as HPV16, have been found in nearly all cases of cervical cancer. Therapies targeted at blocking the HPV16 E6 protein and its deleterious effects on the tumour suppressor pathways of the cell can reverse the malignant phenotype of affected keratinocytes while sparing uninfected cells. Through a strong interdisciplinary collaboration between engineering and biology, a novel, non-invasive intracellular delivery method for the HPV16 E6 antibody, F127-6G6, was developed. The method employs high intensity focused ultrasound (HIFU) in combination with microbubbles, in a process known as sonoporation. In this proof of principle study, it was first demonstrated that sonoporation antibody delivery into the HPV16 positive cervical carcinoma derived cell lines CaSki and SiHa was possible, using chemical transfection as a baseline for comparison. Delivery of the E6 antibody using sonoporation significantly restored p53 expression in these cells, indicating the antibody is able to enter the cells and remains active. This delivery method is targeted, non-cytotoxic, and non-invasive, making it more easily translatable for in vivo experiments than other transfection methods. PMID:23226365

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. 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

  11. 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

  12. CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53

    PubMed Central

    Saldaña-Meyer, Ricardo; González-Buendía, Edgar; Guerrero, Georgina; Narendra, Varun; Bonasio, Roberto; Recillas-Targa, Félix; Reinberg, Danny

    2014-01-01

    The multifunctional CCCTC-binding factor (CTCF) protein exhibits a broad range of functions, including that of insulator and higher-order chromatin organizer. We found that CTCF comprises a previously unrecognized region that is necessary and sufficient to bind RNA (RNA-binding region [RBR]) and is distinct from its DNA-binding domain. Depletion of cellular CTCF led to a decrease in not only levels of p53 mRNA, as expected, but also those of Wrap53 RNA, an antisense transcript originated from the p53 locus. PAR-CLIP-seq (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation [PAR-CLIP] combined with deep sequencing) analyses indicate that CTCF binds a multitude of transcripts genome-wide as well as to Wrap53 RNA. Apart from its established role at the p53 promoter, CTCF regulates p53 expression through its physical interaction with Wrap53 RNA. Cells harboring a CTCF mutant in its RBR exhibit a defective p53 response to DNA damage. Moreover, the RBR facilitates CTCF multimerization in an RNA-dependent manner, which may bear directly on its role in establishing higher-order chromatin structures in vivo. PMID:24696455

  13. Green tea extract reduces induction of p53 and apoptosis in UVB-irradiated human skin independent of transcriptional controls.

    PubMed

    Mnich, Christian D; Hoek, Keith S; Virkki, Leila V; Farkas, Arpad; Dudli, Christa; Laine, Elisabeth; Urosevic, Mirjana; Dummer, Reinhard

    2009-01-01

    Ultraviolet (UV) irradiation plays a pivotal role in human skin carcinongenesis. Preclinically, systemically and topically applied green tea extract (GTE) has shown reduction of UV-induced (i) erythema, (ii) DNA damage, (iii) formation of radical oxygen species and (iv) downregulation of numerous factors related to apoptosis, inflammation, differentiation and carcinogenesis. In humans, topical GTE has so far only been tested in limited studies, with usually very high GTE concentrations and over short periods of time. Both chemical stability of GTE and staining properties of highly concentrated green tea polyphenols limit the usability of highly concentrated green tea extracts in cosmetic products. The present study tested the utility of stabilized low-dose GTE as photochemopreventive agents under everyday conditions. We irradiated with up to 100 mJ/cm(2) of UVB light skin patches which were pretreated with either OM24-containing lotion or a placebo lotion. Biopsies were taken from both irradiated and un-irradiated skin for both immunohistochemistry and DNA microarray analysis. We found that while OM24 treatment did not significantly affect UV-induced erythema and thymidine dimer formation, OM24 treatment significantly reduced UV-induced p53 expression in keratinocytes. We also found that OM24 treatment significantly reduced the number of apoptotic keratinocytes (sunburn cells and TUNEL-positive cells). Carefully controlled DNA microarray analyses showed that OM24 treatment does not induce off-target changes in gene expression, reducing the likelihood of unwanted side-effects. Topical GTE (OM24) reduces UVB-mediated epithelial damage already at low, cosmetically usable concentrations, without tachyphylaxis over 5 weeks, suggesting GTE as suitable everyday photochemopreventive agents.

  14. Arsenite induced poly(ADP-ribosyl)ation of tumor suppressor P53 in human skin keratinocytes as a possible mechanism for carcinogenesis associated with arsenic exposure.

    PubMed

    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(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 microM) 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 microM 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.

  15. 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.

  16. c-myc, ras p21 and p53 expression in pleomorphic adenoma and its malignant form of the human salivary glands.

    PubMed

    Deguchi, H; Hamano, H; Hayashi, Y

    1993-01-01

    Using an immunohistochemical study and an immunoblot analysis, the expression of cellular oncogenes of the human salivary glands such as c-myc, ras p21, and p53 tumor-suppressor gene in pleomorphic adenomas and its malignant form, carcinoma in pleomorphic adenomas was examined to evaluate a differential biological significance, in comparison with that in normal salivary gland tissues. Immunohistochemically, the c-myc product was detected in 42% of the pleomorphic adenomas and in 56% of the carcinomas in pleomorphic adenoma. The ras p21 expression was observed in 24% of pleomorphic adenomas, and in 50% of carcinomas in pleomorphic adenoma. The p53 protein was detected in 18% of the pleomorphic adenomas and in 67% of the carcinomas in pleomorphic adenoma. Although there was no significant difference between the benign and malignant forms for the expression of c-myc, a statistical significance in ras p21 and p53 expression was found between the pleomorphic adenoma and its malignant form (P < 0.05) and P < 0.001, respectively). An immunoblotting assay clearly demonstrated the expression of c-myc and p53 gene products in both the benign and malignant forms of the pleomorphic adenoma, and that of ras p21 in the malignant form. These results indicate that activation of c-myc and ras p21 proto-oncogenes and the involvement of p53 mutation may play important roles in the malignant transformation of salivary gland pleomorphic adenoma.

  17. Uptake, p53 Pathway Activation, and Cytotoxic Responses for Co(II) and Ni(II) in Human Lung Cells: Implications for Carcinogenicity

    PubMed Central

    Luczak, Michal W.; Zhitkovich, Anatoly

    2013-01-01

    Cobalt(II) and nickel(II) ions display similar chemical properties and act as hypoxia mimics in cells. However, only soluble Co(II) but not soluble Ni(II) is carcinogenic by inhalation. To explore potential reasons for these differences, we examined responses of human lung cells to both metals. We found that Co(II) showed almost 8 times higher accumulation than Ni(II) in H460 cells but caused a less efficient activation of the transcriptional factor p53 as measured by its accumulation, Ser15 phosphorylation, and target gene expression. Unlike Ni(II), Co(II) was ineffective in downregulating the p53 inhibitor MDM4 (HDMX). Co(II)-treated cells continued DNA replication at internal doses that caused massive apoptosis by Ni(II). Apoptosis and the overall cell death by Co(II) were delayed and weaker than by Ni(II). Inhibition of caspases but not programmed necrosis pathways suppressed Co(II)-induced cell death. Knockdown of p53 produced 50%–60% decreases in activation of caspases 3/7 and expression of 2 most highly upregulated proapoptotic genes PUMA and NOXA by Co(II). Overall, p53-mediated apoptosis accounted for 55% cell death by Co(II), p53-independent apoptosis for 20%, and p53/caspase-independent mechanisms for 25%. Similar to H460, normal human lung fibroblasts and primary human bronchial epithelial cells had several times higher accumulation of Co(II) than Ni(II) and showed a delayed and weaker caspase activation by Co(II). Thus, carcinogenicity of soluble Co(II) could be related to high survival of metal-loaded cells, which permits accumulation of genetic and epigenetic abnormalities. High cytotoxicity of soluble Ni(II) causes early elimination of damaged cells and is expected to be cancer suppressive. PMID:24068677

  18. Determination of the allelic frequencies of an L-myc and a p53 polymorphism in human lung cancer.

    PubMed

    Weston, A; Ling-Cawley, H M; Caporaso, N E; Bowman, E D; Hoover, R N; Trump, B F; Harris, C C

    1994-04-01

    The L-myc and p53 genes have been implicated in lung cancer. Both of these genes have restriction fragment length polymorphisms (RFLPs) that could account for differential expression or activity of variant forms. An EcoRI restriction site in the L-myc gene was previously reported to be a predictor of poor prognosis in Japanese lung cancer patients. There are several RFLPs in the p53 gene. In exon 4 there is a polymorphism that codes for either an arginine or proline residue at codon 72. We previously reported the frequency of DNA-RFLPs at these gene loci revealed by EcoRI and AccII respectively. Here we report results from a study comparing lung cancer cases (n = 31) with chronic obstructive pulmonary disease controls (n = 49). No association was found between these RFLPs and disease status. Previous observations that the frequencies of these RFLPs varied by race were confirmed. The p53 arginine allele was found to be more common in Caucasians (0.71) than African-Americans (0.50). The EcoRI restriction site present allele in L-myc was more frequent in African-Americans (0.71) than Caucasians (0.49). Thus, the allelic frequency for L-myc was similar in African-Americans to that reported for Japanese, and the allelic frequency for p53 was similar in Caucasians to that reported for Japanese.

  19. Leptin reduces apoptosis triggered by high temperature in human placental villous explants: The role of the p53 pathway.

    PubMed

    Pérez-Pérez, Antonio; Toro, Ayelén R; Vilarino-Garcia, Teresa; Guadix, Pilar; Maymó, Julieta L; Dueñas, José L; Varone, Cecilia L; Sánchez-Margalet, Víctor

    2016-06-01

    Maternal fever is common during pregnancy and has for many years been suspected to harm the developing fetus. Whether increased maternal temperature produces exaggerated apoptosis in trophoblast cells remains unclear. Since p53 is a critical regulator of apoptosis we hypothesized that increased temperature in placenta produces abnormal expression of proteins in the p53 pathway and finally caspase-3 activation. Moreover, leptin, produced by placenta, is known to promote the proliferation and survival of trophoblastic cells. Thus, we aimed to study the possible role of leptin preventing apoptosis triggered by high temperature, as well as the molecular mechanisms underlying this effect. Fresh placental tissue was collected from normal pregnancies. Explants of placental villi were exposed to 37 °C, 40 °C and 42 °C during 3 h in the presence or absence of 10 nM leptin in DMEM-F12 medium. Western blotting and qRT-PCR was performed to analyze the expression of p53 and downstream effector, P53AIP1, Mdm2, p21, BAX and BCL-2 as well as the activated cleaved form of caspase-3 and the fragment of cytokeratin-18 (CK-18) cleaved at Asp396 (neoepitope M30). Phosphorylation of the Ser 46 residue on p53, the expression of P53AIP1, Mdm2, p21, as well as caspase-3 and CK-18 were significantly increased in explants at 40 °C and 42 °C. Conversely, these effects were significantly attenuated by leptin 10 nM at both 40 °C and 42 °C. The BCL2/BAX ratio was also significantly decreased in explants at 40 °C and 42 °C compared with explants incubated at 37 °C, which was prevented by leptin stimulation. These data illustrate the potential role of leptin for reducing apoptosis in trophoblast explants, including trophoblastic cells, triggered by high temperature, by preventing the activation of p53 signaling. PMID:27238720

  20. 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.

  1. 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.

  2. A symphony of regulations centered on p63 to control development of ectoderm-derived structures.

    PubMed

    Guerrini, Luisa; Costanzo, Antonio; Merlo, Giorgio R

    2011-01-01

    The p53-related transcription factor p63 is critically important for basic cellular functions during development of the ectoderm and derived structure and tissues, including skin, limb, palate, and hair. On the one side, p63 is required to sustain the proliferation of keratinocyte progenitors, while on the other side it is required for cell stratification, commitment to differentiate, cell adhesion, and epithelial-mesenchymal signaling. Molecules that are components or regulators of the p63 pathway(s) are rapidly being identified, and it comes with no surprise that alterations in the p63 pathway lead to congenital conditions in which the skin and other ectoderm-derived structures are affected. In this paper, we summarize the current knowledge of the molecular and cellular regulations centered on p63, derived from the comprehension of p63-linked human diseases and the corresponding animal models, as well as from cellular models and high-throughput molecular approaches. We point out common themes and features, that allow to speculate on the possible role of p63 downstream events and their potential exploitation in future attempts to correct the congenital defect in preclinical studies.

  3. 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.

  4. Gain of oncogenic function of p53 mutants induces invasive phenotypes in human breast cancer cells by silencing CCN5/WISP-2.

    PubMed

    Dhar, Gopal; Banerjee, Snigdha; Dhar, Kakali; Tawfik, Ossama; Mayo, Matthew S; Vanveldhuizen, Peter J; Banerjee, Sushanta K

    2008-06-15

    CCN5/WISP-2 is overexpressed in noninvasive breast cancer cells and tissue samples, whereas its expression is minimal or undetected in invasive conditions. CCN5/WISP-2 has been considered as an antiinvasive gene because CCN5/WISP-2 silencing augments the invasive phenotypes in vitro. However, the mechanism of silencing of CCN5 during the progression of the disease has been elusive. Because p53 mutations are associated with breast cancer progression and have been shown to correlate inversely with CCN5/WISP-2 expression in other cancer cell types, the objective of this study was to explore whether p53 mutants suppress CCN5 expression in breast tumor cells resulting in the progression of this disease. We found CCN5 expression is inversely correlated with the mutational activation of p53 in human breast tumor cells. The ectopic expression of p53 mutants in ER-positive noninvasive breast tumor cells silenced the CCN5/WISP-2 expression and enhanced invasive phenotypes, including the induction of morphologic changes from the epithelial-to-mesenchymal type along with the alterations of hallmark proteins of these cell types and an augmentation of the migration of these cells. The suppression of CCN5 by the p53 mutants can be nullified by estrogen signaling in these cells through the transcriptional activation of the CCN5 gene. Moreover, the invasive changes can be imitated by blocking the CCN5/WISP-2 expression through RNA interference or can be reversed by the addition of CCN5/WISP-2 recombinant protein in the culture. Thus, these studies suggest that CCN5 inactivation could be an essential molecular event for p53 mutant-induced invasive phenotypes.

  5. 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.

  6. Status quo of p53 in the treatment of tumors.

    PubMed

    Guan, Yong-Song; He, Qing; Zou, Qing

    2016-10-01

    The p53 gene is pivotal for oncogenesis in a combination of mutations in oncogenes and antioncogenes. The ubiquitous loss of the p53 pathway in human cancers has generated considerable interest in developing p53-targeted cancer therapies, but current ideas and approaches targeting p53 are conflicting. Current researches focus on cancer-selective drugs with therapeutic strategies that both activate and inhibit p53. As p53 is ubiquitously lost in human cancers, the strategy of exogenous p53 addition is reasonable. However, p53 acts not equally in all cell types; thus, individualized p53 therapy is the direction of future research. To clarify the controversies on p53 for improvement of future antitumor studies, the review focuses on the available technological protocols, including their advantages and limitations in terms of future therapeutic use of p53 in the management of tumors.

  7. A p53-like protein from a freshwater mollusc Lamellidens corrianus.

    PubMed

    Mohanty, B P

    2006-08-01

    p53 is the most frequently mutated protein in human cancers and the accumulation of its high levels is a potential novel marker for malignancy. Recently, its homologues such as p63 and p73 have been reported in human, mice and fish. Environmentally induced alterations in p53 protein have been reported to contribute to pathogenesis of leukemia in soft-shell clam Mya arenaria inhabiting polluted water, suggesting that p53 proteins can also be used as pollution markers. In the present study, the presence of p53 protein or its homologues was investigated in tissues of bivalve molluscs Lamellidens corrianus that are predominant in the freshwater riverine environment and are well suited to act as test organisms for evaluation of habitat degradation. The molluscs were collected live from the river Ganga at three sampling sites viz., Kanpur, Allahabad and Varanasi and different tissues (foot, gill and mantle) were collected. Proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). On immunoblot analysis, a 45 kDa protein (p45) was recognized by the monoclonal anti-p53 antibody in the molluscan tissues. The p45 showed immunoreactivity in all the three tissues of molluscs collected at Kanpur, in foot and gill tissues in those collected at Allahabad, and in foot tissue only, in those collected at Varanasi. Since monoclonal anti-p53 recognizes a denaturation-resistant epitope on the p53 (53 kDa) nuclear protein and does not react with other cellular proteins, the molluscan p45 is a p53-homologue or p53-like protein. Further, the differential expression of p45 in the different organs might serve as a useful biomarker that would help in establishing pollution gradient for environmental monitoring in the large aquatic ecosystems.

  8. A p53-like protein from a freshwater mollusc Lamellidens corrianus.

    PubMed

    Mohanty, B P

    2006-08-01

    p53 is the most frequently mutated protein in human cancers and the accumulation of its high levels is a potential novel marker for malignancy. Recently, its homologues such as p63 and p73 have been reported in human, mice and fish. Environmentally induced alterations in p53 protein have been reported to contribute to pathogenesis of leukemia in soft-shell clam Mya arenaria inhabiting polluted water, suggesting that p53 proteins can also be used as pollution markers. In the present study, the presence of p53 protein or its homologues was investigated in tissues of bivalve molluscs Lamellidens corrianus that are predominant in the freshwater riverine environment and are well suited to act as test organisms for evaluation of habitat degradation. The molluscs were collected live from the river Ganga at three sampling sites viz., Kanpur, Allahabad and Varanasi and different tissues (foot, gill and mantle) were collected. Proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). On immunoblot analysis, a 45 kDa protein (p45) was recognized by the monoclonal anti-p53 antibody in the molluscan tissues. The p45 showed immunoreactivity in all the three tissues of molluscs collected at Kanpur, in foot and gill tissues in those collected at Allahabad, and in foot tissue only, in those collected at Varanasi. Since monoclonal anti-p53 recognizes a denaturation-resistant epitope on the p53 (53 kDa) nuclear protein and does not react with other cellular proteins, the molluscan p45 is a p53-homologue or p53-like protein. Further, the differential expression of p45 in the different organs might serve as a useful biomarker that would help in establishing pollution gradient for environmental monitoring in the large aquatic ecosystems. PMID:17133770

  9. Maintaining epithelial stemness with p63.

    PubMed

    Melino, Gerry; Memmi, Elisa Maria; Pelicci, Pier Giuseppe; Bernassola, Francesca

    2015-07-28

    In stratified epithelial and glandular tissues, homeostasis relies on the self-renewing capacity of stem cells, which are within the basal layer. The p53 family member p63 is an indispensable transcription factor for epithelial morphogenesis and stemness. A splice variant of the transcription factor p63 that lacks an amino-terminal domain, ΔNp63, is selectively found in the basal compartments of several ectoderm-derived tissues such as stratified and glandular epithelia, in which it is required for the replenishment of stem cells. Thus far, the transcriptional programs downstream of p63 in stemness regulation remain incompletely defined. Unveiling the molecular basis of stem cell self-renewal may be relevant in understanding how this process may contribute to cancer development. In this review, we specifically highlight experimental investigations, which suggest that p63 is a marker of normal epithelial stem cells and describe p63 transcriptional targets that may be involved in stemness regulation. Finally, we discuss relevant findings implicating p63 in epithelial cancer stem cell biology. PMID:26221054

  10. 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

  11. 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. PMID:25490748

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. Noscapine Induced Apoptosis via Downregulation of Survivin in Human Neuroblastoma Cells Having Wild Type or Null p53

    PubMed Central

    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 Ser15-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. PMID:22848370

  17. 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

  18. 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

    2016-01-01

    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

  19. Cellular Stress and p53-Associated Apoptosis by Juniperus communis L. Berry Extract Treatment in the Human SH-SY5Y Neuroblastoma Cells.

    PubMed

    Lantto, Tiina A; Laakso, Into; Dorman, H J Damien; Mauriala, Timo; Hiltunen, Raimo; Kõks, Sulev; Raasmaja, Atso

    2016-07-13

    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.

  20. Aqueous extract of Curcuma aromatica induces apoptosis and G2/M arrest in human colon carcinoma LS-174-T cells independent of p53.

    PubMed

    Hu, Bing; Shen, Ke-Ping; An, Hong-Mei; Wu, Yang; Du, Qin

    2011-02-01

    Curcuma aromatica is a common Chinese herb for treating diseases with blood stasis and has been regarded as an anticancer herb in modern clinical practice. However, the anticancer effects and related molecular mechanisms of Curcuma aromatica remain unclear. In the present study, human colon carcinoma LS-174-T cell line with wild-type p53 was used as a model cell to evaluate the anticancer effects of aqueous extract of Curcuma aromatica (AECA). AECA inhibits LS-174-T cell proliferation in a dose- and time-dependent manner and colony formation in a dose-dependent manner. AECA treatment induces apoptosis accompanied by caspase-8, -9, and -3 activation in LS-174-T cells. Moreover, blocking the activities of these caspases with a specific inhibitor significantly protected LS-174-T cells from AECA-induced apoptosis. AECA treatment also induces G2/M phase arrest in LS-174-T cells. Expression of p53 was unchanged after AECA treatment; specific silence of p53 did not influence AECA-induced apoptosis and G2/M phase arrest. Further, the expression of cyclin B1 and CDK1 was reduced by AECA. This study suggests that AECA might be effective as an antiproliferative herb for colon carcinoma, the antitumor activity of AECA may involve both extrinsic and intrinsic apoptosis, and AECA induces G2/M phase arrest via downregulation of cyclin B1 and CDK1 and without the participation of p53.

  1. 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

  2. Signalling pathways involved in antitumoral effects of VIP in human renal cell carcinoma A498 cells: VIP induction of p53 expression.

    PubMed

    Vacas, Eva; Muñoz-Moreno, Laura; Fernández-Martínez, Ana B; Bajo, Ana M; Sánchez-Chapado, Manuel; Prieto, Juan C; Carmena, María J

    2014-08-01

    Vasoactive intestinal peptide (VIP) decreases cell proliferation through PI3K signalling and prevents tumour progression in clear renal cell carcinoma (RCC). Here we analyzed the signalling pathways that mediate such VIP effects by using human RCC A498 cells. The effects of treatment with 1 μM VIP and/or specific protein kinase inhibitors such as H89, Wortmannin and PD98059 were studied by cell adhesion assay, ELISA of VEGF165 and ROS production assays. Semiquantitative RT-PCR and western blot were performed to study p53 expression. VIP increased cell adhesion and ROS production, and decreased VEGF165 secretion through PI3K signalling. Moreover, VIP increased nuclear expression of tumour suppressor p53. VIP effects could be blocked by cell incubation with a specific p53 inhibitor, cyclin pifithrin-α hydrobromide (CPFT-αH). In conclusion, this study provides a p53-dependent mechanism by which VIP regulates cell proliferation in RCC development. It supports a potential usefulness of VIP in new therapies of RCC.

  3. 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. PMID:26718026

  4. 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

  5. USP11 regulates p53 stability by deubiquitinating p53*

    PubMed Central

    Ke, Jia-ying; Dai, Cong-jie; Wu, Wen-lin; Gao, Jin-hua; Xia, Ai-juan; Liu, Guang-ping; Lv, Kao-sheng; Wu, Chun-lin

    2014-01-01

    The p53 tumor suppressor protein coordinates the cellular responses to a broad range of cellular stresses, leading to DNA repair, cell cycle arrest or apoptosis. The stability of p53 is essential for its tumor suppressor function, which is tightly controlled by ubiquitin-dependent degradation primarily through its negative regulator murine double minute 2 (Mdm2). To better understand the regulation of p53, we tested the interaction between p53 and USP11 using co-immunoprecipitation. The results show that USP11, an ubiquitin-specific protease, forms specific complexes with p53 and stabilizes p53 by deubiquitinating it. Moreover, down-regulation of USP11 dramatically attenuated p53 induction in response to DNA damage stress. These findings reveal that USP11 is a novel regulator of p53, which is required for p53 activation in response to DNA damage. PMID:25471832

  6. p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

    PubMed Central

    Gurdziel, Katherine; Bell, George W.; Jacks, Tyler; Flores, Elsa R.

    2009-01-01

    The p53 family activates many of the same genes in response to DNA damage. Because p63 and p73 have structural differences from p53 and play distinct biological functions in development and metastasis, it is likely that they activate a unique transcriptional network. Therefore, we performed a genome-wide analysis using cells lacking the p53 family members after treatment with DNA damage. We identified over 100 genes involved in multiple pathways that were uniquely regulated by p63 or p73, and not p53. Further validation indicated that BRCA2, Rad51, and mre11 are direct transcriptional targets of p63 and p73. Additionally, cells deficient for p63 and p73 are impaired in DNA repair and p63+/−;p73+/− mice develop mammary tumors suggesting a novel mechanism whereby p63 and p73 suppress tumorigenesis. PMID:19816568

  7. 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.

  8. 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

    Alves da Costa, Cristine; Paitel, Erwan; Mattson, Mark P; Amson, Robert; Telerman, Adam; Ancolio, Karine; Checler, Frédéric; Mattson, Marc P

    2002-03-19

    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-alpha, 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-alpha 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

  9. The natural toxin juglone causes degradation of p53 and induces rapid H2AX phosphorylation and cell death in human fibroblasts

    SciTech Connect

    Paulsen, Michelle T.; Ljungman, Mats . E-mail: ljungman@umich.edu

    2005-11-15

    Juglone (5-hydroxy-1,4-naphtoquinone) is a natural toxin produced by walnut trees. In this study we show that juglone differentially reduces viability of human cells in culture. Normal fibroblast were found to be especially sensitive to juglone and lost viability primarily through a rapid apoptotic and necrotic response. This response may have been triggered by DNA damage since juglone induced a rapid and strong phosphorylation of H2AX in all phases of the cell cycle. Furthermore, juglone inhibits mRNA synthesis in human fibroblasts in a dose-dependent manner. Surprisingly, juglone caused a drastic reduction of the basal level of p53 in human fibroblasts and this loss could not be fully rescued by proteasome and calpain I inhibitors. However, when cells were pretreated with UV light or ionizing radiation, juglone was not able to reduce the cellular levels of activated p53. Our results show that juglone has multiple effects on cells such as the induction of DNA damage, inhibition of transcription, reduction of p53 protein levels and the induction of cell death.

