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Sample records for p53 cooperative integrators

  1. MIF family members cooperatively inhibit p53 expression and activity.

    PubMed

    Brock, Stephanie E; Rendon, Beatriz E; Xin, Dan; Yaddanapudi, Kavitha; Mitchell, Robert A

    2014-01-01

    The tumor suppressor p53 is induced by genotoxic stress in both normal and transformed cells and serves to transcriptionally coordinate cell cycle checkpoint control and programmed cell death responses. Macrophage migration inhibitory factor (MIF) is an autocrine and paracrine acting cytokine/growth factor that promotes lung adenocarcinoma cell motility, anchorage-independence and neo-angiogenic potential. Several recent studies indicate that the only known homolog of MIF, D-dopachrome tautomerase (D-DT - also referred to as MIF-2), has functionally redundant activities with MIF and cooperatively promotes MIF-dependent pro-tumorigenic phenotypes. We now report that MIF and D-DT synergistically inhibit steady state p53 phosphorylation, stabilization and transcriptional activity in human lung adenocarcinoma cell lines. The combined loss of MIF and D-DT by siRNA leads to dramatically reduced cell cycle progression, anchorage independence, focus formation and increased programmed cell death when compared to individual loss of MIF or D-DT. Importantly, p53 mutant and p53 null lung adenocarcinoma cell lines were only nominally rescued from the cell growth effects of MIF/D-DT combined deficiency suggesting only a minor role for p53 in these transformed cell growth phenotypes. Finally, increased p53 activation was found to be independent of aberrantly activated AMP-activated protein kinase (AMPK) that occurs in response to MIF/D-DT-deficiency but is dependent on reactive oxygen species (ROS) that mediate aberrant AMPK activation in these cells. Combined, these findings suggest that both p53 wildtype and mutant human lung adenocarcinoma tumors rely on MIF family members for maximal cell growth and survival.

  2. p53 isoform Δ133p53 promotes efficiency of induced pluripotent stem cells and ensures genomic integrity during reprogramming

    PubMed Central

    Gong, Lu; Pan, Xiao; Chen, Haide; Rao, Lingjun; Zeng, Yelin; Hang, Honghui; Peng, Jinrong; Xiao, Lei; Chen, Jun

    2016-01-01

    Human induced pluripotent stem (iPS) cells have great potential in regenerative medicine, but this depends on the integrity of their genomes. iPS cells have been found to contain a large number of de novo genetic alterations due to DNA damage response during reprogramming. Thus, to maintain the genetic stability of iPS cells is an important goal in iPS cell technology. DNA damage response can trigger tumor suppressor p53 activation, which ensures genome integrity of reprogramming cells by inducing apoptosis and senescence. p53 isoform Δ133p53 is a p53 target gene and functions to not only antagonize p53 mediated apoptosis, but also promote DNA double-strand break (DSB) repair. Here we report that Δ133p53 is induced in reprogramming. Knockdown of Δ133p53 results 2-fold decrease in reprogramming efficiency, 4-fold increase in chromosomal aberrations, whereas overexpression of Δ133p53 with 4 Yamanaka factors showes 4-fold increase in reprogamming efficiency and 2-fold decrease in chromosomal aberrations, compared to those in iPS cells induced only with 4 Yamanaka factors. Overexpression of Δ133p53 can inhibit cell apoptosis and promote DNA DSB repair foci formation during reprogramming. Our finding demonstrates that the overexpression of Δ133p53 not only enhances reprogramming efficiency, but also results better genetic quality in iPS cells. PMID:27874035

  3. Mutation of Lkb1 and p53 genes exert a cooperative effect on tumorigenesis.

    PubMed

    Wei, Chongjuan; Amos, Christopher I; Stephens, L Clifton; Campos, Imelda; Deng, Jian Min; Behringer, Richard R; Rashid, Asif; Frazier, Marsha L

    2005-12-15

    Peutz-Jeghers syndrome (PJS) is a dominantly inherited disorder characterized by gastrointestinal hamartomatous polyps and mucocutaneous melanin pigmentation. Germ line mutations in LKB1 cause PJS. We have generated mice carrying an Lkb1 exon 2 to 8 deletion by gene targeting in embryonic stem cells. Heterozygotes develop gastric hamartomas that are histologically similar to those found in humans with PJS. LKB1 is also reportedly a mediator of p53-dependent apoptosis. To explore the potential combined effects of p53 and Lkb1 alterations on tumorigenesis, we carried out a series of matings with Lkb1(+/-) and p53 null mice to generate Lkb1(+/-)/p53(+/-) and Lkb1(+/-)/p53(-/-) mice. Similar to the Lkb1(+/-) mice, gastrointestinal hamartomas have also been detected in the mice with these two genotypes. The Lkb1(+/-)/p53(+/-) mice displayed a dramatically reduced life span and increased tumor incidence compared to the mice with either Lkb1 or p53 single gene knockout. The time to onset of polyposis in Lkb1(+/-)/p53(-/-) mice is approximately 2 months earlier than Lkb1(+/-)/p53(+/-) and Lkb1(+/-) mice, whereas the latter two show a similar time to onset which is at approximately 6 months of age. These results strongly suggested that mutations of p53 and Lkb1 gene cooperate in the acceleration of tumorigenesis.

  4. Functional cooperation of RKTG with p53 in tumorigenesis and epithelial-mesenchymal transition.

    PubMed

    Jiang, Yuhui; Xie, Xiaoduo; Li, Zhigang; Wang, Zheng; Zhang, Yixuan; Ling, Zhi-Qiang; Ling, Zhiqiang; Pan, Yi; Wang, Zhenzhen; Chen, Yan

    2011-04-15

    Raf kinase trapping to Golgi (RKTG) is a potential tumor suppressor gene due to its negative roles in regulating Ras/Raf/MEK/ERK (extracellular signal-regulated kinase) pathway and GPCR (G protein-coupled receptor) Gβγ subunit signaling. Interestingly, RKTG-deficient mice are free of tumors, although they are prone to form skin cancer on carcinogen administration. On the other hand, p53 is a well-characterized tumor suppressor gene and p53 heterozygous mice develop sarcoma and other tumors starting from 12 months of age. In RKTG-null mouse embryonic fibroblasts, lypophosphatidic acid (LPA), but not EGF (epidermal growth factor), could stimulate hyperphosphorylation of AKT and GSK3β, accompanied by increases in phosphorylation of p53 at Ser15 and accumulation of p53, as well as its target genes p21 and p16. Spontaneous skin cancer-like tumors were detected in about 25% of RKTG nullizygous and p53 heterozygous mice within 7 months of age. Hyperplasia and epithelial-mesenchymal transition (EMT) were observed in the tumor-overlying epidermis, in which LOH of p53 occurred and EMT features emerged. In p53-mutated A431 epithelial carcinoma cells, knockdown of RKTG led to enhancement of LPA-stimulated AKT and GSK3β phosphorylation, together with increased accumulation of β-catenin and appearance of EMT features that were antagonized by p53 overexpression. In HepG2 epithelial cells, LPA-stimulated AKT phosphorylation and EMT features reached maximum when both RKTG and p53 were simultaneously silenced. In summary, these results not only indicate that RKTG has an in vivo tumor suppressor function to cooperate with p53 in tumorigenesis but also suggest that p53 has an EMT checkpoint function and the loss of this function can combine with loss of RKTG to drive EMT and tumor progression.

  5. ARF and ATM/ATR cooperate in p53-mediated apoptosis upon oncogenic stress

    SciTech Connect

    Pauklin, Siim . E-mail: spauklin@ut.ee; Kristjuhan, Arnold; Maimets, Toivo; Jaks, Viljar

    2005-08-26

    Induction of apoptosis is pivotal for eliminating cells with damaged DNA or deregulated proliferation. We show that tumor suppressor ARF and ATM/ATR kinase pathways cooperate in the induction of apoptosis in response to elevated expression of c-myc, {beta}-catenin or human papilloma virus E7 oncogenes. Overexpression of oncogenes leads to the formation of phosphorylated H2AX foci, induction of Rad51 protein levels and ATM/ATR-dependent phosphorylation of p53. Inhibition of ATM/ATR kinases abolishes both induction of Rad51 and phosphorylation of p53, and remarkably reduces the level of apoptosis induced by co-expression of oncogenes and ARF. However, the induction of apoptosis is downregulated in p53-/- cells and does not depend on activities of ATM/ATR kinases, indicating that efficient induction of apoptosis by oncogene activation depends on coordinated action of ARF and ATM/ATR pathways in the regulation of p53.

  6. Cooperative action of germ-line mutations in decorin and p53 accelerates lymphoma tumorigenesis.

    PubMed

    Iozzo, R V; Chakrani, F; Perrotti, D; McQuillan, D J; Skorski, T; Calabretta, B; Eichstetter, I

    1999-03-16

    Ectopic expression of decorin in a wide variety of transformed cells results in growth arrest and the inability to generate tumors in nude mice. This process is caused by a decorin-mediated activation of the epidermal growth factor receptor, which leads to a sustained induction of endogenous p21(WAF1/CIP1) (the cyclin-dependent kinase inhibitor p21) and growth arrest. However, mice harboring a targeted disruption of the decorin gene do not develop spontaneous tumors. To test the role of decorin in tumorigenesis, we generated mice lacking both decorin and p53, an established tumor-suppressor gene. Mice lacking both genes showed a faster rate of tumor development and succumbed almost uniformly to thymic lymphomas within 6 months [mean survival age (T50) approximately 4 months]. Mice harboring one decorin allele and no p53 gene developed the same spectrum of tumors as the double knockout animals, but had a survival rate similar to the p53 null animals (T50 approximately 6 months). Ectopic expression of decorin in thymic lymphoma cells isolated from double mutant animals markedly suppressed their colony-forming ability. When these lymphoma cells were cocultured with fibroblasts derived from either wild-type or decorin null embryos, the cells grew faster in the absence of decorin. Moreover, exogenous decorin proteoglycan or its protein core significantly retarded their growth in vitro. These results indicate that the lack of decorin is permissive for lymphoma tumorigenesis in a mouse model predisposed to cancer and suggest that germ-line mutations in decorin and p53 may cooperate in the transformation of lymphocytes and ultimately lead to a more aggressive phenotype by shortening the tumor latency.

  7. Suppression of p53 activity through the cooperative action of Ski and histone deacetylase SIRT1.

    PubMed

    Inoue, Yasumichi; Iemura, Shun-ichiro; Natsume, Tohru; Miyazawa, Keiji; Imamura, Takeshi

    2011-02-25

    Ski was originally identified as an oncogene based on the fact that Ski overexpression transformed chicken and quail embryo fibroblasts. Consistent with these proposed oncogenic roles, Ski is overexpressed in various human tumors. However, whether and how Ski functions in mammalian tumorigenesis has not been fully investigated. Here, we show that Ski interacts with p53 and attenuates the biological functions of p53. Ski overexpression attenuated p53-dependent transactivation, whereas Ski knockdown enhanced the transcriptional activity of p53. Interestingly, Ski bound to the histone deacetylase SIRT1 and stabilized p53-SIRT1 interaction to promote p53 deacetylation, which subsequently decreased the DNA binding activity of p53. Consistent with the ability of Ski to inactivate p53, overexpressing Ski desensitized cells to genotoxic drugs and Nutlin-3, a small-molecule antagonist of Mdm2 that stabilizes p53 and activates the p53 pathway, whereas knocking down Ski increased the cellular sensitivity to these agents. These results indicate that Ski negatively regulates p53 and suggest that the p53-Ski-SIRT1 axis is an attractive target for cancer therapy.

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

    PubMed

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

    2001-10-05

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

  9. Heterogeneous biomedical database integration using a hybrid strategy: a p53 cancer research database.

    PubMed

    Bichutskiy, Vadim Y; Colman, Richard; Brachmann, Rainer K; Lathrop, Richard H

    2007-02-20

    Complex problems in life science research give rise to multidisciplinary collaboration, and hence, to the need for heterogeneous database integration. The tumor suppressor p53 is mutated in close to 50% of human cancers, and a small drug-like molecule with the ability to restore native function to cancerous p53 mutants is a long-held medical goal of cancer treatment. The Cancer Research DataBase (CRDB) was designed in support of a project to find such small molecules. As a cancer informatics project, the CRDB involved small molecule data, computational docking results, functional assays, and protein structure data. As an example of the hybrid strategy for data integration, it combined the mediation and data warehousing approaches. This paper uses the CRDB to illustrate the hybrid strategy as a viable approach to heterogeneous data integration in biomedicine, and provides a design method for those considering similar systems. More efficient data sharing implies increased productivity, and, hopefully, improved chances of success in cancer research. (Code and database schemas are freely downloadable, http://www.igb.uci.edu/research/research.html.).

  10. Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells.

    PubMed

    Pfister, Neil T; Fomin, Vitalay; Regunath, Kausik; Zhou, Jeffrey Y; Zhou, Wen; Silwal-Pandit, Laxmi; Freed-Pastor, William A; Laptenko, Oleg; Neo, Suat Peng; Bargonetti, Jill; Hoque, Mainul; Tian, Bin; Gunaratne, Jayantha; Engebraaten, Olav; Manley, James L; Børresen-Dale, Anne-Lise; Neilsen, Paul M; Prives, Carol

    2015-06-15

    Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.

  11. Spatially- and temporally-controlled postnatal p53 knockdown cooperates with embryonic Schwann cell precursor Nf1 gene loss to promote malignant peripheral nerve sheath tumor formation.

    PubMed

    Hirbe, Angela C; Dahiya, Sonika; Friedmann-Morvinski, Dinorah; Verma, Inder M; Clapp, D Wade; Gutmann, David H

    2016-02-16

    Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that arise sporadically or in association with the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome. In individuals with NF1, MPNSTs are hypothesized to arise from Nf1-deficient Schwann cell precursor cells following the somatic acquisition of secondary cooperating genetic mutations (e.g., p53 loss). To model this sequential genetic cooperativity, we coupled somatic lentivirus-mediated p53 knockdown in the adult right sciatic nerve with embryonic Schwann cell precursor Nf1 gene inactivation in two different Nf1 conditional knockout mouse strains. Using this approach, ~60% of mice with Periostin-Cre-mediated Nf1 gene inactivation (Periostin-Cre; Nf1(flox/flox) mice) developed tumors classified as low-grade MPNSTs following p53 knockdown (mean, 6 months). Similarly, ~70% of Nf1+/- mice with GFAP-Cre-mediated Nf1 gene inactivation (GFAP-Cre; Nf1(flox/null) mice) developed low-grade MPNSTs following p53 knockdown (mean, 3 months). In addition, wild-type and Nf1+/- mice with GFAP-Cre-mediated Nf1 loss develop MPNSTs following somatic p53 knockout with different latencies, suggesting potential influences of Nf1+/- stromal cells in MPNST pathogenesis. Collectively, this new MPNST model system permits the analysis of somatically-acquired events as well as tumor microenvironment signals that potentially cooperate with Nf1 loss in the development and progression of this deadly malignancy.

  12. Spontaneous and irradiation-induced tumor susceptibility in BRCA2 germline mutant mice and cooperative effects with a p53 germline mutation.

    PubMed

    McAllister, Kimberly A; Houle, Christopher D; Malphurs, Jason; Ward, Toni; Collins, N Keith; Gersch, William; Wharey, Laura; Seely, John C; Betz, Laura; Bennett, L Michelle; Wiseman, Roger W; Davis, Barbara J

    2006-01-01

    Mutations in both p53 and BRCA2 are commonly seen together in human tumors suggesting that the loss of both genes enhances tumor development. To elucidate this interaction in an animal model, mice lacking the carboxy terminal domain of Brca2 were crossed with p53 heterozygous mice. Females from this intercross were then irradiated with an acute dose of 5 Gy ionizing radiation at 5 weeks of age and compared to nonirradiated controls. We found decreased survival and timing of tumor onsets, and significantly higher overall tumor incidences and prevalence of particular tumors, including stomach tumors and squamous cell carcinomas, associated with the homozygous loss of Brca2, independent of p53 status. The addition of a p53 mutation had a further impact on overall survival, incidence of osteosarcomas and stomach tumors, and tumor latency. The spectrum of tumors observed for this Brca2 germline mouse model suggest that it faithfully recapitulates some human disease phenotypes associated with BRCA2 loss. In addition, these findings include extensive in vivo data demonstrating that germline Brca2 and p53 mutations cooperatively affect animal survivals, tumor susceptibilities, and tumor onsets.

  13. ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies

    PubMed Central

    Ishizawa, Jo; Kojima, Kensuke; Chachad, Dhruv; Ruvolo, Peter; Ruvolo, Vivian; Jacamo, Rodrigo O.; Borthakur, Gautam; Mu, Hong; Zeng, Zhihong; Tabe, Yoko; Allen, Joshua E.; Wang, Zhiqiang; Ma, Wencai; Lee, Hans C.; Orlowski, Robert; Sarbassov, Dos D.; Lorenzi, Philip L.; Huang, Xuelin; Neelapu, Sattva S.; McDonnell, Timothy; Miranda, Roberto N.; Wang, Michael; Kantarjian, Hagop; Konopleva, Marina; Davis, R. Eric.; Andreeff, Michael

    2016-01-01

    The clinical challenge posed by p53 abnormalities in hematological malignancies requires therapeutic strategies other than standard genotoxic chemotherapies. ONC201 is a first-in-class small molecule that activates p53-independent apoptosis, has a benign safety profile, and is in early clinical trials. We found that ONC201 caused p53-independent apoptosis and cell cycle arrest in cell lines and in mantle cell lymphoma (MCL) and acute myeloid leukemia (AML) samples from patients; these included samples from patients with genetic abnormalities associated with poor prognosis or cells that had developed resistance to the nongenotoxic agents ibrutinib and bortezomib. Moreover, ONC201 caused apoptosis in stem and progenitor AML cells and abrogated the engraftment of leukemic stem cells in mice while sparing normal bone marrow cells. ONC201 caused changes in gene expression similar to those caused by the unfolded protein response (UPR) and integrated stress responses (ISRs), which increase the translation of the transcription factor ATF4 through an increase in the phosphorylation of the translation initiation factor eIF2α. However, unlike the UPR and ISR, the increase in ATF4 abundance in ONC201-treated hematopoietic cells promoted apoptosis and did not depend on increased phosphorylation of eIF2α. ONC201 also inhibited mammalian target of rapamycin complex 1 (mTORC1) signaling, likely through ATF4-mediated induction of the mTORC1 inhibitor DDIT4. Overexpression of BCL-2 protected against ONC201-induced apoptosis, and the combination of ONC201 and the BCL-2 antagonist ABT-199 synergistically increased apoptosis. Thus, our results suggest that by inducing an atypical ISR and p53-independent apoptosis, ONC201 has clinical potential in hematological malignancies. PMID:26884599

  14. Selenoprotein H is an essential regulator of redox homeostasis that cooperates with p53 in development and tumorigenesis

    PubMed Central

    Cox, Andrew G.; Tsomides, Allison; Kim, Andrew J.; Saunders, Diane; Hwang, Katie L.; Evason, Kimberley J.; Heidel, Jerry; Brown, Kristin K.; Yuan, Min; Lien, Evan C.; Lee, Byung Cheon; Nissim, Sahar; Dickinson, Bryan; Chhangawala, Sagar; Chang, Christopher J.; Asara, John M.; Houvras, Yariv; Gladyshev, Vadim N.; Goessling, Wolfram

    2016-01-01

    Selenium, an essential micronutrient known for its cancer prevention properties, is incorporated into a class of selenocysteine-containing proteins (selenoproteins). Selenoprotein H (SepH) is a recently identified nucleolar oxidoreductase whose function is not well understood. Here we report that seph is an essential gene regulating organ development in zebrafish. Metabolite profiling by targeted LC-MS/MS demonstrated that SepH deficiency impairs redox balance by reducing the levels of ascorbate and methionine, while increasing methionine sulfoxide. Transcriptome analysis revealed that SepH deficiency induces an inflammatory response and activates the p53 pathway. Consequently, loss of seph renders larvae susceptible to oxidative stress and DNA damage. Finally, we demonstrate that seph interacts with p53 deficiency in adulthood to accelerate gastrointestinal tumor development. Overall, our findings establish that seph regulates redox homeostasis and suppresses DNA damage. We hypothesize that SepH deficiency may contribute to the increased cancer risk observed in cohorts with low selenium levels. PMID:27588899

  15. MG132 plus apoptosis antigen-1 (APO-1) antibody cooperate to restore p53 activity inducing autophagy and p53-dependent apoptosis in HPV16 E6-expressing keratinocytes.

    PubMed

    Lagunas-Martínez, Alfredo; García-Villa, Enrique; Arellano-Gaytán, Magaly; Contreras-Ochoa, Carla O; Dimas-González, Jisela; López-Arellano, María E; Madrid-Marina, Vicente; Gariglio, Patricio

    2017-01-01

    The E6 oncoprotein can interfere with the ability of infected cells to undergo programmed cell death through the proteolytic degradation of proapoptotic proteins such as p53, employing the proteasome pathway. Therefore, inactivation of the proteasome through MG132 should restore the activity of several proapoptotic proteins. We investigated whether in HPV16 E6-expressing keratinocytes (KE6 cells), the restoration of p53 levels mediated by MG132 and/or activation of the CD95 pathway through apoptosis antigen-1 (APO-1) antibody are responsible for the induction of apoptosis. We found that KE6 cells underwent apoptosis mainly after incubation for 24 h with MG132 alone or APO-1 plus MG132. Both treatments activated the extrinsic and intrinsic apoptosis pathways. Autophagy was also activated, principally by APO-1 plus MG132. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in the elevation of p53 protein levels and its phosphorylation in Ser46 and Ser20; the p53 protein was localized mainly at nucleus after treatment with MG132 or APO-1 plus MG132. In addition, induction of its transcriptional target genes such as p21, Bax and TP53INP was observed 3 and 6 h after treatment. Also, LC3 mRNA was induced after 3 and 6 h, which correlates with lipidation of LC3B protein and induction of autophagy. Finally, using pifithrin alpha we observed a decrease in apoptosis induced by MG132, and by APO-1 plus MG132, suggesting that restoration of APO-1 sensitivity occurs in part through an increase in both the levels and the activity of p53. The use of small molecules to inhibit the proteasome pathway might permit the activation of cell death, providing new opportunities for CC treatment.

  16. Hypoxia and hypoxia mimetic cooperate to counteract tumor cell resistance to glucose starvation preferentially in tumor cells with mutant p53.

    PubMed

    Chavez-Perez, Valery A; Strasberg-Rieber, Mary; Rieber, Manuel

    2014-01-03

    We demonstrated that exogenous pyruvate promotes survival under glucose depletion in aerobic mutant p53 (R175H) human melanoma cells. Others subsequently indicated that mutant p53 tumor cells undergo p53 degradation and cell death under aerobic glucose-free conditions. Since glucose starvation occurs in hypoxic gradients of poorly vascularized tumors, we investigated the role of p53 siRNA under hypoxia in wt p53 C8161 melanoma using glucose starvation or 5mM physiological glucose. p53 Silencing decreased survival of glucose-starved C8161 melanoma with pyruvate supplementation under hypoxia (≤1% oxygen), but increased resistance to glycolytic inhibitors oxamate and 2-deoxyglucose in 5mM glucose, preferentially under normoxia. Aiming to counteract hypoxic tumor cell survival irrespective of p53 status, genetically-matched human C8161 melanoma harboring wt p53 or mutant p53 (R175H) were used combining true hypoxia (≤1% oxygen) and hypoxia mimetic CoCl2. No significant decrease in metabolic activity was evidenced in C8161 melanoma irrespective of p53 status in 2.5mM glucose after 48h of physical hypoxia. However, combining the latter with 100μM CoCl2 was preferentially toxic for mutant p53 C8161 melanoma, and was enhanced by catalase in wt p53 C8161 cells. Downregulation of MnSOD and LDHA accompanied the toxicity induced by hypoxia and CoCl2 in 5mM glucose, and these changes were enhanced by oxamate or 2-deoxyglucose. Our results show for the first time that survival of malignant cells in a hypoxic microenvironment can be counteracted by hypoxia mimetic co-treatment in a p53 dependent manner.

  17. The Enigma of p53.

    PubMed

    Lozano, Guillermina

    2016-12-08

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

  18. Integrated Stochastic Model of DNA Damage Repair by Non-homologous End Joining and p53/p21- Mediated Early Senescence Signalling

    PubMed Central

    Nelson, Glyn; Hall, Philip; Miwa, Satomi; Kirkwood, Thomas B. L.; Shanley, Daryl P.

    2015-01-01

    Unrepaired or inaccurately repaired DNA damage can lead to a range of cell fates, such as apoptosis, cellular senescence or cancer, depending on the efficiency and accuracy of DNA damage repair and on the downstream DNA damage signalling. DNA damage repair and signalling have been studied and modelled in detail separately, but it is not yet clear how they integrate with one another to control cell fate. In this study, we have created an integrated stochastic model of DNA damage repair by non-homologous end joining and of gamma irradiation-induced cellular senescence in human cells that are not apoptosis-prone. The integrated model successfully explains the changes that occur in the dynamics of DNA damage repair after irradiation. Simulations of p53/p21 dynamics after irradiation agree well with previously published experimental studies, further validating the model. Additionally, the model predicts, and we offer some experimental support, that low-dose fractionated irradiation of cells leads to temporal patterns in p53/p21 that lead to significant cellular senescence. The integrated model is valuable for studying the processes of DNA damage induced cell fate and predicting the effectiveness of DNA damage related medical interventions at the cellular level. PMID:26020242

  19. Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression.

    PubMed

    Valenti, Fabio; Ganci, Federica; Fontemaggi, Giulia; Sacconi, Andrea; Strano, Sabrina; Blandino, Giovanni; Di Agostino, Silvia

    2015-03-20

    Genomic instability (IN) is a common feature of many human cancers. The TP53 tumour suppressor gene is mutated in approximately half of human cancers. Here, we show that BRCA1 and RAD17 genes, whose derived proteins play a pivotal role in DNA damage repair, are transcriptional targets of gain-of-function mutant p53 proteins. Indeed, high levels of mutp53 protein facilitate DNA damage accumulation and severely impair BRCA1 and RAD17 expression in proliferating cancer cells. The recruitment of mutp53/E2F4 complex onto specific regions of BRCA1 and RAD17 promoters leads to the inhibition of their expression. BRCA1 and RAD17 mRNA expression is reduced in HNSCC patients carrying TP53 mutations when compared to those bearing wt-p53 gene. Furthermore, the analysis of gene expression databases for breast cancer patients reveals that low expression of DNA repair genes correlates significantly with reduced relapse free survival of patients carrying TP53 gene mutations. Collectively, these findings highlight the direct involvement of transcriptionally active gain of function mutant p53 proteins in genomic instability through the impairment of DNA repair mechanisms.

  20. Annexin A2 and PSF proteins interact with p53 IRES and regulate translation of p53 mRNA.

    PubMed

    Sharathchandra, Arandkar; Lal, Ridhima; Khan, Debjit; Das, Saumitra

    2012-12-01

    p53 mRNA has been shown to be translated into two isoforms, full-length p53 (FL-p53) and a truncated isoform ΔN-p53, which modulates the functions of FL-p53 and also has independent functions. Previously, we have shown that translation of p53 and ΔN-p53 can be initiated at Internal Ribosome Entry Sites (IRES). These two IRESs were shown to regulate the translation of p53 and ΔN-p53 in a distinct cell-cycle phase-dependent manner. Earlier observations from our laboratory also suggest that the structural integrity of the p53 RNA is critical for IRES function and is compromised by mutations that affect the structure as well as RNA protein interactions. In the current study, using RNA affinity approach we have identified Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) as novel ITAFs for p53 IRESs. We have showed that the purified Annexin A2 and PSF proteins specifically bind to p53 IRES elements. Interestingly, in the presence of calcium ions Annexin A2 showed increased binding with p53 IRES. Immunopulldown experiments suggest that these two proteins associate with p53 mRNA ex vivo as well. Partial knockdown of Annexin A2 and PSF showed decrease in p53 IRES activity and reduced levels of both the p53 isoforms. More importantly the interplay between Annexin A2, PSF and PTB proteins for binding to p53mRNA appears to play a crucial role in IRES function. Taken together, our observations suggest pivotal role of two new trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.

  1. Quantitative proteomics analysis integrated with microarray data reveals that extracellular matrix proteins, catenins, and p53 binding protein 1 are important for chemotherapy response in ovarian cancers.

    PubMed

    Pan, Sheng; Cheng, Lihua; White, James T; Lu, Wei; Utleg, Angelita G; Yan, Xiaowei; Urban, Nicole D; Drescher, Charles W; Hood, Leroy; Lin, Biaoyang

    2009-08-01

    Chemotherapy with carboplatin and paclitaxel is the standard treatment for ovarian cancer patients. Although most patients initially respond to this treatment, few are cured. Resistance to chemotherapy is the major cause of treatment failure. We applied a quantitative proteomic approach based on ICAT/MS/MS technology to analyze tissues harvested at primary debulking surgery before the initiation of combination chemotherapy in order to identify potential naive or intrinsic chemotherapy response proteins in ovarian cancers. We identified 44 proteins that are overexpressed, and 34 proteins that are underexpressed in the chemosensitive tissue compared to the chemoresistant tissue. The overexpressed proteins identified in the chemoresistant tissue include 10 proteins (25.6%) belonging to the extracellular matrix (ECM), including decorin, versican, basigin (CD147), fibulin-1, extracellular matrix protein 1, biglycan, fibronectin 1, dermatopontin, alpha-cardiac actin (smooth muscle actin), and an EGF-containing fibulin-like extracellular matrix protein 1. Interesting proteins identified as overexpressed in the chemosensitive tissue include gamma-catenin (junction plakoglobin) and delta-catenin, tumor suppressor p53-binding protein 1 (53BP1), insulin-like growth factor-binding protein 2 (IGFBP2), proliferating cell nuclear antigen (PCNA), annexin A11, and 53 kDa selenium binding protein 1. Integrative analysis with expression profiling data of eight chemoresistant tissues and 13 chemosensitive tissues revealed that 16 proteins showed consistent changes at both the protein and the RNA levels. These include P53 binding protein 1, catenin delta 1 and plakoglobin, EGF-containing fibulin-like extracellular matrix protein 1 and voltage-dependent anion-selective channel protein 1. Our results suggest that chemotherapy response may be determined by multiple and complex system properties involving extracellular-matrix, cell adhesion and junction proteins.

  2. Rap2b, a novel p53 target, regulates p53-mediated pro-survival function

    PubMed Central

    Zhang, Xinyue; He, Yunlong; Lee, Kyoung-Hwa; Dubois, Wendy; Li, Ziqing; Wu, Xiaolin; Kovalchuk, Alexander; Zhang, Weimin; Huang, Jing

    2013-01-01

    The tumor suppressor p53 is a critical regulator of apoptosis and cell cycle arrest/pro-survival. Upon DNA damage, p53 evokes both cell cycle arrest/pro-survival and apoptosis transcriptional programs. The ultimate cellular outcome depends on the balance of these two programs. However, the p53 downstream targets that mediate this cell fate decision remain to be identified. Using an integrative genomic approach, we identify Rap2b as a conserved p53-activated gene that counters p53-mediated apoptosis after DNA damage. Upon DNA damage, p53 directly binds to the promoter of Rap2b and activates its transcription. The reduction of Rap2b levels by small interference RNA sensitizes cells to DNA damage-induced apoptosis in a p53-dependent manner. Consistent with its pro-survival function, analysis of cancer genomic data reveals that Rap2b is overexpressed in many types of tumors. Anchorage-independent growth assays show that Rap2b has only weak transformation activity, suggesting that it is not an oncogene by itself. Together, our results identify Rap2b as a new player in the pro-survival program conducted by p53 and raise the possibility that targeting Rap2b could sensitize tumor cells to apoptosis in response to DNA damage. PMID:23535297

  3. p53-Regulated Networks of Protein, mRNA, miRNA, and lncRNA Expression Revealed by Integrated Pulsed Stable Isotope Labeling With Amino Acids in Cell Culture (pSILAC) and Next Generation Sequencing (NGS) Analyses*

    PubMed Central

    Hünten, Sabine; Kaller, Markus; Drepper, Friedel; Oeljeklaus, Silke; Bonfert, Thomas; Erhard, Florian; Dueck, Anne; Eichner, Norbert; Friedel, Caroline C.; Meister, Gunter; Zimmer, Ralf; Warscheid, Bettina; Hermeking, Heiko

    2015-01-01

    We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics (pulsed stable isotope labeling with amino acids in cell culture/pSILAC) in the colorectal cancer cell line SW480. This was combined with mRNA and noncoding RNA expression analyses by next generation sequencing (RNA-, miR-Seq). Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated proteins (542 up, 569 down), mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down) and lncRNAs (270 up, 123 down). Changes in protein and mRNA expression levels showed a positive correlation (r = 0.50, p < 0.0001). In total, we detected 133 direct p53 target genes that were differentially expressed and displayed p53 occupancy in the vicinity of their promoter. More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the down-regulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3′-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed up-regulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibits proliferation in SW480 cells. Furthermore, KLF12, HMGB1 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486–5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of KLF12, HMGB1 and CIT was detected in advanced stages of cancer. In conclusion, the integration of multiple omics methods allowed the comprehensive identification of direct and indirect effectors of p53 that provide new insights and leads into the

  4. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53

    PubMed Central

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1–393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using “hot-spot” p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  5. p53 as an Effector or Inhibitor of Therapy Response.

    PubMed

    Ablain, Julien; Poirot, Brigitte; Esnault, Cécile; Lehmann-Che, Jacqueline; de Thé, Hugues

    2015-12-04

    Although integrity of the p53 signaling pathway in a given tumor was expected to be a critical determinant of response to therapies, most clinical studies failed to link p53 status and treatment outcome. Here, we present two opposite situations: one in which p53 is an essential effector of cure by targeted leukemia therapies and another one in advanced breast cancers in which p53 inactivation is required for the clinical efficacy of dose-dense chemotherapy. If p53 promotes or blocks therapy response, therapies must be tailored on its status in individual tumors.

  6. p53: its mutations and their impact on transcription.

    PubMed

    Vaughan, Catherine; Pearsall, Isabella; Yeudall, Andrew; Deb, Swati Palit; Deb, Sumitra

    2014-01-01

    p53 is a tumor suppressor protein whose key function is to maintain the integrity of the cell. Mutations in p53 have been found in up to 50 % of all human cancers and cause an increase in oncogenic phenotypes such as proliferation and tumorigenicity. Both wild-type and mutant p53 have been shown to transactivate their target genes, either through directly binding to DNA, or indirectly through protein-protein interactions. This review discusses possible mechanisms behind both wild-type and mutant p53-mediated transactivation and touches on the concept of addiction to mutant p53 of cancer cells and how that may be used for future therapies.

  7. Overexpression of PDGFRA cooperates with loss of NF1 and p53 to accelerate the molecular pathogenesis of malignant peripheral nerve sheath tumors.

    PubMed

    Ki, D H; He, S; Rodig, S; Look, A T

    2017-02-23

    Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, frequently metastatic sarcomas that are associated with neurofibromatosis type 1 (NF1), a prominent inherited genetic disease in humans. Although loss of the NF1 gene predisposes to MPNST induction, relatively long tumor latency in NF1 patients suggests that additional genetic or epigenetic abnormalities are needed for the development of these nerve sheath malignancies. To study the molecular pathways contributing to the formation of MPNSTs in NF1 patients, we used a zebrafish tumor model defined by nf1 loss in a p53-deficient background together with the overexpression of either wild-type or constitutively activated PDGFRA (platelet-derived growth factor receptor-α) under control of the sox10 neural crest-specific promoter. Here we demonstrate the accelerated onset and increased penetrance of MPNST formation in fish overexpressing both the wild-type and the mutant PDGFRA transgenes in cells of neural crest origin. Interestingly, overexpression of the wild-type PDGFRA was even more potent in promoting transformation than the mutant PDGFRA, which is important because ~78% of human MPNSTs have expression of wild-type PDGFRA, whereas only 5% harbor activating mutations of the gene encoding this receptor. Further analysis revealed the induction of cellular senescence in zebrafish embryos overexpressing mutant, but not wild-type, PDGFRA, suggesting a mechanism through which the oncogenic activity of the mutant receptor is tempered by the activation of premature cellular senescence in an NF1-deficient background. Taken together, our study suggests a model in which overexpression of wild-type PDGFRA associated with NF1 deficiency leads to aberrant activation of downstream RAS signaling and thus contributes importantly to MPNST development-a prediction supported by the ability of the kinase inhibitor sunitinib alone and in combination with the MEK inhibitor trametinib to retard MPNST progression in

  8. Overexpression of PDGFRA cooperates with loss of NF1 and p53 to accelerate the molecular pathogenesis of malignant peripheral nerve sheath tumors

    PubMed Central

    Ki, D H; He, S; Rodig, S; Look, A T

    2017-01-01

    Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, frequently metastatic sarcomas that are associated with neurofibromatosis type 1 (NF1), a prominent inherited genetic disease in humans. Although loss of the NF1 gene predisposes to MPNST induction, relatively long tumor latency in NF1 patients suggests that additional genetic or epigenetic abnormalities are needed for the development of these nerve sheath malignancies. To study the molecular pathways contributing to the formation of MPNSTs in NF1 patients, we used a zebrafish tumor model defined by nf1 loss in a p53-deficient background together with the overexpression of either wild-type or constitutively activated PDGFRA (platelet-derived growth factor receptor-α) under control of the sox10 neural crest-specific promoter. Here we demonstrate the accelerated onset and increased penetrance of MPNST formation in fish overexpressing both the wild-type and the mutant PDGFRA transgenes in cells of neural crest origin. Interestingly, overexpression of the wild-type PDGFRA was even more potent in promoting transformation than the mutant PDGFRA, which is important because ~78% of human MPNSTs have expression of wild-type PDGFRA, whereas only 5% harbor activating mutations of the gene encoding this receptor. Further analysis revealed the induction of cellular senescence in zebrafish embryos overexpressing mutant, but not wild-type, PDGFRA, suggesting a mechanism through which the oncogenic activity of the mutant receptor is tempered by the activation of premature cellular senescence in an NF1-deficient background. Taken together, our study suggests a model in which overexpression of wild-type PDGFRA associated with NF1 deficiency leads to aberrant activation of downstream RAS signaling and thus contributes importantly to MPNST development—a prediction supported by the ability of the kinase inhibitor sunitinib alone and in combination with the MEK inhibitor trametinib to retard MPNST progression in

  9. Regulation of p53 during senescence in normal human keratinocytes

    PubMed Central

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

    2015-01-01

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

  10. p53: out of Africa.

    PubMed

    Lane, David

    2016-04-15

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

  11. p53 regulation upon genotoxic stress: intricacies and complexities

    PubMed Central

    Kumari, Rajni; Kohli, Saishruti; Das, Sanjeev

    2014-01-01

    p53, the revered savior of genomic integrity, receives signals from diverse stress sensors and strategizes to maintain cellular homeostasis. However, the predominance of p53 overshadows the fact that this herculean task is no one-man show; rather, there is a huge army of regulators that reign over p53 at various levels to avoid an unnecessary surge in its levels and sculpt it dynamically to favor one cellular outcome over another. This governance starts right at the time of p53 translation, which is gated by proteins that bind to p53 mRNA and keep a stringent check on p53 protein levels. The same effect is also achieved by ubiquitylases and deubiquitylases that fine-tune p53 turnover and miRNAs that modulate p53 levels, adding precision to this entire scheme. In addition, extensive covalent modifications and differential protein interactions allow p53 to trigger a tailor-made response for a given circumstance. To magnify the marvel, these various tiers of regulation operate simultaneously and in various combinations. In this review, we have tried to provide a glimpse into this bewildering labyrinth. We believe that further studies will result in a better understanding of p53 regulation and that new insights will help unravel many aspects of cancer biology. PMID:27308356

  12. C-Abl as a modulator of p53

    SciTech Connect

    Levav-Cohen, Yaara; Goldberg, Zehavit; Zuckerman, Valentina; Grossman, Tamar; Haupt, Sue; Haupt, Ygal . E-mail: haupt@md.huji.ac.il

    2005-06-10

    P53 is renowned as a cellular tumor suppressor poised to instigate remedial responses to various stress insults that threaten DNA integrity. P53 levels and activities are kept under tight regulation involving a complex network of activators and inhibitors, which determine the type and extent of p53 growth inhibitory signaling. Within this complexity, the p53-Mdm2 negative auto-regulatory loop serves as a major route through which intra- and extra-cellular stress signals are channeled to appropriate p53 responses. Mdm2 inhibits p53 transcriptional activities and through its E3 ligase activity promotes p53 proteasomal degradation either within the nucleus or following nuclear export. Upon exposure to stress signals these actions of Mdm2 have to be moderated, or even interrupted, in order to allow sufficient p53 to accumulate in an active form. Multiple mechanisms involving a variety of factors have been demonstrated to mediate this interruption. C-Abl is a critical factor that under physiological conditions is required for the maximal and efficient accumulation of active p53 in response to DNA damage. C-Abl protects p53 by antagonizing the inhibitory effect of Mdm2, an action that requires a direct interplay between c-Abl and Mdm2. In addition, c-Abl protects p53 from other inhibitors of p53, such as the HPV-E6/E6AP complex, that inhibits and degrades p53 in HPV-infected cells. Surprisingly, the oncogenic form of c-Abl, the Bcr-Abl fusion protein in CML cells, also promotes the accumulation of wt p53. However, in contrast to the activation of p53 by c-Abl, its oncogenic form, Bcr-Abl, counteracts the growth inhibitory activities of p53 by modulating the p53-Mdm2 loop. Thus, it appears that by modulating the p53-Mdm2 loop, c-Abl and its oncogenic forms critically determine the type and extent of the cellular response to DNA damage.

  13. p53 facilitates pRb cleavage in IL-3-deprived cells: novel pro-apoptotic activity of p53.

    PubMed Central

    Gottlieb, E; Oren, M

    1998-01-01

    In the interleukin-3 (IL-3)-dependent lymphoid cell line DA-1, functional p53 is required for efficient apoptosis in response to IL-3 withdrawal. Activation of p53 in these cells, by either DNA damage or p53 overexpression, results in a vital growth arrest in the presence of IL-3 and in accelerated apoptosis in its absence. Thus, IL-3 can control the choice between p53-dependent cell-cycle arrest and apoptosis. Here we report that the cross-talk between p53 and IL-3 involves joint control of pRb cleavage and degradation. Depletion of IL-3 results in caspase-mediated pRb cleavage, occurring preferentially within cells which express functional p53. Moreover, pRb can be cleaved efficiently by extracts prepared from DA-1 cells but not from their derivatives which lack p53 function. Inactivation of pRb through expression of the human papillomavirus (HPV) E7 oncogene overrides the effect of IL-3 in a p53-dependent manner. Our data suggest a novel role for p53 in the regulation of cell death and a novel mechanism for the cooperation between p53 and survival factor deprivation. Thus, p53 makes cells permissive to pRb cleavage, probably by controlling the potential activity of a pRb-cleaving caspase, whereas IL-3 withdrawal provides signals that turn on this potential activity and lead to the actual cleavage and subsequent degradation of pRb. Elimination of a presumptive anti-apoptotic effect of pRb may then facilitate conversion of p53-mediated growth arrest into apoptosis. PMID:9649429

  14. Integration of Genomic, Biologic, and Chemical Approaches to Target p53 Loss and Gain-of-Function in Triple Negative Breast Cancer

    DTIC Science & Technology

    2014-09-01

    in this renewal: p53 triple negative breast cancer subtypes gene expression somatic cell genetics CRISPR /Cas 3. OVERALL PROJECT SUMMARY...to the efficacy of the synthetic lethality screen. In addition, we have optimized the use of CRISPR /Cas, a novel somatic cell recombination...completing this stage of the research within the upcoming Year 2 of the award period. Figure 1. CRISPR /Cas-mediated in vitro somatic cell

  15. Discovery of Novel Isatin-Based p53 Inducers

    PubMed Central

    2015-01-01

    A series of isatin Schiff base derivatives were identified during in silico screening of the small molecule library for novel activators of p53. The compounds selected based on molecular docking results were further validated by a high-content screening assay using U2OS human osteosarcoma cells with an integrated EGFP-expressing p53-dependent reporter. The hit compounds activated and stabilized p53, as shown by Western blotting, at higher rates than the well-known positive control Nutlin-3. Thus, the p53-activating compounds identified by this approach represent useful molecular probes for various cancer studies. PMID:26288684

  16. On p53 revival using system oriented drug dosage design.

    PubMed

    Haseeb, Muhammad; Azam, Shumaila; Bhatti, A I; Azam, Rizwan; Ullah, Mukhtar; Fazal, Sahar

    2017-02-21

    We propose a new paradigm in the drug design for the revival of the p53 pathway in cancer cells. It is shown that the current strategy of using small molecule based Mdm2 inhibitors is not enough to adequately revive p53 in cancerous cells, especially when it comes to the extracting pulsating behavior of p53. This fact has come to notice when a novel method for the drug dosage design is introduced using system oriented concepts. As a test case, small molecule drug Mdm2 repressor Nutlin 3a is considered. The proposed method determines the dose of Nutlin to revive p53 pathway functionality. For this purpose, PBK dynamics of Nutlin have also been integrated with p53 pathway model. The p53 pathway is the focus of researchers for the last thirty years for its pivotal role as a frontline cancer suppressant protein due to its effect on cell cycle checkpoints and cell apoptosis in response to a DNA strand break. That is the reason for finding p53 being absent in more than 50% of tumor cancers. Various drugs have been proposed to revive p53 in cancer cells. Small molecule based drugs are at the foremost and are the subject of advanced clinical trials. The dosage design of these drugs is an important issue. We use control systems concepts to develop the drug dosage so that the cancer cells can be treated in appropriate time. We investigate by using a computational model how p53 protein responds to drug Nutlin 3a, an agent that interferes with the MDM2-mediated p53 regulation. The proposed integrated model describes in some detail the regulation network of p53 including the negative feedback loop mediated by MDM2 and the positive feedback loop mediated by Mdm2 mRNA as well as the reversible represses of MDM2 caused by Nutlin. The reported PBK dynamics of Nutlin 3a are also incorporated to see the full effect. It has been reported that p53 response to stresses in two ways. Either it has a sustained (constant) p53 response, or there are oscillations in p53 concentration. The

  17. INGN 201: Ad-p53, Ad5CMV-p53, Adenoviral p53, INGN 101, p53 gene therapy--Introgen, RPR/INGN 201.

    PubMed

    2003-01-01

    undergoing phase I trials for the potential treatment of lung, breast, ovarian, bladder, liver and brain cancers. Introgen and Aventis Pharma had signed a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute (NCI). NCI will sponsor clinical trials to evaluate and develop RPR/INGN 201 as a potential anticancer agent for these cancer indications. The trials conducted under a NCI-sponsored IND will evaluate RPR/INGN 201 alone and in combination with other anticancer agents. This agreement was originally signed by Rhône-Poulenc Rorer's Gencell. Introgen has completed three phase I clinical trials with INGN 201 in patients with bronchioalveolar cell lung carcinoma, ovarian cancer and recurrent glioblastomas, respectively. Intratumoural injection of RPR/INGN 201 in patients with recurrent glioblastomas was well tolerated and resulted in expression of the p53 protein. Direct administration of RPR/INGN 201 to the lower airways of patients with bronchioalveolar cell lung carcinoma resulted in symptomatic improvement and improved lung function in some patients. In February 2003, Introgen announced that the US Patent and Trademark Office has issued to The Board of Regents of The University of Texas System, patent No. 6,511,847 entitled "Recombinant p53 Adenovirus Methods and Compositions". Introgen Therapeutics is the exclusive licensee of this patent. The patent covers any adenoviral DNA molecules that encode the p53 gene positioned under the control of a promoter. Such a DNA molecule forms the genetic core of Introgen's ADVEXIN cancer therapy. Introgen's ADVEXIN therapy is now covered by up to ten separate US patents relevant to the product including compositions, therapeutic methods of administering the product in virtually any form, alone and in conjunction with the most widely used chemotherapeutic and radiation treatments, as well as its production. Introgen has a number of US patents that relate to the clinical use of ADVEXIN in cancer as

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

    PubMed Central

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

    2012-01-01

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

  19. Regulation of Mutant p53 Protein Expression.

    PubMed

    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.

  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. Energetic Landscape of MDM2-p53 Interactions by Computational Mutagenesis of the MDM2-p53 Interaction.

    PubMed

    Thayer, Kelly M; Beyer, George A

    2016-01-01

    The ubiquitin ligase MDM2, a principle regulator of the tumor suppressor p53, plays an integral role in regulating cellular levels of p53 and thus a prominent role in current cancer research. Computational analysis used MUMBO to rotamerize the MDM2-p53 crystal structure 1YCR to obtain an exhaustive search of point mutations, resulting in the calculation of the ΔΔG comprehensive energy landscape for the p53-bound regulator. The results herein have revealed a set of residues R65-E69 on MDM2 proximal to the p53 hydrophobic binding pocket that exhibited an energetic profile deviating significantly from similar residues elsewhere in the protein. In light of the continued search for novel competitive inhibitors for MDM2, we discuss possible implications of our findings on the drug discovery field.

  2. p53 Acetylation: Regulation and Consequences

    PubMed Central

    Reed, Sara M.; Quelle, Dawn E.

    2014-01-01

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer. PMID:25545885

  3. Benzyl Isothiocyanate potentiates p53 signaling and antitumor effects against breast cancer through activation of p53-LKB1 and p73-LKB1 axes

    PubMed Central

    Xie, Bei; Nagalingam, Arumugam; Kuppusamy, Panjamurthy; Muniraj, Nethaji; Langford, Peter; Győrffy, Balázs; Saxena, Neeraj K.; Sharma, Dipali

    2017-01-01

    Functional reactivation of p53 pathway, although arduous, can potentially provide a broad-based strategy for cancer therapy owing to frequent p53 inactivation in human cancer. Using a phosphoprotein-screening array, we found that Benzyl Isothiocynate, (BITC) increases p53 phosphorylation in breast cancer cells and reveal an important role of ERK and PRAS40/MDM2 in BITC-mediated p53 activation. We show that BITC rescues and activates p53-signaling network and inhibits growth of p53-mutant cells. Mechanistically, BITC induces p73 expression in p53-mutant cells, disrupts the interaction of p73 and mutant-p53, thereby releasing p73 from sequestration and allowing it to be transcriptionally active. Furthermore, BITC-induced p53 and p73 axes converge on tumor-suppressor LKB1 which is transcriptionally upregulated by p53 and p73 in p53-wild-type and p53-mutant cells respectively; and in a feed-forward mechanism, LKB1 tethers with p53 and p73 to get recruited to p53-responsive promoters. Analyses of BITC-treated xenografts using LKB1-null cells corroborate in vitro mechanistic findings and establish LKB1 as the key node whereby BITC potentiates as well as rescues p53-pathway in p53-wild-type as well as p53-mutant cells. These data provide first in vitro and in vivo evidence of the integral role of previously unrecognized crosstalk between BITC, p53/LKB1 and p73/LKB1 axes in breast tumor growth-inhibition. PMID:28071670

  4. Lactose binding to human galectin-7 (p53-induced gene 1) induces long-range effects through the protein resulting in increased dimer stability and evidence for positive cooperativity

    PubMed Central

    Ermakova, Elena; Miller, Michelle C; Nesmelova, Irina V; López-Merino, Lara; Berbís, Manuel Alvaro; Nesmelov, Yuri; Tkachev, Yaroslav V; Lagartera, Laura; Daragan, Vladimir A; André, Sabine; Cañada, F Javier; Jiménez-Barbero, Jesús; Solís, Dolores; Gabius, Hans-Joachim; Mayo, Kevin H

    2013-01-01

    The product of p53-induced gene 1 is a member of the galectin family, i.e., galectin-7 (Gal-7). To move beyond structural data by X-ray diffraction, we initiated the study of the lectin by nuclear magnetic resonance (NMR) and circular dichroism spectroscopies, and molecular dynamics (MD) simulations. In concert, our results indicate that lactose binding to human Gal-7 induces long-range effects (minor conformational shifts and changes in structural dynamics) throughout the protein that result in stabilization of the dimer state, with evidence for positive cooperativity. Monte Carlo fits of 15N-Gal-7 HSQC titrations with lactose using a two-site model yield K1 = 0.9 ± 0.6 × 103 M−1 and K2 = 3.4 ± 0.8 × 103 M−1. Ligand binding-induced stabilization of the Gal-7 dimer was supported by several lines of evidence: MD-based calculations of interaction energies between ligand-loaded and ligand-free states, gel filtration data and hetero-FRET spectroscopy that indicate a highly reduced tendency for dimer dissociation in the presence of lactose, CD-based thermal denaturation showing that the transition temperature of the lectin is significantly increased in the presence of lactose, and saturation transfer difference (STD) NMR using a molecular probe of the monomer state whose presence is diminished in the presence of lactose. MD simulations with the half-loaded ligand-bound state also provided insight into how allosteric signaling may occur. Overall, our results reveal long-range effects on Gal-7 structure and dynamics, which factor into entropic contributions to ligand binding and allow further comparisons with other members of the galectin family. PMID:23376190

  5. Mechanisms of p53-Mediated Apoptosis

    DTIC Science & Technology

    2007-03-01

    See Figure 4 and Figure 5 in Appendix, Harms and Chen, 2007). Specifically the p53 target genes p21, Mdm2, FDXR, and DKK1 are induced to a greater...repression of c-Myc in a manner that partly depends on p53 • Knockdown of HDAC2 augments the induction of p53 target genes p21, Mdm2, FDXR, and DKK1 ...induction of p21, Mdm2, ferrodoxin reductase (FDXR), and dickkopf-1 ( DKK1 ) by p53. The enhancement of p53 trans-repression and trans-activation was

  6. The critical role of catalase in prooxidant and antioxidant function of p53.

    PubMed

    Kang, M Y; Kim, H-B; Piao, C; Lee, K H; Hyun, J W; Chang, I-Y; You, H J

    2013-01-01

    The tumor suppressor p53 is an important regulator of intracellular reactive oxygen species (ROS) levels, although downstream mediators of p53 remain to be elucidated. Here, we show that p53 and its downstream targets, p53-inducible ribonucleotide reductase (p53R2) and p53-inducible gene 3 (PIG3), physically and functionally interact with catalase for efficient regulation of intracellular ROS, depending on stress intensity. Under physiological conditions, the antioxidant functions of p53 are mediated by p53R2, which maintains increased catalase activity and thereby protects against endogenous ROS. After genotoxic stress, high levels of p53 and PIG3 cooperate to inhibit catalase activity, leading to a shift in the oxidant/antioxidant balance toward an oxidative status, which could augment apoptotic cell death. These results highlight the essential role of catalase in p53-mediated ROS regulation and suggest that the p53/p53R2-catalase and p53/PIG3-catalase pathways are critically involved in intracellular ROS regulation under physiological conditions and during the response to DNA damage, respectively.

  7. The p53 core domain is a molten globule at low pH: functional implications of a partially unfolded structure.

    PubMed

    Bom, Ana Paula D Ano; Freitas, Monica S; Moreira, Flavia S; Ferraz, Danielly; Sanches, Daniel; Gomes, Andre M O; Valente, Ana Paula; Cordeiro, Yraima; Silva, Jerson L

    2010-01-22

    p53 is a transcription factor that maintains genome integrity, and its function is lost in 50% of human cancers. The majority of p53 mutations are clustered within the core domain. Here, we investigate the effects of low pH on the structure of the wild-type (wt) p53 core domain (p53C) and the R248Q mutant. At low pH, the tryptophan residue is partially exposed to the solvent, suggesting a fluctuating tertiary structure. On the other hand, the secondary structure increases, as determined by circular dichroism. Binding of the probe bis-ANS (bis-8-anilinonaphthalene-1-sulfonate) indicates that there is an increase in the exposure of hydrophobic pockets for both wt and mutant p53C at low pH. This behavior is accompanied by a lack of cooperativity under urea denaturation and decreased stability under pressure when p53C is in acidic pH. Together, these results indicate that p53C acquires a partially unfolded conformation (molten-globule state) at low pH (5.0). The hydrodynamic properties of this conformation are intermediate between the native and denatured conformation. (1)H-(15)N HSQC NMR spectroscopy confirms that the protein has a typical molten-globule structure at acidic pH when compared with pH 7.2. Human breast cells in culture (MCF-7) transfected with p53-GFP revealed localization of p53 in acidic vesicles, suggesting that the low pH conformation is present in the cell. Low pH stress also tends to favor high levels of p53 in the cells. Taken together, all of these data suggest that p53 may play physiological or pathological roles in acidic microenvironments.

