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Sample records for cell expressed p53

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

    SciTech Connect

    Abdelalim, Essam Mohamed; Tooyama, Ikuo

    2014-01-10

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2012-02-01

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

  4. p53 tumour suppressor gene expression in pancreatic neuroendocrine tumour cells.

    PubMed Central

    Bartz, C; Ziske, C; Wiedenmann, B; Moelling, K

    1996-01-01

    Neuroendocrine pancreatic tumours grow slower and metastasise later than ductal and acinar carcinomas. The expression of the p53 tumour suppressor gene in pancreatic neuroendocrine tumour cells is unknown. Pancreatic neuroendocrine cell lines (n = 5) and human tumour tissues (n = 19) were studied for changed p53 coding sequence, transcription, and translation. Proliferative activity of tumour cells was determined analysing Ki-67 expression. No mutation in the p53 nucleotide sequence of neuroendocrine tumour cell was found. However, an overexpression of p53 could be detected in neuroendocrine pancreatic tumour cell lines at a protein level. As no p53 mutations were seen, it is suggested that post-translational events can also lead to an overexpression of p53. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8675094

  5. Jasmonates induce nonapoptotic death in high-resistance mutant p53-expressing B-lymphoma cells

    PubMed Central

    Fingrut, Orit; Reischer, Dorit; Rotem, Ronit; Goldin, Natalia; Altboum, Irit; Zan-Bar, Israel; Flescher, Eliezer

    2005-01-01

    Mutations in p53, a tumor suppressor gene, occur in more than half of human cancers. Therefore, we tested the hypothesis that jasmonates (novel anticancer agents) can induce death in mutated p53-expressing cells. Two clones of B-lymphoma cells were studied, one expressing wild-type (wt) p53 and the other expressing mutated p53. Jasmonic acid and methyl jasmonate (0.25–3 mM) were each equally cytotoxic to both clones, whereas mutant p53-expressing cells were resistant to treatment with the radiomimetic agent neocarzinostatin and the chemotherapeutic agent bleomycin. Neocarzinostatin and bleomycin induced an elevation in the p53 levels in wt p53-expressing cells, whereas methyl jasmonate did not. Methyl jasmonate induced mostly apoptotic death in the wt p53-expressing cells, while no signs of early apoptosis were detected in mutant p53-expressing cells. In contrast, neocarzinostatin and bleomycin induced death only in wt p53-expressing cells, in an apoptotic mode. Methyl jasmonate induced a rapid depletion of ATP in both clones. In both clones, oligomycin (a mitochondrial ATP synthase inhibitor) did not increase ATP depletion induced by methyl jasmonate, whereas inhibition of glycolysis with 2-deoxyglucose did. High glucose levels protected both clones from methyl jasmonate-induced ATP depletion (and reduced methyl jasmonate-induced cytotoxicity), whereas high levels of pyruvate did not. These results suggest that methyl jasmonate induces ATP depletion mostly by compromising oxidative phosphorylation in the mitochondria. In conclusion, jasmonates can circumvent the resistance of mutant p53-expressing cells towards chemotherapy by inducing a nonapoptotic cell death. PMID:16170329

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

    SciTech Connect

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

    2008-12-15

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

  7. Wild-type p53 controls cell motility and invasion by dual regulation of MET expression

    PubMed Central

    Hwang, Chang-Il; Matoso, Andres; Corney, David C.; Flesken-Nikitin, Andrea; Körner, Stefanie; Wang, Wei; Boccaccio, Carla; Thorgeirsson, Snorri S.; Comoglio, Paolo M.; Hermeking, Heiko; Nikitin, Alexander Yu.

    2011-01-01

    Recent observations suggest that p53 mutations are responsible not only for growth of primary tumors but also for their dissemination. However, mechanisms involved in p53-mediated control of cell motility and invasion remain poorly understood. By using the primary ovarian surface epithelium cell culture, we show that conditional inactivation of p53 or expression of its mutant forms results in overexpression of MET receptor tyrosine kinase, a crucial regulator of invasive growth. At the same time, cells acquire increased MET-dependent motility and invasion. Wild-type p53 negatively regulates MET expression by two mechanisms: (i) transactivation of MET-targeting miR-34, and (ii) inhibition of SP1 binding to MET promoter. Both mechanisms are not functional in p53 absence, but mutant p53 proteins retain partial MET promoter suppression. Accordingly, MET overexpression, cell motility, and invasion are particularly high in p53-null cells. These results identify MET as a critical effector of p53 and suggest that inhibition of MET may be an effective antimetastatic approach to treat cancers with p53 mutations. These results also show that the extent of advanced cancer traits, such as invasion, may be determined by alterations in individual components of p53/MET regulatory network. PMID:21831840

  8. Mutant p53 expression in fallopian tube epithelium drives cell migration.

    PubMed

    Quartuccio, Suzanne M; Karthikeyan, Subbulakshmi; Eddie, Sharon L; Lantvit, Daniel D; Ó hAinmhire, Eoghainín; Modi, Dimple A; Wei, Jian-Jun; Burdette, Joanna E

    2015-10-01

    Ovarian cancer is the fifth leading cause of cancer death among US women. Evidence supports the hypothesis that high-grade serous ovarian cancers (HGSC) may originate in the distal end of the fallopian tube. Although a heterogeneous disease, 96% of HGSC contain mutations in p53. In addition, the "p53 signature," or overexpression of p53 protein (usually associated with mutation), is a potential precursor lesion of fallopian tube derived HGSC suggesting an essential role for p53 mutation in early serous tumorigenesis. To further clarify p53-mutation dependent effects on cells, murine oviductal epithelial cells (MOE) were stably transfected with a construct encoding for the R273H DNA binding domain mutation in p53, the most common mutation in HGSC. Mutation in p53 was not sufficient to transform MOE cells but did significantly increase cell migration. A similar p53 mutation in murine ovarian surface epithelium (MOSE), another potential progenitor cell for serous cancer, was not sufficient to transform the cells nor change migration suggesting tissue specific effects of p53 mutation. Microarray data confirmed expression changes of pro-migratory genes in p53(R273H) MOE compared to parental cells, which could be reversed by suppressing Slug expression. Combining p53(R273H) with KRAS(G12V) activation caused transformation of MOE into high-grade sarcomatoid carcinoma when xenografted into nude mice. Elucidating the specific role of p53(R273H) in the fallopian tube will improve understanding of changes at the earliest stage of transformation. This information can help develop chemopreventative strategies to prevent the accumulation of additional mutations and reverse progression of the "p53 signature" thereby, improving survival rates.

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2008-01-25

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

  11. Wild-type p53 induces diverse effects in 32D cells expressing different oncogenes.

    PubMed Central

    Soddu, S; Blandino, G; Scardigli, R; Martinelli, R; Rizzo, M G; Crescenzi, M; Sacchi, A

    1996-01-01

    Expression of exogenous wild-type (wt) p53 in different leukemia cell lines can induce growth arrest, apoptotic cell death, or cell differentiation. The hematopoietic cell lines that have been used so far to study wt p53 functions have in common the characteristic of not expressing endogenous p53. However, the mechanisms involved in the transformation of these cells are different, and the cells are at different stages of tumor progression. It can be postulated that each type of neoplastic cell offers a particular environment in which p53 might generate different effects. To test this hypothesis, we introduced individual oncogenes into untransformed, interleukin-3 (IL-3)-dependent myeloid precursor 32D cells to have a single transforming agent at a time. The effects induced by wt p53 overexpression were subsequently evaluated in each oncogene-expressing 32D derivative. We found that in not fully transformed, v-ras-expressing 32D cells, as already shown for the parental 32D cells, overexpression of the wt p53 gene caused no phenotypic changes and no reduction of the proliferative rate as long as the cells were maintained in their normal culture conditions (presence of IL-3 and serum). An accelerated rate of apoptosis was observed after IL-3 withdrawal. In contrast, in transformed, IL-3-independent 32D cells, wt p53 overexpression induced different effects. The v-abl-transformed cells manifested a reduction in growth rate, while the v-src-transformed cells underwent monocytic differentiation. These results show that the phenotype effects of wt p53 action(s) can vary as a function of the cellular environment. PMID:8552075

  12. Alpha-particle-induced p53 protein expression in a rat lung epithelial cell strain.

    PubMed

    Hickman, A W; Jaramillo, R J; Lechner, J F; Johnson, N F

    1994-11-15

    Other investigators have shown that both sparsely ionizing and UV radiation cause cell cycle arrest that is associated with increased expression of wild-type p53 protein. The effect of exposure to alpha-particles from 238Pu on the induction of the p53 protein has now been examined in cultured lung epithelial cells derived from male F344 rats. The number of cells having increased levels of p53 protein was determined by flow cytometry after the cells had been stained with a monoclonal antibody to p53. alpha-Particle irradiation caused a dose-dependent increase in p53 protein levels detectable at doses as low as 0.6 cGy, with no evidence of a threshold. An increase in p53 protein also occurred in X-irradiated cells. However, no increase was seen in cells exposed to less than 10 cGy of X-rays, indicating the existence of a relatively higher DNA damage threshold for sparsely ionizing radiation. In addition, more cells exposed to low doses of alpha radiation had increased p53 protein levels than would be predicted based on the number of nuclei expected to be traversed by an alpha-particle, suggesting that alpha-particles cause genetic damage by mechanisms in addition to direct interactions with DNA.

  13. Regulation of Mutant p53 Protein Expression.

    PubMed

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

    2015-01-01

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

  14. p53-dependent expression of CXCR5 chemokine receptor in MCF-7 breast cancer cells.

    PubMed

    Mitkin, Nikita A; Hook, Christina D; Schwartz, Anton M; Biswas, Subir; Kochetkov, Dmitry V; Muratova, Alisa M; Afanasyeva, Marina A; Kravchenko, Julia E; Bhattacharyya, Arindam; Kuprash, Dmitry V

    2015-03-19

    Elevated expression of chemokine receptors in tumors has been reported in many instances and is related to a number of survival advantages for tumor cells including abnormal activation of prosurvival intracellular pathways. In this work we demonstrated an inverse correlation between expression levels of p53 tumor suppressor and CXCR5 chemokine receptor in MCF-7 human breast cancer cell line. Lentiviral transduction of MCF-7 cells with p53 shRNA led to elevated CXCR5 at both mRNA and protein levels. Functional activity of CXCR5 in p53-knockdown MCF-7 cells was also increased as shown by activation of target gene expression and chemotaxis in response to B-lymphocyte chemoattractant CXCL13. Using deletion analysis and site-directed mutagenesis of the cxcr5 gene promoter and enhancer elements, we demonstrated that p53 appears to act upon cxcr5 promoter indirectly, by repressing the activity of NFκB transcription factors. Using chromatin immunoprecipitation and reporter gene analysis, we further demonstrated that p65/RelA was able to bind the cxcr5 promoter in p53-dependent manner and to directly transactivate it when overexpressed. Through the described mechanism, elevated CXCR5 expression may contribute to abnormal cell survival and migration in breast tumors that lack functional p53.

  15. Che-1 gene silencing induces osteosarcoma cell apoptosis by inhibiting mutant p53 expression.

    PubMed

    Liu, Ming; Wang, Dan; Li, Ning

    2016-04-22

    The transcriptional cofactor Che-1 is an RNA polymerase II (Pol II) which is involved in tumorigenesis, such as breast cancer and multiple myeloma. Che-1 can also regulate mutant p53 expression, which plays roles in many types of cancer. In this study, we aimed to investigate the effects and specific mechanism of Che-1 in the regulation of osteosarcoma (OS) cell growth. We found that Che-1 is highly expressed in several kinds of OS cells compared with osteoblast hFOB1.19 cells. MTT and flow cytometry assays showed that Che-1 depletion by siRNA markedly suppressed MG-63 and U2OS cell proliferation and promoted apoptosis. The chromatin immunoprecipitation (ChIP) assay verified the presence of Che-1 on the p53 promoter in MG-63 and U2OS cells carrying mutant p53. Further studies showed that Che-1 depletion inhibited mutant p53 expression. Notably, our study showed that the loss of Che-1 inhibits proliferation and promotes apoptosis in MG-63 cells by decreasing the level of mutant p53. Therefore, these findings open the possibility that silencing of Che-1 will have therapeutic benefit in OS.

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

    PubMed Central

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

    2015-01-01

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

  17. Effect of Thymoquinone on P53 Gene Expression and Consequence Apoptosis in Breast Cancer Cell Line

    PubMed Central

    Dastjerdi, Mehdi Nikbakht; Mehdiabady, Ebrahim Momeni; Iranpour, Farhad Golshan; Bahramian, Hamid

    2016-01-01

    Background: Nigella sativa has been a nutritional flavoring factor and natural treatment for many ailments for so many years in medical science. Earlier studies have been reported that thymoquinone (TQ), an active compound of its seed, contains anticancer properties. Previous studies have shown that TQ induces apoptosis in breast cancer cells but it is unclear the role of P53 in the apoptotic pathway. Hereby, this study reports the potency of TQ on expression of tumor suppressor gene P53 and apoptosis induction in breast cancer cell line Michigan Cancer Foundation-7 (MCF-7). Methods: MCF-7 cell line was cultured and treated with TQ, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out for evaluating the half-maximal inhibitory concentration (IC50) values after 24 h of treatment. The percentage of apoptotic cells was measured by flow cytometry. Real-time polymerase chain reaction (PCR) was performed to estimate the messenger RNA expression of P53 in MCF-7 cell line at different times. Results: The IC50 value for the TQ in MCF-7 cells was 25 μM that determined using MTT assay. The flow cytometry and real-time PCR results showed that TQ could induce apoptosis in MCF-7 cells, and the P53 gene expression was dramatically up-regulated by ascending time, respectively. Hence, there was significant difference in 48 and 72 h. Conclusions: Our results demonstrated that TQ could induce apoptosis in MCF-7 cells through up-regulation of P53 expression in breast cancer cell line (MCF-7) by time-dependent manner. PMID:27141285

  18. Interleukin-13 interferes with activation-induced t-cell apoptosis by repressing p53 expression

    PubMed Central

    Yang, Li; Xu, Ling-Zhi; Liu, Zhi-Qiang; Yang, Gui; Geng, Xiao-Rui; Mo, Li-Hua; Liu, Zhi-Gang; Zheng, Peng-Yuan; Yang, Ping-Chang

    2016-01-01

    The etiology and the underlying mechanism of CD4+ T-cell polarization are unclear. This study sought to investigate the mechanism by which interleukin (IL)-13 prevents the activation-induced apoptosis of CD4+ T cells. Here we report that CD4+ T cells expressed IL-13 receptor α2 in the intestine of sensitized mice. IL-13 suppressed both the activation-induced apoptosis of CD4+ T cells and the expression of p53 and FasL. Exposure to recombinant IL-13 inhibited activation-induced cell death (AICD) along with the expression of p53, caspase 3, and tumor necrosis factor-α in CD4+ T cells. Administration of an anti-IL-13 antibody enhanced the effect of specific immunotherapy on allergic inflammation in the mouse intestine, enforced the expression of p53 in intestinal CD4+ T cells, and enhanced the frequency of CD4+ T-cell apoptosis upon challenge with specific antigens. In summary, blocking IL-13 enhances the therapeutic effect of antigen-specific immunotherapy by regulating apoptosis and thereby enforcing AICD in CD4+ T cells. PMID:26189367

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

    PubMed Central

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

    2012-01-01

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

  20. Senescence Process in Primary Wilms' Tumor Cell Culture Induced by p53 Independent p21 Expression

    PubMed Central

    Theerakitthanakul, Korkiat; Saetang, Jirakrit; Kruatong, Jirasak; Graidist, Potchanapond; Raungrut, Pritsana; Kayasut, Kanita; Sangkhathat, Surasak

    2016-01-01

    Wilms tumor (WT) is an embryonal tumor occurring in developing kidney tissue. WT cells showing invasive cancer characteristics, also retain renal stem cell behaviours. In-vitro culture of WT is hampered by limited replicative potential. This study aimed to establish a longterm culture of WT cells to enable the study of molecular events to attempt to explain its cellular senescence. Methods: Primary cell cultures from fresh WT tumor specimen were established. Of 5 cultures tried, only 1 could be propagated for more than 7 passages. One culture, identified as PSU-SK-1, could be maintained > 35 passages and was then subjected to molecular characterization and evaluation for cancer characteristics. The cells consistently harbored concomitant mutations of CTNNB1 (Ser45Pro) and WT1 (Arg413Stop) thorough the cultivation. On Transwell invasion assays, the cells exhibited migration and invasion at 55% and 27% capability of the lung cancer cells, A549. On gelatin zymography, PSU-SK-1 showed high expression of the matrix metaloproteinase. The cells exhibited continuous proliferation with 24-hour doubling time until passages 28-30 when the growth slowed, showing increased cell size, retention of cells in G1/S proportion and positive β-galactosidase staining. As with those evidence of senescence in advanced cell passages, expression of p21 and cyclin D1 increased when the expression of β-catenin and its downstream protein, TCF, declined. There was also loss-of-expression of p53 in this cell line. In conclusion, cellular senescence was responsible for limited proliferation in the primary culture of WT, which was also associated with increased expression of p21 and was independent of p53 expression. Decreased activation of the Wnt signalling might explain the induction of p21 expression. PMID:27698927

  1. p53-dependent NDRG1 expression induces inhibition of intestinal epithelial cell proliferation but not apoptosis after polyamine depletion.

    PubMed

    Zhang, Ai-Hong; Rao, Jaladanki N; Zou, Tongtong; Liu, Lan; Marasa, Bernard S; Xiao, Lan; Chen, Jie; Turner, Douglas J; Wang, Jian-Ying

    2007-07-01

    Normal intestinal mucosal growth requires polyamines that regulate expression of various genes involved in cell proliferation, growth arrest, and apoptosis. Our previous studies have shown that polyamine depletion stabilizes p53, resulting in inhibition of intestinal epithelial cell (IEC) proliferation, but the exact downstream targets of induced p53 are still unclear. The NDRG1 (N-myc downregulated gene-1) gene encodes a growth-related protein, and its transcription can be induced in response to stress. The current study tests the hypothesis that induced p53 inhibits IEC proliferation by upregulating NDRG1 expression following polyamine depletion. Depletion of cellular polyamines by inhibiting ornithine decarboxylase (ODC) with alpha-difluoromethylornithine not only induced p53 but also increased NDRG1 transcription as indicated by induction of the NDRG1 promoter activity and increased levels of NDRG1 mRNA and protein, all of which were prevented by using specific p53 siRNA and in cells with a targeted deletion of p53. In contrast, increased levels of cellular polyamines by ectopic expression of the ODC gene decreased p53 and repressed expression of NDRG1. Consistently, polyamine depletion-induced activation of the NDRG1-promoter was decreased when p53-binding sites within the NDRG1 proximal promoter region were deleted. Ectopic expression of the wild-type NDRG1 gene inhibited DNA synthesis and decreased final cell numbers regardless of the presence or absence of endogenous p53, whereas silencing NDRG1 promoted cell growth. However, overexpression of NDRG1 failed to directly induce cell death and to alter susceptibility to apoptosis induced by tumor necrosis factor-alpha/cycloheximide. These results indicate that NDRG1 is one of the direct mediators of induced p53 following polyamine depletion and that p53-dependent NDRG1 expression plays a critical role in the negative control of IEC proliferation.

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

    PubMed

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

    2004-10-01

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

  3. Concordant p53 and mdm-2 protein expression in vulvar squamous cell carcinoma and adjacent lichen sclerosus.

    PubMed

    Carlson, J A; Amin, S; Malfetano, J; Tien, A T; Selkin, B; Hou, J; Goncharuk, V; Wilson, V L; Rohwedder, A; Ambros, R; Ross, J S

    2001-06-01

    To determine if carcinogenic events in vulvar skin precede the onset of morphologic atypia, the authors investigated for derangements in DNA content, cell proliferation, and cell death in vulvar carcinomas and surrounding skin in 140 samples of tumor and surrounding skin collected from 35 consecutive vulvectomy specimen for squamous cell carcinoma (SCC) or vulvar intraepithelial neoplasia (VIN) 3. Vulvar non-cancer excisions were used as controls. Investigations consisted of histologic classification and measurement of 9 variables--epidermal thickness (acanthosis and rete ridge length), immunolabeling index (LI) for 3 proteins (p53 protein, Ki-67, and mdm-2), pattern of p53 expression (dispersed vs. compact), DNA content index, and presence of aneuploidy by image analysis and apoptotic rate by Apotag labeling. Significant positive correlations were found for all nine variables studied versus increasing histologic severity in two proposed histologic stepwise models of vulvar carcinogenesis (lichen sclerosus (LS) and VIN 3 undifferentiated associated SCC groups). High p53 LI (>25) and the compact pattern of p53 expression (suspected oncoprotein) significantly correlated with LS and its associated vulvar samples compared with samples not associated with LS (P < or = 0.001). Furthermore, p53 LI, mdm-2 LI, and pattern of p53 expression were concordant between patient matched samples of LS and SCC. In addition, mdm-2 LI significantly correlated with dispersed pattern p53 LI suggesting a response to wild-type p53 protein accumulation. These findings support the hypothesis that neoplastic transformation occurs in sequential steps and compromises proteins involved in the cell cycle control. Concordance of p53 and mdm-2 protein expression in LS and adjacent SCC provides evidence that LS can act as a precursor lesion in the absence of morphologic atypia. Overexpression of mdm-2 with stabilization and inactivation of p53 protein may provide an alternate pathway for vulvar

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

    PubMed

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

    2016-10-01

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

  5. p53 and bcl-2 expression in high-grade B-cell lymphomas: correlation with survival time.

    PubMed Central

    Piris, M. A.; Pezzella, F.; Martinez-Montero, J. C.; Orradre, J. L.; Villuendas, R.; Sanchez-Beato, M.; Cuena, R.; Cruz, M. A.; Martinez, B.; Pezella F [corrected to Pezzella, F. ].

    1994-01-01

    B-cell high-grade lymphomas are heterogeneous in terms of histology, clinical presentation, treatment response and prognosis. As bcl-2 and p53 gene deregulations are frequently involved in several types of lymphoid malignancies, we aimed our investigation at the study of the relation between bcl-2 and p53 expression and survival probability in a group of 119 patients with B-cell high-grade lymphoma. These were obtained from the Virgen de la Salud Hospital, Toledo, Spain (73 cases), John Radcliffe Hospital, Oxford, UK (31 cases), and the Istituto Nazionale dei Tumori, Milan, Italy (15 cases). The relation between bcl-2 protein expression and survival was small, depending on the primary localisation of the tumour (in lymph node of mucosae), and lacked a significant correlation with overall survival. In contrast with this, p53 expression was related to survival probability in our series, this relation being both significant and independent of histological diagnosis. p53-positive patients showed a sudden decrease in life expectancy in the first months after diagnosis. Multivariant regression analysis confirmed that the only parameters significantly related with survival were extranodal origin, which is associated with a better prognosis, and p53 expression, which indicates a poor prognosis. Simultaneous expression of bcl-2 and p53 was associated with a poorer prognosis than p53 alone. This is particularly significant for large B-cell lymphomas presenting in lymph nodes. The cumulative poor effect of both p53 and bcl-2 in large B-cell lymphomas, which is more significant in nodal tumours, could confirm the existence of a multistep genetic deregulation in non-Hodgkin's lymphoma. This indicates that the genetic mechanisms controlling apoptosis and their disregulation are critical steps in the progression of lymphomas. PMID:8297731

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

    PubMed

    Nakano, Hisako; Yonekawa, Hiromichi; Shinohara, Kunio

    2003-06-01

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

  7. KAI-1 and p53 expression in oral squamous cell carcinomas: Markers of significance in future diagnostics and possibly therapeutics

    PubMed Central

    Patil, Namrata N; Wadhwan, Vijay; Chaudhary, Minal; Nayyar, Abhishek Singh

    2016-01-01

    Context: KAI-1/CD82 is a tumor suppressor gene with decreased gene expression being associated with increased invasive ability of oral squamous cell carcinomas (OSCCs). p53 protein functions in the G1-S phase of the cell cycle to allow repair of damaged DNA. In the present study, p53 and KAI-1 expression was investigated using monoclonal antibodies in OSCC. Aims: The aim of this study was to detect KAI-1 and p53 expression in OSCCs and to assess the relation between both in OSCCs. Materials and Methods: The present study included histopathologically diagnosed thirty cases of well- and moderately differentiated OSCCs to study the expression of KAI-1 and p53 antibodies. Statistical Analysis: The results obtained were tabulated and statistically analyzed using descriptive statistical analysis; one-way ANOVA; least square difference method and independent t-test. Results: OSCCs exhibited 41.62% positivity for KAI-1 while p53 positive cells were recorded to an extent of 60.82%. A significant positive correlation was observed between KAI-1 and p53 expression in OSCCs. Conclusions: Although a significant amount of work is still required to uncover the mechanisms of action and regulation of KAI-1 and p53 expression, control of the complex metastatic processes would be of interest in controlling the tumor biology in OSCCs as well as other types of malignancies to enhance prognosis in the affected patients and to help protect against future metastasis in the going to be treated and treated patients. PMID:27721601

  8. p53, c-myc p62 and proliferating cell nuclear antigen (PCNA) expression in non-Hodgkin's lymphomas.

    PubMed Central

    Korkolopoulou, P; Oates, J; Kittas, C; Crocker, J

    1994-01-01

    AIMS--To investigate the immunohistochemical expression of p53 protein in non-Hodgkin's lymphomas (NHL) and its relation to that of c-myc p62 oncoprotein and proliferating cell nuclear antigen (PCNA). METHODS--Paraffin wax embedded tissue from 90 non-Hodgkin's lymphomas (72 B cell and 18 T cell) was stained immunohistochemically for p53 protein, c-myc p62 oncoprotein, and PCNA using the monoclonal antibodies DO7, c-myc 1-9 E10, and PC-10, respectively. RESULTS--Of the non-Hodgkin's lymphomas studied, 55 (61%) stained positively for p53 protein. The proportion of positive cases increased from low grade non-Hodgkin's lymphoma and was higher in tumours of T cell origin. The percentage of positive cells (labelling index or LI) was significantly lower in low grade non-Hodgkin's lymphoma, but no difference was established between intermediate and high grade non-Hodgkin's lymphoma. In a large proportion of low grade non-Hodgkin's lymphoma the LI was below 1%. c-myc p62 immunoreactivity was identified in all cases. A significant positive correlation was established between p53 LI and c-myc p62 LI (rs = 0.453) as well as between p53 LI and PCNA LI (rs = 0.338). CONCLUSIONS--p53 immunoreactivity was present in about half the cases of non-Hodgkin's lymphoma and was related to the grade of malignancy and possibly to the B or T cell origin of the tumour. It was also associated with the proliferation state as expressed by PCNA LI and c-myc p62 expression, indicating that the expression of these three cell cycle-related genes might be interrelated. Images PMID:7907610

  9. Connection between Cell Phone use, p53 Gene Expression in Different Zones of Glioblastoma Multiforme and Survival Prognoses

    PubMed Central

    Akhavan-Sigari, Reza; Baf, Morteza Mazloum Farsi; Ariabod, Vahid; Rohde, Veit; Rahighi, Saeed

    2014-01-01

    The aim of this paper is to investigate p53 gene expression in the central and peripheral zones of glioblastoma multiforme using a real-time reverse transcription polymerase chain reaction (RT-PCR) technique in patients who use cell phones ≥3 hours a day and determine its relationship to clinicopathological findings and overall survival. Sixty-three patients (38 males and 25 females), diagnosed with glioblastoma multiforme (GBM), underwent tumor resection between 2008 and 2011. Patient ages ranged from 25 to 88 years, with a mean age of 55. The levels of expression of p53 in the central and peripheral zone of the GBM were quantified by RT-PCR. Data on p53 gene expression from the central and peripheral zone, the related malignancy and the clinicopatholagical findings (age, gender, tumor location and size), as well as overall survival, were analyzed. Forty-one out of 63 patients (65%) with the highest level of cell phone use (≥3 hours/day) had higher mutant type p53 expression in the peripheral zone of the glioblastoma; the difference was statistically significant (P=0.034). Results from the present study on the use of mobile phones for ≥3 hours a day show a consistent pattern of increased risk for the mutant type of p53 gene expression in the peripheral zone of the glioblastoma, and that this increase was significantly correlated with shorter overall survival time. The risk was not higher for ipsilateral exposure. We found that the mutant type of p53 gene expression in the peripheral zone of the glioblastoma was increased in 65% of patients using cell phones ≥3 hours a day. PMID:25276320

  10. Expression profiling of cutaneous squamous cell carcinoma with perineural invasion implicates the p53 pathway in the process

    PubMed Central

    Warren, Timothy A.; Broit, Natasa; Simmons, Jacinta L.; Pierce, Carly J.; Chawla, Sharad; Lambie, Duncan L. J.; Quagliotto, Gary; Brown, Ian S.; Parsons, Peter G.; Panizza, Benedict J.; Boyle, Glen M.

    2016-01-01

    Squamous cell carcinoma (SCC) is the second most common cancer worldwide and accounts for approximately 30% of all keratinocyte cancers. The vast majority of cutaneous SCCs of the head and neck (cSCCHN) are readily curable with surgery and/or radiotherapy unless high-risk features are present. Perineural invasion (PNI) is recognized as one of these high-risk features. The molecular changes during clinical PNI in cSCCHN have not been previously investigated. In this study, we assessed the global gene expression differences between cSCCHN with or without incidental or clinical PNI. The results of the analysis showed signatures of gene expression representative of activation of p53 in tumors with PNI compared to tumors without, amongst other alterations. Immunohistochemical staining of p53 showed cSCCHN with clinical PNI to be more likely to exhibit a diffuse over-expression pattern, with no tumors showing normal p53 staining. DNA sequencing of cSCCHN samples with clinical PNI showed no difference in mutation number or position with samples without PNI, however a significant difference was observed in regulators of p53 degradation, stability and activity. Our results therefore suggest that cSCCHN with clinical PNI may be more likely to contain alterations in the p53 pathway, compared to cSCCHN without PNI. PMID:27665737

  11. In vivo expression of p53 and Bcl-2 and their role in programmed cell death in premalignant and malignant lung lesions.

    PubMed

    Koty, Patrick P; Zhang, Haifan; Franklin, Wilbur A; Yousem, Samuel A; Landreneau, Rodney; Levitt, Mark L

    2002-02-01

    Forty-four specimens of non-malignant and malignant human lung tissue, taken from patients with non-small cell lung cancer (NSCLC), were examined for the expression of wild-type p53, mutant p53, and bcl-2 and the occurrence of programmed cell death (apoptosis). Wild-type p53 expression peaked in peritumoral and metaplastic samples, whereas mutant p53, bcl-2 and apoptosis were first detected in metaplasia and increased with progression to carcinoma. Bcl-2 positive samples had lower levels of apoptosis than bcl-2 negative samples and was independent of wild-type or mutant p53 expression. These results suggest that the over-expression of wild-type p53 may be an early cellular response to an alteration in normal cellular homeostasis. The ensuing increase in apoptosis appears to be relatively independent of mutant or wild-type p53 expression, but does not occur in cells expressing bcl-2.

  12. Expression of mouse Fbxw7 isoforms is regulated in a cell cycle- or p53-dependent manner

    SciTech Connect

    Matsumoto, Akinobu; Onoyama, Ichiro; Nakayama, Keiichi I. . E-mail: nakayak1@bioreg.kyushu-u.ac.jp

    2006-11-10

    Fbxw7 is the F-box protein component of an SCF-type ubiquitin ligase that contributes to the ubiquitin-dependent degradation of cell cycle activators and oncoproteins. Three isoforms ({alpha}, {beta}, and {gamma}) of Fbxw7 are produced from mRNAs with distinct 5' exons. We have now investigated regulation of Fbxw7 expression in mouse tissues. Fbxw7{alpha} mRNA was present in all tissues examined, whereas Fbxw7{beta} mRNA was detected only in brain and testis, and Fbxw7{gamma} mRNA in heart and skeletal muscle. The amount of Fbxw7{alpha} mRNA was high during quiescence (G phase) in mouse embryonic fibroblasts (MEFs) and T cells, but it decreased markedly as these cells entered the cell cycle. The abundance of Fbxw7{alpha} mRNA was unaffected by cell irradiation or p53 status. In contrast, X-irradiation increased the amount of Fbxw7{beta} mRNA in wild-type MEFs but not in those from p53-deficient mice, suggesting that radiation-induced up-regulation of p53 leads to production of Fbxw7{beta} mRNA. Our results thus indicate that expression of Fbxw7 isoforms is differentially regulated in a cell cycle- or p53-dependent manner.

  13. Changes in expression of p53, proliferating cell nuclear antigen and bcl-2 in recurrent laryngeal cancer after radiotherapy.

    PubMed

    Lee, B-J; Wang, S-G; Roh, H-J; Goh, E-K; Chon, K-M; Park, D-Y

    2006-07-01

    The biological changes in recurrent laryngeal cancer following radiotherapy are not fully understood. The authors investigated differences in the expression of p53, proliferating cell nuclear antigen (PCNA) and bcl-2 in laryngeal cancer specimens before radiotherapy and in recurrent laryngeal cancer specimens following radiotherapy in the same patients. The authors investigated the expression of p53, PCNA and bcl-2 by immunohistochemical stain in 30 specimens from 15 patients with primary laryngeal cancer and recurrent laryngeal cancer after radiotherapy. The expression of p53 protein was significantly different in laryngeal cancer before radiotherapy (4/15, 26.7 per cent) compared with recurrent laryngeal cancer after radiotherapy (8/15, 53.3 per cent) (p<0.05). The PCNA index was also significantly different in laryngeal cancer specimens before radiotherapy (mean, 11.9 per cent) compared with recurrent laryngeal cancer after radiotherapy (mean, 18.0 per cent) (p<0.05). However, there was no statistically significant alteration of bcl-2 expression in primary compared with recurrent laryngeal cancer. The expression of p53 and PCNA increased in recurrent laryngeal cancers after radiotherapy, compared with that in laryngeal cancers before radiotherapy. Recurrent laryngeal cancers arising following radiotherapy became biologically aggressive.

  14. Over-expression of p53 mutants in LNCaP cells alters tumor growth and angiogenesis in vivo

    SciTech Connect

    Perryman, L.A.; Blair, J.M.; Kingsley, E.A.; Szymanska, B.; Ow, K.T.; Wen, V.W.; MacKenzie, K.L.; Vermeulen, P.B.; Jackson, P.; Russell, P.J. . E-mail: p.russell@unsw.edu.au

    2006-07-07

    This study has investigated the impact of three specific dominant-negative p53 mutants (F134L, M237L, and R273H) on tumorigenesis by LNCaP prostate cancer cells. Mutant p53 proteins were associated with an increased subcutaneous 'take rate' in NOD-SCID mice, and increased production of PSA. Tumors expressing F134L and R273H grew slower than controls, and were associated with decreased necrosis and apoptosis, but not hypoxia. Interestingly, hypoxia levels were increased in tumors expressing M237L. There was less proliferation in F134L-bearing tumors compared to control, but this was not statistically significant. Angiogenesis was decreased in tumors expressing F134L and R273H compared with M237L, or controls. Conditioned medium from F134L tumors inhibited growth of normal human umbilical-vein endothelial cells but not telomerase-immortalized bone marrow endothelial cells. F134L tumor supernatants showed lower levels of VEGF and endostatin compared with supernatants from tumors expressing other mutants. Our results support the possibility that decreased angiogenesis might account for reduced growth rate of tumor cells expressing the F134L p53 mutation.

  15. 11R-P53 and GM-CSF Expressing Oncolytic Adenovirus Target Cancer Stem Cells with Enhanced Synergistic Activity

    PubMed Central

    Lv, Sai-qun; Ye, Zhen-long; Liu, Pin-yi; Huang, Yao; Li, Lin-fang; Liu, Hui; Zhu, Hai-li; Jin, Hua-jun; Qian, Qi-jun

    2017-01-01

    Targeting cancer stem cells with oncolytic virus (OV) holds great potential for thorough elimination of cancer cells. Based on our previous studies, we here established 11R-P53 and mGM-CSF carrying oncolytic adenovirus (OAV) SG655-mGMP and investigated its therapeutic effect on hepatocellular carcinoma stem cells Hep3B-C and teratoma stem cells ECCG5. Firstly, the augmenting effect of 11R in our construct was tested and confirmed by examining the expression of EGFP with Fluorescence and FCM assays after transfecting Hep3B-C and ECCG5 cells with OVA SG7605-EGFP and SG7605-11R-EGFP. Secondly, the expressions of 11R-P53 and GM-CSF in Hep3B-C and ECCG5 cells after transfection with OAV SG655-mGMP were detected by Western blot and Elisa assays, respectively. Thirdly, the enhanced growth inhibitory and augmented apoptosis inducing effects of OAV SG655-mGMP on Hep3B-C and ECCG5 cells were tested with FCM assays by comparing with the control, wild type 5 adenovirus, 11R-P53 carrying OVA in vitro. Lastly, the in vivo therapeutic effect of OAV SG655-mGMP toward ECCG5 cell-formed xenografts was studied by measuring tumor volumes post different treatments with PBS, OAV SG655-11R-P53, OAV SG655-mGM-CSF and OAV SG655-mGMP. Treatment with OAV SG655-mGMP induced significant xenograft growth inhibition, inflammation factor AIF1 expression and immune cells infiltration. Therefore, our OAV SG655-mGMP provides a novel platform to arm OVs to target cancer stem cells. PMID:28243324

  16. Regulation of p53 expression and apoptosis by vault RNA2-1-5p in cervical cancer cells

    PubMed Central

    Kong, Lu; Hao, Qi; Wang, Ying; Zhou, Ping; Zou, Binbin; Zhang, Yu-xiang

    2015-01-01

    nc886 or VRNA2-1 has recently been identified as a noncoding RNA instead of a vault RNA or a pre-microRNA. Several studies have reported that pre-miR-886 plays a tumor-suppressive role in a wide range of cancer cells through its activity as a cellular protein kinase RNA-activated (PKR) ligand and repressor. However, by sequencing stem-PCR products, we found that a microRNA originating from this precursor, vault RNA2-1-5p (VTRNA2-1-5p), occurs in cervical cancer cells. The expression levels of the predicted targets of VTRNA2-1-5p are negatively correlated with VTRNA2-1-5p levels by quantitative reversion transcription PCR (qRT-PCR). Previous results have shown that VTRNA2-1-5p is overexpressed in human cervical squamous cell carcinomas (CSCCs) compared with adjacent healthy tissues. Inhibition of VTRNA2-1-5p increases Bax protein expression and apoptotic cell death in cervical cancer cells. Our findings suggest that VTRNA2-1-5p has oncogenic activity related to the progression of cervical cancer. Here, we report that VTRNA2-1-5p directly targeted p53 expression and functioned as an oncomir in cervical cancer. VTRNA2-1-5p inhibition decreased cervical cancer cell invasion, proliferation, and tumorigenicity while increasing apoptosis and p53 expression. Interestingly, VTRNA2-1-5p inhibition also increased cisplatin-induced apoptosis of HeLa and SiHa cells. In human clinical cervical cancer specimens, low p53 expression and high VTRNA2-1-5p expression were positively associated. In addition, VTRNA2-1-5p was found to directly target the 5′ and 3′ untranslated regions (UTRs) of p53. We propose that VTRNA2-1-5p is a direct regulator of p53 and suggest that it plays an essential role in the apoptosis and proliferation of cervical cancer cells. PMID:26318295

  17. Mitochondrial STAT3 contributes to transformation of Barrett's epithelial cells that express oncogenic Ras in a p53-independent fashion.

    PubMed

    Yu, Chunhua; Huo, Xiaofang; Agoston, Agoston T; Zhang, Xi; Theiss, Arianne L; Cheng, Edaire; Zhang, Qiuyang; Zaika, Alexander; Pham, Thai H; Wang, David H; Lobie, Peter E; Odze, Robert D; Spechler, Stuart J; Souza, Rhonda F

    2015-08-01

    Metaplastic epithelial cells of Barrett's esophagus transformed by the combination of p53-knockdown and oncogenic Ras expression are known to activate signal transducer and activator of transcription 3 (STAT3). When phosphorylated at tyrosine 705 (Tyr705), STAT3 functions as a nuclear transcription factor that can contribute to oncogenesis. STAT3 phosphorylated at serine 727 (Ser727) localizes in mitochondria, but little is known about mitochondrial STAT3's contribution to carcinogenesis in Barrett's esophagus, which is the focus of this study. We introduced a constitutively active variant of human STAT3 (STAT3CA) into the following: 1) non-neoplastic Barrett's (BAR-T) cells; 2) BAR-T cells with p53 knockdown; and 3) BAR-T cells that express oncogenic H-Ras(G12V). STAT3CA transformed only the H-Ras(G12V)-expressing BAR-T cells (evidenced by loss of contact inhibition, formation of colonies in soft agar, and generation of tumors in immunodeficient mice), and did so in a p53-independent fashion. The transformed cells had elevated levels of both mitochondrial (Ser727) and nuclear (Tyr705) phospho-STAT3. Introduction of a STAT3CA construct with a mutated tyrosine phosphorylation site into H-Ras(G12V)-expressing Barrett's cells resulted in high levels of mitochondrial phospho-STAT3 (Ser727) with little or no nuclear phospho-STAT3 (Tyr705), and the cells still formed tumors in immunodeficient mice. Thus tyrosine phosphorylation of STAT3 is not required for tumor formation in Ras-expressing Barrett's cells. We conclude that mitochondrial STAT3 (Ser727) can contribute to oncogenesis in Barrett's cells that express oncogenic Ras. These findings suggest that agents targeting STAT3 might be useful for chemoprevention in patients with Barrett's esophagus.

  18. Lysine methylation represses p53 activity in teratocarcinoma cancer cells

    PubMed Central

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

    2016-01-01

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

  19. Y-box-binding protein-1 expression is not correlated with p53 expression but with proliferating cell nuclear antigen expression in non-small cell lung cancer.

    PubMed

    Yoshimatsu, Takashi; Uramoto, Hidetaka; Oyama, Tsunehiro; Yashima, Yasunori; Gu, Chundong; Morita, Masaru; Sugio, Kenji; Kohno, Kimitoshi; Yasumoto, Kosei

    2005-01-01

    Transcription factor Y-box-binding protein 1 (YB-1), which binds to the inverted CCAAT box, is not only involved in the transcription of various genes, but also in cell proliferation and DNA repair. The aim of this study was to detect YB-1 and p53 expression and their relationship to proliferating cell nuclear antigen (PCNA) in non-small cell lung cancer (NSCLC) using immunohistochemical (IHC) staining, and to evaluate the relationship between their expression levels and the prognosis of patients with NSCLC. Positive expressions of YB-1, p53 and PCNA were detected in NSCLC cells in 43 (45.7%), 33 (35.0%) and 45 (47.9%) out of 94 patients, respectively. No significant differences were observed between YB-1 expression and the patients' gender, age at surgery, pathological stage, pathological T status, pathological N status, or pathological M status. The mean PCNA-labelling index (LI) for cells was 40.7+/-2.6. Also, a significant correlation between YB-1 and PCNA-LI was found (p<0.01), but none was found between p53 expression and PCNA. The positive expression of YB-1 was associated with squamous cell carcinoma and large cell carcinoma, compared with adenocarcinomas (p<0.01), and higher levels of PCNA-LI were associated with large cell carcinoma compared with adenocarcinomas and squamous cell carcinoma (p<0.01). These results suggest that YB-1 expression is correlated with PCNA expression in NSCLC. In addition, the DNA repair pathway and tumor proliferation mediated by YB-1 linking to PCNA may be responsible for controlling the growth of NSCLC.

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

    SciTech Connect

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

    2009-07-24

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

  1. Expression of P53 protein after exposure to ionizing radiation

    NASA Astrophysics Data System (ADS)

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

    2001-10-01

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

  2. p53 status is a major determinant of effects of decreasing peroxiredoxin I expression on tumor growth and response of lung cancer cells to treatment

    SciTech Connect

    Chen, M.-F. . E-mail: miaofen@adm.cgmh.org.tw; Chen, W.-C.; Wu, C.-T.; Lin, P.-Y.; Shau Hungyi; Liao, S.-K.; Yang, C.-T.; Lee, K.-D.

    2006-12-01

    Purpose: The potential roles of peroxiredoxin (Prx) I in carcinogenesis and treatment have been explored. Our previous study revealed differences between A549 (functional p53) and H1299 (null p53) Prx I antisense transfectants. The discrepancy might have resulted from the p53 status. In this study, we further investigated the role of Prx I and p53 on lung cancer growth and the response to treatment in vitro and in vivo. Methods: We established stable A549 and H1299 transfectants with Prx I antisense and p53, respectively. We then examined their characteristics in vitro and used nude mice xenografts of these cell lines to compare their capacity for tumor invasion and spontaneous metastasis and their sensitivity to radiotherapy. Results: Increased reactive oxygen species caused by lower Prx I activity induced p53 expression. In lethal stress, the augmentation of reactive oxygen species was partially reversed by blocking p53 in A549 with Prx I antisense. We demonstrated the potential contribution of p53-dependent mechanisms to inhibit lung tumor growth and increase radiosensitization using H1299 transfected with p53 in vitro and in vivo. An increased p53 level attenuated the capacity of the cells for metastasis by decreasing vascular endothelial growth factor and induced radiosensitization by increased apoptosis and cell senescence and by regulating intracellular reactive oxygen species. Conclusion: These results suggest that p53 status has an important role in the tumor-inhibiting and radiosensitizing effects of decreasing Prx I. Both Prx I and p53 may be powerful prognosticators for lung cancer.