  10. Analysis of p53 mutants for transcriptional activity.

    PubMed Central

    Raycroft, L; Schmidt, J R; Yoas, K; Hao, M M; Lozano, G

    1991-01-01

    The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GAL4. Human and mouse wild-type p53/GAL4 specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 proteins, including a mouse p53 mutant which is temperature sensitive for suppression, were also analyzed. A p53/GAL4 fusion protein with this mutation was also transcriptionally active only at the permissive temperature. Another mutant p53/GAL4 fusion protein analyzed mimics the mutation inherited in Li-Fraumeni patients. This fusion protein was as active as wild-type p53/GAL4 in our assay. Two human p53 mutants that arose from alterations of the p53 gene in colorectal carcinomas were 30- to 40-fold less effective at activating transcription than wild-type p53/GAL4 fusion proteins. Thus, functional wild-type p53/GAL4 fusion proteins activate transcription, while several transformation competent mutants do so poorly or not at all. Only one mutant p53/GAL4 fusion protein remained transcriptionally active. Images PMID:1944276

  11. Sedanolide induces autophagy through the PI3K, p53 and NF-κB signaling pathways in human liver cancer cells.

    PubMed

    Hsieh, Shu-Ling; Chen, Chi-Tsai; Wang, Jyh-Jye; Kuo, Yu-Hao; Li, Chien-Chun; Hsieh, Lan-Chi; Wu, Chih-Chung

    2015-12-01

    Sedanolide (SN), a phthalide-like compound from celery seed oil, possesses antioxidant effects. However, the effect of SN on cell death in human liver cancer cells has yet to be determined. In this study, cell viability determination, monodansylcadaverine (MDC) fluorescent staining and immunoblot analysis were performed to determine autophagy induction and autophagy-induced protein expression changes via molecular examination after human liver cancer (J5) cells were treated with SN. Our studies demonstrate that SN suppressed J5 cell viability by inducing autophagy. Phosphoinositide 3-kinase (PI3K)-I, mammalian target of rapamycin (mTOR) and Akt protein levels decreased, whereas PI3K-III, LC3-II and Beclin-1 protein levels increased following SN treatment in J5 cells. In addition, SN treatment upregulated nuclear p53 and damage-regulated autophagy modulator (DRAM) and downregulated cytosolic p53 and Tp53-induced glycolysis and apoptosis regulator (TIGAR) expression in J5 cells. Furthermore, the cytosolic phosphorylation of inhibitor of kappa B (IκB) and nuclear p65 and the DNA-binding activity of NF-κB increased after SN treatment. These results suggest that SN induces J5 cell autophagy by regulating PI3K, p53 and NF-κB autophagy-associated signaling pathways in J5 cells. PMID:26500073

  12. 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

  13. A new semisynthetic 1-O-acetyl-6-O-lauroylbritannilactone induces apoptosis of human laryngocarcinoma cells through p53-dependent pathway.

    PubMed

    Han, Yang-Yang; Tang, Jiang-Jiang; Gao, Rong-Fang; Guo, Xin; Lei, Ming; Gao, Jin-Ming

    2016-09-01

    Initiation of apoptosis is an important event for chemoprevention and chemotherapy of cancer. Naturally derived products had drawn growing attention as lead compounds for anticancer drug discovery. ABL-L, a semisynthetic analogue of natural sesquiterpenoid 1-O-acetylbritannilactone (ABL) isolated from Inula britannica, showed stronger suppression against three solid tumor cell lines with 4-10 fold improvement than ABL. However, its molecular mechanism of cell death induction has still not been determined. The present study evaluated the anticancer efficacy of ABL-L and its biological activities mechanism on human laryngocarcinoma cells HEp-2 in vitro. We found that ABL-L-induced inhibition of cell proliferation was associated with an increase in G1-phase arrest. Typical apoptotic morphological and biochemical features were also observed in treated cells. Furthermore, the levels of the anti-apoptotic Bcl-2, pro-caspase 3/8/9 and poly(ADP-ribose) polymerase PARP decreased, and the level of pro-apoptotic Bax increased. Involvement of the caspase-mediated apoptosis was confirmed using caspase inhibitor Z-VAD-FMK pretreatment. In addition, ABL-L induced a tumor suppressor p53 and its target genes expression p21, fas, noxa and puma. The results of p53 knockdown suggest that caspase-mediated apoptosis induced by ABL-L was in p53-dependent pathway on HEp-2 cells. Our data indicate that the cytotoxicity of the novel semisynthetic analogue ABL-L involved G1 cell cycle arrest and apoptosis via a p53-dependent, caspase-mediated pathway on human laryngocarcinoma cells. PMID:27262408

  14. Targeting Oncogenic Mutant p53 for Cancer Therapy

    PubMed Central

    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 mutant p53 levels. PMID:26732534

  15. Crosstalk between p53 and TGF-β Signalling

    PubMed Central

    Elston, Rebecca; Inman, Gareth J.

    2012-01-01

    Wild-type p53 and TGF-β are key tumour suppressors which regulate an array of cellular responses. TGF-β signals in part via the Smad signal transduction pathway. Wild-type p53 and Smads physically interact and coordinately induce transcription of a number of key tumour suppressive genes. Conversely mutant p53 generally subverts tumour suppressive TGF-β responses, diminishing transcriptional activation of key TGF-β target genes. Mutant p53 can also interact with Smads and this enables complex formation with the p53 family member p63 and blocks p63-mediated activation of metastasis suppressing genes to promote tumour progression. p53 and Smad function may also overlap during miRNA biogenesis as they can interact with the same components of the Drosha miRNA processing complex to promote maturation of specific subsets of miRNAs. This paper investigates the crosstalk between p53 and TGF-β signalling and the potential roles this plays in cancer biology. PMID:22545213

  16. 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.

  17. Human papillomavirus tumor-infiltrating T-regulatory lymphocytes and P53 codon 72 polymorphisms correlate with clinical staging and prognosis of oropharyngeal cancer.

    PubMed

    Rittà, Massimo; Landolfo, Vincenzo; Mazibrada, Jasenka; De Andrea, Marco; Dell'Oste, Valentina; Caneparo, Valeria; Peretti, Alberto; Giordano, Carlo; Pecorari, Giancarlo; Garzaro, Massimiliano; Landolfo, Santo

    2013-04-01

    The association between human papillomavirus (HPV) DNA positivity, p53 codon 72 polymorphisms, and the type of leukocyte infiltration in head and neck squamous cell carcinomas (HNSCC) and their combined impact upon patient survival is poorly investigated. For this reason, leukocyte infiltration profile and p53 codon 72 polymorphisms were assessed in freshly removed HNSCC specimens (N=71 patients). HPV detection was performed by nested-PCR followed by DNA sequencing. Viral loads were determined by quantitative RT-PCR. The choice to investigate fresh instead of archive paraffin-embedded specimens was privileged to avoid possible artifacts due to sample processing. HPV DNA was detected in 14% of cases. Oropharyngeal carcinomas were the most frequently associated with the presence of HPV16 DNA (41%) and were associated with p53 Pro/Pro or Pro/Arg polymorphisms. In HPV16-positive oropharyngeal carcinomas increased infiltrations of CD3+ and FoxP3+ T-cells correlated with higher HPV16 copy numbers. The presence of HPV may trigger a stronger immune response and may be considered a reliable marker for clinical staging and a more favorable prognosis of oropharyngeal carcinoma. PMID:23686119

  18. Treatment with a Small Synthetic Compound, KMU-193, induces Apoptosis in A549 Human Lung Carcinoma Cells through p53 Up-Regulation.

    PubMed

    Choi, Eun Young; Shin, Kyeong-Cheol; Lee, Jinho; Kwon, Taeg Kyu; Kim, Shin; Park, Jong-Wook

    2015-01-01

    Despite recent advances in therapeutic strategies for lung cancer, mortality still is increasing. In the present study, we investigated the anti-cancer effects of KMU-193, 2-(4-Ethoxy-phenyl)-N-{5-[2-fluoro-4-(4-methyl- piperazine-1-carbonyl)-phenylamino]-1H-indazol-3-yl}-acetamide in a human non-small cell lung cancer cell line A549. KMU-193 strongly inhibited the proliferation of A549 cells, but it did not have anti-proliferative effect in other types of cancer cell lines. KMU-193 further induced apoptosis in association with activation of caspase-3 and cleavage of PLC-γ1. However, KMU-193 had no apoptotic effect in untransformed cells such as TMCK-1 and BEAS-2B. Interestingly, pretreatment with z-VAD-fmk, a pan-caspase inhibitor, strongly abrogated KMU- 193-induced apoptosis. KMU-193 treatment enhanced the expression levels of p53 and PUMA. Importantly, p53 siRNA transfection attenuated KMU-193-induced apoptosis. Collectively, these results for the first time demonstrate that KMU-193 has strong apoptotic effects on A549 cells and these are largely mediated through caspase-3- and p53-dependent pathways. PMID:26320467

  19. 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.

  20. Cellular localization of NRF2 determines the self-renewal and osteogenic differentiation potential of human MSCs via the P53–SIRT1 axis

    PubMed Central

    Yoon, D S; Choi, Y; Lee, J W

    2016-01-01

    NRF2 (nuclear factor erythroid-derived 2-like 2) plays an important role in defense against oxidative stress at the cellular level. Recently, the roles of NRF2 in embryonic and adult stem cells have been reported, but its role in maintaining self-renewal and differentiation potential remains unknown. We studied the mechanisms of NRF2 action in mesenchymal stem cells (MSCs) derived from human bone marrow. We found that the cellular localization of NRF2 changed during prolonged cell passage and osteogenic differentiation. Blocking the nuclear import of NRF2 using ochratoxin A (OTA) induced the loss of the self-renewal and osteogenic potential of early-passage (EP) MSCs. Conversely, reinforcing the nuclear import of NRF2 using tert-butylhydroquinone (t-BHQ) improved the self-renewal capacity and maintained the differentiation potential in the osteogenic lineage of EP MSCs. Real-time quantitative PCR and western blot analysis showed that NRF2 positively regulates sirtuin 1 (SIRT1) at the mRNA and protein levels via the negative regulation of p53. The self-renewal and osteogenic potential suppressed in OTA-treated or NRF2-targeting small hairpin RNA (shRNA)-infected EP MSCs were rescued by introducing small interfering RNA (siRNA) targeting p53. t-BHQ treatment in late-passage (LP) MSCs, which lost their self-renewal and osteogenic potential, reversed these effects. In LP MSCs treated with t-BHQ for ∼7 days, the phosphorylation and nuclear localization of NRF2 improved and SIRT1 protein level increased, whereas p53 protein levels decreased. Therefore, our results suggest that NRF2 plays an important role in regulating p53 and SIRT1 to maintain MSC stemness. This study is the first to establish a functional link between NRF2 and SIRT1 expression in the maintenance of MSC self-renewal and differentiation potential. PMID:26866273

  1. Inhibitor of p53-p21 pathway induces the differentiation of human umbilical cord derived mesenchymal stem cells into cardiomyogenic cells.

    PubMed

    Ruan, Zhong-Bao; Zhu, Li; Yin, Yi-Gang; Chen, Ge-Cai

    2016-08-01

    P53 is shown recently to play an important role in the proliferation and differentiation of mesenchymal stem cells. In this study, human umbilical cord derived mesenchymal stem cells (hUCMSCs) were isolated and purified from the umbilical cords of normal or cesarean term deliveries, after treatment with 20 μmol/L PFT-α for 24 h, hUCMSCs were continued to be cultured for 4 weeks, cardiac-specific protein expression of cTnI, Desmin and Nkx2.5 was determined using immunofluorescence assay and RT-PCR. The expression of p53 and p21 was detected by western blot. Results showed that no expression of cTnI, Desmin or Nkx2.5 was observed in the control and the PFT-α group at 1 week after induction. However, after 4 weeks, while control group still had little expression of cTnI, Desmin and Nkx2.5, the PFT-α group demonstrated strong expression of cTnI, Desmin and Nkx2.5 (P < 0.001). At 4 weeks after induction, differentiation rate of cardiomyogenic cells in the PFT-α group (36.98 %) was significantly higher than that in the control group (4.41 %) (P < 0.01). Western blot analysis show that downregulation of p53 and p21 was seen in the PFT-α group at 4 weeks. The difference compared with the control group was statistically significant (P < 0.01). In conclusion, PFT-α can promote the differentiation of hUCMSCs into cardiomyogenic cells by modulating the p53-p21 pathway.

  2. MicroRNA-34a induces a senescence-like change via the down-regulation of SIRT1 and up-regulation of p53 protein in human esophageal squamous cancer cells with a wild-type p53 gene background.

    PubMed

    Ye, Zhimin; Fang, Jun; Dai, Shujun; Wang, Yuezhen; Fu, Zhenfu; Feng, Wei; Wei, Qichun; Huang, Pintong

    2016-01-28

    MiR-34a has been reported as a non-coding RNA universally expressed in normal old cells and a probable suppressor of diverse cancer cells; however, this miRNA's expression and anti-tumor mechanism in esophageal squamous cancer cells (ESCC) remains unclear. We explored these questions in three human ESCC lines, KYSE-450, KYSE-410, and ECa-109, with wild-type p53 and mutant p53 backgrounds. Through a specific stem-loop RT primer for miR-34a, we examined the relevant expression level of miR-34a in these three cell lines using real-time reverse transcription PCR (qRT-PCR). We found that the expression level of miR-34a induced by the DNA damage agent adrmycin (ADR) was both p53- and time-dependent. Following incubation with miR-34a, cellular growth inhibition was exhibited differently in the three cell lines harbored with different p53 backgrounds. Furthermore, the MTT assay demonstrated an miR-34a-related cytotoxic effect in cell growth. Senescence-associated β-galactosidase (SA-β-Gal) staining was used to examine senescence-like phenotypes induced by miR-34a. Mechanistic investigation suggested that the down-regulation of Sirtuin1 (SIRT1) and up-regulation of p53/p21 contributed to the anti-tumor mechanism of miR-34a in wild-type p53 ECa-109 cells, while neither of the apoptosis-related proteins PARP and caspase-3 caused significant changes. In summary, our findings indicated that the intrinsic expression of miR-34a was relatively low and was expressed differently among different p53 backgrounds and ADR treatment times. The anti-tumor effect of miR-34a was primarily dependent on the regulation of SIRT1 and p53/p21 protein, not apoptosis-associated proteins.

  3. Curcumin improves the paclitaxel-induced apoptosis of HPV-positive human cervical cancer cells via the NF-κB-p53-caspase-3 pathway

    PubMed Central

    DANG, YU-PING; YUAN, XIAO-YING; TIAN, RONG; LI, DONG-GUANG; LIU, WEI

    2015-01-01

    Paclitaxel, isolated from Taxus brevifolia, is considered to be an efficacious agent against a wide spectrum of human cancers, including human cervical cancer. However, dose-limiting toxicity and high cost limit its clinical application. Curcumin, a nontoxic food additive, has been reported to improve paclitaxel chemotherapy in mouse models of cervical cancer. However, the underlying mechanisms remain unclear. In this study, two human cervical cancer cell lines, CaSki [human papilloma virus (HPV)16-positive] and HeLa (HPV18-positive), were selected in which to investigate the effect of curcumin on the anticancer action of paclitaxel and further clarify the mechanisms. Flow cytometry and MTT analysis demonstrated that curcumin significantly promoted paclitaxel-induced apoptosis and cytotoxicity in the two cervical cell lines compared with that observed with paclitaxel alone (P<0.05). Reverse transcription-polymerase chain reaction indicated that the decline of HPV E6 and E7 gene expression induced by paclitaxel was also assisted by curcumin. The expression levels of p53 protein and cleaved caspase-3 were increased significantly in the curcumin plus paclitaxel-treated HeLa and CaSki cells compared with those in the cells treated with paclitaxel alone (P<0.01). Significant reductions in the levels of phosphorylation of IκBα and the p65-NF-κB subunit in CaSki cells treated with curcumin and paclitaxel were observed compared with those in cells treated with paclitaxel alone (P<0.05). This suggests that the combined effect of curcumin and paclitaxel was associated with the NF-κB-p53-caspase-3 pathway. In conclusion, curcumin has the ability to improve the paclitaxel-induced apoptosis of HPV-positive human cervical cancer cell lines via the NF-κB-p53-caspase-3 pathway. Curcumin in combination with paclitaxel may provide a superior therapeutic effect on human cervical cancer. PMID:25780454

  4. 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.

  5. p63 Inhibits Extravillous Trophoblast Migration and Maintains Cells in a Cytotrophoblast Stem Cell-Like State

    PubMed Central

    Li, Yingchun; Moretto-Zita, Matteo; Leon-Garcia, Sandra; Parast, Mana M.

    2015-01-01

    Proper differentiation of placental epithelial cells, called trophoblast, is required for implantation. Early during placentation, trophoblast cell columns help anchor the developing embryo in the uterine wall. Although proximally continuous with villous cytotrophoblast (CTB) distally, these cells differentiate into invasive extravillous trophoblast. We previously reported that p63, a p53 family member, is highly expressed in proliferative villous CTB and required for induction of the trophoblast lineage in human pluripotent stem cells. We now further explore its function in human trophoblast by using both primary CTB from the early placenta and established trophoblast cell lines. We show that p63 is expressed in epidermal growth factor receptor-positive CTB and that its expression decreases with differentiation into HLA-G+ extravillous trophoblast. In trophoblast cell lines, p63 is expressed in JEG3 cells but absent from HTR8 cells. Overexpression of p63 in both cell lines enhances cell proliferation and significantly reduces cell migration; conversely, down-regulation of p63 in JEG3 cells reduces cell proliferation and restores cell migration. Analysis of epithelial-to-mesenchymal transition, cell adhesion, and matrix degradation pathways shows that p63 blocks epithelial-to-mesenchymal transition, promotes a CTB-specific cell adhesion profile, and inhibits expression of matrix metalloproteinases. Taken together, these data show that p63 maintains the proliferative CTB state, at least partially through regulation of epithelial-to-mesenchymal transition, cell adhesion, and matrix degradation pathways. PMID:25307348

  6. P53 mutations in Ewing's sarcoma.

    PubMed

    Park, Y K; Chi, S G; Kim, Y W; Park, H R; Unni, K K

    2001-01-01

    The p53 tumor suppressor gene is one of the most frequently altered genes in human malignancies. To explore the implication of p53 alteration in Ewing's sarcoma, we analyzed the deletion and sequence alterations of p53 and abnormal amplification of MDM2, which acts as a functional inhibitor of p53, in 35 tissue specimens. Quantitative genomic PCR analysis showed that 2 of 35 tumors have extremely low levels of the p53 gene, indicating a homozygous deletion of the gene. Mutational analysis of exons 4 to 9 of p53 by PCR-SSCP revealed that 3 of 35 tumors carry sequence alterations in exons 5 or 8, and DNA sequencing analysis identified missense point mutations at codon 132 (AAG-->ATG, lysine-->methionine) and codon 135 (TGC-->TCC, cystein-->serine) in exon 5, and codon 287 (GAG-->GTG, glutamic acid-->valine) in exon 8 from these tumors. No abnormal amplification of the MDM2 gene was recognized. Taken together, our data demonstrate that p53 is genetically altered in a small fraction of Ewing's sarcoma.

  7. p63 expression in benign and malignant breast lesions.

    PubMed

    Stefanou, D; Batistatou, A; Nonni, A; Arkoumani, E; Agnantis, N J

    2004-04-01

    The p63 gene encodes six protein isoforms. The transactivating isoforms have similar actions with p53, while the N-isoforms inhibit transcription activation by p53 and transactivating isoforms. p63 is expressed in stratified epithelia and in basal cells of the prostate and salivary glands. In mammary epithelium p63 has been shown to be expressed only in the myoepithelial layer. In the present study we investigated the immunohistochemical expression of p63, in benign and malignant breast lesions, and compared it with known myoepithelial cell markers. Our material consisted of 140 benign and 126 malignant breast lesions. We used the antibodies anti-p63, anti-alpha-smooth muscle actin, anti-S-100 protein and anti-cytokeratin 14. In all benign lesions, p63 immunoreactivity was noted in the myoepithelial cell layer surrounding the luminal epithelial cells. A less continuous peripheral rim of myoepithelial cells was also highlighted with p63-staining in all situ carcinomas. All invasive breast carcinomas were devoided of peripheral p63 staining. Interestingly, strong nuclear p63 immunoreactivity was noted in a small fraction (5-15%) of epithelial cells in all cases of papillomatosis, in 62.5% of in situ ductal papillary-type carcinomas and in 33.3% of invasive papillary carcinomas. Comparable staining was observed with S-100. The stromal cells were unreactive to p63. Our findings suggest that p63 is a sensitive and specific myoepithelial marker, and may be included in immunohistochemical panels aiming to identify myoepithelial cells in problematic breast lesions. Regarding papillary neoplasms, it is possible that tumor cells acquire and exhibit at least in part a myoepithelial differentiation program.

  8. Simvastatin rises reactive oxygen species levels and induces senescence in human melanoma cells by activation of p53/p21 pathway

    SciTech Connect

    Guterres, Fernanda Augusta de Lima Barbosa; Martinez, Glaucia Regina; Rocha, Maria Eliane Merlin; Winnischofer, Sheila Maria Brochado

    2013-11-15

    Recent studies demonstrated that simvastatin has antitumor properties in several types of cancer cells, mainly by inducing apoptosis and inhibiting growth. The arrest of proliferation is a feature of cellular senescence; however, the occurrence of senescence in melanoma cells upon simvastatin treatment has not been investigated until now. Our results demonstrated that exposure of human metastatic melanoma cells (WM9) to simvastatin induces a senescent phenotype, characterized by G1 arrest, positive staining for senescence-associated β-galactosidase assay, and morphological changes. Also, the main pathways leading to cell senescence were examined in simvastatin-treated human melanoma cells, and the expression levels of phospho-p53 and p21 were upregulated by simvastatin, suggesting that cell cycle regulators and DNA damage pathways are involved in the onset of senescence. Since simvastatin can act as a pro-oxidant agent, and oxidative stress may be related to senescence, we measured the intracellular ROS levels in WM9 cells upon simvastatin treatment. Interestingly, we found an increased amount of intracellular ROS in these cells, which was accompanied by elevated expression of catalase and peroxiredoxin-1. Collectively, our results demonstrated that simvastatin can induce senescence in human melanoma cells by activation of p53/p21 pathway, and that oxidative stress may be related to this process. - Highlights: • Lower concentrations of simvastatin can induce senescent phenotype in melanoma cells. • Simvastatin induces senescence in human melanoma cells via p53/p21 pathway. • Senescent phenotype is related with increased intracellular ROS. • Partial detoxification of ROS by catalase/peroxiredoxin-1 could lead cells to senescence rather than apoptosis.

  9. Nucleolar stress with and without p53

    PubMed Central

    James, Allison; Wang, Yubo; Raje, Himanshu; Rosby, Raphyel; DiMario, Patrick

    2014-01-01

    A veritable explosion of primary research papers within the past 10 years focuses on nucleolar and ribosomal stress, and for good reason: with ribosome biosynthesis consuming ~80% of a cell’s energy, nearly all metabolic and signaling pathways lead ultimately to or from the nucleolus. We begin by describing p53 activation upon nucleolar stress resulting in cell cycle arrest or apoptosis. The significance of this mechanism cannot be understated, as oncologists are now inducing nucleolar stress strategically in cancer cells as a potential anti-cancer therapy. We also summarize the human ribosomopathies, syndromes in which ribosome biogenesis or function are impaired leading to birth defects or bone narrow failures; the perplexing problem in the ribosomopathies is why only certain cells are affected despite the fact that the causative mutation is systemic. We then describe p53-independent nucleolar stress, first in yeast which lacks p53, and then in other model metazoans that lack MDM2, the critical E3 ubiquitin ligase that normally inactivates p53. Do these presumably ancient p53-independent nucleolar stress pathways remain latent in human cells? If they still exist, can we use them to target >50% of known human cancers that lack functional p53? PMID:25482194

  10. Nucleolar stress with and without p53.

    PubMed

    James, Allison; Wang, Yubo; Raje, Himanshu; Rosby, Raphyel; DiMario, Patrick

    2014-01-01

    A veritable explosion of primary research papers within the past 10 years focuses on nucleolar and ribosomal stress, and for good reason: with ribosome biosynthesis consuming ~80% of a cell's energy, nearly all metabolic and signaling pathways lead ultimately to or from the nucleolus. We begin by describing p53 activation upon nucleolar stress resulting in cell cycle arrest or apoptosis. The significance of this mechanism cannot be understated, as oncologists are now inducing nucleolar stress strategically in cancer cells as a potential anti-cancer therapy. We also summarize the human ribosomopathies, syndromes in which ribosome biogenesis or function are impaired leading to birth defects or bone narrow failures; the perplexing problem in the ribosomopathies is why only certain cells are affected despite the fact that the causative mutation is systemic. We then describe p53-independent nucleolar stress, first in yeast which lacks p53, and then in other model metazoans that lack MDM2, the critical E3 ubiquitin ligase that normally inactivates p53. Do these presumably ancient p53-independent nucleolar stress pathways remain latent in human cells? If they still exist, can we use them to target >50% of known human cancers that lack functional p53? PMID:25482194

  11. AMPK/p53 Axis Is Essential for α-Lipoic Acid-Regulated Metastasis in Human and Mouse Colon Cancer Cells.

    PubMed

    Park, Sunmi; Choi, Seung Kug; Choi, Yura; Moon, Hyun-Seuk

    2015-10-01

    α-Lipoic acid (ALA) has an anticancer property of lung, cervix, and prostate cancer cells. However, direct evidence that ALA contributes to the development of colon cancer has not been fully elucidated. In addition, no previous studies have evaluated whether ALA may regulate malignant potential, such as adhesion, invasion, and colony formation of colon cancer cells. To address the aforementioned questions, we conducted in vitro ALA signaling studies using human (HT29) and mouse (MCA38) colon cancer cell lines. We observed that cell proliferation is reduced by ALA administration in a dose-dependent manner in human and mouse colon cancer cell lines. Specifically, 0.5 to 1 mM concentration of ALA significantly decreased cell proliferation when compared with control. Similarly, we found that ALA downregulates adhesion, invasion, and colony formation. Finally, we observed that ALA activates p53 and AMPK signaling pathways in human and mouse colon cancer cells. We found for the first time that ALA suppresses cell proliferation and malignant potential via p53 and AMPK signaling pathways in human and mouse colon cancer cells. These new and early mechanistic studies provide a causal role of ALA in colon cancer, suggesting that ALA might be a useful agent in the management or chemoprevention of colon cancer.