  8. Regulation of autophagy by cytoplasmic p53.

    PubMed

    Tasdemir, Ezgi; Maiuri, M Chiara; Galluzzi, Lorenzo; Vitale, Ilio; Djavaheri-Mergny, Mojgan; D'Amelio, Marcello; Criollo, Alfredo; Morselli, Eugenia; Zhu, Changlian; Harper, Francis; Nannmark, Ulf; Samara, Chrysanthi; Pinton, Paolo; Vicencio, José Miguel; Carnuccio, Rosa; Moll, Ute M; Madeo, Frank; Paterlini-Brechot, Patrizia; Rizzuto, Rosario; Szabadkai, Gyorgy; Pierron, Gérard; Blomgren, Klas; Tavernarakis, Nektarios; Codogno, Patrice; Cecconi, Francesco; Kroemer, Guido

    2008-06-01

    Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

  9. p53 regulates the cardiac transcriptome

    PubMed Central

    Mak, Tak W.; Hauck, Ludger; Grothe, Daniela; Billia, Filio

    2017-01-01

    The tumor suppressor Trp53 (p53) inhibits cell growth after acute stress by regulating gene transcription. The mammalian genome contains hundreds of p53-binding sites. However, whether p53 participates in the regulation of cardiac tissue homeostasis under normal conditions is not known. To examine the physiologic role of p53 in adult cardiomyocytes in vivo, Cre-loxP–mediated conditional gene targeting in adult mice was used. Genome-wide transcriptome analyses of conditional heart-specific p53 knockout mice were performed. Genome-wide annotation and pathway analyses of >5,000 differentially expressed transcripts identified many p53-regulated gene clusters. Correlative analyses identified >20 gene sets containing more than 1,000 genes relevant to cardiac architecture and function. These transcriptomic changes orchestrate cardiac architecture, excitation-contraction coupling, mitochondrial biogenesis, and oxidative phosphorylation capacity. Interestingly, the gene expression signature in p53-deficient hearts confers resistance to acute biomechanical stress. The data presented here demonstrate a role for p53, a previously unrecognized master regulator of the cardiac transcriptome. The complex contributions of p53 define a biological paradigm for the p53 regulator network in the heart under physiological conditions. PMID:28193895

  10. 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.

  11. 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.

  12. Constant rate of p53 tetramerization in response to DNA damage controls the p53 response

    PubMed Central

    Gaglia, Giorgio; Lahav, Galit

    2014-01-01

    The dynamics of the tumor suppressor protein p53 have been previously investigated in single cells using fluorescently tagged p53. Such approach reports on the total abundance of p53 but does not provide a measure for functional p53. We used fluorescent protein-fragment complementation assay (PCA) to quantify in single cells the dynamics of p53 tetramers, the functional units of p53. We found that while total p53 increases proportionally to the input strength, p53 tetramers are formed in cells at a constant rate. This breaks the linear input–output relation and dampens the p53 response. Disruption of the p53-binding protein ARC led to a dose-dependent rate of tetramers formation, resulting in enhanced tetramerization and induction of p53 target genes. Our work suggests that constraining the p53 response in face of variable inputs may protect cells from committing to terminal outcomes and highlights the importance of quantifying the active form of signaling molecules in single cells. Quantification of the dynamics of p53 tetramers in single cells using a fluorescent protein-fragment complementation assay reveals that, while total p53 increases proportionally to the DNA damage strength, p53 tetramers are formed at a constant rate. PMID:25344068

  13. Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2.

    PubMed

    Heyne, Kristina; Förster, Juliane; Schüle, Roland; Roemer, Klaus

    2014-04-01

    NIR (novel INHAT repressor) can bind to p53 at promoters and inhibit p53-mediated gene transactivation by blocking histone acetylation carried out by p300/CBP. Like NIR, the E3 ubiquitin ligase MDM2 can also bind and inhibit p53 at promoters. Here, we present data indicating that NIR, which shuttles between the nucleolus and nucleoplasm, not only binds to p53 but also directly to MDM2, in part via the central acidic and zinc finger domain of MDM2 that is also contacted by several other nucleolus-based MDM2/p53-regulating proteins. Like some of these, NIR was able to inhibit the ubiquitination of MDM2 and stabilize MDM2; however, unlike these nucleolus-based MDM2 regulators, NIR did not inhibit MDM2 to activate p53. Rather, NIR cooperated with MDM2 to repress p53-induced transactivation. This cooperative repression may at least in part involve p300/CBP. We show that NIR can block the acetylation of p53 and MDM2. Non-acetylated p53 has been documented previously to more readily associate with inhibitory MDM2. NIR may thus help to sustain the inhibitory p53:MDM2 complex, and we present evidence suggesting that all three proteins can indeed form a ternary complex. In sum, our findings suggest that NIR can support MDM2 to suppress p53 as a transcriptional activator.

  14. Regulation of autophagy by cytoplasmic p53

    PubMed Central

    Tasdemir, Ezgi; Maiuri, M. Chiara; Galluzzi, Lorenzo; Vitale, Ilio; Djavaheri-Mergny, Mojgan; D'Amelio, Marcello; Criollo, Alfredo; Morselli, Eugenia; Zhu, Changlian; Harper, Francis; Nannmark, Ulf; Samara, Chrysanthi; Pinton, Paolo; Vicencio, José Miguel; Carnuccio, Rosa; Moll, Ute M.; Madeo, Frank; Paterlini-Brechot, Patrizia; Rizzuto, Rosario; Szabadkai, Gyorgy; Pierron, Gérard; Blomgren, Klas; Tavernarakis, Nektarios; Codogno, Patrice; Cecconi, Francesco; Kroemer, Guido

    2009-01-01

    Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that knockout, knockdown or pharmacological inhibition of p53 can induce autophagy in human, mouse and nematode cells. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53-/- cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53. PMID:18454141

  15. The E7 protein of the cottontail rabbit papillomavirus immortalizes normal rabbit keratinocytes and reduces pRb levels, while E6 cooperates in immortalization but neither degrades p53 nor binds E6AP

    SciTech Connect

    Ganzenmueller, Tina; Matthaei, Markus; Muench, Peter; Scheible, Michael; Iftner, Angelika; Hiller, Thomas; Leiprecht, Natalie; Probst, Sonja; Stubenrauch, Frank; Iftner, Thomas

    2008-03-15

    Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19{sup ARF} induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53.

  16. Mdm2 links genotoxic stress and metabolism to p53.

    PubMed

    Wang, Zhongfeng; Li, Baojie

    2010-12-01

    Mouse double minute 2 (Mdm2) gene was isolated from a cDNA library derived from transformed mouse 3T3 cells, and was classified as an oncogene as it confers 3T3 and Rat2 cells tumorigenicity when overexpressed. It encodes a nucleocytoplasmic shuttling ubiquitin E3 ligase, with its main target being tumor suppressor p53, which is mutated in more than 50% of human primary tumors. Mdm2's oncogenic activity is mainly mediated by p53, which is activated by various stresses, especially genotoxic stress, via Atm (ataxia telangiectasia mutated) and Atr (Atm and Rad3-related). Activated p53 inhibits cell proliferation, induces apoptosis or senescence, and maintains genome integrity. Mdm2 is also a target gene of p53 transcription factor. Thus, Mdm2 and p53 form a feedback regulatory loop. External and internal cues, through multiple signaling pathways, can act on Mdm2 to regulate p53 levels and cell proliferation, death, and senescence. This review will focus on how Mdm2 is regulated under genotoxic stress, and by the Akt1-mTOR-S6K1 pathway that is activated by insulin, growth factors, amino acids, or energy status.

  17. Mitochondrial death functions of p53

    PubMed Central

    Marchenko, N D; Moll, U M

    2014-01-01

    The p53 tumor suppressor network plays a fundamental surveillance role in both homeostatic and adaptive cell biology. p53 is one of the most important barriers against malignant derailment of normal cells, orchestrating growth arrest, senescence, or cell death by linking many different pathways in response to genotoxic and non-genotoxic insults. p53 is the key broadband sensor for numerous cellular stresses such as DNA damage, hypoxia, oxidative stress, oncogenic signaling, and nucleolar stress. The crucial tumor suppressive and tissue homeostasis activity of p53 is its ability to activate cell death via multiple different pathways. A well-characterized biochemical function of p53 in the regulation of apoptosis is its role as a potent transcriptional regulator. p53 activates a panel of proapoptotic genes from the mitochondrial apoptotic and death receptor programs while repressing antiapoptotic Bcl2 family genes. In addition, over the last 10 y a growing body of evidence has also defined direct extranuclear non-transcriptional p53 activities within mitochondria-mediated cell death pathways that are based on p53 protein accumulation in cytosolic and mitochondrial compartments and protein-protein interactions. To date, transcription-independent p53-mediated cell death regulation has been described for apoptosis, necrosis, and autophagy. Because mitochondrial dysregulation is central to the development of a number of pathologic processes such as cancer and neurodegenerative and age-related diseases, understanding the direct roles of p53 protein in mitochondria has high translational impact and could facilitate the development of novel drug targets to combat these diseases. In this review we will mainly focus on mechanisms of p53-mediated transcription-independent cell death pathways at mitochondria. PMID:27308326

  18. The p53-dependent radioadaptive response

    NASA Astrophysics Data System (ADS)

    Ohnishi, Takeo

    We already reported that conditioning exposures at low doses, or at low dose-rates, lowered radiation-induced p53-dependent apoptosis in cultured cells in vitro and in the spleens of mice in vivo. In this study, the aim was to characterize the p53-dependent radioadaptive response at the molecular level. We used wild-type (wt) p53 and mutated (m) p53 containing cells derived from the human lung cancer H1299 cell line, which is p53-null. Cellular radiation sensitivities were determined with a colony-forming assay. The accumulation of p53, Hdm2, and iNOS was analyzed with Western blotting. The quantification of chromosomal aberrations was estimated by scoring dicentrics per cell. In wtp53 cells, it was demonstrated that the lack of p53 accumulation was coupled with the activation of Hdm2 after low dose irradiation (0.02 Gy). Although NO radicals were only minimally induced in wtp53 cells irradiated with a challenging irradiation (6 Gy) alone, NO radicals were seen to increase about 2-4 fold after challenging irradiation following a priming irradiation (0.02 Gy). Under similar irradiation conditions with a priming and challenging irradiation in wtp53 cells, induction of radioresistance and a depression of chromosomal aberrations were observed only in the absence of Pifithrin-α (a p53 inhibitor), RITA or Nutlin-3 (p53-Hdm2 interaction inhibitors), aminoguanidine (an iNOS inhibitor) and c-PTIO (an NO radical scavenger). On the other hand, in p53 dysfunctional cells, a radioadaptive response was not observed in the presence or absence of those inhibitors. Moreover, radioresistance developed when wtp53 cells were treated with ISDN (an NO generating agent) alone. These findings suggest that NO radicals are an initiator of the radioadaptive response acting through the activation of Hdm2 and the depression of p53 accumulations.

  19. p53 responsive elements in human retrotransposons.

    PubMed

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

    2009-11-05

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

  20. SUMOylation of p53 mediates interferon activities

    PubMed Central

    Marcos-Villar, Laura; Pérez-Girón, José V; Vilas, Jéssica M; Soto, Atenea; de la Cruz-Hererra, Carlos F; Lang, Valerie; Collado, Manuel; Vidal, Anxo; Rodríguez, Manuel S; Muñoz-Fontela, César; Rivas, Carmen

    2013-01-01

    There is growing evidence that many host proteins involved in innate and intrinsic immunity are regulated by SUMOylation, and that SUMO contributes to the regulatory process that governs the initiation of the type I interferon (IFN) response. The tumor suppressor p53 is a modulator of the IFN response that plays a role in virus-induced apoptosis and in IFN-induced senescence. Here we demonstrate that IFN treatment increases the levels of SUMOylated p53 and induces cellular senescence through a process that is partially dependent upon SUMOylation of p53. Similarly, we show that vesicular stomatitis virus (VSV) infection induces p53 SUMOylation, and that this modification favors the control of VSV replication. Thus, our study provides evidence that IFN signaling induces p53 SUMOylation, which results in the activation of a cellular senescence program and contributes to the antiviral functions of interferon. PMID:23966171

  1. Dial 9-1-1 for p53: Mechanisms of p53 Activation by Cellular Stress

    PubMed Central

    Ljungman, Mats

    2000-01-01

    Abstract The tumor suppressor protein, p53, is part of the cell's emergency team that is called upon following cellular insult. How do cells sense DNA damage and other cellular stresses and what signal transduction pathways are used to alert p53? How is the resulting nuclear accumulation of p53 accomplished and what determines the outcome of p53 induction? Many posttranslational modifications of p53, such as phosphorylation, dephosphorylation, acetylation and ribosylation, have been shown to occur following cellular stress. Some of these modifications may activate the p53 protein, interfere with MDM2 binding and/or dictate cellular localization of p53. This review will focus on recent findings about how the p53 response may be activated following cellular stress. PMID:10935507

  2. Two faces of p53: aging and tumor suppression

    PubMed Central

    Rodier, Francis; Campisi, Judith; Bhaumik, Dipa

    2007-01-01

    The p53 tumor suppressor protein, often termed guardian of the genome, integrates diverse physiological signals in mammalian cells. In response to stress signals, perhaps the best studied of which is the response to DNA damage, p53 becomes functionally active and triggers either a transient cell cycle arrest, cell death (apoptosis) or permanent cell cycle arrest (cellular senescence). Both apoptosis and cellular senescence are potent tumor suppressor mechanisms that irreversibly prevent damaged cells from undergoing neoplastic transformation. However, both processes can also deplete renewable tissues of proliferation-competent progenitor or stem cells. Such depletion, in turn, can compromise the structure and function of tissues, which is a hallmark of aging. Moreover, whereas apoptotic cells are by definition eliminated from tissues, senescent cells can persist, acquire altered functions, and thus alter tissue microenvironments in ways that can promote both cancer and aging phenotypes. Recent evidence suggests that increased p53 activity can, at least under some circumstances, promote organismal aging. Here, we discuss the role of p53 as a key regulator of the DNA damage responses, and discuss how p53 integrates the outcome of the DNA damage response to optimally balance tumor suppression and longevity. PMID:17942417

  3. The transcription factor CREBZF is a novel positive regulator of p53

    PubMed Central

    López-Mateo, Irene; Villaronga, M. Ángeles; Llanos, Susana; Belandia, Borja

    2012-01-01

    CREBZF is a member of the mammalian ATF/CREB family of transcription factors. Here, we describe a novel functional interaction between CREBZF and the tumor suppressor p53. CREBZF was identified in a yeast two-hybrid screen using HEY1, recently characterized as an indirect p53 activator, as bait. CREBZF interacts in vitro with both HEY1 and p53, and CREBZF expression stabilizes and activates p53. Moreover, CREBZF cooperates synergistically with HEY1 to enhance p53 transcriptional activity. On the other hand, partial depletion of endogenous CREBZF diminishes p53 protein levels and inhibits HEY1-mediated activation of p53. CREBZF-positive effects on p53 signaling may reflect, at least in part, an observed induction of posttranslational modifications in p53 known to prevent its degradation. CREBZF expression protects HCT116 cells from UV radiation-induced cell death. In addition, CREBZF expression confers sensitivity to 5-fluorouracil, a p53-activating chemotherapeutic drug. Our study suggests that CREBZF may participate in the modulation of p53 tumor suppressor function. PMID:22983008

  4. Mutant p53 Promotes Tumor Cell Malignancy by Both Positive and Negative Regulation of the Transforming Growth Factor β (TGF-β) Pathway*

    PubMed Central

    Ji, Lei; Xu, Jinjin; Liu, Jian; Amjad, Ali; Zhang, Kun; Liu, Qingwu; Zhou, Lei; Xiao, Jianru; Li, Xiaotao

    2015-01-01

    Specific p53 mutations abrogate tumor-suppressive functions by gaining new abilities to promote tumorigenesis. Inactivation of p53 is known to distort TGF-β signaling, which paradoxically displays both tumor-suppressive and pro-oncogenic functions. The molecular mechanisms of how mutant p53 simultaneously antagonizes the tumor-suppressive and synergizes the tumor-promoting function of the TGF-β pathway remain elusive. Here we demonstrate that mutant p53 differentially regulates subsets of TGF-β target genes by enhanced binding to the MH2 domain in Smad3 upon the integration of ERK signaling, therefore disrupting Smad3/Smad4 complex formation. Silencing Smad2, inhibition of ERK, or introducing a phosphorylation-defective mutation at Ser-392 in p53 abrogates the R175H mutant p53-dependent regulation of these TGF-β target genes. Our study shows a mechanism to reconcile the seemingly contradictory observations that mutant p53 can both attenuate and cooperate with the TGF-β pathway to promote cancer cell malignancy in the same cell type. PMID:25767119

  5. p53, Oxidative Stress, and Aging

    PubMed Central

    Liu, Dongping

    2011-01-01

    Abstract Mammalian aging is associated with elevated levels of oxidative damage of DNA, proteins, and lipids as a result of unbalanced prooxidant and antioxidant activities. Accumulating evidence indicates that oxidative stress is a major physiological inducer of aging. p53, the guardian of the genome that is important for cellular responses to oxidative stresses, might be a key coordinator of oxidative stress and aging. In response to low levels of oxidative stresses, p53 exhibits antioxidant activities to eliminate oxidative stress and ensure cell survival; in response to high levels of oxidative stresses, p53 exhibits prooxidative activities that further increase the levels of stresses, leading to cell death. p53 accomplishes these context-dependent roles by regulating the expression of a panel of genes involved in cellular responses to oxidative stresses and by modulating other pathways important for oxidative stress responses. The mechanism that switches p53 function from antioxidant to prooxidant remains unclear, but could account for the findings that increased p53 activities have been linked to both accelerated aging and increased life span in mice. Therefore, a balance of p53 antioxidant and prooxidant activities in response to oxidative stresses could be important for longevity by suppressing the accumulation of oxidative stresses and DNA damage. Antioxid. Redox Signal. 15, 1669–1678. PMID:21050134

  6. p53 in the game of transposons.

    PubMed

    Wylie, Annika; Jones, Amanda E; Abrams, John M

    2016-11-01

    Throughout the animal kingdom, p53 genes function to restrain mobile elements and recent observations indicate that transposons become derepressed in human cancers. Together, these emerging lines of evidence suggest that cancers driven by p53 mutations could represent "transpospoathies," i.e. disease states linked to eruptions of mobile elements. The transposopathy hypothesis predicts that p53 acts through conserved mechanisms to contain transposon movement, and in this way, prevents tumor formation. How transposon eruptions provoke neoplasias is not well understood but, from a broader perspective, this hypothesis also provides an attractive framework to explore unrestrained mobile elements as inciters of late-onset idiopathic disease. Also see the video abstract here.

  7. Effects of chronic deoxynivalenol exposure on p53 heterozygous and p53 homozygous mice.

    PubMed

    Bondy, G S; Coady, L; Curran, I; Caldwell, D; Armstrong, C; Aziz, S A; Nunnikhoven, A; Gannon, A M; Liston, V; Shenton, J; Mehta, R

    2016-10-01

    Deoxynivalenol (DON) is a secondary metabolite associated with Fusarium species pathogenic to important food crops. A two-year feeding study reported that DON was non-carcinogenic in B6C3F1 mice. The present study was conducted to further characterize the chronic effects of DON by exposing cancer-prone transgenic p53 heterozygous (p53+/-) male mice and p53 homozygous (p53+/+) male mice to 0, 1, 5, or 10 mg DON/kg in diet for 26 weeks. Gross and microscopic organ-specific neoplastic and non-neoplastic changes and expression profiles of key hepatic and renal genes were assessed. Few toxicologic differences between p53+/+ and p53+/- mice were observed, and no tumours were observed due to DON. The results indicated that DON was non-carcinogenic and that reduced expression of the p53 gene did not play a key role in responses to DON toxicity. The lack of inflammatory and proliferative lesions in mice may be attributed to the anorectic effects of DON, which resulted in dose-dependent reductions in body weight in p53+/+ and p53+/- mice. Hepatic and renal gene expression analyses confirmed that chronic exposure to DON was noninflammatory. The effects of 26-week DON exposure on p53+/+ and p53+/-mice were consistent with those previously seen in B6C3F1 mice exposed to DON for two years.

  8. The PIDDosome activates p53 in response to supernumerary centrosomes

    PubMed Central

    Fava, Luca L.; Schuler, Fabian; Sladky, Valentina; Haschka, Manuel D.; Soratroi, Claudia; Eiterer, Lisa; Demetz, Egon; Weiss, Guenter; Geley, Stephan; Nigg, Erich A.; Villunger, Andreas

    2017-01-01

    Centrosomes, the main microtubule-organizing centers in animal cells, are replicated exactly once during the cell division cycle to form the poles of the mitotic spindle. Supernumerary centrosomes can lead to aberrant cell division and have been causally linked to chromosomal instability and cancer. Here, we report that an increase in the number of mature centrosomes, generated by disrupting cytokinesis or forcing centrosome overduplication, triggers the activation of the PIDDosome multiprotein complex, leading to Caspase-2-mediated MDM2 cleavage, p53 stabilization, and p21-dependent cell cycle arrest. This pathway also restrains the extent of developmentally scheduled polyploidization by regulating p53 levels in hepatocytes during liver organogenesis. Taken together, the PIDDosome acts as a first barrier, engaging p53 to halt the proliferation of cells carrying more than one mature centrosome to maintain genome integrity. PMID:28130345

  9. Smoking, p53 mutation, and lung cancer.

    PubMed

    Gibbons, Don L; Byers, Lauren A; Kurie, Jonathan M

    2014-01-01

    This issue marks the 50th anniversary of the release of the U.S. Surgeon General's Report on Smoking and Health. Perhaps no other singular event has done more to highlight the effects of smoking on the development of cancer. Tobacco exposure is the leading cause of cancers involving the oral cavity, conductive airways, and the lung. Owing to the many carcinogens in tobacco smoke, smoking-related malignancies have a high genome-wide burden of mutations, including in the gene encoding for p53. The p53 protein is the most frequently mutated tumor suppressor in cancer, responsible for a range of critical cellular functions that are compromised by the presence of a mutation. Herein, we review the epidemiologic connection between tobacco exposure and cancer, the molecular basis of p53 mutation in lung cancer, and the normal molecular and cellular roles of p53 that are abrogated during lung tumor development and progression as defined by in vitro and in vivo studies. We also consider the therapeutic potential of targeting mutant p53 in a clinical setting based upon the cellular role of mutant p53 and data from genetic murine models.

  10. 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

  11. p53 regulates thymic Notch1 activation.

    PubMed

    Laws, Amy M; Osborne, Barbara A

    2004-03-01

    Notch is crucial for multiple stages of T cell development, including the CD4+CD8+ double positive (DP)/CD8+ single positive (SP) transition, but regulation of Notchactivation is not well understood. p53 regulates Presenilin1 (PS1) expression, and PS1 cleaves Notch, releasing its intracellular domain (NIC), leading to the expression of downstream targets, e.g. the HES1 gene. We hypothesize that p53 regulates Notch activity during T cell development. We found that Notch1 expression and activation were negatively regulated by p53in several thymoma lines. Additionally, NIC was elevated in Trp53(-/-) thymocytes as compared to Trp53(+/+) thymocytes. To determine if elevated Notch1 activation in Trp53(-/-) thymocytes had an effect on T cell development, CD4 and CD8 expression were analyzed. The CD4+ SP/CD8+ SP T cell ratio was decreased in Trp53(-/-) splenocytes and thymocytes. This alteration in T cell development correlated with the increased Notch1 activation observed in the absence of p53. These data indicate that p53 negatively regulates Notch1 activation during T cell development. Skewing of T cell development toward CD8+SP T cells in Trp53(-/-) mice is reminiscent of the phenotype of NIC-overexpressing mice. Thus, we suggest that p53 plays a role in T cell development, in part by regulating Notch1 activation.

  12. p53 loss promotes acute myeloid leukemia by enabling aberrant self-renewal

    PubMed Central

    Zhao, Zhen; Zuber, Johannes; Diaz-Flores, Ernesto; Lintault, Laura; Kogan, Scott C.; Shannon, Kevin; Lowe, Scott W.

    2010-01-01

    The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. Here, we test whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML), an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. Our approach combined mosaic mouse models, Cre-lox technology, and in vivo RNAi to disable p53 and simultaneously activate endogenous KrasG12D—a common AML lesion that promotes proliferation but not self-renewal. We show that p53 inactivation strongly cooperates with oncogenic KrasG12D to induce aggressive AML, while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells, such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently, myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells, resembling cancer stem cells capable of maintaining AML in vivo. Our results establish an efficient new strategy for interrogating oncogene cooperation, and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity. PMID:20595231

  13. Development of an adenoviral vector with robust expression driven by p53

    SciTech Connect

    Bajgelman, Marcio C.; Strauss, Bryan E.

    2008-02-05

    Here we introduce a new adenoviral vector where transgene expression is driven by p53. We first developed a synthetic promoter, referred to as PGTx{beta}, containing a p53-responsive element, a minimal promoter and the first intron of the rabbit {beta}-globin gene. Initial assays using plasmid-based vectors indicated that expression was tightly controlled by p53 and was 5-fold stronger than the constitutive CMV immediate early promoter/enhancer. The adenoviral vector, AdPG, was also shown to offer p53-responsive expression in prostate carcinoma cells LNCaP (wt p53), DU-145 (temperature sensitive mutant of p53) and PC3 (p53-null, but engineered to express temperature-sensitive p53 mutants). AdPG served as a sensor of p53 activity in LNCaP cells treated with chemotherapeutic agents. Since p53 can be induced by radiotherapy and chemotherapy, this new vector could be further developed for use in combination with conventional therapies to bring about cooperation between the genetic and pharmacologic treatment modalities.

  14. Calcium and S100B Regulation of p53-Dependent Cell Growth Arrest and Apoptosis

    PubMed Central

    Scotto, Christian; Deloulme, Jean Christophe; Rousseau, Denis; Chambaz, Edmond; Baudier, Jacques

    1998-01-01

    In glial C6 cells constitutively expressing wild-type p53, synthesis of the calcium-binding protein S100B is associated with cell density-dependent inhibition of growth and apoptosis in response to UV irradiation. A functional interaction between S100B and p53 was first demonstrated in p53-negative mouse embryo fibroblasts (MEF cells) by sequential transfection with the S100B and the temperature-sensitive p53Val135 genes. We show that in MEF cells expressing a low level of p53Val135, S100B cooperates with p53Val135 in triggering calcium-dependent cell growth arrest and cell death in response to UV irradiation at the nonpermissive temperature (37.5°C). Calcium-dependent growth arrest of MEF cells expressing S100B correlates with specific nuclear accumulation of the wild-type p53Val135 conformational species. S100B modulation of wild-type p53Val135 nuclear translocation and functions was confirmed with the rat embryo fibroblast (REF) cell line clone 6, which is transformed by oncogenic Ha-ras and overexpression of p53Val135. Ectopic expression of S100B in clone 6 cells restores contact inhibition of growth at 37.5°C, which also correlates with nuclear accumulation of the wild-type p53Val135 conformational species. Moreover, a calcium ionophore mediates a reversible G1 arrest in S100B-expressing REF (S100B-REF) cells at 37.5°C that is phenotypically indistinguishable from p53-mediated G1 arrest at the permissive temperature (32°C). S100B-REF cells proceeding from G1 underwent apoptosis in response to UV irradiation. Our data support a model in which calcium signaling and S100B cooperate with the p53 pathways of cell growth inhibition and apoptosis. PMID:9632811

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

    PubMed Central

    Ponchel, F.; Milner, J.

    1998-01-01

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

  16. Interferons alpha and gamma induce p53-dependent and p53-independent apoptosis, respectively.

    PubMed

    Porta, Chiara; Hadj-Slimane, Reda; Nejmeddine, Mohamed; Pampin, Mathieu; Tovey, Michael G; Espert, Lucile; Alvarez, Sandra; Chelbi-Alix, Mounira K

    2005-01-20

    Type I interferon (IFN) enhances the transcription of the tumor suppressor gene p53. To elucidate the molecular mechanism mediating IFN-induced apoptosis, we analysed programmed cell death in response to type I (IFNalpha) or type II (IFNgamma) treatment in relation to p53 status. In two cell lines (MCF-7, SKNSH), IFNalpha, but not IFNgamma, enhanced apoptosis in a p53-dependent manner. Furthermore, only IFNalpha upregulated p53 as well as p53 target genes (Noxa, Mdm2 and CD95). The apoptotic response to IFNalpha decreased in the presence of ZB4, an anti-CD95 antibody, suggesting that CD95 is involved in this process. When p53 was inactivated by the E6 viral protein or the expression of a p53 mutant, IFNalpha-induced apoptosis and p53 target genes upregulation were abrogated. Altogether these results demonstrate that p53 plays a pivotal role in the IFNalpha-induced apoptotic response. IFNalpha-induced PML was unable to recruit p53 into nuclear bodies and its downregulation by siRNA did not alter CD95 expression. In contrast, IFNgamma-induced apoptosis is p53-independent. CD95 and IFN-regulatory factor 1 (IRF1) are directly upregulated by this cytokine. Apoptotic response to IFNgamma is decreased in the presence of ZB4 and strongly diminished by IRF1 siRNA, implicating both CD95 and IRF1 in IFNgamma-induced apoptotic response. Taken together, these results show that in two different cell lines, IFNalpha and IFNgamma, induce p53-dependent -independent apoptosis, respectively.

  17. DJ-1 restores p53 transcription activity inhibited by Topors/p53BP3.

    PubMed

    Shinbo, Yumi; Taira, Takahiro; Niki, Takeshi; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2005-03-01

    DJ-1 is a multi-functional protein that plays roles in transcriptional regulation and anti-oxidative stress, and loss of its function is thought to result in onset of Parkinson's disease. Here, we report that DJ-1 bound to Topors/p53BP3, a ring finger protein binding to both topoisomerase I and p53, in vitro and in vivo and that both proteins were colocalized in cells. DJ-1 and p53 were then found to be sumoylated by Topors in cells. It was also found that DJ-1 bound to p53 in vitro and in vivo and that colocalization with and its binding to p53 were stimulated by UV irradiation of cells. Transcription activity of p53 was found to be abrogated by Topors concomitant with sumoylation of p53 in a dose-dependent manner, and DJ-1 restored its repressed activity by releasing the sumoylated form of p53. These findings suggest that DJ-1 positively regulates p53 through Topors-mediated sumoylation.

  18. p53 Suppresses Tetraploid Development in Mice

    PubMed Central

    Horii, Takuro; Yamamoto, Masamichi; Morita, Sumiyo; Kimura, Mika; Nagao, Yasumitsu; Hatada, Izuho

    2015-01-01

    Mammalian tetraploid embryos die in early development because of defects in the epiblast. Experiments with diploid/tetraploid chimeric mice, obtained via the aggregation of embryonic stem cells, clarified that while tetraploid cells are excluded from epiblast derivatives, diploid embryos with tetraploid extraembryonic tissues can develop to term. Today, this method, known as tetraploid complementation, is usually used for rescuing extraembryonic defects or for obtaining completely embryonic stem (ES) cell-derived pups. However, it is still unknown why defects occur in the epiblast during mammalian development. Here, we demonstrated that downregulation of p53, a tumour suppressor protein, rescued tetraploid development in the mammalian epiblast. Tetraploidy in differentiating epiblast cells triggered p53-dependent cell-cycle arrest and apoptosis, suggesting the activation of a tetraploidy checkpoint during early development. Finally, we found that p53 downregulation rescued tetraploid embryos later in gestation. PMID:25752699

  19. p53 and rapamycin are additive

    PubMed Central

    Campisi, Judith; Huang, Jing; Jones, Diane; Dodds, Sherry G.; Williams, Charnae; Hubbard, Gene; Livi, Carolina B.; Gao, Xiaoli; Weintraub, Susan; Curiel, Tyler; Sharp, Z. Dave; Hasty, Paul

    2015-01-01

    Mechanistic target of rapamycin (mTOR) is a kinase found in a complex (mTORC1) that enables macromolecular synthesis and cell growth and is implicated in cancer etiology. The rapamycin-FK506 binding protein 12 (FKBP12) complex allosterically inhibits mTORC1. In response to stress, p53 inhibits mTORC1 through a separate pathway involving cell signaling and amino acid sensing. Thus, these different mechanisms could be additive. Here we show that p53 improved the ability of rapamycin to: 1) extend mouse life span, 2) suppress ionizing radiation (IR)-induced senescence-associated secretory phenotype (SASP) and 3) increase the levels of amino acids and citric acid in mouse embryonic stem (ES) cells. This additive effect could have implications for cancer treatment since rapamycin and p53 are anti-oncogenic. PMID:26158292

  20. p53 suppresses tetraploid development in mice.

    PubMed

    Horii, Takuro; Yamamoto, Masamichi; Morita, Sumiyo; Kimura, Mika; Nagao, Yasumitsu; Hatada, Izuho

    2015-03-10

    Mammalian tetraploid embryos die in early development because of defects in the epiblast. Experiments with diploid/tetraploid chimeric mice, obtained via the aggregation of embryonic stem cells, clarified that while tetraploid cells are excluded from epiblast derivatives, diploid embryos with tetraploid extraembryonic tissues can develop to term. Today, this method, known as tetraploid complementation, is usually used for rescuing extraembryonic defects or for obtaining completely embryonic stem (ES) cell-derived pups. However, it is still unknown why defects occur in the epiblast during mammalian development. Here, we demonstrated that downregulation of p53, a tumour suppressor protein, rescued tetraploid development in the mammalian epiblast. Tetraploidy in differentiating epiblast cells triggered p53-dependent cell-cycle arrest and apoptosis, suggesting the activation of a tetraploidy checkpoint during early development. Finally, we found that p53 downregulation rescued tetraploid embryos later in gestation.

  1. p53 suppresses hyper-recombination by modulating BRCA1 function.

    PubMed

    Dong, Chao; Zhang, Fengmei; Luo, Yue; Wang, Hui; Zhao, Xipeng; Guo, Gongshe; Powell, Simon N; Feng, Zhihui

    2015-09-01

    Both p53 and BRCA1 are tumor suppressors and are involved in a number of cellular processes including cell cycle arrest, apoptosis, transcriptional regulation, and DNA damage repair. Some studies have suggested that the association of BRCA1 and p53 is required for transcriptional regulation of genes involved in cell replication and DNA repair pathways. However, the relationship between the two proteins in molecular mechanisms of DNA repair is still not clear. Therefore, we sought to determine whether there is a functional link between p53 and BRCA1 in DNA repair. Firstly, using a plasmid recombination substrate, pDR-GFP, integrated into the genome of breast cancer cell line MCF7, we have demonstrated that p53 suppressed Rad51-mediated hyper-recombinational repair by two independent cell models of HPV-E6 induced p53 inactivation and p53 knockdown assay. Our study further indicated that p53 mediated homologous recombination (HR) through inhibiting BRCA1 over-function via mechanism of transcription regulation in response to DNA repair. Since it was found p53 and BRCA1 existed in a protein complex, indicating both proteins may be associated at post-transcriptional level. Moreover, defective p53-induced hyper-recombination was associated with cell radioresistance and chromosomal stability, strongly supporting the involvement of p53 in the inhibition of hyper-recombination, which led to genetic stability and cellular function in response to DNA damage. In addition, it was found that p53 loss rescued BRCA1 deficiency via recovering HR and chromosomal stability, suggesting that p53 is also involved in the HR-inhibition independently of BRCA1. Thus, our data indicated that p53 was involved in inhibiting recombination by both BRCA1-dependent and -independent mechanisms, and there is a functional link between p53-suppression and BRCA1-promotion in regulation of HR activity at transcription level and possible post-transcription level.

  2. Regulation of p53 tetramerization and nuclear export by ARC

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2014-01-01

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

  4. p53-Dependent suppression of genome instability in germ cells.

    PubMed

    Otozai, Shinji; Ishikawa-Fujiwara, Tomoko; Oda, Shoji; Kamei, Yasuhiro; Ryo, Haruko; Sato, Ayuko; Nomura, Taisei; Mitani, Hiroshi; Tsujimura, Tohru; Inohara, Hidenori; Todo, Takeshi

    2014-02-01

    Radiation increases mutation frequencies at tandem repeat loci. Germline mutations in γ-ray-irradiated medaka fish (Oryzias latipes) were studied, focusing on the microsatellite loci. Mismatch-repair genes suppress microsatellite mutation by directly removing altered sequences at the nucleotide level, whereas the p53 gene suppresses genetic alterations by eliminating damaged cells. The contribution of these two defense mechanisms to radiation-induced microsatellite instability was addressed. The spontaneous mutation frequency was significantly higher in msh2(-/-) males than in wild-type fish, whereas there was no difference in the frequency of radiation-induced mutations between msh2(-/-) and wild-type fish. By contrast, irradiated p53(-/-) fish exhibited markedly increased mutation frequencies, whereas their spontaneous mutation frequency was the same as that of wild-type fish. In the spermatogonia of the testis, radiation induced a high level of apoptosis both in wild-type and msh2(-/-) fish, but negligible levels in p53(-/-) fish. The results demonstrate that the msh2 and p53 genes protect genome integrity against spontaneous and radiation-induced mutation by two different pathways: direct removal of mismatches and elimination of damaged cells.

  5. S100A4 interacts with p53 in the nucleus and promotes p53 degradation.

    PubMed

    Orre, L M; Panizza, E; Kaminskyy, V O; Vernet, E; Gräslund, T; Zhivotovsky, B; Lehtiö, J

    2013-12-05

    S100A4 is a small calcium-binding protein that is commonly overexpressed in a range of different tumor types, and it is widely accepted that S100A4 has an important role in the process of cancer metastasis. In vitro binding assays has shown that S100A4 interacts with the tumor suppressor protein p53, indicating that S100A4 may have additional roles in tumor development. In the present study, we show that endogenous S100A4 and p53 interact in complex samples, and that the interaction increases after inhibition of MDM2-dependent p53 degradation using Nutlin-3A. Further, using proximity ligation assay, we show that the interaction takes place in the cell nucleus. S100A4 knockdown experiments in two p53 wild-type cell lines, A549 and HeLa, resulted in stabilization of p53 protein, indicating that S100A4 is promoting p53 degradation. Finally, we demonstrate that S100A4 knockdown leads to p53-dependent cell cycle arrest and increased cisplatin-induced apoptosis. Thus, our data add a new layer to the oncogenic properties of S100A4 through its inhibition of p53-dependent processes.

  6. Mechanisms of p53-Mediated Apoptosis

    DTIC Science & Technology

    2005-03-01

    exclusion assay and under control of the IGFBP3 promoter and 1 pig of empty pcDNA3 or pcDNA3 vector expressing p53 and various mutants. (A) The BD found that...C. C. Harris, and P. p73-deficient mice have neurological, pheromonal and inflammatory defects Hainaut. 2002. The IARC TP53 database: new online

  7. 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.

  8. EBNA3C regulates p53 through induction of Aurora kinase B.

    PubMed

    Jha, Hem C; Yang, Karren; El-Naccache, Darine W; Sun, Zhiguo; Robertson, Erle S

    2015-03-20

    In multicellular organisms p53 maintains genomic integrity through activation of DNA repair, and apoptosis. EBNA3C can down regulate p53 transcriptional activity. Aurora kinase (AK) B phosphorylates p53, which leads to degradation of p53. Aberrant expression of AK-B is a hallmark of numerous human cancers. Therefore changes in the activities of p53 due to AK-B and EBNA3C expression is important for understanding EBV-mediated cell transformation. Here we show that the activities of p53 and its homolog p73 are dysregulated in EBV infected primary cells which can contribute to increased cell transformation. Further, we showed that the ETS-1 binding site is crucial for EBNA3C-mediated up-regulation of AK-B transcription. Further, we determined the Ser 215 residue of p53 is critical for functional regulation by AK-B and EBNA3C and that the kinase domain of AK-B which includes amino acid residues 106, 111 and 205 was important for p53 regulation. AK-B with a mutation at residue 207 was functionally similar to wild type AK-B in terms of its kinase activities and knockdown of AK-B led to enhanced p73 expression independent of p53. This study explores an additional mechanism by which p53 is regulated by AK-B and EBNA3C contributing to EBV-induced B-cell transformation.

  9. Emerging Non-Canonical Functions and Regulation by p53: p53 and Stemness

    PubMed Central

    Olivos, David J.; Mayo, Lindsey D.

    2016-01-01

    Since its discovery nearly 40 years ago, p53 has ascended to the forefront of investigated genes and proteins across diverse research disciplines and is recognized most exclusively for its role in cancer as a tumor suppressor. Levine and Oren (2009) reviewed the evolution of p53 detailing the significant discoveries of each decade since its first report in 1979. In this review, we will highlight the emerging non-canonical functions and regulation of p53 in stem cells. We will focus on general themes shared among p53’s functions in non-malignant stem cells and cancer stem-like cells (CSCs) and the influence of p53 on the microenvironment and CSC niche. We will also examine p53 gain of function (GOF) roles in stemness. Mutant p53 (mutp53) GOFs that lead to survival, drug resistance and colonization are reviewed in the context of the acquisition of advantageous transformation processes, such as differentiation and dedifferentiation, epithelial-to-mesenchymal transition (EMT) and stem cell senescence and quiescence. Finally, we will conclude with therapeutic strategies that restore wild-type p53 (wtp53) function in cancer and CSCs, including RING finger E3 ligases and CSC maintenance. The mechanisms by which wtp53 and mutp53 influence stemness in non-malignant stem cells and CSCs or tumor-initiating cells (TICs) are poorly understood thus far. Further elucidation of p53’s effects on stemness could lead to novel therapeutic strategies in cancer research. PMID:27898034

  10. Induction of p53-dependent activation of the human proliferating cell nuclear antigen gene in chromatin by ionizing radiation.

    PubMed

    Shan, Bin; Xu, Jin; Zhuo, Ying; Morris, Cindy A; Morris, Gilbert F

    2003-11-07

    A human fibroblast cell line with conditional p53 expression displayed a p53-dependent increase in both the protein and mRNA levels of proliferating cell nuclear antigen (PCNA) after exposure to ionizing radiation (IR). The combination of p53 induction and IR cooperated to activate a transiently expressed human PCNA promoter-reporter gene via a p53-responsive element. Chromatin immunoprecipitation assays with antibodies specific for p53 or p300/CREB-binding protein revealed specific p53-dependent enrichment of PCNA promoter sequences in immunoprecipitates of sheared chromatin prepared from irradiated cells. Maximal and specific association of acetylated histone H4 with the PCNA promoter also depended on p53 induction and exposure to IR. These data demonstrate p53 binding to a target site in the PCNA promoter, recruitment of p300/CREB-binding protein, and localized acetylation of histone H4 in an IR-dependent manner. These molecular events are likely to play a role in mediating activation of PCNA gene expression by p53 during the cellular response to DNA damage. The analyses indicate that the combination of p53 induction and IR activate the PCNA gene via mechanisms similar to that of p21/wild-type p53-activated factor but to a lesser extent. This differential regulation of PCNA and p21/wild-type p53-activated factor may establish the proper ratio of the two proteins to coordinate DNA repair with cell cycle arrest.

  11. Mutant p53: Multiple Mechanisms Define Biologic Activity in Cancer

    PubMed Central

    Kim, Michael Paul; Zhang, Yun; Lozano, Guillermina

    2015-01-01

    The functional importance of p53 as a tumor suppressor gene is evident through its pervasiveness in cancer biology. The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of alterations involve p53 missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may concomitantly gain novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in various model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 in various organ systems are reviewed and their limitations discussed. PMID:26618142

  12. Mutant p53: Multiple Mechanisms Define Biologic Activity in Cancer.

    PubMed

    Kim, Michael Paul; Zhang, Yun; Lozano, Guillermina

    2015-01-01

    The functional importance of p53 as a tumor suppressor gene is evident through its pervasiveness in cancer biology. The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of alterations involve p53 missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may concomitantly gain novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in various model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 in various organ systems are reviewed and their limitations discussed.

  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. SCH529074, a small molecule activator of mutant p53, which binds p53 DNA binding domain (DBD), restores growth-suppressive function to mutant p53 and interrupts HDM2-mediated ubiquitination of wild type p53.

    PubMed

    Demma, Mark; Maxwell, Eugene; Ramos, Robert; Liang, Lianzhu; Li, Cheng; Hesk, David; Rossman, Randall; Mallams, Alan; Doll, Ronald; Liu, Ming; Seidel-Dugan, Cynthia; Bishop, W Robert; Dasmahapatra, Bimalendu

    2010-04-02

    Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 microm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

  15. A DNA damage signal is required for p53 to activate gadd45.

    PubMed

    Xiao, G; Chicas, A; Olivier, M; Taya, Y; Tyagi, S; Kramer, F R; Bargonetti, J

    2000-03-15

    We provide direct evidence that overexpression of p53 is not sufficient for robust p53-dependent activation of the endogenous gadd45 gene. When p53 was induced in TR9-7 cells in the absence of DNA damage, waf1/p21 and mdm2 mRNA levels were increased, but a change in gadd45 mRNA was barely detectable. Activation of the gadd45 gene was observed when camptothecin was added to cells containing p53 in the absence of a further increase in the p53 level. Phosphorylation of p53 at serine 15 and acetylation at lysine 382 were detected after drug treatment. It has been suggested that p53 posttranslational modification is critical during activation. However, inhibition of these modifications by wortmannin was not sufficient to block the transactivation of gadd45. Interestingly, after camptothecin treatment, increased DNase I sensitivity was detected at the gadd45 promoter, suggesting that an undetermined DNA damage signal is involved in inducing chromatin remodeling at the gadd45 promoter while cooperating with p53 to activate gadd45 transcription.

  16. p53 and ribosome biogenesis stress: the essentials.

    PubMed

    Golomb, Lior; Volarevic, Sinisa; Oren, Moshe

    2014-08-19

    Cell proliferation and cell growth are two tightly linked processes, as the proliferation program cannot be executed without proper accumulation of cell mass, otherwise endangering the fate of the two daughter cells. It is therefore not surprising that ribosome biogenesis, a key element in cell growth, is regulated by many cell cycle regulators. This regulation is exerted transcriptionally and post-transcriptionally, in conjunction with numerous intrinsic and extrinsic signals. Those signals eventually converge at the nucleolus, the cellular compartment that is not only responsible for executing the ribosome biogenesis program, but also serves as a regulatory hub, responsible for integrating and transmitting multiple stress signals to the omnipotent cell fate gatekeeper, p53. In this review we discuss when, how and why p53 is activated upon ribosomal biogenesis stress, and how perturbation of this critical regulatory interplay may impact human disease.

  17. Necdin, a p53-Target Gene, Is an Inhibitor of p53-Mediated Growth Arrest

    PubMed Central

    Lafontaine, Julie; Rodier, Francis; Ouellet, Véronique; Mes-Masson, Anne-Marie

    2012-01-01

    In vitro, cellular immortalization and transformation define a model for multistep carcinogenesis and current ongoing challenges include the identification of specific molecular events associated with steps along this oncogenic pathway. Here, using NIH3T3 cells, we identified transcriptionally related events associated with the expression of Polyomavirus Large-T antigen (PyLT), a potent viral oncogene. We propose that a subset of these alterations in gene expression may be related to the early events that contribute to carcinogenesis. The proposed tumor suppressor Necdin, known to be regulated by p53, was within a group of genes that was consistently upregulated in the presence of PyLT. While Necdin is induced following p53 activation with different genotoxic stresses, Necdin induction by PyLT did not involve p53 activation or the Rb-binding site of PyLT. Necdin depletion by shRNA conferred a proliferative advantage to NIH3T3 and PyLT-expressing NIH3T3 (NIHLT) cells. In contrast, our results demonstrate that although overexpression of Necdin induced a growth arrest in NIH3T3 and NIHLT cells, a growing population rapidly emerged from these arrested cells. This population no longer showed significant proliferation defects despite high Necdin expression. Moreover, we established that Necdin is a negative regulator of p53-mediated growth arrest induced by nutlin-3, suggesting that Necdin upregulation could contribute to the bypass of a p53-response in p53 wild type tumors. To support this, we characterized Necdin expression in low malignant potential ovarian cancer (LMP) where p53 mutations rarely occur. Elevated levels of Necdin expression were observed in LMP when compared to aggressive serous ovarian cancers. We propose that in some contexts, the constitutive expression of Necdin could contribute to cancer promotion by delaying appropriate p53 responses and potentially promote genomic instability. PMID:22355404

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

    PubMed Central

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

    2009-01-01

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

  19. Dynamics of p53: A Master Decider of Cell Fate

    PubMed Central

    Luo, Qingyin; Beaver, Jill M.; Liu, Yuan; Zhang, Zunzhen

    2017-01-01

    Cellular stress-induced temporal alterations—i.e., dynamics—are typically exemplified by the dynamics of p53 that serve as a master to determine cell fate. p53 dynamics were initially identified as the variations of p53 protein levels. However, a growing number of studies have shown that p53 dynamics are also manifested in variations in the activity, spatial location, and posttranslational modifications of p53 proteins, as well as the interplay among all p53 dynamical features. These are essential in determining a specific outcome of cell fate. In this review, we discuss the importance of the multifaceted features of p53 dynamics and their roles in the cell fate decision process, as well as their potential applications in p53-based cancer therapy. The review provides new insights into p53 signaling pathways and their potentials in the development of new strategies in p53-based cancer therapy. PMID:28208785

  20. Ferroptosis: A missing puzzle piece in the p53 blueprint?

    PubMed

    Wang, Shang-Jui; Ou, Yang; Jiang, Le; Gu, Wei

    2016-05-01

    Recent evidence indicates that canonical functions of p53 (i.e., apoptosis and growth arrest) are dispensable for p53-mediated tumor suppression. We have uncovered a novel function of p53 that contributes to tumor suppression through regulation of cystine metabolism, reactive oxygen species responses, and ferroptosis. The p53-mediated ferroptotic response via SLC7A11 denotes an extra layer of defense against tumorigenesis in conjunction with other p53 functions.

  1. Overcoming immunosuppression to enhance a p53MVA vaccine.

    PubMed

    Hardwick, Nicola; Chung, Vincent; Cristea, Mihaela; Ellenhorn, Joshua DI; Diamond, Don J

    2014-11-01

    A Phase I trial of a p53-targeting modified vaccinia Ankara (p53MVA) vaccine in patients afflicted with refractory gastrointestinal cancers demonstrated enhanced T-cell recognition of p53 following vaccination. However, this effect was transient suggesting that p53MVA requires combination with immunomodulatory agents to deliver clinical benefit. Here, we outline our rationale for combining p53MVA with immunomodulatory chemotherapy in a forthcoming trial.

  2. Targeting Oncogenic Mutant p53 for Cancer Therapy.

    PubMed

    Parrales, Alejandro; Iwakuma, Tomoo

    2015-01-01

    Among genetic alterations in human cancers, mutations in the tumor suppressor p53 gene are the most common, occurring in over 50% of human cancers. The majority of p53 mutations are missense mutations and result in the accumulation of dysfunctional p53 protein in tumors. These mutants frequently have oncogenic gain-of-function activities and exacerbate malignant properties of cancer cells, such as metastasis and drug resistance. Increasing evidence reveals that stabilization of mutant p53 in tumors is crucial for its oncogenic activities, while depletion of mutant p53 attenuates malignant properties of cancer cells. Thus, mutant p53 is an attractive druggable target for cancer therapy. Different approaches have been taken to develop small-molecule compounds that specifically target mutant p53. These include compounds that restore wild-type conformation and transcriptional activity of mutant p53, induce depletion of mutant p53, inhibit downstream pathways of oncogenic mutant p53, and induce synthetic lethality to mutant p53. In this review article, we comprehensively discuss the current strategies targeting oncogenic mutant p53 in cancers, with special focus on compounds that restore wild-type p53 transcriptional activity of mutant p53 and those reducing mutant p53 levels.

  3. Patterns of Proteins that Associate with p53 or with p53 Binding Sites Present in the Ribosomal Gene Cluster and MDM2 (P2) Promoter

    DTIC Science & Technology

    2000-08-01

    factor receptor (efr) (Ludes-Meyers, et al., 1996), proliferating cell nuclear antigen (pCna) (Deb et al., 1992), the multi drug resistant gene (mdr-1...by the HlV -LTR (Subler et al., 1994; Gualberto and Baldwin, 1995). 4 Mutant p53 proteins may activate transcription of the HIV-LTR by cooperation

  4. Hitting cancers' weak spots: vulnerabilities imposed by p53 mutation.

    PubMed

    Gurpinar, Evrim; Vousden, Karen H

    2015-08-01

    The tumor suppressor protein p53 plays a critical role in limiting malignant development and progression. Almost all cancers show loss of p53 function, through either mutation in the p53 gene itself or defects in the mechanisms that activate p53. While reactivation of p53 can effectively limit tumor growth, this is a difficult therapeutic goal to achieve in the many cancers that do not retain wild type p53. An alternative approach focuses on identifying vulnerabilities imposed on cancers by virtue of the loss of or alterations in p53, to identify additional pathways that can be targeted to specifically kill or inhibit the growth of p53 mutated cells. These indirect ways of exploiting mutations in p53 - which occur in more than half of all human cancers - provide numerous exciting therapeutic possibilities.

  5. Deconstructing p53 transcriptional networks in tumor suppression.

    PubMed

    Bieging, Kathryn T; Attardi, Laura D

    2012-02-01

    p53 is a pivotal tumor suppressor that induces apoptosis, cell-cycle arrest and senescence in response to stress signals. Although p53 transcriptional activation is important for these responses, the mechanisms underlying tumor suppression have been elusive. To date, no single or compound mouse knockout of specific p53 target genes has recapitulated the dramatic tumor predisposition that characterizes p53-null mice. Recently, however, analysis of knock-in mice expressing p53 transactivation domain mutants has revealed a group of primarily novel direct p53 target genes that may mediate tumor suppression in vivo. We present here an overview of well-known p53 target genes and the tumor phenotypes of the cognate knockout mice, and address the recent identification of new p53 transcriptional targets and how they enhance our understanding of p53 transcriptional networks central for tumor suppression.

  6. Nuclear inclusion bodies of mutant and wild-type p53 in cancer: a hallmark of p53 inactivation and proteostasis remodelling by p53 aggregation.

    PubMed

    De Smet, Frederik; Saiz Rubio, Mirian; Hompes, Daphne; Naus, Evelyne; De Baets, Greet; Langenberg, Tobias; Hipp, Mark S; Houben, Bert; Claes, Filip; Charbonneau, Sarah; Delgado Blanco, Javier; Plaisance, Stephane; Ramkissoon, Shakti; Ramkissoon, Lori; Simons, Colinda; van den Brandt, Piet; Weijenberg, Matty; Van England, Manon; Lambrechts, Sandrina; Amant, Frederic; D'Hoore, André; Ligon, Keith L; Sagaert, Xavier; Schymkowitz, Joost; Rousseau, Frederic

    2016-12-30

    Although p53 protein aggregates have been observed in cancer cell lines and tumour tissue, their impact in cancer remains largely unknown. Here, we extensively screened for p53 aggregation phenotypes in tumour biopsies, and identified nuclear inclusion bodies (nIBs) of transcriptionally inactive mutant or wild-type p53 as the most frequent aggregation-like phenotype across six different cancer types. p53-positive nIBs co-stained with nuclear aggregation markers, and shared molecular hallmarks of nIBs commonly found in neurodegenerative disorders. In cell culture, tumour-associated stress was a strong inducer of p53 aggregation and nIB formation. This was most prominent for mutant p53, but could also be observed in wild-type p53 cell lines, for which nIB formation correlated with the loss of p53's transcriptional activity. Importantly, protein aggregation also fuelled the dysregulation of the proteostasis network in the tumour cell by inducing a hyperactivated, oncogenic heat-shock response, to which tumours are commonly addicted, and by overloading the proteasomal degradation system, an observation that was most pronounced for structurally destabilized mutant p53. Patients showing tumours with p53-positive nIBs suffered from a poor clinical outcome, similar to those with loss of p53 expression, and tumour biopsies showed a differential proteostatic expression profile associated with p53-positive nIBs. p53-positive nIBs therefore highlight a malignant state of the tumour that results from the interplay between (1) the functional inactivation of p53 through mutation and/or aggregation, and (2) microenvironmental stress, a combination that catalyses proteostatic dysregulation. This study highlights several unexpected clinical, biological and therapeutically unexplored parallels between cancer and neurodegeneration. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  7. Mechanisms That Enhance Sustainability of p53 Pulses

    PubMed Central

    Kim, Jae Kyoung; Jackson, Trachette L.

    2013-01-01

    The tumor suppressor p53 protein shows various dynamic responses depending on the types and extent of cellular stresses. In particular, in response to DNA damage induced by γ-irradiation, cells generate a series of p53 pulses. Recent research has shown the importance of sustaining repeated p53 pulses for recovery from DNA damage. However, far too little attention has been paid to understanding how cells can sustain p53 pulses given the complexities of genetic heterogeneity and intrinsic noise. Here, we explore potential molecular mechanisms that enhance the sustainability of p53 pulses by developing a new mathematical model of the p53 regulatory system. This model can reproduce many experimental results that describe the dynamics of p53 pulses. By simulating the model both deterministically and stochastically, we found three potential mechanisms that improve the sustainability of p53 pulses: 1) the recently identified positive feedback loop between p53 and Rorα allows cells to sustain p53 pulses with high amplitude over a wide range of conditions, 2) intrinsic noise can often prevent the dampening of p53 pulses even after mutations, and 3) coupling of p53 pulses in neighboring cells via cytochrome-c significantly reduces the chance of failure in sustaining p53 pulses in the presence of heterogeneity among cells. Finally, in light of these results, we propose testable experiments that can reveal important mechanisms underlying p53 dynamics. PMID:23755198

  8. Interference with p53 protein inhibits hematopoietic and muscle differentiation

    PubMed Central

    1996-01-01

    The involvement of p53 protein in cell differentiation has been recently suggested by some observations made with tumor cells and the correlation found between differentiation and increased levels of p53. However, the effect of p53 on differentiation is in apparent contrast with the normal development of p53-null mice. To test directly whether p53 has a function in cell differentiation, we interfered with the endogenous wt-p53 protein of nontransformed cells of two different murine histotypes: 32D myeloid progenitors, and C2C12 myoblasts. A drastic inhibition of terminal differentiation into granulocytes or myotubes, respectively, was observed upon expression of dominant- negative p53 proteins. This inhibition did not alter the cell cycle withdrawal typical of terminal differentiation, nor p21(WAF1/CIP1) upregulation, indicating that interference with endogenous p53 directly affects cell differentiation, independently of the p53 activity on the cell cycle. We also found that the endogenous wt-p53 protein of C2C12 cells becomes transcriptionally active during myogenesis, and this activity is inhibited by p53 dominant-negative expression. Moreover, we found that p53 DNA-binding and transcriptional activities are both required to induce differentiation in p53-negative K562 cells. Taken together, these data strongly indicate that p53 is a regulator of cell differentiation and it exerts this role, at least in part, through its transcriptional activity. PMID:8698814

  9. p53 modulation of TFIIH-associated nucleotide excision repair activity.