  3. Targeting cancer stem cells with p53 modulators

    PubMed Central

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

    2016-01-01

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

  4. Basal and copper-induced expression of metallothionein isoform 1,2 and 3 genes in epithelial cancer cells: The role of tumor suppressor p53.

    PubMed

    Ostrakhovitch, E A; Song, Y P; Cherian, M G

    2016-05-01

    Metallothioneins (MTs) are a ubiquitous low-molecular weight, cysteine rich proteins with a high affinity for metal ions. The expression and induction of MTs have been associated with protection against DNA damage, oxidative stress, and apoptosis. Our past research had shown that p53 is an important factor in metal regulation of MTs. The present study was undertaken to explore further the interrelationship between p53 and MTs. We investigated whether silencing of p53 could affect expression pattern of basal and copper induced metallothioneins. The silencing of wild-type p53 (wt-p53) in epithelial breast cancer MCF7 cells affected the basal level of MT-2A RNA, whereas the levels of MT-1A and MT-1X RNA remained largely unchanged. The expression of MT-3 was undetectable in MCF7 with either functional or silenced p53. MCF7 cells with silenced wt-p53 failed to upregulate MT-2A in response to copper and showed a reduced sensitivity toward copper induced cell apoptotic death. Similarly in MCF7-E6 and MDA-MB-231 cells, the presence of inactive/mutated p53 halted MT-1A and MT-2A gene expression in response to copper. Constitutive expression of MT-3 RNA was detectable in the presence of mutated p53 (mtp53). Transient transfection of MDA-MB-231 cells with wt-p53 enabled copper induced upregulation of both MT-1A and MT-2A but not basal level of MT-2A, MT-1E, MT-1X and MT-3. Inactivation of p53 in HepG2 cells amplified the basal expression of studied MT isoforms, including MT-3, as well as copper-induced mRNA expression of MTs except MT-1H and MT-3. Presented data demonstrate a direct relation between p53 and MT-1A and MT-2A and they also indicate that wt-p53 might be a negative regulator of MT-3 in epithelial cancer cells.

  5. Association of p53/p21 expression and cigarette smoking with tumor progression and poor prognosis in non-small cell lung cancer patients.

    PubMed

    Xie, Deyao; Lan, Linhua; Huang, Kate; Chen, Lin; Xu, Cuicui; Wang, Rongrong; Shi, Yang; Wu, Xiaoyi; Wang, Lu; Liu, Yongzhang; Lu, Bin

    2014-12-01

    Non-small cell lung cancer (NSCLC) accounts for approximately 80-85% of all lung cancer cases. Cigarette smoking is the number one risk factor which is attributed to more than four out of five cases of lung cancers. The prognostic impact of cell cycle regulation-associated tumor suppressors including p53 and p21 for NSCLC is still controversial. In the present study, we examined p53 and p21 expression using immunoblotting in tumor and adjacent non-cancerous tissues from NSCLC patients. Moreover, tissue microarrays (TMAs) including 150 specimens was used to examine p53 and p21 expression by immunohistochemical staining (IHC). The association between p53/p21 and various clinicopathological characteristics was evaluated. Kaplan-Meier overall survival was used to analyze the association between p53/p21 expression and prognosis of NSCLC patients, as well as the association of cigarette smoking with p53/p21 expression and prognosis. The results of the immunoblotting showed that expression of p53 and p21 in tumor tissues was significantly higher than that in the matched adjacent non-cancerous tissues (P<0.001 and P<0.05, respectively). The IHC results showed that 50.67% of the cases had high expression of p21; however, the percentage of patients having high expression of p53 was 31.3%. Univariate and Cox regression models were used to evaluate the factors related to prognosis with p53 and p21 expression. Multivariate analysis indicated that p53 expression was an independent prognostic factor for NSCLC (P=0.005), while p21 could not serve as an independent prognostic factor (P=0.123). In addition, smoking history was closely related to lung cancer risk (P=0.041), but could not be an independent assessment factor (P=0.740). In this study, we further demonstrated the association of p53/p21 expression and cigarette smoking. Our results suggest that cigarette smoking and overexpression of p53 or p21 are associated with poor prognosis. The combination of p53/p21 expression and

  6. Immunohistochemistry and scoring of Ki-67 proliferative index and p53 expression in gastric B cell lymphoma from Northern African population: a pilot study

    PubMed Central

    Zeggai, Soumia; Tou, Abdelnacer; Sellam, Feriel; Mrabent, Meriem N.; Salah, Rachida

    2016-01-01

    Background This study aimed to clarify the Ki-67 distribution, p53 expression and their relationship with clinico-pathologic features of gastric B cell lymphoma from Northern African population. Methods Twenty paraffin blocks of gastric lymphoma were retrieved from the archival materials of Department of Pathology, Central University Hospital of Sidi Bel Abbes (Western Algeria) from 2007 to 2013. Four µm section specimens were stained by immunohistochemical (IHC) technique with Ki-67 and p53 tumor markers. P values <0.05 were considered statistically significant. Results Expression of p53 proteins and the mean proliferative index (PI) were compared between high grade gastric B cell lymphomas (DLBCL) and low grade gastric B cell lymphomas (gastric MALTs). p53 overexpression (P=0.007) and a high proliferation index Ki-67 (P=0.001) were significantly associated with gastric DLBCL. We found also a statistically significant correlation between p53 and Ki-67 (P=0.007) but no obvious relationships were found between Ki-67 PI and p53 expression as well as clinico-pathological features (age, sex, location, macroscopic type). Conclusions The IHC studies of Ki-67 and p53 expression in gastric B cell lymphoma can help in monitoring of patients at risk, and to give suitable treatment and management of patients. PMID:27284480

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

    PubMed

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

    2015-07-01

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

  8. Arecoline-induced phosphorylated p53 and p21(WAF1) protein expression is dependent on ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells.

    PubMed

    Chou, Wen-Wen; Guh, Jinn-Yuh; Tsai, Jung-Fa; Hwang, Chi-Ching; Chiou, Shean-Jaw; Chuang, Lea-Yea

    2009-06-01

    Betel-quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53-dependent p21(WAF1) protein expression in Clone-9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3-related (ATR)-p53-p21(WAF1) and the phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline-induced p53 and p21(WAF1) protein expression in Clone-9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline-activated ATM/ATR substrate contained p-p53Ser15. Moreover, arecoline only increased the levels of the p-p53Ser6, p-p53Ser15, and p-p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline-induced p-p53Ser15 and p21(WAF1) at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30-60 min. Dominant-negative PI3K plasmids attenuated arecoline-induced p21(WAF1), but not p-p53Ser15, at 24 h. Rapamycin attenuated arecoline-induced phosphrylated p-p53Ser15, but not p21(WAF1), at 24 h. ATM shRNA, but not dominant-negative PI3K plasmids, attenuated arecoline-induced p21(WAF1) gene transcription. We conclude that arecoline activates the ATM/ATR-p53-p21(WAF1) and the PI3K/Akt-mTOR-p53 pathways in Clone-9 cells. Arecoline-induced phosphorylated p-p53Ser15 expression is dependent on ATM whereas arecoline-induced p21(WAF1) protein expression is dependent on ATM and PI3K. Moreover, p21(WAF1) gene is transcriptionally induced by arecoline-activated ATM.

  9. p53 and MDM2 protein expression in actinic cheilitis.

    PubMed

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

    2008-01-01

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

  10. Ectopic AP4 expression induces cellular senescence via activation of p53 in long-term confluent retinal pigment epithelial cells.

    PubMed

    Wang, Yiping; Wong, Matthew Man-Kin; Zhang, Xiaojian; Chiu, Sung-Kay

    2015-11-15

    When cells are grown to confluence, cell-cell contact inhibition occurs and drives the cells to enter reversible quiescence rather than senescence. Confluent retinal pigment epithelial (RPE) cells exhibiting contact inhibition was used as a model in this study to examine the role of overexpression of transcription factor AP4, a highly expressed transcription factor in many types of cancer, in these cells during long-term culture. We generated stable inducible RPE cell clones expressing AP4 or AP4 without the DNA binding domain (DN-AP4) and observed that, when cultured for 24 days, RPE cells with a high level of AP4 exhibit a large, flattened morphology and even cease proliferating; these changes were not observed in DN-AP4-expressing cells or non-induced cells. In addition, AP4-expressing cells exhibited senescence-associated β-galactosidase activity and the senescence-associated secretory phenotype. We demonstrated that the induced cellular senescence was mediated by enhanced p53 expression and that AP4 regulates the p53 gene by binding directly to two of the three E-boxes present on the promoter of the p53 gene. Moreover, we showed that serum is essential for AP4 in inducing p53-associated cellular senescence. Collectively, we showed that overexpression of AP4 mediates cellular senescence involving in activation of p53 in long-term post-confluent RPE cells.

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

    PubMed Central

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

    2017-01-01

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

  12. FBXW7-mutated colorectal cancer cells exhibit aberrant expression of phosphorylated-p53 at Serine-15

    PubMed Central

    Normatova, Makhliyo; Babaei-Jadidi, Roya; Tomlinson, Ian; Nateri, Abdolrahman S.

    2015-01-01

    FBXW7 mutations occur in a variety of human cancers including colorectal cancer (CRC). Elucidating its mechanism of action has become crucial for cancer therapy; however, it is also complicated by the fact that FBXW7 can influence many pathways due to its role as an E3-ubiquitin ligase in proteasome degradation. FBXW7 and TP53 are tumour suppressors intensively implicated in colorectal carcinogenesis. Deletion mutations in these two genes in animal models mark the progression from adenoma to carcinoma. Although still largely unknown, the last defense mechanism against CRC at the molecular level could be through a synergistic effect of the two genes. The underlying mechanism requires further investigation. In our laboratory, we have used a phospho-kinase profiler array to illustrate a potential molecular link between FBXW7 and p53 in CRC cells. In vitro and in vivo assessments demonstrated aberrant induction of phosphorylated p53 at Serine 15 [phospho-p53(Ser15)] in human FBXW7-deficient CRC cells as compared to their FBXW7-wild-type counterparts. FBXW7 loss in HCT116 cells promoted resistance to oxaliplatin. Immunoblotting data further confirmed that reduction of phospho-p53(Ser15) may contribute to the decreased efficacy of therapy in FBXW7-mutated CRC cells. The findings may suggest the applicability of phospho-p53(Ser15) as an indicative marker of FBXW7-mutations. Phospho-p53(Ser15) regulation by FBXW7 E3-ligase activity could provide important clues for understanding FBXW7 behavior in tumour progression and grounds for its clinical applicability thereafter. PMID:25860929

  13. Activated p53 with Histone Deacetylase Inhibitor Enhances L-Fucose-Mediated Drug Delivery through Induction of Fucosyltransferase 8 Expression in Hepatocellular Carcinoma Cells

    PubMed Central

    Arihara, Yohei; Kikuchi, Shohei; Osuga, Takahiro; Nakamura, Hajime; Kamihara, Yusuke; Hayasaka, Naotaka; Usami, Makoto; Murase, Kazuyuki; Miyanishi, Koji; Kobune, Masayoshi; Kato, Junji

    2016-01-01

    Background The prognosis of advanced hepatocellular carcinoma (HCC) is dismal, underscoring the need for novel effective treatments. The α1,6-fucosyltransferase (fucosyltransferase 8, FUT8) has been reported to accelerate malignant potential in HCC. Our study aimed to investigate the regulation of FUT8 expression by p53 and develop a novel therapeutic strategy for targeting HCC cells using L-fucose-mediated drug delivery. Methods Binding sites for p53 were searched for within the FUT8 promoter region. FUT8 expression was assessed by immunoblotting. Chromatin immunoprecipitation (ChIP) assays were performed to analyze p53 binding to the FUT8 promoter. The delivery of Cy5.5-encapsulated L-fucose-liposomes (Fuc-Lip-Cy5.5) to a Lens Culinaris agglutinin-reactive fraction of α-fetoprotein (AFP-L3)-expressing HCC cells was analyzed by flow cytometry. The induction of FUT8 by histone deacetylase inhibitor (HDACi) -inducing acetylated -p53 was evaluated by immunoblotting. Flow cytometric analysis was performed to assess whether the activation of p53 by HDACi affected the uptake of Fuc-Lip-Cy5.5 by HCC cells. The cytotoxicity of an L-fucose-bound liposome carrying sorafenib (Fuc-Lip-sorafenib) with HDACi was assessed in vivo and in vitro. Results The knock down of p53 with siRNA led to decreased FUT8 expression. ChIP assays revealed p53 binds to the FUT8 promoter region. Flow cytometric analyses demonstrated the specific uptake of Fuc-Lip-Cy5.5 into AFP-L3-expressing HCC cells in a p53- and FUT8-dependent manner. HDACi upregulated the uptake of Fuc-Lip-Cy5.5 by HCC cells by increasing FUT8 via acetylated -p53. The addition of a HDACi increased apoptosis induced by Fuc-Lip-sorafenib in HCC cells. Conclusions Our findings reveal that FUT8 is a p53 target gene and suggest that p53 activated by HDACi induces Fuc-Lip-sorafenib uptake by HCC cells, highlighting this pathway as a promising therapeutic intervention for HCC. PMID:27977808

  14. [Effect of Glycyrrhizae Radix et Rhizoma combined with Atractylodis Macrocephalae Rhizoma on p53 and p21 gene expression of IEC-6 cells].

    PubMed

    Zheng, Fang; Jiang, Ze-bo; Zhang, Xian; Hu, Jin-ping; Li, Si-ming; Zhao, Jin; Zeng, Xing

    2015-05-01

    To study the effect of the combined administration of different doses of Glycyrrhizae Radix et Rhizoma and Atractylodis Macrocephalae Rhizoma on the proliferation of DFMO-treated intestinal epithelial cells (IEC-6) and p53, p21 mRNA and protein expressions, in order to define the molecular basis for the effect of the combined administration of different doses of Glycyrrhizae Radix et Rhizoma and Atractylodis Macrocephalae Rhizoma on the cell proliferation. The effect of the drugs on the cell division rate and cell cycle of IEC-6 cells was detected by FCM. Quantitative Real-time PCR (qRT-PCR) was used to analyze the effect of the drugs on mRNA of p2l and p53 related to IEC-6 proliferation. Western blot was used to analyze the effect of the drugs on p2l and p53 protein expressions of IEC-6 cells. Atractylodis Macrocephalae Rhizoma could increase p53, p21 mRNA and proteins expression in DFMO-treated IEC-6 cells. The combined administration of different ratios of Atractylodis Macrocephalae Rhizoma and Glycyrrhizae Radix et Rhizoma could significantly down-regulate Atractylodis Macrocephalae Rhizoma's effect on p53, p21 mRNA and proteins expression in DFMO-treated IEC-6 cells and promote the proliferation of IEC-6 cells. The combined administration of Atractylodis Macrocephalae Rhizoma and Glycyrrhizae Radix et Rhizoma could down-regulate Atractylodis Macrocephalae Rhizoma's effect on DFMO-treated intestinal epithelial cells (IEC-6).

  15. p21WAF1/Cip1 expression is associated with cell differentiation but not with p53 mutations in squamous cell carcinomas of the larynx.

    PubMed

    Nadal, A; Jares, P; Cazorla, M; Fernández, P L; Sanjuan, X; Hernandez, L; Pinyol, M; Aldea, M; Mallofré, C; Muntané, J; Traserra, J; Campo, E; Cardesa, A

    1997-10-01

    p21WAF1/Cip1 is a recently identified gene involved in cell cycle regulation through cyclin-CDK-complex inhibition. The expression of this gene in several cell lines seems to be induced by wild-type, but not mutant, p53. p21WAF1/Cip1 expression has been studied at both mRNA and protein levels in a series of 49 normal mucosae and squamous cell carcinomas of the larynx. A significant association was found between mRNA and protein expression in tumours (P < 0.0001). p21WAF1/Cip1 expression was strongly associated with squamous cell differentiation of carcinomas, because six of seven (86 per cent) undifferentiated carcinomas (grade 4) showed very low levels of p21WAF1/Cip1 expression, whereas 41 out of 42 (98 per cent) carcinomas with squamous cell differentiation (grades 1-3) had normal or high levels of p21WAF1/Cip1 expression (P < 0.0001). In addition, p21WAF1/Cip1 expression was topologically related to the squamous differentiation of tumour cells with a distribution similar to that seen in normal squamous epithelium. No correlation was found between p21WAF1/Cip1 expression and the global S-phase of the carcinomas. p53 mutations (exons 5-9) were found in ten carcinomas with p21WAF1/Cip1 expression, but no p53 mutations were detected in three p21WAF1/Cip1-negative tumours. In conclusion, p21WAF1/Cip1 expression is frequently upregulated in squamous cell carcinomas of the larynx and is associated with tumour cell differentiation. p21WAF1/Cip1 expression in these tumours is independent of p53 gene mutations.

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

    PubMed Central

    Gottlieb, E; Oren, M

    1998-01-01

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

  17. The expression of p21 is upregulated by forkhead box A1/2 in p53-null H1299 cells.

    PubMed

    An, Joo-Hee; Jang, Sang-Min; Kim, Jung-Woong; Kim, Chul-Hong; Song, Peter I; Choi, Kyung-Hee

    2014-11-03

    The expression of the cell cycle inhibitor p21 is increased in response to various stimuli and stress signals through p53-dependent and independent pathways. We demonstrate in this study that forkhead box A1/2 (FOXA1/2) is a crucial transcription factor in the activation of p21 transcription via direct binding to the p21 promoter in p53-null H1299 lung carcinoma cells. In addition, histone deacetylase inhibitor trichostatin A (TSA)-mediated upregulation of p21 expression was repressed by knockdown of FOXA1/2 in H1299 cells. Consequently, these results suggest that FOXA1/2 is required for p53-independent p21 expression.

  18. Comparative analysis of the expression of proliferating cell nuclear antigen, p53, bax, and bcl-2 in oral lichen planus and oral squamous cell carcinoma.

    PubMed

    de Sousa, Fernando Augusto Cervantes Garcia; Paradella, Thaís Cachuté; Carvalho, Yasmin Rodarte; Rosa, Luiz Eduardo Blumer

    2009-10-01

    Several epidemiologic studies have shown the malignant transformation potential of oral lichen planus; however, this potential is subject of much controversy. To evaluate the expression of proteins related to the cell proliferation and apoptosis processes in oral lichen planus, we compared oral lichen planus with oral squamous cell carcinoma. Twenty-four cases of each lesion were submitted according to streptavidin-biotin technique to evaluate the immunohistochemical expression of proliferating cell nuclear antigen, p53, bax, and bcl-2 proteins. chi(2) test showed no statistically significant differences between the expression of p53, bax, and bcl-2 in oral lichen planus and oral squamous cell carcinoma (P > .05). However, the expression of proliferating cell nuclear antigen was significantly lower in oral lichen planus than in oral squamous cell carcinoma (P < .05). No statistically significant differences between the expression of p53, bax, and bcl-2 in oral lichen planus and oral squamous cell carcinoma were observed, which may be an evidence of the potential of malignant transformation of oral lichen planus.

  19. Method for lipidomic analysis: p53 expression modulation of sulfatide, ganglioside, and phospholipid composition of U87 MG glioblastoma cells.

    PubMed

    He, Huan; Conrad, Charles A; Nilsson, Carol L; Ji, Yongjie; Schaub, Tanner M; Marshall, Alan G; Emmett, Mark R

    2007-11-15

    Lipidomics can complement genomics and proteomics by providing new insight into dynamic changes in biomembranes; however, few reports in the literature have explored, on an organism-wide scale, the functional link between nonenzymatic proteins and cellular lipids. Here, we report changes induced by adenovirus-delivered wild-type p53 gene and chemotherapy of U87 MG glioblastoma cells, a treatment known to trigger apoptosis and cell cycle arrest. We compare polar lipid changes in treated cells and control cells by use of a novel, sensitive method that employs lipid extraction, one-step liquid chromatography separation, high-resolution mass analysis, and Kendrick mass defect analysis. Nano-LC FT-ICR MS and quadrupole linear ion trap MS/MS analysis of polar lipids yields hundreds of unique assignments of glyco- and phospholipids at sub-ppm mass accuracy and high resolving power (m/Deltam50% = 200 000 at m/z 400) at 1 s/scan. MS/MS data confirm molecular structures in many instances. Sulfatides are most highly modulated by wild-type p53 treatment. The treatment also leads to an increase in phospholipids such as phosphatidyl inositols, phosphatidyl serines, phosphatidyl glycerols, and phosphatidyl ethanolamines. An increase in hydroxylated phospholipids is especially noteworthy. Also, a decrease in the longer chain gangliosides, GD1 and GM1b, is observed in wild-type p53 (treated) cells.

  20. Differential association of S100A9, an inflammatory marker, and p53, a cell cycle marker, expression with epicardial adipocyte size in patients with cardiovascular disease.

    PubMed

    Agra, Rosa María; Fernández-Trasancos, Ángel; Sierra, Juan; González-Juanatey, José Ramón; Eiras, Sonia

    2014-10-01

    S100A9 (calgranulin B) has inflammatory and oxidative stress properties and was found to be associated with atherosclerosis and obesity. One of the proteins that can regulate S100A9 transcription is p53, which is involved in cell cycle, apoptosis and adipogenesis. Thus, it triggers adipocyte enlargement and finally obesity. Because epicardial adipose tissue (EAT) volume and thickness is related to coronary artery disease (CAD), we studied the gene expression of this pathway in patients with cardiovascular disease and its association with obesity. Adipocytes and stromal cells from EAT and subcutaneous adipose tissue (SAT) from 48 patients who underwent coronary artery bypass graft and/or valve replacement were obtained after collagenase digestion and differential centrifugation. The expression levels of the involved genes on adipogenesis and cell cycle like fatty acid-binding protein (FABP) 4, retinol-binding protein (RBP)4, p53 and S100A9 were determined by real-time polymerase chain reaction (PCR). Adipocyte diameter was measured by optical microscopy. We found that epicardial adipocytes expressed significantly lower levels of adipogenic genes (FABP4 and RBP4) and cell cycle-related genes (S100A9 and p53) than subcutaneous adipocytes. However, in obese patients, upregulation of adipogenic and cell cycle-related genes in subcutaneous and epicardial adipocytes, respectively, was observed. The enlargement of adipocyte size was related to FABP4, S100A9 and p53 expression levels in stromal cells. But only the p53 association was maintained in epicardial stromal cells from obese patients (p=0.003). The expression of p53, but not S100A9, in epicardial stromal cells is related to adipocyte enlargement in obese patients with cardiovascular disease. These findings suggest new mechanisms for understanding the relationship between epicardial fat thickness, obesity and cardiovascular disease.

  1. Expression of the apoptosis inducer gene head involution defective in primordial germ cells of the Drosophila embryo requires eiger, p53, and loki function.

    PubMed

    Maezawa, Takanobu; Arita, Kayo; Shigenobu, Shuji; Kobayashi, Satoru

    2009-05-01

    Nanos (Nos) is an evolutionarily conserved protein essential for the maintenance of primordial germ cells (PGCs). In Drosophila, the PGCs or pole cells express head involution defective (hid), which is required for caspase activation, but its translation is repressed by maternal Nos. In the absence of Nos activity, translation of hid mRNA into protein induces apoptosis in pole cells. However, it remains unclear how hid mRNA is regulated in pole cells. Here, we report that hid expression requires eiger (egr), a tumor necrosis factor ligand (TNF) homologue, which is induced in pole cells by decapentaplegic (dpp). In addition, we demonstrate that p53 and loki (lok), a damage-activated kinase known to be required for p53 phosphorylation, are both required for hid expression in pole cells. Since maternal lok mRNA is enriched in pole cells, it is possible that ubiquitously distributed p53 is activated in pole cells by maternal Lok. We propose that hid expression is activated in a pole cell-specific manner by loki/p53 and dpp/egr during embryogenesis.

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

    PubMed

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

    2015-02-10

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

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

    PubMed Central

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

    2004-01-01

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

  4. The role of p53 in cell metabolism

    PubMed Central

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

    2010-01-01

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

  5. Serum starvation and thymidine double blocking achieved efficient cell cycle synchronization and altered the expression of p27, p53, bcl-2 in canine breast cancer cells.

    PubMed

    Tong, Jinjin; Sun, Dongdong; Yang, Chao; Wang, Yingxue; Sun, Sichao; Li, Qing; Bao, Jun; Liu, Yun

    2016-04-01

    Cell synchronization is an approach to obtain cell populations of the same stage, which is a prerequisite to studying the regulation of cell cycle progression in vivo. Serum starvation and thymidine double blocking (TdR) are two important practices in studying cell cycle synchronization. However, their effects on canine cancer cells as well as the regulatory mechanisms by these two methods are poorly understood. In this study, we determined the optimum conditions of serum starvation and TdR and their effects on cell cycle synchronization. We further explored the involvement of PI3K/Akt signaling pathway in the cell cycle synchronization by investigating the expression of three key genes (p27, p53 and bcl-2). Serum starvation resulted in a reversible cell cycle arrest and synchronously progress through G0/G1. The highest percentage of CHMm cells (87.47%) in G0/G1 stage was obtained after 42 h incubation with 0.5% fetal bovine serum (FBS). TdR double blocking could arrest 98.9% of CHMm cells in G1/S phase (0 h of release), and could arrest 93.74% of CHMm cells in S phase after 4h of release. We also found that the p27, p53, bcl-2 genes were most highly expressed in G0/G1 phase. Our current work revealed that serum starvation and TdR methods could achieve sufficient synchronization of CHMm cells. Moreover, the expression of p27, p53 and bcl-2 genes was related to cyclical movements and apoptosis. Our results will provide a new insight into cell cycle regulation and reprogramming of canine cancer cells induced by serum starvation and TdR blocking.

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

    PubMed Central

    Ghosh, Anirban; Stewart, Deborah; Matlashewski, Greg

    2004-01-01

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

  7. Combined Expression of c-jun, c-fos, and p53 Improves Estimation of Prognosis in Oral Squamous Cell Carcinoma.

    PubMed

    Wang, Shan; Xu, Xin; Xu, Fei; Meng, Yan; Sun, Changsheng; Shi, Lei; Zhao, Eryang

    2016-09-13

    To identify the prognostic value of c-jun, c-fos, and p53 in oral cancer, we examined the impact of immunohistochemical expression of these markers on tumor progression in 157 oral squamous cell carcinoma (OSCC). We found that c-jun or c-fos was significantly associated with lymph node metastasis, and coexpression of c-jun/c-fos, or c-jun/c-fos/p53 were significantly associated with lymph node metastasis, poor differentiation and clinical stage. The coexpression of c-jun/c-fos/p53 was identified as independent prognostic factors for overall survival. Simultaneous coexpression of these markers in OSCCs might prove to be a useful indicator for differentiation of low and high-risk patients.

  8. Plakoglobin Reduces the in vitro Growth, Migration and Invasion of Ovarian Cancer Cells Expressing N-Cadherin and Mutant p53

    PubMed Central

    Alaee, Mahsa; Danesh, Ghazal; Pasdar, Manijeh

    2016-01-01

    Aberrant expression of cadherins and catenins plays pivotal roles in ovarian cancer development and progression. Plakoglobin (PG, γ-catenin) is a paralog of β-catenin with dual adhesive and signaling functions. While β-catenin has known oncogenic function, PG generally acts as a tumor/metastasis suppressor. We recently showed that PG interacted with p53 and that its growth/metastasis inhibitory function may be mediated by this interaction. Very little is known about the role of PG in ovarian cancer. Here, we investigated the in vitro tumor/metastasis suppressor effects of PG in ovarian cancer cell lines with mutant p53 expression and different cadherin profiles. We showed that the N-cadherin expressing and E-cadherin and PG deficient ES-2 cells were highly migratory and invasive, whereas OV-90 cells that express E-cadherin, PG and very little/no N-cadherin were not. Exogenous expression of PG or E-cadherin or N-cadherin knockdown in ES-2 cells (ES-2-E-cad, ES-2-PG and ES-2-shN-cad) significantly reduced their migration and invasion. Also, PG expression or N-cadherin knockdown significantly decreased ES-2 cells growth. Furthermore, PG interacted with both cadherins and with wild type and mutant p53 in normal ovarian and ES-2-PG cell lines, respectively. PMID:27144941

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

    PubMed Central

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

    2017-01-01

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

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

    SciTech Connect

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

    2013-02-15

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

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

    PubMed Central

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

    2017-01-01

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

  12. Wild-type and mutated presenilins 2 trigger p53-dependent apoptosis and down-regulate presenilin 1 expression in HEK293 human cells and in murine neurons

    PubMed Central

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

    2002-01-01

    Presenilins 1 and 2 are two homologous proteins that, when mutated, account for most early onset Alzheimer's disease. Several lines of evidence suggest that, among various functions, presenilins could modulate cell apoptotic responses. Here we establish that the overexpression of presenilin 2 (PS2) and its mutated form Asn-141-Ile-PS2 alters the viability of human embryonic kidney (HEK)293 cells as established by combined trypan blue exclusion, sodium 3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate assay, and propidium iodide incorporation FACS analyses. The two parent proteins increase the acetyl-DEVD-al-sensitive caspase-3-like activity in both HEK293 cells and Telencephalon specific murine neurons, modulate Bax and bcl-2 expressions, and enhance cytochrome C translocation into the cytosol. We show that overexpression of both wild-type and mutated PS2 increases p53-like immunoreactivity and transcriptional activity. We also establish that wild-type- and mutated PS2-induced caspase activation is reduced by p53 antisense approach and by pifithrin-α, a chemical inhibitor of p53. Furthermore, mouse fibroblasts in which the PS2 gene has been knocked out exhibited strongly reduced p53-transcriptional activity. Finally, we establish that the overexpression of both wild-type and mutated PS2 is accompanied by a drastic reduction of endogenous presenilin 1 (PS1) expression. Interestingly, pifithrin-α diminished endogenous PS2 immunoreactivity, whereas the inhibitor increases PS1 expression. Altogether, our data demonstrate that wild-type and familial Alzheimer's disease-linked PS2 trigger apoptosis and down-regulate PS1 expression through p53-dependent mechanisms. PMID:11904448

  13. Anomalous retinoblastoma protein expression in Sternberg-Reed cells in Hodgkin's disease: a comparative study with p53 and Ki67 expression.

    PubMed Central

    Sánchez-Beato, M.; Martínez-Montero, J. C.; Doussis-Anagnostopoulou, T. A.; Gatter, K. C.; García, J.; García, J. F.; LLoret, E.; Piris, M. A.

    1996-01-01

    Retinoblastoma (Rb) tumour-suppressor protein plays a critical role in cell cycle control. Rb inactivation is a frequent phenomenon in tumours of different cell lineages, in which the absence of Rb protein has been considered to be a marker of Rb disregulation. We used modern immunohistochemical techniques to study the expression of Rb protein in a large series of 130 patients with Hodgkin's disease. Simultaneously, Western blot was used to analyse a more restricted group (12 patients) to confirm the immunohistochemical results and to clarify the phosphorylation status of Rb protein. As the level of Rb expression varied according to cell cycle stage, we also performed immunostaining for Ki67, a protein present in proliferating cells. To make comparison possible, we first characterised the amount and phosphorylation status of Rb protein in reactive lymphoid tissue and phytohaemagglutinin (PHA)-stimulated lymphocytes. The presence of p53 in Sternberg-Reed cells was also included in the study, as both proteins (p53 and Rb) have been found to be closely associated in cell cycle control. PHA-stimulated peripheral blood lymphocytes showed a parallel increase in Rb and cell cycle progression, together with progressive Rb phosphorylation. In reactive lymphoid tissue there was also a clear correlation between Rb expression and the Ki67 proliferation index (R = 0.96, P = 0.038). When analysing Hodgkin's disease samples, a clear difference emerges between cases of nodular lymphocyte predominance, which preserve the relationship between Rb and Ki67 expression (r = 0.8727, P = 0.000), and classical forms of Hodgkin's disease (nodular sclerosis and mixed cellularity), which display a strong deviation from this pattern. Two main anomalies were found: (1) One group of 21/130 cases with partial or total loss of Rb protein expression, which could reflect the existence of genetic alterations, or an altered transcriptional or translational regulation of Rb gene. (2) Another group with

  14. Restoration of microRNA-214 expression reduces growth of myeloma cells through positive regulation of P53 and inhibition of DNA replication

    PubMed Central

    Misiewicz-Krzeminska, Irena; Sarasquete, María E.; Quwaider, Dalia; Krzeminski, Patryk; Ticona, Fany V.; Paíno, Teresa; Delgado, Manuel; Aires, Andreia; Ocio, Enrique M.; García-Sanz, Ramón; San Miguel, Jesús F.; Gutiérrez, Norma C.

    2013-01-01

    MicroRNA have been demonstrated to be deregulated in multiple myeloma. We have previously reported that miR-214 is down-regulated in multiple myeloma compared to in normal plasma cells. The functional role of miR-214 in myeloma pathogenesis was explored by transfecting myeloma cell lines with synthetic microRNA followed by gene expression profiling. Putative miR-214 targets were validated by luciferase reporter assay. Ectopic expression of miR-214 reduced cell growth and induced apoptosis of myeloma cells. In order to identify the potential direct target genes of miR-214 which could be involved in the biological pathways regulated by this microRNA, gene expression profiling of the H929 myeloma cell line transfected with precursor miR-214 was carried out. Functional analysis revealed significant enrichment for DNA replication, cell cycle phase and DNA binding. miR-214 directly down-regulated the expression of PSMD10, which encodes the oncoprotein gankyrin, and ASF1B, a histone chaperone required for DNA replication, by binding to their 3'-untranslated regions. In addition, gankyrin inhibition induced an increase of P53 mRNA levels and subsequent up-regulation of CDKN1A (p21Waf1/Cip1) and BAX transcripts, which are direct transcriptional targets of p53. In conclusion, MiR-214 functions as a tumor suppressor in myeloma by positive regulation of p53 and inhibition of DNA replication. PMID:23100276

  15. Modification of tumor cell exosome content by transfection with wt-p53 and microRNA-125b expressing plasmid DNA and its effect on macrophage polarization

    PubMed Central

    Trivedi, M; Talekar, M; Shah, P; Ouyang, Q; Amiji, M

    2016-01-01

    Exosomes are responsible for intercellular communication between tumor cells and others in the tumor microenvironment. These microvesicles promote oncogensis and can support towards metastasis by promoting a pro-tumorogenic environment. Modifying the exosomal content and exosome delivery are emerging novel cancer therapies. However, the clinical translation is limited due to feasibility of isolating and delivery of treated exosomes as well as an associated immune response in patients. In this study, we provide proof-of-concept for a novel treatment approach for manipulating exosomal content by genetic transfection of tumor cells using dual-targeted hyaluronic acid-based nanoparticles. Following transfection with plasmid DNA encoding for wild-type p53 (wt-p53) and microRNA-125b (miR-125b), we evaluate the transgene expression in the SK-LU-1 cells and in the secreted exosomes. Furthermore, along with modulation of wt-p53 and miR-125b expression, we also show that the exosomes (i.e., wt-p53/exo, miR-125b/exo and combination/exo) have a reprogramed global miRNA profile. The miRNAs in the exosomes were mainly related to the activation of genes associated with apoptosis as well as p53 signaling. More importantly, these altered miRNA levels in the exosomes could mediate macrophage repolarization towards a more pro-inflammatory/antitumor M1 phenotype. However, further studies, especially in vivo studies, are warranted to assess the direct influence of such macrophage reprogramming on cancer cells and oncogenesis post-treatment. The current study provides a novel platform enabling the development of therapeutic strategies affecting not only the cancer cells but also the tumor microenvironment by utilizing the ‘bystander effect' through genetic transfer with secreted exosomes. Such modification could also support antitumor environment leading to decreased oncogenesis. PMID:27500388

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

    PubMed Central

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

    2016-01-01

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

  17. Ovotoxic Effects of Galactose Involve Attenuation of Follicle-Stimulating Hormone Bioactivity and Up-Regulation of Granulosa Cell p53 Expression

    PubMed Central

    Banerjee, Sayani; Chakraborty, Pratip; Saha, Piyali; Bandyopadhyay, Soma Aditya; Banerjee, Sutapa; Kabir, Syed N.

    2012-01-01

    Clinical evidence suggests an association between galactosaemia and premature ovarian insufficiency (POI); however, the mechanism still remains unresolved. Experimental galactose toxicity in rats produces an array of ovarian dysfunction including ovarian development with deficient follicular reserve and follicular resistance to gonadotrophins that characterize the basic tenets of human POI. The present investigation explores if galactose toxicity in rats attenuates the bioactivity of gonadotrophins or interferes with their receptor competency, and accelerates the rate of follicular atresia. Pregnant rats were fed isocaloric food-pellets supplemented with or without 35% D-galactose from day-3 of gestation and continuing through weaning of the litters. The 35-day old female litters were autopsied. Serum galactose-binding capacity, galactosyltransferase (GalTase) activity, and bioactivity of FSH and LH together with their receptor competency were assessed. Ovarian follicular atresia was evaluated in situ by TUNEL. The in vitro effects of galactose were studied in isolated whole follicles in respect of generation of reactive oxygen species (ROS) and expression of caspase 3, and in isolated granulosa cells in respect of mitochondrial membrane potential, expression of p53, and apoptosis. The rats prenatally exposed to galactose exhibited significantly decreased serum GalTase activity and greater degree of galactose-incorporation capacity of sera proteins. LH biopotency and LH-FSH receptor competency were comparable between the control and study population, but the latter group showed significantly attenuated FSH bioactivity and increased rate of follicular atresia. In culture, galactose increased follicular generation of ROS and expression of caspase 3. In isolated granulosa cells, galactose disrupted mitochondrial membrane potential, stimulated p53 expression, and induced apoptosis in vitro; however co-treatment with either FSH or estradiol significantly prevented

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

    PubMed Central

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

    1997-01-01

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

  19. Repeated PM2.5 exposure inhibits BEAS-2B cell P53 expression through ROS-Akt-DNMT3B pathway-mediated promoter hypermethylation.

    PubMed

    Zhou, Wei; Tian, Dongdong; He, Jun; Wang, Yimei; Zhang, Lijun; Cui, Lan; Jia, Li; Zhang, Li; Li, Lizhong; Shu, Yulei; Yu, Shouzhong; Zhao, Jun; Yuan, Xiaoyan; Peng, Shuangqing

    2016-04-12

    Long-term exposure to fine particulate matter (PM2.5) has been reported to be closely associated with the increased lung cancer risk in populations, but the mechanisms underlying PM-associated carcinogenesis are not yet clear. Previous studies have indicated that aberrant epigenetic alterations, such as genome-wide DNA hypomethylation and gene-specific DNA hypermethylation contribute to lung carcinogenesis. And silence or mutation of P53 tumor suppressor gene is the most prevalent oncogenic driver in lung cancer development. To explore the effects of PM2.5 on global and P53 promoter methylation changes and the mechanisms involved, we exposed human bronchial epithelial cells (BEAS-2B) to low concentrations of PM2.5 for 10 days. Our results indicated that PM2.5-induced global DNA hypomethylation was accompanied by reduced DNMT1 expression. PM2.5 also induced hypermethylation of P53 promoter and inhibited its expression by increasing DNMT3B protein level. Furthermore, ROS-induced activation of Akt was involved in PM2.5-induced increase in DNMT3B. In conclusion, our results strongly suggest that repeated exposure to PM2.5 induces epigenetic silencing of P53 through ROS-Akt-DNMT3B pathway-mediated promoter hypermethylation, which not only provides a possible explanation for PM-induced lung cancer, but also may help to identify specific interventions to prevent PM-induced lung carcinogenesis.

  20. Enhanced radiosensitization of p53 mutant cells by oleamide

    SciTech Connect

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

    2006-04-01

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

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

    PubMed

    Slovackova, J; Smarda, J; Smardova, J

    2012-01-01

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

  2. Mitochondrial matrix P53 sensitizes cells to oxidative stress☆

    PubMed Central

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

    2013-01-01

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

  3. Gleditsia sinensis thorn extract inhibits human colon cancer cells: the role of ERK1/2, G2/M-phase cell cycle arrest and p53 expression.

    PubMed

    Lee, Se-Jung; Park, Keerang; Ha, Sang-Do; Kim, Wun-Jae; Moon, Sung-Kwon

    2010-12-01

    The thorns of Gleditsia sinensis are used as a medicinal herb in China and Korea. However, the mechanisms responsible for the antitumor effects of the water extract of Gleditsia sinensis thorns (WEGS) remain unknown. HCT116 cells treated with the WEGS at a dose of 800 μg/mL (IC₅₀) showed a significant decrease in cell growth and an increase in cell cycle arrest during the G2/M-phase. G2/M-phase arrest was correlated with increased p53 levels and down-regulation of the check-point proteins, cyclinB1, Cdc2 and Cdc25c. In addition, treatment with WEGS induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 MAP kinase and JNK (c-Jun N-terminal kinases). Moreover, inhibition of ERK by treatment of cells with the ERK-specific inhibitor PD98059 blocked WEGS-mediated p53 expression. Similarly, blockage of ERK function in the WEGS-treated cells reversed cell-growth inhibition and decreased cell cycle proteins. Finally, in vivo WEGS treatment significantly inhibited the growth of HCT116 tumor cell xenografts in nude mice with no negative side effects, including loss of body weight. These results describe the molecular mechanisms whereby the WEGS might inhibit proliferation of colon cancer both in vitro and in vivo, suggesting that WEGS has potential as an anticancer agent for the treatment of malignancies.

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

    PubMed

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

    2015-03-01

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

  5. Radiation and SN38 treatments modulate the expression of microRNAs, cytokines and chemokines in colon cancer cells in a p53-directed manner.

    PubMed

    Pathak, Surajit; Meng, Wen-Jian; Nandy, Suman Kumar; Ping, Jie; Bisgin, Atil; Helmfors, Linda; Waldmann, Patrik; Sun, Xiao-Feng

    2015-12-29

    Aberrant expression of miRNAs, cytokines and chemokines are involved in pathogenesis of colon cancer. However, the expression of p53 mediated miRNAs, cyto- and chemokines after radiation and SN38 treatment in colon cancer remains elusive. Here, human colon cancer cells, HCT116 with wild-type, heterozygous and a functionally null p53, were treated by radiation and SN38. The expression of 384 miRNAs was determined by using the TaqMan® miRNA array, and the expression of cyto- and chemokines was analyzed by Meso-Scale-Discovery instrument. Up- or down-regulations of miRNAs after radiation and SN38 treatments were largely dependent on p53 status of the cells. Cytokines, IL-6, TNF-α, IL-1β, Il-4, IL-10, VEGF, and chemokines, IL-8, MIP-1α were increased, and IFN-γ expression was decreased after radiation, whereas, IL-6, IFN-γ, TNF-α, IL-1β, Il-4, IL-10, IL-8 were decreased, and VEGF and MIP-1α were increased after SN38 treatment. Bioinformatic analysis pointed out that the highly up-regulated miRNAs, let-7f-5p, miR-455-3p, miR-98, miR-155-5p and the down-regulated miRNAs, miR-1, miR-127-5p, miR-142-5p, miR-202-5p were associated with colon cancer pathways and correlated with cyto- or chemokine expression. These miRNAs have the potential for use in colon cancer therapy as they are related to p53, pro- or anti-inflammatory cyto- or chemokines after the radiation and SN38 treatment.

  6. Radiation and SN38 treatments modulate the expression of microRNAs, cytokines and chemokines in colon cancer cells in a p53-directed manner

    PubMed Central

    Pathak, Surajit; Meng, Wen-Jian; Nandy, Suman Kumar; Ping, Jie; Bisgin, Atil; Helmfors, Linda; Waldmann, Patrik; Sun, Xiao-Feng

    2015-01-01

    Aberrant expression of miRNAs, cytokines and chemokines are involved in pathogenesis of colon cancer. However, the expression of p53 mediated miRNAs, cyto- and chemokines after radiation and SN38 treatment in colon cancer remains elusive. Here, human colon cancer cells, HCT116 with wild-type, heterozygous and a functionally null p53, were treated by radiation and SN38. The expression of 384 miRNAs was determined by using the TaqMan® miRNA array, and the expression of cyto- and chemokines was analyzed by Meso-Scale-Discovery instrument. Up- or down-regulations of miRNAs after radiation and SN38 treatments were largely dependent on p53 status of the cells. Cytokines, IL-6, TNF-α, IL-1β, Il-4, IL-10, VEGF, and chemokines, IL-8, MIP-1α were increased, and IFN-γ expression was decreased after radiation, whereas, IL-6, IFN-γ, TNF-α, IL-1β, Il-4, IL-10, IL-8 were decreased, and VEGF and MIP-1α were increased after SN38 treatment. Bioinformatic analysis pointed out that the highly up-regulated miRNAs, let-7f-5p, miR-455-3p, miR-98, miR-155-5p and the down-regulated miRNAs, miR-1, miR-127-5p, miR-142-5p, miR-202-5p were associated with colon cancer pathways and correlated with cyto- or chemokine expression. These miRNAs have the potential for use in colon cancer therapy as they are related to p53, pro- or anti-inflammatory cyto- or chemokines after the radiation and SN38 treatment. PMID:26556872

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

    PubMed

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

    2010-10-22

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

  8. Hepatic expression of the proliferative marker Ki-67 and p53 protein in HBV or HCV cirrhosis in relation to dysplastic liver cell changes and hepatocellular carcinoma.