  12. Human amniotic epithelial cell niche enhances the functional properties of human corneal endothelial cells via inhibiting P53-survivin-mitochondria axis.

    PubMed

    Sha, Xiangyin; Liu, Zhiping; Song, Li; Wang, Zhonghao; Liang, Xuanwei

    2013-11-01

    The particular microenvironment or niche plays an important role in determining the fate of stem cells and adult cells. The objective of this study was to explore the potential role of the niche of human amniotic epithelial cells in enhancing the functional properties of human corneal endothelial cells (HCECs). The HCECs were cultured in different media, including corneal endothelium medium (CEM), 20% human amniotic epithelial cell culture medium (20% HAEC-Me), and 20% human amniotic epithelial cell-conditioned medium (20% HAEC-CM). We observed that the HCECs cultured in the 20% HAEC-CM had an increased proliferative capacity, higher colony-forming efficiency (CFE), fewer apoptotic cells, and similar cell-junction formation capabilities and pump functionality compared with primary HCECs. Compared with CEM and 20% HAEC-Me, the 20% HAEC-CM system enhanced the functional properties of HCECs by reducing the generation of reactive oxygen species (ROS), maintaining the membrane potential (Δψm) at higher levels, reducing the expression of the p53 protein, and increasing the level of survivin protein expression. This study may shed light on the expansion of HCECs and the clinical applications of these cells in regenerative medicine, especially in corneal tissue engineering.

  13. Discovery of Azurin-Like Anticancer Bacteriocins from Human Gut Microbiome through Homology Modeling and Molecular Docking against the Tumor Suppressor p53

    PubMed Central

    Nguyen, Chuong; Nguyen, Van Duy

    2016-01-01

    Azurin from Pseudomonas aeruginosa is known anticancer bacteriocin, which can specifically penetrate human cancer cells and induce apoptosis. We hypothesized that pathogenic and commensal bacteria with long term residence in human body can produce azurin-like bacteriocins as a weapon against the invasion of cancers. In our previous work, putative bacteriocins have been screened from complete genomes of 66 dominant bacteria species in human gut microbiota and subsequently characterized by subjecting them as functional annotation algorithms with azurin as control. We have qualitatively predicted 14 putative bacteriocins that possessed functional properties very similar to those of azurin. In this work, we perform a number of quantitative and structure-based analyses including hydrophobic percentage calculation, structural modeling, and molecular docking study of bacteriocins of interest against protein p53, a cancer target. Finally, we have identified 8 putative bacteriocins that bind p53 in a same manner as p28-azurin and azurin, in which 3 peptides (p1seq16, p2seq20, and p3seq24) shared with our previous study and 5 novel ones (p1seq09, p2seq05, p2seq08, p3seq02, and p3seq17) discovered in the first time. These bacteriocins are suggested for further in vitro tests in different neoplastic line cells. PMID:27239476

  14. The combination of 5-fluorouracil plus p53 pathway restoration is associated with depletion of p53-deficient or mutant p53-expressing putative colon cancer stem cells.

    PubMed

    Huang, Catherine; Zhang, Xiang M; Tavaluc, Raluca T; Hart, Lori S; Dicker, David T; Wang, Wenge; El-Deiry, Wafik S

    2009-11-01

    The cancer stem cell hypothesis suggests that rare populations of tumor-initiating cells may be resistant to therapy, lead to tumor relapse and contribute to poor prognosis for cancer patients. We previously demonstrated the feasibility of p53 pathway restoration in p53-deficient tumor cell populations using small molecules including ellipticine or its derivatives. We now establish a single cell p53-regulated green fluorescent protein (EGFP)-reporter system in human DLD1 colon tumor cells expressing mutant p53 protein. We use these p53-EGFP reporter DLD1 cells to investigate the status of p53 transcriptional activity in putative colon cancer stem cell populations following exposure to p53 pathway-restoring drugs and/or classical chemotherapy. We demonstrate induction of p53-specific EGFP reporter fluorescence following overexpression of p53 family member p73 by an Adenovirus vector. We further show that p53-reporter activity is induced in DLD1 putative cancer stem cell side-populations analyzed by their Hoechst dye efflux properties following treatment with the p53 pathway restoring drug ellipticine. Combination of ellipticine with the cytotoxic agent 5-fluorouracil resulted in increased cytotoxicity as compared to either agent alone and this was associated with depletion of putative cancer stem cell populations as compared with 5-FU alone treatment. Our results support the feasibility of therapeutic targeting of mutant p53 in putative cancer stem cells as well as the potential to enhance cytotoxic chemotherapy. PMID:19923910

  15. 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. PMID:26181229

  16. Cochinchina momordica seed extract induces apoptosis and cell cycle arrest in human gastric cancer cells via PARP and p53 signal pathways.

    PubMed

    Liu, Hong-Rui; Meng, Lin-Yi; Lin, Zhi-Yan; Shen, Yang; Yu, Yun-Qiu; Zhu, Yi-Zhun

    2012-01-01

    Cochinchina momordica seed is the dried ripe seed of Momordica cochinchinensis (Lour.) Spreng, which is a kind of fruit and consumed for dietary as well as medicinal uses. In this study, using the human SGC7901 and MKN-28 gastric cancer cell lines, we explored the anticancer activity of the extract from cochinchina momordica seed (ECMS). ECMS inhibited significantly the survival rates of SGC7901 and MKN-28 cells in concentration- and time-dependent manners by MTT assay. The typical apoptotic morphological changes were observed by Hoechst 33258 dye assay after SGC7901 and MKN-28 cells were treated with ECMS for 48 h. Flow cytometry analysis revealed that ECMS-treatment blocked the cells at the S phase of cell cycle. Furthermore, the protein expression levels of poly (ADP-ribose) polymerase (PARP) and Bcl-2 were downregulated notably by ECMS-treatment, whereas those of Fas/Fas-associated death domain, p53, and Bax were upregulated in SGC7901 cells. ECMS dramatically enhanced the enzymatic activities of caspase-3 and caspase-9 whilst slightly increased caspase-8 activity. Taken together, this study demonstrated that ECMS exerted cytotoxic activities via PARP and p53 signal pathways in the human gastric cancer cells. PMID:23020228

  17. SIRT1 alleviates senescence of degenerative human intervertebral disc cartilage endo-plate cells via the p53/p21 pathway

    PubMed Central

    Zhou, Nian; Lin, Xin; Dong, Wen; Huang, Wei; Jiang, Wei; Lin, Liangbo; Qiu, Quanhe; Zhang, Xiaojun; Shen, Jieliang; Song, Zhaojun; Liang, Xi; Hao, Jie; Wang, Dawu; Hu, Zhenming

    2016-01-01

    Cartilage end plates (CEP) degeneration plays an integral role in intervertebral disc (IVD) degeneration resulting from nutrient diffusion disorders. Although cell senescence resulting from oxidative stress is known to contribute to degeneration, no studies concerning the role of senescence in CEP degeneration have been conducted. SIRT1 is a longevity gene that plays a pivotal role in many cellular functions, including cell senescence. Therefore, the aim of this study was to investigate whether senescence is more prominent in human degenerative CEP and whether SIRT1-regulated CEP cells senescence in degenerative IVD as well as identify the signaling pathways that control that cell fate decision. In this study, the cell senescence phenotype was found to be more prominent in the CEP cells obtained from disc degenerative disease (DDD) patients than in the CEP cells obtained from age-matched lumbar vertebral fractures (LVF) patients. In addition, the results indicated that p53/p21 pathway plays an important role in the senescence of CEP cells in vivo and vitro. Furthermore, SIRT1 was found to be capable of alleviating the oxidative stress-induced senescence of CEP cells in humans via p53/p21 pathway. Thus, the information presented in this study could be used to further investigate the underlying mechanisms of CEP. PMID:26940203

  18. Mutational analysis of the N-ras, p53, p16INK4a, CDK4, and MC1R genes in human congenital melanocytic naevi.

    PubMed

    Papp, T; Pemsel, H; Zimmermann, R; Bastrop, R; Weiss, D G; Schiffmann, D

    1999-08-01

    Eighteen human congenital melanocytic naevi (CMN) from 17 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 and for sequence variants in the MC1R gene by combined RFLP-PCR/SSCP analysis. In addition, all lesions were screened for deletions and point mutations in the tumour suppressor genes p53 and p16INK4a (CDKN2A) by combined multiplex PCR/SSCP analysis. Positive screening data were specified by sequencing of the corresponding PCR product. Activating point mutations in the N-ras gene (nine CAA (Gln) to AAA (Lys) transversions and one CAA (Gln) to CGA (Arg) transition at codon 61) were detected at high frequency (56%). Furthermore, three missense mutations (V92M) and two silent mutations (CGA (Arg) to CGG (Arg), codon 213, exon 6) were found in the MC1R and p53 genes, respectively. No mutations were found in p16 or CDK4. The activated N-ras oncogene, which is also found in human cutaneous melanomas, may constitute a potential risk factor for melanoma formation within CMN.

  19. Cochinchina momordica seed extract induces apoptosis and cell cycle arrest in human gastric cancer cells via PARP and p53 signal pathways.

    PubMed

    Liu, Hong-Rui; Meng, Lin-Yi; Lin, Zhi-Yan; Shen, Yang; Yu, Yun-Qiu; Zhu, Yi-Zhun

    2012-01-01

    Cochinchina momordica seed is the dried ripe seed of Momordica cochinchinensis (Lour.) Spreng, which is a kind of fruit and consumed for dietary as well as medicinal uses. In this study, using the human SGC7901 and MKN-28 gastric cancer cell lines, we explored the anticancer activity of the extract from cochinchina momordica seed (ECMS). ECMS inhibited significantly the survival rates of SGC7901 and MKN-28 cells in concentration- and time-dependent manners by MTT assay. The typical apoptotic morphological changes were observed by Hoechst 33258 dye assay after SGC7901 and MKN-28 cells were treated with ECMS for 48 h. Flow cytometry analysis revealed that ECMS-treatment blocked the cells at the S phase of cell cycle. Furthermore, the protein expression levels of poly (ADP-ribose) polymerase (PARP) and Bcl-2 were downregulated notably by ECMS-treatment, whereas those of Fas/Fas-associated death domain, p53, and Bax were upregulated in SGC7901 cells. ECMS dramatically enhanced the enzymatic activities of caspase-3 and caspase-9 whilst slightly increased caspase-8 activity. Taken together, this study demonstrated that ECMS exerted cytotoxic activities via PARP and p53 signal pathways in the human gastric cancer cells.

  20. Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide.

    PubMed

    Röpke, M; Hald, J; Guldberg, P; Zeuthen, J; Nørgaard, L; Fugger, L; Svejgaard, A; Van der Burg, S; Nijman, H W; Melief, C J; Claesson, M H

    1996-12-10

    A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant p53 genes, in a L9V/HLA-A2 specific and restricted fashion. Thus, the normal tolerance against endogenously processed p53 protein-derived self-epitopes can be broken by peptide-specific in vitro priming. p53 protein-derived wild-type peptides might thus represent tumor associated target molecules for immunotherapeutical approaches. PMID:8962118

  1. The p53 circuit board

    PubMed Central

    Sullivan, Kelly D.; Gallant-Behm, Corrie L.; Henry, Ryan E.; Fraikin, Jean-Luc; Espinosa, Joaquín M.

    2012-01-01

    The p53 tumor suppressor is embedded in a large gene network controlling diverse cellular and organismal phenotypes. Multiple signaling pathways converge onto p53 activation, mostly by relieving the inhibitory effects of its repressors, MDM2 and MDM4. In turn, signals originating from increased p53 activity diverge into distinct effector pathways to deliver a specific cellular response to the activating stimuli. Much attention has been devoted to dissecting how the various input pathways trigger p53 activation and how the activity of the p53 protein itself can be modulated by a plethora of co-factors and post-translational modifications. In this review we will focus instead on the multiple configurations of the effector pathways. We will discuss how p53-generated signals are transmitted, amplified, resisted and eventually integrated by downstream gene circuits operating at the transcriptional, post-transcriptional and post-translational level. We will also discuss how context-dependent variations in these gene circuits define the cellular response to p53 activation and how they may impact the clinical efficacy of p53-based targeted therapies. PMID:22333261

  2. Aggregation tendencies in the p53 family are modulated by backbone hydrogen bonds

    PubMed Central

    Cino, Elio A.; Soares, Iaci N.; Pedrote, Murilo M.; de Oliveira, Guilherme A. P.; Silva, Jerson L.

    2016-01-01

    The p53 family of proteins is comprised of p53, p63 and p73. Because the p53 DNA binding domain (DBD) is naturally unstable and possesses an amyloidogenic sequence, it is prone to form amyloid fibrils, causing loss of functions. To develop p53 therapies, it is necessary to understand the molecular basis of p53 instability and aggregation. Light scattering, thioflavin T (ThT) and high hydrostatic pressure (HHP) assays showed that p53 DBD aggregates faster and to a greater extent than p63 and p73 DBDs, and was more susceptible to denaturation. The aggregation tendencies of p53, p63, and p73 DBDs were strongly correlated with their thermal stabilities. Molecular Dynamics (MD) simulations indicated specific regions of structural heterogeneity unique to p53, which may be promoted by elevated incidence of exposed backbone hydrogen bonds (BHBs). The results indicate regions of structural vulnerability in the p53 DBD, suggesting new targetable sites for modulating p53 stability and aggregation, a potential approach to cancer therapy. PMID:27600721

  3. Aggregation tendencies in the p53 family are modulated by backbone hydrogen bonds

    NASA Astrophysics Data System (ADS)

    Cino, Elio A.; Soares, Iaci N.; Pedrote, Murilo M.; de Oliveira, Guilherme A. P.; Silva, Jerson L.

    2016-09-01

    The p53 family of proteins is comprised of p53, p63 and p73. Because the p53 DNA binding domain (DBD) is naturally unstable and possesses an amyloidogenic sequence, it is prone to form amyloid fibrils, causing loss of functions. To develop p53 therapies, it is necessary to understand the molecular basis of p53 instability and aggregation. Light scattering, thioflavin T (ThT) and high hydrostatic pressure (HHP) assays showed that p53 DBD aggregates faster and to a greater extent than p63 and p73 DBDs, and was more susceptible to denaturation. The aggregation tendencies of p53, p63, and p73 DBDs were strongly correlated with their thermal stabilities. Molecular Dynamics (MD) simulations indicated specific regions of structural heterogeneity unique to p53, which may be promoted by elevated incidence of exposed backbone hydrogen bonds (BHBs). The results indicate regions of structural vulnerability in the p53 DBD, suggesting new targetable sites for modulating p53 stability and aggregation, a potential approach to cancer therapy.

  4. Aggregation tendencies in the p53 family are modulated by backbone hydrogen bonds.

    PubMed

    Cino, Elio A; Soares, Iaci N; Pedrote, Murilo M; de Oliveira, Guilherme A P; Silva, Jerson L

    2016-01-01

    The p53 family of proteins is comprised of p53, p63 and p73. Because the p53 DNA binding domain (DBD) is naturally unstable and possesses an amyloidogenic sequence, it is prone to form amyloid fibrils, causing loss of functions. To develop p53 therapies, it is necessary to understand the molecular basis of p53 instability and aggregation. Light scattering, thioflavin T (ThT) and high hydrostatic pressure (HHP) assays showed that p53 DBD aggregates faster and to a greater extent than p63 and p73 DBDs, and was more susceptible to denaturation. The aggregation tendencies of p53, p63, and p73 DBDs were strongly correlated with their thermal stabilities. Molecular Dynamics (MD) simulations indicated specific regions of structural heterogeneity unique to p53, which may be promoted by elevated incidence of exposed backbone hydrogen bonds (BHBs). The results indicate regions of structural vulnerability in the p53 DBD, suggesting new targetable sites for modulating p53 stability and aggregation, a potential approach to cancer therapy. PMID:27600721

  5. Mutant p53: One, No One, and One Hundred Thousand

    PubMed Central

    Walerych, Dawid; Lisek, Kamil; Del Sal, Giannino

    2015-01-01

    Encoded by the mutated variants of the TP53 tumor suppressor gene, mutant p53 proteins are getting an increased experimental support as active oncoproteins promoting tumor growth and metastasis. p53 missense mutant proteins are losing their wild-type tumor suppressor activity and acquire oncogenic potential, possessing diverse transforming abilities in cell and mouse models. Whether various mutant p53s differ in their oncogenic potential has been a matter of debate. Recent discoveries are starting to uncover the existence of mutant p53 downstream programs that are common to different mutant p53 variants. In this review, we discuss a number of studies on mutant p53, underlining the advantages and disadvantages of alternative experimental approaches that have been used to describe the numerous mutant p53 gain-of-function activities. Therapeutic possibilities are also discussed, taking into account targeting either individual or multiple mutant p53 proteins in human cancer. PMID:26734571

  6. Induction of wild-type p53, Bax, and acidic endonuclease during somatostatin-signaled apoptosis in MCF-7 human breast cancer cells.

    PubMed

    Sharma, K; Srikant, C B

    1998-04-13

    Somatostatin (SST) analogs inhibit tumor cell growth by exerting direct anti-proliferative effects with cytostatic (growth arrest) or cytotoxic (apoptosis) consequences. The SST analog SMS 201-995 (octreotide, OCT) inhibits growth of MCF-7 human breast adenocarcinoma cells, which express multiple SSTRs. Its action has been reported to result in either apoptosis or growth arrest, but the underlying mechanisms have not been elucidated in this tumor cell model. Here, we report that OCT elicits cytotoxic response in these cells, leading to apoptosis, which is associated with a rapid, time-dependent induction of wild-type p53 and an increase in Bax. There was no G1 cell-cycle arrest in these cells during OCT treatment as suggested by the decrease in G1/S ratio and the lack of induction of pRb and p21. Additionally, we demonstrate that OCT-induced DNA fragmentation in this cell line is due to selective activation of a cation-insensitive acidic endonuclease. Our data provide a rationale for utilizing SST analogs to treat SSTR-positive breast cancer cells expressing wild-type p53.

  7. Wild-type and mutant p53 differentially regulate NADPH oxidase 4 in TGF-β-mediated migration of human lung and breast epithelial cells

    PubMed Central

    Boudreau, H E; Casterline, B W; Burke, D J; Leto, T L

    2014-01-01

    Background: Transforming growth factor-beta (TGF-β) induces the epithelial-to-mesenchymal transition (EMT) leading to increased cell plasticity at the onset of cancer cell invasion and metastasis. Mechanisms involved in TGF-β-mediated EMT and cell motility are unclear. Recent studies showed that p53 affects TGF-β/SMAD3-mediated signalling, cell migration, and tumorigenesis. We previously demonstrated that Nox4, a Nox family NADPH oxidase, is a TGF-β/SMAD3-inducible source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. Our present study investigates the involvement of p53 in TGF-β-regulated Nox4 expression and cell migration. Methods: We investigated the effect of wild-type p53 (WT-p53) and mutant p53 proteins on TGF-β-regulated Nox4 expression and cell migration. Nox4 mRNA and protein, ROS production, cell migration, and focal adhesion kinase (FAK) activation were examined in three different cell models based on their p53 mutational status. H1299, a p53-null lung epithelial cell line, was used for heterologous expression of WT-p53 or mutant p53. In contrast, functional studies using siRNA-mediated knockdown of endogenous p53 were conducted in MDA-MB-231 metastatic breast epithelial cells that express p53-R280K and MCF-10A normal breast cells that have WT-p53. Results: We found that WT-p53 is a potent suppressor of TGF-β-induced Nox4, ROS production, and cell migration in p53-null lung epithelial (H1299) cells. In contrast, tumour-associated mutant p53 proteins (R175H or R280K) caused enhanced Nox4 expression and cell migration in both TGF-β-dependent and TGF-β-independent pathways. Moreover, knockdown of endogenous mutant p53 (R280K) in TGF-β-treated MDA-MB-231 metastatic breast epithelial cells resulted in decreased Nox4 protein and reduced phosphorylation of FAK, a key regulator of cell motility. Expression of WT-p53 or dominant-negative Nox4

  8. Effects of Chronic Ochratoxin A Exposure on p53 Heterozygous and p53 Homozygous Mice.

    PubMed

    Bondy, Genevieve S; Caldwell, Donald S; Aziz, Syed A; Coady, Laurie C; Armstrong, Cheryl L; Curran, Ivan H A; Koffman, Robyn L; Kapal, Kamla; Lefebvre, David E; Mehta, Rekha

    2015-07-01

    Exposure to the mycotoxin ochratoxin A (OTA) causes nephropathy in domestic animals and rodents and renal tumors in rodents and poultry. Humans are exposed to OTA by consuming foods made with contaminated cereal grains and other commodities. Management of human health risks due to OTA exposure depends, in part, on establishing a mode of action (MOA) for OTA carcinogenesis. To further investigate OTA's MOA, p53 heterozygous (p53+/-) and p53 homozygous (p53+/+) mice were exposed to OTA in diet for 26 weeks. The former are susceptible to tumorigenesis upon chronic exposure to genotoxic carcinogens. OTA-induced renal damage but no tumors were observed in either strain, indicating that p53 heterozygosity conferred little additional sensitivity to OTA. Renal changes included dose-dependent increases in cellular proliferation, apoptosis, karyomegaly, and tubular degeneration in proximal tubules, which were consistent with ochratoxicosis. The lowest observed effect level for renal changes in p53+/- and p53+/+ mice was 200 μg OTA/kg bw/day. Based on the lack of tumors and the severity of renal and body weight changes at a maximum tolerated dose, the results were interpreted as suggestive of a primarily nongenotoxic (epigenetic) MOA for OTA carcinogenesis in this mouse model.

  9. p53 genes function to restrain mobile elements.

    PubMed

    Wylie, Annika; Jones, Amanda E; D'Brot, Alejandro; Lu, Wan-Jin; Kurtz, Paula; Moran, John V; Rakheja, Dinesh; Chen, Kenneth S; Hammer, Robert E; Comerford, Sarah A; Amatruda, James F; Abrams, John M

    2016-01-01

    Throughout the animal kingdom, p53 genes govern stress response networks by specifying adaptive transcriptional responses. The human member of this gene family is mutated in most cancers, but precisely how p53 functions to mediate tumor suppression is not well understood. Using Drosophila and zebrafish models, we show that p53 restricts retrotransposon activity and genetically interacts with components of the piRNA (piwi-interacting RNA) pathway. Furthermore, transposon eruptions occurring in the p53(-) germline were incited by meiotic recombination, and transcripts produced from these mobile elements accumulated in the germ plasm. In gene complementation studies, normal human p53 alleles suppressed transposons, but mutant p53 alleles from cancer patients could not. Consistent with these observations, we also found patterns of unrestrained retrotransposons in p53-driven mouse and human cancers. Furthermore, p53 status correlated with repressive chromatin marks in the 5' sequence of a synthetic LINE-1 element. Together, these observations indicate that ancestral functions of p53 operate through conserved mechanisms to contain retrotransposons. Since human p53 mutants are disabled for this activity, our findings raise the possibility that p53 mitigates oncogenic disease in part by restricting transposon mobility.

  10. p53 genes function to restrain mobile elements.

    PubMed

    Wylie, Annika; Jones, Amanda E; D'Brot, Alejandro; Lu, Wan-Jin; Kurtz, Paula; Moran, John V; Rakheja, Dinesh; Chen, Kenneth S; Hammer, Robert E; Comerford, Sarah A; Amatruda, James F; Abrams, John M

    2016-01-01

    Throughout the animal kingdom, p53 genes govern stress response networks by specifying adaptive transcriptional responses. The human member of this gene family is mutated in most cancers, but precisely how p53 functions to mediate tumor suppression is not well understood. Using Drosophila and zebrafish models, we show that p53 restricts retrotransposon activity and genetically interacts with components of the piRNA (piwi-interacting RNA) pathway. Furthermore, transposon eruptions occurring in the p53(-) germline were incited by meiotic recombination, and transcripts produced from these mobile elements accumulated in the germ plasm. In gene complementation studies, normal human p53 alleles suppressed transposons, but mutant p53 alleles from cancer patients could not. Consistent with these observations, we also found patterns of unrestrained retrotransposons in p53-driven mouse and human cancers. Furthermore, p53 status correlated with repressive chromatin marks in the 5' sequence of a synthetic LINE-1 element. Together, these observations indicate that ancestral functions of p53 operate through conserved mechanisms to contain retrotransposons. Since human p53 mutants are disabled for this activity, our findings raise the possibility that p53 mitigates oncogenic disease in part by restricting transposon mobility. PMID:26701264

  11. p53 genes function to restrain mobile elements

    PubMed Central

    Wylie, Annika; Jones, Amanda E.; D'Brot, Alejandro; Lu, Wan-Jin; Kurtz, Paula; Moran, John V.; Rakheja, Dinesh; Chen, Kenneth S.; Hammer, Robert E.; Comerford, Sarah A.; Amatruda, James F.; Abrams, John M.