    PubMed

    Wang, X W; Yeh, H; Schaeffer, L; Roy, R; Moncollin, V; Egly, J M; Wang, Z; Freidberg, E C; Evans, M K; Taffe, B G

    1995-06-01

    p53 has pleiotropic functions including control of genomic plasticity and integrity. Here we report that p53 can bind to several transcription factor IIH-associated factors, including transcription-repair factors, XPD (Rad3) and XPB, as well as CSB involved in strand-specific DNA repair, via its C-terminal domain. We also found that wild-type, but not Arg273His mutant p53 inhibits XPD (Rad3) and XPB DNA helicase activities. Moreover, repair of UV-induced dimers is slower in Li-Fraumeni syndrome cells (heterozygote p53 mutant) than in normal human cells. Our findings indicate that p53 may play a direct role in modulating nucleotide excision repair pathways.

  10. Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis

    PubMed Central

    Prokesch, Andreas; Graef, Franziska A.; Madl, Tobias; Kahlhofer, Jennifer; Heidenreich, Steffi; Schumann, Anne; Moyschewitz, Elisabeth; Pristoynik, Petra; Blaschitz, Astrid; Knauer, Miriam; Muenzner, Matthias; Bogner-Strauss, Juliane G.; Dohr, Gottfried; Schulz, Tim J.; Schupp, Michael

    2017-01-01

    The ability to adapt cellular metabolism to nutrient availability is critical for survival. The liver plays a central role in the adaptation to starvation by switching from glucose-consuming processes and lipid synthesis to providing energy substrates like glucose to the organism. Here we report a previously unrecognized role of the tumor suppressor p53 in the physiologic adaptation to food withdrawal. We found that starvation robustly increases p53 protein in mouse liver. This induction was posttranscriptional and mediated by a hepatocyte-autonomous and AMP-activated protein kinase-dependent mechanism. p53 stabilization was required for the adaptive expression of genes involved in amino acid catabolism. Indeed, acute deletion of p53 in livers of adult mice impaired hepatic glycogen storage and induced steatosis. Upon food withdrawal, p53-deleted mice became hypoglycemic and showed defects in the starvation-associated utilization of hepatic amino acids. In summary, we provide novel evidence for a p53-dependent integration of acute changes of cellular energy status and the metabolic adaptation to starvation. Because of its tumor suppressor function, p53 stabilization by starvation could have implications for both metabolic and oncological diseases of the liver.—Prokesch, A., Graef, F. A., Madl, T., Kahlhofer, J., Heidenreich, S., Schumann, A., Moyschewitz, E., Pristoynik, P., Blaschitz, A., Knauer, M., Muenzner, M., Bogner-Strauss, J. G., Dohr, G., Schulz, T. J., Schupp, M. Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis. PMID:27811061

  11. Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis.

    PubMed

    Prokesch, Andreas; Graef, Franziska A; Madl, Tobias; Kahlhofer, Jennifer; Heidenreich, Steffi; Schumann, Anne; Moyschewitz, Elisabeth; Pristoynik, Petra; Blaschitz, Astrid; Knauer, Miriam; Muenzner, Matthias; Bogner-Strauss, Juliane G; Dohr, Gottfried; Schulz, Tim J; Schupp, Michael

    2017-02-01

    The ability to adapt cellular metabolism to nutrient availability is critical for survival. The liver plays a central role in the adaptation to starvation by switching from glucose-consuming processes and lipid synthesis to providing energy substrates like glucose to the organism. Here we report a previously unrecognized role of the tumor suppressor p53 in the physiologic adaptation to food withdrawal. We found that starvation robustly increases p53 protein in mouse liver. This induction was posttranscriptional and mediated by a hepatocyte-autonomous and AMP-activated protein kinase-dependent mechanism. p53 stabilization was required for the adaptive expression of genes involved in amino acid catabolism. Indeed, acute deletion of p53 in livers of adult mice impaired hepatic glycogen storage and induced steatosis. Upon food withdrawal, p53-deleted mice became hypoglycemic and showed defects in the starvation-associated utilization of hepatic amino acids. In summary, we provide novel evidence for a p53-dependent integration of acute changes of cellular energy status and the metabolic adaptation to starvation. Because of its tumor suppressor function, p53 stabilization by starvation could have implications for both metabolic and oncological diseases of the liver.-Prokesch, A., Graef, F. A., Madl, T., Kahlhofer, J., Heidenreich, S., Schumann, A., Moyschewitz, E., Pristoynik, P., Blaschitz, A., Knauer, M., Muenzner, M., Bogner-Strauss, J. G., Dohr, G., Schulz, T. J., Schupp, M. Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis.

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

    PubMed

    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.

  13. Mitochondrial matrix P53 sensitizes cells to oxidative stress☆

    PubMed Central

    Koczor, Christopher A.; Torres, Rebecca A.; Fields, Earl J.; Boyd, Amy; Lewis, William

    2013-01-01

    A mitochondrial matrix-specific p53 construct (termed p53–290) in HepG2 cells was utilized to determine the impact of p53 in the mitochondrial matrix following oxidative stress. H2O2 exposure reduced cellular proliferation similarly in both p53–290 and vector cells, and p53–290 cells demonstrating decreased cell viability at 1 mM H2O2 (~85% viable). Mitochondrial DNA (mtDNA) abundance was decreased in a dose-dependent manner in p53–290 cells while no change was observed in vector cells. Oximetric analysis revealed reduced maximal respiration and reserve capacity in p53–290 cells. Our results demonstrate that mitochondrial matrix p53 sensitizes cells to oxidative stress by reducing mtDNA abundance and mitochondrial function. PMID:23499753

  14. Activation and activities of the p53 tumour suppressor protein

    PubMed Central

    Bálint, É; Vousden, K H

    2001-01-01

    The p53 tumour suppressor protein inhibits malignant progression by mediating cell cycle arrest, apoptosis or repair following cellular stress. One of the major regulators of p53 function is the MDM2 protein, and multiple forms of cellular stress activate p53 by inhibiting the MDM2-mediated degradation of p53. Mutations in p53, or disruption of the pathways that allow activation of p53, seem to be a general feature of all cancers. Here we review recent advances in our understanding of the pathways that regulate p53 and the pathways that are induced by p53, as well as their implications for cancer therapy. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11747320

  15. p53-dependent ceramide response to genotoxic stress.

    PubMed Central

    Dbaibo, G S; Pushkareva, M Y; Rachid, R A; Alter, N; Smyth, M J; Obeid, L M; Hannun, Y A

    1998-01-01

    Both p53 and ceramide have been implicated in the regulation of growth suppression. p53 has been proposed as the "guardian of the genome" and ceramide has been suggested as a "tumor suppressor lipid. " Both molecules appear to regulate cell cycle arrest, senescence, and apoptosis. In this study, we investigated the relationship between p53 and ceramide. We found that treatment of Molt-4 cells with low concentrations of actinomycin D or gamma-irradiation, which activate p53-dependent apoptosis, induces apoptosis only in cells expressing normal levels of p53. In these cells, p53 activation was followed by a dose- and time-dependent increase in endogenous ceramide levels which was not seen in cells lacking functional p53 and treated similarly. Similar results were seen in irradiated L929 cells whereby the p53-deficient clone was significantly more resistant to irradiation and exhibited no ceramide response. However, in p53-independent systems, such as growth suppression induced by TNF-alpha or serum deprivation, ceramide accumulated irrespective of the upregulation of p53, indicating that p53 regulates ceramide accumulation in only a subset of growth-suppressive pathways. Finally, ceramide did not increase p53 levels when used at growth-suppressive concentrations. Also, when cells lacking functional p53, either due to mutation or the expression of the E6 protein of human papilloma virus, were treated with exogenous ceramide, there was equal growth suppression, cell cycle arrest, and apoptosis as compared with cells expressing normal p53. These results indicate that p53 is unlikely to function "downstream" of ceramide. Instead, they suggest that, in situations where p53 performs a critical regulatory role, such as the response to genotoxic stress, it functions "upstream" of ceramide. These studies begin to define a relationship between these two pathways of growth inhibition. PMID:9664074

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

    PubMed

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

    2014-12-19

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

  17. Identification of p53-target genes in Danio rerio

    PubMed Central

    Mandriani, Barbara; Castellana, Stefano; Rinaldi, Carmela; Manzoni, Marta; Venuto, Santina; Rodriguez-Aznar, Eva; Galceran, Juan; Nieto, M. Angela; Borsani, Giuseppe; Monti, Eugenio; Mazza, Tommaso; Merla, Giuseppe; Micale, Lucia

    2016-01-01

    To orchestrate the genomic response to cellular stress signals, p53 recognizes and binds to DNA containing specific and well-characterized p53-responsive elements (REs). Differences in RE sequences can strongly affect the p53 transactivation capacity and occur even between closely related species. Therefore, the identification and characterization of a species-specific p53 Binding sistes (BS) consensus sequence and of the associated target genes may help to provide new insights into the evolution of the p53 regulatory networks across different species. Although p53 functions were studied in a wide range of species, little is known about the p53-mediated transcriptional signature in Danio rerio. Here, we designed and biochemically validated a computational approach to identify novel p53 target genes in Danio rerio genome. Screening all the Danio rerio genome by pattern-matching-based analysis, we found p53 RE-like patterns proximal to 979 annotated Danio rerio genes. Prioritization analysis identified a subset of 134 candidate pattern-related genes, 31 of which have been investigated in further biochemical assays. Our study identified runx1, axin1, traf4a, hspa8, col4a5, necab2, and dnajc9 genes as novel direct p53 targets and 12 additional p53-controlled genes in Danio rerio genome. The proposed combinatorial approach resulted to be highly sensitive and robust for identifying new p53 target genes also in additional animal species. PMID:27581768

  18. Mutant p53 protein localized in the cytoplasm inhibits autophagy.

    PubMed

    Morselli, Eugenia; Tasdemir, Ezgi; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Vicencio, José Miguel; Soussi, Thierry; Kroemer, Guido

    2008-10-01

    The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

  19. P53 mutations and cancer: a tight linkage

    PubMed Central

    Pisconti, Salvatore; Della Vittoria Scarpati, Giuseppina

    2016-01-01

    P53 is often mutated in solid tumors, in fact, somatic changes involving the gene encoding for p53 (TP53) have been discovered in more than 50% of human malignancies and several data confirmed that p53 mutations represent an early event in cancerogenesis. Main p53 functions consist in cell cycle arrest, DNA repair, senescence and apoptosis induction in response to mutagenic stimuli, and, to exert those functions, p53 acts as transcriptional factor. Recent data have highlighted another very important role of p53, consisting in regulate cell metabolism and cell response to oxidative stress. Majority of tumor suppressor genes, such as adenomatous polyposis coli (APC), retinoblastoma-associated protein (RB) and Von-Hippel-Lindau (VHL) are inactivated by deletion or early truncation mutations in tumors, resulting in the decreased or loss of expression of their proteins. Differently, most p53 mutations in human cancer are missense mutations, which result in the production of full-length mutant p53 proteins. It has been reported that mutant p53 proteins and wild type p53 proteins often regulate same cellular biological processes with opposite effects. So, mutant p53 has been reported to supply the cancer cells of glucose and nutrients, and, to avoid reactive oxygen species (ROS) mediated damage during oxidative stress. These last features are able to render tumor cells resistant to ionizing radiations and chemotherapy. A future therapeutic approach in tumors bearing p53 mutations may be to deplete cancer cells of their energy reserves and antioxidants. PMID:28149884

  20. 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

  1. Emerging roles of p53 and other tumour-suppressor genes in immune regulation

    PubMed Central

    Muñoz-Fontela, César; Mandinova, Anna; Aaronson, Stuart A.; Lee, Sam W.

    2017-01-01

    Tumour-suppressor genes are indispensable for the maintenance of genomic integrity. Recently, several of these genes, including p53, PTEN, RB1 and ARF, have been implicated in immune responses and inflammatory diseases. In particular, the p53 tumour-suppressor pathway is involved in crucial aspects of tumour immunology and in homeostatic regulation of immune responses. Other studies have identified roles for p53 in various cellular processes, including metabolism and stem cell maintenance. Here, we discuss the emerging roles of p53 and other tumour-suppressor genes in tumour immunology as well as in additional immunological settings, such as virus infection. This relatively unexplored area could yield important insights into the homeostatic control of immune cells in health and disease, and facilitate the development of more effective immunotherapies. Consequently, tumour-suppressor genes are emerging as potential guardians of immune integrity. PMID:27667712

  2. 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

  3. Lysine methylation represses p53 activity in teratocarcinoma cancer cells

    PubMed Central

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

    2016-01-01

    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

  4. Chemical Variations on the p53 Reactivation Theme.

    PubMed

    Ribeiro, Carlos J A; Rodrigues, Cecília M P; Moreira, Rui; Santos, Maria M M

    2016-05-13

    Among the tumor suppressor genes, p53 is one of the most studied. It is widely regarded as the "guardian of the genome", playing a major role in carcinogenesis. In fact, direct inactivation of the TP53 gene occurs in more than 50% of malignancies, and in tumors that retain wild-type p53 status, its function is usually inactivated by overexpression of negative regulators (e.g., MDM2 and MDMX). Hence, restoring p53 function in cancer cells represents a valuable anticancer approach. In this review, we will present an updated overview of the most relevant small molecules developed to restore p53 function in cancer cells through inhibition of the p53-MDMs interaction, or direct targeting of wild-type p53 or mutated p53. In addition, optimization approaches used for the development of small molecules that have entered clinical trials will be presented.

  5. Chemical Variations on the p53 Reactivation Theme

    PubMed Central

    Ribeiro, Carlos J. A.; Rodrigues, Cecília M. P.; Moreira, Rui; Santos, Maria M. M.

    2016-01-01

    Among the tumor suppressor genes, p53 is one of the most studied. It is widely regarded as the “guardian of the genome”, playing a major role in carcinogenesis. In fact, direct inactivation of the TP53 gene occurs in more than 50% of malignancies, and in tumors that retain wild-type p53 status, its function is usually inactivated by overexpression of negative regulators (e.g., MDM2 and MDMX). Hence, restoring p53 function in cancer cells represents a valuable anticancer approach. In this review, we will present an updated overview of the most relevant small molecules developed to restore p53 function in cancer cells through inhibition of the p53-MDMs interaction, or direct targeting of wild-type p53 or mutated p53. In addition, optimization approaches used for the development of small molecules that have entered clinical trials will be presented. PMID:27187415

  6. Rb and p53 Liver Functions Are Essential for Xenobiotic Metabolism and Tumor Suppression.

    PubMed

    Nantasanti, Sathidpak; Toussaint, Mathilda J M; Youssef, Sameh A; Tooten, Peter C J; de Bruin, Alain

    2016-01-01

    The tumor suppressors Retinoblastoma (Rb) and p53 are frequently inactivated in liver diseases, such as hepatocellular carcinomas (HCC) or infections with Hepatitis B or C viruses. Here, we discovered a novel role for Rb and p53 in xenobiotic metabolism, which represent a key function of the liver for metabolizing therapeutic drugs or toxins. We demonstrate that Rb and p53 cooperate to metabolize the xenobiotic 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). DDC is metabolized mainly by cytochrome P450 (Cyp)3a enzymes resulting in inhibition of heme synthesis and accumulation of protoporphyrin, an intermediate of heme pathway. Protoporphyrin accumulation causes bile injury and ductular reaction. We show that loss of Rb and p53 resulted in reduced Cyp3a expression decreased accumulation of protoporphyrin and consequently less ductular reaction in livers of mice fed with DDC for 3 weeks. These findings provide strong evidence that synergistic functions of Rb and p53 are essential for metabolism of DDC. Because Rb and p53 functions are frequently disabled in liver diseases, our results suggest that liver patients might have altered ability to remove toxins or properly metabolize therapeutic drugs. Strikingly the reduced biliary injury towards the oxidative stress inducer DCC was accompanied by enhanced hepatocellular injury and formation of HCCs in Rb and p53 deficient livers. The increase in hepatocellular injury might be related to reduce protoporphyrin accumulation, because protoporphrin is well known for its anti-oxidative activity. Furthermore our results indicate that Rb and p53 not only function as tumor suppressors in response to carcinogenic injury, but also in response to non-carcinogenic injury such as DDC.

  7. The function of Drosophila p53 isoforms in apoptosis

    PubMed Central

    Zhang, B; Rotelli, M; Dixon, M; Calvi, B R

    2015-01-01

    The p53 protein is a major mediator of the cellular response to genotoxic stress and is a crucial suppressor of tumor formation. In a variety of organisms, p53 and its paralogs, p63 and p73, each encode multiple protein isoforms through alternative splicing, promoters, and translation start sites. The function of these isoforms in development and disease are still being defined. Here, we evaluate the apoptotic potential of multiple isoforms of the single p53 gene in the genetic model Drosophila melanogaster. Most previous studies have focused on the p53A isoform, but it has been recently shown that a larger p53B isoform can induce apoptosis when overexpressed. It has remained unclear, however, whether one or both isoforms are required for the apoptotic response to genotoxic stress. We show that p53B is a much more potent inducer of apoptosis than p53A when overexpressed. Overexpression of two newly identified short isoforms perturbed development and inhibited the apoptotic response to ionizing radiation. Analysis of physiological protein expression indicated that p53A is the most abundant isoform, and that both p53A and p53B can form a complex and co-localize to sub-nuclear compartments. In contrast to the overexpression results, new isoform-specific loss-of-function mutants indicated that it is the shorter p53A isoform, not full-length p53B, that is the primary mediator of pro-apoptotic gene transcription and apoptosis after ionizing radiation. Together, our data show that it is the shorter p53A isoform that mediates the apoptotic response to DNA damage, and further suggest that p53B and shorter isoforms have specialized functions. PMID:25882045

  8. Hyperglycemia promotes p53-Mdm2 interaction but reduces p53 ubiquitination in RINm5F cells.

    PubMed

    Barzalobre-Gerónimo, R; Raúl, Barzalobre-Gerónimo; Flores-López, L A; Antonio, Flores-López Luis; Baiza-Gutman, L A; Arturo, Baiza-Gutman Luis; Cruz, M; Miguel, Cruz; García-Macedo, R; Rebeca, García-Macedo; Ávalos-Rodríguez, A; Alejandro, Ávalos-Rodríguez; Contreras-Ramos, A; Alejandra, Contreras-Ramos; Díaz-Flores, A; Margarita, Díaz-Flores; Ortega-Camarillo, C; Clara, Ortega-Camarillo

    2015-07-01

    The apoptosis of β cells induced by hyperglycemia has been associated with p53 mobilization to mitochondria and p53 phosphorylation. Murine double minute 2 (Mdm2) induces the degradation of p53 and thereby protects cells from apoptosis. We studied the effect of glucose at high concentration on the ability of Mdm2 to ubiquitinate p53 and promote its degradation. RINm5F cells were grown in RPMI-1640 medium with 5 or 30 mM glucose for varying periods of time. After this treatment, the expression of Mdm2 was measured using real-time PCR. The phosphorylation of Mdm2 at Ser166, p53 at Ser15, and the kinases Akt and ATM were measured by Western blotting. The formation of the p53-Mdm2 complex and p53 ubiquitination was assessed by p53 immunoprecipitation and immunofluorescence. Our results showed that high glucose reduced Mdm2 mRNA expression and protein concentration and increased Mdm2 and Akt phosphorylation, albeit with slower kinetics for Akt. It also promoted p53-Mdm2 complex formation, whereas p53 ubiquitination was suppressed. Furthermore, phosphorylation of both p53 Ser15 and ATM was increased in the presence of 30 mM glucose. These data indicate that high concentration glucose decrease the mRNA expression and cytosolic concentration of Mdm2. However, although the increase in glucose promoted the phosphorylation of Mdm2, it also decreased p53 ubiquitination, thus avoiding p53 degradation. In hyperglycemic conditions, such as diabetes mellitus, the reduction of pancreatic β cells mass is favored by stabilization of p53 in association with low p53 ubiquitination and reduced expression of Mdm2.

  9. ErbB2 inhibition by lapatinib promotes degradation of mutant p53 protein in cancer cells

    PubMed Central

    Li, Dun; Marchenko, Natalia D

    2017-01-01

    Mutations in the p53 tumor suppressor gene are the most prevalent genetic events in human Her2-positive breast cancer and are associated with poor prognosis and survival. Human clinical data and our in vitro and in vivo studies strongly suggest potent oncogenic cooperation between mutant p53 and Her2 (ErbB2). Yet, the translational significance of mutant p53 in Her2 positive breast cancer, especially with respect to Her2-targeted therapies, has not been evaluated. Our previous work identified novel oncogenic activity of mutant p53 whereby mutp53 amplifies ErbB2 signaling via the mutp53-HSF1-ErbB2 feed-forward loop. Here we report that pharmacological interception of this circuit by ErbB2 inhibitor lapatinib downregulates mutant p53 in vitro and in vivo. We found that ErbB2 inhibition by lapatinib inhibits transcription factor HSF1, and its target Hsp90, followed by mutant p53 degradation in MDM2 dependent manner. Thus, our data suggest that mutant p53 sensitizes cancer cells to lapatinib via two complementary mechanisms: mutant p53 mediated amplification of ErbB2 signaling, and simultaneous annihilation of both potent oncogenic drivers, ErbB2 and mutant p53. Hence, our study could provide valuable information for the optimization of therapeutic protocols to achieve superior clinical effects in the treatment of Her2 positive breast cancer. PMID:27791982

  10. The physical interaction of p53 and plakoglobin is necessary for their synergistic inhibition of migration and invasion

    PubMed Central

    Mehrabani, Vahedah; Churchill, Lucas; Pasdar, Manijeh

    2016-01-01

    Plakoglobin (PG) is a paralog of β-catenin with similar adhesive, but contrasting signalling functions. Although β-catenin has well-known oncogenic function, PG generally acts as a tumor/metastasis suppressor by mechanisms that are just beginning to be deciphered. Previously, we showed that PG interacted with wild type (WT) and a number of mutant p53s, and that its tumor/metastasis suppressor activity may be mediated, at least partially, by this interaction. Here, carcinoma cell lines deficient in both p53 and PG (H1299), or expressing mutant p53 in the absence of PG (SCC9), were transfected with expression constructs encoding WT and different fragments and deletions of p53 and PG, individually or in pairs. Transfectants were characterized for their in vitro growth, migratory and invasive properties and for mapping the interacting domain of p53 and PG. We showed that when coexpressed, p53-WT and PG-WT cooperated to decrease growth, and acted synergistically to significantly reduce cell migration and invasion. The DNA-binding domain of p53 and C-terminal domain of PG mediated p53/PG interaction, and furthermore, the C-terminus of PG played a central role in the inhibition of invasion in association with p53. PMID:27058623

  11. ErbB2 inhibition by lapatinib promotes degradation of mutant p53 protein in cancer cells.

    PubMed

    Li, Dun; Marchenko, Natalia D

    2017-01-24

    Mutations in the p53 tumor suppressor gene are the most prevalent genetic events in human Her2-positive breast cancer and are associated with poor prognosis and survival. Human clinical data and our in vitro and in vivo studies strongly suggest potent oncogenic cooperation between mutant p53 and Her2 (ErbB2). Yet, the translational significance of mutant p53 in Her2 positive breast cancer, especially with respect to Her2-targeted therapies, has not been evaluated. Our previous work identified novel oncogenic activity of mutant p53 whereby mutp53 amplifies ErbB2 signaling via the mutp53-HSF1-ErbB2 feed-forward loop. Here we report that pharmacological interception of this circuit by ErbB2 inhibitor lapatinib downregulates mutant p53 in vitro and in vivo. We found that ErbB2 inhibition by lapatinib inhibits transcription factor HSF1, and its target Hsp90, followed by mutant p53 degradation in MDM2 dependent manner. Thus, our data suggest that mutant p53 sensitizes cancer cells to lapatinib via two complementary mechanisms: mutant p53 mediated amplification of ErbB2 signaling, and simultaneous annihilation of both potent oncogenic drivers, ErbB2 and mutant p53. Hence, our study could provide valuable information for the optimization of therapeutic protocols to achieve superior clinical effects in the treatment of Her2 positive breast cancer.

  12. Analyses of p53 antibodies in sera of patients with lung carcinoma define immunodominant regions in the p53 protein.

    PubMed Central

    Schlichtholz, B.; Trédaniel, J.; Lubin, R.; Zalcman, G.; Hirsch, A.; Soussi, T.

    1994-01-01

    Antibodies specific for human p53 were analysed in sera of lung cancer patients. We detected p53 antibodies in the sera of 24% (10/42) of patients with lung carcinoma. The distribution was as follows: 4/9 small-cell lung carcinomas (SCLCs), 2/18 squamous cell lung carcinomas (SCCs), 2/10 adenocarcinomas (ADCs) and 2/5 large-cell lung carcinomas (LCCs). p53 antibodies were always present at the time of diagnosis and did not appear during progression of the disease. Using an original peptide-mapping procedure, we precisely localised the p53 epitopes recognised by p53 antibodies. Immunodominant epitopes reacting with antibodies were localised in the amino and carboxy termini of the protein, similar to those found in breast carcinoma patients or in animals immunised with p53. In light of these data, we suggest that p53 antibodies occur via a self-immunisation process that is the consequence of p53 accumulation in tumour cells. p53 antibodies were also detected in two patients without detected malignant disease. One of these patients died 6 months later of lung carcinoma, suggesting that p53 antibodies may be a precocious marker of p53 alteration. Images Figure 2 PMID:7514026

  13. The p53 isoform delta133p53ß regulates cancer cell apoptosis in a RhoB-dependent manner

    PubMed Central

    Arsic, Nikola; Ho-Pun-Cheung, Alexandre; Evelyne, Crapez; Assenat, Eric; Jarlier, Marta; Anguille, Christelle; Colard, Manon; Pezet, Mikaël

    2017-01-01

    The TP53 gene plays essential roles in cancer. Conventionally, wild type (WT) p53 is thought to prevent cancer development and metastasis formation, while mutant p53 has transforming abilities. However, clinical studies failed to establish p53 mutation status as an unequivocal predictive or prognostic factor of cancer progression. The recent discovery of p53 isoforms that can differentially regulate cell cycle arrest and apoptosis suggests that their expression, rather than p53 mutations, could be a more clinically relevant biomarker in patients with cancer. In this study, we show that the p53 isoform delta133p53ß is involved in regulating the apoptotic response in colorectal cancer cell lines. We first demonstrate delta133p53ß association with the small GTPase RhoB, a well-described anti-apoptotic protein. We then show that, by inhibiting RhoB activity, delta133p53ß protects cells from camptothecin-induced apoptosis. Moreover, we found that high delta133p53 mRNA expression levels are correlated with higher risk of recurrence in a series of patients with locally advanced rectal cancer (n = 36). Our findings describe how a WT TP53 isoform can act as an oncogene and add a new layer to the already complex p53 signaling network. PMID:28212429

  14. Mitofusin-2 is a novel direct target of p53

    SciTech Connect

    Wang, Weilin; Cheng, Xiaofei; Lu, Jianju; Wei, Jianfeng; Fu, Guanghou; Zhu, Feng; Jia, Changku; Zhou, Lin; Xie, Haiyang; Zheng, Shusen

    2010-10-01

    Research highlights: {yields} Mfn2 is a novel target gene of p53. {yields} Mfn2 mRNA and protein levels can be up-regulated in a p53-dependent manner. {yields} Mfn2 promoter activity can be elevated by the p53 protein. {yields} P53 protein binds the Mfn2 promoter directly both in vitro and in vivo. -- Abstract: The tumor suppressor p53 modulates transcription of a number of target genes involved in cell cycle arrest, apoptosis, DNA repair, and other important cellular responses. Mitofusin-2 (Mfn2) is a novel suppressor of cell proliferation that may also exert apoptotic effects via the mitochondrial apoptotic pathway. Through bioinformatics analysis, we identified a p53 binding site in the Mfn2 promoter. Consistent with this, we showed that the p53 protein binds the Mfn2 promoter directly both in vitro and in vivo. Additionally, we found that Mfn2 mRNA and protein levels are up-regulated in a p53-dependent manner. Furthermore, luciferase assays revealed that the activity of the wild-type Mfn2 promoter, but not a mutated version of the promoter, was up-regulated by p53. These results indicate that Mfn2 is a novel p53-inducible target gene, which provides insight into the regulation of Mfn2 and its associated activities in the inhibition of cell proliferation, promotion of apoptosis, and modulation of tumor suppression.

  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. p53 isoform profiling in glioblastoma and injured brain.

    PubMed

    Takahashi, R; Giannini, C; Sarkaria, J N; Schroeder, M; Rogers, J; Mastroeni, D; Scrable, H

    2013-06-27

    The tumor suppressor p53 has been found to be the most commonly mutated gene in human cancers; however, the frequency of p53 mutations varies from 10 to 70% across different cancer types. This variability can partly be explained by inactivating mechanisms aside from direct genomic polymorphisms. The p53 gene encodes 12 isoforms, some of which can modulate full-length p53 activity in cancer. In this study, we characterized p53 isoform expression patterns in glioblastoma, gliosis, non-tumor brain and neural progenitor cells by SDS-PAGE, immunoblot, mass spectrometry and reverse transcription-PCR. We found that the most consistently expressed isoform in glioblastoma, Δ40p53, was uniquely expressed in regenerative processes, such as those involving neural progenitor cells and gliosis compared with tumor samples. Isoform profiling of glioblastoma tissues revealed the presence of both Δ40p53 and full-length p53, neither of which were detected in non-tumor cerebral cortex. Upon xenograft propagation of tumors, p53 levels increased. The variability of overall p53 expression and relative levels of isoforms suggest fluctuations in subpopulations of cells with greater or lesser capacity for proliferation, which can change as the tumor evolves under different growth conditions.

  17. p53 nuclear protein accumulation correlates with mutations in the p53 gene, tumor grade, and stage in bladder cancer.

    PubMed

    Esrig, D; Spruck, C H; Nichols, P W; Chaiwun, B; Steven, K; Groshen, S; Chen, S C; Skinner, D G; Jones, P A; Cote, R J

    1993-11-01

    Seventy-three transitional cell carcinomas of the bladder were analyzed by immunohistochemistry for p53 nuclear accumulation, and the results were compared to mutations detected in the p53 gene by single strand conformational polymorphism analysis (SSCP) and DNA sequence analysis. Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded tissue sections. A highly significant association between the presence of p53 mutations and p53 nuclear reactivity as detected by immunohistochemistry was found (P = 0.0001). Of 32 tumors that demonstrated p53 mutations by SSCP, 27 (84%) showed p53 nuclear reactivity. Of the five cases that did not demonstrate p53 nuclear reactivity, four had mutations in exon 5. However, of 41 tumors with no evidence of p53 mutation by molecular analysis, 12 (29%) showed p53 immunoreactivity. This indicates that immunohistochemical methods may be more sensitive than SSCP in detecting p53 mutations or that discordant cases represent tumors with accumulation of wild type p53 protein, without mutations at the p53 locus. Of the 15 tumors that were found to have mutations at exon 8, 13 demonstrated high-intensity homogeneous p53 nuclear reactivity by immunohistochemistry, and all mutations located at codon 280 demonstrated high-intensity homogeneous immunoreactivity. However, three of three tumors with exon 6 mutations demonstrated low-level p53 immunoreactivity, and four of six tumors with mutations in exon 5 showed no detectable p53 nuclear reactivity. This indicates that the heterogeneity of immunoreactivity observed when analyzing p53 nuclear accumulation may be related to the site of the p53 gene mutation. Information on tumor grade, stage, lymph node status, disease-free interval, and overall survival were available in 54 patients who had undergone cystectomy. A significant association was observed between p53 alterations (detected by immunohistochemistry and SSCP) and histological tumor grade (P = 0.003) and stage (P = 0

  18. The Transactivation Domains of the p53 Protein.

    PubMed

    Raj, Nitin; Attardi, Laura D

    2017-01-03

    The p53 tumor suppressor is a transcriptional activator, with discrete domains that participate in sequence-specific DNA binding, tetramerization, and transcriptional activation. Mutagenesis and reporter studies have delineated two distinct activation domains (TADs) and specific hydrophobic residues within these TADs that are critical for their function. Knockin mice expressing p53 mutants with alterations in either or both of the two TADs have revealed that TAD1 is critical for responses to acute DNA damage, whereas both TAD1 and TAD2 participate in tumor suppression. Biochemical and structural studies have identified factors that bind either or both TADs, including general transcription factors (GTFs), chromatin modifiers, and negative regulators, helping to elaborate a model through which p53 activates transcription. Posttranslational modifications (PTMs) of the p53 TADs through phosphorylation also regulate TAD activity. Together, these studies on p53 TADs provide great insight into how p53 serves as a tumor suppressor.

  19. Targeting cancer stem cells with p53 modulators

    PubMed Central

    Hayashi, Ryo; Appella, Ettore; Kopelovich, Levy; DeLeo, Albert B.

    2016-01-01

    Cancer stem cells (CSC) typically over-express aldehyde dehydrogenase (ALDH). Thus, ALDHbright tumor cells represent targets for developing novel cancer prevention/treatment interventions. Loss of p53 function is a common genetic event during cancer development wherein small molecular weight compounds (SMWC) that restore p53 function and reverse tumor growth have been identified. Here, we focused on two widely studied p53 SMWC, CP-31398 and PRIMA-1, to target ALDHbright CSC in human breast, endometrial and pancreas carcinoma cell lines expressing mutant or wild type (WT) p53. CP-31398 and PRIMA-1 significantly reduced CSC content and sphere formation by these cell lines in vitro. In addition, these agents were more effective in vitro against CSC compared to cisplatin and gemcitabine, two often-used chemotherapeutic agents. We also tested a combinatorial treatment in methylcholantrene (MCA)-treated mice consisting of p53 SMWC and p53-based vaccines. Yet using survival end-point analysis, no increased efficacy in the presence of either p53 SMWC alone or with vaccine compared to vaccine alone was observed. These results may be due, in part, to the presence of immune cells, such as activated lymphocytes expressing WT p53 at levels comparable to some tumor cells, wherein further increase of p53 expression by p53 SMWC may alter survival of these immune cells and negatively impact an effective immune response. Continuous exposure of mice to MCA may have also interfered with the action of these p53 SMWC, including potential direct interaction with MCA. Nonetheless, the effect of p53 SMWC on CSC and cancer treatment remains of great interest. PMID:27074569

  20. The Transcription Factor p53 Influences Microglial Activation Phenotype

    PubMed Central

    Jayadev, Suman; Nesser, Nicole K.; Hopkins, Stephanie; Myers, Scott J.; Case, Amanda; Lee, Rona J.; Seaburg, Luke A.; Uo, Takuma; Murphy, Sean P.; Morrison, Richard S.; Garden, Gwenn A.

    2011-01-01

    Several neurodegenerative diseases are influenced by the innate immune response in the central nervous system (CNS). Microglia, have pro-inflammatory and subsequently neurotoxic actions as well as anti-inflammatory functions that promote recovery and repair. Very little is known about the transcriptional control of these specific microglial behaviors. We have previously shown that in HIV associated neurocognitive disorders (HAND), the transcription factor p53 accumulates in microglia and that microglial p53 expression is required for the in vitro neurotoxicity of the HIV coat glycoprotein gp120. These findings suggested a novel function for p53 in regulating microglial activation. Here we report that in the absence of p53, microglia demonstrate a blunted response to interferon-γ, failing to increase expression of genes associated with classical macrophage activation or secrete pro-inflammatory cytokines. Microarray analysis of global gene expression profiles revealed increased expression of genes associated with anti-inflammatory functions, phagocytosis and tissue repair in p53 knockout (p53−/−) microglia compared with those cultured from strain matched p53 expressing (p53+/+) mice. We further observed that p53−/− microglia demonstrate increased phagocytic activity in vitro and expression of markers for alternative macrophage activation both in vitro and in vivo. In HAND brain tissue, the alternative activation marker CD163 was expressed in a separate subset of microglia than those demonstrating p53 accumulation. These data suggest that p53 influences microglial behavior, supporting the adoption of a pro-inflammatory phenotype, while p53 deficiency promotes phagocytosis and gene expression associated with alternative activation and anti-inflammatory functions. PMID:21598312

  1. Regulation of neuronal P53 activity by CXCR4

    PubMed Central

    Khan, Muhammad Z.; Shimizu, Saori; Patel, Jeegar P.; Nelson, Autumn; Le, My-Thao; Mullen-Przeworski, Anna; Brandimarti, Renato; Fatatis, Alessandro; Meucci, Olimpia

    2009-01-01

    Abnormal activation of CXCR4 during inflammatory/infectious states may lead to neuronal dysfunction or damage. The major goal of this study was to determine the coupling of CXCR4 to p53-dependent survival pathways in primary neurons. Neurons were stimulated with the HIV envelope protein gp120IIIB or the endogenous CXCR4 agonist, SDF-1α. We found that gp120 stimulates p53 activity and induces expression of the p53 pro-apoptotic target Apaf-1 in cultured neurons. Inhibition of CXCR4 by AMD3100 abrogates the effect of gp120 on both p53 and Apaf-1. Moreover, gp120 neurotoxicity is markedly reduced by the p53-inhibitor, pifithrin-α. The viral protein also regulates p53 phosphorylation and expression of other p53-responsive genes, such as MDM2 and p21. Conversely, SDF-1α, which can promote neuronal survival, increases p53 acetylation and p21 expression in neurons. Thus, the stimulation of different p53 targets could be instrumental in determining the outcome of CXCR4 activation on neuronal survival in neuroinflammatory disorders. PMID:16005638

  2. Recognition of Local DNA Structures by p53 Protein.

    PubMed

    Brázda, Václav; Coufal, Jan

    2017-02-10

    p53 plays critical roles in regulating cell cycle, apoptosis, senescence and metabolism and is commonly mutated in human cancer. These roles are achieved by interaction with other proteins, but particularly by interaction with DNA. As a transcription factor, p53 is well known to bind consensus target sequences in linear B-DNA. Recent findings indicate that p53 binds with higher affinity to target sequences that form cruciform DNA structure. Moreover, p53 binds very tightly to non-B DNA structures and local DNA structures are increasingly recognized to influence the activity of wild-type and mutant p53. Apart from cruciform structures, p53 binds to quadruplex DNA, triplex DNA, DNA loops, bulged DNA and hemicatenane DNA. In this review, we describe local DNA structures and summarize information about interactions of p53 with these structural DNA motifs. These recent data provide important insights into the complexity of the p53 pathway and the functional consequences of wild-type and mutant p53 activation in normal and tumor cells.

  3. Anoikis triggers Mdm2-dependent p53 degradation

    PubMed Central

    Ghosh, Abhijit; Chen, Tina Chunyuan; Kapila, Yvonne L.

    2010-01-01

    The extracellular matrix (ECM) plays a key role in cell–cell communication and signaling, and the signals it propagates are important for tissue remodeling and survival. However, signals from disease-altered ECM may lead to anoikis—apoptotic cell death triggered by loss of ECM contacts. Previously, we found that an altered fibronectin matrix triggers anoikis in human primary ligament cells via a pathway that requires p53 transcriptional downregulation. Here we show that this p53 reduction is suppressed by transfecting cells with Mdm2 antisense oligonucleotides or small interfering RNA. Similar results were found in cells treated to prevent p53 and Mdm2 interactions. When p53 was overexpressed in cells lacking Mdm2 and p53, p53 levels were unaffected by anoikis conditions. However, cells cotransfected with p53 and wild type Mdm2, but not a mutant Mdm2, exhibited decreased p53 levels in response to anoikis conditions. Thus, cells under anoikis conditions undergo p53 degradation that is mediated by Mdm2. PMID:20577896

  4. Recognition of Local DNA Structures by p53 Protein

    PubMed Central

    Brázda, Václav; Coufal, Jan

    2017-01-01

    p53 plays critical roles in regulating cell cycle, apoptosis, senescence and metabolism and is commonly mutated in human cancer. These roles are achieved by interaction with other proteins, but particularly by interaction with DNA. As a transcription factor, p53 is well known to bind consensus target sequences in linear B-DNA. Recent findings indicate that p53 binds with higher affinity to target sequences that form cruciform DNA structure. Moreover, p53 binds very tightly to non-B DNA structures and local DNA structures are increasingly recognized to influence the activity of wild-type and mutant p53. Apart from cruciform structures, p53 binds to quadruplex DNA, triplex DNA, DNA loops, bulged DNA and hemicatenane DNA. In this review, we describe local DNA structures and summarize information about interactions of p53 with these structural DNA motifs. These recent data provide important insights into the complexity of the p53 pathway and the functional consequences of wild-type and mutant p53 activation in normal and tumor cells. PMID:28208646

  5. Regulation of p53 and MDM2 activity by MTBP.

    PubMed

    Brady, Mark; Vlatkovic, Nikolina; Boyd, Mark T

    2005-01-01

    p53 is a critical coordinator of a wide range of stress responses. To facilitate a rapid response to stress, p53 is produced constitutively but is negatively regulated by MDM2. MDM2 can inhibit p53 in multiple independent ways: by binding to its transcription activation domain, inhibiting p53 acetylation, promoting nuclear export, and probably most importantly by promoting proteasomal degradation of p53. The latter is achieved via MDM2's E3 ubiquitin ligase activity harbored within the MDM2 RING finger domain. We have discovered that MTBP promotes MDM2-mediated ubiquitination and degradation of p53 and also MDM2 stabilization in an MDM2 RING finger-dependent manner. Moreover, using small interfering RNA to down-regulate endogenous MTBP in unstressed cells, we have found that MTBP significantly contributes to MDM2-mediated regulation of p53 levels and activity. However, following exposure of cells to UV, but not gamma-irradiation, MTBP is destabilized as part of the coordinated cellular response. Our findings suggest that MTBP differentially regulates the E3 ubiquitin ligase activity of MDM2 towards two of its most critical targets (itself and p53) and in doing so significantly contributes to MDM2-dependent p53 homeostasis in unstressed cells.

  6. REGgamma modulates p53 activity by regulating its cellular localization.

    PubMed

    Liu, Jian; Yu, Guowu; Zhao, Yanyan; Zhao, Dengpan; Wang, Ying; Wang, Lu; Liu, Jiang; Li, Lei; Zeng, Yu; Dang, Yongyan; Wang, Chuangui; Gao, Guang; Long, Weiwen; Lonard, David M; Qiao, Shanlou; Tsai, Ming-Jer; Zhang, Bianhong; Luo, Honglin; Li, Xiaotao

    2010-12-01

    The proteasome activator REGγ mediates a shortcut for the destruction of intact mammalian proteins. The biological roles of REGγ and the underlying mechanisms are not fully understood. Here we provide evidence that REGγ regulates cellular distribution of p53 by facilitating its multiple monoubiquitylation and subsequent nuclear export and degradation. We also show that inhibition of p53 tetramerization by REGγ might further enhance cytoplasmic relocation of p53 and reduce active p53 in the nucleus. Furthermore, multiple monoubiquitylation of p53 enhances its physical interaction with HDM2 and probably facilitates subsequent polyubiquitylation of p53, suggesting that monoubiquitylation can act as a signal for p53 degradation. Depletion of REGγ sensitizes cells to stress-induced apoptosis, validating its crucial role in the control of apoptosis, probably through regulation of p53 function. Using a mouse xenograft model, we show that REGγ knockdown results in a significant reduction of tumor growth, suggesting an important role for REGγ in tumor development. Our study therefore demonstrates that REGγ-mediated inactivation of p53 is one of the mechanisms involved in cancer progression.

  7. Podocyte p53 Limits the Severity of Experimental Alport Syndrome

    PubMed Central

    Fukuda, Ryosuke; Suico, Mary Ann; Kai, Yukari; Omachi, Kohei; Motomura, Keishi; Koga, Tomoaki; Komohara, Yoshihiro; Koyama, Kosuke; Yokota, Tsubasa; Taura, Manabu; Shuto, Tsuyoshi

    2016-01-01

    Alport syndrome (AS) is one of the most common types of inherited nephritis caused by mutation in one of the glomerular basement membrane components. AS is characterized by proteinuria at early stage of the disease and glomerular hyperplastic phenotype and renal fibrosis at late stage. Here, we show that global deficiency of tumor suppressor p53 significantly accelerated AS progression in X-linked AS mice and decreased the lifespan of these mice. p53 protein expression was detected in 21-week-old wild-type mice but not in age-matched AS mice. Expression of proinflammatory cytokines and profibrotic genes was higher in p53+/− AS mice than in p53+/+ AS mice. In vitro experiments revealed that p53 modulates podocyte migration and positively regulates the expression of podocyte-specific genes. We established podocyte-specific p53 (pod-p53)-deficient AS mice, and determined that pod-p53 deficiency enhanced the AS-induced renal dysfunction, foot process effacement, and alteration of gene-expression pattern in glomeruli. These results reveal a protective role of p53 in the progression of AS and in maintaining glomerular homeostasis by modulating the hyperplastic phenotype of podocytes in AS. PMID:25967122

  8. p53 family interactions and yeast: together in anticancer therapy.

    PubMed

    Gomes, Sara; Leão, Mariana; Raimundo, Liliana; Ramos, Helena; Soares, Joana; Saraiva, Lucília

    2016-04-01

    The p53 family proteins are among the most appealing targets for cancer therapy. A deeper understanding of the complex interplay that these proteins establish with murine double minute (MDM)2, MDMX, and mutant p53 could reveal new exciting therapeutic opportunities in cancer treatment. Here, we summarize the most relevant advances in the biology of p53 family protein-protein interactions (PPIs), and the latest pharmacological developments achieved from targeting these interactions. We also highlight the remarkable contributions of yeast-based assays to this research. Collectively, we emphasize promising strategies, based on the inhibition of p53 family PPIs, which have expedited anticancer drug development.

  9. Free Radicals Generated by Ionizing Radiation Signal Nuclear Translocation of p53

    NASA Technical Reports Server (NTRS)

    Martinez, J. D.; Pennington, M. E.; Craven, M. T.; Warters, R. L.

    1997-01-01

    The p53 tumor suppressor is a transcription factor that regulates several pathways, which function collectively to maintain the integrity of the genome. Nuclear localization is critical for wild-type function. However, the signals that regulate subcellular localization of p53 have not been identified. Here, we examine the effect of ionizing radiation on the subcellular localization of p53 in two cell lines in which p63 is normally sequestered in the cytoplasm and found that ionizing radiation caused a biphasic translocation response. p53 entered the nucleus 1-2 hours postirradiation (early response), subsequently emerged from the nucleus, and then again entered the nucleus 12-24 hours after the cells had been irradiated (delayed response). These changes in subcellular localization could be completely blocked by the free radical scavenger, WR1065. By comparison, two DNA-damaging agents that do not generate free radicals, mitomycin C and doxorubicin, caused translocation only after 12-24 h of exposure to the drugs, and this effect could not be inhibited by WR1065. Hence, although all three DNA-damaging agents induced relocalization of p53 to the nucleus, only the translocation caused by radiation was sensitive to free radical scavenging. We suggest that the free radicals generated by ionizing radiation can signal p53 translocation to the nucleus.

  10. Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function.

    PubMed

    de Vries, Annemieke; Flores, Elsa R; Miranda, Barbara; Hsieh, Harn-Mei; van Oostrom, Conny Th M; Sage, Julien; Jacks, Tyler

    2002-03-05

    The p53 tumor suppressor gene is the most frequently mutated gene in human cancers, and germ-line p53 mutations cause a familial predisposition for cancer. Germ-line or sporadic p53 mutations are usually missense and typically affect the central DNA-binding domain of the protein. Because p53 functions as a tetrameric transcription factor, mutant p53 is thought to inhibit the function of wild-type p53 protein. Here, we studied the possible dominant-negative inhibition of wild-type p53 protein by two different, frequently occurring point mutations. The R270H and P275S mutations were targeted into the genome of mouse embryonic stem cells to allow the analysis of the effects of the mutant proteins expressed in normal cells at single-copy levels. In embryonic stem cells, the presence of a heterozygous point-mutated allele resulted in delayed transcriptional activation of several p53 downstream target genes on exposure to gamma irradiation. Doxorubicin-induced apoptosis was severely affected in the mutant embryonic stem cells compared with wild-type cells. Heterozygous mutant thymocytes had a severe defect in p53-dependent apoptotic pathways after treatment with gamma irradiation or doxorubicin, whereas p53-independent apoptotic pathways were intact. Together these data demonstrate that physiological expression of point-mutated p53 can strongly limit overall cellular p53 function, supporting the dominant-negative action of such mutants. Also, cells heterozygous for such mutations may be compromised in terms of tumor suppression and response to chemotherapeutic agents.

  11. Mechanisms of p53 Functional De-Regulation: Role of the IκB-α/p53 Complex

    PubMed Central

    Carrà, Giovanna; Crivellaro, Sabrina; Taulli, Riccardo; Guerrasio, Angelo; Saglio, Giuseppe; Morotti, Alessandro

    2016-01-01

    TP53 is one of the most frequently-mutated and deleted tumor suppressors in cancer, with a dramatic correlation with dismal prognoses. In addition to genetic inactivation, the p53 protein can be functionally inactivated in cancer, through post-transductional modifications, changes in cellular compartmentalization, and interactions with other proteins. Here, we review the mechanisms of p53 functional inactivation, with a particular emphasis on the interaction between p53 and IκB-α, the NFKBIA gene product. PMID:27916821

  12. Quantifying levels of p53 mutation in mouse skin tumors.

    PubMed

    Verkler, Tracie L; Couch, Letha H; Howard, Paul C; Parsons, Barbara L

    2005-06-01

    Allele-specific competitive blocker PCR (ACB-PCR) amplification and quantification was developed for mouse p53 codon 270 CGT-->TGT base substitution and codon 244/245 AAC/CGC-->AAT/TGC tandem mutation. PCR products corresponding to p53 mutant and wild-type DNA sequences were generated. These DNAs were mixed in known proportions to construct samples with defined mutant fractions and the allele-specific detection of each mutation was systematically optimized. Each assay was used to analyze eight simulated solar light (SSL)-induced tumors. By analyzing mutant fraction (MF) standards in parallel with PCR products generated from tumor samples, p53 mutants could be quantified as subpopulations within the tumors. All eight tumors contained detectable levels of p53 codon 270 CGT-->TGT mutation. Three tumors had p53 MFs between 10(-4) and 10(-3). Five tumors had p53 MFs between 10(-3) and 10(-2). None of the eight mouse skin tumors had measurable levels of p53 codon 244/245 tandem mutation. Frequent detection of p53 codon 270 CGT-->TGT mutation provides additional evidence that a pyrimidine dinucleotide overlapping a methylated CpG site (Pyr(me)CG) is a susceptible target for SSL-induced mutagenesis. The absence of p53 codon 244/245 mutation in tumors may be explained by its mutant p53 phenotype and/or indicate that this site is not methylated. These initial results indicate that p53 codon 270 CGT-->TGT mutation may be a sensitive biomarker for SSL- or UV-induced mutagenesis. This mutational endpoint may be useful for evaluating the co-carcinogenicity of compounds administered in combination with UV or SSL.

  13. 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

  14. Crocetin exploits p53-induced death domain (PIDD) and FAS-associated death domain (FADD) proteins to induce apoptosis in colorectal cancer

    PubMed Central

    Ray, Pallab; Guha, Deblina; Chakraborty, Juni; Banerjee, Shuvomoy; Adhikary, Arghya; Chakraborty, Samik; Das, Tanya; Sa, Gaurisankar

    2016-01-01

    Tumor suppressor p53 preserves the genomic integrity by restricting anomaly at the gene level. The hotspots for mutation in half of all colon cancers reside in p53. Hence, in a p53-mutated cellular milieu targeting cancer cells may be achievable by targeting the paralogue(s) of p53. Here we have shown the effectiveness of crocetin, a dietary component, in inducing apoptosis of colon cancer cells with varying p53 status. In wild-type p53-expressing cancer cells, p53 in one hand transactivates BAX and in parallel up-regulates p53-induced death domain protein (PIDD) that in turn cleaves and activates BID through caspase-2. Both BAX and t-BID converge at mitochondria to alter the transmembrane potential thereby leading to caspase-9 and caspase-3-mediated apoptosis. In contrast, in functional p53-impaired cells, this phytochemical exploits p53-paralogue p73, which up-regulates FAS to cleave BID through FAS-FADD-caspase-8-pathway. These findings not only underline the phenomenon of functional switch-over from p53 to p73 in p53-impaired condition, but also validate p73 as a promising and potential target for cancer therapy in absence of functional p53. PMID:27622714

  15. Repression of p53 activity by Smyd2-mediated methylation.

    PubMed

    Huang, Jing; Perez-Burgos, Laura; Placek, Brandon J; Sengupta, Roopsha; Richter, Mario; Dorsey, Jean A; Kubicek, Stefan; Opravil, Susanne; Jenuwein, Thomas; Berger, Shelley L

    2006-11-30

    Specific sites of lysine methylation on histones correlate with either activation or repression of transcription. The tumour suppressor p53 (refs 4-7) is one of only a few non-histone proteins known to be regulated by lysine methylation. Here we report a lysine methyltransferase, Smyd2, that methylates a previously unidentified site, Lys 370, in p53. This methylation site, in contrast to the known site Lys 372, is repressing to p53-mediated transcriptional regulation. Smyd2 helps to maintain low concentrations of promoter-associated p53. We show that reducing Smyd2 concentrations by short interfering RNA enhances p53-mediated apoptosis. We find that Set9-mediated methylation of Lys 372 inhibits Smyd2-mediated methylation of Lys 370, providing regulatory cross-talk between post-translational modifications. In addition, we show that the inhibitory effect of Lys 372 methylation on Lys 370 methylation is caused, in part, by blocking the interaction between p53 and Smyd2. Thus, similar to histones, p53 is subject to both activating and repressing lysine methylation. Our results also predict that Smyd2 may function as a putative oncogene by methylating p53 and repressing its tumour suppressive function.

  16. 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

  17. p53 aerobics: the major tumor suppressor fuels your workout.

    PubMed

    Kruse, Jan-Philipp; Gu, Wei

    2006-07-01

    In addition to its role as the central regulator of the cellular stress response, p53 can regulate aerobic respiration via the novel transcriptional target SCO2, a critical regulator of the cytochrome c oxidase complex (Matoba et al., 2006). Loss of p53 results in decreased oxygen consumption and aerobic respiration and promotes a switch to glycolysis, thereby reducing endurance during physical exercise.

  18. Unfolded p53: a potential biomarker for Alzheimer's disease.

    PubMed

    Lanni, Cristina; Uberti, Daniela; Racchi, Marco; Govoni, Stefano; Memo, Maurizio

    2007-08-01

    The identification of biological markers of AD can improve diagnostic accuracy and therapy follow-up as well as provide information on the pathogenesis of the disease. We recently found that fibroblasts derived from AD patients expressed an altered conformational status of p53 and were less sensitive to p53-dependent apoptosis compared to fibroblasts from non-AD subjects. When investigating the mechanism of such alteration, we found that the exposure to nanomolar concentrations of amyloid-beta (Abeta) 1-40 peptide induced the expression of an unfolded p53 protein isoform in fibroblasts derived from non-AD subjects. These data suggest that the tertiary structure of p53 and the sensitivity to p53-dependent apoptosis is influenced by low concentrations of soluble Abeta. On this basis, we hypothesized that low amounts of soluble Abeta induce early pathological changes at cellular level that may precede the amyloidogenic cascade. One of these changes is the induction of a novel conformational state of p53. If low amounts of Abeta peptide, not resulting in cytotoxic effects, are responsible for p53 structure changes, it could be possible to consider the unfolded p53 both as an agent participating to the early pathogenesis and as a specific marker of the early stage of AD.

  19. 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

  20. 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.

  1. Interaction of p53 with the human Rad51 protein.

    PubMed Central

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

    1997-01-01

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

  2. p53 prevents neurodegeneration by regulating synaptic genes.

    PubMed

    Merlo, Paola; Frost, Bess; Peng, Shouyong; Yang, Yawei J; Park, Peter J; Feany, Mel

    2014-12-16

    DNA damage has been implicated in neurodegenerative disorders, including Alzheimer's disease and other tauopathies, but the consequences of genotoxic stress to postmitotic neurons are poorly understood. Here we demonstrate that p53, a key mediator of the DNA damage response, plays a neuroprotective role in a Drosophila model of tauopathy. Further, through a whole-genome ChIP-chip analysis, we identify genes controlled by p53 in postmitotic neurons. We genetically validate a specific pathway, synaptic function, in p53-mediated neuroprotection. We then demonstrate that the control of synaptic genes by p53 is conserved in mammals. Collectively, our results implicate synaptic function as a central target in p53-dependent protection from neurodegeneration.

  3. 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

  4. p53-independent death and p53-induced protection against apoptosis in fibroblasts treated with chemotherapeutic drugs.