    PubMed

    Koskinas, J; Petraki, K; Kavantzas, N; Rapti, I; Kountouras, D; Hadziyannis, S

    2005-11-01

    To evaluate hepatic expression of the nuclear proliferative marker Ki-67 and the p53 oncoprotein in hepatitis B virus (HBV)/HCV cirrhosis in relation to dysplastic liver cell changes and hepatocellular carcinoma (HCC). We studied needle liver biopsies from 107 patients with cirrhosis and no HCC (52 HBV, 55 HCV) who had been assessed for protocol studies, and 57 cirrhotic patients with HCC (40 HBV, 17 HCV). We evaluated small and large cell dysplastic changes along with the expression of Ki-67 and p53 by immunohistochemistry. The labelling index (LI) was defined as the proportion (%) of positive-stained nuclei of the 500 measured. Large and small cell dysplastic changes were observed in 12 and 9% of specimens respectively. Only small cell changes were associated with Ki-67 expression. Ki-67 LI was 5.50 +/- 5.7 in cirrhosis (13.90 +/- 3.84 in those with small cell dysplastic changes vs 4.64 +/- 4.98 in those without, P < 0.01), 10.2 +/- 5.95 in cirrhosis with HCC (P < 0.05) and 18.56 +/- 10 in HCC (P < 0.01). Neither the presence of small cell dysplastic changes nor the expression of Ki-67 was related to severity or aetiology of cirrhosis. Expression of p53 was observed in 30% of the non-tumorous and in 53% of the neoplastic tissue obtained from patients with HCC, with no differences between HCV and HBV. Ki-67 and p53 expression was associated with the tumour grade (P < 0.001). Our observations clearly demonstrate the association between the proliferation activity and the morphological changes in the cirrhotic liver from the non-dysplastic to dysplastic lesion to HCC. They also support the hypothesis that p53 alterations are a rather late event in carcinogenesis and related to HCC grade. And finally, they suggest that the final steps of hepatocarcinogenesis are common and independent of the aetiology of the chronic viral infection.

  9. Clinical and pathological correlations of marrow PUMA and P53 expressions in myelodysplastic syndromes.

    PubMed

    Bektas, Ozlen; Uner, Aysegul; Buyukasik, Yahya; Uz, Burak; Bozkurt, Sureyya; Eliacik, Eylem; Işik, Ayse; Haznedaroglu, Ibrahim Celalettin; Goker, Hakan; Demiroglu, Haluk; Aksu, Salih; Ozcebe, Osman Ilhami; Sayinalp, Nilgun

    2015-05-01

    p53 is a key regulator of apoptosis. p53 upregulated modulator of apoptosis (PUMA) is a critical mediator of p53-dependent and independent apoptosis. The objective of this study was to evaluate the relationship of p53 and PUMA to the prognosis of MDS. Bone marrow biopsies of MDS patients at the time of diagnosis (n = 76) and at the time of transformation (n = 19) were included in the study group. The expression of p53 and PUMA was evaluated using immunohistochemistry. When compared to the control group, both p53 (p < 0.001) and PUMA (p = 0.012) expression levels were significantly higher in MDS group. In MDS group, there was a moderate positive correlation between p53 and PUMA expressions. PUMA expression was not correlated with event free and overall survival. However, overall survival was significantly lower in cases with p53 expression in more than 50% of the cells. There was an increase in PUMA expression in cases that showed transformation as compared to the initial diagnostic bone marrows but was not statistically significant. The correlation that existed between p53 and PUMA was lost in transformed cases. Our results showed that PUMA and p53 expressions are increased in MDS marrows compared to normal marrows. PUMA expression increases further during transformation while the expression of p53 is not significantly altered which suggests that PUMA alterations might be a late event during the evolution of MDS.

  10. BAC transgenic mice provide evidence that p53 expression is highly regulated in vivo.

    PubMed

    Chen, L; Zhang, G X; Zhou, Y; Zhang, C X; Xie, Y Y; Xiang, C; He, X Y; Zhang, Q; Liu, G

    2015-09-17

    p53 is an important tumor suppressor and stress response mediator. Proper control of p53 level and activity is tightly associated with its function. Posttranslational modifications and the interactions with Mdm2 and Mdm4 are major mechanisms controlling p53 activity and stability. As p53 protein is short-lived and hardly detectable in unstressed situations, less is known on its basal level expression and the corresponding controlling mechanisms in vivo. In addition, it also remains obscure how p53 expression might contribute to its functional regulation. In this study, we established bacterial artificial chromosome transgenic E.coli β-galactosidase Z gene reporter mice to monitor p53 expression in mouse tissues and identify important regulatory elements critical for the expression in vivo. We revealed preferentially high level of p53 reporter expressions in the proliferating, but not the differentiated compartments of the majority of tissues during development and tissue homeostasis. In addition, tumors as well as regenerating tissues in the p53 reporter mice also expressed high level of β-gal. Furthermore, both the enhancer box sequence (CANNTG) in the p53 promoter and the 3' terminal untranslated region element were critical in mediating the high-level expression of the reporter. We also provided evidence that cellular myelocytomatosis oncogene was a critical player regulating p53 mRNA expression in proliferating cells and tissues. Finally, we found robust p53 activation preferentially in the proliferating compartment of mouse tissues upon DNA damage and the proliferating cells exhibited an enhanced p53 response as compared with cells in a quiescent state. Together, these results suggested a highly regulated expression pattern of p53 in the proliferating compartment controlled by both transcriptional and posttranscriptional mechanisms, and such regulated p53 expression may impose functional significance upon stress by setting up a precautionary mode in defense

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

    DTIC Science & Technology

    2005-09-01

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

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

    SciTech Connect

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

    2008-04-04

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

  13. p53 gene product expression in resected non-small cell carcinoma of the lung, with studies of concurrent cytological preparations and microwave antigen retrieval.

    PubMed Central

    Binks, S; Clelland, C A; Ronan, J; Bell, J

    1997-01-01

    AIM: To document the frequency and extent of p53 gene product expression in paraffin sections of resected non-small cell carcinoma of the lung and in cytological preparations of the same tumours; to determine the effect of microwave antigen retrieval on antigen detection. METHODS: Representative paraffin sections of 50 non-small cell carcinomas were stained with an antibody to p53 gene product (DO-7) both with and without prior microwave antigen retrieval. Cytoblocks and cell smears obtained from 19 cases were similarly stained. RESULTS: Using a histochemical scoring system (0-300) which takes into account staining intensity and extent, 78% (n = 39) of microwave pretreated paraffin sections and 52% (n = 26) of non-pretreated sections scored between 5 and 300; p = 0.001; 56% (n = 28) of microwave pretreated sections and only 2% (n = 1) of non-pretreated sections scored between 100 and 300 (p = 0.0001); 75% of direct smears of tumours and 80% of cytoblocks stained similarly to the paraffin sections of the resected specimens. No smears or cytoblocks stained positively when the sections of the resected specimen were negative. CONCLUSIONS: As up to 78% of non-small cell lung carcinomas overexpress p53 gene product, this may prove to be a valuable diagnostic method in biopsy or cytological material when the morphological diagnosis is uncertain. Microwave antigen retrieval is effective on formalin fixed tissue. Images PMID:9215149

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

    PubMed

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

    2009-02-01

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

  15. p53 shapes genome-wide and cell type-specific changes in microRNA expression during the human DNA damage response

    PubMed Central

    Hattori, Hiroyoshi; Janky, Rekin’s; Nietfeld, Wilfried; Aerts, Stein; Madan Babu, M; Venkitaraman, Ashok R

    2014-01-01

    The human DNA damage response (DDR) triggers profound changes in gene expression, whose nature and regulation remain uncertain. Although certain micro-(mi)RNA species including miR34, miR-18, miR-16 and miR-143 have been implicated in the DDR, there is as yet no comprehensive description of genome-wide changes in the expression of miRNAs triggered by DNA breakage in human cells. We have used next-generation sequencing (NGS), combined with rigorous integrative computational analyses, to describe genome-wide changes in the expression of miRNAs during the human DDR. The changes affect 150 of 1523 miRNAs known in miRBase v18 from 4–24 h after the induction of DNA breakage, in cell-type dependent patterns. The regulatory regions of the most-highly regulated miRNA species are enriched in conserved binding sites for p53. Indeed, genome-wide changes in miRNA expression during the DDR are markedly altered in TP53-/- cells compared to otherwise isogenic controls. The expression levels of certain damage-induced, p53-regulated miRNAs in cancer samples correlate with patient survival. Our work reveals genome-wide and cell type-specific alterations in miRNA expression during the human DDR, which are regulated by the tumor suppressor protein p53. These findings provide a genomic resource to identify new molecules and mechanisms involved in the DDR, and to examine their role in tumor suppression and the clinical outcome of cancer patients. PMID:25486198

  16. p53 shapes genome-wide and cell type-specific changes in microRNA expression during the human DNA damage response.

    PubMed

    Hattori, Hiroyoshi; Janky, Rekin's; Nietfeld, Wilfried; Aerts, Stein; Madan Babu, M; Venkitaraman, Ashok R

    2014-01-01

    The human DNA damage response (DDR) triggers profound changes in gene expression, whose nature and regulation remain uncertain. Although certain micro-(mi)RNA species including miR34, miR-18, miR-16 and miR-143 have been implicated in the DDR, there is as yet no comprehensive description of genome-wide changes in the expression of miRNAs triggered by DNA breakage in human cells. We have used next-generation sequencing (NGS), combined with rigorous integrative computational analyses, to describe genome-wide changes in the expression of miRNAs during the human DDR. The changes affect 150 of 1523 miRNAs known in miRBase v18 from 4-24 h after the induction of DNA breakage, in cell-type dependent patterns. The regulatory regions of the most-highly regulated miRNA species are enriched in conserved binding sites for p53. Indeed, genome-wide changes in miRNA expression during the DDR are markedly altered in TP53-/- cells compared to otherwise isogenic controls. The expression levels of certain damage-induced, p53-regulated miRNAs in cancer samples correlate with patient survival. Our work reveals genome-wide and cell type-specific alterations in miRNA expression during the human DDR, which are regulated by the tumor suppressor protein p53. These findings provide a genomic resource to identify new molecules and mechanisms involved in the DDR, and to examine their role in tumor suppression and the clinical outcome of cancer patients.

  17. Comparative effects of histone deacetylase inhibitors on p53 target gene expression, cell cycle and apoptosis in MCF-7 breast cancer cells.

    PubMed

    Knutson, Andrew Kekapa'a; Welsh, Jennifer; Taylor, Travis; Roy, Somdutta; Wang, Wei-Lin Winnie; Tenniswood, Martin

    2012-03-01

    Histone deacetylase inhibitors are currently being evaluated for their therapeutic potential and have shown considerable promise as adjuvant therapies for a number of cancers. This study compared the effects of 2 hydroxamic acid based inhibitors, CG-1521 and SAHA, on gene expression, cell cycle and cell death in MCF-7 human breast cancer cells. Both compounds show a dose- and time-dependent effect on cell number (evaluated using crystal violet), however CG-1521 exerts its effects significantly earlier than SAHA, and CG-1521 induces apoptosis (assessed by Apo-BrdU staining and flow cytometry) more rapidly than SAHA. qPCR of cell cycle regulatory and apoptotic genes shows that CG-1521 and SAHA modulate similar cohorts of p53-responsive genes, however, the levels of induction and the timing of the induction differs significantly between the 2 inhibitors. In particular SAHA downregulates cell cycle-associated genes that modulate the G1/S transition (including cyclin D1 and cdc25a) and the G2/M transition [cyclin B1, Plk1, Stk6 (serine-threonine kinase 6, Aurora kinase A) and Kntc2] more significantly than CG-1521. In contrast, CG-1521 significantly induces the expression of several p53 target genes associated with apoptosis including Bnip3/Bnip3L, p21/p21B and Gdf15. The differential levels of gene induction provide molecular evidence of both cell cycle arrest and apoptosis, and suggest a molecular mechanism that explains the difference in the biological effects of the 2 histone deacetylase inhibitors.

  18. Combined HDAC1 and HDAC2 Depletion Promotes Retinal Ganglion Cell Survival After Injury Through Reduction of p53 Target Gene Expression

    PubMed Central

    Suter, Ueli

    2015-01-01

    Histones deacetylases (HDACs), besides their function as epigenetic regulators, deacetylate and critically regulate the activity of nonhistone targets. In particular, HDACs control partially the proapoptotic activity of p53 by balancing its acetylation state. HDAC inhibitors have revealed neuroprotective properties in different models, but the exact mechanisms of action remain poorly understood. We have generated a conditional knockout mouse model targeting retinal ganglion cells (RGCs) to investigate specifically the functional role of HDAC1 and HDAC2 in an acute model of optic nerve injury. Our results demonstrate that combined HDAC1 and HDAC2 ablation promotes survival of axotomized RGCs. Based on global gene expression analyses, we identified the p53-PUMA apoptosis-inducing axis to be strongly activated in axotomized mouse RGCs. Specific HDAC1/2 ablation inhibited this apoptotic pathway by impairing the crucial acetylation status of p53 and reducing PUMA expression, thereby contributing to the ensuing enhanced neuroprotection due to HDAC1/2 depletion. HDAC1/2 inhibition and the affected downstream signaling components emerge as specific targets for developing therapeutic strategies in neuroprotection. PMID:26129908

  19. Endopolyploidy in irradiated p53-deficient tumour cell lines: Persistence of cell division activity in giant cells expressing Aurora B- kinase

    PubMed Central

    Erenpreisa, Jekaterina; Ivanov, Andrei; Wheatley, Sally P; Kosmacek, Elizabeth A; Ianzini, Fiorenza; Anisimov, Alim P; Mackey, Michael; Davis, Paul J; Plakhins, Grigorijs; Illidge, Timothy M

    2008-01-01

    Recent findings including computerized live imaging suggest that polyploidy cells transiently emerging after severe genotoxic stress (and named ‘endopolyploid cells’) may have a role in tumour regrowth after anti-cancer treatment. Until now, mostly the factors enabling metaphase were studied in them. Here we investigate the mitotic activities and the role of Aurora B, in view of potential de-polyploidisation of these cells, because Aurora B- kinase is responsible for coordination and completion of mitosis. We observed that endopolyploid giant cells are formed in irradiated p53 tumours in several ways: (1) by division/fusion of daughter cells creating early multi-nucleated cells; (2) by asynchronous division/fusion of sub-nuclei of these multinucleated cells; (3) by a series of polyploidising mitoses reverting replicative interphase from aborted metaphase and forming giant cells with a single nucleus; (4) by micronucleation of arrested metaphases enclosing genome fragments; or (5) by incomplete division in the multipolar mitoses forming late multi-nucleated giant cells. We also observed that these activities are able to release para-diploid cells, although they do so infrequently. Although after a substantial delay, apoptosis typically occurs in these cells, we also found that roughly 2% of endopolyploid cells evade apoptosis and senescence arrest and continue mitotic activities. In this article we describe that catalytically active aurora B-kinase is expressed in the nuclei of many interphase endopolyploid cells, as well as being present at the centromeres, mitotic spindle and cleavage furrow during their mitotic efforts. The totally micronucleated giant cells (containing subgenomic fragments in multiple micronuclei) represented the only minor fraction, which failed to undergo mitosis and Aurora B was absent from it. These observations suggest that most endopolyploid tumour cells are not reproductively inert and that aurora B may contribute to the establishment

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

    PubMed

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

    2003-04-18

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

  1. High expression of fibronectin is associated with poor prognosis, cell proliferation and malignancy via the NF-κB/p53-apoptosis signaling pathway in colorectal cancer

    PubMed Central

    Yi, Wenzhong; Xiao, Enhua; Ding, Ru; Luo, Ping; Yang, Yi

    2016-01-01

    Fibronectin is a glycoprotein of the extracellular matrix, and regulates the processes of self-renewal and cell cycle progression. This study aimed to investigate fibronectin expression in colorectal cancer (CRC) and elucidate the effects of fibronectin on CRC by using a knockdown approach. Immunohistochemistry was used to evaluate the expression of fibronectin in 107 CRC patient tissues and gene expression was detected by real-time quantitative PCR (qPCR) and western blot analysis. Based on the above findings, the association among fibronectin expression, clinicopathological features and prognosis was analyzed. Next, fibronectin expression was silenced by small-interfering RNAs (siRNAs) and the effects of fibronectin siRNA transfection on CRC cells and tumor growth in nude mice were assessed. Expression of genes in the NF-κB/p53-apoptosis signaling pathway were analyzed after fibronectin siRNA transfection both in vitro and in vivo. Based on the results, high expression of fibronectin was observed both in the CRC tissues and CRC cell lines. The expression level was positively correlated with TNM stage (P=0.0025) and distant metastasis (P=0.0013). By Kaplan-Meier analysis, the patients with low fibronectin expression had a longer survival time comparing to those with relatively high expression. Knockdown of fibronectin suppressed SW480 cell proliferation, migration and invasion. In addition, knockdown of fibronectin led to S phase cell cycle arrest. The following study showed that the NF-κB/p53-apoptosis signaling pathway in CRC was affected by fibronectin knockdown. Tumor formation was also depressed by fibronectin siRNA transfection of CRC cells. These results showed the significant role of fibronectin in CRC tissues and cell lines. Therefore, fibronectin may be regarded as a potential target for CRC treatment. PMID:27748871

  2. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

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

  4. Regulation of hTERT Expression and Function in Newly Immortalized p53(+) Human Mammary Epithelial Cell Lines

    DTIC Science & Technology

    2008-06-01

    generation of multiple errors that permit telomerase reactivation.2 In contrast with post-selection HMEC, we show here that GSE22-mediated abrogation of...can also be readily overcome by multiple types of errors that inactivate an Rb-mediated barrier. Agonescence is characterized by a moderate LI...such as radiation, might also employ p53‑dependent p21 to enforce stasis. Multiple types of errors that can inactivate a stress‑induced Rb‑mediated

  5. The long non-coding RNA maternally expressed gene 3 activates p53 and is downregulated in esophageal squamous cell cancer.

    PubMed

    Lv, Desheng; Sun, Run; Yu, Qian; Zhang, Xuefei

    2016-10-24

    Esophageal squamous cell cancer (ESCC) is an aggressive malignancy with poor survival. Long non-coding RNAs (lncRNAs) play important roles in tumorigenesis and cancer progression; hence, lncRNAs are also involved in the development and progression of ESCC. In this study, we used quantitative real-time polymerase chain reaction (qRT-PCR) to investigate expression of lncRNA, maternally expressed gene 3 (MEG3) in ESCC. Ectopic expression of MEG3 was performed in ESCC cell lines. Proliferation and apoptosis of ESCC cell lines were analyzed after ectopic expression of MEG3. We found MEG3 was significantly downregulated in ESCC tissues compared with normal tissues by qRT-PCR. Low expression of MEG3 was correlated with lymph node metastasis and advanced TNM stages of ESCC patients and indicated shorter survival (HR = 0.471, 95 % CI 0.234-0.950, P = 0.035), which was confirmed by The Cancer Genome Atlas (TCGA) esophageal cancer dataset. DNA-demethylating agent (5-aza-2-deoxy-cytidine (5-aza-CdR)) treatment significantly increased MEG3 expression level in ESCC cells, and TCGA esophageal cancer dataset also showed that DNA methylation of MEG3 predicted survival. Ectopic expression of MEG3 in ESCC cells inhibited cell proliferation, promoted apoptosis, and suppressed metastasis. Further investigation showed enforced expression of MEG3 activated p53 and its target genes by downregulation of mouse double minute 2 homolog (MDM2). Overall, our study indicated that MEG3 expression loss is common in ESCC and MEG3 could activate p53 and predict prognosis in ESCC.

  6. Cisplatin modulates B-cell translocation gene 2 to attenuate cell proliferation of prostate carcinoma cells in both p53-dependent and p53-independent pathways.

    PubMed

    Chiang, Kun-Chun; Tsui, Ke-Hung; Chung, Li-Chuan; Yeh, Chun-Nan; Feng, Tsui-Hsia; Chen, Wen-Tsung; Chang, Phei-Lang; Chiang, Hou-Yu; Juang, Horng-Heng

    2014-07-01

    Cisplatin is a widely used anti-cancer drug. The B-cell translocation gene 2 (BTG2) is involved in the cell cycle transition regulation. We evaluated the cisplatin effects on prostate cancer cell proliferation and the expressions of BTG2, p53, androgen receptor (AR) and prostate specific antigen (PSA) in prostate carcinoma, p53 wild-type LNCaP or p53-null PC-3, cells. Cisplatin treatments attenuated cell prostate cancer cell growth through inducing Go/G1 cell cycle arrest in lower concentration and apoptosis at higher dosage. Cisplatin treatments enhanced p53 and BTG2 expression, repressed AR and PSA expression, and blocked the activation of androgen on the PSA secretion in LNCaP cells. BTG2 knockdown in LNCaP cells attenuated cisplatin-mediated growth inhibition. Cisplatin enhanced BTG2 gene expression dependent on the DNA fragment located within -173 to -82 upstream of BTG2 translation initiation site in prostate cancer cells. Mutation of the p53 response element from GGGCAGAGCCC to GGGCACC or mutation of the NFκB response element from GGAAAGTCC to GGAAAGGAA by site-directed mutagenesis abolished the stimulation of cisplatin on the BTG2 promoter activity in LNCaP or PC-3 cells, respectively. Our results indicated that cisplatin attenuates prostate cancer cell proliferation partly mediated by upregulation of BTG2 through the p53-dependent pathway or p53-independent NFκB pathway.

  7. Development of a novel recombinant adenovirus containing gfp-zeocin fusion expression cassette for conditional replication in p53-deficient human tumor cells.

    PubMed

    Hu, Baoli; Joshua, Mallam Nock; Dong, Changyuan; Qi, Yipeng

    2004-05-01

    Two obstacles limiting the efficacy of nearly all cancer gene therapy trails are low gene transduction efficiency and the lack of tumor specificity. Fortunately, a replication-competent, E1B-deficient adenovirus (dl1520) was developed that could overcome these limitations, because it was capable of efficiently and selectively destroying tumor cells lacking functional p53. In an attempt to appraise the efficiency and safety of this approach, a novel recombinant adenovirus, r3/Ad, containing a gfp-zeocin expression cassette was constructed in this work. The study in vitro demonstrated that r3/Ad has the ability to replicate in and lyse only the p53-deficient human tumor cells such as the human glioblastoma cells (U251) and human bladder cells (EJ) but not in the human fibroblast cells (MRC-5) with functional p53. Importantly, this gfp-zeocin fusion gene driven by the bipromoter (CMV and EM-7) could be used as an effective selective marker and reporter in prokaryotic and eukaryotic cells; and also zeocin as a selective marker could minimize contamination of the recombinant virus by the wt-Ad5. Additionally, it was found that the r3/Ad could be useful for studying the selective replication of E1B-deficient adenovirus in vivo, it could be used as a "guide" to study the ability of the recombinant adenovirus to spread and to infect distant tumor cells in any tumor bearing animal model by GFP as a reporter. This may help determine the safety of using any E1B-deficient adenovirus in cancer gene therapy.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-07-20

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

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

    SciTech Connect

    Bajgelman, Marcio C.; Strauss, Bryan E.

    2008-02-05

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

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

    PubMed

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

    2016-09-01

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

  12. Liver p53 expression in patients with HCV-related chronic hepatitis.

    PubMed

    Loguercio, C; Cuomo, A; Tuccillo, C; Gazzerro, P; Cioffi, M; Molinari, A M; Del Vecchio Blanco, C

    2003-07-01

    Mutated p53 acts as a dominant oncogene and alterations in the p53 gene are described in a large number of patients with hepatocellular carcinoma (HCC). It has been demonstrated that hepatitis C virus (HCV)-core protein regulates transcriptionally cellular genes, as well as cell growth and apoptosis. This study was undertaken to evaluate whether p53 may be expressed also in a precocious stage of HCV-related liver damage. We studied p53 expression by immunoluminometric assay on liver samples from 40 patients (M/F 18/ 22, median age 44 years, range 13-64 years) with biopsy-proven HCV-related chronic hepatitis. We considered the following factors: degree of liver damage, liver iron content and HCV-RNA titre. We also evaluated as possible co-factors alcohol and food intake in the last 3 years. p53 was over-expressed in seven of 40 (17.5%) patients. Liver histology documented the presence of unexpected cirrhosis in two patients among the p53 positive subjects. The p53 positive group had a daily ethanol intake significantly higher in respect to that of the p53 negative group (P < 0.05). Alimentary history documented that patients with a p53 over-expression had a lower intake of total calories, monounsaturated fatty acids, vitamin C and riboflavin. Data indicate that p53 over-expression can occur even in initial stages of HCV-related liver disease.

  13. Expression of p21(WAF1/CIP1/SDI1) and p53 in apoptotic cells in the adrenal cortex and induction by ischemia/reperfusion injury.

    PubMed Central

    Didenko, V V; Wang, X; Yang, L; Hornsby, P J

    1996-01-01

    p21(WAF1/CIP1/SDI1), an inhibitor of cyclin-dependent kinases, is expressed at varying levels in human adrenal glands removed during surgery or organ recovery. In glands with p21 mRNA, nuclear p21 immunoreactivity, which was occasionally extensive, colocalized with p53 immunoreactivity and DNA damage, as evidenced by in situ end-labeling. Many cells showed morphological features of apoptosis when observed by fluorescent DNA dye staining and electron microscopy. This pattern was also associated with high levels of cytoplasmic heat shock protein 70. To address the question of the origin of p21 expression in some human adrenal glands, rat adrenal glands were subjected to 30 min of ischemia followed by 8 h of reperfusion. Cells with nuclear p21 and p53 appeared in the adrenal cortex together with DNA damage detected by in situ end-labeling. Nuclear p21 immunoreactivity was also produced in adrenal tissue fragments incubated at 37 degrees C in vitro. However, in this case, p21 expression was confined to the cut edge of the tissue. In contrast, p21 in human adrenal glands, as in ischemic rat glands, was within the inner regions of the cortex, supporting an origin of the protein in vivo rather than postmortem. The p53/p21 pathway of reaction to cellular injury, potentially leading to apoptosis, may play a role in tissue damage such as that resulting from ischemia/reperfusion. In the human adrenal cortex this process may be a precursor of adrenal failure. PMID:8601638

  14. Radiation response and cell cycle regulation of p53 rescued malignant keratinocytes

    SciTech Connect

    Niemantsverdriet, Maarten; Jongmans, Wim; Backendorf, Claude . E-mail: backendo@chem.leidenuniv.nl

    2005-10-15

    Mutations in the tumor suppressor gene p53 were found in more than 90% of all human squamous cell carcinomas (SCC). To study the function of p53 in a keratinocyte background, a tetracycline-controlled p53 transgene was introduced into a human SCC cell line (SCC15), lacking endogenous p53. Conditional expression of wild-type p53 protein upon withdrawal of tetracycline was accompanied with increased expression of p21{sup WAF1/Cip1} resulting in reduced cell proliferation. Flow-cytometric analysis revealed that these cells were transiently arrested in the G1/S phase of the cell cycle. However, when SCC15 cells expressing p53 were exposed to ionizing radiation (IR), a clear shift from a G1/S to a G2/M cell cycle arrest was observed. This effect was greatly depending on the presence of wild-type p53, as it was not observed to the same extent in SCC15 cells lacking p53. Unexpectedly, the p53- and IR-dependent G2/M cell cycle arrest in the keratinocyte background was not depending on increased expression or stabilization of 14-3-3{sigma}, a p53-regulated effector of G2/M progression in colorectal cancer cells. In keratinocytes, 14-3-3{sigma} (stratifin) is involved in terminal differentiation and its cell cycle function in this cell type might diverge from the one it fulfills in other cellular backgrounds.

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

    PubMed

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

    2014-01-01

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

  16. Prognostic value of microvessel density and p53 expression on the locoregional metastasis and survival of the patients with head and neck squamous cell carcinoma.

    PubMed

    de Oliveira, Marcos Vinícius M; Pereira Gomes, Erika P; Pereira, Camila S; de Souza, Ludmilla R; Barros, Lucas O; Mendes, Danilo C; Guimarães, André L S; De Paula, Alfredo M B

    2013-10-01

    Cancer cells need to develop microvessels in order to grow and to establish metastatic foci. A role for the p53 protein in the regulation of the angiogenic process is suggested. This study aimed to investigate the relationship between immunohistochemical expression of microvessel density (MVD), measured by CD31 staining, and p53 protein with clinicopathologic factors, and survival in head and neck squamous cell carcinoma (n=70). Tumor angiogenesis was estimated by determining MVD in areas with the highest number of stained microvessels (hot spots). Clinicopathologic factors and immunohistochemical data were evaluated by χ statistical test and were submitted to binary logistic regression to analyze the risk of presence of lymph node metastasis. Factors that might predict survival were investigated using Cox proportional hazards tests. Differences were considered statistically significant when P<0.05. The percentage of p53-positive cells showed no association with clinicopathologic parameters and MVD. Patients with locoregional metastasis presented statistically significant higher MVD (P=0.043). Individuals presenting head and neck squamous cell carcinoma in posterior sites (P=0.022; OR=3.644) and higher MVD (P=0.039; OR=3.247) had a significant increase in risk of metastasis occurrence. Multivariate analysis showed that presence of lymph node metastasis was statistically significant for overall survival of head and neck carcinoma patients (P=0.006; OR =2.917). The present data suggest that MVD represents a promising diagnostic tool to identify individuals with increased risk for the development of metastatic disease, which is very indicative of poor prognosis.

  17. P53 tumor suppressor gene and protein expression is altered in cell lines derived from spontaneous and alpha-radiation-induced canine lung tumors

    SciTech Connect

    Tierney, L.A.; Johnson, N.F.; Lechner, J.F.

    1994-11-01

    Mutations in the p53 tumor suppressor gene are the most frequently occurring gene alterations in malignant human cancers, including lung cancer. In lung cancer, common point mutations within conserved exons of the p53 gene result in a stabilized form of mutant protein which is detectable in most cases by immunohistochemistry. In addition to point mutations, allelic loss, rearrangements, and deletions of the p53 gene have also been detected in both human and rodent tumors. It has been suggested that for at least some epithelial neoplasms, the loss of expression of wild-type p53 protein may be more important for malignant transformation than the acquisition of activating mutations. Mechanisms responsible for the loss of expression of wild-type protein include gene deletion or rearrangement, nonsense or stop mutations, mutations within introns or upstream regulatory regions of the gene, and accelerated rates of degradation of the protein by DNA viral oncoproteins.

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

    PubMed

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

    2002-12-01

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

  19. Y14 governs p53 expression and modulates DNA damage sensitivity

    PubMed Central

    Lu, Chia-Chen; Lee, Chi-Chieh; Tseng, Ching-Tzu; Tarn, Woan-Yuh

    2017-01-01

    Y14 is a core component of the exon junction complex (EJC), while it also exerts cellular functions independent of the EJC. Depletion of Y14 causes G2/M arrest, DNA damage and apoptosis. Here we show that knockdown of Y14 induces the expression of an alternative spliced isoform of p53, namely p53β, in human cells. Y14, in the context of the EJC, inhibited aberrant exon inclusion during the splicing of p53 pre-mRNA, and thus prevent p53β expression. The anti-cancer agent camptothecin specifically suppressed p53β induction. Intriguingly, both depletion and overexpression of Y14 increased overall p53 protein levels, suggesting that Y14 governs the quality and quantity control of p53. Moreover, Y14 depletion unexpectedly reduced p21 protein levels, which in conjunction with aberrant p53 expression accordingly increased cell sensitivity to genotoxic agents. This study establishes a direct link between Y14 and p53 expression and suggests a function for Y14 in DNA damage signaling. PMID:28361991

  20. Mutant p53 upregulates alpha-1 antitrypsin expression and promotes invasion in lung cancer.

    PubMed

    Shakya, R; Tarulli, G A; Sheng, L; Lokman, N A; Ricciardelli, C; Pishas, K I; Selinger, C I; Kohonen-Corish, M R J; Cooper, W A; Turner, A G; Neilsen, P M; Callen, D F

    2017-04-03

    Missense mutations in the TP53 tumor-suppressor gene inactivate its antitumorigenic properties and endow the incipient cells with newly acquired oncogenic properties that drive invasion and metastasis. Although the oncogenic effect of mutant p53 transcriptome has been widely acknowledged, the global influence of mutant p53 on cancer cell proteome remains to be fully elucidated. Here, we show that mutant p53 drives the release of invasive extracellular factors (the ‘secretome’) that facilitates the invasion of lung cancer cell lines. Proteomic characterization of the secretome from mutant p53-inducible H1299 human non-small cell lung cancer cell line discovered that the mutant p53 drives its oncogenic pathways through modulating the gene expression of numerous targets that are subsequently secreted from the cells. Of these genes, alpha-1 antitrypsin (A1AT) was identified as a critical effector of mutant p53 that drives invasion in vitro and in vivo, together with induction of epithelial–mesenchymal transition markers expression. Mutant p53 upregulated A1AT transcriptionally through the involvement with its family member p63. Conditioned medium containing secreted A1AT enhanced cell invasion, while an A1AT-blocking antibody attenuated the mutant p53-driven migration and invasion. Importantly, high A1AT expression correlated with increased tumor stage, elevated p53 staining and shorter overall survival in lung adenocarcinoma patients. Collectively, these findings suggest that A1AT is an indispensable target of mutant p53 with prognostic and therapeutic potential in mutant p53-expressing tumors. Oncogene advance online publication, 3 April 2017; doi:10.1038/onc.2017.66.

  1. Recombinant adeno-associated virus expressing a p53-derived apoptotic peptide (37AA) inhibits HCC cells growth in vitro and in vivo.

    PubMed

    Zhang, Hongyong; Wang, Yufeng; Bai, Yanxia; Shao, Yuan; Bai, Jigang; Ma, Zhenhua; Liu, Qingguang; Wu, Shengli

    2017-02-06

    Recent studies have confirmed that a p53-derived apoptotic peptide (37AA) could act as a tumor suppressor inducing apoptosis in multiple tumor cells through derepressing p73. However, the tumor suppressive effects of recombinant adeno-associated virus (rAAV) expressing 37AA on HCC cells are still unknown. In this study, we successfully constructed a recombinant rAAV expressing 37AA. In vitro and in vivo assays showed that transfection of NT4-37AA/rAAV in HCC cells strongly suppressed cell proliferation, induced apoptosis, and up-regulated the cellular expression of p73. NT4-37AA/rAAV transfection markedly slowed Huh-7 xenografted tumor growth in murine. Pretreatment of HCC cells with p73 siRNA abrogated these effects of NT4-37AA/rAAV. Furthermore, we found that expression of p73 was upregulated and the formation of P73/iASSP complex was prevented when 37AA was introduced into HCC cells. Taken together, these results indicate that introduction of 37AA into HCC cells with a rAAV vector may lead to the development of broadly applicable agents for the treatment of HCC, and the mechanism may, at least in part, be associated with the upregulation of p73 expression and reduced level of P73/iASSP complex.

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

    PubMed Central

    2001-01-01

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

  3. Gene expression in the lung of p53 mutant mice exposed to cigarette smoke.

    PubMed

    Izzotti, Alberto; Cartiglia, Cristina; Longobardi, Mariagrazia; Bagnasco, Maria; Merello, Andrea; You, Ming; Lubet, Ronald A; De Flora, Silvio

    2004-12-01

    We showed previously that p53 mutations play a role in cigarette smoke-related carcinogenesis not only in humans but also in A/J mice. In fact, (UL53-3 x A/J)F(1) mice, carrying a dominant-negative germ-line p53 mutation, responded to exposure to environmental cigarette smoke more efficiently than their wild-type (wt) littermate controls in terms of molecular alterations, cytogenetic damage, and lung tumor yield. To clarify the mechanisms involved, we analyzed by cDNA array the expression of 1,185 cancer-related genes in the lung of the same mice. Neither environmental cigarette smoke nor the p53 status affected the expression of the p53 gene, but the p53 mutation strikingly increased the basal levels of p53 nuclear protein in the lung. Environmental cigarette smoke increased p53 protein levels in wt mice only. The p53 mutation enhanced the expression of positive cell cycle regulators in sham-exposed mice, which suggests a physiologic protective role of p53. In environmental cigarette smoke-exposed mice, the p53 mutation resulted in a lack of induction of proapoptotic genes and in overexpression of genes involved in cell proliferation, signal transduction, angiogenesis, inflammation, and immune response. Mutant mice and wt mice reacted to environmental cigarette smoke in a similar manner regarding genes involved in metabolism of xenobiotics, multidrug resistance, and protein repair. Irrespective of the p53 status, environmental cigarette smoke poorly affected the expression of oncogenes, tumor suppressor genes, and DNA repair genes. Taken together, these findings may explain the increased susceptibility of p53 mutant mice to smoke-related alterations of intermediate biomarkers and lung carcinogenesis.

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

    SciTech Connect

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

    2007-12-01

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

  5. The traditional Chinese medical compound Rocaglamide protects nonmalignant primary cells from DNA damage-induced toxicity by inhibition of p53 expression

    PubMed Central

    Becker, M S; Schmezer, P; Breuer, R; Haas, S F; Essers, M A; Krammer, P H; Li-Weber, M

    2014-01-01

    One of the main obstacles of conventional anticancer therapy is the toxicity of chemotherapeutics to normal tissues. So far, clinical approaches that aim to specifically reduce chemotherapy-mediated toxicities are rare. Recently, a number of studies have demonstrated that herbal extracts derived from traditional Chinese medicine (TCM) may reduce chemotherapy-induced side effects. Thus, we screened a panel of published cancer-inhibiting TCM compounds for their chemoprotective potential and identified the phytochemical Rocaglamide (Roc-A) as a candidate. We show that Roc-A significantly reduces apoptotic cell death induced by DNA-damaging anticancer drugs in primary human and murine cells. Investigation of the molecular mechanism of Roc-A-mediated protection revealed that Roc-A specifically blocks DNA damage-induced upregulation of the transcription factor p53 by inhibiting its protein synthesis. The essential role of p53 in Roc-A-mediated protection was confirmed by siRNA knockdown of p53 and by comparison of the effects of Roc-A on chemoprotection of splenocytes isolated from wild-type and p53-deficient mice. Importantly, Roc-A did not protect p53-deficient or -mutated cancer cells. Our data suggest that Roc-A may be used as an adjuvant to reduce the side effects of chemotherapy in patients with p53-deficient or -mutated tumors. PMID:24434508

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

    PubMed Central

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

    1998-01-01

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

  7. p53, Cip1, and Gadd153 expression following treatment of A549 cells with natural and man-made vitreous fibers.

    PubMed

    Johnson, N F; Jaramillo, R J

    1997-09-01

    DNA damage induced by chemicals and ionizing radiation is associated with the expression of negative regulators of the cell cycle. The arrest of cells in G1 and G2 phases of the cell cycle provides time for DNA repair. Asbestos fibers are carcinogenic when inhaled by both humans and animals; however, the mechanism by which the fibers exert their effect is unknown. This work was undertaken to determine whether the expression of DNA damage-inducible genes differs between crocidolite, a fiber positive for lung tumors, and JM 100 glass microfiber, which is negative for lung tumors when inhaled by rats. Temporal and dose-related expressions of p53, Cip1, and Gadd153 proteins were determined in cultured A549 cells treated with either Union Internationale Contre le Cancer crocidolite or JM 100 for 20 hr and cultured in fresh media. Immunolabeled cells were analyzed by flow cytometry, and the increased number of protein-expressing cells was determined by subtracting the expression in unexposed cells from exposed cells. Crocidolite induced the expression of all three proteins with a maximum expression after approximately 18 hr in fresh media. At a similar time point, JM 100 did not markedly induce the three proteins. Crocidolite also induced a dose-dependent increase in the number of cells in the G2 phase of the cell cycle. These results show that asbestos behaves like ionizing radiation and genotoxic chemicals by inducing proteins associated with DNA damage and cell-cycle arrest. The clear difference in response between crocidolite and JM 100 may help elucidate the mechanism of action of toxic and nontoxic fibers.

  8. p53, Cip1, and Gadd153 expression following treatment of A549 cells with natural and man-made vitreous fibers.

    PubMed Central

    Johnson, N F; Jaramillo, R J

    1997-01-01

    DNA damage induced by chemicals and ionizing radiation is associated with the expression of negative regulators of the cell cycle. The arrest of cells in G1 and G2 phases of the cell cycle provides time for DNA repair. Asbestos fibers are carcinogenic when inhaled by both humans and animals; however, the mechanism by which the fibers exert their effect is unknown. This work was undertaken to determine whether the expression of DNA damage-inducible genes differs between crocidolite, a fiber positive for lung tumors, and JM 100 glass microfiber, which is negative for lung tumors when inhaled by rats. Temporal and dose-related expressions of p53, Cip1, and Gadd153 proteins were determined in cultured A549 cells treated with either Union Internationale Contre le Cancer crocidolite or JM 100 for 20 hr and cultured in fresh media. Immunolabeled cells were analyzed by flow cytometry, and the increased number of protein-expressing cells was determined by subtracting the expression in unexposed cells from exposed cells. Crocidolite induced the expression of all three proteins with a maximum expression after approximately 18 hr in fresh media. At a similar time point, JM 100 did not markedly induce the three proteins. Crocidolite also induced a dose-dependent increase in the number of cells in the G2 phase of the cell cycle. These results show that asbestos behaves like ionizing radiation and genotoxic chemicals by inducing proteins associated with DNA damage and cell-cycle arrest. The clear difference in response between crocidolite and JM 100 may help elucidate the mechanism of action of toxic and nontoxic fibers. PMID:9400714

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

    PubMed Central

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

    2009-01-01

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

  10. Polypodium leucotomos decreases UV-induced epidermal cell proliferation and enhances p53 expression and plasma antioxidant capacity in hairless mice.

    PubMed

    Rodríguez-Yanes, Esperanza; Juarranz, Ángeles; Cuevas, Jesús; Gonzalez, Salvador; Mallol, Jordi

    2012-08-01

    A single dose of ultraviolet radiation (UVR) induces significant changes in blood and skin of hairless mice. Oral administration of a hydrophilic extract of the fern Polypodium leucotomos (PL, 300 mg/kg during 5 days before UVR and for two additional days after irradiation) modulates some of the effects of UVR. Most significantly, PL administration reduced the number of proliferating cells by 13%, increased the number of p53(+) cells by 63%, enhanced the antioxidant plasma capacity (ORAC) by 30% and reinforced the network of dermal elastic fibres. Western blot analysis of skin antioxidant-related enzymes failed to demonstrate significant changes caused by PL. Thus, the beneficial effect of PL likely owes to its antioxidant and anti-ROS properties rather than its modulation of the expression of endogenous antioxidant systems. These data provide mechanistic clues for its efficacy as a systemic photoprotective agent with antioxidant and anti-photo-ageing properties.

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

    PubMed Central

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

    1998-01-01

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

  12. p53, PCNA and Ki-67 expression in oral squamous cell carcinomas: the vagaries of fixation and microwave enhancement of immunocytochemistry.

    PubMed

    Allison, R T; Best, T

    1998-10-01

    Proliferation markers are widely used as indicators of tumour progression and aggression. Fixation and antigen retrieval methods may enhance the immunocytochemical sensitivity of these markers but may also lead to loss of specificity. As these methods are often used quantitatively, standardisation of internal and external methodology is paramount. This study aimed to compare the effects of alcohol and formalin fixation and of microwaving on the immunocytochemical demonstration of p53, PCNA and Ki-67 in oral squamous cell carcinoma using duplicate tissue blocks from 24 cases. Both qualitative and quantitative differences in antigen expression were revealed. Whilst alcohol fixation alone at least maintained and usually increased the strength of positive staining, microwaving alcohol-fixed sections often gave rise to non-specific staining. p53 staining following microwave enhancement of alcohol-fixed tissue showed a significant incidence of conversion of negative results to positive and of positive staining in unexpected tissue components. Alcohol fixation increased the sensitivity of PCNA detection with a far less dramatic loss of specificity. The results emphasise the need for careful standardisation of immunocytochemical methods, particularly when used quantitatively and for inter-laboratory comparisons.

  13. p53 in cell invasion, podosomes, and invadopodia

    PubMed Central

    Mak, Alan S

    2014-01-01

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

  14. Modulation of p53, c-fos, RARE, cyclin A, and cyclin D1 expression in human leukemia (HL-60) cells exposed to arsenic trioxide

    PubMed Central

    Yedjou, Clement G.; Tchounwou, Paul B.