    2016-01-01

    Throughout the animal kingdom, p53 genes govern stress response networks by specifying adaptive transcriptional responses. The human member of this gene family is mutated in most cancers, but precisely how p53 functions to mediate tumor suppression is not well understood. Using Drosophila and zebrafish models, we show that p53 restricts retrotransposon activity and genetically interacts with components of the piRNA (piwi-interacting RNA) pathway. Furthermore, transposon eruptions occurring in the p53− germline were incited by meiotic recombination, and transcripts produced from these mobile elements accumulated in the germ plasm. In gene complementation studies, normal human p53 alleles suppressed transposons, but mutant p53 alleles from cancer patients could not. Consistent with these observations, we also found patterns of unrestrained retrotransposons in p53-driven mouse and human cancers. Furthermore, p53 status correlated with repressive chromatin marks in the 5′ sequence of a synthetic LINE-1 element. Together, these observations indicate that ancestral functions of p53 operate through conserved mechanisms to contain retrotransposons. Since human p53 mutants are disabled for this activity, our findings raise the possibility that p53 mitigates oncogenic disease in part by restricting transposon mobility. PMID:26701264

  12. Cysteine-Conjugated Metabolites of Ginger Components, Shogaols, Induce Apoptosis through Oxidative Stress-Mediated p53 Pathway in Human Colon Cancer Cells

    PubMed Central

    2015-01-01

    Shogaols, the major constituents of thermally processed ginger, have been proven to be highly effective anticancer agents. Our group has identified cysteine-conjugated shogaols (M2, M2′, and M2″) as the major metabolites of [6]-, [8]-, and [10]-shogaol in human and found that M2 is a carrier of its parent molecule [6]-shogaol in cancer cells and in mice, while being less toxic to normal colon fibroblast cells. The objectives of this study are to determine whether M2′ and M2″ behave in a similar manner to M2, in both metabolism and efficacy as anticancer agents, and to further explore the biological pro-apoptotic mechanisms of the cysteine-conjugated shogaols against human colon cancer cells HCT-116 and HT-29. Our results show that [8]- and [10]-shogaol have similar metabolic profiles to [6]-shogaol and exhibit similar toxicity toward human colon cancer cells. M2′ and M2″ both show low toxicity against normal colon cells but retain potency against colon cancer cells, suggesting that they have similar activity to M2. We further demonstrate that the cysteine-conjugated shogaols can cause cancer cell death through the activation of the mitochondrial apoptotic pathway. Our results show that oxidative stress activates a p53 pathway that ultimately leads to p53 up-regulated modulator of apoptosis (PUMA) induction and down-regulation of B-cell lymphoma 2 (Bcl-2), followed by cytochrome c release, perturbation of inhibitory interactions of X-linked inhibitor of apoptosis protein (XIAP) with caspases, and finally caspase 9 and 3 activation and cleavage. A brief screen of the markers attenuated by the proapoptotic activity of M2 revealed similar results for [8]- and [10]-shogaol and their respective cysteine-conjugated metabolites M2′ and M2″. This study highlights the cysteine-conjugated metabolites of shogaols as novel dietary colon cancer preventive agents. PMID:24786146

  13. Transcriptional activation of p21(WAF¹/CIP¹) is mediated by increased DNA binding activity and increased interaction between p53 and Sp1 via phosphorylation during replicative senescence of human embryonic fibroblasts.

    PubMed

    Kim, Hyun-Seok; Heo, Jee-In; Park, Seong-Hoon; Shin, Jong-Yeon; Kang, Hong-Jun; Kim, Min-Ju; Kim, Sung Chan; Kim, Jaebong; Park, Jae-Bong; Lee, Jae-Yong

    2014-01-01

    Although p21(WAF1/CIP1) is known to be elevated during replicative senescence of human embryonic fibroblasts (HEFs), the mechanism for p21 up-regulation has not been elucidated clearly. In order to explore the mechanism, we analyzed expression of p21 mRNA and protein and luciferase activity of full-length p21 promoter. The result demonstrated that p21 up-regulation was accomplished largely at transcription level. The promoter assay using serially-deleted p21 promoter constructs revealed that p53 binding site was the most important site and Sp1 binding sites were necessary but not sufficient for transcriptional activation of p21. In addition, p53 protein was shown to interact with Sp1 protein. The interaction was increased in aged fibroblasts and was regulated by phosphorylation of p53 and Sp1. DNA binding activity of p53 was significantly elevated in aged fibroblasts but that of Sp1 was not. DNA binding activities of p53 and Sp1 were also regulated by phosphorylation. Phosphorylation of p53 at serine-15 and of Sp1 at serines appears to be involved. Taken together, the result demonstrated that p21 transcription during replicative senescence of HEFs is up-regulated by increase in DNA binding activity and interaction between p53 and Sp1 via phosphorylation.

  14. p53 mutations increase resistance to ionizing radiation

    SciTech Connect

    Lee, J.M. ); Bernstein, A. Univ. of Toronto, Ontario )

    1993-06-15

    Mouse and human tumors of diverse origin frequently have somatically acquired mutations or rearrangements of the p53 gene, or they have lost one or both copies of the gene. Although wild-type p53 protein is believed to function as a tumor-suppressor gene, it is as yet unclear how p53 mutations lead to neoplastic development. Wild-type p53 has been postulated to play a role in DNA repair, suggesting that expression of mutant forms of p53 might alter cellular resistance to the DNA damage caused by [gamma] radiation. Moreover, p53 is thought to function as a cell cycle checkpoint after irradiation, also suggesting that mutant p53 might change the cellular proliferative response to radiation. The authors have used transgenic mice expressing one of two mutant alleles of p53 to test this prediction. Their results show that expression of both mutant variants of the mouse p53 gene significantly increases the cellular resistance of a variety of hematopoietic cell lineages to [gamma] radiation. These observations provide direct evidence that p53 mutations affect the cellular response to DNA damage, either by increasing DNA repair processes or, possibly, by increasing cellular tolerance to DNA damage. The association of p53 mutations with increased radioresistance suggests possible mechanisms through which alterations in the p53 gene might lead to oncogenic transformation. 53 refs., 5 figs.

  15. Tumor suppressor p53 protects mice against Listeria monocytogenes infection.

    PubMed

    Wang, Shaohui; Liu, Pingping; Wei, Jianchao; Zhu, Zixiang; Shi, Zixue; Shao, Donghua; Ma, Zhiyong

    2016-01-01

    Tumor suppressor p53 is involved in regulating immune responses, which contribute to antitumor and antiviral activity. However, whether p53 has anti-bacterial functions remains unclear. Listeria monocytogenes (LM) causes listeriosis in humans and animals, and it is a powerful model for studying innate and adaptive immunity. In the present study, we illustrate an important regulatory role of p53 during LM infection. p53 knockout (p53KO) mice were more susceptible to LM infection, which was manifested by a shorter survival time and lower survival rate. p53KO mice showed significant impairments in LM eradication. Knockdown of p53 in RAW264.7 and HeLa cells resulted in increased invasion and intracellular survival of LM. Furthermore, the invasion and intracellular survival of LM was inhibited in p53-overexpressing RAW264.7 and HeLa cells. LM-infected p53KO mice exhibited severe clinical symptoms and organ injury, presumably because of the abnormal production of the pro-inflammatory cytokines TNF-α, IL-6, IL-12, and IL-18. Decreased IFN-γ and GBP1 productions were observed in LM-infected p53-deficient mice or cells. The combination of these defects likely resulted in the overwhelming LM infection in the p53KO mice. These observations indicate that p53 serves as an important regulator of the host innate immune that protects against LM infection. PMID:27644341

  16. Tumor suppressor p53 protects mice against Listeria monocytogenes infection

    PubMed Central

    Wang, Shaohui; Liu, Pingping; Wei, Jianchao; Zhu, Zixiang; Shi, Zixue; Shao, Donghua; Ma, Zhiyong

    2016-01-01

    Tumor suppressor p53 is involved in regulating immune responses, which contribute to antitumor and antiviral activity. However, whether p53 has anti-bacterial functions remains unclear. Listeria monocytogenes (LM) causes listeriosis in humans and animals, and it is a powerful model for studying innate and adaptive immunity. In the present study, we illustrate an important regulatory role of p53 during LM infection. p53 knockout (p53KO) mice were more susceptible to LM infection, which was manifested by a shorter survival time and lower survival rate. p53KO mice showed significant impairments in LM eradication. Knockdown of p53 in RAW264.7 and HeLa cells resulted in increased invasion and intracellular survival of LM. Furthermore, the invasion and intracellular survival of LM was inhibited in p53-overexpressing RAW264.7 and HeLa cells. LM-infected p53KO mice exhibited severe clinical symptoms and organ injury, presumably because of the abnormal production of the pro-inflammatory cytokines TNF-α, IL-6, IL-12, and IL-18. Decreased IFN-γ and GBP1 productions were observed in LM-infected p53-deficient mice or cells. The combination of these defects likely resulted in the overwhelming LM infection in the p53KO mice. These observations indicate that p53 serves as an important regulator of the host innate immune that protects against LM infection. PMID:27644341

  17. Tumor suppressor p53 protects mice against Listeria monocytogenes infection.

    PubMed

    Wang, Shaohui; Liu, Pingping; Wei, Jianchao; Zhu, Zixiang; Shi, Zixue; Shao, Donghua; Ma, Zhiyong

    2016-01-01

    Tumor suppressor p53 is involved in regulating immune responses, which contribute to antitumor and antiviral activity. However, whether p53 has anti-bacterial functions remains unclear. Listeria monocytogenes (LM) causes listeriosis in humans and animals, and it is a powerful model for studying innate and adaptive immunity. In the present study, we illustrate an important regulatory role of p53 during LM infection. p53 knockout (p53KO) mice were more susceptible to LM infection, which was manifested by a shorter survival time and lower survival rate. p53KO mice showed significant impairments in LM eradication. Knockdown of p53 in RAW264.7 and HeLa cells resulted in increased invasion and intracellular survival of LM. Furthermore, the invasion and intracellular survival of LM was inhibited in p53-overexpressing RAW264.7 and HeLa cells. LM-infected p53KO mice exhibited severe clinical symptoms and organ injury, presumably because of the abnormal production of the pro-inflammatory cytokines TNF-α, IL-6, IL-12, and IL-18. Decreased IFN-γ and GBP1 productions were observed in LM-infected p53-deficient mice or cells. The combination of these defects likely resulted in the overwhelming LM infection in the p53KO mice. These observations indicate that p53 serves as an important regulator of the host innate immune that protects against LM infection.

  18. Effect of pH on the Interaction of Gold Nanoparticles with DNA and Application in the Detection of Human p53 Gene Mutation

    NASA Astrophysics Data System (ADS)

    Sun, Liping; Zhang, Zhaowu; Wang, Shuang; Zhang, Jianfeng; Li, Hui; Ren, Lei; Weng, Jian; Zhang, Qiqing

    2009-03-01

    Gold nanoparticles (GNPs) are widely used to detect DNA. We studied the effect of pH on the assembly/disassembly of single-stranded DNA functionalized GNPs. Based on the different binding affinities of DNA to GNPs, we present a simple and fast way that uses HCl to drive the assembly of GNPs for detection of DNA sequences with single nucleotide differences. The assembly is reversible and can be switched by changing the solution pH. No covalent modification of DNA or GNP surface is needed. Oligonucleotide derived from human p53 gene with one-base substitution can be distinguished by a color change of the GNPs solution or a significant difference of the maximum absorption wavelength (λmax), compared with wildtype sequences. This method enables detection of 10 picomole quantities of target DNA.

  19. Phytometabolite Dehydroleucodine Induces Cell Cycle Arrest, Apoptosis, and DNA Damage in Human Astrocytoma Cells through p73/p53 Regulation

    PubMed Central

    Bailon-Moscoso, Natalia; González-Arévalo, Gabriela; Velásquez-Rojas, Gabriela; Malagon, Omar; Vidari, Giovanni; Zentella-Dehesa, Alejandro; Ratovitski, Edward A.; Ostrosky-Wegman, Patricia

    2015-01-01

    Accumulating evidence supports the idea that secondary metabolites obtained from medicinal plants (phytometabolites) may be important contributors in the development of new chemotherapeutic agents to reduce the occurrence or recurrence of cancer. Our study focused on Dehydroleucodine (DhL), a sesquiterpene found in the provinces of Loja and Zamora-Chinchipe. In this study, we showed that DhL displayed cytostatic and cytotoxic activities on the human cerebral astrocytoma D384 cell line. With lactone isolated from Gynoxys verrucosa Wedd, a medicinal plant from Ecuador, we found that DhL induced cell death in D384 cells by triggering cell cycle arrest and inducing apoptosis and DNA damage. We further found that the cell death resulted in the increased expression of CDKN1A and BAX proteins. A marked induction of the levels of total TP73 and phosphorylated TP53, TP73, and γ-H2AX proteins was observed in D384 cells exposed to DhL, but no increase in total TP53 levels was detected. Overall these studies demonstrated the marked effect of DhL on the diminished survival of human astrocytoma cells through the induced expression of TP73 and phosphorylation of TP73 and TP53, suggesting their key roles in the tumor cell response to DhL treatment. PMID:26309132

  20. Phytometabolite Dehydroleucodine Induces Cell Cycle Arrest, Apoptosis, and DNA Damage in Human Astrocytoma Cells through p73/p53 Regulation.

    PubMed

    Bailon-Moscoso, Natalia; González-Arévalo, Gabriela; Velásquez-Rojas, Gabriela; Malagon, Omar; Vidari, Giovanni; Zentella-Dehesa, Alejandro; Ratovitski, Edward A; Ostrosky-Wegman, Patricia

    2015-01-01

    Accumulating evidence supports the idea that secondary metabolites obtained from medicinal plants (phytometabolites) may be important contributors in the development of new chemotherapeutic agents to reduce the occurrence or recurrence of cancer. Our study focused on Dehydroleucodine (DhL), a sesquiterpene found in the provinces of Loja and Zamora-Chinchipe. In this study, we showed that DhL displayed cytostatic and cytotoxic activities on the human cerebral astrocytoma D384 cell line. With lactone isolated from Gynoxys verrucosa Wedd, a medicinal plant from Ecuador, we found that DhL induced cell death in D384 cells by triggering cell cycle arrest and inducing apoptosis and DNA damage. We further found that the cell death resulted in the increased expression of CDKN1A and BAX proteins. A marked induction of the levels of total TP73 and phosphorylated TP53, TP73, and γ-H2AX proteins was observed in D384 cells exposed to DhL, but no increase in total TP53 levels was detected. Overall these studies demonstrated the marked effect of DhL on the diminished survival of human astrocytoma cells through the induced expression of TP73 and phosphorylation of TP73 and TP53, suggesting their key roles in the tumor cell response to DhL treatment. PMID:26309132

  1. The ethanol extract of Scutellaria baicalensis and the active compounds induce cell cycle arrest and apoptosis including upregulation of p53 and Bax in human lung cancer cells

    SciTech Connect

    Gao Jiayu; Morgan, Winston A.; Sanchez-Medina, Alberto; Corcoran, Olivia

    2011-08-01

    Despite a lack of scientific authentication, Scutellaria baicalensis is clinically used in Chinese medicine as a traditional adjuvant to chemotherapy of lung cancer. In this study, cytotoxicity assays demonstrated that crude ethanolic extracts of S. baicalensis were selectively toxic to human lung cancer cell lines A549, SK-LU-1 and SK-MES-1 compared with normal human lung fibroblasts. The active compounds baicalin, baicalein and wogonin did not exhibit such selectivity. Following exposure to the crude extracts, cellular protein expression in the cancer cell lines was assessed using 2D gel electrophoresis coupled with MALDI-TOF-MS/Protein Fingerprinting. The altered protein expression indicated that cell growth arrest and apoptosis were potential mechanisms of cytotoxicity. These observations were supported by PI staining cell cycle analysis using flow cytometry and Annexin-V apoptotic analysis by fluorescence microscopy of cancer cells treated with the crude extract and pure active compounds. Moreover, specific immunoblotting identification showed the decreased expression of cyclin A results in the S phase arrest of A549 whereas the G{sub 0}/G{sub 1} phase arrest in SK-MES-1 cells results from the decreased expression of cyclin D1. Following treatment, increased expression in the cancer cells of key proteins related to the enhancement of apoptosis was observed for p53 and Bax. These results provide further insight into the molecular mechanisms underlying the clinical use of this herb as an adjuvant to lung cancer therapy. - Research Highlights: > Scutellaria baicalensis is a clinical adjuvant to lung cancer chemotherapy in China. > Scutellaria ethanol extracts selectively toxic to A549, SK-LU-1 and SK-MES-1. > Baicalin, baicalein and wogonin were toxic to all lung cancer cell lines. > Proteomics identified increased p53 and BAX in response to Scutellaria extracts.

  2. Newly synthesized quinazolinone HMJ-38 suppresses angiogenetic responses and triggers human umbilical vein endothelial cell apoptosis through p53-modulated Fas/death receptor signaling

    SciTech Connect

    Chiang, Jo-Hua; Yang, Jai-Sing; Lu, Chi-Cheng; Hour, Mann-Jen; Chang, Shu-Jen; Lee, Tsung-Han; Chung, Jing-Gung

    2013-06-01

    The current study aims to investigate the antiangiogenic responses and apoptotic death of human umbilical vein endothelial cells (HUVECs) by a newly synthesized compound named 2-(3′-methoxyphenyl)-6-pyrrolidinyl-4-quinazolinone (HMJ-38). This work attempted to not only explore the effects of angiogenesis on in vivo and ex vivo studies but also hypothesize the implications for HUVECs (an ideal cell model for angiogenesis in vitro) and further undermined apoptotic experiments to verify the underlying molecular signaling by HMJ-38. Our results demonstrated that HMJ-38 significantly inhibited blood vessel growth and microvessel formation by the mouse Matrigel plug assay of angiogenesis, and the suppression of microsprouting from the rat aortic ring assay was observed after HMJ-38 exposure. In addition, HMJ-38 disrupted the tube formation and blocked the ability of HUVECs to migrate in response to VEGF. We also found that HMJ-38 triggered cell apoptosis of HUVECs in vitro. HMJ-38 concentration-dependently suppressed viability and induced apoptotic damage in HUVECs. HMJ-38-influenced HUVECs were performed by determining the oxidative stress (ROS production) and ATM/p53-modulated Fas and DR4/DR5 signals that were examined by flow cytometry, Western blotting, siRNA and real-time RT-PCR analyses, respectively. Our findings demonstrate that p53-regulated extrinsic pathway might fully contribute to HMJ-38-provoked apoptotic death in HUVECs. In view of these observations, we conclude that HMJ-38 reduces angiogenesis in vivo and ex vivo as well as induces apoptosis of HUVECs in vitro. Overall, HMJ-38 has a potent anti-neovascularization effect and could warrant being a vascular targeting agent in the future. - Highlights: • HMJ-38 suppresses angiogenic actions in vivo and ex vivo. • Inhibitions of blood vessel and microvessel formation by HMJ-38 are acted. • Cytotoxic effects of HUVECs occur by HMJ-38 challenge. • p53-modulated extrinsic pathway contributes to HMJ-38

  3. p53 mutant human glioma cells are sensitive to UV-C-induced apoptosis due to impaired cyclobutane pyrimidine dimer removal.

    PubMed

    Batista, Luis F Z; Roos, Wynand P; Kaina, Bernd; Menck, Carlos F M

    2009-02-01

    The p53 protein is a key regulator of cell responses to DNA damage, and it has been shown that it sensitizes glioma cells to the alkylating agent temozolomide by up-regulating the extrinsic apoptotic pathway, whereas it increases the resistance to chloroethylating agents, such as ACNU and BCNU, probably by enhancing the efficiency of DNA repair. However, because these agents induce a wide variety of distinct DNA lesions, the direct importance of DNA repair is hard to access. Here, it is shown that the induction of photoproducts by UV light (UV-C) significantly induces apoptosis in a p53-mutated glioma background. This is caused by a reduced level of photoproduct repair, resulting in the persistence of DNA lesions in p53-mutated glioma cells. UV-C-induced apoptosis in p53 mutant glioma cells is preceded by strong transcription and replication inhibition due to blockage by unrepaired photolesions. Moreover, the results indicate that UV-C-induced apoptosis of p53 mutant glioma cells is executed through the intrinsic apoptotic pathway, with Bcl-2 degradation and sustained Bax and Bak up-regulation. Collectively, the data indicate that unrepaired DNA lesions induce apoptosis in p53 mutant gliomas despite the resistance of these gliomas to temozolomide, suggesting that efficiency of treatment of p53 mutant gliomas might be higher with agents that induce the formation of DNA lesions whose global genomic repair is dependent on p53.

  4. Combined treatment with vitamin C and sulindac synergistically induces p53- and ROS-dependent apoptosis in human colon cancer cells.

    PubMed

    Gong, Eun-Yeung; Shin, Yu Jin; Hwang, Ih-Yeon; Kim, Jeong Hee; Kim, Seung-Mi; Moon, Jai-Hee; Shin, Jae-Sik; Lee, Dae-Hee; Hur, Dae Young; Jin, Dong-Hoon; Hong, Seung-Woo; Lee, Won Keun; Lee, Wang-Jae

    2016-09-01

    Sulindac has anti-neoplastic properties against colorectal cancers; however, its use as a chemopreventive agent has been limited due to toxicity and efficacy concerns. Combinatorial treatment of colorectal cancers has been attempted to maximize anti-cancer efficacy with minimal side effects by administrating NSAIDs in combination with other inhibitory compounds or drugs such as l-ascorbic acid (vitamin C), which is known to exhibit cytotoxicity towards various cancer cells at high concentrations. In this study, we evaluated a combinatorial strategy utilizing sulindac and vitamin C. The death of HCT116 cells upon combination therapy occurred via a p53-mediated mechanism. The combination therapeutic resistance developed in isogenic p53 null HCT116 cells and siRNA-mediated p53 knockdown HCT116 cells, but the exogenous expression of p53 in p53 null isogenic cells resulted in the induction of cell death. In addition, we investigated an increased level of intracellular ROS (reactive oxygen species), which was preceded by p53 activation. The expression level of PUMA (p53-upregulated modulator of apoptosis), but not Bim, was significantly increased in HCT116 cells in response to the combination treatment. Taken together, our results demonstrate that combination therapy with sulindac and vitamin C could be a novel anti-cancer therapeutic strategy for p53 wild type colon cancers.

  5. Combined treatment with vitamin C and sulindac synergistically induces p53- and ROS-dependent apoptosis in human colon cancer cells.

    PubMed

    Gong, Eun-Yeung; Shin, Yu Jin; Hwang, Ih-Yeon; Kim, Jeong Hee; Kim, Seung-Mi; Moon, Jai-Hee; Shin, Jae-Sik; Lee, Dae-Hee; Hur, Dae Young; Jin, Dong-Hoon; Hong, Seung-Woo; Lee, Won Keun; Lee, Wang-Jae

    2016-09-01

    Sulindac has anti-neoplastic properties against colorectal cancers; however, its use as a chemopreventive agent has been limited due to toxicity and efficacy concerns. Combinatorial treatment of colorectal cancers has been attempted to maximize anti-cancer efficacy with minimal side effects by administrating NSAIDs in combination with other inhibitory compounds or drugs such as l-ascorbic acid (vitamin C), which is known to exhibit cytotoxicity towards various cancer cells at high concentrations. In this study, we evaluated a combinatorial strategy utilizing sulindac and vitamin C. The death of HCT116 cells upon combination therapy occurred via a p53-mediated mechanism. The combination therapeutic resistance developed in isogenic p53 null HCT116 cells and siRNA-mediated p53 knockdown HCT116 cells, but the exogenous expression of p53 in p53 null isogenic cells resulted in the induction of cell death. In addition, we investigated an increased level of intracellular ROS (reactive oxygen species), which was preceded by p53 activation. The expression level of PUMA (p53-upregulated modulator of apoptosis), but not Bim, was significantly increased in HCT116 cells in response to the combination treatment. Taken together, our results demonstrate that combination therapy with sulindac and vitamin C could be a novel anti-cancer therapeutic strategy for p53 wild type colon cancers. PMID:27339904

  6. An early function of the adenoviral E1B 55 kDa protein is required for the nuclear relocalization of the cellular p53 protein in adenovirus-infected normal human cells

    SciTech Connect

    Cardoso, F.M.; Kato, Sayuri E.M.; Huang Wenying; Flint, S. Jane; Gonzalez, Ramon A.

    2008-09-01

    It is well established that the human subgroup C adenovirus type 5 (Ad5) E1B 55 kDa protein can regulate the activity and concentration of the cellular tumor suppressor, p53. However, the contribution(s) of these functions of the E1B protein to viral reproduction remains unclear. To investigate this issue, we examined properties of p53 in normal human cells infected by E1B mutant viruses that display defective entry into the late phase or viral late mRNA export. The steady-state concentrations of p53 were significantly higher in cells infected by the E1B 55 kDa null mutant Hr6 or three mutants carrying small insertions in the E1B 55 kDa protein coding sequence than in Ad5-infected cells. Nevertheless, none of the mutants induced apoptosis in infected cells. Rather, the localization of p53 to E1B containing nuclear sites observed during infection by Ad5 was prevented by mutations that impair interaction of the E1B protein with p53 and/or with the E4 Orf6 protein. These results indicate that the E1B protein fulfills an early function that correlates efficient entry into the late phase with the localization of E1B and p53 in the nucleus of Ad5-infected normal human cells.

  7. Lysine methylation represses p53 activity in teratocarcinoma cancer cells.

    PubMed

    Zhu, Jiajun; Dou, Zhixun; Sammons, Morgan A; Levine, Arnold J; Berger, Shelley L

    2016-08-30

    TP53 (which encodes the p53 protein) is the most frequently mutated gene among all human cancers, whereas tumors that retain the wild-type TP53 gene often use alternative mechanisms to repress the p53 tumor-suppressive function. Testicular teratocarcinoma cells rarely contain mutations in TP53, yet the transcriptional activity of wild-type p53 is compromised, despite its high expression level. Here we report that in the teratocarcinoma cell line NTera2, p53 is subject to lysine methylation at its carboxyl terminus, which has been shown to repress p53's transcriptional activity. We show that reduction of the cognate methyltransferases reactivates p53 and promotes differentiation of the NTera2 cells. Furthermore, reconstitution of methylation-deficient p53 mutants into p53-depleted NTera2 cells results in elevated expression of p53 downstream targets and precocious loss of pluripotent gene expression compared with re-expression of wild-type p53. Our results provide evidence that lysine methylation of endogenous wild-type p53 represses its activity in cancer cells and suggest new therapeutic possibilities of targeting testicular teratocarcinoma. PMID:27535933

  8. Lysine methylation represses p53 activity in teratocarcinoma cancer cells.

    PubMed

    Zhu, Jiajun; Dou, Zhixun; Sammons, Morgan A; Levine, Arnold J; Berger, Shelley L

    2016-08-30

    TP53 (which encodes the p53 protein) is the most frequently mutated gene among all human cancers, whereas tumors that retain the wild-type TP53 gene often use alternative mechanisms to repress the p53 tumor-suppressive function. Testicular teratocarcinoma cells rarely contain mutations in TP53, yet the transcriptional activity of wild-type p53 is compromised, despite its high expression level. Here we report that in the teratocarcinoma cell line NTera2, p53 is subject to lysine methylation at its carboxyl terminus, which has been shown to repress p53's transcriptional activity. We show that reduction of the cognate methyltransferases reactivates p53 and promotes differentiation of the NTera2 cells. Furthermore, reconstitution of methylation-deficient p53 mutants into p53-depleted NTera2 cells results in elevated expression of p53 downstream targets and precocious loss of pluripotent gene expression compared with re-expression of wild-type p53. Our results provide evidence that lysine methylation of endogenous wild-type p53 represses its activity in cancer cells and suggest new therapeutic possibilities of targeting testicular teratocarcinoma.