    PubMed Central

    Malcomson, R. D.; Oren, M.; Wyllie, A. H.; Harrison, D. J.

    1995-01-01

    Many recent studies have implicated p53 in the cellular response to injury and induction of cell death by apoptosis. In a rat embryonal fibroblast cell line transformed with c-Ha-ras and a mutant temperature-sensitive p53 (val135), cells were G1 arrested at the permissive temperature of 32 degrees C when overexpressed p53 was in wild-type conformation. In this state cells were resistant to apoptosis induced by etoposide (at up to 50 microM) or bleomycin (15 microU ml-1). Cells at 37 degrees C with overexpressed p53 in mutant conformation were freed from this growth arrest, continued proliferating and showed dose-dependent increases in apoptosis. This death is independent of wild-type p53 function. Control cells containing a non-temperature-sensitive mutant p53 (phe132) were sensitive to both etoposide and bleomycin after 24 h at 32 degrees C and 37 degrees C, indicating that the results are not simply due to temperature effects on pharmacokinetics or DNA damage. Our data show that induction of a stable p53-mediated growth arrest renders these cells much less likely to undergo apoptosis in response to certain anti-cancer drugs, and we conclude that the regulatory role of p53 in apoptosis is influenced by the particular cellular context in which this gene is expressed. PMID:7547247

  5. The Fusion Protein of Respiratory Syncytial Virus Triggers p53-Dependent Apoptosis▿

    PubMed Central

    Eckardt-Michel, Julia; Lorek, Markus; Baxmann, Diane; Grunwald, Thomas; Keil, Günther M.; Zimmer, Gert

    2008-01-01

    Infection with respiratory syncytial virus (RSV) frequently causes inflammation and obstruction of the small airways, leading to severe pulmonary disease in infants. We show here that the RSV fusion (F) protein, an integral membrane protein of the viral envelope, is a strong elicitor of apoptosis. Inducible expression of F protein in polarized epithelial cells triggered caspase-dependent cell death, resulting in rigorous extrusion of apoptotic cells from the cell monolayer and transient loss of epithelial integrity. A monoclonal antibody directed against F protein inhibited apoptosis and was also effective if administered to A549 lung epithelial cells postinfection. F protein expression in epithelial cells caused phosphorylation of tumor suppressor p53 at serine 15, activation of p53 transcriptional activity, and conformational activation of proapoptotic Bax. Stable expression of dominant-negative p53 or p53 knockdown by RNA interference inhibited the apoptosis of RSV-infected A549 cells. HEp-2 tumor cells with low levels of p53 were not sensitive to RSV-triggered apoptosis. We propose a new model of RSV disease with the F protein as an initiator of epithelial cell shedding, airway obstruction, secondary necrosis, and consequent inflammation. This makes the RSV F protein a key target for the development of effective postinfection therapies. PMID:18216092

  6. Restoring expression of wild-type p53 suppresses tumor growth but does not cause tumor regression in mice with a p53 missense mutation.

    PubMed

    Wang, Yongxing; Suh, Young-Ah; Fuller, Maren Y; Jackson, James G; Xiong, Shunbin; Terzian, Tamara; Quintás-Cardama, Alfonso; Bankson, James A; El-Naggar, Adel K; Lozano, Guillermina

    2011-03-01

    The transcription factor p53 is a tumor suppressor. As such, the P53 gene is frequently altered in human cancers. However, over 80% of the P53 mutations found in human cancers are missense mutations that lead to expression of mutant proteins that not only lack p53 transcriptional activity but exhibit new functions as well. Recent studies show that restoration of p53 expression leads to tumor regression in mice carrying p53 deletions. However, the therapeutic efficacy of restoring p53 expression in tumors containing p53 missense mutations has not been evaluated. Here we demonstrate that restoring wild-type p53 expression halted tumor growth in mice inheriting a p53(R172H) missense mutation that is equivalent to a P53 missense mutation detected in approximately 6% of human cancers. However, it did not lead to tumor regression, as was observed in mice lacking p53. We further showed that the dominant-negative effect of the mutant p53 encoded by p53(R172H) dampened the activity of the restored wild-type p53. We therefore conclude that in a mutant p53 background, p53 restoration has the therapeutic potential to suppress tumor progression. Our findings support using p53 restoration as a strategy to treat human cancers with P53 missense mutations and provide direction for optimizing p53 restoration in cancer therapy.

  7. Restoring expression of wild-type p53 suppresses tumor growth but does not cause tumor regression in mice with a p53 missense mutation

    PubMed Central

    Wang, Yongxing; Suh, Young-Ah; Fuller, Maren Y.; Jackson, James G.; Xiong, Shunbin; Terzian, Tamara; Quintás-Cardama, Alfonso; Bankson, James A.; El-Naggar, Adel K.; Lozano, Guillermina

    2011-01-01

    The transcription factor p53 is a tumor suppressor. As such, the P53 gene is frequently altered in human cancers. However, over 80% of the P53 mutations found in human cancers are missense mutations that lead to expression of mutant proteins that not only lack p53 transcriptional activity but exhibit new functions as well. Recent studies show that restoration of p53 expression leads to tumor regression in mice carrying p53 deletions. However, the therapeutic efficacy of restoring p53 expression in tumors containing p53 missense mutations has not been evaluated. Here we demonstrate that restoring wild-type p53 expression halted tumor growth in mice inheriting a p53R172H missense mutation that is equivalent to a P53 missense mutation detected in approximately 6% of human cancers. However, it did not lead to tumor regression, as was observed in mice lacking p53. We further showed that the dominant-negative effect of the mutant p53 encoded by p53R172H dampened the activity of the restored wild-type p53. We therefore conclude that in a mutant p53 background, p53 restoration has the therapeutic potential to suppress tumor progression. Our findings support using p53 restoration as a strategy to treat human cancers with P53 missense mutations and provide direction for optimizing p53 restoration in cancer therapy. PMID:21285512

  8. Genotoxic stress-induced expression of p53 and apoptosis in leukemic clam hemocytes with cytoplasmically sequestered p53.

    PubMed

    Böttger, Stefanie; Jerszyk, Emily; Low, Ben; Walker, Charles

    2008-02-01

    In nature, the soft shell clam, Mya arenaria, develops a fatal blood cancer in which a highly conserved homologue for wild-type human p53 protein is rendered nonfunctional by cytoplasmic sequestration. In untreated leukemic clam hemocytes, p53 is complexed throughout the cytoplasm with overexpressed variants for both clam homologues (full-length variant, 1,200-fold and truncated variant, 620-fold above normal clam hemocytes) of human mortalin, an Hsp70 family protein. In vitro treatment with etoposide only and in vivo treatment with either etoposide or mitoxantrone induces DNA damage, elevates expression (600-fold) and promotes nuclear translocation of p53, and results in apoptosis of leukemic clam hemocytes. Pretreatment with wheat germ agglutinin followed by etoposide treatment induces DNA damage and elevates p53 expression (893-fold) but does not overcome cytoplasmic sequestration of p53 or induce apoptosis. We show that leukemic clam hemocytes have an intact p53 pathway, and that maintenance of this tumor phenotype requires nuclear absence of p53, resulting from its localization in the cytoplasm of leukemic clam hemocytes. The effects of these topoisomerase II poisons may result as mortalin-based cytoplasmic tethering is overwhelmed by de novo expression of p53 protein after DNA damage induced by genotoxic stress. Soft shell clam leukemia provides excellent in vivo and in vitro models for developing genotoxic and nongenotoxic cancer therapies for reactivating p53 transcription in human and other animal cancers displaying mortalin-based cytoplasmic sequestration of the p53 tumor suppressor, such as colorectal cancers and primary and secondary glioblastomas, though not apparently leukemias or lymphomas.

  9. The role of p53 in cell metabolism

    PubMed Central

    Zhang, Xing-ding; Qin, Zheng-hong; Wang, Jin

    2010-01-01

    The p53 tumor suppressor gene has recently been shown to mediate metabolic changes in cells under physiological and pathological conditions. It has been revealed that p53 regulates energy metabolism, oxidative stress, and amino acid metabolism through balancing glycolysis and oxidative phosphorylation (OXPHOS) as well as the autophagy pathway. p53 is activated by metabolic stress through AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR) signaling pathways. p53 regulates OXPHOS through the transcriptional regulation of fructose-2,6-bisphosophatase, TP53-induced glycolysis regulator (TIGAR) and synthesis of cytochrome c oxidase (SCO2) subunit of complex IV of the electron transport chain. p53 also indirectly influences the energy metabolism through regulating glucose transporter (GLUT) expression, glutaminase 2 (GLS2) and fatty acid synthase (FAS). In addition, p53 regulates autophagy to provide cell metabolites for surviving through damage regulated autophagy modulator (DRAM1). Here we review the recent findings to elucidate the important role of p53 in cell metabolism. PMID:20729871

  10. p53 and the pathogenesis of skin cancer

    SciTech Connect

    Benjamin, Cara L.; Ananthaswamy, Honnavara N.

    2007-11-01

    The p53 tumor suppressor gene and gene product are among the most diverse and complex molecules involved in cellular functions. Genetic alterations within the p53 gene have been shown to have a direct correlation with cancer development and have been shown to occur in nearly 50% of all cancers. p53 mutations are particularly common in skin cancers and UV irradiation has been shown to be a primary cause of specific 'signature' mutations that can result in oncogenic transformation. There are certain 'hot-spots' in the p53 gene where mutations are commonly found that result in a mutated dipyrimidine site. This review discusses the role of p53 from normal function and its dysfunction in pre-cancerous lesions and non-melanoma skin cancers. Additionally, special situations are explored, such as Li-Fraumeni syndrome in which there is an inherited p53 mutation, and the consequences of immune suppression on p53 mutations and the resulting increase in non-melanoma skin cancer in these patients.

  11. Ferroptosis as a p53-mediated activity during tumour suppression.

    PubMed

    Jiang, Le; Kon, Ning; Li, Tongyuan; Wang, Shang-Jui; Su, Tao; Hibshoosh, Hanina; Baer, Richard; Gu, Wei

    2015-04-02

    Although p53-mediated cell-cycle arrest, senescence and apoptosis serve as critical barriers to cancer development, emerging evidence suggests that the metabolic activities of p53 are also important. Here we show that p53 inhibits cystine uptake and sensitizes cells to ferroptosis, a non-apoptotic form of cell death, by repressing expression of SLC7A11, a key component of the cystine/glutamate antiporter. Notably, p53(3KR), an acetylation-defective mutant that fails to induce cell-cycle arrest, senescence and apoptosis, fully retains the ability to regulate SLC7A11 expression and induce ferroptosis upon reactive oxygen species (ROS)-induced stress. Analysis of mutant mice shows that these non-canonical p53 activities contribute to embryonic development and the lethality associated with loss of Mdm2. Moreover, SLC7A11 is highly expressed in human tumours, and its overexpression inhibits ROS-induced ferroptosis and abrogates p53(3KR)-mediated tumour growth suppression in xenograft models. Our findings uncover a new mode of tumour suppression based on p53 regulation of cystine metabolism, ROS responses and ferroptosis.

  12. 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

  13. p53 and the Pathogenesis of Skin Cancer

    PubMed Central

    Benjamin, Cara L.

    2007-01-01

    The p53 tumor suppressor gene and gene product are among the most diverse and complex molecules involved in cellular functions. Genetic alterations within the p53 gene have been shown to have a direct correlation with cancer development and have been shown to occur in nearly 50% of all cancers. p53 mutations are particularly common in skin cancers and UV irradiation has been shown to be a primary cause of specific ‘signature’ mutations that can result in oncogenic transformation. There are certain ‘hot-spots’ in the p53 gene where mutations are commonly found that result in a mutated dipyrimidine site. This review discusses the role of p53 from normal function and its dysfunction in pre-cancerous lesions and non-melanoma skin cancers. Additionally, special situations are explored, such as Li-Fraumeni syndrome in which there is an inherited p53 mutation, and the consequences of immune suppression on p53 mutations and the resulting increase in non-melanoma skin cancer in these patients. PMID:17270229

  14. 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

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

    PubMed Central

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

    1994-01-01

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

  16. Dendrosomal nanocurcumin and p53 overexpression synergistically trigger apoptosis in glioblastoma cells

    PubMed Central

    Keshavarz, Reihaneh; Bakhshinejad, Babak; Babashah, Sadegh; Baghi, Narges; Sadeghizadeh, Majid

    2016-01-01

    Objective(s): Glioblastoma is the most lethal tumor of the central nervous system. Here, we aimed to evaluate the effects of exogenous delivery of p53 and a nanoformulation of curcumin called dendrosomal curcumin (DNC), alone and in combination, on glioblastoma tumor cells. Materials and Methods: MTT assay was exploited to measure the viability of U87-MG cells against DNC treatment. Cells were separately subjected to DNC treatment and transfected with p53-containing vector and then were co-exposed to DNC and p53 overexpression[A GA1][B2]. Annexin-V-FLUOS staining followed by flow cytometry and real-time PCR were applied to examine apoptosis and analyze the expression levels of the genes involved in cell cycle and oncogenesis, respectively. Results: The results of cell viability assay through MTT indicated that DNC inhibits the proliferation of U87-MG cells in a time- and dose-dependent manner. Apoptosis evaluation revealed that p53 overexpression accompanied by DNC treatment can act in a synergistic manner to significantly enhance the number of apoptotic cells (90%) compared with their application alone (15% and 38% for p53 overexpression and DNC, respectively). Also, real-time PCR data showed that the concomitant exposure of cells to both DNC and p53 overexpression leads to an enhanced expression of GADD45 and a reduced expression of NF-κB and c-Myc. Conclusion: The findings of the current study suggest that our combination strategy, which merges two detached gene (p53) and drug (curcumin) delivery systems into an integrated platform, may represent huge potential as a novel and efficient modality for glioblastoma treatment. PMID:28096969

  17. POSTRANSLATIONAL MODIFICATIONS OF P53: UPSTREAM SIGNALING PATHWAYS.

    SciTech Connect

    ANDERSON,C.W.APPELLA,E.

    2003-10-23

    The p53 tumor suppressor is a tetrameric transcription factor that is posttranslational modified at >20 different sites by phosphorylation, acetylation, or sumoylation in response to various cellular stress conditions. Specific posttranslational modifications, or groups of modifications, that result from the activation of different stress-induced signaling pathways are thought to modulate p53 activity to regulate cell fate by inducing cell cycle arrest, apoptosis, or cellular senescence. Here we review recent progress in characterizing the upstream signaling pathways whose activation in response to various genotoxic and non-genotoxic stresses result in p53 posttranslational modifications.

  18. Robustness of the p53 network and biological hackers.

    PubMed

    Dartnell, Lewis; Simeonidis, Evangelos; Hubank, Michael; Tsoka, Sophia; Bogle, I David L; Papageorgiou, Lazaros G

    2005-06-06

    The p53 protein interaction network is crucial in regulating the metazoan cell cycle and apoptosis. Here, the robustness of the p53 network is studied by analyzing its degeneration under two modes of attack. Linear Programming is used to calculate average path lengths among proteins and the network diameter as measures of functionality. The p53 network is found to be robust to random loss of nodes, but vulnerable to a targeted attack against its hubs, as a result of its architecture. The significance of the results is considered with respect to mutational knockouts of proteins and the directed attacks mounted by tumour inducing viruses.

  19. Pivotal roles of p53 transcription-dependent and -independent pathways in manganese-induced mitochondrial dysfunction and neuronal apoptosis.

    PubMed

    Wan, Chunhua; Ma, Xa; Shi, Shangshi; Zhao, Jianya; Nie, Xiaoke; Han, Jingling; Xiao, Jing; Wang, Xiaoke; Jiang, Shengyang; Jiang, Junkang

    2014-12-15

    Chronic exposure to excessive manganese (Mn) has been known to lead to neuronal loss and a clinical syndrome resembling idiopathic Parkinson's disease (IPD). p53 plays an integral role in the development of various human diseases, including neurodegenerative disorders. However, the role of p53 in Mn-induced neuronal apoptosis and neurological deficits remains obscure. In the present study, we showed that p53 was critically involved in Mn-induced neuronal apoptosis in rat striatum through both transcription-dependent and -independent mechanisms. Western blot and immunohistochemistrical analyses revealed that p53 was remarkably upregulated in the striatum of rats following Mn exposure. Coincidentally, increased level of cleaved PARP, a hallmark of apoptosis, was observed. Furthermore, using nerve growth factor (NGF)-differentiated PC12 cells as a neuronal cell model, we showed that Mn exposure decreased cell viability and induced apparent apoptosis. Importantly, p53 was progressively upregulated, and accumulated in both the nucleus and the cytoplasm. The cytoplasmic p53 had a remarkable distribution in mitochondria, suggesting an involvement of p53 mitochondrial translocation in Mn-induced neuronal apoptosis. In addition, Mn-induced impairment of mitochondrial membrane potential (ΔΨm) could be partially rescued by pretreatment with inhibitors of p53 transcriptional activity and p53 mitochondrial translocation, Pifithrin-α (PFT-α) and Pifithrin-μ (PFT-μ), respectively. Moreover, blockage of p53 activities with PFT-α and PFT-μ significantly attenuated Mn-induced reactive oxidative stress (ROS) generation and mitochondrial H₂O₂ production. Finally, we observed that pretreatment with PFT-α and PFT-μ ameliorated Mn-induced apoptosis in PC12 cells. Collectively, these findings implicate that p53 transcription-dependent and -independent pathways may play crucial roles in the regulation of Mn-induced neuronal death.

  20. 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

  1. A naturally occurring 4-bp deletion in the intron 4 of p53 creates a spectrum of novel p53 isoforms with anti-apoptosis function.

    PubMed

    Shi, Hui; Tao, Ting; Huang, Delai; Ou, Zhao; Chen, Jun; Peng, Jinrong

    2015-01-01

    p53 functions as a tumor suppressor by transcriptionally regulating the expression of genes involved in controlling cell proliferation or apoptosis. p53 and its isoform Δ133p53/Δ113p53 form a negative regulation loop in that p53 activates the expression of Δ133p53/Δ113p53 while Δ133p53/Δ113p53 specifically antagonizes p53 apoptotic activity. This pathway is especially important to safeguard the process of embryogenesis because sudden activation of p53 by DNA damage signals or developmental stress is detrimental to a developing embryo. Here we report the identification of five novel p53 isoforms. p53β is generated due to alternative splicing of the intron 8 of p53 while the other four, namely, TA2p53, TA3p53, TA4p53 and TA5p53, result from the combination of alternative splicing of intron 1 (within intron 4 of the p53 gene) of the Δ113p53 gene and a naturally occurring CATT 4 bp deletion within the alternative splicing product in zebrafish. The CATT 4 bp deletion creates four translation start codons which are in-frame to the open reading frame of Δ113p53. We also show that TAp53 shares the same promoter with Δ113p53 and functions to antagonize p53 apoptotic activity. The identification of Δ113p53/TA2/3/4/5p53 reveals a pro-survival mechanism which operates robustly during embryogenesis in response to the DNA-damage condition.

  2. Proteasome machinery is instrumental in a common gain-of-function program of the p53 missense mutants in cancer.

    PubMed

    Walerych, Dawid; Lisek, Kamil; Sommaggio, Roberta; Piazza, Silvano; Ciani, Yari; Dalla, Emiliano; Rajkowska, Katarzyna; Gaweda-Walerych, Katarzyna; Ingallina, Eleonora; Tonelli, Claudia; Morelli, Marco J; Amato, Angela; Eterno, Vincenzo; Zambelli, Alberto; Rosato, Antonio; Amati, Bruno; Wiśniewski, Jacek R; Del Sal, Giannino

    2016-08-01

    In cancer, the tumour suppressor gene TP53 undergoes frequent missense mutations that endow mutant p53 proteins with oncogenic properties. Until now, a universal mutant p53 gain-of-function program has not been defined. By means of multi-omics: proteome, DNA interactome (chromatin immunoprecipitation followed by sequencing) and transcriptome (RNA sequencing/microarray) analyses, we identified the proteasome machinery as a common target of p53 missense mutants. The mutant p53-proteasome axis globally affects protein homeostasis, inhibiting multiple tumour-suppressive pathways, including the anti-oncogenic KSRP-microRNA pathway. In cancer cells, p53 missense mutants cooperate with Nrf2 (NFE2L2) to activate proteasome gene transcription, resulting in resistance to the proteasome inhibitor carfilzomib. Combining the mutant p53-inactivating agent APR-246 (PRIMA-1MET) with the proteasome inhibitor carfilzomib is effective in overcoming chemoresistance in triple-negative breast cancer cells, creating a therapeutic opportunity for treatment of solid tumours and metastasis with mutant p53.

  3. p53-dependent delayed effects of radiation vary according to time of irradiation of p53 + / - mice.

    PubMed

    Okazaki, Ryuji; Ootsuyama, Akira

    2014-01-01

    We previously reported that in p53 (+ / -) mice that had been given a whole-body dose of 3 Gy at 8 weeks of age, p53-dependent delayed effects of radiation, as manifested in T-cell receptor (TCR) variant fractions (VF) instability in mouse splenocytes, were biphasic, namely, induction of TCR-VF mutation reappeared at 44 weeks. The manifestation of the delayed effects and the measures of biological markers varied according to the timing of irradiation. We also reported that the decrease in function of the p53 gene was related to the effects of a delayed mutation. In the present study, we investigated the functions and mutations of the p53 gene in old age for p53 (+ / -) mice following irradiation at various ages. p53 (+ / -) mice were given a whole-body dose of 3 Gy at 8, 28 or 40 weeks of age. There were significant differences for all variables tested at 8 weeks of age. This was similarly the case for mice irradiated at 28 weeks of age, in which there were also significant differences in TCR VF and the percentage of apoptosis. In mice irradiated at 40 weeks of age, there were significant differences for all considered variables except for the p53 allele. We demonstrated that the different patterns of delayed mutation of the p53 gene at 56 weeks of age depended on the age at which mice had undergone 3-Gy whole-body irradiation. Our conclusions are limited to variation in p53-dependent delayed effects according to the time of irradiation.

  4. 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

  5. Oncogenomic Approaches in Exploring Gain of Function of Mutant p53

    PubMed Central

    Donzelli, Sara; Biagioni, Francesca; Fausti, Francesca; Strano, Sabrina; Fontemaggi, Giulia; Blandino, Giovanni

    2008-01-01

    Cancer is caused by the spatial and temporal accumulation of alterations in the genome of a given cell. This leads to the deregulation of key signalling pathways that play a pivotal role in the control of cell proliferation and cell fate. The p53 tumor suppressor gene is the most frequent target in genetic alterations in human cancers. The primary selective advantage of such mutations is the elimination of cellular wild type p53 activity. In addition, many evidences in vitro and in vivo have demonstrated that at least certain mutant forms of p53 may possess a gain of function, whereby they contribute positively to cancer progression. The fine mapping and deciphering of specific cancer phenotypes is taking advantage of molecular-profiling studies based on genome-wide approaches. Currently, high-throughput methods such as array-based comparative genomic hybridization (CGH array), single nucleotide polymorphism array (SNP array), expression arrays and ChIP-on-chip arrays are available to study mutant p53-associated alterations in human cancers. Here we will mainly focus on the integration of the results raised through oncogenomic platforms that aim to shed light on the molecular mechanisms underlying mutant p53 gain of function activities and to provide useful information on the molecular stratification of tumor patients. PMID:19440517

  6. Structure of the stapled p53 peptide bound to Mdm2.

    PubMed

    Baek, Sohee; Kutchukian, Peter S; Verdine, Gregory L; Huber, Robert; Holak, Tad A; Lee, Ki Won; Popowicz, Grzegorz M

    2012-01-11

    Mdm2 is a major negative regulator of the tumor suppressor p53 protein, a protein that plays a crucial role in maintaining genome integrity. Inactivation of p53 is the most prevalent defect in human cancers. Inhibitors of the Mdm2-p53 interaction that restore the functional p53 constitute potential nongenotoxic anticancer agents with a novel mode of action. We present here a 2.0 Å resolution structure of the Mdm2 protein with a bound stapled p53 peptide. Such peptides, which are conformationally and proteolytically stabilized with all-hydrocarbon staples, are an emerging class of biologics that are capable of disrupting protein-protein interactions and thus have broad therapeutic potential. The structure represents the first crystal structure of an i, i + 7 stapled peptide bound to its target and reveals that rather than acting solely as a passive conformational brace, a staple can intimately interact with the surface of a protein and augment the binding interface.

  7. p53 in cell invasion, podosomes, and invadopodia

    PubMed Central

    Mak, Alan S

    2014-01-01

    Cell invasion of the extracellular matrix is prerequisite to cross tissue migration of tumor cells in cancer metastasis, and vascular smooth muscle cells in atherosclerosis. The tumor suppressor p53, better known for its roles in the regulation of cell cycle and apoptosis, has ignited much interest in its function as a suppressor of cell migration and invasion. How p53 and its gain-of-function mutants regulate cell invasion remains a puzzle and a challenge for future studies. In recent years, podosomes and invadopodia have also gained center stage status as veritable apparatus specialized in cell invasion. It is not clear, however, whether p53 regulates cell invasion through podosomes and invadopodia. In this review, evidence supporting a negative role of p53 in podosomes formation in vascular smooth muscle cells will be surveyed, and signaling nodes that may mediate this regulation in other cell types will be explored. PMID:24714032

  8. The p53-Deficient Mouse as a Breast Cancer Model

    DTIC Science & Technology

    1995-10-01

    mammary tumor progression in a relatively controlled fashion within a reasonable length of time. Elucidation of the biological processes affected by p53...role of p53 loss in the mammary tumorigenesis process . The primary goals of the remaining years will be to extend these studies by looking at other...regulation and cell proliferation; (2) angiogenesis; and (3) invasiveness and metastases. The particular genes which regulate these biological processes will

  9. Gulf Cooperation Council: Arabia's model of integration

    SciTech Connect

    Etaibi, G.T.

    1984-01-01

    This study is an analysis of the foundations and emergence in 1981 of the Gulf Cooperation Council (GCC), which consists of six traditional Arab Gulf states (the United Arab Emirates, Bahrain, Saudi Arabia, Oman, Qatar, and Kuwait). It finds the GCC to be a unique case among twentieth-century integrative schemes. The study also identifies and analyzes relevant local, regional, and international forces. Among the local forces are traditional religio-political systems, economic dependence on a depletable resource, and the presence of a large number of foreign residents. On the regional level, this study takes into consideration such issues as the Arab League, Arab Nationalism, and the Islamic revolutionary movement in Iran. On the international level, the influence of the superpowers and the major industrialized nations on the emergence and future of the GCC Community are analyzed. Throughout the past decade there has been a growing scholarly interest in the Gulf region. In preparation for this study, the author relied heavily on the literature generated by this new research, as well as on documents and official publications, mostly in Arabic. A survey was conducted among a limited number of GCC graduate students during the summer of 1983. In addition, interviews with selected members of the GCC Secretariat-General and various member-state officials were conducted during a research trip in the region in the spring of 1984.

  10. Ribosomal Proteins Control or Bypass p53 during Nucleolar Stress

    PubMed Central

    Russo, Annapina; Russo, Giulia

    2017-01-01

    The nucleolus is the site of ribosome biogenesis, a complex process that requires the coordinate activity of all three RNA polymerases and hundreds of non-ribosomal factors that participate in the maturation of ribosomal RNA (rRNA) and assembly of small and large subunits. Nevertheless, emerging studies have highlighted the fundamental role of the nucleolus in sensing a variety of cellular stress stimuli that target ribosome biogenesis. This condition is known as nucleolar stress and triggers several response pathways to maintain cell homeostasis, either p53-dependent or p53-independent. The mouse double minute (MDM2)-p53 stress signaling pathways are activated by multiple signals and are among the most important regulators of cellular homeostasis. In this review, we will focus on the role of ribosomal proteins in p53-dependent and p53-independent response to nucleolar stress considering novel identified regulators of these pathways. We describe, in particular, the role of ribosomal protein uL3 (rpL3) in p53-independent nucleolar stress signaling pathways. PMID:28085118

  11. 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

  12. UHRF2, another E3 ubiquitin ligase for p53

    SciTech Connect

    Bai, Lu; Wang, Xiaohui; Jin, Fangmin; Yang, Yan; Qian, Guanhua; Duan, Changzhu

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer UHRF2 associates with p53 in vivo and in vitro. Black-Right-Pointing-Pointer UHRF2 interacts with p53 through its SRA/YDG domain. Black-Right-Pointing-Pointer UHRF2 ubiquitinates p53 in vivo and in vitro. -- Abstract: UHRF2, ubiquitin-like with PHD and ring finger domains 2, is a nuclear E3 ubiquitin ligase, which is involved in cell cycle and epigenetic regulation. UHRF2 interacts with multiple cell cycle proteins, including cyclins (A2, B1, D1, and E1), CDK2, and pRb; moreover, UHRF2 could ubiquitinate cyclin D1 and cyclin E1. Also, UHRF2 has been shown to be implicated in epigenetic regulation by associating with DNMTs, G9a, HDAC1, H3K9me2/3 and hemi-methylated DNA. We found that UHRF2 associates with tumor suppressor protein p53, and p53 is ubiquitinated by UHRF2 in vivo and in vitro. Given that both UHRF2 and p53 are involved in cell cycle regulation, this study may suggest a novel signaling pathway on cell proliferation.

  13. Tumourigenesis associated with the p53 tumour suppressor gene.

    PubMed Central

    Chang, F.; Syrjänen, S.; Tervahauta, A.; Syrjänen, K.

    1993-01-01

    The p53 gene is contained within 16-20 kb of cellular DNA located on the short arm of human chromosome 17 at position 17p13.1. This gene encodes a 393-amino-acid nuclear phosphoprotein involved in the regulation of cell proliferation. Current evidence suggests that loss of normal p53 function is associated with cell transformation in vitro and development of neoplasms in vivo. More than 50% of human malignancies of epithelial, mesenchymal, haematopoietic, lymphoid, and central nervous system origin analysed thus far, were shown to contain an altered p53 gene. The oncoproteins derived from several tumour viruses, including the SV40 large T antigen, the adenovirus E1B protein and papillomavirus E6 protein, as well as specific cellular gene products, e.g. murine double minute-2 (MDM2), were found to bind to the wild-type p53 protein and presumably lead to inactivation of this gene product. Therefore, the inactivation of p53 tumour suppressor gene is currently regarded as an almost universal step in the development of human cancers. The current data on p53-associated tumourigenesis are briefly discussed in this minireview. PMID:8398688

  14. Post-translational regulation enables robust p53 regulation

    PubMed Central

    2013-01-01

    Background The tumor suppressor protein p53 plays important roles in DNA damage repair, cell cycle arrest and apoptosis. Due to its critical functions, the level of p53 is tightly regulated by a negative feedback mechanism to increase its tolerance towards fluctuations and disturbances. Interestingly, the p53 level is controlled by post-translational regulation rather than transcriptional regulation in this feedback mechanism. Results We analyzed the dynamics of this feedback to understand whether post-translational regulation provides any advantages over transcriptional regulation in regard to disturbance rejection. When a disturbance happens, even though negative feedback reduces the steady-state error, it can cause a system to become less stable and transiently overshoots, which may erroneously trigger downstream reactions. Therefore, the system needs to balance the trade-off between steady-state and transient errors. Feedback control and adaptive estimation theories revealed that post-translational regulation achieves a better trade-off than transcriptional regulation, contributing to a more steady level of p53 under the influence of noise and disturbances. Furthermore, post-translational regulation enables cells to respond more promptly to stress conditions with consistent amplitude. However, for better disturbance rejection, the p53- Mdm2 negative feedback has to pay a price of higher stochastic noise. Conclusions Our analyses suggest that the p53-Mdm2 feedback favors regulatory mechanisms that provide the optimal trade-offs for dynamic control. PMID:23992617

  15. p53 and MDM2 protein expression in actinic cheilitis.

    PubMed

    de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

    2008-01-01

    Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  16. The p53 Transcriptional Network Influences Microglia Behavior and Neuroinflammation.

    PubMed

    Aloi, Macarena S; Su, Wei; Garden, Gwenn A

    2015-01-01

    The tumor-suppressor protein p53 belongs to a family of proteins that play pivotal roles in multiple cellular functions including cell proliferation, cell death, genome stability, and regulation of inflammation. Neuroinflammation is a common feature of central nervous system (CNS) pathology, and microglia are the specialized resident population of CNS myeloid cells that initiate innate immune responses. Microglia maintain CNS homeostasis through pathogen containment, phagocytosis of debris, and initiation of tissue-repair cascades. However, an unregulated pro-inflammatory response can lead to tissue injury and dysfunction in both acute and chronic inflammatory states. Therefore, regulation of the molecular signals that control the induction, magnitude, and resolution of inflammation are necessary for optimal CNS health. We and others have described a novel mechanism by which p53 transcriptional activity modulates microglia behaviors in vitro and in vivo. Activation of p53 induces expression of microRNAs (miRNAs) that support microglia pro-inflammatory functions and suppress anti-inflammatory and tissue repair behaviors. In this review, we introduce the previously described roles of the p53 signaling network and discuss novel functions of p53 in the microglia-mediated inflammatory response in CNS health and disease. Ultimately, improved understanding of the molecular regulators modulated by p53 transcriptional activity in microglia will enhance the development of rational therapeutic strategies to harness the homeostatic and tissue repair functions of microglia.

  17. Dynamics of Delayed p53 Mutations in Mice Given Whole-Body Irradiation at 8 Weeks

    SciTech Connect

    Okazaki, Ryuji; Ootsuyama, Akira; Kakihara, Hiroyo; Mabuchi, Yo; Matsuzaki, Yumi; Michikawa, Yuichi; Imai, Takashi; Norimura, Toshiyuki

    2011-01-01

    Purpose: Ionizing irradiation might induce delayed genotoxic effects in a p53-dependent manner. However, a few reports have shown a p53 mutation as a delayed effect of radiation. In this study, we investigated the p53 gene mutation by the translocation frequency in chromosome 11, loss of p53 alleles, p53 gene methylation, p53 nucleotide sequence, and p53 protein expression/phosphorylation in p53{sup +/+} and p53{sup +/-} mice after irradiation at a young age. Methods and Materials: p53{sup +/+} and p53{sup +/-} mice were exposed to 3 Gy of whole-body irradiation at 8 weeks of age. Chromosome instability was evaluated by fluorescence in situ hybridization analysis. p53 allele loss was evaluated by polymerase chain reaction, and p53 methylation was evaluated by methylation-specific polymerase chain reaction. p53 sequence analysis was performed. p53 protein expression was evaluated by Western blotting. Results: The translocation frequency in chromosome 11 showed a delayed increase after irradiation. In old irradiated mice, the number of mice that showed p53 allele loss and p53 methylation increased compared to these numbers in old non-irradiated mice. In two old irradiated p53{sup +/-} mice, the p53 sequence showed heteromutation. In old irradiated mice, the p53 and phospho-p53 protein expressions decreased compared to old non-irradiated mice. Conclusion: We concluded that irradiation at a young age induced delayed p53 mutations and p53 protein suppression.

  18. p53 protein, EGF receptor, and anti-p53 antibodies in serum from patients with occupationally derived lung cancer

    PubMed Central

    Schneider, J; Presek, P; Braun, A; Bauer, P; Konietzko, N; Wiesner, B; Woitowitz, H-J

    1999-01-01

    The oncogene product epidermal growth factor receptor (EGF-R), the tumour suppressor gene product p53 and anti-p53 antibodies are detectable in the serum of certain cancer patients. Increased levels of some of these products were reported in lung cancer patients after occupational asbestos exposure and after exposure to polycyclic aromatic hydrocarbons or vinylchloride. In the first step, this study investigated the possible diagnostic value of serum EGF-R, p53-protein and anti-p53 antibodies, measured by an enzyme-linked immunosorbent assay, in lung tumour patients. In addition to being investigated on a molecular epidemiological basis, these parameters were examined as biomarkers of carcinogenesis, especially with regard to asbestos incorporation effects or of radon-induced lung cancers. Also, a possible effect of cigarette smoking and age dependence were studied. A total of 116 male patients with lung or pleural tumours were examined. The histological classification was four small-cell cancers, six large-cell cancers, 32 adenocarcinomas, 47 squamous carcinomas, 12 mixed lung carcinomas, five diffuse malignant mesotheliomas and ten lung metastasis of extrapulmonary tumours. Twenty-two lung cancers and all mesotheliomas were related to asbestos, 22 lung cancers were related to ionizing radiation and 61 patients had cigarette smoke-related lung cancer. Besides these patients 50 male patients with non-malignant lung or pleural diseases were included; of the latter eight subjects suffered from asbestosis. Controls were 129 male subjects without any lung disease. No significantly elevated or decreased serum values for p53 protein, EGF-R, or anti-p53 antibodies as a function of histological tumour type, age, or degree and type of exposure (asbestos, smoking, ionizing radiation) could be found. The utility of p53-protein, EGF-R and anti-p53 antibodies as routine biomarkers for screening occupationally derived lung cancers is limited. © 1999 Cancer Research Campaign

  19. p53 pathway determines the cellular response to alcohol-induced DNA damage in MCF-7 breast cancer cells

    PubMed Central

    Zhao, Ming; Howard, Erin W.; Guo, Zhiying; Parris, Amanda B.; Yang, Xiaohe

    2017-01-01

    Alcohol consumption is associated with increased breast cancer risk; however, the underlying mechanisms that contribute to mammary tumor initiation and progression are unclear. Alcohol is known to induce oxidative stress and DNA damage; likewise, p53 is a critical modulator of the DNA repair pathway and ensures genomic integrity. p53 mutations are frequently detected in breast and other tumors. The impact of alcohol on p53 is recognized, yet the role of p53 in alcohol-induced mammary carcinogenesis remains poorly defined. In our study, we measured alcohol-mediated oxidative DNA damage in MCF-7 cells using 8-OHdG and p-H2AX foci formation assays. p53 activity and target gene expression after alcohol exposure were determined using p53 luciferase reporter assay, qPCR, and Western blotting. A mechanistic study delineating the role of p53 in DNA damage response and cell cycle arrest was based on isogenic MCF-7 cells stably transfected with control (MCF-7/Con) or p53-targeting siRNA (MCF-7/sip53), and MCF-7 cells that were pretreated with Nutlin-3 (Mdm2 inhibitor) to stabilize p53. Alcohol treatment resulted in significant DNA damage in MCF-7 cells, as indicated by increased levels of 8-OHdG and p-H2AX foci number. A p53-dependent signaling cascade was stimulated by alcohol-induced DNA damage. Moderate to high concentrations of alcohol (0.1–0.8% v/v) induced p53 activation, as indicated by increased p53 phosphorylation, reporter gene activity, and p21/Bax gene expression, which led to G0/G1 cell cycle arrest. Importantly, compared to MCF-7/Con cells, alcohol-induced DNA damage was significantly enhanced, while alcohol-induced p21/Bax expression and cell cycle arrest were attenuated in MCF-7/sip53 cells. In contrast, inhibition of p53 degradation via Nutlin-3 reinforced G0/G1 cell cycle arrest in MCF-7 control cells. Our study suggests that functional p53 plays a critical role in cellular responses to alcohol-induced DNA damage, which protects the cells from DNA damage

  20. Pivotal roles of p53 transcription-dependent and -independent pathways in manganese-induced mitochondrial dysfunction and neuronal apoptosis

    SciTech Connect

    Wan, Chunhua; Ma, Xa; Shi, Shangshi; Zhao, Jianya; Nie, Xiaoke; Han, Jingling; Xiao, Jing; Wang, Xiaoke; Jiang, Shengyang; Jiang, Junkang

    2014-12-15

    Chronic exposure to excessive manganese (Mn) has been known to lead to neuronal loss and a clinical syndrome resembling idiopathic Parkinson's disease (IPD). p53 plays an integral role in the development of various human diseases, including neurodegenerative disorders. However, the role of p53 in Mn-induced neuronal apoptosis and neurological deficits remains obscure. In the present study, we showed that p53 was critically involved in Mn-induced neuronal apoptosis in rat striatum through both transcription-dependent and -independent mechanisms. Western blot and immunohistochemistrical analyses revealed that p53 was remarkably upregulated in the striatum of rats following Mn exposure. Coincidentally, increased level of cleaved PARP, a hallmark of apoptosis, was observed. Furthermore, using nerve growth factor (NGF)-differentiated PC12 cells as a neuronal cell model, we showed that Mn exposure decreased cell viability and induced apparent apoptosis. Importantly, p53 was progressively upregulated, and accumulated in both the nucleus and the cytoplasm. The cytoplasmic p53 had a remarkable distribution in mitochondria, suggesting an involvement of p53 mitochondrial translocation in Mn-induced neuronal apoptosis. In addition, Mn-induced impairment of mitochondrial membrane potential (ΔΨm) could be partially rescued by pretreatment with inhibitors of p53 transcriptional activity and p53 mitochondrial translocation, Pifithrin-α (PFT-α) and Pifithrin-μ (PFT-μ), respectively. Moreover, blockage of p53 activities with PFT-α and PFT-μ significantly attenuated Mn-induced reactive oxidative stress (ROS) generation and mitochondrial H{sub 2}O{sub 2} production. Finally, we observed that pretreatment with PFT-α and PFT-μ ameliorated Mn-induced apoptosis in PC12 cells. Collectively, these findings implicate that p53 transcription-dependent and -independent pathways may play crucial roles in the regulation of Mn-induced neuronal death. - Highlights: • p53 is robustly

  1. Phosphorylation of p53 by TAF1 inactivates p53-dependent transcription in the DNA damage response

    PubMed Central

    Wu, Yong; Lin, Joy C.; Piluso, Landon G.; Dhahbi, Joseph M.; Bobadilla, Selene; Spindler, Stephen R.; Liu, Xuan

    2014-01-01

    Summary While p53 activation has long been studied, the mechanisms by which its targets genes are restored to their pre-activation state are less clear. We report here that TAF1 phosphorylates p53 at Thr55, leading to dissociation of p53 from the p21 promoter and inactivation of transcription late in the DNA damage response. We further show that cellular ATP level might act as a molecular switch for Thr55 phosphorylation on the p21 promoter, indicating that TAF1 is a cellular ATP sensor. Upon DNA damage, cells undergo PARP-1-dependent ATP depletion, which is correlated with reduced TAF1 kinase activity and Thr55 phosphorylation, resulting in p21 activation. As cellular ATP levels recover, TAF1 is able to phosphorylate p53 on Thr55, which leads to dissociation of p53 from the p21 promoter. ChIP-sequencing analysis reveals p53 dissociates from promoters genome-wide as cells recover from DNA damage, suggesting the general nature of this mechanism. PMID:24289924

  2. R248Q mutation--Beyond p53-DNA binding.

    PubMed

    Ng, Jeremy W K; Lama, Dilraj; Lukman, Suryani; Lane, David P; Verma, Chandra S; Sim, Adelene Y L

    2015-12-01

    R248 in the DNA binding domain (DBD) of p53 interacts directly with the minor groove of DNA. Earlier nuclear magnetic resonance (NMR) studies indicated that the R248Q mutation resulted in conformation changes in parts of DBD far from the mutation site. However, how information propagates from the mutation site to the rest of the DBD is still not well understood. We performed a series of all-atom molecular dynamics (MD) simulations to dissect sterics and charge effects of R248 on p53-DBD conformation: (i) wild-type p53 DBD; (ii) p53 DBD with an electrically neutral arginine side-chain; (iii) p53 DBD with R248A; (iv) p53 DBD with R248W; and (v) p53 DBD with R248Q. Our results agree well with experimental observations of global conformational changes induced by the R248Q mutation. Our simulations suggest that both charge- and sterics are important in the dynamics of the loop (L3) where the mutation resides. We show that helix 2 (H2) dynamics is altered as a result of a change in the hydrogen bonding partner of D281. In turn, neighboring L1 dynamics is altered: in mutants, L1 predominantly adopts the recessed conformation and is unable to interact with the major groove of DNA. We focused our attention the R248Q mutant that is commonly found in a wide range of cancer and observed changes at the zinc-binding pocket that might account for the dominant negative effects of R248Q. Furthermore, in our simulations, the S6/S7 turn was more frequently solvent exposed in R248Q, suggesting that there is a greater tendency of R248Q to partially unfold and possibly lead to an increased aggregation propensity. Finally, based on the observations made in our simulations, we propose strategies for the rescue of R248Q mutants.

  3. Regulation of p53 is critical for vertebrate limb regeneration.

    PubMed

    Yun, Maximina H; Gates, Phillip B; Brockes, Jeremy P

    2013-10-22

    Extensive regeneration of the vertebrate body plan is found in salamander and fish species. In these organisms, regeneration takes place through reprogramming of differentiated cells, proliferation, and subsequent redifferentiation of adult tissues. Such plasticity is rarely found in adult mammalian tissues, and this has been proposed as the basis of their inability to regenerate complex structures. Despite their importance, the mechanisms underlying the regulation of the differentiated state during regeneration remain unclear. Here, we analyzed the role of the tumor-suppressor p53 during salamander limb regeneration. The activity of p53 initially decreases and then returns to baseline. Its down-regulation is required for formation of the blastema, and its up-regulation is necessary for the redifferentiation phase. Importantly, we show that a decrease in the level of p53 activity is critical for cell cycle reentry of postmitotic, differentiated cells, whereas an increase is required for muscle differentiation. In addition, we have uncovered a potential mechanism for the regulation of p53 during limb regeneration, based on its competitive inhibition by ΔNp73. Our results suggest that the regulation of p53 activity is a pivotal mechanism that controls the plasticity of the differentiated state during regeneration.

  4. The evolution of thymic lymphomas in p53 knockout mice

    PubMed Central

    Dudgeon, Crissy; Chan, Chang; Kang, Wenfeng; Sun, Yvonne; Emerson, Ryan; Robins, Harlan

    2014-01-01

    Germline deletion of the p53 gene in mice gives rise to spontaneous thymic (T-cell) lymphomas. In this study, the p53 knockout mouse was employed as a model to study the mutational evolution of tumorigenesis. The clonality of the T-cell repertoire from p53 knockout and wild-type thymic cells was analyzed at various ages employing TCRβ sequencing. These data demonstrate that p53 knockout thymic lymphomas arose in an oligoclonal fashion, with tumors evolving dominant clones over time. Exon sequencing of tumor DNA revealed that all of the independently derived oligoclonal mouse tumors had a deletion in the Pten gene prior to the formation of the TCRβ rearrangement, produced early in development. This was followed in each independent clone of the thymic lymphoma by the amplification or overexpression of cyclin Ds and Cdk6. Alterations in the expression of Ikaros were common and blocked further development of CD-4/CD-8 T cells. While the frequency of point mutations in the genome of these lymphomas was one per megabase, there were a tremendous number of copy number variations producing the tumors’ driver mutations. The initial inherited loss of p53 functions appeared to delineate an order of genetic alterations selected for during the evolution of these thymic lymphomas. PMID:25452272

  5. The expanding regulatory universe of p53 in gastrointestinal cancer.

    PubMed

    Fesler, Andrew; Zhang, Ning; Ju, Jingfang

    2016-01-01

    Tumor suppresser gene TP53 is one of the most frequently deleted or mutated genes in gastrointestinal cancers. As a transcription factor, p53 regulates a number of important protein coding genes to control cell cycle, cell death, DNA damage/repair, stemness, differentiation and other key cellular functions. In addition, p53 is also able to activate the expression of a number of small non-coding microRNAs (miRNAs) through direct binding to the promoter region of these miRNAs.  Many miRNAs have been identified to be potential tumor suppressors by regulating key effecter target mRNAs. Our understanding of the regulatory network of p53 has recently expanded to include long non-coding RNAs (lncRNAs). Like miRNA, lncRNAs have been found to play important roles in cancer biology.  With our increased understanding of the important functions of these non-coding RNAs and their relationship with p53, we are gaining exciting new insights into the biology and function of cells in response to various growth environment changes. In this review we summarize the current understanding of the ever expanding involvement of non-coding RNAs in the p53 regulatory network and its implications for our understanding of gastrointestinal cancer.

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

    PubMed

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

    2012-08-15

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

  7. Mapping of UV photoproducts along the human P53 gene

    SciTech Connect

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

    1994-12-31

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

  8. p53 on the crossroad between regeneration and cancer

    PubMed Central

    Charni, Meital; Aloni-Grinstein, Ronit; Molchadsky, Alina; Rotter, Varda

    2017-01-01

    Regeneration and tumorigenesis share common molecular pathways, nevertheless the outcome of regeneration is life, whereas tumorigenesis leads to death. Although the process of regeneration is strictly controlled, malignant transformation is unrestrained. In this review, we discuss the involvement of TP53, the major tumor-suppressor gene, in the regeneration process. We point to the role of p53 as coordinator assuring that regeneration will not shift to carcinogenesis. The fluctuation in p53 activity during the regeneration process permits a tight control. On one hand, its inhibition at the initial stages allows massive proliferation, on the other its induction at advanced steps of regeneration is essential for preservation of robustness and fidelity of the regeneration process. A better understanding of the role of p53 in regulation of regeneration may open new opportunities for implementation of TP53-based therapies, currently available for cancer patients, in regenerative medicine. PMID:27768121

  9. p53 as a Regulator of Lipid Metabolism in Cancer

    PubMed Central

    Parrales, Alejandro; Iwakuma, Tomoo

    2016-01-01

    Enhanced proliferation and survival are common features of cancer cells. Cancer cells are metabolically reprogrammed which aids in their survival in nutrient-poor environments. Indeed, changes in metabolism of glucose and glutamine are essential for tumor progression. Thus, metabolic reprogramming is now well accepted as a hallmark of cancer. Recent findings suggest that reprogramming of lipid metabolism also occurs in cancer cells, since lipids are used for biosynthesis of membranes, post-translational modifications, second messengers for signal transduction, and as a source of energy during nutrient deprivation. The tumor suppressor p53 is a transcription factor that controls the expression of proteins involved in cell cycle arrest, DNA repair, apoptosis, and senescence. p53 also regulates cellular metabolism, which appears to play a key role in its tumor suppressive activities. In this review article, we summarize non-canonical functions of wild-type and mutant p53 on lipid metabolism and discuss their association with cancer progression. PMID:27973397

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

    PubMed Central

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

    2017-01-01

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

  11. Lung cancer stem cells, p53 mutations and MDM2.

    PubMed

    Gadepalli, Venkat Sundar; Deb, Swati Palit; Deb, Sumitra; Rao, Raj R

    2014-01-01

    Over the past few decades, advances in cancer research have enabled us to understand the different mechanisms that contribute to the aberrant proliferation of normal cells into abnormal cells that result in tumors. In the pursuit to find cures, researchers have primarily focused on various molecular level changes that are unique to cancerous cells. In humans, about 50 % or more cancers have a mutated tumor suppressor p53 gene thereby resulting in accumulation of p53 protein and losing its function to activate the target genes that regulate cell cycle and apoptosis. Extensive research conducted in murine cancer models with activated p53, loss of p53, or p53 missense mutations have facilitated researchers to understand the role of this key protein. Despite the identification of numerous triggers that causes lung cancer specific cure still remain elusive. One of the primary reasons attributed to this is due to the fact that the tumor tissue is heterogeneous and contains numerous sub-populations of cells. Studies have shown that a specific sub-population of cells termed as cancer stem cells (CSCs) drive the recurrence of cancer in response to standard chemotherapy. These CSCs are mutated cells with core properties similar to those of adult stem cells. They reside in a microenvironment within the tumor tissue that supports their growth and make them less susceptible to drug treatment. These cells possess properties of symmetric self-renewal and migration thus driving tumor formation and metastasis. Therefore, research specifically targeting these cells has gained prominence towards developing new therapeutic agents against cancer. This chapter focuses on lung cancer stem cells, p53 mutations noted in these cells, and importance of MDM2 interactions. Further, research approaches for better understanding of molecular mechanisms that drive CSC function and developing appropriate therapies are discussed.

  12. p53 gene alterations and protein accumulation in colorectal cancer

    PubMed Central

    Bertorelle, R; Esposito, G; Belluco, C; Bonaldi, L; Del Mistro, A; Nitti, D; Lise, M; Chieco-Bianchi, L

    1996-01-01

    Aim—To correlate immunohistochemical staining with single strand conformation polymorphism (SSCP) analysis of the p53 gene in colorectal cancer in order to understand how the findings provided by the two techniques complement each other in defining p53 functional status. Methods—Frozen tumour tissue from 94 patients with colorectal cancer was studied for p53 protein accumulation and gene mutations. Accumulation of p53 protein was detected by immunohistochemistry using PAb1801 and BP53-12-1 monoclonal antibodies. The findings were then compared with SSCP analysis of exons 5 to 8 of the p53 gene. All cases with a positive result by SSCP analysis were confirmed by sequencing. Results—Nuclear staining was observed in 51 (54.2%) cases. SSCP analysis of the DNA amplified by PCR revealed that the electrophoretic pattern had shifted in 30 cases; sequence analysis confirmed the occurrence of a mutation in 29 cases and of a polymorphism in one. In 27 cases both assays gave a positive result, and in 40 both were negative; therefore, concordance between PCR-SSCP and immunohistochemistry was seen in 72% of cases. Conclusion—The data indicate that positive immunostaining corresponds with the presence of a mutation in most, but not all, cases studied; other mechanisms could be responsible for stabilisation and accumulation of p53 protein in the nucleus. Nonsense mutations which do not confer stability on the protein will not be detected by immunohistochemistry and false negative results can also occur with SSCP analysis. Images PMID:16696056

  13. Enhanced radiosensitization of p53 mutant cells by oleamide

    SciTech Connect

    Lee, Yoon-Jin; Chung, Da Yeon; Lee, Su-Jae; Ja Jhon, Gil; Lee, Yun-Sil . E-mail: yslee@kcch.re.kr

    2006-04-01

    Purpose: Effect of oleamide, an endogenous fatty-acid primary amide, on tumor cells exposed to ionizing radiation (IR) has never before been explored. Methods and Materials: NCI H460, human lung cancer cells, and human astrocytoma cell lines, U87 and U251, were used. The cytotoxicity of oleamide alone or in combination with IR was determined by clonogenic survival assay, and induction of apoptosis was estimated by FACS analysis. Protein expressions were confirmed by Western blotting, and immunofluorescence analysis of Bax by use of confocal microscopy was also performed. The combined effect of IR and oleamide to suppress tumor growth was studied by use of xenografts in the thighs of nude mice. Results: Oleamide in combination with IR had a synergistic effect that decreased clonogenic survival of lung-carcinoma cell lines and also sensitized xenografts in nude mice. Enhanced induction of apoptosis of the cells by the combined treatment was mediated by loss of mitochondrial membrane potential, which resulted in the activation of caspase-8, caspase-9, and caspase-3 accompanied by cytochrome c release and Bid cleavage. The synergistic effects of the combined treatment were more enhanced in p53 mutant cells than in p53 wild-type cells. In p53 wild-type cells, both oleamide and radiation induced Bax translocation to mitochondria. On the other hand, in p53 mutant cells, radiation alone slightly induced Bax translocation to mitochondria, whereas oleamide induced a larger translocation. Conclusions: Oleamide may exhibit synergistic radiosensitization in p53 mutant cells through p53-independent Bax translocation to mitochondria.

  14. SET1 and p300 Act Synergistically, through Coupled Histone Modifications, in Transcriptional Activation by p53

    PubMed Central

    Tang, Zhanyun; Chen, Wei-Yi; Shimada, Miho; Nguyen, Uyen T.T.; Kim, Jaehoon; Sun, Xiao-Jian; Sengoku, Toru; McGinty, Robert K.; Fernandez, Joseph P.; Muir, Tom W.; Roeder, Robert G.

    2014-01-01

    SUMMARY The H3K4me3 mark in chromatin is closely correlated with actively transcribed genes, although the mechanisms involved in its generation and function are not fully understood. In vitro studies with recombinant chromatin and purified human factors demonstrate a robust SET1 complex (SET1C)-mediated H3K4 trimethylation that is dependent upon p53- and p300-mediated H3 acetylation, a corresponding SET1C-mediated enhancement of p53- and p300-dependent transcription that reflects a primary effect of SET1C through H3K4 trimethylation, and direct SET1C-p53 and SET1C-p300 interactions indicative of a targeted recruitment mechanism. Complementary cell-based assays demonstrate a DNA-damage-induced p53-SET1C interaction, a corresponding enrichment of SET1C and H3K4me3 on a p53 target gene (p21/WAF1), and a corresponding codependency of H3K4 trimethylation and transcription upon p300 and SET1C. These results establish a mechanism in which SET1C and p300 act cooperatively, through direct interactions and coupled histone modifications, to facilitate the function of p53. PMID:23870121

  15. TGF-β induces p53/Smads complex formation in the PAI-1 promoter to activate transcription

    PubMed Central

    Kawarada, Yuki; Inoue, Yasumichi; Kawasaki, Fumihiro; Fukuura, Keishi; Sato, Koichi; Tanaka, Takahito; Itoh, Yuka; Hayashi, Hidetoshi

    2016-01-01

    Transforming growth factor β (TGF-β) signaling facilitates tumor development during the advanced stages of tumorigenesis, but induces cell-cycle arrest for tumor suppression during the early stages. However, the mechanism of functional switching of TGF-β is still unknown, and it is unclear whether inhibition of TGF-β signaling results amelioration or exacerbation of cancers. Here we show that the tumor suppressor p53 cooperates with Smad proteins, which are TGF-β signal transducers, to selectively activate plasminogen activator inhibitor type-1 (PAI-1) transcription. p53 forms a complex with Smad2/3 in the PAI-1 promoter to recruit histone acetyltransferase CREB-binding protein (CBP) and enhance histone H3 acetylation, resulting in transcriptional activation of the PAI-1 gene. Importantly, p53 is required for TGF-β-induced cytostasis and PAI-1 is involved in the cytostatic activity of TGF-β in several cell lines. Our results suggest that p53 enhances TGF-β-induced cytostatic effects by activating PAI-1 transcription, and the functional switching of TGF-β is partially caused by p53 mutation or p53 inactivation during cancer progression. It is expected that these findings will contribute to optimization of TGF-β-targeting therapies for cancer. PMID:27759037

  16. Tumor-suppressive p53 Signaling Empowers Metastatic Inhibitor KLF17-dependent Transcription to Overcome Tumorigenesis in Non-small Cell Lung Cancer.