    2010-01-01

    Arsenic trioxide (As2O3) has recently been successfully used to treat all-trans retinoic acid (ATRA) resistant relapsing acute promyelocytic leukemia. However, its molecular mechanisms of action are poorly understood. In the present study, we used the human leukemia (HL-60) cell line as a test model to study the cellular and molecular mechanisms of anti-cancer properties of As2O3. We hypothesized that As2O3-induced expression of stress genes and related proteins may play a role in the cellular and molecular events leading to cell cycle modulation in leukemic cells. To test this hypothesis, we performed Western blot analysis to assess the expression of specific cellular response proteins including p53, c-fos, RARE, Cyclin A, and Cyclin D1. Densitometric analysis was performed to determine the relative abundance of these proteins. Western Blot and densitometric analyses demonstrated a strong dose-response relationship with regard to p53 and RARE expression within the dose range of 0-8μg/mL. Expression of c-fos was slightly up-regulated at 2μg/mL, and down-regulated within the dose-range of 4-8 μg/mL. A statistically significant down-regulation of this protein was detected at the 6 and 8 μg/mL dose levels. No statistically significant differences (p>0.05) in Cyclin D1 expression was found between As2O3-treated cells and the control. Cyclin A expression in As2O3-treated HL-60 cells was up-regulated at 6μg/mL, suggesting that it is required for S phase and passage through G2 phase in cell cycle progression. Taken together, these results indicate that As2O3 has the potential to induce cell cycle arrest through activation of the 53-kDa tumor suppressor protein and repression of the c-fos transcription factor. Up-regulation of RARE by As2O3 indicates that its cytotoxicity may be mediated through interaction/binding with the retinoic acid receptor, and subsequent inhibition of growth and differentiation. PMID:19444595

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

    PubMed Central

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

    2016-01-01

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

  16. p53 contributes to T cell homeostasis through the induction of pro-apoptotic SAP.

    PubMed

    Madapura, Harsha S; Salamon, Daniel; Wiman, Klas G; Lain, Sonia; Klein, George; Klein, Eva; Nagy, Noémi

    2012-12-15

    Lack of functional SAP protein, due to gene deletion or mutation, is the cause of X-linked lymphoproliferative disease (XLP), characterized by functionally impaired T and NK cells and a high risk of lymphoma development. We have demonstrated earlier that SAP has a pro-apoptotic function in T and B cells. Deficiency of this function might contribute to the pathogenesis of XLP. We have also shown that SAP is a target of p53 in B cell lines. In the present study, we show that activated primary T cells express p53, which induces SAP expression. p53 is functional as a transcription factor in activated T cells and induces the expression of p21, PUMA and MDM2. PARP cleavage in the late phase of activation indicates that T cells expressing high levels of SAP undergo apoptosis. Modifying p53 levels using Nutlin-3, which specifically dissociates the MDM2-p53 interaction, was sufficient to upregulate SAP expression, indicating that SAP is a target of p53 in T cells. We also demonstrated p53's role as a transcription factor for SAP in activated T cells by ChIP assays. Our result suggests that p53 contributes to T cell homeostasis through the induction of the pro-apoptotic SAP. A high level of SAP is necessary for the activation-induced cell death that is pivotal in termination of the T cell response.

  17. Regulation of ES cell differentiation by functional and conformational modulation of p53.

    PubMed Central

    Sabapathy, K; Klemm, M; Jaenisch, R; Wagner, E F

    1997-01-01

    Embryonic stem (ES) cell lines were used to examine the role of p53 during in vitro differentiation. Undifferentiated ES cells express high levels of p53 exclusively in the wild-type conformation, immunoprecipitable by monoclonal antibody PAb246, and p53 was found to be functionally active as determined by its ability to bind DNA specifically and to activate transcription of target genes. Differentiation in vitro resulted in a decrease in the levels of p53 and in a shift in its conformational status to the mutant form, detectable by monoclonal antibody PAb240, with a concomitant loss of functional activity. The presence of functional p53 in the undifferentiated ES cells renders them hypersensitive to UV irradiation, whereas the differentiated cells were resistant to UV treatment. ES cells lacking p53 exhibit enhanced proliferation in both the undifferentiated and differentiated state, and apoptosis accompanying differentiation was found to be reduced. Furthermore, wild-type ES cells undergoing apoptosis expressed functional p53. Expression of the temperature-sensitive p53val135 mutant in wild-type ES cells resulted in a reduction of apoptosis accompanying differentiation when it adopted a mutant conformation at 39 degrees C. These data demonstrate that functional inactivation of p53 allows differentiating cells to escape from apoptosis, and suggest that the conformational switch could regulate the inactivation process. PMID:9321401

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

    PubMed

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

    2008-02-01

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

  19. Immunohistochemical expression of the p53, mdm2, p21/Waf-1, Rb, p16, Ki67, cyclin D1, cyclin A and cyclin B1 proteins and apoptotic index in T-cell lymphomas.

    PubMed

    Kanavaros, P; Bai, M; Stefanaki, K; Poussias, G; Rontogianni, D; Zioga, E; Gorgoulis, V; Agnantis, N J

    2001-04-01

    Fifty-seven cases of T-cell lymphomas (TCL) including 5 lymphoblastic (T-LBL) and 52 peripheral TCL (PTCL) were analyzed by immunohistochemistry for the expression of p53, mdm2, p21, Rb, cyclin D1, cyclin A, cyclin B1, and Ki67/MIB1 proteins and 39/52 PTCL were also analyzed for the expression of p16 protein and for the presence of apoptotic cells by the TUNEL method. The aim was to search for abnormal immunoprofiles of p53 and Rb growth control pathways and to determine the proliferative activity and the apoptotic index of TCL. Abnormal overexpression of p53, p21 and mdm2, in comparison to normal lymph nodes, was found in 12/57, 10/57 and 2/57 cases of TCL, respectively. Abnormal loss of Rb and p16 expression was found in 1/57 and 2/39 cases, respectively, whereas abnormal overexpression of cyclin D1 was not detected in any of the 57 cases. Our data revealed entity-related p53/p21/mdm2 phenotypes. Indeed, most nodal and cutaneous CD30+ anaplastic large cell lymphomas (ALCL) showed concomitant overexpression of p53 and p21 proteins (7/8 cases), and mdm2 was overexpressed in 2 p53-positive nodal ALCL. In contrast, overexpression of p53 was found in 3/17 cases of nodal peripheral TCL unspecified (PTCL-UC) and 2/7 non-ALCL cutaneous pleomorphic TCL. Overexpression of p21 protein was detected in 2/3 p53-positive PTCL-UC and in 1/2 p53-positive non-ALCL cutaneous pleomorphic TCL. Finally, all the remaining 25 cases of TCL did not show p53 and p21 overexpression. Overall, the p53+/p21+ phenotype in 10/57 TCL suggests wild-type p53 capable of inducing p21 expression. The highest apoptotic index (AI) was found in ALCL and a positive correlation between apoptotic index and Ki67 index (p<0.001) was detected. Ki67, cyclin A and cyclin B1 expression was found in all 57 TCL and on the basis of the combined use of these 3 variables, 3 groups of proliferative activity could be determined: a) high in ALCL and T-LBL, b) low in mycosis fungoides (MF) and gammadelta hepatosplenic TCL

  20. Mutant p53 stimulates chemoresistance of pancreatic adenocarcinoma cells to gemcitabine.

    PubMed

    Fiorini, Claudia; Cordani, Marco; Padroni, Chiara; Blandino, Giovanni; Di Agostino, Silvia; Donadelli, Massimo

    2015-01-01

    Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths worldwide; PDAC is characterized by poor prognosis, resistance to conventional chemotherapy and high mortality rate. TP53 tumor suppressor gene is frequently mutated in PDAC, resulting in the accumulation of mutated protein with potential gain-of-function (GOF) activities, such as genomic instability, hyperproliferation and chemoresistance. The purpose of this study was to assess the relevance of the p53 status on the PDAC cells response to the standard drug gemcitabine. We also examined the potential therapeutic effect of p53-reactivating molecules to restore the mutant p53 function in GEM treated PDAC cells. We showed that gemcitabine stabilized mutant p53 protein in the nuclei and induced chemoresistance, concurrent with the mutant p53-dependent expression of Cdk1 and CCNB1 genes, resulting in a hyperproliferation effect. Despite the adverse activation of mutant p53 by gemcitabine, simultaneous treatment of PDAC cells with gemcitabine and p53-reactivating molecules (CP-31398 and RITA) reduced growth rate and induced apoptosis. This synergistic effect was observed in both wild-type and mutant p53 cell lines and was absent in p53-null cells. The combination drug treatment induced p53 phosphorylation on Ser15, apoptosis and autophagosome formation. Furthermore, pharmacological inhibition of autophagy further increased apoptosis stimulated by gemcitabine/CP-31398 treatment. Together, our results show that gemcitabine aberrantly stimulates mutant p53 activity in PDAC cells identifying key processes with potential for therapeutic targeting. Our data also support an anti-tumoral strategy based on inhibition of autophagy combined with p53 activation and standard chemotherapy for both wild-type and mutant p53 expressing PDACs.

  1. Regulation of hTERT Expression and Function in Newly Immortalized p53(+) Human Mammary Epithelial Cell Lines

    DTIC Science & Technology

    2007-06-01

    considered crucial for human carcinogenesis, in order for a single cell to accumulate the multiple errors necessary for malignancy. In human...prior to encountering stasis. Multiple types of single changes that prevent Rb-mediated growth inhibition will overcome stasis. Loss of CDKN2A...stringent barrier to human cellular immor- talization; in post-selection HMEC multiple errors appear to be necessary for telomerase reactivation, and

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. Chrysin abrogates cisplatin-induced oxidative stress, p53 expression, goblet cell disintegration and apoptotic responses in the jejunum of Wistar rats.

    PubMed

    Khan, Rehan; Khan, Abdul Quaiyoom; Qamar, Wajhul; Lateef, Abdul; Ali, Farrah; Rehman, Muneeb U; Tahir, Mir; Sharma, Swati; Sultana, Sarwat

    2012-11-14

    Cisplatin (cis-diamminedichloroplatinum (II) (CDDP)) is a commonly used chemotherapeutic drug for the treatment of numerous forms of cancer, but it has pronounced adverse effects, namely nephrotoxicity, ototoxicity, neurotoxicity, hepatotoxicity, diarrhoea and nausea. CDDP-induced emesis and diarrhoea are also marked toxicities that may be due to intestinal injury. Chrysin (5,7-dihydroxyflavone), a natural flavone commonly found in many plants, possesses multiple biological activities, such as antioxidant and anti-inflammatory properties. In the present study, we investigated the protective effect of chrysin against CDDP-induced jejunal toxicity. The plausible mechanism of CDDP-induced jejunal toxicity includes oxidative stress, p53 and apoptosis via up-regulating the expression of caspase-6 and -3. Chrysin was administered to Wistar rats orally in maize oil. A single intraperitoneal injection of CDDP was given and the animals were killed after 24 h of CDDP injection. Chrysin ameliorated CDDP-induced lipid peroxidation, increase in xanthine oxidase activity, glutathione depletion, decrease in antioxidant (catalase, glutathione reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase) and phase-II detoxifying (glutathione-S-transferase and quinone reductase) enzyme activities. Chrysin attenuated CDDP-induced goblet cell disintegration, enhanced expression of p53 and apoptotic tissue damage. Histological findings further substantiated the protective effects of chrysin against CDDP-induced damage in the jejunum. The results of the present study demonstrate that oxidative stress and apoptosis are closely associated with CDDP-induced toxicity and chrysin shows the protective efficacy against CDDP-induced jejunum toxicity possibly via attenuating the oxidative stress and apoptotic tissue damage.

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

    PubMed

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

    2011-03-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  9. p53 causes butein-mediated apoptosis of chronic myeloid leukemia cells

    PubMed Central

    WOO, SANG-MI; CHOI, YOUN KYNUG; KIM, AH JEONG; CHO, SUNG-GOOK; KO, SEONG-GYU

    2016-01-01

    Progression of chronic myeloid leukemia, marked by the oncogenic Bcr-Abl mutation, is tightly associated with an alteration of the p53 pathway. It is known that butein extracted from various plants represses cancer growth. Although the anticancer effects of butein are widely accepted, the mechanisms by which butein induces apoptosis of chronic myeloid leukemia cells remains to be elucidated. The present study demonstrated that butein-induced apoptosis was mediated by p53. KBM5 chronic myeloid leukemia (CML) cells expressing wild-type p53 were more sensitive to butein compared with p53-null K562 CML cells in terms of apoptotic cell death. In addition, butein arrested KBM5 cells at S-phase and altered the expression levels of certain cyclins and the p53-downstream targets, MDM2 and p21. In addition, while butein reduced the protein expression of MDM2 in the KBM5 and K562 cells, it resulted in proteasome-independent MDM2 degradation in p53-expressing KBM5 cells, however, not in p53-null K562 cells. Therefore, the present study suggested that p53 causes the butein-mediated apoptosis of leukemic cells. PMID:26676515

  10. Silymarin modulates doxorubicin-induced oxidative stress, Bcl-xL and p53 expression while preventing apoptotic and necrotic cell death in the liver

    SciTech Connect

    Patel, Nirav; Joseph, Cecil; Corcoran, George B.; Ray, Sidhartha D.

    2010-06-01

    The emergence of silymarin (SMN) as a natural remedy for liver diseases, coupled with its entry into NIH clinical trial, signifies its hepatoprotective potential. SMN is noted for its ability to interfere with apoptotic signaling while acting as an antioxidant. This in vivo study was designed to explore the hepatotoxic potential of Doxorubicin (Dox), the well-known cardiotoxin, and in particular whether pre-exposures to SMN can prevent hepatotoxicity by reducing Dox-induced free radical mediated oxidative stress, by modulating expression of apoptotic signaling proteins like Bcl-xL, and by minimizing liver cell death occurring by apoptosis or necrosis. Groups of male ICR mice included Control, Dox alone, SMN alone, and Dox with SMN pre/co-treatment. Control and Dox groups received saline i.p. for 14 days. SMN was administered p.o. for 14 days at 16 mg/kg/day. An approximate LD{sub 50} dose of Dox, 60 mg/kg, was administered i.p. on day 12 to animals receiving saline or SMN. Animals were euthanized 48 h later. Dox alone induced frank liver injury (> 50-fold increase in serum ALT) and oxidative stress (> 20-fold increase in malondialdehyde [MDA]), as well as direct damage to DNA (> 15-fold increase in DNA fragmentation). Coincident genomic damage and oxidative stress influenced genomic stability, reflected in increased PARP activity and p53 expression. Decreases in Bcl-xL protein coupled with enhanced accumulation of cytochrome c in the cytosol accompanied elevated indexes of apoptotic and necrotic cell death. Significantly, SMN exposure reduced Dox hepatotoxicity and associated apoptotic and necrotic cell death. The effects of SMN on Dox were broad, including the ability to modulate changes in both Bcl-xL and p53 expression. In animals treated with SMN, tissue Bcl-xL expression exceeded control values after Dox treatment. Taken together, these results demonstrated that SMN (i) reduced, delayed onset, or prevented toxic effects of Dox which are typically associated

  11. ATM Expression Predicts Veliparib and Irinotecan Sensitivity in Gastric Cancer by Mediating P53-Independent Regulation of Cell Cycle and Apoptosis.

    PubMed

    Subhash, Vinod Vijay; Tan, Shi Hui; Yeo, Mei Shi; Yan, Fui Leng; Peethala, Praveen C; Liem, Natalia; Krishnan, Vaidehi; Yong, Wei Peng

    2016-12-01

    Identification of synthetically lethal cellular targets and synergistic drug combinations is important in cancer chemotherapy as they help to overcome treatment resistance and increase efficacy. The Ataxia Telangiectasia Mutated (ATM) kinase is a nuclear protein that plays a major role in the initiation of DNA repair signaling and cell-cycle check points during DNA damage. Although ATM was shown to be associated with poor prognosis in gastric cancer, its implications as a predictive biomarker for cancer chemotherapy remain unexplored. The present study evaluated ATM-induced synthetic lethality and its role in sensitization of gastric cancer cells to PARP and TOP1 inhibitors, veliparib (ABT-888) and irinotecan (CPT-11), respectively. ATM expression was detected in a panel of gastric cell lines, and the IC50 against each inhibitors was determined. The combinatorial effect of ABT-888 and CPT-11 in gastric cancer cells was also determined both in vitro and in vivo ATM deficiency was found to be associated with enhanced sensitivity to ABT-888 and CPT-11 monotherapy, hence suggesting a mechanism of synthetic lethality. Cells with high ATM expression showed reduced sensitivity to monotherapy; however, they showed a higher therapeutic effect with ABT-888 and CPT-11 combinatorial therapy. Furthermore, ATM expression was shown to play a major role in cellular homeostasis by regulating cell-cycle progression and apoptosis in a P53-independent manner. The present study highlights the clinical utility of ATM expression as a predictive marker for sensitivity of gastric cancer cells to PARP and TOP1 inhibition and provides a deeper mechanistic insight into ATM-dependent regulation of cellular processes. Mol Cancer Ther; 15(12); 3087-96. ©2016 AACR.

  12. Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

    PubMed Central

    Forrester, K; Ambs, S; Lupold, S E; Kapust, R B; Spillare, E A; Weinberg, W C; Felley-Bosco, E; Wang, X W; Geller, D A; Tzeng, E; Billiar, T R; Harris, C C

    1996-01-01

    The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage. Images Fig. 1 Fig. 2 Fig. 3 PMID:8637893

  13. The repair capacity of lung cancer cell lines A549 and H1299 depends on HMGB1 expression level and the p53 status.

    PubMed

    Yusein-Myashkova, Shazie; Stoykov, Ivan; Gospodinov, Anastas; Ugrinova, Iva; Pasheva, Evdokia

    2016-07-01

    Elucidation of the cellular components responsive to chemotherapeutic agents as cisplatin rationalizes the strategy for anticancer chemotherapy. The removal of the cisplatin/DNA lesions gives the chance to the cancer cells to survive and compromises the chemotherapeutical treatment. Therefore, the cell repair efficiency is substantial for the clinical outcome. High mobility group box 1 (HMGB1) protein is considered to be involved in the removal of the lesions as it binds with high affinity to cisplatin/DNA adducts. We demonstrated that overexpression of HMGB1 protein inhibited cis-platinated DNA repair in vivo and the effect strongly depended on its C-terminus. We registered increased levels of DNA repair after HMGB1 silencing only in p53 defective H1299 lung cancer cells. Next, introduction of functional p53 resulted in DNA repair inhibition. H1299 cells overexpressing HMGB1 were significantly sensitized to treatment with cisplatin demonstrating the close relation between the role of HMGB1 in repair of cis-platinated DNA and the efficiency of the anticancer drug, the process being modulated by the C-terminus. In A549 cells with functional p53, the repair of cisplatin/DNA adducts is determined by а complex action of HMGB1 and p53 as an increase of DNA repair capacity was registered only after silencing of both proteins.

  14. Negative Regulation of Tumor Suppressor p53 Transcription in Breast Cancer Cells

    DTIC Science & Technology

    2003-07-01

    suppression. The region -96 to -41 contains the NF-kB and c-myc binding sites, and a newly identified UV-inducible element PE21. Mutations to disrupt NF-kB...binding or c-myc binding to the p53 promoter decreased the basal promoter activity without affecting the OM-mediated suppression, whereas mutation at...of the p53 gene contributes to the change in expression of wildtype p53 during the cell cycle and to the elevated expression of mutated p53 in tumor

  15. P53 mediates amosite asbestos-induced alveolar epithelial cell mitochondria-regulated apoptosis.

    PubMed

    Panduri, Vijayalakshmi; Surapureddi, Sailesh; Soberanes, Saul; Weitzman, Sigmund A; Chandel, Navdeep; Kamp, David W

    2006-04-01

    Asbestos causes pulmonary toxicity in part by generating reactive oxygen species that cause DNA damage. We previously showed that the mitochondria-regulated (intrinsic) death pathway mediates alveolar epithelial cell (AEC) DNA damage and apoptosis. Because p53 regulates the DNA damage response in part by inducing intrinsic cell death, we determined whether p53-dependent transcriptional activity mediates asbestos-induced AEC mitochondrial dysfunction and apoptosis. We show that inhibitors of p53-dependent transcriptional activation (pifithrin and type 16-E6 protein) block asbestos-induced AEC mitochondrial membrane potential change (DeltaPsim), caspase 9 activation, and apoptosis. We demonstrate that asbestos activates p53 promoter activity, mRNA levels, protein expression, and Bax and p53 mitochondrial translocation. Further, pifithrin, E6, phytic acid, or rho(0)-A549 cells (cells incapable of mitochondrial reactive oxygen species production) block asbestos-induced p53 activation. Finally, we show that asbestos augments p53 expression in cells at the bronchoalveolar duct junctions of rat lungs and that phytic acid prevents this. These data suggest that p53-dependent transcription pathways mediate asbestos-induced AEC mitochondria-regulated apoptosis. This suggests an important interactive effect between p53 and the mitochondria in the pathogenesis of asbestos-induced pulmonary toxicity that may have broader implications for our understanding of pulmonary fibrosis and lung cancer.

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

    PubMed

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

    2016-05-01

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

  17. p53 Dependent Centrosome Clustering Prevents Multipolar Mitosis in Tetraploid Cells

    PubMed Central

    Yi, Qiyi; Zhao, Xiaoyu; Huang, Yun; Ma, Tieliang; Zhang, Yingyin; Hou, Heli; Cooke, Howard J.; Yang, Da-Qing; Wu, Mian; Shi, Qinghua

    2011-01-01

    Background p53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy. Principal Findings Live cell imaging was performed to determine the fates and mitotic behaviors of several human and mouse tetraploid cells with different p53 status, and centrosome and spindle immunostaining was used to investigate centrosome behaviors. We found that p53 dominant-negative mutation, point mutation, or knockout led to a 2∼ 33-fold increase of multipolar mitosis in N/TERT1, 3T3 and mouse embryonic fibroblasts (MEFs), while mitotic entry and cell death were not significantly affected. In p53-/- tetraploid MEFs, the ability of centrosome clustering was compromised, while centrosome inactivation was not affected. Suppression of RhoA/ROCK activity by specific inhibitors in p53-/- tetraploid MEFs enhanced centrosome clustering, decreased multipolar mitosis from 38% to 20% and 16% for RhoA and ROCK, respectively, while expression of constitutively active RhoA in p53+/+ tetraploid 3T3 cells increased the frequency of multipolar mitosis from 15% to 35%. Conclusions p53 could not prevent tetraploid cells entering mitosis or induce tetraploid cell death. However, p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway. PMID:22076149

  18. Cell-Specific Modulation of Papovavirus Replication by Tumor Suppressor Protein p53

    PubMed Central

    Lepik, Dina; Ustav, Mart

    2000-01-01

    Small DNA tumor viruses like human papillomaviruses, simian virus 40, and adenoviruses modulate the activity of cellular tumor suppressor proteins p53 and/or pRB. These viruses replicate as nuclear multicopy extrachromosomal elements during the S phase of the cell cycle, and it has been suggested that inactivation of p53 and pRb is necessary for directing the cells to the S phase. Mouse polyomavirus (Py), however, modulates only the pRB protein activity without any obvious interference with the action of p53. We show here that Py replication was not suppressed by the p53 protein indeed in all tested different mouse cell lines. In addition, E1- and E2-dependent papillomavirus origin replication was insensitive to the action of p53 in mouse cells. We show that in hamster (Chinese hamster ovary) or human (osteosarcoma 143) cell lines the replication of both Py and papillomavirus origins was efficiently blocked by p53. The block of Py replication in human and hamster cells is not caused by the downregulation of large T-antigen expression. The deletion analysis of the p53 protein shows that the RPA binding, proline-rich regulatory, DNA-binding, and oligomerization domains are necessary for p53 action in both replication systems. These results indicate that in mouse cells the p53 protein could be inactive for the suppression of papovavirus replication. PMID:10775606

  19. Loss of p21{sup Sdi1} expression in senescent cells after DNA damage accompanied with increase of miR-93 expression and reduced p53 interaction with p21{sup Sdi1} gene promoter

    SciTech Connect

    Choi, Ok Ran; Lim, In Kyoung

    2011-04-08

    Highlights: {yields} Reduced p21 expression in senescent cells treated with DNA damaging agents. {yields} Increase of [{sup 3}H]thymidine and BrdU incorporations in DNA damaged-senescent cells. {yields} Upregulation of miR-93 expression in senescent cells in response to DSB. {yields} Failure of p53 binding to p21 promoter in senescent cells in response to DSB. {yields} Molecular mechanism of increased cancer development in aged than young individuals. -- Abstract: To answer what is a critical event for higher incidence of tumor development in old than young individuals, primary culture of human diploid fibroblasts were employed and DNA damage was induced by doxorubicin or X-ray irradiation. Response to the damage was different between young and old cells; loss of p21{sup sdi1} expression in spite of p53{sup S15} activation in old cells along with [{sup 3}H]thymidine and BrdU incorporation, but not in young cells. The phenomenon was confirmed by other tissue fibroblasts obtained from different donor ages. Induction of miR-93 expression and reduced p53 binding to p21 gene promoter account for loss of p21{sup sdi1} expression in senescent cells after DNA damage, suggesting a mechanism of in vivo carcinogenesis in aged tissue without repair arrest.

  20. Microenvironment influence on human colon adenocarcinoma phenotypes and matrix metalloproteinase-2, p53 and β-catenin tumor expressions from identical monoclonal cell tumor in the orthotopic model in athymic nude rats.

    PubMed

    Priolli, Denise Gonçalves; Abrantes, Ana Margarida; Neves, Silvia; Gonçalves, Ana Cristina; Lopes, Camila Oliveira; Martinez, Natalia Peres; Cardinalli, Izilda Aparecida; Ribeiro, Ana Bela Sarmento; Botelho, Maria Filomena

    2014-03-01

    The present study aims to identify differences between left and right colon adenocarcinoma arising from identical clonal cell and to find out if microenvironment has any influence on matrix metalloproteinase-2 (MMP2), p53 and β-catenin tumor expressions. MATERIAL AND METHODS. Rats (RNU) were submitted to cecostomy to obtain the orthotopic model of right colon tumor (n = 10), while for the left colon model (n = 10), a colon diversion and distal mucous fistula in the descending colon was used. Cultivated human colon adenocarcinoma cells (WiDr) were inoculated in stomas submucosa. Histopathological analysis, real-time reverse transcription-PCR for β-catenin, p53 and MMP2, as well as immunohistochemical analysis for p53 and β-catenin expression were conducted. Central tendency, variance analysis and the Livak delta-delta-CT method were used for statistical analysis, adopting a 5% significance level. RESULTS. All tumors from the left colon exhibited infiltrative ulceration, while in the right colon tumor growth was predominantly exophytic (67%). In the left colon, tumor growth was undifferentiated (100%), while it was moderately differentiated in the right colon (83%). In right colon tumors, MMP2, p53, and β-catenin gene expressions were higher than compared to left colon (p = 4.59354E-05, p = 0.0035179, p = 0.00093798, respectively, for MMP2, p53 and β-catenin). β-catenin and p53 results obtained by real-time polymerase chain reaction were confirmed by immunohistochemistry assay (p = 0.01 and p = 0.001, respectively, for β-catenin and p53). CONCLUSION. Left and right human colon adenocarcinomas developed in animal models have distinct phenotypes even when they have the same clonal origin. Microenvironment has influenced p53, β-catenin, and MMP2 expression in animal models of colon cancer.

  1. Expression of p53 in preneoplastic and early neoplastic bronchial lesions.

    PubMed

    Martin, B; Verdebout, J-M; Mascaux, C; Paesmans, M; Rouas, G; Verhest, A; Ninane, V; Sculier, J-P

    2002-01-01

    p53 alteration has been reported to be an early event in bronchial carcinogenesis. Our study purpose was to determine the rate of p53 expression in the various preneoplastic and early neoplastic bronchial lesions obtained by biopsy during fluorescence bronchoscopy and to analyse its association with patients characteristics. Various stages of preneoplastic lesions as well as radio-occult lung cancer were studied in biopsies obtained by fluorescence bronchoscopy. We assessed the expression of p53 by immunohistochemistry using monoclonal antibody clone DO7. The p53 expression was considered as positive if > or = 1% of cells were positive and the level of positivity was expressed in percentage of positive cells. Fourteen patients were included in each category of preneoplastic lesions. At the threshold of 1% of positive cells p53 expression was observed in 28.5% of the patients with a histologically normal epithelium. This number of positive patients increased with the severity of preneoplastic lesions and reached 100% in the mild dysplasia. The mean rates of p53 positive cells for normal epithelium, hyperplasia, metaplasia, mild and severe dysplasia, carcinoma in situ and invasive radio-occult carcinoma were respectively 0.9, 3.4, 9.1, 20.5, 50.2, 34.7 and 42.5%. There was no statistically significant correlation between p53 expression and patient characteristics such as sex, age, smoking habits and indication for fluorescence bronchoscopy. The alteration of p53 expression in patients with high risk of lung cancer was an early event: this abnormality increased with the severity of the lesions, without significant correlation with patient characteristics.

  2. Immunohistochemical expression of protein p53 in neoplasms of the mammary gland in bitches.

    PubMed

    Rodo, A; Malicka, E

    2008-01-01

    The aim of the study was to investigate the presence of protein p53 in correlation with other tumor traits: histological type, tumor grade and proliferative activity. Material for the investigation comprised mammary gland tumours collected from dogs, the patients of veterinary clinics, during surgical procedures, and archival samples. Alltogether 21 adenomas, 31 complex carcinomas, 35 simple carcinomas and 12 solid carcinomas were qualified for further investigation. No protein p53 expression was found in adenomas. Cancers show positive reaction in 32.5%. The highest percent of p53 positive neoplasms was observed in solid carcinomas and neoplasms with the highest degree of histological malignancy. The smallest number showing this expression was observed in adenomas and the highest was characteristic for solid carcinomas. Considering the tumour grading, it was found that an increase in neoplasm malignancy was positively correlated with the number of the cells showing the expression of protein p53. The differences were statistically significant. Statistically significant positive correlations were observed between the proliferative activity and protein p53 expression. Higher accumulation of protein p53 in more malignant neoplasms suggests that mutations of protein p53 can be responsible for higher proliferation in neoplasms with advanced progression of malignancy.

  3. TP53 drives invasion through expression of its Δ133p53β variant

    PubMed Central

    Gadea, Gilles; Arsic, Nikola; Fernandes, Kenneth; Diot, Alexandra; Joruiz, Sébastien M; Abdallah, Samer; Meuray, Valerie; Vinot, Stéphanie; Anguille, Christelle; Remenyi, Judit; Khoury, Marie P; Quinlan, Philip R; Purdie, Colin A; Jordan, Lee B; Fuller-Pace, Frances V; de Toledo, Marion; Cren, Maïlys; Thompson, Alastair M

    2016-01-01

    TP53 is conventionally thought to prevent cancer formation and progression to metastasis, while mutant TP53 has transforming activities. However, in the clinic, TP53 mutation status does not accurately predict cancer progression. Here we report, based on clinical analysis corroborated with experimental data, that the p53 isoform Δ133p53β promotes cancer cell invasion, regardless of TP53 mutation status. Δ133p53β increases risk of cancer recurrence and death in breast cancer patients. Furthermore Δ133p53β is critical to define invasiveness in a panel of breast and colon cell lines, expressing WT or mutant TP53. Endogenous mutant Δ133p53β depletion prevents invasiveness without affecting mutant full-length p53 protein expression. Mechanistically WT and mutant Δ133p53β induces EMT. Our findings provide explanations to 2 long-lasting and important clinical conundrums: how WT TP53 can promote cancer cell invasion and reciprocally why mutant TP53 gene does not systematically induce cancer progression. DOI: http://dx.doi.org/10.7554/eLife.14734.001 PMID:27630122

  4. Differential programming of p53-deficient embryonic cells during rotenone block

    EPA Science Inventory

    Mitochondrial dysfunction has been implicated in chemical toxicities. The present study used an in vitro model to investigate the differential expression of metabolic pathways during cellular stress in p53- efficient embryonic fibroblasts compared to p53-deficient cells. These c...

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

    PubMed

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

    2014-01-01

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

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

    SciTech Connect

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

    2013-11-15

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

  7. Ubiquitin-specific protease 2 decreases p53-dependent apoptosis in cutaneous T-cell lymphoma.

    PubMed

    Wei, Tianling; Biskup, Edyta; Gjerdrum, Lise Mette Rahbek; Niazi, Omid; Ødum, Niels; Gniadecki, Robert

    2016-07-26

    Treatment of advanced cutaneous T-cell lymphomas (CTCL) is challenging because they are resistant to conventional chemotherapy. USP2 has been shown to promote resistance to chemotherapeutic agents in several cancer models.We show here USP2 is expressed in quiescent and activated T-cells and its expression is 50% lower in CTCL cell lines (MyLa2000, SeAx and Hut-78) than in normal T-cells. USP2 is expressed in neoplastic cells in early, plaque-stage mycosis fungoides (MF) and is downregulated in advanced tumor stages. Upon treatment with psoralen with UVA (PUVA) or a p53 activator, nutlin3a, USP2 expression is significantly increased in MyLa2000 (p53wt/wt), but not in SeAx (p53mut) or Hut-78 (p53-/-). USP2 knockdown decreases MyLa2000 cell viability after PUVA by 50% but not Hut-78, suggesting that the function of USP2 in CTCL cells is p53-dependent. Furthermore, USP2 knockdown results in a decreased Mdm2 expression and upregulation of p53. Taken together, our findings suggest that USP2 stabilizes Mdm2 which antagonizes pro-apoptotic activity of p53 and possibly contributes to therapeutic resistance in CTCL.

  8. Ubiquitin-specific protease 2 decreases p53-dependent apoptosis in cutaneous T-cell lymphoma

    PubMed Central

    Wei, Tianling; Biskup, Edyta; Rahbek Gjerdrum, Lise Mette; Niazi, Omid; Ødum, Niels; Gniadecki, Robert

    2016-01-01

    Treatment of advanced cutaneous T-cell lymphomas (CTCL) is challenging because they are resistant to conventional chemotherapy. USP2 has been shown to promote resistance to chemotherapeutic agents in several cancer models. We show here USP2 is expressed in quiescent and activated T-cells and its expression is 50% lower in CTCL cell lines (MyLa2000, SeAx and Hut-78) than in normal T-cells. USP2 is expressed in neoplastic cells in early, plaque-stage mycosis fungoides (MF) and is downregulated in advanced tumor stages. Upon treatment with psoralen with UVA (PUVA) or a p53 activator, nutlin3a, USP2 expression is significantly increased in MyLa2000 (p53wt/wt), but not in SeAx (p53mut) or Hut-78 (p53−/−). USP2 knockdown decreases MyLa2000 cell viability after PUVA by 50% but not Hut-78, suggesting that the function of USP2 in CTCL cells is p53-dependent. Furthermore, USP2 knockdown results in a decreased Mdm2 expression and upregulation of p53. Taken together, our findings suggest that USP2 stabilizes Mdm2 which antagonizes pro-apoptotic activity of p53 and possibly contributes to therapeutic resistance in CTCL. PMID:27351221

  9. Characterization and expression pattern of p53 during spermatogenesis in the Chinese mitten crab Eriocheir sinensis.

    PubMed

    Hou, Cong-Cong; Yang, Wan-Xi

    2013-02-01

    p53, as a "Guardian of the Genome", plays an important role in cell cycle arrest, apoptosis, DNA repair and inhibition of angiogenesis in different tissues including testis. p53 gene and its protein perform many essential roles for mammalian spermatogenesis. To explore its functions during spermatogenesis in Eriocheir sinensis, we have cloned and sequenced the cDNA (1,218 bp) of p53 from the testis by degenerating primer PCR and rapid-amplification of cDNA ends. The protein alignment of p53 shows the conserved DNA binding domain, dimerization site and zinc binding site consisted of the predicted structures. Phylogenetic analysis revealed that p53 was more closer to Marsupenaeus japonicus and Tigriopus japonicus than other examined species. Tissue expression analysis of p53 mRNA showed p53 was distinctly expressed in accessory sexual gland, muscle, gill, heart, hepatopancreas and testis. In situ hybridization revealed that the p53 mRNA was weakly distributed around the nucleus, but stronger in the invaginated acrosomal tubule at the early stage. At the middle stage, p53 mRNA signal was increased than the early stage and the signal displayed dot-like pattern on the surface of cup-like nucleus. The signal on acrosomal cap is stronger than on the acrosomal tubule, despite acrosomal tubule signal was also distinct. At the late stage, the signal was still mainly located in acrosomal cap and acrosomal tubule. Sporadic signal were found surrounding the cup-like nucleus, but they were very weak. In the mature sperm, the signal was dramatically decreased. Even though the signal on cup-like nucleus and acrosomal tubule were distinct, they were weaker than those in middle stage. Based on these results, we concluded that p53 may play an important role in formation of acrosome biogenesis and nuclear shaping during spermiogenesis of E. sinensis.

  10. Substrate Stiffness Influences Doxorubicin-Induced p53 Activation via ROCK2 Expression

    PubMed Central

    Ebata, Takahiro; Mitsui, Yasumasa; Sugimoto, Wataru; Maeda, Miho; Machiyama, Hiroaki; Harada, Ichiro; Sawada, Yasuhiro; Fujita, Hideaki; Hirata, Hiroaki

    2017-01-01

    The physical properties of the extracellular matrix (ECM), such as stiffness, are involved in the determination of the characteristics of cancer cells, including chemotherapy sensitivity. Resistance to chemotherapy is often linked to dysfunction of tumor suppressor p53; however, it remains elusive whether the ECM microenvironment interferes with p53 activation in cancer cells. Here, we show that, in MCF-7 breast cancer cells, extracellular stiffness influences p53 activation induced by the antitumor drug doxorubicin. Cell growth inhibition by doxorubicin was increased in response to ECM rigidity in a p53-dependent manner. The expression of Rho-associated coiled coil-containing protein kinase (ROCK) 2, which induces the activation of myosin II, was significantly higher when cells were cultured on stiffer ECM substrates. Knockdown of ROCK2 expression or pharmacological inhibition of ROCK decreased doxorubicin-induced p53 activation. Our results suggest that a soft ECM causes downregulation of ROCK2 expression, which drives resistance to chemotherapy by repressing p53 activation. PMID:28191463

  11. Lack of p53 Affects the Expression of Several Brain Mitochondrial Proteins: Insights from Proteomics into Important Pathways Regulated by p53

    PubMed Central

    Fiorini, Ada; Sultana, Rukhsana; Barone, Eugenio; Cenini, Giovanna; Perluigi, Marzia; Mancuso, Cesare; Cai, Jian; Klein, Jon B.; St. Clair, Daret; Butterfield, D. Allan

    2012-01-01

    The tumor suppressor protein p53 has been described “as the guardian of the genome” for its crucial role in regulating the transcription of numerous genes responsible for cells cycle arrest, senescence, or apoptosis in response to various stress signals. Although p53 promotes longevity by decreasing the risk of cancer through activation of apoptosis or cellular senescence, several findings suggest that an increase of its activity may have deleterious effects leading to selected aspects of the aging phenotype and neurodegenerative diseases. There is the link between p53 and oxidative stress, the latter a crucial factor that contributes to neurodegenerative processes like Alzheimer disease (AD). In the present study, using a proteomics approach, we analyzed the impact of lack of p53 on the expression of several brain mitochondrial proteins involved in different pathways, and how lack of p53 may present a target to restore neuronal impairments. Our investigation on isolated brain mitochondria from p53(−/−) mice also provides a better understanding of the p53-mitochondria relationship and its involvement in the development of many diseases. PMID:23209608

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

    PubMed

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

    2012-01-01

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

  13. Proposed megakaryocytic regulon of p53: the genes engaged to control cell cycle and apoptosis during megakaryocytic differentiation.

    PubMed

    Apostolidis, Pani A; Lindsey, Stephan; Miller, William M; Papoutsakis, Eleftherios T

    2012-06-15

    During endomitosis, megakaryocytes undergo several rounds of DNA synthesis without division leading to polyploidization. In primary megakaryocytes and in the megakaryocytic cell line CHRF, loss or knock-down of p53 enhances cell cycling and inhibits apoptosis, leading to increased polyploidization. To support the hypothesis that p53 suppresses megakaryocytic polyploidization, we show that stable expression of wild-type p53 in K562 cells (a p53-null cell line) attenuates the cells' ability to undergo polyploidization during megakaryocytic differentiation due to diminished DNA synthesis and greater apoptosis. This suggested that p53's effects during megakaryopoiesis are mediated through cell cycle- and apoptosis-related target genes, possibly by arresting DNA synthesis and promoting apoptosis. To identify candidate genes through which p53 mediates these effects, gene expression was compared between p53 knock-down (p53-KD) and control CHRF cells induced to undergo terminal megakaryocytic differentiation using microarray analysis. Among substantially downregulated p53 targets in p53-KD megakaryocytes were cell cycle regulators CDKN1A (p21) and PLK2, proapoptotic FAS, TNFRSF10B, CASP8, NOTCH1, TP53INP1, TP53I3, DRAM1, ZMAT3 and PHLDA3, DNA-damage-related RRM2B and SESN1, and actin component ACTA2, while antiapoptotic CKS1B, BCL2, GTSE1, and p53 family member TP63 were upregulated in p53-KD cells. Additionally, a number of cell cycle-related, proapoptotic, and cytoskeleton-related genes with known functions in megakaryocytes but not known to carry p53-responsive elements were differentially expressed between p53-KD and control CHRF cells. Our data support a model whereby p53 expression during megakaryopoiesis serves to control polyploidization and the transition from endomitosis to apoptosis by impeding cell cycling and promoting apoptosis. Furthermore, we identify a putative p53 regulon that is proposed to orchestrate these effects.

  14. Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells.

    PubMed Central

    Deb, S; Jackson, C T; Subler, M A; Martin, D W

    1992-01-01

    Wild-type p53 has recently been shown to repress transcription from several cellular and viral promoters. Since p53 mutations are the most frequently reported genetic defects in human cancers, it becomes important to study the effects of mutations of p53 on promoter functions. We, therefore, have studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen (PCNA) promoter and on several viral promoters, including the herpes simplex virus type 1 UL9 promoter, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and a plasmid containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. As expected, expression of the wild-type p53 inhibited promoter function. Expression of a p53 with a mutation at any one of the four amino acid positions 175, 248, 273, or 281, however, correlated with a significant increase of the PCNA promoter activity (2- to 11-fold). The viral promoters were also activated, although to a somewhat lesser extent. We also showed that activation by a mutant p53 requires a minimal promoter containing a lone TATA box. A more significant increase (25-fold) in activation occurs when the promoter contains a binding site for the activating transcription factor or cyclic AMP response element-binding protein. Using Saos-2 cells that do not express p53, we showed that activation by a mutant p53 was a direct enhancement. The mutant forms of p53 used in this study are found in various cancer cells. The activation of PCNA by mutant p53s may indicate a way to increase cell proliferation by the mutant p53s. Thus, our data indicate a possible functional role for the mutants of p53 found in cancer cells in activating several important loci, including PCNA. Images PMID:1356162

  15. Zn(II)-curc targets p53 in thyroid cancer cells

    PubMed Central

    GARUFI, ALESSIA; D'ORAZI, VALERIO; CRISPINI, ALESSANDRA; D'ORAZI, GABRIELLA

    2015-01-01

    TP53 mutation is a common event in many cancers, including thyroid carcinoma. Defective p53 activity promotes cancer resistance to therapies and a more malignant phenotype, acquiring oncogenic functions. Rescuing the function of mutant p53 (mutp53) protein is an attractive anticancer therapeutic strategy. Zn(II)-curc is a novel small molecule that has been shown to target mutp53 protein in several cancer cells, but its effect in thyroid cancer cells remains unclear. Here, we investigated whether Zn(II)-curc could affect p53 in thyroid cancer cells with both p53 mutation (R273H) and wild-type p53. Zn(II)-curc induced mutp53H273 downregulation and reactivation of wild-type functions, such as binding to canonical target promoters and target gene transactivation. This latter effect was similar to that induced by PRIMA-1. In addition, Zn(II)-curc triggered p53 target gene expression in wild-type p53-carrying cells. In combination treatments, Zn(II)-curc enhanced the antitumor activity of chemotherapeutic drugs, in both mutant and wild-type-carrying cancer cells. Taken together, our data indicate that Zn(II)-curc promotes the reactivation of p53 in thyroid cancer cells, providing in vitro evidence for a potential therapeutic approach in thyroid cancers. PMID:26314369

  16. Effect of topical tretinoin, chemical peeling and dermabrasion on p53 expression in facial skin.

    PubMed

    El-Domyati, Moetaz M; Attia, Sameh K; Saleh, Fatma Y; Ahmad, Hesham M; Gasparro, Frances P; Uitto, Jouni J

    2003-01-01

    The tumour suppressor protein p53 is a phosphoprotein that is activated by DNA damage. It is involved in the decision whether the cells should stop replication and proceed to repair their DNA, or to die by apoptosis. In the present study, we evaluate the effect of some treatment modalities on the expression of p53 in facial skin. Biopsy specimens were obtained from the facial skin of 20 patients before and after treatment using topical tretinoin (11 cases), TCA chemical peeling (5 cases) and dermabrasion (4 cases). Biopsy specimens were also obtained from 12 control subjects representing the same age groups of the patients. Topical tretinoin therapy was found to induce a significant decrease in the expression of p53 up to 6 months of therapy followed by a significant increase after 10 months of therapy. On the contrary, superficial TCA peeling did not induce any statistically significant change in the expression of p53. On the other hand dermabrasion was found to induce a significant decrease in the level of expression of p53 in biopsies obtained after complete re-epithelialization followed by a significant increase. These changes in the expression of p53 may play a role in mediating the effects of such treatment modalities on the epidermis, as well as prevention of actinic neoplasia by adjusting any disturbance in the proliferation/apoptosis balance observed in photoaged facial skin.