  9. Controlling the Mdm2-Mdmx-p53 Circuit

    PubMed Central

    Waning, David L.; Lehman, Jason A.; Batuello, Christopher N.; Mayo, Lindsey D.

    2010-01-01

    The p53 tumor suppressor is a key protein in maintaining the integrity of the genome by inducing either cell cycle arrest or apoptosis following cellular stress signals. Two human family members, Mdm2 and Mdmx, are primarily responsible for inactivating p53 transcription and targeting p53 protein for ubiquitin-mediated degradation. In response to genotoxic stress, post-translational modifications to p53, Mdm2 and Mdmx stabilize and activate p53. The role that phosphorylation of these molecules plays in the cellular response to genotoxic agents has been extensively studied with respect to cancer biology. In this review, we discuss the main phosphorylation events of p53, Mdm2 and Mdmx in response to DNA damage that are important for p53 stability and activity. In tumors that harbor wild-type p53, reactivation of p53 by modulating both Mdm2 and Mdmx signaling is well suited as a therapeutic strategy. However, the rationale for development of kinase inhibitors that target the Mdm2-Mdmx-p53 axis must be carefully considered since modulation of certain kinase signaling pathways has the potential to destabilize and inactivate p53. PMID:20651945

  10. The microRNA feedback regulation of p63 in cancer progression

    PubMed Central

    Lin, Changwei; Li, Xiaorong; Zhang, Yi; Guo, Yihang; Zhou, Jianyu; Gao, Kai; Dai, Jing; Hu, Gui; Lv, Lv; Du, Juan; Zhang, Yi

    2015-01-01

    The transcription factor p63 is a member of the p53 gene family that plays a complex role in cancer due to its involvement in epithelial differentiation, cell cycle arrest and apoptosis. MicroRNAs are a class of small, non-coding RNAs with an important regulatory role in various cellular processes, as well as in the development and progression of cancer. A number of microRNAs have been shown to function as transcriptional targets of p63. Conversely, microRNAs also can modulate the expression and activity of p63. However, the p63–microRNA regulatory circuit has not been addressed in depth so far. Here, computational genomic analysis was performed using miRtarBase, Targetscan, microRNA.ORG, DIANA-MICROT, RNA22-HSA and miRDB to analyze miRNA binding to the 3′UTR of p63. JASPAR (profile score threshold 80%) and TFSEARCH datasets were used to search transcriptional start sites for p53/p63 response elements. Remarkably, these data revealed 63 microRNAs that targeted p63. Furthermore, there were 39 microRNAs targeting p63 that were predicted to be regulated by p63. These analyses suggest a crosstalk between p63 and microRNAs. Here, we discuss the crosstalk between p63 and the microRNA network, and the role of their interactions in cancer. PMID:25726529

  11. Interactions of the p53 protein family in cellular stress response in gastrointestinal tumors

    PubMed Central

    Vilgelm, Anna E.; Washington, Mary K.; Wei, Jinxiong; Chen, Heidi; Prassolov, Vladimir S.; Zaika, Alexander I.

    2010-01-01

    p53, p63 and p73 are members of the p53 protein family involved in regulation of cell cycle, apoptosis, differentiation and other critical cellular processes. Here we investigated the contribution of the entire p53 family in chemotherapeutic drug response in gastrointestinal tumors. Real-time PCR and immunohistochemistry revealed complexity and variability of expression profiles of the p53 protein family. Using colon and esophageal cancer cells, we found that the integral transcription activity of the entire p53 family, as measured by the reporter analysis, associated with response to drug treatment in studied cells. We also found that p53 and p73, as well as p63 and p73, bind simultaneously to the promoters of p53 target genes. Taken together, our results support the view that the p53 protein family functions as an interacting network of proteins and show that cellular responses to chemotherapeutic drug treatment are determined by the total activity of the entire p53 family, rather than p53 alone. PMID:20197393

  12. Interactions of the p53 protein family in cellular stress response in gastrointestinal tumors.

    PubMed

    Vilgelm, Anna E; Washington, Mary K; Wei, Jinxiong; Chen, Heidi; Prassolov, Vladimir S; Zaika, Alexander I

    2010-03-01

    p53, p63, and p73 are members of the p53 protein family involved in regulation of cell cycle, apoptosis, differentiation, and other critical cellular processes. Here, we investigated the contribution of the entire p53 family in chemotherapeutic drug response in gastrointestinal tumors. Real-time PCR and immunohistochemistry revealed complexity and variability of expression profiles of the p53 protein family. Using colon and esophageal cancer cells, we found that the integral transcription activity of the entire p53 family, as measured by the reporter analysis, associated with response to drug treatment in studied cells. We also found that p53 and p73, as well as p63 and p73, bind simultaneously to the promoters of p53 target genes. Taken together, our results support the view that the p53 protein family functions as an interacting network of proteins and show that cellular responses to chemotherapeutic drug treatment are determined by the total activity of the entire p53 family rather than p53 alone.

  13. NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2.

    PubMed

    Liu, Xiaofeng; Tan, Yuqin; Zhang, Chunfeng; Zhang, Ying; Zhang, Liangliang; Ren, Pengwei; Deng, Hongkui; Luo, Jianyuan; Ke, Yang; Du, Xiaojuan

    2016-03-01

    As a genome guardian, p53 maintains genome stability by arresting cells for damage repair or inducing cell apoptosis to eliminate the damaged cells in stress response. Several nucleolar proteins stabilize p53 by interfering Mdm2-p53 interaction upon cellular stress, while other mechanisms by which nucleolar proteins activate p53 remain to be determined. Here, we identify NAT10 as a novel regulator for p53 activation. NAT10 acetylates p53 at K120 and stabilizes p53 by counteracting Mdm2 action. In addition, NAT10 promotes Mdm2 degradation with its intrinsic E3 ligase activity. After DNA damage, NAT10 translocates to nucleoplasm and activates p53-mediated cell cycle control and apoptosis. Finally, NAT10 inhibits cell proliferation and expression of NAT10 decreases in human colorectal carcinomas. Thus, our data demonstrate that NAT10 plays a critical role in p53 activation via acetylating p53 and counteracting Mdm2 action, providing a novel pathway by which nucleolar protein activates p53 as a cellular stress sensor. PMID:26882543

  14. Role of p53 isoforms and aggregations in cancer.

    PubMed

    Kim, SeJin; An, Seong Soo A

    2016-06-01

    p53 is a master regulatory protein that is involved in diverse cellular metabolic processes such as apoptosis, DNA repair, and cell cycle arrest. The protective function of p53 (in its homotetrameric form) as a tumor suppressor is lost in more than 50% of human cancers.Despite considerable experimental evidence suggesting the presence of multiple p53 states, it has been difficult to correlate the status of p53 with cancer response to treatments and clinical outcomes, which suggest the importance of complex but essential p53 regulatory pathways.Recent studies have indicated that the expression pattern of p53 isoforms may play a crucial role in regulating normal and cancer cell fates in response to diverse stresses. The human TP53 gene encodes at least 12 p53 isoforms, which are produced in normal tissue through alternative initiation of translation, usage of alternative promoters, and alternative splicing. Furthermore, some researchers have suggested that the formation of mutant p53 aggregates may be associated with cancer pathogenesis due to loss-of function (LoF), dominant-negative (DN), and gain-of function (GoF) effects.As different isoforms or the aggregation state of p53 may influence tumorigenesis, this review aims to examine the correlation of p53 isoforms and aggregation with cancer. PMID:27368003

  15. Role of p53 isoforms and aggregations in cancer

    PubMed Central

    Kim, SeJin; An, Seong Soo A.

    2016-01-01

    Abstract p53 is a master regulatory protein that is involved in diverse cellular metabolic processes such as apoptosis, DNA repair, and cell cycle arrest. The protective function of p53 (in its homotetrameric form) as a tumor suppressor is lost in more than 50% of human cancers. Despite considerable experimental evidence suggesting the presence of multiple p53 states, it has been difficult to correlate the status of p53 with cancer response to treatments and clinical outcomes, which suggest the importance of complex but essential p53 regulatory pathways. Recent studies have indicated that the expression pattern of p53 isoforms may play a crucial role in regulating normal and cancer cell fates in response to diverse stresses. The human TP53 gene encodes at least 12 p53 isoforms, which are produced in normal tissue through alternative initiation of translation, usage of alternative promoters, and alternative splicing. Furthermore, some researchers have suggested that the formation of mutant p53 aggregates may be associated with cancer pathogenesis due to loss-of function (LoF), dominant-negative (DN), and gain-of function (GoF) effects. As different isoforms or the aggregation state of p53 may influence tumorigenesis, this review aims to examine the correlation of p53 isoforms and aggregation with cancer. PMID:27368003

  16. Convolvulus galaticus, Crocus antalyensis, and Lilium candidum extracts show their antitumor activity through induction of p53-mediated apoptosis on human breast cancer cell line MCF-7 cells.

    PubMed

    Tokgun, Onur; Akca, Hakan; Mammadov, Ramazan; Aykurt, Candan; Deniz, Gökhan

    2012-11-01

    Conventional and newly emerging treatment procedures such as chemotherapy, catalytic therapy, photodynamic therapy, and radiotherapy have not succeeded in reversing the outcome of cancer diseases to any drastic extent, which has led researchers to investigate alternative treatment options. The extensive repertoire of traditional medicinal knowledge systems from various parts of the world are being re-investigated for their healing properties. It has been reported that several members of the Convolvulaceae, Iridaceae, and Liliaceae families have antitumor activity against some tumor cell lines. Here we first report that Convolvulus galaticus, Crocus antalyensis, and Lilium candidum species have cytotoxic activity on human breast cancer cell line MCF-7 cells. Plant samples were collected and identified, and their cytotoxic effects on the MCF-7 cell line were examined at different concentrations of methanol extracts. We found that all three plants have cytotoxic effects on MCF-7 cells but that C. galaticus has the strongest cytotoxic effect even in the lowest extract concentration tested (0.32 μg/mL). Our results indicate that these plant extracts have cytotoxic effects on human breast carcinoma cell line MCF-7 cells and that this cytotoxic effect comes from p53-mediated stimulation of apoptosis.

  17. Regulation of p53-dependent apoptosis, transcriptional repression, and cell transformation by phosphorylation of the 55-kilodalton E1B protein of human adenovirus type 5.

    PubMed Central

    Teodoro, J G; Branton, P E

    1997-01-01

    The adenovirus type 5 55-kDa E1B protein (E1B-55kDa) cooperates with E1A gene products to induce cell transformation. E1A proteins stimulate DNA synthesis and cell proliferation; however, they also cause rapid cell death by p53-dependent and p53-independent apoptosis. It is believed that the role of the E1B-55kDa protein in transformation is to protect against p53-dependent apoptosis by binding to and inactivating p53. It has been shown previously that the 55-kDa polypeptide abrogates p53-mediated transactivation and that mutants defective in p53 binding are unable to cooperate with E1A in transformation. We have previously mapped phosphorylation sites near the carboxy terminus of the E1B-55kDa protein at Ser-490 and Ser-491, which lie within casein kinase II consensus sequences. Conversion of these sites to alanine residues greatly reduced transforming activity, and although the mutant 55-kDa protein was found to interact with p53 at normal levels, it was somewhat defective for suppression of p53 transactivation activity. We now report that a nearby residue, Thr-495, also appears to be phosphorylated. We demonstrate directly that the wild-type 55-kDa protein is able to block E1A-induced p53-dependent apoptosis, whereas cells infected by mutant pm490/1/5A, which contains alanine residues at all three phosphorylation sites, exhibited extensive DNA fragmentation and classic apoptotic cell death. The E1B-55kDa product has been shown to exhibit intrinsic transcriptional repression activity when localized to promoters, such as by fusion with the GAL4 DNA-binding domain, even in the absence of p53. Such repression activity was totally absent with mutant pm490/1/5A. These data suggested that inhibition of p53-dependent apoptosis may depend on the transcriptional repression function of the 55-kDa protein, which appears to be regulated be phosphorylation at the carboxy terminus. PMID:9094635

  18. Targeting Mortalin by Embelin Causes Activation of Tumor Suppressor p53 and Deactivation of Metastatic Signaling in Human Breast Cancer Cells

    PubMed Central

    Nigam, Nupur; Grover, Abhinav; Goyal, Sukriti; Katiyar, Shashank P.; Bhargava, Priyanshu; Wang, Pi-Chao; Sundar, Durai; Kaul, Sunil C.; Wadhwa, Renu

    2015-01-01

    Embelin, a natural quinone found in the fruits of Embelia ribes, is commonly used in Ayurvedic home medicine for a variety of therapeutic potentials including anti-inflammation, anti-fever, anti-bacteria and anti-cancer. Molecular mechanisms of these activities and cellular targets have not been clarified to-date. We demonstrate that the embelin inhibits mortalin-p53 interactions, and activates p53 protein in tumor cells. We provide bioinformatics, molecular docking and experimental evidence to the binding affinity of embelin with mortalin and p53. Binding of embelin with mortalin/p53 abrogates their complex resulted in nuclear translocation and transcriptional activation function of p53 causing growth arrest in cancer cells. Furthermore, analyses of growth factors and metastatic signaling using antibody membrane array revealed their downregulation in embelin-treated cells. We also found that the embelin causes transcriptional attenuation of mortalin and several other proteins involved in metastatic signaling in cancer cells. Based on these molecular dynamics and experimental data, it is concluded that the anticancer activity of embelin involves targeting of mortalin, activation of p53 and inactivation of metastatic signaling. PMID:26376435

  19. Autoantibody recognition mechanisms of p53 epitopes

    NASA Astrophysics Data System (ADS)

    Phillips, J. C.

    2016-06-01

    There is an urgent need for economical blood based, noninvasive molecular biomarkers to assist in the detection and diagnosis of cancers in a cost-effective manner at an early stage, when curative interventions are still possible. Serum autoantibodies are attractive biomarkers for early cancer detection, but their development has been hindered by the punctuated genetic nature of the ten million known cancer mutations. A landmark study of 50,000 patients (Pedersen et al., 2013) showed that a few p53 15-mer epitopes are much more sensitive colon cancer biomarkers than p53, which in turn is a more sensitive cancer biomarker than any other protein. The function of p53 as a nearly universal "tumor suppressor" is well established, because of its strong immunogenicity in terms of not only antibody recruitment, but also stimulation of autoantibodies. Here we examine dimensionally compressed bioinformatic fractal scaling analysis for identifying the few sensitive epitopes from the p53 amino acid sequence, and show how it could be used for early cancer detection (ECD). We trim 15-mers to 7-mers, and identify specific 7-mers from other species that could be more sensitive to aggressive human cancers, such as liver cancer. Our results could provide a roadmap for ECD.

  20. An intact sequence-specific DNA-binding domain is required for human cytomegalovirus-mediated sequestration of p53 and may promote in vivo binding to the viral genome during infection

    SciTech Connect

    Rosenke, Kyle; Samuel, Melanie A.; McDowell, Eric T.; Toerne, Melissa A.; Fortunato, Elizabeth A. . E-mail: lfort@uidaho.edu

    2006-04-25

    The p53 protein is stabilized during infection of primary human fibroblasts with human cytomegalovirus (HCMV). However, the p53 in HCMV-infected cells is unable to activate its downstream targets. HCMV accomplishes this inactivation, at least in part, by sequestering p53 into viral replication centers within the cell's nucleus soon after they are established. In order to better understand the interplay between HCMV and p53 and the mechanism of sequestration, we constructed a panel of mutant p53-GFP fusion constructs for use in transfection/infection experiments. These mutants affected several post-translational modification sites and several sites within the central sequence-specific DNA-binding domain of the protein. Two categories of p53 sequestration were observed when the mutant constructs were transfected into primary fibroblasts and then infected at either high or low multiplicity. The first category, including all of the post-translational modification mutants, showed sequestration comparable to a wild-type (wt) control, while the second category, mutants affecting the DNA-binding core, were not specifically sequestered above control GFP levels. This suggested that the DNA-binding ability of the protein was required for sequestration. When the HCMV genome was analyzed for p53 consensus binding sites, 21 matches were found, which localized either to the promoters or the coding regions of viral proteins involved in DNA replication and processing as well as structural proteins. An analysis of in vivo binding to these identified sites via chromatin immunoprecipitation assays revealed differential binding to several of the sites over the course of infection.

  1. p53: a molecular marker for the detection of cancer

    PubMed Central

    Boyd, Mark T; Vlatkovic, Nikolina

    2013-01-01

    Background The p53 gene is the most frequently mutated gene in cancer and accordingly has been the subject of intensive investigation for almost 30 years. Loss of p53 function due to mutations has been unequivocally demonstrated to promote cancer in both humans and in model systems. As a consequence, there exists an enormous body of information regarding the function of normal p53 in biology and the pathobiological consequences of p53 mutation. It has long been recognised that analysis of p53 has considerable potential as a tool for use in both diagnostic and, to a greater extent, prognostic settings and some significant progress has been made in both of these arenas. Objective To provide an overview of the biology of p53, particularly in the context of uses of p53 as a diagnostic tool. Methods A literature review focused upon the methods and uses of p53 analysis in the diagnosis of sporadic cancers, rare genetic disorders and in detection of residual disease. Conclusion p53 is currently an essential diagnostic for the rare inherited cancer prone syndrome (Li-Fraumeni) and is an important diagnostic in only a limited number of settings in sporadic disease. Research in specific cancers indicates that the uses of increasingly well informed p53 mutational analysis are likely to expand to other cancers. PMID:23495923

  2. Targeting the p53 Pathway in Ewing Sarcoma

    PubMed Central

    Neilsen, Paul M.; Pishas, Kathleen I.; Callen, David F.; Thomas, David M.

    2011-01-01

    The p53 tumour suppressor plays a pivotal role in the prevention of oncogenic transformation. Cancers frequently evade the potent antitumour surveillance mechanisms of p53 through mutation of the TP53 gene, with approximately 50% of all human malignancies expressing dysfunctional, mutated p53 proteins. Interestingly, genetic lesions in the TP53 gene are only observed in 10% of Ewing Sarcomas, with the majority of these sarcomas expressing a functional wild-type p53. In addition, the p53 downstream signaling pathways and DNA-damage cell cycle checkpoints remain functionally intact in these sarcomas. This paper summarizes recent insights into the functional capabilities and regulation of p53 in Ewing Sarcoma, with a particular focus on the cross-talk between p53 and the EWS-FLI1 gene rearrangement frequently associated with this disease. The development of several activators of p53 is discussed, with recent evidence demonstrating the potential of small molecule p53 activators as a promising systemic therapeutic approach for the treatment of Ewing Sarcomas with wild-type p53. PMID:21197471

  3. Curcumin suppresses human papillomavirus oncoproteins, restores p53, Rb, and PTPN13 proteins and inhibits benzo[a]pyrene-induced upregulation of HPV E7.

    PubMed

    Maher, Diane M; Bell, Maria C; O'Donnell, Emmylu A; Gupta, Brij K; Jaggi, Meena; Chauhan, Subhash C

    2011-01-01

    Curcumin has great potential as a chemopreventive and chemotherapeutic agent; however, its effects on human papillomavirus (HPV)-associated molecular events are inadequately explored. This study examined the effects of curcumin on HPV-associated pathways involved in developing cervical cancer. We demonstrate for the first time that curcumin treatment suppresses cervical cancer cell growth in a three-dimensional raft culture system. Curcumin also inhibits tumorigenic characteristics as shown by decreases in both clonogenic potential and cell motility. Additionally, our findings show that curcumin treatment inhibits the transcription of HPV16 E6/E7 as early as 6 h posttreatment and restores the expression of tumor suppressor proteins p53, retinoblastoma protein, and PTPN13. While smoking is a recognized risk factor for cervical cancer, the molecular effects of smoke carcinogens on the expression of HPV E6/E7 oncogenes are not well known. We show for the first time that exposure to benzo[a]pyrene (BaP), a tobacco carcinogen, increases the expression of HPV E7 oncoprotein suggesting a molecular link between smoking and cervical cancer. Importantly, curcumin decreases the BaP induced increase in the expression of HPV E7 oncoprotein. The results of this study clearly demonstrate that curcumin alters HPV-associated molecular pathways in cervical cancer cells. These novel findings imply that curcumin may be an effective chemopreventive and therapeutic agent for cervical cancer prevention and treatment.

  4. p38α MAPK-mediated induction and interaction of FOXO3a and p53 contribute to the inhibited-growth and induced-apoptosis of human lung adenocarcinoma cells by berberine

    PubMed Central

    2014-01-01

    Background Berberine (BBR), a component from traditional Chinese medicine, has been shown to possess anti-tumor activity against a wide spectrum of cancer cells including human lung cancer, but the detailed mechanism underlining this has not been well elucidated. Methods In this study, the effect of berberine on cell growth and apoptosis were assessed by MTT, flow cytometry and Hoechst 33258 staining assays. The phosphorylation of p38 MAPK and ERK1/2, and expressions of p38 MAPK isoforms α and β, total ERK1/2, p53, FOXO3a and p21 protein were evaluated by Western Blot analysis. Silencing of p38 MAPK isoform α and β, p53, FOXO3a and p21 were performed by siRNA methods. Exogenous expression of FOXO3a was carried out by electroporated transfection assays. Results We showed that BBR significantly inhibited growth and induced cell cycle arrest of non small cell lung cancer (NSCLC) cells in the G0/G1 phase in a dose-dependent manner. Furthermore, we found that BBR increased phosphorylation of p38 MAPK and ERK1/2 in a time-dependent and induced protein expression of tumor suppressor p53 and transcription factor FOXO3a in a dose-dependent fashion. The specific inhibitor of p38 MAPK (SB203580), and silencing of p38α MAPK by small interfering RNAs (siRNAs), but not ERK1/2 inhibitor (PD98059) blocked the stimulatory effects of BBR on protein expression of p53 and FOXO3a. Interestingly, inhibition of p53 using one specific inhibitor (Pifithrin-α) and silencing of p53 using siRNAs overcome the inhibitory effect of BBR on cell growth. Silencing of FOXO3a appeared to attenuate the effect of BBR on p53 expression, cell proliferation and apoptosis. Furthermore, BBR induces the protein expression of cell cycle inhibitor p21 (CIP1/WAF1), which was not observed in cells silencing of p53 or FOXO3α gene. Intriguingly, exogenous expression of FOXO3a enhanced the expression of p21 (CIP1/WAF1) and strengthened BBR-induced apoptosis. Conclusion Our results show that BBR inhibits

  5. Transcriptional regulation of the human glycoprotein hormone common alpha subunit gene by cAMP-response-element-binding protein (CREB)-binding protein (CBP)/p300 and p53.

    PubMed Central

    Zhang, Xian; Grand, Roger J A; McCabe, Christopher J; Franklyn, Jayne A; Gallimore, Phillip H; Turnell, Andrew S

    2002-01-01

    We have investigated the functional interactions between adenovirus early region 1A (AdE1A) protein, the co-activators cAMP-response-element-binding protein (CREB)-binding protein (CBP)/p300 and SUG1, and the transcriptional repressor retinoblastoma (Rb) in mediating T3-dependent repression. Utilizing the human glycoprotein hormone common alpha-subunit (alpha-subunit) promoter and AdE1A mutants with selective binding capacity to these molecules we have determined an essential role for CBP/p300. In normal circumstances, wild-type 12 S AdE1A inhibited alpha-subunit activity. In contrast, adenovirus mutants that retain both the SUG1- and Rb-binding sites, but lack the CBP/p300-binding site, were unable to repress promoter activity. We have also identified a role for the tumour-suppressor gene product p53 in regulation of the alpha-subunit promoter. Akin to 12 S AdE1A, exogenous p53 expression repressed alpha-subunit activity. This function resided in the ability of p53 to interact with CBP/p300; an N-terminal mutant incapable of interacting with CBP/p300 did not inhibit alpha-subunit activity. Stabilization of endogenous p53 by UV irradiation also correlated positively with reduced alpha-subunit activity. Intriguingly, T3 stimulated endogenous p53 transcriptional activity, implicating p53 in T3-dependent signalling pathways. These data indicate that CBP/p300 and p53 are key regulators of alpha-subunit activity. PMID:12164786

  6. Prostaglandin EP2 receptor signaling protects human trabecular meshwork cells from apoptosis induced by ER stress through down-regulation of p53.

    PubMed

    Kalouche, Georges; Boucher, Céline; Coste, Annick; Debussche, Laurent; Orsini, Cécile; Baudouin, Christophe; Debeir, Thomas; Vigé, Xavier; Rostène, William

    2016-09-01

    E-prostanoid receptor subtype 2 (EP2) agonists are currently under clinical development as hypotensive agents for the treatment of ocular hypertension. However, the effects of EP2 receptor agonists on trabecular meshwork (TM) alterations leading to primary open-angle glaucoma (POAG) are still unknown. Here, we evaluated whether EP2 receptor activation exhibits protective functions on TM cell death induced by endoplasmic reticulum (ER) stress. We show that the EP2 receptor agonist butaprost protects TM cell death mediated by the ER stress inducer tunicamycin through a cyclic AMP (cAMP)-dependent mechanism, but independent of the classical cAMP sensors, protein kinase A and exchange proteins activated by cAMP. The ER stress-induced intrinsic apoptosis inhibited by the EP2 receptor agonist was correlated with a decreased accumulation of the cellular stress sensor p53. In addition, p53 down-regulation was associated with inhibition of its transcriptional activity, which led to decreased expression of the pro-apoptotic p53-upregulated modulator of apoptosis (PUMA). The stabilization of p53 by nutlin-3a abolished butaprost-mediated cell death protection. In conclusion, we showed that EP2 receptor activation protects against ER stress-dependent mitochondrial apoptosis through down-regulation of p53. The specific inhibition of this pathway could reduce TM alterations observed in POAG patients. PMID:27321910

  7. p63 protein is essential for the embryonic development of vibrissae and teeth

    SciTech Connect

    Rufini, Alessandro; McKeon, Frank; Barlattani, Alberto; Candi, Eleonora . E-mail: candi@uniroma2.it

    2006-02-17

    Development of skin appendages strongly depends on epithelial-mesenchymal interactions. One of the genes involved in this process is p63, a member of the p53 family of transcription factors, essential for ectodermal development, as elucidated by the phenotype of p63 knock-out mice. Surprisingly, no information on p63 expression in tooth and hair is yet available. Here, we show p63 expression during teeth and vibrissae morphogenesis in mouse embryos and we also show a correlation with the expression patterns of the epithelial marker keratin 5 and the proliferation marker Ki67. Our results show that p63 colocalizes with both K5 and Ki67 in the epithelium of developing vibrissae, while in teeth p63 is expressed, together with K5, in the undifferentiated ectoderm (enamel organ), and in ameloblasts, a subpopulation of differentiated ectodermal cells. Moreover, p63 expression in tooth seems not to be fully colocalized with nuclear Ki67 expression.