    PubMed

    Ali, Amjad; Bhatti, Muhammad Zeeshan; Shah, Abdus Saboor; Duong, Hong-Quan; Alkreathy, Huda Mohammad; Mohammad, Shah Faisal; Khan, Rahmat Ali; Ahmad, Ayaz

    2015-08-28

    Metastasis, which is controlled by concerted action of multiple genes, is a complex process and is an important cause of cancer death. Krüppel-like factor 17 (KLF17) is a negative regulator of metastasis and epithelial-mesenchymal transition (EMT) during cancer progression. However, the underlying molecular mechanism and biological relevance of KLF17 in cancer cells are poorly understood. Here, we show that tumor suppressor protein p53 plays an integral role to induce KLF17 expression in non-small cell lung cancer (NSCLC). p53 is recruited to the KLF17 promoter and results in the formation of p53-DNA complex. p53 enhances binding of p300 and favors histone acetylation on the KLF17 promoter. Mechanistically, p53 physically interacts with KLF17 and thereby enhances the anti-metastatic function of KLF17. p53 empowers KLF17-mediated EMT genes transcription via enhancing physical association of KLF17 with target gene promoters. Nutlin-3 recruits KLF17 to EMT target gene promoters and results in the formation of KLF17-DNA complex via a p53-dependent pathway. p53 depletion abrogates DNA binding affinity of KLF17 to EMT target gene promoters. KLF17 is critical for p53 cellular activities in NSCLC. Importantly, KLF17 enhances p53 transcription to generate a novel positive feedback loop. KLF17 depletion accelerates lung cancer cell growth in response to chemotherapy. Mechanistically, we found that KLF17 increases the expression of tumor suppressor genes p53, p21, and pRB. Functionally, KLF17 required p53 to suppress cancer cell invasion and migration in NSCLC. In conclusion, our study highlights a novel insight into the anti-EMT effect of KLF17 via a p53-dependent pathway in NSCLC, and KLF17 may be a new therapeutic target in NSCLC with p53 status.

  17. p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage.

    PubMed

    Gong, Lu; Gong, Hongjian; Pan, Xiao; Chang, Changqing; Ou, Zhao; Ye, Shengfan; Yin, Le; Yang, Lina; Tao, Ting; Zhang, Zhenhai; Liu, Cong; Lane, David P; Peng, Jinrong; Chen, Jun

    2015-03-01

    The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.

  18. HZE particle radiation induces tissue-specific and p53-dependent mutagenesis in transgenic animals

    NASA Technical Reports Server (NTRS)

    Chang, P. Y.; Kanazawa, N.; Lutze-Mann, L.; Winegar, R.

    2001-01-01

    Transgenic animals, with the integrated target gene, provide a unique approach for measuring and characterizing mutations in any tissue of the animal. We are using the plasmid-based lacZ transgenic mice with different p53 genetic background to examine radiation-induced genetic damage resulting from exposure to heavy particle radiation. We measured lacZ mutation frequencies (MF) in the brain and spleen tissues at various times after exposing animals to an acute dose of 1 Gy of 1GeV/amu iron particles. MF in the spleen of p53+/+ animals increased up to 2.6-fold above spontaneous levels at 8 weeks post irradiation. In contrast, brain MF from the same animals increased 1.7-fold above controls in the same period. In the p53-/- animals, brain MF increased to 2.2-fold above spontaneous levels at 1 week after treatment, but returned to control levels thereafter. Radiation also induced alterations in the spectrum of mutants in both tissues, accompanied by changes in the frequency of mutants with deletions extending past the transgene into mouse genomic DNA. Our results indicate that the accumulation of transgene MF after radiation exposure is dependant on the tissue examined as well as the p53 genetic background of the animals.

  19. 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

  20. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro

    PubMed Central

    Xing, Fuqiang; Zhan, Qiuqiang; He, Yiduo; Cui, Jiesheng; He, Sailing; Wang, Guanyu

    2016-01-01

    Recent studies have reported that exposure of mammalian cells to microwave radiation may have adverse effects such as induction of cell apoptosis. However, the molecular mechanisms underlying microwave induced mammalian cell apoptosis are not fully understood. Here, we report a novel mechanism: exposure to 1800MHz microwave radiation induces p53-dependent cell apoptosis through cytochrome c-mediated caspase-3 activation pathway. We first measured intensity of microwave radiation from several electronic devices with an irradiation detector. Mouse NIH/3T3 and human U-87 MG cells were then used as receivers of 1800MHz electromagnetic radiation (EMR) at a power density of 1209 mW/m2. Following EMR exposure, cells were analyzed for viability, intracellular reactive oxygen species (ROS) generation, DNA damage, p53 expression, and caspase-3 activity. Our analysis revealed that EMR exposure significantly decreased viability of NIH/3T3 and U-87 MG cells, and increased caspase-3 activity. ROS burst was observed at 6 h and 48 h in NIH/3T3 cells, while at 3 h in U-87 MG cells. Hoechst 33258 staining and in situ TUNEL assay detected that EMR exposure increased DNA damage, which was significantly restrained in the presence of N-acetyl-L-cysteine (NAC, an antioxidant). Moreover, EMR exposure increased the levels of p53 protein and p53 target gene expression, promoted cytochrome c release from mitochondrion, and increased caspase-3 activity. These events were inhibited by pretreatment with NAC, pifithrin-α (a p53 inhibitor) and caspase inhibitor. Collectively, our findings demonstrate, for the first time, that 1800MHz EMR induces apoptosis-related events such as ROS burst and more oxidative DNA damage, which in turn promote p53-dependent caspase-3 activation through release of cytochrome c from mitochondrion. These findings thus provide new insights into physiological mechanisms underlying microwave-induced cell apoptosis. PMID:27689798

  1. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro.

    PubMed

    Xing, Fuqiang; Zhan, Qiuqiang; He, Yiduo; Cui, Jiesheng; He, Sailing; Wang, Guanyu

    Recent studies have reported that exposure of mammalian cells to microwave radiation may have adverse effects such as induction of cell apoptosis. However, the molecular mechanisms underlying microwave induced mammalian cell apoptosis are not fully understood. Here, we report a novel mechanism: exposure to 1800MHz microwave radiation induces p53-dependent cell apoptosis through cytochrome c-mediated caspase-3 activation pathway. We first measured intensity of microwave radiation from several electronic devices with an irradiation detector. Mouse NIH/3T3 and human U-87 MG cells were then used as receivers of 1800MHz electromagnetic radiation (EMR) at a power density of 1209 mW/m2. Following EMR exposure, cells were analyzed for viability, intracellular reactive oxygen species (ROS) generation, DNA damage, p53 expression, and caspase-3 activity. Our analysis revealed that EMR exposure significantly decreased viability of NIH/3T3 and U-87 MG cells, and increased caspase-3 activity. ROS burst was observed at 6 h and 48 h in NIH/3T3 cells, while at 3 h in U-87 MG cells. Hoechst 33258 staining and in situ TUNEL assay detected that EMR exposure increased DNA damage, which was significantly restrained in the presence of N-acetyl-L-cysteine (NAC, an antioxidant). Moreover, EMR exposure increased the levels of p53 protein and p53 target gene expression, promoted cytochrome c release from mitochondrion, and increased caspase-3 activity. These events were inhibited by pretreatment with NAC, pifithrin-α (a p53 inhibitor) and caspase inhibitor. Collectively, our findings demonstrate, for the first time, that 1800MHz EMR induces apoptosis-related events such as ROS burst and more oxidative DNA damage, which in turn promote p53-dependent caspase-3 activation through release of cytochrome c from mitochondrion. These findings thus provide new insights into physiological mechanisms underlying microwave-induced cell apoptosis.

  2. Structures of oncogenic, suppressor and rescued p53 core-domain variants: mechanisms of mutant p53 rescue

    SciTech Connect

    Wallentine, Brad D.; Wang, Ying; Tretyachenko-Ladokhina, Vira; Tan, Martha; Senear, Donald F.; Luecke, Hartmut

    2013-10-01

    X-ray crystallographic structures of four p53 core-domain variants were determined in order to gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53. To gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53, X-ray crystallographic structures of four p53 core-domain variants were determined. These include an oncogenic mutant, V157F, two single-site suppressor mutants, N235K and N239Y, and the rescued cancer mutant V157F/N235K/N239Y. The V157F mutation substitutes a smaller hydrophobic valine with a larger hydrophobic phenylalanine within strand S4 of the hydrophobic core. The structure of this cancer mutant shows no gross structural changes in the overall fold of the p53 core domain, only minor rearrangements of side chains within the hydrophobic core of the protein. Based on biochemical analysis, these small local perturbations induce instability in the protein, increasing the free energy by 3.6 kcal mol{sup −1} (15.1 kJ mol{sup −1}). Further biochemical evidence shows that each suppressor mutation, N235K or N239Y, acts individually to restore thermodynamic stability to V157F and that both together are more effective than either alone. All rescued mutants were found to have wild-type DNA-binding activity when assessed at a permissive temperature, thus pointing to thermodynamic stability as the critical underlying variable. Interestingly, thermodynamic analysis shows that while N239Y demonstrates stabilization of the wild-type p53 core domain, N235K does not. These observations suggest distinct structural mechanisms of rescue. A new salt bridge between Lys235 and Glu198, found in both the N235K and rescued cancer mutant structures, suggests a rescue mechanism that relies on stabilizing the

  3. 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.

  4. Super p53 for Treatment of Ovarian Cancer

    DTIC Science & Technology

    2016-07-01

    therapy, carboplatin, paclitaxel, polymeric drug delivery, polymer -adenovirus hybrid 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18...modified p53, tumor suppressor, high grade serous carcinoma, combination therapy, carboplatin, paclitaxel, polymeric drug delivery, polymer ... ORGANIZATION : University of Utah Salt Lake City, UT 84112 REPORT DATE: July 2016 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and

  5. p53 and MDM2: antagonists or partners in crime?

    PubMed

    Eischen, Christine M; Lozano, Guillermina

    2009-03-03

    Therapeutics that disrupt the p53-MDM2 interaction show promise for cancer treatment but surprisingly have different biological outcomes. A study by Enge et al. in this issue of Cancer Cell shows that the ability of MDM2 to target hnRNP K for degradation contributes to the decision to induce apoptosis rather than cell-cycle arrest.

  6. NRF2 and p53: Januses in cancer?

    PubMed Central

    Rotblat, Barak; Melino, Gerry; Knight, Richard A.

    2012-01-01

    The transcription factor nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or NRF2, is a master regulator of the anti-oxidative stress response and positively controls the expression of a battery of anti-oxidative stress response proteins and enzymes implicated in detoxification and glutathione generation. Although its detoxifying activity is important in cancer prevention, it has recently been shown that cancer cells also exploit its protective functions to thrive and resist chemotherapy. NRF2 was also shown to the pentose phosphate pathway and glutaminolysis, which promotes purine synthesis for supporting rapid proliferation and glutathione for providing anti-oxidative stress protection. Evidence obtained from cancer patients and cell lines suggest that NRF2 is highly active in a variety of human cancers and is associated with aggressiveness. p53 is a tumor suppressor that also promotes an anti-oxidative stress metabolic program and glutaminolysis. Here we will discuss the similarities between NRF2 and p53 and review evidence that p53 might be exploited by cancer cells to gain protection against oxidative stress, as is the case for NRF2. We discuss findings of co-regulation between these transcription factors and propose possible therapeutic strategies that can be used for treatment of cancers that harbor WT p53 and express high levels of NRF2. PMID:23174755

  7. A role for p53 in selenium-induced senescence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The tumor suppressor p53 and the ataxia-telangiectasia mutated (ATM) kinase play important roles in the senescence response to oncogene activation and DNA damage. We have previously shown that selenium-containing compounds can activate an ATM-dependent senescence response in MRC-5 normal fibroblasts...

  8. Immunohistochemical detection of P53 and Mdm2 in vitiligo

    PubMed Central

    Bakry, Ola A.; Hammam, Mostafa A.; Wahed, Moshira M. Abdel

    2012-01-01

    Background: Vitiligo is a common depigmented skin disorder that is caused by selective destruction of melanocytes. It is generally accepted that the main function of melanin resides in the protection of skin cells against the deleterious effect of ultraviolet rays (UVRs). Association of vitiligo and skin cancer has been a subject of controversy. Occurrence of skin cancer in long-lasting vitiligo is rare despite multiple evidences of DNA damage in vitiliginous skin. Aim: To detect the expression of P53 and Mdm2 proteins in both depigmented and normally pigmented skin of vitiligo patients and to compare it to control subjects suffering from nonmelanoma skin cancer (NMSC). Materials and Methods: Thirty-four patients with vitiligo and 30 age and sex-matched patients with nodulo-ulcerative basal cell carcinoma (BCC) as a control group were selected. Both patients and control subjects had outdoor occupations. Skin biopsies were taken from each case and control subjects. Histopathological examination of Hematoxylin and eosin-stained sections was done. Expression of P53 and Mdm2 proteins were examined immunohistochemically. Results: Both P53 and Mdm2 were strongly expressed in depigmented as well as normally pigmented skin of vitiligo patients. This expression involved the epidermis, skin adnexa and blood vessels with significant differences between cases and controls. Conclusions: The overexpression of P53 and Mdm2 proteins in both normally pigmented and depigmented skin of patients with vitiligo could contribute to the decreased occurrence of actinic damage and NMSC in these patients. PMID:23189248

  9. Functional repair of p53 mutation in colorectal cancer cells using trans-splicing.

    PubMed

    He, Xingxing; Liao, Jiazhi; Liu, Fang; Yan, Junwei; Yan, Jingjun; Shang, Haitao; Dou, Qian; Chang, Ying; Lin, Jusheng; Song, Yuhu

    2015-02-10

    Mutation in the p53 gene is arguably the most frequent type of gene-specific alterations in human cancers. Current p53-based gene therapy contains the administration of wt-p53 or the suppression of mutant p53 expression in p53-defective cancer cells. . We hypothesized that trans-splicing could be exploited as a tool for the correction of mutant p53 transcripts in p53-mutated human colorectal cancer (CRC) cells. In this study, the plasmids encoding p53 pre-trans-splicing molecules (PTM) were transfected into human CRC cells carrying p53 mutation. The plasmids carrying p53-PTM repaired mutant p53 transcripts in p53-mutated CRC cells, which resulted in a reduction in mutant p53 transcripts and an induction of wt-p53 simultaneously. Intratumoral administration of adenovirus vectors carrying p53 trans-splicing cassettes suppressed the growth of tumor xenografts. Repair of mutant p53 transcripts by trans-splicing induced cell-cycle arrest and apoptosis in p53-defective colorectal cancer cells in vitro and in vivo. In conclusion, the present study demonstrated for the first time that trans-splicing was exploited as a strategy for the repair of mutant p53 transcripts, which revealed that trans-splicing would be developed as a new therapeutic approach for human colorectal cancers carrying p53 mutation.

  10. Epimorphic regeneration in mice is p53-independent.

    PubMed

    Arthur, L Matthew; Demarest, Renee M; Clark, Lise; Gourevitch, Dmitri; Bedelbaeva, Kamila; Anderson, Rhonda; Snyder, Andrew; Capobianco, Anthony J; Lieberman, Paul; Feigenbaum, Lionel; Heber-Katz, E

    2010-09-15

    The process of regeneration is most readily studied in species of sponge, hydra, planarian and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a mammalian model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G₂/M "arrest," showed that p21(Cip1/Waf1) was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL. p53(-/-) mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21(-/-) mice showed chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cycle-related mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfβ/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfβ/Smad pathway.

  11. 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

  12. Chk2 and p53 regulate the transmission of healed chromosomes in the Drosophila male germline.

    PubMed

    Titen, Simon W A; Lin, Ho-Chen; Bhandari, Jayaram; Golic, Kent G

    2014-02-01

    When a dicentric chromosome breaks in mitosis, the broken ends cannot be repaired by normal mechanisms that join two broken ends since each end is in a separate daughter cell. However, in the male germline of Drosophila melanogaster, a broken end may be healed by de novo telomere addition. We find that Chk2 (encoded by lok) and P53, major mediators of the DNA damage response, have strong and opposite influences on the transmission of broken-and-healed chromosomes: lok mutants exhibit a large increase in the recovery of healed chromosomes relative to wildtype control males, but p53 mutants show a strong reduction. This contrasts with the soma, where mutations in lok and p53 have the nearly identical effect of allowing survival and proliferation of cells with irreparable DNA damage. Examination of testes revealed a transient depletion of germline cells after dicentric chromosome induction in the wildtype controls, and further showed that P53 is required for the germline to recover. Although lok mutant males transmit healed chromosomes at a high rate, broken chromosome ends can also persist through spermatogonial divisions without healing in lok mutants, giving rise to frequent dicentric bridges in Meiosis II. Cytological and genetic analyses show that spermatid nuclei derived from such meiotic divisions are eliminated during spermiogenesis, resulting in strong meiotic drive. We conclude that the primary responsibility for maintaining genome integrity in the male germline lies with Chk2, and that P53 is required to reconstitute the germline when cells are eliminated owing to unrepaired DNA damage.

  13. A novel mutant p53 binding partner BAG5 stabilizes mutant p53 and promotes mutant p53 GOFs in tumorigenesis

    PubMed Central

    Yue, Xuetian; Zhao, Yuhan; Huang, Grace; Li, Jun; Zhu, Junlan; Feng, Zhaohui; Hu, Wenwei

    2016-01-01

    Tumor suppressor p53 is the most frequently mutated gene in human tumors. Many tumor-associated mutant p53 (mutp53) proteins gain new tumor-promoting activities, including increased proliferation, metastasis and chemoresistance of tumor cells, which are defined as gain-of-functions (GOFs). Mutp53 proteins often accumulate at high levels in human tumors, which is important for mutp53 to exert their GOFs. The mechanism underlying mutp53 proteins accumulation in tumors is not fully understood. Here, we report that BAG5, a member of Bcl-2-associated athanogene (BAG) family proteins, promotes mutp53 accumulation in tumors, which in turn enhances mutp53 GOFs. Mechanistically, BAG5 interacts with mutp53 proteins to protect mutp53 from ubiquitination and degradation by E3 ubiquitin ligases MDM2 and CHIP, which in turn promotes mutp53 protein accumulation and therefore GOFs in promoting cell proliferation, tumor growth, cell migration and chemoresistance. BAG5 is frequently overexpressed in many human tumors and the overexpression of BAG5 is associated with poor prognosis of cancer patients. Altogether, this study revealed that inhibition of mutp53 degradation by BAG5 is a novel and critical mechanism underlying mutp53 protein accumulation and GOFs in cancer. Furthermore, our results also uncovered that promoting mutp53 accumulation and GOFs is a novel mechanism of BAG5 in tumorigenesis. PMID:27807478

  14. Depression of p53-independent Akt survival signals after high-LET radiation in mutated p53 cells

    NASA Astrophysics Data System (ADS)

    Ohnishi, Takeo; Takahashi, Akihisa; Nakagawa, Yosuke

    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. 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 the activities of 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 signals were analyzed with Western blotting analysis 1 h, 2 h, 3 h and 6 h after irradiation. Cell cycle distributions after irradiation were assayed with flow cytometric analysis.Akt-related protein levels were decreased when cells were irradiated with high-LET radiation. High-LET radiation increased G _{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 depresses cell growth by suppressing Akt-related signals, even in the mp53 cells.

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

    PubMed

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

    1987-09-01

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

  16. Science Integrating Learning Objectives: A Cooperative Learning Group Process

    ERIC Educational Resources Information Center

    Spindler, Matt

    2015-01-01

    The integration of agricultural and science curricular content that capitalizes on natural and inherent connections represents a challenge for secondary agricultural educators. The purpose of this case study was to create information about the employment of Cooperative Learning Groups (CLG) to enhance the science integrating learning objectives…

  17. p53's choice of myocardial death or survival: Oxygen protects infarct myocardium by recruiting p53 on NOS3 promoter through regulation of p53-Lys(118) acetylation.

    PubMed

    Gogna, Rajan; Madan, Esha; Khan, Mahmood; Pati, Uttam; Kuppusamy, Periannan

    2013-11-01

    Myocardial infarction, an irreversible cardiac tissue damage, involves progressive loss of cardiomyocytes due to p53-mediated apoptosis. Oxygenation is known to promote cardiac survival through activation of NOS3 gene. We hypothesized a dual role for p53, which, depending on oxygenation, can elicit apoptotic death signals or NOS3-mediated survival signals in the infarct heart. p53 exhibited a differential DNA-binding, namely, BAX-p53RE in the infarct heart or NOS3-p53RE in the oxygenated heart, which was regulated by oxygen-induced, post-translational modification of p53. In the infarct heart, p53 was heavily acetylated at Lys(118) residue, which was exclusively reversed in the oxygenated heart, apparently regulated by oxygen-dependent expression of TIP60. The inhibition of Lys(118) acetylation promoted the generation of NOS3-promoting prosurvival form of p53. Thus, oxygenation switches p53-DNA interaction by regulating p53 core-domain acetylation, promoting a prosurvival transcription activity of p53. Understanding this novel oxygen-p53 survival pathway will open new avenues in cardioprotection molecular therapy.

  18. p53 mutation, but not p53 overexpression, correlates with survival in head and neck squamous cell carcinoma.

    PubMed Central

    Mineta, H.; Borg, A.; Dictor, M.; Wahlberg, P.; Akervall, J.; Wennerberg, J.

    1998-01-01

    Survival in squamous cell carcinoma of the head and neck (HNSCC) was compared with overexpression and mutation of the p53 gene. Archival tissue from 77 tumours was analysed for protein expression using immunohistochemistry (IHC) with the monoclonal antibody Do-7, and for the presence of mutation in exons 5-8 using single-stranded conformation polymorphism (SSCP), followed by DNA sequencing in SSCP-positive cases. p53 expression was scored as high (>70% nuclei stained) in 25 (32%) tumours, as intermediate (10-70% nuclei stained) in 19 (25%) tumours and as low (<10% nuclei stained) in 33 (43%) tumours. Twelve (18%) tumours exhibited gene mutation (ten missense and two nonsense mutations) and an additional five tumours contained changes that could not result in amino acid substitution or protein truncation. There was no correlation between gene expression and mutation, mutations being equally frequent in tumours with either high (4/25), intermediate (4/19) or low protein expression (4/33). Fifty-eight patients were eligible for survival analysis. There was a strong correlation between p53 mutation and cause-specific survival; median survival among mutated cases was 12.5 months compared with >160 months among non-mutated patients (P < 0.005). There was no correlation between p53 overexpression and survival. The results suggest that p53 mutation status is an important prognostic factor in HNSCC, and that IHC analysis of protein overexpression is an inadequate measure of gene mutation in these tumours. Images Figure 1 PMID:9792155

  19. Gain of cellular adaptation due to prolonged p53 impairment leads to functional switchover from p53 to p73 during DNA damage in acute myeloid leukemia cells.

    PubMed

    Chakraborty, Juni; Banerjee, Shuvomoy; Ray, Pallab; Hossain, Dewan Md Sakib; Bhattacharyya, Sankar; Adhikary, Arghya; Chattopadhyay, Sreya; Das, Tanya; Sa, Gaurisankar

    2010-10-22

    Tumor suppressor p53 plays the central role in regulating apoptosis in response to genotoxic stress. From an evolutionary perspective, the activity of p53 has to be backed up by other protein(s) in case of any functional impairment of this protein, to trigger DNA damage-induced apoptosis in cancer cells. We adopted multiple experimental approaches to demonstrate that in p53-impaired cancer cells, DNA damage caused accumulation of p53 paralogue p73 via Chk-1 that strongly impacted Bax expression and p53-independent apoptosis. On the contrary, when p53 function was restored by ectopic expression, Chk-2 induced p53 accumulation that in turn overshadowed p73 activity, suggesting an antagonistic interaction between p53 family members. To understand such interaction better, p53-expressing cells were impaired differentially for p53 activity. In wild-type p53-expressing cancer cells that were silenced for p53 for several generations, p73 was activated, whereas no such trend was observed when p53 was transiently silenced. Prolonged p53 interference, even in functional p53 settings, therefore, leads to the "gain of cellular adaptation" in a way that alters the cellular microenvironment in favor of p73 activation by altering p73-regulatory proteins, e.g. Chk1 activation and dominant negative p73 down-regulation. These findings not only unveil a hitherto unexplained mechanism underlying the functional switchover from p53 to p73, but also validate p73 as a promising and potential target for cancer therapy in the absence of functional p53.

  20. HDACi inhibits liposarcoma via targeting of the MDM2-p53 signaling axis and PTEN, irrespective of p53 mutational status.

    PubMed

    Ou, Wen-Bin; Zhu, Jiaqing; Eilers, Grant; Li, Xuhui; Kuang, Ye; Liu, Li; Mariño-Enríquez, Adrián; Yan, Ziqin; Li, Hailong; Meng, Fanguo; Zhou, Haimeng; Sheng, Qing; Fletcher, Jonathan A

    2015-04-30

    The MDM2-p53 pathway plays a prominent role in well-differentiated liposarcoma (LPS) pathogenesis. Here, we explore the importance of MDM2 amplification and p53 mutation in LPS independently, to determine whether HDACi are therapeutically useful in LPS. We demonstrated that simultaneous knockdown of MDM2 and p53 in p53-mutant LPS lines resulted in increased apoptosis, anti-proliferative effects, and cell cycle arrest, as compared to either intervention alone. HDACi treatment resulted in the dephosphorylation and depletion of MDM2 and p53 without affecting CDK4 and JUN expression, irrespective of p53 mutational status in MDM2-amplified LPS. In control mesothelioma cell lines, HDACi treatment resulted in down-regulation of p53 in the p53 mutant cell line JMN1B, but resulted in no changes of MDM2 and p53 in two mesothelioma lines with normal MDM2 and wild-type p53. HDACi treatment substantially decreased LPS and mesothelioma proliferation and survival, and was associated with upregulation of PTEN and p21, and inactivation of AKT. Our findings indicate that wild-type p53 depletion by HDACi is MDM2 amplification-dependent. These findings underscore the importance of targeting both MDM2 and p53 in LPS and other cancers harboring p53 mutations. Moreover, the pro-apoptotic and anti-proliferative effect of HDACi warrants further evaluation as a therapeutic strategy in MDM2-amplified LPS.

  1. Adenovirus-mediated wild-type p53 transfer radiosensitizes H1299 cells to subclinical-dose carbon-ion irradiation through the restoration of p53 function.

    PubMed

    Liu, Bing; Zhang, Hong; Duan, Xin; Hao, Jifang; Xie, Yi; Zhou, Qingming; Wang, Yanling; Tian, Yuan; Wang, Tao

    2009-02-01

    To determine whether adenovirus-mediated wild-type p53 transfer after radiotherapy could radiosensitize non-small-cell lung cancer (NSCLC) cells to subclinical-dose carbon-ion beam (C-beam), H1299 cells were exposed to a C-beam or gamma-ray and then infected with 5 MOI of AdCMV-p53 or GFP (C-beam or gamma-ray with p53 or GFP). Cell cycle was detected by flow cytometric analysis. The apoptosis was examined by a fluorescent microscope with DAPI staining. DNA fragmentation was monitored by the TUNEL assay. P53 mRNA was detected by reverse-transcriptase polymerase chain reaction. The expression of p53, MDM(2), and p21 was monitored by Western blot. Survival fractions were determined by colony-forming assay. The percentages of G(1)-phase cells in C-beam with p53 increased by 8.2%-16.0%, 5.2%-7.0%, and 5.8%-18.9%, respectively, compared with C-beam only, gamma-ray with p53, or p53 only. The accumulation of G(2)-phase cells in C-beam with p53 increased by 5.7%-8.9% and 8.8%-14.8%, compared with those in gamma-ray with p53 or p53 only, respectively. The percentage of apoptosis for C-beam with p53 increased by 7.4%-19.1%, 5.8%-11.7%, and 5.2 %-19.2%, respectively, compared with C-beam only, gamma-ray with p53, or p53 only. The level of p53 mRNA in C-beam with p53 was significantly higher than that in p53 only. The expression level of p53 and p21 in C-beam with p53 was significantly higher than that in both C-beam with GFP and p53 only. The survival fractions for C-beam with p53 were significantly less than those for the other groups (p < 0.05). The data suggested that AdCMV-p53 transfer could more efficiently radiosensitize H1299 cells to subclinical-dose C-beam irradiation through the restoration of p53 function.

  2. C23 promotes tumorigenesis via suppressing p53 activity

    PubMed Central

    Wang, Juan; Hu, Guilin; Fang, Xing; Hu, Yamin; Tao, Tingting; Wei, Xin; Tang, Haitao; Huang, Baojun; Hu, Wanglai

    2016-01-01

    C23 is an abundant and multi-functional protein, which plays an important role in various biological processes, including ribosome biogenesis and maturation, cell cycle checkpoints and transcriptional regulation [1, 2]. However, the role of C23 in controlling tumorigenesis has not been well defined. Here we report that C23 is highly expressed in cancer cells and the elevated expression of C23 facilitates cancer cell proliferation in vitro and tumor xenograft growth in vivo. Notably, C23 binds to p53 through its GAR domain and suppresses the transcriptional activity of p53 under DNA damage and hypoxia. Moreover, the GAR domain is critical for C23-mediated tumor cell proliferation both in vitro and in vivo. Our findings reveal a novel role of C23 in tumorigenesis and suggest that C23 may represent a potential therapeutic target for treating malignancy. PMID:27506938

  3. Targeting p53 as a general tumor antigen.

    PubMed Central

    Theobald, M; Biggs, J; Dittmer, D; Levine, A J; Sherman, L A

    1995-01-01

    A major barrier to the design of immunotherapeutics and vaccines for cancer is the idiosyncratic nature of many tumor antigens and the possibility that T cells may be tolerant of broadly distributed antigens. We have devised an experimental strategy that exploits species differences in protein sequences to circumvent tolerance of high-affinity T cells. HLA transgenic mice were used to obtain cytotoxic T lymphocytes specific for peptides from the human p53 tumor-suppressor molecule presented in association with HLA-A2.1. Although such p53-specific cytotoxic T cells did not recognize nontransformed human cells, they were able to lyse a wide variety of human tumor cells lines, thus confirming the existence of broadly distributed determinants that may serve as targets for immunotherapy. PMID:8618830

  4. Mdm2 inhibition confers protection of p53-proficient cells from the cytotoxic effects of Wee1 inhibitors.

    PubMed

    Li, Yizhu; Saini, Priyanka; Sriraman, Anusha; Dobbelstein, Matthias

    2015-10-20

    Pharmacological inhibition of the cell cycle regulatory kinase Wee1 represents a promising strategy to eliminate cancer cells. Wee1 inhibitors cooperate with chemotherapeutics, e. g. nucleoside analogues, pushing malignant cells from S phase towards premature mitosis and death. However, considerable toxicities are observed in preclinical and clinical trials. A high proportion of tumor cells can be distinguished from all other cells of a patient's body by inactivating mutations in the tumor suppressor p53. Here we set out to develop an approach for the selective protection of p53-proficient cells against the cytotoxic effects of Wee1 inhibitors. We pretreated such cells with Nutlin-3a, a prototype inhibitor of the p53-antagonist Mdm2. The resulting transient cell cycle arrest effectively increased the survival of cells that were subsequently treated with combinations of the Wee1 inhibitor MK-1775 and/or the nucleoside analogue gemcitabine. In this constellation, Nutlin-3a reduced caspase activation and diminished the phosphorylation of Histone 2AX, an indicator of the DNA damage response. Both effects were strictly dependent on the presence of p53. Moreover, Nutlin pre-treatment reduced the fraction of cells that were undergoing premature mitosis in response to Wee1 inhibition. We conclude that the pre-activation of p53 through Mdm2 antagonists serves as a viable option to selectively protect p53-proficient cells against the cytotoxic effects of Wee1 inhibitors, especially when combined with a nucleoside analogue. Thus, Mdm2 antagonists might prove useful to avoid unwanted side effects of Wee1 inhibitors. On the other hand, when a tumor contains wild type p53, care should be taken not to induce its activity before applying Wee1 inhibitors.

  5. Regulation of Mammary Progenitor Cells by p53 and Parity

    DTIC Science & Technology

    2010-01-01

    SUPPLEMENTARY NOTES 14. ABSTRACT Breast cancer is the most frequent cancer among women in the United States. A full term pregnancy early in...reproductive life can reduce breast cancer incidence in women by up to 50% and p53, an important tumor suppressor gene, was shown to be a major effector...Introduction Breast cancer is the most frequent cancer among women in the United States1. Understanding the

  6. Axis of ageing: telomeres, p53 and mitochondria

    PubMed Central

    Sahin, Ergün; DePinho, Ronald A.

    2013-01-01

    Progressive DNA damage and mitochondrial decline are both considered to be prime instigators of natural ageing. Traditionally, these two pathways have been viewed largely in isolation. However, recent studies have revealed a molecular circuit that directly links DNA damage to compromised mitochondrial biogenesis and function via p53. This axis of ageing may account for both organ decline and disease development associated with advanced age and could illuminate a path for the development of relevant therapeutics. PMID:22588366

  7. E3 ubiquitin ligase Hades negatively regulates the exonuclear function of p53

    PubMed Central

    Jung, J H; Bae, S; Lee, J Y; Woo, S R; Cha, H J; Yoon, Y; Suh, K-S; Lee, S-J; Park, I-C; Jin, Y-W; Lee, K-H; An, S; Lee, J H

    2011-01-01

    Following DNA damage, p53 translocates to the cytoplasm and mitochondria, where it triggers transcription-independent apoptosis by binding to Bcl-2 family proteins. However, little is known about how this exonuclear function of p53 is regulated. Here, we identify and characterize a p53-interacting protein called Hades, an E3 ligase that interacts with p53 in the mitochondria. Hades reduces p53 stability via a mechanism that requires its RING-finger domain with ubiquitin ligase activity. Hades polyubiquitinates p53 in vitro independent of Mdm2 and targets a critical lysine residue in p53 (lysine 24) distinct from those targeted by Mdm2. Hades inhibits a p53-dependent mitochondrial cell death pathway by inhibiting p53 and Bcl-2 interactions. These findings show that Hades-mediated p53 ubiquitination is a novel mechanism for negatively regulating the exonuclear function of p53. PMID:21597459

  8. E3 ubiquitin ligase Hades negatively regulates the exonuclear function of p53.

    PubMed

    Jung, J H; Bae, S; Lee, J Y; Woo, S R; Cha, H J; Yoon, Y; Suh, K-S; Lee, S-J; Park, I-C; Jin, Y-W; Lee, K-H; An, S; Lee, J H

    2011-12-01

    Following DNA damage, p53 translocates to the cytoplasm and mitochondria, where it triggers transcription-independent apoptosis by binding to Bcl-2 family proteins. However, little is known about how this exonuclear function of p53 is regulated. Here, we identify and characterize a p53-interacting protein called Hades, an E3 ligase that interacts with p53 in the mitochondria. Hades reduces p53 stability via a mechanism that requires its RING-finger domain with ubiquitin ligase activity. Hades polyubiquitinates p53 in vitro independent of Mdm2 and targets a critical lysine residue in p53 (lysine 24) distinct from those targeted by Mdm2. Hades inhibits a p53-dependent mitochondrial cell death pathway by inhibiting p53 and Bcl-2 interactions. These findings show that Hades-mediated p53 ubiquitination is a novel mechanism for negatively regulating the exonuclear function of p53.

  9. Che-1/AATF: A Critical Cofactor for Both Wild-Type- and Mutant-p53 Proteins

    PubMed Central

    Bruno, Tiziana; Iezzi, Simona; Fanciulli, Maurizio

    2016-01-01

    The p53 protein is a key player in a wide range of protein networks that allow the state of “good health” of the cell. Not surprisingly, mutations of the TP53 gene are one of the most common alterations associated to cancer cells. Mutated forms of p53 (mtp53) not only lose the ability to protect the integrity of the genetic heritage of the cell but also acquire pro-oncogenic functions, behaving like dangerous accelerators of transformation and tumor progression. In recent years, many studies focused on investigating possible strategies aiming to counteract this mutant p53 “gain of function” but the results have not always been satisfactory. Che-1/AATF is a nuclear protein that binds to RNA polymerase II and plays a role in multiple fundamental processes, including control of transcription, cell cycle regulation, DNA damage response, and apoptosis. Several studies showed Che-1/AATF as an important endogenous regulator of p53 expression and activity in a variety of biological processes. Notably, this same regulation was more recently observed also on mtp53. The depletion of Che-1/AATF strongly reduces the expression of mutant p53 in several tumors in vitro and in vivo, making the cells an easier target for chemotherapy treatments. In this mini review, we report an overview of Che-1/AATF functions and discuss a possible role of Che-1/AATF in cancer therapy, with particular regard to its action on p53/mtp53. PMID:26913241

  10. The Stress-responsive Gene ATF3 Mediates Dichotomous UV Responses by Regulating the Tip60 and p53 Proteins.

    PubMed

    Cui, Hongmei; Li, Xingyao; Han, Chunhua; Wang, Qi-En; Wang, Hongbo; Ding, Han-Fei; Zhang, Junran; Yan, Chunhong

    2016-05-13

    The response to UV irradiation is important for a cell to maintain its genetic integrity when challenged by environmental genotoxins. An immediate early response to UV irradiation is the rapid induction of activating transcription factor 3 (ATF3) expression. Although emerging evidence has linked ATF3 to stress pathways regulated by the tumor suppressor p53 and the histone acetyltransferase Tip60, the role of ATF3 in the UV response remains largely unclear. Here, we report that ATF3 mediated dichotomous UV responses. Although UV irradiation enhanced the binding of ATF3 to Tip60, knockdown of ATF3 expression decreased Tip60 stability, thereby impairing Tip60 induction by UV irradiation. In line with the role of Tip60 in mediating UV-induced apoptosis, ATF3 promoted the death of p53-defective cells in response to UV irradiation. However, ATF3 could also activate p53 and promote p53-mediated DNA repair, mainly through altering histone modifications that could facilitate recruitment of DNA repair proteins (such as DDB2) to damaged DNA sites. As a result, ATF3 rather protected the p53 wild-type cells from UV-induced apoptosis. Our results thus indicate that ATF3 regulates cell fates upon UV irradiation in a p53-dependent manner.

  11. Yin Yang 1 Promotes Thymocyte Survival by Downregulating p53.

    PubMed

    Chen, Liang; Foreman, Daniel P; Sant'Angelo, Derek B; Krangel, Michael S

    2016-03-15

    Yin Yang 1 (YY1) is a zinc finger protein that functions as a transcriptional activator or repressor and participates in multiple biological processes, including development and tumorigenesis. To investigate the role of YY1 in developing T cells, we used mouse models that depleted YY1 at two distinct stages of thymocyte development. When YY1 was depleted in CD4(-)CD8(-) double-negative thymocytes, development to the CD4(+)CD8(+) double-positive stage was impaired, due to increased apoptosis that prevented expansion of post-β-selection thymocytes. When YY1 was depleted in double-positive thymocytes, they underwent increased cell-autonomous apoptosis in vitro and displayed a shorter lifespan in vivo, as judged by their ability to undergo secondary Vα-to-Jα recombination. Mechanistically, we found that the increased apoptosis in YY1-deficient thymocytes was attributed to overexpression of p53, because concurrent loss of p53 completely rescued the developmental defects of YY1-deficient thymocytes. These results indicated that YY1 functions as a critical regulator of thymocyte survival and that it does so by suppressing the expression of p53.

  12. Non-Canonical Cell Death Induced by p53

    PubMed Central

    Ranjan, Atul; Iwakuma, Tomoo

    2016-01-01

    Programmed cell death is a vital biological process for multicellular organisms to maintain cellular homeostasis, which is regulated in a complex manner. Over the past several years, apart from apoptosis, which is the principal mechanism of caspase-dependent cell death, research on non-apoptotic forms of programmed cell death has gained momentum. p53 is a well characterized tumor suppressor that controls cell proliferation and apoptosis and has also been linked to non-apoptotic, non-canonical cell death mechanisms. p53 impacts these non-canonical forms of cell death through transcriptional regulation of its downstream targets, as well as direct interactions with key players involved in these mechanisms, in a cell type- or tissue context-dependent manner. In this review article, we summarize and discuss the involvement of p53 in several non-canonical modes of cell death, including caspase-independent apoptosis (CIA), ferroptosis, necroptosis, autophagic cell death, mitotic catastrophe, paraptosis, and pyroptosis, as well as its role in efferocytosis which is the process of clearing dead or dying cells. PMID:27941671

  13. Non-Canonical Cell Death Induced by p53.

    PubMed

    Ranjan, Atul; Iwakuma, Tomoo

    2016-12-09

    Programmed cell death is a vital biological process for multicellular organisms to maintain cellular homeostasis, which is regulated in a complex manner. Over the past several years, apart from apoptosis, which is the principal mechanism of caspase-dependent cell death, research on non-apoptotic forms of programmed cell death has gained momentum. p53 is a well characterized tumor suppressor that controls cell proliferation and apoptosis and has also been linked to non-apoptotic, non-canonical cell death mechanisms. p53 impacts these non-canonical forms of cell death through transcriptional regulation of its downstream targets, as well as direct interactions with key players involved in these mechanisms, in a cell type- or tissue context-dependent manner. In this review article, we summarize and discuss the involvement of p53 in several non-canonical modes of cell death, including caspase-independent apoptosis (CIA), ferroptosis, necroptosis, autophagic cell death, mitotic catastrophe, paraptosis, and pyroptosis, as well as its role in efferocytosis which is the process of clearing dead or dying cells.

  14. 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

  15. p53 gene mutations in asbestos associated cancers.

    PubMed

    Liu, B C; Fu, D C; Miao, Q; Wang, H H; You, B R

    1998-09-01

    The accumulation of mutant p53 protein in cancer cells was observed by immunohistochemistry analysis. DNA was extracted from paraffin-embedded tissue. Exons 5, 7 and 8 were amplified and studied by PCR-SSCP and sequencing analysis. Ten cases of asbestos associated cancer tissue were studied, of which five cases had adenocarcinoma, and the other five had mesothelioma, squamous carcinoma, small cell lung cancer, adenosquamous carcinoma and malignant lymphoma respectively. Employing monoclonal antibody PAb1801, five cases were found to be mutant p53 protein positive. Seven cases were found to have mutations by PCR-SSCP. A total of 7 cases (8 mutations) were found to be positive and 4 cases were found to be positive by both of these analyses. Of the 8 mutations found by SSCP analysis, 4(50%, 4/8) were clustered in exon 8. A high mutation frequency was noticed in adenocarcinoma (80%, 4/5). Sequencing analysis on two specimens revealed two hotspot mutations. In codon 234, TAC for tyrosin was mutated to AAC for asparagine by a T to A transversion of the first letter. In codon 273, CGT for arginine was mutated to AGT for serine by a C to A transversion of the first letter. In conclusion, the mutation of p53 gene is common in asbestos associated cancers. However, the mutational spectrum of asbestos associated cancers might be different from that of non-asbestos associated cancers.

  16. Green Tea Polyphenols Induce p53-Dependent and p53-Independent Apoptosis in Prostate Cancer Cells through Two Distinct Mechanisms

    PubMed Central

    Gupta, Karishma; Thakur, Vijay S.; Bhaskaran, Natarajan; Nawab, Akbar; Babcook, Melissa A.; Jackson, Mark W.; Gupta, Sanjay

    2012-01-01

    Inactivation of the tumor suppressor gene p53 is commonly observed in human prostate cancer and is associated with therapeutic resistance. We have previously demonstrated that green tea polyphenols (GTP) induce apoptosis in prostate cancer cells irrespective of p53 status. However, the molecular mechanisms underlying these observations remain elusive. Here we investigated the mechanisms of GTP-induced apoptosis in human prostate cancer LNCaP cells stably-transfected with short hairpin-RNA against p53 (LNCaPshp53) and control vector (LNCaPshV). GTP treatment induced p53 stabilization and activation of downstream targets p21/waf1 and Bax in a dose-dependent manner specifically in LNCaPshV cells. However, GTP-induced FAS upregulation through activation of c-jun-N-terminal kinase resulted in FADD phosphorylation, caspase-8 activation and truncation of BID, leading to apoptosis in both LNCaPshV and LNCaPshp53 cells. In parallel, treatment of cells with GTP resulted in inhibition of survival pathway, mediated by Akt deactivation and loss of BAD phosphorylation more prominently in LNCaPshp53 cells. These distinct routes of cell death converged to a common pathway, leading to loss of mitochondrial transmembrane potential, cytochrome c release and activation of terminal caspases, resulting in PARP-cleavage. GTP-induced apoptosis was attenuated with JNK inhibitor, SP600125 in both cell lines; whereas PI3K-Akt inhibitor, LY294002 resulted in increased cell death prominently in LNCaPshp53 cells, establishing the role of two distinct pathways of GTP-mediated apoptosis. Furthermore, GTP exposure resulted in inhibition of class I HDAC protein, accumulation of acetylated histone-H3 in total cellular chromatin, resulting in increased accessibility of transcription factors to bind with the promoter sequences of p21/waf1 and Bax, regardless of the p53 status of cells, consistent with effects elicited by an HDAC inhibitor, trichostatin A. These results demonstrate that GTP induces

  17. Identification of Two Reactive Cysteine Residues in the Tumor Suppressor Protein p53 Using Top-Down FTICR Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Scotcher, Jenna; Clarke, David J.; Weidt, Stefan K.; Mackay, C. Logan; Hupp, Ted R.; Sadler, Peter J.; Langridge-Smith, Pat R. R.

    2011-05-01

    The tumor suppressor p53 is a redox-regulated transcription factor involved in cell cycle arrest, apoptosis and senescence in response to multiple forms of stress, as well as many other cellular processes such as DNA repair, glycolysis, autophagy, oxidative stress and differentiation. The discovery of cysteine-targeting compounds that cause re-activation of mutant p53 and the death of tumor cells in vivo has emphasized the functional importance of p53 thiols. Using a combination of top-down and middle-down FTICR mass spectrometry, we show that of the 10 Cys residues in the core domain of wild-type p53, Cys182 and Cys277 exhibit a remarkable preference for modification by the alkylating reagent N-ethylmaleimide. The assignment of Cys182 and Cys277 as the two reactive Cys residues was confirmed by site-directed mutagenesis. Further alkylation of p53 beyond Cys182 and Cys277 was found to trigger co-operative modification of the remaining seven Cys residues and protein unfolding. This study highlights the power of top-down FTICR mass spectrometry for analysis of the cysteine reactivity and redox chemistry in multiple cysteine-containing proteins.

  18. Identification of antipsychotic drug fluspirilene as a potential p53-MDM2 inhibitor: a combined computational and experimental study

    NASA Astrophysics Data System (ADS)

    Patil, Sachin P.; Pacitti, Michael F.; Gilroy, Kevin S.; Ruggiero, John C.; Griffin, Jonathan D.; Butera, Joseph J.; Notarfrancesco, Joseph M.; Tran, Shawn; Stoddart, John W.

    2015-02-01

    The inhibition of tumor suppressor p53 protein due to its direct interaction with oncogenic murine double minute 2 (MDM2) protein, plays a central role in almost 50 % of all human tumor cells. Therefore, pharmacological inhibition of the p53-binding pocket on MDM2, leading to p53 activation, presents an important therapeutic target against these cancers expressing wild-type p53. In this context, the present study utilized an integrated virtual and experimental screening approach to screen a database of approved drugs for potential p53-MDM2 interaction inhibitors. Specifically, using an ensemble rigid-receptor docking approach with four MDM2 protein crystal structures, six drug molecules were identified as possible p53-MDM2 inhibitors. These drug molecules were then subjected to further molecular modeling investigation through flexible-receptor docking followed by Prime/MM-GBSA binding energy analysis. These studies identified fluspirilene, an approved antipsychotic drug, as a top hit with MDM2 binding mode and energy similar to that of a native MDM2 crystal ligand. The molecular dynamics simulations suggested stable binding of fluspirilene to the p53-binding pocket on MDM2 protein. The experimental testing of fluspirilene showed significant growth inhibition of human colon tumor cells in a p53-dependent manner. Fluspirilene also inhibited growth of several other human tumor cell lines in the NCI60 cell line panel. Taken together, these computational and experimental data suggest a potentially novel role of fluspirilene in inhibiting the p53-MDM2 interaction. It is noteworthy here that fluspirilene has a long history of safe human use, thus presenting immediate clinical potential as a cancer therapeutic. Furthermore, fluspirilene could also serve as a structurally-novel lead molecule for the development of more potent, small-molecule p53-MDM2 inhibitors against several types of cancer. Importantly, the combined computational and experimental screening protocol

  19. p53 loss-of-heterozygosity is a necessary prerequisite for mutant p53 stabilization and gain-of-function in vivo.

    PubMed

    Alexandrova, Evguenia M; Mirza, Safia A; Xu, Sulan; Schulz-Heddergott, Ramona; Marchenko, Natalia D; Moll, Ute M

    2017-03-09

    Missense mutations in TP53 comprise >75% of all p53 alterations in cancer, resulting in highly stabilized mutant p53 proteins that not only lose their tumor-suppressor activity, but often acquire oncogenic gain-of-functions (GOFs). GOF manifests itself in accelerated tumor onset, increased metastasis, increased drug resistance and shortened survival in patients and mice. A known prerequisite for GOF is mutant p53 protein stabilization, which itself is linked to aberrant protein conformation. However, additional determinants for mutant p53 stabilization likely exist. Here we show that in initially heterozygous mouse tumors carrying the hotspot GOF allele R248Q (p53Q/+), another necessary prerequisite for mutant p53 stabilization and GOF in vivo is loss of the remaining wild-type p53 allele, termed loss-of-heterozygosity (LOH). Thus, in mouse tumors with high frequency of p53 LOH (osteosarcomas and fibrosarcomas), we find that mutant p53 protein is stabilized (16/17 cases, 94%) and tumor onset is significantly accelerated compared with p53+/- tumors (GOF). In contrast, in mouse tumors with low frequency of p53 LOH (MMTV-Neu breast carcinomas), mutant p53 protein is not stabilized (16/20 cases, 80%) and GOF is not observed. Of note, human genomic databases (TCGA, METABRIC etc.) show a high degree of p53 LOH in all examined tumor types that carry missense p53 mutations, including sarcomas and breast carcinomas (with and without HER2 amplification). These data - while cautioning that not all genetic mouse models faithfully represent the human situation - demonstrate for the first time that p53 LOH is a critical prerequisite for missense mutant p53 stabilization and GOF in vivo.

  20. RGS16, a novel p53 and pRb cross-talk candidate inhibits migration and invasion of pancreatic cancer cells

    PubMed Central

    Carper, Miranda B.; Denvir, James; Boskovic, Goran; Primerano, Donald A.; Claudio, Pier Paolo

    2014-01-01

    Data collected since the discovery of p53 and pRb/RB1 suggests these tumor suppressors cooperate to inhibit tumor progression. Patients who have mutations in both p53 and RB1 genes have increased tumor reoccurrence and decreased survival compared to patients with only one tumor suppressor gene inactivated. It remains unclear how p53 and pRb cooperate toward inhibiting tumorigenesis. Using RNA expression profiling we identified 179 p53 and pRb cross-talk candidates in normal lung fibroblasts (WI38) cells exogenously coexpressing p53 and pRb. Regulator of G protein signaling 16 (RGS16) was among the p53 and pRb cross-talk candidates and has been implicated in inhibiting activation of several oncogenic pathways associated with proliferation, migration, and invasion of cancer cells. RGS16 has been found to be downregulated in pancreatic cancer patients with metastases compared to patients without metastasis. Expression of RGS16 mRNA was decreased in the pancreatic cancer cell lines tested compared to control. Expression of RGS16 inhibited migration of the BxPC-3 and AsPC-1 but not PANC-1 cells and inhibited invasion of BxPC-3 and AsPC-1 cells with no impact on cell viability. We have identified for the first time p53 and pRb cross-talk candidates and a role for RGS16 to inhibit pancreatic cancer migration and invasion. PMID:25568667

  1. Release of targeted p53 from the mitochondrion as an early signal during mitochondrial dysfunction

    EPA Science Inventory

    Increased accumulation of p53 tumor suppressor protein is an early response to low-level stressors. To investigate the fate of mitochondrial-sequestered p53, mouse embryonic fibroblast cells (MEFs) on a p53-deficient genetic background were transfected with p53-EGFP fusion protei...

  2. A SENSITIVE IMMUNOFLUORESCENCE ASSAY FOR DETECTION OF P53 PROTEIN ACCUMULATION IN SPUTUM

    EPA Science Inventory

    p53 mutations are common genetic alterations in lung cancers and usually result in p53 protein accumulation in tumor cells. Sputum is noninvasive to collect and ideal for screening p53 abnormalities. This study was to determine the feasibility of detecting p53 protein accumulatio...

  3. Identification of two novel functional p53 responsive elements in the herpes simplex virus-1 genome.

    PubMed

    Hsieh, Jui-Cheng; Kuta, Ryan; Armour, Courtney R; Boehmer, Paul E

    2014-07-01

    Analysis of the herpes simplex virus-1 (HSV-1) genome reveals two candidate p53 responsive elements (p53RE), located in proximity to the replication origins oriL and oriS, referred to as p53RE-L and p53RE-S, respectively. The sequences of p53RE-L and p53RE-S conform to the p53 consensus site and are present in HSV-1 strains KOS, 17, and F. p53 binds to both elements in vitro and in virus-infected cells. Both p53RE-L and p53RE-S are capable of conferring p53-dependent transcriptional activation onto a heterologous reporter gene. Importantly, expression of the essential immediate early viral transactivator ICP4 and the essential DNA replication protein ICP8, that are adjacent to p53RE-S and p53RE-L, are repressed in a p53-dependent manner. Taken together, this study identifies two novel functional p53RE in the HSV-1 genome and suggests a complex mechanism of viral gene regulation by p53 which may determine progression of the lytic viral replication cycle or the establishment of latency.

  4. Mutant p53 accumulates in cycling and proliferating cells in the normal tissues of p53 R172H mutant mice

    PubMed Central

    Leushacke, Marc; Li, Ling; Wong, Julin S.; Chiam, Poh Cheang; Rahmat, Siti Aishah Binte; Mann, Michael B.; Mann, Karen M.; Barker, Nick; Lozano, Guillermina; Terzian, Tamara; Lane, David P.

    2015-01-01

    The tumour suppressor p53 is regulated primarily at the protein level. In normal tissues its levels are maintained at a very low level by the action of specific E3 ligases and the ubiquitin proteosome pathway. The mutant p53 protein contributes to transformation, metastasis and drug resistance. High levels of mutant p53 can be found in tumours and the accumulation of mutant p53 has previously been reported in pathologically normal cells in human skin. We show for the first time that similarly elevated levels of mutant p53 can be detected in apparently normal cells in a mutant p53 knock-in mouse model. In fact, in the small intestine, mutant p53 spontaneously accumulates in a manner dependent on gene dosage and cell type. Mutant p53 protein is regulated similarly to wild type p53, which can accumulate rapidly after induction by ionising radiation or Mdm2 inhibitors, however, the clearance of mutant p53 protein is much slower than wild type p53. The accumulation of the protein in the murine small intestine is limited to the cycling, crypt base columnar cells and proliferative zone and is lost as the cells differentiate and exit the cell cycle. Loss of Mdm2 results in even higher levels of p53 expression but p53 is still restricted to proliferating cells in the small intestine. Therefore, the small intestine of these p53 mutant mice is an experimental system in which we can dissect the molecular pathways leading to p53 accumulation, which has important implications for cancer prevention and therapy. PMID:26255629

  5. Mutant p53 accumulates in cycling and proliferating cells in the normal tissues of p53 R172H mutant mice.

    PubMed

    Goh, Amanda M; Xue, Yuezhen; Leushacke, Marc; Li, Ling; Wong, Julin S; Chiam, Poh Cheang; Rahmat, Siti Aishah Binte; Mann, Michael B; Mann, Karen M; Barker, Nick; Lozano, Guillermina; Terzian, Tamara; Lane, David P

    2015-07-20

    The tumour suppressor p53 is regulated primarily at the protein level. In normal tissues its levels are maintained at a very low level by the action of specific E3 ligases and the ubiquitin proteosome pathway. The mutant p53 protein contributes to transformation, metastasis and drug resistance. High levels of mutant p53 can be found in tumours and the accumulation of mutant p53 has previously been reported in pathologically normal cells in human skin. We show for the first time that similarly elevated levels of mutant p53 can be detected in apparently normal cells in a mutant p53 knock-in mouse model. In fact, in the small intestine, mutant p53 spontaneously accumulates in a manner dependent on gene dosage and cell type. Mutant p53 protein is regulated similarly to wild type p53, which can accumulate rapidly after induction by ionising radiation or Mdm2 inhibitors, however, the clearance of mutant p53 protein is much slower than wild type p53. The accumulation of the protein in the murine small intestine is limited to the cycling, crypt base columnar cells and proliferative zone and is lost as the cells differentiate and exit the cell cycle. Loss of Mdm2 results in even higher levels of p53 expression but p53 is still restricted to proliferating cells in the small intestine. Therefore, the small intestine of these p53 mutant mice is an experimental system in which we can dissect the molecular pathways leading to p53 accumulation, which has important implications for cancer prevention and therapy.