  17. Clinical effects of p53 overexpression in squamous cell carcinoma of the sinonasal tract

    PubMed Central

    Wang, Xiaowei; Lv, Wei; Qi, Fang; Gao, Zhiqiang; Yang, Hua; Wang, Weiqing; Gao, Yali

    2017-01-01

    Abstract Background: The level of p53 protein expression in sinonasal squamous cell carcinoma (SNSCC) has been estimated, but the results remain inconsistent and the point of consensus has not been reached. This study was first determined to evaluate the clinical effects of p53 expression in SCC of the sinonasal tract. Methods: According to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement criteria, the potential literature was searched from diverse databases. The pooled odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated to assess the strength of association between p53 expression and SNSCC. Results: Final 17 eligible studies were included in a total of 258 cases and 748 controls. The result of p53 expression was shown to be notably higher in SNSCC than in benign sinonasal papillomas and normal sinonasal mucosa (OR = 26.93, P < 0.001; OR = 39.79, P < 0.001; respectively). Subgroup analyses of ethnicity revealed that p53 expression had significant association with SNSCC in Asian and Caucasian populations in cancer versus benign sinonasal papillomas or normal sinonasal mucosa. The expression of p53 was notably higher in moderately or poorly differentiated SNSCC than in well-differentiated SNSCC (OR = 3.51, P = 0.021), while p53 expression was not associated with histological type. Conclusion: The results suggested that p53 overexpression may be correlated with the carcinogenesis and progression of SNSCC. The p53 gene may become a novel drug target of SNSCC. Additional studies on the correlation of p53 expression with clinicopathological features are needed. PMID:28328848

  18. [Prognostic and predictive value of koilocytosis, expression of e6 hpv types 16/18, p16ink4a, p53 in locally advanced squamous cell carcinomas of oral cavity and oropharynx, associated with human papillomavirus].

    PubMed

    Riaboshapka, A N

    2014-11-01

    To determine the predictive and prognostic value of koilocytosis, expression of E6 HPV types 16/18, p16INK4a, p53 in patients with locally advanced HPV-associated squamous cell carcinoma of oral cavity and oropharynx. In biopsy specimens of squamous cell carcinomas of oral cavity and oropharynx from 60 patients performed koylocytes count, immunohistochemical detection of HPV 16/18 types E6 protein, proteins p16INK4a and p53. Koilocytosis was detected in 50 patients (83.3%); in all 60 patients (100%) were simultaneous expression of p16INK4a and E6 HPV types 16/18; p53 expression was found in 37 patients (61.7%). After combined treatment (induction chemotherapy followed by radiotherapy) stable disease (SD) was detected in 11 patients (18.3%), partial response (PR) - in 25 patients (41.7%), complete response (CR) - in 24 patients (40.0%). There were no cases of disease progression. Treatment effect correlated with expression of p16INK4a (ρ = 0.3, p = 0.024) and expression of p53 (ρ = - 0.3, p = 0.019). Patients with a low expression of p16INK4a (2 points) and high expression of p53 (4 "+") had a high level of SD and had no CR. For all patients, the median of overall survival (OS) was 17 months, 1-year cumulative survival rate was 66.7%, 2-year cumulative survival rate - 35.0%. Median of overall survival was correlated with koilocytosis (ρ=0.5, p<0,001) and expression of E6 HPV types 16/18 (ρ=0.9, p<0.001), p16INK4a (ρ=0.9, p=0.037), p53 (ρ=-0.9; p<0.001). Patients with low expression of p53 (0 and 1 "+") had cumulative 1-year survival rates 87% and 90%, respectively (p<0.001), 2-year survival rates - 52% and 80%, respectively (p=0.015). In the Cox proportional hazards model the significant prognostic factors were prevalence of primary tumor (OR 2.2, 95% CI 1.3 - 3.5, p=0.003) and p53 expression (OR 1.3, 95% CI 1.1=1.7, p=0.016). High expression of p16INK4a associated with a high effect of combined treatment, high expression of a p53 - with low effect of

  19. Down-regulation of dihydrofolate reductase inhibits the growth of endothelial EA.hy926 cell through induction of G1 cell cycle arrest via up-regulating p53 and p21(waf1/cip1) expression.

    PubMed

    Fei, Zhewei; Gao, Yong; Qiu, Mingke; Qi, Xianqin; Dai, Yuxin; Wang, Shuqing; Quan, Zhiwei; Liu, Yingbin; Ou, Jingmin

    2016-03-01

    Folic acid supplementation may meliorate cardiovascular disease risk by improving vascular endothelial structure and function. However, the underlying mechanisms are still lack of a global understanding. To be used, folic acid must be converted to 7,8-dihydrofolate by dihydrofolate reductase to generate one-carbon derivatives serving as important cellular cofactors in the synthesis of nucleotides and amino acids required for cell growth. Therefore, this study explored the effect of dihydrofolate reductase knockdown on endothelial EA.hy926 cell growth and the mechanism involved. We found that down-regulation of dihydrofolate reductase inhibited EA.hy926 cell proliferation, and induced G1 phase arrest. Meanwhile, the expression of regulators necessary for G1/S phase transition, such as cyclin-dependent kinases CDK2, CDK4 and CDK6, were remarkably down-regulated; by contrast, the cell cycle inhibitors p21(waf/cip1), p27(Kip1) and p53 were significantly up-regulated after dihydrofolate reductase knockdown. Furthermore, supplementation of 5-methyltetrahydrofolate to the dihydrofolate reductase knockdown cells could weaken the inhibitory effect of dihydrofolate reductase knockdown on cell proliferation, simultaneously, inducing the expression of p53 and p21(waf/cip1) falling back moderately. Our findings suggest that attenuating dihydrofolate reductase may cause imbalanced expression of cell cycle regulators, especially up-regulation of p53-p21(waf/cip1) pathway, leading to G1 cell cycle arrest, thereby inhibiting the growth of endothelial EA.hy926 cells.

  20. The absence of Ser389 phosphorylation in p53 affects the basal gene expression level of many p53-dependent genes and alters the biphasic response to UV exposure in mouse embryonic fibroblasts.

    PubMed

    Bruins, Wendy; Bruning, Oskar; Jonker, Martijs J; Zwart, Edwin; van der Hoeven, Tessa V; Pennings, Jeroen L A; Rauwerda, Han; de Vries, Annemieke; Breit, Timo M

    2008-03-01

    Phosphorylation is important in p53-mediated DNA damage responses. After UV irradiation, p53 is phosphorylated specifically at murine residue Ser389. Phosphorylation mutant p53.S389A cells and mice show reduced apoptosis and compromised tumor suppression after UV irradiation. We investigated the underlying cellular processes by time-series analysis of UV-induced gene expression responses in wild-type, p53.S389A, and p53(-/-) mouse embryonic fibroblasts. The absence of p53.S389 phosphorylation already causes small endogenous gene expression changes for 2,253, mostly p53-dependent, genes. These genes showed basal gene expression levels intermediate to the wild type and p53(-/-), possibly to readjust the p53 network. Overall, the p53.S389A mutation lifts p53-dependent gene repression to a level similar to that of p53(-/-) but has lesser effect on p53-dependently induced genes. In the wild type, the response of 6,058 genes to UV irradiation was strictly biphasic. The early stress response, from 0 to 3 h, results in the activation of processes to prevent the accumulation of DNA damage in cells, whereas the late response, from 12 to 24 h, relates more to reentering the cell cycle. Although the p53.S389A UV gene response was only subtly changed, many cellular processes were significantly affected. The early response was affected the most, and many cellular processes were phase-specifically lost, gained, or altered, e.g., induction of apoptosis, cell division, and DNA repair, respectively. Altogether, p53.S389 phosphorylation seems essential for many p53 target genes and p53-dependent processes.

  1. The presence of carbon nanostructures in bakery products induces metabolic stress in human mesenchymal stem cells through CYP1A and p53 gene expression.

    PubMed

    Al-Hadi, Ahmed M; Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Alshatwi, Ali A

    2016-01-01

    Ingredients commonly present in processed foods are excellent substrates for chemical reactions during modern thermal cooking or processing, which could possibly result in deteriorative carbonization changes mediated by a variety of thermal reactions. Spontaneous self-assembling complexation or polymerization of partially combusted lipids, proteins, and other food macromolecules with synthetic food additives during high temperature food processing or baking (200-250 °C) would result in the formation of carbon nanostructures (CNs). These unknown nanostructures may produce adverse physiological effects or potential health risks. The present work aimed to identify and characterize the nanostructures from the crusts of bread. Furthermore, a toxicological risk assessment of these nanostructures was conducted using human mesenchymal stem cells (hMSCs) as a model for cellular uptake and metabolic oxidative stress, with special reference to induced adipogenesis. CNs isolated from bread crusts were characterized using transmission electron microscopy. The in vitro risk assessment of the CNs was carried out in hMSCs using an MTT assay, cell morphological assessment, a reactive oxygen species assay, a mitochondrial trans-membrane potential assay, cell cycle progression assessment and gene expression analysis. Our results revealed that bread crusts contain CNs, which may form during the bread-making process. The in vitro results indicate that carbon nanostructures have moderately toxic effects in the hMSCs at a high dose (400 μg/mL). The mitochondrial trans-membrane potentials and intracellular ROS levels of the hMSCs were altered at this dose. The levels of the mRNA transcripts of metabolic stress-responsive genes such as CAT, GSR, GSTA4, CYP1A and p53 were significantly altered in response to CNs.

  2. p53 is important for the anti-proliferative effect of ibuprofen in colon carcinoma cells

    SciTech Connect

    Janssen, Astrid; Schiffmann, Susanne; Birod, Kerstin; Maier, Thorsten J.; Wobst, Ivonne; Geisslinger, Gerd

    2008-01-25

    S-ibuprofen which inhibits the cyclooxygenase-1/-2 and R-ibuprofen which shows no COX-inhibition at therapeutic concentrations have anti-carcinogenic effects in human colon cancer cells; however, the molecular mechanisms for these effects are still unknown. Using HCT-116 colon carcinoma cell lines, expressing either the wild-type form of p53 (HCT-116 p53{sup wt}) or being p(HCT-116 p53{sup -/-}), we demonstrated that both induction of a cell cycle block and apoptosis after S- and R-ibuprofen treatment is in part dependent on p53. Also in the in vivo nude mice model HCT-116 p53{sup -/-} xenografts were less sensitive for S- and R-ibuprofen treatment than HCT-116 p53{sup wt} cells. Furthermore, results indicate that induction of apoptosis in HCT-116 p53{sup wt} cells after ibuprofen treatment is in part dependent on a signalling pathway including the neutrophin receptor p75{sup NTR}, p53 and Bax.

  3. c-Abl inhibits breast cancer tumorigenesis through reactivation of p53-mediated p21 expression

    PubMed Central

    Thompson, Cheryl L.; Gilmore, Hannah L.; Chang, Jenny C.; Keri, Ruth A.; Schiemann, William P.

    2016-01-01

    We previously reported that constitutive c-Abl activity (CST-Abl) abrogates the tumorigenicity of triple-negative breast cancer cells through the combined actions of two cellular events: downregulated matrix metalloproteinase (MMP) and upregulated p21Waf1/Cip1 expression. We now find decreased c-Abl expression to be significantly associated with diminished relapse-fee survival in breast cancer patients, particularly those exhibiting invasive and basal phenotypes. Moreover, CST-Abl expression enabled 4T1 cells to persist innocuously in the mammary glands of mice, doing so by exhausting their supply of cancer stem cells. Restoring MMP-9 expression and activity in CST-Abl-expressing 4T1 cells failed to rescue their malignant phenotypes; however, rendering these same cells deficient in p21 expression not only delayed their acquisition of senescent phenotypes, but also partially restored their tumorigenicity in mice. Although 4T1 cells lacked detectable expression of p53, those engineered to express CST-Abl exhibited robust production and secretion of TGF-β1 that engendered the reactivated expression of p53. Mechanistically, TGF-β-mediated p53 expression transpired through the combined actions of Smad1/5/8 and Smad2, leading to the dramatic upregulation of p21 and its stimulation of TNBC senescence. Collectively, we identified a novel c-Abl:p53:p21 signaling axis that functions as a powerful suppressor of mammary tumorigenesis and metastatic progression. PMID:27626309

  4. p53 Restoration in Induction and Maintenance of Senescence: Differential Effects in Premalignant and Malignant Tumor Cells

    PubMed Central

    Harajly, Mohamad; Zalzali, Hasan; Nawaz, Zafar; Ghayad, Sandra E.; Ghamloush, Farah; Basma, Hussein; Zainedin, Samiha; Rabeh, Wissam; Jabbour, Mark; Tawil, Ayman; Badro, Danielle A.; Evan, Gerard I.

    2015-01-01

    The restoration of p53 has been suggested as a therapeutic approach in tumors. However, the timing of p53 restoration in relation to its efficacy during tumor progression still is unclear. We now show that the restoration of p53 in murine premalignant proliferating pineal lesions resulted in cellular senescence, while p53 restoration in invasive pineal tumors did not. The effectiveness of p53 restoration was not dependent on p19Arf expression but showed an inverse correlation with Mdm2 expression. In tumor cells, p53 restoration became effective when paired with either DNA-damaging therapy or with nutlin, an inhibitor of p53-Mdm2 interaction. Interestingly, the inactivation of p53 after senescence resulted in reentry into the cell cycle and rapid tumor progression. The evaluation of a panel of human supratentorial primitive neuroectodermal tumors (sPNET) showed low activity of the p53 pathway. Together, these data suggest that the restoration of the p53 pathway has different effects in premalignant versus invasive pineal tumors, and that p53 activation needs to be continually sustained, as reversion from senescence occurs rapidly with aggressive tumor growth when p53 is lost again. Finally, p53 restoration approaches may be worth exploring in sPNET, where the p53 gene is intact but the pathway is inactive in the majority of examined tumors. PMID:26598601

  5. p53 Specifically Binds Triplex DNA In Vitro and in Cells

    PubMed Central

    Brázdová, Marie; Tichý, Vlastimil; Helma, Robert; Bažantová, Pavla; Polášková, Alena; Krejčí, Aneta; Petr, Marek; Navrátilová, Lucie; Tichá, Olga; Nejedlý, Karel; Bennink, Martin L.; Subramaniam, Vinod; Bábková, Zuzana; Martínek, Tomáš; Lexa, Matej; Adámik, Matej

    2016-01-01

    Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed. PMID:27907175

  6. Novel roles for p53 in the genesis and targeting of tetraploid cancer cells.

    PubMed

    Davaadelger, Batzaya; Shen, Hong; Maki, Carl G

    2014-01-01

    Tetraploid (4N) cells are considered important in cancer because they can display increased tumorigenicity, resistance to conventional therapies, and are believed to be precursors to whole chromosome aneuploidy. It is therefore important to determine how tetraploid cancer cells arise, and how to target them. P53 is a tumor suppressor protein and key regulator of tetraploidy. As part of the "tetraploidy checkpoint", p53 inhibits tetraploid cell proliferation by promoting a G1-arrest in incipient tetraploid cells (referred to as a tetraploid G1 arrest). Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In the current study, Nutlin-3a promoted a p53-dependent tetraploid G1 arrest in two diploid clones of the HCT116 colon cancer cell line. Both clones underwent endoreduplication after Nutlin removal, giving rise to stable tetraploid clones that showed increased resistance to ionizing radiation (IR) and cisplatin (CP)-induced apoptosis compared to their diploid precursors. These findings demonstrate that transient p53 activation by Nutlin can promote tetraploid cell formation from diploid precursors, and the resulting tetraploid cells are therapy (IR/CP) resistant. Importantly, the tetraploid clones selected after Nutlin treatment expressed approximately twice as much P53 and MDM2 mRNA as diploid precursors, expressed approximately twice as many p53-MDM2 protein complexes (by co-immunoprecipitation), and were more susceptible to p53-dependent apoptosis and growth arrest induced by Nutlin. Based on these findings, we propose that p53 plays novel roles in both the formation and targeting of tetraploid cells. Specifically, we propose that 1) transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells, and 2) therapy-resistant tetraploid cells, by virtue of having higher P53 gene copy number and expressing twice as many p53-MDM2

  7. Δ40p53α suppresses tumor cell proliferation and induces cellular senescence in hepatocellular carcinoma cells

    PubMed Central

    Ota, Akinobu; Sawada, Yumi; Karnan, Sivasundaram; Wahiduzzaman, Md; Inoue, Tadahisa; Kobayashi, Yuji; Yamamoto, Takaya; Ishii, Norimitsu; Ohashi, Tomohiko; Nakade, Yukiomi; Sato, Ken; Itoh, Kiyoaki; Konishi, Hiroyuki; Hosokawa, Yoshitaka; Yoneda, Masashi

    2017-01-01

    ABSTRACT Splice variants of certain genes impact on genetic biodiversity in mammals. The tumor suppressor TP53 gene (encoding p53) plays an important role in the regulation of tumorigenesis in hepatocellular carcinoma (HCC). Δ40p53α is a naturally occurring p53 isoform that lacks the N-terminal transactivation domain, yet little is known about the role of Δ40p53α in the development of HCC. Here, we first report on the role of Δ40p53α in HCC cell lines. In the TP53+/Δ40 cell clones, clonogenic activity and cell survival dramatically decreased, whereas the percentage of senescence-associated β-galactosidase (SA-β-gal)-positive cells and p21 (also known as WAF1, CIP1 and CDKN1A) expression significantly increased. These observations were clearly attenuated in the TP53+/Δ40 cell clones after Δ40p53α knockdown. In addition, exogenous Δ40p53 expression significantly suppressed cell growth in HCC cells with wild-type TP53, and in those that were mutant or null for TP53. Notably, Δ40p53α-induced tumor suppressor activity was markedly attenuated in cells expressing the hot-spot mutant Δ40p53α-R175H, which lacks the transcription factor activity of p53. Moreover, Δ40p53α expression was associated with increased full-length p53 protein expression. These findings enhance the understanding of the molecular pathogenesis of HCC and show that Δ40p53α acts as an important tumor suppressor in HCC cells. PMID:27980070

  8. Cholecystokinin attenuates radiation-induced lung cancer cell apoptosis by modulating p53 gene transcription

    PubMed Central

    Han, Yi; Su, Chongyu; Yu, Daping; Zhou, Shijie; Song, Xiaoyun; Liu, Shuku; Qin, Ming; Li, Yunsong; Xiao, Ning; Cao, Xiaoqing; Shi, Kang; Cheng, Xu; Liu, Zhidong

    2017-01-01

    The deregulation of p53 in cancer cells is one of the important factors by which cancer cells escape from the immune surveillance. Cholecystokinin (CCK) has strong bioactivity in the regulation of a number of cell activities. This study tests a hypothesis that CCK interferes with p53 expression to affect the apoptotic process in lung cancer (tumor) cells. In this study, tumor-bearing mice and A549 cells (a tumor cell line) were irradiated. The expression of CCK and p53 in tumor cells was assessed with RT-qPCR and Western blotting. The binding of p300 to the promoter of p53 was evaluated by chromatin immunoprecipitation. We observed that, with a given amount and within a given period, small doses/more sessions of irradiation markedly increased the levels of CCK in the sera and tumor cells, which were positively correlated with the tumor growth in mice and negatively correlated with tumor cell apoptosis. CCK increased the levels of histone acetyltransferase p300 and repressed the levels of nuclear factor-kB at the p53 promoter locus in tumor cells, which suppressed the expression of p53. In conclusion, CCK plays an important role in attenuating the radiation-induced lung cancer cell apoptosis. CCK may be a novel therapeutic target in the treatment of lung cancers. PMID:28337291

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

    PubMed Central

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

    2012-01-01

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

  10. Altered expression of p53, but not Rb, is involved in canine prostatic carcinogenesis.

    PubMed

    Pagliarone, Simone; Frattone, Luca; Pirocchi, Valeria; Della Salda, Leonardo; Palmieri, Chiara

    2016-04-01

    Abnormalities in the retinoblastoma (Rb) and p53 tumour suppressor gene have been frequently detected in human and canine cancers, but never investigated in canine prostate cancer, considered a good model for the advanced and aggressive androgen-resistant prostate cancer in men. Therefore, the aim of this study was to evaluate the immunohistochemical expression of Rb and p53 in 6 normal canine prostates, 15 canine prostates with benign prostatic hyperplasia (BPH) and 10 prostatic carcinomas (PCs). In all normal samples, p53 was expressed in low number of epithelial cells, while a greater number of positive cells were observed in BPH and PC. The mean number of positive cells was statistically significantly higher in PCs than normal and hyperplastic prostates. A cytoplasmic or nucleo-cytoplasmic staining was observed in 5 out of 10 PCs. Rb protein was expressed in high number of normal, hyperplastic and neoplastic cells without a statistically significant differences. Considering that Rb is frequently lost in human prostate cancer, we suggest that Rb is not involved in canine prostatic carcinogenesis. On the other hand, the increased expression of p53 that corresponds to genetic defects in the p53 gene may be associated with the malignant growth of canine prostate cancer, conferring an apoptosis-resistant phenotype.

  11. p53 and p21 determine the sensitivity of noscapine-induced apoptosis in colon cancer cells.

    PubMed

    Aneja, Ritu; Ghaleb, Amr M; Zhou, Jun; Yang, Vincent W; Joshi, Harish C

    2007-04-15

    We have previously discovered the naturally occurring antitussive alkaloid noscapine as a tubulin-binding agent that attenuates microtubule dynamics and arrests mammalian cells at mitosis via activation of the c-Jun NH(2)-terminal kinase pathway. It is well established that the p53 protein plays a crucial role in the control of tumor cell response to chemotherapeutic agents and DNA-damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. In this study, we compared chemosensitivity, cell cycle distribution, and apoptosis on noscapine treatment in four cell lines derived from the colorectal carcinoma HCT116 cells: p53(+/+) (p53-wt), p53(-/-) (p53-null), p21(-/-) (p21-null), and BAX(-/-) (BAX-null). Using these isogenic variants, we investigated the roles of p53, BAX, and p21 in the cellular response to treatment with noscapine. Our results show that noscapine treatment increases the expression of p53 over time in cells with wild-type p53 status. This increase in p53 is associated with an increased apoptotic BAX/Bcl-2 ratio consistent with increased sensitivity of these cells to apoptotic stimuli. Conversely, loss of p53 and p21 alleles had a counter effect on both BAX and Bcl-2 expression and the p53-null and p21-null cells were significantly resistant to the antiproliferative and apoptotic effects of noscapine. All but the p53-null cells displayed p53 protein accumulation in a time-dependent manner on noscapine treatment. Interestingly, despite increased levels of p53, p21-null cells were resistant to apoptosis, suggesting a proapoptotic role of p21 and implying that p53 is a necessary but not sufficient condition for noscapine-mediated apoptosis.

  12. Non-Canonical Cell Death Induced by p53

    PubMed Central

    Ranjan, Atul; Iwakuma, Tomoo

    2016-01-01

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

  13. Non-Canonical Cell Death Induced by p53.

    PubMed

    Ranjan, Atul; Iwakuma, Tomoo

    2016-12-09

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

  14. VHL missense mutations in the p53 binding domain show different effects on p53 signaling and HIFα degradation in clear cell renal cell carcinoma.

    PubMed

    Razafinjatovo, Caroline Fanja; Stiehl, Daniel; Deininger, Eva; Rechsteiner, Markus; Moch, Holger; Schraml, Peter

    2017-02-07

    Clear cell Renal Cell Carcinoma (ccRCC) formation is connected to functional loss of the von Hippel-Lindau (VHL) gene. Recent data identified its gene product, pVHL, as a multifunctional adaptor protein which interacts with HIFα subunits but also with the tumor suppressor p53. p53 is hardly expressed and rarely mutated in most ccRCC. We showed that low and absent p53 expression correlated with the severity of VHL mutations in 262 analyzed ccRCC tissues. In contrast to nonsense and frameshift mutations which abrogate virtually all pVHL functions, missense mutations may rather influence one or few functions. Therefore, we focused on four VHL missense mutations, which affect the overlapping pVHL binding sites of p53 and Elongin C, by investigating their impact on HIFα degradation, p53 expression and signaling, as well as on cellular behavior using ccRCC cell lines and tissues. TP53 mRNA and its effector targets p21, Bax and Noxa, were altered both in engineered cell lines and in tumor tissues which carried the same missense mutations. Two of these mutations were not able to degrade HIFα whereas the remaining two mutations led to HIFα downregulation, suggesting the latter are p53 binding site-specific. The selected VHL missense mutations further enhanced tumor cell survival, but had no effects on cell proliferation. Whereas Sunitinib was able to efficiently reduce cell proliferation, Camptothecin was additionally able to increase apoptotic activity of the tumor cells. It is concluded that systematic characterization of the VHL mutation status may help optimizing targeted therapy for patients with metastatic ccRCC.

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

    PubMed

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

    2014-02-01

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

  16. Addiction of lung cancer cells to GOF p53 is promoted by up-regulation of epidermal growth factor receptor through multiple contacts with p53 transactivation domain and promoter

    PubMed Central

    Vaughan, Catherine A.; Pearsall, Isabella; Singh, Shilpa; Windle, Brad; Deb, Swati P.; Grossman, Steven R.; Yeudall, W. Andrew; Deb, Sumitra

    2016-01-01

    Human lung cancers harboring gain-of-function (GOF) p53 alleles express higher levels of the epidermal growth factor receptor (EGFR). We demonstrate that a number of GOF p53 alleles directly upregulate EGFR. Knock-down of p53 in lung cancer cells lowers EGFR expression and reduces tumorigenicity and other GOF p53 properties. However, addiction of lung cancer cells to GOF p53 can be compensated by overexpressing EGFR, suggesting that EGFR plays a critical role in addiction. Chromatin immunoprecipitation (ChIP) using lung cancer cells expressing GOF p53 alleles showed that GOF p53 localized to the EGFR promoter. The sequence where GOF p53 is found to interact by ChIP seq can act as a GOF p53 response element. The presence of GOF p53 on the EGFR promoter increased histone H3 acetylation, indicating a mechanism whereby GOF p53 enhances chromatin opening for improved access to transcription factors (TFs). ChIP and ChIP-re-ChIP with p53, Sp1 and CBP histone acetylase (HAT) antibodies revealed docking of GOF p53 on Sp1, leading to increased binding of Sp1 and CBP to the EGFR promoter. Up-regulation of EGFR can occur via GOF p53 contact at other novel sites in the EGFR promoter even when TAD-I is inactivated; these sites are used by both intact and TAD-I mutated GOF p53 and might reflect redundancy in GOF p53 mechanisms for EGFR transactivation. Thus, the oncogenic action of GOF p53 in lung cancer is highly dependent on transactivation of the EGFR promoter via a novel transcriptional mechanism involving coordinated interactions of TFs, HATs and GOF p53. PMID:26820293

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

    PubMed

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

    2003-11-07

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

  18. The p53 inhibitor, pifithrin-{alpha}, suppresses self-renewal of embryonic stem cells

    SciTech Connect

    Abdelalim, Essam Mohamed; Tooyama, Ikuo

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer We determine the role of p53 in ES cells under unstressful conditions. Black-Right-Pointing-Pointer PFT-{alpha} suppresses ES cell proliferation. Black-Right-Pointing-Pointer PFT-{alpha} induces ES cell cycle arrest. Black-Right-Pointing-Pointer PFT-{alpha} downregulates Nanog and cyclin D1. -- Abstract: Recent studies have reported the role of p53 in suppressing the pluripotency of embryonic stem (ES) cells after DNA damage and blocking the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. However, to date no evidence has been presented to support the function of p53 in unstressed ES cells. In this study, we investigated the effect of pifithrin (PFT)-{alpha}, an inhibitor of p53-dependent transcriptional activation, on self-renewal of ES cells. Our results revealed that treatment of ES cells with PFT-{alpha} resulted in the inhibition of ES cell propagation in a dose-dependent manner, as indicated by a marked reduction in the cell number and colony size. Also, PFT-{alpha} caused a cell cycle arrest and significant reduction in DNA synthesis. In addition, inhibition of p53 activity reduced the expression levels of cyclin D1 and Nanog. These findings indicate that p53 pathway in ES cells rather than acting as an inactive gene, is required for ES cell proliferation and self-renewal under unstressful conditions.

  19. Mutant p53 proteins counteract autophagic mechanism sensitizing cancer cells to mTOR inhibition.

    PubMed

    Cordani, Marco; Oppici, Elisa; Dando, Ilaria; Butturini, Elena; Dalla Pozza, Elisa; Nadal-Serrano, Mercedes; Oliver, Jordi; Roca, Pilar; Mariotto, Sofia; Cellini, Barbara; Blandino, Giovanni; Palmieri, Marta; Di Agostino, Silvia; Donadelli, Massimo

    2016-08-01

    Mutations in TP53 gene play a pivotal role in tumorigenesis and cancer development. Here, we report that gain-of-function mutant p53 proteins inhibit the autophagic pathway favoring antiapoptotic effects as well as proliferation of pancreas and breast cancer cells. We found that mutant p53 significantly counteracts the formation of autophagic vesicles and their fusion with lysosomes throughout the repression of some key autophagy-related proteins and enzymes as BECN1 (and P-BECN1), DRAM1, ATG12, SESN1/2 and P-AMPK with the concomitant stimulation of mTOR signaling. As a paradigm of this mechanism, we show that atg12 gene repression was mediated by the recruitment of the p50 NF-κB/mutant p53 protein complex onto the atg12 promoter. Either mutant p53 or p50 NF-κB depletion downregulates atg12 gene expression. We further correlated the low expression levels of autophagic genes (atg12, becn1, sesn1, and dram1) with a reduced relapse free survival (RFS) and distant metastasis free survival (DMFS) of breast cancer patients carrying TP53 gene mutations conferring a prognostic value to this mutant p53-and autophagy-related signature. Interestingly, the mutant p53-driven mTOR stimulation sensitized cancer cells to the treatment with the mTOR inhibitor everolimus. All these results reveal a novel mechanism through which mutant p53 proteins promote cancer cell proliferation with the concomitant inhibition of autophagy.

  20. p53-regulated autophagy is controlled by glycolysis and determines cell fate.

    PubMed

    Duan, Lei; Perez, Ricardo E; Davaadelger, Batzaya; Dedkova, Elena N; Blatter, Lothar A; Maki, Carl G

    2015-09-15

    The tumor suppressor p53 regulates downstream targets that determine cell fate. Canonical p53 functions include inducing apoptosis, growth arrest, and senescence. Non-canonical p53 functions include its ability to promote or inhibit autophagy and its ability to regulate metabolism. The extent to which autophagy and/or metabolic regulation determines cell fate by p53 is unclear. To address this, we compared cells resistant or sensitive to apoptosis by the p53 activator Nutlin-3a. In resistant cells, glycolysis was maintained upon Nutlin-3a treatment, and activated p53 promoted prosurvival autophagy. In contrast, in apoptosis sensitive cells activated p53 increased superoxide levels and inhibited glycolysis through repression of glycolytic pathway genes. Glycolysis inhibition and increased superoxide inhibited autophagy by repressing ATG genes essential for autophagic vesicle maturation. Inhibiting glycolysis increased superoxide and blocked autophagy in apoptosis-resistant cells, causing p62-dependent caspase-8 activation. Finally, treatment with 2-DG or the autophagy inhibitors chloroquine or bafilomycin A1 sensitized resistant cells to Nutlin-3a-induced apoptosis. Together, these findings reveal novel links between glycolysis and autophagy that determine apoptosis-sensitivity in response to p53. Specifically, the findings indicate 1) that glycolysis plays an essential role in autophagy by limiting superoxide levels and maintaining expression of ATG genes required for autophagic vesicle maturation, 2) that p53 can promote or inhibit autophagy depending on the status of glycolysis, and 3) that inhibiting protective autophagy can expand the breadth of cells susceptible to Nutlin-3a induced apoptosis.

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

    PubMed

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

    2017-04-11

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

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

    PubMed

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

    2011-12-01

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

  3. MIF Maintains the Tumorigenic Capacity of Brain Tumor-Initiating Cells by Directly Inhibiting p53.

    PubMed

    Fukaya, Raita; Ohta, Shigeki; Yaguchi, Tomonori; Matsuzaki, Yumi; Sugihara, Eiji; Okano, Hideyuki; Saya, Hideyuki; Kawakami, Yutaka; Kawase, Takeshi; Yoshida, Kazunari; Toda, Masahiro

    2016-05-01

    Tumor-initiating cells thought to drive brain cancer are embedded in a complex heterogeneous histology. In this study, we isolated primary cells from 21 human brain tumor specimens to establish cell lines with high tumorigenic potential and to identify the molecules enabling this capability. The morphology, sphere-forming ability upon expansion, and differentiation potential of all cell lines were indistinguishable in vitro However, testing for tumorigenicity revealed two distinct cell types, brain tumor-initiating cells (BTIC) and non-BTIC. We found that macrophage migration inhibitory factor (MIF) was highly expressed in BTIC compared with non-BTIC. MIF bound directly to both wild-type and mutant p53 but regulated p53-dependent cell growth by different mechanisms, depending on glioma cell line and p53 status. MIF physically interacted with wild-type p53 in the nucleus and inhibited its transcription-dependent functions. In contrast, MIF bound to mutant p53 in the cytoplasm and abrogated transcription-independent induction of apoptosis. Furthermore, MIF knockdown inhibited BTIC-induced tumor formation in a mouse xenograft model, leading to increased overall survival. Collectively, our findings suggest that MIF regulates BTIC function through direct, intracellular inhibition of p53, shedding light on the molecular mechanisms underlying the tumorigenicity of certain malignant brain cells. Cancer Res; 76(9); 2813-23. ©2016 AACR.

  4. p53 inhibition by the LANA protein of KSHV protects against cell death.

    PubMed

    Friborg, J; Kong, W; Hottiger, M O; Nabel, G J

    Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, has been implicated in the development of Kaposi's sarcoma (KS) and several B-cell lymphoproliferative diseases. Most cells in lesions derived from these malignancies are latently infected, and different viral gene products have been identified in association with lytic or latent infection by KSHV. The latency-associated nuclear antigen (LANA), encoded by open reading frame 73 of the KSHV genome, is a highly immunogenic protein that is expressed predominantly during viral latency, in most KS spindle cells and in cell lines established from body-cavity-based lymphomas. Antibodies to LANA can be detected in a high percentage of HIV-infected individuals who subsequently develop KS, although its role in disease pathogenesis is not completely understood. p53 is a potent transcriptional regulator of cell growth whose induction leads either to cell-cycle arrest or apoptosis. Loss of p53 function correlates with cell transformation and oncogenesis, and several viral oncoproteins interact with p53 and modulate its biological activity. Here we show that LANA interacts with the tumour suppressor protein p53 and represses its transcriptional activity. This viral gene product further inhibits the ability of p53 to induce cell death. We propose that LANA contributes to viral persistence and oncogenesis in KS through its ability to promote cell survival by altering p53 function.

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

    PubMed

    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.

  6. Transglutaminase 2 Inhibitor KCC009 Induces p53-Independent Radiosensitization in Lung Adenocarcinoma Cells

    PubMed Central

    Huayin, Sheng; Dong, Yao; Chihong, Zhu; Xiaoqian, Qian; Danying, Wan; Jianguo, Feng

    2016-01-01

    Background The expression of transglutaminase 2 (TG2) is correlated to DNA damage repair and apoptosis through the p53 pathway. The present study aimed to investigate the potential radiosensitization effect and possible mechanisms of the TG2 inhibitor KCC009 in lung cancer in vitro. Material/Methods A single hit multi-target model was used to plot survival curves and to calculate the sensitizing enhancement ratios in lung cancer wild-type or mutant p53 of H1299 cells. We performed analyses for changes of cell cycling and apoptotic responses of cells; Western blot analysis and real-time SYBR Green PCR assay were used to determine the changes of mRNA/protein expressions; ELISA assay was used for examination of cytochrome c release in cytoplasm. Results Our results showed that KCC009 induced radiosensitization in both H1299/WT-p53 and H1299/M175H-p53 cells. KCC009+IR induced G0/G1 arrest in H1299/WT cells and G2/M arrest in H1299/M175H-p53 cells. KCC009+IR also induced apoptosis in both cell lines. In addition, KCC009+IR decreased the TG2 expression, and increased the p53 expression in H1299/WT cells but not in H1299/M175H-p53 cells. KCC009+IR also increased the expression of p21, Bax, p-caspase-3, and decreased Bcl-2 and CyclinD expression in H1299/WT cells. While KCC009+IR induced phosphorylation of caspase-3 and increase Cyt-C level in the cytoplasm of, and decreased CyclinB, Bcl-2 expression in H1299/M175H-p53 cells, we noticed that Cyt-C level in the nucleus decreased in the H1299/WT cells. Conclusions KCC009, a TG2 inhibitor, exhibits potent radiosensitization effects in human lung cancer cells expressing wild-type or mutant p53 with different mechanisms. PMID:28002389

  7. Transglutaminase 2 Inhibitor KCC009 Induces p53-Independent Radiosensitization in Lung Adenocarcinoma Cells.

    PubMed

    Huaying, Sheng; Dong, Yao; Chihong, Zhu; Xiaoqian, Qian; Danying, Wan; Jianguo, Feng

    2016-12-21

    BACKGROUND The expression of transglutaminase 2 (TG2) is correlated to DNA damage repair and apoptosis through the p53 pathway. The present study aimed to investigate the potential radiosensitization effect and possible mechanisms of the TG2 inhibitor KCC009 in lung cancer in vitro. MATERIAL AND METHODS A single hit multi-target model was used to plot survival curves and to calculate the sensitizing enhancement ratios in lung cancer wild-type or mutant p53 of H1299 cells. We performed analyses for changes of cell cycling and apoptotic responses of cells; Western blot analysis and real-time SYBR Green PCR assay were used to determine the changes of mRNA/protein expressions; ELISA assay was used for examination of cytochrome c release in cytoplasm. RESULTS Our results showed that KCC009 induced radiosensitization in both H1299/WT-p53 and H1299/M175H-p53 cells. KCC009+IR induced G0/G1 arrest in H1299/WT cells and G2/M arrest in H1299/M175H-p53 cells. KCC009+IR also induced apoptosis in both cell lines. In addition, KCC009+IR decreased the TG2 expression, and increased the p53 expression in H1299/WT cells but not in H1299/M175H-p53 cells. KCC009+IR also increased the expression of p21, Bax, p-caspase-3, and decreased Bcl-2 and CyclinD expression in H1299/WT cells. While KCC009+IR induced phosphorylation of caspase-3 and increase Cyt-C level in the cytoplasm of, and decreased CyclinB, Bcl-2 expression in H1299/M175H-p53 cells, we noticed that Cyt-C level in the nucleus decreased in the H1299/WT cells. CONCLUSIONS KCC009, a TG2 inhibitor, exhibits potent radiosensitization effects in human lung cancer cells expressing wild-type or mutant p53 with different mechanisms.

  8. Functional characterization of a new p53 mutant generated by homozygous deletion in a neuroblastoma cell line

    SciTech Connect

    Nakamura, Yohko; Ozaki, Toshinori; Niizuma, Hidetaka; Ohira, Miki; Kamijo, Takehiko; Nakagawara, Akira . E-mail: akiranak@chiba-cc.jp

    2007-03-23

    p53 is a key modulator of a variety of cellular stresses. In human neuroblastomas, p53 is rarely mutated and aberrantly expressed in cytoplasm. In this study, we have identified a novel p53 mutant lacking its COOH-terminal region in neuroblastoma SK-N-AS cells. p53 accumulated in response to cisplatin (CDDP) and thereby promoting apoptosis in neuroblastoma SH-SY5Y cells bearing wild-type p53, whereas SK-N-AS cells did not undergo apoptosis. We found another p53 (p53{delta}C) lacking a part of oligomerization domain and nuclear localization signals in SK-N-AS cells. p53{delta}C was expressed largely in cytoplasm and lost the transactivation function. Furthermore, a 3'-part of the p53 locus was homozygously deleted in SK-N-AS cells. Thus, our present findings suggest that p53 plays an important role in the DNA-damage response in certain neuroblastoma cells and it seems to be important to search for p53 mutations outside DNA-binding domain.

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

    PubMed Central

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

    1994-01-01

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

  10. Expression of p16 and p53 in Intraepithelial Periocular Sebaceous Carcinoma

    PubMed Central

    Bell, W. Robert; Singh, Kamaljeet; Rajan KD, Anand; Eberhart, Charles G.

    2015-01-01

    Purpose Identifying intraepithelial sebaceous carcinoma cells in small periocular biopsies can be difficult, particularly in the conjunctiva. The goal of this study was to evaluate p53 and p16 immunohistochemistry as potential markers of intraepithelial sebaceous carcinoma. Procedures A total of 25 tumors, including 4 recurrent lesions, were stained for p16 and p53, with intensity scored as negative, weak, moderate or strong. Results Expression of p16 was detected in intraepithelial sebaceous carcinoma cells in 24 of the 25 cases (96%), with only 1 case showing weak immunoreactivity. Intraepithelial p53 immunoreactivity was present in 17 of 25 tumors (68%), but was weak in 3 cases. Expression levels remained relatively stable in primary and recurrent tumors, but varied in a few cases between intraepithelial and subepithelial sites. Conclusions Intraepithelial sebaceous carcinomas stained for p53 and p16 demonstrated moderate to strong immunoreactivity in 100% of cases for at least one of these proteins, suggesting that together they are useful markers for determining the extent of tumor spread. Of the two, p16 was immunoreactive in more cases than p53. PMID:27171611

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

    PubMed

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

    2011-07-01

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

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

    SciTech Connect

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

    1991-07-01

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

  13. p53 Dependent Apoptotic Cell Death Induces Embryonic Malformation in Carassius auratus under Chronic Hypoxia

    PubMed Central

    Dasgupta, Subrata; Sawant, Bhawesh T.; Chadha, Narinder K.; Pal, Asim K.

    2014-01-01

    Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD), leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf) and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD), ultimately resulting in significant (p<0.05) embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos. PMID:25068954

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

    NASA Astrophysics Data System (ADS)

    Ohnishi, Takeo; Takahashi, Akihisa; Nakagawa, Yosuke

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

  15. The Role of p16, p21, p27, p53 and Ki-67 Expression in the Differential Diagnosis of Cutaneous Squamous Cell Carcinomas and Keratoacanthomas: An Immunohistochemical Study

    PubMed Central

    Bedir, Recep; Güçer, Hasan; Şehitoğlu, İbrahim; Yurdakul, Cüneyt; Bağcı, Pelin; Üstüner, Pelin

    2016-01-01

    Background: Distinguishing squamous cell carcinoma (SCC) from keratoacanthoma (KA) by histopathological features may not be sufficient for a differential diagnosis, as KAs may, in some cases, imitate well-differentiated SCCs. Aims: In this study, we investigated whether the expression of the p16, p21, p27, p53 genes and a Ki-67 proliferation index are useful in distinguishing between these two tumors. Study Design: Cross-sectional study. Methods: Immunohistochemistry was used to investigate the expression of the p16, p21, p27, p53 genes and the Ki-67 proliferation index was investigated in well-differentiated SCC with KA-like features (n=40) and KA (n=30). Results: The results of all of the examined markers, except for p27 (p16, p21, p53, and Ki-67) were found to be significantly different between the SCC and KA samples (p<0.05). Conclusion: In well-differentiated SCC with KA-like features and KA cases where the differential diagnosis is difficult from a histopathological perspective, the use of p16, p21, p53 expression and a Ki-67 proliferation index can be useful for the differential diagnosis of SCCs and KAs. PMID:27403379

  16. S100A4 interacts with mutant p53 and affects gastric cancer MKN1 cell autophagy and differentiation.

    PubMed

    Shen, Wei; Chen, Danqi; Liu, Shanshan; Chen, Lisha; Yu, Aiwen; Fu, Hao; Sun, Xiuju

    2015-12-01

    The acquired p53 mutations are the most common genetic alterations in human cancers. Mutant p53 proteins tend to accumulate, augmenting their oncogenic potential. However, the mechanisms for mutant p53 accumulation are not known. Previous studies have shown that S100A4 interacts with wild‑type p53. The present study marks the first time the effect of S100A4 on mutant p53 levels in gastric cancer MKN1 cells, which harbor mutant p53V143A, and the functional consequences have been investigated. S100A4 interacted with mutant p53V143A in the cells, and S100A4 inhibition decreased mutant p53V143A levels, indicating that S100A4 promoted mutant p53 accumulation through their interaction. We also found that S100A4 inhibition altered the expression of the mutant p53V143A target genes [c-Myc and inhibitor of DNA binding 2 (Id2)]. Moreover, we demonstrated that S100A4 knockdown increased mutant p53-related autophagy and cell differentiation. In conclusion, our data suggest a novel mechanism for mutant p53V143A accumulation and add a new facet to the role of S100A4 in cancer.