  8. The p53 activation domain binds the TATA box-binding polypeptide in Holo-TFIID, and a neighboring p53 domain inhibits transcription.

    PubMed Central

    Liu, X; Miller, C W; Koeffler, P H; Berk, A J

    1993-01-01

    Antioncogene product p53 is a transcriptional transactivator. To investigate how p53 stimulates transcription, we examined the interaction of p53 with general transcription factors in vitro. We found that p53 binds directly to the human TATA box-binding polypeptide (TBP). We also observed a direct interaction between p53 and purified holo-TFIID, a complex composed of TBP and a group of TBP-associated polypeptides known as TAFs. The p53 binding domain on TBP was mapped to the conserved region of TBP, including residues 220 to 271. The TBP binding domain on p53 was mapped to the p53 activation domain between residues 20 and 57. To analyze the significance of the p53-TBP interaction in p53 transactivation, we compared the ability of Gal4-p53 fusion proteins to bind to TBP in vitro and to activate transcription in transient transfection assays. Fusion proteins which bound to TBP activated transcription, and those that did not bind to TBP did not activate transcription to a detectable level, suggesting that a direct interaction between TBP and p53 is required for p53 transactivation. We also found that inclusion of residues 93 to 160 of p53 in a Gal4-p53 fusion repressed transcriptional activation 100-fold. Consequently, this region of p53 inhibits transcriptional activation by the minimal p53 activation domain. Highest levels of activation were observed with sequences 1 to 92 of p53 fused to Gal4, even though this construct bound to TBP in vitro with an affinity similar to that of other Gal4-p53 fusion proteins. We conclude that TBP binding is necessary for p53 transcriptional activation and that p53 sequences outside the TBP binding domain modulate the level of activation. Images PMID:8497252

  9. Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code

    PubMed Central

    Ciribilli, Yari; Monti, Paola; Bisio, Alessandra; Nguyen, H. Thien; Ethayathulla, Abdul S.; Ramos, Ana; Foggetti, Giorgia; Menichini, Paola; Menendez, Daniel; Resnick, Michael A.; Viadiu, Hector; Fronza, Gilberto; Inga, Alberto

    2013-01-01

    Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein–DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis. PMID:23892287

  10. Role and interaction of p53, BAX and the stress-activated protein kinases p38 and JNK in benzo(a)pyrene-diolepoxide induced apoptosis in human colon carcinoma cells.

    PubMed

    Donauer, Julia; Schreck, Ilona; Liebel, Urban; Weiss, Carsten

    2012-02-01

    Polycyclic aromatic hydrocarbons are ubiquitous environmental pollutants formed during incomplete combustion of organic material. For example benzo[a]pyrene (B[a]P) is a constituent and contaminant of cigarette smoke, automobile exhaust, industrial waste and even food products. B[a]P is carcinogenic to rodents and humans. B[a]P induces its own metabolism, which generates different metabolites such as the highly reactive electrophilic genotoxin and ultimal carcinogen B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE can bind to nucleophilic macromolecules such as proteins and DNA and causes mutations. Multiple defence mechanisms have evolved to protect the cell from DNA damage. Specific signalling pathways operate to detect and repair different kinds of lesions. In case, the damage is poorly removed expansion of damaged cells can be counteracted, e.g., by the inhibition of proliferation or triggering apoptosis. Examples of damage sensors and transducers are stress-activated protein kinases (SAPKs) and the tumour suppressor protein p53. Here, we studied the role of p53 and the pro-apoptotic protein BAX in BPDE-induced cell death by using wild-type- or knock-out-human colon carcinoma cells. As reported previously, we could reconfirm a critical role of p53 in BPDE-induced apoptosis. Furthermore, induced levels of total p53 and its transcriptional target p21 declined at higher BPDE concentrations correlating with reduced rates of apoptosis. Interestingly, increased phosphorylation of p53 at serine 15 remained elevated at higher BPDE concentrations thus disconnecting p53 phosphorylation from downstream apoptosis. Hence, phosphorylation of p53 seems not only to be a more sensitive biomarker of BPDE exposure but might serve other functions unrelated to apoptosis. In addition, we identify BAX as a novel and essential factor to trigger the intrinsic pathway of apoptosis in response to BPDE. Furthermore, BPDE in parallel activates the SAPKs p38 and JNK, which are as well

  11. A Platform for Interrogating Cancer-Associated p53 Alleles

    PubMed Central

    D’Brot, Alejandro; Kurtz, Paula; Regan, Erin; Jakubowski, Brandon; Abrams, John M

    2016-01-01

    p53 is the most frequently mutated gene in human cancer. Compelling evidence argues that full transformation involves loss of growth suppression encoded by wild-type p53 together with poorly understood oncogenic activity encoded by missense mutations. Furthermore, distinguishing disease alleles from natural polymorphisms is an important clinical challenge. To interrogate the genetic activity of human p53 variants, we leveraged the Drosophila model as an in vivo platform. We engineered strains that replace the fly p53 gene with human alleles, producing a collection of stocks that are, in effect, ‘humanized’ for p53 variants. Like the fly counterpart, human p53 transcriptionally activated a biosensor and induced apoptosis after DNA damage. However, all humanized strains representing common alleles found in cancer patients failed to complement in these assays. Surprisingly, stimulus-dependent activation of hp53 occurred without stabilization, demonstrating that these two processes can be uncoupled. Like its fly counterpart, hp53 formed prominent nuclear foci in germline cells but cancer-associated p53 variants did not. Moreover, these same mutant alleles disrupted hp53 foci and inhibited biosensor activity, suggesting that these properties are functionally linked. Together these findings establish a functional platform for interrogating human p53 alleles and suggest that simple phenotypes could be used to stratify disease variants. PMID:26996664

  12. Loss of p53-regulatory protein IFI16 induces NBS1 leading to activation of p53-mediated checkpoint by phosphorylation of p53 SER37.

    PubMed

    Tawara, Hideyuki; Fujiuchi, Nobuko; Sironi, Juan; Martin, Sarah; Aglipay, Jason; Ouchi, Mutsuko; Taga, Makoto; Chen, Phang-Lang; Ouchi, Toru

    2008-01-01

    Our previous results that IFI16 is involved in p53 transcription activity under conditions of ionizing radiation (IR), and that the protein is frequently lost in human breast cancer cell lines and breast adenocarcinoma tissues suggesting that IFI16 plays a crucial role in controlling cell growth. Here, we show that loss of IFI16 by RNA interference in cell culture causes elevated phosphorylation of p53 Ser37 and accumulated NBS1 (nibrin) and p21WAF1, leading to growth retardation. Consistent with these observations, doxycyclin-induced NBS1 caused accumulation of p21WAF1 and increased phosphorylation of p53 Ser37, leading to cell cycle arrest in G1 phase. Wortmannin treatment was found to decrease p53 Ser37 phosphorylation in NBS-induced cells. These results suggest that loss of IFI16 activates p53 checkpoint through NBS1-DNA-PKcs pathway. PMID:17981542

  13. Phosphorylation at Ser-15 and Ser-392 in mutant p53 molecules from tumors is altered compared to wild-type p53

    SciTech Connect

    Ullrich, S.J.; Sakaguchi, K.; Fiscella, M.; Appella, E. ); Lees-Miller, S.P.; Anderson, C.W. ); Mercer, W.E. )

    1993-07-01

    The product of the p53 gene suppresses cell growth and plays a critical role in suppressing development of human tumors. p53 protein binds DNA, activates transcription, and can be phosphorylated at N- and C-terminal sites. Previously, wild-type p53 was shown to be hyperphosphorylated compared to mutant p53 during p53-mediated growth arrest in vivo. Here we show that Ser-15 and Ser-9 in the N-terminal transactivation domain of wild-type human p53 are phosphorylated in vivo in cells derived from the human glioblastoma line T98G. In [IIe[sup 237

  14. Human spleen tyrosine kinase p72Syk associates with the Src-family kinase p53/56Lyn and a 120-kDa phosphoprotein.

    PubMed Central

    Sidorenko, S P; Law, C L; Chandran, K A; Clark, E A

    1995-01-01

    The 72-kDa spleen tyrosine kinase (Syk) and Src-family kinase p53/56Lyn (Lyn) contribute to signaling via the B-cell antigen receptor complex. Here we show that Syk and Lyn from human B lymphocytes can interact directly. Syk and Lyn coimmunoprecipitated from mature and activated B-cell lines, and gel-purified Syk and Lyn reassociated in vitro, demonstrating their direct interaction. This Syk-Lyn interaction may be dependent on the stage of B-cell differentiation, since Syk-Lyn associations were not detected in pre-B and myeloma cell lines and Syk from an immature B-cell line did not reassociate with Lyn in vitro. Serine/threonine kinase activity was also associated with Syk. Crosslinking of cell surface IgM led to rapid activation of both tyrosine and serine/threonine protein kinase activities that resulted in phosphorylation in vitro of proteins coprecipitating with Syk--in particular, a serine/threonine phosphorylated protein 120 kDa in size (pp120). Several phosphoproteins, including one of 72 kDa and one of 120 kDa, coprecipitated with phospholipase C-gamma 1 (PLC gamma 1). Sequential immunoprecipitation identified the 72-kDa protein associated with PLC gamma 1 as Syk. The 120-kDa serine/threonine phosphorylated protein that coprecipitated with PLC gamma 1 resembled the Syk-associated pp120 by several criteria. Thus, pp120 may serve as a link between Syk and PLC gamma 1, coupling the B-cell antigen receptor to the phosphatidylinositol pathway. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7831290

  15. ZBP-89 Promotes Growth Arrest through Stabilization of p53

    PubMed Central

    Bai, Longchuan; Merchant, Juanita L.

    2001-01-01

    Transcription factor p53 can induce growth arrest and/or apoptosis in cells through activation or repression of downstream target genes. Recently, we reported that ZBP-89 cooperates with histone acetyltransferase coactivator p300 in the regulation of p21waf1, a cyclin-dependent kinase inhibitor whose associated gene is a target gene of p53. Therefore, we examined whether ZBP-89 might also inhibit cell growth by activating p53. In the present study, we demonstrate that elevated levels of ZBP-89 induce growth arrest and apoptosis in human gastrointestinal cell lines. The ZBP-89 protein accumulated within 4 h, and the p53 protein accumulated within 16 h, of serum starvation without changes in p14ARF levels, demonstrating a physiological increase in the cellular levels of these two proteins. Overexpression of ZBP-89 stabilized the p53 protein and enhanced its transcriptional activity through direct protein-protein interactions. The DNA binding and C-terminal domains of p53 and the zinc finger domain of ZBP-89 mediated the interaction. A point mutation in the p53 DNA binding domain, R273H, greatly reduced ZBP-89-mediated stabilization but not their physical interaction. Furthermore, ZBP-89 formed a complex with p53 and MDM2 and therefore did not prevent the MDM2-p53 interaction. However, heterokaryon assays demonstrated that ZBP-89 retained p53 in the nucleus. Collectively, these data indicate that ZBP-89 regulates cell proliferation in part through its ability to directly bind the p53 protein and retard its nuclear export. Our findings further our understanding of how ZBP-89 modulates cell proliferation and reveals a novel mechanism by which the p53 protein is stabilized. PMID:11416144

  16. p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration.

    PubMed

    Turnquist, C; Horikawa, I; Foran, E; Major, E O; Vojtesek, B; Lane, D P; Lu, X; Harris, B T; Harris, C C

    2016-09-01

    Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53β are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53β, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53β overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53β expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases. PMID:27104929

  17. Relationship between DNA double-strand break rejoining and cell survival after exposure to ionizing radiation in human fibroblast strains with differing ATM/p53 status: Implications for evaluation of clinical radiosensitivity

    SciTech Connect

    Mirzayans, Razmik; Severin, Diane; Murray, David . E-mail: davem@cancerboard.ab.ca

    2006-12-01

    Purpose: To better understand the impact of defects in the DNA damage-surveillance network on the various cell-based assays used for the prediction of patient radiosensitivity. Methods and Materials: We examined noncancerous human fibroblast strains from individuals with ataxia telangiectasia (ataxia telangiectasia mutated [ATM] deficient) or Li-Fraumeni syndrome (p53 deficient) using the neutral comet, H2AX phosphorylation, and clonogenic survival assays. Results: Using the comet assay, we found that, compared with normal fibroblasts, cells lacking either ATM or p53 function exhibited a reduced rate of double-strand break (DSB) rejoining early ({<=}4 h) after exposure to 8 Gy of {gamma}-radiation and also exhibited high levels of unrejoined DSBs later after irradiation. ATM-deficient and p53-deficient fibroblasts also exhibited abnormally increased levels of phosphorylated H2AX ({gamma}-H2AX) at later intervals after irradiation. In the clonogenic assay, ATM-deficient cells exhibited marked radiosensitivity and p53-deficient cells had varying degrees of radioresistance compared with normal fibroblasts. Conclusion: Regardless of whether ataxia telangiectasia and Li-Fraumeni syndrome fibroblasts are DSB-repair deficient per se, it is apparent that p53 and ATM defects greatly influence the cellular phenotype as evidenced by the neutral comet and {gamma}-H2AX assays. Our data suggest that the {gamma}-H2AX levels observed at later intervals after irradiation may represent a reliable measure of the overall DSB rejoining capabilities of human fibroblasts. However, it appears that using this parameter as a predictor of radiosensitivity without knowledge of the cells' p53 status could lead to incorrect conclusions.

  18. Mechanisms of p53-induced apoptosis.

    PubMed

    Bennett, M R

    1999-10-01

    The p53 tumour suppressor gene functions in both cell cycle arrest and apoptosis. Despite considerable advances in understanding as to how p53 regulates growth arrest, the mechanisms by which p53 regulates apoptosis are only just emerging. In particular, there appears to be a structural and functional separation between the ability of p53 to induce growth arrest and apoptosis. This review examines the interactions between p53-induced growth arrest and apoptosis, and the mechanisms of p53-induced apoptosis, both via induction of p53 transcriptional targets and via nontranscriptional mechanisms.

  19. THE HEPARIN-BINDING DOMAIN AND V REGION OF FIBRONECTIN REGULATE APOPTOSIS BY SUPPRESSION OF P53 AND C-MYC IN HUMAN PRIMARY CELLS

    EPA Science Inventory

    In apoptosis the tumor suppressor p53 and oncogene c-myc, are usually upregulated. However, we report here an alternate pathway of regulation that is triggered by inflammatory-associated matrix fragments of fibronectin (FN) and leads to apoptosis. It is mediated by transcriptio...

  20. Use of p63, a Myoepithelial Cell Marker, in Determining the Invasiveness of Spontaneous Mammary Neoplasia in a Rhesus Macaque (Macaca mulatta)

    PubMed Central

    Williams-Fritze, Misty J; Scholz, Jodi A Carlson; Bossuyt, Veerle; Booth, Carmen J

    2011-01-01

    Here we describe a case of mammary gland ductal carcinoma in an aged rhesus macaque. Tumors were diagnosed based on routine hematoxylin and eosin staining. Invasiveness was further characterized by p63 immunohistochemistry. p63 is a p53 homolog that strongly and specifically stains nuclei of myoepithelial cells in human and canine mammary tissue. Because p63 has an affinity for the nucleus of myoepithelial cells, it is readily visible. Staining of mammary tissue from the monkey for p63 revealed that multiple foci of neoplastic cells had breached the myoepithelial cell layer surrounding ducts, suggesting the potential for local invasion of the tumor. Regional metastasis was confirmed at necropsy. To our knowledge, this is the first documented use of p63 for effectively determining the invasive nature of a mammary tumor in a nonhuman primate and the first use of p63 as an effective means of staining myoepithelial cells in a mammary ductal carcinoma in a nonhuman primate. Because nonhuman primates are important animal models for human diseases, including neoplasia, this method may prove useful for both diagnostic and research purposes. PMID:21439221

  1. Individual variation in p53 and Cip1 expression profiles in normal human fibroblast strains following exposure to high-let radiation

    SciTech Connect

    Carpenter, T.R.; Johnson, N.F.; Gilliland, F.D.

    1995-12-01

    Exposure to {alpha}-particles emitted by radon progeny appears to be the second-leading cause of lung cancer mortality. However, individual susceptibility to the carcinogenic effects of {alpha}-particles remains poorly characterized. Variation in susceptibility to cancer produced by certian classes of DNA-damaging chemicals is suspected to involve differences in metabolic activation and detoxication. Susceptibility to {alpha}-particle-induced cancer may involve variations in capacity or opportunity to repair DNA damage. Subtle variations in DNA repair capacity would more likely explain radon-related lung cancer susceptibility. The p53 tumor suppressor protein accumulates as a cellular response to DNA damage from ionizing radiation and regulates arrest in the G{sub 1} portion of the cell cycle. Arrest in G{sub 1} portion of the cell cycle. While upstream regulation of p53 protein stability is poorly understood, variations in the ability to accumulate p53 following DNA damage represent potential variations in lung cancer susceptibility related to radon progeny. Further, transcription of the cell-cycle regulatory gene Cip1 is regulated by p53 and increases following ionizing radiation. Therefore, variations in the expression of Cip1 following {alpha}-particle exposure may also be a susceptibility factor in radon-related lung cancers. The purpose of the present investigation was to measure p53 and Cip1 protein induction following {alpha}-particle exposure of fibroblast lines from nine individuals to determine if there were significant variations. The expression of Cip1 protein indicates the differences in response are biologically relevant.

  2. The Human ARF Cell Cycle Regulatory Gene Promoter Is a CpG Island Which Can Be Silenced by DNA Methylation and Down-Regulated by Wild-Type p53

    PubMed Central

    Robertson, Keith D.; Jones, Peter A.

    1998-01-01

    The INK4a/ARF locus encodes two proteins involved in tumor suppression in a manner virtually unique in mammalian cells. Distinct first exons, driven from separate promoters, splice onto a common exon 2 and 3 but utilize different reading frames to produce two completely distinct proteins, both of which play roles in cell cycle control. INK4a, a critical element of the retinoblastoma gene pathway, binds to and inhibits the activities of CDK4 and CDK6, while ARF, a critical element of the p53 pathway, increases the level of functional p53 via interaction with MDM2. Here we clone and characterize the promoter of the human ARF gene and show that it is a CpG island characteristic of a housekeeping gene which contains numerous Sp1 sites. Both ARF and INK4a are coordinately expressed in cells except when their promoter regions become de novo methylated. In one of these situations, ARF transcription could be reactivated by treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine, and the reactivation kinetics of ARF and INK4a were found to differ slightly in a cell line in which both genes were silenced by methylation. The ARF promoter was also found to be highly responsive to E2F1 expression, in keeping with previous results at the RNA level. Lastly, transcription from the ARF promoter was down-regulated by wild-type p53 expression, and the magnitude of the effect correlated with the status of the endogenous p53 gene. This finding points to the existence of an autoregulatory feedback loop between p53, MDM2, and ARF, aimed at keeping p53 levels in check. PMID:9774662

  3. Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells.

    PubMed

    Zhu, Juanjuan; Liu, Shanshan; Ye, Fuqiang; Shen, Yuan; Tie, Yi; Zhu, Jie; Wei, Lixin; Jin, Yinghua; Fu, Hanjiang; Wu, Yongge; Zheng, Xiaofei

    2015-01-01

    Maternally Expressed Gene 3 (MEG3) encodes a lncRNA which is suggested to function as a tumor suppressor. Previous studies suggested that MEG3 functioned through activation of p53, however, the functional properties of MEG3 remain obscure and their relevance to human diseases is under continuous investigation. Here, we try to illuminate the relationship of MEG3 and p53, and the consequence in hepatoma cells. We find that transfection of expression construct of MEG3 enhances stability and transcriptional activity of p53. Deletion analysis of MEG3 confirms that full length and intact structure of MEG3 are critical for it to activate p53-mediated transactivation. Interestingly, our results demonstrate for the first time that MEG3 can interact with p53 DNA binding domain and various p53 target genes are deregulated after overexpression of MEG3 in hepatoma cells. Furthermore, results of qRT-PCR have shown that MEG3 RNA is lost or reduced in the majority of HCC samples compared with adjacent non-tumorous samples. Ectopic expression of MEG3 in hepatoma cells significantly inhibits proliferation and induces apoptosis. In conclusion, our data demonstrates that MEG3 functions as a tumor suppressor in hepatoma cells through interacting with p53 protein to activate p53-mediated transcriptional activity and influence the expression of partial p53 target genes.

  4. Prodigiosin rescues deficient p53 signaling and anti-tumor effects via up-regulating p73 and disrupting its interaction with mutant p53

    PubMed Central

    Hong, Bo; Prabhu, Varun V.; Zhang, Shengliang; van den Heuvel, A. Pieter J.; Dicker, David T.; Kopelovich, Levy; El-Deiry, Wafik S.

    2015-01-01

    p53 reactivation offers a broad-based strategy for cancer therapy. In this study we report the identification of prodigiosin that can reactivate p53 family-dependent transcriptional activity in p53 deficient human colon cancer cells. Prodigiosin and its structural analogue (compound R) induced the expression of p53 target genes accompanied by cell cycle arrest and apoptosis in p53 deficient cancer cells. Prodigiosin restored p53 signaling in cancer cells harboring hotspot p53 mutations, with little to no detectable cytotoxicity in normal human fibroblasts and with no genotoxicity. Prodigiosin induced the expression of p73 and disrupted its interaction with mutant p53, thereby rescuing p53 pathway deficiency and promoting anti-tumor effects. The disruption of mutant p53/p73 interaction was specific to prodigiosin and not related to mTOR inhibition. Our findings suggest that mutant p53 needs to be targeted in the context of p73 stimulation to allow efficient restoration of the p53 pathway. In exhibiting this capability, prodigiosin and its analogue provide lead compounds to rescue deficiencies in the p53 pathway in cancer cells by up-regulating p73 and targeting mutant p53/p73 interaction there. PMID:24247721

  5. Comparisons of IL-8, ROS and p53 responses in human lung epithelial cells exposed to two extracts of PM2.5 collected from an e-waste recycling area, China

    NASA Astrophysics Data System (ADS)

    Yang, Fangxing; Jin, Shiwei; Xu, Ying; Lu, Yuanan

    2011-04-01

    To identify the different effects of organic-soluble and water-soluble pollutants adsorbed on PM2.5 (PM: particulate matter) released from e-waste (electrical/electronic waste) on inflammatory response, oxidative stress and DNA damage, interleukin-8 (IL-8), reactive oxygen species (ROS) and p53 protein levels were determined and compared in human lung epithelial A549 cells exposed to extracts of PM2.5 collected from two sampling sites in an e-waste recycling area in China. It is found that both extracts induced increases of IL-8 release, ROS production and p53 protein expression. The differences between the organic-soluble and water-soluble extracts were determined as of significance for ROS production (p < 0.05) and p53 protein expression (p < 0.01). The ROS production and p53 protein expression induced by the organic-soluble extracts were found to be greater than those induced by the water-soluble extracts, for both sampling sites. The results indicated that PM2.5 collected from the e-waste recycling areas could lead to inflammatory response, oxidative stress and DNA damage, and the organic-soluble extracts had higher potential to induce such adverse effects on human health.

  6. Shallot and licorice constituent isoliquiritigenin arrests cell cycle progression and induces apoptosis through the induction of ATM/p53 and initiation of the mitochondrial system in human cervical carcinoma HeLa cells.

    PubMed

    Hsu, Ya-Ling; Chia, Chun-Chieh; Chen, Ping-Jye; Huang, Su-Er; Huang, Soon-Cen; Kuo, Po-Lin

    2009-07-01

    This study is the first to investigate the anticancer effect of isoliquiritigenin (ISL) in human cervical carcinoma HeLa cells. The results reveal that ISL inhibits HeLa cells by blocking cell cycle progression in the G2/M phase and inducing apoptosis. Blockade of cell cycle is associated with increased activation of ataxia telangiectasia-mutated (ATM). Activation of ATM by ISL phosphorylated p53 at Serine15, resulting in increased stability of p53 by decreasing p53 and murine double minute-2 (MDM2) interaction. In addition, ISL-mediated G2/M phase arrest was also associated with decreases in the amounts of cyclin B, cyclin A, cdc2, and cdc25C, and increases in the phosphorylation of Chk2, cdc25C, and cdc2. The specific ATM inhibitor caffeine significantly decreased ISL-mediated G2/M arrest by inhibiting the phosphorylation of p53 (Serine15) and Chk2. ISL induced apoptotic cell death is associated with changes in the expression of Bax and Bak, decreasing levels of Bcl-2 and Bcl-X(L), and subsequently triggering mitochondrial apoptotic pathway. In addition, pretreatment of cells with caspase-9 inhibitor blocked ISL-induced apoptosis, indicating that caspase-9 activation is involved in ISL-mediated HeLa cell apoptosis. These findings suggest that ISL may be a promising chemopreventive agent against human uterine cervical cancer.

  7. Shallot and licorice constituent isoliquiritigenin arrests cell cycle progression and induces apoptosis through the induction of ATM/p53 and initiation of the mitochondrial system in human cervical carcinoma HeLa cells.

