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Sample records for exogenous wild p53

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

    Science.gov (United States)

    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

    2017-05-01

    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. Copyright © 2016 Pathological Society of Great

  2. Wild-type and mutant p53 mediate cisplatin resistance through interaction and inhibition of active caspase-9

    Science.gov (United States)

    Chee, Jacqueline L.Y.; Saidin, Suzan; Lane, David P.; Leong, Sai Mun; Noll, Jacqueline E.; Neilsen, Paul M.; Phua, Yi Ting; Gabra, Hani; Lim, Tit Meng

    2013-01-01

    The p53 gene has been implicated in many cancers due to its frequent mutations as well as mutations in other genes whose proteins directly affect p53’s functions. In addition, high expression of p53 [wild-type (WT) or mutant] has been found in the cytoplasm of many tumor cells, and studies have associated these observations with more aggressive tumors and poor prognosis. Cytoplasmic mis-localization of p53 subsequently reduced its transcriptional activity and this loss-of-function (LOF) was used to explain the lack of response to chemotherapeutic agents. However, this hypothesis seemed inadequate in explaining the apparent selection for tumor cells with high levels of p53 protein, a phenomenon that suggests a gain-of-function (GOF) of these mis-localized p53 proteins. In this study, we explored whether the direct involvement of p53 in the apoptotic response is via regulation of the caspase pathway in the cytoplasm. We demonstrate that p53, when present at high levels in the cytoplasm, has an inhibitory effect on caspase-9. Concurrently, knockdown of endogenous p53 caused an increase in the activity of caspase-9. p53 was found to interact with the p35 fragment of caspase-9, and this interaction inhibits the caspase-9 activity. In a p53-null background, the high-level expression of both exogenous WT and mutant p53 increased the resistance of these cells to cisplatin, and the data showed a correlation between high p53 expression and caspase-9 inhibition. These results suggest the inhibition of caspase-9 as a potential mechanism in evading apoptosis in tumors with high-level p53 expression that is cytoplasmically localized. PMID:23255126

  3. Evidence for allosteric variants of wild-type p53, a tumour suppressor protein.

    OpenAIRE

    Cook, A.; Milner, J.

    1990-01-01

    A tumour suppressor function for p53 is indicated in human lung cancer and in carcinoma of the colorectum. Loss of suppressor function, by mutation of the p53 gene, is associated with activation of p53 as an oncogene. The suppressor (wild type) and oncogenic (mutant) forms of the murine p53 protein are distinguishable at the molecular level by reactivity with anti-p53 monoclonal antibodies. For example, activated mutant p53 fails to react with PAb246 (p53-246 degrees). We now demonstrate that...

  4. Biological activity and safety of adenoviral vector-expressed wild-type p53 after intratumoral injection in melanoma and breast cancer patients with p53-overexpressing tumors

    NARCIS (Netherlands)

    Dummer, R; Bergh, J; Karlsson, Y; Horovitz, JA; Mulder, NH; Huinin, DT; Burg, G; Hofbauer, G; Osanto, S

    p53 mutations are common genetic alterations in human cancer. Gene transfer of a wild-type (wt) p53 gene reverses the loss of normal p53 function in vitro and in vivo. A phase I dose escalation study of single intratumoral (i.t.) injection of a replication-defective adenoviral expression vector

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

    Science.gov (United States)

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

    2017-09-01

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

  6. Radiation induces p53-dependent cell apoptosis in bladder cancer cells with wild-type- p53 but not in p53-mutated bladder cancer cells.

    Science.gov (United States)

    Hinata, Nobuyuki; Shirakawa, Toshiro; Zhang, Zhujun; Matsumoto, Akira; Fujisawa, Masato; Okada, Hiroshi; Kamidono, Sadao; Gotoh, Akinobu

    2003-12-01

    Purpose. It has been reported in several studies that the absence in cancer cells of the p53 tumor suppressor gene, mutations of which are frequently found in bladder cancer, increases their resistance to ionizing radiation. Other studies, however, suggest that mutations of the p53 gene could increase the radiosensitivity of cancer cells, although the evidence is still inconclusive. In the present study, we investigated the relationship between p53 status and radiation response in five different bladder cancer cell lines. Materials and Methods. Five different human bladder cancer cell lines (KK47: with wt- p53, RT4: with wt- p53, T24: with mutated p53, 5637: with mutated p53, UM-UC-3: with mutated p53) were used in the study. Cells were irradiated with 0, 2, 4, 6 or 8 Gy, then trypsinized and re-plated for clonogenic survival assay, quantitative RT-PCR assay, flow-cytometry analysis and TUNEL assay. Results. The clonogenic assay demonstrated that KK47 and RT4 had significantly higher radiosensitivity than other cell lines. Quantitative RT-PCR analysis showed that radiation induced increased expression of p53, Bax, and p21 mRNA in KK47 and RT4. After irradiation, G1 cell-cycle arrest was observed in KK47 and RT4 under flow cytometry analysis, while T24, 5637, and UM-UC-3 showed an increase in the proportion of G2 cells. Increased cell apoptosis was also observed under TUNEL assay in KK47 and RT4, but not in other cell lines. It was demonstrated that ionizing radiation induces p53-dependent cell apoptosis in bladder cancer cells with wt- p53 but not in those with mutated p53.

  7. Mutant p53 cooperates with knockdown of endogenous wild-type p53 to disrupt tubulogenesis in Madin-Darby canine kidney cells.

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    Yanhong Zhang

    Full Text Available Mutation of the p53 gene is the most common genetic alteration in human malignances and associated clinically with tumor progression and metastasis. To determine the effect of mutant p53 on epithelial differentiation, we developed three-dimensional culture (3-D of Madin-Darby canine kidney (MDCK cells. We found that parental MDCK cells undergo a series of morphological changes and form polarized and growth-arrested cysts with hollow lumen, which resembles branching tubules in vitro. We also found that upon knockdown of endogenous wild-type p53 (p53-KD, MDCK cells still form normal cysts in 3-D culture, indicating that p53-KD alone is not sufficient to disrupt cysts formation. However, we found that ectopic expression of mutant R163H (human equivalent R175H or R261H (human equivalent R273H in MDCK cells leads to disruption of cyst polarity and formation of invasive aggregates, which is further compounded by knockdown of endogenous wild-type p53. Consistently, we found that expression of E-cadherin, β-catenin, and epithelial-to-mesenchymal transition (EMT transcription factors (Snail-1, Slug and Twist is altered by mutant p53, which is also compounded by knockdown of wild-type p53. Moreover, the expression level of c-Met, the hepatocyte growth factor receptor and a key regulator of kidney cell tubulogenesis, is enhanced by combined knockdown of endogenous wild-type p53 and ectopic expression of mutant R163H or R261H but not by each individually. Together, our data suggest that upon inactivating mutation of the p53 gene, mutant p53 acquires its gain of function by altering morphogenesis and promoting cell migration and invasion in part by upregulating EMT and c-Met.

  8. Noncanonical DNA motifs as transactivation targets by wild type and mutant p53.

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    Jennifer J Jordan

    2008-06-01

    Full Text Available Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0-13 nucleotides (nt, originally defined by the consensus RRRCWWGYYY (n = 0-13 RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs by wild type (WT and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi-in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical (1/2-(a single decamer and (3/4-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of (1/2- and (3/4-site REs greatly expands the p53 master regulatory network.

  9. The Role of Wild-Type p53 in Cisplatin-Induced Chk2 Phosphorylation and the Inhibition of Platinum Resistance with a Chk2 Inhibitor

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    Xiaobing Liang

    2011-01-01

    Full Text Available The major obstacle in platinum chemotherapy is the repair of platinum-damaged DNA that results in increased resistance, reduced apoptosis, and finally treatment failure. Our research goal is to determine and block the mechanisms of platinum resistance. Our recent studies demonstrate that several kinases in the DNA-repair pathway are activated after cells are exposed to cisplatin. These include ATM, p53, and Chk2. The increased Chk2 phosphorylation is modulated by p53 in a wild-type p53 model. Overexpression of p53 by cDNA transfection in wt-p53 (but not p53 deficient cells doubled the amount of Chk2 phosphorylation 48 hours after cisplatin treatment. p53 knockdown by specific siRNA greatly reduced Chk2 phosphorylation. We conclude that wild-type p53, in response to cisplatin stimulation, plays a role in the upstream regulation of Chk2 phosphorylation at Thr-68. Cells without normal p53 function survive via an alternative pathway in response to the exogenous influence of cisplatin. We strongly suggest that it is very important to include the p53 mutational status in any p53 involved studies due to the functional differentiation of wt p53 and p53 mutant. Inhibition of Chk2 pathway with a Chk2 inhibitor (C3742 increased cisplatin efficacy, especially those with defective p53. Our findings suggest that inhibition of platinum resistance can be achieved with a small-molecule inhibitor of Chk2, thus improving the therapeutic indices for platinum chemotherapy.

  10. Wild type p53 transcriptionally represses the SALL2 transcription factor under genotoxic stress.

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    Carlos Farkas

    Full Text Available SALL2- a member of the Spalt gene family- is a poorly characterized transcription factor found deregulated in various cancers, which suggests it plays a role in the disease. We previously identified SALL2 as a novel interacting protein of neurotrophin receptors and showed that it plays a role in neuronal function, which does not necessarily explain why or how SALL2 is deregulated in cancer. Previous evidences indicate that SALL2 gene is regulated by the WT1 and AP4 transcription factors. Here, we identified SALL2 as a novel downstream target of the p53 tumor suppressor protein. Bioinformatic analysis of the SALL2 gene revealed several putative p53 half sites along the promoter region. Either overexpression of wild-type p53 or induction of the endogenous p53 by the genotoxic agent doxorubicin repressed SALL2 promoter activity in various cell lines. However R175H, R249S, and R248W p53 mutants, frequently found in the tumors of cancer patients, were unable to repress SALL2 promoter activity, suggesting that p53 specific binding to DNA is important for the regulation of SALL2. Electrophoretic mobility shift assay demonstrated binding of p53 to one of the identified p53 half sites in the Sall2 promoter, and chromatin immunoprecipitation analysis confirmed in vivo interaction of p53 with the promoter region of Sall2 containing this half site. Importantly, by using a p53ER (TAM knockin model expressing a variant of p53 that is completely dependent on 4-hydroxy-tamoxifen for its activity, we show that p53 activation diminished SALL2 RNA and protein levels during genotoxic cellular stress in primary mouse embryo fibroblasts (MEFs and radiosensitive tissues in vivo. Thus, our finding indicates that p53 represses SALL2 expression in a context-specific manner, adding knowledge to the understanding of SALL2 gene regulation, and to a potential mechanism for its deregulation in cancer.

  11. Wild-Type P53 Induces Sodium/Iodide Symporter Expression Allowing Radioiodide Therapy in Anaplastic Thyroid Cancer.

    Science.gov (United States)

    Liu, Lin; Li, Dan; Chen, Zhengqi; Yang, Jian; Ma, Yushui; Cai, Haidong; Shan, Chengxiang; Lv, Zhongwei; Zhang, Xiaoping

    2017-09-29

    Anaplastic thyroid cancer(ATC) is one of the most aggressive solid tumors. Mutations in the p53 gene are common in anaplastic thyroid cancer, but the effects of p53 mutations are yet to be elucidated. Here, we investigated the role of p53 in ATC. p53 mutation was detect by immunohistochemistry in ATC tissues. Expression of NIS were measured using immunohistochemistry, qRT-PCR, western blot, immunofluorescence in ATC tissues and cell line 8505c. Luciferase reporter assay was performed to examine the effect of wild-type p53 on NIS. Radioiodide uptake assay and flow cytometry analysis were used to detect the role of wild-type p53 on radioiodide uptake.and cell apoptosis in ATC cell line. We showed that the p53 mutation can be detected in ATC tissues. Furthermore, we demonstrated that wild-type p53 transactivated the NIS promoter. In 8505c cells transfected with wild-type p53, treatment with radioiodine resulted in increased radioiodine uptake and increased apoptotic cell death compared with 8505c cells harboring the p53 mutation. In summary, transfection with wild-type p53 can increase the therapeutic effect of radioiodine by regulating the expression of the NIS. The Author(s). Published by S. Karger AG, Basel.

  12. Wild-type alternatively spliced p53: binding to DNA and interaction with the major p53 protein in vitro and in cells.

    OpenAIRE

    Wu, Y.; Liu, Y; L. Lee; Miner, Z; Kulesz-Martin, M

    1994-01-01

    A p53 variant protein (p53as) generated from alternatively spliced p53 RNA is expressed in normal and malignant mouse cells and tissues, and p53as antigen activity is preferentially associated with the G2 phase of the cell cycle, suggesting that p53as and p53 protein may have distinct properties. Using p53as and p53 proteins translated in vitro, we now provide evidence that p53as protein has efficient sequence-specific DNA-binding ability. DNA binding by p53 protein is inefficient in comparis...

  13. Restoration of Wild-Type Activity to Mutant p53 in Prostate Cancer: A Novel Therapeutic Approach

    National Research Council Canada - National Science Library

    Manfredi, James

    2006-01-01

    A summary is presented of research performed during the first year of a project to determine feasibility of approaches to restore wild-type transcriptional activity on mutant p53 proteins found in human prostate tumors...

  14. Restoration of Wild-Type Activity to Mutant p53 in Prostate Cancer: A Novel Therapeutic Approach

    National Research Council Canada - National Science Library

    Manfredi, James J

    2008-01-01

    A summary is presented of research performed during three years of a project to determine feasibility of approaches to restore wild-type transcriptional activity on mutant p53 proteins found in human prostate tumors...

  15. Restore Wild-Type Functions to P53 Mutants Using an RNA-Based Combinatorial Approach

    National Research Council Canada - National Science Library

    Green, Christopher

    2000-01-01

    The p53 protein is a transcription factor and a suppressor regulating the expression of a wide range of genes involved in apoptosis, growth control, and inhibiting the proliferation of tumor cells in animal models...

  16. Effects of the Kava Chalcone Flavokawain A Differ in Bladder Cancer Cells with Wild-type versus Mutant p53

    Science.gov (United States)

    Tang, Yaxiong; Simoneau, Anne R.; Xie, Jun; Shahandeh, Babbak; Zi, Xiaolin

    2010-01-01

    Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G1 arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2 and then increased the stability of the p27/KIP1 protein. The accumulation of p21/WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G2-M arrest in six p53 mutant-type, high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation and then led to a G2-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G2-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G2-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer. PMID:19138991

  17. Effects of the kava chalcone flavokawain A differ in bladder cancer cells with wild-type versus mutant p53.

    Science.gov (United States)

    Tang, Yaxiong; Simoneau, Anne R; Xie, Jun; Shahandeh, Babbak; Zi, Xiaolin

    2008-11-01

    Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G(1) arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2, which then increased the stability of the p27/KIP1 protein. The accumulation of p21/WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G(2)-M arrest in six p53 mutant-type, high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation, which then led to a G(2)-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G(2)-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G(2)-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer.

  18. Differential suppression by protease inhibitors and cytokines of apoptosis induced by wild-type p53 and cytotoxic agents.

    OpenAIRE

    Lotem, J; Sachs, L.

    1996-01-01

    Apoptosis induced in myeloid leukemic cells by wild-type p53 was suppressed by different cleavage-site directed protease inhibitors, which inhibit interleukin-1 beta-converting enzyme-like, granzyme B and cathepsins B and L proteases. Apoptosis was also suppressed by the serine and cysteine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone (TPCK) [corrected], but not by other serine or cysteine protease inhibitors including N alpha-p-tosyl-L-lysine chloromethylketone (TLCK), E64, ...

  19. TP53 Mutation Status of Tubo-ovarian and Peritoneal High-grade Serous Carcinoma with a Wild-type p53 Immunostaining Pattern.

    Science.gov (United States)

    Na, Kiyong; Sung, Ji-Youn; Kim, Hyun-Soo

    2017-12-01

    Diffuse and strong nuclear p53 immunoreactivity and a complete lack of p53 expression are regarded as indicative of missense and nonsense mutations, respectively, of the TP53 gene. Tubo-ovarian and peritoneal high-grade serous carcinoma (HGSC) is characterized by aberrant p53 expression induced by a TP53 mutation. However, our experience with some HGSC cases with a wild-type p53 immunostaining pattern led us to comprehensively review previous cases and investigate the TP53 mutational status of the exceptional cases. We analyzed the immunophenotype of 153 cases of HGSC and performed TP53 gene sequencing analysis in those with a wild-type p53 immunostaining pattern. Immunostaining revealed that 109 (71.3%) cases displayed diffuse and strong p53 expression (missense mutation pattern), while 39 (25.5%) had no p53 expression (nonsense mutation pattern). The remaining five cases of HGSC showed a wild-type p53 immunostaining pattern. Direct sequencing analysis revealed that three of these cases harbored nonsense TP53 mutations and two had novel splice site deletions. TP53 mutation is almost invariably present in HGSC, and p53 immunostaining can be used as a surrogate marker of TP53 mutation. In cases with a wild-type p53 immunostaining pattern, direct sequencing for TP53 mutational status can be helpful to confirm the presence of a TP53 mutation. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  20. Effects of the Kava Chalcone Flavokawain A Differ in Bladder Cancer Cells with Wild-type versus Mutant p53

    OpenAIRE

    Tang, Yaxiong; Simoneau, Anne R.; Xie, Jun; Shahandeh, Babbak; Zi, Xiaolin

    2008-01-01

    Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G1 arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated wi...

  1. Efek ekstrak buah delima (Punica Granatum L) terhadap ekspresi wild p53 pada sel ganas rongga mulut mencit strain swiss webster

    OpenAIRE

    Hernawati, Sri; Rantam, Fedik Abdul; Sudiana, I Ketut; Rahayu, Retno Pudji

    2013-01-01

    Background: Squamous cell carcinoma is the most common cancer in the oral cavity. DNA tests showed that almost 90% of cases revealed wild p53 gene mutations. Wild p53 gene mutations cause p53 inactivation so the cell cycle does not stop in G1 phase but continues to S phase and G2 and M, it makes the mutated DNA remains multiplied and apoptosis does not occur. One candidate of the cancer treatment alternatives is pomegranate extract (Punica granatum L – PGL). Purpose: The purpose of study was ...

  2. Histone modifications affect differential regulation of TGFβ- induced NADPH oxidase 4 (NOX4) by wild-type and mutant p53

    Science.gov (United States)

    Boudreau, Howard E.; Ma, Wei Feng; Korzeniowska, Agnieszka; Park, Jonathan J.; Bhagwat, Medha A.; Leto, Thomas L.

    2017-01-01

    Previously, we showed wild-type (WT) and mutant (mut) p53 differentially regulate reactive oxygen species (ROS) generation by NADPH oxidase-4 (NOX4): p53-WT suppresses TGFβ-induced NOX4, ROS and cell migration, whereas tumor-associated mut-p53 proteins enhance NOX4 expression and cell migration. Here, we extended our findings on the effects of p53 on NOX4 in several tumors and examined the basis of NOX4 transcriptional regulation by p53 and SMAD3. Statistical analysis of expression data from primary tumors available from The Cancer Genome Atlas (TCGA) detected correlations between mut-p53 and increased NOX4 expression. Furthermore, by altering p53 levels in cell culture models we showed several common tumor-associated mutant forms support TGFβ/SMAD3-dependent NOX4 expression. Deletion analysis revealed two critical SMAD3 binding elements (SBE) required for mut-p53-dependent NOX4 induction, whereas p53-WT caused dose-dependent suppression of NOX4 transcription. ChIP analysis revealed SMAD3 and p53-WT or mut-p53 associate with SBEs and p53 response elements in a TGFβ-dependent manner. Interestingly, the repressive effects of p53-WT on NOX4 were relieved by mutation of its transactivation domain or histone deacetylase (HDAC) inhibitor treatment. Overexpression of p300, a transcriptional co-regulator and histone acetyltransferase (HAT), enhanced p53-mediated NOX4 induction, whereas HAT-inactive p300 reduced NOX4 expression. Mut-p53 augmented TGFβ-stimulated histone acetylation within the NOX4 promoter. Finally, wound assays demonstrated NOX4 and p300 promote TGFβ/mut-p53-mediated cell migration. Our studies provide new insight into TGFβ/SMAD3 and mut-p53-mediated NOX4 induction involving epigenetic control of NOX4 in tumor cell migration, suggesting NOX4 is a potential therapeutic target to combat tumor progression and metastasis. PMID:28574838

  3. Efek ekstrak buah delima (Punica Granatum L terhadap ekspresi wild p53 pada sel ganas rongga mulut mencit strain swiss webster

    Directory of Open Access Journals (Sweden)

    Sri Hernawati

    2013-09-01

    Full Text Available Background: Squamous cell carcinoma is the most common cancer in the oral cavity. DNA tests showed that almost 90% of cases revealed wild p53 gene mutations. Wild p53 gene mutations cause p53 inactivation so the cell cycle does not stop in G1 phase but continues to S phase and G2 and M, it makes the mutated DNA remains multiplied and apoptosis does not occur. One candidate of the cancer treatment alternatives is pomegranate extract (Punica granatum L – PGL. Purpose: The purpose of study was to examine the effect of PGL on wild p53 expression in oral cavity malignant cell of swiss webster strain mice. Methods: Thirty- two swiss webster strain mice (Balb/c 5 months old were randomly divided into four groups. Two control groups (K0: no benzopirene exposed and untreated; K1: benzopirene exposed and untreated; and 2 treatment groups (P1: benzopirene exposed and given EA; P2: benzopirene exposed and given PGL extract. The expression of wild p53 was determined by immunohistochemical techniques. Results: The results showed that administration of PGL could increase the expression of wild p53 in malignant epithelial cells in the oral mucosa of mice, and the expression was higher than EA. Conclusion: This study suggested that the PGL extract could express wild p53 in the oral cavity malignant cells of swiss Webster strains mice.Latar belakang: Karsinoma sel skuamosa merupakan kanker yang sering terjadi pada rongga mulut. Pemeriksaan DNA menunjukkan hampir 90% kasus dijumpai adanya mutasi gen wild p53. Mutasi gen wild p53 menyebabkan inaktivasi wild p53 sehingga siklus sel tidak berhenti pada fase G1 tetapi berlanjut ke fase S dan G2 dan M, sehingga DNA yang mengalami mutasi tetap dilipatgandakan dan apoptosis tidak terjadi. Salah satu kandidat obat kanker adalah ekstrak buah delima (Punica Granatum L - PGL. Tujuan: Penelitian ini bertujuan untuk meneliti efek ekstrak PGL terhadap ekspresi wild p53 pada sel ganas rongga mulut mencit strain swiss webster

  4. Pharmacological activation of tumor suppressor, wild-type p53 as a promising strategy to fight cancer

    Directory of Open Access Journals (Sweden)

    Alicja Sznarkowska

    2010-08-01

    Full Text Available A powerful tumor suppressor – p53 protein is a transcription factor which plays a critical role in eliciting cellular responses to a variety of stress signals, including DNA damage, hypoxia and aberrant proliferative signals, such as oncogene activation. Since its discovery thirty one years ago, p53 has been connected to tumorigenesis as it accumulates in the transformed tumor cells. Cellular stress induces stabilization of p53 and promotes, depending on the stress level, cell cycle arrest or apoptosis in the irreversibly damaged cells. The p53 protein is found inactive in more than 50�0of human tumors either by enhanced proteasomal degradation or due to the inactivating point mutations in its gene. Numerous data indicate that low molecular weight compounds, identified by molecular modeling or in the functional, cell-based assays, efficiently activate non-mutated p53 in cancer cells which in consequence leads to their elimination due to p53-dependent apoptosis. In this work we describe the structure and cellular function of p53 as well as the latest discoveries on the compounds with high anti-tumor activities aiming at reactivation of the tumor suppressor function of p53.

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

    DEFF Research Database (Denmark)

    Röpke, M; Hald, J; Guldberg, Per

    1996-01-01

    A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant...

  6. p53-based Cancer Therapy

    Science.gov (United States)

    Lane, David P.; Cheok, Chit Fang; Lain, Sonia

    2010-01-01

    Inactivation of p53 functions is an almost universal feature of human cancer cells. This has spurred a tremendous effort to develop p53 based cancer therapies. Gene therapy using wild-type p53, delivered by adenovirus vectors, is now in widespread use in China. Other biologic approaches include the development of oncolytic viruses designed to replicate and kill only p53 defective cells and also the development of siRNA and antisense RNA's that activate p53 by inhibiting the function of the negative regulators Mdm2, MdmX, and HPV E6. The altered processing of p53 that occurs in tumor cells can elicit T-cell and B-cell responses to p53 that could be effective in eliminating cancer cells and p53 based vaccines are now in clinical trial. A number of small molecules that directly or indirectly activate the p53 response have also reached the clinic, of which the most advanced are the p53 mdm2 interaction inhibitors. Increased understanding of the p53 response is also allowing the development of powerful drug combinations that may increase the selectivity and safety of chemotherapy, by selective protection of normal cells and tissues. PMID:20463003

  7. Role of wild type p53 and double suicide genes in interventional therapy of liver cancer in rabbits.

    Science.gov (United States)

    Niu, Hong-xin; Du, Tong; Xu, Zhong-fa; Zhang, Xi-kun; Wang, Ruo-gu

    2012-08-01

    To investigate the feasibility of interventional lipiodol embolism and multigene therapy in combination with focal chemotherapy in the treatment of VX2 liver cancer in rabbits. Forty five rabbits with cancer larger than 2cm in diameter were randomly divided into five groups (n=9 per group). In Group 1, animals were treated with 0.9% sodium chloride. In Group 2, animals received lipiodol embolism. In Group 3, animals received lipiodol embolism and p53 gene therapy. In Group 4, animals received lipiodol embolism and TK/CD gene therapy. In Group 5, animals received lipiodol embolism and p53 and TK/CD gene therapy. Ultrasonography and CT were performed before and at ten days after interventional therapy. The VX2 model of liver cancer was successfully established in rabbits and interventional therapy smoothly performed. At ten days after interventional therapy, significant difference in the tumor volume was noted among five groups (pgene therapy with p53 or TK/CD and lipiodol embolism independently exert significantly inhibitory effect on cancer growth. In addition, the suppression on tumor growth rate was the most obvious in the Group 5. Combination of gene therapy with lipiodol embolism can effectively inhibit the cancer growth and prolong the survival time. These findings demonstrate the effectiveness of multigene therapy in combination with lipiodol embolism in the treatment of liver cancer.

  8. Noscapine induced apoptosis via downregulation of survivin in human neuroblastoma cells having wild type or null p53.

    Directory of Open Access Journals (Sweden)

    Shiwang Li

    Full Text Available Neuroblastoma is the most common extracranial solid tumor of childhood. It accounts for 15% of pediatric cancer deaths. Chemotherapy is the mainstay of treatment in children with advanced neuroblastoma. Noscapine, a nontoxic natural compound, can trigger apoptosis in many cancer types. We now show that p53 is dispensable for Noscapine-induced cell death in neuroblastoma cell lines, proapoptotic response to this promising chemopreventive agent is mediated by suppression of survivin protein expression. The Noscapine treatment increased levels of total and Ser(15-phosphorylated p53 protein in SK-SY5Y cells, but the proapoptotic response to this agent was maintained even after knockdown of the p53 protein level. Exposure of SK-SY5Y and LA1-5S cells to Noscapine resulted in a marked decrease in protein and mRNA level of survivin as early as 12 hours after treatment. Ectopic expression of survivin conferred statistically significant protection against Noscapine-mediated cytoplasmic histone-associated apoptotic DNA fragmentation. Also, the Noscapine-induced apoptosis was modestly but statistically significantly augmented by RNA interference of survivin in both cell lines. Furthermore, Noscapine-induced apoptotic cell death was associated with activation of caspase-3 and cleavage of PARP. In conclusion, the present study provides novel insight into the molecular circuitry of Noscapine-induced apoptosis to indicate suppression of survivin expression as a critical mediator of this process.

  9. Identification of Semaphorin3B as a Direct Target of p53

    OpenAIRE

    Kensuke Ochi; Toshiki Mori; Yoshiaki Toyama; Yusuke Nakamura; Hirofumi Arakawa

    2002-01-01

    A cDNA microarray analysis indicated that Semaphorin3B. (20Sema3B), a gene whose product is involved in axon guidance and axonal repulsion, is inducible by p53. Introduction of exogenous p53 into a glioblastoma cell line lacking wild-type p53. (20U373MG) dramatically induced expression of Sema3B mRNA. An electrophoretic mobility shift assay and a reporter assay confirmed that a potential p53 binding site present in the promoter region had p53-dependent transcriptional activity. Expression of ...

  10. Cyclin K as a Direct Transcriptional Target of the p53 Tumor Suppressor

    OpenAIRE

    Toshiki Mori; Yoshio Anazawa; Kuniko Matsui; Seisuke Fukuda; Yusuke Nakamura; Hirofumi Arakawa

    2002-01-01

    Cyclin K, a newly recognized member of the “transcription” cyclin family, may play a dual role by regulating CDK and transcription. Using cDNA microarray technology, we found that cyclin K mRNA was dramatically increased in U373MG, a glioblastoma cell line deficient in wild-type p53, in the presence of exogenous p53. An electrophoretic mobility-shift assay showed that a potential p53-binding site (p53BS) in intron 1 of the cyclin K gene could indeed bind to p53 protein. Moreover, a heterologo...

  11. Effects of Temperature on the p53-DNA Binding Interactions and Their Dynamical Behavior: Comparing the Wild Type to the R248Q Mutant

    Science.gov (United States)

    Barakat, Khaled; Issack, Bilkiss B.; Stepanova, Maria; Tuszynski, Jack

    2011-01-01

    Background The protein p53 plays an active role in the regulation of cell cycle. In about half of human cancers, the protein is inactivated by mutations located primarily in its DNA-binding domain. Interestingly, a number of these mutations possess temperature-induced DNA-binding characteristics. A striking example is the mutation of Arg248 into glutamine or tryptophan. These mutants are defective for binding to DNA at 310 K although they have been shown to bind specifically to several p53 response elements at sub-physiological temperatures (298–306 K). Methodology/Principal Findings This important experimental finding motivated us to examine the effects of temperature on the structure and configuration of R248Q mutant and compare it to the wild type protein. Our aim is to determine how and where structural changes of mutant variants take place due to temperature changes. To answer these questions, we compared the mutant to the wild-type proteins from two different aspects. First, we investigated the systems at the atomistic level through their DNA-binding affinity, hydrogen bond networks and spatial distribution of water molecules. Next, we assessed changes in their long-lived conformational motions at the coarse-grained level through the collective dynamics of their side-chain and backbone atoms separately. Conclusions The experimentally observed effect of temperature on the DNA-binding properties of p53 is reproduced. Analysis of atomistic and coarse-grained data reveal that changes in binding are determined by a few key residues and provide a rationale for the mutant-loss of binding at physiological temperatures. The findings can potentially enable a rescue strategy for the mutant structure. PMID:22110706

  12. Effects of temperature on the p53-DNA binding interactions and their dynamical behavior: comparing the wild type to the R248Q mutant.

    Directory of Open Access Journals (Sweden)

    Khaled Barakat

    Full Text Available BACKGROUND: The protein p53 plays an active role in the regulation of cell cycle. In about half of human cancers, the protein is inactivated by mutations located primarily in its DNA-binding domain. Interestingly, a number of these mutations possess temperature-induced DNA-binding characteristics. A striking example is the mutation of Arg248 into glutamine or tryptophan. These mutants are defective for binding to DNA at 310 K although they have been shown to bind specifically to several p53 response elements at sub-physiological temperatures (298-306 K. METHODOLOGY/PRINCIPAL FINDINGS: This important experimental finding motivated us to examine the effects of temperature on the structure and configuration of R248Q mutant and compare it to the wild type protein. Our aim is to determine how and where structural changes of mutant variants take place due to temperature changes. To answer these questions, we compared the mutant to the wild-type proteins from two different aspects. First, we investigated the systems at the atomistic level through their DNA-binding affinity, hydrogen bond networks and spatial distribution of water molecules. Next, we assessed changes in their long-lived conformational motions at the coarse-grained level through the collective dynamics of their side-chain and backbone atoms separately. CONCLUSIONS: The experimentally observed effect of temperature on the DNA-binding properties of p53 is reproduced. Analysis of atomistic and coarse-grained data reveal that changes in binding are determined by a few key residues and provide a rationale for the mutant-loss of binding at physiological temperatures. The findings can potentially enable a rescue strategy for the mutant structure.

  13. p53 Isoforms

    Science.gov (United States)

    Khoury, Marie P.; Bourdon, Jean-Christophe

    2011-01-01

    Normal function of the p53 pathway is ubiquitously lost in cancers either through mutation or inactivating interaction with viral or cellular proteins. However, it is difficult in clinical studies to link p53 mutation status to cancer treatment and clinical outcome, suggesting that the p53 pathway is not fully understood. We have recently reported that the human p53 gene expresses not only 1 but 12 different p53 proteins (isoforms) due to alternative splicing, alternative initiation of translation, and alternative promoter usage. p53 isoform proteins thus contain distinct protein domains. They are expressed in normal human tissues but are abnormally expressed in a wide range of cancer types. We have recently reported that p53 isoform expression is associated with breast cancer prognosis, suggesting that they play a role in carcinogenesis. Indeed, the cellular response to damages can be switched from cell cycle arrest to apoptosis by only manipulating p53 isoform expression. This may provide an explanation to the hitherto inconsistent relationship between p53 mutation, treatment response, and outcome in breast cancer. However, the molecular mechanism is still unknown. Recent reports suggest that it involves modulation of gene expression in a p53-dependent and -independent manner. In this review, we summarize our current knowledge about the biological activities of p53 isoforms and propose a molecular mechanism conciliating our current knowledge on p53 and integrating p63 and p73 isoforms in the p53 pathway. PMID:21779513

  14. Study of Bioreductive Anticancer Agent RH-1-Induced Signals Leading the Wild-Type p53-Bearing Lung Cancer A549 Cells to Apoptosis.

    Science.gov (United States)

    Stulpinas, Aurimas; Imbrasaitė, Aušra; Krestnikova, Natalija; Šarlauskas, Jonas; Čėnas, Narimantas; Kalvelytė, Audronė Valerija

    2016-01-19

    Aziridinylquinone RH-1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-cyclohexa-2,5-diene-1,4-dione) is a potential anticancer agent. RH-1 action is associated with quinone oxidoreductase (NQO1) which reduces this diaziridinylbenzoquinone into DNA-alkylating hydroquinone and is overexpressed in many tumors. Another suggested mechanism of RH-1 toxicity is the formation of reactive oxygen species (ROS) arising from its redox cycling. In order to improve anticancer action of this and similar antitumor quinones, we investigated the involvement of different signaling molecules in cytotoxicity induced by RH-1 by using wild-type tumor suppressor p53 bearing nonsmall cell lung carcinoma A549 cells as a model. Gradual and prolonged increase of mitogen-activated protein kinases (MAPK) ERK, P38, and JNK phosphorylation was observed during 24-h RH-1 treatment. In parallel, activation of DNA damage-sensing ATM kinase, upregulation, and phosphorylation of TP53 (human p53) took place. Inhibition studies revealed that RH-1-induced A549 apoptosis involved the NQO1-ATM-p53 signaling pathway and ROS generation. TP53 participated in ROS- and DNA damage-induced cell death differently. Moreover, MAP kinase JNK was another TP53 activator and death inducer in A549 cells. At the same time, rapid and prolonged activation of AKT kinase during RH-1 treatment was found, and it proved to be antiapoptotic kinase in our model system. Therefore, we identified that different and opposite cell death regulating signaling pathways, which may counteract one another, are induced in cancer cells during chemotherapeutic RH-1 treatment.

  15. Spectra of spontaneous and X-ray-induced mutations at the hprt locus in related human lymphoblast cell lines that express wild-type or mutant p53

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, E.N.; Xia, F.; Kelsey, K.T.; Liber, H.L. [Harvard School of Public Health, Boston, MA (United States)

    1995-09-01

    Previous work showed that WTK1 human lymphoblastoid cells are radioresistant but more sensitive to X-ray-induced mutation than the closely related line TK6. In addition, WTK1 cells contain a mutant p53 while in TK6 cells p53 is wild-type. In this work, we examined the spectra of 68 X-ray-induced and 56 spontaneous mutants at the hemizygous hprt locus in WTK1 cells. The induced spectra were classified by Southern blot and multiplex polymerase chain reaction (PCR); there were 19 point mutations (28%) with an unaltered Southern blot or PCR pattern, 26 (38%) partial deletions or rearrangements and 23 (34%) complete gene deletions. The spontaneous spectrum included 25 (45%) point mutations, 22 (39%) partial deletions and 9 (16%) complete gene deletions. These spectra of mutations were compared to those of TK6 cells. Although distinct differences in the spectra of mutations at the tk locus were reported previously, overall there is no significant difference in the spectra of X-ray-induced or spontaneous mutations at the hprt locus in these two cell lines. While there was an increase in the proportion of large-scale changes that occurred at tk after X irradiation, the spectrum of mutations at the hprt locus shows all classes of mutations increasing proportionately in WTK1 cells. However, the proportion of internal partial deletion mutations at the hprt locus was about 2 times more frequent in WTK1 than in TK6 cells. 39 refs., 2 figs., 2 tabs.

  16. Superior anti-tumor activity of the MDM2 antagonist idasanutlin and the Bcl-2 inhibitor venetoclax in p53 wild-type acute myeloid leukemia models

    Directory of Open Access Journals (Sweden)

    Christian Lehmann

    2016-06-01

    Full Text Available Abstract Background Venetoclax, a small molecule BH3 mimetic which inhibits the anti-apoptotic protein Bcl-2, and idasanutlin, a selective MDM2 antagonist, have both shown activity as single-agent treatments in pre-clinical and clinical studies in acute myeloid leukemia (AML. In this study, we deliver the rationale and molecular basis for the combination of idasanutlin and venetoclax for treatment of p53 wild-type AML. Methods The effect of idasanutlin and venetoclax combination on cell viability, apoptosis, and cell cycle progression was investigated in vitro using established AML cell lines. In vivo efficacy was demonstrated in subcutaneous and orthotopic xenograft models generated in female nude or non-obese diabetic/severe combined immunodeficiency (NOD/SCID mice. Mode-of-action analyses were performed by means of cell cycle kinetic studies, RNA sequencing as well as western blotting experiments. Results Combination treatment with venetoclax and idasanutlin results in synergistic anti-tumor activity compared with the respective single-agent treatments in vitro, in p53 wild-type AML cell lines, and leads to strongly superior efficacy in vivo, in subcutaneous and orthotopic AML models. The inhibitory effects of idasanutlin were cell-cycle dependent, with cells arresting in G1 in consecutive cycles and the induction of apoptosis only evident after cells had gone through at least two cell cycles. Combination treatment with venetoclax removed this dependency, resulting in an acceleration of cell death kinetics. As expected, gene expression studies using RNA sequencing showed significant alterations to pathways associated with p53 signaling and cell cycle arrest (CCND1 pathway in response to idasanutlin treatment. Only few gene expression changes were observed for venetoclax treatment and combination treatment, indicating that their effects are mediated mainly at the post-transcriptional level. Protein expression studies demonstrated that

  17. p53 mutations in urinary bladder cancer

    OpenAIRE

    Berggren, P; Steineck, G; Adolfsson, J; Hansson, J; Jansson, O; Larsson, P; Sandstedt, B; Wijkstr?m, H; Hemminki, K

    2001-01-01

    We have screened for mutations in exons 5?8 of the p53 gene in a series consisting of 189 patients with urinary bladder neoplasms. 82 (44%) neoplasms were lowly malignant (Ta, G1?G2a) and 106 (56%) were highly malignant (G2b?G4 or ?T1). Only one mutation was in a lowly malignant urinary bladder neoplasm, in total we found p53 mutations in 26 (14%) of the 189 patients. 30% of the samples had loss of heterozygosity (LOH) for one or both of the p53 exogenic (CA)n repeat and the p53 intragenic (A...

  18. Lysosomal destabilization in p53-induced apoptosis

    OpenAIRE

    Yuan, Xi-Ming; Li, Wei; Dalen, Helge; Lotem, Joseph; Kama, Rachel; Sachs, Leo; Brunk, Ulf T.

    2002-01-01

    The tumor suppressor wild-type p53 can induce apoptosis. M1-t-p53 myeloid leukemic cells have a temperature-sensitive p53 protein that changes its conformation to wild-type p53 after transfer from 37°C to 32°C. We have now found that these cells showed an early lysosomal rupture after transfer to 32°C. Mitochondrial damage, including decreased membrane potential and release of cytochrome c, and the appearance of apoptotic cells occurred later. Lysosomal rupture, mitochondrial damage, and apop...

  19. Nutlin-3 preferentially sensitises wild-type p53-expressing cancer cells to DR5-selective TRAIL over rhTRAIL

    NARCIS (Netherlands)

    Meijer, A.; Kruyt, F. A. E.; van der Zee, A. G. J.; Hollema, H.; Le, P.; ten Hoor, K. A.; Groothuis, G. M. M.; Quax, W. J.; de Vries, E. G. E.; de Jong, S.

    2013-01-01

    BACKGROUND: Tumour cell-selective activation of apoptosis by recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL) is enhanced through co-activation of p53 by chemotherapeutic drugs. The novel anticancer agent nutlin-3 provides a promising alternative for p53 activation by disrupting the

  20. Gene p53 mutations, protein p53, and anti-p53 antibodies as biomarkers of cancer process.

    Science.gov (United States)

    Lutz, Waldemar; Nowakowska-Swirta, Ewa

    2002-01-01

    The finding that gene mutations and changes in their expression form the basis of cancer processes, has prompted molecular epidemiologists to use biomarkers for detecting damaged genes or proteins synthesized under their control in easily available cellular material or systemic liquids. Mutations in the suppressor gen p53 are thought to be essential for cancer development. This gen is one of the most important regulators of transcription, cellular cycle, DNA repair and apoptosis detected till now. Inactivation of gene p53 leads to uncontrolled cell divisions, and further to transformation of normal cells into the carcinous ones. Observations that mutations in gene p53 appear under conditions of occupational and environmental exposures to chemical and physical carcinogens, such as vinyl chloride, radon, or aflatoxin B1, have proved to be of enormous importance for the occupational and environmental health. Changes in expression of gene p53, and also its mutations, cause variations of cellular protein p53 concentration. Higher cellular protein p53 levels are associated with increased protein transfer to the extracellular liquid and to blood. It has been observed that increased blood serum protein p53 concentrations may have a prognostic value in early diagnosis of lung cancer. The results of a number of studies confirm that accumulation of a mutated form of protein p53, and presumably also large quantities of wild forms of that protein in the cells, may be a factor that triggers the production of anti-p53 antibodies. Statistical analysis showed that anti-p53 antibodies can be regarded as a specific biomarker of cancer process. The prevalence of anti-p53 antibodies correlated with the degree of cancer malignancy. The increased incidence of anti-p53 antibodies was also associated with higher frequency of mutations in gene p53. There are some reports confirming that anti-p53 antibodies emerging in blood serum in the subclinical phase of cancer development may be

  1. Binding of Amphipathic Cell Penetrating Peptide p28 to Wild Type and Mutated p53 as studied by Raman, Atomic Force and Surface Plasmon Resonance spectroscopies.

    Science.gov (United States)

    Signorelli, Sara; Santini, Simona; Yamada, Tohru; Bizzarri, Anna Rita; Beattie, Craig W; Cannistraro, Salvatore

    2017-04-01

    Mutations within the DNA binding domain (DBD) of the tumor suppressor p53 are found in >50% of human cancers and may significantly modify p53 secondary structure impairing its function. p28, an amphipathic cell-penetrating peptide, binds to the DBD through hydrophobic interaction and induces a posttranslational increase in wildtype and mutant p53 restoring functionality. We use mutation analyses to explore which elements of secondary structure may be critical to p28 binding. Molecular modeling, Raman spectroscopy, Atomic Force Spectroscopy (AFS) and Surface Plasmon Resonance (SPR) were used to identify which secondary structure of site-directed and naturally occurring mutant DBDs are potentially altered by discrete changes in hydrophobicity and the molecular interaction with p28. We show that specific point mutations that alter hydrophobicity within non-mutable and mutable regions of the p53 DBD alter specific secondary structures. The affinity of p28 was positively correlated with the β-sheet content of a mutant DBD, and reduced by an increase in unstructured or random coil that resulted from a loss in hydrophobicity and redistribution of surface charge. These results help refine our knowledge of how mutations within p53-DBD alter secondary structure and provide insight on how potential structural alterations in p28 or similar molecules improve their ability to restore p53 function. Raman spectroscopy, AFS, SPR and computational modeling are useful approaches to characterize how mutations within the p53DBD potentially affect secondary structure and identify those structural elements prone to influence the binding affinity of agents designed to increase the functionality of p53. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Identification of p53 in mitochondria.

    Science.gov (United States)

    Vaseva, Angelina V; Moll, Ute M

    2013-01-01

    p53 is a master regulator of cell death pathways and has transcription-dependent and transcription-independent modes of action. Mitochondria are major signal transducers in apoptosis and are critical for p53-dependent cell death. Our lab and others have discovered that a fraction of stress-induced wild-type p53 protein rapidly translocates to mitochondria upon various stress stimuli and exerts p53-dependent apoptosis. Suborganellar localization by various methods shows that p53 localizes to the surface of mitochondria. Direct targeting of p53 to mitochondria is sufficient to induce apoptosis in p53-null cells, without requiring further DNA damage. Recently, p53 has been also shown to localize to other mitochondrial compartments such as the mitochondrial matrix where it plays a role in maintaining mitochondrial genome integrity. Here, we describe subcellular fractionation as a classic technique for detecting mitochondrial p53 in cell extracts. It consists of cell homogenization by hypo-osmotic swelling, removal of nuclear components by low-speed centrifugation, and mitochondrial isolation by a discontinuous sucrose density gradient. Additionally, we describe a method for submitochondrial fractionation, performed by phosphate buffer mediated swelling/shrinking. p53 and other mitochondrial proteins can then be detected by standard immunoblotting procedures. The quality of mitochondrial isolates/subfractions can be verified for purity and intactness.

  3. NF-κB p50 activation associated with immune dysregulation confers poorer survival for diffuse large B-cell lymphoma patients with wild-type p53

    DEFF Research Database (Denmark)

    Cai, Qingqing; Tu, Meifeng; Xu-Monette, Zijun Y

    2017-01-01

    -AKT, and CXCR4 expression and less frequent BCL2 translocations. In contrast, in germinal center B-cell-like diffuse large B-cell lymphoma patients with TP53 mutations, p50(+) nuclear expression correlated with significantly better clinical outcomes, and decreased p53, Bcl-2, and Myc expression. Gene expression...... without TP53 mutations. The results also provide insights into the regulation and function of p50 in diffuse large B-cell lymphoma and its cross talk with the p53 pathway with important therapeutic implications....

  4. In vitro and clinical studies of gene therapy with recombinant human adenovirus-p53 injection for oral leukoplakia.

    Science.gov (United States)

    Li, Yi; Li, Long-Jiang; Zhang, Song-Tao; Wang, Li-Juan; Zhang, Zhuang; Gao, Ning; Zhang, Yuan-Yuan; Chen, Qian-Ming

    2009-11-01

    Oral leukoplakia is a well-recognized precancerous lesion of squamous cell carcinoma. When accompanied with abnormal p53 expression, it suffered a higher risk of canceration. The present study was carried out to test whether the recombinant human adenovirus-p53 could introduce wild-type p53 gene to oral leukoplakia cells and induce cell cycle arrest and apoptosis. We select p53(-) oral dysplastic keratinocyte POE-9n, to observe the growth inhibition, cell cycle change, apoptosis-induced effects, and elaborate the corresponding molecular mechanism of recombinant adenovirus-p53 on POE-9n cells. Meanwhile, we evaluate the feasibility, safety, and biological activity of multipoints intraepithelial injections of recombinant adenovirus-p53 in 22 patients with dysplastic oral leukoplakia. Exogenous p53 could be successfully transduced into POE-9n cells by recombinant adenovirus-p53. The optimal infecting titer in this study was multiplicity of infection (MOI) = 100. Recombinant adenovirus-p53 could strongly inhibit cell proliferation, induce apoptosis, and arrest cell cycle in stage G(1) in POE-9n cells by inducing p21(CIP/WAF) and downregulating bcl-2 expression. In the posttreatment patients, p53 protein and p21(CIP/WAF) protein expression were significantly enhanced, yet bcl-2 protein presented low expression. Sixteen patients showed clinical response to the treatment, and 14 patients showed obvious histopathologic improvement. Intraepithelial injections of recombinant human adenovirus-p53 were safe, feasible, and biologically active for patients with dysplastic oral leukoplakia.

  5. CLCA2 as a p53-Inducible Senescence Mediator

    OpenAIRE

    Chizu Tanikawa; Hidewaki Nakagawa; Yoichi Furukawa; Yusuke Nakamura; Koichi Matsuda

    2012-01-01

    p53 is a tumor suppressor gene that is frequently mutated in multiple cancer tissues. Activated p53 protein regulates its downstream genes and subsequently inhibits malignant transformation by inducing cell cycle arrest, apoptosis, DNA repair, and senescence. However, genes involved in the p53-mediated senescence pathway are not yet fully elucidated. Through the screening of two genome-wide expression profile data sets, one for cells in which exogenous p53 was introduced and the other for sen...

  6. OTUD5 regulates p53 stability by deubiquitinating p53.

    Directory of Open Access Journals (Sweden)

    Judong Luo

    Full Text Available The p53 tumour suppressor protein is a transcription factor that prevents oncogenic progression by activating the expression of apoptosis and cell-cycle arrest genes in stressed cells. The stability of p53 is tightly regulated by ubiquitin-dependent degradation, driven mainly by its negative regulators ubiquitin ligase MDM2.In this study, we have identified OTUD5 as a DUB that interacts with and deubiquitinates p53. OTUD5 forms a direct complex with p53 and controls level of ubiquitination. The function of OTUD5 is required to allow the rapid activation of p53-dependent transcription and a p53-dependent apoptosis in response to DNA damage stress.As a novel deubiquitinating enzyme for p53, OTUD5 is required for the stabilization and the activation of a p53 response.

  7. Inhibition of p53-induced apoptosis without affecting expression of p53-regulated genes

    OpenAIRE

    Lotem, Joseph; Gal, Hilah; Kama, Rachel; Amariglio, Ninette; Rechavi, Gideon; Domany, Eytan; Sachs, Leo; Givol, David

    2003-01-01

    Using DNA microarray and clustering of expressed genes we have analyzed the mechanism of inhibition of wild-type p53-induced apoptosis by the cytokine interleukin 6 (IL-6) and the calcium mobilizer thapsigargin (TG). Clustering analysis of 1,786 genes, the expression level of which changed after activation of wild-type p53 in the absence or presence of IL-6 or TG, showed that these compounds did not cause a general inhibition of the ability of p53 to up-regulate or down-regulate gene ex...

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

    Science.gov (United States)

    Olivos, David J; Mayo, Lindsey D

    2016-11-26

    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.

  9. Mutant p53 in Cancer: New Functions and Therapeutic Opportunities

    Science.gov (United States)

    Muller, Patricia A.J.; Vousden, Karen H.

    2014-01-01

    Many different types of cancer show a high incidence of TP53 mutations, leading to the expression of mutant p53 proteins. There is growing evidence that these mutant p53s have both lost wild-type p53 tumor suppressor activity and gained functions that help to contribute to malignant progression. Understanding the functions of mutant p53 will help in the development of new therapeutic approaches that may be useful in a broad range of cancer types. PMID:24651012

  10. Cytotoxic T-lymphocyte clones, established by stimulation with the HLA-A2 binding p5365-73 wild type peptide loaded on dendritic cells In vitro, specifically recognize and lyse HLA-A2 tumour cells overexpressing the p53 protein

    DEFF Research Database (Denmark)

    Barfoed, Annette Malene; Petersen, T R; Kirkin, A F

    2000-01-01

    Mutations in the tumour suppressor gene p53 are among the most frequent genetic alterations in human malignancies, often associated with an accumulation of the p53 protein in the cytoplasm. We have generated a number of cytotoxic T lymphocyte (CTL) clones that specifically recognize the HLA-A*0201...... of recognizing p53 derived wild type (self) peptides. Furthermore, the capacity of R9V specific T cell clones to exert HLA restricted cytotoxicity, argues that the R9V peptide is naturally presented on certain cancer cells. This supports the view that p53 derived wild type peptides might serve as candidate...

  11. The MDM2-p53 pathway revisited

    Science.gov (United States)

    Nag, Subhasree; Qin, Jiangjiang; Srivenugopal, Kalkunte S.; Wang, Minghai; Zhang, Ruiwen

    2013-01-01

    The p53 tumor suppressor is a key transcription factor regulating cellular pathways such as DNA repair, cell cycle, apoptosis, angiogenesis, and senescence. It acts as an important defense mechanism against cancer onset and progression, and is negatively regulated by interaction with the oncoprotein MDM2. In human cancers, the TP53 gene is frequently mutated or deleted, or the wild-type p53 function is inhibited by high levels of MDM2, leading to downregulation of tumor suppressive p53 pathways. Thus, the inhibition of MDM2-p53 interaction presents an appealing therapeutic strategy for the treatment of cancer. However, recent studies have revealed the MDM2-p53 interaction to be more complex involving multiple levels of regulation by numerous cellular proteins and epigenetic mechanisms, making it imperative to reexamine this intricate interplay from a holistic viewpoint. This review aims to highlight the multifaceted network of molecules regulating the MDM2-p53 axis to better understand the pathway and exploit it for anticancer therapy. PMID:23885265

  12. Expression of Androgen Receptor Is Negatively Regulated By p53

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    Fatouma Alimirah

    2007-12-01

    Full Text Available Increased expression of androgen receptor (AR in prostate cancer (PC is associated with transition to androgen independence. Because the progression of PC to advanced stages is often associated with the loss of p53 function, we tested whether the p53 could regulate the expression of AR gene. Here we report that p53 negatively regulates the expression of AR in prostate epithelial cells (PrECs. We found that in LNCaP human prostate cancer cells that express the wild-type p53 and AR and in human normal PrECs, the activation of p53 by genotoxic stress or by inhibition of p53 nuclear export downregulated the expression of AR. Furthermore, forced expression of p53 in LNCaP cells decreased the expression of AR. Conversely, knockdown of p53 expression in LNCaP cells increased the AR expression. Consistent with the negative regulation of AR expression by p53, the p53-null HCT116 cells expressed higher levels of AR compared with the isogenic HCT116 cells that express the wildtype p53. Moreover, we noted that in etoposide treated LNCaP cells p53 bound to the promoter region of the AR gene, which contains a potential p53 DNA-binding consensus sequence, in chromatin immunoprecipitation assays. Together, our observations provide support for the idea that the loss of p53 function in prostate cancer cells contributes to increased expression of AR.

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

    Science.gov (United States)

    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.

  14. Chemical Variations on the p53 Reactivation Theme

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    Carlos J. A. Ribeiro

    2016-05-01

    Full Text Available 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.

  15. Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents

    NARCIS (Netherlands)

    Michaelis, M.; Rothweiler, F.; Agha, B.; Barth, S.; Voges, Y.; Loeschmann, N.; von Deimling, A.; Breitling, R.; Doerr, H. Wilhelm; Roedel, F.; Speidel, D.; Cinatl, J.; Cinatl Jr., J.; Stephanou, A.

    Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3,

  16. NQO1 stabilizes p53 through a distinct pathway

    OpenAIRE

    Asher, Gad; Lotem, Joseph; Kama, Rachel; Sachs, Leo; Shaul, Yosef

    2002-01-01

    Wild-type p53 is a tumor-suppressor gene that encodes a short-lived protein that, upon accumulation, induces growth arrest or apoptosis. Accumulation of p53 occurs mainly by posttranslational events that inhibit its proteosomal degradation. We have reported previously that inhibition of NAD(P)H: quinone oxidoreductase 1 (NQO1) activity by dicoumarol induces degradation of p53, indicating that NQO1 plays a role in p53 stabilization. We now have found that wild-type NQO1, but not the inactive p...

  17. Adenoviral vector-mediated gene transfer: timing of wild-type p53 gene expression in vivo and effect of tumor transduction on survival in a rat glioma brachytherapy model.

    Science.gov (United States)

    Bampoe, J; Glen, J; Hubbard, S L; Salhia, B; Shannon, P; Rutka, J; Bernstein, M

    2000-08-01

    This study sought to investigate modification of the radiation response in a rat 9L brain tumor model in vivo by the wild-type p53 gene (wtp53). Determination of the timing and dose of radiation therapy required the assessment of the duration of the effect of wtp53 expression on 9L tumors after in vivo transfection. Anesthetized male F-344 rats each were stereotactically inoculated with 4 x 10(4) 9L gliosarcoma cells through a skull screw into the cerebrum in the right frontal region. Twelve-day-old tumors were inoculated through the screw with recombinant adenoviral vectors under isoflurane anaesthesia: control rats with Ad5/RSV/GL2 (carrying the luciferase gene), and study rats with Ad5CMV-p53 (carrying the wtp53 gene). Brain tumors removed at specific times after transfection were measured, homogenized, and lysed and wtp53 expression determined by Western blot analysis. Four groups of nine rats were, subsequently, implanted with iodine-125 seeds 15 days post-tumor inoculation to give a minimum tumor dose of 40 or 60 Gy. We demonstrated transfer of wtp53 into rat 9L tumors in vivo using the Ad5CMV-p53 vector. The expression of wtp53 was demonstrated to be maximum between days 1 and 3 post-vector inoculation. Tumors expressing wtp53 were smaller than controls transfected with Ad5/RSV/GL2 but this difference was not statistically significant. Radiation made a significant difference to the survival of tumor-bearing rats. Moreover, wtp53 expression conferred a significant additional survival advantage. The expression of wtp53 significantly improves the survival of irradiated tumor-bearing rats in our model.

  18. Role of p53 in Cell Death and Human Cancers

    Science.gov (United States)

    Ozaki, Toshinori; Nakagawara, Akira

    2011-01-01

    p53 is a nuclear transcription factor with a pro-apoptotic function. Since over 50% of human cancers carry loss of function mutations in p53 gene, p53 has been considered to be one of the classical type tumor suppressors. Mutant p53 acts as the dominant-negative inhibitor toward wild-type p53. Indeed, mutant p53 has an oncogenic potential. In some cases, malignant cancer cells bearing p53 mutations display a chemo-resistant phenotype. In response to a variety of cellular stresses such as DNA damage, p53 is induced to accumulate in cell nucleus to exert its pro-apoptotic function. Activated p53 promotes cell cycle arrest to allow DNA repair and/or apoptosis to prevent the propagation of cells with serious DNA damage through the transactivation of its target genes implicated in the induction of cell cycle arrest and/or apoptosis. Thus, the DNA-binding activity of p53 is tightly linked to its tumor suppressive function. In the present review article, we describe the regulatory mechanisms of p53 and also p53-mediated therapeutic strategies to cure malignant cancers. PMID:24212651

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

    Directory of Open Access Journals (Sweden)

    David J. Olivos

    2016-11-01

    Full Text Available 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.

  20. Targeting the p53 Pathway in Ewing Sarcoma

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    Paul M. Neilsen

    2011-01-01

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

  1. Stability of p53 homologs.

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    Tobias Brandt

    Full Text Available Most proteins have not evolved for maximal thermal stability. Some are only marginally stable, as for example, the DNA-binding domains of p53 and its homologs, whose kinetic and thermodynamic stabilities are strongly correlated. Here, we applied high-throughput methods using a real-time PCR thermocycler to study the stability of several full-length orthologs and paralogs of the p53 family of transcription factors, which have diverse functions, ranging from tumour suppression to control of developmental processes. From isothermal denaturation fluorimetry and differential scanning fluorimetry, we found that full-length proteins showed the same correlation between kinetic and thermodynamic stability as their isolated DNA-binding domains. The stabilities of the full-length p53 orthologs were marginal and correlated with the temperature of their organism, paralleling the stability of the isolated DNA-binding domains. Additionally, the paralogs p63 and p73 were significantly more stable and long-lived than p53. The short half-life of p53 orthologs and the greater persistence of the paralogs may be biologically relevant.