  6. ACCEPT. Academic Cooperative Career Education Program Today. An Integrated Approach to Cooperative Education.

    ERIC Educational Resources Information Center

    Hoskey, Peter, Ed.; Lauten, Michael, Ed.

    An integrated approach to education for special needs students is presented in this curriculum guide for ACCEPT (Academic Cooperative Career Education Program Today). Introductory information covers a definition of special needs students (educationally and socioeconomically disadvantaged and physically and mentally handicapped, including…

  7. The Role of Mutant p53 in Progression of Prostate Cancer

    DTIC Science & Technology

    2005-12-01

    p53 can be inducibly knocked down by siRNA. RNA was purified from MIA - PaCa -1 cells that are uninduced or induced to...mutant p53 is knocked down in MIA - PaCa -1 cells (Fig. 7B). These data suggest that mutant p53 confers cancer cells growth advantages by regulating down...Identification of novel target genes of mutant p53 . (A) MIA - PaCa -1-p53KD-4 and MIA - PaCa -1-p53KD-4 cells were un-induced (-) or induced (+) to express p53

  8. Regulation of p53 Activity by Reversible-Acetylation in Prostate Tumor Suppression

    DTIC Science & Technology

    2006-01-01

    localization of HDAC1. A. The subcellular localization of endogenous HDAC1 was determined in p300 expressing H1299 cells by immunostaining with an antibody...p53 following transfection into p53 (-/-) H1299 cells. Cells were transfected with p53wt alone (a, g, m), p53wt and myc-p300 (b, d, h, j, n, p), p53wt...signal that promotes p53 export to the cytoplasm. Materials and Methods Cell lines and transfection - H1299 human cells, p53(-/-), MDM2(-/-) mouse

  9. Activation of p53-dependent responses in tumor cells treated with a PARC-interacting peptide

    SciTech Connect

    Vitali, Roberta; Cesi, Vincenzo; Tanno, Barbara; Ferrari-Amorotti, Giovanna; Dominici, Carlo; Calabretta, Bruno; Raschella, Giuseppe

    2008-04-04

    We tested the activity of a p53 carboxy-terminal peptide containing the PARC-interacting region in cancer cells with wild type cytoplasmic p53. Peptide delivery was achieved by fusing it to the TAT transduction domain (TAT-p53-C-ter peptide). In a two-hybrid assay, the tetramerization domain (TD) of p53 was necessary and sufficient to bind PARC. The TAT-p53-C-ter peptide disrupted the PARC-p53 complex. Peptide treatment caused p53 nuclear relocation, p53-dependent changes in gene expression and enhancement of etoposide-induced apoptosis. These studies suggest that PARC-interacting peptides are promising candidates for the enhancement of p53-dependent apoptosis in tumors with wt cytoplasmic p53.

  10. Structure-Based Design of Molecules to Reactivate Tumor-Derived p53 Mutations

    DTIC Science & Technology

    2007-06-01

    peptide called FL-CDB3, (2)Use the Multiple Solvent Crystal Structures (MSCS) technique, to identify novel p53 stabilization sites, (3) Use the... peptides for the reactivation of tumor derived p53 mutants, and (4) Functionally characterize the p53-stabilizing and p53- reactivation properties of the...prepared by mixing the p53 core domain with the Fl-CDB3 peptide . Unfortunately, the structure did not reveal ordered electron density for the peptide

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

    PubMed Central

    Ghosh, Anirban; Stewart, Deborah; Matlashewski, Greg

    2004-01-01

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

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

    PubMed Central

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

    1997-01-01

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

  13. Long non-coding RNA NEAT1 is a transcriptional target of p53 and modulates p53-induced transactivation and tumor-suppressor function.

    PubMed

    Idogawa, Masashi; Ohashi, Tomoko; Sasaki, Yasushi; Nakase, Hiroshi; Tokino, Takashi

    2017-03-14

    p53 is one of the most important tumor suppressor genes, and the direct transcriptional targets of p53 must be explored to elucidate its functional mechanisms. Thus far, the p53 targets that have been primarily studied are protein-coding genes. Our previous study revealed that several long non-coding RNAs (lncRNAs) are direct transcriptional targets of p53, and knockdown of specific lncRNAs modulates p53-induced apoptosis. In this study, analysis of next-generation chromatin immunoprecipitation-sequencing (ChIP-seq) data for p53 revealed that the lncRNA NEAT1 is a direct transcriptional target of p53. The suppression of NEAT1 induction by p53 attenuates the inhibitory effect of p53 on cancer cell growth and also modulates gene transactivation, including that of many lncRNAs. Furthermore, low expression of NEAT1 is related to poor prognosis in several cancers. These results indicate that the induction of NEAT1 expression contributes to the tumor-suppressor function of p53 and suggest that p53 and NEAT1 constitute a transcriptional network contributing to various biological functions and tumor suppression.

  14. P53 Suppression of Homologous Recombination and Tumorigenesis

    DTIC Science & Technology

    2011-01-01

    MO, Cat# F6178) and 200 units of penicillin /streptomycin and 0.5 mg amphotericin-B. Transfection was performed using Amaxa Nucleofector technology...Eagle’s medium supplemented with 10 000U/ml penicillin , 10 000 mg/ml streptomycin, 25 mg/ml Amphotericin B (Cellgro). The cells were grown at 37C in the...suggesting that p53 is highly active over this period and must be negatively regulated. In addition, the hypersensitivity of embryos to ionizing radiation

  15. Transcriptional and post-transcriptional regulation of the ionizing radiation response by ATM and p53

    PubMed Central

    Venkata Narayanan, Ishwarya; Paulsen, Michelle T.; Bedi, Karan; Berg, Nathan; Ljungman, Emily A.; Francia, Sofia; Veloso, Artur; Magnuson, Brian; di Fagagna, Fabrizio d’Adda; Wilson, Thomas E.; Ljungman, Mats

    2017-01-01

    In response to ionizing radiation (IR), cells activate a DNA damage response (DDR) pathway to re-program gene expression. Previous studies using total cellular RNA analyses have shown that the stress kinase ATM and the transcription factor p53 are integral components required for induction of IR-induced gene expression. These studies did not distinguish between changes in RNA synthesis and RNA turnover and did not address the role of enhancer elements in DDR-mediated transcriptional regulation. To determine the contribution of synthesis and degradation of RNA and monitor the activity of enhancer elements following exposure to IR, we used the recently developed Bru-seq, BruChase-seq and BruUV-seq techniques. Our results show that ATM and p53 regulate both RNA synthesis and stability as well as enhancer element activity following exposure to IR. Importantly, many genes in the p53-signaling pathway were coordinately up-regulated by both increased synthesis and RNA stability while down-regulated genes were suppressed either by reduced synthesis or stability. Our study is the first of its kind that independently assessed the effects of ionizing radiation on transcription and post-transcriptional regulation in normal human cells. PMID:28256581

  16. Role of DREF in transcriptional regulation of the Drosophila p53 gene.

    PubMed

    Trong-Tue, N; Thao, D T P; Yamaguchi, M

    2010-04-08

    The tumor suppressor protein p53 has a critical role in safeguarding the integrity of the genome. Its functions are well understood but factors responsible for the transcriptional regulation of the p53 gene are almost entirely unknown. The DNA replication-related element (DRE)/DNA replication-related element-binding factor (DREF) transcriptional regulatory system is established as a master key to cell proliferation in Drosophila. DREF binds specifically to DRE sequences in the Drosophila p53 (dmp53) gene promoter as shown using anti-DREF antibodies in chromatin immunoprecipitation assays. Furthermore, a rough eye phenotype because of overexpression of DREF in Drosophila eye imaginal disks could be suppressed by half dose reduction of the dmp53 gene. In addition, the level of mRNA of dmp53 was decreased in DREF-knockdown cells and transient expression of the luciferase gene under control of the wild-type dmp53 gene promoter showed strong promoter activity in S2 cells, but this was almost completely abrogated with a DRE-mutated promoter. Requirement of DREs for dmp53 promoter activity was further confirmed by anti-beta-galactosidase antibody-staining of various tissues from transgenic flies carrying dmp53 promoter-lacZ fusion genes. These results indicate that DREF is necessary for dmp53 gene promoter activity.

  17. A single-molecule characterization of p53 search on DNA

    PubMed Central

    Tafvizi, Anahita; Huang, Fang; Fersht, Alan R.; Mirny, Leonid A.; van Oijen, Antoine M.

    2011-01-01

    The tumor suppressor p53 slides along DNA while searching for its cognate site. Central to this process is the basic C-terminal domain, whose regulatory role and its coordination with the core DNA-binding domain is highly debated. Here we use single-molecule techniques to characterize the search process and disentangle the roles played by these two DNA-binding domains in the search process. We demonstrate that the C-terminal domain is capable of rapid translocation, while the core domain is unable to slide and instead hops along DNA. These findings are integrated into a model, in which the C-terminal domain mediates fast sliding of p53, while the core domain samples DNA by frequent dissociation and reassociation, allowing for rapid scanning of long DNA regions. The model further proposes how modifications of the C-terminal domain can activate “latent” p53 and reconciles seemingly contradictory data on the action of different domains and their coordination. PMID:21178072

  18. Akt phosphorylates myc-associated zinc finger protein (MAZ), releases P-MAZ from the p53 promoter, and activates p53 transcription.

    PubMed

    Lee, Wei-Ping; Lan, Keng-Hsin; Li, Chung-Pin; Chao, Yee; Lin, Han-Chieh; Lee, Shou-Dong

    2016-05-28

    The p53 protein is a cell cycle regulator. When the cell cycle progresses, p53 plays an important role in putting a brake on the G1 phase to prevent unwanted errors during cell division. Akt is a downstream kinase of receptor tyrosine kinase. Upon activation, Akt phorphorylates IKK that then phosphorylates IκB and releases NF-κB, leading to transcriptional activation of Dmp1. Dmp1 is a transcriptional activator of Arf. It has been known that oncogene activation stabilizes p53 through transcriptional activation of Arf, which then binds and inhibits Mdm2. In the current study, we show that myc-associated zinc finger protein (MAZ) is a transcriptional repressor of the p53 promoter. Akt phosphorylates MAZ at Thr385, and the phosphorylated MAZ is released from the p53 promoter, leading to transcriptional activation of p53, a new mechanism that contributes to increased p53 protein pool during oncogene activation.

  19. Detection of the anti-P53 antibodies in dogs with tumors.

    PubMed

    Kanaya, Noriko; Okuda, Masaru; Toyama, Naomi; Oikawa, Tatsuo; Inokuma, Hisashi; Morimoto, Masahiro; Hayashi, Toshiharu; Une, Satoshi; Nakaichi, Munekazu; Taura, Yasuho; Tsujimoto, Hajime; Onishi, Takafumi

    2002-11-01

    To detect the anti-P53 antibodies of dogs with tumors, a GST-recombinant canine (rc) P53 fusion protein was expressed and purified. Immunoblot analysis was performed using this GST-rcP53 fusion protein as an antigen and serum samples from dogs suffering from tumors as primary antibodies. Out of 16 serum samples obtained from various tumor cases, four samples showed reaction with GST-rcP53. In contrast, serum from other 12 dogs with tumors, four dogs with non-neoplastic diseases and two control healthy dogs (as controls) did not show any reaction with GST-rcP53 in immunoblotting. The p53 gene mutation and the P53 protein expression were examined, using the tumor tissues to explore the relationship between the existence of the GST-rcP53 bands, gene mutations of p53 and the accumulation of P53 protein. One case, which showed a clear GST-rcP53 band, had a point mutation of the p53 cDNA and showed nuclear accumulation of P53 protein. These results suggest that the anti-P53 antibodies are also produced in tumor dogs with p53 gene mutations.

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

    PubMed

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

    2015-08-11

    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.

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

    PubMed Central

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

    2016-01-01

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

  2. 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

  3. Mutant p53 in cancer: Accumulation, gain-of-function and therapy.

    PubMed

    Yue, Xuetian; Zhao, Yuhan; Xu, Yang; Zheng, Min; Feng, Zhaohui; Hu, Wenwei

    2017-04-05

    Tumor suppressor p53 plays a central role in tumor suppression. p53 is the most frequently mutated gene in human cancer, and over half of human cancers contain p53 mutations. Majority of p53 mutations in cancer are missense mutations, leading to the expression of full-length mutant p53 protein. While the critical role of wild type p53 in tumor suppression has been firmly established, mounting evidence has demonstrated that many tumor-associated mutant p53 proteins not only lose tumor suppressive function of wild type p53, but also gain new activities to promote tumorigenesis independently of wild type p53, termed gain-of-function. Mutant p53 protein often accumulates to very high levels in tumors, contributing to malignant progression. Recently, mutant p53 has become an attractive target for cancer therapy. Further understanding of the mechanisms underlying mutant p53 protein accumulation and gain-of-function will accelerate the development of targeted therapies for human cancer harboring mutant p53. In this review, we summarize the recent advances in the studies on mutant p53 protein accumulation and gain-of-function as well as targeted therapies for mutant p53 in human cancer.

  4. Novel Implications of DNA Damage Response in Drug Resistance of Malignant Cancers Obtained from the Functional Interaction between p53 Family and RUNX2

    PubMed Central

    Ozaki, Toshinori; Nakamura, Mizuyo; Shimozato, Osamu

    2015-01-01

    During the lifespan of cells, their genomic DNA is continuously exposed to the endogenous and exogenous DNA insults. Thus, the appropriate cellular response to DNA damage plays a pivotal role in maintaining genomic integrity and also acts as a molecular barrier towards DNA legion-mediated carcinogenesis. The tumor suppressor p53 participates in an integral part of proper regulation of DNA damage response (DDR). p53 is frequently mutated in a variety of human cancers. Since mutant p53 displays a dominant-negative behavior against wild-type p53, cancers expressing mutant p53 sometimes acquire drug-resistant phenotype, suggesting that mutant p53 prohibits the p53-dependent cell death pathway following DNA damage, and thereby contributing to the acquisition and/or maintenance of drug resistance of malignant cancers. Intriguingly, we have recently found that silencing of pro-oncogenic RUNX2 enhances drug sensitivity of aggressive cancer cells regardless of p53 status. Meanwhile, cancer stem cells (CSCs) have stem cell properties such as drug resistance. Therefore, the precise understanding of the biology of CSCs is quite important to overcome their drug resistance. In this review, we focus on molecular mechanisms behind DDR as well as the serious drug resistance of malignant cancers and discuss some attractive approaches to improving the outcomes of patients bearing drug-resistant cancers. PMID:26512706

  5. DIMP53-1: A novel small-molecule dual inhibitor of p53-MDM2/X interactions with multifunctional p53-dependent anticancer properties.

    PubMed

    Soares, Joana; Espadinha, Margarida; Raimundo, Liliana; Ramos, Helena; Gomes, Ana Sara; Gomes, Sara; Loureiro, Joana B; Inga, Alberto; Reis, Flávio; Gomes, Célia; Santos, Maria M M; Saraiva, Lucília

    2017-03-10

    The transcription factor p53 plays a crucial role in cancer development and dissemination, and thus p53-targeted therapies are amongst the most encouraging anticancer strategies. In human cancers with wild-type (wt) p53, its inactivation by interaction with murine double minute (MDM)2 and MDMX is a common event. Simultaneous inhibition of the p53 interaction with both MDMs is crucial to restore the tumor suppressor activity of p53. Here we describe the synthesis of the new tryptophanol-derived oxazoloisoindolinone DIMP53-1 and identify its activity as a dual inhibitor of the p53-MDM2/X interactions using a yeast-based assay. DIMP53-1 caused growth inhibition, mediated by p53 stabilization and upregulation of p53 transcriptional targets involved in cell cycle arrest and apoptosis, in wt p53-expressing tumor cells, including MDM2- or MDMX-overexpressing cells. Importantly, DIMP53-1 abolishes the p53-MDM2/X interactions by binding to p53, in human colon adenocarcinoma HCT116 cells. DIMP53-1 also inhibited the migration and invasion of HCT116 cells, and the migration and tube formation of HMVEC-D endothelial cells. Notably, in human tumor xenograft mice models, DIMP53-1 showed a p53-dependent antitumor activity through induction of apoptosis and inhibition of proliferation and angiogenesis. Finally, no genotoxicity or undesirable toxic effects were observed with DIMP53-1. In conclusion, DIMP53-1 is a novel p53 activator, which potentially binds to p53 inhibiting its interaction with MDM2 and MDMX. Although target-directed, DIMP53-1 has a multifunctional activity, targeting major hallmarks of cancer through its anti-proliferative, pro-apoptotic, anti-angiogenic, anti-invasive and anti-migratory properties. DIMP53-1 is a promising anticancer drug candidate and an encouraging starting point to develop improved derivatives for clinical application.

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

    PubMed

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

    2007-12-01

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

  7. MMP13, Birc2 (cIAP1) and Birc3 (cIAP2), Amplified on Chromosome 9, Collaborate with p53 Deficiency in Mouse Osteosarcoma Progression

    PubMed Central

    Ma, Ou; Cai, Wei-Wen; Zender, Lars; Dayaram, Tajhal; Shen, Jianhe; Herron, Alan J.; Lowe, Scott W.; Man, Tsz-Kwong; Lau, Ching C.; Donehower, Lawrence A.

    2009-01-01

    Osteosarcoma is the primary malignant cancer of bone and particularly affects adolescents and young adults, causing debilitation, and sometimes death. As a model for human osteosarcoma we have been studying p53+/− mice, which develop osteosarcoma at high frequency. To discover genes that cooperate with p53 deficiency in osteosarcoma formation we have integrated array comparative genomic hybridization, microarray expression analyses in mouse and human osteosarcomas, and functional assays. In this study we found seven frequent regions of copy number gain and loss in the mouse p53+/− osteosarcomas, but have focused on a recurrent amplification event on mouse chromosome 9A1. This amplicon is syntenic with a similar chromosome 11q22 amplicon identified in a number of human tumor types. Three genes on this amplicon, the matrix metalloproteinase gene MMP13, and the anti-apoptotic genes Birc2 (cIAP1), and Birc3 (cIAP2) show elevated expression in mouse and human osteosarcomas. We developed a functional assay using clonal osteosarcoma cell lines transduced with lentiviral shRNA vectors to show that downregulation of MMP13, Birc2, or Birc3 resulted in reduced tumor growth when transplanted into immunodeficient recipient mice. These experiments revealed that high MMP13 expression enhances osteosarcoma cell survival and that Birc2 and Birc3 also enhance cell survival, but only in osteosarcoma cells with the chromosome 9A1 amplicon. We conclude that the anti-apoptotic genes Birc2 and Birc3 are potential oncogenic drivers in the chromosome 9A1 amplicon. PMID:19276372

  8. Mutant p53 and ETS2, a Tale of Reciprocity.

    PubMed

    Martinez, Luis Alfonso

    2016-01-01

    TP53 is one of the most frequently inactivated tumor suppressor genes in human cancer. However, unlike other tumor suppressor genes whose expression is lost, TP53 is usually inactivated as a result of a single nucleotide change within the coding region. Typically, these single nucleotide mutations result in a codon change that creates an amino acid substitution. Thus, unlike other tumor suppressor genes whose expression is lost due to genetic or epigenetic changes, the p53 gene primarily suffers missense mutations, and therefore, the cells retain and express a mutant form of the p53 protein (mtp53). It is now well established that mtp53 contributes to tumor development through its gain-of-function (GOF) activities. These GOF activities can arise from novel protein-protein interactions that can either disable other tumor suppressors (e.g., p63 and p73) or enable oncogenes such as ETS2, an ETS family member. In this review, I will focus on the identification of the mtp53/ETS2 complex and outline the diverse activities that this transcriptional regulatory complex controls to promote cancer.

  9. ZASP Interacts with the Mechanosensing Protein Ankrd2 and p53 in the Signalling Network of Striated Muscle

    PubMed Central

    Martinelli, Valentina C.; Kyle, W. Buck; Kojic, Snezana; Vitulo, Nicola; Li, Zhaohui; Belgrano, Anna; Maiuri, Paolo; Banks, Lawrence; Vatta, Matteo; Valle, Giorgio; Faulkner, Georgine

    2014-01-01

    ZASP is a cytoskeletal PDZ-LIM protein predominantly expressed in striated muscle. It forms multiprotein complexes and plays a pivotal role in the structural integrity of sarcomeres. Mutations in the ZASP protein are associated with myofibrillar myopathy, left ventricular non-compaction and dilated cardiomyopathy. The ablation of its murine homologue Cypher results in neonatal lethality. ZASP has several alternatively spliced isoforms, in this paper we clarify the nomenclature of its human isoforms as well as their dynamics and expression pattern in striated muscle. Interaction is demonstrated between ZASP and two new binding partners both of which have roles in signalling, regulation of gene expression and muscle differentiation; the mechanosensing protein Ankrd2 and the tumour suppressor protein p53. These proteins and ZASP form a triple complex that appears to facilitate poly-SUMOylation of p53. We also show the importance of two of its functional domains, the ZM-motif and the PDZ domain. The PDZ domain can bind directly to both Ankrd2 and p53 indicating that there is no competition between it and p53 for the same binding site on Ankrd2. However there is competition for this binding site between p53 and a region of the ZASP protein lacking the PDZ domain, but containing the ZM-motif. ZASP is negative regulator of p53 in transactivation experiments with the p53-responsive promoters, MDM2 and BAX. Mutations in the ZASP ZM-motif induce modification in protein turnover. In fact, two mutants, A165V and A171T, were not able to bind Ankrd2 and bound only poorly to alpha-actinin2. This is important since the A165V mutation is responsible for zaspopathy, a well characterized autosomal dominant distal myopathy. Although the mechanism by which this mutant causes disease is still unknown, this is the first indication of how a ZASP disease associated mutant protein differs from that of the wild type ZASP protein. PMID:24647531

  10. Okadaic acid mediates p53 hyperphosphorylation and growth arrest in cells with wild-type p53 but increases aberrant mitoses in cells with non-functional p53.

    PubMed

    Milczarek, G J; Chen, W; Gupta, A; Martinez, J D; Bowden, G T

    1999-06-01

    The protein phosphatase inhibitor and tumor promoting agent okadaic acid (OA), has been shown previously to induce hyperphosphorylation of p53 protein, which in turn correlated with increased transactivation or apoptotic function. However, how the tumor promotion effects of OA relate to p53 tumor supressor function (or dysfunction) remain unclear. Rat embryonic fibroblasts harboring a temperature-sensitive mouse p53 transgene were treated with 50 nM doses of OA. At the wild-type permissive temperature this treatment resulted in: (i) the hyperphosphorylation of sites within tryptic peptides of the transactivation domain of p53; (ii) an increase in p53 affinity for a p21(waf1) promotor oligonucleotide; (iii) an increase in cellular steady state levels of p21(waf1) message; (iv) a G2/M cell cycle blockage in addition to the G1/S arrest previously associated with p53; and (v) no increased incidence of apoptosis. On the other hand, OA treatment at the mutated p53 permissive temperature resulted in a relatively high incidence of aberrant mitosis with no upregulation of p21(waf1) message. These results suggest that while wild-type p53 blocks the proliferative effects of OA through p21(waf1)-mediated growth arrest, cells with non-functional p53 cannot arrest and suffer relatively high levels of OA-mediated aberrant mitoses.

  11. p53 directly activates cystatin D/CST5 to mediate mesenchymal-epithelial transition: a possible link to tumor suppression by vitamin D3

    PubMed Central

    Hünten, Sabine; Hermeking, Heiko

    2015-01-01

    Cystatin D (CST5) encodes an inhibitor of cysteine proteases of the cathepsin family and is directly induced by the vitamin D receptor (VDR). Interestingly, vitamin D3 exerts tumor suppressive effects in a variety of tumor types. In colorectal cancer (CRC) cells CST5 was shown to mediate mesenchymal-epithelial transition (MET). We recently performed an integrated genomic and proteomic screen to identify targets of the p53 tumor suppressor in CRC cells. Thereby, we identified CST5 as a putative p53 target gene. Here, we validated and characterized CST5 as a direct p53 target gene. After activation of a conditional p53 allele, CST5 was upregulated on mRNA and protein levels. Treatment with nutlin-3a or etoposide induced CST5 in a p53-dependent manner. These regulations were direct, since ectopic and endogenous p53 occupied a conserved binding site in the CST5 promoter region. In addition, treatment with calcitriol, the active vitamin D3 metabolite, and simultaneous activation of p53 resulted in enhanced CST5 induction and increased repression of SNAIL, an epithelial-mesenchymal transition (EMT) inducing transcription factor. Furthermore, CST5 inactivation decreased p53-induced mesenchymal-epithelial transition (MET) as evidenced by decreased inhibition of SNAIL and of migration by p53. Furthermore, CST5 expression was directly repressed by SNAIL. In summary, these results imply CST5 as an important mediator of tumor suppression by p53 in colorectal cancer. In addition, they suggest that a combined treatment activating p53 and the vitamin D3 pathway may function via induction of CST5. PMID:26158294

  12. Aurora B interacts with NIR-p53, leading to p53 phosphorylation in its DNA-binding domain and subsequent functional suppression.

    PubMed

    Wu, Liming; Ma, Chi A; Zhao, Yongge; Jain, Ashish

    2011-01-21

    NIR (novel INHAT repressor) is a transcriptional co-repressor with inhibitor of histone acetyltransferase (INHAT) activity and has previously been shown to physically interact with and suppress p53 transcriptional activity and function. However, the mechanism by which NIR suppresses p53 is not completely understood. Using a proteomic approach, we have identified the Aurora kinase B as a novel binding partner of NIR. We show that Aurora B, NIR and p53 exist in a protein complex in which Aurora B binds to NIR, thus also indirectly associates with p53. Functionally, overexpression of Aurora B or NIR suppresses p53 transcriptional activity, and depletion of Aurora B or NIR causes p53-dependent apoptosis and cell growth arrest, due to the up-regulation of p21 and Bax. We then demonstrate that Aurora B phosphorylates multiple sites in the p53 DNA-binding domain in vitro, and this phosphorylation probably also occurs in cells. Importantly, the Aurora B-mediated phosphorylation on Ser(269) or Thr(284) significantly compromises p53 transcriptional activity. Taken together, these results provide novel insight into NIR-mediated p53 suppression and also suggest an additional way for p53 regulation.

  13. Tetramerization-defects of p53 result in aberrant ubiquitylation and transcriptional activity.

    PubMed

    Lang, Valérie; Pallara, Chiara; Zabala, Amaia; Lobato-Gil, Sofia; Lopitz-Otsoa, Fernando; Farrás, Rosa; Hjerpe, Roland; Torres-Ramos, Monica; Zabaleta, Lorea; Blattner, Christine; Hay, Ronald T; Barrio, Rosa; Carracedo, Arkaitz; Fernandez-Recio, Juan; Rodríguez, Manuel S; Aillet, Fabienne

    2014-07-01

    The tumor suppressor p53 regulates the expression of genes involved in cell cycle progression, senescence and apoptosis. Here, we investigated the effect of single point mutations in the oligomerization domain (OD) on tetramerization, transcription, ubiquitylation and stability of p53. As predicted by docking and molecular dynamics simulations, p53 OD mutants show functional defects on transcription, Mdm2-dependent ubiquitylation and 26S proteasome-mediated degradation. However, mutants unable to form tetramers are well degraded by the 20S proteasome. Unexpectedly, despite the lower structural stability compared to WT p53, p53 OD mutants form heterotetramers with WT p53 when expressed transiently or stably in cells wild type or null for p53. In consequence, p53 OD mutants interfere with the capacity of WT p53 tetramers to be properly ubiquitylated and result in changes of p53-dependent protein expression patterns, including the pro-apoptotic proteins Bax and PUMA under basal and adriamycin-induced conditions. Importantly, the patient derived p53 OD mutant L330R (OD1) showed the more severe changes in p53-dependent gene expression. Thus, in addition to the well-known effects on p53 stability, ubiquitylation defects promote changes in p53-dependent gene expression with implications on some of its functions.

  14. Shifting p53-induced senescence to cell death by TIS21(/BTG2/Pc3) gene through posttranslational modification of p53 protein.

    PubMed

    Choi, Ok Ran; Ryu, Min Sook; Lim, In Kyoung

    2016-09-01

    Cellular senescence and apoptosis can be regulated by p53 activity, although the underlying mechanism of the switch between the two events remains largely unknown. Cells exposed to cancer chemotherapy can escape to senescence phenotype rather than undergoing apoptosis. By employing adenoviral transduction of p53 or TIS21 genes, we observed shifting of p53 induced-senescence to apoptosis in EJ bladder cancer cells, which express H-RasV12 and mutant p53; transduction of p53 increased H-RasV12 expression along with senescence phenotypes, whereas coexpression with TIS21 (p53+TIS21) induced cell death rather than senescence. The TIS21-mediated switch of senescence to apoptosis was accompanied by nuclear translocation of p53 protein and its modifications on Ser-15 and Ser-46 phosphorylation and acetylations on Lys-120, -320, -373 and -382 residues. Mechanistically, TIS21(/BTG2) regulated posttranslational modification of p53 via enhancing miR34a and Bax expressions as opposed to inhibiting SIRT1 and Bcl2 expression. At the same time, TIS21 increased APAF-1 and p53AIP1 expressions, but inhibited the interaction of p53 with iASPP. In vitro tumorigenicity was significantly reduced in the p53+TIS21 expresser through inhibiting micro-colony proliferation by TIS21. Effect of TIS21 on the regulation of p53 activity was confirmed by knockdown of TIS21 expression by RNA interference. Therefore, we suggest TIS21 expression as an endogenous cell death inducer at the downstream of p53 gene, which might be useful for intractable cancer chemotherapy.

  15. Combined deletion of Pten and p53 in mammary epithelium accelerates triple-negative breast cancer with dependency on eEF2K

    PubMed Central

    Liu, Jeff C; Voisin, Veronique; Wang, Sharon; Wang, Dong-Yu; Jones, Robert A; Datti, Alessandro; Uehling, David; Al-awar, Rima; Egan, Sean E; Bader, Gary D; Tsao, Ming; Mak, Tak W; Zacksenhaus, Eldad

    2014-01-01

    The tumor suppressors Pten and p53 are frequently lost in breast cancer, yet the consequences of their combined inactivation are poorly understood. Here, we show that mammary-specific deletion of Pten via WAP-Cre, which targets alveolar progenitors, induced tumors with shortened latency compared to those induced by MMTV-Cre, which targets basal/luminal progenitors. Combined Pten-p53 mutations accelerated formation of claudin-low, triple-negative-like breast cancer (TNBC) that exhibited hyper-activated AKT signaling and more mesenchymal features relative to Pten or p53 single-mutant tumors. Twenty-four genes that were significantly and differentially expressed between WAP-Cre:Pten/p53 and MMTV-Cre:Pten/p53 tumors predicted poor survival for claudin-low patients. Kinome screens identified eukaryotic elongation factor-2 kinase (eEF2K) inhibitors as more potent than PI3K/AKT/mTOR inhibitors on both mouse and human Pten/p53-deficient TNBC cells. Sensitivity to eEF2K inhibition correlated with AKT pathway activity. eEF2K monotherapy suppressed growth of Pten/p53-deficient TNBC xenografts in vivo and cooperated with doxorubicin to efficiently kill tumor cells in vitro. Our results identify a prognostic signature for claudin-low patients and provide a rationale for using eEF2K inhibitors for treatment of TNBC with elevated AKT signaling. PMID:25330770

  16. Structure of p53 binding to the BAX response element reveals DNA unwinding and compression to accommodate base-pair insertion

    SciTech Connect

    Chen, Y.; Zhang, X.; Dantas Machado, A. C.; Ding, Y.; Chen, Z.; Qin, P. Z.; Rohs, R.; Chen, L.

    2013-07-08

    The p53 core domain binds to response elements (REs) that contain two continuous half-sites as a cooperative tetramer, but how p53 recognizes discontinuous REs is not well understood. Here we describe the crystal structure of the p53 core domain bound to a naturally occurring RE located at the promoter of the Bcl-2-associated X protein (BAX) gene, which contains a one base-pair insertion between the two half-sites. Surprisingly, p53 forms a tetramer on the BAX-RE that is nearly identical to what has been reported on other REs with a 0-bp spacer. Each p53 dimer of the tetramer binds in register to a half-site and maintains the same protein–DNA interactions as previously observed, and the two dimers retain all the protein–protein contacts without undergoing rotation or translation. To accommodate the additional base pair, the DNA is deformed and partially disordered around the spacer region, resulting in an apparent unwinding and compression, such that the interactions between the dimers are maintained. Furthermore, DNA deformation within the p53-bound BAX-RE is confirmed in solution by site-directed spin labeling measurements. Our results provide a structural insight into the mechanism by which p53 binds to discontinuous sites with one base-pair spacer.

  17. Combined deletion of Pten and p53 in mammary epithelium accelerates triple-negative breast cancer with dependency on eEF2K.

    PubMed

    Liu, Jeff C; Voisin, Veronique; Wang, Sharon; Wang, Dong-Yu; Jones, Robert A; Datti, Alessandro; Uehling, David; Al-awar, Rima; Egan, Sean E; Bader, Gary D; Tsao, Ming; Mak, Tak W; Zacksenhaus, Eldad

    2014-12-01

    The tumor suppressors Pten and p53 are frequently lost in breast cancer, yet the consequences of their combined inactivation are poorly understood. Here, we show that mammary-specific deletion of Pten via WAP-Cre, which targets alveolar progenitors, induced tumors with shortened latency compared to those induced by MMTV-Cre, which targets basal/luminal progenitors. Combined Pten-p53 mutations accelerated formation of claudin-low, triple-negative-like breast cancer (TNBC) that exhibited hyper-activated AKT signaling and more mesenchymal features relative to Pten or p53 single-mutant tumors. Twenty-four genes that were significantly and differentially expressed between WAP-Cre:Pten/p53 and MMTV-Cre:Pten/p53 tumors predicted poor survival for claudin-low patients. Kinome screens identified eukaryotic elongation factor-2 kinase (eEF2K) inhibitors as more potent than PI3K/AKT/mTOR inhibitors on both mouse and human Pten/p53-deficient TNBC cells. Sensitivity to eEF2K inhibition correlated with AKT pathway activity. eEF2K monotherapy suppressed growth of Pten/p53-deficient TNBC xenografts in vivo and cooperated with doxorubicin to efficiently kill tumor cells in vitro. Our results identify a prognostic signature for claudin-low patients and provide a rationale for using eEF2K inhibitors for treatment of TNBC with elevated AKT signaling.

  18. A dual role of p53 in the control of autophagy.

    PubMed

    Tasdemir, Ezgi; Chiara Maiuri, M; Morselli, Eugenia; Criollo, Alfredo; D'Amelio, Marcello; Djavaheri-Mergny, Mojgan; Cecconi, Francesco; Tavernarakis, Nektarios; Kroemer, Guido

    2008-08-01

    Genotoxic stress can induce autophagy in a p53-dependent fashion and p53 can transactivate autophagy-inducing genes. We have observed recently that inactivation of p53 by deletion, depletion or inhibition can trigger autophagy. Thus, human and mouse cells subjected to knockout, knockdown or pharmacological inhibition of p53 manifest signs of autophagy such as depletion of p62/SQSTM1, LC3 lipidation, redistribution of GFP-LC3 in cytoplasmic puncta, and accumulation of autophagosomes and autolysosomes, both in vitro and in vivo. Inhibition of p53 causes autophagy in enucleated cells, indicating that the cytoplasmic, non-nuclear pool of p53 can regulate autophagy. Accordingly, retransfection of p53(-/-) cells with wild-type p53 as well as a p53 mutant that is excluded from the nucleus (due to the deletion of the nuclear localization sequence) can inhibit autophagy, whereas retransfection with a nucleus-restricted p53 mutant (in which the nuclear localization sequence has been deleted) does not inhibit autophagy. Several distinct autophagy inducers (e.g., starvation, rapamycin, lithium, tunicamycin and thapsigargin) stimulate the rapid degradation of p53. In these conditions, inhibition of the p53-specific E3 ubiquitin ligase HDM2 can avoid p53 depletion and simultaneously prevent the activation of autophagy. Moreover, a p53 mutant that lacks the HDM2 ubiquitinylation site and hence is more stable than wild-type p53 is particularly efficient in suppressing autophagy. In conclusion, p53 plays a dual role in the control of autophagy. On the one hand, nuclear p53 can induce autophagy through transcriptional effects. On the other hand, cytoplasmic p53 may act as a master repressor of autophagy.

  19. 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.

  20. ACTIVATION OF p53 IN DOWN SYNDROME AND IN THE Ts65Dn MOUSE BRAIN IS ASSOCIATED WITH A PRO-APOPTOTIC PHENOTYPE

    PubMed Central

    Di Domenico, Fabio; Barone, Eugenio; Arena, Andrea; Lanzillotta, Chiara; Brokeaart, Diede; Blarzino, Carla; Head, Elizabeth; Butterfield, D Allan; Perluigi, Marzia

    2016-01-01

    Down Syndrome (DS) is the most common genetic cause of intellectual disability resulting from trisomy of chromosome 21. The main feature of DS neuropathology includes early onset of Alzheimer's disease, with deposition of senile plaques and tangles. We hypothesized that apoptosis may be activated in the presence of AD neuropathology in DS, thus we measured proteins associated with upstream and downstream pathways of p53 in the frontal cortex from DS cases with and without AD pathology and from Ts65Dn mice, at different ages. We observed increased acetylation and phosphorylation of p53, coupled to reduced MDM2-mediated ubiquitination and lower levels of SIRT1. Activation of p53 was associated with a number of down-stream targets (bax, PARP1, caspase-3, heat shock proteins and PGC1α) that were modulated in both DS and DS/AD compared with age-matched controls. In particular, the most relevant changes (increased p-p53, acetyl-p53 and reduced formation of MDM2/p53 complex) were found to be modified only in the presence of AD pathology in DS. In addition, a similar pattern of alterations in the p53 pathway were found in Ts65Dn mice. These results suggest that p53 may integrate different signals, which can result in a pro-apoptotic-phenotype contributing to AD neuropathology in people with DS. PMID:26967221

  1. Loss of Sparc in p53-null Astrocytes Promotes Macrophage Activation and Phagocytosis Resulting in Decreased Tumor Size and Tumor Cell Survival.

    PubMed

    Thomas, Stacey L; Schultz, Chad R; Mouzon, Ezekiell; Golembieski, William A; El Naili, Reima; Radakrishnan, Archanna; Lemke, Nancy; Poisson, Laila M; Gutiérrez, Jorge A; Cottingham, Sandra; Rempel, Sandra A

    2015-07-01

    Both the induction of SPARC expression and the loss of the p53 tumor suppressor gene are changes that occur early in glioma development. Both SPARC and p53 regulate glioma cell survival by inverse effects on apoptotic signaling. Therefore, during glioma formation, the upregulation of SPARC may cooperate with the loss of p53 to enhance cell survival. This study determined whether the loss of Sparc in astrocytes that are null for p53 would result in reduced cell survival and tumor formation and increased tumor immunogenicity in an in vivo xenograft brain tumor model. In vitro, the loss of Sparc in p53-null astrocytes resulted in an increase in cell proliferation, but a loss of tumorigenicity. At 7 days after intracranial implantation, Sparc-null tumors had decreased tumor cell survival, proliferation and reduced tumor size. The loss of Sparc promoted microglia/macrophage activation and phagocytosis of tumor cells. Our results indicate that the loss of p53 by deletion/mutation in the early stages of glioma formation may cooperate with the induction of SPARC to potentiate cancer cell survival and escape from immune surveillance.

  2. Development of multi-epitope vaccines targeting wild-type sequence p53 peptides.

    PubMed

    DeLeo, Albert B; Whiteside, Theresa L

    2008-09-01

    Loss of p53 tumor-suppressor function is the most common abnormality in human cancer, which can result in enhanced presentation to immune cells of wild-type (wt)-sequence peptides from tumor p53 molecules, thus providing the rationale for wt p53 peptide-based cancer vaccines. We review evidence from preclinical murine tumor models and preclinical studies that led to the clinical introduction of wt p53 peptide-based vaccines for cancer immunotherapy. Overall, this review illustrates the complex process of wt p53 epitope selection and the issues and concerns involved in the application of p53-based vaccines for patients with cancer.

  3. The p53 Protein is an Unusually Shaped Tetramer that Binds Directly to DNA

    NASA Astrophysics Data System (ADS)

    Friedman, Paula N.; Chen, Xinbin; Bargonetti, Jill; Prives, Carol

    1993-04-01

    We have analyzed the size and structure of native immunopurified human p53 protein. By using a combination of chemical crosslinking, gel filtration chromatography, and zonal velocity gradient centrifugation, we have determined that the predominant form of p53 in such preparations is a tetramer. The behavior of purified p53 in gels and sucrose gradients implies that the protein has an extended shape. Wild-type p53 has been shown to bind specifically to sites in cellular and viral DNA. We show in this study by Southwestern ligand blotting and by analysis of DNA-bound crosslinked p53 that p53 monomers, dimers, and tetramers can bind directly to DNA.

  4. No association of the p53 codon 72 polymorphism with malaria in Ghanaian primiparae and Rwandan children.

    PubMed

    Gai, Prabhanjan P; Meese, Stefanie; Bedu-Addo, George; Gahutu, Jean Bosco; Mockenhaupt, Frank P

    2014-06-01

    The p53 protein is a key cell-signaling mediator integrating host responses to various types of stress. A common polymorphism of the encoding TP53 gene (codon 72, Pro > Arg, rs1042522) is associated with susceptibility to virus-related and other cancers. The p53 has also been shown to be central for successful Plasmodium liver stage infection. We examined whether the polymorphism is associated with P. falciparum infection in Ghanaian primiparae and Rwandan children. The allele frequency of TP53 codon 72 Arg was 0.30 among 314 Ghanaian primiparae and 0.31 among 545 Rwandan children, respectively, and it was not associated with infection prevalence or parasite density. This does not exclude p53 to be of pathophysiological relevance in malaria but argues against a major respective role of the TP53 codon 72 polymorphism.

  5. Role of DNA repair machinery and p53 in the testicular germ cell cancer: a review.

    PubMed

    Romano, Francesco Jacopo; Rossetti, Sabrina; Conteduca, Vincenza; Schepisi, Giuseppe; Cavaliere, Carla; Di Franco, Rossella; La Mantia, Elvira; Castaldo, Luigi; Nocerino, Flavia; Ametrano, Gianluca; Cappuccio, Francesca; Malzone, Gabriella; Montanari, Micaela; Vanacore, Daniela; Quagliariello, Vincenzo; Piscitelli, Raffaele; Pepe, Maria Filomena; Berretta, Massimiliano; D'Aniello, Carmine; Perdonà, Sisto; Muto, Paolo; Botti, Gerardo; Ciliberto, Gennaro; Veneziani, Bianca Maria; De Falco, Francesco; Maiolino, Piera; Caraglia, Michele; Montella, Maurizio; De Giorgi, Ugo; Facchini, Gaetano

    2016-12-20

    Notwithstanding the peculiar sensitivity to cisplatin-based treatment, resulting in a very high percentage of cures even in advanced stages of the disease, still we do not know the biological mechanisms that make Testicular Germ Cell Tumor (TGCT) "unique" in the oncology scene. p53 and MDM2 seem to play a pivotal role, according to several in vitro observations, but no correlation has been found between their mutational or expression status in tissue samples and patients clinical outcome. Furthermore, other players seem to be on stage: DNA Damage Repair Machinery (DDR) , especially Homologous Recombination (HR) proteins, above all Ataxia Telangiectasia Mutated (ATM), cooperates with p53 in response to DNA damage, activating apoptotic cascade and contributing to cell "fate". Homologous Recombination deficiency has been assumed to be a Germ Cell Tumor characteristic underlying platinum-sensitivity, whereby Poly(ADP-ribose) polymerase (PARP), an enzyme involved in HR DNA repair, is an intriguing target: PARP inhibitors have already entered in clinical practice of other malignancies and trials are recruiting TGCT patients in order to validate their role in this disease. This paper aims to summarize evidence, trying to outline an overview of DDR implications not only in TGCT curability, but also in resistance to chemotherapy.

  6. Cellular senescence: ex vivo p53-dependent asymmetric cell kinetics

    PubMed Central

    2001-01-01

    Although senescence is a defining property of euploid mammalian cells, its physiologic basis remains obscure. Previously, cell kinetics properties of normal tissue cells have not been considered in models for senescence. We now provide evidence that senescence is in fact the natural consequence of normal in vivo somatic stem cell kinetics extended in culture. This concept of senescence is based on our discovery that cells engineered to conditionally express the well-recognized tumor suppressor protein and senescence factor, p53, exhibit asymmetric cell kinetics. In vivo, asymmetric cell kinetics are essential for maintenance of somatic stem cells; ex vivo, the same cell kinetics yield senescence as a simple kinetic endpoint. This new “asymmetric cell kinetics model” for senescence suggests novel strategies for the isolation and propagation of somatic tissue stem cells in culture. PMID:12488624

  7. Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression

    SciTech Connect

    Yu, Zhendong; Wang, Hao; Zhang, Libin; Tang, Aifa; Zhai, Qinna; Wen, Jianxiang; Yao, Li; Li, Pengfei

    2009-09-04

    CDglyTK fusion suicide gene has been well characterized to effectively kill tumor cells. However, the exact mechanism and downstream target genes are not fully understood. In our study, we found that CDglyTK/prodrug treatment works more efficiently in p53 wild-type (HONE1) cells than in p53 mutant (CNE1) cells. We then used adenovirus-mediated gene delivery system to either knockdown or overexpress p53 and its target genes in these cells. Consistent results showed that both p53-PUMA/NOXA/Bcl2-Bax and p53-p21 pathways contribute to the CDglyTK induced tumor cell suppression. Our work for the first time addressed the role of p53 related genes in the CDglyTK/prodrug system.

  8. p53-repressed miRNAs are involved with E2F in a feed-forward loop promoting proliferation

    PubMed Central

    Brosh, Ran; Shalgi, Reut; Liran, Atar; Landan, Gilad; Korotayev, Katya; Nguyen, Giang Huong; Enerly, Espen; Johnsen, Hilde; Buganim, Yosef; Solomon, Hilla; Goldstein, Ido; Madar, Shalom; Goldfinger, Naomi; Børresen-Dale, Anne-Lise; Ginsberg, Doron; Harris, Curtis C; Pilpel, Yitzhak; Oren, Moshe; Rotter, Varda

    2008-01-01

    Normal cell growth is governed by a complicated biological system, featuring multiple levels of control, often deregulated in cancers. The role of microRNAs (miRNAs) in the control of gene expression is now increasingly appreciated, yet their involvement in controlling cell proliferation is still not well understood. Here we investigated the mammalian cell proliferation control network consisting of transcriptional regulators, E2F and p53, their targets and a family of 15 miRNAs. Indicative of their significance, expression of these miRNAs is downregulated in senescent cells and in breast cancers harboring wild-type p53. These miRNAs are repressed by p53 in an E2F1-mediated manner. Furthermore, we show that these miRNAs silence antiproliferative genes, which themselves are E2F1 targets. Thus, miRNAs and transcriptional regulators appear to cooperate in the framework of a multi-gene transcriptional and post-transcriptional feed-forward loop. Finally, we show that, similarly to p53 inactivation, overexpression of representative miRNAs promotes proliferation and delays senescence, manifesting the detrimental phenotypic consequence of perturbations in this circuit. Taken together, these findings position miRNAs as novel key players in the mammalian cellular proliferation network. PMID:19034270

  9. Gene expression profiling analysis reveals arsenic-induced cell cycle arrest and apoptosis in p53-proficient and p53-deficient cells through differential gene pathways

    SciTech Connect

    Yu Xiaozhong Robinson, Joshua F.; Gribble, Elizabeth; Hong, Sung Woo; Sidhu, Jaspreet S.; Faustman, Elaine M.

    2008-12-15

    Arsenic (As) is a well-known environmental toxicant and carcinogen as well as an effective chemotherapeutic agent. The underlying mechanism of this dual capability, however, is not fully understood. Tumor suppressor gene p53, a pivotal cell cycle checkpoint signaling protein, has been hypothesized to play a possible role in mediating As-induced toxicity and therapeutic efficiency. In this study, we found that arsenite (As{sup 3+}) induced apoptosis and cell cycle arrest in a dose-dependent manner in both p53{sup +/+} and p53{sup -/-} mouse embryonic fibroblasts (MEFs). There was, however, a distinction between genotypes in the apoptotic response, with a more prominent induction of caspase-3 in the p53{sup -/-} cells than in the p53{sup +/+} cells. To examine this difference further, a systems-based genomic analysis was conducted comparing the critical molecular mechanisms between the p53 genotypes in response to As{sup 3+}. A significant alteration in the Nrf2-mediated oxidative stress response pathway was found in both genotypes. In p53{sup +/+} MEFs, As{sup 3+} induced p53-dependent gene expression alterations in DNA damage and cell cycle regulation genes. However, in the p53{sup -/-} MEFs, As{sup 3+} induced a significant up-regulation of pro-apoptotic genes (Noxa) and down-regulation of genes in immune modulation. Our findings demonstrate that As-induced cell death occurs through a p53-independent pathway in p53 deficient cells while apoptosis induction occurs through p53-dependent pathway in normal tissue. This difference in the mechanism of apoptotic responses between the genotypes provides important information regarding the apparent dichotomy of arsenic's dual mechanisms, and potentially leads to further advancement of its utility as a chemotherapeutic agent.

  10. Cycloheximide suppresses radiation-induced apoptosis in MOLT-4 cells with Arg72 variant of p53 through translational inhibition of p53 accumulation.

    PubMed

    Ito, Azusa; Morita, Akinori; Ohya, Soichiro; Yamamoto, Shinichi; Enomoto, Atsushi; Ikekita, Masahiko

    2011-01-01

    The human T-cell leukemia cell line MOLT-4 is highly radiosensitive, and thus it is often used as a model of p53-dependent radiation-induced apoptosis. Two branches of the p53-mediated apoptotic pathway are reported: "transcription-dependent" and "transcription-independent." However, the relative contribution of each in different types of cells is not yet clearly defined. Moreover, recent studies have shown that the codon 72 polymorphic variants of p53 show different sensitivities to apoptosis signals. The Arg72 variant has a more potent apoptosis-inducing activity in mitochondria than the Pro72 variant. Here, we initially investigated the codon 72 polymorphism of p53 in MOLT-4 cells. Analysis of the p53 exon 4 genomic DNA sequence, which includes codon 72, revealed that MOLT-4 cells are homozygous for the allele encoding Arg72. We next investigated the involvement of the transcription-independent function of p53 using an RNA synthesis inhibitor, actinomycin D (ActD), and a protein synthesis inhibitor, cycloheximide (CHX), and found that the apoptosis was suppressed by CHX but not by ActD. We also revealed that the suppressive effect of CHX on apoptosis was specifically mediated by p53, using a p53-knockdown MOLT-4 transfectant. Furthermore, the suppressive effect of CHX on apoptosis was highly correlated with the suppression of p53 protein accumulation, and less correlated with the suppression of p53 target genes expression. These results indicated that p53 transactivation is not necessary to induce apoptosis, and that p53 protein accumulation itself is both necessary and sufficient to do so.

  11. Differentiation-dependent p53 regulation of nucleotide excision repair in keratinocytes.

    PubMed Central

    Li, G.; Ho, V. C.; Mitchell, D. L.; Trotter, M. J.; Tron, V. A.

    1997-01-01

    The role of the tumor suppressor p53 in repair of ultraviolet light (UV)-induced DNA damage was evaluated using a host-cell reactivation (HCR) assay. HCR determines a cell's ability to repair UV-damaged DNA through reactivation of a transfected CAT reported plasmid. Most UV damage is removed through nucleotide excision repair (NER). Primary murine keratinocytes isolated from p53-deficient and wild-type p53 mice were used in the HCR assay. The NER was reduced in p53-/- keratinocytes as compared with p53+/+ keratinocytes. The reduced DNA repair in p53-/- mice was confirmed with a radioimmunoassay comparing cyclobutane dimers (CPDs) and (6-4) photoproducts in p53+/+ and p53-/- keratinocytes after the cells were exposed to UV irradiation. Our results demonstrate that wildtype p53 plays a significant role in regulating NER. Furthermore, as there is evidence that p53 protein levels decrease after keratinocytes become differentiated, we sought to determine whether p53 plays a role in NER in differentiated keratinocytes. Differentiation of the keratinocytes by increasing the Ca2+ concentration in the culture media resulted in a marked reduction in NER equally in both p53+/+ and p53-/- groups. This finding suggests that reduced DNA repair after differentiation is p53 independent. A similar reduction in HCR was confirmed in differentiated human keratinocytes. These data, taken together, indicate that p53 or p53-regulated proteins enhance NER in basal undifferentiated keratinocytes but not in differentiated cells. As nonmelanoma skin cancers originate from the basal keratinocytes, our findings suggest that loss of p53 may contribute to the pathogenesis of this common skin cancer. PMID:9095000

  12. Identification of GRO1 as a critical determinant for mutant p53 gain of function.

    PubMed

    Yan, Wensheng; Chen, Xinbin

    2009-05-01

    Mutant p53 gain of function contributes to cancer progression, increased invasion and metastasis potentials, and resistance to anticancer therapy. The ability of mutant p53 to acquire its gain of function is shown to correlate with increased expression of progrowth genes, such as c-MYC, MDR1, and NF-kappaB2. However, most of the published studies to identify mutant p53 target genes were performed in a cell system that artificially overexpresses mutant p53. Thus, it remains unclear whether such mutant p53 targets can be regulated by endogenous physiological levels of mutant p53. Here, we utilized SW480 and MIA-PaCa-2 cells, in which endogenous mutant p53 can be inducibly knocked down, to identify mutant p53 target genes that potentially mediate mutant p53 gain of function. We found that knockdown of mutant p53 inhibits GRO1 expression, whereas ectopic expression of mutant R175H in p53-null HCT116 cells increases GRO1 expression. In addition, we found that endogenous mutant p53 is capable of binding to and activating the GRO1 promoter. Interestingly, ectopic expression of GRO1 can rescue the proliferative defect in SW480 and MIA-PaCa-2 cells induced by knockdown of mutant p53. Conversely, knockdown of endogenous GRO1 inhibits cell proliferation and thus abrogates mutant p53 gain of function in SW480 cells. Taken together, our findings define a novel mechanism by which mutant p53 acquires its gain of function via transactivating the GRO1 gene in cancer cells. Thus, targeting GRO1 for cancer therapy would be applicable to a large portion of human tumors with mutant p53, but the exploration of GRO1 as a potential target should take the mutation status of p53 into consideration.

  13. UBE4B targets phosphorylated p53 at serines 15 and 392 for degradation

    PubMed Central

    Du, Cheng; Wu, Hong; Leng, Roger P.

    2016-01-01

    Phosphorylation of p53 is a key mechanism responsible for the activation of its tumor suppressor functions in response to various stresses. In unstressed cells, p53 is rapidly turned over and is maintained at a low basal level. After DNA damage or other forms of cellular stress, the p53 level increases, and the protein becomes metabolically stable. However, the mechanism of phosphorylated p53 regulation is unclear. In this study, we studied the kinetics of UBE4B, Hdm2, Pirh2, Cop1 and CHIP induction in response to p53 activation. We show that UBE4B coimmunoprecipitates with phosphorylated p53 at serines 15 and 392. Notably, the affinity between UBE4B and Hdm2 is greatly decreased after DNA damage. Furthermore, we observe that UBE4B promotes endogenous phospho-p53(S15) and phospho-p53(S392) degradation in response to IR. We demonstrate that UBE4B and Hdm2 repress p53S15A, p53S392A, and p53-2A(S15A, S392A) functions, including p53-dependent transactivation and growth inhibition. Overall, our results reveal that UBE4B plays an important role in regulating phosphorylated p53 following DNA damage. PMID:26673821

  14. Inactivation of p53 by HTLV type 1 and HTLV type 2 Tax trans-activators.

    PubMed

    Mahieux, R; Pise-Masison, C A; Nicot, C; Green, P; Hall, W W; Brady, J N

    2000-11-01

    Human T cell lymphotropic virus type II (HTLV-2) was originally isolated from a patient with a hairy T cell leukemia. It has been associated with rare cases of CD8(+) T lymphoproliferative disorders, and has a controversial role as a pathogen. The loss of p53 function, as a consequence of mutation or inactivation, increases the chances of genetic damage. Indeed, the importance of p53 as a tumor suppressor is evident from the fact that over 60% of all human cancers have a mutant or inactive p53. p53 status has been extensively studied in HTLV-1-infected cell lines. Interestingly, despite the fact that p53 mutations have been found in only a minority of cells, the p53 functions were found to be impaired. We have analyzed the functional activity of the p53 tumor suppressor in cells transformed with HTLV-2 subtypes A and B. As with HTLV-1-infected cells, abundant levels of the p53 protein are detected in HTLV-2 virus-infected cell lines. Using p53 reporter plasmid or induction of p53-responsive genes in response to gamma-irradiation, the p53 was found to be transcriptionally inhibited in HTLV-2-infected cells. Interestingly, although Tax-2A and-2B inactivate p53, the Tax-2A protein appears to inhibit p53 function less efficiently than either Tax-1 or Tax-2B in T cells, but not in fibroblasts.