  17. The prognostic implication of the expression of EGFR, p53, cyclin D1, Bcl-2 and p16 in primary locally advanced oral squamous cell carcinoma cases: a tissue microarray study.

    PubMed

    Solomon, Monica Charlotte; Vidyasagar, M S; Fernandes, Donald; Guddattu, Vasudev; Mathew, Mary; Shergill, Ankur Kaur; Carnelio, Sunitha; Chandrashekar, Chetana

    2016-12-01

    Oral squamous cell carcinomas comprise a heterogeneous tumor cell population with varied molecular characteristics, which makes prognostication of these tumors a complex and challenging issue. Thus, molecular profiling of these tumors is advantageous for an accurate prognostication and treatment planning. This is a retrospective study on a cohort of primary locally advanced oral squamous cell carcinomas (n = 178) of an Indian rural population. The expression of EGFR, p53, cyclin D1, Bcl-2 and p16 in a cohort of primary locally advanced oral squamous cell carcinomas was evaluated. A potential biomarker that can predict the tumor response to treatment was identified. Formalin-fixed paraffin-embedded tumor blocks of (n = 178) of histopathologically diagnosed cases of locally advanced oral squamous cell carcinomas were selected. Tissue microarray blocks were constructed with 2 cores of 2 mm diameter from each tumor block. Four-micron-thick sections were cut from these tissue microarray blocks. These tissue microarray sections were immunohistochemically stained for EGFR, p53, Bcl-2, cyclin D1 and p16. In this cohort, EGFR was the most frequently expressed 150/178 (84%) biomarker of the cases. Kaplan-Meier analysis showed a significant association (p = 0.038) between expression of p53 and a poor prognosis. A Poisson regression analysis showed that tumors that expressed p53 had a two times greater chance of recurrence (unadjusted IRR-95% CI 2.08 (1.03, 4.5), adjusted IRR-2.29 (1.08, 4.8) compared with the tumors that did not express this biomarker. Molecular profiling of oral squamous cell carcinomas will enable us to categorize our patients into more realistic risk groups. With biologically guided tumor characterization, personalized treatment protocols can be designed for individual patients, which will improve the quality of life of these patients.

  18. Inhaled asbestos fibers induce p53 expression in the rat lung.

    PubMed

    Mishra, A; Liu, J Y; Brody, A R; Morris, G F

    1997-04-01

    Humans and rodents exposed to an aerosol of asbestos fibers develop lung injury that can lead to a fibroproliferative response culminating in excessive scarring and impaired lung function. To define the early events that precede asbestos-induced fibrotic lung disease, rats were exposed to an aerosol of chrysotile asbestos fibers for 5 h. At various times after exposure, the lungs of the asbestos-exposed animals were evaluated immunohistochemically for expression of the p53 tumor suppressor protein, a growth regulatory protein. p53 became detectable by immunostaining at the predicted sites of fiber deposition (the bronchiolar-alveolar duct bifurcations) by 24 h after exposure. The number of cells positive for p53 immunostaining increased to a maximal level at 8 days after exposure, decreased by 14 days and returned to a low basal level at the 30-day time point. Control groups of rats that were unexposed or exposed to an aerosol of iron beads were negative for p53 immunostaining throughout the 30-day assessment period. Simultaneous detection of the proliferating cell nuclear antigen (PCNA) at the sites of fiber deposition in the asbestos-exposed animals agrees with our previous finding that p53 binds and regulates the PCNA promoter.

  19. Zinc enhances CDKN2A, pRb1 expression and regulates functional apoptosis via upregulation of p53 and p21 expression in human breast cancer MCF-7 cell.

    PubMed

    Al-Saran, Nada; Subash-Babu, Pandurangan; Al-Nouri, Doha M; Alfawaz, Hanan A; Alshatwi, Ali A

    2016-10-01

    Zinc (Zn) is an essential trace elements, its deficiency is associated with increased incidence of human breast cancer. We aimed to study the effect of Zn on human breast cancer MCF-7 cells cultured in Zn depleted and Zn adequate medium. We found increased cancer cell growth in zinc depleted condition, further Zn supplementation inhibits the viability of breast cancer MCF-7 cell cultured in Zn deficient condition and the IC25, IC50 value for Zn is 6.2μM, 15μM, respectively after 48h. Zn markedly induced apoptosis through the characteristic apoptotic morphological changes and DNA fragmentation after 48h. In addition, Zn deficient cells significantly triggered intracellular ROS level and develop oxidative stress induced DNA damage; it was confirmed by elevated expression of CYP1A, GPX, GSK3β and TNF-α gene. Zinc depleted MCF-7 cells expressed significantly (p≤0.001) decreased levels of CDKN2A, pRb1, p53 and increased the level of mdm2 expression. Zn supplementation (IC50=15μM), increased significantly CDKN2A, pRB1 & p53 and markedly reduced mdm2 expression; also protein expression levels of CDKN2A and pRb1 was significantly increased. In addition, intrinsic apoptotic pathway related genes such as Bax, caspase-3, 8, 9 & p21 expression was enhanced and finally induced cell apoptosis. In conclusion, physiological level of zinc is important to prevent DNA damage and MCF-7 cell proliferation via regulation of tumor suppressor gene.

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

    PubMed Central

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

    2016-01-01

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

  1. p53 expression in oral lichenoid lesions and oral lichen planus.

    PubMed

    Arreaza, A; Rivera, H; Correnti, M

    2015-01-01

    The aim of this article was to compare the expression of p53 protein in oral lichen planus (OLP) and oral lichenoid reaction (OLR). The study population consisted of 65 patients--31 diagnosed with OLP and 34 with OLR. The results showed more p53 positive cases in the OLP group than in the OLR group. However, the difference between the 2 groups was not statistically significant (P = 0.114). The most common immunolocalization was observed at the basal cell layer. Due to the chance of potential future malignancy, follow-up for all cases is recommended.

  2. Knockdown of Merm1/Wbscr22 attenuates sensitivity of H460 non-small cell lung cancer cells to SN-38 and 5-FU without alteration to p53 expression levels.

    PubMed

    Yan, Dongmei; Zheng, Xiaoliang; Tu, Linglan; Jia, Jing; Li, Qin; Cheng, Liyan; Wang, Xiaoju

    2015-01-01

    Merm1/Wbscr22 is a novel metastasis promoter that has been shown to be involved in tumor metastasis, viability and apoptosis. To the best of our knowledge, there are currently no studies suggesting the possible correlation between the expression of Merm1/Wbscr22 in tumor cells and chemosensitivity to antitumor agents. In the present study, two human non-small cell lung cancer cell lines, H1299 and H460, were used to investigate whether Merm1/Wbscr22 affects chemosensitivity to antitumor agents, including cisplatin (CDDP), doxorubicin (ADM), paclitaxel (PTX), mitomycin (MMC), 7-Ethyl-10-hydroxycamptothecin (SN-38; the active metabolite of camptothecin) and 5-fluorouracil (5-FU). Merm1/Wbscr22 knockdown cell lines (H1299-shRNA and H460-shRNA) and negative control cell lines (H1299-NC and H460-NC) were established by stable transfection, and the efficiency of Merm1/Wbscr22 knockdown was confirmed by western blotting, immunofluorescence microscopy and quantitative polymerase chain reaction. The results demonstrated that shRNA-mediated knockdown of Merm1/Wbscr22 did not affect cell proliferation in vitro and in vivo. The H460 cells harboring wild type p53 were markedly more sensitive to all six antitumor agents as compared with the p53-null H1299 cells. Downregulation of Merm1/Wbscr22 did not affect H1299 sensitivity to any of the six antitumor agents, whereas attenuated H460 sensitivity to SN-38 and 5-FU, without significant alteration in p53 at both mRNA and protein levels, was identified. The reduced H460 sensitivity to SN-38 was further confirmed in vivo. SN-38 demonstrated significant tumor growth inhibitory activity in both H460 and H460‑NC tumor xenograft models, but only marginally suppressed the H460-shRNA xenograft tumor growth. Furthermore, CDDP (4, 10, 15 µg/ml)-resistant human non-small lung cancer cells A549 (A549-CDDPr-4, 10, 15) expressed significant amounts of Merm1/Wbscr22 protein, as compared with the parental A549 cells. In conclusion, sh

  3. p53 controls colorectal cancer cell invasion by inhibiting the NF-κB-mediated activation of Fascin

    PubMed Central

    Tang, Haimei; Wang, Chan; Zhou, Jichun; Han, Weidong; Wang, Xian; Fang, Yong; Xu, Yinghua; Li, Da; Chen, Rui; Ma, Junhong; Jing, Zhao; Gu, Xidong; Pan, Hongming; He, Chao

    2015-01-01

    p53 mutation is known to contribute to cancer progression. Fascin is an actin-bundling protein and has been recently identified to promote cancer cell migration and invasion through its role in formation of cellular protrusions such as filopodia and invadopodia. However, the relationship between p53 and Fascin is not understood. Here, we have found a new link between them. In colorectal adenocarcinomas, p53 mutation correlated with high NF-κB, Fascin and low E-cadherin expression. Moreover, this expression profile was shown to contribute to poor overall survival in patients with colorectal cancer. Wild-type p53 could inhibit NF-κB activity that repressed the expression of Fascin and cancer cell invasiveness. In contrast, in p53-deficient primary cultured cells, NF-κB activity was enhanced and then activation of NF-κB increased the expression of Fascin. In further analysis, we showed that NF-κB was a key determinant for p53 deletion-stimulated Fascin expression. Inhibition of NF-κB /p65 expression by pharmacological compound or p65 siRNA suppressed Fascin activity in p53-deficient cells. Moreover, restoration of p53 expression decreased the activation of Fascin through suppression of the NF-κB pathway. Taken together, these data suggest that a negative-feedback loop exists, whereby p53 can suppress colorectal cancer cell invasion by inhibiting the NF-κB-mediated activation of Fascin. PMID:26362504

  4. Lonidamine induces apoptosis in drug-resistant cells independently of the p53 gene.

    PubMed Central

    Del Bufalo, D; Biroccio, A; Soddu, S; Laudonio, N; D'Angelo, C; Sacchi, A; Zupi, G

    1996-01-01

    Lonidamine, a dichlorinated derivative of indazole-3-carboxylic acid, was shown to play a significant role in reversing or overcoming multidrug resistance. Here, we show that exposure to 50 microg/ml of lonidamine induces apoptosis in adriamycin and nitrosourea-resistant cells (MCF-7 ADR(r) human breast cancer cell line, and LB9 glioblastoma multiform cell line), as demonstrated by sub-G1 peaks in DNA content histograms, condensation of nuclear chromatin, and internucleosomal DNA fragmentation. Moreover, we find that apoptosis is preceded by accumulation of the cells in the G0/G1 phase of the cell cycle. Interestingly, lonidamine fails to activate the apoptotic program in the corresponding sensitive parental cell lines (ADR-sensitive MCF-7 WT, and nitrosourea-sensitive LI cells) even after long exposure times. The evaluation of bcl-2 protein expression suggests that this different effect of lonidamine treatment in drug-resistant and -sensitive cell lines might not simply be due to dissimilar expression levels of bcl-2 protein. To determine whether the lonidamine-induced apoptosis is mediated by p53 protein, we used cells lacking endogenous p53 and overexpressing either wild-type p53 or dominant-negative p53 mutant. We find that apoptosis by lonidamine is independent of the p53 gene. PMID:8787680

  5. Inhibition of Wild-Type p53-Expressing AML by the Novel Small Molecule HDM2 Inhibitor CGM097.

    PubMed

    Weisberg, Ellen; Halilovic, Ensar; Cooke, Vesselina G; Nonami, Atsushi; Ren, Tao; Sanda, Takaomi; Simkin, Irene; Yuan, Jing; Antonakos, Brandon; Barys, Louise; Ito, Moriko; Stone, Richard; Galinsky, Ilene; Cowens, Kristen; Nelson, Erik; Sattler, Martin; Jeay, Sebastien; Wuerthner, Jens U; McDonough, Sean M; Wiesmann, Marion; Griffin, James D

    2015-10-01

    The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cell-based proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient-derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML.

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

    PubMed Central

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

    2016-01-01

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

  7. Lack of association between p53 expression and betel nut chewing in oral cancers from Thailand.

    PubMed

    Thongsuksai, P; Boonyaphiphat, P

    2001-04-01

    To elucidate whether betel-associated oral squamous cell carcinoma is associated with p53 protein expression, tumor samples from 156 patients with detailed histories of exposures were investigated immunohistochemically using CM1 antibody. The expression of p53 (>10% positive cells) was found in 38.5% of the cases. The frequency of expression in betel chewers alone and betel chewer with tobacco use were 37.9% (11/29) and 25%(9/36), respectively, whereas that in betel chewers with smoking/drinking it was 47.2%(17/36) and in smokers or drinkers without chewing was 42.0% (21/50). However, the differences were not statistically significant. Multivariate analysis also revealed with the no independent association of betel chewing with p53 expression (odds ratio [OR] 1.81, 95% confidence interval 0.50-6.49), whereas alcohol drinking and smokeless tobacco use were significant (OR 7.58, 2.01-28.53 and 0.39, 0.16-0.98, respectively). These results suggested that betel chewing with or without smokeless tobacco use may not induce oral cancers via a p53-dependent pathway. However, since this is an immunohistochemical study, further molecular analysis is needed.

  8. Differential Gene Expression Profiles of Radioresistant Non-Small-Cell Lung Cancer Cell Lines Established by Fractionated Irradiation: Tumor Protein p53-Inducible Protein 3 Confers Sensitivity to Ionizing Radiation

    SciTech Connect

    Lee, Young Sook; Oh, Jung-Hwa; Yoon, Seokjoo; Kwon, Myung-Sang

    2010-07-01

    Purpose: Despite the widespread use of radiotherapy as a local and regional modality for the treatment of cancer, some non-small-cell lung cancers commonly develop resistance to radiation. We thus sought to clarify the molecular mechanisms underlying resistance to radiation. Methods and Materials: We established the radioresistant cell line H460R from radiosensitive parental H460 cells. To identify the radioresistance-related genes, we performed microarray analysis and selected several candidate genes. Results: Clonogenic and MTT assays showed that H460R was 10-fold more resistant to radiation than H460. Microarray analysis indicated that the expression levels of 1,463 genes were altered more than 1.5-fold in H460R compared with parental H460. To evaluate the putative functional role, we selected one interesting gene tumor protein p53-inducible protein 3 (TP53I3), because that this gene was significantly downregulated in radioresistant H460R cells and that it was predicted to link p53-dependent cell death signaling. Interestingly, messenger ribonucleic acid expression of TP53I3 differed in X-ray-irradiated H460 and H460R cells, and overexpression of TP53I3 significantly affected the cellular radiosensitivity of H460R cells. Conclusions: These results show that H460R may be useful in searching for candidate genes that are responsible for radioresistance and elucidating the molecular mechanism of radioresistance.

  9. c-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting p53/Notch1 and breast cancer gene 1 expression

    PubMed Central

    Pfefferle, Adam D.; Perou, Charles M.; Van Den Berg, Carla Lynn

    2015-01-01

    Breast cancer is a heterogeneous disease with several subtypes carrying unique prognoses. Patients with differentiated luminal tumors experience better outcomes, while effective treatments are unavailable for poorly differentiated tumors, including the basal-like subtype. Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments. C-Jun N-terminal kinase 2 (JNK2) is important in mammary tumorigenesis and tumor progression. Using a variety of mouse models, human breast cancer cell lines and tumor expression data, studies herein support that JNK2 inhibits cell differentiation in normal and cancer-derived mammary cells. JNK2 prevents precocious pubertal mammary development and inhibits Notch-dependent expansion of luminal cell populations. Likewise, JNK2 suppresses luminal populations in a p53-competent Polyoma Middle T-antigen tumor model where jnk2 knockout causes p53-dependent upregulation of Notch1 transcription. In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition. JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression. These data suggest that therapies inhibiting JNK2 in breast cancer may promote tumor differentiation, improve endocrine therapy response, and inhibit metastasis. PMID:25970777

  10. c-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting p53/Notch1 and breast cancer gene 1 expression.

    PubMed

    Cantrell, Michael A; Ebelt, Nancy D; Pfefferle, Adam D; Perou, Charles M; Van Den Berg, Carla Lynn

    2015-05-20

    Breast cancer is a heterogeneous disease with several subtypes carrying unique prognoses. Patients with differentiated luminal tumors experience better outcomes, while effective treatments are unavailable for poorly differentiated tumors, including the basal-like subtype. Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments. C-Jun N-terminal kinase 2 (JNK2) is important in mammary tumorigenesis and tumor progression. Using a variety of mouse models, human breast cancer cell lines and tumor expression data, studies herein support that JNK2 inhibits cell differentiation in normal and cancer-derived mammary cells. JNK2 prevents precocious pubertal mammary development and inhibits Notch-dependent expansion of luminal cell populations. Likewise, JNK2 suppresses luminal populations in a p53-competent Polyoma Middle T-antigen tumor model where jnk2 knockout causes p53-dependent upregulation of Notch1 transcription. In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition. JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression. These data suggest that therapies inhibiting JNK2 in breast cancer may promote tumor differentiation, improve endocrine therapy response, and inhibit metastasis.

  11. Polyvinyl pyrrolidone-coated silver nanoparticles in a human lung cancer cells: time- and dose-dependent influence over p53 and caspase-3 protein expression and epigenetic effects.

    PubMed

    Blanco, Jordi; Lafuente, Daisy; Gómez, Mercedes; García, Tánia; Domingo, José L; Sánchez, Domènec J

    2017-02-01

    The present study was aimed at providing a better understanding of the influence of silver nanoparticles (AgNPs) on the p53 tumor suppressor protein. Cell line A549 was exposed to a range of concentrations of AgNPs, and a time course (up to 72 h) of cell viability was determined. We also determined the time course of gene and protein expression of p53, p21, murine double minute 2 (MDM2) and caspase-3. The expression of all of these proteins was also determined after daily exposure of the cells to 10 µg/mL of AgNPs for 7 days, or after discontinuous exposure by treating the cells every 3 days, for 15 or 30 days. Moreover, epigenetic changes in the acetylation of the histone H3 protein and in global DNA methylation patterns were determined after 72 h of exposure. Results showed that daily exposure to low doses of AgNPs, or a single exposure to high concentrations for 72 h, decreased gene and protein expression of p53, p21, MDM2 and caspase-3 in A549 cells. In contrast, a discontinuous exposure to low doses or a single exposure to low concentrations for 72 h increased the levels of the active forms of p53 and caspase-3, as well as the p21 and MDM2 protein levels. In addition, exposure to high concentrations of AgNPs for 72 h induced higher levels of global DNA methylation and global histone H3 deacetylation in A549 cells. These results provide new information on the toxic action of AgNPs.

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

    PubMed

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

    2008-06-15

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

  13. Loss of LSD1 (lysine-specific demethylase 1) suppresses growth and alters gene expression of human colon cancer cells in a p53- and DNMT1(DNA methyltransferase 1)-independent manner

    PubMed Central

    Jin, Lihua; Hanigan, Christin L.; Wu, Yu; Wang, Wei; Park, Ben Ho; Woster, Patrick M.; Casero, Robert A.

    2012-01-01

    Epigenetic silencing of gene expression is important in cancer. Aberrant DNA CpG island hypermethylation and histone modifications are involved in the aberrant silencing of tumour-suppressor genes. LSD1 (lysine-specific demethylase 1) is a H3K4 (histone H3 Lys4) demethylase associated with gene repression and is overexpressed in multiple cancer types. LSD1 has also been implicated in targeting p53 and DNMT1 (DNA methyltransferase 1), with data suggesting that the demethylating activity of LSD1 on these proteins is necessary for their stabilization. To examine the role of LSD1 we generated LSD1 heterozygous (LSD1+/−) and homozygous (LSD1−/−) knockouts in the human colorectal cancer cell line HCT116. The deletion of LSD1 led to a reduced cell proliferation both in vitro and in vivo. Surprisingly, the knockout of LSD1 in HCT116 cells did not result in global increases in its histone substrate H3K4me2 (dimethyl-H3K4) or changes in the stability or function of p53 or DNMT1. However, there was a significant difference in gene expression between cells containing LSD1 and those null for LSD1. The results of the present study suggested that LSD1 is critical in the regulation of cell proliferation, but also indicated that LSD1 is not an absolute requirement for the stabilization of either p53 or DNMT1. PMID:23072722

  14. Loss of LSD1 (lysine-specific demethylase 1) suppresses growth and alters gene expression of human colon cancer cells in a p53- and DNMT1(DNA methyltransferase 1)-independent manner.

    PubMed

    Jin, Lihua; Hanigan, Christin L; Wu, Yu; Wang, Wei; Park, Ben Ho; Woster, Patrick M; Casero, Robert A

    2013-01-15

    Epigenetic silencing of gene expression is important in cancer. Aberrant DNA CpG island hypermethylation and histone modifications are involved in the aberrant silencing of tumour-suppressor genes. LSD1 (lysine-specific demethylase 1) is a H3K4 (histone H3 Lys4) demethylase associated with gene repression and is overexpressed in multiple cancer types. LSD1 has also been implicated in targeting p53 and DNMT1 (DNA methyltransferase 1), with data suggesting that the demethylating activity of LSD1 on these proteins is necessary for their stabilization. To examine the role of LSD1 we generated LSD1 heterozygous (LSD1+/-) and homozygous (LSD1-/-) knockouts in the human colorectal cancer cell line HCT116. The deletion of LSD1 led to a reduced cell proliferation both in vitro and in vivo. Surprisingly, the knockout of LSD1 in HCT116 cells did not result in global increases in its histone substrate H3K4me2 (dimethyl-H3K4) or changes in the stability or function of p53 or DNMT1. However, there was a significant difference in gene expression between cells containing LSD1 and those null for LSD1. The results of the present study suggested that LSD1 is critical in the regulation of cell proliferation, but also indicated that LSD1 is not an absolute requirement for the stabilization of either p53 or DNMT1.

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

    PubMed Central

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

    2012-01-01

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

  16. Inhibitors of histone deacetylase (HDAC) restore the p53 pathway in neuroblastoma cells

    PubMed Central

    Condorelli, F; Gnemmi, I; Vallario, A; Genazzani, A A; Canonico, P L

    2007-01-01

    Background and purpose: Inhibitors of histone deacetylase (HDAC) are emerging as a promising class of anti-cancer drugs, but a generic deregulation of transcription in neoplastic cells cannot fully explain their therapeutic effects. In this study we evaluated alternative molecular mechanisms by which HDAC inhibitors could affect neuroblastoma viability. Experimental approach: Effects of HDAC inhibitors on survival of the I-type SK-N-BE and the N-type NB SH-SY5Y neuroblastoma cell lines were assessed by the MTT assay. Molecular pathways leading to this were examined by western blot, confocal microscopy and cytofluorometry. The mRNA levels of apoptotic mediators were assessed semi-quantitatively by RT-PCR. Tumour-suppressor p53 trans activity was assessed in EMSA experiments. HDAC inhibitors were also studied in cells subjected to plasmid-based p53 interference (p53i). Key results: HDAC inhibitors induced cell death via the mitochondrial pathway of apoptosis with recruitment of Bcl-2 family members. Bcl-2 overexpression rendered neuroblastoma cells resistant to HDAC inhibitor treatment. Low concentrations of HDAC inhibitors (0.9 mM) caused a G2 cell-cycle arrest and a marked upregulation of the p21/Waf1/Cip1 protein. HDAC inhibitors also activate the p53 protein via hyper-acetylation and nuclear re-localization, without affecting its protein expression. Accordingly, HDAC inhibitor-induced cell-killing and p21/Waf1/Cip1 upregulation is impaired in p53i-cells. Conclusions and implications: In neuroblastoma cells, HDAC inhibitors may overcome the resistance to classical chemotherapeutic drugs by restoring the p53 tumour-repressor function via its hyper-acetylation and nuclear migration, events usually impaired in such tumours. In neuroblastoma cells, HDAC inhibitors are not able to induce p21/Waf1/Cip1 in the absence of a functional p53. PMID:18059320

  17. Inhibition of p53 increases chemosensitivity to 5-FU in nutrient-deprived hepatocarcinoma cells by suppressing autophagy.

    PubMed

    Guo, Xian-ling; Hu, Fei; Zhang, Shan-shan; Zhao, Qiu-dong; Zong, Chen; Ye, Fei; Guo, Shi-wei; Zhang, Jian-wei; Li, Rong; Wu, Meng-chao; Wei, Li-xin

    2014-05-01

    Activation of p53 can induce apoptosis, cell cycle arrest, and cell senescence, although some evidence has suggested that p53 could promote cell survival. However, whether p53 plays a positive role in cancer cell survival to chemotherapy remains unknown. In this study, we show that inhibition of p53 enhanced apoptosis and increased chemosensitivity to 5-fluorouracil (5-FU) in nutrient-deprived hepatocarcinoma cells (HCC). Meanwhile, nutrient-deprivation-induced autophagy was inhibited by pifithrin-α or small interfering RNA targeting p53. The expression of p53 was not increased when HCC were incubated under nutrient-deprived conditions. This indicates that the basal level of p53 is important to autophagy activation in nutrient-deprived HCC cells. Furthermore, combining p53 inhibition and nutrient deprivation or 5-FU treatment resulted in a marked increase in reactive oxygen species generation and mitochondrial damage. Antioxidants reduced nutrient deprivation or 5-FU-induced cell death of HCC after p53 inhibition. Our results suggest that p53 contributes to cell survival and chemoresistance in HCC under nutrient-deprived conditions by modulating autophagy activation.

  18. Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53

    PubMed Central

    Yoon, Kyoung Wan; Byun, Sanguine; Kwon, Eunjeong; Hwang, So-Young; Chu, Kiki; Hiraki, Masatsugu; Jo, Seung-Hee; Weins, Astrid; Hakroush, Samy; Cebulla, Angelika; Sykes, David B.; Greka, Anna; Mundel, Peter; Fisher, David E.; Mandinova, Anna; Lee, Sam W.

    2016-01-01

    The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses. PMID:26228159

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

    PubMed

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

    2011-12-01

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

  20. Selective killing of p53-deficient cancer cells by SP600125

    PubMed Central

    Jemaà, Mohamed; Vitale, Ilio; Kepp, Oliver; Berardinelli, Francesco; Galluzzi, Lorenzo; Senovilla, Laura; Mariño, Guillermo; Malik, Shoaib Ahmad; Rello-Varona, Santiago; Lissa, Delphine; Antoccia, Antonio; Tailler, Maximilien; Schlemmer, Frederic; Harper, Francis; Pierron, Gérard; Castedo, Maria; Kroemer, Guido

    2012-01-01

    The genetic or functional inactivation of p53 is highly prevalent in human cancers. Using high-content videomicroscopy based on fluorescent TP53+/+ and TP53−/− human colon carcinoma cells, we discovered that SP600125, a broad-spectrum serine/threonine kinase inhibitor, kills p53-deficient cells more efficiently than their p53-proficient counterparts, in vitro. Similar observations were obtained in vivo, in mice carrying p53-deficient and -proficient human xenografts. Such a preferential cytotoxicity could be attributed to the failure of p53-deficient cells to undergo cell cycle arrest in response to SP600125. TP53−/− (but not TP53+/+) cells treated with SP600125 became polyploid upon mitotic abortion and progressively succumbed to mitochondrial apoptosis. The expression of an SP600125-resistant variant of the mitotic kinase MPS1 in TP53−/− cells reduced SP600125-induced polyploidization. Thus, by targeting MPS1, SP600125 triggers a polyploidization program that cannot be sustained by TP53−/− cells, resulting in the activation of mitotic catastrophe, an oncosuppressive mechanism for the eradication of mitosis-incompetent cells. PMID:22438244

  1. A planarian p53 homolog regulates proliferation and self-renewal in adult stem cell lineages.

    PubMed

    Pearson, Bret J; Sánchez Alvarado, Alejandro

    2010-01-01

    The functions of adult stem cells and tumor suppressor genes are known to intersect. However, when and how tumor suppressors function in the lineages produced by adult stem cells is unknown. With a large population of stem cells that can be manipulated and studied in vivo, the freshwater planarian is an ideal system with which to investigate these questions. Here, we focus on the tumor suppressor p53, homologs of which have no known role in stem cell biology in any invertebrate examined thus far. Planaria have a single p53 family member, Smed-p53, which is predominantly expressed in newly made stem cell progeny. When Smed-p53 is targeted by RNAi, the stem cell population increases at the expense of progeny, resulting in hyper-proliferation. However, ultimately the stem cell population fails to self-renew. Our results suggest that prior to the vertebrates, an ancestral p53-like molecule already had functions in stem cell proliferation control and self-renewal.

  2. Immunohistochemical detection of mutant p53 protein in small-cell lung cancer: relationship to treatment outcome.

    PubMed

    Gemba, K; Ueoka, H; Kiura, K; Tabata, M; Harada, M

    2000-07-01

    We investigated the expression of mutant p53 proteins in small-cell lung cancer (SCLC) immunohistochemically, by identification of stabilized mutant p53 proteins with a much longer half-life than the wild-type protein. Of 103 tumor specimens obtained by transbronchial tumor biopsy for histologic diagnosis, 52 (50%) showed positive staining for p53 protein with a p53 monoclonal antibody, DO-1. Positive staining for p53 protein was not correlated with age, sex, performance status, lifetime cigarette consumption, serum concentration of neuron-specific enolase and extent of disease. Complete response rates in patients with a mutant p53 protein-positive tumor were significantly lower than those in p53-negative patients (25% versus 59%; P=0.0005, by chi-square test). Similarly, survival periods in patients with a mutant p53 protein-positive tumor were significantly shorter than those in mutant p53-protein-negative patients (10.8 months versus 20.6 months; P=0.0001, by generalized Wilcoxon test). Multivariate analysis using Cox's proportional hazards model revealed that the presence of mutant p53 protein is an independent factor associated with differences in overall survival (hazards ratio=2.72; 95% confidence interval, 1.71-4.34; P=0.0001). These observations suggest that the expression of mutant p53 proteins in SCLC may be an important factor predicting poor prognosis.

  3. Mutant p53 promotes ovarian cancer cell adhesion to mesothelial cells via integrin β4 and Akt signals.

    PubMed

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

    2015-07-30

    Missense mutations in the TP53 gene resulting in the accumulation of mutant proteins are extremely common in advanced ovarian cancer, which is characterised by peritoneal metastasis. Attachment of cancer cells to the peritoneal mesothelium is regarded as an initial, key step for the metastatic spread of ovarian cancer. In the present study, we investigated the possible role of a p53 mutant in the mesothelial adhesion of ovarian cancer cells. We found that OVCAR-3 cells with the R248 TP53 mutation (p53(R248)) were more adhesive to mesothelial Met5A cells than were A2780 cells expressing wild-type p53. In addition, ectopic expression of p53(R248) in p53-null SKOV-3 cells significantly increased adhesion to Met5A cells. Knockdown of mutant p53 significantly compromised p53(R248)-induced cell adhesion to Met5A cells. Microarray analysis revealed that several adhesion-related genes, including integrin β4, were markedly up-regulated, and certain signalling pathways, including PI3K/Akt, were activated in p53(R248) transfectants of SKOV-3 cells. Inhibition of integrin β4 and Akt signalling using blocking antibody and the inhibitor LY294002, respectively, significantly attenuated p53(R248)-mediated ovarian cancer-mesothelial adhesion. These data suggest that the p53(R248) mutant endows ovarian cancer cells with increased adhesiveness and that integrin β4 and Akt signalling are associated with the mutation-enhanced ovarian cancer-mesothelial cell adhesion.

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

    PubMed

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

    2015-06-15

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

  5. Single cell monitoring of growth arrest and morphological changes induced by transfer of wild-type p53 alleles to glioblastoma cells.

    PubMed Central

    Van Meir, E G; Roemer, K; Diserens, A C; Kikuchi, T; Rempel, S A; Haas, M; Huang, H J; Friedmann, T; de Tribolet, N; Cavenee, W K

    1995-01-01

    Mutation of the p53 tumor suppressor gene is one of the earliest identified genetic lesions during malignant progression of human astrocytomas. To assess the functional significance of these mutations, wild-type (WT) p53 genes were introduced into glioblastoma cell lines having mutant, WT, or null endogenous p53 alleles. Populations of cells with mutant or null endogenous p53 alleles and exogenous WT p53 were spontaneously selected in culture for cells expressing only mutant p53 or no p53, which then displayed a growth and tumorigenic phenotype identical to the parental cells. To determine the phenotypic consequences of WT p53 expression before the occurrence of mutations, we developed a single cell assay to monitor WT p53-dependent transcription activity. Transfer and expression of exogenous WT p53 genes to cells with endogenous mutant or deleted, but not WT, p53 alleles caused growth arrest and morphological changes, including increased cell size and acquisition of multiple nuclei. This supports the hypothesis that genetic lesions of the p53 gene play an important role in the genesis of astrocytomas. Furthermore, the high sensitivity of the episomal single cell reporter strategy developed here has potential clinical applications in the rapid screening of patients for germ-line mutations of the p53 gene or any other gene with known targets for transcriptional transactivation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7862624

  6. Decreasing CNPY2 Expression Diminishes Colorectal Tumor Growth and Development through Activation of p53 Pathway.

    PubMed

    Yan, Ping; Gong, Hui; Zhai, Xiaoyan; Feng, Yi; Wu, Jun; He, Sheng; Guo, Jian; Wang, Xiaoxia; Guo, Rui; Xie, Jun; Li, Ren-Ke

    2016-04-01

    Neovascularization drives tumor development, and angiogenic factors are important neovascularization initiators. We recently identified the secreted angiogenic factor CNPY2, but its involvement in cancer has not been explored. Herein, we investigate CNPY2's role in human colorectal cancer (CRC) development. Tumor samples were obtained from CRC patients undergoing surgery. Canopy 2 (CNPY2) expression was analyzed in tumor and adjacent normal tissue. Stable lines of human HCT116 cells expressing CNPY2 shRNA or control shRNA were established. To determine CNPY2's effects on tumor xenografts in vivo, human CNPY2 shRNA HCT116 cells and controls were injected into nude mice, separately. Cellular apoptosis, growth, and angiogenesis in the xenografts were evaluated. CNPY2 expression was significantly higher in CRC tissues. CNPY2 knockdown in HCT116 cells inhibited growth and migration and promoted apoptosis. In xenografts, CNPY2 knockdown prevented tumor growth and angiogenesis and promoted apoptosis. Knockdown of CNPY2 in the HCT116 CRC cell line reversibly increased p53 activity. The p53 activation increased cyclin-dependent kinase inhibitor p21 and decreased cyclin-dependent kinase 2, thereby inhibiting tumor cell growth, inducing cell apoptosis, and reducing angiogenesis both in vitro and in vivo. CNPY2 may play a critical role in CRC development by enhancing cell proliferation, migration, and angiogenesis and by inhibiting apoptosis through negative regulation of the p53 pathway. Therefore, CNPY2 may represent a novel CRC therapeutic target and prognostic indicator.

  7. p53 coordinates DNA repair with nucleotide synthesis by suppressing PFKFB3 expression and promoting the pentose phosphate pathway

    PubMed Central

    Franklin, Derek A.; He, Yizhou; Leslie, Patrick L.; Tikunov, Andrey P.; Fenger, Nick; Macdonald, Jeffrey M.; Zhang, Yanping

    2016-01-01

    Activation of p53 in response to DNA damage is essential for tumor suppression. Although previous studies have emphasized the importance of p53-dependent cell cycle arrest and apoptosis for tumor suppression, recent studies have suggested that other areas of p53 regulation, such as metabolism and DNA damage repair (DDR), are also essential for p53-dependent tumor suppression. However, the intrinsic connections between p53-mediated DDR and metabolic regulation remain incompletely understood. Here, we present data suggesting that p53 promotes nucleotide biosynthesis in response to DNA damage by repressing the expression of the phosphofructokinase-2 (PFK2) isoform 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a rate-limiting enzyme that promotes glycolysis. PFKFB3 suppression increases the flux of glucose through the pentose phosphate pathway (PPP) to increase nucleotide production, which results in more efficient DNA damage repair and increased cell survival. Interestingly, although p53-mediated suppression of PFKFB3 could increase the two major PPP products, NADPH and nucleotides, only nucleotide production was essential to promote DDR. By identifying the novel p53 target PFKFB3, we report an important mechanistic connection between p53-regulated metabolism and DDR, both of which play crucial roles in tumor suppression. PMID:27901115

  8. TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

    SciTech Connect

    Ding, Li; Huang, Yong; Du, Qian; Dong, Feng; Zhao, Xiaomin; Zhang, Wenlong; Xu, Xingang; Tong, Dewen

    2014-03-07

    Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.

  9. Pure versus combined Merkel cell carcinomas: immunohistochemical evaluation of cellular proteins (p53, Bcl-2, and c-kit) reveals significant overexpression of p53 in combined tumors.

    PubMed

    Lai, Jonathan H; Fleming, Kirsten E; Ly, Thai Yen; Pasternak, Sylvia; Godlewski, Marek; Doucette, Steve; Walsh, Noreen M

    2015-09-01

    Merkel cell polyomavirus is of oncogenic significance in approximately 80% of Merkel cell carcinomas. Morphological subcategories of the tumor differ in regard to viral status, the rare combined type being uniformly virus negative and the predominant pure type being mainly virus positive. Indications that different biological subsets of the tumor exist led us to explore this diversity. In an Eastern Canadian cohort of cases (75 patients; mean age, 76 years [range, 43-91]; male/female ratio, 43:32; 51 [68%] pure and 24 [34%] combined tumors), we semiquantitatively compared the immunohistochemical expression of 3 cellular proteins (p53, Bcl-2, and c-kit) in pure versus combined groups. Viral status was known in a subset of cases. The significant overexpression of p53 in the combined group (mean [SD], 153.8 [117.8] versus 121.6 [77.9]; P = .01) and the increased epidermal expression of this protein (p53 patches) in the same group lend credence to a primary etiologic role for sun damage in these cases. Expression of Bcl-2 and c-kit did not differ significantly between the 2 morphological groups. A relative increase in c-kit expression was significantly associated with a virus-negative status (median [interquartile range], 100 [60-115] versus 70 [0-100]; P = .03). Emerging data reveal divergent biological pathways in Merkel cell carcinoma, each with a characteristic immunohistochemical profile. Virus-positive tumors (all pure) exhibit high retinoblastoma protein and low p53 expression, whereas virus-negative cases (few pure and all combined) show high p53 and relatively high c-kit expression. The potential biological implications of this dichotomy call for consistent stratification of these tumors in future studies.

  10. Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage

    PubMed Central

    Solomon, Jonathan M.; Pasupuleti, Rao; Xu, Lei; McDonagh, Thomas; Curtis, Rory; DiStefano, Peter S.; Huber, L. Julie

    2006-01-01

    Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells. PMID:16354677

  11. Methylsulfonylmethane Induces p53 Independent Apoptosis in HCT-116 Colon Cancer Cells

    PubMed Central

    Karabay, Arzu Zeynep; Koc, Asli; Ozkan, Tulin; Hekmatshoar, Yalda; Sunguroglu, Asuman; Aktan, Fugen; Buyukbingol, Zeliha

    2016-01-01

    Methylsulfonylmethane (MSM) is an organic sulfur-containing compound which has been used as a dietary supplement for osteoarthritis. MSM has been shown to reduce oxidative stress and inflammation, as well as exhibit apoptotic or anti-apoptotic effects depending on the cell type or activating stimuli. However, there are still a lot of unknowns about the mechanisms of actions of MSM. In this study, MSM was tested on colon cancer cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis revealed that MSM inhibited cell viability and increased apoptotic markers in both HCT-116 p53 +/+ and HCT-116 p53 −/− colon cancer cells. Increased poly (ADP-ribose) polymerase (PARP) fragmentation and caspase-3 activity by MSM also supported these findings. MSM also modulated the expression of various apoptosis-related genes and proteins. Moreover, MSM was found to increase c-Jun N-terminal kinases (JNK) phosphorylation in both cell lines, dose-dependently. In conclusion, our results show for the first time that MSM induces apoptosis in HCT-116 colon cancer cells regardless of their p53 status. Since p53 is defective in >50% of tumors, the ability of MSM to induce apoptosis independently of p53 may offer an advantage in anti-tumor therapy. Moreover, the remarkable effect of MSM on Bim, an apoptotic protein, also suggests its potential use as a novel chemotherapeutic agent for Bim-targeted anti-cancer therapies. PMID:27428957

  12. MicroRNA 203 expression in keratinocytes is dependent on regulation of p53 levels by E6.

    PubMed

    McKenna, Declan J; McDade, Simon S; Patel, Daksha; McCance, Dennis J

    2010-10-01

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miRNA 203 (miR-203), which has previously been shown to play an important role in epithelial cell biology by regulating p63 levels. We investigated how expression of human papillomavirus type 16 (HPV16) oncoproteins E6 and E7 affected miR-203 expression during proliferation and differentiation of HFKs. We demonstrated that miR-203 expression is reduced in HFKs where p53 function is compromised, either by the viral oncoprotein E6 or by knockout of p53 using short hairpin RNAs (p53i). We show that the induction of miR-203 observed during calcium-induced differentiation of HFKs is significantly reduced in HFKs expressing E6 and in p53i HFKs. Induction of miR-203 in response to DNA damage is also reduced in the absence of p53. We report that proliferation of HFKs is dependent on the level of miR-203 expression and that overexpression of miR-203 can reduce overproliferation in E6/E7-expressing and p53i HFKs. In summary, these results indicate that expression of miR-203 is dependent on p53, which may explain how expression of HPV16 E6 can disrupt the balance between proliferation and differentiation, as well as the response to DNA damage, in keratinocytes.

  13. Differential regulated microRNA by wild type and mutant p53 in induced pluripotent stem cells

    PubMed Central

    Grespi, Francesca; Landré, Vivien; Molchadsky, Alina; Di Daniele, Nicola; Marsella, Luigi Tonino; Melino, Gerry; Rotter, Varda

    2016-01-01

    The tumour suppressor p53 plays an important role in somatic cell reprogramming. While wild-type p53 reduces reprogramming efficiency, mutant p53 exerts a gain of function activity that leads to increased reprogramming efficiency. Furthermore, induced pluripotent stem cells expressing mutant p53 lose their pluripotency in vivo and form malignant tumours when injected in mice. It is therefore of great interest to identify targets of p53 (wild type and mutant) that are responsible for this phenotype during reprogramming, as these could be exploited for therapeutic use, that is, formation of induced pluripotent stem cells with high reprogramming efficiency, but no oncogenic potential. Here we studied the transcriptional changes of microRNA in a series of mouse embryonic fibroblasts that have undergone transition to induced pluripotent stem cells with wild type, knock out or mutant p53 status in order to identify microRNAs whose expression during reprogramming is dependent on p53. We identified a number of microRNAs, with known functions in differentiation and carcinogenesis, the expression of which was dependent on the p53 status of the cells. Furthermore, we detected several uncharacterised microRNAs that were regulated differentially in the different p53 backgrounds, suggesting a novel role of these microRNAs in reprogramming and pluripotency. PMID:28032868

  14. 3-MCPD 1-Palmitate Induced Tubular Cell Apoptosis In Vivo via JNK/p53 Pathways.

    PubMed

    Liu, Man; Huang, Guoren; Wang, Thomas T Y; Sun, Xiangjun; Yu, Liangli Lucy

    2016-05-01

    Fatty acid esters of 3-chloro-1, 2-propanediol (3-MCPD esters) are a group of processing induced food contaminants with nephrotoxicity but the molecular mechanism(s) remains unclear. This study investigated whether and how the JNK/p53 pathway may play a role in the nephrotoxic effect of 3-MCPD esters using 3-MCPD 1-palmitate (MPE) as a probe compound in Sprague Dawley rats. Microarray analysis of the kidney from the Sprague Dawley rats treated with MPE, using Gene Ontology categories and KEGG pathways, revealed that MPE altered mRNA expressions of the genes involved in the mitogen-activated protein kinase (JNK and ERK), p53, and apoptotic signal transduction pathways. The changes in the mRNA expressions were confirmed by qRT-PCR and Western blot analyses and were consistent with the induction of tubular cell apoptosis as determined by histopathological, TUNEL, and immunohistochemistry analyses in the kidneys of the Sprague Dawley rats. Additionally, p53 knockout attenuated the apoptosis, and the apoptosis-related protein bax expression and cleaved caspase-3 activation induced by MPE in the p53 knockout C57BL/6 mice, whereas JNK inhibitor SP600125 but not ERK inhibitor U0126 inhibited MPE-induced apoptosis, supporting the conclusion that JNK/p53 might play a critical role in the tubular cell apoptosis induced by MPE and other 3-MCPD fatty acid esters.

  15. A new invertebrate member of the p53 gene family is developmentally expressed and responds to polychlorinated biphenyls.