  2. Blocking of p53-Snail Binding, Promoted by Oncogenic K-Ras, Recovers p53 Expression and function

    Directory of Open Access Journals (Sweden)

    Sun-Hye Lee

    2009-01-01

    Full Text Available Differentially from other kinds of Ras, oncogenic K-Ras, which is mutated approximately 30% of human cancer, does not induce apoptosis and senescence. Here, we provide the evidence that oncogenic K-Ras abrogates p53 function and expression through induction of Ataxia telangiectasia-mutated and Rad3-related mediated Snail stabilization. Snail directly binds to DNA binding domain of p53 and diminishes the tumor-suppressive function of p53. Thus, elimination of Snail through si-RNA can induce p53 in K-Ras-mutated cells, whereas Snail and mutant K-Ras can suppress p53 in regardless of K-Ras status. Chemicals, isolated from inhibitor screening of p53-Snail binding, can block the Snail-mediated p53 suppression and enhance the expression of p53 as well as the transcriptional activity of p53 in an oncogenic K-Ras-dependent manner. Among the chemicals, two are very similar in structure. These results can answer why K-Ras can coexist with wild type p53 and propose the Snail-p53 binding as the new therapeutic target for K-Ras-mutated cancers including pancreatic, lung, and colon cancers.

  3. Expression of Androgen Receptor Is Negatively Regulated By p53

    OpenAIRE

    Fatouma Alimirah; Ravichandran Panchanathan; Jianming Chen; Xiang Zhang; Shuk-Mei Ho; Divaker Choubey

    2007-01-01

    Increased expression of androgen receptor (AR) in prostate cancer (PC) is associated with transition to androgen independence. Because the progression of PC to advanced stages is often associated with the loss of p53 function, we tested whether the p53 could regulate the expression of AR gene. Here we report that p53 negatively regulates the expression of AR in prostate epithelial cells (PrECs). We found that in LNCaP human prostate cancer cells that express the wild-type p53 and AR and in hu...

  4. Combining Oncolytic Virotherapy with p53 Tumor Suppressor Gene Therapy

    Directory of Open Access Journals (Sweden)

    Christian Bressy

    2017-06-01

    Full Text Available Oncolytic virus (OV therapy utilizes replication-competent viruses to kill cancer cells, leaving non-malignant cells unharmed. With the first U.S. Food and Drug Administration-approved OV, dozens of clinical trials ongoing, and an abundance of translational research in the field, OV therapy is poised to be one of the leading treatments for cancer. A number of recombinant OVs expressing a transgene for p53 (TP53 or another p53 family member (TP63 or TP73 were engineered with the goal of generating more potent OVs that function synergistically with host immunity and/or other therapies to reduce or eliminate tumor burden. Such transgenes have proven effective at improving OV therapies, and basic research has shown mechanisms of p53-mediated enhancement of OV therapy, provided optimized p53 transgenes, explored drug-OV combinational treatments, and challenged canonical roles for p53 in virus-host interactions and tumor suppression. This review summarizes studies combining p53 gene therapy with replication-competent OV therapy, reviews preclinical and clinical studies with replication-deficient gene therapy vectors expressing p53 transgene, examines how wild-type p53 and p53 modifications affect OV replication and anti-tumor effects of OV therapy, and explores future directions for rational design of OV therapy combined with p53 gene therapy.

  5. Combining Oncolytic Virotherapy with p53 Tumor Suppressor Gene Therapy.

    Science.gov (United States)

    Bressy, Christian; Hastie, Eric; Grdzelishvili, Valery Z

    2017-06-16

    Oncolytic virus (OV) therapy utilizes replication-competent viruses to kill cancer cells, leaving non-malignant cells unharmed. With the first U.S. Food and Drug Administration-approved OV, dozens of clinical trials ongoing, and an abundance of translational research in the field, OV therapy is poised to be one of the leading treatments for cancer. A number of recombinant OVs expressing a transgene for p53 (TP53) or another p53 family member (TP63 or TP73) were engineered with the goal of generating more potent OVs that function synergistically with host immunity and/or other therapies to reduce or eliminate tumor burden. Such transgenes have proven effective at improving OV therapies, and basic research has shown mechanisms of p53-mediated enhancement of OV therapy, provided optimized p53 transgenes, explored drug-OV combinational treatments, and challenged canonical roles for p53 in virus-host interactions and tumor suppression. This review summarizes studies combining p53 gene therapy with replication-competent OV therapy, reviews preclinical and clinical studies with replication-deficient gene therapy vectors expressing p53 transgene, examines how wild-type p53 and p53 modifications affect OV replication and anti-tumor effects of OV therapy, and explores future directions for rational design of OV therapy combined with p53 gene therapy.

  6. P53 family members modulate the expression of PRODH, but not PRODH2, via intronic p53 response elements.

    Directory of Open Access Journals (Sweden)

    Ivan Raimondi

    Full Text Available The tumor suppressor p53 was previously shown to markedly up-regulate the expression of the PRODH gene, encoding the proline dehydrogenase (PRODH enzyme, which catalyzes the first step in proline degradation. Also PRODH2, which degrades 4-hydroxy-L-proline, a product of protein (e.g. collagen catabolism, was recently described as a p53 target. Here, we confirmed p53-dependent induction of endogenous PRODH in response to genotoxic damage in cell lines of different histological origin. We established that over-expression of TAp73β or TAp63β is sufficient to induce PRODH expression in p53-null cells and that PRODH expression parallels the modulation of endogenous p73 by genotoxic drugs in several cell lines. The p53, p63, and p73-dependent transcriptional activation was linked to specific intronic response elements (REs, among those predicted by bioinformatics tools and experimentally validated by a yeast-based transactivation assay. p53 occupancy measurements were validated in HCT116 and MCF7 human cell lines. Conversely, PRODH2 was not responsive to p63 nor p73 and, at best, could be considered a weak p53 target. In fact, minimal levels of PRODH2 transcript induction by genotoxic stress was observed exclusively in one of four p53 wild-type cell lines tested. Consistently, all predicted p53 REs in PRODH2 were poor matches to the p53 RE consensus and showed very weak responsiveness, only to p53, in the functional assay. Taken together, our results highlight that PRODH, but not PRODH2, expression is under the control of p53 family members, specifically p53 and p73. This supports a deeper link between proteins of the p53-family and metabolic pathways, as PRODH modulates the balance of proline and glutamate levels and those of their derivative alpha-keto-glutarate (α-KG under normal and pathological (tumor conditions.

  7. Transcriptional repression of Aurora-A gene by wild-type p53 through directly binding to its promoter with histone deacetylase 1 and mSin3a.

    Science.gov (United States)

    Yang, Tsung-Ying; Teng, Chieh-Lin Jerry; Lin, Tsung-Chieh Chester; Chen, Kun-Chieh; Hsu, Shih-Lan; Wu, Chun-Chi

    2018-01-01

    In this study, we firstly showed that p53 transcriptionally represses Aurora-A gene expression through directly binding to its promoter. DNA affinity precipitation assay and chromatin immunoprecipitation assay indicated that p53 physically bound to the Aurora-A promoter. Moreover, the in vitro and in vivo assays showed that p53 directly bound to the Aurora-A promoter together with histone deacetylase 1 (HDAC1) and mSin3a as corepressors. Furthermore, we identified that the nucleotides -360 to -354 (CCTGCCC), upstream of the Aurora-A transcriptional start site, was responsible for the p53-mediated repression. Mutation within this site disrupted its interaction with p53, mSin3a and HDAC1, as well as attenuated the repressive effect of p53 on Aurora-A promoter activity. Treatment with trichostatin A (TSA), a HDAC1 inhibitor, disrupted the interaction of p53-HDAC1-mSin3a complex with the nucleotides -365∼-345 region, and enhanced the Aurora-A promoter activity and gene expression. Additionally, knockdown of p53 or mSin3a also drastically blocked the formation of p53-HDAC1-mSin3a repressive complex onto this promoter region and elevated the Aurora-A promoter activity and gene expression. Moreover, the p53-HDAC1-mSin3a repressive complex also involved in the inhibition of Aurora-A gene expression upon cisplatin treatment. Finally, the clinical investigation showed that Aurora-A and p53 exhibited an inverse correlation in both the expression level and prognostic status, and the low p53/high Aurora-A showed the poorest prognosis of NSCLC patients. Our findings showed novel regulatory mechanisms of p53 in regulating Aurora-A gene expression in NSCLC cells. © 2017 UICC.

  8. Restoration of p53 using the novel MDM2-p53 antagonist APG115 suppresses dedifferentiated papillary thyroid cancer cells.

    Science.gov (United States)

    Chen, Haibo; Luo, Dingyuan; Zhang, Lin; Lin, Xiaofeng; Luo, Qiuyun; Yi, Hanjie; Wang, Jing; Yan, Xianglei; Li, Baoxia; Chen, Yuelei; Liu, Xingguang; Zhang, Hong; Liu, Sheng; Qiu, Miaozhen; Yang, Dajun; Jiang, Ningyi

    2017-06-27

    Dedifferentiated papillary thyroid cancer (DePTC) is characterized by aggressive growth, recurrence, distant metastasis, and resistance to radioactive iodine (RAI) therapy. DePTC is also accompanied by poor prognosis and high early-mortality. Nevertheless, most DePTC cells show intact p53 downstream functionality. In cells with wild-type p53, the murine double minute2 (MDM2) protein interacts with p53 and abrogates its activity. Inhibition of the MDM2-p53 interaction restores p53 activity and leads to cell cycle arrest and apoptosis. Restoring p53 function by inhibiting its interaction with p53 suppressors such as MDM2 is thus a promising therapeutic strategy for the treatment of DePTC. The novel MDM2-p53 interaction antagonist APG115 is an analogue of SAR405838, and is being tested in a phase I clinical trial. In this study, we evaluated the efficacy of APG115 as a single-agent to treat DePTC. APG115 diminished the viability of p53 wild-type DePTC cells and induced cell cycle arrest and apoptosis. In a human xenograft mouse model, APG115 elicited robust tumor regression and cell apoptosis. These data demonstrate that further research is warranted to determine whether APG115 can be used to effectively treat DePTC patients.

  9. p53: an overview of over two decades of study.

    Science.gov (United States)

    Cheah, P L; Looi, L M

    2001-06-01

    results achieved in tumour therapy where introduction of wild type p53 gene has resulted in regression of non-small-cell lung cancer (NSCLC). Many other notable developments in this field include description of p53 homologues, "gain of function" mutants, p53 polymorphisms, angiogenesis-inhibiting properties of wild type p53 protein etc.

  10. Battle Against Cancer: An Everlasting Saga of p53

    Directory of Open Access Journals (Sweden)

    Qian Hao

    2014-12-01

    Full Text Available Cancer is one of the most life-threatening diseases characterized by uncontrolled growth and spread of malignant cells. The tumor suppressor p53 is the master regulator of tumor cell growth and proliferation. In response to various stress signals, p53 can be activated and transcriptionally induces a myriad of target genes, including both protein-encoding and non-coding genes, controlling cell cycle progression, DNA repair, senescence, apoptosis, autophagy and metabolism of tumor cells. However, around 50% of human cancers harbor mutant p53 and, in the majority of the remaining cancers, p53 is inactivated through multiple mechanisms. Herein, we review the recent progress in understanding the molecular basis of p53 signaling, particularly the newly identified ribosomal stress—p53 pathway, and the development of chemotherapeutics via activating wild-type p53 or restoring mutant p53 functions in cancer. A full understanding of p53 regulation will aid the development of effective cancer treatments.

  11. p53 isoform Δ113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish

    OpenAIRE

    Chen, Jun; Ng, Sok Meng; Chang, Changqing; Zhang, Zhenhai; Bourdon, Jean-Christophe; Lane, David P; Peng, Jinrong

    2009-01-01

    p53 is a well-known tumor suppressor and is also involved in processes of organismal aging and developmental control. A recent exciting development in the p53 field is the discovery of various p53 isoforms. One p53 isoform is human Δ133p53 and its zebrafish counterpart Δ113p53. These N-terminal-truncated p53 isoforms are initiated from an alternative p53 promoter, but their expression regulation and physiological significance at the organismal level are not well understood. We show here that ...

  12. "Super p53" mice display retinal astroglial changes.

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    Juan J Salazar

    Full Text Available Tumour-suppressor genes, such as the p53 gene, produce proteins that inhibit cell division under adverse conditions, as in the case of DNA damage, radiation, hypoxia, or oxidative stress (OS. The p53 gene can arrest proliferation and trigger death by apoptosis subsequent to several factors. In astrocytes, p53 promotes cell-cycle arrest and is involved in oxidative stress-mediated astrocyte cell death. Increasingly, astrocytic p53 is proving fundamental in orchestrating neurodegenerative disease pathogenesis. In terms of ocular disease, p53 may play a role in hypoxia due to ischaemia and may be involved in the retinal response to oxidative stress (OS. We studied the influence of the p53 gene in the structural and quantitative characteristics of astrocytes in the retina. Adult mice of the C57BL/6 strain (12 months old were distributed into two groups: 1 mice with two extra copies of p53 ("super p53"; n = 6 and 2 wild-type p53 age-matched control, as the control group (WT; n = 6. Retinas from each group were immunohistochemically processed to locate the glial fibrillary acidic protein (GFAP. GFAP+ astrocytes were manually counted and the mean area occupied for one astrocyte was quantified. Retinal-astrocyte distribution followed established patterns; however, morphological changes were seen through the retinas in relation to p53 availability. The mean GFAP+ area occupied by one astrocyte in "super p53" eyes was significantly higher (p<0.05; Student's t-test than in the WT. In addition, astroglial density was significantly higher in the "super p53" retinas than in the WT ones, both in the whole-retina (p<0,01 Student's t-test and in the intermediate and peripheral concentric areas of the retina (p<0.05 Student's t-test. This fact might improve the resistance of the retinal cells against OS and its downstream signalling pathways.

  13. PRIMA-1 and PRIMA-1Met (APR-246: From Mutant/Wild Type p53 Reactivation to Unexpected Mechanisms Underlying Their Potent Anti-Tumor Effect in Combinatorial Therapies

    Directory of Open Access Journals (Sweden)

    Anne Perdrix

    2017-12-01

    Full Text Available p53 protects cells from genetic assaults by triggering cell-cycle arrest and apoptosis. Inactivation of p53 pathway is found in the vast majority of human cancers often due to somatic missense mutations in TP53 or to an excessive degradation of the protein. Accordingly, reactivation of p53 appears as a quite promising pharmacological approach and, effectively, several attempts have been made in that sense. The most widely investigated compounds for this purpose are PRIMA-1 (p53 reactivation and induction of massive apoptosis and PRIMA-1Met (APR-246, that are at an advanced stage of development, with several clinical trials in progress. Based on publications referenced in PubMed since 2002, here we review the reported effects of these compounds on cancer cells, with a specific focus on their ability of p53 reactivation, an overview of their unexpected anti-cancer effects, and a presentation of the investigated drug combinations.

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

    Energy Technology Data Exchange (ETDEWEB)

    Cappadone, C., E-mail: concettina.cappadone@unibo.it [Department of Pharmacy and Biotechnology, University of Bologna, Bologna (Italy); Stefanelli, C. [Department for Life Quality Studies, University of Bologna, Rimini Campus, Rimini (Italy); Malucelli, E. [Department of Pharmacy and Biotechnology, University of Bologna, Bologna (Italy); Zini, M. [Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna (Italy); Onofrillo, C. [Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna (Italy); Locatelli, A.; Rambaldi, M.; Sargenti, A. [Department of Pharmacy and Biotechnology, University of Bologna, Bologna (Italy); Merolle, L. [ELETTRA–Sincrotrone Trieste S.C.p.A., Trieste (Italy); Farruggia, G. [Department of Pharmacy and Biotechnology, University of Bologna, Bologna (Italy); National Institute of Biostructures and Biosystems, Roma (Italy); Graziadio, A. [Department of Pharmacy and Biotechnology, University of Bologna, Bologna (Italy); Montanaro, L. [Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna (Italy); Iotti, S. [Department of Pharmacy and Biotechnology, University of Bologna, Bologna (Italy); National Institute of Biostructures and Biosystems, Roma (Italy)

    2015-11-13

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

  15. Functional four-base A/T gap core sequence CATTAG of P53 response elements specifically bound tetrameric P53 differently than two-base A/T gap core sequence CATG bound both dimeric and tetrameric P53.

    Science.gov (United States)

    Cai, Bi-He; Chen, Jang-Yi; Lu, Mei-Hua; Chang, Li-Tze; Lin, Hwang-Chi; Chang, Yu-Ming; Chao, Chung-Faye

    2009-04-01

    The consensus sequence of p53 is repeated half sites of PuPuPuC(A/T)(A/T)GPyPyPy. GtAGCAttAGCCCAGACATGTCC is a 14-3-3sigma promoter p53 regulation site; the first core sequence is CAttAG, and the second is CATG. Both mutants GtAGgAttAGCCCAGACATGTCC and GtAGCAttAGCCCAGACATcTCC can be activated by p53 as a 1.5-fold half site. The original p53 regulated site on the 14-3-3sigma promoter is a whole site, and CATTAG is a functional core sequence. The p53-binding affinity and the activity of CATTAG were lower than for the mutant CATATG core sequence. Wild-type p53 acts as a tetramer to bind to the whole site; however, it also can bind to a half site by one of its dimers. Wild-type p53 can only bind to a half site with core sequence CATG but not to CATATG. The 1.5-fold half site or whole site with core sequence CATATG can be bound by wild-type p53. A p53 mutant, A344, forms dimeric p53; it can only bind to CATG, and not to CATATG. Therefore, tetrameric and dimeric p53 can bind to a two-base A/T gap core sequence, but only tetrameric p53 can bind to a four-base A/T gap core sequence.

  16. Malign Melanomda Serum P53 Onkoprotein Düzeyleri

    OpenAIRE

    Özbek, Uğur; Şengün, Zeynep; Kurul, Sıdıka; Aydıner, Adnan; Ertürk, Nurcan; Topuz, Erkan

    1994-01-01

    p53, SV 40 trasforme edilmiş hücrelerde bulunmuş ve tanımlanmış bir nukleer fosfoproteindir. P53'ün mutasyona uğramış ya da 'wild-type' formu, hücre bölünmesinde negatif regülatör rolü olan bir tümör-supressör gen olarak kabul edilir. P53 geninin yüksek oranda korunmuş dizilerinden birinde nokta mutasyonu ortaya çıktığında, aktive olmuş onkogen ürünü özellikleri gösteren bir mutant protein eksprese edilir. p53 proteini monoklonal antikorlarla ayırt edilebilir. Mutant p53 serum ...

  17. CLCA2 as a p53-Inducible Senescence Mediator

    Directory of Open Access Journals (Sweden)

    Chizu Tanikawa

    2012-02-01

    Full Text Available p53 is a tumor suppressor gene that is frequently mutated in multiple cancer tissues. Activated p53 protein regulates its downstream genes and subsequently inhibits malignant transformation by inducing cell cycle arrest, apoptosis, DNA repair, and senescence. However, genes involved in the p53-mediated senescence pathway are not yet fully elucidated. Through the screening of two genome-wide expression profile data sets, one for cells in which exogenous p53 was introduced and the other for senescent fibroblasts, we have identified chloride channel accessory 2 (CLCA2 as a p53-inducible senescence-associated gene. CLCA2 was remarkably induced by replicative senescence as well as oxidative stress in a p53-dependent manner. We also found that ectopically expressed CLCA2 induced cellular senescence, and the down-regulation of CLCA2 by small interfering RNA caused inhibition of oxidative stress-induced senescence. Interestingly, the reduced expression of CLCA2 was frequently observed in various kinds of cancers including prostate cancer, whereas its expression was not affected in precancerous prostatic intraepithelial neoplasia. Thus, our findings suggest a crucial role of p53/CLCA2-mediated senescence induction as a barrier for malignant transformation.

  18. Acetylation Is Indispensable for p53 Activation

    OpenAIRE

    Tang, Yi; Zhao, Wenhui; Chen, Yue; Zhao, Yingming; Gu, Wei

    2008-01-01

    The activation of the tumor suppressor p53 facilitates the cellular response to genotoxic stress; however, the p53 response can only be executed if its interaction with its inhibitor Mdm2 is abolished. There have been conflicting reports on the question of whether p53 posttranslational modifications, such as phosphorylation or acetylation, are essential or only play a subtle, fine-tuning role in the p53 response. Thus, it remains unclear whether p53 modification is absolutely required for its...

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

    Directory of Open Access Journals (Sweden)

    Deppert Wolfgang

    2006-10-01

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

  20. Thymidylate synthase inhibition induces p53-dependent and p53-independent apoptotic responses in human urinary bladder cancer cells.

    Science.gov (United States)

    Stravopodis, Dimitrios J; Karkoulis, Panagiotis K; Konstantakou, Eumorphia G; Melachroinou, Sophia; Thanasopoulou, Angeliki; Aravantinos, Gerasimos; Margaritis, Lukas H; Anastasiadou, Ema; Voutsinas, Gerassimos E

    2011-02-01

    In search for more effective clinical protocols, the antimetabolite drug 5-fluorouracil (5-FU) has been successfully included in new regimens of bladder cancer combination chemotherapy. In the present study, we have investigated the effects of 5-FU treatment on apoptosis induction in wild-type and mutant p53 urinary bladder cancer cells. We have used MTT-based assays, FACS analysis, Western blotting and semi-quantitative RT-PCR in RT4 and RT112 (grade I, wild-type p53), as well as in T24 (grade III, mutant p53) and TCCSUP (grade IV, mutant p53) human urinary bladder cancer cell lines. In the urothelial bladder cancer cell lines RT4 and T24, 5-FU-induced TS inhibition proved to be associated with cell type-dependent (a) sensitivity to the drug, (b) Caspase-mediated apoptosis, (c) p53 stabilization and activation, as well as Rb phosphorylation and E2F1 expression and (d) transcriptional regulation of p53 target genes and their cognate proteins, while an E2F-dependent transcriptional network did not seem to be critically engaged in such type of responses. We have shown that in the wild-type p53 context of RT4 cells, 5-FU-triggered apoptosis was prominently efficient and mainly regulated by p53-dependent mechanisms, whereas the mutant p53 environment of T24 cells was able to provide notable levels of resistance to apoptosis, basically ascribed to E2F-independent, and still unidentified, pathways. Nevertheless, the differential vulnerability of RT4 and T24 cells to 5-FU administration could also be associated with cell-type-specific transcriptional expression patterns of certain genes critically involved in 5-FU metabolism.

  1. Synergistic effect of p53 on TSA-induced stanniocalcin 1 expression in human nasopharyngeal carcinoma cells, CNE2.

    Science.gov (United States)

    Ching, L Y; Yeung, Bonnie H Y; Wong, Chris K C

    2012-06-01

    Human stanniocalcin 1 (STC1) has recently been identified as a putative protein factor involved in cellular apoptosis. The use of histone deacetylase inhibitor (i.e. trichostatin A (TSA)) and doxorubicin (Dox) is one of the common treatment methods to induce apoptosis in human cancer cells. A study on TSA and Dox-mediated apoptosis may shed light on the regulation and function of STC1 in cancer treatment. In this study, TSA and Dox cotreatment in human nasopharyngeal carcinoma cells (CNE2) elicited synergistic effects on STC1 gene expression and cellular apoptosis. An activation of p53 (TP53) transcriptional activity in Dox- or Dox+TSA-treated cells was revealed by the increased expression levels of p53 mRNA/protein as well as p53-driven luciferase activities. To elucidate the possible involvement of p53 in STC1 gene transcription, a vector expressing wild-type or dominant negative (DN) p53 was transiently transfected into the cells. Both STC1 promoter luciferase constructs and chromatin immunoprecipitation assays did not support the direct role of p53 in STC1 gene transactivation. However, the synergistic effects of p53 on the induction of NF-κB phosphorylation and the recruitment of acetylated histone H3 in STC1 promoter were observed in TSA-cotreated cells. The overexpression of exogenous STC1 sensitized apoptosis in Dox-treated cells. Taken together, this study provides data to show the cross talk of NF-κB, p53, and histone protein in the regulation of STC1 expression and function.

  2. Pathologies Associated with the p53 Response

    Science.gov (United States)

    Gudkov, Andrei V.; Komarova, Elena A.

    2010-01-01

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

  3. Arginine methylation regulates the p53 response

    DEFF Research Database (Denmark)

    Jansson, Martin; Durant, Stephen T; Cho, Er-Chieh

    2008-01-01

    Activation of the p53 tumour suppressor protein in response to DNA damage leads to apoptosis or cell-cycle arrest. Enzymatic modifications are widely believed to affect and regulate p53 activity. We describe here a level of post-translational control that has an important functional consequence...... on the p53 response. We show that the protein arginine methyltransferase (PRMT) 5, as a co-factor in a DNA damage responsive co-activator complex that interacts with p53, is responsible for methylating p53. Arginine methylation is regulated during the p53 response and affects the target gene specificity...... of p53. Furthermore, PRMT5 depletion triggers p53-dependent apoptosis. Thus, methylation on arginine residues is an underlying mechanism of control during the p53 response....

  4. Role of wild type p53 and double suicide genes in interventional therapy of liver cancer in rabbits Papel do p53 selvagem e do duplo genes suicidas na terapia intervencionista do câncer de fígado em coelhos

    Directory of Open Access Journals (Sweden)

    Hong-xin Niu

    2012-08-01

    Full Text Available PURPOSE: To investigate the feasibility of interventional lipiodol embolism and multigene therapy in combination with focal chemotherapy in the treatment of VX2 liver cancer in rabbits. METHODS: Forty five rabbits with cancer larger than 2cm in diameter were randomly divided into five groups (n=9 per group. In Group 1, animals were treated with 0.9% sodium chloride. In Group 2, animals received lipiodol embolism. In Group 3, animals received lipiodol embolism and p53 gene therapy. In Group 4, animals received lipiodol embolism and TK/CD gene therapy. In Group 5, animals received lipiodol embolism and p53 and TK/CD gene therapy. Ultrasonography and CT were performed before and at ten days after interventional therapy. RESULTS: The VX2 model of liver cancer was successfully established in rabbits and interventional therapy smoothly performed. At ten days after interventional therapy, significant difference in the tumor volume was noted among five groups (pOBJETIVO: Investigar a possibilidade de terapia multigênica e intervenção por embolização com lipiodol em combinação com quimioterapia focal no tratamento de câncer de fígado VX2 em coelhos. MÉTODOS: Quarenta e cinco coelhos com câncer maior do que 2cm de diâmetro foram distribuídos, aleatoriamente, em cinco grupos (n=9 por grupo. Grupo 1: animais foram tratados com cloreto de sódio 0,9% e no grupo 2 os animais receberam embolização com lipidol. Grupo 3: animais receberam embolização com lipiodol e terapia do gene p53 e grupo 4 animais receberam embolização com lipiodol e terapia do gene TK/CD. Grupo 5: animais receberam embolização com lipiodol e terapia do gene p53 e do gene TK/CD. Ultrassonografia e tomografia computadorizada foram realizadas antes e dez dias após a intervenção terapêutica. RESULTADOS: O modelo VX2 de câncer de fígado foi estabelecido com sucesso em coelhos e a terapia intervencionista foi bem executada. Dez dias após a intervenção terap

  5. p53 Acetylation: Regulation and Consequences

    Science.gov (United States)

    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

  6. p53 Acetylation: Regulation and Consequences

    Directory of Open Access Journals (Sweden)

    Sara M. Reed

    2014-12-01

    Full Text Available 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.

  7. p53 Acetylation: Regulation and Consequences

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Sara M. [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Quelle, Dawn E., E-mail: dawn-quelle@uiowa.edu [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Department of Pathology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States)

    2014-12-23

    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.

  8. p53: Biology and role for cellular radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Dahm-Daphi, J. [Hamburg Univ. (Germany). Abt. fuer Strahlentherapie und Onkologie

    2000-06-01

    Purpose: p53 is the most commonly mutated gene in human tumors with large impact on cellular biology and response to radiation. Many excellent reviews are available on various aspects but for several years none about the role of p53 for radiosensitivity. The latter is the aim of the present paper. Methods: Review of the literature. Results: p53 is a regulator of apoptosis mainly in hematopoetic tissue. In normal tissue and solid tumors presumably other functions have more impact on the cellular response. p53 controls cell-cycle progression after irradiation and also DNA-repair, namely homologous and non-homologous recombination. Mutations of p53 alter these functions which may be responsible for an enhanced cellular and tumor radioresistance. At present only few reports were able to show that under tightly controlled conditions loss of p53 wild-type function leads to enhanced radioresistance. A general proof is still lacking. Conclusion: The emerging picture in the year 2000 shows p53 as a central protein in a multi-enzyme multi-function network which is far from being fully understood. Although p53 appears to be a major regulator it is certainly not the unreplacable component the loss of which uniformly determines radioresistance. Only further understanding of modifiers and cooperators in the cell and in the specific tissue context will elucidate p53's role for radiosensitivity and radiotherapy. (orig.) [German] Hintergrund: p53 ist das am haeufigsten mutierte Gen in menschlichen Tumoren mit grossem Einfluss auf die zellulaere Biologie und Strahlenantwort. Viele ausgezeichnete Uebersichten sind verfuegbar, aber seit Jahren keine, die die Rolle von p53 fuer die Strahlenempfindlichkeit beleuchtet. Dies ist das Ziel der vorliegenden Arbeit. Methode: Literaturuebersicht. Ergebnis: p53 ist ein Regulator der Apoptose in haematopoetischen Gewerben. Im uebrigen Normalgewebe und in soliden Tumoren haben andere Funktionen groessere Bedeutung fuer die Zellantwort. Nach

  9. Cytoskeleton-Anchoring of Conformational Mutant-Like p53, but Not Shorter Isoforms p53β and p47 (ΔN40p53) in Senescent Human Fibroblasts.

    Science.gov (United States)

    Nishio, Koji

    2017-01-01

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

  10. Cross-regulation of protein stability by p53 and nuclear receptor SHP.

    Directory of Open Access Journals (Sweden)

    Zhihong Yang

    Full Text Available We report here a novel interplay between tumor suppressor p53 and nuclear receptor SHP that controls p53 and SHP stability. Overexpression of p53 causes rapid SHP protein degradation, which does not require the presence of Mdm2 and is mediated by the proteosome pathway. Overexpressing SHP alone does not affect p53 stability. However, SHP destabilizes p53 by augmentation of Mdm2 ubiquitin ligase activity toward p53. The single amino acid substitution in the SHP protein SHPK170R increases SHP binding to p53 relative to SHP wild-type, whereas SHPG171A variant shows a diminished p53 binding. As a result of the cross-regulation, the tumor suppressor function of p53 and SHP in inhibition of colon cancer growth is compromised. Our findings reveal a unique scenario for a cross-inhibition between two tumor suppressors to keep their expression and function in check.

  11. AdDelta24 and the p53-expressing variant AdDelta24-p53 achieve potent anti-tumor activity in glioma when combined with radiotherapy.

    Science.gov (United States)

    Idema, Sander; Lamfers, Martine L M; van Beusechem, Victor W; Noske, David P; Heukelom, Stan; Moeniralm, Sharif; Gerritsen, Winald R; Vandertop, W Peter; Dirven, Clemens M F

    2007-12-01

    The conditionally replicating adenovirus (CRAd) AdDelta24-p53 replicates selectively in Rb mutant cells, and encodes the p53 suppressor protein. It has shown improved oncolytic potency compared to the parental control AdDelta24. As exogenous p53 has been shown to enhance radiosensitivity, the combination of AdDelta24-p53 and AdDelta24 with radiotherapy was assessed in vitro and in vivo against the therapy resistant gliomas. In glioma cells, multicellular spheroids and animal xenografts the efficacy of combination therapy was assessed. P53 phosphorylation, induction of apoptosis and viral replication were determined following single or combination therapies. In vitro, AdDelta24-p53 was more effective against glioma cells than the control AdDelta24. Addition of irradiation equally increased the efficacy of both AdDelta24-p53 and AdDelta24 resulting in improved oncolysis compared to single agent treatment. Radiotherapy did not significantly change the replication kinetics of AdDelta24-p53 or AdDelta24. No detectable increase in p53 phosphorylation was observed but combination of radiotherapy and AdDelta24-p53 caused an increase in the percentage of apoptotic cells. In vivo, combination therapy with either AdDelta24 or AdDelta24-p53 significantly increased the number of mice demonstrating tumor regression (100%) as well as long-term survival (50%). No differences between viruses were noted. Exogenous p53 expression does not appear to increase the synergistic interaction of CRAds combined with radiotherapy. These results however do indicate that radiotherapy provides the time frame in which AdDelta24 and AdDelta24-p53 can eradicate established tumors that would otherwise escape treatment, and establishes the need to combine these modalities to form an effective anti-cancer treatment.

  12. A Novel p53 Phosphorylation Site within the MDM2 Ubiquitination Signal

    Science.gov (United States)

    Fraser, Jennifer A.; Madhumalar, Arumugam; Blackburn, Elizabeth; Bramham, Janice; Walkinshaw, Malcolm D.; Verma, Chandra; Hupp, Ted R.

    2010-01-01

    The p53 DNA-binding domain harbors a conformationally flexible multiprotein binding site that regulates p53 ubiquitination. A novel phosphorylation site exists within this region at Ser269, whose phosphomimetic mutation inactivates p53. The phosphomimetic p53 (S269D) exhibits characteristics of mutant p53: stable binding to Hsp70 in vivo, elevated ubiquitination in vivo, inactivity in DNA binding and transcription, increased thermoinstability using thermal shift assays, and λmax of intrinsic tryptophan fluorescence at 403 nm rather than 346 nm, characteristic of wild type p53. These data indicate that p53 conformational stability is regulated by a phosphoacceptor site within an exposed flexible surface loop and that this can be destabilized by phosphorylation. To test whether other motifs within p53 have similarly evolved, we analyzed the effect of Ser215 mutation on p53 function because Ser215 is another inactivating phosphorylation site in the conformationally flexible PAb240 epitope. The p53S215D protein is inactive like p53S269D, whereas p53S215A is as active as p53S269A. However, the double mutant p53S215A/S269A was transcriptionally inactive and more thermally unstable than either individual Ser-Ala loop mutant. Molecular dynamics simulations suggest that (i) solvation of phospho-Ser215 and phospho-Ser269 by positive charged residues or solvent water leads to local unfolding, which is accompanied by local destabilization of the N-terminal loop and global destabilization of p53, and (ii) the double alanine 215/269 mutation disrupts hydrogen bonding normally stabilized by both Ser215 and Ser269. These data indicate that p53 has evolved two serine phosphoacceptor residues within conformationally flexible epitopes that normally stabilize the p53 DNA-binding domain but whose phosphorylation induces a mutant conformation to wild type p53. PMID:20847049

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  15. Lung tumors with distinct p53 mutations respond similarly to p53 targeted therapy but exhibit genotype-specific statin sensitivity

    Science.gov (United States)

    Turrell, Frances K.; Kerr, Emma M.; Gao, Meiling; Thorpe, Hannah; Doherty, Gary J.; Cridge, Jake; Shorthouse, David; Speed, Alyson; Samarajiwa, Shamith; Hall, Benjamin A.; Griffiths, Meryl; Martins, Carla P.

    2017-01-01

    Lung adenocarcinoma accounts for ∼40% of lung cancers, the leading cause of cancer-related death worldwide, and current therapies provide only limited survival benefit. Approximately half of lung adenocarcinomas harbor mutations in TP53 (p53), making these mutants appealing targets for lung cancer therapy. As mutant p53 remains untargetable, mutant p53-dependent phenotypes represent alternative targeting opportunities, but the prevalence and therapeutic relevance of such effects (gain of function and dominant-negative activity) in lung adenocarcinoma are unclear. Through transcriptional and functional analysis of murine KrasG12D-p53null, -p53R172H (conformational), and -p53R270H (contact) mutant lung tumors, we identified genotype-independent and genotype-dependent therapeutic sensitivities. Unexpectedly, we found that wild-type p53 exerts a dominant tumor-suppressive effect on mutant tumors, as all genotypes were similarly sensitive to its restoration in vivo. These data show that the potential of p53 targeted therapies is comparable across all p53-deficient genotypes and may explain the high incidence of p53 loss of heterozygosity in mutant tumors. In contrast, mutant p53 gain of function and their associated vulnerabilities can vary according to mutation type. Notably, we identified a p53R270H-specific sensitivity to simvastatin in lung tumors, and the transcriptional signature that underlies this sensitivity was also present in human lung tumors, indicating that this therapeutic approach may be clinically relevant. PMID:28790158

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

    Science.gov (United States)

    1995-10-01

    Vogelstein, B. and Fornace, A.J., Jr. (1992). Cell 71:587-597. (6) Yonish-Rouach, E., Resnitsky, D., Lotem, J., Sachs, L., Kimchi , A., and Oren, M. (1991...Cell 70: 937-948. Yonish-Rouach, E., D. Resnitzky, J. Lotem, L. Sachs, A. Kimchi , and M. Oren. 1991. Wild-type p53 induces apoptosis of my- eloid

  17. Mutant p53 as a target for cancer treatment.

    Science.gov (United States)

    Duffy, Michael J; Synnott, Naoise C; Crown, John

    2017-09-01

    TP53 (p53) is the single most frequently altered gene in human cancers, with mutations being present in approximately 50% of all invasive tumours. However, in some of the most difficult-to-treat cancers such as high-grade serous ovarian cancers, triple-negative breast cancers, oesophageal cancers, small-cell lung cancers and squamous cell lung cancers, p53 is mutated in at least 80% of samples. Clearly, therefore, mutant p53 protein is an important candidate target against which new anticancer treatments could be developed. Although traditionally regarded as undruggable, several compounds such as p53 reactivation and induction of massive apoptosis-1 (PRIMA-1), a methylated derivative and structural analogue of PRIMA-1, i.e. APR-246, 2-sulfonylpyrimidines such as PK11007, pyrazoles such as PK7088, zinc metallochaperone-1 (ZMC1), a third generation thiosemicarbazone developed by Critical Outcome Techonologies Inc. (COTI-2) as well as specific peptides have recently been reported to reactive mutant p53 protein by converting it to a form exhibiting wild-type properties. Consistent with the reactivation of mutant p53, these compounds have been shown to exhibit anticancer activity in preclinical models expressing mutant p53. To date, two of these compounds, i.e. APR-246 and COTI-2 have progressed to clinical trials. A phase I/IIa clinical trial with APR-246 reported no major adverse effect. Currently, APR-246 is undergoing a phase Ib/II trial in patients with advanced serous ovarian cancer, while COTI-2 is being evaluated in a phase I trial in patients with advanced gynaecological cancers. It remains to be shown however, whether any mutant p53 reactivating compound has efficacy for the treatment of human cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Transcriptional inhibition of p21{sup WAF1/CIP1} gene (CDKN1) expression by survivin is at least partially p53-dependent: Evidence for survivin acting as a transcription factor or co-factor

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Lei [Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY (United States); Pre-Doctoral Chinese Fellowship Student, Second West China Hospital, Sichuan University, Sichuan (China); Ling, Xiang; Liu, Wensheng; Das, Gokul M. [Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY (United States); Li, Fengzhi, E-mail: fengzhi.li@roswellpark.org [Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY (United States)

    2012-05-04

    Highlights: Black-Right-Pointing-Pointer Survivin inhibits the expression of p21 protein, mRNA and promoter activity. Black-Right-Pointing-Pointer Survivin neutralizes p53-induced p21 expression and promoter activity. Black-Right-Pointing-Pointer Survivin physically interacts with p53 in cancer cells. Black-Right-Pointing-Pointer Genetic silencing of endogenous survivin upregulates p21 in p53 wild type cancer cells. Black-Right-Pointing-Pointer Both p53 and survivin interacts on the two p53-binding sites in the p21 promoter. -- Abstract: Growing evidence suggests a role for the antiapoptotic protein survivin in promotion of cancer cell G1/S transition and proliferation. However, the underlying mechanism is unclear. Further, although upregulation of p21{sup WAF1/CIP1} by p53 plays an important role in p53-mediated cell G1 arrests in response to various distresses, it is unknown whether survivin plays a role in the regulation of p21{sup WAF1/CIP1} expression. Here, we report that exogenous expression of survivin in p53-wild type MCF-7 breast cancer cells inhibits the expression of p21{sup WAF1/CIP1} protein, mRNA and promoter activity, while the survivin C84A mutant and antisense failed to do so. Cotransfection experiments in the p53 mutant H1650 lung cancer cell line showed that survivin neutralizes p53-induced p21{sup WAF1/CIP1} expression and promoter activity. Importantly, genetically silencing of endogenous survivin using lentiviral survivin shRNA also enhances endogenous p21 in p53 wild type cancer cells, suggesting the physiological relevance of the fining. We further demonstrated that both p53 and survivin interacts on the two p53-binding sites in the p21{sup WAF1/CIP1} promoter (-2313 to -2212; -1452 to -1310), and survivin physically interacts with p53 in cancer cells. Together, we propose that survivin may act as a transcription factor or cofactor to interact with p53 on the p21{sup WAF1/CIP1} promoter leading to the inhibition of p21{sup WAF1/CIP1

  19. Irradiation selects for p53-deficient hematopoietic progenitors.

    Directory of Open Access Journals (Sweden)

    Andriy Marusyk

    2010-03-01

    Full Text Available Identification and characterization of mutations that drive cancer evolution constitute a major focus of cancer research. Consequently, dominant paradigms attribute the tumorigenic effects of carcinogens in general and ionizing radiation in particular to their direct mutagenic action on genetic loci encoding oncogenes and tumor suppressor genes. However, the effects of irradiation are not limited to genetic loci that encode oncogenes and tumor suppressors, as irradiation induces a multitude of other changes both in the cells and their microenvironment which could potentially affect the selective effects of some oncogenic mutations. P53 is a key tumor suppressor, the loss of which can provide resistance to multiple genotoxic stimuli, including irradiation. Given that p53 null animals develop T-cell lymphomas with high penetrance and that irradiation dramatically accelerates lymphoma development in p53 heterozygous mice, we hypothesized that increased selection for p53-deficient cells contributes to the causal link between irradiation and induction of lymphoid malignancies. We sought to determine whether ionizing irradiation selects for p53-deficient hematopoietic progenitors in vivo using mouse models. We found that p53 disruption does not provide a clear selective advantage within an unstressed hematopoietic system or in previously irradiated BM allowed to recover from irradiation. In contrast, upon irradiation p53 disruption confers a dramatic selective advantage, leading to long-term expansion of p53-deficient clones and to increased lymphoma development. Selection for cells with disrupted p53 appears to be attributable to several factors: protection from acute irradiation-induced ablation of progenitor cells, prevention of irradiation-induced loss of clonogenic capacity for stem and progenitor cells, improved long-term maintenance of progenitor cell fitness, and the disabling/elimination of competing p53 wild-type progenitors. These studies

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

    OpenAIRE

    Ljungman, Mats

    2000-01-01

    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 fol...

  1. Hatching time and alevin growth prior to the onset of exogenous feeding in farmed, wild and hybrid Norwegian Atlantic salmon.

    Directory of Open Access Journals (Sweden)

    Monica Favnebøe Solberg

    Full Text Available The onset of exogenous feeding, when juveniles emerge from the gravel, is a critical event for salmonids where early emergence and large size provide a competitive advantage in the wild. Studying 131 farmed, hybrid and wild Norwegian Atlantic salmon families, originating from four wild populations and two commercial strains, we investigated whether approximately 10 generations of selection for faster growth has also resulted in increased somatic growth prior to the onset of exogenous feeding. In addition, we tested whether relaxed selection in farms has allowed for alterations in hatching time between farmed and wild salmon. Across three cohorts, wild salmon families hatched earlier than farmed salmon families, while hybrid families displayed intermediate hatching times. While the observed differences were small, i.e., 1-15 degree-days (0-3 days, as water temperatures were c. 5-6°C, these data suggest additive genetic variation for hatching time. Alevin length prior to exogenous feeding was positively related to egg size. After removal of egg size effects, no systematic differences in alevin length were observed between the wild and farmed salmon families. While these results indicate additive genetic variation for egg development timing, and wild salmon families consistently hatched earlier than farmed salmon families, these differences were so small they are unlikely to significantly influence early life history competition of farmed and wild salmon in the natural environment. This is especially the case given that the timing of spawning among females can vary by several weeks in some rivers. The general lack of difference in size between farmed and wild alevins, strongly suggest that the documented differences in somatic growth rate between wild and farmed Norwegian Atlantic salmon under hatchery conditions are first detectable after the onset of exogenous feeding.

  2. 9-Hydroxyellipticine alters the conformation and DNA binding characteristics of mutated p53 protein.

    Science.gov (United States)

    Sugikawa, E; Tsunoda, S; Nakanishi, N; Ohashi, M

    2001-01-01

    The tumor suppressor protein p53 is a phosphoprotein which shows growth and transformation suppression functions. Mutational loss of p53 function is the most frequently detected genetic event in human cancers. We examined whether 9-hydroxyellipticine (9HE), a cytotoxic agent, affected the tertiary structure of mutant p53 and DNA binding characteristics. Although several types of p53 mutants were resistant to degradation by calpain, the p53 mutants treated with 9HE were markedly sensitive to calpain as well as wild-type p53. Furthermore, mutant p53 proteins isolated from 9HE-treated cells regained the ability to bind a wild-type-specific p53 DNA consensus sequence. Wild-type p53 proteins prepared from both untreated and 9HE-treated cells bound the p53 consensus sequence and were degradaded by calpain equally well. These results suggest that 9HE affects the tertiary structure of mutated p53, which results in the restoration of DNA binding characteristics.

  3. Inhibiting p53 Acetylation Reduces Cancer Chemotoxicity.

    Science.gov (United States)

    Zheng, Shunsheng; Koh, Xin Yu; Goh, Hui Chin; Rahmat, Siti Aishah B; Hwang, Le-Ann; Lane, David P

    2017-08-15

    Chemotoxicity due to unwanted p53 activation in the bone marrow remains an unmet clinical challenge. Doxorubicin, a first-line chemotherapy drug, often causes myelosuppression in patients, thus limiting its effectiveness. In this study, we discovered that C646, a reversible p300 inhibitor, downregulates p53 transcription and selectively protects noncancerous cells from p53-dependent apoptosis. C646 treatment blocked acetylation of specific lysine residues that regulate p53 activity. Exploitation of differential p53 genetic backgrounds between human hematopoietic and colorectal cancer cells improved the therapeutic index of doxorubicin with C646 cotreatment. C646 administration in mice afflicted with p53-mutant tumors protected them from doxorubicin-induced neutropenia and anemia while retaining antitumor efficacy. We deduce that temporary and reversible inhibition of p53 acetylation in cancer subjects, especially those with p53-mutant tumors, may protect them from severe chemotoxicity while allowing treatment regimens to effectively proceed. Cancer Res; 77(16); 4342-54. ©2017 AACR. ©2017 American Association for Cancer Research.

  4. Expression of p53 in oligodendrogliomas

    NARCIS (Netherlands)

    J.M. Kros (Johan); J.J.C.J. Godschalk (J. J C J); K.K. Krishnadath (Kausilia); C.G. van Eden (C.)

    1993-01-01

    textabstractThe expression of the nuclear protein p53 in oligodendrogliomas was investigated by immunohistochemistry, using a monoclonal anti-p53 antibody (DO-7) on formalin-fixed, paraffin-embedded material in 84 histologically verified cases, and compared with the histopathological grade and

  5. Microbial Regulation of p53 Tumor Suppressor.

    Directory of Open Access Journals (Sweden)

    Alexander I Zaika

    2015-09-01

    Full Text Available p53 tumor suppressor has been identified as a protein interacting with the large T antigen produced by simian vacuolating virus 40 (SV40. Subsequent research on p53 inhibition by SV40 and other tumor viruses has not only helped to gain a better understanding of viral biology, but also shaped our knowledge of human tumorigenesis. Recent studies have found, however, that inhibition of p53 is not strictly in the realm of viruses. Some bacterial pathogens also actively inhibit p53 protein and induce its degradation, resulting in alteration of cellular stress responses. This phenomenon was initially characterized in gastric epithelial cells infected with Helicobacter pylori, a bacterial pathogen that commonly infects the human stomach and is strongly linked to gastric cancer. Besides H. pylori, a number of other bacterial species were recently discovered to inhibit p53. These findings provide novel insights into host-bacteria interactions and tumorigenesis associated with bacterial infections.

  6. p53, oxidative stress, and aging.

    Science.gov (United States)

    Liu, Dongping; Xu, Yang

    2011-09-15

    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 pro-oxidative 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.

  7. [Constructing a p53-fused dual luciferase reporter and verifying its function].

    Science.gov (United States)

    Tan, Jing; Jiang, Xianzhen; He, Leye; Jiang, Zhiqiang; Tang, Yuxin; Yao, Kun; Chen, Binghai; Xue, Juan

    2013-11-01

    To construct a p53-fused dual luciferase reporter and to test whether this reporter can mimic wild-type p53 activities in a high-throughput screen. A restriction endonuclease site was added to each terminus and the stop codon of the wild-type full-length p53 open reading frame (ORF) was removed by PCR. A restriction endonuclease site was added to each terminus and the start codon of the firefly luciferase ORF was removed by PCR. The two modified ORFs were inserted upstream of the IRES-induced renilla luciferase ORF in a CMV-derived vector. The p53 fusion protein was expressed in cells to test its MDM2-mediated degradation, subcellular localization, and induction of p53-responsive promoter. The p53-fused dual luciferase reporter was successfully constructed. After transfection into the host cells, the reporter expressing the p53 fusion protein that was degraded by oncoprotein MDM2, was mainly located inside the nucleus, and induced the p53-responsive promoter, respectively. The p53-fused dual luciferase reporter (p53FL/IRES/RL) can identify modulators of P53 protein level in a high-throughput screen of genetic or chemical libraries.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Zhendong, E-mail: zdyu@hotmail.com [Department of Clinical laboratory, Peking University Shenzhen Hospital, Guangdong (China); Wang, Hao [Department of pathology, The Chinese University of Hong Kong, Hong Kong (China); Zhang, Libin; Tang, Aifa; Zhai, Qinna; Wen, Jianxiang; Yao, Li [Department of Clinical laboratory, Peking University Shenzhen Hospital, Guangdong (China); Li, Pengfei, E-mail: lipengfei@cuhk.edu.hk [Department of pathology, The Chinese University of Hong Kong, Hong Kong (China)

    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.

  9. Mechanisms of p53-Mediated Apoptosis

    Science.gov (United States)

    2007-03-01

    terminus. Cell 81:1021–1029. 27. Juan , L. J., W. J. Shia, M. H. Chen, W. M. Yang, E. Seto, Y. S. Lin, and C. W. VOL. 25, 2005 THE C TERMINUS REGULATES p53...functionally resembles p53. Nat. Med. 4:839–843. 45. Pariat, M., S. Carillo, M. Molinari, C. Salvat , L. Debussche, L. Bracco, J. Milner, and M. Piechaczyk. 1997...apoptosis. Nature 2000;408:377-81. 27. Juan LJ, Shia WJ, Chen MH, et al. Histone deacetylases specifically down-regulate p53- dependent gene

  10. Toca-1 is suppressed by p53 to limit breast cancer cell invasion and tumor metastasis.

    Science.gov (United States)

    Chander, Harish; Brien, Colin D; Truesdell, Peter; Watt, Kathleen; Meens, Jalna; Schick, Colleen; Germain, Doris; Craig, Andrew W B

    2014-12-30

    Transducer of Cdc42-dependent actin assembly-1 (Toca-1) recruits actin regulatory proteins to invadopodia, and promotes breast tumor metastasis. Since metastatic breast tumors frequently harbor mutations in the tumor suppressor p53, we tested whether p53 regulates Toca-1 expression. Normal mammary epithelial cells (HBL-100, MCF10A) and breast cancer cell lines expressing wild-type (WT) p53 (DU4475, MTLn3) were treated with camptothecin or Nutlin-3 to stabilize p53 to test effects on Toca-1 mRNA and protein levels. Chromatin immunoprecipitation (ChIP) assays were performed to identify p53 binding site in Toca-1 gene. Stable silencing of p53 and Toca-1 were performed in MTLn3 cells to test effects on invadopodia and cell invasion in vitro, and tumor metastasis in vivo. We observed that breast cancer cell lines with mutant p53 have high levels of Toca-1 compared to those with WT p53. Stabilization of WT p53 led to further reduction in Toca-1 mRNA and protein levels in normal breast epithelial cells and breast cancer cells. ChIP assays revealed p53 binding within intron 2 of toca1, and reduced histone acetylation within its promoter region upon p53 upregulation or activation. Stable silencing of WT p53 in MTLn3 cells led to increased extracellular matrix degradation and cell invasion compared to control cells. Interestingly, the combined silencing of p53 and Toca-1 led to a partial rescue of these effects of p53 silencing in vitro and reduced lung metastases in mice. In human breast tumors, Toca-1 levels were high in subtypes with frequent p53 mutations, and high Toca-1 transcript levels correlated with increased risk of relapse. Based on these findings, we conclude that loss of p53 tumor suppressor function in breast cancers leads to upregulation of Toca-1, and results in enhanced risk of developing metastatic disease.

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

    Science.gov (United States)

    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

  12. Nutlin-3 downregulates p53 phosphorylation on serine392 and induces apoptosis in hepatocellular carcinoma cells.

    Science.gov (United States)

    Shi, Xinli; Liu, Jingli; Ren, Laifeng; Mao, Nan; Tan, Fang; Ding, Nana; Yang, Jing; Li, Mingyuan

    2014-04-01

    Drug-resistance and imbalance of apoptotic regulation limit chemotherapy clinical application for the human hepatocellular carcinoma (HCC) treatment. The reactivation of p53 is an attractive therapeutic strategy in cancer with disrupted-p53 function. Nutlin-3, a MDM2 antagonist, has antitumor activity in various cancers. The post-translational modifications of p53 are a hot topic, but there are some controversy ideas about the function of phospho-Ser392-p53 protein in cancer cell lines in response to Nutlin-3. Therefore, we investigated the relationship between Nutlin-3 and phospho-Ser392-p53 protein expression levels in SMMC-7721 (wild-type TP53) and HuH-7 cells (mutant TP53). We demonstrated that Nutlin-3 induced apoptosis through down-regulation phospho-Ser392-p53 in two HCC cells. The result suggests that inhibition of p53 phosphorylation on Ser392 presents an alternative for HCC chemotherapy.

  13. p53 switches off pluripotency on differentiation.

    Science.gov (United States)

    Lin, Tongxiang; Lin, Yi

    2017-02-28

    The role of p53 as "a guardian of the genome" has been well established in somatic cells. However, its role in pluripotent stem cells remains much more elusive. Here, we discuss research progress in understanding the role of p53 in pluripotent stem cells and in pluripotent stem cell-like cancer stem cells. The p53 protein, which plays a key role in embryonic stem cells, was first discovered in 2005. Landmark studies of p53-related reprogramming elucidated this protein's importance in induced pluripotent stem cells in 2009. The p53-related safety concerns in pluripotent stem cells have been raised in stem cell-based therapy although the use of iPSCs in therapeutic application is promising. Because cancer stem cells have profiles similar to those of pluripotent stem cells, we also describe potential strategies for studies in cancer stem cells and cancer treatments. The new discoveries of p53 family proteins in pluripotent stem cells have made possible stable progress in stem cell transplantation efficiency and safety, as well as treatment strategies targeting cancer stem cells based on pluripotent stem cell technology.

  14. Protecting the hedgerow: p53 and hedgehog pathway interactions.

    Science.gov (United States)

    Ho, Louisa; Alman, Benjamin

    2010-02-01

    A common environment for the Hedgehog (Subfamily: erinaceinae) is a row of shrubs and trees often used on farms for enclosing or separating fields, called a hedgerow. Maintenance of a continuous shrub border is important for shielding crops from weather damage, but also provides an ideal protective habitat for the hedgehog. Similar to its mammalian counterpart, the Hedgehog (Hh) signalling pathway requires a controlled environment to regulate proper functioning of the cell. When allowed to run wild, constitutive activation of the Hh pathway results in tumorigenesis in different tissues types, including brain, skin and cartilage. With an additional loss of p53 tumor suppressor activity, an increase in tumor incidence, size and metastasis have been observed. p53 has a number of functions that can suppress tumor formation and growth in most, if not all Hh-related cancers, such as the inhibition of cell cycle progression and cell survival. Furthermore, increasing evidence of an interaction between p53 and Hedgehog signalling pathways suggests a critical role for the tumor suppressor activity of p53 in "protecting the hedgerow".

  15. Mutant p53 exhibits trivial effects on mitochondrial functions which can be reactivated by ellipticine in lymphoma cells.

    Science.gov (United States)

    Wang, Fei; Liu, Jianfeng; Robbins, Delira; Morris, Kerri; Sit, Amos; Liu, Yong-Yu; Zhao, Yunfeng

    2011-03-01

    Increasing evidence has shown that a fraction of the wild-type (wt) form of the tumor suppressor p53, can translocate to mitochondria due to genotoxic stress. The mitochondrial targets of wt p53 have also been studied. However, whether mutant p53, which exists in 50% of human cancers, translocates to mitochondria and affects mitochondrial functions is unclear. In this study, we used doxorubicin, a chemotherapeutic drug, to treat five human lymphoma cell lines with wt, mutant or deficient in p53, to induce p53 activation and mitochondrial translocation. Our results demonstrated that mutant p53, like wt p53, was induced upon doxorubicin treatment. Similarly, a fraction of mutant p53 also translocated to mitochondria. However, Complex I and II activities in the mitochondria were compromised only in wt p53-bearing cells after doxorubicin treatment, but not in mutant p53-bearing cells. Similarly, doxorubicin treatment caused greater cell death only in wt p53-bearing cells, but not in mutant p53-bearing cells. When p53 deficient Ramos cells were transfected with mutant p53 (249S), the cells showed resistance to doxorubicin-induced cell death and decreases in complex activities. To reactivate mutant p53 and reverse chemoresistance, ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) was used to treat mutant p53 cells. Ellipticine enhanced p53 mitochondrial translocation, decreased Complex I activity, and sensitized p53 mutant cells to doxorubicin-induced apoptosis. In summary, our studies suggest that mutations in p53 may not hinder p53's mitochondrial translocation, but impair its effects on mitochondrial functions. Therefore, restoring mutant p53 by ellipticine may sensitize these cells to chemotherapy.

  16. Lung tumors with distinct p53 mutations respond similarly to p53 targeted therapy but exhibit genotype-specific statin sensitivity.

    Science.gov (United States)

    Turrell, Frances K; Kerr, Emma M; Gao, Meiling; Thorpe, Hannah; Doherty, Gary J; Cridge, Jake; Shorthouse, David; Speed, Alyson; Samarajiwa, Shamith; Hall, Benjamin A; Griffiths, Meryl; Martins, Carla P

    2017-08-08

    Lung adenocarcinoma accounts for ∼40% of lung cancers, the leading cause of cancer-related death worldwide, and current therapies provide only limited survival benefit. Approximately half of lung adenocarcinomas harbor mutations in TP53 (p53), making these mutants appealing targets for lung cancer therapy. As mutant p53 remains untargetable, mutant p53-dependent phenotypes represent alternative targeting opportunities, but the prevalence and therapeutic relevance of such effects (gain of function and dominant-negative activity) in lung adenocarcinoma are unclear. Through transcriptional and functional analysis of murine Kras(G12D) -p53(null) , -p53(R172H) (conformational), and -p53(R270H) (contact) mutant lung tumors, we identified genotype-independent and genotype-dependent therapeutic sensitivities. Unexpectedly, we found that wild-type p53 exerts a dominant tumor-suppressive effect on mutant tumors, as all genotypes were similarly sensitive to its restoration in vivo. These data show that the potential of p53 targeted therapies is comparable across all p53-deficient genotypes and may explain the high incidence of p53 loss of heterozygosity in mutant tumors. In contrast, mutant p53 gain of function and their associated vulnerabilities can vary according to mutation type. Notably, we identified a p53(R270H) -specific sensitivity to simvastatin in lung tumors, and the transcriptional signature that underlies this sensitivity was also present in human lung tumors, indicating that this therapeutic approach may be clinically relevant. © 2017 Turrell et al.; Published by Cold Spring Harbor Laboratory Press.