    PubMed

    Hsu, Ya-Ling; Chia, Chun-Chieh; Chen, Ping-Jye; Huang, Su-Er; Huang, Soon-Cen; Kuo, Po-Lin

    2009-07-01

    This study is the first to investigate the anticancer effect of isoliquiritigenin (ISL) in human cervical carcinoma HeLa cells. The results reveal that ISL inhibits HeLa cells by blocking cell cycle progression in the G2/M phase and inducing apoptosis. Blockade of cell cycle is associated with increased activation of ataxia telangiectasia-mutated (ATM). Activation of ATM by ISL phosphorylated p53 at Serine15, resulting in increased stability of p53 by decreasing p53 and murine double minute-2 (MDM2) interaction. In addition, ISL-mediated G2/M phase arrest was also associated with decreases in the amounts of cyclin B, cyclin A, cdc2, and cdc25C, and increases in the phosphorylation of Chk2, cdc25C, and cdc2. The specific ATM inhibitor caffeine significantly decreased ISL-mediated G2/M arrest by inhibiting the phosphorylation of p53 (Serine15) and Chk2. ISL induced apoptotic cell death is associated with changes in the expression of Bax and Bak, decreasing levels of Bcl-2 and Bcl-X(L), and subsequently triggering mitochondrial apoptotic pathway. In addition, pretreatment of cells with caspase-9 inhibitor blocked ISL-induced apoptosis, indicating that caspase-9 activation is involved in ISL-mediated HeLa cell apoptosis. These findings suggest that ISL may be a promising chemopreventive agent against human uterine cervical cancer. PMID:19536869

  8. Pathologies Associated with the p53 Response

    PubMed Central

    Gudkov, Andrei V.; Komarova, Elena A.

    2010-01-01

    Although p53 is a major cancer preventive factor, under certain extreme stress conditions it may induce severe pathologies. Analyses of animal models indicate that p53 is largely responsible for the toxicity of ionizing radiation or DNA damaging drugs contributing to hematopoietic component of acute radiation syndrome and largely determining severe adverse effects of cancer treatment. p53-mediated damage is strictly tissue specific and occurs in tissues prone to p53-dependent apoptosis (e.g., hematopoietic system and hair follicles); on the contrary, p53 can serve as a survival factor in tissues that respond to p53 activation by cell cycle arrest (e.g., endothelium of small intestine). There are multiple experimental indications that p53 contributes to pathogenicity of acute ischemic diseases. Temporary reversible suppression of p53 by small molecules can be an effective and safe approach to reduce severity of p53-associated pathologies. PMID:20595398

  9. Regulation of Mutant p53 Protein Expression

    PubMed Central

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation. PMID:26734569

  10. P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

    PubMed Central

    Zhang, E-b; Yin, D-d; Sun, M; Kong, R; Liu, X-h; You, L-h; Han, L; Xia, R; Wang, K-m; Yang, J-s; De, W; Shu, Y-q; Wang, Z-x

    2014-01-01

    Recently, a novel class of transcripts, long non-coding RNAs (lncRNAs), is being identified at a rapid pace. These RNAs have critical roles in diverse biological processes, including tumorigenesis. Here we report that taurine-upregulated gene 1 (TUG1), a 7.1-kb lncRNA, recruiting and binding to polycomb repressive complex 2 (PRC2), is generally downregulated in non-small cell lung carcinoma (NSCLC) tissues. In a cohort of 192 NSCLC patients, the lower expression of TUG1 was associated with a higher TNM stage and tumor size, as well as poorer overall survival (P<0.001). Univariate and multivariate analyses revealed that TUG1 expression serves as an independent predictor for overall survival (P<0.001). Further experiments revealed that TUG1 expression was induced by p53, and luciferase and chromatin immunoprecipitation (ChIP) assays confirmed that TUG1 was a direct transcriptional target of p53. TUG1 knockdown significantly promoted the proliferation in vitro and in vivo. Moreover, the lncRNA-mediated regulation of the expression of HOX genes in tumorigenesis and development has been recently receiving increased attention. Interestingly, inhibition of TUG1 could upregulate homeobox B7 (HOXB7) expression; ChIP assays demonstrated that the promoter of HOXB7 locus was bound by EZH2 (enhancer of zeste homolog 2), a key component of PRC2, and was H3K27 trimethylated. This TUG1-mediated growth regulation is in part due to specific modulation of HOXB7, thus participating in AKT and MAPK pathways. Together, these results suggest that p53-regulated TUG1 is a growth regulator, which acts in part through control of HOXB7. The p53/TUG1/PRC2/HOXB7 interaction might serve as targets for NSCLC diagnosis and therapy. PMID:24853421

  11. p38 MAPK-induced MDM2 degradation confers paclitaxel resistance through p53-mediated regulation of EGFR in human lung cancer cells

    PubMed Central

    Park, Shin-Hyung; Seong, Myeong-A; Lee, Ho-Young

    2016-01-01

    Paclitaxel (PTX) is a chemotherapeutic agent that is used to treat a variety of cancers, including non-small cell lung cancer (NSCLC). However, the emergence of drug resistance limits the utility of PTX. This study determined the signaling pathway that contributes to PTX resistance. We first established PTX resistant cell lines (H460/R and 226B/R) using a dose-escalating maintenance of PTX. We found that p38 MAPK and epidermal growth factor receptor (EGFR) were constitutively activated in these cell lines. The inhibition of p38 MAPK activity by SB203580 treatment or the transfection of dominant-negative p38 MAPK sensitized both cell lines to PTX treatment. Erlotinib, an EGFR inhibitor, also increased PTX-induced apoptosis in PTX resistant cells, which suggests a role for p38 MAPK and EGFR in the development of PTX resistance. We demonstrated that p38 MAPK enhanced EGFR expression via the induction of the rapid degradation of mouse double-minute 2 homolog (MDM2) and the consequent stabilization of p53, a transcription factor of EGFR. These results suggest for the first time that the p38 MAPK/p53/EGFR axis is crucial for the facilitation of PTX resistance in NSCLCs. We also propose a mechanism for the role of the tumor-suppressor p53 in drug resistance. These results provide a foundation for the future development of potential therapeutic strategies to regulate the p38 MAPK/p53/EGFR pathway for the treatment of lung cancer patients with PTX resistance. PMID:26799187

  12. Human papillomavirus type 16 E6 inhibits p21{sup WAF1} transcription independently of p53 by inactivating p150{sup Sal2}

    SciTech Connect

    Parroche, Peggy; Touka, Majid; Mansour, Mariam; Bouvard, Veronique; Thepot, Amelie; Accardi, Rosita; Carreira, Christine; Roblot, Guillaume G.; Sylla, Bakary S.; Hasan, Uzma; Tommasino, Massimo

    2011-09-01

    HPV16 E6 deregulates G1/S cell cycle progression through p53 degradation preventing transcription of the CDK inhibitor p21{sup WAF1}. However, additional mechanisms independent of p53 inactivation appear to exist. Here, we report that HPV16 E6 targets the cellular factor p150{sup Sal2}, which positively regulates p21{sup WAF1} transcription. HPV16 E6 associates with p150{sup Sal2}, inducing its functional inhibition by preventing its binding to cis elements on the p21{sup WAF1} promoter. A HPV16 E6 mutant, L110Q, which was unable to bind p150{sup Sal2}, did not affect the ability of the cellular protein to bind p21{sup WAF1} promoter, underlining the linkage between these events. These data describe a novel mechanism by which HPV16 E6 induces cell cycle deregulation with a p53-independent pathway. The viral oncoprotein targets p150{sup Sal2}, a positive transcription regulator of p21{sup WAF1} gene, preventing G1/S arrest and allowing cellular proliferation and efficient viral DNA replication.

  13. Distinct tumor protein p53 mutants in breast cancer subgroups.

    PubMed

    Dumay, Anne; Feugeas, Jean-Paul; Wittmer, Evelyne; Lehmann-Che, Jacqueline; Bertheau, Philippe; Espié, Marc; Plassa, Louis-François; Cottu, Paul; Marty, Michel; André, Fabrice; Sotiriou, Christos; Pusztai, Lajos; de Thé, Hugues

    2013-03-01

    Tumor protein p53 (TP53) is mutated in approximately 30% of breast cancers, but this frequency fluctuates widely between subclasses. We investigated the p53 mutation status in 572 breast tumors, classified into luminal, basal and molecular apocrine subgroups. As expected, the lowest mutation frequency was observed in luminal (26%), and the highest in basal (88%) tumors. Luminal tumors showed significantly higher frequency of substitutions (82 vs. 65%), notably A/T to G/C transitions (31 vs. 15%), whereas molecular apocrine and basal tumors presented much higher frequencies of complex mutations (deletions/insertions) (36 and 33%, respectively, vs. 18%). Accordingly, missense mutations were significantly more frequent in luminal tumors (75 vs. 54%), whereas basal tumors displayed significantly increased rates of TP53 truncations (43 vs. 25%), resulting in loss of function and/or expression. Interestingly, as basal tumors, molecular apocrine tumors presented with a high rate of complex mutations, but paradoxically, these were not associated with increased frequency of p53 truncation. As in luminal tumors, this could reflect a selective pressure for p53 gain of function, possibly through P63/P73 inactivation. Collectively, these observations point not only to different mechanisms of TP53 alterations, but also to different functional consequences in the different breast cancer subtypes.

  14. p53 in the DNA-Damage-Repair Process.

    PubMed

    Williams, Ashley B; Schumacher, Björn

    2016-01-01

    The cells in the human body are continuously challenged by a variety of genotoxic attacks. Erroneous repair of the DNA can lead to mutations and chromosomal aberrations that can alter the functions of tumor suppressor genes or oncogenes, thus causing cancer development. As a central tumor suppressor, p53 guards the genome by orchestrating a variety of DNA-damage-response (DDR) mechanisms. Already early in metazoan evolution, p53 started controlling the apoptotic demise of genomically compromised cells. p53 plays a prominent role as a facilitator of DNA repair by halting the cell cycle to allow time for the repair machineries to restore genome stability. In addition, p53 took on diverse roles to also directly impact the activity of various DNA-repair systems. It thus appears as if p53 is multitasking in providing protection from cancer development by maintaining genome stability.

  15. p53 in the DNA-Damage-Repair Process.

    PubMed

    Williams, Ashley B; Schumacher, Björn

    2016-01-01

    The cells in the human body are continuously challenged by a variety of genotoxic attacks. Erroneous repair of the DNA can lead to mutations and chromosomal aberrations that can alter the functions of tumor suppressor genes or oncogenes, thus causing cancer development. As a central tumor suppressor, p53 guards the genome by orchestrating a variety of DNA-damage-response (DDR) mechanisms. Already early in metazoan evolution, p53 started controlling the apoptotic demise of genomically compromised cells. p53 plays a prominent role as a facilitator of DNA repair by halting the cell cycle to allow time for the repair machineries to restore genome stability. In addition, p53 took on diverse roles to also directly impact the activity of various DNA-repair systems. It thus appears as if p53 is multitasking in providing protection from cancer development by maintaining genome stability. PMID:27048304

  16. p53 in the DNA damage repair process

    PubMed Central

    Williams, Ashley B.; Schumacher, Björn

    2016-01-01

    The cells in the human body are continuously challenged by a variety of genotoxic attacks. Erroneous repair of the DNA can lead to mutations and chromosomal aberrations that can alter the functions of tumor suppressor genes or oncogenes, thus causing cancer development. As a central tumor suppressor, p53 guards the genome by orchestrating a variety of DNA damage response (DDR) mechanisms. Already early in metazoan evolution, p53 started controlling the apoptotic demise of genomically compromised cells. p53 plays a prominent role as a facilitator of DNA repair by halting the cell cycle to allow time for the repair machineries to restore genome stability. In addition, p53 took on diverse roles to also directly impact the activity of various DNA repair systems. It thus appears as if p53 is multitasking in protecting from cancer development by maintaining genome stability. PMID:27048304

  17. Expression of P53 protein after exposure to ionizing radiation

    NASA Astrophysics Data System (ADS)

    Salazar, A. M.; Salvador, C.; Ruiz-Trejo, C.; Ostrosky, P.; Brandan, M. E.

    2001-10-01

    One of the most important tumor suppressor genes is p53 gene, which is involved in apoptotic cell death, cell differentiation and cell cycle arrest. The expression of p53 gene can be evaluated by determining the presence of P53 protein in cells using Western Blot assay with a chemiluminescent method. This technique has shown variabilities that are due to biological factors. Film developing process can influence the quality of the p53 bands obtained. We irradiated tumor cell lines and human peripheral lymphocytes with 137Cs and 60Co gamma rays to standardize irradiation conditions, to compare ionizing radiation with actinomycin D and to reduce the observed variability of P53 protein induction levels. We found that increasing radiation doses increase P53 protein induction while it decreases viability. We also conclude that ionizing radiation could serve as a positive control for Western Blot analysis of protein P53. In addition, our results show that the developing process may play an important role in the quality of P53 protein bands and data interpretation.

  18. p53 downregulates the Fanconi anaemia DNA repair pathway

    PubMed Central

    Jaber, Sara; Toufektchan, Eléonore; Lejour, Vincent; Bardot, Boris; Toledo, Franck

    2016-01-01

    Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53Δ31, a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53Δ31/Δ31 fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53Δ31/Δ31 fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop. PMID:27033104

  19. Prodigiosin rescues deficient p53 signaling and antitumor effects via upregulating p73 and disrupting its interaction with mutant p53.

    PubMed

    Hong, Bo; Prabhu, Varun V; Zhang, Shengliang; van den Heuvel, A Pieter J; Dicker, David T; Kopelovich, Levy; El-Deiry, Wafik S

    2014-02-15

    p53 reactivation offers a broad-based strategy for cancer therapy. In this study, we report the identification of prodigiosin that can reactivate p53 family-dependent transcriptional activity in p53-deficient human colon cancer cells. Prodigiosin and its structural analogue (compound R) induced the expression of p53 target genes accompanied by cell-cycle arrest and apoptosis in p53-deficient cancer cells. Prodigiosin restored p53 signaling in cancer cells harboring hotspot TP53 mutations, with little to no detectable cytotoxicity in normal human fibroblasts and with no genotoxicity. Prodigiosin induced the expression of p73 and disrupted its interaction with mutant p53, thereby rescuing p53 pathway deficiency and promoting antitumor effects. The disruption of mutant p53/p73 interaction was specific to prodigiosin and not related to mTOR inhibition. Our findings suggest that mutant p53 needs to be targeted in the context of p73 stimulation to allow efficient restoration of the p53 pathway. In exhibiting this capability, prodigiosin and its analogue provide lead compounds to rescue deficiencies in the p53 pathway in cancer cells by upregulating p73 and targeting mutant p53/p73 interaction there.

  20. Prospective therapeutic applications of p53 inhibitors

    SciTech Connect

    Gudkov, Andrei V. . E-mail: gudkov@ccf.org; Komarova, Elena A.

    2005-06-10

    p53, in addition to being a key cancer preventive factor, is also a determinant of cancer treatment side effects causing excessive apoptotic death in several normal tissues during cancer therapy. p53 inhibitory strategy has been suggested to protect normal tissues from chemo- and radiotherapy, and to treat other pathologies associated with stress-mediated activation of p53. This strategy was validated by isolation and testing of small molecule p53 inhibitor pifithrin-{alpha} that demonstrated broad tissue protecting capacity. However, in some normal tissues and tumors p53 plays protective role by inducing growth arrest and preventing cells from premature entrance into mitosis and death from mitotic catastrophe. Inhibition of this function of p53 can sensitize tumor cells to chemo- and radiotherapy, thus opening new potential application of p53 inhibitors and justifying the need in pharmacological agents targeting specifically either pro-apoptotic or growth arrest functions of p53.

  1. The isolation of an RNA aptamer targeting to p53 protein with single amino acid mutation

    PubMed Central

    Chen, Liang; Rashid, Farooq; Shah, Abdullah; Awan, Hassaan M.; Wu, Mingming; Liu, An; Wang, Jun; Zhu, Tao; Luo, Zhaofeng; Shan, Ge

    2015-01-01

    p53, known as a tumor suppressor, is a DNA binding protein that regulates cell cycle, activates DNA repair proteins, and triggers apoptosis in multicellular animals. More than 50% of human cancers contain a mutation or deletion of the p53 gene, and p53R175 is one of the hot spots of p53 mutation. Nucleic acid aptamers are short single-stranded oligonucleotides that are able to bind various targets, and they are typically isolated from an experimental procedure called systematic evolution of ligand exponential enrichment (SELEX). Using a previously unidentified strategy of contrast screening with SELEX, we have isolated an RNA aptamer targeting p53R175H. This RNA aptamer (p53R175H-APT) has a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays. p53R175H-APT decreased the growth rate, weakened the migration capability, and triggered apoptosis in human lung cancer cells harboring p53R175H. Further analysis actually indicated that p53R175H-APT might partially rescue or correct the p53R175H to function more like the wild-type p53. In situ injections of p53R175H-APT to the tumor xenografts confirmed the effects of this RNA aptamer on p53R175H mutation in mice. PMID:26216949

  2. Entrance of the Tat protein of HIV-1 into human uterine cervical carcinoma cells causes upregulation of HPV-E6 expression and a decrease in p53 protein levels

    PubMed Central

    Barillari, Giovanni; Palladino, Clelia; Bacigalupo, Ilaria; Leone, Patrizia; Falchi, Mario; Ensoli, Barbara

    2016-01-01

    The infection of uterine cervical epithelial cells by oncogenic, high-risk human papilloma viruses (HR-HPVs) may lead to the development of cervical carcinoma. Of note, the incidence of this tumor is significantly increased in women infected by both HR-HPV and human immunodeficiency virus (HIV)-1. In this regard, previous studies have linked the HIV-1 Tat protein, a trans-activator of viral gene expression, to the pathogenesis of HIV-associated malignancies. In particular, it has been shown that upon its release by acutely infected cells, Tat protein can enter human cells, thus modifying their phenotype. Based on these findings, the present study evaluated whether extracellular Tat protein could be taken up by human uterine cervical carcinoma cells, and whether this could affect the expression of HPV (E6 or E7) or cellular (p16 or p53) molecules, which are key to cervical carcinoma development or progression. The results indicated that extracellular, biologically active HIV-1 Tat protein is taken up by human uterine cervical carcinoma cells, and that this is followed by an increase in the expression of the E6 protein of HPV, and by a reduction in the protein levels of the cellular oncosuppressor p53. Since p53 loss is associated with cell dedifferentiation and immortalization, these findings suggest a possible link between extracellular Tat protein and the high incidence and clinical aggressiveness of uterine cervical carcinoma observed in HIV/HPV doubly infected women.

  3. Training intensity modulates changes in PGC-1α and p53 protein content and mitochondrial respiration, but not markers of mitochondrial content in human skeletal muscle.

    PubMed

    Granata, Cesare; Oliveira, Rodrigo S F; Little, Jonathan P; Renner, Kathrin; Bishop, David J

    2016-02-01

    Exercise training has been associated with increased mitochondrial content and respiration. However, no study to date has compared in parallel how training at different intensities affects mitochondrial respiration and markers of mitochondrial biogenesis. Twenty-nine healthy men performed 4 wk (12 cycling sessions) of either sprint interval training [SIT; 4-10 × 30-s all-out bouts at ∼200% of peak power output (WPeak)], high-intensity interval training (HIIT; 4-7 × 4-min intervals at ∼90% WPeak), or sublactate threshold continuous training (STCT; 20-36 min at ∼65% WPeak). The STCT and HIIT groups were matched for total work. Resting biopsy samples (vastus lateralis) were obtained before and after training. The maximal mitochondrial respiration in permeabilized muscle fibers increased significantly only after SIT (25%). Similarly, the protein content of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α, p53, and plant homeodomain finger-containing protein 20 (PHF20) increased only after SIT (60-90%). Conversely, citrate synthase activity, and the protein content of TFAM and subunits of the electron transport system complexes remained unchanged throughout. Our findings suggest that training intensity is an important factor that regulates training-induced changes in mitochondrial respiration and that there is an apparent dissociation between training-induced changes in mitochondrial respiration and mitochondrial content. Moreover, changes in the protein content of PGC-1α, p53, and PHF20 are more strongly associated with training-induced changes in mitochondrial respiration than mitochondrial content.

  4. Translational approaches targeting the p53 pathway for anti-cancer therapy

    PubMed Central

    Essmann, Frank; Schulze-Osthoff, Klaus

    2012-01-01

    The p53 tumour suppressor blocks cancer development by triggering apoptosis or cellular senescence in response to oncogenic stress or DNA damage. Consequently, the p53 signalling pathway is virtually always inactivated in human cancer cells. This unifying feature has commenced tremendous efforts to develop p53-based anti-cancer therapies. Different strategies exist that are adapted to the mechanisms of p53 inactivation. In p53-mutated tumours, delivery of wild-type p53 by adenovirus-based gene therapy is now practised in China. Also, remarkable progress has been made in the development of p53-binding drugs that can rescue and reactivate the function of mutant or misfolded p53. Other biologic approaches include the development of oncolytic viruses that are designed to specifically replicate in and kill p53-defective cells. Inactivation of wt-p53 frequently results from dysregulation of MDM2, an E3 ligase that regulates p53 levels. Small-molecule drugs that inhibit the interaction of MDM2 and p53 and block p53 degradation are currently tested in clinical trials. This survey highlights the recent developments that attempt to modulate the function of p53 and outlines strategies that are being investigated for pharmacological intervention in the p53 pathway. PMID:21718309

  5. Wild-Type p53 Binds to the TATA-Binding Protein and Represses Transcription

    NASA Astrophysics Data System (ADS)

    Seto, Edward; Usheva, Anny; Zambetti, Gerard P.; Momand, Jamil; Horikoshi, Nobuo; Weinmann, Roberto; Levine, Arnold J.; Shenk, Thomas

    1992-12-01

    p53 activates transcription of genes with a p53 response element, and it can repress genes lacking the element. Here we demonstrate that wild-type but not mutant p53 inhibits transcription in a HeLa nuclear extract from minimal promoters. Wild-type but not mutant p53 binds to human TATA-binding protein (TBP). p53 does not bind to yeast TBP, and it cannot inhibit transcription in a HeLa extract where yeast TBP substitutes for human TBP. These results suggest a model in which p53 binds to TBP and interferes with transcriptional initiation.

  6. p53MVA therapy in patients with refractory gastrointestinal malignancies elevates p53-specific CD8+ T cell responses

    PubMed Central

    Hardwick, Nicola R; Carrol, Mary; Kaltcheva, Teodora; Qian, Dajun; Lim, Dean; Leong, Lucille; Chu, Peiguo; Kim, Joseph; Chao, Joseph; Fakih, Marwan; Yen, Yun; Espenschied, Jonathan; Ellenhorn, Joshua D I; Diamond, Don J; Chung, Vincent

    2014-01-01

    PURPOSE: To conduct a Phase I trial of a Modified Vaccinia Ankara vaccine delivering wild type human p53 (p53MVA) in patients with refractory gastrointestinal cancers. EXPERIMENTAL DESIGN: Three patients were vaccinated with 1.0 × 108 pfu p53MVA followed by nine patients at 5.6 × 108 pfu. Toxicity was classified using the NCI Common Toxicity Criteria and clinical responses were assessed by CT scan. Peripheral blood samples were collected pre- and post-immunization for immunophenotyping, monitoring of p53MVA induced immune response and examination of PD-1 checkpoint inhibition in vitro. RESULTS: p53MVA immunization was well tolerated at both doses, with no adverse events above grade 2. CD4+ and CD8+ T cells showing enhanced recognition of a p53 overlapping peptide library were detectable after the first immunization, particularly in the CD8+ T cell compartment (p=0.03). However in most patients this did not expand further with the second and third immunization. The frequency of PD-1+ T cells detectable in patients PBMC was significantly higher than in healthy controls. Furthermore, the frequency of PD-1+ CD8+ T cells showed an inverse correlation with the peak CD8+ p53 response (p=0.02) and antibody blockade of PD-1 in vitro increased the p53 immune responses detected after the second or third immunizations. Induction of strong T cell and antibody responses to the MVA backbone were also apparent. CONCLUSION: p53MVA was well tolerated and induced robust CD8+ T cell responses. Combination of p53MVA with immune checkpoint inhibition could help sustain immune responses and lead to enhanced clinical benefit. PMID:24987057

  7. Rice bran phytic acid induced apoptosis through regulation of Bcl-2/Bax and p53 genes in HepG2 human hepatocellular carcinoma cells.

    PubMed

    Al-Fatlawi, Atheer Abbas; Al-Fatlawi, Anees Abbas; Irshad, Md; Zafaryab, Md; Rizvi, M Moshahid Alam; Ahmad, Ayaz

    2014-01-01

    Phytic acid (PA) has been reported to have positive nutritional benefits and prevent cancer formation. This study investigated the anticancer activity of rice bran PA against hepatocellular carcinoma (HepG2) cells. Cytotoxicty of PA (0.5 to 4mM) was examined by MTT and LDH assays after 24 and 48 h treatment. Apoptotic activity was evaluated by expression analysis of apoptosis-regulatory genes [i.e. p53, Bcl-2, Bax, Caspase-3 and -9] by reverse transcriptase-PCR and DNA fragmentation assay. The results showed antioxidant activity of PA in Fe3+ reducing power assay (p ≤ 0.03). PA inhibited the growth of HepG2 cells in a concentration dependent manner (p ≤ 0.04). After 48h treatment, cell viability was recorded 84.7, 74.4, 65.6, 49.6, 36.0 and 23.8% in MTT assay and 92.6, 77.0%, 66.8%, 51.2, 40.3 and 32.3% in LDH assay at concentrations of 1, 1.5, 2.0, 2.5, 3.0, and 3.5mM, respectively. Hence, treatment of PA for 24h, recorded viability of cells 93.5, 88.6, 55.5, 34.6 and 24.4% in MTT assay and 94.2, 86.1%, 59.7%, 42.3 and 31.6%, in LDH assay at concentrations of 1, 2.2, 3.0, 3.6 and 4.0mM, respectively. PA treated HepG2 cells showed up-regulation of p53, Bax, Caspase-3 and -9, and down- regulation of Bcl-2 gene (p ≤ 0.01). At the IC50 (2.49 mM) of PA, the p53, Bax, Caspase-3 and-9 genes were up- regulated by 6.03, 7.37, 19.7 and 14.5 fold respectively. Also, the fragmented genomic DNA in PA treated cells provided evidence of apoptosis. Our study confirmed the biological activity of PA and demonstrated growth inhibition and induction of apoptosis in HepG2 cells with modulation of the expression of apoptosis-regulatory genes. PMID:24870784

  8. Crystal structure of a p53 core tetramer bound to DNA

    SciTech Connect

    Malecka, K.A.; Ho, W.C.; Marmorstein, R.