    PubMed Central

    Jessen-Eller, Kathryn; Kreiling, Jill A; Begley, Gail S; Steele, Marjorie E; Walker, Charles W; Stephens, Raymond E; Reinisch, Carol L

    2002-01-01

    The cell-cycle checkpoint protein p53 both directs terminal differentiation and protects embryos from DNA damage. To study invertebrate p53 during early development, we identified three differentially expressed p53 family members (p53, p97, p120) in the surf clam, Spisula solidissima. In these mollusks, p53 and p97 occur in both embryonic and adult tissue, whereas p120 is exclusively embryonic. We sequenced, cloned, and characterized p120 cDNA. The predicted protein, p120, resembles p53 across all evolutionarily conserved regions and contains a C-terminal extension with a sterile alpha motif (SAM) as in p63 and p73. These vertebrate forms of p53 are required for normal inflammatory, epithelial, and neuronal development. Unlike clam p53 and p97, p120 mRNA and protein levels are temporally expressed in embryos, with mRNA levels decreasing with increasing p120 protein (R(2) = 0.97). Highest surf clam p120 mRNA levels coincide with the onset of neuronal growth. In earlier work we have shown that neuronal development is altered by exposure to polychlorinated biphenyls (PCBs), a neurotoxic environmental contaminant. In this study we show that PCBs differentially affect expression of the three surf clam p53 family members. p120 mRNA and protein are reduced the most and earliest in development, p97 protein shows a smaller and later reduction, and p53 protein levels do not change. For the first time we report that unlike p53 and p97, p120 is specifically embryonic and expressed in a time-dependent manner. Furthermore, p120 responds to PCBs by 48 hr when PCB-induced suppression of the serotonergic nervous system occurs. PMID:11940455

  16. Murine Double Minute-2 Prevents p53-Overactivation-Related Cell Death (Podoptosis) of Podocytes

    PubMed Central

    Bruns, Hauke A.; Kretschmer, Victoria; Ebrahim, Martrez; Romoli, Simone; Liapis, Helen; Kotb, Ahmed M.; Endlich, Nicole; Anders, Hans-Joachim

    2015-01-01

    Murine double minute-2 (MDM2), an E3 ligase that regulates the cell cycle and inflammation, is highly expressed in podocytes. In podocyte injury, MDM2 drives podocyte loss by mitotic catastrophe, but the function of MDM2 in resting podocytes has not been explored. Here, we investigated the effects of podocyte MDM2 deletion in vitro and in vivo. In vitro, MDM2 knockdown by siRNA caused increased expression of p53 and podocyte death, which was completely rescued by coknockdown of p53. Apoptosis, pyroptosis, pyronecrosis, necroptosis, ferroptosis, and parthanatos were excluded as modes of occurrence for this p53-overactivation-related cell death (here referred to as podoptosis). Podoptosis was associated with cytoplasmic vacuolization, endoplasmic reticulum stress, and dysregulated autophagy (previously described as paraptosis). MDM2 knockdown caused podocyte loss and proteinuria in a zebrafish model, which was consistent with the phenotype of podocyte-specific MDM2-knockout mice that also showed the aforementioned ultrastructual podocyte abnormalities before and during progressive glomerulosclerosis. The phenotype of both animal models was entirely rescued by codeletion of p53. We conclude that MDM2 maintains homeostasis and long-term survival in podocytes by preventing podoptosis, a p53-regulated form of cell death with unspecific features previously classified as paraptosis. PMID:25349197

  17. Knockdown of FAM3B triggers cell apoptosis through p53-dependent pathway.

    PubMed

    Mou, Haiwei; Li, Zongmeng; Yao, Pengle; Zhuo, Shu; Luan, Wei; Deng, Bo; Qian, Lihua; Yang, Mengmei; Mei, Hong; Le, Yingying

    2013-03-01

    FAM3B, also named PANDER, is a cytokine-like protein identified in 2002. Previous studies showed that FAM3B regulates glucose and lipid metabolism through interaction with liver and endocrine pancreas. FAM3B is also expressed by other tissues but its basic function is unclear. In this study, we found that FAM3B was expressed in mouse colon, intestine, liver and lung tissues and multiple types of cell lines, including murine pancreatic β-cell (Min6), microglia (N9) and muscle cell (C2C12); human colon cancer cells (HCT8, HCT116, HT29), hepatocyte (HL-7702), hepatocellular carcinoma cell (SMMC-7721) and lung carcinoma cell (A549). Inhibition of FAM3B expression by RNA interference induced apoptotic cell death of HCT8, HCT116, A549, N9, C2C12 and Min6 cells and decreased cell viability of HL-7702 and murine primary hepatocytes. Further studies with HCT8 cells showed that knockdown of FAM3B increased the protein levels of membrane-bound Fas and Bax, reduced the expression of Bcl-2, promoted the cleavage of caspases-8, -3, -9 and PARP, and the nuclear translocation of cleaved PARP. These results suggest that FAM3B silencing activates both extrinsic and intrinsic apoptotic pathways. Mechanistic studies showed that neutralizing antibody against Fas or silencing Fas-associated death domain had no effect on, while caspase inhibitors could significantly reverse FAM3B knockdown induced apoptosis, suggesting Fas and death receptor mediated extrinsic apoptotic pathway is not involved in FAM3B silencing induced apoptosis. Further studies showed that p53 was significantly upregulated after FAM3B knockdown. Silencing p53 could almost completely reverse FAM3B knockdown induced upregulation of Bax, downregulation of Bcl-2, cleavage of caspases-8, -9, -3, and apoptotic cell death, suggesting p53-dependent pathway plays critical roles in FAM3B silencing induced apoptosis. Studies with HCT116 cells confirmed that inhibition of FAM3B expression induced apoptosis through p53-dependent

  18. The cell death response to the ROS inducer, cobalt chloride, in neuroblastoma cell lines according to p53 status.

    PubMed

    Stenger, Christophe; Naves, Thomas; Verdier, Mireille; Ratinaud, Marie-Helene

    2011-09-01

    Cobalt chloride (CoCl2), a hypoxia-mimetic agent, induces reactive oxygen species (ROS) generation, leading to cell death. Divergent data have been reported concerning p53 implication in this apoptotic mechanism. In this study, we studied cobalt-induced cell death in neuroblastoma cell lines carrying wild-type (WT) p53 ( SHSY5Y) and a mutated DNA-binding domain p53 [SKNBE(2c)]. CoCl2 induced an upregulation of p53, p21 and PUMA expression in WT cells but not in SKNBE(2c). In SHSY5Y cells, p53 serine-15 phosphorylation appeared early (6 h) in the mitochondria, and also in the nucleus after 12 h. In contrast, in SKNBE(2c) cells, the slight nuclear signal disappeared with CoCl2 treatment. In SHSY5Y cells, a mitochondrial pathway dependent on caspases [collapse of mitochondrial transmembrane potential (∆Ψmt), caspase 3 and 9 activation], was activated in a time-dependent manner. SKNBE(2c) cells exhibited a delay in the cell death executive phase linked to a caspase-independent pathway, involving apoptosis inducing factor nuclear translocation, but also an autophagic process attested by LC3-II expression and cathepsin-B activation. The downregulation of p53 in SHSY5Y cells by siRNA induced a cell death pathway related to the one observed in SKNBE(2c) cells. Finally, CoCl2 induced time-dependent canonical p53 mitochondrial apoptosis in the WT p53 cell line, and caspase-independent cell death in cells with a mutated or KO p53.

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

    PubMed Central

    Ponchel, F.; Milner, J.

    1998-01-01

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

  20. The Ews/Fli-1 fusion gene changes the status of p53 in neuroblastoma tumor cell lines.

    PubMed

    Rorie, Checo J; Weissman, Bernard E

    2004-10-15

    One hallmark of Ewing's sarcoma/peripheral neuroectodermal tumors is the presence of the Ews/Fli-1 chimeric oncogene. Interestingly, infection of neuroblastoma tumor cell lines with Ews/Fli-1 switches the differentiation program of neuroblastomas to Ewing's sarcoma/peripheral neuroectodermal tumors. Here we examined the status of cytoplasmically sequestered wt-p53 in neuroblastomas after stable expression of Ews/Fli-1. Immunofluorescence revealed that in the neuroblastoma-Ews/Fli-1 infectant cell lines, p53 went from a punctate-pattern of cytoplasmic sequestration to increased nuclear localization. Western blot analysis revealed that PARC was down-regulated in one neuroblastoma cell line but not expressed in the second. Therefore, decreased PARC expression could not fully account for relieving p53 sequestration in the neuroblastoma tumor cells. Neuroblastoma-Ews/Fli-1 infectant cell lines showed marked increases in p53 protein expression without transcriptional up-regulation. Interestingly, p53 was primarily phosphorylated, without activation of its downstream target p21(WAF1). Western blot analysis revealed that whereas MDM2 gene expression does not change, p14(ARF), a negative protein regulator of MDM2, increases. These observations suggest that the downstream p53 pathway may be inactivated as a result of abnormal p53. We also found that p53 has an extended half-life in the neuroblastoma-Ews/Fli-1 infectants despite the retention of a wild-type sequence in neuroblastoma-Ews/Fli-1 infectant cell lines. We then tested the p53 response pathway and observed that the neuroblastoma parent cells responded to genotoxic stress, whereas the neuroblastoma-Ews/Fli-1 infectants did not. These results suggest that Ews/Fli-1 can directly abrogate the p53 pathway to promote tumorigenesis. These studies also provide additional insight into the relationship among the p53 pathway proteins.

  1. p53-inducible long non-coding RNA PICART1 mediates cancer cell proliferation and migration.

    PubMed

    Cao, Yu; Lin, Minglin; Bu, Yiwen; Ling, Hongyan; He, Yingchun; Huang, Chenfei; Shen, Yi; Song, Bob; Cao, Deliang

    2017-05-01

    Long non-coding RNAs (lncRNAs) function in the development and progression of cancer, but only a small portion of lncRNAs have been characterized to date. A novel lncRNA transcript, 2.53 kb in length, was identified by transcriptome sequencing analysis, and was named p53-inducible cancer-associated RNA transcript 1 (PICART1). PICART1 was found to be upregulated by p53 through a p53-binding site at -1808 to -1783 bp. In breast and colorectal cancer cells and tissues, PICART1 expression was found to be decreased. Ectopic expression of PICART1 suppressed the growth, proliferation, migration, and invasion of MCF7, MDA-MB-231 and HCT116 cells whereas silencing of PICART1 stimulated cell growth and migration. In these cells, the expression of PICART1 suppressed levels of p-AKT (Thr308 and Ser473) and p-GSK3β (Ser9), and accordingly, β-catenin, cyclin D1 and c-Myc expression were decreased, while p21Waf/cip1 expression was increased. Together these data suggest that PICART1 is a novel p53-inducible tumor-suppressor lncRNA, functioning through the AKT/GSK3β/β-catenin signaling cascade.

  2. p53 Mediates Colistin-Induced Autophagy and Apoptosis in PC-12 Cells

    PubMed Central

    Zhang, Ling; Xie, Daoyuan; Chen, Xueping; Hughes, Maria L. R.; Jiang, Guozheng; Lu, Ziyin; Xia, Chunli; Li, Li; Wang, Jinli; Xu, Wei; Sun, Yuan; Li, Rui; Wang, Rui; Qian, Feng

    2016-01-01

    The mechanism of colistin-induced neurotoxicity is still unknown. Our recent study (L. Zhang, Y. H. Zhao, W. J. Ding, G. Z. Jiang, Z. Y. Lu, L. Li, J. L. Wang, J. Li, and J. C. Li, Antimicrob Agents Chemother 59:2189–2197, 2015, http://dx.doi.org/10.1128/AAC.04092-14; H. Jiang, J. C. Li, T. Zhou, C. H. Wang, H. Zhang, and H. Wang, Int J Mol Med 33:1298–1304, 2014, http://dx.doi.org/10.3892/ijmm.2014.1684) indicates that colistin induces autophagy and apoptosis in rat adrenal medulla PC-12 cells, and there is interplay between both cellular events. As an important cellular stress sensor, phosphoprotein p53 can trigger cell cycle arrest and apoptosis and regulate autophagy. The aim of the present study was to investigate the involvement of the p53 pathway in colistin-induced neurotoxicity in PC-12 cells. Specifically, cells were treated with colistin (125 μg/ml) in the absence and presence of a p53 inhibitor, pifithrin-α (PFT-α; 20 nM), for 12 h and 24 h, and the typical hallmarks of autophagy and apoptosis were examined by fluorescence/immunofluorescence microscopy and electron microscopy, real-time PCR, and Western blotting. The results indicate that colistin had a stimulatory effect on the expression levels of the target genes and proteins involved in autophagy and apoptosis, including LC3-II/I, p53, DRAM (damage-regulated autophagy modulator), PUMA (p53 upregulated modulator of apoptosis), Bax, p-AMPK (activated form of AMP-activated protein kinase), and caspase-3. In contrast, colistin appeared to have an inhibitory effect on the expression of p-mTOR (activated form of mammalian target of rapamycin), which is another target protein in autophagy. Importantly, analysis of the levels of p53 in the cells treated with colistin revealed an increase in nuclear p53 at 12 h and cytoplasmic p53 at 24 h. Pretreatment of colistin-treated cells with PFT-α inhibited autophagy and promoted colistin-induced apoptosis. This is the first study to demonstrate that colistin

  3. Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1, and Caspase-9/-3 activation

    PubMed Central

    Sørensen, Belinda Halling; Nielsen, Dorthe; Thorsteinsdottir, Unnur Arna; Hoffmann, Else Kay

    2016-01-01

    The leucine-rich repeat containing 8A (LRRC8A) protein is an essential component of the volume-sensitive organic anion channel (VSOAC), and using pharmacological anion channel inhibitors (NS3728, DIDS) and LRRC8A siRNA we have investigated its role in development of Cisplatin resistance in human ovarian (A2780) and alveolar (A549) carcinoma cells. In Cisplatin-sensitive cells Cisplatin treatment increases p53-protein level as well as downstream signaling, e.g., expression of p21Waf1/Cip1, Bax, Noxa, MDM2, and activation of Caspase-9/-3. In contrast, Cisplatin-resistant cells do not enter apoptosis, i.e., their p53 and downstream signaling are reduced and caspase activity unaltered following Cisplatin exposure. Reduced LRRC8A expression and VSOAC activity are previously shown to correlate with Cisplatin resistance, and here we demonstrate that pharmacological inhibition and transient knockdown of LRRC8A reduce the protein level of p53, MDM2, and p21Waf1/Cip1 as well as Caspase-9/-3 activation in Cisplatin-sensitive cells. Cisplatin resistance is accompanied by reduction in total LRRC8A expression (A2780) or LRRC8A expression in the plasma membrane (A549). Activation of Caspase-3 dependent apoptosis by TNFα-exposure or hyperosmotic cell shrinkage is almost unaffected by pharmacological anion channel inhibition. Our data indicate 1) that expression/activity of LRRC8A is essential for Cisplatin-induced increase in p53 protein level and its downstream signaling, i.e., Caspase-9/-3 activation, expression of p21Waf1/Cip1 and MDM2; and 2) that downregulation of LRRC8A-dependent osmolyte transporters contributes to acquirement of Cisplatin resistance in ovarian and lung carcinoma cells. Activation of LRRC8A-containing channels is upstream to apoptotic volume decrease as hypertonic cell shrinkage induces apoptosis independent of the presence of LRRC8A. PMID:26984736

  4. AB112. Expression of brain-specific angiogenesis inhibitor 1 and association with p53, microvessel density and vascular endothelial growth factor in the tissue of human bladder transitional cell carcinoma

    PubMed Central

    Tian, Dawei; Hu, Hailong; Wu, Changli

    2016-01-01

    Objective Brain-specific angiogenesis inhibitor 1 (BAI1) was initially described in 1997, and there have since been a number of studies on its expression in different types of cancer. The aim of the present study was to investigate the expression levels of BAI1 in bladder transitional cell carcinoma (BTCC) at different stages and the mechanism by which it inhibits tumor endothelial cell proliferation. Methods Normal bladder mucosa biopsy specimens were obtained as the control group, and human BTCC biopsy specimens were used as the study group. Immunohistochemical assays were used to detect the expression levels of BAI1, vascular endothelial growth factor (VEGF) and mutant p53, in addition to microvessel density (MVD) in the tissues. Western blotting was used to analyze the differential expression of BAI1 in the two samples. Results Statistical analysis was performed, which indicated that BAI1 expression levels in the normal bladder mucosa group were significantly higher than those in the BTCC group and were associated with clinical staging. BAI1 levels in the T1 stage BTCC tissues were higher than those in the T2–4 stage BTCC tissues (P<0.05). BAI1 expression levels were negatively correlated with those of VEGF (r=−0.661, P<0.001), mutant p53 (r=−0.406, P=0.002) and with the MVD (r=−0.675, P<0.001). Conclusions BAI1 may be involved in the negative regulation of BTCC microvascular proliferation, and its expression may be associated with a reduction in p53 mutations.

  5. TEL/ETV6 induces apoptosis in 32D cells through p53-dependent pathways

    SciTech Connect

    Yamagata, Tetsuya; Maki, Kazuhiro; Waga, Kazuo; Mitani, Kinuko . E-mail: kinukom-tky@umin.ac.jp

    2006-08-25

    TEL is an ETS family transcription factor that is critical for maintaining hematopoietic stem cells in adult bone marrow. To investigate the roles of TEL in myeloid proliferation and differentiation, we introduced TEL cDNA into mouse myeloid 32Dcl3 cells. Overexpression of TEL repressed interleukin-3-dependent proliferation through blocking cell cycle progression. Also, the presence of TEL triggered apoptosis through the mitochondrial intrinsic pathway on exposure to granulocyte colony-stimulating factor. We found an increase in p53 protein and its DNA binding in the TEL-overexpressing cells. Forced expression of TEL stimulated transcription via the p53-responsive element and increased the expression of cellular target genes for p53 such as cell cycle regulator p21 and apoptosis inducer Puma. Consistently, induction of apoptosis was delayed by pifithrin-{alpha} treatment and completely blocked by increased expression of Bcl-2 in the TEL-overexpressing cells. These data collectively suggest that TEL exerts a tumor suppressive function through augmenting the p53 pathway and facilitates normal development of myelopoiesis.

  6. Naphthoquinone derivative PPE8 induces endoplasmic reticulum stress in p53 null H1299 cells.

    PubMed

    Lien, Jin-Cherng; Huang, Chien-Chun; Lu, Te-Jung; Tseng, Chih-Hsiang; Sung, Ping-Jyun; Lee, Hong-Zin; Bao, Bo-Ying; Kuo, Yueh-Hsiung; Lu, Te-Ling

    2015-01-01

    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.

  7. CP-31398 prevents the growth of p53-mutated colorectal cancer cells in vitro and in vivo.

    PubMed

    He, Xingxing; Kong, Xinjuan; Yan, Junwei; Yan, Jingjun; Zhang, Yunan; Wu, Qian; Chang, Ying; Shang, Haitao; Dou, Qian; Song, Yuhu; Liu, Fang

    2015-03-01

    Rescuing the function of mutant p53 protein is an attractive cancer therapeutic strategy. Small molecule CP-31398 was shown to restore mutant p53 tumor suppressor functions in cancer cells. Here, we determined the effects of CP-31398 on the growth of p53-mutated colorectal cancer (CRC) cells in vitro and in vivo. CRC cells which carry p53 mutation in codon 273 were treated with CP-31398 and the control, and the effects of CP-31398 on cell cycle, cell apoptosis, and proliferation were determined. The expression of p53-responsive downstream genes was evaluated by quantitative reverse transcriptase PCR (RT-PCR) and Western blot. CP-31398 was administrated into xenograft tumors created by the inoculation of HT-29 cells, and then the effect of CP-31398 on the growth of xenograft tumors was examined. CP-31398 induced p53 downstream target molecules in cultured HT-29 cells, which resulted in the inhibition of CRC cell growth assessed by the determination of cell cycle, apoptosis, and cell proliferation. In xenograft tumors, CP-31398 modulated the expression of Bax, Bcl-2, caspase 3, cyclin D, and Mdm2 and then blocked the growth of xenograft tumors. CP-31398 would be developed as a therapeutic candidate for p53-mutated CRC due to the restoration of mutant p53 tumor suppressor functions.

  8. FUSE Binding Protein 1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53

    PubMed Central

    Dixit, Updesh; Pandey, Ashutosh K.; Liu, Zhihe; Kumar, Sushil; Neiditch, Matthew B.; Klein, Kenneth M.

    2015-01-01

    ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic hepatitis C (CHC), liver cirrhosis, and hepatocellular carcinoma (HCC). Immunohistochemistry of archived HCC tumors showed abundant FBP1 expression in HCC tumors with the CHC background. Oncomine data analysis of normal versus HCC tumors with the CHC background indicated a 4-fold increase in FBP1 expression with a concomitant 2.5-fold decrease in the expression of p53. We found that FBP1 promotes HCV replication by inhibiting p53 and regulating BCCIP and TCTP, which are positive and negative regulators of p53, respectively. The severe inhibition of HCV replication in FBP1-knockdown Huh7.5 cells was restored to a normal level by downregulation of either p53 or BCCIP. Although p53 in Huh7.5 cells is transcriptionally inactive as a result of Y220C mutation, we found that the activation and DNA binding ability of Y220C p53 were strongly suppressed by FBP1 but significantly activated upon knockdown of FBP1. Transient expression of FBP1 in FBP1 knockdown cells fully restored the control phenotype in which the DNA binding ability of p53 was strongly suppressed. Using electrophoretic mobility shift assay (EMSA) and isothermal titration calorimetry (ITC), we found no significant difference in in vitro target DNA binding affinity of recombinant wild-type p53 and its Y220C mutant p53. However, in the presence of recombinant FBP1, the DNA binding ability of p53 is strongly inhibited. We confirmed that FBP1 downregulates BCCIP, p21, and p53 and upregulates TCTP under radiation-induced stress. Since FBP1 is overexpressed in most HCC tumors with an HCV background, it may have a role in promoting persistent virus infection and tumorigenesis. IMPORTANCE It is our novel finding that FUSE binding protein 1 (FBP1) strongly inhibits the function of tumor suppressor p53 and is an essential host cell factor required for HCV replication. Oncomine data analysis of a large number of samples has revealed that overexpression of

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

    PubMed Central

    2013-01-01

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

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

    PubMed Central

    JEDINAK, ANDREJ; SLIVA, DANIEL

    2009-01-01

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

  11. Focal adhesion kinase and p53 synergistically decrease neuroblastoma cell survival.

    PubMed

    Gillory, Lauren A; Stewart, Jerry E; Megison, Michael L; Waters, Alicia M; Beierle, Elizabeth A

    2015-06-15

    Neuroblastoma is the most common extracranial solid tumor of childhood and is responsible for over 15% of pediatric cancer deaths. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of neuroblastoma tumor development and progression. The p53 oncogene, although wild type in most neuroblastomas, lacks significant function as a tumor suppressor in these tumors. Recent reports have found that FAK and p53 interact in some tumor types. We have hypothesized FAK and p53 coordinately control each other's expression and also interact in neuroblastoma. In the present study, we showed that not only do FAK and p53 interact but each one controls the expression of the other. In addition, we also examined the effects of FAK inhibition combined with p53 activation in neuroblastoma and showed that these two, in combination, had a synergistic effect on neuroblastoma cell survival. The findings from this present study help to further our understanding of the regulation of neuroblastoma tumorigenesis and may provide novel therapeutic strategies and targets for neuroblastoma and other pediatric solid tumors.

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

    PubMed Central

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

    2015-01-01

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

  13. The maternal genes Ci-p53/p73-a and Ci-p53/p73-b regulate zygotic ZicL expression and notochord differentiation in Ciona intestinalis embryos.

    PubMed

    Noda, Takeshi

    2011-12-01

    I isolated a Ciona intestinalis homolog of p53, Ci-p53/p73-a, in a microarray screen of rapidly degraded maternal mRNA by comparing the transcriptomes of unfertilized eggs and 32-cell stage embryos. Higher expression of the gene in eggs and lower expression in later embryonic stages were confirmed by whole-mount in situ hybridization (WISH) and quantitative reverse transcription-PCR (qRT-PCR); expression was ubiquitous in eggs and early embryos. Knockdown of Ci-p53/p73-a by injection of antisense morpholino oligonucleotides (MOs) severely perturbed gastrulation cell movements and expression of notochord marker genes. A key regulator of notochord differentiation in Ciona embryos is Brachyury (Ci-Bra), which is directly activated by a zic-like gene (Ci-ZicL). The expression of Ci-ZicL and Ci-Bra in A-line notochord precursors was downregulated in Ci-p53/p73-a knockdown embryos. Maternal expression of Ci-p53/p73-b, a homolog of Ci-p53/p73-a, was also detected. In Ci-p53/p73-b knockdown embryos, gastrulation cell movements, expression of Ci-ZicL and Ci-Bra in A-line notochord precursors, and expression of notochord marker gene at later stages were perturbed. The upstream region of Ci-ZicL contains putative p53-binding sites. Cis-regulatory analysis of Ci-ZicL showed that these sites are involved in expression of Ci-ZicL in A-line notochord precursors at the 32-cell and early gastrula stages. These results suggest that p53 genes are maternal factors that play a crucial role in A-line notochord differentiation in C. intestinalis embryos by regulating Ci-ZicL expression.

  14. A p53-independent role of Mdm2 in estrogen-mediated activation of breast cancer cell proliferation

    PubMed Central

    2011-01-01

    Introduction Estrogen receptor positive breast cancers often have high levels of Mdm2. We investigated if estrogen signaling in such breast cancers occurred through an Mdm2 mediated pathway with subsequent inactivation of p53. Methods We examined the effect of long-term 17β-estradiol (E2) treatment (five days) on the p53-Mdm2 pathway in estrogen receptor alpha (ERα) positive breast cancer cell lines that contain wild-type p53 (MCF-7 and ZR75-1). We assessed the influence of estrogen by examining cell proliferation changes, activation of transcription of p53 target genes, p53-chromatin interactions and cell cycle profile changes. To determine the effects of Mdm2 and p53 knockdown on the estrogen-mediated proliferation signals we generated MCF-7 cell lines with inducible shRNA for mdm2 or p53 and monitored their influence on estrogen-mediated outcomes. To further address the p53-independent effect of Mdm2 in ERα positive breast cancer we generated cell lines with inducible shRNA to mdm2 using the mutant p53 expressing cell line T-47D. Results Estrogen increased the Mdm2 protein level in MCF-7 cells without decreasing the p53 protein level. After estrogen treatment of MCF-7 cells, down-regulation of basal transcription of p53 target genes puma and p21 was observed. Estrogen treatment also down-regulated etoposide activated transcription of puma, but not p21. Mdm2 knockdown in MCF-7 cells increased p21 mRNA and protein, decreased cell growth in 3D matrigel and also decreased estrogen-induced cell proliferation in 2D culture. In contrast, knockdown of p53 had no effect on estrogen-induced cell proliferation. In T-47D cells with mutant p53, the knockdown of Mdm2 decreased estrogen-mediated cell proliferation but did not increase p21 protein. Conclusions Estrogen-induced breast cancer cell proliferation required a p53-independent role of Mdm2. The combined influence of genetic and environmental factors on the tumor promoting effects of estrogen implicated Mdm2 as a

  15. Comparison of proliferating cell nuclear antigen, thyroid transcription factor-1, Ki-67, p63, p53 and high-molecular weight cytokeratin expressions in papillary thyroid carcinoma, follicular carcinoma, and follicular adenoma.

    PubMed

    Tan, Ayca; Etit, Demet; Bayol, Umit; Altinel, Deniz; Tan, Sedat

    2011-04-01

    The searching of the reliable and repeatable immunohistochemical markers in the differential diagnosis of the thyroid's differentiated follicular epithelial neoplasms has been continuing. Recently, the studies have majored on immunohistochemical markers such as high-molecular weight cytokeratin (HMW-CK), galectin-3, cytokeratin 19, and p27. We aimed to evaluate the differences of the expressions of the proliferating cell nuclear antigen (PCNA), thyroid transcription factor-1 (TTF-1), Ki-67, p63, p53, and HMW-CK among the papillary thyroid carcinomas (PTCs), follicular carcinomas (FCs), and follicular adenomas (FAs). Thirty-nine patients with the diagnoses of the PTC, FC, and FA in the archives of the Izmir Tepecik Training and Research Hospital Pathology Laboratory registries in between 2004 and 2009 were included in the study. Immunohistochemical stains for PCNA, TTF-1, Ki-67, p63, p53, and HMW-CK were applied. The results were analyzed statistically by using Statistical Package for the Social Sciences (SPSS) for Windows 16.0 program (SPSS Inc., IBM, Somers, New York, USA). In all 3 groups, all tumors showed PCNA and TTF-1 positivity. Ki-67 proliferation index varied in a wide range in all groups. Although it was not statistically significant, 19 of 39 tumors (7 PTCs, 2 FCs, and 10 FAs) were stained with p63. The results of the immunoreactivity seen in PTCs with p53 (41.2%) and HMW-CK (52.9%) were statistically significant. The tumors in the other 2 groups (FC and FA) showed no reactivity with HMW-CK. Although the differential diagnosis of the thyroid follicular neoplasms are based on the histologic and cytomorphological criteria, p53 and HMW-CK positivity might be undertaken in favor of the diagnosis of the PTC.

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

    PubMed

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

    2004-01-01

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

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

    SciTech Connect

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

    2009-10-02

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

  18. HSP25 down-regulation enhanced p53 acetylation by dissociation of SIRT1 from p53 in doxorubicin-induced H9c2 cell apoptosis.

    PubMed

    Zhang, Chi; Qu, Shunlin; Wei, Xing; Feng, Yansheng; Zhu, Honglin; Deng, Jia; Wang, Kangkai; Liu, Ke; Liu, Meidong; Zhang, Huali; Xiao, Xianzhong

    2016-03-01

    Heat shock proteins (HSPs) play important roles in cellular stress resistance. Previous reports had already suggested that HSP27 played multiple roles in preventing doxorubicin-induced cardiotoxicity. Although HSP25 might have biological functions similar to its human homolog HSP27, the mechanism of HSP25 is still unclear in doxorubicin-induced cardiomyocyte apoptosis. To investigate HSP25 biological function on doxorubicin-induced apoptosis, flow cytometry was employed to analyze cell apoptosis in over-expressing HSP25 H9c2 cells in presence of doxorubicin. Unexpectedly, the H9c2 cells of over-expressing HSP25 have no protective effect on doxorubicin-induced apoptosis. Moreover, no detectable interactions were detected by coimmunoprecipitation between HSP25 and cytochrome c, and HSP25 over-expression failed in preventing cytochrome c release induced by doxorubicin. However, down-regulation of endogenous HSP25 by a specific small hairpin RNA aggravates apoptosis in H9c2 cells. Subsequent studies found that HSP25, but not HSP90, HSP70, and HSP20, interacted with SIRT1. Knockdown of HSP25 decreased the interaction between SIRT1 and p53, leading to increased p53 acetylation on K379, up-regulated pro-apoptotic Bax protein expression, induced cytochrome c release, and triggered caspase-3 and caspase-9 activation. These findings indicated a novel mechanism by which HSP25 regulated p53 acetylation through dissociation of SIRT1 from p53 in doxorubicin-induced H9c2 cell apoptosis.

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

    PubMed

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

    2013-08-01

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

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed

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

    1999-06-01

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

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

    PubMed Central

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

    2017-01-01

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

  3. Induction of apoptosis in cancer cells through N-acetyl-l-leucine-modified polyethylenimine-mediated p53 gene delivery.

    PubMed

    Li, Zhiyuan; Zhang, Liu; Li, Quanshun

    2015-11-01

    Herein, N-acetyl-L-leucine-modified polyethylenimine was successfully constructed through the EDC/NHS-mediated coupling reaction and employed as vectors to accomplish p53 gene delivery using HeLa (p53wt) and PC-3 cells (p53null) as models. Compared with PEI25K, the derivatives exhibited lower cytotoxicity, protein adsorption and hemolytic activity, together with satisfactory pDNA condensation capability and gene transfection efficiency. After p53 transfection, MTT analysis confirmed that the cell proliferation was inhibited. Flow cytometric analysis showed that the derivative-mediated p53 delivery could induce stronger early apoptosis than PEI25K and Lipofectamine(2000). Further, PC-3 cells showed higher sensitivity to the exogenous p53 transfection than HeLa cells. The mechanism for inducing apoptosis was determined to be up-regulation of p53 expression at both mRNA and protein levels using RT-PCR and western blotting analysis. Expression level and activity analysis of caspase-3, -8 and -9, and mitochondrial membrane potential measurement revealed that p53 transfection mediated by these derivatives facilitated early apoptosis of tumor cells via a mitochondria-dependent apoptosis pathway. Thus, the derivatives showed potential as biocompatible carriers for realizing effective tumor gene therapy.

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

    PubMed

    Lazo, Pedro A

    2017-05-01

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

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

    SciTech Connect

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

    2009-09-04

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

  6. Cadherin-6 type 2, K-cadherin (CDH6) is regulated by mutant p53 in the fallopian tube but is not expressed in the ovarian surface.

    PubMed

    Karthikeyan, Subbulakshmi; Lantvit, Daniel D; Chae, Dam Hee; Burdette, Joanna E

    2016-10-25

    High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy and may arise in either the fallopian tube epithelium (FTE) or ovarian surface epithelium (OSE). A mutation in p53 is reported in 96% of HGSOC, most frequently at R273 and R248. The goal of this study was to identify specific gene targets in the FTE that are altered by mutant p53, but not in the OSE. Gene analysis revealed that both R273 and R248 mutant p53 reduces CDH6 expression in the oviduct, but CDH6 was not detected in murine OSE cells. p53R273H induced SLUG and FOXM1 while p53R248W did not induce SLUG and only modestly increased FOXM1, which correlated with less migration as compared to p53R273H. An oviduct specific PAX8Cre/+/p53R270H/+ mouse model was created and confirmed that in vivo mutant p53 repressed CDH6 but was not sufficient to stabilize p53 expression alone. Overexpression of mutant p53 in the p53 null OVCAR5 cells decreased CDH6 levels indicating this was a gain-of-function. SLUG knockdown in murine oviductal cells with p53R273H restored CDH6 repression and a ChIP analysis revealed direct binding of mutant p53 on the CDH6 promoter. NSC59984, a small molecule that degrades mutant p53R273H, rescued CDH6 expression. In summary, CDH6 is expressed in the oviduct, but not the ovary, and is repressed by mutant p53. CDH6 expression with further validations may aide in establishing markers that inform upon the cell of origin of high grade serous tumors.

  7. Copper uptake is required for pyrrolidine dithiocarbamate-mediated oxidation and protein level increase of p53 in cells.

    PubMed Central

    Furuta, Saori; Ortiz, Fausto; Zhu Sun, Xiu; Wu, Hsiao-Huei; Mason, Andrew; Momand, Jamil

    2002-01-01

    The p53 tumour-suppressor protein is a transcription factor that activates the expression of genes involved in cell cycle arrest, apoptosis and DNA repair. The p53 protein is vulnerable to oxidation at cysteine thiol groups. The metal-chelating dithiocarbamates, pyrrolidine dithiocarbamate (PDTC), diethyldithiocarbamate, ethylene(bis)dithiocarbamate and H(2)O(2) were tested for their oxidative effects on p53 in cultured human breast cancer cells. Only PDTC oxidized p53, although all oxidants tested increased the p53 level. Inductively coupled plasma MS analysis indicated that the addition of 60 microM PDTC increased the cellular copper concentration by 4-fold, which was the highest level of copper accumulated amongst all the oxidants tested. Bathocuproinedisulphonic acid, a membrane-impermeable Cu(I) chelator inhibited the PDTC-mediated copper accumulation. Bathocuproinedisulphonic acid as well as the hydroxyl radical scavenger d-mannitol inhibited the PDTC-dependent increase in p53 protein and oxidation. Our results show that a low level of copper accumulation in the range of 25-40 microg/g of cellular protein increases the steady-state levels of p53. At copper accumulation levels higher than 60 microg/g of cellular protein, p53 is oxidized. These results suggest that p53 is vulnerable to free radical-mediated oxidation at cysteine residues. PMID:11964141

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

    PubMed Central

    Chandrangsu, Soranun

    2016-01-01

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

  9. Establishment of a novel clonal murine bone marrow stromal cell line for assessment of p53 responses to genotoxic stress

    SciTech Connect

    Gorbunov, Nikolai V.; Morris, James E.; Greenberger, J S.; Thrall, Brian D.

    2002-10-15

    The p53 protein is widely regarded as an important sensor of genotoxic damage in cells, and mutations in p53 are the most frequent observed in human cancers. Rapid assays for evaluating the potential of a chemical or physical agent to alter the transcriptional regulatory role of p53 may therefore serve as useful tools in toxicological research. In this study, the use of enhanced green fluorescent protein (EGFP) as a live cell reporter to assess the transactivation response of p53 to chemical and physical agents was evaluated. A stable murine bone marrow stromal cell line (D2XRIIGFP24) expressing EGFP under control of p53 response elements was established. D2XRIIGFP24 cells displayed low constitutive background fluorescence which was significantly enhanced in response to exposure to agents that induced of p53 protein levels. Increases in EGFP fluorescence in response to oxidative and nitrosative stress as well as UVC irradiation were dose-dependent, detectable within 3 hours of expo sure and correlated closely with the amount of p53 protein accumulated within the cell. The results demonstrate the potential for rapid and early detection of p53 transactivation using the EGFP reporter approach and indicate this approach is adaptable to a variety of fluorescent assay techniques and a useful cell model for molecular toxicology research.

  10. Surgical resection and radiofrequency ablation initiate cancer in cytokeratin-19+- liver cells deficient for p53 and Rb.

    PubMed

    Matondo, Ramadhan B; Toussaint, Mathilda Jm; Govaert, Klaas M; van Vuuren, Luciel D; Nantasanti, Sathidpak; Nijkamp, Maarten W; Pandit, Shusil K; Tooten, Peter Cj; Koster, Mirjam H; Holleman, Kaylee; Schot, Arend; Gu, Guoqiang; Spee, Bart; Roskams, Tania; Rinkes, Inne Borel; Schotanus, Baukje; Kranenburg, Onno; de Bruin, Alain

    2016-08-23

    The long term prognosis of liver cancer patients remains unsatisfactory because of cancer recurrence after surgical interventions, particularly in patients with viral infections. Since hepatitis B and C viral proteins lead to inactivation of the tumor suppressors p53 and Retinoblastoma (Rb), we hypothesize that surgery in the context of p53/Rb inactivation initiate de novo tumorigenesis.We, therefore, generated transgenic mice with hepatocyte and cholangiocyte/liver progenitor cell (LPC)-specific deletion of p53 and Rb, by interbreeding conditional p53/Rb knockout mice with either Albumin-cre or Cytokeratin-19-cre transgenic mice.We show that liver cancer develops at the necrotic injury site after surgical resection or radiofrequency ablation in p53/Rb deficient livers. Cancer initiation occurs as a result of specific migration, expansion and transformation of cytokeratin-19+-liver (CK-19+) cells. At the injury site migrating CK-19+ cells formed small bile ducts and adjacent cells strongly expressed the transforming growth factor β (TGFβ). Isolated cytokeratin-19+ cells deficient for p53/Rb were resistant against hypoxia and TGFβ-mediated growth inhibition. CK-19+ specific deletion of p53/Rb verified that carcinomas at the injury site originates from cholangiocytes or liver progenitor cells.These findings suggest that human liver patients with hepatitis B and C viral infection or with mutations for p53 and Rb are at high risk to develop tumors at the surgical intervention site.

  11. Surgical resection and radiofrequency ablation initiate cancer in cytokeratin-19+- liver cells deficient for p53 and Rb

    PubMed Central

    Govaert, Klaas M; van Vuuren, Luciel D; Nantasanti, Sathidpak; Nijkamp, Maarten W; Pandit, Shusil K; Tooten, Peter CJ; Koster, Mirjam H; Holleman, Kaylee; Schot, Arend; Gu, Guoqiang; Spee, Bart; Roskams, Tania; Rinkes, Inne Borel; Schotanus, Baukje; Kranenburg, Onno; de Bruin, Alain

    2016-01-01

    The long term prognosis of liver cancer patients remains unsatisfactory because of cancer recurrence after surgical interventions, particularly in patients with viral infections. Since hepatitis B and C viral proteins lead to inactivation of the tumor suppressors p53 and Retinoblastoma (Rb), we hypothesize that surgery in the context of p53/Rb inactivation initiate de novo tumorigenesis. We, therefore, generated transgenic mice with hepatocyte and cholangiocyte/liver progenitor cell (LPC)-specific deletion of p53 and Rb, by interbreeding conditional p53/Rb knockout mice with either Albumin-cre or Cytokeratin-19-cre transgenic mice. We show that liver cancer develops at the necrotic injury site after surgical resection or radiofrequency ablation in p53/Rb deficient livers. Cancer initiation occurs as a result of specific migration, expansion and transformation of cytokeratin-19+-liver (CK-19+) cells. At the injury site migrating CK-19+ cells formed small bile ducts and adjacent cells strongly expressed the transforming growth factor β (TGFβ). Isolated cytokeratin-19+ cells deficient for p53/Rb were resistant against hypoxia and TGFβ-mediated growth inhibition. CK-19+ specific deletion of p53/Rb verified that carcinomas at the injury site originates from cholangiocytes or liver progenitor cells. These findings suggest that human liver patients with hepatitis B and C viral infection or with mutations for p53 and Rb are at high risk to develop tumors at the surgical intervention site. PMID:27323406

  12. Eurycomanone induce apoptosis in HepG2 cells via up-regulation of p53

    PubMed Central

    Zakaria, Yusmazura; Rahmat, Asmah; Pihie, Azimahtol Hawariah Lope; Abdullah, Noor Rain; Houghton, Peter J

    2009-01-01

    Background Eurycomanone is a cytotoxic compound found in Eurycoma longifolia Jack. Previous studies had noted the cytotoxic effect against various cancer cell lines. The aim of this study is to investigate the cytotoxicity against human hepato carcinoma cell in vitro and the mode of action. The cytotoxicity of eurycomanone was evaluated using MTT assay and the mode of cell death was detected by Hoechst 33258 nuclear staining and flow cytometry with Annexin-V/propidium iodide double staining. The protein expression Bax, Bcl-2, p53 and cytochrome C were studied by flow cytometry using a spesific antibody conjugated fluorescent dye to confirm the up-regulation of p53 and Bax in cancer cells. Results The findings suggested that eurycomanone was cytotoxic on cancerous liver cell, HepG2 and less toxic on normal cells Chang's liver and WLR-68. Furthermore, various methods proved that apoptosis was the mode of death in eurycomanone-treated HepG2 cells. The characteristics of apoptosis including chromatin condensation, DNA fragmentation and apoptotic bodies were found following eurycomanone treatment. This study also found that apoptotic process triggered by eurycomanone involved the up-regulation of p53 tumor suppressor protein. The up-regulation of p53 was followed by the increasing of pro-apoptotic Bax and decreasing of anti-apoptotic Bcl-2. The increased of cytochrome C levels in cytosol also results in induction of apoptosis. Conclusion The data suggest that eurycomanone was cytotoxic on HepG2 cells by inducing apoptosis through the up-regulation of p53 and Bax, and down-regulation of Bcl-2. PMID:19508737

  13. Co-expression of ING4 and P53 enhances hypopharyngeal cancer chemosensitivity to cisplatin in vivo

    PubMed Central

    Ren, Xin; Liu, Hao; Zhang, Mingjie; Wang, Mengjun; Ma, Shiyin

    2016-01-01

    Hypopharyngeal cancer is a distinct type of malignant head and neck tumor, which exhibits low sensitivity to anti-cancer drugs. The importance of developing methods for reducing chemotherapy resistance, and improving and enhancing prognosis has previously been emphasized and is considered a challenge for effective clinical treatment of hypopharyngeal cancer. The current study investigated the effects of co-expression of inhibitor of growth protein 4 (ING4) and P53, a tumor suppressor gene, on chemosensitivity to cisplatin in human hypopharyngeal cancer xenografts in vivo, and the potential molecular mechanisms involved. A tumor model was established by injecting athymic nude mice with FADU human hypopharyngeal cancer cells. Five days after intratumoral and peritumoral injections of an empty adenoviral vector (Ad), Ad-ING4-P53, cisplatin, or a combination of Ad-ING4-P53 and cisplatin (Ad-ING4-P53 + cisplatin) every other day for 5 days, the mice were euthanized and their tumors, livers, and kidneys were removed. The tumor weights were used to calculate the inhibition rate, and the expression levels of ING4 and P53 were detected by reverse transcription-polymerase chain reaction. Additionally, apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunohistochemistry determined the levels ING4, P53, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) protein expression. The results demonstrated increased expression of ING4 and P53 in the Ad-ING4-P53 groups compared with PBS and Ad groups, indicating successful introduction of the genes into the tumor cells. Notably, the Ad-ING4-P53 + cisplatin group exhibited a higher inhibition rate compared with the four other groups. The results of immunohistochemistry analysis demonstrated that Bax expression was increased and Bcl-2 was decreased in the Ad-ING4-P53 + cisplatin group. This suggested that the enhanced cisplatin chemosensitivity with Ad-ING4-P53 gene therapy

  14. Feedbacks, Bifurcations, and Cell Fate Decision-Making in the p53 System

    PubMed Central

    Bogdał, Marta N.; Lipniacki, Tomasz

    2016-01-01

    The p53 transcription factor is a regulator of key cellular processes including DNA repair, cell cycle arrest, and apoptosis. In this theoretical study, we investigate how the complex circuitry of the p53 network allows for stochastic yet unambiguous cell fate decision-making. The proposed Markov chain model consists of the regulatory core and two subordinated bistable modules responsible for cell cycle arrest and apoptosis. The regulatory core is controlled by two negative feedback loops (regulated by Mdm2 and Wip1) responsible for oscillations, and two antagonistic positive feedback loops (regulated by phosphatases Wip1 and PTEN) responsible for bistability. By means of bifurcation analysis of the deterministic approximation we capture the recurrent solutions (i.e., steady states and limit cycles) that delineate temporal responses of the stochastic system. Direct switching from the limit-cycle oscillations to the “apoptotic” steady state is enabled by the existence of a subcritical Neimark—Sacker bifurcation in which the limit cycle loses its stability by merging with an unstable invariant torus. Our analysis provides an explanation why cancer cell lines known to have vastly diverse expression levels of Wip1 and PTEN exhibit a broad spectrum of responses to DNA damage: from a fast transition to a high level of p53 killer (a p53 phosphoform which promotes commitment to apoptosis) in cells characterized by high PTEN and low Wip1 levels to long-lasting p53 level oscillations in cells having PTEN promoter methylated (as in, e.g., MCF-7 cell line). PMID:26928575

  15. Mutant p53-Notch1 Signaling Axis Is Involved in Curcumin-Induced Apoptosis of Breast Cancer Cells.

    PubMed

    Bae, Yun-Hee; Ryu, Jong Hyo; Park, Hyun-Joo; Kim, Kwang Rok; Wee, Hee-Jun; Lee, Ok-Hee; Jang, Hye-Ock; Bae, Moon-Kyoung; Kim, Kyu-Won; Bae, Soo-Kyung

    2013-08-01

    Notch1 has been reported to be highly expressed in triple-negative and other subtypes of breast cancer. Mutant p53 (R280K) is overexpressed in MDA-MB-231 triple-negative human breast cancer cells. The present study aimed to determine whether the mutant p53 can be a potent transcriptional activator of the Notch1 in MDA-MB-231 cells, and explore the role of this mutant p53-Notch1 axis in curcumin-induced apoptosis. We found that curcumin treatment resulted in an induction of apoptosis in MDA-MB-231 cells, together with downregulation of Notch1 and its downstream target, Hes1. This reduction in Notch1 expression was determined to be due to the decreased activity of endogenous mutant p53. We confirmed the suppressive effect of curcumin on Notch1 transcription by performing a Notch1 promoter-driven reporter assay and identified a putative p53-binding site in the Notch1 promoter by EMSA and chromatin immunoprecipitation analysis. Overexpression of mutant p53 increased Notch1 promoter activity, whereas knockdown of mutant p53 by small interfering RNA suppressed Notch1 expression, leading to the induction of cellular apoptosis. Moreover, curcumin-induced apoptosis was further enhanced by the knockdown of Notch1 or mutant p53, but it was decreased by the overexpression of active Notch1. Taken together, our results demonstrate, for the first time, that Notch1 is a transcriptional target of mutant p53 in breast cancer cells and suggest that the targeting of mutant p53 and/or Notch1 may be combined with a chemotherapeutic strategy to improve the response of breast cancer cells to curcumin.