  15. Knockdown of p53 suppresses Nanog expression in embryonic stem cells

    SciTech Connect

    Abdelalim, Essam Mohamed; Tooyama, Ikuo

    2014-01-10

    Highlights: •We investigate the role of p53 in ESCs in the absence of DNA damage. •p53 knockdown suppresses ESC proliferation. •p53 knockdown downregulates Nanog expression. •p53 is essential for mouse ESC self-renewal. -- Abstract: Mouse embryonic stem cells (ESCs) express high levels of cytoplasmic p53. Exposure of mouse ESCs to DNA damage leads to activation of p53, inducing Nanog suppression. In contrast to earlier studies, we recently reported that chemical inhibition of p53 suppresses ESC proliferation. Here, we confirm that p53 signaling is involved in the maintenance of mouse ESC self-renewal. RNA interference-mediated knockdown of p53 induced downregulation of p21 and defects in ESC proliferation. Furthermore, p53 knockdown resulted in a significant downregulation in Nanog expression at 24 and 48 h post-transfection. p53 knockdown also caused a reduction in Oct4 expression at 48 h post-transfection. Conversely, exposure of ESCs to DNA damage caused a higher reduction of Nanog expression in control siRNA-treated cells than in p53 siRNA-treated cells. These data show that in the absence of DNA damage, p53 is required for the maintenance of mouse ESC self-renewal by regulating Nanog expression.

  16. MEF/ELF4 transactivation by E2F1 is inhibited by p53.

    PubMed

    Taura, Manabu; Suico, Mary Ann; Fukuda, Ryosuke; Koga, Tomoaki; Shuto, Tsuyoshi; Sato, Takashi; Morino-Koga, Saori; Okada, Seiji; Kai, Hirofumi

    2011-01-01

    Myeloid elf-1-like factor (MEF) or Elf4 is an E-twenty-six (ETS)-related transcription factor with strong transcriptional activity that influences cellular senescence by affecting tumor suppressor p53. MEF downregulates p53 expression and inhibits p53-mediated cellular senescence by transcriptionally activating MDM2. However, whether p53 reciprocally opposes MEF remains unexplored. Here, we show that MEF is modulated by p53 in human cells and mice tissues. MEF expression and promoter activity were suppressed by p53. While we found that MEF promoter does not contain p53 response elements, intriguingly, it contains E2F consensus sites. Subsequently, we determined that E2F1 specifically binds to MEF promoter and transactivates MEF. Nevertheless, E2F1 DNA binding and transactivation of MEF promoter was inhibited by p53 through the association between p53 and E2F1. Furthermore, we showed that activation of p53 in doxorubicin-induced senescent cells increased E2F1 and p53 interaction, diminished E2F1 recruitment to MEF promoter and reduced MEF expression. These observations suggest that p53 downregulates MEF by associating with and inhibiting the binding activity of E2F1, a novel transcriptional activator of MEF. Together with previous findings, our present results indicate that a negative regulatory mechanism exists between p53 and MEF.

  17. Acetylation Is Crucial for p53-Mediated Ferroptosis and Tumor Suppression.

    PubMed

    Wang, Shang-Jui; Li, Dawei; Ou, Yang; Jiang, Le; Chen, Yue; Zhao, Yingming; Gu, Wei

    2016-10-04

    Although previous studies indicate that loss of p53-mediated cell cycle arrest, apoptosis, and senescence does not completely abrogate its tumor suppression function, it is unclear how the remaining activities of p53 are regulated. Here, we have identified an acetylation site at lysine K98 in mouse p53 (or K101 for human p53). Whereas the loss of K98 acetylation (p53(K98R)) alone has very modest effects on p53-mediated transactivation, simultaneous mutations at all four acetylation sites (p53(4KR): K98R+ 3KR[K117R+K161R+K162R]) completely abolish its ability to regulate metabolic targets, such as TIGAR and SLC7A11. Notably, in contrast to p53(3KR), p53(4KR) is severely defective in suppressing tumor growth in mouse xenograft models. Moreover, p53(4KR) is still capable of inducing the p53-Mdm2 feedback loop, but p53-dependent ferroptotic responses are markedly abrogated. Together, these data indicate the critical role of p53 acetylation in ferroptotic responses and its remaining tumor suppression activity.

  18. Insulin Receptor Tyrosine Kinase Substrate Enhances Low Levels of MDM2-Mediated p53 Ubiquitination

    PubMed Central

    Wang, Ke-Sheng; Chen, Gang; Shen, Hai-Lian; Li, Ting-Ting; Chen, Fei; Wang, Qin-Wan; Wang, Zhi-Qin; Han, Ze-Guang; Zhang, Xin

    2011-01-01

    The tumor suppressor p53 controls multiple cellular functions including DNA repair, cell cycle arrest and apoptosis. MDM2-mediated p53 ubiquitination affects both degradation and cytoplasmic localization of p53. Several cofactors are known to modulate MDM2-mediated p53 ubiquitination and proteasomal degradation. Here we show that IRTKS, a novel IRSp53-like protein inhibited p53-induced apoptosis and depressed its transcription activity. IRTKS bound directly to p53 and increased p53 ubiquitination and cytoplasmic localization. Further studies revealed that IRTKS interacted with MDM2 and promoted low levels of MDM2-mediated p53 ubiquitination in vitro and in vivo. In unstressed cells with low levels of MDM2, IRTKS was found to stabilize the interaction of p53 and MDM2. In stressed cells, IRTKS dissociated from p53, and high levels of MDM2 induced by p53 activation mediate IRTKS poly-ubiquitination and subsequent proteasomal degradation. These data suggest that IRTKS is a novel regulator of p53, modulating low level of MDM2-mediated p53 ubiquitination in unstressed cells. PMID:21887275

  19. Impact of the p53 status of tumor cells on extrinsic and intrinsic apoptosis signaling

    PubMed Central

    2013-01-01

    Background The p53 protein is the best studied target in human cancer. For decades, p53 has been believed to act mainly as a tumor suppressor and by transcriptional regulation. Only recently, the complex and diverse function of p53 has attracted more attention. Using several molecular approaches, we studied the impact of different p53 variants on extrinsic and intrinsic apoptosis signaling. Results We reproduced the previously published results within intrinsic apoptosis induction: while wild-type p53 promoted cell death, different p53 mutations reduced apoptosis sensitivity. The prediction of the impact of the p53 status on the extrinsic cell death induction was much more complex. The presence of p53 in tumor cell lines and primary xenograft tumor cells resulted in either augmented, unchanged or reduced cell death. The substitution of wild-type p53 by mutant p53 did not affect the extrinsic apoptosis inducing capacity. Conclusions In summary, we have identified a non-expected impact of p53 on extrinsic cell death induction. We suggest that the impact of the p53 status of tumor cells on extrinsic apoptosis signaling should be studied in detail especially in the context of therapeutic approaches that aim to restore p53 function to facilitate cell death via the extrinsic apoptosis pathway. PMID:23594441

  20. Roscovitine-induced apoptosis of H1299 cells depends on functional status of p53.

    PubMed

    Slovackova, J; Smarda, J; Smardova, J

    2012-01-01

    Roscovitine, an inhibitor of cyclin-dependent kinases, is promising anticancer agent. Its antiproliferative and cytotoxic effects can be mediated by the p53 signaling pathway. To define the role of p53 in roscovitine-induced cell response, we prepared H1299/p53 cell lines inducibly expressing specific variants of p53 (p53wt and hotspot R175H, temperature-dependent P98A, A159V, S215G, Y220C, Y234C mutants). In the presence of roscovitine, each cell line variant behaved in specific way reflecting activity of the p53 protein. Roscovitine decreased production of the cell cycle inhibitor p21 and induced apoptosis. This effect was the most efficient in cells expressing p53wt protein with full activity. The cell expressing partially and conditionally active p53 mutants responded to roscovitine less efficiently. The cells expressing p53 mutants A159V and Y234C were very sensitive to roscovitine but their response was clearly temperature-dependent. The cells expressing P98A, S215G and Y220C p53 mutants exhibited only weak sensitivity to roscovitine and underwent apoptosis in low frequency. In principle, each td p53 mutant responded to roscovitine in distinct way. We showed clearly that the impact of roscovitine on H1299 cells depends on functional status of p53 they produce. This suggests that patients with tumors exhibiting specific p53 variants can benefit from the roscovitine therapy.

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

    PubMed

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

    2017-03-01

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

  2. Acetylation of Lysine 382 and Phosphorylation of Serine 392 in p53 Modulate the Interaction between p53 and MDC1 In Vitro

    PubMed Central

    Shahar, Or David; Gabizon, Ronen; Feine, Oren; Alhadeff, Raphael; Ganoth, Assaf; Argaman, Liron; Shimshoni, Elee; Friedler, Assaf; Goldberg, Michal

    2013-01-01

    Occurrence of DNA damage in a cell activates the DNA damage response, a survival mechanism that ensures genomics stability. Two key members of the DNA damage response are the tumor suppressor p53, which is the most frequently mutated gene in cancers, and MDC1, which is a central adaptor that recruits many proteins to sites of DNA damage. Here we characterize the in vitro interaction between p53 and MDC1 and demonstrate that p53 and MDC1 directly interact. The p53-MDC1 interaction is mediated by the tandem BRCT domain of MDC1 and the C-terminal domain of p53. We further show that both acetylation of lysine 382 and phosphorylation of serine 392 in p53 enhance the interaction between p53 and MDC1. Additionally, we demonstrate that the p53-MDC1 interaction is augmented upon the induction of DNA damage in human cells. Our data suggests a new role for acetylation of lysine 382 and phosphorylation of serine 392 in p53 in the cellular stress response and offers the first evidence for an interaction involving MDC1 that is modulated by acetylation. PMID:24194938

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

    PubMed Central

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

    2015-01-01

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

  4. Microvessel density and p53 mutations in advanced-stage epithelial ovarian cancer.

    PubMed

    Nadkarni, Niyati J; Geest, Koen De; Neff, Traci; Young, Barry De; Bender, David P; Ahmed, Amina; Smith, Brian J; Button, Anna; Goodheart, Michael J

    2013-04-30

    We planned to determine the relationship between angiogenesis and p53 mutational status in advanced-stage epithelial ovarian cancer. Using 190 tumor samples from patients with stage III and IV ovarian cancer we performed p53 sequencing, immunohistochemistry, and CD31 microvessel density (MVD) determination. MVD was elevated in tumors with p53 null mutations compared to p53 missense mutation or no mutation. Disease recurrence was increased with higher MVD in both unadjusted and adjusted analyses. In adjusted analysis, p53 null mutation was associated with increased recurrence and worse overall survival. Worse overall survival and increased recurrence risk were also associated with the combination of CD31 MVD values >25 vessels/HPF and any p53 mutation. P53 mutation status and MVD may have prognostic significance in patients with advanced-stage ovarian cancer. Tumors with p53 null mutations are likely to be more vascular, contributing to decreased survival and increased recurrence probability.

  5. Mitochondrial localization of the low level p53 protein in proliferative cells

    SciTech Connect

    Ferecatu, Ioana; Bergeaud, Marie; Rodriguez-Enfedaque, Aida; Le Floch, Nathalie; Oliver, Lisa; Rincheval, Vincent; Renaud, Flore; Vallette, Francois M.; Mignotte, Bernard; Vayssiere, Jean-Luc

    2009-10-02

    p53 protein plays a central role in suppressing tumorigenesis by inducing cell cycle arrest or apoptosis through transcription-dependent and -independent mechanisms. Emerging publications suggest that following stress, a fraction of p53 translocates to mitochondria to induce cytochrome c release and apoptosis. However, the localization of p53 under unstressed conditions remains largely unexplored. Here we show that p53 is localized at mitochondria in absence of apoptotic stimuli, when cells are proliferating, localization observed in various cell types (rodent and human). This is also supported by acellular assays in which p53 bind strongly to mitochondria isolated from rat liver. Furthermore, the mitochondria subfractionation study and the alkaline treatment of the mitochondrial p53 revealed that the majority of mitochondrial p53 is present in the membranous compartments. Finally, we identified VDAC, a protein of the mitochondrial outer-membrane, as a putative partner of p53 in unstressed/proliferative cells.

  6. Nerve growth factor receptor negates the tumor suppressor p53 as a feedback regulator

    PubMed Central

    Zhou, Xiang; Hao, Qian; Liao, Peng; Luo, Shiwen; Zhang, Minhong; Hu, Guohui; Liu, Hongbing; Zhang, Yiwei; Cao, Bo; Baddoo, Melody; Flemington, Erik K; Zeng, Shelya X; Lu, Hua

    2016-01-01

    Cancer develops and progresses often by inactivating p53. Here, we unveil nerve growth factor receptor (NGFR, p75NTR or CD271) as a novel p53 inactivator. p53 activates NGFR transcription, whereas NGFR inactivates p53 by promoting its MDM2-mediated ubiquitin-dependent proteolysis and by directly binding to its central DNA binding domain and preventing its DNA-binding activity. Inversely, NGFR ablation activates p53, consequently inducing apoptosis, attenuating survival, and reducing clonogenic capability of cancer cells, as well as sensitizing human cancer cells to chemotherapeutic agents that induce p53 and suppressing mouse xenograft tumor growth. NGFR is highly expressed in human glioblastomas, and its gene is often amplified in breast cancers with wild type p53. Altogether, our results demonstrate that cancers hijack NGFR as an oncogenic inhibitor of p53. DOI: http://dx.doi.org/10.7554/eLife.15099.001 PMID:27282385

  7. Bcl-2/Bax protein ratio predicts 5-fluorouracil sensitivity independently of p53 status

    PubMed Central

    Mirjolet, J-F; Barberi-Heyob, M; Didelot, C; Peyrat, J-P; Abecassis, J; Millon, R; Merlin, J-L

    2000-01-01

    p53 tumour-suppressor gene is involved in cell growth control, arrest and apoptosis. Nevertheless cell cycle arrest and apoptosis induction can be observed in p53-defective cells after exposure to DNA-damaging agents such as 5-fluorouracil (5-FU) suggesting the importance of alternative pathways via p53-independent mechanisms. In order to establish relationship between p53 status, cell cycle arrest, Bcl-2/Bax regulation and 5-FU sensitivity, we examined p53 mRNA and protein expression and p53 protein functionality in wild-type (wt) and mutant (mt) p53 cell lines. p53 mRNA and p53 protein expression were determined before and after exposure to equitoxic 5-FU concentration in six human carcinoma cell lines differing in p53 status and displaying marked differences in 5-FU sensitivity, with IC 50 values ranging from 0.2–22.6 mM. 5-FU induced a rise in p53 mRNA expression in mt p53 cell lines and in human papilloma virus positive wt p53 cell line, whereas significant decrease in p53 mRNA expression was found in wt p53 cell line. Whatever p53 status, 5-FU altered p53 transcriptional and translational regulation leading to up-regulation of p53 protein. In relation with p53 functionality, but independently of p53 mutational status, after exposure to 5-FU equitoxic concentration, all cell lines were able to arrest in G1. No relationship was evidenced between G1 accumulation ability and 5-FU sensitivity. Moreover, after 5-FU exposure, Bax and Bcl-2 proteins regulation was under p53 protein control and a statistically significant relationship (r= 0.880,P= 0.0097) was observed between Bcl-2/Bax ratio and 5-FU sensitivity. In conclusion, whatever p53 status, Bcl-2 or Bax induction and Bcl-2/Bax protein ratio were correlated to 5-FU sensitivity. © 2000 Cancer Research Campaign PMID:11044365

  8. Regulation of Drug Sensitivity by Functional Status of p53 in Human Prostate Cancer

    DTIC Science & Technology

    2005-01-01

    We also determined the effect of compounds that alter p53 function on MRP1 expression. We found that chlorpromazine , promazine, and trans...flupenthixol caused a 2-3-fold increase in wild-type p53 conformation and CP-31398 increased wild-type p53 conformation 6-10- fold. Promazine and chlorpromazine ...the p53 wild-type conformation, we incubated the cells with phenothiazines and performed an ELISA. Figure 1 lb shows that promazine and chlorpromazine

  9. Identification of HEXIM1 as a Positive Regulator of p53*

    PubMed Central

    Lew, Qiao Jing; Chia, Yi Ling; Chu, Kai Ling; Lam, Yuen Ting; Gurumurthy, Meera; Xu, Shengli; Lam, Kong Peng; Cheong, Nge; Chao, Sheng-Hao

    2012-01-01

    Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which regulates the transcription elongation of RNA polymerase II and controls 60–70% of mRNA synthesis. Our previous studies show that HEXIM1 interacts with two key p53 regulators, nucleophosmin and human double minute-2 protein (HDM2), implying a possible connection between HEXIM1 and the p53 signaling pathway. Here we report the interaction between p53 and HEXIM1 in breast cancer, acute myeloid leukemia, and colorectal carcinoma cells. The C-terminal regions of p53 and HEXIM1 are required for the protein-protein interaction. Overexpression of HEXIM1 prevents the ubiquitination of p53 by HDM2 and enhances the protein stability of p53, resulting in up-regulation of p53 target genes, such as Puma and p21. Induction of p53 can be achieved by several means, such as UV radiation and treatment with anti-cancer agents (including doxorubicin, etoposide, roscovitine, flavopiridol, and nutlin-3). Under all the conditions examined, elevated protein levels of p53 are found to associate with the increased p53-HEXIM1 interaction. In addition, knockdown of HEXIM1 significantly inhibits the induction of p53 and releases the cell cycle arrest caused by p53. Finally, the transcription of the p53 target genes is regulated by HEXIM1 in a p53-dependent fashion. Our results not only identify HEXIM1 as a positive regulator of p53, but also propose a novel molecular mechanism of p53 activation caused by the anti-cancer drugs and compounds. PMID:22948151

  10. Targeting the p53 signaling pathway in cancer therapy - The promises, challenges, and perils

    PubMed Central

    Stegh, Alexander H.

    2012-01-01

    Introduction Research over the past three decades has identified p53 as a multifunctional transcription factor, which regulates the expression of >2,500 target genes. p53 impacts myriad, highly diverse cellular processes, including the maintenance of genomic stability and fidelity, metabolism, longevity, and represents one of the most important and extensively studied tumor suppressors. Activated by various stresses, foremost genotoxic damage, hypoxia, heat shock and oncogenic assault, p53 blocks cancer progression by provoking transient or permanent growth arrest, by enabling DNA repair or by advancing cellular death programs. This potent and versatile anti-cancer activity profile, together with genomic and mutational analyses documenting inactivation of p53 in more than 50% of human cancers, motivated drug development efforts to (re-) activate p53 in established tumors. Areas covered In this review the complexities of p53 signaling in cancer are summarized. Current strategies and challenges to restore p53’s tumor suppressive function in established tumors, i.e. adenoviral gene transfer and small molecules to activate p53, to inactivate p53 inhibitors and to restore wild type function of p53 mutant proteins are discussed. Expert opinion It is indubitable that p53 represents an attractive target for the development of anti-cancer therapies. Whether p53 is ‘druggable’, however, remains an area of active research and discussion, as p53 has pro-survival functions and chronic p53 activation accelerates aging, which may compromise the long-term homeostasis of an organism. Thus, the complex biology and dual functions of p53 in cancer prevention and age-related cellular responses pose significant challenges on the development of p53-targeting cancer therapies. PMID:22239435

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

    PubMed

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

    1995-06-01

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

  12. Divergence between the high rate of p53 mutations in skin carcinomas and the low prevalence of anti-p53 antibodies

    PubMed Central

    Moch, C; Moysan, A; Lubin, R; Salmonière, P de La; Soufir, N; Galisson, F; Vilmer, C; Venutolo, E; Pelletier, F Le; Janin, A; Basset-Séguin, N

    2001-01-01

    Circulating anti-p53 antibodies have been described and used as tumoural markers in patients with various cancers and strongly correlate with the p53 mutated status of the tumours. No study has yet looked at the prevalence of such antibodies in skin carcinoma patients although these tumours have been shown to be frequently p53 mutated. Most skin carcinoma can be diagnosed by examination or biopsy, but aggressive, recurrent and/or non-surgical cases' follow up would be helped by a biological marker of residual disease. We performed a prospective study looking at the prevalence of anti-p53 antibodies using an ELISA technique in a series of 105 skin carcinoma patients in comparison with a sex- and age-matched control skin carcinoma-free group (n = 130). Additionally, p53 accumulation was studied by immunohistochemistry to confirm p53 protein altered expression in a sample of tumours. Anti-p53 antibodies were detected in 2.9% of the cases, with a higher prevalence in patients suffering from the more aggressive squamous cell type (SCC) of skin carcinoma (8%) than for the more common and slowly growing basal cell carcinoma type or BCC (1.5%). p53 protein stabilization could be confirmed in 80% of tumours studied by IHC. This low level of anti-p53 antibody detection contrasts with the high rate of p53 mutations reported in these tumours. This observation shows that the anti-p53 humoral response is a complex and tissue-specific mechanism. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11747330

  13. p53 directly suppresses BNIP3 expression to protect against hypoxia-induced cell death

    PubMed Central

    Feng, Xi; Liu, Xing; Zhang, Wei; Xiao, Wuhan

    2011-01-01

    Hypoxia stabilizes the tumour suppressor p53, allowing it to function primarily as a transrepressor; however, the function of p53 during hypoxia remains unclear. In this study, we showed that p53 suppressed BNIP3 expression by directly binding to the p53-response element motif and recruiting corepressor mSin3a to the BNIP3 promoter. The DNA-binding site of p53 must remain intact for the protein to suppress the BNIP3 promoter. In addition, taking advantage of zebrafish as an in vivo model, we confirmed that zebrafish nip3a, a homologous gene of mammalian BNIP3, was indeed induced by hypoxia and p53 mutation/knockdown enhanced nip3a expression under hypoxia resulted in cell death enhancement in p53 mutant embryos. Furthermore, p53 protected against hypoxia-induced cell death mediated by p53 suppression of BNIP3 as illustrated by p53 knockdown/loss assays in both human cell lines and zebrafish model, which is in contrast to the traditional pro-apoptotic role of p53. Our results suggest a novel function of p53 in hypoxia-induced cell death, leading to the development of new treatments for ischaemic heart disease and cerebral stoke. PMID:21792176

  14. Reverting p53 activation after recovery of cellular stress to resume with cell cycle progression.

    PubMed

    Lazo, Pedro A

    2017-05-01

    The activation of p53 in response to different types of cellular stress induces several protective reactions including cell cycle arrest, senescence or cell death. These protective effects are a consequence of the activation of p53 by specific phosphorylation performed by several kinases. The reversion of the cell cycle arrest, induced by p53, is a consequence of the phosphorylated and activated p53, which triggers its own downregulation and that of its positive regulators. The different down-regulatory processes have a sequential and temporal order of events. The mechanisms implicated in p53 down-regulation include phosphatases, deacetylases, and protein degradation by the proteasome or autophagy, which also affect different p53 protein targets and functions. The necessary first step is the dephosphorylation of p53 to make it available for interaction with mdm2 ubiquitin-ligase, which requires the activation of phosphatases targeting both p53 and p53-activating kinases. In addition, deacetylation of p53 is required to make lysine residues accessible to ubiquitin ligases. The combined action of these downregulatory mechanisms brings p53 protein back to its basal levels, and cell cycle progression can resume if cells have overcome the stress or damage situation. The specific targeting of these down-regulatory mechanisms can be exploited for therapeutic purposes in cancers harbouring wild-type p53.

  15. Cell cycle regulation and p53 activation by protein phosphatase 2C alpha.

    PubMed

    Ofek, Paula; Ben-Meir, Daniella; Kariv-Inbal, Zehavit; Oren, Moshe; Lavi, Sara

    2003-04-18

    Protein phosphatase 2C (PP2C) dephosphorylates a broad range of substrates, regulating stress response and growth-related pathways in both prokaryotes and eukaryotes. We now demonstrate that PP2C alpha, a major mammalian isoform, inhibits cell growth and activates the p53 pathway. In 293 cell clones, in which PP2C alpha expression is regulated by a tetracycline-inducible promoter, PP2C alpha overexpression led to G(2)/M cell cycle arrest and apoptosis. Furthermore, PP2C alpha induced the expression of endogenous p53 and the p53-responsive gene p21. Activation of the p53 pathway by PP2C alpha took place both in cells harboring endogenous p53, as well as in p53-null cells transfected with exogenous p53. Induction of PP2C alpha resulted in an increase in the overall levels of p53 protein as well as an augmentation of p53 transcription activity. The dephosphorylation activity of PP2C alpha is essential to the described phenomena, as none of these effects was detected when an enzymatically inactive PP2C alpha mutant was overexpressed. p53 plays an important role in PP2C alpha-directed cell cycle arrest and apoptosis because perturbation of p53 expression in human 293 cells by human papillomavirus E6 led to a significant increase in cell survival. The role of PP2C alpha in p53 activation is discussed.

  16. Oscillations of the p53-Akt Network: Implications on Cell Survival and Death

    PubMed Central

    Wee, Keng Boon; Surana, Uttam; Aguda, Baltazar D.

    2009-01-01

    Intracellular protein levels of p53 and MDM2 have been shown to oscillate in response to ionizing radiation (IR), but the physiological significance of these oscillations remains unclear. The p53-MDM2 negative feedback loop – the putative cause of the oscillations – is embedded in a network involving a mutual antagonism (or positive feedback loop) between p53 and AKT. We have shown earlier that this p53-AKT network predicts an all-or-none switching behavior between a pro-survival cellular state (low p53 and high AKT levels) and a pro-apoptotic state (high p53 and low AKT levels). Here, we show that upon exposure to IR, the p53-AKT network can also reproduce the experimentally observed p53 and MDM2 oscillations. The present work is based on the hypothesis that the physiological significance of the experimentally observed oscillations could be found in their role in regulating the switching behavior of the p53-AKT network between pro-survival and pro-apoptotic states. It is shown here that these oscillations are associated with a significant decrease in the threshold level of IR at which switching from a pro-survival to a pro-apoptotic state occurs. Moreover, oscillations in p53 protein levels induce higher levels of expression of p53-target genes compared to non-oscillatory p53, and thus influence cell-fate decisions between cell cycle arrest/DNA damage repair versus apoptosis. PMID:19197384

  17. Release of targeted p53 from the mitochondrion as an early signal during mitochondrial dysfunction.

    PubMed

    Green, M L; Pisano, M M; Prough, R A; Knudsen, T B

    2013-12-01

    Increased accumulation of p53 tumor suppressor protein is an early response to low-level stressors. To investigate the fate of mitochondrial-sequestered p53, mouse embryonic fibroblast cells (MEFs) on a p53-deficient genetic background were transfected with p53-EGFP fusion protein led by a sense (m53-EGFP) or antisense (c53-EGFP) mitochondrial import signal. Rotenone exposure (100nM, 1h) triggered the translocation of m53-EGFP from the mitochondrion to the nucleus, thus shifting the transfected cells from a mitochondrial p53 to a nuclear p53 state. Antibodies for p53 serine phosphorylation or lysine acetylation indicated a different post-translational status of recombinant p53 in the nucleus and mitochondrion, respectively. These data suggest that cycling of p53 through the mitochondria may establish a direct pathway for p53 signaling from the mitochondria to the nucleus during mitochondrial dysfunction. PK11195, a pharmacological ligand of mitochondrial TSPO (formerly known as the peripheral-type benzodiazepine receptor), partially suppressed the release of mitochondria-sequestered p53. These findings support the notion that p53 function mediates a direct signaling pathway from the mitochondria to nucleus during mitochondrial dysfunction.

  18. Indispensable role of Mdm2/p53 interaction during the embryonic and postnatal inner ear development

    PubMed Central

    Laos, M.; Sulg, M.; Herranen, A.; Anttonen, T.; Pirvola, U.

    2017-01-01

    p53 is a key component of a signaling network that protects cells against various stresses. As excess p53 is detrimental to cells, its levels are tightly controlled by several mechanisms. The E3 ubiquitin ligase Mdm2 is a major negative regulator of p53. The significance of balanced p53 levels in normal tissues, at different stages of lifetime, is poorly understood. We have studied in vivo how the disruption of Mdm2/p53 interaction affects the early-embryonic otic progenitor cells and their descendants, the auditory supporting cells and hair cells. We found that p53 accumulation, as a consequence of Mdm2 abrogation, is lethal to both proliferative progenitors and non-proliferating, differentiating cells. The sensitivity of postmitotic supporting cells to excess p53 decreases along maturation, suggesting that maturation-related mechanisms limit p53′s transcriptional activity towards pro-apoptotic factors. We have also investigated in vitro whether p53 restricts supporting cell’s regenerative capacity. Unlike in several other regenerative cellular models, p53 inactivation did not alter supporting cell’s proliferative quiescence nor transdifferentiation capacity. Altogether, the postmitotic status of developing hair cells and supporting cells does not confer protection against the detrimental effects of p53 upregulation. These findings might be linked to auditory disturbances observed in developmental syndromes with inappropriate p53 upregulation. PMID:28181574

  19. In Vivo p53 Signaling in Breast Epithelial Cells After Oncogenic Stimulus

    DTIC Science & Technology

    2005-09-01

    cell line, a derivative of the H1299 p53 null lung carcinoma cell line that contains ponasterone-inducible p53, and the isogenic colon carcinoma...induc- ible H1299 cell line in which p53 expression was under the control of the ecdysone promoter and induced by ponasterone A addition (HIp53), and (iii

  20. The PTTG1-binding factor (PBF/PTTG1IP) regulates p53 activity in thyroid cells.

    PubMed

    Read, Martin L; Seed, Robert I; Fong, Jim C W; Modasia, Bhavika; Ryan, Gavin A; Watkins, Rachel J; Gagliano, Teresa; Smith, Vicki E; Stratford, Anna L; Kwan, Perkin K; Sharma, Neil; Dixon, Olivia M; Watkinson, John C; Boelaert, Kristien; Franklyn, Jayne A; Turnell, Andrew S; McCabe, Christopher J

    2014-04-01

    The PTTG1-binding factor (PBF/PTTG1IP) has an emerging repertoire of roles, especially in thyroid biology, and functions as a protooncogene. High PBF expression is independently associated with poor prognosis and lower disease-specific survival in human thyroid cancer. However, the precise role of PBF in thyroid tumorigenesis is unclear. Here, we present extensive evidence demonstrating that PBF is a novel regulator of p53, a tumor suppressor protein with a key role in maintaining genetic stability, which is infrequently mutated in differentiated thyroid cancer. By coimmunoprecipitation and proximity-ligation assays, we show that PBF binds specifically to p53 in thyroid cells and significantly represses transactivation of responsive promoters. Further, we identify that PBF decreases p53 stability by enhancing ubiquitination, which appears dependent on the E3 ligase activity of Mdm2. Impaired p53 function was evident in a transgenic mouse model with thyroid-specific PBF overexpression (transgenic PBF mice), which had significantly increased genetic instability as indicated by fluorescent inter simple sequence repeat-PCR analysis. Consistent with this, approximately 40% of all DNA repair genes examined were repressed in transgenic PBF primary cultures, including genes with critical roles in maintaining genomic integrity such as Mgmt, Rad51, and Xrcc3. Our data also revealed that PBF induction resulted in up-regulation of the E2 enzyme Rad6 in murine thyrocytes and was associated with Rad6 expression in human thyroid tumors. Overall, this work provides novel insights into the role of the protooncogene PBF as a negative regulator of p53 function in thyroid tumorigenesis, in which PBF is generally overexpressed and p53 mutations are rare compared with other tumor types.

  1. The PTTG1-Binding Factor (PBF/PTTG1IP) regulates p53 activity in thyroid cells

    PubMed Central

    Read, Martin L.; Seed, Robert I.; Fong, Jim C.W.; Modasia, Bhavika; Ryan, Gavin A.; Watkins, Rachel J; Gagliano, Teresa; Smith, Vicki E.; Stratford, Anna L.; Kwan, Perkin K; Sharma, Neil; Dixon, Olivia M.; Watkinson, John C.; Boelaert, Kristien; Franklyn, Jayne A.; Turnell, Andrew S.; McCabe, Christopher J.

    2016-01-01

    The PTTG1-Binding Factor (PBF/PTTG1IP) has an emerging repertoire of roles, especially in thyroid biology, and functions as a proto-oncogene. High PBF expression is independently associated with poor prognosis and lower disease-specific survival in human thyroid cancer. However, the precise role of PBF in thyroid tumorigenesis is unclear. Here, we present extensive evidence demonstrating that PBF is a novel regulator of p53, a tumor suppressor protein with a key role in maintaining genetic stability, which is infrequently mutated in differentiated thyroid cancer. By coimmunoprecipitation and proximity ligation assays, we show that PBF binds specifically to p53 in thyroid cells, and significantly represses transactivation of responsive promoters. Further, we identify that PBF decreases p53 stability by enhancing ubiquitination, which appears dependent on the E3 ligase activity of Mdm2. Impaired p53 function was evident in a transgenic mouse model with thyroid-specific PBF over-expression (PBF-Tg), which had significantly increased genetic instability as indicated by FISSR-PCR analysis. Consistent with this, ~40% of all DNA repair genes examined were repressed in PBF-Tg primary cultures, including genes with critical roles in maintaining genomic integrity such as Mgmt, Rad51 and Xrcc3. Our data also revealed that PBF induction resulted in upregulation of the E2 enzyme Rad6 in murine thyrocytes, and was associated with Rad6 expression in human thyroid tumors. Overall, this work provides novel insights into the role of the proto-oncogene PBF as a negative regulator of p53 function in thyroid tumorigenesis, where PBF is generally over-expressed and p53 mutations are rare compared to other tumor types. PMID:24506068

  2. Aspirin acetylates wild type and mutant p53 in colon cancer cells: identification of aspirin acetylated sites on recombinant p53.

    PubMed

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D Ramesh; Marimuthu, Srinivasan; Alfonso, Lloyd F; Bhat, G Jayarama

    2016-05-01

    Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anti-cancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. We demonstrate that aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21(CIP1). Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anti-cancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin.

  3. PPM1D phosphatase, a target of p53 and RBM38 RNA-binding protein, inhibits p53 mRNA translation via dephosphorylation of RBM38.

    PubMed

    Zhang, M; Xu, E; Zhang, J; Chen, X

    2015-11-26

    PPM1D phosphatase, also called wild-type p53-induced phosphatase 1, promotes tumor development by inactivating the p53 tumor suppressor pathway. RBM38 RNA-binding protein, also called RNPC1 and a target of p53, inhibits p53 messenger RNA (mRNA) translation, which can be reversed by GSK3 protein kinase via phosphorylation of RBM38 at serine 195. Here we showed that ectopic expression of RBM38 increases, whereas knockdown of RBM38 inhibits, PPM1D mRNA translation. Consistent with this, we found that RBM38 directly binds to PPM1D 3'-untranslated region (3'-UTR) and promotes expression of a heterologous reporter gene that carries PPM1D 3'-UTR in a dose-dependent manner. Interestingly, we showed that PPM1D directly interacts with and dephosphorylates RBM38 at serine 195. Furthermore, we showed that PPM1D modulates p53 mRNA translation and p53-dependent growth suppression through dephosphorylation of RBM38. These findings provide evidence that the crosstalk between PPM1D and RBM38, both of which are targets and modulators of p53, has a critical role in p53 expression and activity.

  4. PPM1D phosphatase, a target of p53 and RBM38 RNA-binding protein, inhibits p53 mRNA translation via dephosphorylation of RBM38

    PubMed Central

    Zhang, Min; Xu, Enshun; Zhang, Jin; Chen, Xinbin

    2015-01-01

    PPM1D phosphatase, also called wild-type p53-induced phosphatase 1 (Wip1), promotes tumor development by inactivating the p53 tumor suppressor pathway. RBM38 RNA-binding protein, also called RNPC1 and a target of p53, inhibits p53 mRNA translation, which can be reversed by GSK3 protein kinase via phosphorylation of RBM38 at serine 195. Here we showed that ectopic expression of RBM38 increases, whereas knockdown of RBM38 inhibits, PPM1D mRNA translation. Consistent with this, we found that RBM38 directly binds to PPM1D 3' untranslated region (3’UTR) and promotes expression of a heterologous reporter gene that carries PPM1D 3’UTR in a dose-dependent manner. Interestingly, we showed that PPM1D directly interacts with and dephosphorylates RBM38 at serine 195. Furthermore, we showed that PPM1D modulates p53 mRNA translation and p53-dependent growth suppression through dephosphorylation of RBM38. These findings provide evidence that the crosstalk between PPM1D and RBM38, both of which are targets and modulators of p53, plays a critical role in p53 expression and activity. PMID:25823026

  5. Construction and expression of a bispecific single-chain antibody that penetrates mutant p53 colon cancer cells and binds p53.

    PubMed

    Weisbart, Richard H; Wakelin, Rika; Chan, Grace; Miller, Carl W; Koeffler, Phillip H

    2004-10-01

    A bispecific, single-chain antibody Fv fragment (Bs-scFv) was constructed from a single-chain Fv fragment of mAb 3E10 that penetrates living cells and localizes in the nucleus, and a single-chain Fv fragment of a non-penetrating antibody, mAb PAb421 that binds the C-terminal of p53. PAb421 binding restores wild-type functions of some p53 mutants, including those of SW480 human colon cancer cells. The Bs-scFv penetrated SW480 cells and was cytotoxic, suggesting an ability to restore activity to mutant p53. COS-7 cells (monkey kidney cells with wild-type p53) served as a control since they are unresponsive to PAb421 due to the presence of SV40 large T antigen that inhibits binding of PAb421 to p53. Bs-scFv penetrated COS-7 cells but was not cytotoxic, thereby eliminating non-specific toxicity of Bs-scFv unrelated to binding p53. A single mutation in CDR1 of PAb421 VH eliminated binding of the Bs-scFv to p53 and abrogated cytotoxicity for SW480 cells without altering cellular penetration, further supporting the requirement of PAb421 binding to p53 for cytotoxicity. Our study demonstrates the use of an antibody that penetrates living cells in the design of a bispecific single chain antibody to target and restore the function of an intracellular protein.

  6. Evidence for a latent precursor (p53 signature) that may precede serous endometrial intraepithelial carcinoma.

    PubMed

    Jarboe, Elke A; Pizer, Ellen S; Miron, Alexander; Monte, Nick; Mutter, George L; Crum, Christopher P

    2009-03-01

    Both serous intraepithelial carcinoma and endometrial glandular dysplasia are associated with uterine serous carcinoma. Recently a candidate serous cancer precursor containing p53 mutations (p53 signature) was described in the fallopian tube. We analyzed normal and neoplastic endometrium for a similar entity. In total 10 endometrial polyps involved by intraepithelial and/or invasive carcinoma and 137 benign polyps were studied. All were stained for p53 and MIB-1. A subset of p53 signatures and carcinomas were analyzed for gamma-H2AX and p53 mutations. p53 signatures were identified in 7 of 10 cases intraepithelial carcinoma and were multicentric in 2. In one case, the signature was in continuity with intraepithelial carcinoma. Of 137 benign polyps (4%), 6 contained p53 signatures. The MIB-1 fraction in most signatures was less than 5%, and ranged from 50 to 90% in carcinomas. DNA damage (gamma-H2AX) was demonstrated in both p53 signatures and adjacent carcinomas but not in benign polyps. Shared identical p53 mutations were found in paired signatures and carcinomas in two of three cases analyzed, including one case with multiple signatures. In one, a coexistent invasive serous cancer was not found to contain a p53 mutation. In a third, a p53 signature and an invasive cancer harbored two different p53 mutations. This is the first description of p53 signatures adjacent to carcinoma, suggesting a role for this entity in the genesis of serous malignancy. The significance of p53 signatures in benign conditions (polyps) remains to be determined. The role of the p53 signature in early serous neoplasia is discussed.

  7. Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90.

    PubMed Central

    Blagosklonny, M V; Toretsky, J; Bohen, S; Neckers, L

    1996-01-01

    The p53 mutant, 143ala, was translated in vitro in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE). In RRL, p53-143ala protein of both mutant and wild-type conformation, as detected immunologically with conformation-specific antibodies, was translated. The chaperone protein HSP90, present in RRL, was found to coprecipitate only with the mutated conformation of p53. Geldanamycin, shown previously to bind to HSP90 and destabilize its association with other proteins, decreased the amount of immunologically detectable mutated p53 and increased the amount of detectable wild-type protein, without affecting the total translation of p53. When translated in WGE, known to contain functionally deficient HSP90, p53-143ala produced p53 protein, which was not recognized by a mutated conformation-specific antibody. In contrast, the synthesis of conformationally detectable wild-type p53 in this system was not compromised. Reconstitution of HSP90 function in WGE permitted synthesis of conformationally detectable mutated p53, and this was abrogated by geldanamycin. Finally, when p53-143ala was stably tansfected into yeast engineered to be defective for HSP90 function, conformational recognition of mutated p53 was impaired. When stable transfectants of p53-143ala were prepared in yeast expressing wild-type HSP90, conformational recognition of mutated p53 was antagonized by macbecin I, a geldanamycin analog also known to bind HSP90. Taken together, these data demonstrate a role for HSP90 in the achievement and/or stabilization of the mutated conformation of p53-143ala. Furthermore, we show that the mutated conformation of p53 can be pharmacologically antagonized by drugs targeting HSP90. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8710879

  8. Nuclear interactor of ARF and Mdm2 regulates multiple pathways to activate p53

    PubMed Central

    Reed, Sara M; Hagen, Jussara; Tompkins, Van S; Thies, Katie; Quelle, Frederick W; Quelle, Dawn E

    2014-01-01

    The p53 tumor suppressor is controlled by an interactive network of factors that stimulate or inhibit its transcriptional activity. Within that network, Mdm2 functions as the major antagonist of p53 by promoting its ubiquitylation and degradation. Conversely, Tip60 activates p53 through direct association on target promoters as well as acetylation of p53 at lysine 120 (K120). This study examines the functional relationship between Mdm2 and Tip60 with a novel p53 regulator, NIAM (nuclear interactor of ARF and Mdm2). Previous work showed NIAM can suppress proliferation and activate p53 independently of ARF, indicating that other factors mediate those activities. Here, we demonstrate that NIAM is a chromatin-associated protein that binds Tip60. NIAM can promote p53 K120 acetylation, although that modification is not required for NIAM to inhibit proliferation or induce p53 transactivation of the p21 promoter. Notably, Tip60 silencing showed it contributes to but is not sufficient for NIAM-mediated p53 activation, suggesting other mechanisms are involved. Indeed, growth-inhibitory forms of NIAM also bind to Mdm2, and increased NIAM expression levels disrupt p53–Mdm2 association, inhibit p53 polyubiquitylation, and prevent Mdm2-mediated inhibition of p53 transcriptional activity. Importantly, loss of NIAM significantly impairs p53 activation. Together, these results show that NIAM activates p53 through multiple mechanisms involving Tip60 association and Mdm2 inhibition. Thus, NIAM regulates 2 critical pathways that control p53 function and are altered in human cancers, implying an important role for NIAM in tumorigenesis. PMID:24621507

  9. A cooperative model for integration in a cardiology outpatient clinic.

    PubMed Central

    Cornet, R.; van Mulligen, E. M.; Timmers, T.

    1994-01-01

    With the increasing amount of digitally stored patient information, such as images and findings, the possibility and need arise for a system which is able to both store and display this information in a structured, user-friendly way. In the common situation where different information systems are used within one department, this means that the various information systems have to be integrated. However, integration requires more complex management of data, processes and windows. This management can be handled by a Workspace Manager, which controls inter-application tasks, and interaction with the user. Furthermore, this Workspace Manager supports the user with the definition of complex tasks such as collecting problem-related patient information from the various remote systems. In an outpatient clinic for cardiology this architecture is used to achieve cooperative integration of an open Computer Patient Record with a range of information-specific services. Images Figure 4 PMID:7949997

  10. miR-380-5p represses p53 to control cellular survival and is associated with poor outcome in MYCN amplified neuroblastoma

    PubMed Central

    Swarbrick, Alexander; Woods, Susan L.; Shaw, Alexander; Balakrishnan, Asha; Phua, Yuwei; Nguyen, Akira; Chanthery, Yvan; Lim, Lionel; Ashton, Lesley J.; Judson, Robert L.; Huskey, Noelle; Blelloch, Robert; Haber, Michelle; Norris, Murray D.; Lengyel, Peter; Hackett, Christopher S.; Preiss, Thomas; Chetcuti, Albert; Sullivan, Christopher S.; Marcusson, Eric G.; Weiss, William; L'Etoile, Noelle; Goga, Andrei

    2010-01-01

    Inactivation of the p53 tumor-suppressor pathway occurs in many human cancers, however some cancers such as neuroblastoma and normal stem cells maintain wild-type p53. Here we describe a microRNA, miR-380-5p, that represses p53 expression via a conserved sequence in the p53 3′UTR. miR-380-5p is highly expressed in embryonic stem cells and neuroblastomas and high expression correlates with poor outcome in neuroblastomas with MYCN amplification. miR-380 overexpression cooperates with activated RAS to transform primary cells, form tumors in mice, and block oncogene induced senescence. In contrast, inhibition of endogenous miR-380-5p in embryonic stem or neuroblastoma cells results in induction of miR-380-5p targets including p53 and extensive apoptotic cell death. In vivo delivery of a miR-380-5p antagonist decreases tumor size in an orthotopic mouse model of neuroblastoma. We demonstrate a new mechanism of p53 regulation in cancer and stem cells and uncover a potential therapeutic target for neuroblastoma. PMID:20871609

  11. Characteristics and survival of patients with advanced cancer and p53 mutations.

    PubMed

    Said, Rabih; Ye, Yang; Hong, David S; Janku, Filip; Fu, Siqing; Naing, Aung; Wheler, Jennifer J; Kurzrock, Razelle; Thomas, Christoforos; Palmer, Gary A; Hess, Kenneth R; Aldape, Kenneth; Tsimberidou, Apostolia M

    2014-06-15

    P53 mutations are associated with invasive tumors in mouse models. We assessed the p53mutations and survival in patients with advanced cancer treated in the Phase I Program. Of 691 tested patients, 273 (39.5%) had p53 mutations. Patients with p53 mutations were older (p<.0001) and had higher numbers of liver metastases (p=.005). P53 mutations were associated with higher numbers of other aberrations; PTEN (p=.0005) and HER2 (p=.003)aberrations were more common in the p53 mutation group. No survival difference was observed between patients with p53 mutations and those with wild-type p53. In patients with wild-type p53 and other aberrations, patients treated with matched-therapy against the additional aberrations had longer survival compared to those treated with non-matched-therapy or those who received no therapy (median survival, 26.0 vs. 11.8 vs. 9.8 months, respectively; p= .0007). Results were confirmed in a multivariate analysis (p= .0002). In the p53 mutation group with additional aberrations, those who received matched-therapy against the additional aberrations had survival similar to those treated with non-matched-therapy or those who received no therapy (p=.15). In conclusion, our results demonstrated resistance to matched-targeted therapy to the other aberrations in patients with p53 mutations and emphasize the need to overcome this resistance.

  12. Cytoplasmic sequestration of p53 promotes survival in leukocytes transformed by Theileria.

    PubMed

    Haller, D; Mackiewicz, M; Gerber, S; Beyer, D; Kullmann, B; Schneider, I; Ahmed, J S; Seitzer, U

    2010-05-27

    The function of the p53 protein as the central effector molecule of the p53 apoptotic pathway was investigated in a reversible model of epigenetic transformation. The infection of bovine leukocytes by the intracellular protozoan parasite Theileria annulata results in parasite-dependent transformation and proliferation of the host cells. We found p53 to be largely localized in the host cell cytoplasm and associated with the parasite membrane of isolated schizonts. Curing infected cells of the parasite with the theilericidal drug buparvaquone resulted in a time-dependent translocation of p53 into the host cell nucleus and the upregulation of the proapoptotic Bax and Apaf-1 and the downregulation of the anti-apoptotic Bcl-2 proteins. Although buparvaquone treatment led to apoptosis of the host cell, inhibition of either p53 or Bax significantly reduced buparvaquone-induced apoptosis of the transformed cells. Thus, the p53 apoptotic pathway of host cells is not induced by infection and transformation with Theileria by a mechanism involving cytoplasmic sequestration of p53. The close association of host cell p53 with the parasite membrane implies that the parasite either interacts directly with p53 or mediates cytoplasmic sequestration of p53 by interacting with other host cell proteins regulating p53 localization.

  13. BRCA1 regulates PIG3-mediated apoptosis in a p53-dependent manner.

    PubMed

    Zhang, Wenwen; Luo, Jiayan; Chen, Fengxia; Yang, Fang; Song, Wei; Zhu, Aiyu; Guan, Xiaoxiang

    2015-04-10

    BRCA1 plays a key role in the regulation of p53-dependent target gene transcription activation. Meanwhile, the p53 inducible gene 3 (PIG3) is a downstream target of p53 and is involved in p53-initiated apoptosis. However, little is known about whether BRCA1 can regulate PIG3-mediated apoptosis. Using a tissue microarray containing 149 breast cancer patient samples, we found that BRCA1 and PIG3 expression status were significantly positively correlated (r = 0.678, P < 0.001) and identified a significant positive correlation between high expression of BRCA1 and/or PIG3 and overall survival (OS). Moreover, we reveal that overexpression of BRCA1 significantly increased expression of PIG3 in cells with intact p53, whereas no increase in PIG3 was observed in p53-null MDA-MB-157 cells and p53-depleted HCT116p53-/- cells. Meanwhile, ectopic expression of BRCA1 could not lead to an increase expression level of prohibitin (PHB), which we have previously identified to induce PIG3-mediated apoptosis. Finally, ChIP analysis revealed that PHB can bind to the PIG3 promoter and activate PIG3 transcription independent of p53, although p53 presence did enhance this process. Taken together, our findings suggest that BRCA1 regulates PIG3-mediated apoptosis in a p53-dependent manner, and that PIG3 expression is associated with a better OS in breast cancer patients.

  14. Stimulation of autophagy by the p53 target gene Sestrin2.

    PubMed

    Maiuri, Maria Chiara; Malik, Shoaib Ahmad; Morselli, Eugenia; Kepp, Oliver; Criollo, Alfredo; Mouchel, Pierre-Luc; Carnuccio, Rosa; Kroemer, Guido

    2009-05-15

    The oncosuppressor protein p53 regulates autophagy in a dual fashion. The pool of cytoplasmic p53 protein represses autophagy in a transcription-independent fashion, while the pool of nuclear p53 stimulates autophagy through the transactivation of specific genes. Here we report the discovery that Sestrin2, a novel p53 target gene, is involved in the induction of autophagy. Depletion of Sestrin2 by RNA interference reduced the level of autophagy in a panel of p53-sufficient human cancer cell lines responding to distinct autophagy inducers. In quantitative terms, Sestrin2 depletion was as efficient in preventing autophagy induction as was the depletion of Dram, another p53 target gene. Knockout of either Sestrin2 or Dram reduced autophagy elicited by nutrient depletion, rapamycin, lithium or thapsigargin. Moreover, autophagy induction by nutrient depletion or pharmacological stimuli led to an increase in Sestrin2 expression levels in p53-proficient cells. In strict contrast, the depletion of Sestrin2 or Dram failed to affect autophagy in p53-deficient cells and did not modulate the inhibition of baseline autophagy by a cytoplasmic p53 mutant that was reintroduced into p53-deficient cells. We conclude that Sestrin2 acts as a positive regulator of autophagy in p53-proficient cells.

  15. 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

    PubMed Central

    Bauer, Matthias R.; Joerger, Andreas C.; Fersht, Alan R.

    2016-01-01

    The tumor suppressor p53 has the most frequently mutated gene in human cancers. Many of p53’s oncogenic mutants are just destabilized and rapidly aggregate, and are targets for stabilization by drugs. We found certain 2-sulfonylpyrimidines, including one named PK11007, to be mild thiol alkylators with anticancer activity in several cell lines, especially those with mutationally compromised p53. PK11007 acted by two routes: p53 dependent and p53 independent. PK11007 stabilized p53 in vitro via selective alkylation of two surface-exposed cysteines without compromising its DNA binding activity. Unstable p53 was reactivated by PK11007 in some cancer cell lines, leading to up-regulation of p53 target genes such as p21 and PUMA. More generally, there was cell death that was independent of p53 but dependent on glutathione depletion and associated with highly elevated levels of reactive oxygen species and induction of endoplasmic reticulum (ER) stress, as also found for the anticancer agent PRIMA-1MET(APR-246). PK11007 may be a lead for anticancer drugs that target cells with nonfunctional p53 or impaired reactive oxygen species (ROS) detoxification in a wide variety of mutant p53 cells. PMID:27551077

  16. Mutant p53 forms a complex with Sp1 on HIV-LTR DNA.

    PubMed

    Chicas, A; Molina, P; Bargonetti, J

    2000-12-20

    Many mutants of p53 activate HIV-LTR driven transcription and promote HIV replication. The region of the HIV-LTR containing Sp1-binding sites is important for this effect. In this study we test the hypothesis that mutant p53 interacts with DNA-bound Sp1 and in this way can increase transcription from Sp1-dependent promoters. We have used the breast cancer cell line MDA-MB-468 that expresses endogenous mutant p53(His273) as our source of p53 protein. First, we demonstrated that this mutant p53 participates in activating transcription from the HIV-LTR by showing that HIV-LTR-directed transcription in MDA-MB-468 cells is inhibited in a dominant-negative manner by p53(Val135). Using HIV-LTR DNA affinity chromatography, we detected coelution of p53(His273) and Sp1. We also demonstrated that this mutant p53 binds sequence specifically to the super consensus sequence (SCS) and that Sp1 coeluted with p53(His273) from a column containing this site. These data indicate that p53(His273) can associate with DNA-bound Sp1 suggesting that activated HIV-LTR transcription associated with mutant p53 occurs through a DNA driven multi-protein complex.

  17. Clinical and pathological correlations of marrow PUMA and P53 expressions in myelodysplastic syndromes.

    PubMed

    Bektas, Ozlen; Uner, Aysegul; Buyukasik, Yahya; Uz, Burak; Bozkurt, Sureyya; Eliacik, Eylem; Işik, Ayse; Haznedaroglu, Ibrahim Celalettin; Goker, Hakan; Demiroglu, Haluk; Aksu, Salih; Ozcebe, Osman Ilhami; Sayinalp, Nilgun

    2015-05-01

    p53 is a key regulator of apoptosis. p53 upregulated modulator of apoptosis (PUMA) is a critical mediator of p53-dependent and independent apoptosis. The objective of this study was to evaluate the relationship of p53 and PUMA to the prognosis of MDS. Bone marrow biopsies of MDS patients at the time of diagnosis (n = 76) and at the time of transformation (n = 19) were included in the study group. The expression of p53 and PUMA was evaluated using immunohistochemistry. When compared to the control group, both p53 (p < 0.001) and PUMA (p = 0.012) expression levels were significantly higher in MDS group. In MDS group, there was a moderate positive correlation between p53 and PUMA expressions. PUMA expression was not correlated with event free and overall survival. However, overall survival was significantly lower in cases with p53 expression in more than 50% of the cells. There was an increase in PUMA expression in cases that showed transformation as compared to the initial diagnostic bone marrows but was not statistically significant. The correlation that existed between p53 and PUMA was lost in transformed cases. Our results showed that PUMA and p53 expressions are increased in MDS marrows compared to normal marrows. PUMA expression increases further during transformation while the expression of p53 is not significantly altered which suggests that PUMA alterations might be a late event during the evolution of MDS.

  18. ID4 regulates transcriptional activity of wild type and mutant p53 via K373 acetylation.

    PubMed

    Morton, Derrick J; Patel, Divya; Joshi, Jugal; Hunt, Aisha; Knowell, Ashley E; Chaudhary, Jaideep

    2017-01-10

    Given that mutated p53 (50% of all human cancers) is over-expressed in many cancers, restoration of mutant p53 to its wild type biological function has been sought after as cancer therapy. The conformational flexibility has allowed to restore the normal biological function of mutant p53 by short peptides and small molecule compounds. Recently, studies have focused on physiological mechanisms such as acetylation of lysine residues to rescue the wild type activity of mutant p53. Using p53 null prostate cancer cell line we show that ID4 dependent acetylation promotes mutant p53 DNA-binding capabilities to its wild type consensus sequence, thus regulating p53-dependent target genes leading to subsequent cell cycle arrest and apoptosis. Specifically, by using wild type, mutant (P223L, V274F, R175H, R273H), acetylation mimics (K320Q and K373Q) and non-acetylation mimics (K320R and K373R) of p53, we identify that ID4 promotes acetylation of K373 and to a lesser extent K320, in turn restoring p53-dependent biological activities. Together, our data provides a molecular understanding of ID4 dependent acetylation that suggests a strategy of enhancing p53 acetylation at sites K373 and K320 that may serve as a viable mechanism of physiological restoration of mutant p53 to its wild type biological function.

  19. 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells.