  17. Mdm2 RING mutation enhances p53 transcriptional activity and p53-p300 interaction.

    Directory of Open Access Journals (Sweden)

    Hilary V Clegg

    Full Text Available The p53 transcription factor and tumor suppressor is regulated primarily by the E3 ubiquitin ligase Mdm2, which ubiquitinates p53 to target it for proteasomal degradation. Aside from its ubiquitin ligase function, Mdm2 has been believed to be capable of suppressing p53's transcriptional activity by binding with and masking the transactivation domain of p53. The ability of Mdm2 to restrain p53 activity by binding alone, without ubiquitination, was challenged by a 2007 study using a knockin mouse harboring a single cysteine-to-alanine point mutation (C462A in Mdm2's RING domain. Mouse embryonic fibroblasts with this mutation, which abrogates Mdm2's E3 ubiquitin ligase activity without affecting its ability to bind with p53, were unable to suppress p53 activity. In this study, we utilized the Mdm2(C462A mouse model to characterize in further detail the role of Mdm2's RING domain in the control of p53. Here, we show in vivo that the Mdm2(C462A protein not only fails to suppress p53, but compared to the complete absence of Mdm2, Mdm2(C462A actually enhances p53 transcriptional activity toward p53 target genes p21/CDKN1A, MDM2, BAX, NOXA, and 14-3-3σ. In addition, we found that Mdm2(C462A facilitates the interaction between p53 and the acetyltransferase CBP/p300, and it fails to heterodimerize with its homolog and sister regulator of p53, Mdmx, suggesting that a fully intact RING domain is required for Mdm2's inhibition of the p300-p53 interaction and for its interaction with Mdmx. These findings help us to better understand the complex regulation of the Mdm2-p53 pathway and have important implications for chemotherapeutic agents targeting Mdm2, as they suggest that inhibition of Mdm2's E3 ubiquitin ligase activity may be sufficient for increasing p53 activity in vivo, without the need to block Mdm2-p53 binding.

  18. Sulforaphane increases the efficacy of doxorubicin in mouse fibroblasts characterized by p53 mutations

    Energy Technology Data Exchange (ETDEWEB)

    Fimognari, Carmela [Department of Pharmacology, University of Bologna, Bologna (Italy)]. E-mail: carmela.fimognari@unibo.it; Nuesse, Michael [GSF-Flow Cytometry Group, Neuherberg (Germany); Lenzi, Monia [Department of Pharmacology, University of Bologna, Bologna (Italy); Sciuscio, Davide [Department of Pharmacology, University of Bologna, Bologna (Italy); Cantelli-Forti, Giorgio [Department of Pharmacology, University of Bologna, Bologna (Italy); Hrelia, Patrizia [Department of Pharmacology, University of Bologna, Bologna (Italy)

    2006-10-10

    One novel strategy for increasing cancer chemotherapy efficacy and reversing chemoresistance involves co-administration of natural chemopreventive compounds alongside standard chemotherapeutic protocols. Sulforaphane is a particularly promising chemopreventive agent, which has been shown to exert proapoptotic effects on tumor cells containing p53 mutations. The p53{sup Ser220} mutation has been implicated in reduced efficacy and drug resistance in the context of osteosarcomas and breast tumors treated with doxorubicin-based protocols. We investigated the effects of a combination of doxorubicin and sulforaphane on cell viability and apoptosis induction in fibroblasts characterized by different p53 status (p53 wild-type, p53 knock-out, and p53{sup Ser220} mutation), and identified some of the molecular pathways triggered by the drug combination. Very high concentrations of doxorubicin were necessary to decrease the viability of p53{sup Ser220} and p53 knock-out (but not wild-type) cells. Treatment of p53{sup Ser220} and p53 knock-out cells with doxorubicin did not induce apoptosis, also at very high concentrations (10 {mu}M). Sulforaphane restored chemosensitivity and induced apoptosis in doxorubicin-resistant p53{sup Ser220} and p53 knock-out cells, irrespective of p53 status. The induction of apoptosis was caspase-3 dependent and caspase-8 independent. Bongkrekic acid, a mitochondrial membrane stabilizer, partially prevented the effects of doxorubicin plus sulforaphane on mitochondrial permeability but was unable to prevent the induction of apoptosis. N-acetyl-cysteine, a glutathione precursor, blocked the induction of apoptosis by doxorubicin plus sulforaphane. Considering the negligible safety profile of sulforaphane, our findings could prompt innovative clinical studies designed to investigate whether its coadministration can enhance the efficacy of doxorubicin-based regimens.

  19. Pro- and anti-apoptotic effects of p53 in cisplatin-treated human testicular cancer are cell context-dependent

    NARCIS (Netherlands)

    di Pietro, Alessandra; Koster, Roelof; Boersma-van Eck, Wytske; Dam, Wendy A.; Mulder, Nanno H.; Gietema, Jourik A.; de Vries, Elisabeth G. E.; de Jong, Steven

    2012-01-01

    In murine testicular cancer (TC) cells wild-type p53 contributes to sensitivity to DNA-damaging drugs in a dose-dependent way. In human TC, however, the role of wild-type p53 functionality in chemotherapeutic response remains elusive. We analyzed functionality of wild-type p53 in cisplatin

  20. HPV and p53 in cervical cancer.

    OpenAIRE

    Ngan, H Y; Stanley, M; Liu, S S; Ma, H K

    1994-01-01

    Objective - To determine the prevalence of HPV 16 and 18 E6 by DNA detection and p53 abnormal protein expression in cervical cancers in Hong Kong. Materials and methods - Seventy-three squamous cell cervical cancer biopsy were analysed. Detection of HPV DNA was carried out by the polymerase chain reaction and Southern blotting (PCR/SB) technique using primers to the HPV16 and 18 E6 region and consensus primers to the L1 region. Abnormal expression of the p53 protein was detected by immunohist...

  1. The p53HMM algorithm: using profile hidden markov models to detect p53-responsive genes

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    Yu Xin

    2009-04-01

    Full Text Available Abstract Background A computational method (called p53HMM is presented that utilizes Profile Hidden Markov Models (PHMMs to estimate the relative binding affinities of putative p53 response elements (REs, both p53 single-sites and cluster-sites. These models incorporate a novel "Corresponded Baum-Welch" training algorithm that provides increased predictive power by exploiting the redundancy of information found in the repeated, palindromic p53-binding motif. The predictive accuracy of these new models are compared against other predictive models, including position specific score matrices (PSSMs, or weight matrices. We also present a new dynamic acceptance threshold, dependent upon a putative binding site's distance from the Transcription Start Site (TSS and its estimated binding affinity. This new criteria for classifying putative p53-binding sites increases predictive accuracy by reducing the false positive rate. Results Training a Profile Hidden Markov Model with corresponding positions matching a combined-palindromic p53-binding motif creates the best p53-RE predictive model. The p53HMM algorithm is available on-line: http://tools.csb.ias.edu Conclusion Using Profile Hidden Markov Models with training methods that exploit the redundant information of the homotetramer p53 binding site provides better predictive models than weight matrices (PSSMs. These methods may also boost performance when applied to other transcription factor binding sites.

  2. Urodele p53 tolerates amino acid changes found in p53 variants linked to human cancer

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    Villiard Éric

    2007-09-01

    Full Text Available Abstract Background Urodele amphibians like the axolotl are unique among vertebrates in their ability to regenerate and their resistance to develop cancers. It is unknown whether these traits are linked at the molecular level. Results Blocking p53 signaling in axolotls using the p53 inhibitor, pifithrin-α, inhibited limb regeneration and the expression of p53 target genes such as Mdm2 and Gadd45, suggesting a link between tumor suppression and regeneration. To understand this relationship we cloned the p53 gene from axolotl. When comparing its sequence with p53 from other organisms, and more specifically human we observed multiple amino acids changes found in human tumors. Phylogenetic analysis of p53 protein sequences from various species is in general agreement with standard vertebrate phylogeny; however, both mice-like rodents and teleost fishes are fast evolving. This leads to long branch attraction resulting in an artefactual basal emergence of these groups in the phylogenetic tree. It is tempting to assume a correlation between certain life style traits (e.g. lifespan and the evolutionary rate of the corresponding p53 sequences. Functional assays of the axolotl p53 in human or axolotl cells using p53 promoter reporters demonstrated a temperature sensitivity (ts, which was further confirmed by performing colony assays at 37°C. In addition, axolotl p53 was capable of efficient transactivation at the Hmd2 promoter but has moderate activity at the p21 promoter. Endogenous axolotl p53 was activated following UV irradiation (100 j/m2 or treatment with an alkylating agent as measured using serine 15 phosphorylation and the expression of the endogenous p53 target Gadd45. Conclusion Urodele p53 may play a role in regeneration and has evolved to contain multiple amino acid changes predicted to render the human protein defective in tumor suppression. Some of these mutations were probably selected to maintain p53 activity at low temperature. However

  3. Hsf1 Is Required for the Nuclear Translocation of p53 Tumor Suppressor

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    Qiang Li

    2008-10-01

    Full Text Available Although the p53 tumor suppressor is most frequently inactivated by genetic mutations, exclusion from the nucleus is also seen in human tumors. We have begun to examine p53 nuclear importation by isolating a series of mutant cells in which the temperature-sensitive murine p53Val135 mutant is sequestered in the cytoplasm. We previously showed that that three of them (ALTR12, ALTR19, and ALTR25 constituted a single complementation group. Here, we found that ALTR12 cells are more sensitive to heat stress than either ALTR19 or ALTR25 and that there was a complete lack of induction of Hsp70 in response to heat shock. Western blot analysis showed no expression of the Hsf1 transcription factor, and neither heat shock nor azetidine could induce p53 nuclear localization in ALTR12 cells but did in parental A1–5 cells. Suppression of Hsf1 in A1–5 cells with quercetin or an Hsf1 siRNA reduced p53 nuclear importation and inhibited p53-mediated activation of a p21 reporter. Most convincingly, p53 nuclear importation could be restored in ALTR12 cells by introducing an exogenous Hsf1 gene. Collectively, our result suggests that Hsf1 is required for p53 nuclear importation and activation and implies that heat shock factors play a role in the regulation of p53.

  4. Aurora A mediates cross-talk between N- and C-terminal post-translational modifications of p53

    Science.gov (United States)

    Raines, Sally Anne; Milner, Jo

    2011-01-01

    The serine/threonine protein kinase Aurora A is known to interact with and phosphorylate tumor suppressor p53 at Serine 215 (S215), inhibiting the transcriptional activity of p53. We show that Aurora A positively regulates human p53 protein levels and, using isogenic p53 wild-type and p53-null colorectal carcinoma cells, further show that p53 regulates human Aurora A protein expression. S215 is located in the DNA-binding core of p53 and at the center of the cryptic epitope for PAb240 antibody, which is used to detect mutant and denatured p53. Following denaturing SDS PAGE, the PAb240 epitope was detectable by immunoblotting in only two out of eight cell lines. The efficacy of novel p53-targeted anticancer therapies may be influenced by the conformational state of p53, therefore, the initial determination of p53 status may be relevant. We found no correlation between phosphorylation of p53 at S215 and PAb240 antibody recognition. However, phosphorylation at S37 was positively associated with PAb240 reactivity. More importantly, we provide the first evidence of Aurora A-mediated cross-talk between N- and C-terminal p53 post-translational modifications. As p53 and Aurora A are targets for anticancer therapy the impact of their reciprocal relationship and Aurora A-induced post-translational modification of p53 should be considered. PMID:22157150

  5. Ensemble-based computational approach discriminates functional activity of p53 cancer and rescue mutants.

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    Özlem Demir

    2011-10-01

    Full Text Available The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain ("cancer mutants". Activity can be restored by second-site suppressor mutations ("rescue mutants". This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD, without focusing on local structural details. A novel global measure of protein flexibility for the p53 core DNA-binding domain, the number of clusters at a certain RMSD cutoff, was computed by clustering over 0.7 µs of explicitly solvated all-atom MD simulations. For wild-type p53 and a sample of p53 cancer or rescue mutants, the number of clusters was a good predictor of in vivo p53 functional activity in cell-based assays. This number-of-clusters (NOC metric was strongly correlated (r(2 = 0.77 with reported values of experimentally measured ΔΔG protein thermodynamic stability. Interpreting the number of clusters as a measure of protein flexibility: (i p53 cancer mutants were more flexible than wild-type protein, (ii second-site rescue mutations decreased the flexibility of cancer mutants, and (iii negative controls of non-rescue second-site mutants did not. This new method reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants.

  6. Diverse p53/DNA binding modes expand the repertoire of p53 response elements.

    Science.gov (United States)

    Vyas, Pratik; Beno, Itai; Xi, Zhiqun; Stein, Yan; Golovenko, Dmitrij; Kessler, Naama; Rotter, Varda; Shakked, Zippora; Haran, Tali E

    2017-10-03

    The tumor suppressor protein p53 acts as a transcription factor, binding sequence-specifically to defined DNA sites, thereby activating the expression of genes leading to diverse cellular outcomes. Canonical p53 response elements (REs) are made of two decameric half-sites separated by a variable number of base pairs (spacers). Fifty percent of all validated p53 REs contain spacers between 1 and 18 bp; however, their functional significance is unclear at present. Here, we show that p53 forms two different tetrameric complexes with consensus or natural REs, both with long spacers: a fully specific complex where two p53 dimers bind to two specific half-sites, and a hemispecific complex where one dimer binds to a specific half-site and the second binds to an adjacent spacer sequence. The two types of complexes have comparable binding affinity and specificity, as judged from binding competition against bulk genomic DNA. Structural analysis of the p53 REs in solution shows that these sites are not bent in both their free and p53-bound states when the two half-sites are either abutting or separated by spacers. Cell-based assay supports the physiological relevance of our findings. We propose that p53 REs with long spacers comprise separate specific half-sites that can lead to several different tetrameric complexes. This finding expands the universe of p53 binding sites and demonstrates that even isolated p53 half-sites can form tetrameric complexes. Moreover, it explains the manner in which p53 binds to clusters of more than one canonical binding site, common in many natural REs.

  7. WDR5 positively regulates p53 stability by inhibiting p53 ubiquitination.

    Science.gov (United States)

    Xie, Qingqing; Li, Zengpeng; Chen, Jianming

    2017-05-27

    WD40 repeat protein WDR5 is a core component of the Set/MLL histone methyltransferase complex which catalyzes histone H3 Lys4 trimethylation and activates gene transcription in human cells. WDR5 promotes Set/MLL complex assembly and mediates the complex binding to Lys4-dimethylated histone H3 tail. Most earlier studies report that WDR5 exerts profound effects on various cellular and organismal processes mainly through epigenetic regulation of gene transcription. However, the functions of WDR5 in lung cancer remain largely unknown. Here, we report that WDR5 positively regulates p53 stability by inhibiting p53 ubiquitination in human lung cancer A549 cells. Overexpression of WDR5 dramatically increases p53 protein levels and its half-life in A549 cells, while depletion of WDR5 with WDR5-specific siRNAs significantly decreases p53 protein levels. We also observe that WDR5 is required for p53 induction in response to cisplatin treatment. Mechanistically, WDR5 colocalizes with p53 and inhibits p53 ubiquitination, resulting in p53 stabilization. Consequently, overexpression of WDR5 induces G1 phase arrest in A549 cells, and knocking down WDR5 by siRNAs reduces the population at G1 phase. Furthermore, p53 expression levels is at least in part determined by the p53 positive regulator WDR5 in some cancer cells. Taken together, these data suggest that WDR5 is directly involved in p53 signaling pathway. Our studies provide a new insight into WDR5 functions in A549 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. p53 Codon 72 Gene Polymorphism Studies and p53 Expression by Immunohistochemistry in Oral Lesions as Risk Factor for Malignancy

    Science.gov (United States)

    Tandon, Nishi; Srivastava, Anand Narain; Fatima, Naseem; Raza, Syed Tasleem; Kumar, Vijay

    2017-01-01

    Background: Wild-type p53 nuclear phosphoproteins are critical cell cycle regulatory tumor-suppressor gene. Genetic mutation of p53 gene is common in several head–neck cancers, usually associated with smoking and human papillomavirus (HPV) infection. In India, instead of HPV, tobacco/pan masala chewing is more commonly associated with oral cancer. Aim: The aim of this study was to investigate p53 codon 72 gene polymorphism and expression of p53 by immunohistochemistry (IHC) in oral lesions as a risk factor for its association with malignancy. Materials and Methods: A total of 41 cases of oral lesions comprising 6 cases of leukoplakia and 35 cases of oral squamous cell carcinoma (OSCC), between 30 and 60 years age and tobacco/pan masala chewers were taken. Molecular analysis of p53 codon 72 gene polymorphism was performed by polymerase chain reaction – restriction fragment length polymorphism for Arg/Arg, Arg/Pro, and Pro/Pro. Tissue expression of p53 was done by IHC. Results: Genotype frequencies of 35 carcinoma cases of p53 Arg/Arg, Arg/Pro, and Pro/Pro were 23%, 57%, and 20%, respectively, and six leukoplakia cases of p53 Arg/Arg and Arg/Pro genotype were 50% and 50%, respectively. By IHC for expression of p53 out of 35 cases of OSCC biopsies, 17 (48.57%) had weak staining, 14 cases (40%) showed evidence of p53 protein staining, and four cases (11.42%) showed negative staining. Among six cases of leukoplakia, 3 (50%) showed weak staining and 3 (50%) showed negative results. Conclusion: The findings of the study indicate that there is no significant association between p53 codon 72 gene polymorphism with OSCC and leukoplakia associated with tobacco/pan masala chewing. PMID:29308362

  9. Genistein induces apoptosis by stabilizing intracellular p53 protein through an APE1-mediated pathway.

    Science.gov (United States)

    Zhu, Jianwu; Zhang, Chong; Qing, Yi; Cheng, Yi; Jiang, Xiaolin; Li, Mengxia; Yang, Zhenzhou; Wang, Dong

    2015-09-01

    Genistein (GEN) has been previously shown to have a proapoptotic effect on cancer cells through a p53-dependent pathway, the mechanism of which remains unclear. One of its intracellular targets, APE1, protects against apoptosis under genotoxic stress and interacts with p53. In this current study, we explored the mechanism of the proapoptotic effect of GEN by examining the APE1-p53 protein-protein interaction. We initially showed that the p53 protein level was elevated in GEN-treated human non-small lung cancer A549 cells and cervical cancer HeLa cells. By examining both protein synthesis and degradation, we found that GEN enhances p53 intracellular stability by interfering with the interaction of APE1 and p53, which provided a plausible explanation for how GEN initiates apoptosis. Furthermore, we found that the interaction between APE1 and p53 is important for the degradation of p53 and is dependent on the redox domain of APE1 by utilizing the redox domain mutant APE1 C65A. Our data suggest that the degradation of wild-type p53 is blocked when the redox domain of APE1 is masked or interrupted. Based on this evidence, we hereby report a novel mechanism of p53 degradation through an APE1-mediated, redox-dependent pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Discrimination of p53 immunohistochemistry-positive tumors by its staining pattern in gastric cancer

    Science.gov (United States)

    Ando, Koji; Oki, Eiji; Saeki, Hiroshi; Yan, Zhao; Tsuda, Yasuo; Hidaka, Gen; Kasagi, Yuta; Otsu, Hajime; Kawano, Hiroyuki; Kitao, Hiroyuki; Morita, Masaru; Maehara, Yoshihiko

    2015-01-01

    Immunohistochemistry staining of p53 is a cheap and simple method to detect aberrant function of p53. However, there are some discrepancies between the result of immunohistochemistry staining and mutation analysis. This study attempted to find a new definition of p53 staining by its staining pattern. Immunohistochemistry staining of p53 and TP53 gene mutation analysis were performed in 148 gastric cancer patients. Also SNP-CGH array analysis was conducted to four cases. Positive staining of p53 was observed in 88 (59.5%) tumors. Tumors with positive p53 staining showed malignant features compared to negative tumors. Mutation of TP53 gene was observed in 29 (19.6%) tumors with higher age and differentiated type. In positive p53 tumors, two types could be distinguished; aberrant type and scattered type. With comparison to TP53 gene mutation analysis, all the scattered type had wild-type TP53 gene (P = 0.0003). SNP-CGH array showed that scattered-type tumors had no change in the structure of chromosome 17. P53-scattered-type staining tumors may reflect a functionally active nonmutated TP53 gene. In interpretation of p53 immunohistochemistry staining, distinguishing p53-positive tumors by their staining pattern may be important in gastric cancer. PMID:25354498

  11. An N-terminal Region of Mot-2 Binds to p53 In Vitro

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    Sunil C. Kaul

    2001-01-01

    Full Text Available 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 Nterminal 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.

  12. p53 regulation and activity in mouse embryonic stem cells

    OpenAIRE

    Solozobova, Valeriya

    2010-01-01

    P53 is a tumour development p53. The aim of this work was to study the regulation of p53 in embryonic stem cells and its activation in response to DNA damage. p53 was found that p53 becomes transcriptionally active in ES cells after DNA damage. Embryonic stem cells contain a relatively high amount of p53 protein and p53 RNA. After differentiation p53 level is rapidly downregulated. The high abundance of p53 in undifferentiated ES cells is a result of enhanced translation.

  13. Corellation of p53 expressions and histopathological grading in oral cavity squamous cell carcinoma

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    Silvi Kintawati

    2016-09-01

    Full Text Available Background: Squamous cell carcinoma is a malignancy of oral cavity mostly occurred and can also metastasize. p53 gene is a tumor suppressor gene that plays an important role in carcinogenesis. The role of wild-type p53 is very important in suppressing the formation of a malignancy. p53 also has many other important functions. p53 is a suppressor of tumor/ cancer progression through the response of cell cycle to DNA damage and by giving time to repair DNA prior to replication of genes. p53 mutation, mostly occurs in a malignancy, so earlier histopathological transformation can be detected by observing p53 mutation. The prognosis of squamous cell carcinoma in oral cavity, therefore, depends on histopathological grading and clinical staging of the tumor. To enforce the histopathological grading, in addition based on histopathology differentiation, the earlier histopathological transformation can also be assessed. Purpose: This study aimed to determine the correlation of p53 expressions and histopathological grading in oral cavity squamous cell carcinoma. Method: This study was a retrospective study on 20 cases of oral cavity squamous cell carcinoma examined at Department of Pathology Anatomy in Hasan Sadikin Hospital in Bandung. Immunohistochemical examination was then performed using p53 antibodies to determine the correlation of p53 expression and histopathological grading in oral cavity squamous cell carcinoma to predict prognosis. Result: The overall results showed that there was no correlation between p53 expression and histopathological grading in oral cavity squamous cell carcinoma of the oral cavity although there was a very strong correlation between p53 expression and histopathological grading I (p<0.01. Conclusion: It can be concluded that there was no correlation between p53 expression and histopathological grading in oral cavity squamous cell carcinoma. Thus, p53 expression cannot be used to predict a prognosis.

  14. p53 Deletion promotes myeloma cells invasion by upregulating miR19a/CXCR5.

    Science.gov (United States)

    Yue, Zhijie; Zhou, Yongxia; Zhao, Pan; Chen, Yafang; Yuan, Ying; Jing, Yaoyao; Wang, Xiaofang

    2017-09-01

    P53 deletion has been identified as one of the few factors that defined high risk and poor prognosis in MM. It has been reported p53 deletion is associated with resistance to chemotherapy and organ infiltrations of MM. However, p53 deletion in the migration and dissemination of MM cells has not been totally elucidated. In this research, first, we investigated whether p53 is associated with migration of MM cells. We found that p53 regulates the migration of NCI-H929 cells with wild-type p53 but not U266 cells with mutated-type p53. Next, we investigated the related mechanism by which p53 regulates the migration. We found that down-regulation of p53 reduced adhesion of NCI-H929 cells to the BM stroma via decreased expression of E-cadherin and increased EMT-regulating proteins. Further study have identified the miR-19a/CXCR5 pathway as a candidate p53-induced migration mechanism. In conclusion, we have demonstrated for the first time the critical value of p53 deletion in MM cell migration and dissemination, as well as the acquisition of an EMT-like phenotype. Our research provides new insights into the function of p53 in migration of MM and suggests p53/miRNA19a/CXCR5 may provide potentially therapeutic targets for the treatment of myeloma with p53 deletion. Copyright © 2017. Published by Elsevier Ltd.

  15. p53 inactivation upregulates p73 expression through E2F-1 mediated transcription.

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    Chaitali Tophkhane

    Full Text Available While p73 overexpression has been associated with increased apoptosis in cancer tissues, p73 overexpressing tumors appear to be of high grade malignancy. Why this putative tumor suppressor is overexpressed in cancer cells and what the function of overexpressed p73 is in breast cancers are critical questions to be addressed. By investigating the effect of p53 inactivation on p73 expression, we found that both protein and mRNA levels of TAp73 were increased in MCF-7/p53siRNA cells, MCF-7/p53mt135 cells and HCT-116/p53-/- cells, as compared to wild type control, suggesting that p53 inactivation by various forms upregulates p73. We showed that p53 knockdown induced p73 was mainly regulated at the transcriptional level. However, although p53 has a putative binding site in the TAp73 promoter, deletion of this binding site did not affect p53 knockdown mediated activation of TAp73 promoter. Chromatin immuno-precipitation (ChIP data demonstrated that loss of p53 results in enhanced occupancy of E2F-1 in the TAp73 promoter. The responsive sequence of p53 inactivation mediated p73 upregulation was mapped to the proximal promoter region of the TAp73 gene. To test the role of E2F-1 in p53 inactivation mediated regulation of p73 transcription, we found that p53 knockdown enhanced E2F-1 dependent p73 transcription, and mutations in E2F-1 binding sites in the TAp73 promoter abrogated p53 knockdown mediated activation of TAp73 promoter. Moreover, we demonstrated that p21 is a mediator of p53-E2F crosstalk in the regulation of p73 transcription. We concluded that p53 knockdown/inactivation may upregulate TAp73 expression through E2F-1 mediated transcriptional regulation. p53 inactivation mediated upregulation of p73 suggests an intrinsic rescuing mechanism in response to p53 mutation/inactivation. These findings support further analysis of the correlation between p53 status and p73 expression and its prognostic/predictive significance in human cancers.

  16. The p53 Isoform Δ133p53β Promotes Cancer Stem Cell Potential

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    Nikola Arsic

    2015-04-01

    Full Text Available Cancer stem cells (CSC are responsible for cancer chemoresistance and metastasis formation. Here we report that Δ133p53β, a TP53 splice variant, enhanced cancer cell stemness in MCF-7 breast cancer cells, while its depletion reduced it. Δ133p53β stimulated the expression of the key pluripotency factors SOX2, OCT3/4, and NANOG. Similarly, in highly metastatic breast cancer cells, aggressiveness was coupled with enhanced CSC potential and Δ133p53β expression. Like in MCF-7 cells, SOX2, OCT3/4, and NANOG expression were positively regulated by Δ133p53β in these cells. Finally, treatment of MCF-7 cells with etoposide, a cytotoxic anti-cancer drug, increased CSC formation and SOX2, OCT3/4, and NANOG expression via Δ133p53, thus potentially increasing the risk of cancer recurrence. Our findings show that Δ133p53β supports CSC potential. Moreover, they indicate that the TP53 gene, which is considered a major tumor suppressor gene, also acts as an oncogene via the Δ133p53β isoform.

  17. The p53 Isoform Δ133p53β Promotes Cancer Stem Cell Potential

    Science.gov (United States)

    Arsic, Nikola; Gadea, Gilles; Lagerqvist, E. Louise; Busson, Muriel; Cahuzac, Nathalie; Brock, Carsten; Hollande, Frederic; Gire, Veronique; Pannequin, Julie; Roux, Pierre

    2015-01-01

    Summary Cancer stem cells (CSC) are responsible for cancer chemoresistance and metastasis formation. Here we report that Δ133p53β, a TP53 splice variant, enhanced cancer cell stemness in MCF-7 breast cancer cells, while its depletion reduced it. Δ133p53β stimulated the expression of the key pluripotency factors SOX2, OCT3/4, and NANOG. Similarly, in highly metastatic breast cancer cells, aggressiveness was coupled with enhanced CSC potential and Δ133p53β expression. Like in MCF-7 cells, SOX2, OCT3/4, and NANOG expression were positively regulated by Δ133p53β in these cells. Finally, treatment of MCF-7 cells with etoposide, a cytotoxic anti-cancer drug, increased CSC formation and SOX2, OCT3/4, and NANOG expression via Δ133p53, thus potentially increasing the risk of cancer recurrence. Our findings show that Δ133p53β supports CSC potential. Moreover, they indicate that the TP53 gene, which is considered a major tumor suppressor gene, also acts as an oncogene via the Δ133p53β isoform. PMID:25754205

  18. FTIR Microspectroscopy Probes Particle-Radiation Effect on HCT116 cells (p53+/+, p53-/-).

    Science.gov (United States)

    Yan, Jingwen; Zhang, Fengqiu; Huang, Qing

    2018-02-01

    p53 is a crucial tumor suppressor and plays an important role in cell cycle arrest, DNA damage repair, promotion of cell senescence and apoptosis, prevention of DNA damage and maintaining genomic stability and integrity. It has been reported that p53 might also be related to radiation sensitivity, for which the involved effects and processes could be further examined biochemically at the molecular level. In this study, we explored a new spectroscopic approach to probe the radiation-induced biological effects related to p53. Infrared microspectroscopy was used to detect the metabolic changes related to p53 under particle radiation. After alpha-particle irradiation of HCT116 cells (p53+/+, p53-/-), cell cycle arrest, DNA damage and lipid peroxidation in the cancer cells were observed using Fourier-transform infrared (FTIR) spectroscopy and microspectroscopy imaging. A remarkable difference in radiosensitivity between the two genotypes of cells was observed as well. This work provides a biochemical analysis of the p53-related radiation effects in cells and demonstrates the potential usefulness of FTIR microspectroscopy in the field of radiation research.

  19. p53 Aggregates penetrate cells and induce the co-aggregation of intracellular p53.

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    Karolyn J Forget

    Full Text Available Prion diseases are unique pathologies in which the infectious particles are prions, a protein aggregate. The prion protein has many particular features, such as spontaneous aggregation, conformation transmission to other native PrP proteins and transmission from an individual to another. Protein aggregation is now frequently associated to many human diseases, for example Alzheimer's disease, Parkinson's disease or type 2 diabetes. A few proteins associated to these conformational diseases are part of a new category of proteins, called prionoids: proteins that share some, but not all, of the characteristics associated with prions. The p53 protein, a transcription factor that plays a major role in cancer, has recently been suggested to be a possible prionoid. The protein has been shown to accumulate in multiple cancer cell types, and its aggregation has also been reproduced in vitro by many independent groups. These observations suggest a role for p53 aggregates in cancer development. This study aims to test the «prion-like» features of p53. Our results show in vitro aggregation of the full length and N-terminally truncated protein (p53C, and penetration of these aggregates into cells. According to our findings, the aggregates enter cells using macropinocytosis, a non-specific pathway of entry. Lastly, we also show that once internalized by the cell, p53C aggregates can co-aggregate with endogenous p53 protein. Together, these findings suggest prion-like characteristics for p53 protein, based on the fact that p53 can spontaneously aggregate, these aggregates can penetrate cells and co-aggregate with cellular p53.

  20. Interaction between transactivation domain of p53 and middle part of TBP-like protein (TLP is involved in TLP-stimulated and p53-activated transcription from the p21 upstream promoter.

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    Ryo Maeda

    Full Text Available TBP-like protein (TLP is involved in transcriptional activation of an upstream promoter of the human p21 gene. TLP binds to p53 and facilitates p53-activated transcription from the upstream promoter. In this study, we clarified that in vitro affinity between TLP and p53 is about one-third of that between TBP and p53. Extensive mutation analyses revealed that the TLP-stimulated function resides in transcription activating domain 1 (TAD1 in the N-terminus of p53. Among the mutants, #22.23, which has two amino acid substitutions in TAD1, exhibited a typical mutant phenotype. Moreover, #22.23 exhibited the strongest mutant phenotype for TLP-binding ability. It is thus thought that TLP-stimulated and p53-dependent transcriptional activation is involved in TAD1 binding of TLP. #22.23 had a decreased transcriptional activation function, especially for the upstream promoter of the endogenous p21 gene, compared with wild-type p53. This mutant did not facilitate p53-dependent growth repression and etoposide-mediated cell-death as wild-type p53 does. Moreover, mutation analysis revealed that middle part of TLP, which is requited for p53 binding, is involved in TLP-stimulated and p53-dependent promoter activation and cell growth repression. These results suggest that activation of the p21 upstream promoter is mediated by interaction between specific regions of TLP and p53.

  1. Abrogation of p53-mediated transactivation by SV40 large T antigen.

    Science.gov (United States)

    Segawa, K; Minowa, A; Sugasawa, K; Takano, T; Hanaoka, F

    1993-03-01

    p53 is known to bind specifically to the 44-bp human DNA sequence in an immunoprecipitation assay. We show here that the transcription of the reporter CAT gene linked with the herpesvirus thymidine kinase (tk) promoter containing the 44-base sequence is enhanced by mouse wild-type but not mutant-type p53 in F9 and p53-null Saos-2 cells. The p53-mediated transactivation was dramatically abrogated by introduction of SV40 large T antigen (SVLT) in Saos-2 cells in which p53 was clearly associated with SVLT. Furthermore, the p53-SVLT complex did not bind to the 44-base sequence at all. Thus, SVLT sequesters the transactivation function of the wild-type p53 by inhibiting the binding of p53 to the 44-base sequence. This is good evidence to show 'loss of functions' in the product of a tumor-suppressor oncogene by a dominant oncogene product at a molecular level.

  2. Grape Seed Proanthocyanidins Induce Apoptosis through p53, Bax, and Caspase 3 Pathways

    Directory of Open Access Journals (Sweden)

    Anshu M. Roy

    2005-01-01

    Full Text Available Grape seed proanthocyanidins (GSP have been shown to inhibit skin chemical carcinogenesis and photocarcinogenesis in mice. The mechanisms responsible for the anticarcinogenic effects of GSP are not clearly understood. Here, we report that treatment of JB6 C141 cells (a well-developed cell culture model for studying tumor promotion in keratinocytes and p53+/+ fibroblasts with GSP resulted in a dose-dependent induction of apoptosis. GSP-induced (20–80 g/ml apoptosis was observed by using immunofluorescence (27–90% apoptosis and flow cytometry (18–87% apoptosis. The induction of apoptosis by GSP was p53-dependent because it occurred mainly in cells expressing wild-type p53 (p53+/+; 15–80% to a much greater extent than in p53-deficient cells (p53-/-; 6–20%. GSP-induced apoptosis in JB6 C141 cells was associated with increased expression of the tumorsuppressor protein, p53, and its phosphorylation at Ser15. The antiapoptotic proteins, Bcl-2 and Bcl-xl, were downregulated by GSP, whereas the expression of the pro-apoptotic protein, Bax, and the levels of cytochrome c release, Apaf-1, caspase-9, and cleaved caspase 3 (p19 and p17 were markedly increased in JB6 C141 cells. The downregulation of Bcl-2 and upregulation of Bax were also observed in wild-type p53 (p53+/+ fibroblasts but was not observed in their p53-deficient counterparts. These data clearly demonstrate that GSP-induced apoptosis is p53-dependent and mediated through the Bcl-2, Bax, and caspase 3 pathways.

  3. Driving p53 Response to Bax Activation Greatly Enhances Sensitivity to Taxol by Inducing Massive Apoptosis

    Directory of Open Access Journals (Sweden)

    Paola De Feudis

    2000-05-01

    Full Text Available The proapoptotic gene bax is one of the downstream effectors of p53. The p53 binding site in the bax promoter is less responsive to p53 than the one in the growth arrest mediating gene p21. We introduced the bax gene under the control of 13 copies of a strong p53 responsive element into two ovarian cancer cell lines. The clones expressing bax under the control of p53 obtained from the wild-type (wt p53-expressing cell line A2780 were much more sensitive (500- to 1000-fold to the anticancer agent taxol than the parent cell line, with a higher percentage of cells undergoing apoptosis after drug treatment that was clearly p53-dependent and bax-mediated. Xenografts established in nude mice from one selected clone (A2780/C3 were more responsive to taxol than the parental line and the apoptotic response of A2780/C3 tumors was also increased after treatment. Introduction of the same plasmid into the p53 null SKOV3 cell line did not alter the sensitivity to taxol or the induction of apoptosis. In conclusion, driving the p53 response (after taxol treatment by activating the bax gene rather than the p21 gene results in induction of massive apoptosis, in vitro and in vivo, and greatly enhances sensitivity to the drug.

  4. Evidence for allosteric effects on p53 oligomerization induced by phosphorylation.

    Science.gov (United States)

    Muller, Petr; Chan, Juliana M; Simoncik, Oliver; Fojta, Miroslav; Lane, David P; Hupp, Ted; Vojtesek, Borivoj

    2017-11-10

    p53 is a tetrameric protein with a thermodynamically unstable deoxyribonucleic acid (DNA)-binding domain flanked by intrinsically disordered regulatory domains that control its activity. The unstable and disordered segments of p53 allow high flexibility as it interacts with binding partners and permits a rapid on/off switch to control its function. The p53 tetramer can exist in multiple conformational states, any of which can be stabilized by a particular modification. Here, we apply the allostery model to p53 to ask whether evidence can be found that the "activating" C-terminal phosphorylation of p53 stabilizes a specific conformation of the protein in the absence of DNA. We take advantage of monoclonal antibodies for p53 that measure indirectly the following conformations: unfolded, folded, and tetrameric. A double antibody capture enzyme linked-immunosorbent assay was used to observe evidence of conformational changes of human p53 upon phosphorylation by casein kinase 2 in vitro. It was demonstrated that oligomerization and stabilization of p53 wild-type conformation results in differential exposure of conformational epitopes PAb1620, PAb240, and DO12 that indicates a reduction in the "unfolded" conformation and increases in the folded conformation coincide with increases in its oligomerization state. These data highlight that the oligomeric conformation of p53 can be stabilized by an activating enzyme and further highlight the utility of the allostery model when applied to understanding the regulation of unstable and intrinsically disordered proteins. © 2017 The Protein Society.

  5. Driving p53 Response to Bax Activation Greatly Enhances Sensitivity to Taxol by Inducing Massive Apoptosis

    Science.gov (United States)

    De Feudis, Paola; Vignati, Sara; Rossi, Cosmo; Mincioni, Tatiana; Giavazzi, Raffaella; D'Incalci, Maurizio; Broggini, Massimo

    2000-01-01

    Abstract The proapoptotic gene bax is one of the downstream effectors of p53. The p53 binding site in the bax promoter is less responsive to p53 than the one in the growth arrest mediating gene p21. We introduced the bax gene under the control of 13 copies of a strong p53 responsive element into two ovarian cancer cell lines. The clones expressing bax under the control of p53 obtained from the wild-type (wt) p53-expressing cell line A2780 were much more sensitive (500- to 1000-fold) to the anticancer agent taxol than the parent cell line, with a higher percentage of cells undergoing apoptosis after drug treatment that was clearly p53-dependent and bax-mediated. Xenografts established in nude mice from one selected clone (A2780/C3) were more responsive to taxol than the parental line and the apoptotic response of A2780/C3 tumors was also increased after treatment. Introduction of the same plasmid into the p53 null SKOV3 cell line did not alter the sensitivity to taxol or the induction of apoptosis. In conclusion, driving the p53 response (after taxol treatment) by activating the bax gene rather than the p21 gene results in induction of massive apoptosis, in vitro and in vivo, and greatly enhances sensitivity to the drug. PMID:10935506

  6. Aciculatin induces p53-dependent apoptosis via MDM2 depletion in human cancer cells in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Chin-Yu Lai

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

  7. Induction of cell proliferation arrest and apoptosis in hepatoma cells through adenoviral-mediated transfer of p53 gene.

    Science.gov (United States)

    Reiser, M; Neumann, I; Schmiegel, W; Wu, P C; Lau, J Y

    2000-05-01

    Loss of p53 function is common in hepatocellular carcinoma and is associated with an extremely poor prognosis. The aim of the study was to evaluate the biologic effect of adenoviral-mediated gene transfer of wild-type p53 gene in four hepatoma cell lines with different p53 genetic makeup. Recombinant adenovirus expressing wild-type p53 was used. Recombinant adenoviruses with either an empty expression cassette or expressing beta-galactosidase gene served as controls. High-level expression of wild-type p53 was achieved with adenoviral-mediated gene transfer. The expressed p53 protein showed nuclear localization and its expression was associated with an induction of p21 and bax expression. Expression of the p53 gene was associated with inhibition of tumor cell proliferation and induction of apoptosis. Expression of p53 was also associated with an upregulation of CD95 (Apo-1/Fas) gene expression, which may predispose the tumor cells to undergo apoptosis induced by the Fas Ligand/Fas cytolytic pathway. An additional anti-tumor effect, in terms of allowing the replication-defective adenovirus to replicate, was observed in hepatoma cells with homozygous deletion of p53 genes and to a lesser extent, hepatoma cells with mutated p53 genes. These data showed that adenoviral-mediated gene transfer is effective in delivering p53 gene to tumor cells, and the multiple pathways involved in their antitumor activities.

  8. Tumor Suppressor p53 Stimulates the Expression of Epstein-Barr Virus Latent Membrane Protein 1.

    Science.gov (United States)

    Wang, Qianli; Lingel, Amy; Geiser, Vicki; Kwapnoski, Zachary; Zhang, Luwen

    2017-10-15

    Epstein-Barr virus (EBV) is associated with multiple human malignancies. EBV latent membrane protein 1 (LMP1) is required for the efficient transformation of primary B lymphocytes in vitro and possibly in vivo The tumor suppressor p53 plays a seminal role in cancer development. In some EBV-associated cancers, p53 tends to be wild type and overly expressed; however, the effects of p53 on LMP1 expression is not clear. We find LMP1 expression to be associated with p53 expression in EBV-transformed cells under physiological and DNA damaging conditions. DNA damage stimulates LMP1 expression, and p53 is required for the stimulation. Ectopic p53 stimulates endogenous LMP1 expression. Moreover, endogenous LMP1 blocks DNA damage-mediated apoptosis. Regarding the mechanism of p53-mediated LMP1 expression, we find that interferon regulatory factor 5 (IRF5), a direct target of p53, is associated with both p53 and LMP1. IRF5 binds to and activates a LMP1 promoter reporter construct. Ectopic IRF5 increases the expression of LMP1, while knockdown of IRF5 leads to reduction of LMP1. Furthermore, LMP1 blocks IRF5-mediated apoptosis in EBV-infected cells. All of the data suggest that cellular p53 stimulates viral LMP1 expression, and IRF5 may be one of the factors for p53-mediated LMP1 stimulation. LMP1 may subsequently block DNA damage- and IRF5-mediated apoptosis for the benefits of EBV. The mutual regulation between p53 and LMP1 may play an important role in EBV infection and latency and its related cancers.IMPORTANCE The tumor suppressor p53 is a critical cellular protein in response to various stresses and dictates cells for various responses, including apoptosis. This work suggests that an Epstein-Bar virus (EBV) principal viral oncogene is activated by cellular p53. The viral oncogene blocks p53-mediated adverse effects during viral infection and transformation. Therefore, the induction of the viral oncogene by p53 provides a means for the virus to cope with infection and DNA

  9. Stathmin is overexpressed and regulated by mutant p53 in oral squamous cell carcinoma.

    Science.gov (United States)

    Ma, Hai-Long; Jin, Shu-Fang; Ju, Wu-Tong; Fu, Yong; Tu, Yao-Yao; Wang, Li-Zhen; Jiang-Li; Zhang, Zhi-Yuan; Zhong, Lai-Ping

    2017-08-14

    The aim of this study was to investigate the oncogenic function and regulatory mechanism of stathmin in oral squamous cell carcinoma (OSCC). Two-dimensional electrophoresis and liquid chromatography-tandem mass chromatography were applied to screen differentiated proteins during carcinogenesis in OSCC. Cell Counting Kit-8 (CCK-8) assays, colony formation, migration, flow cytometry, immunofluorescence and a xenograft model were used to detect the function of stathmin. The correlation between stathmin and p53 expression was analyzed using immunohistochemistry. Mutant/wild type p53 plasmids and small interfering RNA were used to examine the regulation of stathmin. Chromatin immunoprecipitation assays and luciferase assays were performed to detect the transcriptional activation of stathmin by p53. Overexpression of stathmin was screened and confirmed in OSCC patients and cell lines. Silencing expression of stathmin inhibited proliferation, colony formation and migration and promoted apoptosis. Poly ADP ribose polymerase (PARP) and cyclin-dependent kinase 1 (cdc2) were activated after silencing the expression of stathmin. Suppression of tumorigenicity was also confirmed in vivo. Mutant p53 transcriptionally activated the expression of stathmin in HN6 and HN13 cancer cells, but not in HN30 cells harboring wild type p53. These results suggest that stathmin acts as an oncogene and is transcriptionally regulated by mutant p53, but not by wild-type p53. Stathmin could be a potential anti-tumor therapeutic target in OSCC.

  10. Inhibition of NAMPT pathway by FK866 activates the function of p53 in HEK293T cells

    Energy Technology Data Exchange (ETDEWEB)

    Thakur, Basant Kumar, E-mail: thakur.basant@mh-hannover.de [Department of Pediatrics, Hematology and Oncology, Hannover Medical School, Carl Neuberg Str-1, 30625 Hannover (Germany); Department of Molecular Hematopoiesis, Hannover Medical School, Carl Neuberg Str-1, 30625 Hannover (Germany); Dittrich, Tino [Department of Pediatrics, Hematology and Oncology, Hannover Medical School, Carl Neuberg Str-1, 30625 Hannover (Germany); Department of Molecular Hematopoiesis, Hannover Medical School, Carl Neuberg Str-1, 30625 Hannover (Germany); Chandra, Prakash [Frankfurt University Medical School, Molecular Biology, Building 57, Theodor-Stern-Kai-7, 60590 Frankfurt (Germany); Becker, Annette [Department of Biology, Technical University of Darmstadt, 64287 Darmstadt (Germany); Lippka, Yannick [Department of Molecular Hematopoiesis, Hannover Medical School, Carl Neuberg Str-1, 30625 Hannover (Germany); Selvakumar, Divakarvel [Department of Molecular Hematopoiesis, Hannover Medical School, Carl Neuberg Str-1, 30625 Hannover (Germany); Rutgers, The State University of New Jersey, NJ 08901 (United States); Klusmann, Jan-Henning; Reinhardt, Dirk [Department of Pediatrics, Hematology and Oncology, Hannover Medical School, Carl Neuberg Str-1, 30625 Hannover (Germany); Welte, Karl, E-mail: Welte.Karl.H@mh-hannover.de [Department of Molecular Hematopoiesis, Hannover Medical School, Carl Neuberg Str-1, 30625 Hannover (Germany)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer In 293T cells, p53 is considered to be inactive due to its interaction with the large T-antigen. Black-Right-Pointing-Pointer Acetylation of p53 at lysine 382 is important for its functional activation. Black-Right-Pointing-Pointer First evidence to document the presence of a functional p53 in 293T cells. Black-Right-Pointing-Pointer Inhibition of NAMPT/SIRT pathway by FK866 in 293T cells increases the functional activity of p53. Black-Right-Pointing-Pointer This activation of p53 involves reversible acetylation of p53 at lysine 382. -- Abstract: Inactivation of p53 protein by endogenous and exogenous carcinogens is involved in the pathogenesis of different human malignancies. In cancer associated with SV-40 DNA tumor virus, p53 is considered to be non-functional mainly due to its interaction with the large T-antigen. Using the 293T cell line (HEK293 cells transformed with large T antigen) as a model, we provide evidence that p53 is one of the critical downstream targets involved in FK866-mediated killing of 293T cells. A reduced rate of apoptosis and an increased number of cells in S-phase was accompanied after knockdown of p53 in these cells. Inhibition of NAMPT by FK866, or inhibition of SIRT by nicotinamide decreased proliferation and triggered death of 293T cells involving the p53 acetylation pathway. Additionally, knockdown of p53 attenuated the effect of FK866 on cell proliferation, apoptosis, and cell cycle arrest. The data presented here shed light on two important facts: (1) that p53 in 293T cells is active in the presence of FK866, an inhibitor of NAMPT pathway; (2) the apoptosis induced by FK866 in 293T cells is associated with increased acetylation of p53 at Lys382, which is required for the functional activity of p53.

  11. The p53-MDM2 network: from oscillations to apoptosis

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    developed a class of small molecules known as nutlins which occupy the p53 binding pocket in MDM2, thus preventing the binding of MDM2 to p53 and facilitating the activation of the p53 pathways in human cancer cell lines. The efficacy of the strategy has been demonstrated in experiments paving. Figure 4. Pulses of p53 ...

  12. Loss of p53 Induces Tumorigenesis in p21-Deficient Mesenchymal Stem Cells

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    Rene Rodriguez

    2009-04-01

    Full Text Available There is growing evidence about the role of mesenchymal stem cells (MSCs as cancer stem cells in many sarcomas. Nevertheless, little is still known about the cellular and molecular mechanisms underlying MSCs transformation. We aimed at investigating the role of p53 and p21, two important regulators of the cell cycle progression and apoptosis normally involved in protection against tumorigenesis. Mesenchymal stem cells from wild-type, p21-/-p53+/+, and p21-/-p53+/- mice were cultured in vitro and analyzed for the appearance of tumoral transformation properties after low, medium, and high number of passages both in vitro and in vivo. Wild-type or p21-/-p53+/+ MSCs did not show any sign of tumoral transformation. Indeed, after short-term in vitro culture, wild-type MSCs became senescent, and p21-/-p53+/+ MSCs showed an elevated spontaneous apoptosis rate. Conversely, MSCs carrying a mutation in one allele of the p53 gene (p21-/-p53+/- MSCs completely lost p53 expression after in vitro long-term culture. Loss of p53 was accompanied by a significant increase in the growth rate, gain of karyotypic instability, loss of p16 expression, and lack of senescence response. Finally, these cells were able to form fibrosarcomas partially differentiated into different mesenchymal lineages when injected in immunodeficient mice both after subcutaneous and intrafemoral injection. These findings show that MSCs are very sensitive to mutations in genes involved in cell cycle control and that these deficiencies can be at the origin of some mesodermic tumors.

  13. USP3 stabilizes p53 protein through its deubiquitinase activity.

    Science.gov (United States)

    Fu, Song; Shao, Shize; Wang, Longqiang; Liu, Haijun; Hou, Haitao; Wang, Yanan; Wang, Huan; Huang, Xiangpeng; Lv, Renhua

    2017-10-14

    p53 is the guardian of the genome integrity and the degradation of p53 protein is mediated by MDM2. Here we report that USP3 interacts with p53 and regulates p53 stability. Depletion of USP3 lead to accelerated degradation of p53 in normal cells thereby enhanced cell proliferation and transformation. Reconstitution of wildtype USP3, but not the USP3 C168S mutant, restored the stability of p53 protein and inhibited cell proliferation and transformation. These findings suggest that USP3 is an important regulator of p53 and regulates normal cell transformation. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Reprogramming pancreatic stellate cells via p53 activation: A putative target for pancreatic cancer therapy.

    Directory of Open Access Journals (Sweden)

    Maya Saison-Ridinger

    Full Text Available Pancreatic ductal adenocarcinoma (PDAC is characterized by an extremely dense fibrotic stroma, which contributes to tumor growth, metastasis, and drug resistance. During tumorigenesis, quiescent pancreatic stellate cells (PSCs are activated and become major contributors to fibrosis, by increasing growth factor signaling and extracellular matrix deposition. The p53 tumor suppressor is known to restrict tumor initiation and progression through cell autonomous mechanisms including apoptosis, cell cycle arrest, and senescence. There is growing evidence that stromal p53 also exerts anti-tumor activity by paracrine mechanisms, though a role for stromal p53 in PDAC has not yet been described. Here, we demonstrate that activation of stromal p53 exerts anti-tumor effects in PDAC. We show that primary cancer-associated PSCs (caPSCs isolated from human PDAC express wild-type p53, which can be activated by the Mdm2 antagonist Nutlin-3a. Our work reveals that p53 acts as a major regulator of PSC activation and as a modulator of PDAC fibrosis. In vitro, p53 activation by Nutlin-3a induces profound transcriptional changes, which reprogram activated PSCs to quiescence. Using immunofluorescence and lipidomics, we have also found that p53 activation induces lipid droplet accumulation in both normal and tumor-associated fibroblasts, revealing a previously undescribed role for p53 in lipid storage. In vivo, treatment of tumor-bearing mice with the clinical form of Nutlin-3a induces stromal p53 activation, reverses caPSCs activation, and decreases fibrosis. All together our work uncovers new functions for stromal p53 in PDAC.

  15. Differential Sensitivity of Cells to Radiation Mediated by p53 Pulses

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    Kim, Jin Kyu; Kang, Mi Young; Kawala, Remigius A.; Ryu, Tae Ho; Kim, Jin-Hong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2015-10-15

    Exposure of cells to ionizing radiation activates protein genes related cell cycle arrest and cell death (apoptosis or autophagy). The tumor suppressor p53 participates not only in regulation of apoptosis, but also in autophagy mechanism. Apoptosis (type I cell death) is characterized by the activation of caspases and the formation of apoptotic bodies, and plays essential roles in all multicellular organisms. On the other hand, autophagy (type II cell death) is characterized by the presence of cytoplasmic engulfing vesicles, alias autophagosomes, and is a major intracellular pathway for degradation and recycling of proteins, ribosomes and entire organelles. The purpose of this study was to determine whether ionizing radiation treatment induces autophagy depending on the p53 expression levels. RKO (wild-type p53) and RKO E6 (null-type p53) cells were used to evaluate the effects of p53 on the sensitivity of cells to ionizing radiation. In the RKO E6 cells, the function of p53 was disabled with human papillomavirus E6 oncoprotein. These results indicated that p53 and p21 were required to block apoptosis and induce autophagy in RKO cells. The expression of p21 by a p53-dependent mechanism is required to develop autophagic properties after DNA damage. Results in this study suggest that the radioresistance of the RKO cells was associated with the increased p21 expression, resulting in autophagy induction. The tumor suppressor p53 could regulate radiosensitivity by inhibiting autophagy and activating apoptosis; the ionizing radiation-induced expression of p53 in the RKO cells regulated autophagy, suggesting the significance of the level of p53 in determining the radiosensitivity by regulating autophagy and apoptosis.

  16. Oncogenic intra-p53 family member interactions in human cancers

    Directory of Open Access Journals (Sweden)

    Maria eFerraiuolo

    2016-03-01

    Full Text Available 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 homologues 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 (GOF 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 (VDR, 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 (DDR, DNA double-strand breaks (DSBs 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.

  17. Chemotherapy-Induced Apoptosis in a Transgenic Model of Neuroblastoma Proceeds Through p53 Induction

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    Louis Chesler

    2008-11-01

    Full Text Available Chemoresistance in neuroblastoma is a significant issue complicating treatment of this common pediatric solid tumor. MYCN-amplified neuroblastomas are infrequently mutated at p53 and are chemosensitive at diagnosis but acquire p53 mutations and chemoresistance with relapse. Paradoxically, Myc-driven transformation is thought to require apoptotic blockade. We used the TH-MYCN transgenic murine model to examine the role of p53-driven apoptosis on neuroblastoma tumorigenesis and the response to chemotherapy. Tumors formed with high penetrance and low latency in p53-haploinsufficient TH-MYCN mice. Cyclophosphamide (CPM induced a complete remission in p53 wild type TH-MYCN tumors, mirroring the sensitivity of childhood neuroblastoma to this agent. Treated tumors showed a prominent proliferation block, induction of p53 protein, and massive apoptosis proceeding through induction of the Bcl-2 homology domain-3-only proteins PUMA and Bim, leading to the activation of Bax and cleavage of caspase-3 and -9. Apoptosis induced by CPM was reduced in p53-haploinsufficient tumors. Treatment of MYCN-expressing human neuroblastoma cell lines with CPM induced apoptosis that was suppressible by siRNA to p53. Taken together, the results indicate that the p53 pathway plays a significant role in opposing MYCN-driven oncogenesis in a mouse model of neuroblastoma and that basal inactivation of the pathway is achieved in progressing tumors. This, in part, explains the striking sensitivity of such tumors to chemotoxic agents that induce p53-dependent apoptosis and is consistent with clinical observations that therapy-associated mutations in p53 are a likely contributor to the biology of tumors at relapse and secondarily mediate resistance to therapy.

  18. Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats

    Science.gov (United States)

    Botcheva, Krassimira; McCorkle, Sean R.

    2014-01-01

    The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We report distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). Our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways. PMID:25415302

  19. Correlation between osteosarcoma and the expression of WWOX and p53.

    Science.gov (United States)

    Liu, Pingtao; Wang, Mingyue; Li, Li; Jin, Tao

    2017-10-01

    The objective of this study was to analyze the effect of the expression of WWOX and p53 on the growth of MG-63 osteosarcoma cells and to explore the correlation between osteosarcoma and the expression of WWOX and p53. WWOX and p53-overexpressing MG-63 osteosarcoma cell lines were established by transfection and named the MW and MP cell lines, respectively. Untransfected MG-63 cells (blank control) were used as control. Quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of WWOX and wild-type p53 mRNA and protein, respectively. The effects of WWOX and p53 (wild-type) on the activity of MG-63 cells were determined by MTT assay and flow cytometry. The expression of mutant p53 protein in 65 cases of osteosarcoma was detected by immunohistochemistry to analyze the correlation between p53 and the development of osteosarcoma. qPCR showed that WWOX and p53 mRNA was overexpressed in MW and MP cells, respectively. Western blot analysis showed that the levels of WWOX and p53 protein in MW and MP cells were higher than in the blank control group. MTT assay showed that the cell proliferation ability of MW and MP cells was significantly lower than in the blank control group. Flow cytometry showed that 78.49% of MW and 66.76% of MP cells were arrested in the G0/G1 phase. Immunohistochemistry showed that mutant p53 was highly expressed in osteosarcoma, with a positive expression rate of 47.7%. The expression rate was positively correlated with the pathological grade of cancer. In conclusion, WWOX can affect the cell cycle of MG-63 osteosarcoma cells to inhibit cell proliferation, which provides new insights into gene therapy for osteosarcoma. The two types of the p53 gene have different functions in the development of osteosarcoma. Wild-type p53 acts as a tumor suppressor, while mutant p53, which is overexpressed in malignant osteosarcoma, has a carcinogenic effect associated with the degree of osteosarcoma.

  20. p53 activation contributes to patulin-induced nephrotoxicity via modulation of reactive oxygen species generation.

    Science.gov (United States)

    Jin, Huan; Yin, Shutao; Song, Xinhua; Zhang, Enxiang; Fan, Lihong; Hu, Hongbo

    2016-04-13

    Patulin is a major mycotoxin found in fungal contaminated fruits and their derivative products. Previous studies showed that patulin was able to induce increase of reactive oxygen species (ROS) generation and oxidative stress was suggested to play a pivotal role in patulin-induced multiple toxic signaling. The objective of the present study was to investigate the functional role of p53 in patulin-induced oxidative stress. Our study demonstrated that higher levels of ROS generation and DNA damage were induced in wild-type p53 cell lines than that found in either knockdown or knockout p53 cell lines in response to patulin exposure, suggesting p53 activation contributed to patulin-induced ROS generation. Mechanistically, we revealed that the pro-oxidant role of p53 in response to patulin was attributed to its ability to suppress catalase activity through up-regulation of PIG3. Moreover, these in vitro findings were further validated in the p53 wild-type/knockout mouse model. To the best of our knowledge, this is the first report addressing the functional role of p53 in patulin-induced oxidative stress. The findings of the present study provided novel insights into understanding mechanisms behind oxidative stress in response to patulin exposure.

  1. The tobacco carcinogen NNK disturbs mitotic chromosome alignment by interrupting p53 targeting to the centrosome.

    Science.gov (United States)

    Park, Ji Eun; Jang, Yu Lim; Jang, Chang-Young

    2017-11-05

    The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent risk factor among tobacco-related carcinogens in lung cancer progression and outcomes. Although genetic mutations and chromosome instability have been detected in NNK-induced lung tumors, the oncogenic mechanisms of NNK are not fully understood. Here, we show that NNK increases chromosomal instability by disrupting spindle microtubule (MT) attachment to the kinetochore (KT) and spindle dynamics. Mechanistically, NNK blocks the targeting of p53 to the centrosome during mitosis, leading to chromosome alignment defects in metaphase. Therefore, lung cancer cells with wild-type p53, such as A594 and H226B, are more resistant to the NNK treatment than p53-mutant lung cancer cells, such as A1299 and H226Br. Although NNK does not affect the levels or transcriptional activity of p53, the reduction of the p53 level at the centrosome exacerbates the NNK-induced chromosome alignment defect in A549 and H226B cells. Therefore, p53 protects against NNK-induced chromosome instability by modulating the function of centrosome-localized p53 and not by modulating transcriptional activity. We conclude that NNK may increase the risk of lung cancer progression and poorer outcomes in patients with p53 mutations by perturbing proper mitotic progression and chromosome integrity. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. A novel dysfunctional p53 mutation in the human neuroblastoma cell line TGW.