    2009-09-02

    The tumor suppressor p53 regulates downstream genes in response to many cellular stresses and is frequently mutated in human cancers. Here, we report the use of a crosslinking strategy to trap a tetrameric p53 DNA-binding domain (p53DBD) bound to DNA and the X-ray crystal structure of the protein/DNA complex. The structure reveals that two p53DBD dimers bind to B form DNA with no relative twist and that a p53 tetramer can bind to DNA without introducing significant DNA bending. The numerous dimer-dimer interactions involve several strictly conserved residues, thus suggesting a molecular basis for p53DBD-DNA binding cooperativity. Surface residue conservation of the p53DBD tetramer bound to DNA highlights possible regions of other p53 domain or p53 cofactor interactions.

  9. The DR6 protein from human herpesvirus-6B induces p53-independent cell cycle arrest in G{sub 2}/M

    SciTech Connect

    Schleimann, Mariane H.; Hoberg, Søren; Solhøj Hansen, Aida; Bundgaard, Bettina; Witt, Christoffer T.; Kofod-Olsen, Emil; Höllsberg, Per

    2014-03-15

    HHV-6B infection inhibits cell proliferation in G{sub 2}/M, but no protein has so far been recognized to exert this function. Here we identify the protein product of direct repeat 6, DR6, as an inhibitor of G{sub 2}/M cell-cycle progression. Transfection of DR6 reduced the total number of cells compared with mock-transfected cells. Lentiviral transduction of DR6 inhibited host cell DNA synthesis in a p53-independent manner, and this inhibition was DR6 dose-dependent. A deletion of 66 amino acids from the N-terminal part of DR6 prevented efficient nuclear translocation and the ability to inhibit DNA synthesis. DR6-induced accumulation of cells in G{sub 2}/M was accompanied by an enhanced expression of cyclin B1 that accumulated predominantly in the cytoplasm. Pull-down of cyclin B1 brought down pCdk1 with the inactivating phosphorylation at Tyr15. Together, DR6 delays cell cycle with an accumulation of cells in G{sub 2}/M and thus might be involved in HHV-6B-induced cell-cycle arrest. - Highlights: • HHV-6B-encoded DR6 protein inhibits cell proliferation. • DR6 inhibits host cell DNA synthesis independent of p53. • DR6 delays the cell cycle in G{sub 2}/M. • An N-terminal sequence is necessary for DR6 function. • DR6 induces cytoplasmic accumulation of cyclin B1.

  10. CuO nanoparticles induce cytotoxicity and apoptosis in human K562 cancer cell line via mitochondrial pathway, through reactive oxygen species and P53

    PubMed Central

    Shafagh, Maryam; Rahmani, Fatemeh; Delirezh, Norouz

    2015-01-01

    Objective(s): This study focused on determining cytotoxic effects of copper oxide nanoparticles (CuO NPs) on chronic myeloid leukemia (CML) K562 cell line in a cell-specific manner and its possible mechanism of cell death. We investigated the cytotoxicity of CuO NPs against K562 cell line (cancerous cell) and peripheral blood mononuclear cell (normal cell). Materials and Methods: The toxicity was evaluated using cell viability, oxidative stress and apoptosis detection. In addition, the expression levels of P53, Caspase 3, Bcl-2, and Bax genes in K562 cells were studied by reverse transcription polymerase chain reaction (RT-PCR) analysis. Results: CuO NPs exerted distinct effects on cell viability via selective killing of cancer cells in a dose-dependent manner while not impacting normal cells in MTT assay. The dose-dependent cytotoxicity of CuO NPs against K562 cells was shown through reactive oxygen species (ROS) generation. The CuO NPs induced apoptosis was confirmed through acridine orange and propidium iodide double staining. Tumor suppressor gene P53 was up regulated due to CuO NPs exposure, and increase in Bax/Bcl-2 ratio suggested mitochondria-mediated pathway is involved in CuO NPs induced apoptosis. We also observed that Caspase 3 gene expression remained unchanged up to 24 hr exposure. Conclusion: These molecular alterations provide an insight into CuO NPs-caused inhibition of growth, generation of ROS, and apoptotic death of K562 cells. PMID:26730334

  11. Mouse models of p53 functions.

    PubMed

    Lozano, Guillermina

    2010-04-01

    Studies in mice have yielded invaluable insight into our understanding of the p53 pathway. Mouse models with activated p53, no p53, and mutant p53 have queried the role of p53 in development and tumorigenesis. In these models, p53 is activated and stabilized via redundant posttranslational modifications. On activation, p53 initiates two major responses: inhibition of proliferation (via cell-cycle arrest, quiescence, senescence, and differentiation) and induction of apoptosis. Importantly, these responses are cell-type and tumor-type-specific. The analysis of mutant p53 alleles has established a gain-of-function role for p53 mutants in metastasis. The development of additional models that can precisely time the oncogenic events in single cells will provide further insight into the evolution of tumors, the importance of the stroma, and the cooperating events that lead to disruption of the p53 pathway. Ultimately, these models should serve to study the effects of novel drugs on tumor response as well as normal homeostasis.

  12. Silver nanoparticles defeat p53-positive and p53-negative osteosarcoma cells by triggering mitochondrial stress and apoptosis

    PubMed Central

    Kovács, Dávid; Igaz, Nóra; Keskeny, Csilla; Bélteky, Péter; Tóth, Tímea; Gáspár, Renáta; Madarász, Dániel; Rázga, Zsolt; Kónya, Zoltán; Boros, Imre M.; Kiricsi, Mónika

    2016-01-01

    Loss of function of the tumour suppressor p53 observed frequently in human cancers challenges the drug-induced apoptotic elimination of cancer cells from the body. This phenomenon is a major concern and provides much of the impetus for current attempts to develop a new generation of anticancer drugs capable of provoking apoptosis in a p53-independent manner. Since silver nanoparticles (AgNPs) possess unique cytotoxic features, we examined, whether their activity could be exploited to kill tumour suppressor-deficient cancer cells. Therefore, we investigated the effects of AgNPs on osteosarcoma cells of different p53 genetic backgrounds. As particle diameters might influence the molecular mechanisms leading to AgNP-induced cell death we applied 5 nm and 35 nm sized citrate-coated AgNPs. We found that both sized AgNPs targeted mitochondria and induced apoptosis in wild-type p53-containing U2Os and p53-deficient Saos-2 cells. According to our findings AgNPs are able to kill osteosarcoma cells independently from their actual p53 status and induce p53-independent cancer cell apoptosis. This feature renders AgNPs attractive candidates for novel chemotherapeutic approaches. PMID:27291325

  13. Adenovirus-mediated p53 and ING4 gene co-transfer elicits synergistic antitumor effects through enhancement of p53 acetylation in breast cancer.

    PubMed

    Wu, Jie; Zhu, Yanbo; Xu, Chun; Xu, Hong; Zhou, Xiumin; Yang, Jicheng; Xie, Yufeng; Tao, Min

    2016-01-01

    Multigene-based combination therapy may be an effective practice in cancer gene therapy. Substantial studies have demonstrated that tumor suppressor p53 acetylation is indispensable for p53 activation. Inhibitor of growth 4 (ING4), as a novel tumor suppressor, is capable of remarkably enhancing p53 acetylation and its transcriptional activity. Hence, we assumed that combined treatment of p53 and ING4 double tumor suppressors would exhibit enhanced antitumor effects. The combined therapeutic efficacy of p53 and ING4 for human cancers has not been previously reported. We thus generated multiple promoter expression cassette-based recombinant adenovirus-co-expressing ING4 and p53 double tumor suppressor genes (AdVING4/p53), evaluated the combined effects of AdVING4/p53 on breast cancer using the MDA-MB-231 (mutant p53) human breast cancer cell line, and also elucidated its underlying molecular mechanisms. We demonstrated that AdVING4/p53-mediated p53 and ING4 co-expression induced synergistic growth inhibition and apoptosis as well as enhanced effects on upregulation of acetylated p53, P21, Bax, PUMA, Noxa, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, and downregulation of Bcl-2, CD31 and microvessel density (MVD) in MDA-MB-231 breast cancer in vitro and/or in vivo subcutaneous (s.c.) xenografted tumors. The synergistic antitumor activity elicited by AdVING4/p53 was closely associated with the enhanced activation of the intrinsic apoptotic pathway and synergistic inhibition of tumor angiogenesis, very possibly via ING4-mediated enhancement of p53 acetylation and activity. Thus, our results indicate that cancer gene therapy combining two or more tumor suppressors such as p53 and ING4 may constitute a novel and effective therapeutic modality for human breast cancer and other cancers.

  14. p63 steps into the limelight: crucial roles in the suppression of tumorigenesis and metastasis

    PubMed Central

    Su, Xiaohua; Chakravarti, Deepavali; Flores, Elsa R.

    2014-01-01

    The role of p63 in cancer has been an area of intense debate and controversy. Is TP63 (which encodes p63) a tumour suppressor gene or an oncogene? This debate is partly due to the complexity of the gene. There are several p63 isoforms — some with tumour suppressive functions and others with oncogenic functions. In this Opinion article, we focus on the recent advances in understanding p63 biology and its roles in cancer. In this regard, we discuss the role of p63 in multiple stem cell compartments, ageing, in the response to DNA damage and in DNA repair. Finally, we highlight the importance of understanding the interactions between all three p53 family members and the potential impact of this knowledge on cancer therapy and regenerative medicine. PMID:23344544

  15. Differential regulation of the REGγ–proteasome pathway by p53/TGF-β signalling and mutant p53 in cancer cells

    PubMed Central

    Ali, Amjad; Wang, Zhuo; Fu, Junjiang; Ji, Lei; Liu, Jiang; Li, Lei; Wang, Hui; Chen, Jiwu; Caulin, Carlos; Myers, Jeffrey N.; Zhang, Pei; Xiao, Jianru; Zhang, Bianhong; Li, Xiaotao

    2013-01-01

    Proteasome activity is frequently enhanced in cancer to accelerate metastasis and tumorigenesis. REGγ, a proteasome activator known to promote p53/p21/p16 degradation, is often overexpressed in cancer cells. Here we show that p53/TGF-β signalling inhibits the REGγ–20S proteasome pathway by repressing REGγ expression. Smad3 and p53 interact on the REGγ promoter via the p53RE/SBE region. Conversely, mutant p53 binds to the REGγ promoter and recruits p300. Importantly, mutant p53 prevents Smad3/N-CoR complex formation on the REGγ promoter, which enhances the activity of the REGγ–20S proteasome pathway and contributes to mutant p53 gain of function. Depletion of REGγ alters the cellular response to p53/TGF-β signalling in drug resistance, proliferation, cell cycle progression and proteasome activity. Moreover, p53 mutations show a positive correlation with REGγ expression in cancer samples. These findings suggest that targeting REGγ–20S proteasome for cancer therapy may be applicable to human tumours with abnormal p53/Smad protein status. Furthermore, this study demonstrates a link between p53/TGF-β signalling and the REGγ–20S proteasome pathway, and provides insight into the REGγ/p53 feedback loop. PMID:24157709

  16. The emerging role of p53 in exercise metabolism.

    PubMed

    Bartlett, Jonathan D; Close, Graeme L; Drust, Barry; Morton, James P

    2014-03-01

    The major tumour suppressor protein, p53, is one of the most well-studied proteins in cell biology. Often referred to as the Guardian of the Genome, the list of known functions of p53 include regulatory roles in cell cycle arrest, apoptosis, angiogenesis, DNA repair and cell senescence. More recently, p53 has been implicated as a key molecular player regulating substrate metabolism and exercise-induced mitochondrial biogenesis in skeletal muscle. In this context, the study of p53 therefore has obvious implications for both human health and performance, given that impaired mitochondrial content and function is associated with the pathology of many metabolic disorders such as ageing, type 2 diabetes, obesity and cancer, as well as reduced exercise performance. Studies on p53 knockout (KO) mice collectively demonstrate that ablation of p53 content reduces intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondrial yield, reduces cytochrome c oxidase (COX) activity and peroxisome proliferator-activated receptor gamma co-activator 1-α protein content whilst also reducing mitochondrial respiration and increasing reactive oxygen species production during state 3 respiration in IMF mitochondria. Additionally, p53 KO mice exhibit marked reductions in exercise capacity (in the magnitude of 50 %) during fatiguing swimming, treadmill running and electrical stimulation protocols. p53 may regulate contractile-induced increases in mitochondrial content via modulating mitochondrial transcription factor A (Tfam) content and/or activity, given that p53 KO mice display reduced skeletal muscle mitochondrial DNA, Tfam messenger RNA and protein levels. Furthermore, upon muscle contraction, p53 is phosphorylated on serine 15 and subsequently translocates to the mitochondria where it forms a complex with Tfam to modulate expression of mitochondrial-encoded subunits of the COX complex. In human skeletal muscle, the exercise-induced phosphorylation of p53(Ser15) is enhanced in conditions

  17. Oxazoloisoindolinones with in vitro antitumor activity selectively activate a p53-pathway through potential inhibition of the p53-MDM2 interaction.

    PubMed

    Soares, Joana; Pereira, Nuno A L; Monteiro, Ângelo; Leão, Mariana; Bessa, Cláudia; Dos Santos, Daniel J V A; Raimundo, Liliana; Queiroz, Glória; Bisio, Alessandra; Inga, Alberto; Pereira, Clara; Santos, Maria M M; Saraiva, Lucília

    2015-01-23

    One of the most appealing targets for anticancer treatment is the p53 tumor suppressor protein. In half of human cancers, this protein is inactivated due to endogenous negative regulators such as MDM2. Actually, restoring the p53 activity, particularly through the inhibition of its interaction with MDM2, is considered a valuable therapeutic strategy against cancers with a wild-type p53 status. In this work, we report the synthesis of nine enantiopure phenylalaninol-derived oxazolopyrrolidone lactams and the evaluation of their biological effects as p53-MDM2 interaction inhibitors. Using a yeast-based screening assay, two oxazoloisoindolinones, compounds 1b and 3a, were identified as potential p53-MDM2 interaction inhibitors. The molecular mechanism of oxazoloisoindolinone 3a was further validated in human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53(+/+)) and in its isogenic derivative without p53 (HCT116 p53(-/-)). Indeed, using these cells, we demonstrated that oxazoloisoindolinone 3a exhibited a p53-dependent in vitro antitumor activity through induction of G0/G1-phase cell cycle arrest and apoptosis. The selective activation of a p53-apoptotic pathway by oxazoloisoindolinone 3a was further supported by the occurrence of PARP cleavage only in p53-expressing HCT116 cells. Moreover, oxazoloisoindolinone 3a led to p53 protein stabilization and to the up-regulation of p53 transcriptional activity with increased expression levels of several p53 target genes, as p21(WAF1/CIP1), MDM2, BAX and PUMA, in p53(+/+) but not in p53(-/-) HCT116 cells. Additionally, the ability of oxazoloisoindolinone 3a to block the p53-MDM2 interaction in HCT116 p53(+/+) cells was confirmed by co-immunoprecipitation. Finally, the molecular docking analysis of the interactions between the synthesized compounds and MDM2 revealed that oxazoloisoindolinone 3a binds to MDM2. Altogether, this work adds, for the first time, the oxazoloisoindolinone scaffold to the list of

  18. Expression of full-length p53 and its isoform Δp53 in breast carcinomas in relation to mutation status and clinical parameters

    PubMed Central

    Baumbusch, Lars O; Myhre, Simen; Langerød, Anita; Bergamaschi, Anna; Geisler, Stephanie B; Lønning, Per E; Deppert, Wolfgang; Dornreiter, Irene; Børresen-Dale, Anne-Lise

    2006-01-01

    Background The tumor suppressor gene p53 (TP53) controls numerous signaling pathways and is frequently mutated in human cancers. Novel p53 isoforms suggest alternative splicing as a regulatory feature of p53 activity. Results In this study we have analyzed mRNA expression of both wild-type and mutated p53 and its respective Δp53 isoform in 88 tumor samples from breast cancer in relation to clinical parameters and molecular subgroups. Three-dimensional structure differences for the novel internally deleted p53 isoform Δp53 have been predicted. We confirmed the expression of Δp53 mRNA in tumors using quantitative real-time PCR technique. The mRNA expression levels of the two isoforms were strongly correlated in both wild-type and p53-mutated tumors, with the level of the Δp53 isoform being approximately 1/3 of that of the full-length p53 mRNA. Patients expressing mutated full-length p53 and non-mutated (wild-type) Δp53, "mutational hybrids", showed a slightly higher frequency of patients with distant metastasis at time of diagnosis compared to other patients with p53 mutations, but otherwise did not differ significantly in any other clinical parameter. Interestingly, the p53 wild-type tumors showed a wide range of mRNA expression of both p53 isoforms. Tumors with mRNA expression levels in the upper or lower quartile were significantly associated with grade and molecular subtypes. In tumors with missense or in frame mutations the mRNA expression levels of both isoforms were significantly elevated, and in tumors with nonsense, frame shift or splice mutations the mRNA levels were significantly reduced compared to those expressing wild-type p53. Conclusion Expression of p53 is accompanied by the functionally different isoform Δp53 at the mRNA level in cell lines and human breast tumors. Investigations of "mutational hybrid" patients highlighted that wild-type Δp53 does not compensates for mutated p53, but rather may be associated with a worse prognosis. In tumors

  19. Oxazoloisoindolinones with in vitro antitumor activity selectively activate a p53-pathway through potential inhibition of the p53-MDM2 interaction.

    PubMed

    Soares, Joana; Pereira, Nuno A L; Monteiro, Ângelo; Leão, Mariana; Bessa, Cláudia; Dos Santos, Daniel J V A; Raimundo, Liliana; Queiroz, Glória; Bisio, Alessandra; Inga, Alberto; Pereira, Clara; Santos, Maria M M; Saraiva, Lucília

    2015-01-23

    One of the most appealing targets for anticancer treatment is the p53 tumor suppressor protein. In half of human cancers, this protein is inactivated due to endogenous negative regulators such as MDM2. Actually, restoring the p53 activity, particularly through the inhibition of its interaction with MDM2, is considered a valuable therapeutic strategy against cancers with a wild-type p53 status. In this work, we report the synthesis of nine enantiopure phenylalaninol-derived oxazolopyrrolidone lactams and the evaluation of their biological effects as p53-MDM2 interaction inhibitors. Using a yeast-based screening assay, two oxazoloisoindolinones, compounds 1b and 3a, were identified as potential p53-MDM2 interaction inhibitors. The molecular mechanism of oxazoloisoindolinone 3a was further validated in human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53(+/+)) and in its isogenic derivative without p53 (HCT116 p53(-/-)). Indeed, using these cells, we demonstrated that oxazoloisoindolinone 3a exhibited a p53-dependent in vitro antitumor activity through induction of G0/G1-phase cell cycle arrest and apoptosis. The selective activation of a p53-apoptotic pathway by oxazoloisoindolinone 3a was further supported by the occurrence of PARP cleavage only in p53-expressing HCT116 cells. Moreover, oxazoloisoindolinone 3a led to p53 protein stabilization and to the up-regulation of p53 transcriptional activity with increased expression levels of several p53 target genes, as p21(WAF1/CIP1), MDM2, BAX and PUMA, in p53(+/+) but not in p53(-/-) HCT116 cells. Additionally, the ability of oxazoloisoindolinone 3a to block the p53-MDM2 interaction in HCT116 p53(+/+) cells was confirmed by co-immunoprecipitation. Finally, the molecular docking analysis of the interactions between the synthesized compounds and MDM2 revealed that oxazoloisoindolinone 3a binds to MDM2. Altogether, this work adds, for the first time, the oxazoloisoindolinone scaffold to the list of

  20. Activations of Both Extrinsic and Intrinsic Pathways in HCT 116 Human Colorectal Cancer Cells Contribute to Apoptosis through p53-Mediated ATM/Fas Signaling by Emilia sonchifolia Extract, a Folklore Medicinal Plant.

    PubMed

    Lan, Yu-Hsuan; Chiang, Jo-Hua; Huang, Wen-Wen; Lu, Chi-Cheng; Chung, Jing-Gung; Wu, Tian-Shung; Jhan, Jia-Hua; Lin, Kuei-Li; Pai, Shu-Jen; Chiu, Yu-Jen; Tsuzuki, Minoru; Yang, Jai-Sing

    2012-01-01

    Emilia sonchifolia (L.) DC (Compositae), an herbaceous plant found in Taiwan and India, is used as folk medicine. The clinical applications include inflammation, rheumatism, cough, cuts fever, dysentery, analgesic, and antibacteria. The activities of Emilia sonchifolia extract (ESE) on colorectal cancer cell death have not been fully investigated. The purpose of this study explored the induction of apoptosis and its molecular mechanisms in ESE-treated HCT 116 human colorectal cancer cells in vitro. The methanolic ESE was characterized, and γ-humulene was formed as the major constituent (63.86%). ESE induced cell growth inhibition in a concentration- and time-dependent response by MTT assay. Apoptotic cells (DNA fragmentation, an apoptotic catachrestic) were found after ESE treatment by TUNEL assay and DNA gel electrophoresis. Alternatively, ESE stimulated the activities of caspase-3, -8, and -9 and their specific caspase inhibitors protected against ESE-induced cytotoxicity. ESE promoted the mitochondria-dependent and death-receptor-associated protein levels. Also, ESE increased ROS production and upregulated the levels of ATM, p53, and Fas in HCT 116 cells. Strikingly, p53 siRNA reversed ESE-reduced viability involved in p53-mediated ATM/Fas signaling in HCT 116 cells. In summary, our result is the first report suggesting that ESE may be potentially efficacious in the treatment of colorectal cancer.

  1. Synthetic Bichalcone TSWU-BR23 Induces Apoptosis of Human Colon Cancer HT-29 Cells by p53-Mediated Mitochondrial Oligomerization of BAX/BAK and Lipid Raft Localization of CD95/FADD.

    PubMed

    Lin, Meng-Liang; Chen, Shih-Shun; Wu, Tian-Shung

    2015-10-01

    A synthetic bichalcone analog, (E)-1-(3-((4-(4-acetylphenyl)piperazin-1-yl)methyl)-4-hydroxy-5-methoxyphenyl)-3-(pyridin-3-yl)prop-2-en-1-one (TSWU-BR23), has been shown to induce apoptosis in human colon cancer HT-29 cells involving the induction of CD95 and FAS-associated protein death domain (FADD), but its precise mechanism of action has not been fully elucidated. Using cell-surface biotinylation and sucrose density-gradient-based membrane flotation techniques, we showed that the disruption of TSWU-BR23-induced lipid raft localization of CD95/FADD by cholesterol-depleting agent (methyl-β-cyclodextrin) was reversed by cholesterol replenishment. Blockade of p53 expression by short-hairpin RNA (shRNA) suppressed oligomeric Bcl-2-associated x protein (BAX)/Bcl-2 antagonist killer 1 (BAK)-mediated mitochondrial apoptosis but did not inhibit lipid raft localization of CD95/FADD and pro-caspase-8 cleavage induced by TSWU-BR23. Co-expression of p53 shRNA and dominant-negative mutant of FADD completely inhibited TSWU-BR32-induced mitochondrial apoptotic cell death. Collectively, these data demonstrate that TSWU-BR23 leads to HT-29 cell apoptosis by inducing p53-mediated mitochondrial oligomerization of BAX/BAK and the localization of CD95/FADD with lipid rafts at the cell surface.

  2. p53 E3 ubiquitin protein ligase homolog regulates p53 in vivo in the adult mouse eye lens

    PubMed Central

    Jaramillo-Rangel, Gilberto; Ortega-Martínez, Marta; Sepúlveda-Saavedra, Julio; Saucedo-Cárdenas, Odila; Montes-de-Oca-Luna, Roberto

    2013-01-01

    Purpose p53 is a transcription factor that plays an important role in preventing cancer development. p53 participates in relevant aspects of cell biology, including apoptosis and cell cycle control and must be strictly regulated to maintain normal tissue homeostasis. p53 E3 ubiquitin protein ligase homolog (Mdm2) is an important negative regulator of p53. The purpose of this study was to determine if Mdm2 regulates p53 in vivo in the adult lens. Methods We analyzed mice expressing human p53 transgene (Tgp53) selectively in the lens in the presence or absence of Mdm2. Mice with the required genotypes were obtained by crossing transgenic, mdm2+/−, and p53−/− mice. Eye phenotype and lens histology and ultrastructure were analyzed in adult mice. Results In a wild-type genetic background (mdm2+/+), lens damage and microphthalmia were observed only in mice homozygous for Tgp53 (t/t). However, in an mdm2 null background, just one allele of Tgp53 (mdm2−/−/Tgp53t/0 mice) was sufficient to cause lens damage and microphthalmia. Furthermore, Mdm2 in only one allele was sufficient to rescue these deleterious effects, since the mdm2+/−/Tgp53t/0 mice had eye size and lens morphology similar to the control mice. Conclusions Mdm2 regulates p53 in the adult lens in vivo. This information may have relevance for analyzing normal and pathological conditions of the lens, and designing cancer therapies targeting Mdm2–p53 interaction. PMID:24339722

  3. The expanding universe of p53 targets.

    PubMed

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

    2009-10-01

    The p53 tumour suppressor is modified through mutation or changes in expression in most cancers, leading to the altered regulation of hundreds of genes that are directly influenced by this sequence-specific transcription factor. Central to the p53 master regulatory network are the target response element (RE) sequences. The extent of p53 transactivation and transcriptional repression is influenced by many factors, including p53 levels, cofactors and the specific RE sequences, all of which contribute to the role that p53 has in the aetiology of cancer. This Review describes the identification and functionality of REs and highlights the inclusion of non-canonical REs that expand the universe of genes and regulation mechanisms in the p53 tumour suppressor network.

  4. The RP-Mdm2-p53 Pathway and Tumorigenesis

    P