    PubMed

    Bauer, Matthias R; Joerger, Andreas C; Fersht, Alan R

    2016-09-06

    The tumor suppressor p53 has the most frequently mutated gene in human cancers. Many of p53's oncogenic mutants are just destabilized and rapidly aggregate, and are targets for stabilization by drugs. We found certain 2-sulfonylpyrimidines, including one named PK11007, to be mild thiol alkylators with anticancer activity in several cell lines, especially those with mutationally compromised p53. PK11007 acted by two routes: p53 dependent and p53 independent. PK11007 stabilized p53 in vitro via selective alkylation of two surface-exposed cysteines without compromising its DNA binding activity. Unstable p53 was reactivated by PK11007 in some cancer cell lines, leading to up-regulation of p53 target genes such as p21 and PUMA. More generally, there was cell death that was independent of p53 but dependent on glutathione depletion and associated with highly elevated levels of reactive oxygen species and induction of endoplasmic reticulum (ER) stress, as also found for the anticancer agent PRIMA-1(MET)(APR-246). PK11007 may be a lead for anticancer drugs that target cells with nonfunctional p53 or impaired reactive oxygen species (ROS) detoxification in a wide variety of mutant p53 cells.

  20. PML IV/ARF interaction enhances p53 SUMO-1 conjugation, activation, and senescence.

    PubMed

    Ivanschitz, Lisa; Takahashi, Yuki; Jollivet, Florence; Ayrault, Olivier; Le Bras, Morgane; de Thé, Hugues

    2015-11-17

    Promyelocytic leukemia protein (PML) nuclear bodies (NBs) recruit multiple partners, including p53 and many of its regulators. NBs are believed to facilitate several posttranslational modifications and are key regulators of senescence. PML, the organizer of NBs, is expressed as a number of splice variants that all efficiently recruit p53 partners. However, overexpression of only one of them, PML IV, triggers p53-driven senescence. Here, we show that PML IV specifically binds ARF, a key p53 regulator. Similar to ARF, PML IV enhances global SUMO-1 conjugation, particularly that of p53, resulting in p53 stabilization and activation. ARF interacts with and stabilizes the NB-associated UBC9 SUMO-conjugating enzyme, possibly explaining PML IV-enhanced SUMOylation. These results unexpectedly link two key tumor suppressors, highlighting their convergence for global control of SUMO conjugation, p53 activation, and senescence induction.

  1. 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.

  2. TAp73 promotes cell survival upon genotoxic stress by inhibiting p53 activity

    PubMed Central

    Chen, Dongshi; Ming, Lihua; Zou, Fangdong; Peng, Ye; Houten, Bennett Van; Yu, Jian; Zhang, Lin

    2014-01-01

    p53 plays a key role in regulating DNA damage response by suppressing cell cycle progression or inducing apoptosis depending on extent of DNA damage. However, it is not clear why mild genotoxic stress favors growth arrest, whereas excessive lesions signal cells to die. Here we showed that TAp73, a p53 homologue thought to have a similar function as p53, restrains the transcriptional activity of p53 and prevents excessive activation of its downstream targets upon low levels of DNA damage, which results in cell cycle arrest. Extensive DNA damage triggers TAp73 depletion through ubiquitin/proteasome-mediated degradation of E2F1, leading to enhanced transcriptional activation by p53 and subsequent induction of apoptosis. These findings provide novel insights into the regulation of p53 function and suggest that TAp73 keeps p53 activity in check in regulating cell fate decisions upon genotoxic stress. PMID:25237903

  3. Targeting of p53 and its homolog p73 by protoporphyrin IX.

    PubMed

    Sznarkowska, Alicja; Maleńczyk, Katarzyna; Kadziński, Leszek; Bielawski, Krzysztof P; Banecki, Bogdan; Zawacka-Pankau, Joanna

    2011-01-03

    The p53 tumor suppressor is recognized as a promising target for anti-cancer therapies. We previously reported that protoporphyrin IX (PpIX) disrupts the p53/murine double minute 2 (MDM2) complex and leads to p53 accumulation and activation of apoptosis in HCT 116 cells. Here we show the direct binding of PpIX to the N-terminal domain of p53. Furthermore, we addressed the induction of apoptosis in HCT 116 p53-null cells by PpIX and revealed interactions between PpIX and p73. We propose that PpIX disrupts the p53/MDM2 or MDMX and p73/MDM2 complexes and thereby activates the p53- or p73-dependent cancer cell death.

  4. p53, a potential predictor of Helicobacter pylori infection-associated gastric carcinogenesis?

    PubMed Central

    Lu, Nong-Hua

    2016-01-01

    Helicobacter pylori (H. pylori) is an ancient and persistent inhabitant of the human stomach that is closely linked to the development of gastric cancer (GC). . Emerging evidence suggests that H. pylori strain interactions with gastric epithelial cells subvert the best- characterized p53 tumour suppressor pathway. A high prevalence of p53 mutations is related to H. pylori infection. H. pylori also accelerates p53 protein degradation by disturbing the MDM2-P53 feedback loop. Additionally, H. pylori triggers the alteration of other p53 isoforms. Dysregulation of p53 by H. pylori infection contributes to gastric carcinogenesis by mediating cell proliferation and apoptosis. This review focuses on the regulation of p53 in H. pylori infection-associated GC. PMID:27556187

  5. BAC transgenic mice provide evidence that p53 expression is highly regulated in vivo.

    PubMed

    Chen, L; Zhang, G X; Zhou, Y; Zhang, C X; Xie, Y Y; Xiang, C; He, X Y; Zhang, Q; Liu, G

    2015-09-17

    p53 is an important tumor suppressor and stress response mediator. Proper control of p53 level and activity is tightly associated with its function. Posttranslational modifications and the interactions with Mdm2 and Mdm4 are major mechanisms controlling p53 activity and stability. As p53 protein is short-lived and hardly detectable in unstressed situations, less is known on its basal level expression and the corresponding controlling mechanisms in vivo. In addition, it also remains obscure how p53 expression might contribute to its functional regulation. In this study, we established bacterial artificial chromosome transgenic E.coli β-galactosidase Z gene reporter mice to monitor p53 expression in mouse tissues and identify important regulatory elements critical for the expression in vivo. We revealed preferentially high level of p53 reporter expressions in the proliferating, but not the differentiated compartments of the majority of tissues during development and tissue homeostasis. In addition, tumors as well as regenerating tissues in the p53 reporter mice also expressed high level of β-gal. Furthermore, both the enhancer box sequence (CANNTG) in the p53 promoter and the 3' terminal untranslated region element were critical in mediating the high-level expression of the reporter. We also provided evidence that cellular myelocytomatosis oncogene was a critical player regulating p53 mRNA expression in proliferating cells and tissues. Finally, we found robust p53 activation preferentially in the proliferating compartment of mouse tissues upon DNA damage and the proliferating cells exhibited an enhanced p53 response as compared with cells in a quiescent state. Together, these results suggested a highly regulated expression pattern of p53 in the proliferating compartment controlled by both transcriptional and posttranscriptional mechanisms, and such regulated p53 expression may impose functional significance upon stress by setting up a precautionary mode in defense

  6. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways.

    PubMed Central

    Nelson, W G; Kastan, M B

    1994-01-01

    The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA

  7. Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53

    PubMed Central

    Tao, Weikang; Levine, Arnold J.

    1999-01-01

    The Hdm2 oncoprotein inhibits p53 functions by two means: (i) it blocks p53’s transactivation activity and (ii) it targets p53 for degradation in a proteasome-dependent manner. Recent data indicate that Hdm2 shuttles between the nucleus and the cytoplasm and that the regulation of p53 levels by Hdm2 requires its nuclear export activity. Two different models are consistent with these observations. In the first, Hdm2 binds to p53 in the nucleus and shuttles p53 from the nucleus to the cytoplasm, and then it targets p53 to the cytoplasmic proteasome. Alternatively, Hdm2 and p53 could be exported separately from the nucleus and then associate in the cytoplasm, where Hdm2 promotes the degradation of p53. To distinguish between these two models, several Hdm2 mutants were employed. Hdm2NLS lacks the ability to enter the nucleus, whereas Hdm2NES is deficient in nuclear export. Hdm2NLS, Hdm2NES, or the combination of both mutants were unable to promote p53 degradation in the cotransfected 2KO cells (which were null for both the p53 and mdm2 genes), although wild-type Hdm2 efficiently reduced p53 levels under the same conditions. This observation is not a result of the differences in expression levels or stability between Hdm2 and these mutants. Moreover, coexpression of these mutants had no effect on wild-type Hdm-2-induced p53 destabilization. Thus, Hdm2 must shuttle p53 from the nucleus to the cytoplasm to target it for degradation in the cytoplasm. PMID:10077639

  8. Identifying p53 Transactivation Domain 1-Specific Inhibitors to Alleviate the Side Effects of Prostate Cancer Therapy

    DTIC Science & Technology

    2015-12-01

    cancer. 15. SUBJECT TERMS p53 inhibitor, p53 transactivation mutants 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES...gene regulatory elements. Keywords p53 inhibitor, p53 transactivation mutants , p53 transcriptional activation domain inhibitor Overall Project

  9. Neem oil limonoids induces p53-independent apoptosis and autophagy.

    PubMed

    Srivastava, Pragya; Yadav, Neelu; Lella, Ravi; Schneider, Andrea; Jones, Anthony; Marlowe, Timothy; Lovett, Gabrielle; O'Loughlin, Kieran; Minderman, Hans; Gogada, Raghu; Chandra, Dhyan

    2012-11-01

    Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.

  10. Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response

    PubMed Central

    Nantajit, Danupon; Fan, Ming; Duru, Nadire; Wen, Yunfei; Reed, John C.; Li, Jian Jian

    2010-01-01

    The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53−/− cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53−/− cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53. PMID:20808790

  11. DNA repair and aging: the impact of the p53 family

    PubMed Central

    Nicolai, Sara; Rossi, Antonello; Di Daniele, Nicola; Melino, Gerry; Annicchiarico-Petruzzelli, Margherita; Raschellà, Giuseppe

    2015-01-01

    Cells are constantly exposed to endogenous and exogenous factors that threaten the integrity of their DNA. The maintenance of genome stability is of paramount importance in the prevention of both cancer and aging processes. To deal with DNA damage, cells put into operation a sophisticated and coordinated mechanism, collectively known as DNA damage response (DDR). The DDR orchestrates different cellular processes, such as DNA repair, senescence and apoptosis. Among the key factors of the DDR, the related proteins p53, p63 and p73, all belonging to the same family of transcription factors, play multiple relevant roles. Indeed, the members of this family are directly involved in the induction of cell cycle arrest that is necessary to allow the cells to repair. Alternatively, they can promote cell death in case of prolonged or irreparable DNA damage. They also take part in a more direct task by modulating the expression of core factors involved in the process of DNA repair or by directly interacting with them. In this review we will analyze the fundamental roles of the p53 family in the aging process through their multifaceted function in DDR. PMID:26668111

  12. DNA repair and aging: the impact of the p53 family.

    PubMed

    Nicolai, Sara; Rossi, Antonello; Di Daniele, Nicola; Melino, Gerry; Annicchiarico-Petruzzelli, Margherita; Raschellà, Giuseppe

    2015-12-01

    Cells are constantly exposed to endogenous and exogenous factors that threaten the integrity of their DNA. The maintenance of genome stability is of paramount importance in the prevention of both cancer and aging processes. To deal with DNA damage, cells put into operation a sophisticated and coordinated mechanism, collectively known as DNA damage response (DDR). The DDR orchestrates different cellular processes, such as DNA repair, senescence and apoptosis. Among the key factors of the DDR, the related proteins p53, p63 and p73, all belonging to the same family of transcription factors, play multiple relevant roles. Indeed, the members of this family are directly involved in the induction of cell cycle arrest that is necessary to allow the cells to repair. Alternatively, they can promote cell death in case of prolonged or irreparable DNA damage. They also take part in a more direct task by modulating the expression of core factors involved in the process of DNA repair or by directly interacting with them. In this review we will analyze the fundamental roles of the p53 family in the aging process through their multifaceted function in DDR.

  13. Hyperactivation of ATM upon DNA-PKcs inhibition modulates p53 dynamics and cell fate in response to DNA damage

    PubMed Central

    Finzel, Ana; Grybowski, Andrea; Strasen, Jette; Cristiano, Elena; Loewer, Alexander

    2016-01-01

    A functional DNA damage response is essential for maintaining genome integrity in the presence of DNA double-strand breaks. It is mainly coordinated by the kinases ATM, ATR, and DNA-PKcs, which control the repair of broken DNA strands and relay the damage signal to the tumor suppressor p53 to induce cell cycle arrest, apoptosis, or senescence. Although many functions of the individual kinases have been identified, it remains unclear how they act in concert to ensure faithful processing of the damage signal. Using specific inhibitors and quantitative analysis at the single-cell level, we systematically characterize the contribution of each kinase for regulating p53 activity. Our results reveal a new regulatory interplay in which loss of DNA-PKcs function leads to hyperactivation of ATM and amplification of the p53 response, sensitizing cells for damage-induced senescence. This interplay determines the outcome of treatment regimens combining irradiation with DNA-PKcs inhibitors in a p53-dependent manner. PMID:27280387

  14. Cisplatin modulates B-cell translocation gene 2 to attenuate cell proliferation of prostate carcinoma cells in both p53-dependent and p53-independent pathways.

    PubMed

    Chiang, Kun-Chun; Tsui, Ke-Hung; Chung, Li-Chuan; Yeh, Chun-Nan; Feng, Tsui-Hsia; Chen, Wen-Tsung; Chang, Phei-Lang; Chiang, Hou-Yu; Juang, Horng-Heng

    2014-07-01

    Cisplatin is a widely used anti-cancer drug. The B-cell translocation gene 2 (BTG2) is involved in the cell cycle transition regulation. We evaluated the cisplatin effects on prostate cancer cell proliferation and the expressions of BTG2, p53, androgen receptor (AR) and prostate specific antigen (PSA) in prostate carcinoma, p53 wild-type LNCaP or p53-null PC-3, cells. Cisplatin treatments attenuated cell prostate cancer cell growth through inducing Go/G1 cell cycle arrest in lower concentration and apoptosis at higher dosage. Cisplatin treatments enhanced p53 and BTG2 expression, repressed AR and PSA expression, and blocked the activation of androgen on the PSA secretion in LNCaP cells. BTG2 knockdown in LNCaP cells attenuated cisplatin-mediated growth inhibition. Cisplatin enhanced BTG2 gene expression dependent on the DNA fragment located within -173 to -82 upstream of BTG2 translation initiation site in prostate cancer cells. Mutation of the p53 response element from GGGCAGAGCCC to GGGCACC or mutation of the NFκB response element from GGAAAGTCC to GGAAAGGAA by site-directed mutagenesis abolished the stimulation of cisplatin on the BTG2 promoter activity in LNCaP or PC-3 cells, respectively. Our results indicated that cisplatin attenuates prostate cancer cell proliferation partly mediated by upregulation of BTG2 through the p53-dependent pathway or p53-independent NFκB pathway.

  15. Lack of p53 Affects the Expression of Several Brain Mitochondrial Proteins: Insights from Proteomics into Important Pathways Regulated by p53

    PubMed Central

    Fiorini, Ada; Sultana, Rukhsana; Barone, Eugenio; Cenini, Giovanna; Perluigi, Marzia; Mancuso, Cesare; Cai, Jian; Klein, Jon B.; St. Clair, Daret; Butterfield, D. Allan

    2012-01-01

    The tumor suppressor protein p53 has been described “as the guardian of the genome” for its crucial role in regulating the transcription of numerous genes responsible for cells cycle arrest, senescence, or apoptosis in response to various stress signals. Although p53 promotes longevity by decreasing the risk of cancer through activation of apoptosis or cellular senescence, several findings suggest that an increase of its activity may have deleterious effects leading to selected aspects of the aging phenotype and neurodegenerative diseases. There is the link between p53 and oxidative stress, the latter a crucial factor that contributes to neurodegenerative processes like Alzheimer disease (AD). In the present study, using a proteomics approach, we analyzed the impact of lack of p53 on the expression of several brain mitochondrial proteins involved in different pathways, and how lack of p53 may present a target to restore neuronal impairments. Our investigation on isolated brain mitochondria from p53(−/−) mice also provides a better understanding of the p53-mitochondria relationship and its involvement in the development of many diseases. PMID:23209608

  16. Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

    PubMed Central

    Forrester, K; Ambs, S; Lupold, S E; Kapust, R B; Spillare, E A; Weinberg, W C; Felley-Bosco, E; Wang, X W; Geller, D A; Tzeng, E; Billiar, T R; Harris, C C

    1996-01-01

    The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage. Images Fig. 1 Fig. 2 Fig. 3 PMID:8637893

  17. Suppressing activity of tributyrin on hepatocarcinogenesis is associated with inhibiting the p53-CRM1 interaction and changing the cellular compartmentalization of p53 protein

    PubMed Central

    Ortega, Juliana F.; de Conti, Aline; Tryndyak, Volodymyr; Furtado, Kelly S.; Heidor, Renato; Horst, Maria Aderuza; Fernandes, Laura Helena Gasparini; Tavares, Paulo Eduardo Latorre Martins; Pogribna, Marta; Shpyleva, Svitlana; Beland, Frederick A.; Pogribny, Igor P.; Moreno, Fernando Salvador

    2016-01-01

    Hepatocellular carcinoma (HCC), an aggressive and the fastest growing life-threatening cancer worldwide, is often diagnosed at intermediate or advanced stages of the disease, which substantially limits therapeutic approaches for its successful treatment. This indicates that the prevention of hepatocarcinogenesis is probably the most promising approach to reduce both the HCC incidence and cancer-related mortality. In previous studies, we demonstrated a potent chemopreventive effect of tributyrin, a butyric acid prodrug, on experimental hepatocarcinogenesis. The cancer-inhibitory effect of tributyrin was linked to the suppression of sustained cell proliferation and induction of apoptotic cell death driven by an activation of the p53 apoptotic signaling pathway. The goal of the present study was to investigate the underlying molecular mechanisms linked to tributyrin-mediated p53 activation. Using in vivo and in vitro models of liver cancer, we demonstrate that an increase in the level of p53 protein in nuclei, a decrease in the level of cytoplasmic p53, and, consequently, an increase in the ratio of nuclear/cytoplasmic p53 in rat preneoplastic livers and in rat and human HCC cell lines caused by tributyrin or sodium butyrate treatments was associated with a marked increase in the level of nuclear chromosome region maintenance 1 (CRM1) protein. Mechanistically, the increase in the level of nuclear p53 protein was associated with a substantially reduced binding interaction between CRM1 and p53. The results demonstrate that the cancer-inhibitory activity of sodium butyrate and its derivatives on liver carcinogenesis may be attributed to retention of p53 and CRM1 proteins in the nucleus, an event that may trigger activation of p53-mediated apoptotic cell death in neoplastic cells. PMID:27013579

  18. Oxidized DJ-1 Inhibits p53 by Sequestering p53 from Promoters in a DNA-Binding Affinity-Dependent Manner

    PubMed Central

    Kato, Izumi; Maita, Hiroshi; Takahashi-Niki, Kazuko; Saito, Yoshiro; Noguchi, Noriko; Iguchi-Ariga, Sanae M. M.

    2013-01-01

    DJ-1 is an oncogene and the causative gene for familial Parkinson's disease. Although the oxidative status of DJ-1 at cysteine 106 (C106) is thought to affect all of the activities of DJ-1 and excess oxidation leads to the onset of various diseases, the precise molecular mechanisms underlying the effects of oxidation of DJ-1 on protein-protein interactions of DJ-1 remain unclear. In this study, we found that DJ-1 bound to the DNA-binding region of p53 in a manner dependent on the oxidation of C106. Of the p53 target genes, the expression level and promoter activity of the DUSP1 gene, but not those of the p21 gene, were increased in H2O2-treated DJ-1−/− cells and were decreased in wild-type DJ-1- but not C106S DJ-1-transfected H1299 cells through sequestration of p53 from the DUSP1 promoter by DJ-1. DUSP1 downregulated by oxidized DJ-1 activated extracellular signal-regulated kinase (ERK) and decreased apoptosis. The DUSP1 and p21 promoters harbor nonconsensus and consensus p53 recognition sequences, respectively, which have low affinity and high affinity for p53. However, DJ-1 inhibited p21 promoter activity exhibited by p53 mutants harboring low DNA-binding affinity but not by wild-type p53. These results indicate that DJ-1 inhibits the expression of p53 target genes and depend on p53 DNA-binding affinity and oxidation of DJ-1 C106. PMID:23149933

  19. Oxidized DJ-1 inhibits p53 by sequestering p53 from promoters in a DNA-binding affinity-dependent manner.

    PubMed

    Kato, Izumi; Maita, Hiroshi; Takahashi-Niki, Kazuko; Saito, Yoshiro; Noguchi, Noriko; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2013-01-01

    DJ-1 is an oncogene and the causative gene for familial Parkinson's disease. Although the oxidative status of DJ-1 at cysteine 106 (C106) is thought to affect all of the activities of DJ-1 and excess oxidation leads to the onset of various diseases, the precise molecular mechanisms underlying the effects of oxidation of DJ-1 on protein-protein interactions of DJ-1 remain unclear. In this study, we found that DJ-1 bound to the DNA-binding region of p53 in a manner dependent on the oxidation of C106. Of the p53 target genes, the expression level and promoter activity of the DUSP1 gene, but not those of the p21 gene, were increased in H(2)O(2)-treated DJ-1(-/-) cells and were decreased in wild-type DJ-1- but not C106S DJ-1-transfected H1299 cells through sequestration of p53 from the DUSP1 promoter by DJ-1. DUSP1 downregulated by oxidized DJ-1 activated extracellular signal-regulated kinase (ERK) and decreased apoptosis. The DUSP1 and p21 promoters harbor nonconsensus and consensus p53 recognition sequences, respectively, which have low affinity and high affinity for p53. However, DJ-1 inhibited p21 promoter activity exhibited by p53 mutants harboring low DNA-binding affinity but not by wild-type p53. These results indicate that DJ-1 inhibits the expression of p53 target genes and depend on p53 DNA-binding affinity and oxidation of DJ-1 C106.

  20. Suppressing activity of tributyrin on hepatocarcinogenesis is associated with inhibiting the p53-CRM1 interaction and changing the cellular compartmentalization of p53 protein.

    PubMed

    Ortega, Juliana F; de Conti, Aline; Tryndyak, Volodymyr; Furtado, Kelly S; Heidor, Renato; Horst, Maria Aderuza; Fernandes, Laura Helena Gasparini; Tavares, Paulo Eduardo Latorre Martins; Pogribna, Marta; Shpyleva, Svitlana; Beland, Frederick A; Pogribny, Igor P; Moreno, Fernando Salvador

    2016-04-26

    Hepatocellular carcinoma (HCC), an aggressive and the fastest growing life-threatening cancer worldwide, is often diagnosed at intermediate or advanced stages of the disease, which substantially limits therapeutic approaches for its successful treatment. This indicates that the prevention of hepatocarcinogenesis is probably the most promising approach to reduce both the HCC incidence and cancer-related mortality. In previous studies, we demonstrated a potent chemopreventive effect of tributyrin, a butyric acid prodrug, on experimental hepatocarcinogenesis. The cancer-inhibitory effect of tributyrin was linked to the suppression of sustained cell proliferation and induction of apoptotic cell death driven by an activation of the p53 apoptotic signaling pathway. The goal of the present study was to investigate the underlying molecular mechanisms linked to tributyrin-mediated p53 activation. Using in vivo and in vitro models of liver cancer, we demonstrate that an increase in the level of p53 protein in nuclei, a decrease in the level of cytoplasmic p53, and, consequently, an increase in the ratio of nuclear/cytoplasmic p53 in rat preneoplastic livers and in rat and human HCC cell lines caused by tributyrin or sodium butyrate treatments was associated with a marked increase in the level of nuclear chromosome region maintenance 1 (CRM1) protein. Mechanistically, the increase in the level of nuclear p53 protein was associated with a substantially reduced binding interaction between CRM1 and p53. The results demonstrate that the cancer-inhibitory activity of sodium butyrate and its derivatives on liver carcinogenesis may be attributed to retention of p53 and CRM1 proteins in the nucleus, an event that may trigger activation of p53-mediated apoptotic cell death in neoplastic cells.

  1. Complex formation between p53 and replication protein A inhibits the sequence-specific DNA binding of p53 and is regulated by single-stranded DNA.

    PubMed Central

    Miller, S D; Moses, K; Jayaraman, L; Prives, C

    1997-01-01

    Human replication protein A (RP-A) (also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. Potentially important to both these functions, it is also capable of complex formation with the tumor suppressor protein p53. Here we show that although p53 is unable to prevent RP-A from associating with a range of single-stranded DNAs in solution, RP-A is able to strongly inhibit p53 from functioning as a sequence-specific DNA binding protein when the two proteins are complexed. This inhibition, in turn, can be regulated by the presence of various lengths of single-stranded DNAs, as RP-A, when bound to these single-stranded DNAs, is unable to interact with p53. Interestingly, the lengths of single-stranded DNA capable of relieving complex formation between the two proteins represent forms that might be introduced through repair and replicative events. Increasing p53 concentrations can also overcome the inhibition by steady-state levels of RP-A, potentially mimicking cellular points of balance. Finally, it has been shown previously that p53 can itself be stimulated for site-specific DNA binding when complexed through the C terminus with short single strands of DNA, and here we show that p53 stays bound to these short strands even after binding a physiologically relevant site. These results identify a potential dual role for single-stranded DNA in the regulation of DNA binding by p53 and give insights into the p53 response to DNA damage. PMID:9121469

  2. Combined loss of PUMA and p21 accelerates c-MYC-driven lymphoma development considerably less than loss of one allele of p53.

    PubMed

    Valente, L J; Grabow, S; Vandenberg, C J; Strasser, A; Janic, A

    2016-07-21

    The tumor suppressor p53 is mutated in ~50% of human cancers. P53 is activated by a range of stimuli and regulates several cellular processes, including apoptotic cell death, cell cycle arrest, senescence and DNA repair. P53 induces apoptosis via transcriptional induction of the BH3-only proteins PUMA (p53-upregulated modulator of apoptosis) and NOXA, and cell cycle arrest via p21. Induction of these processes was proposed to be critical for p53-mediated tumor suppression. It is therefore surprising that mice lacking PUMA, NOXA and p21, as well as mice bearing mutations in p53 that impair the transcriptional activation of these genes, are not tumor prone, unlike mice lacking p53 function, which spontaneously develop tumors with 100% incidence. These p53 target genes and the processes they regulate may, however, impact differently on tumor development depending on the oncogenic drivers. For example, loss of PUMA enhances c-MYC-driven lymphoma development in mice, but, interestingly, the acceleration was less impressive compared with that caused by the loss of even a single p53 allele. Different studies have reported that loss of p21 can accelerate, delay or have no impact on tumorigenesis. In an attempt to resolve this controversy, we examined whether loss of p21-mediated cell cycle arrest cooperates with PUMA deficiency in accelerating lymphoma development in Eμ-Myc mice (overexpressing c-MYC in B-lymphoid cells). We found that Eμ-Myc mice lacking both p21 and PUMA (Eμ-Myc;Puma(-/-);p21(-/-)) developed lymphoma at a rate comparable to Eμ-Myc;Puma(-/-) animals, notably with considerably longer latency than Eμ-Myc;p53(+/-)mice. Loss of p21 had no impact on the numbers, cycling or survival of pre-leukemic Eμ-Myc B-lymphoid cells, even when PUMA was lost concomitantly. These results demonstrate that even in the context of deregulated c-MYC expression, p53 must suppress tumor development by activating processes apart from, or in addition to, PUMA

  3. Prevention of mammalian DNA reduplication, following the release from the mitotic spindle checkpoint, requires p53 protein, but not p53-mediated transcriptional activity.

    PubMed

    Notterman, D; Young, S; Wainger, B; Levine, A J

    1998-11-26

    The tumor suppressor p53 has been identified as a component of a mitotic spindle checkpoint. When exposed to a spindle-disrupting drug such as nocodazole, fibroblasts derived from mice having wild-type p53 are blocked with a 4N content of DNA. Conversely, fibroblasts from p53-deficient mice become polyploid. To learn if transcriptional activation of downstream genes by p53 plays a role in this putative checkpoint, three cell lines were exposed to nocodazole. In one line, p53 protein is not expressed, while the other two cell lines over-express p53. In one of these two lines, the N-terminal transactivation domain is wild-type and in the second, this region contains a mutation that eliminates the ability of the protein to act as a transcription factor. Incubation with nocodazole of cells containing wild-type p53 results in accumulation of both 2N and 4N populations of cells. Under the same conditions, cells containing a transactivation-deficient mutant of p53 accumulate a 4N population of cells, but not a 2N population of cells. Cells entirely deficient in p53 protein become hyperdiploid, and display 8N to 16N DNA content. In all three cell lines, nocodazole elicited an initial increase in mitotic cells, but within 24 h the mitotic index returned to baseline. Expression patterns of cyclins B and D indicated that following entry into mitosis, the cells returned to a G1 state but with 4N DNA content. Subsequent re-duplication of DNA beyond 4N is prevented in cells containing either wild-type or transcriptionally inactive p53 protein. In cells entirely lacking p53 protein, DNA is re-duplicated (without an intervening mitosis) and the cells become hyperdiploid. These experiments indicate that p53 does not participate in the transient mitotic arrest that follows spindle disruption, but is essential to prevent subsequent reduplication of DNA and the resulting hyperdiploid state. This function is intact in a mutant that is transcriptionally inactive.

  4. Wip1 inhibitor GSK2830371 inhibits neuroblastoma growth by inducing Chk2/p53-mediated apoptosis

    PubMed Central

    Chen, Zhenghu; Wang, Long; Yao, Dayong; Yang, Tianshu; Cao, Wen-Ming; Dou, Jun; Pang, Jonathan C.; Guan, Shan; Zhang, Huiyuan; Yu, Yang; Zhao, Yanling; Wang, Yongfeng; Xu, Xin; Shi, Yan; Patel, Roma; Zhang, Hong; Vasudevan, Sanjeev A.; Liu, Shangfeng; Yang, Jianhua; Nuchtern, Jed G.

    2016-01-01

    Neuroblastoma (NB) is the most common extracranial tumor in children. Unlike in most adult tumors, tumor suppressor protein 53 (p53) mutations occur with a relatively low frequency in NB and the downstream function of p53 is intact in NB cell lines. Wip1 is a negative regulator of p53 and hindrance of Wip1 activity by novel inhibitor GSK2830371 is a potential strategy to activate p53’s tumor suppressing function in NB. Yet, the in vivo efficacy and the possible mechanisms of GSK2830371 in NB have not yet been elucidated. Here we report that novel Wip1 inhibitor GSK2830371 induced Chk2/p53-mediated apoptosis in NB cells in a p53-dependent manner. In addition, GSK2830371 suppressed the colony-formation potential of p53 wild-type NB cell lines. Furthermore, GSK2830371 enhanced doxorubicin- (Dox) and etoposide- (VP-16) induced cytotoxicity in a subset of NB cell lines, including the chemoresistant LA-N-6 cell line. More importantly, GSK2830371 significantly inhibited tumor growth in an orthotopic xenograft NB mouse model by inducing Chk2/p53-mediated apoptosis in vivo. Taken together, this study suggests that GSK2830371 induces Chk2/p53-mediated apoptosis both in vitro and in vivo in a p53 dependent manner. PMID:27991505

  5. Decrease of mitochondrial p53 during late apoptosis is linked to its dephosphorylation on serine 20

    PubMed Central

    Castrogiovanni, Cédric; Vandaudenard, Marie; Waterschoot, Béranger; De Backer, Olivier; Dumont, Patrick

    2015-01-01

    Following a genotoxic stress, the tumor suppressor p53 translocates to mitochondria to take part in direct induction of apoptosis, via interaction with BCL-2 family members such as BAK and BAX. We determined the kinetics of the mitochondrial translocation of p53 in HCT-116 and PA-1 cells exposed to different genotoxic stresses (doxorubicin, camptothecin, UVB). This analysis revealed an early escalation in the amount of mitochondrial p53, followed by a peak amount and a decrease of mitochondrial p53 at later time points. We show that the serine 20 phosphorylated form of p53 is present at the mitochondria and that the decrease of p53 mitochondrial level during late apoptosis correlates with a decrease of Ser-20 phosphorylation. Moreover, the S20A p53 mutant translocates well to mitochondria after a genotoxic stress but its mitochondrial localization is very low during late apoptosis when compared to wt p53. The S20A mutant also appears to be compromised for interaction with BAK. We propose here that the level of serine 20 phosphorylation is influential on p53 mitochondrial localization during late apoptosis. Additionally, we report the presence of a new ≃45 kDa caspase-cleaved fragment of p53 in the cytosolic and mitochondrial fractions of apoptotic cells. PMID:26252178

  6. An N-terminal region of mot-2 binds to p53 in vitro.

    PubMed

    Kaul, S C; Reddel, R R; Mitsui, Y; Wadhwa, R

    2001-01-01

    The mouse mot-2 protein was earlier shown to bind to the tumor suppressor protein, p53. The mot-2 binding site of p53 was mapped to C-terminal amino acid residues 312-352, which includes the cytoplasmic sequestration domain. In the present study, we have found that both mot-1 and mot-2 bind to p53 in vitro. By using His-tagged deletion mutant proteins, the p53-binding domain of mot-2 was mapped to its N-terminal amino acid residues 253-282, which are identical in mot-1 and mot-2 proteins. Some peptides containing the p53-binding region of mot-2 were able to compete with the full-length protein for p53 binding. The data provided rationale for in vitro binding of mot-1 and mot-2 proteins to p53 and supported the conclusion that inability of mot-1 protein to bind p53 in vivo depends on secondary structure or its binding to other cellular factors. Most interestingly, the p53-binding region of mot-2 was common to its MKT-077, a cationic dye that exhibits antitumor activity, binding region. Therefore it is most likely that MKT-077-induced nuclear translocation and restoration of wild-type p53 function in transformed cells takes place by a competitional mechanism.

  7. Liver p53 expression in patients with HCV-related chronic hepatitis.

    PubMed

    Loguercio, C; Cuomo, A; Tuccillo, C; Gazzerro, P; Cioffi, M; Molinari, A M; Del Vecchio Blanco, C

    2003-07-01

    Mutated p53 acts as a dominant oncogene and alterations in the p53 gene are described in a large number of patients with hepatocellular carcinoma (HCC). It has been demonstrated that hepatitis C virus (HCV)-core protein regulates transcriptionally cellular genes, as well as cell growth and apoptosis. This study was undertaken to evaluate whether p53 may be expressed also in a precocious stage of HCV-related liver damage. We studied p53 expression by immunoluminometric assay on liver samples from 40 patients (M/F 18/ 22, median age 44 years, range 13-64 years) with biopsy-proven HCV-related chronic hepatitis. We considered the following factors: degree of liver damage, liver iron content and HCV-RNA titre. We also evaluated as possible co-factors alcohol and food intake in the last 3 years. p53 was over-expressed in seven of 40 (17.5%) patients. Liver histology documented the presence of unexpected cirrhosis in two patients among the p53 positive subjects. The p53 positive group had a daily ethanol intake significantly higher in respect to that of the p53 negative group (P < 0.05). Alimentary history documented that patients with a p53 over-expression had a lower intake of total calories, monounsaturated fatty acids, vitamin C and riboflavin. Data indicate that p53 over-expression can occur even in initial stages of HCV-related liver disease.

  8. Recurrent Initiation: A Mechanism for Triggering p53 Pulses in Response to DNA Damage

    PubMed Central

    Batchelor, Eric; Mock, Caroline; Bhan, Irun; Loewer, Alexander; Lahav, Galit

    2008-01-01

    SUMMARY DNA damage initiates a series of p53 pulses. Although much is known about the interactions surrounding p53, little is known about which interactions contribute to p53’s dynamical behavior. The simplest explanation is that these pulses are oscillations intrinsic to the p53/Mdm2 negative feedback loop. Here we present evidence that this simple mechanism is insufficient to explain p53 pulses; we show that p53 pulses are externally driven by pulses in the upstream signaling kinases, ATM and Chk2, and that the negative feedback between p53 and ATM, via Wip1, is essential for maintaining the uniform shape of p53 pulses. We propose that p53 pulses result from repeated initiation by ATM which is re-activated by persistent DNA damage. Our study emphasizes the importance of collecting quantitative dynamic information at high temporal resolution for understanding the regulation of signaling pathways and opens new ways to manipulate p53 pulses to ask questions about their function in response to DNA damage. PMID:18471974

  9. 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

  10. JMJD6 Promotes Colon Carcinogenesis through Negative Regulation of p53 by Hydroxylation

    PubMed Central

    Wang, Feng; He, Lin; Huangyang, Peiwei; Liang, Jing; Si, Wenzhe; Yan, Ruorong; Han, Xiao; Liu, Shumeng; Gui, Bin; Li, Wanjin; Miao, Di; Jing, Chao; Liu, Zhihua; Pei, Fei; Sun, Luyang; Shang, Yongfeng

    2014-01-01

    Jumonji domain-containing 6 (JMJD6) is a member of the Jumonji C domain-containing family of proteins. Compared to other members of the family, the cellular activity of JMJD6 is still not clearly defined and its biological function is still largely unexplored. Here we report that JMJD6 is physically associated with the tumor suppressor p53. We demonstrated that JMJD6 acts as an α-ketoglutarate– and Fe(II)-dependent lysyl hydroxylase to catalyze p53 hydroxylation. We found that p53 indeed exists as a hydroxylated protein in vivo and that the hydroxylation occurs mainly on lysine 382 of p53. We showed that JMJD6 antagonizes p53 acetylation, promotes the association of p53 with its negative regulator MDMX, and represses transcriptional activity of p53. Depletion of JMJD6 enhances p53 transcriptional activity, arrests cells in the G1 phase, promotes cell apoptosis, and sensitizes cells to DNA damaging agent-induced cell death. Importantly, knockdown of JMJD6 represses p53-dependent colon cell proliferation and tumorigenesis in vivo, and significantly, the expression of JMJD6 is markedly up-regulated in various types of human cancer especially in colon cancer, and high nuclear JMJD6 protein is strongly correlated with aggressive clinical behaviors of colon adenocarcinomas. Our results reveal a novel posttranslational modification for p53 and support the pursuit of JMJD6 as a potential biomarker for colon cancer aggressiveness and a potential target for colon cancer intervention. PMID:24667498

  11. Dynamic roles of p53-mediated metabolic activities in ROS-induced stress responses.

    PubMed

    Jiang, Le; Hickman, Justin H; Wang, Shang-Jui; Gu, Wei

    2015-01-01

    The p53 tumor suppressor is a multifaceted polypeptide that impedes tumorigenesis by regulating a diverse array of cellular processes. Triggered by a wide variety of stress stimuli, p53 transcriptionally regulates genes involved in the canonical tumor suppression pathways of apoptosis, cell-cycle arrest, and senescence. We recently discovered a novel mechanism whereby p53 inhibits cystine uptake through repression of the SLC7A11 gene to mediate ferroptosis. Importantly, this p53-SLC7A11 axis is preserved in the p53(3KR) mutant, and contributes to its ability to suppress tumorigenesis in the absence of the classical tumor suppression mechanisms. Here, we report that wild type p53 can induce both apoptosis and ferroptosis upon reactive oxygen species (ROS)-induced stress. Furthermore, we demonstrate that p53's functional N-terminal domain is required for its capacity to regulate oxidative stress responses and ferroptosis. Notably, activated p53 dynamically modulates intracellular ROS, causing an initial reduction and a subsequent increase of ROS levels. Taken together, these data implicate ferroptosis as an additional component of the cell death program induced by wild type p53 in human cancer cells, and reveal a complex and dynamic role of p53 in oxidative stress responses.

  12. Wild-type p53 controls cell motility and invasion by dual regulation of MET expression

    PubMed Central

    Hwang, Chang-Il; Matoso, Andres; Corney, David C.; Flesken-Nikitin, Andrea; Körner, Stefanie; Wang, Wei; Boccaccio, Carla; Thorgeirsson, Snorri S.; Comoglio, Paolo M.; Hermeking, Heiko; Nikitin, Alexander Yu.

    2011-01-01

    Recent observations suggest that p53 mutations are responsible not only for growth of primary tumors but also for their dissemination. However, mechanisms involved in p53-mediated control of cell motility and invasion remain poorly understood. By using the primary ovarian surface epithelium cell culture, we show that conditional inactivation of p53 or expression of its mutant forms results in overexpression of MET receptor tyrosine kinase, a crucial regulator of invasive growth. At the same time, cells acquire increased MET-dependent motility and invasion. Wild-type p53 negatively regulates MET expression by two mechanisms: (i) transactivation of MET-targeting miR-34, and (ii) inhibition of SP1 binding to MET promoter. Both mechanisms are not functional in p53 absence, but mutant p53 proteins retain partial MET promoter suppression. Accordingly, MET overexpression, cell motility, and invasion are particularly high in p53-null cells. These results identify MET as a critical effector of p53 and suggest that inhibition of MET may be an effective antimetastatic approach to treat cancers with p53 mutations. These results also show that the extent of advanced cancer traits, such as invasion, may be determined by alterations in individual components of p53/MET regulatory network. PMID:21831840

  13. Immunomodulatory Function of the Tumor Suppressor p53 in Host Immune Response and the Tumor Microenvironment

    PubMed Central

    Cui, Yan; Guo, Gang

    2016-01-01

    The tumor suppressor p53 is the most frequently mutated gene in human cancers. Most of the mutations are missense leading to loss of p53 function in inducing apoptosis and senescence. In addition to these autonomous effects of p53 inactivation/dysfunction on tumorigenesis, compelling evidence suggests that p53 mutation/inactivation also leads to gain-of-function or activation of non-autonomous pathways, which either directly or indirectly promote tumorigenesis. Experimental and clinical results suggest that p53 dysfunction fuels pro-tumor inflammation and serves as an immunological gain-of-function driver of tumorigenesis via skewing immune landscape of the tumor microenvironment (TME). It is now increasingly appreciated that p53 dysfunction in various cellular compartments of the TME leads to immunosuppression and immune evasion. Although our understanding of the cellular and molecular processes that link p53 activity to host immune regulation is still incomplete, it is clear that activating/reactivating the p53 pathway in the TME also represents a compelling immunological strategy to reverse immunosuppression and enhance antitumor immunity. Here, we review our current understanding of the potential cellular and molecular mechanisms by which p53 participates in immune regulation and discuss how targeting the p53 pathway can be exploited to alter the immunological landscape of tumors for maximizing therapeutic outcome. PMID:27869779

  14. Effects of p53-knockout in vascular smooth muscle cells on atherosclerosis in mice

    PubMed Central

    Jia, Lilly; Funk, Colin D.; Jia, Zongchao; Mak, Alan S.

    2017-01-01

    In vitro and in vivo evidence has indicated that the tumor suppressor, p53, may play a significant role in the regulation of atherosclerotic plaque formation. In vivo studies using global knockout mice models, however, have generated inconclusive results that do not address the roles of p53 in various cell types involved in atherosclerosis. In this study, we have specifically ablated p53 in vascular smooth muscle cells (VSMC) in the ApoE-/- mouse model to investigate the roles of p53 in VSMC in atherosclerotic plaque formation and stability. We found that p53 deficiency in VSMC alone did not affect the overall size of atherosclerotic lesions. However, there was a significant increase in the number of p53-/- VSMC in the fibrous caps of atherosclerotic plaques in the early stages of plaque development. Loss of p53 results in migration of VSMC at a faster rate using wound healing assays and augments PDGF-induced formation of circular dorsal ruffles (CDR), known to be involved in cell migration and internalization of surface receptors. Furthermore, aortic VSMC from ApoE-/- /p53-/- mice produce significantly more podosomes and are more invasive. We conclude that p53-/- VSMC are enriched in the fibrous caps of lesions at early stages of plaque formation, which is caused in part by an increase in VSMC migration and invasion as shown by p53-/- VSMC in culture having significantly higher rates of migration and producing more CDRs and invasive podosomes. PMID:28362832

  15. p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner.

    PubMed Central

    Miller, S D; Farmer, G; Prives, C

    1995-01-01

    The p53 tumor suppressor gene product is a sequence-specific DNA-binding protein that is necessary for the G1 arrest of many cell types. Consistent with its role as a cell cycle checkpoint factor, p53 has been shown to be capable of both transcriptional activation and repression. Here we show a new potential role for p53 as a DNA-binding-dependent regulator of DNA replication. Constructs containing multiple copies of the ribosomal gene cluster (RGC) p53 binding site cloned on the late side of the polyomavirus origin were used in in vitro replication assays. In the presence of p53, the replication of these constructs was strongly inhibited, while the replication of constructs containing a mutant version of the RGC site was not affected by p53. Several tumor-derived mutant p53 proteins were unable to inhibit replication of the construct with wild-type RGC sites. Additionally, the transactivator GAL4-VP16 was unable to inhibit replication of a construct containing GAL4 binding sites adjacent to the polyomavirus origin. We also show that the inhibition by p53 can occur from sites cloned as far as 600 bp from the origin. Preincubation experiments suggest that p53 inhibits replication at a step mediated by ATP, possibly by inhibiting the binding of polyomavirus T antigen to the core origin. The presence of an endogenous p53 binding site in the polyomavirus origin suggests potential mechanisms for the observed inhibition. PMID:8524220

  16. Radiation response and cell cycle regulation of p53 rescued malignant keratinocytes

    SciTech Connect

    Niemantsverdriet, Maarten; Jongmans, Wim; Backendorf, Claude . E-mail: backendo@chem.leidenuniv.nl

    2005-10-15

    Mutations in the tumor suppressor gene p53 were found in more than 90% of all human squamous cell carcinomas (SCC). To study the function of p53 in a keratinocyte background, a tetracycline-controlled p53 transgene was introduced into a human SCC cell line (SCC15), lacking endogenous p53. Conditional expression of wild-type p53 protein upon withdrawal of tetracycline was accompanied with increased expression of p21{sup WAF1/Cip1} resulting in reduced cell proliferation. Flow-cytometric analysis revealed that these cells were transiently arrested in the G1/S phase of the cell cycle. However, when SCC15 cells expressing p53 were exposed to ionizing radiation (IR), a clear shift from a G1/S to a G2/M cell cycle arrest was observed. This effect was greatly depending on the presence of wild-type p53, as it was not observed to the same extent in SCC15 cells lacking p53. Unexpectedly, the p53- and IR-dependent G2/M cell cycle arrest in the keratinocyte background was not depending on increased expression or stabilization of 14-3-3{sigma}, a p53-regulated effector of G2/M progression in colorectal cancer cells. In keratinocytes, 14-3-3{sigma} (stratifin) is involved in terminal differentiation and its cell cycle function in this cell type might diverge from the one it fulfills in other cellular backgrounds.

  17. Mutant p53 expression in fallopian tube epithelium drives cell migration.

    PubMed

    Quartuccio, Suzanne M; Karthikeyan, Subbulakshmi; Eddie, Sharon L; Lantvit, Daniel D; Ó hAinmhire, Eoghainín; Modi, Dimple A; Wei, Jian-Jun; Burdette, Joanna E

    2015-10-01

    Ovarian cancer is the fifth leading cause of cancer death among US women. Evidence supports the hypothesis that high-grade serous ovarian cancers (HGSC) may originate in the distal end of the fallopian tube. Although a heterogeneous disease, 96% of HGSC contain mutations in p53. In addition, the "p53 signature," or overexpression of p53 protein (usually associated with mutation), is a potential precursor lesion of fallopian tube derived HGSC suggesting an essential role for p53 mutation in early serous tumorigenesis. To further clarify p53-mutation dependent effects on cells, murine oviductal epithelial cells (MOE) were stably transfected with a construct encoding for the R273H DNA binding domain mutation in p53, the most common mutation in HGSC. Mutation in p53 was not sufficient to transform MOE cells but did significantly increase cell migration. A similar p53 mutation in murine ovarian surface epithelium (MOSE), another potential progenitor cell for serous cancer, was not sufficient to transform the cells nor change migration suggesting tissue specific effects of p53 mutation. Microarray data confirmed expression changes of pro-migratory genes in p53(R273H) MOE compared to parental cells, which could be reversed by suppressing Slug expression. Combining p53(R273H) with KRAS(G12V) activation caused transformation of MOE into high-grade sarcomatoid carcinoma when xenografted into nude mice. Elucidating the specific role of p53(R273H) in the fallopian tube will improve understanding of changes at the earliest stage of transformation. This information can help develop chemopreventative strategies to prevent the accumulation of additional mutations and reverse progression of the "p53 signature" thereby, improving survival rates.

  18. Targeting Mdmx to treat breast cancers with wild-type p53.

    PubMed

    Haupt, S; Buckley, D; Pang, J-M B; Panimaya, J; Paul, P J; Gamell, C; Takano, E A; Lee, Y Ying; Hiddingh, S; Rogers, T-M; Teunisse, A F A S; Herold, M J; Marine, J-C; Fox, S B; Jochemsen, A; Haupt, Y

    2015-07-16

    The function of the tumor suppressor p53 is universally compromised in cancers. It is the most frequently mutated gene in human cancers (reviewed). In cases where p53 is not mutated, alternative regulatory pathways inactivate its tumor suppressive functions. This is primarily achieved through elevation in the expression of the key inhibitors of p53: Mdm2 or Mdmx (also called Mdm4) (reviewed). In breast cancer (BrCa), the frequency of p53 mutations varies markedly between the different subtypes, with basal-like BrCas bearing a high frequency of p53 mutations, whereas luminal BrCas generally express wild-type (wt) p53. Here we show that Mdmx is unexpectedly highly expressed in normal breast epithelial cells and its expression is further elevated in most luminal BrCas, whereas p53 expression is generally low, consistent with wt p53 status. Inducible knockdown (KD) of Mdmx in luminal BrCa MCF-7 cells impedes the growth of these cells in culture, in a p53-dependent manner. Importantly, KD of Mdmx in orthotopic xenograft transplants resulted in growth inhibition associated with prolonged survival, both in a preventative model and also in a treatment model. Growth impediment in response to Mdmx KD was associated with cellular senescence. The growth inhibitory capacity of Mdmx KD was recapitulated in an additional luminal BrCa cell line MPE600, which expresses wt p53. Further, the growth inhibitory capacity of Mdmx KD was also demonstrated in the wt p53 basal-like cell line SKBR7 line. These results identify Mdmx growth dependency in wt p53 expressing BrCas, across a range of subtypes. Based on our findings, we propose that Mdmx targeting is an attractive strategy for treating BrCas harboring wt p53.

  19. Correlation of p53 Overexpression with the Clinicopathological Prognostic Factors in Colorectal Adenocarcinoma

    PubMed Central

    Akshatha C; Mysorekar, Vijaya; Arundhathi S; Raj, Adithi; Shetty, Smitha

    2016-01-01

    Introduction Mutation in p53 gene and accumulation of p53 protein is a common genetic event in colorectal carcinomas. p53 mutation can be detected by various techniques such as DNA sequencing, polymerase chain reaction and immunohistochemistry (IHC). However, IHC is simple and is consistent with other techniques. Aim To establish a correlation between overexpression of p53 with the clinical features, tumour histopathology and stage of Colorectal Carcinoma (CRC). Materials and Methods This prospective and retrospective study of clinical, histopathological and IHC features of CRC was conducted on colectomy and abdomino-perineal resection specimens received from January 2008 to June 2013. For each case, the clinical features, tumour morphology and p53 status (by IHC) were evaluated. Results The most common histologic type of CRC was Non-Specific Type (NST) and grade II tumours were seen predominantly (60%). Overall, 67.5% of CRCs showed p53 positivity on IHC. Intense p53 positivity was observed in 37.5% of CRCs of NST type and 33.3% of mucinous adenocarcinomas showed moderate positivity. Grade III tumours showed variable p53 positivity and those with lymph node metastasis showed moderate (55.6%) or intense positivity (53.8%). But there was no statistically significant correlation of p53 status and various clinicopathological prognostic factors. Conclusion As p53 protein overexpression is seen in a relatively high percentage of CRCs, it seems that p53 mutation plays an important role in development of CRC. However, no direct correlation could be established between p53 results and the patients’ age, sex, tumour site, size, histological type, grade, lymph node status, or TNM stage. A prolonged follow up is necessary to conclude whether p53 status has any influence on the long, term prognosis and patient survival. PMID:28208862

  20. Regulation of p53 in NIH3T3 mouse fibroblasts following hyperosmotic stress

    PubMed Central

    Lambert, Ian Henry; Enghoff, Maria Stine; Brandi, Marie-Luise; Hoffmann, Else Kay

    2015-01-01

    The aim of this project was to analyze the regulation of p53 expression in NIH3T3 fibroblasts under the influence of increasing hyperosmotic stress. Expression of p53 showed a biphasic response pattern in NIH3T3 cells under increasing osmotic stress (337 mOsm to 737 mOsm) with a maximum at 587 mOsm. Under isotonic conditions p53 expression increased after addition of the proteasome inhibitor MG132 indicating that cellular p53 levels in unperturbed cells is kept low by proteasomal degradation. However, under hypertonic conditions p53 synthesis as well as p53 degradation were significantly reduced and it is demonstrated that the increase in p53 expression observed when tonicity is increased from 337 to 587 mOsm reflects that degradation is more inhibited than synthesis, whereas the decrease in p53 expression at higher tonicities reflects that synthesis is more inhibited than degradation. The activity of the p53 regulating proteins p38 MAP kinase and the ubiquitin ligase MDM2 were studied as a function of increasing osmolarity. MDM2 protein expression was unchanged at all osmolarities, whereas MDM2 phosphorylation (Ser166) increased at osmolarities up to 537 mOsm and remained constant at higher osmolarities. Phosphorylation of p38 increased at osmolarities up to 687 mOsm which correlated with an increased phosphorylation of p53 (Ser15) and the decreased p53 degradation. Caspase-3 activity increased gradually with hypertonicity and at 737 mOsm both Caspase-3 activity and annexin V binding are high even though p53 expression and activity are low, indicating that initiation of apoptosis under severe hypertonic conditions is not strictly controlled by p53. PMID:26056062

  1. Mutant p53 stimulates chemoresistance of pancreatic adenocarcinoma cells to gemcitabine.

    PubMed

    Fiorini, Claudia; Cordani, Marco; Padroni, Chiara; Blandino, Giovanni; Di Agostino, Silvia; Donadelli, Massimo

    2015-01-01

    Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths worldwide; PDAC is characterized by poor prognosis, resistance to conventional chemotherapy and high mortality rate. TP53 tumor suppressor gene is frequently mutated in PDAC, resulting in the accumulation of mutated protein with potential gain-of-function (GOF) activities, such as genomic instability, hyperproliferation and chemoresistance. The purpose of this study was to assess the relevance of the p53 status on the PDAC cells response to the standard drug gemcitabine. We also examined the potential therapeutic effect of p53-reactivating molecules to restore the mutant p53 function in GEM treated PDAC cells. We showed that gemcitabine stabilized mutant p53 protein in the nuclei and induced chemoresistance, concurrent with the mutant p53-dependent expression of Cdk1 and CCNB1 genes, resulting in a hyperproliferation effect. Despite the adverse activation of mutant p53 by gemcitabine, simultaneous treatment of PDAC cells with gemcitabine and p53-reactivating molecules (CP-31398 and RITA) reduced growth rate and induced apoptosis. This synergistic effect was observed in both wild-type and mutant p53 cell lines and was absent in p53-null cells. The combination drug treatment induced p53 phosphorylation on Ser15, apoptosis and autophagosome formation. Furthermore, pharmacological inhibition of autophagy further increased apoptosis stimulated by gemcitabine/CP-31398 treatment. Together, our results show that gemcitabine aberrantly stimulates mutant p53 activity in PDAC cells identifying key processes with potential for therapeutic targeting. Our data also support an anti-tumoral strategy based on inhibition of autophagy combined with p53 activation and standard chemotherapy for both wild-type and mutant p53 expressing PDACs.

  2. Extensive Post-translational Modification of Active and Inactivated Forms of Endogenous p53*

    PubMed Central

    DeHart, Caroline J.; Chahal, Jasdave S.; Flint, S. J.; Perlman, David H.

    2014-01-01

    The p53 tumor suppressor protein accumulates to very high concentrations in normal human fibroblasts infected by adenovirus type 5 mutants that cannot direct assembly of the viral E1B 55-kDa protein-containing E3 ubiquitin ligase that targets p53 for degradation. Despite high concentrations of nuclear p53, the p53 transcriptional program is not induced in these infected cells. We exploited this system to examine select post-translational modifications (PTMs) present on a transcriptionally inert population of endogenous human p53, as well as on p53 activated in response to etoposide treatment of normal human fibroblasts. These forms of p53 were purified from whole cell lysates by means of immunoaffinity chromatography and SDS-PAGE, and peptides derived from them were subjected to nano-ultra-high-performance LC-MS and MS/MS analyses on a high-resolution accurate-mass MS platform (data available via ProteomeXchange, PXD000464). We identified an unexpectedly large number of PTMs, comprising phosphorylation of Ser and Thr residues, methylation of Arg residues, and acetylation, ubiquitinylation, and methylation of Lys residues—for example, some 150 previously undescribed modifications of p53 isolated from infected cells. These modifications were distributed across all functional domains of both forms of the endogenous human p53 protein, as well as those of an orthologous population of p53 isolated from COS-1 cells. Despite the differences in activity, including greater in vitro sequence-specific DNA binding activity exhibited by p53 isolated from etoposide-treated cells, few differences were observed in the location, nature, or relative frequencies of PTMs on the two populations of human p53. Indeed, the wealth of PTMs that we have identified is consistent with a far greater degree of complex, combinatorial regulation of p53 by PTM than previously anticipated. PMID:24056736

  3. Involvement of p53 mutation and mismatch repair proteins dysregulation in NNK-induced mali