    Science.gov (United States)

    Sugiyama, Hisahiko; Arita, Michitsune; Min, Zhenghua; Zhong, Xioling; Iwasaki, Iwao; Hirano, Keihachiro; Shimatake, Hiroyuki; Hemmi, Hiromichi

    2003-12-01

    Mutations of p53 are rare in primary and advanced neuroblastomas. The p53 gene was studied in a TGW cell line established from a TNB1 xenograft, derived from metastasized neuroblastoma. The p53 protein level in TGW was elevated at baseline. Treatment with doxorubicin to induce genotoxic stress neither altered the p53 protein level nor induced p21 protein within 24 hours. DNA sequencing analysis revealed a novel triplet deletion mutation at codon 282 (R282del) of the p53 gene, a mutation also found in TNB1, indicating that the mutation occurred in the relapsed tumor. The mutant was incapable of transactivation and had no effect on the transactivational activity of the wild-type p53 gene product in reporter assays using a plasmid possessing a p53 responsive element of p21, bax or mdm2. These results suggest that the mutant p53R282del found in TGW is a non-functional mutant and has no dominant negative nature.

  3. High Mdm4 levels suppress p53 activity and enhance its half-life in acute myeloid leukaemia

    Science.gov (United States)

    Tan, Ban Xiong; Khoo, Kian Hoe; Lim, Tit Meng; Lane, David Philip

    2014-01-01

    Although p53 is found mutated in almost 50% of all cancers, p53 mutations in leukaemia are relatively rare. Acute myeloid leukaemia (AML) cells employ other strategies to inactivate their wild type p53 (WTp53), like the overexpression of the p53 negative regulators Mdm2 and Mdm4. As such, AMLs are excellent candidates for therapeutics involving the reactivation of their WTp53 to restrict and destroy cancer cells, and the Mdm2 antagonist nutlin-3 is one such promising agent. Using AML cell lines with WTp53, we identified stable and high levels of p53 in the OCI/AML-2 cell lines. We demonstrate that this nutlin-3 sensitive cell line overexpressed Mdm4 to sequester, stabilise and inhibit p53 in the cytoplasm. We also show that elevated Mdm4 competed with Mdm2-p53 interaction and therefore extended p53 half-life while preventing p53 transcriptional activity. Our results provide biochemical evidence on the dynamics of the p53-Mdm2-Mdm4 interactions in affecting p53 levels and activity, and unlike previously reported findings derived from genetically manipulated systems, AML cells with naturally high levels of Mdm4 remain sensitive to nutlin treatment. Key Points Endogenously high levels of Mdm4 inhibit and sequester p53 in AML. High levels of Mdm4 do not block function of Mdm2 inhibitors in AML. PMID:24659749

  4. p53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system.

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    Virginia Andreotti

    Full Text Available BACKGROUND: The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4. METHODOLOGY/PRINCIPAL FINDINGS: Given the many factors that might influence p53 function, including expression levels, mutations, cofactor proteins and small molecules, we expanded our previously described yeast-based system to provide the opportunity for efficient investigation of their individual and combined impacts in a miniaturized format. The system integrates i variable expression of p53 proteins under the finely tunable GAL1,10 promoter, ii single copy, chromosomally located p53-responsive and control luminescence reporters, iii enhanced chemical uptake using modified ABC-transporters, iv small-volume formats for treatment and dual-luciferase assays, and v opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1. CONCLUSIONS/SIGNIFICANCE: We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins.

  5. The p53 target Wig-1 regulates p53 mRNA stability through an AU-rich element

    DEFF Research Database (Denmark)

    Vilborg, Anna; Glahder, Jacob-Andreas Harald; Wilhelm, Margareta T

    2009-01-01

    The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3' UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells...... exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link...... between the tumor suppressor p53 and posttranscriptional gene regulation via AREs in mRNA....

  6. Isg15 controls p53 stability and functions

    Science.gov (United States)

    Huang, Yi-Fu; Wee, Sheena; Gunaratne, Jayantha; Lane, David P; Bulavin, Dmitry V

    2014-01-01

    Degradation of p53 is a cornerstone in the control of its functions as a tumor suppressor. This process is attributed to ubiquitin-dependent modification of p53. In addition to polyubiquitination, we found that p53 is targeted for degradation through ISGylation. Isg15, a ubiquitin-like protein, covalently modifies p53 at 2 sites in the N and C terminus, and ISGylated p53 can be degraded by the 20S proteasome. ISGylation primarily targets a misfolded, dominant-negative p53, and Isg15 deletion in normal cells results in suppression of p53 activity and functions. We propose that Isg15-dependent degradation of p53 represents an alternative mechanism of controlling p53 protein levels, and, thus, it is an attractive pathway for drug discovery. PMID:24844324

  7. Impact of p53 status on heavy-ion radiation-induced micronuclei in circulating erythrocytes

    Science.gov (United States)

    Chang, P. Y.; Torous, D.; Lutze-Mann, L.; Winegar, R.

    2000-01-01

    Transgenic mice that differed in their p53 genetic status were exposed to an acute dose of highly charged and energetic (HZE) iron particle radiation. Micronuclei (MN) in two distinct populations of circulating peripheral blood erythrocytes, the immature reticulocytes (RETs) and the mature normochromatic erythrocytes (NCEs), were measured using a simple and efficient flow cytometric procedure. Our results show significant elevation in the frequency of micronucleated RETs (%MN-RETs) at 2 and 3 days post-radiation. At 3 days post-irradiation, the magnitude of the radiation-induced MN-RET was 2.3-fold higher in the irradiated p53 wild-type animals compared to the unirradiated controls, 2.5-fold higher in the p53 hemizygotes and 4.3-fold higher in the p53 nullizygotes. The persistence of this radiation-induced elevation of MN-RETs is dependent on the p53 genetic background of the animal. In the p53 wild-type and p53 hemizygotes, %MN-RETs returned to control levels by 9 days post-radiation. However, elevated levels of %MN-RETs in p53 nullizygous mice persisted beyond 56 days post-radiation. We also observed elevated MN-NCEs in the peripheral circulation after radiation, but the changes in radiation-induced levels of MN-NCEs appear dampened compared to those of the MN-RETs for all three strains of animals. These results suggest that the lack of p53 gene function may play a role in the iron particle radiation-induced genomic instability in stem cell populations in the hematopoietic system.

  8. p53 mutations occur in aggressive breast cancer.

    Science.gov (United States)

    Mazars, R; Spinardi, L; BenCheikh, M; Simony-Lafontaine, J; Jeanteur, P; Theillet, C

    1992-07-15

    Using a polymerase chain reaction-single strand conformation polymorphism approach we analyzed 96 human primary breast tumors for the presence of mutations in exons 2, 5, 6, 7, 8, and 9 of the p53 gene. These exons have been shown to comprise highly conserved sequences and the portion including exons 5 through 9 is believed to be the target for over 90% of the acquired mutations in human cancer. Eighteen tumors of the 96 (18.7%) tested showed reproducibly a variant band indicative of a mutation. Most (15 tumors) of the mutations were single nucleotide substitutions and G:C to A:T transitions were prevalent (6 tumors), G:C to T:A transversions came next (4 tumors), and guanines were always on the nontranscribed strand. Concomitant loss of the wild type allele and mutation of the other copy was observed in only 3 of 18 mutated cases; this is consistent with the heterogeneous cellular composition of breast tumors. Furthermore p53 mutations were correlated to estrogen and/or progesterone receptor negative tumors, thus indicating their relationships to aggressive breast cancer. No association could be observed with DNA amplification events in these tumors.

  9. Prospective virtual screening for novel p53-MDM2 inhibitors using ultrafast shape recognition

    Science.gov (United States)

    Patil, Sachin P.; Ballester, Pedro J.; Kerezsi, Cassidy R.

    2014-02-01

    The p53 protein, known as the guardian of genome, is mutated or deleted in approximately 50 % of human tumors. In the rest of the cancers, p53 is expressed in its wild-type form, but its function is inhibited by direct binding with the murine double minute 2 (MDM2) protein. Therefore, inhibition of the p53-MDM2 interaction, leading to the activation of tumor suppressor p53 protein presents a fundamentally novel therapeutic strategy against several types of cancers. The present study utilized ultrafast shape recognition (USR), a virtual screening technique based on ligand-receptor 3D shape complementarity, to screen DrugBank database for novel p53-MDM2 inhibitors. Specifically, using 3D shape of one of the most potent crystal ligands of MDM2, MI-63, as the query molecule, six compounds were identified as potential p53-MDM2 inhibitors. These six USR hits were then subjected to molecular modeling investigations through flexible receptor docking followed by comparative binding energy analysis. These studies suggested a potential role of the USR-selected molecules as p53-MDM2 inhibitors. This was further supported by experimental tests showing that the treatment of human colon tumor cells with the top USR hit, telmisartan, led to a dose-dependent cell growth inhibition in a p53-dependent manner. It is noteworthy that telmisartan has a long history of safe human use as an approved anti-hypertension drug and thus may present an immediate clinical potential as a cancer therapeutic. Furthermore, it could also serve as a structurally-novel lead molecule for the development of more potent, small-molecule p53-MDM2 inhibitors against variety of cancers. Importantly, the present study demonstrates that the adopted USR-based virtual screening protocol is a useful tool for hit identification in the domain of small molecule p53-MDM2 inhibitors.

  10. Hypoxia regulates human lung fibroblast proliferation via p53-dependent and -independent pathways

    Directory of Open Access Journals (Sweden)

    Ameshima Shingo

    2009-03-01

    Full Text Available Abstract Background Hypoxia induces the proliferation of lung fibroblasts in vivo and in vitro. However, the subcellular interactions between hypoxia and expression of tumor suppressor p53 and cyclin-dependent kinase inhibitors p21 and p27 remain unclear. Methods Normal human lung fibroblasts (NHLF were cultured in a hypoxic chamber or exposed to desferroxamine (DFX. DNA synthesis was measured using bromodeoxyuridine incorporation, and expression of p53, p21 and p27 was measured using real-time RT-PCR and Western blot analysis. Results DNA synthesis was increased by moderate hypoxia (2% oxygen but was decreased by severe hypoxia (0.1% oxygen and DFX. Moderate hypoxia decreased p21 synthesis without affecting p53 synthesis, whereas severe hypoxia and DFX increased synthesis of both p21 and p53. p27 protein expression was decreased by severe hypoxia and DFX. Gene silencing of p21 and p27 promoted DNA synthesis at ambient oxygen concentrations. p21 and p53 gene silencing lessened the decrease in DNA synthesis due to severe hypoxia or DFX exposure. p21 gene silencing prevented increased DNA synthesis in moderate hypoxia. p27 protein expression was significantly increased by p53 gene silencing, and was decreased by wild-type p53 gene transfection. Conclusion These results indicate that in NHLF, severe hypoxia leads to cell cycle arrest via the p53-p21 pathway, but that moderate hypoxia enhances cell proliferation via the p21 pathway in a p53-independent manner. In addition, our results suggest that p27 may be involved in compensating for p53 in cultured NHLF proliferation.

  11. P53 suppresses expression of the 14-3-3gamma oncogene

    Directory of Open Access Journals (Sweden)

    Qi Wenqing

    2011-08-01

    Full Text Available Abstract Background 14-3-3 proteins are a family of highly conserved proteins that are involved in a wide range of cellular processes. Recent evidence indicates that some of these proteins have oncogenic activity and that they may promote tumorigenesis. We previously showed that one of the 14-3-3 family members, 14-3-3gamma, is over expressed in human lung cancers and that it can induce transformation of rodent cells in vitro. Methods qRTPCR and Western blot analysis were performed to examine 14-3-3gamma expression in non-small cell lung cancers (NSCLC. Gene copy number was analyzed by qPCR. P53 mutations were detected by direct sequencing and also by western blot. CHIP and yeast one hybrid assays were used to detect p53 binding to 14-3-3gamma promoter. Results Quantitative rtPCR results showed that the expression level of 14-3-3gamma was elevated in the majority of NSCLC that we examined which was also consistent with protein expression. Further analysis of the expression pattern of 14-3-3gamma in lung tumors showed a correlation with p53 mutations suggesting that p53 might suppress 14-3-3 gamma expression. Analysis of the gamma promoter sequence revealed the presence of a p53 consensus binding motif and in vitro assays demonstrated that wild-type p53 bound to this motif when activated by ionizing radiation. Deletion of the p53 binding motif eliminated p53's ability to suppress 14-3-3gamma expression. Conclusion Increased expression of 14-3-3gamma in lung cancer coincides with loss of functional p53. Hence, we propose that 14-3-3gamma's oncogenic activities cooperate with loss of p53 to promote lung tumorigenesis.

  12. Glycogen synthase kinase3 beta phosphorylates serine 33 of p53 and activates p53's transcriptional activity

    Directory of Open Access Journals (Sweden)

    Price Brendan D

    2001-07-01

    Full Text Available Abstract Background The p53 protein is activated by genotoxic stress, oncogene expression and during senescence, p53 transcriptionally activates genes involved in growth arrest and apoptosis. p53 activation is regulated by post-translational modification, including phosphorylation of the N-terminal transactivation domain. Here, we have examined how Glycogen Synthase Kinase (GSK3, a protein kinase involved in tumorigenesis, differentiation and apoptosis, phosphorylates and regulates p53. Results The 2 isoforms of GSK3, GSK3α and GSK3β, phosphorylate the sequence Ser-X-X-X-Ser(P when the C-terminal serine residue is already phosphorylated. Several p53 kinases were examined for their ability to create GSK3 phosphorylation sites on the p53 protein. Our results demonstrate that phosphorylation of serine 37 of p53 by DNA-PK creates a site for GSK3β phosphorylation at serine 33 in vitro. GSK3α did not phosphorylate p53 under any condition. GSK3β increased the transcriptional activity of the p53 protein in vivo. Mutation of either serine 33 or serine 37 of p53 to alanine blocked the ability of GSK3β to regulate p53 transcriptional activity. GSK3β is therefore able to regulate p53 function in vivo. p53's transcriptional activity is commonly increased by DNA damage. However, GSK3β kinase activity was inhibited in response to DNA damage, suggesting that GSK3β regulation of p53 is not involved in the p53-DNA damage response. Conclusions GSK3β can regulate p53's transcriptional activity by phosphorylating serine 33. However, GSK3β does not appear to be part of the p53-DNA damage response pathway. Instead, GSK3β may provide the link between p53 and non-DNA damage mechanisms for p53 activation.

  13. C. elegans CEP-1/p53 and BEC-1 are involved in DNA repair.

    Directory of Open Access Journals (Sweden)

    Sandy Hoffman

    Full Text Available p53 is a transcription factor that regulates the response to cellular stress. Mammalian p53 functions as a tumor suppressor. The C. elegans p53, cep-1, regulates DNA-damage induced germline cell death by activating the transcription of egl-1 and ced-13. We used the C. elegans model to investigate how, in the whole animal, different forms of DNA damage can induce p53-dependent versus p53-independent cell death and DNA repair. DNA damage was induced by ultraviolet type C (UVC radiation, or 10-decarbamoyl mitomycin C (DMC, an agent known to induce mammalian p53-independent cell death. Wild-type or cep-1 loss-of-function mutant animals were assayed for germline cell death and DNA lesions. Wild-type animals displayed greater removal of UVC-lesions over time, whereas cep-1 mutant animals displayed increased UVC-lesion retention. The cep-1 mutation increased UVC-lesion retention directly correlated with a reduction of progeny viability. Consistent with DMC inducing p53-independent cell death in mammalian cells DMC induced a C. elegans p53-independent germline cell death pathway. To examine the influence of wild-type CEP-1 and DNA damage on C. elegans tumors we used glp-1(ar202gf/Notch germline tumor mutants. UVC treatment of glp-1 mutant animals activated the CEP-1 target gene egl-1 and reduced tumor size. In cep-1(gk138;glp-1(ar202gf animals, UVC treatment resulted in increased susceptibility to lesions and larger tumorous germlines. Interestingly, the partial knockdown of bec-1 in adults resulted in a CEP-1-dependent increase in germline cell death and an increase in DNA damage. These results strongly support cross-talk between BEC-1 and CEP-1 to protect the C. elegans genome.

  14. Targeting the Prion-like Aggregation of Mutant p53 to Combat Cancer.

    Science.gov (United States)

    Silva, Jerson L; Cino, Elio A; Soares, Iaci N; Ferreira, Vitor F; A P de Oliveira, Guilherme

    2018-01-16

    Prion-like behavior of several amyloidogenic proteins has been demonstrated in recent years. Despite having functional roles in some cases, irregular aggregation can have devastating consequences. The most commonly known amyloid diseases are Alzheimer's, Parkinson's, and Transmissible Spongiform Encephalopathies (TSEs). The pathophysiology of prion-like diseases involves the structural transformation of wild-type (wt) proteins to transmissible forms that can convert healthy proteins, generating aggregates. The mutant form of tumor suppressor protein, p53, has recently been shown to exhibit prion-like properties. Within the context of p53 aggregation and the search for ways to avert it, this review emphasizes discoveries, approaches, and research from our laboratory and others. Although its standard functions are strongly connected to tumor suppression, p53 mutants and aggregates are involved in cancer progression. p53 aggregates are heterogeneous assemblies composed of amorphous aggregates, oligomers, and amyloid-like fibrils. Evidence of these structures in tumor tissues, the in vitro capability for p53 mutants to coaggregate with wt protein, and the detection of cell-to-cell transmission indicate that cancer has the basic characteristics of prion and prion-like diseases. Various approaches aim to restore p53 functions in cancer. Methods include the use of small-molecule and peptide stabilizers of mutant p53, zinc administration, gene therapy, alkylating and DNA intercalators, and blockage of p53-MDM2 interaction. A primary challenge in developing small-molecule inhibitors of p53 aggregation is the large number of p53 mutations. Another issue is the inability to recover p53 function by dissociating mature fibrils. Consequently, efforts have emerged to target the intermediate species of the aggregation reaction. Φ-value analysis has been used to characterize the kinetics of the early phases of p53 aggregation. Our experiments using high hydrostatic pressure (HHP

  15. TBP-like Protein (TLP) Disrupts the p53-MDM2 Interaction and Induces Long-lasting p53 Activation.

    Science.gov (United States)

    Maeda, Ryo; Tamashiro, Hiroyuki; Takano, Kazunori; Takahashi, Hiro; Suzuki, Hidefumi; Saito, Shinta; Kojima, Waka; Adachi, Noritaka; Ura, Kiyoe; Endo, Takeshi; Tamura, Taka-Aki

    2017-02-24

    Stress-induced activation of p53 is an essential cellular response to prevent aberrant cell proliferation and cancer development. The ubiquitin ligase MDM2 promotes p53 degradation and limits the duration of p53 activation. It remains unclear, however, how p53 persistently escapes MDM2-mediated negative control for making appropriate cell fate decisions. Here we report that TBP-like protein (TLP), a member of the TBP family, is a new regulatory factor for the p53-MDM2 interplay and thus for p53 activation. We found that TLP acts to stabilize p53 protein to ensure long-lasting p53 activation, leading to potentiation of p53-induced apoptosis and senescence after genotoxic stress. Mechanistically, TLP interferes with MDM2 binding and ubiquitination of p53. Moreover, single cell imaging analysis shows that TLP depletion accelerates MDM2-mediated nuclear export of p53. We further show that a cervical cancer-derived TLP mutant has less p53 binding ability and lacks a proliferation-repressive function. Our findings uncover a role of TLP as a competitive MDM2 blocker, proposing a novel mechanism by which p53 escapes the p53-MDM2 negative feedback loop to modulate cell fate decisions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. The role of p53 gene family in reproduction.

    Science.gov (United States)

    Hu, Wenwei

    2009-12-01

    The p53 family of genes (p53, p63, and p73) is conserved over evolutionary time scales. Although the functions of p53 gene and its protein as a tumor suppressor have been firmly established, the earliest functions for the p53 ancestral genes in worms and flies are to ensure germ-line genomic integrity and the fidelity of the developmental process. In vertebrates, the p53 family of genes retains those functions in germ-line genomic integrity but have added important functions in regulation of reproduction. Loss of the p53, p63, or p73 genes in female mice leads to a significant decrease of fertility. The p53 gene product regulates maternal reproduction at the implantation stage of the embryo. p63 and p73 play important roles in monitoring the genomic quality of oocytes. The p53 pathway appears to play a similar role in human fertility. In humans, certain alleles containing a functional single-nucleotide polymorphism (SNP) in the p53 pathway are under positive evolutionary selection. Selected alleles of these SNPs in the p53 pathway are associated with decreased fertility. This important function of the p53 pathway in reproduction provides a plausible explanation for the evolution of p53 as a tumor suppressor gene and the positive selection of some alleles in the p53 gene and its pathway. These observations provide a good possible example of antagonistic pleiotrophy for fertility, tumor suppression, and longevity.

  17. Assessing mutant p53 in primary high-grade serous ovarian cancer using immunohistochemistry and massively parallel sequencing.

    Science.gov (United States)

    Cole, Alexander J; Dwight, Trisha; Gill, Anthony J; Dickson, Kristie-Ann; Zhu, Ying; Clarkson, Adele; Gard, Gregory B; Maidens, Jayne; Valmadre, Susan; Clifton-Bligh, Roderick; Marsh, Deborah J

    2016-05-18

    The tumour suppressor p53 is mutated in cancer, including over 96% of high-grade serous ovarian cancer (HGSOC). Mutations cause loss of wild-type p53 function due to either gain of abnormal function of mutant p53 (mutp53), or absent to low mutp53. Massively parallel sequencing (MPS) enables increased accuracy of detection of somatic variants in heterogeneous tumours. We used MPS and immunohistochemistry (IHC) to characterise HGSOCs for TP53 mutation and p53 expression. TP53 mutation was identified in 94% (68/72) of HGSOCs, 62% of which were missense. Missense mutations demonstrated high p53 by IHC, as did 35% (9/26) of non-missense mutations. Low p53 was seen by IHC in 62% of HGSOC associated with non-missense mutations. Most wild-type TP53 tumours (75%, 6/8) displayed intermediate p53 levels. The overall sensitivity of detecting a TP53 mutation based on classification as 'Low', 'Intermediate' or 'High' for p53 IHC was 99%, with a specificity of 75%. We suggest p53 IHC can be used as a surrogate marker of TP53 mutation in HGSOC; however, this will result in misclassification of a proportion of TP53 wild-type and mutant tumours. Therapeutic targeting of mutp53 will require knowledge of both TP53 mutations and mutp53 expression.

  18. Expression of TP53 isoforms p53β or p53γ enhances chemosensitivity in TP53(null cell lines.

    Directory of Open Access Journals (Sweden)

    Elisabeth Silden

    Full Text Available The carboxy-terminal truncated p53 alternative spliced isoforms, p53β and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53(null background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53β and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53β and p53γ congenic H1299 was accompanied by increased p21((CIP1/WAF1, Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53β protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(PH quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53β and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53β and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level.

  19. [Biochemical characterization of the optic nerve in mice overexpressing the P53 gen. Oxidative stress assays].

    Science.gov (United States)

    Gallego-Pinazo, R; Zanón-Moreno, V; Sanz, S; Andrés, V; Serrano, M; García-Cao, I; Pinazo-Durán, M D

    2008-02-01

    The tumour inhibitor p53 gene has the ability of triggering proliferation arrest and cellular death by apoptosis subsequent to several factors, among them oxidative stress. The p53 protein is a major regulator of gene expression. Using genetically manipulated mice carrying an extra copy of gene p53 (transgenic mice super p53) versus control mice, we have investigated the generation of reactive oxygen species and antioxidant activity in the optic nerve of mice in relation to p53 availability. We studied two groups of 12-month-old mice of the strain C57BL/6: 1) super p53 group (Sp53) and 2) wild-type control group (CG). Mice were anesthetized in ether atmosphere and the eyeball and retrobulbar optic nerves were excised, washed, soaked in PBS, and stored in liquid nitrogen at -85 degrees C until processing. Three-four optic nerves from the same group were placed in an eppendorf tube, homogenized and enzymatic-colorimetric methods used to determine oxidative and antioxidant activities and the nitric oxide synthesis. A significant increase in free radical formation (via lipid peroxidation; pp53 mice compared to respective controls. The presence of an extra copy of the p53 gene correlated with redox status in the mouse optic nerve. This transgenic mouse could be useful as an experimental model to study cell resistance to neurodegenerative processes in relation to oxidative stress and to apoptosis induction, such as glaucomatous optic neuropathy or age-related macular degeneration.

  20. Restriction of human herpesvirus 6B replication by p53

    DEFF Research Database (Denmark)

    Øster, Bodil; Kofod-Olsen, Emil; Bundgaard, Bettina

    2008-01-01

    Human herpesvirus 6B (HHV-6B) induces significant accumulation of p53 in both the nucleus and cytoplasm during infection. Activation of p53 by DNA damage is known to induce either growth arrest or apoptosis; nevertheless, HHV-6B-infected cells are arrested in their cell cycle independently of p53......, and only a minor fraction of the infected cells undergoes apoptosis. Using pifithrin-alpha, a p53 inhibitor, and p53-null cells, this study showed that infected epithelial cells accumulated viral transcripts and proteins to a significantly higher degree in the absence of active p53. Moreover, HHV-6B......-induced cytopathic effects were greatly enhanced in the absence of p53. This suggests that, in epithelial cells, some of the functions of p53 leading to cell-cycle arrest and apoptosis are restrained by HHV-6B infection, whereas other cellular defences, causing inhibition of virus transcription, are partially...

  1. [P53 protein in adenocarcinoma of the large intestine].

    Science.gov (United States)

    Paluszkiewicz, P; Pawłowska-Wakowicz, B; Cybulski, M; Berbeć, H

    1997-01-01

    P53 gen mutations play significant role in neoplastic transformation of colorectal mucosa. We investigated p53 immunostaining in 80 cases of spontaneous human colorectal adenocarcinomas (with monoclonal DO7 antibody and LSAB+ kit). We found positive, nuclear p53 immunostaining in 64% of nonmucinous adenocarcinoma tissues and in 19% of mucinous adenocarcinomas tissues. P53 protein deposits were most often found in colorectal adenocarcinomas localised in rectum (66.67%) and in advanced (Dukes C, D) colorectal adenocarcinomas (59.38%) as well. There was no statistical significance between the p53 positive immunostaining and the histological differentiation of the colorectal adenocarcinomas. The overall survival of patients with tumours positive for p53 protein was significantly shorter than that of patients with colorectal cancers negative for p53 protein. We conclude that p53 immunohistochemical analysis may be treated as a supplementary prognostic marker for patients with colorectal adenocarcinoma, especially it may be useful for adjuvant therapy selection.

  2. Modeling the basal dynamics of p53 system

    National Research Council Canada - National Science Library

    Sun, Tingzhe; Yang, Weiwei; Liu, Jing; Shen, Pingping

    2011-01-01

    .... Most previous models have ignored the basal dynamics of p53 under nonstressed conditions. To explore the basal dynamics of p53, we constructed a stochastic delay model by incorporating two negative feedback loops...

  3. p53 expression in human rectal tissue after radiotherapy: upregulation in normal mucosa versus functional loss in rectal carcinomas.

    NARCIS (Netherlands)

    Marijnen, C.A.; Kapiteijn, E.; Nagtegaal, I.D.; Mulder-Stapel, A.A.; Veld, C.J.H. van de; Schrier, P.I.; Peltenburg, L.T.; Krieken, J.H.J.M. van

    2002-01-01

    PURPOSE: In vitro, ionizing radiation of epithelial cells leads to upregulation of wild-type p53 and subsequent induction of p21(waf1). The effect of radiotherapy (RT) on the expression of these proteins in patients is unknown. We assessed the influence of RT on the expression of p53 and p21(waf1)

  4. T cell-mediated cytotoxicity against p53-protein derived peptides in bulk and limiting dilution cultures of healthy donors

    DEFF Research Database (Denmark)

    Röpke, M; Regner, M; Claesson, M H

    1995-01-01

    wild type and mutated peptides derived from the p53 sequence with a binding motif for HLA-A2.1 molecules. Peripheral blood mononuclear cells (PBMC) from healthy HLA-A2 donors were stimulated in vitro in bulk cultures as well as in limiting dilution cultures using autologous cells pulsed with p53...

  5. Small-molecule stabilization of the p53 - 14-3-3 protein-protein interaction.

    Science.gov (United States)

    Doveston, Richard G; Kuusk, Ave; Andrei, Sebastian A; Leysen, Seppe; Cao, Qing; Castaldi, Maria P; Hendricks, Adam; Brunsveld, Luc; Chen, Hongming; Boyd, Helen; Ottmann, Christian

    2017-08-01

    14-3-3 proteins are positive regulators of the tumor suppressor p53, the mutation of which is implicated in many human cancers. Current strategies for targeting of p53 involve restoration of wild-type function or inhibition of the interaction with MDM2, its key negative regulator. Despite the efficacy of these strategies, the alternate approach of stabilizing the interaction of p53 with positive regulators and, thus, enhancing tumor suppressor activity, has not been explored. Here, we report the first example of small-molecule stabilization of the 14-3-3 - p53 protein-protein interaction (PPI) and demonstrate the potential of this approach as a therapeutic modality. We also observed a disconnect between biophysical and crystallographic data in the presence of a stabilizing molecule, which is unusual in 14-3-3 PPIs. © 2017 Federation of European Biochemical Societies.

  6. p53 Suppresses Metabolic Stress-Induced Ferroptosis in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Amy Tarangelo

    2018-01-01

    Full Text Available How cancer cells respond to nutrient deprivation remains poorly understood. In certain cancer cells, deprivation of cystine induces a non-apoptotic, iron-dependent form of cell death termed ferroptosis. Recent evidence suggests that ferroptosis sensitivity may be modulated by the stress-responsive transcription factor and canonical tumor suppressor protein p53. Using CRISPR/Cas9 genome editing, small-molecule probes, and high-resolution, time-lapse imaging, we find that stabilization of wild-type p53 delays the onset of ferroptosis in response to cystine deprivation. This delay requires the p53 transcriptional target CDKN1A (encoding p21 and is associated with both slower depletion of intracellular glutathione and a reduced accumulation of toxic lipid-reactive oxygen species (ROS. Thus, the p53-p21 axis may help cancer cells cope with metabolic stress induced by cystine deprivation by delaying the onset of non-apoptotic cell death.

  7. The role of the tumor suppressor p53 in spermatogenesis

    NARCIS (Netherlands)

    Beumer, T. L.; Roepers-Gajadien, H. L.; Gademan, I. S.; van Buul, P. P.; Gil-Gomez, G.; Rutgers, D. H.; de rooij, D. G.

    1998-01-01

    The p53 protein appeared to be involved in both spermatogonial cell proliferation and radiation response. During normal spermatogenesis in the mouse, spermatogonia do not express p53, as analyzed by immunohistochemistry. However, after a dose of 4 Gy of X-rays, a distinct p53 staining was present in

  8. p53 specific (auto)immunity in mice

    NARCIS (Netherlands)

    Lauwen, Marjolein Monique

    2008-01-01

    Self-tolerance to p53 is a major potential limitation for the activation of the endogenous T-cell repertoire. So far, p53 specific CD8+ and CD4+ T-cell immunity has been described in cancer patients and healthy individuals. However, the restrictions of tolerance on the recruitment of p53 specific T

  9. P53 MUTATIONS IN HUMAN LUNG-TUMORS

    NARCIS (Netherlands)

    MILLER, CW; ASLO, A; KOK, K; YOKOTA, J; BUYS, CHCM; TERADA, M; KOEFFLER, HP; Simon, K.

    1992-01-01

    Mutation of one p53 allele and loss of the normal p53 allele [loss of heterozygosity (LOH)] occur in many tumors including lung cancers. These alterations apparently contribute to development of cancer by interfering with the tumor suppressor activity of p53. We directly sequenced amplified DNA in

  10. p53 expression in colorectal carcinoma in relation to ...

    African Journals Online (AJOL)

    Background: It has been shown that colorectal carcinoma is increasing in incidence in African countries. This could be due to change in life style. Molecular pathogenesis of colorectal cancer commonly involves mutation in p53 gene which leads to expression of p53 protein in tumor cells. Expression of p53 protein has been ...

  11. Oncogenicity evaluation of resveratrol in p53(+/-) (p53 knockout) mice.

    Science.gov (United States)

    Horn, T L; Cwik, M J; Morrissey, R L; Kapetanovic, I; Crowell, J A; Booth, T D; McCormick, D L

    2007-01-01

    A six-month study was conducted in p53(+/-) mice to evaluate the possible oncogenicity of resveratrol (3,5,4'-trihydroxy-trans-stilbene), a cancer chemopreventive agent present in grapes and other foods. p53(+/-) mice (25/sex/group) received daily gavage exposure to vehicle only (negative control), resveratrol doses of 1000, 2000, or 4000 mg/kg/day, or p-cresidine (400 mg/kg/day; positive control). No mortality was seen in mice receiving the low dose of resveratrol. However, the mid and high doses induced mortality associated with impaction of the test article in the gastrointestinal tract. Resveratrol had no effect on body weight, food consumption, or clinical signs in surviving mice in any dose group, but induced dose-related increases in liver weight and serum cholesterol in both sexes. Mild anemia was seen in male mice at the high dose only; hematologic effects were not seen in females. Histopathology identified the kidney (hydronephrosis) and urinary bladder (epithelial hyperplasia) as target tissues for resveratrol toxicity. The incidences of both benign and malignant tumors in mice exposed to resveratrol were comparable to those in vehicle controls. By contrast, the positive control article, p-cresidine, induced urinary bladder cancer in both sexes. When administered to p53(+/-) mice at its maximum tolerated dose, resveratrol demonstrates no evidence of oncogenicity.

  12. Adaptation of cancer cells from different entities to the MDM2 inhibitor nutlin-3 results in the emergence of p53-mutated multi-drug-resistant cancer cells

    NARCIS (Netherlands)

    Michaelis, M.; Rothweiler, F.; Barth, S.; Cinatl, J.; van Rikxoort, M.; Loeschmann, N.; Voges, Y.; Breitling, R.; von Deimling, A.; Roedel, F.; Weber, K.; Fehse, B.; Mack, E.; Stiewe, T.; Doerr, H. W.; Speidel, D.; Cinatl, J.; Cinatl jr., J.; Stephanou, A.

    2011-01-01

    Six p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, and melanoma) were continuously exposed to increasing concentrations of the murine double minute 2 inhibitor nutlin-3, resulting in the emergence of nutlin-3-resistant, p53-mutated sublines

  13. Infection with E1B-mutant adenovirus stabilizes p53 but blocks p53 acetylation and activity through E1A

    DEFF Research Database (Denmark)

    Savelyeva, I.; Dobbelstein, M.

    2011-01-01

    Wild-type adenovirus type 5 eliminates p53 through the E1B-55 kDa and E4-34 kDa gene products. Deletion or mutation of E1B-55 kDa has long been thought to confer p53-selective replication of oncolytic viruses. We show here that infection with E1B-defective adenovirus mutants induces massive...... acetylation in infected cells. Mutating either of these E1A regions, in addition to E1B, partially restored p21 mRNA levels. Our findings argue that adenovirus attenuates p53-mediated p21 induction, through at least two E1B-independent mechanisms. Other virus species and cancer cells may employ analogous...

  14. Endogenous and exogenous constraints in the population changes of wild boar (sus scrofa Linnaeus, 1758)

    OpenAIRE

    Uzal Fernandez, Antonio; Nores, Carlos

    2004-01-01

    The population dynamics of wild boar (Sus scrofa) was studied in a time series over 26 years using\\ud data from the Regional Hunting Reserve of Somiedo (northern Spain). This population is controlled\\ud by a complex negative feedback system that acts with one (main) and two (secondary) years of delay\\ud (lags). The primary feedback might be explained by intraspecific competition for food resulting from\\ud fluctuations in mast production (acorns and beech), and the secondary feedback might be ...

  15. A p53 Super-tumor Suppressor Reveals a Tumor Suppressive p53-Ptpn14-Yap Axis in Pancreatic Cancer.

    Science.gov (United States)

    Mello, Stephano S; Valente, Liz J; Raj, Nitin; Seoane, Jose A; Flowers, Brittany M; McClendon, Jacob; Bieging-Rolett, Kathryn T; Lee, Jonghyeob; Ivanochko, Danton; Kozak, Margaret M; Chang, Daniel T; Longacre, Teri A; Koong, Albert C; Arrowsmith, Cheryl H; Kim, Seung K; Vogel, Hannes; Wood, Laura D; Hruban, Ralph H; Curtis, Christina; Attardi, Laura D

    2017-10-09

    The p53 transcription factor is a critical barrier to pancreatic cancer progression. To unravel mechanisms of p53-mediated tumor suppression, which have remained elusive, we analyzed pancreatic cancer development in mice expressing p53 transcriptional activation domain (TAD) mutants. Surprisingly, the p5353,54 TAD2 mutant behaves as a "super-tumor suppressor," with an enhanced capacity to both suppress pancreatic cancer and transactivate select p53 target genes, including Ptpn14. Ptpn14 encodes a negative regulator of the Yap oncoprotein and is necessary and sufficient for pancreatic cancer suppression, like p53. We show that p53 deficiency promotes Yap signaling and that PTPN14 and TP53 mutations are mutually exclusive in human cancers. These studies uncover a p53-Ptpn14-Yap pathway that is integral to p53-mediated tumor suppression. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  16. p53 Mutation suppresses adult neurogenesis in medaka fish (Oryzias latipes)

    Energy Technology Data Exchange (ETDEWEB)

    Isoe, Yasuko; Okuyama, Teruhiro [Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033 (Japan); Taniguchi, Yoshihito [Department of Preventive Medicine and Public Health, School of Medicine, Keio University, 35, Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Kubo, Takeo [Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033 (Japan); Takeuchi, Hideaki, E-mail: takeuchi@biol.s.u-tokyo.ac.jp [Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer Progenitor migration is accompanied by an increase in their numbers in the adult brain. Black-Right-Pointing-Pointer p53 Mutation suppressed an increase in the number of the migrated progenitors. Black-Right-Pointing-Pointer The decreased progenitor number is not due to enhanced cell death. Black-Right-Pointing-Pointer p53 Mutation did not affect proliferation of stem cells. -- Abstract: Tumor suppressor p53 negatively regulates self-renewal of neural stem cells in the adult murine brain. Here, we report that the p53 null mutation in medaka fish (Oryzias latipes) suppressed neurogenesis in the telencephalon, independent of cell death. By using 5-bromo-29-deoxyuridine (BrdU) immunohistochemistry, we identified 18 proliferation zones in the brains of young medaka fish; in situ hybridization showed that p53 was expressed selectively in at least 12 proliferation zones. We also compared the number of BrdU-positive cells present in the whole telencephalon of wild-type (WT) and p53 mutant fish. Immediately after BrdU exposure, the number of BrdU-positive cells did not differ significantly between them. One week after BrdU-exposure, the BrdU-positive cells migrated from the proliferation zone, which was accompanied by an increased number in the WT brain. In contrast, no significant increase was observed in the p53 mutant brain. Terminal deoxynucleotidyl transferase (dUTP) nick end-labeling revealed that there was no significant difference in the number of apoptotic cells in the telencephalon of p53 mutant and WT medaka, suggesting that the decreased number of BrdU-positive cells in the mutant may be due to the suppression of proliferation rather than the enhancement of neural cell death. These results suggest that p53 positively regulates neurogenesis via cell proliferation.

  17. Prima-1 induces apoptosis in bladder cancer cell lines by activating p53

    Directory of Open Access Journals (Sweden)

    Camila B. Piantino

    2013-01-01

    Full Text Available OBJECTIVES: Bladder cancer represents 3% of all carcinomas in the Brazilian population and ranks second in incidence among urological tumors, after prostate cancer. The loss of p53 function is the main genetic alteration related to the development of high-grade muscle-invasive disease. Prima-1 is a small molecule that restores tumor suppressor function to mutant p53 and induces cancer cell death in various cancer types. Our aim was to investigate the ability of Prima-1 to induce apoptosis after DNA damage in bladder cancer cell lines. METHOD: The therapeutic effect of Prima-1 was studied in two bladder cancer cell lines: T24, which is characterized by a p53 mutation, and RT4, which is the wild-type for the p53 gene. Morphological features of apoptosis induced by p53, including mitochondrial membrane potential changes and the expression of thirteen genes involved in apoptosis, were assessed by microscopic observation and quantitative real-time PCR (qRT-PCR. RESULTS: Prima-1 was able to reactivate p53 function in the T24 (p53 mt bladder cancer cell line and promote apoptosis via the induction of Bax and Puma expression, activation of the caspase cascade and disruption of the mitochondrial membrane in a BAK-independent manner. CONCLUSION: Prima-1 is able to restore the transcriptional activity of p53. Experimental studies in vivo may be conducted to test this molecule as a new therapeutic agent for urothelial carcinomas of the bladder, which characteristically harbor p53 mutations.

  18. Inhibition of NAMPT pathway by FK866 activates the function of p53 in HEK293T cells.

    Science.gov (United States)

    Thakur, Basant Kumar; Dittrich, Tino; Chandra, Prakash; Becker, Annette; Lippka, Yannick; Selvakumar, Divakarvel; Klusmann, Jan-Henning; Reinhardt, Dirk; Welte, Karl

    2012-08-03

    Inactivation of p53 protein by endogenous and exogenous carcinogens is involved in the pathogenesis of different human malignancies. In cancer associated with SV-40 DNA tumor virus, p53 is considered to be non-functional mainly due to its interaction with the large T-antigen. Using the 293T cell line (HEK293 cells transformed with large T antigen) as a model, we provide evidence that p53 is one of the critical downstream targets involved in FK866-mediated killing of 293T cells. A reduced rate of apoptosis and an increased number of cells in S-phase was accompanied after knockdown of p53 in these cells. Inhibition of NAMPT by FK866, or inhibition of SIRT by nicotinamide decreased proliferation and triggered death of 293T cells involving the p53 acetylation pathway. Additionally, knockdown of p53 attenuated the effect of FK866 on cell proliferation, apoptosis, and cell cycle arrest. The data presented here shed light on two important facts: (1) that p53 in 293T cells is active in the presence of FK866, an inhibitor of NAMPT pathway; (2) the apoptosis induced by FK866 in 293T cells is associated with increased acetylation of p53 at Lys382, which is required for the functional activity of p53. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Census and evaluation of p53 target genes

    Science.gov (United States)

    Fischer, M

    2017-01-01

    The tumor suppressor p53 functions primarily as a transcription factor. Mutation of the TP53 gene alters its response pathway, and is central to the development of many cancers. The discovery of a large number of p53 target genes, which confer p53’s tumor suppressor function, has led to increasingly complex models of p53 function. Recent meta-analysis approaches, however, are simplifying our understanding of how p53 functions as a transcription factor. In the survey presented here, a total set of 3661 direct p53 target genes is identified that comprise 3509 potential targets from 13 high-throughput studies, and 346 target genes from individual gene analyses. Comparison of the p53 target genes reported in individual studies with those identified in 13 high-throughput studies reveals limited consistency. Here, p53 target genes have been evaluated based on the meta-analysis data, and the results show that high-confidence p53 target genes are involved in multiple cellular responses, including cell cycle arrest, DNA repair, apoptosis, metabolism, autophagy, mRNA translation and feedback mechanisms. However, many p53 target genes are identified only in a small number of studies and have a higher likelihood of being false positives. While numerous mechanisms have been proposed for mediating gene regulation in response to p53, recent advances in our understanding of p53 function show that p53 itself is solely an activator of transcription, and gene downregulation by p53 is indirect and requires p21. Taking into account the function of p53 as an activator of transcription, recent results point to an unsophisticated means of regulation. PMID:28288132

  20. CP-31398 inhibits the growth of p53-mutated liver cancer cells in vitro and in vivo.

    Science.gov (United States)

    He, Xing-Xing; Zhang, Yu-Nan; Yan, Jun-Wei; Yan, Jing-Jun; Wu, Qian; Song, Yu-Hu

    2016-01-01

    The tumor suppressor p53 is one of the most frequently mutated genes in hepatocellular carcinoma (HCC). Previous studies demonstrated that CP-31398 restored the native conformation of mutant p53 and trans-activated p53 downstream genes in tumor cells. However, the research on the application of CP-31398 to liver cancer has not been reported. Here, we investigated the effects of CP-31398 on the phenotype of HCC cells carrying p53 mutation. The effects of CP-31398 on the characteristic of p53-mutated HCC cells were evaluated through analyzing cell cycle, cell apoptosis, cell proliferation, and the expression of p53 downstream genes. In tumor xenografts developed by PLC/PRF/5 cells, the inhibition of tumor growth by CP-31398 was analyzed through gross morphology, growth curve, and the expression of p53-related genes. Firstly, we demonstrated that CP-31398 inhibited the growth of p53-mutated liver cancer cells in a dose-dependent and p53-dependent manner. Then, further study showed that CP-31398 re-activated wild-type p53 function in p53-mutated HCC cells, which resulted in inhibitive response of cell proliferation and an induction of cell-cycle arrest and apoptosis. Finally, in vivo data confirmed that CP-31398 blocked the growth of xenografts tumors through transactivation of p53-responsive downstream molecules. Our results demonstrated that CP-31398 induced desired phenotypic change of p53-mutated HCC cells in vitro and in vivo, which revealed that CP-31398 would be developed as a therapeutic candidate for HCC carrying p53 mutation.

  1. p53 Over-expression and p53 mutations in colon carcinomas: Relation to dietary risk factors

    NARCIS (Netherlands)

    Voskuil, D.W.; Kampman, E.; Kraats, A.A. van; Balder, H.F.; Muijen, G.N.P. van; Goldbohm, R.A.; Veer, P. van 't

    1999-01-01

    Epidemiological studies have suggested that dietary factors may differently affect p53-dependent and p53-independent pathways to colon cancer. Results of such studies may depend on the method used to assess p53 status. This case-control study of 185 colon-cancer cases and 259 controls examines this

  2. Screening of medicinal plant phytochemicals as natural antagonists of p53-MDM2 interaction to reactivate p53 functioning.

    Science.gov (United States)

    Riaz, Muhammad; Ashfaq, Usman A; Qasim, Muhammad; Yasmeen, Erum; Ul Qamar, Muhammad T; Anwar, Farooq

    2017-10-01

    In most types of cancer, overexpression of murine double minute 2 (MDM2) often leads to inactivation of p53. The crystal structure of MDM2, with a 109-residue amino-terminal domain, reveals that MDM2 has a core hydrophobic region to which p53 binds as an amphipathic α helix. The interface depends on the steric complementarity between MDM2 and the hydrophobic region of p53. Especially, on p53's triad, amino acids Phe19, Trp23 and Leu26 bind to the MDM2 core. Results from studies suggest that the structural motif of both p53 and MDM2 can be attributed to similarities in the amphipathic α helix. Thus, in the current investigation it is hypothesized that the similarity in the structural motif might be the cause of p53 inactivation by MDM2. Hence, molecular docking and phytochemical screening approaches are appraised to inhibit the hydrophobic cleft of MDM2 and to stop p53-MDM2 interaction, resulting in reactivation of p53 activity. For this purpose, a library of 2295 phytochemicals were screened against p53-MDM2 to find potential candidates. Of these, four phytochemicals including epigallocatechin gallate, alvaradoin M, alvaradoin E and nordihydroguaiaretic acid were found to be potential inhibitors of p53-MDM2 interaction. The screened phytochemicals, derived from natural extracts, may have negligible side effects and can be explored as potent antagonists of p53-MDM2 interactions, resulting in reactivation of the normal transcription of p53.

  3. Molecularly targeted therapies for p53-mutant cancers.

    Science.gov (United States)

    Zhao, Dekuang; Tahaney, William M; Mazumdar, Abhijit; Savage, Michelle I; Brown, Powel H

    2017-11-01

    The tumor suppressor p53 is lost or mutated in approximately half of human cancers. Mutant p53 not only loses its anti-tumor transcriptional activity, but also often acquires oncogenic functions to promote tumor proliferation, invasion, and drug resistance. Traditional strategies have been taken to directly target p53 mutants through identifying small molecular compounds to deplete mutant p53, or to restore its tumor suppressive function. Accumulating evidence suggest that cancer cells with mutated p53 often exhibit specific functional dependencies on secondary genes or pathways to survive, providing alternative targets to indirectly treat p53-mutant cancers. Targeting these genes or pathways, critical for survival in the presence of p53 mutations, holds great promise for cancer treatment. In addition, mutant p53 often exhibits novel gain-of-functions to promote tumor growth and metastasis. Here, we review and discuss strategies targeting mutant p53, with focus on targeting the mutant p53 protein directly, and on the progress of identifying genes and pathways required in p53-mutant cells.

  4. miR-300 promotes proliferation and EMT-mediated colorectal cancer migration and invasion by targeting p53.

    Science.gov (United States)

    Wang, Lin; Yu, Peiwu

    2016-12-01

    p53 mutations in tumors can induce the loss of wild-type tumor-suppressing p53 function, which results in the increase in proliferation, migration and invasion ability in cancer cells. Studies have shown that the expression of p53 is regulated by several microRNAs (miRNAs). In the present study, we found that miR-300 and p53 were significantly increased in colorectal cancer (CRC) tissues when compared with levels noted in adjacent colorectal tissues. Both miR-300 and p53 were significantly correlated with lymphatic metastasis and TNM stage. Both miR-300 and p53 promoted CRC cell (SW480 and HT29) proliferation, migration, and invasion, respectively, in vitro. In addition, we found that miR-300 is a direct positive regulator of p53 through binding to the binding site in the 3'UTR of the p53 gene in human CRC cells. Moreover, both miR-300 and p53 induced CRC cell epithelial‑mesenchymal transition (EMT) respectively. Taken together, we demonstrated that miR-300 promoted proliferation and EMT-mediated CRC migration and invasion by targeting p53. These findings provide a new theoretical basis and potential therapeutic targets, and thus lays the foundation for exploring the pathogenesis of CRC.

  5. [6]-Gingerol Induces Cell Cycle Arrest and Cell Death of Mutant p53-expressing Pancreatic Cancer Cells

    Science.gov (United States)

    Park, Yon Jung; Wen, Jing; Bang, Seungmin; Park, Seung Woo

    2006-01-01

    [6]-Gingerol, a major phenolic compound derived from ginger, has anti-bacterial, anti-inflammatory and anti-tumor activities. While several molecular mechanisms have been described to underlie its effects on cells in vitro and in vivo, the underlying mechanisms by which [6]-gingerol exerts anti-tumorigenic effects are largely unknown. The purpose of this study was to investigate the action of [6]-gingerol on two human pancreatic cancer cell lines, HPAC expressing wild-type (wt) p53 and BxPC-3 expressing mutated p53. We found that [6]-gingerol inhibited the cell growth through cell cycle arrest at G1 phase in both cell lines. Western blot analyses indicated that [6]-gingerol decreased both Cyclin A and Cyclin-dependent kinase (Cdk) expression. These events led to reduction in Rb phosphorylation followed by blocking of S phase entry. p53 expression was decreased by [6]-gingerol treatment in both cell lines suggesting that the induction of Cyclin-dependent kinase inhibitor, p21cip1, was p53-independent. [6]-Gingerol induced mostly apoptotic death in the mutant p53-expressing cells, while no signs of early apoptosis were detected in wild type p53-expressing cells and this was related to the increased phosphorylation of AKT. These results suggest that [6]-gingerol can circumvent the resistance of mutant p53-expressing cells towards chemotherapy by inducing apoptotic cell death while it exerts cytostatic effect on wild type p53-expressing cells by inducing temporal growth arrest. PMID:17066513

  6. Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity

    DEFF Research Database (Denmark)

    Danovi, Davide; Meulmeester, Erik; Pasini, Diego

    2004-01-01

    Human tumors are believed to harbor a disabled p53 tumor suppressor pathway, either through direct mutation of the p53 gene or through aberrant expression of proteins acting in the p53 pathway, such as p14(ARF) or Mdm2. A role for Mdmx (or Mdm4) as a key negative regulator of p53 function in vivo...... has been established. However, a direct contribution of Mdmx to tumor formation remains to be demonstrated. Here we show that retrovirus-mediated Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with HRas(V12......). Furthermore, the human Mdmx ortholog, Hdmx, was found to be overexpressed in a significant percentage of various human tumors and amplified in 5% of primary breast tumors, all of which retained wild-type p53. Hdmx was also amplified and highly expressed in MCF-7, a breast cancer cell line harboring wild...

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Scheffner, M.; Muenger, K.; Byrne, J.C.; Howley, P.M. (National Cancer Inst., Bethesda, MD (United States))

    1991-07-01

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

  9. Inactivation and inducible oncogenic mutation of p53 in gene targeted pigs.

    Directory of Open Access Journals (Sweden)

    Simon Leuchs

    Full Text Available Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs and live pigs carrying a latent TP53(R167H mutant allele, orthologous to oncogenic human mutant TP53(R175H and mouse Trp53(R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53(R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53(R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53(R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.

  10. Family feud in chemosensitvity: p73 and mutant p53.

    Science.gov (United States)

    Irwin, Meredith S

    2004-03-01

    The importance of p53 in chemotherapy-induced apoptosis of cancer cells is well established. p53 plays a critical role in the cellular response to DNA damage by regulating genes involved in cell cycle progression, apoptosis, and genomic stability. As a result, p53 tumor status is a critical determinant of both responses to anti-cancer treatment and clinical prognosis. Interestingly, tumors expressing certain mutant forms of p53 ("gain of function") are particularly resistant to chemotherapy, even when compared to cells that lack any detectable p53. Until recently, the explanation for this enhanced chemoresistance was not clear. Recent studies have shown that the p53 homologues, p73 and p63, are also activated by chemotherapies, leading to tumor cell death. Now the discovery that mutant p53 interacts with p73, and that regulation of this interaction by a p53 polymorphism can modulate chemosensitvity provide a new model for how p53-family interactions can influence the response of tumors to anti-cancer therapies. Since p53 mutations are found in more than 50% of human tumors, strategies aimed at manipulating these interactions may prove useful in enhancing the chemotherapy response, and perhaps, overcoming chemoresistance.

  11. p53: key conductor of all anti-acne therapies.

    Science.gov (United States)

    Melnik, Bodo C

    2017-09-19

    This review based on translational research predicts that the transcription factor p53 is the key effector of all anti-acne therapies. All-trans retinoic acid (ATRA) and isotretinoin (13-cis retinoic acid) enhance p53 expression. Tetracyclines and macrolides via inhibiting p450 enzymes attenuate ATRA degradation, thereby increase p53. Benzoyl peroxide and hydrogen peroxide elicit oxidative stress, which upregulates p53. Azelaic acid leads to mitochondrial damage associated with increased release of reactive oxygen species inducing p53. p53 inhibits the expression of androgen receptor and IGF-1 receptor, and induces the expression of IGF binding protein 3. p53 induces FoxO1, FoxO3, p21 and sestrin 1, sestrin 2, and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), the key inducer of isotretinoin-mediated sebocyte apoptosis explaining isotretinoin's sebum-suppressive effect. Anti-androgens attenuate the expression of miRNA-125b, a key negative regulator of p53. It can thus be concluded that all anti-acne therapies have a common mode of action, i.e., upregulation of the guardian of the genome p53. Immortalized p53-inactivated sebocyte cultures are unfortunate models for studying acne pathogenesis and treatment.

  12. p53 RNA interactions: new clues in an old mystery.

    Science.gov (United States)

    Riley, Kasandra J-L; Maher, L James

    2007-11-01

    The p53 tumor suppressor protein is typically considered to be a sequence-specific DNA-binding transcription factor. However, reports over the last 15 years have described RNA binding by p53 in a variety of contexts, suggesting the possibility of new p53 functions. It is clear that p53-RNA interactions are mediated by a nucleic acid-binding domain of p53 independent of the sequence-specific core domain responsible for DNA recognition. Reports disagree on several aspects of the putative RNA interaction, including sequence specificity and biological relevance. Here we review the history and recent advances in the study of p53-RNA interactions. We argue that p53-RNA interactions are sequence nonspecific and depend on incomplete post-translational modification of the p53 C-terminal domain when the protein is expressed in heterologous systems. It is unknown what fraction of p53 protein exists in a state competent for RNA binding in vivo. Thus, potential physiological roles of p53-RNA interactions remain mysterious.

  13. NAD+ Modulates p53 DNA Binding Specificity and Function

    Science.gov (United States)

    McLure, Kevin G.; Takagi, Masatoshi; Kastan, Michael B.

    2004-01-01

    DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al., Cell 71:587-597, 1992; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD+ binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B3) increases the rate of intracellular NAD+ synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD+ on p53, as a molecule structurally related to part of NAD+, TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B1), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53. PMID:15509798

  14. TPX2-p53-GLIPR1 regulatory circuitry in cell proliferation, invasion, and tumor growth of bladder cancer.

    Science.gov (United States)

    Yan, Liang; Li, Qi; Yang, Juan; Qiao, Baoping

    2017-08-11

    The targeting protein for Xenopus kinesin-like protein 2 (TPX2) is associated with the metastasis and prognosis of bladder cancer. p53 is closely related to the progression of bladder cancer. Human glioma pathogenesis-related protein 1 (GLIPR1) is a p53 target gene with antitumor activity. This study aims to explore the interplay between TPX2, p53, and GLIPR1 and its correlation with cell proliferation, invasion, and tumor growth in bladder cancer. Here, Western blot and qRT-PCR analysis revealed that TPX2 at both mRNA and protein levels was up-regulated in bladder carcinoma tissues compared to their paired adjacent normal tissues. Additionally, tissues expressing high TPX2 level exhibited high p53 level and low GLIPR1 level. The expressions of TPX2 and p53 in non-muscle-invasive bladder cancer cells (KK47 and RT4) were lower than those in muscle-invasive bladder cancer cells (T24, 5637, and UM-UC-3), while GLIPR1 showed the converse expression pattern. Further investigation revealed that TPX2 activated the synthesis of p53; and GLIPR1 is up-regulated by wild-type (wt)-p53 but not affected by mutated p53; Additionally, GLIPR1 inhibited TPX2. These data suggested a TPX2-p53-GLIPR1 regulatory circuitry. Meanwhile, TPX2 overexpression promoted while overexpression of GLIPR1 or p53 inhibited bladder cancer growth. Interestingly, in T24 cells with mutated p53, p53 silence suppressed bladder cancer growth. This study identified a novel TPX2-p53-GLIPR1 regulatory circuitry which modulated cell proliferation, migration, invasion, and tumorigenicity of bladder cancer. Our findings provide new insight into underlying mechanisms of tumorigenesis and novel therapeutic options in bladder cancer. © 2017 Wiley Periodicals, Inc.

  15. HDAC6 deficiency induces apoptosis in mesenchymal stem cells through p53 K120 acetylation.

    Science.gov (United States)

    Park, Song-Yi; Phorl, Sophors; Jung, Suna; Sovannarith, Korm; Lee, Se-In; Noh, Solhee; Han, Miae; Naskar, Rema; Kim, Jae-Young; Choi, Yun-Jaie; Lee, Joo-Yong

    2017-12-09

    The acetylation of p53 is critical in modulating its pro-apoptotic roles. However, its regulatory mechanism and physiological significance are unclear. Here, we show HDAC6 negatively regulates pro-apoptotic acetylation of p53 at lysine residue 120 (K120) in mesenchymal stem cells (MSCs). The loss of HDAC6 expression in MSCs increases K120 acetylation of p53, which is successfully reversed by the wild-type but not by catalytically dead HDAC6. Deletion of HDAC6 induces caspase-dependent apoptosis by promoting transactivation of Bax and suppression of Bcl-2. Moreover, HDAC6 deficiency leads to mitochondrial dysfunction characterized by aberrant reactive oxygen species production and defective oxidative phosphorylation, which is reversed by ectopic expression of wild-type or acetylation mimetic p53. This study demonstrates that HDAC6 is a critical regulator of a pro-apoptotic p53 K120 acetylation and mitochondrial function in MSCs, suggesting that the modulation of HDAC6 activity could be a novel approach to improve MSC- based therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Synthesis and evaluation of modified chalcone based p53 stabilizing agents

    KAUST Repository

    Iftikhar, Sunniya

    2017-07-15

    Tumor suppressor protein p53 induces cell cycle arrest and apoptotic cell death in response to various cellular stresses thereby preventing cancer development. Activation and stabilization of p53 through small organic molecules is, therefore, an attractive approach for the treatment of cancers retaining wild-type p53. In this context, a series of nineteen chalcones with various substitution patterns of functional groups including chloro, fluoro, methoxy, nitro, benzyloxy, 4-methyl benzyloxy was prepared using Claisen-Schmidt condensation. The compounds were characterized using NMR, HRMS, IR and melting points. Evaluation of synthesized compounds against human colorectal (HCT116) and breast (Cal-51) cancer cell lines revealed potent antiproliferative activities. Nine compounds displayed GI50 values in the low micromolar to submicromolar range; for example (E)-1-phenyl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (SSE14108) showed GI50 of 0.473 ± 0.043 µM against HCT116 cells. Further analysis of these compounds revealed that (E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (SSE14105) and (E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (SSE14106) caused rapid (4 and 8-hour post-treatment) accumulation of p53 in HCT116 cells similar to its induction by positive control, Nutlin-3. Such activities were absent in 3-(4-methoxyphenyl)propiophenone (SSE14106H2) demonstrating the importance of conjugated ketone for antiproliferative and p53 stabilizing activity of the chalcones. We further evaluated p53 levels in the presence of cycloheximide (CHX) and the results showed that the p53 stabilization was regulated at post-translational level through blockage of its degradation. These chalcones can, therefore, act as fragment leads for further structure optimization to obtain more potent p53 stabilizing agents with enhanced anti-proliferative activities.

  17. Synthesis and evaluation of modified chalcone based p53 stabilizing agents.

    Science.gov (United States)

    Iftikhar, Sunniya; Khan, Sardraz; Bilal, Aishah; Manzoor, Safia; Abdullah, Muhammad; Emwas, Abdel-Hamid; Sioud, Salim; Gao, Xin; Chotana, Ghayoor Abbas; Faisal, Amir; Saleem, Rahman Shah Zaib

    2017-09-01

    Tumor suppressor protein p53 induces cell cycle arrest and apoptotic cell death in response to various cellular stresses thereby preventing cancer development. Activation and stabilization of p53 through small organic molecules is, therefore, an attractive approach for the treatment of cancers retaining wild-type p53. In this context, a series of nineteen chalcones with various substitution patterns of functional groups including chloro, fluoro, methoxy, nitro, benzyloxy, 4-methyl benzyloxy was prepared using Claisen-Schmidt condensation. The compounds were characterized using NMR, HRMS, IR and melting points. Evaluation of synthesized compounds against human colorectal (HCT116) and breast (CAL-51) cancer cell lines revealed potent antiproliferative activities. Nine compounds displayed GI50 values in the low micromolar to submicromolar range; for example (E)-1-phenyl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (SSE14108) showed GI50 of 0.473±0.043µM against HCT116 cells. Further analysis of these compounds revealed that (E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (SSE14105) and (E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (SSE14106) caused rapid (4 and 8-h post-treatment) accumulation of p53 in HCT116 cells similar to its induction by positive control, Nutlin-3. Such activities were absent in 3-(4-methoxyphenyl)propiophenone (SSE14106H2) demonstrating the importance of conjugated ketone for antiproliferative and p53 stabilizing activity of the chalcones. We further evaluated p53 levels in the presence of cycloheximide (CHX) and the results showed that the p53 stabilization was regulated at post-translational level through blockage of its degradation. These chalcones can, therefore, act as fragment leads for further structure optimization to obtain more potent p53 stabilizing agents with enhanced anti-proliferative activities. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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

  19. Human bladder cancer cells undergo cisplatin-induced apoptosis that is associated with p53-dependent and p53-independent responses.

    Science.gov (United States)

    Konstantakou, Eumorphia G; Voutsinas, Gerassimos E; Karkoulis, Panagiotis K; Aravantinos, Gerasimos; Margaritis, Lukas H; Stravopodis, Dimitrios J

    2009-08-01

    Cisplatin is a first-line chemotherapeutic agent and a powerful component of standard treatment regimens for several human malignancies including bladder cancer. DNA-Pt adducts produced by cisplatin are mainly responsible for cellular toxicity and induction of apoptosis. Identification of the mechanisms that control sensitivity to cisplatin is central to improving its therapeutic index and to successfully encountering the acquired resistance frequently emerging during therapy. In the present study, using MTT-based assays, Western blotting and semi-quantitative RT-PCR, we examined the apoptosis-related cellular responses to cisplatin exposure in two human urinary bladder cancer cell lines characterized by different malignancy grade and p53 genetic status. Both RT4 (grade I; wild-type p53) and T24 (grade III; mutant p53) cell types proved to be vulnerable to cisplatin apoptotic activity, albeit in a grade-dependent and drug dose-specific manner, as demonstrated by the proteolytic processing profiles of Caspase-8, Caspase-9, Caspase-3, and the Caspase repertoire characteristic substrates PARP and Lamin A/C, as well. The differential resistance of RT4 and T24 cells to cisplatin-induced apoptosis was associated with an RT4-specific phosphorylation (Ser15; Ser392) pattern of p53, together with structural amputations of the Akt and XIAP anti-apoptotic regulators. Furthermore, cisplatin administration resulted in a Granzyme B-mediated proteolytic cleavage of Hsp90 molecular chaperone, exclusively occurring in RT4 cells. To generate functional networks, expression analysis of a number of genes, including Bik, Bim, Bcl-2, FAP-1, Fas, FasL, TRAIL, Puma, Caspase-10, ATP7A, ATP7B and MRP1, was performed, strongly supporting the role of p53-dependent and p53-independent transcriptional responses in cisplatin-induced apoptosis of bladder cancer cells.

  20. Senescence and aging: the critical roles of p53.

    Science.gov (United States)

    Rufini, A; Tucci, P; Celardo, I; Melino, G

    2013-10-24

    p53 functions as a transcription factor involved in cell-cycle control, DNA repair, apoptosis and cellular stress responses. However, besides inducing cell growth arrest and apoptosis, p53 activation also modulates cellular senescence and organismal aging. Senescence is an irreversible cell-cycle arrest that has a crucial role both in aging and as a robust physiological antitumor response, which counteracts oncogenic insults. Therefore, via the regulation of senescence, p53 contributes to tumor growth suppression, in a manner strictly dependent by its expression and cellular context. In this review, we focus on the recent advances on the contribution of p53 to cellular senescence and its implication for cancer therapy, and we will discuss p53's impact on animal lifespan. Moreover, we describe p53-mediated regulation of several physiological pathways that could mediate its role in both senescence and aging.

  1. TRIM65 negatively regulates p53 through ubiquitination

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yang [Department of Respiration, The First Hospital of Jilin University, Changchun 130021 (China); Ma, Chengyuan [Department of Neurosurgery, The First Hospital of Jilin University, Changchun 130021 (China); Zhou, Tong [Department of Endocrinology, The First Hospital of Jilin University, Changchun 130021 (China); Liu, Ying [Department of Respiration, The First Hospital of Jilin University, Changchun 130021 (China); Sun, Luyao [Department of Infectious Diseases, The First Hospital of Jilin University, Changchun 130021 (China); Yu, Zhenxiang, E-mail: zhenxiangyu2015@gmail.com [Department of Respiration, The First Hospital of Jilin University, Changchun 130021 (China)

    2016-04-22

    Tripartite-motif protein family member 65 (TRIM65) is an important protein involved in white matter lesion. However, the role of TRIM65 in human cancer remains less understood. Through the Cancer Genome Atlas (TCGA) gene alteration database, we found that TRIM65 is upregulated in a significant portion of non-small cell lung carcinoma (NSCLC) patients. Our cell growth assay revealed that TRIM65 overexpression promotes cell proliferation, while knockdown of TRIM65 displays opposite effect. Mechanistically, TRIM65 binds to p53, one of the most critical tumor suppressors, and serves as an E3 ligase toward p53. Consequently, TRIM65 inactivates p53 through facilitating p53 poly-ubiquitination and proteasome-mediated degradation. Notably, chemotherapeutic reagent cisplatin induction of p53 is markedly attenuated in response to ectopic expression of TRIM65. Cell growth inhibition by TRIM65 knockdown is more significant in p53 positive H460 than p53 negative H1299 cells, and knockdown of p53 in H460 cells also shows compromised cell growth inhibition by TRIM65 knockdown, indicating that p53 is required, at least in part, for TRIM65 function. Our findings demonstrate TRIM65 as a potential oncogenic protein, highly likely through p53 inactivation, and provide insight into development of novel approaches targeting TRIM65 for NSCLC treatment, and also overcoming chemotherapy resistance. - Highlights: • TRIM65 expression is elevated in NSCLC. • TRIM65 inactivates p53 through mediating p53 ubiquitination and degradation. • TRIM65 attenuates the response of NSCLC cells to cisplatin.

  2. Analysis of p53- immunoreactivity in astrocytic brain tumors

    Directory of Open Access Journals (Sweden)

    Shinkarenko T.V.

    2016-12-01

    Full Text Available P53 is an antioncogene with the frequently occured mutations in human tumor cells, leading to corresponding protein overexpression which can be detected by immunohistochemistry. Researches dedicated to the investigation of possibilities of using this technique gave controversial results. The authors investigated features of p53 protein expression in astrocytic brain tumors with different degrees of malignancy. Analyzed the relationship of the expression level of p53 by tumor cells with clinical parameters and Ki-67 proliferation index (PI as well. Tissues were collected from 52 cases with diagnosed astrocytic brain tumors. The sections were immunohistochemically stained with p53 and Ki-67. For each marker, 1000 tumor cells were counted and the ratio of positive tumor cells was calculated using software package ImageJ 1,47v. In normal brain tissue p53- expression was not identified. p53-immunoreactive tumor cells were detected in 25% (1/4 pilocytic astrocytomas, 33.3% (2/6 of diffuse astrocytomas, 53.8% (7/13 anaplastic astrocytomas, 58.6% (17/29 glioblastomas. A high proportion of p53-immunoreactive cells (> 30% was observed only in glioblastomas. The level of p53-imunoreactivity was not related to the age, gender and Grade WHO (p> 0,05. Spearman correlation coefficient between the relative quantity of ki-67- and p53-immunoreactive nuclei showed weak direct correlation (0.023, but the one was not statistically significant (p> 0,05. The level of p53-imunoreactivity is not dependent from age and sex of patients, Grade (WHO and proliferative activity (p>0,05 but the high level of p53-immunoreactive cells (>30% is found in glioblastoma specimens only, that may be due to the accumulation of mutations in DNA of tumor cells. There is insignificant weak relationship between relative quantities of ki-67- and p53-immunoreactive tumor cells (p>0,05.

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

    Science.gov (United States)

    2016-09-01

    LOF) of wild-type p53 through mutation, gene silencing, or amplification of negative p53 regulators , and gain of function (GOF) displayed by some...LOF-adapted state. We will continue our analysis with three main goals : 1) To corroborate our differential gene expression findings with additional...majority of TNBC cases and produces two adaptive states: loss of function (LOF) of wild-type p53 through mutation, gene silencing, or amplification

  4. Evolution of the p53-MDM2 pathway.

    Science.gov (United States)

    Åberg, Emma; Saccoccia, Fulvio; Grabherr, Manfred; Ore, Wai Ying Josefin; Jemth, Per; Hultqvist, Greta

    2017-08-03

    The p53 signalling pathway, which controls cell fate, has been extensively studied due to its prominent role in tumor development. The pathway includes the tumor supressor protein p53, its vertebrate paralogs p63 and p73, and their negative regulators MDM2 and MDM4. The p53/p63/p73-MDM system is ancient and can be traced in all extant animal phyla. Despite this, correct phylogenetic trees including both vertebrate and invertebrate species of the p53/p63/p73 and MDM families have not been published. Here, we have examined the evolution of the p53/p63/p73 protein family with particular focus on the p53/p63/p73 transactivation domain (TAD) and its co-evolution with the p53/p63/p73-binding domain (p53/p63/p73BD) of MDM2. We found that the TAD and p53/p63/p73BD share a strong evolutionary connection. If one of the domains of the protein is lost in a phylum, then it seems very likely to be followed by loss of function by the other domain as well, and due to the loss of function it is likely to eventually disappear. By focusing our phylogenetic analysis to p53/p63/p73 and MDM proteins from phyla that retain the interaction domains TAD and p53/p63/p73BD, we built phylogenetic trees of p53/p63/p73 and MDM based on both vertebrate and invertebrate species. The trees follow species evolution and contain a total number of 183 and 98 species for p53/p63/p73 and MDM, respectively. We also demonstrate that the p53/p63/p73 and MDM families result from whole genome duplications. The signaling pathway of the TAD and p53/p63/p73BD in p53/p63/p73 and MDM, respectively, dates back to early metazoan time and has since then tightly co-evolved, or disappeared in distinct lineages.

  5. Super p53 for Treatment of Ovarian Cancer

    Science.gov (United States)

    2017-09-01

    AWARD NUMBER: W81XWH-15-1-0036 TITLE: Super p53 for Treatment of Ovarian Cancer PRINCIPAL INVESTIGATOR: Carol S. Lim University of Utah...Super p53 for Treatment of Ovarian Cancer 5b. GRANT NUMBER W81XWH-15-1-0036 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Carol S. Lim...ABSTRACT In this final report, we show gene therapy using re-engineered super p53 (p53-CC constructs) kills some ovarian cancer cell lines in vitro

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

    OpenAIRE

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

    2014-01-01

    p53 functions as a tumor suppressor by transcriptionally regulating the expression of genes involved in controlling cell proliferation or apoptosis. p53 and its isoform ?133p53/?113p53 form a negative regulation loop in that p53 activates the expression of ?133p53/?113p53 while ?133p53/?113p53 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 st...

  7. Therapeutic targeting of p53: all mutants are equal, but some mutants are more equal than others.

    Science.gov (United States)

    Sabapathy, Kanaga; Lane, David P

    2017-09-26

    TP53, which encodes the tumour-suppressor protein p53, is the most frequently mutated gene across all cancer types. The presence of mutant p53 predisposes to cancer development, promotes the survival of cancer cells, and is associated with ineffective therapeutic responses and unfavourable prognoses. Despite these effects, no drug that abrogates the oncogenic functions of mutant p53 has yet been approved for the treatment of cancer. Current investigational therapeutic strategies are mostly aimed at restoring the wild-type activity of mutant p53, based on the assumption that all p53 mutants are functionally equal. Our increasing knowledge of mutant forms of p53, however, supports the antithetical hypothesis that not all p53 mutants have equivalent cellular effects; hence, a judicious approach to therapeutic targeting of mutant p53 is required. In this Review, we propose a categorization of the major classes of p53 mutants based on their functionality in tumour suppression and response to therapy. The emerging picture is that the mutations across TP53 form a 'rainbow of mutants', with varying degrees of functionality and different pathobiological consequences, necessitating the use of diverse therapeutic strategies to selectively target specific classes of mutation. The utility of this knowledge of TP53 mutations in developing selective therapeutic options, and in facilitating clinical decision-making is discussed.

  8. Probing the interaction of the p53 C-terminal domain to the histone demethylase LSD1.

    Science.gov (United States)

    Speranzini, Valentina; Ciossani, Giuseppe; Marabelli, Chiara; Mattevi, Andrea

    2017-10-15

    The p53 transcription factor plays a central role in the regulation of the expression of several genes, and itself is post-translationally regulated through its different domains. Of particular relevance for p53 function is its intrinsically disordered C-terminal domain (CTD), representing a hotspot for post-translational modifications and a docking site for transcriptional regulators. For example, the histone H3 lysine demethylase 1 (LSD1) interacts with p53 via the p53-CTD for mutual regulation. To biochemically and functionally characterize this complex, we evaluated the in vitro interactions of LSD1 with several p53-CTD peptides differing in length and modifications. Binding was demonstrated through thermal shift, enzymatic and fluorescence polarization assays, but no enzymatic activity could be detected on methylated p53-CTD peptides in vitro. These experiments were performed using the wild-type enzyme and LSD1 variants that are mutated on three active-site residues. We found that LSD1 demethylase activity is inhibited by p53-CTD. We also noted that the association between the two proteins is mediated by mostly non-specific electrostatic interactions involving conserved active-site residues of LSD1 and a highly charged segment of the p53-CTD. We conclude that p53-CTD inhibits LSD1 activity and that the direct association between the two proteins can contribute to their functional cross-talk. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2017-08-01

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

  10. Cancer cell sensitivity to bortezomib is associated with survivin expression and p53 status but not cancer cell types

    Directory of Open Access Journals (Sweden)

    Chanan-Khan Asher A

    2010-01-01

    Full Text Available Abstract Background Survivin is known playing a role in drug resistance. However, its role in bortezomib-mediated inhibition of growth and induction of apoptosis is unclear. There are conflicting reports for the effect of bortezomib on survivin expression, which lacks of a plausible explanation. Methods: In this study, we tested cancer cells with both p53 wild type and mutant/null background for the relationship of bortezomib resistance with survivin expression and p53 status using MTT assay, flow cytometry, DNA fragmentation, caspase activation, western blots and RNAi technology. Results We found that cancer cells with wild type p53 show a low level expression of survivin and are sensitive to treatment with bortezomib, while cancer cells with a mutant or null p53 show a high level expression of survivin and are resistant to bortezomib-mediated apoptosis induction. However, silencing of survivin expression utilizing survivin mRNA-specific siRNA/shRNA in p53 mutant or null cells sensitized cancer cells to bortezomib mediated apoptosis induction, suggesting a role for survivin in bortezomib resistance. We further noted that modulation of survivin expression by bortezomib is dependent on p53 status but independent of cancer cell types. In cancer cells with mutated p53 or p53 null, bortezomib appears to induce survivin expression, while in cancer cells with wild type p53, bortezomib downregulates or shows no significant effect on survivin expression, which is dependent on the drug concentration, cell line and exposure time. Conclusions Our findings, for the first time, unify the current inconsistent findings for bortezomib treatment and survivin expression, and linked the effect of bortezomib on survivin expression, apoptosis induction and bortezomib resistance in the relationship with p53 status, which is independent of cancer cell types. Further mechanistic studies along with this line may impact the optimal clinical application of bortezomib in

  11. Tobacco, alcohol, and p53 overexpression in early colorectal neoplasia

    Directory of Open Access Journals (Sweden)

    Mansukhani Mahesh

    2003-11-01

    Full Text Available Abstract Background The p53 tumor suppressor gene is commonly mutated in colorectal cancer. While the effect of p53 mutations on colorectal cancer prognosis has been heavily studied, less is known about how epidemiologic risk factors relate to p53 status, particularly in early colorectal neoplasia prior to clinically invasive colorectal cancer (including adenomas, carcinoma in situ (CIS, and intramucosal carcinoma. Methods We examined p53 status, as measured by protein overexpression, in 157 cases with early colorectal neoplasia selected from three New York City colonoscopy clinics. After collecting paraffin-embedded tissue blocks, immunohistochemistry was performed using an anti-p53 monoclonal mouse IgG2a [BP53-12-1] antibody. We analyzed whether p53 status was different for risk factors for colorectal neoplasia relative to a polyp-free control group (n = 508. Results p53 overexpression was found in 10.3%, 21.7%, and 34.9%, of adenomatous polyps, CIS, and intramucosal cases, respectively. Over 90% of the tumors with p53 overexpression were located in the distal colon and rectum. Heavy cigarette smoking (30+ years was associated with cases not overexpressing p53 (OR = 1.8, 95% CI = 1.1–2.9 but not with those cases overexpressing p53 (OR = 1.0, 95% CI = 0.4–2.6. Heavy beer consumption (8+ bottles per week was associated with cases overexpressing p53 (OR = 4.0, 95% CI = 1.3–12.0 but not with cases without p53 overexpression (OR = 1.6, 95% CI = 0.7–3.7. Conclusion Our findings that p53 overexpression in early colorectal neoplasia may be positively associated with alcohol intake and inversely associated with cigarette smoking are consistent with those of several studies of p53 expression and invasive cancer, and suggest that there may be relationships of smoking and alcohol with p53 early in the adenoma to carcinoma sequence.

  12. Rbm24, a target of p53, is necessary for proper expression of p53 and heart development.

    Science.gov (United States)

    Zhang, Min; Zhang, Yanhong; Xu, Enshun; Mohibi, Shakur; de Anda, Danielle Michelle; Jiang, Yuqian; Zhang, Jin; Chen, Xinbin

    2018-01-22

    Activation of p53-dependent apoptosis is critical for tumor suppression but aberrant activation of p53 also leads to developmental defects and heart failure. Here, we found that Rbm24 RNA-binding protein, a target of p53, regulates p53 mRNA translation. Mechanistically, we found that through binding to p53 mRNA and interaction with translation initiation factor eIF4E, Rbm24 prevents eIF4E from binding to p53 mRNA and inhibits the assembly of translation initiation complex. Importantly, we showed that mice deficient in Rbm24 die in utero due to the endocardial cushion defect in the heart at least in part due to aberrant activation of p53-dependent apoptosis. We also showed that the heart developmental defect in Rbm24-null mice can be partially rescued by p53 deficiency through decreased apoptosis in the heart. Together, we postulate that the p53-Rbm24 loop is critical for the heart development and may be explored for mitigating congenital heart diseases and heart failure.

  13. Overexpression of p53 in Nigerian breast cancers and its ...

    African Journals Online (AJOL)

    Background: Mutation of the tumour suppressor gene, p53, is implicated in most cancers. This gene has also been associated with high tumour grade in breast cancers. African women are known to generally have high grade tumours. This study sought to determine the expression of p53 protein as well as the relationship ...

  14. Plk1-mediated phosphorylation of Topors regulates p53 stability.

    Science.gov (United States)

    Yang, Xiaoming; Li, Hongchang; Zhou, Zinan; Wang, Wen-Horng; Deng, Anping; Andrisani, Ourania; Liu, Xiaoqi

    2009-07-10

    Polo-like kinase 1 (Plk1) overexpression is associated with tumorigenesis by an unknown mechanism. Likewise, Plk1 was suggested to act as a negative regulator of tumor suppressor p53, but the mechanism remains to be determined. Herein, we have identified topoisomerase I-binding protein (Topors), a p53-binding protein, as a Plk1 target. We show that Plk1 phosphorylates Topors on Ser(718) in vivo. Significantly, expression of a Plk1-unphosphorylatable Topors mutant (S718A) leads to a dramatic accumulation of p53 through inhibition of p53 degradation. Topors is an ubiquitin and small ubiquitin-like modifier ubiquitin-protein isopeptide ligase (SUMO E3) ligase. Plk1-mediated phosphorylation of Topors inhibits Topors-mediated sumoylation of p53, whereas p53 ubiquitination is enhanced, leading to p53 degradation. These results demonstrate that Plk1 modulates Topors activity in suppressing p53 function and identify a likely mechanism for the tumorigenic potential of Plk1.

  15. Plk1-mediated Phosphorylation of Topors Regulates p53 Stability*

    Science.gov (United States)

    Yang, Xiaoming; Li, Hongchang; Zhou, Zinan; Wang, Wen-Horng; Deng, Anping; Andrisani, Ourania; Liu, Xiaoqi

    2009-01-01

    Polo-like kinase 1 (Plk1) overexpression is associated with tumorigenesis by an unknown mechanism. Likewise, Plk1 was suggested to act as a negative regulator of tumor suppressor p53, but the mechanism remains to be determined. Herein, we have identified topoisomerase I-binding protein (Topors), a p53-binding protein, as a Plk1 target. We show that Plk1 phosphorylates Topors on Ser718 in vivo. Significantly, expression of a Plk1-unphosphorylatable Topors mutant (S718A) leads to a dramatic accumulation of p53 through inhibition of p53 degradation. Topors is an ubiquitin and small ubiquitin-like modifier ubiquitin-protein isopeptide ligase (SUMO E3) ligase. Plk1-mediated phosphorylation of Topors inhibits Topors-mediated sumoylation of p53, whereas p53 ubiquitination is enhanced, leading to p53 degradation. These results demonstrate that Plk1 modulates Topors activity in suppressing p53 function and identify a likely mechanism for the tumorigenic potential of Plk1. PMID:19473992

  16. Fermentative metabolism impedes p53-dependent apoptosis in a ...

    Indian Academy of Sciences (India)

    It is known that the Warburg effect and Crabtree effect aredisplayed by Saccharomyces cerevisiae, during growth on abundant glucose. Beyond this similarity, it was also demonstratedthat expression of human pro-apoptotic proteins in S. cerevisiae such as Bax and p53 caused apoptosis. Here, wedemonstrate that p53 ...

  17. EGFR, p53, IDH-1 and MDM2 immunohistochemical analysis in glioblastoma: therapeutic and prognostic correlation

    Directory of Open Access Journals (Sweden)

    Richard Murdoch Montgomery

    2015-07-01

    Full Text Available We studied 36 glioblastoma cases at HC-UNICAMP from 2008 to 2012 and classified the immunohistochemical distribution of the wild-type epidermal growth factor receptor (EGFR, mutated forms of p53 protein and isocitrate dehydrogenase-1 (IDH-1 and murine double protein 2 (MDM2. Immunostaining findings were correlated with clinical data and response to treatment (surgery, chemotherapy and radiotherapy. About 97% of the tumors were primary, most of them localized in the frontal lobe. Mean time free of clinical or symptomatic disease and free time of radiological disease were 7.56 and 7.14 months, respectively. We observed a significant positive correlation between expressions of p53 and MDM2, EGFR and MDM2. Clinical, radiological and overall survivals also showed a significant positive correlation. p53 staining and clinical survival showed a significant negative correlation. The current series provides clinical and histopathological data that contribute to knowledge on glioblastoma in Brazilians.

  18. The p53 protein plays a central role in the mechanism of action of epigentic drugs that alter the methylation of cytosine residues in DNA.

    Science.gov (United States)

    Levine, Arnold J

    2017-01-31

    Both normal and cancerous cells, treated with drugs that block cytosine methylation of DNA, are preferentially killed by these drugs when they have p53 mutations and survive if they have a wild type protein. It appears that the wild type p53 protein functions to eliminate cells that undergo large epigenetic alterations and save other cells from death by this drug treatment. This has now been observed in cancerous cells in culture, tumors in animals and tumors in humans. AML cells with p53 mutations in humans treated with decitabine are killed by differentiation or senescense, but then relapse at a high rate becoming drug resistant. The mechanism of resistance to epigenetic drugs in p53 mutant cells, by possibly restoring a wild type p53 gene or restoring a defective p53 pathway, is now an interesting hypothesis to explore.

  19. COX-2 and p53 in human sinonasal cancer

    DEFF Research Database (Denmark)

    Holmila, Reetta; Cyr, Diane; Luce, Danièle

    2008-01-01

    to development of cancer. Many signals that activate COX-2 also induce tumor suppressor p53, a transcription factor central in cellular stress response. We investigated COX-2 and p53 expressions by immunohistochemistry in 50 SNCs (23 adenocarcinomas, and 27 squamous cell carcinomas (SCC); 48 analyzed for COX-2......; 41 for p53). Occupational histories and smoking habits were available for majority of the cases. Most of the adenocarcinoma cases with exposure history data had been exposed to wood dust at work in the past (88%, 14/16). For smokers, 63% (12/19) presented with SSC, whereas 64% (7/11) of nonsmokers...... the exposures and p53 accumulation were found; however, the p53 accumulation pattern (p = 0.062 for wood dust exposure) resembled that of COX-2 expression. In summary, our findings show increased COX-2 expression in SNC adenocarcinoma with wood dust exposure, suggesting a role for inflammatory components...

  20. p53 in the DNA damage repair process

    Science.gov (United States)

    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

  1. p53 immunohistochemistry in high-grade urothelial carcinoma of the bladder is prognostically significant.

    Science.gov (United States)

    Hodgson, Anjelica; Xu, Bin; Downes, Michelle R

    2017-08-01

    TP53 mutations are characteristic of the high-grade pathway in the dual pathway of urothelial carcinogenesis. These mutations have been correlated with aberrant accumulation of p53 protein; however the definition and significance of this vary in the literature. The aim of this study was to assess p53 immunostaining in a cohort of high-grade urothelial carcinomas by using standard published cut-offs and a novel binarized method that included assessment of the null phenotype. Each scoring method was correlated with oncological outcome. A triplicate core tissue microarray was constructed from 207 cases of high-grade urothelial carcinoma treated by cystectomy, and was stained with p53. The percentage nuclear staining was recorded for each core and averaged for every case (206 cases were evaluable). Cases were categorized as positive/negative according to published cut-offs (10%, 40%) or by binarizing them as abnormal (null phenotype or >50% positivity) and wild type (1-49% positivity). Correlation with disease-specific survival was not significant according to standard definitions of p53 positivity. When a 40% cut-off was used, a correlation with relapse-free survival was significant on univariate analysis (P = 0.038) but not on multivariate analysis (P = 0.079). Abnormal p53 expression showed a near-significant trend for association with disease-specific survival (P = 0.052) and was a significant predictor for relapse-free survival on both univariate analysis (P = 0.047) and multivariate analysis (P = 0.035). Prior to this study, the p53 null phenotype was not well described in urothelial carcinoma of the bladder. Abnormal p53 immunoexpression (null staining pattern or staining in >50% of cells) is prognostic in terms of oncological outcome. © 2017 John Wiley & Sons Ltd.

  2. Functional consequence of the p53 codon 72 polymorphism in colorectal cancer.

    Science.gov (United States)

    Katkoori, Venkat R; Manne, Upender; Chaturvedi, Lakshmi S; Basson, Marc D; Haan, Pam; Coffey, Daniel; Bumpers, Harvey L

    2017-09-29

    The codon 72 polymorphism in p53 has been implicated in colorectal cancer (CRC) risk, prognosis and CRC health disparities. We examined the functional consequence of this polymorphism in CRC. Plasmids (pCMV6) that express different phenotypes of p53 [p53 wild type (wt) at codon 72 (R72wt), R72wt with mutation at codon 273 cysteine (R72273Cys), p53 mutation at codon 72 (P72wt) and P72wt with mutation at codon 273 (P72273Cys)] were constructed. The CRC cell line Caco2, which does not express p53 for in vitro studies, was used as host. CRC xenografts were established in severe combined immunodeficient (SCID) mice using established cell lines. CRC surgical specimens, corresponding normal colon, and tumor xenografts were sequenced for codon 72 polymorphism of p53. Proteins signaling mechanisms were evaluated to assess the functional consequence of P72 phenotype of p53. This study demonstrated a significantly increased survival of cells expressing P72wt, mutant phenotype, versus R72wt phenotype. WB analyses revealed that P72wt induced activation of p38 and RAF/MEK/ extracellular signal-regulated kinase (ERK) MAP kinases. Activation of CREB was found to be higher in tumors that exhibit P72 phenotype. Metastatic lesions of CRC expressed more phospho-CREB than non-metastatic lesions. The expression of P72wt promoted CRC metastasis. P72 contributes to the aggressiveness of CRC. Because P72 is over-expressed in CRC, specifically in African-American patients, this suggests a role for P72 in cancer health disparities. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251 & U54CA118948.

  3. p53 as the focus of gene therapy: past, present and future.

    Science.gov (United States)

    Valente, Joana Fa; Queiroz, Joao A; Sousa, Fani

    2018-01-15

    Several gene deviations can be responsible for triggering oncogenic processes. However, mutations in tumour suppressor genes are usually more associated to malignant diseases, being p53 one of the most affected and studied element. p53 is implicated in a number of known cellular functions, including DNA damage repair, cell cycle arrest in G1/S and G2/M and apoptosis, being an interesting target for cancer treatment. Considering these facts, the development of gene therapy approaches focused on p53 expression and regulation seems to be a promising strategy for cancer therapy. Several studies have shown that transfection of cancer cells with wild-type p53 expressing plasmids could directly drive cells into apoptosis and/or growth arrest, suggesting that a gene therapy approach for cancer treatment can be based on the re-establishment of the normal p53 expression levels and function. Up until now, several clinical research studies using viral and non-viral vectors delivering p53 genes, isolated or combined with other therapeutic agents, have been accomplished and there are already in the market therapies based on the use of this gene. This review summarizes the different methods used to deliver and/or target the p53 as well as the main results of therapeutic effect obtained with the different strategies applied. Finally, the ongoing approaches are described, also focusing the combinatorial therapeutics to show the increased therapeutic potential of combining gene therapy vectors with chemo or radiotherapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  4. The p53 inhibitor MDM2 facilitates Sonic Hedgehog-mediated tumorigenesis and influences cerebellar foliation.

    Directory of Open Access Journals (Sweden)

    Reem Malek

    Full Text Available Disruption of cerebellar granular neuronal precursor (GNP maturation can result in defects in motor coordination and learning, or in medulloblastoma, the most common childhood brain tumor. The Sonic Hedgehog (Shh pathway is important for GNP proliferation; however, the factors regulating the extent and timing of GNP proliferation, as well as GNP differentiation and migration are poorly understood. The p53 tumor suppressor has been shown to negatively regulate the activity of the Shh effector, Gli1, in neural stem cells; however, the contribution of p53 to the regulation of Shh signaling in GNPs during cerebellar development has not been determined. Here, we exploited a hypomorphic allele of Mdm2 (Mdm2(puro, which encodes a critical negative regulator of p53, to alter the level of wild-type MDM2 and p53 in vivo. We report that mice with reduced levels of MDM2 and increased levels of p53 have small cerebella with shortened folia, reminiscent of deficient Shh signaling. Indeed, Shh signaling in Mdm2-deficient GNPs is attenuated, concomitant with decreased expression of the Shh transducers, Gli1 and Gli2. We also find that Shh stimulation of GNPs promotes MDM2 accumulation and enhances phosphorylation at serine 166, a modification known to increase MDM2-p53 binding. Significantly, loss of MDM2 in Ptch1(+/- mice, a model for Shh-mediated human medulloblastoma, impedes cerebellar tumorigenesis. Together, these results place MDM2 at a major nexus between the p53 and Shh signaling pathways in GNPs, with key roles in cerebellar development, GNP survival, cerebellar foliation, and MB tumorigenesis.

  5. p53 selectively regulates developmental apoptosis of rod photoreceptors.

    Directory of Open Access Journals (Sweden)

    Linda Vuong

    Full Text Available Retinal cells become post-mitotic early during post-natal development. It is likely that p53, a well-known cell cycle regulator, is involved in regulating the genesis, differentiation and death of retinal cells. Furthermore, retinal cells are under constant oxidative stress that can result in DNA damage, due to the extremely high level of metabolic activity associated with phototransduction. If not repaired, this damage may result in p53-dependent cell death and ensuing vision loss. In this study, the role of p53 during retinal development and in the post-mitotic retina is investigated. A previously described super p53 transgenic mouse that expresses an extra copy of the mouse p53 gene driven by its endogenous promoter is utilized. Another transgenic mouse (HIP that expresses the p53 gene in rod and cone photoreceptors driven by the human interphotoreceptor retinoid binding protein promoter was generated. The electroretinogram (ERG of the super p53 mouse exhibited reduced rod-driven scotopic a and b wave and cone-driven photopic b wave responses. This deficit resulted from a reduced number of rod photoreceptors and inner nuclear layer cells. However, the reduced photopic signal arose only from lost inner retinal neurons, as cone numbers did not change. Furthermore, cell loss was non-progressive and resulted from increased apoptosis during retinal developmental as determined by TUNEL staining. In contrast, the continuous and specific expression of p53 in rod and cone photoreceptors in the mature retinas of HIP mice led to the selective loss of both rods and cones. These findings strongly support a role for p53 in regulating developmental apoptosis in the retina and suggest a potential role, either direct or indirect, for p53 in the degenerative photoreceptor loss associated with human blinding disorders.

  6. MDM2 inhibitor nutlin-3a induces apoptosis and senescence in cutaneous T-cell lymphoma: Role of p53

    DEFF Research Database (Denmark)

    Manfé, Valentina; Biskup, Edyta Urszula; Johansen, Peter

    2012-01-01

    53. To investigate the potential therapeutic use of nutlin-3a in CTCL, we screened CTCL lines Hut-78, SeAx, MyLa2000, Mac1, and Mac2a by measuring p53 levels after nutlin-3a treatment. In MyLa2000, Mac1, and Mac2a, we observed the increase in p53, indicating the fully functional p53. In the remaining...... cell lines, P53 mutation analysis identified a homozygous nonsense mutation (R196Stop in Hut-78) and a homozygous missense mutation (G245S in SeAx). In MyLa2000, Mac1, and Mac2a carrying wild-type P53, nutlin-3a induced apoptosis and senescence demonstrated by permanent G0/G1 cell-cycle block...

  7. Expression of Fbxo7 in haematopoietic progenitor cells cooperates with p53 loss to promote lymphomagenesis.

    Directory of Open Access Journals (Sweden)

    Mikhail Lomonosov

    Full Text Available Fbxo7 is an unusual F box protein that augments D-type cyclin complex formation with Cdk6, but not Cdk4 or Cdk2, and its over-expression has been demonstrated to transform immortalised fibroblasts in a Cdk6-dependent manner. Here we present new evidence in vitro and in vivo on the oncogenic potential of this regulatory protein in primary haematopoietic stem and progenitor cells (HSPCs. Increasing Fbxo7 expression in HSPCs suppressed their colony forming ability in vitro, specifically decreasing CD11b (Mac1 expression, and these effects were dependent on an intact p53 pathway. Furthermore, increased Fbxo7 levels enhanced the proliferative capacity of p53 null HSPCs when they were grown in reduced concentrations of stem cell factor. Finally, irradiated mice reconstituted with p53 null, but not wild-type, HSPCs expressing Fbxo7 showed a statistically significant increase in the incidence of T cell lymphoma in vivo. These data argue that Fbxo7 negatively regulates the proliferation and differentiation of HSPCs in a p53-dependent manner, and that in the absence of p53, Fbxo7 expression can promote T cell lymphomagenesis.

  8. MDM2 expression during mouse embryogenesis and the requirement of p53.

    Science.gov (United States)

    Léveillard, T; Gorry, P; Niederreither, K; Wasylyk, B

    1998-06-01

    We compared mouse embryonic expression of the MDM2 proto-oncogene, p21WAF1/CIP1 and their transcriptional regulator, p53. MDM2 expression is ubiquitous from 7.5 to 11.5 days post coitum (dpc) and more restricted from 12.5 dpc, with the highest levels in the testes and neural tube. From 14.5 to 18.5 dpc, the nasal respiratory epithelium expresses high levels of MDM2 RNA and protein and p21WAF1/CIP1 RNA, in both wild type and p53 null embryos. MDM2 expression during development is tissue-specific and, like p21WAF1/CIP1, is independent of p53. MDM2 may have a developmental role after 6.5 dpc, when MDM2 null mice die (Jones, S.N., Roe, A.E., Donehower, L.A., Bradley, A., 1995. Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206-208; Montes de Oca Luna, R., Wagner, D.S., Lozano, G., 1995. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203-206). Copyright 1998 Elsevier Science Ireland Ltd. All rights reserved

  9. Small Molecule Modulator of p53 Signaling Pathway: Application for Radiosensitizing or Radioprotection Agents

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Sang Taek; Cho, Mun Ju; Gwak, Jung Sug; Ryu, Min Jung [PharmacoGenomics Research Center, Inje University, Busan (Korea, Republic of); Song, Jie Young; Yun, Yeon Sook [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2009-05-15

    The tumor suppressor p53 is key molecule to protect the cell against genotoxic stress and..the most frequently mutated..protein..in cancer cells. Lack of functional p53..is accompanied by high rate of genomic instability, rapid tumor progression, resistance to anticancer therapy, and increased angiogenesis. In response to DNA damage, p53 protein rapidly accumulated through attenuated proteolysis and is also activated as transcription factor. Activated p53 up-regulates target genes involved in cell cycle arrest and/or apoptosis and then lead to suppression of malignant transformation and the maintenance of genomic integrity. Chemical genetics is a new technology to uncover the signaling networks that regulated biological phenotype using exogenous reagents such as small molecules. Analogous to classical forward genetic screens in model organism, this approach makes use of high throughput, phenotypic assay to identify small molecules that disrupt gene product function in a way that alters a phenotype of interest. Recently, interesting small molecules were identified from cell based high throughput screening and its target protein or mechanism of action were identified by various methods including affinity chromatography, protein array profiling, mRNA or phage display, transcription profiling, and RNA interference.

  10. Transcription pattern of p53-targeted DNA repair genes in the hypoxia-tolerant subterranean mole rat Spalax.

    Science.gov (United States)

    Shams, Imad; Malik, Assaf; Manov, Irena; Joel, Alma; Band, Mark; Avivi, Aaron

    2013-04-12

    The tumor suppressor gene p53 induces growth arrest and/or apoptosis in response to DNA damage/hypoxia. Inactivation of p53 confers a selective advantage to tumor cells under a hypoxic microenvironment during tumor progression. The subterranean blind mole rat, Spalax, spends its life underground at low-oxygen tensions, hence developing a wide range of respiratory/molecular adaptations to hypoxic stress, including critical changes in p53 structure and signaling pathway. The highly conserved p53 Arg(R)-172 is substituted by lysine (K) in Spalax, identical with a tumor-associated mutation. Functionality assays revealed that Spalax p53 is unable to activate apoptotic target genes but is still capable of activating cell cycle arrest genes. Furthermore, we have shown that the transcription patterns of representative p53-induced genes (Apaf1 and Mdm2) in Spalax are influenced by hypoxia. Cell cycle arrest allows the cells to repair DNA damage via different DNA repair genes. We tested the transcription pattern of three p53-related DNA repair genes (p53R2, Mlh1, and Msh2) under normoxia and short-acute hypoxia in Spalax, C57BL/6 wild-type mice, and two strains of mutant C57BL/6 mice, each carrying a different mutation at the R172 position. Our results show that while wild-type/mutant mice exhibit strong hypoxia-induced reductions of repair gene transcript levels, no such inhibition is found in Spalax under hypoxia. Moreover, unlike mouse p53R2, Spalax p53R2 transcript levels are strongly elevated under hypoxia. These results suggest that critical repair functions, which are known to be inhibited under hypoxia in mice, remain active in Spalax, as part of its unique hypoxia tolerance mechanisms. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. A p53-bound enhancer region controls a long intergenic noncoding RNA required for p53 stress response

    NARCIS (Netherlands)

    Melo, C A; Léveillé, N; Rooijers, K; Wijchers, P J; Geeven, G; Tal, A; Melo, S A; de Laat, W; Agami, R

    2016-01-01

    Genome-wide chromatin studies identified the tumor suppressor p53 as both a promoter and an enhancer-binding transcription factor. As an enhancer factor, p53 can induce local production of enhancer RNAs, as well as transcriptional activation of distal neighboring genes. Beyond the regulation of

  12. Generation of a selectively cytotoxic fusion protein against p53 mutated cancers

    Directory of Open Access Journals (Sweden)

    Kousparou Christina A

    2012-08-01

    Full Text Available Abstract Background A significant number of cancers are caused by defects in p21 causing functional defects in p21 or p53 tumour-suppressor proteins. This has led to many therapeutic approaches including restoration by gene therapy with wild-type p53 or p21 using viral or liposomal vectors, which have toxicity or side-effect limitations. We set out to develop a safer, novel fusion protein which has the ability to reconstitute cancer cell lines with active p21 by protein transduction. Methods The fusion protein was produced from the cell-translocating peptide Antennapedia (Antp and wild-type, full-length p21 (Antp-p21. This was expressed and refolded from E. coli and tested on a variety of cell lines and tumours (in a BALB/c nude xenograft model with differing p21 or p53 status. Results Antp-p21 penetrated and killed cancer cells that do not express wild type p53 or p21. This included cells that were matched to cogenic parental cell lines. Antp-p21 killed cancer cells selectively that were malignant as a result of mutations or nuclear exclusion of the p53 and p21 genes and over-expression of MDM2. Non-specific toxicity was excluded by showing that Antp-p21 penetrated but did not kill p53- or p21- wild-type cells. Antp-p21 was not immunogenic in normal New Zealand White rabbits. Recombinant Antp peptide alone was not cytotoxic, showing that killing was due to the transduction of the p21 component of Antp-p21. Antp-p21 was shown to penetrate cancer cells engrafted in vivo and resulted in tumour eradication when administered with conventionally-used chemotherapeutic agents, which alone were unable to produce such an effect. Conclusions Antp-p21 may represent a new and promising targeted therapy for patients with p53-associated cancers supporting the concept that rational design of therapies directed against specific cancer mutations will play a part in the future of medical oncology.

  13. Fuzzy tandem repeats containing p53 response elements may define species-specific p53 target genes.

    Directory of Open Access Journals (Sweden)

    Iva Simeonova

    2012-06-01

    Full Text Available Evolutionary forces that shape regulatory networks remain poorly understood. In mammals, the Rb pathway is a classic example of species-specific gene regulation, as a germline mutation in one Rb allele promotes retinoblastoma in humans, but not in mice. Here we show that p53 transactivates the Retinoblastoma-like 2 (Rbl2 gene to produce p130 in murine, but not human, cells. We found intronic fuzzy tandem repeats containing perfect p53 response elements to be important for this regulation. We next identified two other murine genes regulated by p53 via fuzzy tandem repeats: Ncoa1 and Klhl26. The repeats are poorly conserved in evolution, and the p53-dependent regulation of the murine genes is lost in humans. Our results indicate a role for the rapid evolution of tandem repeats in shaping differences in p53 regulatory networks between mammalian species.

  14. Fuzzy tandem repeats containing p53 response elements may define species-specific p53 target genes.

    Science.gov (United States)

    Simeonova, Iva; Lejour, Vincent; Bardot, Boris; Bouarich-Bourimi, Rachida; Morin, Aurélie; Fang, Ming; Charbonnier, Laure; Toledo, Franck

    2012-06-01

    Evolutionary forces that shape regulatory networks remain poorly understood. In mammals, the Rb pathway is a classic example of species-specific gene regulation, as a germline mutation in one Rb allele promotes retinoblastoma in humans, but not in mice. Here we show that p53 transactivates the Retinoblastoma-like 2 (Rbl2) gene to produce p130 in murine, but not human, cells. We found intronic fuzzy tandem repeats containing perfect p53 response elements to be important for this regulation. We next identified two other murine genes regulated by p53 via fuzzy tandem repeats: Ncoa1 and Klhl26. The repeats are poorly conserved in evolution, and the p53-dependent regulation of the murine genes is lost in humans. Our results indicate a role for the rapid evolution of tandem repeats in shaping differences in p53 regulatory networks between mammalian species.

  15. Cisplatinum and Taxol Induce Different Patterns of p53 Phosphorylation

    Directory of Open Access Journals (Sweden)

    Giovanna Damia

    2001-01-01

    Full Text Available Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20, only DDP treatment induced p53 phosphorylation at serine 15 (Ser15. Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM cell lines, the role of ATM in druginduced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.

  16. Absence of a p53 allele delays nitrogen mustard-induced early apoptosis and inflammation of murine skin.

    Science.gov (United States)

    Inturi, Swetha; Tewari-Singh, Neera; Jain, Anil K; Roy, Srirupa; White, Carl W; Agarwal, Rajesh

    2013-09-15

    Bifunctional alkylating agent sulfur mustard (SM) and its analog nitrogen mustard (NM) cause DNA damage leading to cell death, and potentially activating inflammation. Transcription factor p53 plays a critical role in DNA damage by regulating cell cycle progression and apoptosis. Earlier studies by our laboratory demonstrated phosphorylation of p53 at Ser15 and an increase in total p53 in epidermal cells both in vitro and in vivo following NM exposure. To elucidate the role of p53 in NM-induced skin toxicity, we employed SKH-1 hairless mice harboring wild type (WT) or heterozygous p53 (p53+/-). Exposure to NM (3.2mg) caused a more profound increase in epidermal thickness and apoptotic cell death in WT relative to p53+/- mice at 24h. However, by 72h after exposure, there was a comparable increase in NM-induced epidermal cell death in both WT and p53+/- mice. Myeloperoxidase activity data showed that neutrophil infiltration was strongly enhanced in NM-exposed WT mice at 24h persisting through 72h of exposure. Conversely, robust NM-induced neutrophil infiltration (comparable to WT mice) was seen only at 72h after exposure in p53+/- mice. Similarly, NM-exposure strongly induced macrophage and mast cell infiltration in WT, but not p53+/- mice. Together, these data indicate that early apoptosis and inflammation induced by NM in mouse skin are p53-dependent. Thus, targeting this pathway could be a novel strategy for developing countermeasures against vesicants-induced skin injury. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  17. Targeted expression of MDM2 uncouples S phase from mitosis and inhibits mammary gland development independent of p53.

    Science.gov (United States)

    Lundgren, K; Montes de Oca Luna, R; McNeill, Y B; Emerick, E P; Spencer, B; Barfield, C R; Lozano, G; Rosenberg, M P; Finlay, C A

    1997-03-15

    MDM2 is a cellular protein that binds to and inactivates the p53 tumor suppressor protein. Although mdm2 has been shown to function as an oncogene in vitro, all studies to date have assessed MDM2 activities in the presence of p53, implicating p53 inactivation in MDM2-directed transformation. To determine the role of MDM2 in the cell cycle and in tumorigenesis and whether or not this role is dependent on p53, an MDM2 minigene was expressed during gestation and lactation in the mammary gland of both wild-type p53 (p53+/+) and p53 knockout (p53-/-) mice using the bovine beta-lactoglobulin promoter. In six different transgenic mouse lines, deregulated expression of MDM2 inhibited normal development and morphogenesis of the mammary gland, and caused cellular hypertrophy and nuclear abnormalities. These abnormalities included both multinucleated cells and enlarged cells with giant nuclei. Although there were fewer epithelial cells present in the transgenic mammary gland, no apoptosis was observed. Instead, BrdU incorporation and PCNA staining showed that 12%-27% of the transgenic mammary epithelial cells were in S phase at a time when normal cells were terminally differentiated. Analysis of DNA content showed that 30%-45% of the cells were polyploid, with DNA contents up to 16N, indicating that overexpression of MDM2 caused mammary epithelial cells to undergo multiple rounds of S phase without cell division. This phenotype was similar in the p53+/+ and p53-/- background, demonstrating a role for MDM2 in the regulation of DNA synthesis that is independent of the ability of MDM2 to inhibit p53 activity. Additionally, multiple lines of BLGMDM2 transgenic mice developed mammary tumors, confirming that overproduction of MDM2 contributes to tumorigenesis in epithelial cells in vivo.

  18. Restoration of p53 Expression in Human Cancer Cell Lines Upregulates the Expression of Notch1: Implications for Cancer Cell Fate Determination after Genotoxic Stress

    Directory of Open Access Journals (Sweden)

    Fatouma Alimirah

    2007-05-01

    Full Text Available Following genotoxic stress, transcriptional activation of target genes by p53 tumor suppressor is critical in cell fate determination. Here we report that the restoration of p53 function in human cancer cell lines that are deficient in p53 function upregulated the expression of Notch1. Interestingly, the expression of wild-type p53 in human prostate and breast cancer cell lines correlated well with increased expression of Notch1. Furthermore, knockdown of p53 expression in cancer cells that express wild-type p53 resulted in reduced expression of Notch1. Importantly, genotoxic stress to cancer cells that resulted in activation of p53 also upregulated the expression of Notch1. Moreover, p53mediated induction of Notch1 expression was associated with stimulation of the activity of Notch-responsive reporters. Notably, p53 differentially regulated the expression of Notch family members: expression of Notch2 and Notch4 was not induced by p53. Significantly, treatment of cells with gamma secretase inhibitor, an inhibitor of Notch signaling, increased susceptibility to apoptosis in response to genotoxic stress. Together, our observations suggest that p53mediated upregulation of Notch1 expression in human cancer cell lines contributes to cell fate determination after genotoxic stress.

  19. ZNF509S1 downregulates PUMA by inhibiting p53K382 acetylation and p53-DNA binding.

    Science.gov (United States)

    Jeon, Bu-Nam; Yoon, Jae-Hyeon; Han, Dohyun; Kim, Min-Kyeong; Kim, Youngsoo; Choi, Seo-Hyun; Song, Jiyang; Kim, Kyung-Sup; Kim, Kunhong; Hur, Man-Wook

    2017-09-01

    Expression of the POK family protein ZNF509L, and -its S1 isoform, is induced by p53 upon exposure to genotoxic stress. Due to alternative splicing of the ZNF509 primary transcript, ZNF509S1 lacks the 6 zinc-fingers and C-terminus of ZNF509L, resulting in only one zinc-finger. ZNF509L and -S1 inhibit cell proliferation by activating p21/CDKN1A and RB transcription, respectively. When cells are exposed to severe DNA damage, p53 activates PUMA (p53-upregulated modulator of apoptosis) transcription. Interestingly, apoptosis due to transcriptional activation of PUMA by p53 is attenuated by ZNF509S1. Thus we investigated the molecular mechanism(s) underlying the transcriptional attenuation and anti-apoptotic effects of ZNF509S1. We show that ZNF509S1 modulation of p53 activity is important in PUMA gene transcription by modulating post-translational modification of p53 by p300. ZNF509S1 directly interacts with p53 and inhibits p300-mediated acetylation of p53 lysine K382, with deacetylation of p53 K382 leading to decreased DNA binding at the p53 response element 1 of the PUMA promoter. ZNF509S1 may play a role not only in cell cycle arrest, by activating RB expression, but also in rescuing cells from apoptotic death by repressing PUMA expression in cells exposed to severe DNA damage. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. p53 immunohistochemical staining patterns in oral squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    G Dundy

    2016-09-01

    Full Text Available Background: Mutation of p53 gene is one of the most common events in oral carcinogenesis. Accumulation of p53 protein has also been detected in premalignant lesions.Materials and Methods:  This study included 40 biopsy samples, which were received in department of pathology, Sarojini Naidu Medical College, Agra, to ascertain p53 expression by immunohistochemically, in patients with oral squamous cell carcinomas and to correlate its expression with histological grade, different sites in oral cavity and tobacco intake/smoking habits.Results: Out of 40 biopsies of oral mucosa, 03 showed normal oral mucosa and 37 were diagnosed as squamous cell carcinoma (SCC, most patients were in 5th and 6th decade and majority (86.5% of oral SCC were males with buccal mucosa being the most common site. There was a statistically significant difference in p53 expression between oral SCC and normal oral mucosa (p value <0.05. Of total 37 cases, 12 cases were well differentiated type, 16 moderately differentiated and 09 of poorly differentiated type of SCC. In each category, about two thirds were positive for p53 staining. Out of total 37 cases of oral SCC, 64.9% were positive and 35.1% were negative for p53 expression, 34 cases had positive history of tobacco intake/smoking habits, of which 23 cases were positive while 11 cases were negative for p53 staining.Conclusion: Abnormal p53 protein was detected in 64.9% of oral squamous cell carcinoma, but not in normal oral mucosa. p53 expression was associated with malignant transformation of oral mucosa. 

  1. p53 mutation is common in microsatellite stable, BRAF mutant colorectal cancers.

    Science.gov (United States)

    Bond, Catherine E; Umapathy, Aarti; Ramsnes, Ingunn; Greco, Sonia A; Zhen Zhao, Zhen; Mallitt, Kylie-Ann; Buttenshaw, Ron L; Montgomery, Grant W; Leggett, Barbara A; Whitehall, Vicki L J

    2012-04-01

    The majority of "serrated pathway" colorectal cancers have mutation of the BRAF oncogene and display the CpG island methylator phenotype (CIMP). Half these cancers have microsatellite instability (MSI) and an excellent prognosis. In the absence of MSI (microsatellite stable, MSS), BRAF mutation has been associated with a particularly poor prognosis. "Traditional pathway" cancers are BRAF wild type. Mutation of p53 is common and this correlates with advanced stage. We therefore hypothesized that p53 mutation would be common in MSS/BRAF mutant colorectal cancer. One thousand and eighty-one colorectal cancers were screened for BRAF mutation to identify two BRAF mutant study groups (MSI: n = 77; MSS: n = 69) and a BRAF wild type control group (n = 101). These were screened for p53 mutation by high resolution melt analysis and classified for CIMP and MGMT methylation by quantitative methylation specific PCR. Molecular data were compared to patient age, gender, tumor location and stage. p53 was mutated significantly more frequently in MSS/BRAF mutant (28/69, 40.6%) compared to MSI/BRAF mutant cancers (13/77, 16.9%), but this mutation rate did not differ from MSS/BRAF wild type cancers (47/101, 46.5%)(p cancers (41/54, 75.9%), but was more common than in MSS/BRAF wild type cancers (3/85, 3.5%) (p cancers were more commonly proximal (38/54, 70.3%), but were similar to MSS/BRAF wild type cancers in terms of patient age, gender distribution and stage at presentation. MSS/BRAF mutant cancers share molecular and clinical features of both the serrated and traditional pathways of colorectal tumorigenesis. Copyright © 2011 UICC.

  2. Detection of genotoxic and non-genotoxic carcinogens in Xpc{sup −/−}p53{sup +/−} mice

    Energy Technology Data Exchange (ETDEWEB)

    Melis, Joost P.M. [Laboratory for Health Protection Research, National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Leiden University Medical Center, Department of Toxicogenetics, Leiden (Netherlands); Speksnijder, Ewoud N. [Leiden University Medical Center, Department of Toxicogenetics, Leiden (Netherlands); Kuiper, Raoul V. [Laboratory for Health Protection Research, National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Dutch Molecular Pathology Center, Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht (Netherlands); Salvatori, Daniela C.F. [Leiden University Medical Center, Central Animal Facility, Leiden (Netherlands); Schaap, Mirjam M. [Laboratory for Health Protection Research, National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Leiden University Medical Center, Department of Toxicogenetics, Leiden (Netherlands); Maas, Saskia [Leiden University Medical Center, Central Animal Facility, Leiden (Netherlands); Robinson, Joke; Verhoef, Aart; Benthem, Jan van; Luijten, Mirjam [Laboratory for Health Protection Research, National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Steeg, Harry van, E-mail: Harry.van.Steeg@rivm.nl [Laboratory for Health Protection Research, National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Leiden University Medical Center, Department of Toxicogenetics, Leiden (Netherlands)

    2013-01-15

    An accurate assessment of the carcinogenic potential of chemicals and pharmaceutical drugs is essential to protect humans and the environment. Therefore, substances are extensively tested before they are marketed to the public. Currently, the rodent two-year bioassay is still routinely used to assess the carcinogenic potential of substances. However, over time it has become clear that this assay yields false positive results and also has several economic and ethical drawbacks including the use of large numbers of animals, the long duration, and the high cost. The need for a suitable alternative assay is therefore high. Previously, we have proposed the Xpa*p53 mouse model as a very suitable alternative to the two-year bioassay. We now show that the Xpc*p53 mouse model preserves all the beneficial traits of the Xpa*p53 model for sub-chronic carcinogen identification and can identify both genotoxic and non-genotoxic carcinogens. Moreover, Xpc*p53 mice appear to be more responsive than Xpa*p53 mice towards several genotoxic and non-genotoxic carcinogens. Furthermore, Xpc*p53 mice are far less sensitive than Xpa*p53 mice for the toxic activity of DNA damaging agents and as such clearly respond in a similar way as wild type mice do. These advantageous traits of the Xpc*p53 model make it a better alternative for in vivo carcinogen testing than Xpa*p53. This pilot study suggests that Xpc*p53 mice are suited for routine sub-chronic testing of both genotoxic and non-genotoxic carcinogens and as such represent a suitable alternative to possibly replace the murine life time cancer bioassay. Highlights: ► The Xpc*p53 mouse model is able to identify genotoxic and non-genotoxic carcinogens. ► Time, animals and cost can be significantly reduced compared to the 2-year bioassay. ► Xpc*p53 mice are more advantageous for carcinogen identification than Xpa*p53 mice. ► Xpc*p53 mice exhibit a wild type response upon exposure to genotoxicants.

  3. Ensemble-based virtual screening reveals dual-inhibitors for the p53-MDM2/MDMX interactions.

    Science.gov (United States)

    Barakat, Khaled; Mane, Jonathan; Friesen, Douglas; Tuszynski, Jack

    2010-02-26

    The p53 protein, a guardian of the genome, is inactivated by mutations or deletions in approximately half of human tumors. While in the rest of human tumors, p53 is expressed in wild-type form, yet it is inhibited by over-expression of its cellular regulators MDM2 and MDMX proteins. Although the p53-binding sites within the MDMX and MDM2 proteins are closely related, known MDM2 small-molecule inhibitors have been shown experimentally not to bind to its homolog, MDMX. As a result, the activity of these inhibitors including Nutlin3 is compromised in tumor cells over-expressing MDMX, preventing these compounds from fully activating the p53 protein. Here, we applied the relaxed complex scheme (RCS) to allow for the full receptor flexibility in screening for dual-inhibitors that can mutually antagonize the two p53-regulator proteins. First, we filtered the NCI diversity set, DrugBank compounds and a derivative library for MDM2-inhibitors against 28 dominant MDM2-conformations. Then, we screened the MDM2 top hits against the binding site of p53 within the MDMX target. Results described herein identify a set of compounds that have been computationally predicted to ultimately activate the p53 pathway in tumor cells retaining the wild-type protein. Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.

  4. DGKζ ablation engenders upregulation of p53 level in the spleen upon whole-body ionizing radiation.

    Science.gov (United States)

    Tanaka, Toshiaki; Iseki, Ken; Tanaka, Ken; Nakano, Tomoyuki; Iino, Mitsuyoshi; Goto, Kaoru

    2017-09-28

    The tumor suppressor gene product p53, which coordinates the cellular response to various stresses, is subject to tight regulation by a complex network of signal transduction. The DGK family metabolizes lipidic second messenger diacylglycerol to produce phosphatidic acid. Our earlier studies showed that one isozyme, DGKζ, is involved in the regulatory mechanism of p53. In a cellular model of doxorubicin-induced DNA damage, overexpression of wild-type DGKζ suppresses p53 protein induction and reduces apoptosis, whereas knockdown of DGKζ upregulates p53 protein level and promotes apoptosis. Further examination reveals that DGKζ facilitates p53 degradation via ubiquitin-proteasome system in the cytoplasm. However, it remains undetermined whether the regulatory mechanism of DGKζ on p53 function found in cell-based experiments is also functional at the animal level. This study was conducted to elucidate this point using an experiment with DGKζ-KO mice under DNA damage induced by whole-body ionizing radiation. Our results reveal that p53 protein is induced robustly in the spleen of DGKζ-KO mice upon exposure to ionizing radiation, thereby promoting apoptosis in this organ. Taken together, the results demonstrate that DGKζ plays a sentinel role in p53 expression at the cellular and organismal levels after DNA damaging stress conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Epigenetic silencing of ASPP1 confers 5-FU resistance in clear cell renal cell carcinoma by preventing p53 activation.

    Science.gov (United States)

    Wang, Xingwen; Cheng, Yiwei; Zhu, YiFu; Li, Huayi; Ge, Wenjie; Wu, Xiaoliang; Zhao, Kunming; Yuan, Jinyang; Li, Zhenglin; Jiang, Shijian; Han, Zhengbin; Jiang, Qinghua; Wu, Qiong; Liu, Tao; Zhang, Cheng; Yu, Miao; Hu, Ying

    2017-10-01

    Inactivation of p53 has been shown to correlate with drug resistance in tumors. However, in clear cell renal cell carcinoma (ccRCC), p53 is rarely mutated, yet the tumors remain highly insensitive to the conventional chemotherapeutic drugs. The underlying mechanisms responsible for the non-genetic p53 inactivation remain obscure. Here, we report, for the first time, that Apoptosis Stimulating of P53 Protein 1 (ASPP1) was remarkably downregulated at both mRNA (about 3.9-fold) and protein (about 4.9-fold) levels in ccRCC human specimens in comparison with the paired normal controls. In addition, lower ASPP1 was closely related to the higher grade of tumors and shorter life expectancy of ccRCC patients, both with p p53 targets expression and p53 target PIG3 luciferase reporter activation. In contrast, ASPP1 knockdown promoted cell growth and prevent 5-FU-induced p53 activation and apoptosis. In conclusion, our results suggest that ASPP1 silencing is one of dominate mechanisms in inhibiting wild type p53 in ccRCC. ASPP1, therefore, may be potentially used as a promising biomarker for prognosis and therapeutic intervention in ccRCC. © 2017 UICC.

  6. The MDM2 Inhibitor AMG 232 Demonstrates Robust Antitumor Efficacy and Potentiates the Activity of p53-Inducing Cytotoxic Agents.

    Science.gov (United States)

    Canon, Jude; Osgood, Tao; Olson, Steven H; Saiki, Anne Y; Robertson, Rebecca; Yu, Dongyin; Eksterowicz, John; Ye, Qiuping; Jin, Lixia; Chen, Ada; Zhou, Jing; Cordover, David; Kaufman, Stephen; Kendall, Richard; Oliner, Jonathan D; Coxon, Angela; Radinsky, Robert

    2015-03-01

    p53 is a critical tumor suppressor and is the most frequently inactivated gene in human cancer. Inhibition of the interaction of p53 with its negative regulator MDM2 represents a promising clinical strategy to treat p53 wild-type tumors. AMG 232 is a potential best-in-class inhibitor of the MDM2-p53 interaction and is currently in clinical trials. We characterized the activity of AMG 232 and its effect on p53 signaling in several preclinical tumor models. AMG 232 binds the MDM2 protein with picomolar affinity and robustly induces p53 activity, leading to cell-cycle arrest and inhibition of tumor cell proliferation. AMG 232 treatment inhibited the in vivo growth of several tumor xenografts and led to complete and durable regression of MDM2-amplified SJSA-1 tumors via growth arrest and induction of apoptosis. Therapeutic combination studies of AMG 232 with chemotherapies that induce DNA damage and p53 activity resulted in significantly superior antitumor efficacy and regression, and markedly increased activation of p53 signaling in tumors. These preclinical data support the further evaluation of AMG 232 in clinical trials as both a monotherapy and in combination with standard-of-care cytotoxics. ©2015 American Association for Cancer Research.

  7. Tumor-promoting phorbol ester transiently down-modulates the p53 level and blocks the cell cycle

    DEFF Research Database (Denmark)

    Skouv, J.; Jensen, P O; Forchhammer, J

    1994-01-01

    Activation of the protein kinase C signaling pathway by tumor-promoting phorbol esters, such as 4 beta-phorbol 12-myristate 13-acetate (PMA), induced a decrease in the level of p53 mRNA in several serum-starved human cell lines. Also, the tumor-promoting phosphatase inhibitor okadaic acid induced...... a decrease in the p53 mRNA level in the cell lines. Normal diploid as well as various tumor cell lines were tested. Two tumor cell lines, HeLa and A549, both containing the wild-type p53 gene, but very different levels of p53 protein, were studied in detail. In both cell lines, the level of p53 m......RNA was minimal after 9 h of exposure to PMA. After approximately 120 h, the p53 mRNA level was similar to the pretreatment level. PMA induced a similar transient decrease in the level of p53 protein in the A549 cell line. The decrease in the p53 mRNA level could not be explained by changes in the transcriptional...

  8. POSTRANSLATIONAL MODIFICATIONS OF P53: UPSTREAM SIGNALING PATHWAYS.

    Energy Technology Data Exchange (ETDEWEB)

    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.

  9. Targeting p53-MDM2-MDMX Loop for Cancer Therapy

    OpenAIRE

    Zhang, Qi; Zeng, Shelya X.; Lu, Hua

    2014-01-01

    The tumor suppressor p53 plays a central role in anti-tumorigenesis and cancer therapy. It has been described as “the guardian of the genome”, because it is essential for conserving genomic stability by preventing mutation, and its mutation and inactivation are highly related to all human cancers. Two important p53 regulators, MDM2 and MDMX, inactivate p53 by directly inhibiting its transcriptional activity and mediating its ubiquitination in a feedback fashion, as their genes are also the tr...

  10. p53 and survival in early onset breast cancer

    DEFF Research Database (Denmark)

    Gentile, M; Bergman Jungeström, M; Olsen, K E

    1999-01-01

    The p53 protein has proven to be central in tumorigenesis by its cell cycle regulatory properties and both gene mutations and protein accumulation have been associated with poor prognosis in breast cancer. The present study was undertaken to investigate the prognostic significance of gene mutations......, p53 protein accumulation and of loss of heterozygosity (LOH) at the TP53 locus in young (age ... (46%). Log rank analysis revealed no significant association between survival and TP53 mutations (in general), p53 protein accumulation or LOH. However, missense mutations localised to the zinc binding domain were significantly (P = 0.0007) associated with poorer prognosis. As indicated...

  11. p53/HMGB1 Complexes Regulate Autophagy and Apoptosis

    OpenAIRE

    Livesey, Kristen M.; Kang, Rui; Vernon, Philip; Buchser, William; Loughran, Patricia; Watkins, Simon C.; Zhang, Lin; Manfredi, James J.; Zeh, Herbert J.; Li, Luyuan; Lotze, Michael T.; Tang, Daolin

    2012-01-01

    The balance between apoptosis (“programmed cell death”) and autophagy (“programmed cell survival”) is important in tumor development and response to therapy. Here we show that HMGB1 and p53 form a complex which regulates the balance between tumor cell death and survival. We demonstrate that knockout of p53 inHCT116 cells increases expression of cytosolic HMGB1 and induces autophagy. Conversely, knockout of HMGB1 in mouse embryonic fibroblasts increases p53 cytosolic localization and decreases...

  12. Robustness of the p53 network and biological hackers.

    Science.gov (United States)

    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.

  13. Preliminary report: the short-term effects of direct p53 DNA injection in primary hepatocellular carcinomas.

    Science.gov (United States)

    Habib, N A; Ding, S F; el-Masry, R; Mitry, R R; Honda, K; Michail, N E; Dalla Serra, G; Izzi, G; Greco, L; Bassyouni, M; el-Toukhy, M; Abdel-Gaffar, Y

    1996-01-01

    A pilot study, to assess the therapeutic potential of percutaneous injection of wild-type p53 (wt-p53) in five patients with primary hepatocellular carcinoma is reported. Three of the five patients showed objective tumor response with reduction of the tumor volume on computed tomographic (CT) scan measurements as well as a significant fall of serum alphafetoprotein. Much further work will be needed to elucidate the mechanism of action.

  14. Targeting p53 via JNK pathway: a novel role of RITA for apoptotic signaling in multiple myeloma.

    Directory of Open Access Journals (Sweden)

    Manujendra N Saha

    Full Text Available The low frequency of p53 alterations e.g., mutations/deletions (∼10% in multiple myeloma (MM makes this tumor type an ideal candidate for p53-targeted therapies. RITA is a small molecule which can induce apoptosis in tumor cells by activating the p53 pathway. We previously showed that RITA strongly activates p53 while selectively inhibiting growth of MM cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy in MM. However, the molecular mechanisms underlying the pro-apoptotic effect of RITA are largely undefined. Gene expression analysis by microarray identified a significant number of differentially expressed genes associated with stress response including c-Jun N-terminal kinase (JNK signaling pathway. By Western blot analysis we further confirmed that RITA induced activation of p53 in conjunction with up-regulation of phosphorylated ASK-1, MKK-4 and c-Jun. These results suggest that RITA induced the activation of JNK signaling. Chromatin immunoprecipitation (ChIP analysis showed that activated c-Jun binds to the activator protein-1 (AP-1 binding site of the p53 promoter region. Disruption of the JNK signal pathway by small interfering RNA (siRNA against JNK or JNK specific inhibitor, SP-600125 inhibited the activation of p53 and attenuated apoptosis induced by RITA in myeloma cells carrying wild type p53. On the other hand, p53 transcriptional inhibitor, PFT-α or p53 siRNA not only inhibited the activation of p53 transcriptional targets but also blocked the activation of c-Jun suggesting the presence of a positive feedback loop between p53 and JNK. In addition, RITA in combination with dexamethasone, known as a JNK activator, displays synergistic cytotoxic responses in MM cell lines and patient samples. Our study unveils a previously undescribed mechanism of RITA-induced p53-mediated apoptosis through JNK signaling pathway and provides the rationale for combination of p53 activating drugs with

  15. Critical roles of DMP1 in HER2/neu-Arf-p53 signaling and breast cancer development

    Science.gov (United States)

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

    2010-01-01

    HER2 overexpression stimulates cell growth in p53-mutated cells while it inhibits cell proliferation in those with wild-type p53, but the molecular mechanism is unknown. The Dmp1 promoter was activated by HER2/neu through the PI3K-Akt-NF-κB pathway, which in turn stimulated Arf transcription. Binding of p65 and p52 subunits of NF-κB was demonstrated to the Dmp1 promoter and that of Dmp1 to the Arf promoter upon HER2/neu overexpression. Both Dmp1 and p53 were induced in pre-malignant lesions from MMTV-neu mice and mammary tumorigenesis was significantly accelerated in both Dmp1+/− and Dmp1−/− mice. Selective deletion of Dmp1 and/or overexpression of Tbx2/Pokemon was found in >50 % of wild-type HER2/neu carcinomas while the involvement of Arf, Mdm2, or p53 was rare. Tumors from Dmp1+/−, Dmp1−/−, and wild-type neu mice with hemizygous Dmp1 deletion showed significant downregulation of Arf and p21Cip1/WAF1, showing p53 inactivity and more aggressive phenotypes than tumors without Dmp1 deletion. Notably, endogenous hDMP1 mRNA decreased when HER2 was depleted in human breast cancer cells. Our study demonstrates the pivotal roles of Dmp1 in HER2/neu-p53 signaling and breast carcinogenesis. PMID:21062982

  16. A dynamic P53-MDM2 model with time delay

    Energy Technology Data Exchange (ETDEWEB)

    Mihalas, Gh.I. [Department of Biophysics and Medical Informatics, University of Medicine and Pharmacy, Piata Eftimie Murgu, nr. 3, 300041 Timisoara (Romania)]. E-mail: mihalas@medinfo.umft.ro; Neamtu, M. [Department of Forecasting, Economic Analysis, Mathematics and Statistics, West University of Timisoara, Str. Pestalozzi, nr. 14A, 300115 Timisoara (Romania)]. E-mail: mihaela.neamtu@fse.uvt.ro; Opris, D. [Department of Applied Mathematics, West University of Timisoara, Bd. V. Parvan, nr. 4, 300223 Timisoara (Romania)]. E-mail: opris@math.uvt.ro; Horhat, R.F. [Department of Biophysics and Medical Informatics, University of Medicine and Pharmacy, Piata Eftimie Murgu, nr. 3, 300041 Timisoara (Romania)]. E-mail: rhorhat@yahoo.com

    2006-11-15

    Specific activator and repressor transcription factors which bind to specific regulator DNA sequences, play an important role in gene activity control. Interactions between genes coding such transcription factors should explain the different stable or sometimes oscillatory gene activities characteristic for different tissues. Starting with the model P53-MDM2 described into [Mihalas GI, Simon Z, Balea G, Popa E. Possible oscillatory behaviour in P53-MDM2 interaction computer simulation. J Biol Syst 2000;8(1):21-9] and the process described into [Kohn KW, Pommier Y. Molecular interaction map of P53 and MDM2 logic elements, which control the off-on switch of P53 in response to DNA damage. Biochem Biophys Res Commun 2005;331:816-27] we enveloped a new model of this interaction. Choosing the delay as a bifurcation parameter we study the direction and stability of the bifurcating periodic solutions. Some numerical examples are finally given for justifying the theoretical results.

  17. p53 Abnormalities and Potential Therapeutic Targeting in Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    P. J. Teoh

    2014-01-01

    Full Text Available p53 abnormalities are regarded as an independent prognostic marker in multiple myeloma. Patients harbouring this genetic anomaly are commonly resistant to standard therapy. Thus, various p53 reactivating agents have been developed in order to restore its tumour suppressive abilities. Small molecular compounds, especially, have gained popularity in its efficacy against myeloma cells. For instance, promising preclinical results have steered both nutlin-3 and PRIMA-1 into phase I/II clinical trials. This review summarizes different modes of p53 inactivation in myeloma and highlights the current p53-based therapies that are being utilized in the clinic. Finally, we discuss the potential and promise that the novel small molecules possess for clinical application in improving the treatment outcome of myeloma.

  18. p53 and disease: when the guardian angel fails.

    Science.gov (United States)

    Royds, J A; Iacopetta, B

    2006-06-01

    The p53 tumor suppressor gene (TP53) is mutated more often in human cancers than any other gene yet reported. Of importance, it is mutated frequently in the common human malignancies of the breast and colorectum and also, but less frequently, in other significant human cancers such as glioblastomas. There is also one inherited cancer predisposing syndrome called Li-Fraumeni that is caused by TP53 mutations. In this review, we discuss the significance of p53 mutations in some of the above tumors with a view to outlining how p53 contributes to malignant progression. We also discuss the usefulness of TP53 status as a prognostic marker and its role as a predictor of response to therapy. Finally, we outline some evidence that abnormalities in p53 function contribute to the etiology of other non-neoplastic diseases.

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

    Science.gov (United States)

    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.

  20. Regulation of Mammary Progenitor Cells by p53 and Parity

    Science.gov (United States)

    2011-01-01

    pregnancy early in reproductive life can reduce breast cancer incidence in women by up to 50% 4. The research in our lab has shown that the p53 tum or...13. SUPPLEMENTARY NOTES 14. ABSTRACT Breast cancer is the most common tumor among women with inherited mutations in the p53 gene (Li-Fraumeni...4 Introduction Breast cancer is the m ost frequent cancer am ong women in the United S tates1. Understanding the biological

  1. Super p53 for Treatment of Ovarian Cancer

    Science.gov (United States)

    2017-09-01

    ABSTRACT In this final report, we show gene therapy using re-engineered super p53 (p53-CC constructs) kills some ovarian cancer cell lines in vitro ...a lead construct. Technical skills gained in the proposal include cell culture , transfections, microscopy, apoptosis assays, transcriptional assays...able to create ovarian tumors in mice. Our polymer-adenovirus constructs were optimized in vitro and in vivo, and did not show gross signs of toxicity

  2. Super p53 for Treatment of Ovarian Cancer

    Science.gov (United States)

    2016-07-01

    as demonstrated by fluorescence microscopy. Preliminary studies indicate that p53-CC causes robust apoptosis in Kuramochi and ID8 cells as well...measured using the 7AAD assay (late stage apoptosis ). IC50 values for taxol have been determined in ID8 (and SKOV3) cells. In ID8 cells (which will...be used to implant into mice for the syngeneic animal study), p53-CCmut causes the highest levels of apoptosis regardless of whether taxol is added

  3. Blocking of p53-Snail Binding, Promoted by Oncogenic K-Ras, Recovers p53 Expression and function

    OpenAIRE

    Lee, Sun-Hye; Lee, Su-Jin; Jung, Yeon Sang; Xu, Yongbin; Kang, Ho Sung; Ha, Nam-Chul; Park, Bum-Joon

    2009-01-01

    Differentially from other kinds of Ras, oncogenic K-Ras, which is mutated approximately 30% of human cancer, does not induce apoptosis and senescence. Here, we provide the evidence that oncogenic K-Ras abrogates p53 function and expression through induction of Ataxia telangiectasia-mutated and Rad3-related mediated Snail stabilization. Snail directly binds to DNA binding domain of p53 and diminishes the tumor-suppressive function of p53. Thus, elimination of Snail through si-RNA can induce p5...

  4. Expression of p53 protein in pituitary adenomas

    Directory of Open Access Journals (Sweden)

    Oliveira M.C.

    2002-01-01

    Full Text Available Inactivating mutations of TP53, a tumor suppressor gene, are associated with abnormal cell proliferation. Although p53 expression is common in many human malignancies, p53 protein has seldom been evaluated in pituitary tumors. When detected, the percentage of p53-positive cells is low, and, in general, it is exclusive for invasive lesions. The aim of the present study was to use immunohistochemistry to determine the presence of p53 protein in pituitary adenomas from tumor samples of 163 surgeries performed in 148 patients (40% male, 60% female. In 35% of the cases the adenoma was nonfunctional, while in the others it was associated with PRL, GH and/or ACTH endocrine hypersecretion syndrome. Macroadenomas were observed in 83.2% of the cases with available neuroimage evaluation, of which 28% invaded the cavernous, sphenoid and/or ethmoidal sinus, bone, third ventricle or subfrontal lobe. p53 protein was detected in 2/148 patients (1.3%. Immunohistochemistry was positive for PRL and GH in these cases. Due to the high percentage of invasive pituitary adenomas found in our study, the low frequency of p53 detection suggests that it is inadequate as a routine marker for aggressiveness and as a predictive factor of tumor behavior.

  5. Stimulus-Specific Transcriptional Regulation Within the p53 Network

    Science.gov (United States)

    Donner, Aaron Joseph; Hoover, Jennifer Michelle; Szostek, Stephanie Aspen; Espinosa, Joaquín Maximiliano

    2010-01-01

    The p53 transcriptional network is composed of hundreds of effector genes involved in varied stress-response pathways, including cell cycle arrest and apoptosis. It is not clear how distinct p53 target genes are differentially activated to trigger stress-specific biological responses. We analyzed the p53 transcriptional program upon activation by two DNA-damaging agents, UVC and doxorubicin, versus the non-genotoxic molecule Nutlin-3. In colorectal cancer cells, UVC triggers apoptosis, doxorubicin induces transient cell cycle arrest followed by apoptosis, and Nutlin-3 leads to cell cycle arrest with no significant apoptosis. Quantitative gene expression analysis allowed us to group p53 target genes into three main classes according to their activation profiles in each scenario. The CDK-inhibitor p21 was classified as a Class I gene, being significantly activated under cell cycle arrest conditions (i.e., doxorubicin and Nutlin-3) but not during UVC-induced apoptosis. Chromatin immunoprecipitation analysis of the p21 locus indicates that the level of p53-dependent transcription is determined by the effects of stimulus-specific transcriptional coregulators acting downstream of p53 binding and histone acetylation. In particular, our analysis indicates that the subunits of the CDK-module of the human Mediator complex function as stimulus-specific positive coregulators of p21 transcription. PMID:17957141

  6. Characterization of p53 expression in rainbow trout.

    Science.gov (United States)

    Liu, Michelle; Tee, Catherine; Zeng, Fanxing; Sherry, James P; Dixon, Brian; Bols, Niels C; Duncker, Bernard P

    2011-11-01

    The tumour suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. Given the high incidence of p53 mutations in human cancers, it has been extensively studied, though only a small fraction of these investigations have been in non-mammalian systems. For the present study, an anti-rainbow trout p53 polyclonal antibody was generated. A variety of rainbow trout (Oncorhynchus mykiss) tissues and cell lines were examined through western blot analysis of cellular protein extracts, which revealed relatively high p53 levels in brain and gills. To evaluate the checkpoint response of rainbow trout p53, RTbrain-W1 and RTgill-W1 cell lines were exposed to varying concentrations of the DNA damaging agent bleomycin and ribonucleotide reductase inhibitor hydroxyurea. In contrast to mammals, these checkpoint-inducing agents provoked no apparent increase in rainbow trout p53 levels. These results infer the presence of alternate DNA damage checkpoint mechanisms in rainbow trout cells. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. A High-Throughput Cell-Based Screen Identified a 2-[(E)-2-Phenylvinyl]-8-Quinolinol Core Structure That Activates p53.

    Science.gov (United States)

    Bechill, John; Zhong, Rong; Zhang, Chen; Solomaha, Elena; Spiotto, Michael T

    2016-01-01

    p53 function is frequently inhibited in cancer either through mutations or by increased degradation via MDM2 and/or E6AP E3-ubiquitin ligases. Most agents that restore p53 expression act by binding MDM2 or E6AP to prevent p53 degradation. However, fewer compounds directly bind to and activate p53. Here, we identified compounds that shared a core structure that bound p53, caused nuclear localization of p53 and caused cell death. To identify these compounds, we developed a novel cell-based screen to redirect p53 degradation to the Skip-Cullin-F-box (SCF) ubiquitin ligase complex in cells expressing high levels of p53. In a multiplexed assay, we coupled p53 targeted degradation with Rb1 targeted degradation in order to identify compounds that prevented p53 degradation while not inhibiting degradation through the SCF complex or other proteolytic machinery. High-throughput screening identified several leads that shared a common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that stabilized p53. Surface plasmon resonance analysis indicated that these compounds bound p53 with a KD of 200 ± 52 nM. Furthermore, these compounds increased p53 nuclear localization and transcription of the p53 target genes PUMA, BAX, p21 and FAS in cancer cells. Although p53-null cells had a 2.5±0.5-fold greater viability compared to p53 wild type cells after treatment with core compounds, loss of p53 did not completely rescue cell viability suggesting that compounds may target both p53-dependent and p53-independent pathways to inhibit cell proliferation. Thus, we present a novel, cell-based high-throughput screen to identify a 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that bound to p53 and increased p53 activity in cancer cells. These compounds may serve as anti-neoplastic agents in part by targeting p53 as well as other potential pathways.

  8. Giant cell glioblastoma is associated with altered aurora b expression and concomitant p53 mutation.

    Science.gov (United States)

    Temme, Achim; Geiger, Kathrin D; Wiedemuth, Ralf; Conseur, Katharina; Pietsch, Torsten; Felsberg, Jörg; Reifenberger, Guido; Tatsuka, Masaaki; Hagel, Christian; Westphal, Manfred; Berger, Hilmar; Simon, Matthias; Weller, Michael; Schackert, Gabriele

    2010-06-01

    Giant cell glioblastoma (gcGB), a subtype of GB, is characterized by the presence of numerous multinucleated giant cells. The prognosis for gcGB is poor, but it may have a better clinical outcome compared with classic GB. The molecular alterations that lead to the multinucleated cell phenotype of gcGB have not been elucidated. Giant cell GB has a higher frequency of the tumor suppressor protein p53 mutations than GB, however, and a role for the mitotic Aurora B kinase has been suggested. We analyzed Aurora B expression in gcGB (n = 28) and GB (n = 54) patient tumor samples by immunohistochemistry; 17 gcGB and 22 GB samples were analyzed at the DNA and mRNA levels. No mutations in the Aurora B gene (AURKB) were found, but its mRNA and protein levels were significantly higher in gcGB than in GB. Fifty-nine percent of gcGB samples but only 18% of the GB samples showed p53 mutations. Ectopic overexpression of Aurora B induced a significant increase inthe proportion of multinucleated cells in p53 mutant U373-MG, but not in p53 wild-type U87-MG, glioma cells. RNAi of p53 in U87-MG cells led to an increase in the fraction of multinucleated cells that was further augmented by ectopic overexpression of Aurora B. These results suggest that loss of p53 function and dysregulated Aurora B protein levels might represent factors that drive the development of multinucleated cells in gcGB.

  9. Characterisation of the p53-mediated cellular responses evoked in primary mouse cells following exposure to ultraviolet radiation.

    Directory of Open Access Journals (Sweden)

    Gillian D McFeat

    Full Text Available Exposure to ultraviolet (UV light can cause significant damage to mammalian cells and, although the spectrum of damage produced varies with the wavelength of UV, all parts of the UV spectrum are recognised as being detrimental to human health. Characterising the cellular response to different wavelengths of UV therefore remains an important aim so that risks and their moderation can be evaluated, in particular in relation to the initiation of skin cancer. The p53 tumour suppressor protein is central to the cellular response that protects the genome from damage by external agents such as UV, thus reducing the risk of tumorigenesis. In response to a variety of DNA damaging agents including UV light, wild-type p53 plays a role in mediating cell-cycle arrest, facilitating apoptosis and stimulating repair processes, all of which prevent the propagation of potentially mutagenic defects. In this study we examined the induction of p53 protein and its influence on the survival of primary mouse fibroblasts exposed to different wavelengths of UV light. UVC was found to elevate p53 protein and its sequence specific DNA binding capacity. Unexpectedly, UVA treatment failed to induce p53 protein accumulation or sequence specific DNA binding. Despite this, UVA exposure of wild-type cells induced a p53 dependent G1 cell cycle arrest followed by a wave of p53 dependent apoptosis, peaking 12 hours post-insult. Thus, it is demonstrated that the elements of the p53 cellular response evoked by exposure to UV radiation are wavelength dependent. Furthermore, the interrelationship between various endpoints is complex and not easily predictable. This has important implications not only for understanding the mode of action of p53 but also for the use of molecular endpoints in quantifying exposure to different wavelengths of UV in the context of human health protection.

  10. p16 and p53 in HPV-positive versus HPV-negative oral squamous cell carcinoma: do pathways differ?

    Science.gov (United States)

    Singh, Vineeta; Husain, Nuzhat; Akhtar, Naseem; Khan, Mohammad Yahia; Sonkar, Abhinav A; Kumar, Vijay

    2017-10-01

    p16 overexpression and wild-type p53 expression are associated with human papilloma virus (HPV) in cervical and oropharyngeal cancer. Role of HPV-related carcinogenesis in the etiology of oral squamous cell carcinoma (OSCC) is still vague in Indian population. We aimed to explore the expression pattern of p16 and p53 in HPV-positive and HPV-negative OSCC to elicit differences, if any. Further their effect on survival of patients was studied. Thirty-one consecutive HPV-positive as well as 31 age and sex-matched HPV-negative OSCC cases from a case series of 369 histologically diagnosed cases of OSCC were included in this study. HPV was detected by two methods, viz. real-time PCR and conventional PCR in biopsy samples. p16 and p53 protein expression was assessed by immunohistochemistry, and p16 mRNA expression was quantified with real-time PCR using SYBR Green assay. p16 was expressed in six (19.4%) HPV-positive and in four (12.9%) HPV-negative cases. Overall mutant-type p53 expression in 62 OSCC cases was 54.8%. Out of ten p16-positive cases, eight expressed mutant-type p53 and only two cases expressed wild-type p53. Risk factors including oral tobacco consumption and alcohol were present in all these ten p16-positive cases. Survival of patients was not affected by HPV, p16 and p53 status. Presence of mutant-type p53 and exposure to tobacco-related risk factors in both HPV-positive and negative cases suggest existence of p53-related carcinogenesis in HPV-positive cases in Indian population. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. The role of p53-microRNA 200-Moesin axis in invasion and drug resistance of breast cancer cells.

    Science.gov (United States)

    Alam, Farheen; Mezhal, Fatima; El Hasasna, Hussain; Nair, Vidhya A; Aravind, S R; Saber Ayad, Maha; El-Serafi, Ahmed; Abdel-Rahman, Wael M

    2017-09-01

    This study aimed to analyze the expression of microRNAs in relation to p53 status in breast cancer cells and to delineate the role of Moesin in this axis. We used three isogenic breast carcinoma cell lines MCF7 (with wild-type p53), 1001 (MCF7 with mutated p53), and MCF7-E6 (MCF7 in which p53 function was disrupted). MicroRNA expression was analyzed using microarray analysis and confirmed by real-time polymerase chain reaction. The 1001 clone with mutant p53 showed 22 upregulated and 25 downregulated microRNAs. The predicted targets of these 47 microRNAs were >700 human genes belonging to interesting functional groups such as stem cell development and maintenance. The most significantly downregulated microRNAs in the p53-mutant cell line were from the miR-200 family. We focused on miR-200c which targets many transcripts involved in epithelial-to-mesenchymal transition including Moesin. We found that Moesin was expressed in 1001 but not in its p53 wild-type parental MCF7 consistent with the observed mesenchymal features in the 1001, such as vimentin positivity, E-cadherin negativity, and ZEB1 positivity in addition to the morphological changes. After Moesin silencing, the p53-mutant cells 1001 reverted from mesenchymal-to-epithelial phenotype and showed subtle reduction in migration and invasion and loss of ZEB1 and SNAIL expression. Interestingly, Moesin silencing restored the 1001 sensitivity to Doxorubicin. These results indicate that loss of miR-200c, as a consequence of p53 mutation, can upregulate Moesin oncogene and thus promote carcinogenesis. Moesin may play a role in metastasis and drug resistance of breast cancer.

  12. The rare nonsense mutation in p53 triggers alternative splicing to produce a protein capable of inducing apoptosis.

    Directory of Open Access Journals (Sweden)

    Evgeny M Makarov

    Full Text Available P53 protein is more frequently mutated in human tumours compared with the other proteins. While the majority of the p53 mutations, especially within its DNA-binding domain, lead to the loss of the wild-type function, there are accumulating data demonstrating that the p53 mutants gain tumour promoting activities; the latter triggers a revitalised interest in functional analysis of the p53 mutants. A systematic screening for p53 mutations in surgical materials from patients with glioma revealed a 378C>G mutation that creates a stop codon at the position of amino acid residue 126. The mutation eliminates the recognition site for the restriction endonuclease Sca I that allowed us to carry out RFLP analysis of DNA extracted from the clinical samples and suggests that this mutation is more frequent than is documented in the p53 databases. Both the ECV-304 and EJ cell lines, that probably originate from the bladder carcinoma T24 cell line, were confirmed to contain the homozygous 378C>G mutation but were shown to produce the p53 protein of expected full-length size detected by Western blotting. We provide evidence that the 378C>G mutation generates an alternative 3' splice site (ss which is more often used instead of the authentic upstream 3' ss, driving the production of mRNA encoding the protein with the single amino acid deletion (p53ΔY126. Using endogenous expression, we demonstrated that the p53ΔY126 protein is nearly as active as the wild type protein in inducing the p21/Waf1 expression and apoptosis.

  13. BAK overexpression mediates p53-independent apoptosis inducing effects on human gastric cancer cells

    Directory of Open Access Journals (Sweden)

    Liu Jun

    2004-07-01

    Full Text Available Abstract Background BAK (Bcl-2 homologous antagonist/killer is a novel pro-apoptotic gene of the Bcl-2 family. It has been reported that gastric tumors have reduced BAK levels when compared with the normal mucosa. Moreover, mutations of the BAK gene have been identified in human gastrointestinal cancers, suggesting that a perturbation of BAK-mediated apoptosis may contribute to the pathogenesis of gastric cancer. In this study, we explored the therapeutic effects of gene transfer mediated elevations in BAK expression on human gastric cancer cells in vitro. Methods Eukaryotic expression vector for the BAK gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53 and MKN-28 (mutant-type p53. RT-PCR and Western Blotting detected cellular BAK gene expression. Cell growth activities were detected by MTT colorimetry and flow cytometry, while apoptosis was assayed by electronic microscopy and TUNEL. Western Blotting and colorimetry investigated cellular caspase-3 activities. Results BAK gene transfer could result in significant BAK overexpression, decreased in vitro growth, cell cycle G0/G1 arrest, and induced apoptosis in gastric cancer cells. In transferred cells, inactive caspase-3 precursor was cleaved into the active subunits p20 and p17, during BAK overexpression-induced apoptosis. In addition, this process occurred equally well in p53 wild-type (MKN-45, or in p53 mutant-type (MKN-28 gastric cancer cells. Conclusions The data presented suggests that overexpression of the BAK gene can lead to apoptosis of gastric cancer cells in vitro, which does not appear to be dependent on p53 status. The action mechanism of BAK mediated apoptosis correlates with activation of caspase-3. This could be served as a potential strategy for further development of gastric cancer therapies.

  14. Targeting polo-like kinase 1, a regulator of p53, in the treatment of adrenocortical carcinoma.

    Science.gov (United States)

    Bussey, Kimberly J; Bapat, Aditi; Linnehan, Claire; Wandoloski, Melissa; Dastrup, Erica; Rogers, Erik; Gonzales, Paul; Demeure, Michael J

    2016-03-01

    Adrenocortical carcinoma (ACC) is an aggressive cancer with a 5 year survival rate of 20-30 %. Various factors have been implicated in the pathogenesis of ACC including dysregulation of the G2/M transition and aberrant activity of p53 and MDM2. Polo-like kinase 1 (PLK-1) negatively modulates p53 functioning, promotes MDM2 activity through its phosphorylation, and is involved in the G2/M transition. Gene expression profiling of 44 ACC samples showed that increased expression of PLK-1 in 29 % of ACC. Consequently, we examined PLK-1's role in the modulation of the p53 signaling pathway in adrenocortical cancer. We used siRNA knock down PLK-1 and pharmacological inhibition of PLK-1 and MDM2 ACC cell lines SW-13 and H295R. We examined viability, protein expression, p53 transactivation, and induction of apoptosis. Knocking down expression of PLK-1 with siRNA or inhibition of PLK-1 by a small molecule inhibitor, BI-2536, resulted in a loss of viability of up to 70 % in the ACC cell lines H295R and SW-13. In xenograft models, BI-2536 demonstrated marked inhibition of growth of SW-13 with less inhibition of H295R. BI-2536 treatment resulted in a decrease in mutant p53 protein in SW-13 cells but had no effect on wild-type p53 protein levels in H295R cells. Additionally, inhibition of PLK-1 restored wild-type p53's transactivation and apoptotic functions in H295R cells, while these functions of mutant p53 were restored only to a smaller extent. Furthermore, inhibition of MDM2 with nutlin-3 reduced the viability of both the ACC cells and also reactivated wild-type p53's apoptotic function. Inhibition of PLK-1 sensitized the ACC cell lines to MDM2 inhibition and this dual inhibition resulted in an additive apoptotic response in H295R cells with wild-type p53. These preclinical studies suggest that targeting p53 through PLK-1 is an attractive chemotherapy strategy warranting further investigation in adrenocortical cancer.

  15. Oncolytic E1B 55KDa-deleted adenovirus replication is independent of p53 levels in cancer cells.

    Science.gov (United States)

    Abbas, B M; El-Mogy, M A; Haj-Ahmad, Y

    2017-08-15

    Oncolytic adenoviruses represent a new approach for cancer therapy due to its tumor specificity. E1B 55kDa-deleted adenovirus type 5 (Ad5dlE1B 55kDa) is a promising therapeutic agent that can selectively replicate in and lyse p53 defective cancer cells. However, the overall efficacy has shown varying degrees of success with raised doubts about the correlation between p53 status and E1B-deleted adenovirus replication ability. In this study, we investigated the relationship between the efficiency of Ad5dlE1B 55kDa replication and p53 levels in cancer cells. Five transient p53 expression vectors were engineered to expresses different p53 levels in transfected cells. Then, the effect of the variable p53 levels and cellular backgrounds on the replication efficiency of oncolytic Ad5dlE1B 55kDa was evaluated in H1299 and HeLa cell lines. We found that the replication efficiency of these oncolytic viruses is dependent on the status, but not the expression levels, of p53. Ad5dlE1B 55kDa was shown to have selective replication activity in H1299 cells (p53-null) and decreased viral replication in HeLa cells (p53-positive), relative to the wild-type adenovirus in both cell lines. Our findings suggest that there is a relation between the E1B-deleted adenovirus replication and the presence as well as the activity of p53, independent of its quantity.

  16. p53-competent cells and p53-deficient cells display different susceptibility to oxygen functionalized graphene cytotoxicity and genotoxicity.

    Science.gov (United States)

    Petibone, Dayton M; Mustafa, Thikra; Bourdo, Shawn E; Lafont, Andersen; Ding, Wei; Karmakar, Alokita; Nima, Zeid A; Watanabe, Fumiya; Casciano, Daniel; Morris, Suzanne M; Dobrovolsky, Vasily N; Biris, Alexandru S

    2017-11-01

    Due to the distinctive physical, electrical, and chemical properties of graphene nanomaterials, numerous efforts pursuing graphene-based biomedical and industrial applications are underway. Oxidation of pristine graphene surfaces mitigates its otherwise hydrophobic characteristic thereby improving its biocompatibility and functionality. Yet, the potential widespread use of oxidized graphene derivatives raises concern about adverse impacts on human health. The p53 tumor suppressor protein maintains cellular and genetic stability after toxic exposures. Here, we show that p53 functional status correlates with oxygen functionalized graphene (f-G) cytotoxicity and genotoxicity in vitro. The f-G exposed p53-competent cells, but not p53-deficient cells, initiated G0 /G1 phase cell cycle arrest, suppressed reactive oxygen species, and entered apoptosis. There was p53-dependent f-G genotoxicity evident as increased structural chromosome damage, but not increased gene mutation or chromatin loss. In conclusion, the cytotoxic and genotoxic potential for f-G in exposed cells was dependent on the p53 functional status. These findings have broad implications for the safe and effective implementation of oxidized graphene derivatives into biomedical and industrial applications. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

  17. Regulation of miR-146a by RelA/NFkB and p53 in STHdh(Q111/Hdh(Q111 cells, a cell model of Huntington's disease.

    Directory of Open Access Journals (Sweden)

    Jayeeta Ghose

    Full Text Available Huntington's disease (HD is caused by the expansion of N-terminal polymorphic poly Q stretch of the protein huntingtin (HTT. Deregulated microRNAs and loss of function of transcription factors recruited to mutant HTT aggregates could cause characteristic transcriptional deregulation associated with HD. We observed earlier that expressions of miR-125b, miR-146a and miR-150 are decreased in STHdh(Q111/Hdh(Q111 cells, a model for HD in comparison to those of wild type STHdh(Q7/Hdh(Q7 cells. In the present manuscript, we show by luciferase reporter assays and real time PCR that decreased miR-146a expression in STHdh(Q111/Hdh(Q111 cells is due to decreased expression and activity of p65 subunit of NFkB (RelA/NFkB. By reporter luciferase assay, RT-PCR and western blot analysis, we also show that both miR-150 and miR-125b target p53. This partially explains the up regulation of p53 observed in HD. Elevated p53 interacts with RelA/NFkB, reduces its expression and activity and decreases the expression of miR-146a, while knocking down p53 increases RelA/NFkB and miR-146a expressions. We also demonstrate that expression of p53 is increased and levels of RelA/NFkB, miR-146a, miR-150 and miR-125b are decreased in striatum of R6/2 mice, a mouse model of HD and in cell models of HD. In a cell model, this effect could be reversed by exogenous expression of chaperone like proteins HYPK and Hsp70. We conclude that (i miR-125b and miR-150 target p53, which in turn regulates RelA/NFkB and miR-146a expressions; (ii reduced miR-125b and miR-150 expressions, increased p53 level and decreased RelA/NFkB and miR-146a expressions originate from mutant HTT (iii p53 directly or indirectly regulates the expression of miR-146a. Our observation of interplay between transcription factors and miRNAs using HD cell model provides an important platform upon which further work is to be done to establish if such regulation plays any role in HD pathogenesis.

  18. AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis.

    Science.gov (United States)

    Höpker, Katja; Hagmann, Henning; Khurshid, Safiya; Chen, Shuhua; Hasskamp, Pia; Seeger-Nukpezah, Tamina; Schilberg, Katharina; Heukamp, Lukas; Lamkemeyer, Tobias; Sos, Martin L; Thomas, Roman K; Lowery, Drew; Roels, Frederik; Fischer, Matthias; Liebau, Max C; Resch, Ulrike; Kisner, Tülay; Röther, Fabian; Bartram, Malte P; Müller, Roman Ulrich; Fabretti, Francesca; Kurschat, Peter; Schumacher, Björn; Gaestel, Matthias; Medema, René H; Yaffe, Michael B; Schermer, Bernhard; Reinhardt, H Christian; Benzing, Thomas

    2012-10-17

    Following genotoxic stress, cells activate a complex signalling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumour suppressor p53 lies at the heart of this DNA damage response. However, it remains incompletely understood, which signalling molecules dictate the choice between these different cellular outcomes. Here, we identify the transcriptional regulator apoptosis-antagonizing transcription factor (AATF)/Che-1 as a critical regulator of the cellular outcome of the p53 response. Upon genotoxic stress, AATF is phosphorylated by the checkpoint kinase MK2. Phosphorylation results in the release of AATF from cytoplasmic MRLC3 and subsequent nuclear translocation where AATF binds to the PUMA, BAX and BAK promoter regions to repress p53-driven expression of these pro-apoptotic genes. In xenograft experiments, mice exhibit a dramatically enhanced response of AATF-depleted tumours following genotoxic chemotherapy with adriamycin. The exogenous expression of a phospho-mimicking AATF point mutant results in marked adriamycin resistance in vivo. Nuclear AATF enrichment appears to be selected for in p53-proficient endometrial cancers. Furthermore, focal copy number gains at the AATF locus in neuroblastoma, which is known to be almost exclusively p53-proficient, correlate with an adverse prognosis and reduced overall survival. These data identify the p38/MK2/AATF signalling module as a critical repressor of p53-driven apoptosis and commend this pathway as a target for DNA damage-sensitizing therapeutic regimens.

  19. Alterations in p53-specific T cells and other lymphocyte subsets in breast cancer patients during vaccination with p53-peptide loaded dendritic cells and low-dose interleukin-2

    DEFF Research Database (Denmark)

    Svane, Inge Marie; Pedersen, Anders E; Nikolajsen, Kirsten

    2008-01-01

    We have previously established a cancer vaccine using autologous DCs, generated by in vitro stimulation with IL-4 and GM-CSF, and pulsed with six HLA-A*0201 binding wild-type p53 derived peptides. This vaccine was used in combination with low-dose interleukin-2 in a recently published clinical...... Phase II trial where 26 HLA-A2+ patients with progressive late-stage metastatic breast cancer (BC) were included. Almost 1/3rd of the patients obtained stable disease or minor regression during treatment with a positive correlation to tumour over-expression of p53. In the present study, we performed...... a comprehensive analysis of the effector stage of the p53-specific CD8+ T cells by the use of Dextramer Technology and multicolour FACS. Pre- and post-treatment blood samples from eight BC patients were analysed. Independent of clinical outcome p53-specific T cells were phenotypic distinctly antigen experienced...

  20. p53 Regulates Bone Differentiation and Osteosarcoma Formation | Center for Cancer Research

    Science.gov (United States)

    Osteosarcoma is an uncommon cancer that usually begins in the large bones of the arm or leg, but is the second leading cause of cancer-related death in children and young adults. The tumor suppressor protein, p53, appears to be an important player in osteosarcomagenesis in part because these cancers are one of the most common to develop in patients with Li-Fraumeni syndrome, which is caused by an inherited mutation in p53. However, the precise role of p53 in osteosarcoma development has not been established. To begin investigating its importance to the formation of normal bone and osteosarcomas, Jing Huang, Ph.D., of CCR’s Laboratory of Cancer Biology and Genetics, and his colleagues, isolated bone marrow-derived mesenchymal stem cells (BMSCs) from p53 wild type (WT) and knock out (KO) mice using a recently validated approach. Because BMSCs are one of the cells-of-origin of osteosarcoma, they serve as a useful model system. BMSCs contain a subset of multipotent stem cells that can differentiate into several cell types, including osteoblasts, and are important mediators of bone homeostasis.

  1. Retrotransposition-Competent Human LINE-1 Induces Apoptosis in Cancer Cells With Intact p53

    Directory of Open Access Journals (Sweden)

    Abdelali Haoudi

    2004-01-01

    Full Text Available Retrotransposition of human LINE-1 (L1 element, a major representative non-LTR retrotransposon in the human genome, is known to be a source of insertional mutagenesis. However, nothing is known about effects of L1 retrotransposition on cell growth and differentiation. To investigate the potential for such biological effects and the impact that human L1 retrotransposition has upon cancer cell growth, we examined a panel of human L1 transformed cell lines following a complete retrotransposition process. The results demonstrated that transposition of L1 leads to the activation of the p53-mediated apoptotic pathway in human cancer cells that possess a wild-type p53. In addition, we found that inactivation of p53 in cells, where L1 was undergoing retrotransposition, inhibited the induction of apoptosis. This suggests an association between active retrotransposition and a competent p53 response in which induction of apoptosis is a major outcome. These data are consistent with a model in which human retrotransposition is sensed by the cell as a “genetic damaging event” and that massive retrotransposition triggers signaling pathways resulting in apoptosis.

  2. Harnessing the p53-PUMA Axis to Overcome DNA Damage Resistance in Renal Cell Carcinoma

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    Xiaoguang Zhou

    2014-12-01

    Full Text Available Resistance to DNA damage–induced apoptosis is a hallmark of cancer and a major cause of treatment failure and lethal disease outcome. A tumor entity that is largely resistant to DNA-damaging therapies including chemo- or radiotherapy is renal cell carcinoma (RCC. This study was designed to explore the underlying molecular mechanisms of DNA damage resistance in RCC to develop strategies to resensitize tumor cells to DNA damage–induced apoptosis. Here, we show that apoptosis-resistant RCC cells have a disconnect between activation of p53 and upregulation of the downstream proapoptotic protein p53 upregulated modulator of apoptosis (PUMA. We demonstrate that this disconnect is not caused by gene-specific repression through CCCTC-binding factor (CTCF but instead by aberrant chromatin compaction. Treatment with an HDAC inhibitor was found to effectively reactivate PUMA expression on the mRNA and protein level and to revert resistance to DNA damage–induced cell death. Ectopic expression of PUMA was found to resensitize a panel of RCC cell lines to four different DNA-damaging agents tested. Remarkably, all RCC cell lines analyzed were wild-type for p53, and a knockdown was likewise able to sensitize RCC cells to acute genotoxic stress. Taken together, our results indicate that DNA damage resistance in RCC is reversible, involves the p53-PUMA axis, and is potentially targetable to improve the oncological outcomes of RCC patients.

  3. Naphthoquinone Derivative PPE8 Induces Endoplasmic Reticulum Stress in p53 Null H1299 Cells

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    Jin-Cherng Lien

    2015-01-01

    Full Text Available Endoplasmic reticulum (ER plays a key role in synthesizing secretory proteins and sensing signal function in eukaryotic cells. Responding to calcium disturbance, oxidation state change, or pharmacological agents, ER transmembrane protein, inositol-regulating enzyme 1 (IRE1, senses the stress and triggers downstream signals. Glucose-regulated protein 78 (GRP78 dissociates from IRE1 to assist protein folding and guard against cell death. In prolonged ER stress, IRE1 recruits and activates apoptosis signal-regulating kinase 1 (ASK1 as well as downstream JNK for cell death. Naphthoquinones are widespread natural phenolic compounds. Vitamin K3, a derivative of naphthoquinone, inhibits variant tumor cell growth via oxygen uptake and oxygen stress. We synthesized a novel naphthoquinone derivative PPE8 and evaluated capacity to induce ER stress in p53 null H1299 and p53 wild-type A549 cells. In H1299 cells, PPE8 induced ER enlargement, GRP78 expression, and transient IER1 activation. Activated IRE1 recruited ASK1 for downstream JNK phosphorylation. IRE1 knockdown by siRNA attenuated PPE8-induced JNK phosphorylation and cytotoxicity. Prolonged JNK phosphorylation may be involved in PPE8-induced cytotoxicity. Such results did not arise in A549 cells, but p53 knockdown by siRNA restored PPE8-induced GRP78 expression and JNK phosphorylation. We offer a novel compound to induce ER stress and cytotoxicity in p53-deficient cancer cells, presenting an opportunity for treatment.

  4. Tumor-promoting phorbol ester transiently down-modulates the p53 level and blocks the cell cycle

    DEFF Research Database (Denmark)

    Skouv, J; Jensen, P O; Forchhammer, J

    1994-01-01

    Activation of the protein kinase C signaling pathway by tumor-promoting phorbol esters, such as 4 beta-phorbol 12-myristate 13-acetate (PMA), induced a decrease in the level of p53 mRNA in several serum-starved human cell lines. Also, the tumor-promoting phosphatase inhibitor okadaic acid induced...... rate or the p53 mRNA stability. The protein synthesis inhibitor cycloheximide completely abolished the PMA-induced down-modulation of the p53 mRNA, suggesting that a short-lived protein was involved in the down-modulation. Flow cytometric cell cycle analysis showed that the phorbol ester treatment...... a decrease in the p53 mRNA level in the cell lines. Normal diploid as well as various tumor cell lines were tested. Two tumor cell lines, HeLa and A549, both containing the wild-type p53 gene, but very different levels of p53 protein, were studied in detail. In both cell lines, the level of p53 m...

  5. Microsatellite instability and p53 mutations in hepatocellular carcinoma.

    Science.gov (United States)

    Karachristos, A; Liloglou, T; Field, J K; Deligiorgi, E; Kouskouni, E; Spandidos, D A

    1999-01-01

    We have studied 27 hepatocellular carcinomas (HCCs) to identify possible relationships between microsatellite instability (MSI), p53 mutations, and HBV infection in hepatocarcinogenesis. MSI was assessed using 19 polymorphic markers and the poly(A) tract BAT-26. All coding regions of p53 were examined for mutations. Tumors were also examined for presence of hepatitis B virus (HBV) DNA sequences; 66.6% of the samples exhibit MSI in at least one microsatellite locus and 44% in two or three loci. None of the tumors examined showed alterations in BAT-26. Moreover, 73.3% of samples with indication of HBV infection showed instability in at least one marker. No association between MSI and pathological profile was found. Five (18.5%) samples harbored mutations in p53, three missense, and two insertions, all in exons 5 and 8 not previously reported. No mutations were detected in codon 249, which has been linked with dietary intake of aflatoxins. Our results support the hypothesis that HCC is a "low" MSI tumor. Only 1/5 samples with MSI in more than two markers harbored a mutation in p53. Although the number of samples is too small to support a statistical significance, this finding may indicate an inverse relationship between p53 mutations and MSI in HCC.

  6. p53 as a retrovirus-induced oxidative stress modulator.

    Science.gov (United States)

    Kim, Soo Jin; Wong, Paul K Y

    2015-01-01

    Infection of astrocytes by the neuropathogenic mutant of Moloney murine leukemia virus, ts1, exhibits increased levels of reactive oxygen species (ROS) and signs of oxidative stress compared with uninfected astrocytes. Previously, we have demonstrated that ts1 infection caused two separate events of ROS upregulation. The first upregulation occurs during early viral establishment in host cells and the second during the virus-mediated apoptotic process. In this study, we show that virus-mediated ROS upregulation activates the protein kinase, ataxia telangiectasia mutated, which in turn phosphorylates serine 15 on p53. This activation of p53 however, is unlikely associated with ts1-induced cell death. Rather p53 appears to be involved in suppressing intracellular ROS levels in astrocytes under oxidative stress. The activated p53 appears to delay retroviral gene expression by suppressing NADPH oxidase, a superoxide-producing enzyme. These results suggest that p53 plays a role as a retrovirus-mediated oxidative stress modulator. © 2015 The Authors.

  7. PERAN MUTASI GEN p53 PADA KARSINOGENESIS SEL BASAL KULIT

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    Kadek Pramesti Dewi

    2015-01-01

    Full Text Available Karsinoma sel basal (KSB merupakan keganasan kulit non-melanotik tersering dan mempunyai kaitan erat dengan paparan sinar ultra violet (UV. Keganasan ini berasal dari sel-sel pluripotensial stratum basalis epidermis maupun selubung akar folikel rambut. Gambaran klinis dan histopatologis terdiri dari KSB tipe klasik (noduler dan KSB varian (tipe superfisial, fibroepithelial, KSB dengan diferensiasi adneksal, basoskuamous, infiltrating, morpheaform.Kanker pada tubuh manusia muncul karena adanya mutasi genetik pada gen-gen yang terlibat dalamkontrol pertumbuhan sel, seperti onkogen, tumor suppressor gene, gen apoptosis, dan DNA repair gene.Pada kebanyakan kasus KSB, gen yang tersering mengalami mutasi adalah tumor suppressor genep53. Mutasi ini timbul akibat paparan langsung sinar UV, bergantung pada dosis, durasi dan intensitas paparan.Gen p53 dikenal dengan sebutan guardian of the genome, karena fungsinya sebagai sensor terhadapterjadinya kerusakan DNA. Adanya kerusakan DNA menginduksi aktivasi p53 untuk menghentikan siklus sel saat memasuki fase G1, sehingga memberikan kesempatan kepada DNA repair proteinbekerja memperbaiki kerusakan DNA. Lebih dari itu, p53 juga mengaktivasi gen GADD45 (growth arrest and DNA damage untuk membantu perbaikan DNA. Jika perbaikan gagal, p53 akanmengarahkan sel dengan DNA yang rusak ke mesin apoptosis.Pada sel-sel basal terpapar UV, gen p53 mengalami mutasi dan inaktivasi. Karena itu, sel-sel dengan DNA yang mengalami kerusakan non-lethal akan mengalami ekspansi klonal sehingga tumbuh menjadilesi pra kanker dan akhirnya kanker (KSB. [MEDICINA 2014;45:38-42

  8. Prognostic significance and gene expression profiles of p53 mutations in microsatellite-stable stage III colorectal adenocarcinomas.

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    Venkat R Katkoori

    Full Text Available Although the prognostic value of p53 abnormalities in Stage III microsatellite stable (MSS colorectal cancers (CRCs is known, the gene expression profiles specific to the p53 status in the MSS background are not known. Therefore, the current investigation has focused on identification and validation of the gene expression profiles associated with p53 mutant phenotypes in MSS Stage III CRCs. Genomic DNA extracted from 135 formalin-fixed paraffin-embedded tissues, was analyzed for microsatellite instability (MSI and p53 mutations. Further, mRNA samples extracted from five p53-mutant and five p53-wild-type MSS-CRC snap-frozen tissues were profiled for differential gene expression by Affymetrix Human Genome U133 Plus 2.0 arrays. Differentially expressed genes were further validated by the high-throughput quantitative nuclease protection assay (qNPA, and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR and by immunohistochemistry (IHC. Survival rates were estimated by Kaplan-Meier and Cox regression analyses. A higher incidence of p53 mutations was found in MSS (58% than in MSI (30% phenotypes. Both univariate (log-rank, P = 0.025 and multivariate (hazard ratio, 2.52; 95% confidence interval, 1.25-5.08 analyses have demonstrated that patients with MSS-p53 mutant phenotypes had poor CRC-specific survival when compared to MSS-p53 wild-type phenotypes. Gene expression analyses identified 84 differentially expressed genes. Of 49 down-regulated genes, LPAR6, PDLIM3, and PLAT, and, of 35 up-regulated genes, TRIM29, FUT3, IQGAP3, and SLC6A8 were confirmed by qNPA, qRT-PCR, and IHC platforms. p53 mutations are associated with poor survival of patients with Stage III MSS CRCs and p53-mutant and wild-type phenotypes have distinct gene expression profiles that might be helpful in identifying aggressive subsets.

  9. p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes

    Science.gov (United States)

    Kawahara, Misako; Malhotra, Gautam K.; Schaum, Nicholas; Huang, Jiahao; Ved, Urvi; Beausejour, Christian M.; Coppe, Jean-Philippe; Rodier, Francis

    2013-01-01

    Cellular senescence irreversibly arrests proliferation in response to potentially oncogenic stress. Senescent cells also secrete inflammatory cytokines such as IL-6, which promote age-associated inflammation and pathology. HMGB1 (high mobility group box 1) modulates gene expression in the nucleus, but certain immune cells secrete HMGB1 as an extracellular Alarmin to signal tissue damage. We show that nuclear HMGB1 relocalized to the extracellular milieu in senescent human and mouse cells in culture and in vivo. In contrast to cytokine secretion, HMGB1 redistribution required the p53 tumor suppressor, but not its activator ATM. Moreover, altered HMGB1 expression induced a p53-dependent senescent growth arrest. Senescent fibroblasts secreted oxidized HMGB1, which stimulated cytokine secretion through TLR-4 signaling. HMGB1 depletion, HMGB1 blocking antibody, or TLR-4 inhibition attenuated senescence-associated IL-6 secretion, and exogenous HMGB1 stimulated NF-κB activity and restored IL-6 secretion to HMGB1-depleted cells. Our findings identify senescence as a novel biological setting in which HMGB1 functions and link HMGB1 redistribution to p53 activity and senescence-associated inflammation. PMID:23649808

  10. p53 regulates cytoskeleton remodeling to suppress tumor progression.

    Science.gov (United States)

    Araki, Keigo; Ebata, Takahiro; Guo, Alvin Kunyao; Tobiume, Kei; Wolf, Steven John; Kawauchi, Keiko

    2015-11-01

    Cancer cells possess unique characteristics such as invasiveness, the ability to undergo epithelial-mesenchymal transition, and an inherent stemness. Cell morphology is altered during these processes and this is highly dependent on actin cytoskeleton remodeling. Regulation of the actin cytoskeleton is, therefore, important for determination of cell fate. Mutations within the TP53 (tumor suppressor p53) gene leading to loss or gain of function (GOF) of the protein are often observed in aggressive cancer cells. Here, we highlight the roles of p53 and its GOF mutants in cancer cell invasion from the perspective of the actin cytoskeleton; in particular its reorganization and regulation by cell adhesion molecules such as integrins and cadherins. We emphasize the multiple functions of p53 in the regulation of actin cytoskeleton remodeling in response to the extracellular microenvironment, and oncogene activation. Such an approach provides a new perspective in the consideration of novel targets for anti-cancer therapy.

  11. The Small Molecule AU14022 Promotes Colorectal Cancer Cell Death via p53-mediated G2/M-phase Arrest and Mitochondria-mediated Apoptosis.

    Science.gov (United States)

    Ryu, Hwani; Nam, Ky-Youb; Kim, Jae Sung; Hwang, Sang-Gu; Song, Jie-Young; Ahn, Jiyeon

    2017-10-14

    The p53 tumor suppressor plays critical roles in cell cycle regulation and apoptotic cell death, with its activation capable of sensitizing cancer cells to radiotherapy or chemotherapy. To identify small molecules that induce apoptosis via increased p53 transcriptional activity, we used a novel in-house library containing 96 small-molecule compounds. Using a cell-based screening method with a p53-responsive luciferase-reporter assay system involving benzoxazole derivatives, we found that AU14022 administration significantly increased p53 transcriptional activity in a concentration-dependent manner. Treatment with AU14022 increased p53 protein expression, p53 Ser15 phosphorylation, p53-mediated expression of downstream target genes, and apoptosis in p53-wild-type HCT116 human colon cancer cells, but not in p53-knockout HCT116 cells. Additionally, p53-wild-type HCT116 cells treated with AU14022 exhibited mitochondrial dysfunction, including modulated expression of B-cell lymphoma-2 family proteins and cytochrome c release. Combination treatment with AU14022 and ionizing radiation (IR) synergistically induced apoptosis as compared with IR or AU14022 treatment alone, with further investigation demonstrating that cell cycle progression was significantly arrested at the G2/M phase following AU14022 treatment. Furthermore, in a mouse p53-wild-type HCT116 colon cancer xenograft model, combined treatment with AU14022 and IR inhibited tumor growth more effectively than radiation alone. Therefore, AU14022 treatment induced apoptosis through p53-mediated cell cycle arrest involving mitochondrial dysfunction, leading to enhanced radiosensitivity in colon cancer cells. These results provide a basis for further assessments of AU14022 as a promising anticancer agent. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  12. The cyclin-dependent kinase inhibitor flavopiridol potentiates the effects of topoisomerase I poisons by suppressing Rad51 expression in a p53-dependent manner.

    Science.gov (United States)

    Ambrosini, Grazia; Seelman, Sharon L; Qin, Li-Xuan; Schwartz, Gary K

    2008-04-01

    The results of a phase I clinical trial of the topoisomerase I (Topo I) poison CPT-11 followed by the cyclin-dependent kinase inhibitor flavopiridol in patients with advanced solid tumors indicate that patients whose tumors were wild-type, but not mutant, for p53 obtained the most clinical benefit from this combination therapy. We elected to elucidate the mechanistic basis for this effect in isogenic-paired HCT116 colon cancer cells that were either wild-type (+/+) or null (-/-) for p53. With the combination therapy of SN-38 (the active metabolite of CPT-11) followed by flavopiridol, the induction of apoptosis was 5-fold greater in the p53+/+ cells compared with the p53-/- cells. This sequential treatment induced phosphorylation of p53 at Ser(15), which interacted with Rad51, a DNA repair protein involved in homologous recombination. Rad51 bound to p53-Ser(15) within the first 5 hours of combination therapy, and then was transcriptionally suppressed at 24 hours by flavopiridol only in p53+/+ cells. Microarray analysis also revealed suppression of Rad51 in a p53-dependent manner. Depletion of Rad51 by small interfering RNA (siRNA) sensitized both p53+/+ and p53-/- cells to SN-38-induced apoptosis with increase of gamma H2AX, a marker of DNA damage. Conversely, overexpression of Rad51 rescued p53+/+ cells from SN-->F-induced apoptosis. Because flavopiridol inhibits Cdk9, we found that inhibition of Cdk9 by DRB or by siRNA could recapitulate the flavopiridol effects, with suppression of Rad51 and induction of apoptosis only in p53+/+ cells. In conclusion, after DNA damage by Topo I poisons, flavopiridol targets homologous recombination through a p53-dependent down-regulation of Rad51, resulting in enhancement of apoptosis.

  13. Combining oncolytic virotherapy with p53 tumor suppressor gene therapy

    OpenAIRE

    Bressy, Christian; Hastie, Eric; Grdzelishvili, Valery Z.

    2017-01-01

    Oncolytic virus (OV) therapy utilizes replication-competent viruses to kill cancer cells, leaving non-malignant cells unharmed. With the first U.S. Food and Drug Administration-approved OV, dozens of clinical trials ongoing, and an abundance of translational research in the field, OV therapy is poised to be one of the leading treatments for cancer. A number of recombinant OVs expressing a transgene for p53 (TP53) or another p53 family member (TP63 or TP73) were engineered with the goal of gen...

  14. Synergistic antitumor interaction between valproic acid, capecitabine and radiotherapy in colorectal cancer: critical role of p53.

    Science.gov (United States)

    Terranova-Barberio, Manuela; Pecori, Biagio; Roca, Maria Serena; Imbimbo, Serena; Bruzzese, Francesca; Leone, Alessandra; Muto, Paolo; Delrio, Paolo; Avallone, Antonio; Budillon, Alfredo; Di Gennaro, Elena

    2017-12-06

    Recurrence with distant metastases has become the predominant pattern of failure in locally advanced rectal cancer (LARC), thus the integration of new antineoplastic agents into preoperative fluoropyrimidine-based chemo-radiotherapy represents a clinical challenge to implement an intensified therapeutic strategy. The present study examined the combination of the histone deacetylase inhibitor (HDACi) valproic acid (VPA) with fluoropyrimidine-based chemo-radiotherapy on colorectal cancer (CRC) cells. HCT-116 (p53-wild type), HCT-116 p53-/- (p53-null), SW620 and HT29 (p53-mutant) CRC cell lines were used to assess the antitumor interaction between VPA and capecitabine metabolite 5'-deoxy-5-fluorouridine (5'-DFUR) in combination with radiotherapy and to evaluate the role of p53 in the combination treatment. Effects on proliferation, clonogenicity and apoptosis were evaluated, along with γH2AX foci formation as an indicator for DNA damage. Combined treatment with equipotent doses of VPA and 5'-DFUR resulted in synergistic effects in CRC lines expressing p53 (wild-type or mutant). In HCT-116 p53-/- cells we observed antagonist effects. Radiotherapy further potentiated the antiproliferative, pro-apoptotic and DNA damage effects induced by 5'-DFUR/VPA combination in p53 expressing cells. These results highlighted the role of VPA as valuable candidate to be added to preoperative chemo-radiotherapy in LARC. On these bases we launched the ongoing phase I/II study of VPA and short-course radiotherapy plus capecitabine as preoperative treatment in low-moderate risk rectal cancer (V-shoRT-R3).

  15. Different headspace profiles in wild crucifer species in response to Plutella xylostella herbivory and exogenous jasmonic acid application

    NARCIS (Netherlands)

    Zhang, P.J.; Shu, J.P.; Dicke, M.; Liu, S.S.

    2010-01-01

    Although exogenous treatment of plants with jasmonic acid (JA) may result in induced responses similar to plant defences induced by herbivory, few studies have compared the details of insect herbivory and JA-mimicked responses. We compared volatiles of two crucifer species, Cardamine impatiens and

  16. Mutant p53: a novel target for the treatment of patients with triple-negative breast cancer?

    Science.gov (United States)

    Synnott, N C; Murray, A; McGowan, P M; Kiely, M; Kiely, P A; O'Donovan, N; O'Connor, D P; Gallagher, W M; Crown, J; Duffy, M J

    2017-01-01

    The identification and validation of a targeted therapy for patients with triple-negative breast cancer (TNBC) is currently one of the most urgent needs in breast cancer therapeutics. One of the key reasons for the failure to develop a new therapy for this subgroup of breast cancer patients has been the difficulty in identifying a highly prevalent, targetable molecular alteration in these tumors. Recently however, the p53 gene was found to be mutated in approximately 80% of basal/TNBC, raising the possibility that targeting the mutant p53 protein product might be a new approach for the treatment of this form of breast cancer. In this study, we investigated the anti-cancer activity of PRIMA-1 and PRIMA-1MET (APR-246), two compounds which were previously reported to reactivate mutant p53 and convert it to a form with wild-type (WT) properties. Using a panel of 18 breast cancer cell lines and 2 immortalized breast cell lines, inhibition of proliferation by PRIMA-1 and PRIMA-1MET was found to be cell-line dependent, but independent of cell line molecular subtype. Although response was independent of molecular subtype, p53 mutated cell lines were significantly more sensitive to PRIMA-1MET than p53 WT cells (p = 0.029). Furthermore, response (measured as IC50 value) correlated significantly with p53 protein level as measured by ELISA (p = 0.0089, r=-0.57, n = 19). In addition to inhibiting cell proliferation, PRIMA-1MET induced apoptosis and inhibited migration in a p53 mutant-dependent manner. Based on our data, we conclude that targeting mutant p53 with PRIMA-1MET is a potential new approach for treating p53-mutated breast cancer, including the subgroup with triple-negative (TN) disease. © 2016 UICC.

  17. High Resolution Melting Analysis for Detecting p53 Gene Mutations in Patients with Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Zhihong CHEN

    2011-10-01

    Full Text Available Background and objective It has been proven that p53 gene was related to many human cancers. The mutations in p53 gene play an important role in carcinogensis and mostly happened in exon 5-8. The aim of this study is to establish a high resolution melting (HRM assay to detect p53 mutations from patients with non-small cell lung cancer (NSCLC, to investigate the characteristics of p53 gene mutations, and to analyze the relationship between p53 mutations and evolution regularity of pathogenesis. Methods p53 mutations in exon 5-8 were detected by HRM assay on DNA insolated from 264 NSCLC samples derived from tumor tissues and 54 control samples from pericancerous pulmonary tissues. The mutation samples by the HRM assay were confirmed by sequencing technique. Samples which were positive by HRM but wild type by sequencing were further confirmed by sub-clone and sequencing. Results No mutation was found in 54 pericancerous pulmonary samples by HRM assay. 104 of the 264 tumor tissues demonstrated mutation curves by HRM assay, 102 samples were confirmed by sequencing, including 95 point mutations and 7 frame shift mutations by insertion or deletion. The mutation rate of p53 gene was 39.4%. The mutation rate from exon 5-8 were 11.7%, 8%, 12.5% and 10.6%, respectively and there was no statistically significant difference between them (P=0.35. p53 mutations were significantly more frequent in males than that in females, but not related to the other clinicopathologic characteristics. Conclusion The results indicate that HRM is a sensitive in-tube methodology to detect for mutations in clinical samples. The results suggest that the arising p53 mutations in NSCLC may be due to spontaneous error in DNA synthesis and repair.

  18. G2-block after irradiation of cells with different p53 status

    Energy Technology Data Exchange (ETDEWEB)

    Zoelzer, Friedo [University of South Bohemia in Ceske Budejovice, Department of Radiology, Toxicology and Civil Protection, Faculty of Health and Social Studies, Ceske Budejovice (Czech Republic); University Duisburg-Essen, Institute of Medical Radiobiology, Medical Faculty, Essen (Germany); Jagetia, Ganesh [University Duisburg-Essen, Institute of Medical Radiobiology, Medical Faculty, Essen (Germany); Mizoram University, Department of Zoology, School of Life Sciences, Aizawl (India); Streffer, Christian [University Duisburg-Essen, Institute of Medical Radiobiology, Medical Faculty, Essen (Germany)

    2014-11-15

    Although it is clear that functional p53 is not required for radiation-induced G{sub 2} block, certain experimental findings suggest a role for p53 in this context. For instance, as we also confirm here, the maximum accumulation in the G{sub 2} compartment after X-ray exposure occurs much later in p53 mutants than in wild types. It remains to be seen, however, whether this difference is due to a longer block in the G{sub 2} phase itself. We observed the movement of BrdU-labeled cells through G{sub 2} and M into G{sub 1}. From an analysis of the fraction of labeled cells that entered the second posttreatment cell cycle, we were able to determine the absolute duration of the G{sub 2} and M phases in unirradiated and irradiated cells. Our experiments with four cell lines, two melanomas and two squamous carcinomas, showed that the radiation-induced delay of transition through the G{sub 2} and M phases did not correlate with p53 status. We conclude that looking at the accumulation of cells in the G{sub 2} compartment alone is misleading when differences in the G{sub 2} block are investigated and that the G{sub 2} block itself is indeed independent of functional p53. (orig.) [German] Obwohl klar ist, dass ein funktionelles p53-Protein fuer die Ausbildung des strahleninduzierten G{sub 2}-Blocks nicht zwingend erforderlich ist, gibt es experimentelle Befunde, die nahe legen, dass p53 in diesem Zusammenhang doch eine gewisse Rolle spielt. Zum Beispiel bestaetigen wir hier fruehere Berichte, dass die Akkumulation von Zellen im G{sub 2}-Kompartiment bei p53-Mutanten deutlich spaeter nach Bestrahlung ihr Maximum erreicht als bei p53-Wildtypen. Es bleibt jedoch zu klaeren, ob dieser Unterschied seinen Grund in einem laengeren Block der G{sub 2}-Phase selbst hat. Beobachtet wurde die Bewegung von BrdU-markierten Zellen durch G{sub 2} und M nach G{sub 1}. Aus der zeitlichen Veraenderung des Anteils markierter Zellen im G{sub 1}-Kompartiment des naechsten Zellzyklus konnte die

  19. Lunatic Fringe and p53 Cooperatively Suppress Mesenchymal Stem-Like Breast Cancer

    Directory of Open Access Journals (Sweden)

    Wen-Cheng Chung

    2017-11-01

    Full Text Available Claudin-low breast cancer (CLBC is a poor prognosis molecular subtype showing stemness and mesenchymal features. We previously discovered that deletion of a Notch signaling modulator, Lunatic Fringe (Lfng, in the mouse mammary gland induced a subset of tumors resembling CLBC. Here we report that deletion of one copy of p53 on this background not only accelerated mammary tumor development but also led to a complete penetrance of the mesenchymal stem-like phenotype. All mammary tumors examined in the Lfng/p53 compound mutant mice displayed a mesenchymal/spindloid pathology. These tumors showed high level expressions of epithelial-to-mesenchymal transition (EMT markers including Vimentin, Twist, and PDGFRα, a gene known to be enriched in CLBC. Prior to tumor onset, Lfng/p53 mutant mammary glands exhibited increased levels of Vimentin and E-cadherin, but decreased expressions of cytokeratin 14 and cytokeratin 8, accompanied by elevated basal cell proliferation and an expanded mammary stem cell-enriched population. Lfng/p53 mutant glands displayed increased accumulation of Notch3 intracellular fragment, up-regulation of Hes5 and down-regulation of Hes1. Analysis in human breast cancer datasets found the lowest HES1 and second lowest LFNG expressions in CLBC among molecular subtypes, and low level of LFNG is associated with poor survival. Immunostaining of human breast cancer tissue array found correlation between survival and LFNG immunoreactivity. Finally, patients carrying TP53 mutations express lower LFNG than patients with wild type TP53. Taken together, these data revealed genetic interaction between Lfng and p53 in mammary tumorigenesis, established a new mouse model resembling CLBC, and may suggest targeting strategy for this disease.

  20. Systematic and comprehensive analysis of mutant p53 proteins in ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    The gene p53 is a well-known tumour suppressor gene that prevents cancer formation. It is the most commonly mutated gene among individuals with a diagnosis of cancer. Through recent advances in DNA sequencing abilities, researchers are now in a position to take a patient's tumour and identify the exact mutation in ...

  1. A role for p53 in selenium-induced senescence

    Science.gov (United States)

    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...

  2. Cellular inactivation of nitric oxide induces p53-dependent ...

    African Journals Online (AJOL)

    Results: c-PTIO and 1400W, alone or in combination, inhibited cell growth and promoted apoptosis via sub-G1 cell cycle arrest mediated by decrease in NO•. Apoptosis was delayed and greatly reduced in magnitude in SK mel-28 cells, underscoring the importance of p53 modulation of the response. In both cell types, ...

  3. Immunohistochemical detection of P53 and Mdm2 in vitiligo

    Directory of Open Access Journals (Sweden)

    Ola A Bakry

    2012-01-01

    Full Text Available 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.

  4. Artificial Macrocycles as Potent p53-MDM2 Inhibitors

    NARCIS (Netherlands)

    Estrada-Ortiz, Natalia; Neochoritis, Constantinos G.; Twarda-Clapa, Aleksandra; Musielak, Bogdan; Holak, Tad A.; Dömling, Alexander

    2017-01-01

    Based on a combination of an Ugi four component reaction and a ring closing metathesis, a library of novel artificial macrocyclic inhibitors of the p53-MDM2 interaction was designed and synthesized. These macrocycles, alternatively to stapled peptides, target for the first time the large hydrophobic

  5. The p53-MDM2 network: from oscillations to apoptosis

    Indian Academy of Sciences (India)

    2007-07-05

    Jul 5, 2007 ... Keywords. Apoptosis; cancer; cell cycle; MDM2 overexpression; tumour suppressor ... In this paper, we give an overview of our studies on the p53-MDM2 module and the associated pathways from a systems biology perspective. We discuss a number of key predictions, related to some specific aspects of ...

  6. Morphological Heterogeneity of p53 Positive and p53 Negative Nuclei in Breast Cancers Stratified by Clinicopathological Variables

    Directory of Open Access Journals (Sweden)

    Katrin Friedrich

    1997-01-01

    Full Text Available The study was aimed to detect differences in nuclear morphology between nuclear populations as well as between tumours with different p53 expression in breast cancers with different clinicopathological features, which also reflect the stage of tumour progression. The p53 immunohistochemistry was performed on paraffin sections from 88 tumour samples. After the cells had been localised by means of an image cytometry workstation and their immunostaining had been categorised visually, the sections were destained and stained by the Feulgen protocol. The nuclei were relocated and measured cytometrically by the workstation.

  7. Concomitant inactivation of p53 and Chk2 in breast cancer.

    Science.gov (United States)

    Sullivan, Alexandra; Yuille, Martin; Repellin, Claire; Reddy, Archana; Reelfs, Olivier; Bell, Alexandra; Dunne, Barbara; Gusterson, Barry A; Osin, Peter; Farrell, Paul J; Yulug, Isik; Evans, Abigail; Ozcelik, Tayfun; Gasco, Milena; Crook, Tim

    2002-02-21

    The structure and expression of the human Rad53 homologue Chk2 was analysed in breast cancer. The previously described silent polymorphism at nucleotide 252 in codon 84 (GAA>GAG) was observed in 5/141 cases. Somatic Chk2 coding mutations were detected in 7/141 cases, these occurring in 4/18 BRCA1-associated breast cancers, 1/78 sporadic breast cancers and 2/25 typical medullary carcinomas. Each of the BRCA1-associated cancers with Chk2 mutations also contained mutations in p53, whereas the single sporadic cancer with Chk2 mutation was wild-type for p53. Expression of Chk2 was ubiquitously detected in normal ductal epithelium of the breast, but there was loss of expression in a significant proportion of breast carcinomas, and this occurred in cancers both with and without p53 mutation. A CpG island was identified 5' of the Chk2 transcriptional start site, but there was no evidence of cytosine methylation in any of the cancers with down-regulated Chk2 expression. Analysis of the germ-line of 45 individuals with hereditary or early onset breast cancer revealed wild-type Chk2 sequence in all cases. Thus, despite the rarity of somatic mutations in Chk2 in sporadic breast carcinomas, our results nevertheless reveal that concomitant loss of function in Chk2 (via down-regulation of expression) and p53 (via mutation) occurs in a proportion of sporadic cases. However, consistent with other studies, we show that germ-line mutations in Chk2 are unlikely to account for a significant proportion of non BRCA1-, non BRCA2-associated hereditary breast cancers.

  8. Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein–DNA interactions

    Science.gov (United States)

    Eldar, Amir; Rozenberg, Haim; Diskin-Posner, Yael; Rohs, Remo; Shakked, Zippora

    2013-01-01

    A p53 hot-spot mutation found frequently in human cancer is the replacement of R273 by histidine or cysteine residues resulting in p53 loss of function as a tumor suppressor. These mutants can be reactivated by the incorporation of second-site suppressor mutations. Here, we present high-resolution crystal structures of the p53 core domains of the cancer-related proteins, the rescued proteins and their complexes with DNA. The structures show that inactivation of p53 results from the incapacity of the mutated residues to form stabilizing interactions with the DNA backbone, and that reactivation is achieved through alternative interactions formed by the suppressor mutations. Detailed structural and computational analysis demonstrates that the rescued p53 complexes are not fully restored in terms of DNA structure and its interface with p53. Contrary to our previously studied wild-type (wt) p53-DNA complexes showing non-canonical Hoogsteen A/T base pairs of the DNA helix that lead to local minor-groove narrowing and enhanced electrostatic interactions with p53, the current structures display Watson–Crick base pairs associated with direct or water-mediated hydrogen bonds with p53 at the minor groove. These findings highlight the pivotal role played by R273 residues in supporting the unique geometry of the DNA target and its sequence-specific complex with p53. PMID:23863845

  9. p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription.

    Directory of Open Access Journals (Sweden)

    Robert Lipski

    Full Text Available DNA damage induced by the topoisomerase I inhibitor SN38 activates cell cycle checkpoints which promote cell cycle arrest. This arrest can be abrogated in p53-defective cells by the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01. Previously, we compared p53 wild-type MCF10A cells with derivatives whose p53 function was inhibited by over-expression of the tetramerization domain (MCF10A/OD or expression of shRNA against p53 (MCF10A/Δp53. Treatment of SN38-arrested MCF10A/OD cells with UCN-01 abrogated S, but not G2 arrest, while the MCF10A/Δp53 cells abrogated both S and G2 arrest. The MCF10A/OD cells had reduced levels of cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B gene expression. In the present study, we analyzed p53 oligomerization status using glutaraldehyde cross-linking. Following SN38 treatment, MCF10A cells contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. However, MCF10A/OD cells possessed only monomers and dimers suggesting that these complexes may be involved in repression of cyclin B. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-head orientation, the cyclin B promoter contains similar elements oriented head-to-tail. Chromatin immunoprecipitation (ChIP assays revealed that p53 associates with this head-to-tail element in both MCF10A and MCF10A/OD. Electrophoretic mobility shift assays (EMSA using a biotin-labeled probe containing the head-to-tail element showed a shift in mobility consistent with the molecular weight of tetramers and dimers in MCF10A nuclear extract, but only the dimer in MCF10A/OD nuclear extract. Taken together, these results suggest a novel mechanism whereby p53 dimers associate with the head-to-tail element to repress cyclin B transcription.

  10. Topotecan triggers apoptosis in p53-deficient cells by forcing degradation of XIAP and survivin thereby activating caspase-3-mediated Bid cleavage.

    Science.gov (United States)

    Tomicic, Maja T; Christmann, Markus; Kaina, Bernd

    2010-01-01

    The topoisomerase I inhibitor topotecan (TPT) is used in the therapy of different tumors including high-grade gliomas. We previously showed that TPT-induced apoptosis depends on p53 with p53 wild-type (wt) cells being more resistant because of p53-controlled degradation of topoisomerase I. Here, we show that p53-deficient (p53(-/-)) fibroblasts undergo excessive mitochondrial apoptosis featuring H2AX phosphorylation, Bcl-x(L) decline, cytochrome c release, caspase-9/-3/-2 activation, and cleavage of Bid. In wt and apaf-1(-/-) cells, caspase-2 did not become activated and Bid was not cleaved. In addition, p53(-/-) cells cotreated with TPT and caspase-3 inhibitor showed neither caspase-2 activation nor Bid cleavage, implying that caspase-2 is processed downstream of the apoptosome by caspase-3. Although processing of caspase-9/-3 was similar in wt and p53(-/-) cells, only p53(-/-) cells displayed active caspase-3. This was due to the proteasomal degradation of X-chromosome-linked inhibitor of apoptosis (XIAP) and survivin that inhibits caspase-3 activity. Accordingly, TPT-induced apoptosis in wt cells was increased after XIAP/survivin knockdown. Silencing of Bid led to reduction of TPT-triggered apoptosis. Data obtained with mouse fibroblasts could be extended to human glioma cells. In U87MG (p53wt) cells cotreated with TPT and pifithrin-alpha, or transfected with p53-siRNA, caspase-2 and Bid were significantly cleaved and XIAP/survivin was degraded. Furthermore, the knockdown of XIAP and survivin led to increased TPT-triggered apoptosis. Overall, the data show that p53-deficient/depleted cells are hypersensitive to TPT because they down-regulate XIAP and survivin, and thus amplify the intrinsic apoptotic pathway via caspase-3-mediated Bid cleavage. Therefore, in gliomas harboring wild-type p53, TPT-based therapy might be improved by targeted down-regulation of XIAP and survivin.

  11. The structure formed by inverted repeats in p53 response elements determines the transactivation activity of p53 protein

    Czech Academy of Sciences Publication Activity Database

    Brázda, Václav; Čechová, Jana; Battistin, M.; Coufal, Jan; Jagelská, Eva; Raimondi, I.; Inga, A.

    2017-01-01

    Roč. 483, č. 1 (2017), s. 516-521 ISSN 0006-291X R&D Projects: GA ČR GA15-21855S Institutional support: RVO:68081707 Keywords : tumor-suppressor p53 * cruciform structures * dna-conformation Subject RIV: CE - Biochemistry Impact factor: 2.466, year: 2016

  12. P53 mutation analysis of colorectal liver metastases : Relation to actual survival, angiogenic status, and p53 overexpression

    NARCIS (Netherlands)

    de Jong, KP; Gouw, ASH; Peeters, PMJG; Bulthuis, M; Menkema, L; Porte, RJ; Slooff, MJH; van Goor, H; van den Berg, Anke

    2005-01-01

    Purpose: To correlate TP53 mutations with angiogenic status of the tumor and prognosis after liver surgery in patients with colorectal liver metastases and to correlate immunohistochemical staining of p53 protein with TP53 gene mutations. Experimental Design: Tumors of 44 patients with surgically

  13. Construction of a triple modified p53 containing DNA vaccine to enhance processing and presentation of the p53 antigen

    NARCIS (Netherlands)

    Hospers, Geke A. P.; Meijer, Coby; Dam, Wendy A.; Roossink, Frank; Mulder, Nanno H.

    2009-01-01

    More effective and less toxic treatments are urgently needed in the treatment of patients with cancer. The turnout suppressor protein p53 is a tumour-associated antigen that could serve that purpose when applied in an immunologic approval to cancer. It is mutated in similar to 50% of the tumours

  14. Perturbation of ribosome biogenesis drives cells into senescence through 5S RNP-mediated p53 activation.

    Science.gov (United States)

    Nishimura, Kazuho; Kumazawa, Takuya; Kuroda, Takao; Katagiri, Naohiro; Tsuchiya, Mai; Goto, Natsuka; Furumai, Ryohei; Murayama, Akiko; Yanagisawa, Junn; Kimura, Keiji

    2015-03-03

    The 5S ribonucleoprotein particle (RNP) complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Perturbation of Ribosome Biogenesis Drives Cells into Senescence through 5S RNP-Mediated p53 Activation

    Directory of Open Access Journals (Sweden)

    Kazuho Nishimura

    2015-03-01

    Full Text Available The 5S ribonucleoprotein particle (RNP complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses.

  16. CONVERGENCE OF P53 AND TGFβ SIGNALING ON ACTIVATING EXPRESSION OF THE TUMOR SUPPRESSOR GENE MASPIN IN MAMMARY EPITHELIAL CELLS

    Science.gov (United States)

    Wang, Shizhen Emily; Narasanna, Archana; Whitell, Corbin W.; Wu, Frederick Y.; Friedman, David B.; Arteaga, Carlos L.

    2014-01-01

    Using two-dimensional difference gel electrophoresis, we identified the tumor suppressor gene maspin as a TGFβ target gene in human mammary epithelial cells. TGFβ upregulates maspin expression both at the RNA and protein levels. This upregulation required Smad2/3 function and intact p53 binding elements in the maspin promoter. DNA affinity immunoblot and chromatin immunoprecipitation (ChIP) revealed the presence of both Smads and p53 at the maspin promoter in TGFβ-treated cells, suggesting that both transcription factors cooperate to induce maspin transcription. TGFβ did not activate maspin-luciferase reporter in p53-mutant MDA-MB-231 breast cancer cells, which exhibit methylation of the endogenous maspin promoter. Expression of ectopic p53, however, restored ligand-induced association of Smad2/3 with a transfected maspin promoter. Stable transfection of maspin inhibited basal and TGFβ-stimulated MDA-MB-231 cell motility. Finally, knockdown of endogenous maspin in p53 wild-type MCF10A/HER2 cells enhanced basal and TGFβ-stimulated motility. Taken together, these data support cooperation between the p53 and TGFβ tumor suppressor pathways in the induction of maspin expression, thus leading to inhibition of cell migration. PMID:17204482

  17. The p53-inducible long noncoding RNA TRINGS protects cancer cells from necrosis under glucose starvation.

    Science.gov (United States)

    Khan, Muhammad Riaz; Xiang, Shaoxun; Song, Zhiyin; Wu, Mian

    2017-12-01

    The tumor suppressor p53 is activated in response to cellular stress to prevent malignant transformation. However, several recent studies have shown that p53 can play protective roles in tumor cell survival under adversity. Whether p53-regulated long noncoding RNAs are involved in this process remains to be fully understood. Here, we show that under glucose starvation condition, p53 directly upregulates a novel lncRNA named TRINGS (Tp53-regulated inhibitor of necrosis under glucose starvation) in human tumor cells. TRINGS binds to STRAP and inhibits STRAP-GSK3β-NF-κB necrotic signaling to protect tumor cells from cell death. Interestingly, TRINGS appears to respond to glucose starvation specifically, as it is not activated by serum, serine, or glutamine deprivation. Collectively, our findings reveal that p53-induced lncRNA TRINGS controls the necrotic pathway and contributes to the survival of cancer cells harboring wild-type p53 under glucose stress. © 2017 The Authors.

  18. Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction.

    Directory of Open Access Journals (Sweden)

    Mariell Pettersson

    Full Text Available The transcription factor p53 is the main tumour suppressor in cells and many cancer types have p53 mutations resulting in a loss of its function. In tumours that retain wild-type p53 function, p53 activity is down-regulated by MDM2 (human murine double minute 2 via a direct protein-protein interaction. We have designed and synthesised two series of 2,5-diketopiperazines as inhibitors of the MDM2-p53 interaction. The first set was designed to directly mimic the α-helical region of the p53 peptide, containing key residues in the i, i+4 and i+7 positions of a natural α-helix. Conformational analysis indicated that 1,3,6-trisubstituted 2,5-diketopiperazines were able to place substituents in the same spatial orientation as an α-helix template. The key step of the synthesis involved the cyclisation of substituted dipeptides. The other set of tetrasubstituted 2,5-diketopiperazines were designed based on structure-based docking studies and the Ugi multicomponent reaction was used for the synthesis. This latter set comprised the most potent inhibitors which displayed micromolar IC50-values in a biochemical fluorescence polarisation assay.

  19. A HLA-A2 restricted human CTL line recognizes a novel tumor cell expressed p53 epitope

    DEFF Research Database (Denmark)

    Würtzen, Peter A; Claesson, Mogens H

    2002-01-01

    was characterized with respect to specificity, affinity and killing of cell lines derived from p53 mutated spontaneous tumors. The CTL line demonstrated lysis of p53(139-147) pulsed target cells and cold target inhibition experiments as well as antibody blocking confirmed that the killing was epitope-specific, HLA......-A2 restricted and dependent on CD8-binding. Interestingly, the affinity of the CTL line was only in the micromole per liter range and target cells pulsed with less than 0.01 microM peptide were not recognized. Furthermore, 3 HLA-A2(+) p53 mutated tumor cell lines were efficiently lysed by the CTL...... line, indicating that this novel p53 peptide epitope is endogenously processed and presented by the HLA-A2 molecules of the tumor cells. In conclusion, CTL reactivity towards a wild-type p53 peptide was revealed through induction with DC pulsed with a pool of HLA-A2 binding p53 peptides. In addition...

  20. Role for p53 in the Recovery of Transcription and Protection Against Apoptosis Induced by Ultraviolet Light

    Directory of Open Access Journals (Sweden)

    Bruce C. McKay

    1999-08-01

    Full Text Available We have previously suggested that the inhibition of RNA polymerase II-mediated transcription after exposure to UV light promotes the accumulation of p53 and the induction of apoptosis (Oncogene 13, 823–831. However, it was not clear whether p53 induction was contributing to apoptosis. Here we report that apoptosis is triggered at lower UV doses in p53-deficient Li-Fraumeni syndrome (LFS and human papillomavirus (HPV E6 expressing fibroblasts than in normal cells, suggesting that p53 can be protective against UVinduced apoptosis. There is no significant difference in the effect of UV-irradiation on the cell cycle distribution of normal and primary LFS fibroblasts. Importantly, the recovery of nascent mRNA synthesis in all p53-deficient fibroblasts is significantly impaired compared with control cells after exposure to relevant doses of UV light. Taken together, our results suggest that wild-type p53 can protect cells against UV-induced apoptosis by facilitating the recovery of transcription. Furthermore, we suggest that the capacity of cells to recover transcription after genotoxic damage is an important determinant of sensitivity to apoptosis.

  1. Differential effects of p53 on bystander phenotypes induced by gamma ray and high LET heavy ion radiation

    Science.gov (United States)

    He, Mingyuan; Dong, Chen; Konishi, Teruaki; Tu, Wenzhi; Liu, Weili; Shiomi, Naoko; Kobayashi, Alisa; Uchihori, Yukio; Furusawa, Yoshiya; Hei, Tom K.; Dang, Bingrong; Shao, Chunlin

    2014-04-01

    High LET particle irradiation has several potential advantages over γ-rays such as p53-independent response. The purpose of this work is to disclose the effect of p53 on the bystander effect induced by different LET irradiations and underlying mechanism. Lymphocyte cells of TK6 (wild type p53) and HMy2.CIR (mutated p53) were exposed to either low or high LET irradiation, then their mitochondrial dysfunction and ROS generation were detected. The micronuclei (MN) induction in HL-7702 hepatocytes co-cultured with irradiated lymphocytes was also measured. It was found that the mitochondrial dysfunction, p66Shc activation, and intracellular ROS were enhanced in TK6 but not in HMy2.CIR cells after γ-ray irradiation, but all of them were increased in both cell lines after carbon and iron irradiation. Consistently, the bystander effect of MN formation in HL-7702 cells was only triggered by γ-irradiated TK6 cells but not by γ-irradiated HMy2.CIR cells. But this bystander effect was induced by both lymphocyte cell lines after heavy ion irradiation. PFT-μ, an inhibitor of p53, only partly inhibited ROS generation and bystander effect induced by 30 keV/μm carbon-irradiated TK6 cells but failed to suppress the bystander effect induced by the TK6 cells irradiated with either 70 keV/μm carbon or 180 keV/μm iron. The mitochondrial inhibitors of rotenone and oligomycin eliminated heavy ion induced ROS generation in TK6 and HMy2.CIR cells and hence diminished the bystander effect on HL-7702 cells. These results clearly demonstrate that the bystander effect is p53-dependent for low LET irradiation, but it is p53-independent for high LET irradiation which may be because of p53-independent ROS generation due to mitochondrial dysfunction.

  2. A synthetic form of frizzled 8-associated antiproliferative factor enhances p53 stability through USP2a and MDM2.

    Directory of Open Access Journals (Sweden)

    Jayoung Kim

    Full Text Available Frizzled 8-associated Antiproliferative Factor (APF is a sialoglycopeptide urinary biomarker of interstitial cystitis/painful bladder syndrome (IC/PBS, a chronic condition of unknown etiology with variable symptoms that generally include pelvic and/or perineal pain, urinary frequency, and urgency. We previously reported that native human APF suppresses the proliferation of normal bladder epithelial cells through a mechanism that involves increased levels of p53. The goal of this study was to delineate the regulatory mechanism whereby p53 expression is regulated by APF. Two APF-responsive cell lines (T24 bladder carcinoma cells and the immortalized human bladder epithelial cell line, TRT-HU1 were treated with asialo-APF (as-APF, a chemically synthesized form of APF. Biochemical analysis revealed that as-APF increased p53 levels in two ways: by decreasing ubiquitin specific protease 2a (USP2a expression leading to enhanced ubiquitination of murine double minute 2 E3 ubiquitin ligase (MDM2, and by suppressing association of p53 with MDM2, thus impairing p53 ubiquitination. Biological responses to as-APF were suppressed by increased expression of wild type, but not mutant USP2a, which enhanced cell growth via upregulation of a cell cycle mediator, cyclin D1, at both transcription and protein levels. Consistent with this, gene silencing of USP2a with siRNA arrested cell proliferation. Our findings suggest that APF upregulates cellular p53 levels via functional attenuation of the USP2a-MDM2 pathway, resulting in p53 accumulation and growth arrest. These data also imply that targeting USP2a, MDM2, p53 and/or complex formation by these molecules may be relevant in the development of novel therapeutic approaches to IC/PBS.

  3. Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Youfeng Shen

    2017-11-01

    Full Text Available Abstract Background Pigs have many features that make them attractive as biomedical models for various diseases, including cancer. P53 is an important tumor suppressor gene that exerts a central role in protecting cells from oncogenic transformation and is mutated in a large number of human cancers. P53 mutations occur in almost every type of tumor and in over 50% of all tumors. In a recent publication, pigs with a mutated P53 gene were generated that resulted in lymphoma and renal and osteogenic tumors. However, approximately 80% of human tumors have dysfunctional P53. A P53-deficient pig model is still required to elucidate. Methods Transcription activator-like effector nucleases (TALENs were designed to target porcine P53 exon 4. The targeting activity was evaluated using a luciferase SSA recombination assay. P53 biallelic knockout (KO cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs followed by electroporation with TALENs plasmids. One cell line was selected as the donor cell line for somatic cell nuclear transfer (SCNT for the generation of P53 KO pigs. P53 KO stillborn fetuses and living piglets were obtained. Gene typing of the collected cloned individuals was performed by T7EI assay and sequencing. Fibroblast cells from Diannan miniature piglets with a P53 biallelic knockout or wild type were analyzed for the P53 response to doxorubicin treatment by confocal microscopy and western blotting. Results The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 55.35-fold higher than those of the control. Eight cell lines (8/19 were mutated for P53, and five of them were biallelic knockouts. One of the biallelic knockout cell lines was selected as nuclear donor cells for SCNT. The cloned embryos were transferred into five recipient gilts, three of them becoming pregnant. Five live fetuses were obtained from one surrogate by caesarean

  4. Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer.

    Science.gov (United States)

    Shen, Youfeng; Xu, Kaixiang; Yuan, Zaimei; Guo, Jianxiong; Zhao, Heng; Zhang, Xuezeng; Zhao, Lu; Qing, Yubo; Li, Honghui; Pan, Weirong; Jia, Baoyu; Zhao, Hong-Ye; Wei, Hong-Jiang

    2017-11-03

    Pigs have many features that make them attractive as biomedical models for various diseases, including cancer. P53 is an important tumor suppressor gene that exerts a central role in protecting cells from oncogenic transformation and is mutated in a large number of human cancers. P53 mutations occur in almost every type of tumor and in over 50% of all tumors. In a recent publication, pigs with a mutated P53 gene were generated that resulted in lymphoma and renal and osteogenic tumors. However, approximately 80% of human tumors have dysfunctional P53. A P53-deficient pig model is still required to elucidate. Transcription activator-like effector nucleases (TALENs) were designed to target porcine P53 exon 4. The targeting activity was evaluated using a luciferase SSA recombination assay. P53 biallelic knockout (KO) cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs followed by electroporation with TALENs plasmids. One cell line was selected as the donor cell line for somatic cell nuclear transfer (SCNT) for the generation of P53 KO pigs. P53 KO stillborn fetuses and living piglets were obtained. Gene typing of the collected cloned individuals was performed by T7EI assay and sequencing. Fibroblast cells from Diannan miniature piglets with a P53 biallelic knockout or wild type were analyzed for the P53 response to doxorubicin treatment by confocal microscopy and western blotting. The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 55.35-fold higher than those of the control. Eight cell lines (8/19) were mutated for P53, and five of them were biallelic knockouts. One of the biallelic knockout cell lines was selected as nuclear donor cells for SCNT. The cloned embryos were transferred into five recipient gilts, three of them becoming pregnant. Five live fetuses were obtained from one surrogate by caesarean section after 38 days of gestation for genotyping

  5. Mutasi Gen P53: Faktor Prediktif Kanker Payudara?

    OpenAIRE

    Suyanto, Putri Y; Utomo, Ahmad R; Sandra, Ferry

    2008-01-01

    Farmakogenetik dalam terapi kanker adalah pemberian terapi kepada pasien berdasarkan status atau profil genetik dari sel kanker. Dengan demikian, pemberian kemoterapi diharapkan bisa lebih efektif dan meningkatkan kualitas hidup pasien dengan menghindari terapi yang diketahui secara genetik tidak memberikan keuntungan klinis. Pemeriksaan DNA gen p53 sebagai salah satu tumor suppresor gene yang termutasi pada hampir sebagian besar tumor diharapkan mampu menjadi faktor prediktif ketika dilakuka...

  6. Pu-erh Tea Inhibits Tumor Cell Growth by Down-Regulating Mutant p53

    Directory of Open Access Journals (Sweden)

    Ying Luo

    2011-11-01

    Full Text Available Pu-erh tea is a kind of fermented tea with the incorporation of microorganisms’ metabolites. Unlike green tea, the chemical characteristics and bioactivities of Pu-erh tea are still not well understood. Using water extracts of Pu-erh tea, we analyzed the tumor cell growth inhibition activities on several genetically engineered mouse tumor cell lines. We found that at the concentration that did not affect wild type mouse embryo fibroblasts (MEFs growth, Pu-erh tea extracts could inhibit tumor cell growth by down-regulated S phase and cause G1 or G2 arrest. Further study showed that Pu-erh tea extracts down-regulated the expression of mutant p53 in tumor cells at the protein level as well as mRNA level. The same concentration of Pu-erh tea solution did not cause p53 stabilization or activation of its downstream pathways in wild type cells. We also found that Pu-erh tea treatment could slightly down-regulate both HSP70 and HSP90 protein levels in tumor cells. These data revealed the action of Pu-erh tea on tumor cells and provided the possible mechanism for Pu-erh tea action, which explained its selectivity in inhibiting tumor cells without affecting wild type cells. Our data sheds light on the application of Pu-erh tea as an anti-tumor agent with low side effects.

  7. Pu-erh Tea Inhibits Tumor Cell Growth by Down-Regulating Mutant p53

    Science.gov (United States)

    Zhao, Lanjun; Jia, Shuting; Tang, Wenru; Sheng, Jun; Luo, Ying

    2011-01-01

    Pu-erh tea is a kind of fermented tea with the incorporation of microorganisms’ metabolites. Unlike green tea, the chemical characteristics and bioactivities of Pu-erh tea are still not well understood. Using water extracts of Pu-erh tea, we analyzed the tumor cell growth inhibition activities on several genetically engineered mouse tumor cell lines. We found that at the concentration that did not affect wild type mouse embryo fibroblasts (MEFs) growth, Pu-erh tea extracts could inhibit tumor cell growth by down-regulated S phase and cause G1 or G2 arrest. Further study showed that Pu-erh tea extracts down-regulated the expression of mutant p53 in tumor cells at the protein level as well as mRNA level. The same concentration of Pu-erh tea solution did not cause p53 stabilization or activation of its downstream pathways in wild type cells. We also found that Pu-erh tea treatment could slightly down-regulate both HSP70 and HSP90 protein levels in tumor cells. These data revealed the action of Pu-erh tea on tumor cells and provided the possible mechanism for Pu-erh tea action, which explained its selectivity in inhibiting tumor cells without affecting wild type cells. Our data sheds light on the application of Pu-erh tea as an anti-tumor agent with low side effects. PMID:22174618

  8. p53's choice of myocardial death or survival: Oxygen protects infarct myocardium by recruiting p53 on NOS3 promoter through regulation of p53-Lys118 acetylation

    Science.gov (United States)

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

    2013-01-01

    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 Lys118 residue, which was exclusively reversed in the oxygenated heart, apparently regulated by oxygen-dependent expression of TIP60. The inhibition of Lys118 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. PMID:24096875

  9. 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.

    Science.gov (United States)

    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. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

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

    Science.gov (United States)

    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

  11. Studying p53 family proteins in yeast: Induction of autophagic cell death and modulation by interactors and small molecules

    Energy Technology Data Exchange (ETDEWEB)

    Leão, Mariana; Gomes, Sara; Bessa, Cláudia; Soares, Joana; Raimundo, Liliana [REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto (Portugal); Monti, Paola; Fronza, Gilberto [Mutagenesis Unit, Istituto di Ricerca e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino-IST-Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa (Italy); Pereira, Clara [REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto (Portugal); Saraiva, Lucília, E-mail: lucilia.saraiva@ff.up.pt [REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto (Portugal)

    2015-01-01

    In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions. - Highlights: • p53, p63 and p73 are individually studied in the yeast S. cerevisiae. • p53 family members induce ROS production, cell cycle arrest and autophagy in yeast. • p53 family members increase actin depolarization and expression levels in yeast. • MDM2 and MDMX inhibit the activity of p53 family members in yeast. • Yeast can be a useful tool to study the biology and drugability of p53, p63 and p73.

  12. Haploinsufficiency of Def activates p53-dependent TGFβ signalling and causes scar formation after partial hepatectomy.

    Directory of Open Access Journals (Sweden)

    Zhihui Zhu

    Full Text Available The metazoan liver exhibits a remarkable capacity to regenerate lost liver mass without leaving a scar following partial hepatectomy (PH. Whilst previous studies have identified components of several different signaling pathways that are essential for activation of hepatocyte proliferation during liver regeneration, the mechanisms that enable such regeneration to occur without accompanying scar formation remain poorly understood. Here we use the adult zebrafish liver, which can regenerate within two weeks following PH, as a new genetic model to address this important question. We focus on the role of Digestive-organ-expansion-factor (Def, a nucleolar protein which has recently been shown to complex with calpain3 (Capn3 to mediate p53 degradation specifically in the nucleolus, in liver regeneration. Firstly, we show that Def expression is up-regulated in the wild-type liver following amputation, and that the defhi429/+ heteroozygous mutant (def+/- suffers from haploinsufficiency of Def in the liver. We then show that the expression of pro-inflammatory cytokines is up-regulated in the def+/- liver, which leads to distortion of the migration and the clearance of leukocytes after PH. Transforming growth factor β (TGFβ signalling is thus activated in the wound epidermis in def+/- due to a prolonged inflammatory response, which leads to fibrosis at the amputation site. Fibrotic scar formation in def+/- is blocked by the over-expression of Def, by the loss-of-function of p53, and by treatment with anti-inflammation drug dexamethasone or TGFβ-signalling inhibitor SB431542. We finally show that the Def- p53 pathway suppresses fibrotic scar formation, at least in part, through the regulation of the expression of the pro-inflammatory factor, high-mobility group box 1. We conclude that the novel Def- p53 nucleolar pathway functions specifically to prevent a scar formation at the amputation site in a normal amputated liver.

  13. Haploinsufficiency of Def activates p53-dependent TGFβ signalling and causes scar formation after partial hepatectomy.

    Science.gov (United States)

    Zhu, Zhihui; Chen, Jun; Xiong, Jing-Wei; Peng, Jinrong

    2014-01-01

    The metazoan liver exhibits a remarkable capacity to regenerate lost liver mass without leaving a scar following partial hepatectomy (PH). Whilst previous studies have identified components of several different signaling pathways that are essential for activation of hepatocyte proliferation during liver regeneration, the mechanisms that enable such regeneration to occur without accompanying scar formation remain poorly understood. Here we use the adult zebrafish liver, which can regenerate within two weeks following PH, as a new genetic model to address this important question. We focus on the role of Digestive-organ-expansion-factor (Def), a nucleolar protein which has recently been shown to complex with calpain3 (Capn3) to mediate p53 degradation specifically in the nucleolus, in liver regeneration. Firstly, we show that Def expression is up-regulated in the wild-type liver following amputation, and that the defhi429/+ heteroozygous mutant (def+/-) suffers from haploinsufficiency of Def in the liver. We then show that the expression of pro-inflammatory cytokines is up-regulated in the def+/- liver, which leads to distortion of the migration and the clearance of leukocytes after PH. Transforming growth factor β (TGFβ) signalling is thus activated in the wound epidermis in def+/- due to a prolonged inflammatory response, which leads to fibrosis at the amputation site. Fibrotic scar formation in def+/- is blocked by the over-expression of Def, by the loss-of-function of p53, and by treatment with anti-inflammation drug dexamethasone or TGFβ-signalling inhibitor SB431542. We finally show that the Def- p53 pathway suppresses fibrotic scar formation, at least in part, through the regulation of the expression of the pro-inflammatory factor, high-mobility group box 1. We conclude that the novel Def- p53 nucleolar pathway functions specifically to prevent a scar formation at the amputation site in a normal amputated liver.

  14. Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

    Directory of Open Access Journals (Sweden)

    Zanatta Daniela B

    2010-06-01

    p53 was further activated by the combination of p19Arf and nutlin-3. Conclusions To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53-activating agents, for the treatment of tumors that retain wild-type p53.

  15. Probing the functional impact of sequence variation on p53-DNA interactions using a novel microsphere assay for protein-DNA binding with human cell extracts.

    Directory of Open Access Journals (Sweden)

    Maher A Noureddine

    2009-05-01

    Full Text Available The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs. Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation -- including polymorphisms -- and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks.

  16. Paracrine Apoptotic Effect of p53 Mediated by Tumor Suppressor Par-4

    Directory of Open Access Journals (Sweden)

    Ravshan Burikhanov

    2014-01-01

    Full Text Available The guardian of the genome, p53, is often mutated in cancer and may contribute to therapeutic resistance. Given that p53 is intact and functional in normal tissues, we harnessed its potential to inhibit the growth of p53-deficient cancer cells. Specific activation of p53 in normal fibroblasts selectively induced apoptosis in p53-deficient cancer cells. This paracrine effect was mediated by p53-dependent secretion of the tumor suppressor Par-4. Accordingly, the activation of p53 in normal mice, but not p53−/− or Par-4−/− mice, caused systemic elevation of Par-4, which induced apoptosis of p53-deficient tumor cells. Mechanistically, p53 induced Par-4 secretion by suppressing the expression of its binding partner, UACA, which sequesters Par-4. Thus, normal cells can be empowered by p53 activation to induce Par-4 secretion for the inhibition of therapy-resistant tumors.

  17. Vaccination with p53 peptide-pulsed dendritic cells is associated with disease stabilization in patients with p53 expressing advanced breast cancer; monitoring of serum YKL-40 and IL-6 as response biomarkers

    DEFF Research Database (Denmark)

    Svane, Inge Marie; Pedersen, Anders E; Johansen, Julia S

    2007-01-01

    II trial including 26 patients with verified progressive breast cancer are presented. Seven patients discontinued treatment after only 2-3 vaccination weeks due to rapid disease progression or death. Nineteen patients were available for first evaluation after 6 vaccinations; 8/19 evaluable patients......p53 Mutations are found in up to 30% of breast cancers and peptides derived from over-expressed p53 protein are presented by class I HLA molecules and may act as tumor-associated epitopes in cancer vaccines. A dendritic cell (DC) based p53 targeting vaccine was analyzed in HLA-A2+ patients...... with progressive advanced breast cancer. DCs were loaded with 3 wild-type and 3 P2 anchor modified HLA-A2 binding p53 peptides. Patients received up to 10 sc vaccinations with 5 x 10(6) p53-peptide loaded DC with 1-2 weeks interval. Concomitantly, 6 MIU/m(2) interleukine-2 was administered sc. Results from a phase...

  18. A systematic review of p53 regulation of oxidative stress in skeletal muscle.

    Science.gov (United States)

    Beyfuss, Kaitlyn; Hood, David A

    2018-12-01

    p53 is a tumor suppressor protein involved in regulating a wide array of signaling pathways. The role of p53 in the cell is determined by the type of imposed oxidative stress, its intensity and duration. The last decade of research has unravelled a dual nature in the function of p53 in mediating the oxidative stress burden. However, this is dependent on the specific properties of the applied stress and thus requires further analysis. A systematic review was performed following an electronic search of Pubmed, Google Scholar, and ScienceDirect databases. Articles published in the English language between January 1, 1990 and March 1, 2017 were identified and isolated based on the analysis of p53 in skeletal muscle in both animal and cell culture models. Literature was categorized according to the modality of imposed oxidative stress including exercise, diet modification, exogenous oxidizing agents, tissue manipulation, irradiation, and hypoxia. With low to moderate levels of oxidative stress, p53 is involved in activating pathways that increase time for cell repair, such as cell cycle arrest and autophagy, to enhance cell survival. However, with greater levels of stress intensity and duration, such as with irradiation, hypoxia, and oxidizing agents, the role of p53 switches to facilitate increased cellular stress levels by initiating DNA fragmentation to induce apoptosis, thereby preventing aberrant cell proliferation. Current evidence confirms that p53 acts as a threshold regulator of cellular homeostasis. Therefore, within each modality, the intensity and duration are parameters of the oxidative stressor that must be analyzed to determine the role p53 plays in regulating signaling pathways to maintain cellular health and function in skeletal muscle. Acadl: acyl-CoA dehydrogenase, long chain; Acadm: acyl-CoA dehydrogenase, C-4 to C-12 straight chain; AIF: apoptosis-inducing factor; Akt: protein kinase B (PKB); AMPK: AMP-activated protein kinase; ATF-4: activating

  19. Distinct pattern of p53 mutations in bladder cancer

    DEFF Research Database (Denmark)

    Spruck, C H; Rideout, W M; Olumi, A F

    1993-01-01

    double mutations, four of which were tandem mutations on the same allele. No double mutations were found in tumors from nonsmoking patients. None of the mutations in smokers were G:C-->T:A transversions, which would be anticipated for exposure to the suspected cigarette smoke carcinogen 4-aminobiphenyl....... The results suggest that, although cigarette smoke exposure may not significantly alter the kinds of mutations sustained in the p53 gene, it may act to increase the extent of DNA damage per mutagenic event....

  20. A dynamic p53-mdm2 model with distributed delay

    Science.gov (United States)

    Horhat, Raluca; Horhat, Raul Florin

    2014-12-01

    Specific activator and repressor transcription factors which bind to specific regulator DNA sequences, play an important role in gene activity control. Interactions between genes coding such transcripion factors should explain the different stable or sometimes oscillatory gene activities characteristic for different tissues. In this paper, the dynamic P53-Mdm2 interaction model with distributed delays is investigated. Both weak and Dirac kernels are taken into consideration. For Dirac case, the Hopf bifurcation is investigated. Some numerical examples are finally given for justifying the theoretical results.

  1. In vivo footprinting and DNA affinity chromatography for analysis of p53 DNA binding ability.

    Science.gov (United States)

    Molina, Maria Patricia; Cain, Christine; Bargonetti, Jill

    2003-01-01

    p53 is a sequence-specific DNA binding protein. The p53 consensus is two copies of 5'- RRRC(A/T)(T/A)GYYY-3'. The interaction of p53 with specific DNA binding sites (DBS) has been analyzed extensively using electrophoretic mobility shift analysis (EMSA). These studies do not address the interaction of p53 with nuclear chromatin or the stability of p53-DBS interactions. In vivo footprinting examines the dynamic interactions of p53 protein in nuclear chromatin. p53 DBS affinity chromatography compares the stability of p53 from different cellular extracts with different DBS. Isogenic strains expressing high p53 levels, and deleted for p53, are required for controlled experiments using both methods. Different systems can be used to generate sufficient p53 protein (including DNA damage), and this results in the analysis of different forms of p53. A comparison of different cellular sources of high levels of p53 (in the presence and absence of DNA damage) vs different p53 DBS is required to appreciate the complexity of the regulation. Methods for comparing p53 from three different cellular sources with different DBS are presented here. The p53 research community needs to expand this analysis to complete the picture of how p53 differentially regulates transcription of target genes in nuclear chromatin.

  2. Induction of p53-Specific Immunity by a p53 Synthetic Long Peptide Vaccine in Patients Treated for Metastatic Colorectal Cancer

    NARCIS (Netherlands)

    Speetjens, Frank M.; Kuppen, PeterJ. K.; Welters, Marij. J. P.; Essahsah, Farah; van den Brink, Anne Marie E. G. Voet; Lantrua, M. Graziella Kallenberg; Valentijn, A. Rob P. M.; Oostendorp, Jaap; Fathers, Lorraine M.; Nijman, Hans W.; Drijfhout, Jan W.; van de Velde, Cornelis J. H.; Melief, Cornelis J. M.; van der Burg, Sjoerd H.

    2009-01-01

    Purpose: The tumor-associated self-antigen p53 is commonly overexpressed in cancer, including colorectal cancer, and can serve as a target for immunotherapy. The safety and immunogenicity of a p53 synthetic long peptide (p53-SLP) vaccine were investigated in patients treated for metastatic

  3. The p53/miR-34a/SIRT1 Positive Feedback Loop in Quercetin-Induced Apoptosis

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    Guohua Lou

    2015-04-01

    Full Text Available Background: The anti-tumor effects of quercetin have been reported, but the underlying molecular mechanisms remain to be elucidated. The aim of present study was to explore the role of miRNA in the anticancer effects of quercetin. Methods: The differential miRNAs expression between the HepG2 and Huh7 cells treated by quercetin were detected by microarray. The xCELLigence, Flow cytometry, RT-PCR and Western blot were used to analyze the cell proliferation, cell apoptosis, cell cycle arrest, anti-tumor genes, and protein expression. Results: miR-34a was up-regulated in HepG2 cells treated by quercetin exhibiting wild-type p53. When inhibiting the miR-34a, the sensitivity of the cells to quercetin decreased and the expression of the SIRT1 was up-regulated, but the acetylation of p53 and the expression of some genes related to p53 down-regulated. Conclusion: miR-34a plays an important role in the anti-tumor effects of querctin in HCC, miR-34a may be a tiemolecule between the p53 and SIRT1 and is composed of a p53/miR-34a/SIRT1 signal feedback loop, which could enhance apoptosis signal and significantly promote cell apoptosis.

  4. Heterogeneous Hydration of p53/MDM2 Complex

    Science.gov (United States)

    2015-01-01

    Water-mediated interactions play critical roles in biomolecular recognition processes. Explicit solvent molecular dynamics (MD) simulations and the variational implicit-solvent model (VISM) are used to study those hydration properties during binding for the biologically important p53/MDM2 complex. Unlike simple model solutes, in such a realistic and heterogeneous solute–solvent system with both geometrical and chemical complexity, the local water distribution sensitively depends on nearby amino acid properties and the geometric shape of the protein. We show that the VISM can accurately describe the locations of high and low density solvation shells identified by the MD simulations and can explain them by a local coupling balance of solvent–solute interaction potentials and curvature. In particular, capillary transitions between local dry and wet hydration states in the binding pocket are captured for interdomain distance between 4 to 6 Å, right at the onset of binding. The underlying physical connection between geometry and polarity is illustrated and quantified. Our study offers a microscopic and physical insight into the heterogeneous hydration behavior of the biologically highly relevant p53/MDM2 system and demonstrates the fundamental importance of hydrophobic effects for biological binding processes. We hope our study can help to establish new design rules for drugs and medical substances. PMID:24803860

  5. The Inherited p53 Mutation in the Brazilian Population.

    Science.gov (United States)

    Achatz, Maria Isabel; Zambetti, Gerard P

    2016-12-01

    A common criticism of studying rare diseases is the often-limited relevance of the findings to human health. Here, we review ∼15 years of research into an unusual germline TP53 mutation (p.R337H) that began with its detection in children with adrenocortical carcinoma (ACC), a remarkably rare childhood cancer that is associated with poor prognosis. We have come to learn that the p.R337H mutation exists at a very high frequency in Southern and Southeastern Brazil, occurring in one of 375 individuals within a total population of ∼100 million. Moreover, it has been determined that carriers of this founder mutation display variable tumor susceptibility, ranging from isolated cases of pediatric ACC to Li-Fraumeni or Li-Fraumeni-like (LFL) syndromes, thus representing a significant medical issue for this country. Studying the biochemical and molecular consequences of this mutation on p53 tumor-suppressor activity, as well as the putative additional genetic alterations that cooperate with this mutation, is advancing our understanding of how p53 functions in tumor suppression in general. These studies, which originated with a rare childhood tumor, are providing important information for guiding genetic counselors and physicians in treating their patients and are already providing clinical benefit. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

  6. Evidence supporting a role for dihydroorotate dehydrogenase, bioenergetics, and p53 in selective teriflunomide-induced apoptosis in transformed versus normal human keratinocytes.

    Science.gov (United States)

    Hail, Numsen; Chen, Ping; Kepa, Jadwiga J; Bushman, Lane R

    2012-03-01

    We have demonstrated previously that the dihydroorotate dehydrogenase (DHODH) inhibitor teriflunomide (TFN) encourages apoptosis in transformed human keratinocytes. Here we sought to determine if this cytotoxic effect could be restricted to transformed keratinocytes relative to their normal human epidermal keratinocyte (NHEK) counterparts, and ascertain a potential mechanistic basis for the selectivity. The NHEK cells proliferated much slower than the premalignant HaCaT and malignant COLO 16 keratinocytes, and exogenous uridine added to the culture medium did not affect this growth. Similarly, DHODH expression and the bioenergetic characteristics of the normal cells were markedly dissimilar from those observed in the transformed cells indicating that de novo pyrimidine synthesis was involved with keratinocyte proliferation. Moreover, a short-term exposure to TFN caused a wild-type p53 response in the NHEK cells illustrating that pyrimidine metabolic stress could regulate this tumor suppressor protein in the normal cells. TFN-induced apoptosis occurred primarily in S phase HaCaT cells. This cell death was sensitive to uridine, an antioxidant, and a caspase inhibitor, and the suppression of Bcl-X(L) and the induction of Mn superoxide dismutase preceded it. These events suggested that mitochondrial/redox stress was involved with the cytotoxic effect of TFN. Conversely, a long-term exposure to TFN caused G(0)/G(1) arrest in the NHEK cells, which supported a cytoprotective role for p53 against TFN-induced apoptosis. Together, these results propose that TFN could be useful in the prevention or therapy of non-melanoma skin cancers and possibly other hyperproliferative keratinocytic diseases.

  7. Phenotype Specific Analyses Reveal Distinct Regulatory Mechanism for Chronically Activated p53

    Science.gov (United States)

    Cairns, Jonathan M.; Menon, Suraj; Pérez-Mancera, Pedro A.; Tomimatsu, Kosuke; Bermejo-Rodriguez, Camino; Ito, Yoko; Chandra, Tamir; Narita, Masako; Lyons, Scott K.; Lynch, Andy G.; Kimura, Hiroshi; Ohbayashi, Tetsuya; Tavaré, Simon; Narita, Masashi

    2015-01-01

    The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage) and chronically activated (in senescent or pro-apoptotic conditions) p53. Compared to the classical ‘acute’ p53 binding profile, ‘chronic’ p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory ‘p53 hubs’ where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the ‘lipogenic phenotype’, a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms. PMID:25790137

  8. Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes

    NARCIS (Netherlands)

    Simeonova, I.; Jaber, S.; Draskovic, I.; Bardot, B.; Fang, M.; Bouarich-Bourimi, R.; Lejour, V.; Charbonnier, L.; Soudais, C.; Bourdon, J.C.; Huerre, M.; Londono-Vallejo, A.; Toledo, F.

    2013-01-01

    Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53(Delta31), a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis,

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

    Science.gov (United States)

    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...

  10. Pharmacological and Genetic Manipulation of p53 in Brown Fat at Adult But Not Embryonic Stages Regulates Thermogenesis and Body Weight in Male Mice.

    Science.gov (United States)

    Al-Massadi, Omar; Porteiro, Begoña; Kuhlow, Doreen; Köhler, Markus; Gonzalez-Rellan, María J; Garcia-Lavandeira, Montserrat; Díaz-Rodríguez, Esther; Quiñones, Mar; Senra, Ana; Alvarez, Clara V; López, Miguel; Diéguez, Carlos; Schulz, Tim J; Nogueiras, Rubén

    2016-07-01

    p53 is a well-known tumor suppressor that plays multiple biological roles, including the capacity to modulate metabolism at different levels. However, its metabolic role in brown adipose tissue (BAT) remains largely unknown. Herein we sought to investigate the physiological role of endogenous p53 in BAT and its implication on BAT thermogenic activity and energy balance. To this end, we generated and characterized global p53-null mice and mice lacking p53 specifically in BAT. Additionally we performed gain-and-loss-of-function experiments in the BAT of adult mice using virogenetic and pharmacological approaches. BAT was collected and analyzed by immunohistochemistry, thermography, real-time PCR, and Western blot. p53-deficient mice were resistant to diet-induced obesity due to increased energy expenditure and BAT activity. However, the deletion of p53 in BAT using a Myf5-Cre driven p53 knockout did not show any changes in body weight or the expression of thermogenic markers. The acute inhibition of p53 in the BAT of adult mice slightly increased body weight and inhibited BAT thermogenesis, whereas its overexpression in the BAT of diet-induced obese mice reduced body weight and increased thermogenesis. On the other hand, pharmacological activation of p53 improves body weight gain due to increased BAT thermogenesis by sympathetic nervous system in obese adult wild-type mice but not in p53(-/-) animals. These results reveal that p53 regulates BAT metabolism by coordinating body weight and thermogenesis, but these metabolic actions are tissue specific and also dependent on the developmental stage.

  11. Integrative genome analysis of somatic p53 mutant osteosarcomas identifies Ets2-dependent regulation of small nucleolar RNAs by mutant p53 protein.

    Science.gov (United States)

    Pourebrahim, Rasoul; Zhang, Yun; Liu, Bin; Gao, Ruli; Xiong, Shunbin; Lin, Patrick P; McArthur, Mark J; Ostrowski, Michael C; Lozano, Guillermina

    2017-09-15

    TP53 is the most frequently mutated gene in human cancer. Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties that contribute to metastasis, but the mechanisms mediating these functions remain poorly defined in vivo. To elucidate how mutant p53 GOF drives metastasis, we developed a traceable somatic osteosarcoma mouse model that is initiated with either a single p53 mutation (p53R172H) or p53 loss in osteoblasts. Our study confirmed that p53 mutant mice developed osteosarcomas with increased metastasis as compared with p53-null mice. Comprehensive transcriptome RNA sequencing (RNA-seq) analysis of 16 tumors identified a cluster of small nucleolar RNAs (snoRNAs) that are highly up-regulated in p53 mutant tumors. Regulatory element analysis of these deregulated snoRNA genes identified strong enrichment of a common Ets2 transcription factor-binding site. Homozygous deletion of Ets2 in p53 mutant mice resulted in strong down-regulation of snoRNAs and reversed the prometastatic phenotype of mutant p53 but had no effect on osteosarcoma development, which remained 100% penetrant. In summary, our studies identify Ets2 inhibition as a potential therapeutic vulnerability in p53 mutant osteosarcomas. © 2017 Pourebrahim et al.; Published by Cold Spring Harbor Laboratory Press.

  12. Inhibition of p53-Dependent, but Not p53-Independent, Cell Death by U19 Protein from Human Herpesvirus 6B

    Science.gov (United States)

    Kofod-Olsen, Emil; Møller, Janni M. L.; Schleimann, Mariane H.; Bundgaard, Bettina; Bak, Rasmus O.; Øster, Bodil; Mikkelsen, Jacob G.; Hupp, Ted; Höllsberg, Per

    2013-01-01

    Infection with human herpesvirus (HHV)-6B alters cell cycle progression and stabilizes tumor suppressor protein p53. In this study, we have analyzed the activity of p53 after stimulation with p53-dependent and -independent DNA damaging agents during HHV-6B infection. Microarray analysis, Western blotting and confocal microscopy demonstrated that HHV-6B-infected cells were resistant to p53-dependent arrest and cell death after γ irradiation in both permissive and non-permissive cell lines. In contrast, HHV-6B-infected cells died normally through p53-independet DNA damage induced by UV radiation. Moreover, we identified a viral protein involved in inhibition of p53 during HHV-6B-infection. The protein product from the U19 ORF was able to inhibit p53-dependent signaling following γ irradiation in a manner similar to that observed during infection. Similar to HHV-6B infection, overexpression of U19 failed to rescue the cells from p53-independent death induced by UV radiation. Hence, infection with HHV-6B specifically blocks DNA damage-induced cell death associated with p53 without inhibiting the p53-independent cell death response. This block in p53 function can in part be ascribed to the activities of the viral U19 protein. PMID:23555634

  13. p53-independent ibrutinib responses in an Eμ-TCL1 mouse model demonstrates efficacy in high-risk CLL.

    Science.gov (United States)

    Lee, H J; Gallardo, M; Ma, H; Zhang, X; Larsson, C A; Mejia, A; Hornbaker, M J; Qi, Y; Su, X; Pageon, L R; Quintas-Cardama, A; Post, S M

    2016-06-10

    Deletion of the short-arm of chromosome 17 (17p-) is one of the most critical genetic alterations used in chronic lymphocytic leukemia (CLL) risk stratification. The tumor suppressor TP53 maps to this region, and its loss or mutation accelerates CLL progression, hampers response to chemotherapy and shortens survival. Although florescent in situ hybridization analyses for 17p deletions are routinely performed during clinical diagnoses, p53 mutational status is often unexamined. Given the limited clinical data that exists for frontline treatment of patients with CLL harboring TP53 mutations, there is a need to understand the biology of CLL with TP53 mutations and identify treatment strategies for this subset of patients. Herein, we used a CLL mouse model (Eμ-TCL1) harboring one of the most common TP53 hot-spot mutations observed in CLL (p53(R172H), corresponding to p53(R175H) in humans) to evaluate its impact on disease progression, survival, response to therapy and loss of the remaining wild-type Trp53 allele following ibrutinib treatment. We show that ibrutinib was effective in increasing survival, activating cellular programs outside the p53 pathway and did not place selective pressure on the remaining wild-type Trp53 allele. These data provide evidence that ibrutinib acts as an effective treatment for aggressive forms of CLL with TP53 mutations.

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

    Science.gov (United States)

    Armitage, Mark

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

  15. Insights into the Effect of the G245S Single Point Mutation on the Structure of p53 and the Binding of the Protein to DNA.

    Science.gov (United States)

    Lepre, Marco Gaetano; Omar, Sara Ibrahim; Grasso, Gianvito; Morbiducci, Umberto; Deriu, Marco Agostino; Tuszynski, Jack A

    2017-08-16

    The transcription factor p53 is a potent tumor suppressor dubbed as the "guardian of the genome" because of its ability to orchestrate protective biological outputs in response to a variety of oncogenic stresses. Mutation and thus inactivation of p53 can be found in 50% of human tumors. The majority are missense mutations located in the DNA binding region. Among them, G245S is known to be a structural hotspot mutation. To understand the behaviors and differences between the wild-type and mutant, both a dimer of the wild type p53 (wt-p53) and its G245S mutant (G245S-mp53), complexed with DNA, were simulated using molecular dynamics for more than 1 μs. wt-p53 and G245S-mp53 apo monomers were simulated for 1 μs as well. Conformational analyses and binding energy evaluations performed underline important differences and therefore provide insights to understand the G245S-mp53 loss of function. Our results indicate that the G245S mutation destabilizes several structural regions in the protein that are crucial for DNA binding when found in its apo form and highlight differences in the mutant-DNA complex structure compared to the wt protein. These findings not only provide means that can be applied to other p53 mutants but also serve as structural basis for further studies aimed at the development of cancer therapies based on restoring the function of p53.

  16. The sirtuin 1/2 inhibitor tenovin-1 induces a nonlinear apoptosis-inducing factor-dependent cell death in a p53 null Ewing's sarcoma cell line.

    Science.gov (United States)

    Marx, Christian; Marx-Blümel, Lisa; Lindig, Nora; Thierbach, René; Hoelzer, Doerte; Becker, Sabine; Wittig, Susan; Lehmann, Roland; Slevogt, Hortense; Heinzel, Thorsten; Wang, Zhao-Qi; Beck, James F; Sonnemann, Jürgen

    2017-11-18

    The sirtuin 1/2 inhibitor tenovin-1 activates p53 and may have potential in the management of cancer. Here, we investigated the responsiveness of Ewing's sarcoma cells to tenovin-1. We examined its effects in two Ewing's sarcoma cell lines with different p53 status, i.e. in p53 wild-type and p53 null cells. Effects were assessed by flow cytometric analyses of cell death, mitochondrial membrane depolarization and reactive oxygen species (ROS) generation, by caspase 3/7 activity measurement, by mRNA expression profiling and by immunoblotting. Tenovin-1 elicited caspase-mediated cell death in p53 wild-type cells, but caspase-independent cell death in p53 null cells. Remarkably, it induced a nonlinear concentration response in the latter: low concentrations of tenovin-1 were much more effective than were higher concentrations. Tenovin-1's effects in p53 null cells involved gene expression changes of Bcl-2 family members, mitochondrial membrane depolarization, nuclear translocation of apoptosis-inducing factor, ROS formation and DNA damage; all these effects followed a bell-shaped pattern. In conclusion, our results provide new insights into tenovin-1's mode of action by demonstrating that it can induce different pathways of cell death.

  17. AG490 influences UCN-01-induced cytotoxicity in glioma cells in a p53-dependent fashion, correlating with effects on BAX cleavage and BAD phosphorylation.

    Science.gov (United States)

    Jane, Esther P; Premkumar, Daniel R; Pollack, Ian F

    2007-11-08

    We determined the cytotoxicity of AG490 as a single agent and in combination with 7-hydroxystaurosporine (UCN-01) in a panel of malignant human glioma cell lines. Because p53 has important roles in cell cycle checkpoints, it has been anticipated that modulation of checkpoint pathways should sensitize p53 defective cells while sparing the normal cells. Cell proliferation was determined from dose-response curves. AG490 was effective as a cytotoxic agent alone regardless of p53 status. Combining the Chk1 inhibitor UCN-01 dramatically enhanced the response to AG490 in p53-mutated or deleted glioma cells. An opposite effect was noted in p53-wild type cells, in which UCN-01 and AG490 had antagonistic effects on cell proliferation and viability. We found that AG490 enhanced BAD phosphorylation in p53 wild type glioma cells, which appeared to protect against UCN-01-induced cytotoxicity, whereas AG490 enhanced UCN-01-induced cytotoxicity in p53 defective cell lines by suppression of BAD phosphorylation and induction of BAX and PARP cleavage. These observations highlight the potential for genotype-dependent factors to strongly influence response to signaling-targeted therapies in malignant gliomas and the importance of considering such factors in correlative response analyses for these agents.

  18. Tumor-specific signaling to p53 is mimicked by Mdm2 inactivation in zebrafish: insights from mdm2 and mdm4 mutant zebrafish

    Science.gov (United States)

    Chua, J S; Liew, H P; Guo, L; Lane, D P

    2015-01-01

    In mice, the deletion of either Mdm2 or Mdm4 results in a p53-dependent embryonic lethality. We used zinc-finger nucleases to construct mutations in the mdm2 and mdm4 genes of zebrafish. Although the loss of mdm2 results in a p53-dependent early embryonic lethality, mdm4 mutant fish are viable and grow to adulthood. We also found that an in-frame five-amino acid deletion in mdm2 creates a novel hypomorphic allele. The lethal phenotype observed in the mdm2 mutant fish could be partially rescued by injecting mRNA encoding functional Mdm2, and this required the E3 ligase activity of the protein. Complete rescue was obtained by crossing the mdm2 mutant fish onto a p53M214K mutant background. Although p53 mutant fish on a wild-type mdm2 background were shown to accumulate high levels of p53 protein specifically in tumor tissues, we detected extensive staining of p53 in many normal tissues of the mdm2–p53M214K double-mutant fish. Our results are suggestive of the hypothesis that p53 protein accumulates during tumor formation as a result of tumor-specific inactivation of the Mdm2 pathway. PMID:25746004

  19. Gene expression profiles resulting from stable loss of p53 mirrors its role in tissue differentiation.

    Directory of Open Access Journals (Sweden)

    Oliver Couture

    Full Text Available The tumor suppressor gene p53 is involved in a variety of cellular activities such as cellular stress responses, cell cycle regulation and differentiation. In our previous studies we have shown p53's transcription activating role to be important in osteoblast differentiation. There is still a debate in the literature as to whether p53 inhibits or promotes differentiation. We have found p53 heterozygous mice to show a p53 dependency on some bone marker gene expression that is absent in knockout mice. Mice heterozygous for p53 also show a higher incidence of osteosarcomas than p53 knockout mice. This suggests that p53 is able to modify the environment within osteoblasts. In this study we compare changes in gene expression resulting after either a transient or stable reduction in p53. Accordingly we reduced p53 levels transiently and stably in C2C12 cells, which are capable of both myoblast and osteoblast differentiation, and compared the changes in gene expression of candidate genes regulated by the p53 pathway. Using a PCR array to assay for p53 target genes, we have found different expression profiles when comparing stable versus transient knockdown of p53. As expected, several genes with profound changes after transient p53 loss were related to apoptosis and cell cycle regulation. In contrast, stable p53 loss produced a greater change in MyoD and other transcription factors with tissue specific roles, suggesting that long term loss of p53 affects tissue homeostasis to a greater degree than changes resulting from acute loss of p53. These differences in gene expression were validated by measuring promoter activity of different pathway specific genes involved in differentiation. These studies suggest that an important role for p53 is context dependent, with a stable reduction in p53 expression affecting normal tissue physiology more than acute loss of p53.

  20. p53 gene mutations and expression of p53 and mdm2 proteins in invasive breast carcinoma. A comparative analysis with clinico-pathological factors.

    Science.gov (United States)

    Günther, T; Schneider-Stock, R; Rys, J; Niezabitowski, A; Roessner, A

    1997-01-01

    The aim of the study was to analyze p53 gene mutations and the expression of p53 and mdm2 proteins in 31 randomly selected invasive breast carcinomas. The results were then correlated with tumor grade, stage, estrogen receptor status, nodal status, and DNA ploidy. The expression of the proteins p53 and mdm2 was determined immunohistochemically using formalin-fixed, paraffin-embedded material. Screening for p53 mutation involved analysis of the highly conserved regions of the p53 gene (exons 5-9) by the polymerase chain reaction/ single-strand conformation polymorphism (PCR-SSCP) technique. PCR products with band shifts were directly sequenced. Immunohistochemical staining of p53 was positive in 9 cases (29.0%), only 2 of which showed a p53 gene mutation. These were identified as a C-->G transversion at the second position of codon 278 in exon 8 and an A-->G transition at the second position of codon 205 in exon 6. A third case with a mutation was observed (C-->T transition, position 1 of codon 250 in exon 7) that did not show p53 immunohistochemically. Of the 9 p53-positive tumors, 2 were moderately differentiated (grade II). The remaining tumors were poorly differentiated (7/9). By contrast, p53-negative carcinomas were well differentiated (grade I) in most cases (P = 0.02). DNA cytometry in 8 of the 9 p53-positive carcinomas revealed an aneuploid stem line. The majority of the p53-negative tumors were diploid (P = 0.01). Mdm2 oncoprotein was detected in 10 tumors (32.2%), 4 of which were p53-positive, including the 3 with mutations. The grading of the mdm2-positive tumors was moderate or poor, G1 carcinomas were always noted to be mdm2-negative (P = 0.04). Overexpression of p53 protein is a complex mechanism and does not merely indicate the detection of mutations in the p53 gene. This study has shown that p53 expression correlates with tumor grade and DNA ploidy. Mdm2 expression was also associated with the tumor grade. Immunohistological demonstration of the p53

  1. Vaccination with p53-peptide-pulsed dendritic cells, of patients with advanced breast cancer: report from a phase I study

    DEFF Research Database (Denmark)

    Svane, Inge Marie; Pedersen, Anders E; Johnsen, Hans E

    2004-01-01

    . Our preclinical studies have shown that wild-type p53-derived HLA-A2-binding peptides are able to activate human T cells and that the generated effector T cells are cytotoxic to human HLA-A2+, p53+ tumour cells. In this phase I pilot study, the toxicity and efficacy of autologous dendritic cells (DCs...... not of course be excluded; further studies are necessary to answer these questions....

  2. Radiosensitivity in lung cancer with focus on p53

    CERN Document Server

    Bergqvist, M

    2002-01-01

    In Sweden approximately 2800 new lung cancer patients are diagnosed every year. Radiotherapy is used with curative intention in certain groups of patients. The aim of this thesis is to study the basis of differences in radioresistance and the possibility to predict response to radiotherapy. In the first study we investigated, using the comet assay, four lung cancer cell lines with different sensitivity towards radiation. A clear dose-response relationship for radiation-induced DNA single strand and double strand breaks were found. All cell lines showed a remarkably efficient repair of both the DNA single strand and double strand breaks one hour after irradiation. However, further studies in one radioresistant and one radiosensitive cell line demonstrated that repair during the first 15 min had the best accordance with radiosensitivity measured as surviving fraction. In the second and third study, sequencing studies of the p53 gene were performed on cell lines as well as on tumour material. Cell lines that wer...

  3. Ninjurin 1 has two opposing functions in tumorigenesis in a p53-dependent manner.

    Science.gov (United States)

    Yang, Hee Jung; Zhang, Jin; Yan, Wensheng; Cho, Seong-Jun; Lucchesi, Christopher; Chen, Mingyi; Huang, Eric C; Scoumanne, Ariane; Zhang, Weici; Chen, Xinbin

    2017-10-24

    WT p53 is critical for tumor suppression, whereas mutant p53 promotes tumor progression. Nerve injury-induced protein 1 (Ninj1) is a target of p53 and forms a feedback loop with p53 by repressing p53 mRNA translation. Here, we show that loss of Ninj1 increased mutant p53 expression and, subsequently, enhanced cell growth and migration in cells carrying a mutant p53. In contrast, loss of Ninj1 inhibited cell growth and migration in cells carrying a WT p53. To explore the biological significance of Ninj1, we generated a cohort of Ninj1-deficient mice and found that Ninj1+/- mice were prone to systemic inflammation and insulitis, but not to spontaneous tumors. We also found that loss of Ninj1 altered the tumor susceptibility in both mutant p53 and p53-null background. Specifically, in a mutant p53(R270H) background, Ninj1 deficiency shortened the lifespan, altered the tumor spectrum, and increased tumor burden, likely via enhanced expression of mutant p53. In a p53-null background, Ninj1 deficiency significantly increased the incidence of T-lymphoblastic lymphoma. Taken together, our data suggest that depending on p53 genetic status, Ninj1 has two opposing functions in tumorigenesis and that the Ninj1-p53 loop may be targeted to manage inflammatory diseases and cancer. Published under the PNAS license.

  4. Sirt1 overexpression suppresses fluoride-induced p53 acetylation to alleviate fluoride toxicity in ameloblasts responsible for enamel formation.

    Science.gov (United States)

    Suzuki, Maiko; Ikeda, Atsushi; Bartlett, John D

    2017-11-28

    Low-dose fluoride is an effective caries prophylactic, but high-dose fluoride is an environmental health hazard that causes skeletal and dental fluorosis. Treatments to prevent fluorosis and the molecular pathways responsive to fluoride exposure remain to be elucidated. Previously we showed that fluoride activates SIRT1 as an adaptive response to protect cells. Here, we demonstrate that fluoride induced p53 acetylation (Ac-p53) [Lys379], which is a SIRT1 deacetylation target, in ameloblast-derived LS8 cells in vitro and in enamel organ in vivo. Here we assessed SIRT1 function on fluoride-induced Ac-p53 formation using CRISPR/Cas9-mediated Sirt1 knockout (LS8Sirt/KO) cells or CRISPR/dCas9/SAM-mediated Sirt1 overexpressing (LS8Sirt1/over) cells. NaF (5 mM) induced Ac-p53 formation and increased cell cycle arrest via Cdkn1a/p21 expression in Wild-type (WT) cells. However, fluoride-induced Ac-p53 was suppressed by the SIRT1 activator resveratrol (50 µM). Without fluoride, Ac-p53 persisted in LS8Sirt/KO cells, whereas it decreased in LS8Sirt1/over. Fluoride-induced Ac-p53 formation was also suppressed in LS8Sirt1/over cells. Compared to WT cells, fluoride-induced Cdkn1a/p21 expression was elevated in LS8Sirt/KO and these cells were more susceptible to fluoride-induced growth inhibition. In contrast, LS8Sirt1/over cells were significantly more resistant. In addition, fluoride-induced cytochrome-c release and caspase-3 activation were suppressed in LS8Sirt1/over cells. Fluoride induced expression of the DNA double strand break marker γH2AX in WT cells and this was augmented in LS8Sirt1/KO cells, but was attenuated in LS8Sirt1/over cells. Our results suggest that SIRT1 deacetylates Ac-p53 to mitigate fluoride-induced cell growth inhibition, mitochondrial damage, DNA damage and apoptosis. This is the first report implicating Ac-p53 in fluoride toxicity.

  5. Exogenous PTHrP Repairs the Damaged Fracture Healing of PTHrP+/− Mice and Accelerates Fracture Healing of Wild Mice

    Directory of Open Access Journals (Sweden)

    Yinhe Wang

    2017-02-01

    Full Text Available Bone fracture healing is a complicated physiological regenerative process initiated in response to injury and is similar to bone development. To demonstrate whether an exogenous supply of parathyroid hormone–related protein (PTHrP helps in bone fracture healing, closed mid-diaphyseal femur fractures were created and stabilized with intramedullary pins in eight-week-old wild-type (WT PTHrP+/+ and PTHrP+/− mice. After administering PTHrP for two weeks, callus tissue properties were analyzed at one, two, and four weeks post-fracture (PF by various methods. Bone formation–related genes and protein expression levels were evaluated by real-time reverse transcriptase–polymerase chain reaction and Western blots. At two weeks PF, mineral density of callus, bony callus areas, mRNA levels of alkaline phosphatase (ALP, type I collagen, Runt-related transcription factor 2 (Runx-2, and protein levels of Runx-2 and insulin-like growth factor-1 decreased in PTHrP+/− mice compared with WT mice. At four weeks PF, total collagen-positive bony callus areas, osteoblast number, ALP-positive areas, and type I collagen-positive areas all decreased in PTHrP+/− mice. At both two and four weeks PF, tartrate-resistant acid phosphatase–positive osteoclast number and surface decreased a little in PTHrP+/− mice. The study indicates that exogenous PTHrP provided by subcutaneous injection could redress impaired bone fracture healing, leading to mutation of activated PTHrP by influencing callus areas, endochondral bone formation, osteoblastic bone formation, and bone turnover.

  6. Exogenous PTHrP Repairs the Damaged Fracture Healing of PTHrP+/− Mice and Accelerates Fracture Healing of Wild Mice

    Science.gov (United States)

    Wang, Yinhe; Fang, Xin; Wang, Chun; Ding, Congzhu; Lin, Hua; Liu, Anlong; Wang, Lei; Cao, Yang

    2017-01-01

    Bone fracture healing is a complicated physiological regenerative process initiated in response to injury and is similar to bone development. To demonstrate whether an exogenous supply of parathyroid hormone–related protein (PTHrP) helps in bone fracture healing, closed mid-diaphyseal femur fractures were created and stabilized with intramedullary pins in eight-week-old wild-type (WT) PTHrP+/+ and PTHrP+/− mice. After administering PTHrP for two weeks, callus tissue properties were analyzed at one, two, and four weeks post-fracture (PF) by various methods. Bone formation–related genes and protein expression levels were evaluated by real-time reverse transcriptase–polymerase chain reaction and Western blots. At two weeks PF, mineral density of callus, bony callus areas, mRNA levels of alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx-2), and protein levels of Runx-2 and insulin-like growth factor-1 decreased in PTHrP+/− mice compared with WT mice. At four weeks PF, total collagen-positive bony callus areas, osteoblast number, ALP-positive areas, and type I collagen-positive areas all decreased in PTHrP+/− mice. At both two and four weeks PF, tartrate-resistant acid phosphatase–positive osteoclast number and surface decreased a little in PTHrP+/− mice. The study indicates that exogenous PTHrP provided by subcutaneous injection could redress impaired bone fracture healing, leading to mutation of activated PTHrP by influencing callus areas, endochondral bone formation, osteoblastic bone formation, and bone turnover. PMID:28178186

  7. Serum p53 antibody as a potential tumor marker in extrahepatic cholangiocarcinoma.

    Science.gov (United States)

    Okada, Rei; Shimada, Hideaki; Otsuka, Yuichiro; Tsuchiya, Masaru; Ishii, Jun; Katagiri, Toshio; Maeda, Tetsuya; Kubota, Yoshihisa; Nemoto, Tetsuo; Kaneko, Hironori

    2017-12-01

    Only a few studies have evaluated the clinicopathological significance of the p53 protein expression and s-p53-Abs level in patients with cholangiocarcinoma. We therefore analyzed the clinicopathological and prognostic significance of s-p53-Abs in patients with extrahepatic cholangiocarcinoma. We prospectively evaluated s-p53-Abs levels before and after surgery in 61 patients with extrahepatic cholangiocarcinoma to determine the relationship between clinicopathological factors and the prognostic significance of s-p53-Abs. Among a total of 61 primary extrahepatic cholangiocarcinoma cases, 23% were positive for s-p53-Abs. Combination of s-p53-Abs with the conventional serum markers carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) significantly increased the rate of positive extrahepatic cholangiocarcinoma cases (57% for CEA and/or CA19-9 vs. 75% for CEA and/or CA19-9 and/or s-p53-Abs, P = 0.035). There were no significant differences in clinicopathological factors between the p53-seropositive and p53-seronegative patients. An immunohistochemical analysis showed the presence of significant associations between the intensity (P = 0.003) and extent (P = 0.001) of p53 immunoreactivity and p53-seropositivitly. Although s-p53-Abs was not a significant prognostic factor for the survival in either univariate or multivariate analyses, p53 immunoreactivity was independently associated with a poor survival. Among patients positive for s-p53-Abs before surgery, the s-p53-Abs levels were reduced after surgery in most. These findings suggested that s-p53-Abs might be associated with p53 immunoreactivity. In addition, s-p53-Abs may be useful for a diagnosis, but was not useful for predicting tumor recurrence or the survival. This study was registered as UMIN000014530.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chiu, Shu-Jun, E-mail: chiusj@mail.tcu.edu.tw [Department of Life Science, Tzu Chi University, Hualien, Taiwan (China); Institute of Radiation Sciences, Tzu Chi Technology College, Hualien, Taiwan (China); Hsaio, Ching-Hui; Tseng, Ho-Hsing; Su, Yu-Han [Department of Life Science, Tzu Chi University, Hualien, Taiwan (China); Shih, Wen-Ling [Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung, Taiwan (China); Lee, Jeng-Woei; Chuah, Jennifer Qiu-Yu [Department of Life Science, Tzu Chi University, Hualien, Taiwan (China)

    2010-04-09

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

  9. Cytotoxic effects of MGI 114 are independent of tumor p53 or p21 expression.

    Science.gov (United States)

    Izbicka, E; Davidson, K; Lawrence, R; Cote, R; MacDonald, J R; Von Hoff, D D

    1999-01-01

    MGI 114, an analog of illudin S, shows potent activity against a broad range of human tumors in vitro and in vivo, including drug resistant tumors. In this study we examined cytotoxicity of MGI 114 against human tumor cell lines (MCF7, MDA.MB.468, EJ1, J82, SCaBER, KG-1, HL60, and IMR-90) with differing expression of p53 and/or p21 (WAF1) tumor suppressor genes. Only MCF7 and IMR-90 express the wild type p53, WAF1 is present in high levels in MCF7 and SCaBER. WAF1 expression can be induced in KG-1, HL60, and IMR-90. The cells were treated with MGI 114 at 0.1, 1.0 and 10 micrograms/ml in 1 h exposure and with 0.01, 0.1 and 1.0 microgram/ml MGI 114 in a continuous exposure. Cell numbers were measured at days 2, 4, and 7. MGI 114 suppressed growth in all cell lines at day 2 after 1 h exposure at the two highest concentrations and at all concentrations in a continuous exposure. Some cells partly recovered from the inhibition by day 4. Expression of WAF1 had no apparent effect on growth suppression by MGI 114, however, cells with inducible WAF1 showed slower recovery from MGI 114 inhibition in comparison with the cells under non-permissive conditions. Overall, MGI 114 effectively inhibited growth of human cancer cells regardless of their p53 and WAF1 status.

  10. Proteasomal degradation of p53 by human papillomavirus E6 oncoprotein relies on the structural integrity of p53 core domain.

    Directory of Open Access Journals (Sweden)

    Xavier Bernard

    Full Text Available The E6 oncoprotein produced by high-risk mucosal HPV stimulates ubiquitinylation and proteasome-dependent degradation of the tumour suppressor p53 via formation of a trimeric complex comprising E6, p53, and E6-AP. p53 is also degraded by its main cellular regulator MDM2. The main binding site of p53 to MDM2 is situated in the natively unfolded N-terminal region of p53. By contrast, the regions of p53 implicated in the degradation by viral E6 are not fully identified to date. Here we generated a series of mutations (Y103G, Y107G, T155A, T155V, T155D, L264A, L265A targeting the central folded core domain of p53 within a region opposite to its DNA-binding site. We analysed by in vitro and in vivo assays the impact of these mutations on p53 degradation mediated by viral E6 oncoprotein. Whereas all mutants remained susceptible to MDM2-mediated degradation, several of them (Y103G, Y107G, T155D, L265A became resistant to E6-mediated degradation, confirming previous works that pointed to the core domain as an essential region for the degradation of p53. In parallel, we systematically checked the impact of the mutations on the transactivation activity of p53 as well as on the conformation of p53, analysed by Nuclear Magnetic Resonance (NMR, circular dichroism (CD, and antibody probing. These measurements suggested that the conformational integrity of the core domain is an essential parameter for the degradation of p53 by E6, while it is not essential for the degradation of p53 by MDM2. Thus, the intracellular stability of a protein may or may not rely on its biophysical stability depending on the degradation pathway taken into consideration.

  11. The differentially mutational spectra of the APC, K-ras, and p53 genes in sporadic colorectal cancers from Taiwanese patients.

    Science.gov (United States)

    Wang, Jaw-Yuan; Hsieh, Jan-Sing; Lu, Chien-Yu; Yu, Fang-Jung; Wu, Jeng-Yih; Chen, Fang-Ming; Huang, Che-Jen; Lin, Shiu-Ru

    2007-12-01

    Adenomatous polyposis coli (APC), K-ras and p53 gene mutations are the most common genetic alterations present in colorectal cancer (CRC). The aim of this study was to analyze tumor mutation frequencies and spectra in a large cohort of Taiwanese patients with CRC. APC, K-ras, and p53 gene mutations in primary tumor tissues and their paired normal tissues of 123 CRC patients were detected by polymerase chain reaction-single strand conformation polymorphism analysis, followed by direct sequencing. Of these 123 CRC patients, 43.1%, 44.7%, 35% of tumor tissue specimens presented mutations in APC, K-ras, and p53 genes, respectively. Overall, gene mutations in APC, K-ras and/or p53 were present in 78% (96/123) of tumor tissues. Among 96 CRC patients harboring gene mutations, 49 (51%) contained mutations of at least two different genes and 47 (49%) contained mutations of one gene only. The most common combination of gene mutations was APC and K-ras mutations (21.9%), followed by K-ras and p53 mutations (12.5%) and then APC and p53 mutations (10.4%). In addition, there were only 6.3% (6/96) of tumor tissues from CRC patients simultaneously containing mutations of APC, K-ras and p53 genes. The most common mutation spectrum of these genes was missense mutations, at a frequency of 38.8%, 92.7% and 70.5% for APC, K-ras and p53 genes, respectively. These data support that the frequencies and patterns of somatic mutation of the APC, Kras and p53 genes in CRCs are considerably variable and distinct among populations, for which the interaction between exogenous environmental factors and endogenous gene alterations may be important determinants.

  12. The induction of tumour suppressor protein P53 limits the entry of cells into the pluripotent inner cell mass lineage in the mouse embryo.

    Science.gov (United States)

    Ganeshan, L; Jin, X L; O'Neill, C

    2017-09-15

    The early preimplantation embryo is susceptible to a range of exogenous stresses which result in their reduced long-term developmental potential. The P53 tumour suppressor protein is normally held at low levels in the preimplantation embryo and we show that culture stress induces the expression of a range of canonical P53-response genes (Mdm2, Bax and Cdkn1a). Culture stress caused a P53-dependent loss of cells from resulting blastocysts, and this was most evident within the inner cell mass population. Culture stress increased the proportion of cells expressing active caspase-3 and undergoing apoptosis, while inhibition of caspase-3 increased the number of cells within the inner cell mass. The P53-dependent loss of cells from the inner cell mass was accompanied by a loss of NANOG-positive epiblast progenitors. Pharmacological activation of P53 by the MDM2 inhibitor, Nutlin-3, also caused increased P53-dependent transcription and the loss of cells from the inner cell mass. This loss of cells could be ameliorated by simultaneous treatment with the P53 inhibitor, Pifithrin-α. Culture stress causes reduced signalling via the phosphatidylinositol-3-kinase signalling pathway, and blocking this pathway caused P53-dependent loss of cells from the inner cell mass. These results point to P53 acting to limit the accumulation and survival of cells within the pluripotent lineage of the blastocyst and provide a molecular framework for the further investigation of the factors determining the effects of stressors on the embryo's developmental potential. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  13. Macrophage heme oxygenase-1-SIRT1-p53 axis regulates sterile inflammation in liver ischemia-reperfusion injury.

    Science.gov (United States)

    Nakamura, Kojiro; Zhang, Min; Kageyama, Shoichi; Ke, Bibo; Fujii, Takehiro; Sosa, Rebecca A; Reed, Elaine F; Datta, Nakul; Zarrinpar, Ali; Busuttil, Ronald W; Araujo, Jesus A; Kupiec-Weglinski, Jerzy W

    2017-12-01

    Hepatic ischemia-reperfusion injury (IRI), characterized by exogenous antigen-independent local inflammation and hepatocellular death, represents a risk factor for acute and chronic rejection in liver transplantation. We aimed to investigate the molecular communication involved in the mechanism of liver IRI. We analyzed human liver transplants, primary murine macrophage cell cultures and IR-stressed livers in myeloid-specific heme oxygenase-1 (HO-1) gene mutant mice, for anti-inflammatory and cytoprotective functions of macrophage-specific HO-1/SIRT1 (sirtuin 1)/p53 (tumor suppressor protein) signaling. Decreased HO-1 expression in human post-reperfusion liver transplant biopsies correlated with a deterioration in hepatocellular function (serum ALT; pp53/MDM2 (murine double minute 2) expression levels decreased (pp53, which in turn attenuated macrophage activation. In a murine model of hepatic warm IRI, myeloid-specific HO-1 deletion lacked SIRT1/p53, exacerbated liver inflammation and IR-hepatocellular death, whereas adjunctive SIRT1 activation restored p53 signaling and rescued livers from IR-damage. This bench-to-bedside study identifies a new class of macrophages activated via the HO-1-SIRT1-p53 signaling axis in the mechanism of hepatic sterile inflammation. This mechanism could be a target for novel therapeutic strategies in liver transplant recipients. Post-transplant low macrophage HO-1 expression in human liver transplants correlates with reduced hepatocellular function and survival. HO-1 regulates macrophage activation via the SIRT1-p53 signaling network and regulates hepatocellular death in liver ischemia-reperfusion injury. Thus targeting this pathway in liver transplant recipients could be of therapeutic benefit. Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  14. Liver-specific expressions of HBx and src in the p53 mutant trigger hepatocarcinogenesis in zebrafish.

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    Jeng-Wei Lu

    Full Text Available Hepatocarcinogenesis is a multistep process that starts from fatty liver and transitions to fibrosis and, finally, into cancer. Many etiological factors, including hepatitis B virus X antigen (HBx and p53 mutations, have been implicated in hepatocarcinogenesis. However, potential synergistic effects between these two factors and the underlying mechanisms by which they promote hepatocarcinogenesis are still unclear. In this report, we show that the synergistic action of HBx and p53 mutation triggers progressive hepatocellular carcinoma (HCC formation via src activation in zebrafish. Liver-specific expression of HBx in wild-type zebrafish caused steatosis, fibrosis and glycogen accumulation. However, the induction of tumorigenesis by HBx was only observed in p53 mutant fish and occurred in association with the up-regulation and activation of the src tyrosine kinase pathway. Furthermore, the overexpression of src in p53 mutant zebrafish also caused hyperplasia, HCC, and sarcomatoid HCC, which were accompanied by increased levels of the signaling proteins p-erk, p-akt, myc, jnk1 and vegf. Increased expression levels of lipogenic factors and the genes involved in lipid metabolism and glycogen storage were detected during the early stages of hepatocarcinogenesis in the HBx and src transgenic zebrafish. The up-regulation of genes involved in cell cycle regulation, tumor progression and other molecular hallmarks of human liver cancer were found at later stages in both HBx and src transgenic, p53 mutant zebrafish. Together, our study demonstrates that HBx and src overexpression induced hepatocarcinogenesis in p53 mutant zebrafish. This phenomenon mimics human HCC formation and provides potential in vivo platforms for drug screening for therapies for human liver cancer.

  15. Role of p53-fibrinolytic system cross-talk in the regulation of quartz-induced lung injury.

    Science.gov (United States)

    Bhandary, Yashodhar P; Shetty, Shwetha K; Marudamuthu, Amarnath S; Fu, Jian; Pinson, Barbara M; Levin, Jeffrey; Shetty, Sreerama

    2015-03-01

    Silica is the major component of airborne dust generated by wind, manufacturing and/or demolition. Chronic occupational inhalation of silica dust containing crystalline quartz is by far the predominant form of silicosis in humans. Silicosis is a progressive lung disease that typically arises after a very long latency and is a major occupational concern with no known effective treatment. The mechanism of silicosis is not clearly understood. However, silicosis is associated with increased cell death, expression of redox enzymes and pro-fibrotic cytokines and chemokines. Since alveolar epithelial cell (AEC) death and disruption of alveolar fibrinolysis is often associated with both acute and chronic lung injuries, we explored whether p53-mediated changes in the urokinase-type plasminogen activator (uPA) system contributes to silica-induced lung injury. We further sought to determine whether caveolin-1 scaffolding domain peptide (CSP), which inhibits p53 expression, mitigates lung injury associated with exposure to silica. Lung tissues and AECs isolated from wild-type (WT) mice exposed to silica exhibit increased apoptosis, p53 and PAI-1, and suppression of uPA expression. Treatment of WT mice with CSP inhibits PAI-1, restores uPA expression and prevents AEC apoptosis by suppressing p53, which is otherwise induced in mice exposed to silica. The process involves CSP-mediated inhibition of serine-15 phosphorylation of p53 by inhibition of protein phosphatase 2A-C (PP2A-C) interaction with silica-induced caveolin-1 in AECs. These observations suggest that changes in the p53-uPA fibrinolytic system cross-talk contribute to lung injury caused by inhalation of silica dust containing crystalline quartz and is protected by CSP by targeting this pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Long Noncoding RNA PURPL Suppresses Basal p53 Levels and Promotes Tumorigenicity in Colorectal Cancer.

    Science.gov (United States)

    Li, Xiao Ling; Subramanian, Murugan; Jones, Matthew F; Chaudhary, Ritu; Singh, Deepak K; Zong, Xinying; Gryder, Berkley; Sindri, Sivasish; Mo, Min; Schetter, Aaron; Wen, Xinyu; Parvathaneni, Swetha; Kazandjian, Dickran; Jenkins, Lisa M; Tang, Wei; Elloumi, Fathi; Martindale, Jennifer L; Huarte, Maite; Zhu, Yuelin; Robles, Ana I; Frier, Susan M; Rigo, Frank; Cam, Maggie; Ambs, Stefan; Sharma, Sudha; Harris, Curtis C; Dasso, Mary; Prasanth, Kannanganattu V; Lal, Ashish

    2017-09-05

    Basal p53 levels are tightly suppressed under normal conditions. Disrupting this regulation results in elevated p53 levels to induce cell cycle arrest, apoptosis, and tumor suppression. Here, we report the suppression of basal p53 levels by a nuclear, p53-regulated long noncoding RNA that we termed PURPL (p53 upregulated regulator of p53 levels). Targeted depletion of PURPL in colorectal cancer cells results in elevated basal p53 levels and induces growth defects in cell culture and in mouse xenografts. PURPL associates with MYBBP1A, a protein that binds to and stabilizes p53, and inhibits the formation of the p53-MYBBP1A complex. In the absence of PURPL, MYBBP1A interacts with and stabilizes p53. Silencing MYBBP1A significantly rescues basal p53 levels and proliferation in PURPL-deficient cells, suggesting that MYBBP1A mediates the effect of PURPL in regulating p53. These results reveal a p53-PURPL auto-regulatory feedback loop and demonstrate a role for PURPL in maintaining basal p53 levels. Published by Elsevier Inc.

  17. Identification of p53 and Its Isoforms in Human Breast Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Zorka Milićević

    2014-01-01

    Full Text Available In breast carcinoma, disruption of the p53 pathway is one of the most common genetic alterations. The observation that the p53 can express multiple protein isoforms adds a novel level of complexity to the outcome of p53 mutations. p53 expression was analysed by Western immunoblotting and immunohistochemistry using monoclonal antibodies DO-7, Pab240, and polyclonal antiserum CM-1. The more frequently p53-positive nuclear staining has been found in the invasive breast tumors. One of the most intriguing findings is that mutant p53 appears as discrete dot-shaped regions within the nucleus of breast cancer cells. In many malignant cells, the nucleolar sequestration of p53 is evident. These observations support the view that the nucleolus is involved directly in the mediation of p53 function or indirectly by the control of the localization of p53 interplayers. p53 expressed in the nuclear fraction of breast cancer cells revealed a wide spectrum of isoforms. p53 isoforms ΔNp53 (47 kDa and Δ133p53β (35 kDa, known as dominant-negative repressors of p53 function, were detected as the most predominant variants in nuclei of invasive breast carcinoma cells. The isoforms expressed also varied between individual tumors, indicating potential roles of these p53 variants in human breast cancer.

  18. Reactivating p53 and Inducing Tumor Apoptosis (RITA Enhances the Response of RITA-Sensitive Colorectal Cancer Cells to Chemotherapeutic Agents 5-Fluorouracil and Oxaliplatin

    Directory of Open Access Journals (Sweden)

    Armin Wiegering

    2017-04-01

    Full Text Available Colorectal carcinoma (CRC is the most common cancer of the gastrointestinal tract with frequently dysregulated intracellular signaling pathways, including p53 signaling. The mainstay of chemotherapy treatment of CRC is 5-fluorouracil (5FU and oxaliplatin. The two anticancer drugs mediate their therapeutic effect via DNA damage-triggered signaling. The small molecule reactivating p53 and inducing tumor apoptosis (RITA is described as an activator of wild-type and reactivator of mutant p53 function, resulting in elevated levels of p53 protein, cell growth arrest, and cell death. Additionally, it has been shown that RITA can induce DNA damage signaling. It is expected that the therapeutic benefits of 5FU and oxaliplatin can be increased by enhancing DNA damage signaling pathways. Therefore, we highlighted the antiproliferative response of RITA alone and in combination with 5FU or oxaliplatin in human CRC cells. A panel of long-term established CRC cell lines (n = 9 including p53 wild-type, p53 mutant, and p53 null and primary patient-derived, low-passage cell lines (n = 5 with different p53 protein status were used for this study. A substantial number of CRC cells with pronounced sensitivity to RITA (IC50< 3.0 μmol/l were identified within established (4/9 and primary patient-derived (2/5 CRC cell lines harboring wild-type or mutant p53 protein. Sensitivity to RITA appeared independent of p53 status and was associated with an increase in antiproliferative response to 5FU and oxaliplatin, a transcriptional increase of p53 targets p21 and NOXA, and a decrease in MYC mRNA. The effect of RITA as an inducer of DNA damage was shown by a strong elevation of phosphorylated histone variant H2A.X, which was restricted to RITA-sensitive cells. Our data underline the primary effect of RITA, inducing DNA damage, and demonstrate the differential antiproliferative effect of RITA to CRC cells independent of p53 protein status. We found a substantial number

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

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Shi-Wei [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Wu, Chun-Ying [Division of Gastroenterology and Hepatology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Wang, Yen-Ting [Department of Medical Research and Education, Cheng Hsin General Hospital, Taipei, Taiwan (China); Kao, Jun-Kai [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Department of Pediatrics, Children' s Hospital, Changhua Christian Hospital, Changhua, Taiwan (China); Lin, Chi-Chen; Chang, Chia-Che; Mu, Szu-Wei; Chen, Yu-Yu [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Chiu, Husan-Wen [Institute of Biotechnology, National Cheng-Kung University, Tainan, Taiwan (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan (China); Chang, Chuan-Hsun [Department of Surgical Oncology, Cheng Hsin General Hospital, Taipei, Taiwan (China); Department of Nutrition Therapy, Cheng Hsin General Hospital, Taipei, Taiwan (China); School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan (China); Liang, Shu-Mei [Institute of Biotechnology, National Cheng-Kung University, Tainan, Taiwan (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan (China); Chen, Yi-Ju [Department of Dermatology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Huang, Jau-Ling [Department of Bioscience Technology, Chang Jung Christian University, Tainan, Taiwan (China); Shieh, Jeng-Jer, E-mail: shiehjj@vghtc.gov.tw [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan (China)

    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. Inhibition of p53 by lentiviral mediated shRNA abrogates G1 arrest and apoptosis in retinal pigmented epithelial cell line.

    Science.gov (United States)

    Nair, Ayyappan R; Schliekelman, Mark; Thomas, Mary Beth; Wakefield, John; Jurgensen, Stewart; Ramabhadran, Ram

    2005-05-01

    We silenced p53 gene expression in ARPE-19, a human retinal pigmented epithelial cell line using RNA interference. The effect of silencing the p53 gene in proliferating ARPE-19 cells was studied. Four short hairpin RNAs (shRNAs) targeting different regions of human p53 mRNA were delivered individually into ARPE-19 cells using lentiviral vector to produce stable cell lines. p53 mRNA and protein levels were reduced to varying extents in the four shRNA-transduced ARPE-19 cell lines. The cell line that showed greatest reduction (85-90%) of p53 expression showed decreased p21 promoter activation after DNA damage with camptothecin, etoposide and MMS. Whereas treatment of wild type ARPE-19 cells with camptothecin resulted in apoptosis, silencing p53 expression increased their survival. Cell cycle analyses indicated that irradiation resulted in a G(1) arrest in ARPE-19 cells, and that the arrest was significantly reduced in p53-silenced cells. Thus, p53 plays a central role in the response of ARPE-19 cells to DNA damaging agents that act via different mechanisms. Additionally, ARPE-19 cells with reduced p53 expression behave similar to tumor cell lines with mutated or non-functional p53. The present data demonstrate the utility of lentiviral vectors to create stable isogenic cell lines with reduced expression of a specific gene, thereby permitting the study of the function of a gene, the pathways controlled by it, and the effect of therapeutics on a cell with altered genetic makeup in a pair-wise fashion.

  1. P53 Mutations Change Phosphatidylinositol Acyl Chain Composition

    Directory of Open Access Journals (Sweden)

    Adam Naguib

    2015-01-01

    Full Text Available Phosphatidylinositol phosphate (PIP second messengers relay extracellular growth cues through the phosphorylation status of the inositol sugar, a signal transduction system that is deregulated in cancer. In stark contrast to PIP inositol head-group phosphorylation, changes in phosphatidylinositol (PI lipid acyl chains in cancer have remained ill-defined. Here, we apply a mass-spectrometry-based method capable of unbiased high-throughput identification and quantification of cellular PI acyl chain composition. Using this approach, we find that PI lipid chains represent a cell-specific fingerprint and are unperturbed by serum-mediated signaling in contrast to the inositol head group. We find that mutation of Trp53 results in PIs containing reduced-length fatty acid moieties. Our results suggest that the anchoring tails of lipid second messengers form an additional layer of PIP signaling in cancer that operates independently of PTEN/PI3-kinase activity but is instead linked to p53.

  2. Mutant p53 promotes cell spreading and migration via ARHGAP44.

    Science.gov (United States)

    Xu, Jinjin; Jiao, Jian; Xu, Wei; Ji, Lei; Jiang, Dongjie; Xie, Shaofang; Kubra, Syeda; Li, Xiaotao; Fu, Junjiang; Xiao, Jianru; Zhang, Bianhong

    2017-09-01

    The tumor suppressor p53 protein is either lost or mutated in about half of all human cancers. Loss of p53 function is well known to influence cell spreading, migration and invasion. While expression of mutant p53 is not equivalent to p53 loss, mutant p53 can acquire new functions to drive cell spreading and migration via different mechanisms. In our study, we found that mutant p53 significantly increased cell spreading and migration when comparing with p53-null cells. RNA-Seq analysis suggested that Rho GTPase activating protein 44 (ARHGAP44) is a new target of mutant p53, which suppressed ARHGAP44 transcription. ARHGAP44 has GAP activity and catalyze GTP hydrolysis on Cdc42. Higher level of GTP-Cdc42 was correlated with increase expression of mutant p53 and reduced ARHGAP44. Importantly, wt-ARHGAP44 but not mutant ARHGAP44 (R291A) suppressed mutant p53 mediated cell spreading and migration. Bioinformatics analysis indicated lower expression of ARHGAP44 in lung carcinoma compared with normal tissues, which was verified by RT-qPCR using specimens from patients. More interestingly, ARHGAP44 mRNA level was lower in tumors with mutant p53 than those with normal p53. Collectively, our results disclose a new mechanism by which mutant p53 stimulates cell spreading and migration.

  3. An adaptive molecular timer in p53-meidated cell fate decision

    Science.gov (United States)

    Zhang, Xiao-Peng; Wang, Ping; Liu, Feng; Wang, Wei

    The tumor suppressor p53 decides cellular outcomes in the DNA damage response. It is intriguing to explore the link between p53 dynamics and cell fates. We developed a theoretical model of p53 signaling network to clarify the mechanism of cell fate decision mediated by its dynamics. We found that the interplay between p53-Mdm2 negative feedback loop and p53-PTEN-Mdm2 positive feedback loop shapes p53 dynamics. Depending on the intensity of DNA damage, p53 shows three modes of dynamics: persistent pulses, two-phase dynamics with pulses followed by sustained high levels and straightforward high levels. Especially, p53 shows two-phase dynamics upon moderated damage and the required number of p53 pulses before apoptosis induction decreases with increasing DNA damage. Our results suggested there exists an adaptive molecular timer that determines whether and when the apoptosis switch should be triggered. We clarified the mechanism behind the switching of p53 dynamical modes by bifurcation analysis. Moreover, we reproduced the experimental results that drug additions alter p53 pulses to sustained p53 activation and leads to senescence. Our work may advance the understanding the significance of p53 dynamics in tumor suppression. This work was supported by National Natural Science Foundation of China (Nos. 11175084, 11204126 and 31361163003).

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

    Science.gov (United States)

    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. PMID:22918438

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

    Energy Technology Data Exchange (ETDEWEB)

    Abdelalim, Essam Mohamed, E-mail: emohamed@qf.org.qa [Qatar Biomedical Research Institute, Qatar Foundation, Doha 5825 (Qatar); Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan); Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia (Egypt); Tooyama, Ikuo [Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan)

    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.

  6. Role of p53–fibrinolytic system cross-talk in the regulation of quartz-induced lung injury

    Energy Technology Data Exchange (ETDEWEB)

    Bhandary, Yashodhar P.; Shetty, Shwetha K.; Marudamuthu, Amarnath S. [Texas Lung Injury Institute, Department of Medicine, University of Texas Health Science Center at Tyler, 11937 US Highway 271, Tyler, TX 75708 (United States); Fu, Jian [Texas Lung Injury Institute, Department of Medicine, University of Texas Health Science Center at Tyler, 11937 US Highway 271, Tyler, TX 75708 (United States); Center for Research on Environmental Disease and Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Pinson, Barbara M. [Occupational Medicine, Department of Medicine, University of Texas Health Science Center at Tyler, 11937 US Highway 271, Tyler, TX 75708 (United States); Center for Research on Environmental Disease and Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Levin, Jeffrey [Occupational Medicine, Department of Medicine, University of Texas Health Science Center at Tyler, 11937 US Highway 271, Tyler, TX 75708 (United States); Shetty, Sreerama, E-mail: sreerama.shetty@uthct.edu [Texas Lung Injury Institute, Department of Medicine, University of Texas Health Science Center at Tyler, 11937 US Highway 271, Tyler, TX 75708 (United States)

    2015-03-01

    Silica is the major component of airborne dust generated by wind, manufacturing and/or demolition. Chronic occupational inhalation of silica dust containing crystalline quartz is by far the predominant form of silicosis in humans. Silicosis is a progressive lung disease that typically arises after a very long latency and is a major occupational concern with no known effective treatment. The mechanism of silicosis is not clearly understood. However, silicosis is associated with increased cell death, expression of redox enzymes and pro-fibrotic cytokines and chemokines. Since alveolar epithelial cell (AEC) death and disruption of alveolar fibrinolysis is often associated with both acute and chronic lung injuries, we explored whether p53-mediated changes in the urokinase-type plasminogen activator (uPA) system contributes to silica-induced lung injury. We further sought to determine whether caveolin-1 scaffolding domain peptide (CSP), which inhibits p53 expression, mitigates lung injury associated with exposure to silica. Lung tissues and AECs isolated from wild-type (WT) mice exposed to silica exhibit increased apoptosis, p53 and PAI-1, and suppression of uPA expression. Treatment of WT mice with CSP inhibits PAI-1, restores uPA expression and prevents AEC apoptosis by suppressing p53, which is otherwise induced in mice exposed to silica. The process involves CSP-mediated inhibition of serine-15 phosphorylation of p53 by inhibition of protein phosphatase 2A-C (PP2A-C) interaction with silica-induced caveolin-1 in AECs. These observations suggest that changes in the p53–uPA fibrinolytic system cross-talk contribute to lung injury caused by inhalation of silica dust containing crystalline quartz and is protected by CSP by targeting this pathway. - Highlights: • Chronic exposure to quartz dusts is a major cause of lung injury and silicosis. • The survival of patients with silicosis is bleak due to lack of effective treatments. • This study defines a new role of

  7. The chemoadjuvant potential of grape seed procyanidins on p53-related cell death in oral cancer cells.

    Science.gov (United States)

    Lin, Yaoh-Shiang; Chen, Su-Feng; Liu, Chia-Lin; Nieh, Shin

    2012-04-01

    To clarify the efficacy of grape seed procyanidin (GSP) on antiproliferative effects related to p53 functional status of oral squamous cell carcinoma (OSCC) for its chemoadjuvant potential. We used GSP to investigate SCC-25 cells with wild-type p53 gene and OEC-M1 cells with mutant p53 gene for the assessment of antiproliferative effects including cell viability, cell cycle, apoptosis, migration and invasion potential, and alterations of associated oncoproteins involved in cellular and molecular events. The findings suggest that GSP on OEC-M1 cells leads to cell cycle arrest by increasing the expression of p21(Cip1) /p27(Kip1) protein without functioning mitochondria-mediated apoptosis, whereas GSP on SCC-25 cells inhibits cell proliferation via both G1-phase arrest and mitochondria-mediated apoptosis in a dose-dependent manner as a result of alterations of Bcl-2. GSP also inhibits the migration and invasion of both cells, which are associated with the suppression of matrix metalloproteinases (MMPs), MMP-2 and MMP-9. Antiproliferative effectiveness of GSP is closely associated with the p53 status of OSCC cells. GSP displays chemoadjuvant potential via cell cycle blockage and apoptotic induction. Our findings clearly suggest that GSP may play a role as a novel chemopreventive or therapeutic agent for OSCC. © 2011 John Wiley & Sons A/S.

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

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    Ferecatu, Ioana; Bergeaud, Marie; Rodriguez-Enfedaque, Aida; Le Floch, Nathalie [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France); Oliver, Lisa [INSERM U601, Universite de Nantes, Faculte de Medecine, Nantes Cedex (France); Rincheval, Vincent; Renaud, Flore [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France); Vallette, Francois M. [INSERM U601, Universite de Nantes, Faculte de Medecine, Nantes Cedex (France); Mignotte, Bernard [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France); Vayssiere, Jean-Luc, E-mail: jean-luc.vayssiere@uvsq.fr [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France)

    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.