  16. Influence of p53 and bcl-2 on chemosensitivity in benign and malignant prostatic cell lines.

    PubMed

    Serafin, Antonio M; Bohm, Lothar

    2005-01-01

    The administration of cancer chemotherapeutic agents results in an increase in the apoptotic cells in the tumor: therefore, it has been assumed that anticancer drugs exhibit their cytotoxic effects via apoptotic signaling pathways. Characteristics that confer sensitivity to drug-induced apoptosis are, a functional p53 protein and expression of the apoptosis-promoting protein, bax. The role of p53 and bax/bcl-2 in drug-induced apoptosis was assessed in six prostate cell lines, 1532T, 1535T, 1542T, 1542N, BPH-1 and LNCaP using TD(50) concentrations of etoposide, vinblastine and estramustine. Cell death was monitored morphologically by fluorescent microscopy, and by flow cytometry (Annexin-V assay). Apoptotic morphology was rather low and ranged from 0.1% to 12.1%, 3.0% to 6.0% and 0.1% to 8.5% for etoposide, estramustine and vinblastine, respectively. Annexin-V binding and flow cytometry indicated apoptotic propensities of 0% to 4%, 0% to 3% and 0% to 5%, respectively. The percentage of cells responding to drug-induced apoptosis was, on average, higher in the tumor cell lines than in the normal cell lines, but showed no correlation with p53 status. The percentage of cells showing necrosis, assessed by Annexin binding and Propidium Iodide permeability in aqueous medium, tended to be much higher, and was found to be at the level of 5% to 30%. Immunoblotting demonstrated that bax and bcl-2 proteins were expressed at a basal level in all cell lines, but did not increase after exposure to TD(50) doses of the three drugs. The ratio of bax and bcl-2, measured by laser scanning densitometry, was not altered by the drug-induced DNA damage. The results suggest that apoptosis is not a major mechanism of drug-induced cell death in prostate cell lines and appears to be independent of p53 status and bax/bcl-2 expression.

  17. Loss of VHL promotes progerin expression, leading to impaired p14/ARF function and suppression of p53 activity

    PubMed Central

    Jung, Youn-Sang; Lee, Su-Jin; Lee, Sun-Hye; Chung, Ji-Yun; Jung, Youn Jin; Hwang, Sang Hyun; Ha, Nam-Chul; Park, Bum-Joon

    2013-01-01

    Renal cell carcinomas (RCCs) are frequently occurring genitourinary malignancies in the aged population. A morphological characteristic of RCCs is an irregular nuclear shape, which is used to index cancer grades. Other features of RCCs include the genetic inactivation of the von Hippel-Lindau gene, VHL, and p53 genetic-independent inactivation. An aberrant nuclear shape or p53 suppression has not yet been demonstrated. We examined the effect of progerin (an altered splicing product of the LMNA gene linked to Hutchinson Gilford progeria syndrome; HGPS) on the nuclear deformation of RCCs in comparison to that of HGPS cells. In this study, we showed that progerin was suppressed by pVHL and was responsible for nuclear irregularities as well as p53 inactivation. Thus, progerin suppression can ameliorate nuclear abnormalities and reactivate p53 in response to genotoxic addition. Furthermore, we found that progerin was a target of pVHL E3 ligase and suppressed p53 activity by p14/ARF inhibition. Our findings indicate that the elevated expression of progerin in RCCs results from the loss of pVHL and leads to p53 inactivation through p14/ARF suppression. Interestingly, we showed that progerin was expressed in human leukemia and primary cell lines, raising the possibility that the expression of this LMNA variant may be a common event in age-related cancer progression. PMID:24067370

  18. HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53.

    PubMed

    Hallgren, O; Gustafsson, L; Irjala, H; Selivanova, G; Orrenius, S; Svanborg, C

    2006-02-01

    HAMLET (Human alpha-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.

  19. TAp73 promotes cell survival upon genotoxic stress by inhibiting p53 activity

    PubMed Central

    Chen, Dongshi; Ming, Lihua; Zou, Fangdong; Peng, Ye; Houten, Bennett Van; Yu, Jian; Zhang, Lin

    2014-01-01

    p53 plays a key role in regulating DNA damage response by suppressing cell cycle progression or inducing apoptosis depending on extent of DNA damage. However, it is not clear why mild genotoxic stress favors growth arrest, whereas excessive lesions signal cells to die. Here we showed that TAp73, a p53 homologue thought to have a similar function as p53, restrains the transcriptional activity of p53 and prevents excessive activation of its downstream targets upon low levels of DNA damage, which results in cell cycle arrest. Extensive DNA damage triggers TAp73 depletion through ubiquitin/proteasome-mediated degradation of E2F1, leading to enhanced transcriptional activation by p53 and subsequent induction of apoptosis. These findings provide novel insights into the regulation of p53 function and suggest that TAp73 keeps p53 activity in check in regulating cell fate decisions upon genotoxic stress. PMID:25237903

  20. Ki67, p27 and p53 Expression in Squamous Epithelial Lesions of Larynx.

    PubMed

    Mondal, Debashri; Saha, Kaushik; Datta, Chhanda; Chatterjee, Uttara; Sengupta, Arunabho

    2013-04-01

    Precise assessment of the biological behavior and progression of squamous epithelial lesions of the larynx with a view to predict the prognosis and therapeutic challenges remains an elusive goal. The knowledge and data regarding the expression of proliferative markers indicating the biological activity in different histological grades of squamous epithelial lesions are lacking till date. To evaluate the relationship between Ki67, p27 and p53 expression as well as topographic distribution of Ki67 with the histological subtypes or grades of laryngeal squamous intraepithelial and invasive lesions. Sixty-two consecutive cases with histologically documented intraepithelial and invasive squamous lesion were studied for Ki67, p27 and p53 expression. Mann-Whitney U, Kruskal-Wallis and Spearman's correlation tests were used for statistical analysis. The mean Ki67 labeling index in hyperplasia, dysplasia and carcinoma were 12.15, 22.03 and 35.53 % respectively and this difference was statistically significant (P < 0.05). There was strong positive correlation between Ki67 labeling index and increasing grades of squamous lesions. p27 expression was progressively decreased and p53 expression was progressively increased as the lesions progressed from hyperplasia to dysplasia and dysplasia to carcinoma. The topographic distribution of Ki67 positive cells increased with progressive grades of dysplasia. The Ki67 labeling index correlates well with the histological grade of both intraepithelial and invasive lesions of the larynx. And the topographic distribution of Ki67 expression depends on the grade of the dysplasia. Hence, Ki67 expression has a definite role in predicting the biological behavior of the lesions.

  1. Role of wild-type p53 in apoptotic and non-apoptotic cell death induced by X-irradiation and heat treatment in p53-mutated mouse M10 cells.

    PubMed

    Ito, Atsushi; Nakano, Hisako; Shinohara, Kunio

    2010-01-01

    The sensitizing effects of wild-type p53 on X-ray-induced cell death and on heat-induced apoptosis in M10, a radiosensitive and Trp53 (mouse p53 gene)-mutated lymphoma cell line which dies through necrosis by X-irradiation, were investigated using three M10 derived transfectants with wild-type TP53 (human p53 gene). Cell death was determined by colony formation and/or dye exclusion test, and apoptosis was detected as the changes in nuclear morphology by Giemsa staining. Expression of wild-type p53 protein increased radiosensitivity of cell death as determined by both clonogenic and dye exclusion assays. This increase in radiosensitivity was attributable largely to apoptosis induction in addition to a small enhancement of necrosis. Interestingly neither pathway to cell death was accompanied by caspase-3 activation. On the other hand, heat-induced caspase-3 dependent apoptotic cell death without transfection was further increased by the transfection of wild-type p53. In conclusion, the introduction of wild-type p53 enhanced apoptotic cell death by X-rays or heat via different mechanisms that do or do not activate caspase-3, respectively. In addition, p53 also enhanced the X-ray-induced necrosis in M10 cells.

  2. Role of p53 in the cellular response following oleic acid accumulation in Chang liver cells.

    PubMed

    Park, Eun-Jung; Lee, Ah Young; Chang, Seung-Hee; Yu, Kyeong-Nam; Kim, Jae-Ho; Cho, Myung-Haing

    2014-01-03

    Abnormal accumulation of fatty acids triggers the harmful cellular response called lipotoxicity. In this study, we investigated the cellular response following accumulation of oleic acid (OA), a monounsaturated fatty acid, in human Chang liver cells. OA droplets were distributed freely in the cytoplasm and/or degraded within lysosomes. OA exposure increased ATP production and concomitantly dilated mitochondria. At 24h after OA exposure, cell viability decreased slightly and was coupled with a reduction in mitochondrial Ca(2+) concentration, the alteration in cell viability was also associated with the generation of reactive oxygen species and changes in the cell cycle. Moreover, OA treatment increased the expression of autophagy- and apoptotic cell death-related proteins in a dose-dependent manner. Furthermore, we investigated the role of p53, a tumor suppressor protein, in the cellular response elicited by OA accumulation. OA-induced changes in cell viability and ATP production were rescued to control levels when cells were pretreated with pifithrin-alpha (PTA), a p53 inhibitor. By contrast, the expressions of LC3-II and perilipin, proteins required for lipophagy, were down-regulated by PTA pretreatment. Taken together, our results suggest that p53 plays a key role in the cellular response elicited by OA accumulation in Chang liver cells.

  3. 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

    PubMed Central

    Bauer, Matthias R.; Joerger, Andreas C.; Fersht, Alan R.

    2016-01-01

    The tumor suppressor p53 has the most frequently mutated gene in human cancers. Many of p53’s oncogenic mutants are just destabilized and rapidly aggregate, and are targets for stabilization by drugs. We found certain 2-sulfonylpyrimidines, including one named PK11007, to be mild thiol alkylators with anticancer activity in several cell lines, especially those with mutationally compromised p53. PK11007 acted by two routes: p53 dependent and p53 independent. PK11007 stabilized p53 in vitro via selective alkylation of two surface-exposed cysteines without compromising its DNA binding activity. Unstable p53 was reactivated by PK11007 in some cancer cell lines, leading to up-regulation of p53 target genes such as p21 and PUMA. More generally, there was cell death that was independent of p53 but dependent on glutathione depletion and associated with highly elevated levels of reactive oxygen species and induction of endoplasmic reticulum (ER) stress, as also found for the anticancer agent PRIMA-1MET(APR-246). PK11007 may be a lead for anticancer drugs that target cells with nonfunctional p53 or impaired reactive oxygen species (ROS) detoxification in a wide variety of mutant p53 cells. PMID:27551077

  4. 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells.

    PubMed

    Bauer, Matthias R; Joerger, Andreas C; Fersht, Alan R

    2016-09-06

    The tumor suppressor p53 has the most frequently mutated gene in human cancers. Many of p53's oncogenic mutants are just destabilized and rapidly aggregate, and are targets for stabilization by drugs. We found certain 2-sulfonylpyrimidines, including one named PK11007, to be mild thiol alkylators with anticancer activity in several cell lines, especially those with mutationally compromised p53. PK11007 acted by two routes: p53 dependent and p53 independent. PK11007 stabilized p53 in vitro via selective alkylation of two surface-exposed cysteines without compromising its DNA binding activity. Unstable p53 was reactivated by PK11007 in some cancer cell lines, leading to up-regulation of p53 target genes such as p21 and PUMA. More generally, there was cell death that was independent of p53 but dependent on glutathione depletion and associated with highly elevated levels of reactive oxygen species and induction of endoplasmic reticulum (ER) stress, as also found for the anticancer agent PRIMA-1(MET)(APR-246). PK11007 may be a lead for anticancer drugs that target cells with nonfunctional p53 or impaired reactive oxygen species (ROS) detoxification in a wide variety of mutant p53 cells.

  5. Mesothelin regulates growth and apoptosis in pancreatic cancer cells through p53-dependent and -independent signal pathway.

    PubMed

    Zheng, Chunning; Jia, Wei; Tang, Yong; Zhao, HuiLiang; Jiang, Yingsheng; Sun, Shaochuan

    2012-10-03

    Mesothelin, a secreted protein, is overexpressed in some cancers, including pancreatic cancer. Rescent studies have shown that overexpression of mesothelin significantly increased tumor cell proliferation, and downregulation of mesothelin inhibited cell proliferation in pancreatic cancer cells, but its exact function and mechanism remains unclear. The aim of the present study was to evaluate the effects of mesothelin on proliferation and apoptosis in pancreatic cancer cells with different p53 status and to explore its signal pathway. Mesothelin levels were detected by western blot and RT-PCR assay in human pancreatic cancer AsPC-1, HPAC and Capan-2, Capan-1 and MIA PaCa-2 cell lines. Mesothelin was slienced by shRNA in AsPC-1, Capan-2 and Capan-1 cells with rich mesothelin level, and mesothelin was overexpressed in the HPAC and Capan-2 cells with less mesothelin level. We observed that in the AsPC-1 and Capan-1cells with mt-p53, and Capan-2 cells with wt-p53, shRNA mediated sliencing of the mesothelin significantly increased PUMA and Bax expression and caspase-3 activity, and decreased bcl-2 expression, followed by the reduced proliferation and colony forming capability and increased cell apoptosis. When PUMA was slienced by siRNA in the stable mesothelin shRNA transfected cells, proliferative capability was significantly increased, and apoptosis was decreased. However, in the Capan-2 cells with wt-p53, suppression of the mesothelin significantly increased wt-p53 levels. When p53 was blocked by siRNA in the stable mesothelin shRNA transfected Capan-2 cells, PUMA was inhibited, followed by increased proliferative capability and decreased cell apoptosis. In the HPAC and Capan-2 cells with wt-p53 and in the MIA PaCa-2 cells with mt-p53, overexpression of the mesothelin significantly decreased bax levels and increased bcl-2 levels, followed by increased proliferative and colony forming capability. Furthermore, mesothelin-shRNA-transfected cells exhibited a reduced rate

  6. Molecular Signature of HPV-Induced Carcinogenesis: pRb, p53 and Gene Expression Profiling

    PubMed Central

    Buitrago-Pérez, Águeda; Garaulet, Guillermo; Vázquez-Carballo, Ana; Paramio, Jesús M; García-Escudero, Ramón

    2009-01-01

    The infection by mucosal human papillomavirus (HPV) is causally associated with tumor development in cervix and oropharynx. The mechanisms responsible for this oncogenic potential are mainly due to the product activities of two early viral oncogenes: E6 and E7. Although a large number of cellular targets have been described for both oncoproteins, the interaction with tumor suppressors p53 and retinoblastoma protein (pRb) emerged as the key functional activities. E6 degrades tumor suppressor p53, thus inhibiting p53-dependent functions, whereas E7 binds and degrades pRb, allowing the transcription of E2F-dependent genes. Since these two tumor suppressors exert their actions through transcriptional modulation, functional genomics has provided a large body of data that reflects the altered gene expression of HPVinfected cells or tissues. Here we will review the similarities and differences of these findings, and we also compare them with those obtained with transgenic mouse models bearing the deletion of some of the viral oncogene targets. The comparative analysis supports molecular evidences about the role of oncogenes E6 and E7 in the interference with the mentioned cellular functions, and also suggests that the mentioned transgenic mice can be used as models for HPV-associated diseases such as human cervical, oropharynx, and skin carcinomas. PMID:19721808

  7. Molecular Signature of HPV-Induced Carcinogenesis: pRb, p53 and Gene Expression Profiling.

    PubMed

    Buitrago-Pérez, Agueda; Garaulet, Guillermo; Vázquez-Carballo, Ana; Paramio, Jesús M; García-Escudero, Ramón

    2009-03-01

    The infection by mucosal human papillomavirus (HPV) is causally associated with tumor development in cervix and oropharynx. The mechanisms responsible for this oncogenic potential are mainly due to the product activities of two early viral oncogenes: E6 and E7. Although a large number of cellular targets have been described for both oncoproteins, the interaction with tumor suppressors p53 and retinoblastoma protein (pRb) emerged as the key functional activities. E6 degrades tumor suppressor p53, thus inhibiting p53-dependent functions, whereas E7 binds and degrades pRb, allowing the transcription of E2F-dependent genes. Since these two tumor suppressors exert their actions through transcriptional modulation, functional genomics has provided a large body of data that reflects the altered gene expression of HPVinfected cells or tissues. Here we will review the similarities and differences of these findings, and we also compare them with those obtained with transgenic mouse models bearing the deletion of some of the viral oncogene targets. The comparative analysis supports molecular evidences about the role of oncogenes E6 and E7 in the interference with the mentioned cellular functions, and also suggests that the mentioned transgenic mice can be used as models for HPV-associated diseases such as human cervical, oropharynx, and skin carcinomas.

  8. Evaluation of p53 protein expression as a marker for long-term prognosis in colorectal carcinoma.

    PubMed Central

    Mulder, J. W.; Baas, I. O.; Polak, M. M.; Goodman, S. N.; Offerhaus, G. J.

    1995-01-01

    Mutation of the p53 gene is reported to be of prognostic importance in colorectal carcinomas. Immunohistochemical staining of the accumulated p53 gene product may be a simple alternative for p53 mutation analysis. Previous studies addressing the prognostic importance of p53 expression, however, yielded contradictory results. Therefore, we evaluated the importance of p53 expression as a marker for long-term prognosis in a well-characterised study population of 109 colorectal carcinomas. After antigen retrieval with target unmasking fluid (TUF), immunostaining of p53 was performed with both monoclonal antibody DO7 and polyclonal antibody CM1. Objective quantification of the p53 signal was assessed by a computerised image analyser. p53 expression was higher in non-mucinous tumours than in mucinous tumours (p53 labelling index = 30% and 17% respectively, P = 0.05), and in metastatic tumours compared with non-metastatic tumours (p53 labelling index = 37% and 22% respectively, P = 0.05). Other histopathological features were not related to p53 expression. In multivariate analysis, Dukes' stage (P = 0.02) and histological grade (P = 0.05) stood out as independent markers for prognosis. p53 expression was not an independent marker for prognosis. At present, p53 expression is not a useful marker for long-term prognosis. Further insight into the relationship between p53 mutations and p53 expression is needed to elucidate more precisely the clinical relevance of p53 alterations. PMID:7779721

  9. Activated AKT regulates NF-kappaB activation, p53 inhibition and cell survival in HTLV-1-transformed cells.

    PubMed

    Jeong, Soo-Jin; Pise-Masison, Cynthia A; Radonovich, Michael F; Park, Hyeon Ung; Brady, John N

    2005-10-06

    AKT activation enhances resistance to apoptosis and induces cell survival signaling through multiple downstream pathways. We now present evidence that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to NF-kappaB activation, p53 inhibition and cell survival. Overexpression of AKT wild type (WT), but not a kinase dead (KD) mutant, resulted in increased Tax-mediated NF-kappaB activation. Blocking AKT with the PI3K/AKT inhibitor LY294002 or AKT SiRNA prevented NF-kappaB activation and inhibition of p53. Treatment of C81 cells with LY294002 resulted in an increase in the p53-responsive gene MDM2, suggesting a role for AKT in the Tax-mediated regulation of p53 transcriptional activity. Further, we show that LY294002 treatment of C81 cells abrogates in vitro IKKbeta phosphorylation of p65 and causes a reduction of p65 Ser-536 phosphorylation in vivo, steps critical to p53 inhibition. Interestingly, blockage of AKT function did not affect IKKbeta phosphorylation of IkappaBalpha in vitro suggesting selective activity of AKT on the IKKbeta complex. Finally, AKT prosurvival function in HTLV-1-transformed cells is linked to expression of Bcl-xL. We suggest that AKT plays a role in the activation of prosurvival pathways in HTLV-1-transformed cells, possibly through NF-kappaB activation and inhibition of p53 transcription activity.

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

    PubMed Central

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

    2012-01-01

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

  11. High prevalence of expression of p53 oncoprotein in oral carcinomas from India associated with betel and tobacco chewing.

    PubMed

    Kuttan, N A; Rosin, M P; Ambika, K; Priddy, R W; Bhakthan, N M; Zhang, L

    1995-05-01

    A recent study reported a low prevalence of p53 expression (11%) in oral squamous cell carcinomas (SCCs) from South Asia, in contrast to a high prevalence (averaging 52%) in other studies. It was proposed that the different aetiologies for oral SCCs in the South Asia population, i.e. betel and tobacco chewing in combination with smoking and alcohol consumption as compared to smoking and alcohol consumption alone in other populations, may account for the low prevalence of p53 expression. To confirm this hypothesis, we examined p53 expression immunohistochemically in 23 cases of oral SCC from patients in Southern India. Thirteen of the 23 SCCs (56.5%) demonstrated nuclear p53 staining. The expression of p53 was strongly correlated with the number of tobacco-containing quids chewed per day (r = 0.8). These data support the hypothesis that carcinogens derived from tobacco and betel chewing may induce p53 mutations, which in turn are involved in the development of oral cancer.

  12. Expression of p21 is dependent on or independent of p53 in carcinoma ex pleomorphic adenoma (undifferentiated and adenocarcinoma types).

    PubMed

    Tarakji, Bassel; Baroudi, Kusai; Hanouneh, Salah; Nassani, Mohammad Z; Alotaibi, Abdullah M; Kharma, M Yaser; Azzeghaiby, Saleh N

    2012-12-01

    Our study is aimed to characterize alteration in the immunohistochemical expression of p21 and p53 in normal tissue of the salivary gland surrounding carcinoma arising in pleomorphic adenoma, and the tumor cells of carcinoma arising in pleomorphic adenoma as well as to identify whether the induction of expression p21 is dependent on or independent of p53 in carcinoma arising in pleomorphic adenoma. A selected series of 27 cases of carcinoma ex pleomorphic adenoma (undifferentiated and adenocarcinoma types) was examined. The results showed that p21 and p53 expression was negative in the most components of normal tissue of the salivary gland surrounding carcinoma arising in pleomorphic adenoma. p21 was strongly expressed in carcinoma cells in 9 (33.3%) cases out of 27. p53 was strongly expressed in carcinoma cells in 10 (37%) cases out of 27. Also a co-expression of p21 and p53 showed negative nuclear staining in 9 cases, while 8 cases expressed positive staining. p21 expressed negative nuclear staining in 4 cases but p53 expressed positive staining in the same cases. p21 expressed positive nuclear staining in 6 cases but p53 expressed negative nuclear staining in the same cases. Our data suggest that inactivation of p53 and p21 may play an important role in the evolution of carcinoma ex pleomorphic adenoma. Also p21 behaves as dependent on or independent of p53 in carcinoma arising in pleomorphic adenoma.

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

    PubMed Central

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

    2016-01-01

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

  14. Induction of p53-independent growth inhibition in lung carcinoma cell A549 by gypenosides

    PubMed Central

    Liu, Jung-Sen; Chiang, Tzu-Hsuan; Wang, Jinn-Shyan; Lin, Li-Ju; Chao, Wei-Chih; Inbaraj, Baskaran Stephen; Lu, Jyh-Feng; Chen, Bing-Huei

    2015-01-01

    The objectives of this study are to investigate antiproliferative effect and mechanisms of bioactive compounds from Gynostemma pentaphyllum (G. pentaphyllum) on lung carcinoma cell A549. Saponins, carotenoids and chlorophylls were extracted and fractionated by column chromatography, and were subjected to high-performance liquid chromatography-mass spectrometry analyses. The saponin fraction, which consisted mainly of gypenoside (Gyp) XXII and XXIII, rather than the carotenoid and chlorophyll ones, was effective in inhibiting A549 cell growth in a concentration- and a time-dependent manner as evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The estimated half maximal inhibitory concentration (IC50) of Gyp on A549 cells was 30.6 μg/ml. Gyp was further demonstrated to induce an apparent arrest of the A549 cell cycle at both the S phase and the G2/M phase, accompanied by a concentration- and a time-dependent increase in the proportions of both the early and late apoptotic cells. Furthermore, Gyp down-regulated cellular expression of cyclin A and B as well as BCL-2, while up-regulated the expression of BAX, DNA degradation factor 35 KD, poly [ADP-ribose] polymerase 1, p53, p21 and caspase-3. Nevertheless, both the treatment of a p53 inhibitor, pifithrin-α, and the small hairpin RNA-mediated p53 knockdown in the A549 cells did not alter the growth inhibition effect induced by Gyp. As a result, the cell cycle arrest and apoptosis of A549 cells induced by Gyp would most likely proceed through p53-independent pathway(s). PMID:25781909

  15. Epoxy clerodane diterpene inhibits MCF-7 human breast cancer cell growth by regulating the expression of the functional apoptotic genes Cdkn2A, Rb1, mdm2 and p53.

    PubMed

    Subash-Babu, P; Alshammari, Ghedeir M; Ignacimuthu, S; Alshatwi, Ali A

    2017-03-01

    Systematic analyses of plants that are used in traditional medicine may lead to the discovery of novel cytotoxic secondary metabolites. Diterpene possesses multiple bioactivities; here, epoxy clerodane diterpene (ECD) was isolated from Tinospora cordifolia (Willd.) stem and shown potential antiproliferative effect in MCF-7 human breast cancer cells. The antiproliferative effect of ECD on MCF-7 cells was systematically analyzed by cell and nuclear morphology, alterations in oxidative stress, and the expression of tumor suppressor and mitochondria-mediated apoptosis-related genes. We found that the IC50 value of ECD was 3.2μM at 24h and 2.4μM at 48h. We observed that the cytotoxicity of ECD was specific to MCF-7 cells, whereas ECD was nontoxic to normal Vero and V79 cells. ECD significantly triggered intracellular ROS generation even from the lower doses of 0.6 and 1.2μM; and it is relative to higher dose of 2.4μM. Further, we used 0.6μM, 1.2μM and 2.4μM as experimental doses to analyze the relative dose-dependent effects. Nuclear staining revealed that cells treated with the 2.4μM dose exhibited characteristic apoptotic morphological changes and that 46% of the cells were apoptotic and 4% were necrotic after 48h. ECD significantly increased the expression of mitochondria-dependent apoptotic pathway-related genes after 48h; we observed significantly (p≤0.05) increased expression of CYP1A, GPX, GSK3β and TNF-α and downregulated expression of NF-κB. ECD also increased the expression of tumor suppressor genes such as Cdkn2A, Rb1 and p53. In addition, we observed that ECD treatment significantly (p≤0.001) upregulated the expression of apoptotic genes such as Bax, cas-3, cas-8, cas-9 and p21 and downregulated the expression of BCL-2, mdm2 and PCNA. In conclusion, ECD regulates the expression of Cdkn2A, p53 and mdm2 and induces apoptosis via the mitochondrial pathway in MCF-7 human breast cancer cells.

  16. Wild-type p53 is not a negative regulator of simian virus 40 DNA replication in infected monkey cells.

    PubMed Central

    von der Weth, A; Deppert, W

    1993-01-01

    To analyze the proposed growth-inhibitory function of wild-type p53, we compared simian virus 40 (SV40) DNA replication in primary rhesus monkey kidney (PRK) cells, which express wild-type p53, and in the established rhesus monkey kidney cell line LLC-MK2, which expresses a mutated p53 that does not complex with large T antigen. SV40 DNA replication proceeded identically in both cell types during the course of infection. Endogenously expressed wild-type p53 thus does not negatively modulate SV40 DNA replication in vivo. We suggest that inhibition of SV40 DNA replication by wild-type p53 in in vitro replication assays is due to grossly elevated ratios of p53 to large T antigen, thus depleting the replication-competent free large T antigen in the assay mixtures by complex formation. In contrast, the ratio of p53 to large T antigen in in vivo replication is low, leaving the majority of large T antigen in a free, replication-competent state. Images PMID:8380470

  17. Dysfunctional p53 deletion mutants in cell lines derived from Hodgkin's lymphoma.

    PubMed

    Feuerborn, Alexander; Möritz, Constanze; Von Bonin, Frederike; Dobbelstein, Matthias; Trümper, Lorenz; Stürzenhofecker, Benjamin; Kube, Dieter

    2006-09-01

    Classical Hodgkin's lymphoma (cHL) is a distinct malignancy of the immune system. Despite the progress made in the understanding of the pathology of cHL, the transforming events remain to be elucidated. It has been proposed that mutations in the TP53 gene in biopsy material as well as cell lines derived from cHL are rare and therefore not notably involved in the pathogenesis of the malignant H&RS cells. Re-evaluating the expression in cHL-derived cell lines, we found that in 3/6 of these cell lines, TP53 transcripts are characterized by deletions within exon 4 (L428 cells) and nearly a complete loss of exons 10 - 11 (L1236) or exons 8 - 11 (HDLM-2), respectively. These changes were found in otherwise rarely mutated regions of TP53. Cell lines L1236 and HDLM-2 harbour fusions with alu-repeats in their TP53 mRNA 3'-ends, resulting in the carboxyterminal truncation and loss of the transcriptional activity of p53. Transcriptional inactivity was also found for p53 in L428 cells. This study characterizes mutations in TP53 transcripts within cHL cell lines with associated functional defects in the resulting p53 proteins and therefore reintroduces the concept that mutations of TP53 might be involved in the pathogenesis of Hodgkin's lymphoma.

  18. Regulation of Mammary Progenitor Cells by p53 and Parity

    DTIC Science & Technology

    2010-01-01

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

  19. Increases in apoptosis, caspase activity and expression of p53 and bax, and the transition between two types of mitochondrion-rich cells, in the gills of the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater

    PubMed Central

    Ching, Biyun; Chen, Xiu L.; Yong, Jing H. A.; Wilson, Jonathan M.; Hiong, Kum C.; Sim, Eugene W. L.; Wong, Wai P.; Lam, Siew H.; Chew, Shit F.; Ip, Yuen K.

    2013-01-01

    This study aimed to test the hypothesis that branchial osmoregulatory acclimation involved increased apoptosis and replacement of mitochdonrion-rich cells (MRCs) in the climbing perch, Anabas testudineus, during a progressive acclimation from freshwater to seawater. A significant increase in branchial caspase-3/-7 activity was observed on day 4 (salinity 20), and an extensive TUNEL-positive apoptosis was detected on day 5 (salinity 25), indicating salinity-induced apoptosis had occurred. This was further supported by an up-regulation of branchial mRNA expression of p53, a key regulator of cell cycle arrest and apoptosis, between day 2 (salinity 10) and day 6 (seawater), and an increase in branchial p53 protein abundance on day 6. Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities. The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater). Western blotting results revealed the presence of a freshwater Na+/K+-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation. Immunofluorescence microscopy revealed the presence of two types of MRCs distinctly different in sizes, and confirmed that the reduction in Nka α1a expression, and the prominent increases in expression of Nka α1b, Na+:K+:2Cl− cotransporter 1, and cystic fibrosis transmembrane conductance regulator Cl− channel coincided with the salinity-induced apoptotic event. Since modulation of existing MRCs alone could not have led to extensive salinity-induced apoptosis, it is probable that some, if not all, freshwater-type MRCs could have been removed through increased apoptosis and subsequently replaced by seawater-type MRCs in the gills of A. testudineus during seawater

  20. p53 deficiency induces cancer stem cell pool expansion in a mouse model of triple-negative breast tumors.

    PubMed

    Chiche, A; Moumen, M; Romagnoli, M; Petit, V; Lasla, H; Jézéquel, P; de la Grange, P; Jonkers, J; Deugnier, M-A; Glukhova, M A; Faraldo, M M

    2016-10-24

    Triple-negative breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Triple-negative tumors often display activated Wnt/β-catenin signaling and most have impaired p53 function. We studied the interplay between p53 loss and Wnt/β-catenin signaling in stem cell function and tumorigenesis, by deleting p53 from the mammary epithelium of K5ΔNβcat mice displaying a constitutive activation of Wnt/β-catenin signaling in basal cells. K5ΔNβcat transgenic mice present amplification of the basal stem cell pool and develop triple-negative mammary carcinomas. The loss of p53 in K5ΔNβcat mice led to an early expansion of mammary stem/progenitor cells and accelerated the formation of triple-negative tumors. In particular, p53-deficient tumors expressed high levels of integrins and extracellular matrix components and were enriched in cancer stem cells. They also overexpressed the tyrosine kinase receptor Met, a feature characteristic of human triple-negative breast tumors. The inhibition of Met kinase activity impaired tumorsphere formation, demonstrating the requirement of Met signaling for cancer stem cell growth in this model. Human basal-like breast cancers with predicted mutated p53 status had higher levels of MET expression than tumors with wild-type p53. These results connect p53 loss and β-catenin activation to stem cell regulation and tumorigenesis in triple-negative cancer and highlight the role of Met signaling in maintaining cancer stem cell properties, revealing new cues for targeted therapies.Oncogene advance online publication, 24 October 2016; doi:10.1038/onc.2016.396.

  1. Cisplatin induced apoptosis of ovarian cancer A2780s cells by activation of ERK/p53/PUMA signals.

    PubMed

    Song, Hao; Wei, Mei; Liu, Wenfen; Shen, Shulin; Li, Jiaqun; Wang, Liming

    2017-03-13

    Cisplatin (CDDP) is one of the most effective anticancer agents widely used in the treatment of solid tumors, including ovarian cancer. It is generally considered as a cytotoxic drug which kills cancer cells by causing DNA damage, and subsequently inducing apoptosis in cancer cells. However, the underlying mechanisms leading to cell apoptosis remain obscure. In this study, the signaling pathways involved in CDDP -induced apoptosis were examined using CDDP-sensitive ovarian cancer A2780s cells. A2780s cells were treated with CDDP (1.5-3 μg/ml) for 6 h, 12 h and 24 h. Using siRNA targeting P53 and PUMA, and a selective MEK inhibitor, PD98059 to examine the relation between ERK1/2 activation, p53 and PUMA expression after exposure to CDDP, and the effect on CDDP-induced apoptosis. The results shown that treatment of A2780s cells with CDDP (3 μg/ml) for 6-24 h induced apoptosis, resulting in the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and accumulation of p53 and PUMA (p53 upregulated modulator of apoptosis) protein. Knockdown of P53 or PUMA by siRNA transfection blocked CDDP-induced apoptosis. Inhibition of ERK1/2 using PD98059, a selective MEK inhibitor, blocked the apoptotic cell death but prevented CDDP-induced accumulation of p53 and PUMA. Knockdown of P53 by siRNA transfection also blocked CDDP-induced accumulation of PUMA. We therefore concluded that CDDP activated ERK1/2 and induced-p53-dependent PUMA upregulation, resulting in triggering apoptosis in A2780s cells. Our study clearly demonstrates that the ERK1/2/p53/PUMA axis is related to CDDP-induced cell death in A2780s cells.

  2. Wt p53 impairs response to chemotherapy: make lemonade to spare normal cells

    PubMed Central

    Blagosklonny, Mikhail V.

    2012-01-01

    As published recently in Cancer Cell, p53 impairs the apoptotic response to chemotherapy and clinical outcome in breast cancer. I discuss that, while treating tumors lacking wt p53, this phenomenon can be exploited to protect normal cells from chemotherapy because all normal cells have wt p53. Also, several therapeutic paradigms can be reassessed, including the role of cellular senescence in cancer therapy. PMID:22802145

  3. p53 promotes cellular survival in a context-dependent manner by directly inducing the expression of haeme-oxygenase-1.

    PubMed

    Nam, S Y; Sabapathy, K

    2011-11-03

    A variety of cellular insults activate the tumour suppressor p53, leading generally to cell-cycle arrest or apoptosis. However, it is not inconceivable that cellular protective mechanisms may be required to keep cells alive while cell-fate decisions are made. In this respect, p53 has been suggested to perform functions that allow cells to survive, by halting of the cell-cycle, and thus preventing immediate cell death. Nonetheless, the existence of direct pro-survival p53 target genes regulating cellular survival is lacking. We show here evidence for p53-dependent cellular survival in a context-dependent manner. Both mouse and human cells lacking p53 are hypersensitive to hydrogen peroxide (H(2)O(2))-induced cell death compared with their isogenic wild-type counterparts. By contrast, p53(-/-) cells are expectedly resistant to cell death upon exposure to DNA-damaging agents such as cisplatin (CDDP) and etoposide. Although p53 and its classical targets such as p21 and Mdm2 are activated by both H(2)O(2) and CDDP, we found that the expression of haeme-oxygenase-1 (HO-1)-an antioxidant and antiapoptotic protein-was directly induced only upon H(2)O(2) treatment in a p53-dependent manner. Consistently, p53, but not its homologue p73, activated HO-1 expression and was bound to the HO-1 promoter specifically only upon H(2)O(2) treatment. Moreover, silencing HO-1 expression enhanced cell death upon H(2)O(2) treatment only in p53-proficient cells. Finally, H(2)O(2)-mediated cell death was rescued significantly in p53-deficient cells by antioxidant treatment, as well as by bilirubin, a by-product of HO-1 metabolism. Taken together, these data demonstrate a direct role for p53 in promoting cellular survival in a context-specific manner through the activation of a direct transcriptional target, HO-1.

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

    SciTech Connect

    Gestl, Erin E.; Anne Boettger, S.

    2012-06-29

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

  5. p53 expression and its relationship to DNA alterations in bone and soft tissue sarcomas.

    PubMed Central

    Wadayama, B.; Toguchida, J.; Yamaguchi, T.; Sasaki, M. S.; Yamamuro, T.

    1993-01-01

    The p53 gene is one of the best studied tumour suppressor genes. Recently we performed mutation analysis on the p53 gene in a large number of bone and soft tissue sarcomas, and found that approximately one-third of the sarcomas have some type of DNA alteration at the p53 locus (Toguchida et al., 1992). However, the expression of the p53 protein resulting from these alterations still remains to be clarified. In this study, p53 expression in the sarcoma tissues was analysed immunohistochemically using antibody PAb421 (Oncogene Science) and its relationship to DNA alterations was examined. Of 113 tumours, 29 (25.7%) showed positive staining for the p53 protein. These included 19 of 67 osteosarcomas, five of 20 chondrosarcomas, four of 11 malignant fibrous histiocytomas (MFHs) and one Ewing's sarcoma. In chondrosarcomas, most of the p53-positive tumours belonged to highly malignant and atypical tumour types (dedifferentiated or mesenchymal type), suggesting a role for p53 mutation in the progression of cartilaginous tumours. All the cases with a missense mutation showed strongly positive staining, while no immunoreactivity was observed in the remaining three-quarters with DNA alterations including gross rearrangement, frame-shift mutation, nonsense mutation or mutation at splicing site except in one case. These results demonstrated the dominance of the p53 mutations with null protein expression in bone and soft tissue sarcomas, showing a unique characteristic of these types of tumours compared with other malignancies such as colon carcinomas. Images Figure 1 Figure 2 Figure 3 PMID:8260365

  6. Mitochondrial p53 mediates a transcription-independent regulation of cell respiration and interacts with the mitochondrial F₁F0-ATP synthase.

    PubMed

    Bergeaud, Marie; Mathieu, Lise; Guillaume, Arnaud; Moll, Ute M; Mignotte, Bernard; Le Floch, Nathalie; Vayssière, Jean-Luc; Rincheval, Vincent

    2013-09-01

    We and others previously reported that endogenous p53 can be located at mitochondria in the absence of stress, suggesting that p53 has a role in the normal physiology of this organelle. The aim of this study was to characterize in unstressed cells the intramitochondrial localization of p53 and identify new partners and functions of p53 in mitochondria. We find that the intramitochondrial pool of p53 is located in the intermembrane space and the matrix. Of note, unstressed HCT116 p53(+/+) cells simultaneously show increased O₂ consumption and decreased mitochondrial superoxide production compared with their p53-null counterpart. This data was confirmed by stable H1299 cell lines expressing low levels of p53 specifically targeted to the matrix. Using immunoprecipitation and mass spectrometry, we identified the oligomycin sensitivity-conferring protein (OSCP), a subunit of the F₁F₀-ATP synthase complex, as a new partner of endogenous p53, specifically interacting with p53 localized in the matrix. Interestingly, this interaction seems implicated in mitochondrial p53 localization. Moreover, p53 localized in the matrix promotes the assembly of F₁F₀-ATP synthase. Taking into account that deregulations of mitochondrial respiration and reactive oxygen species production are tightly linked to cancer development, we suggest that mitochondrial p53 may be an important regulator of normal mitochondrial and cellular physiology, potentially exerting tumor suppression activity inside mitochondria.

  7. Requirement for MLL3 in p53 regulation of hepatic expression of small heterodimer partner and bile acid homeostasis.

    PubMed

    Kim, Dae-Hwan; Kim, Juhee; Lee, Jae W

    2011-12-01

    The histone H3-lysine-4 methyltransferase mixed-lineage leukemia 3 (MLL3) belongs to a large complex that functions as a coactivator of multiple transcription factors, including the bile acid (BA)-activated nuclear receptor, farnesoid X receptor (FXR), a critical player in BA homeostasis. BA-activated FXR induces hepatic expression of small heterodimer partner (SHP), which in turn suppresses expression of BA synthesis genes, Cyp7a1 and Cyp8b1. Thus, MLL3(Δ/Δ) mice that express a catalytically inactive mutant form of MLL3 display increased BA levels. Recently, we have discovered a distinct regulatory pathway for BA homeostasis, in which p53 independently up-regulates SHP expression in the liver. Here, we show that the MLL3 complex is also essential for p53 transactivation of SHP. Although activated p53 signaling in MLL3(+/+) mice results in decreased BA levels through hepatic up-regulation of SHP, these changes are abolished in MLL3(Δ/Δ) mice. For both HepG2 cells and mouse liver, we also demonstrate that p53 directs the recruitment of different components of the MLL3 complex to the p53-response elements of SHP and that p53-dependent H3-lysine-4-trimethylation of SHP requires MLL3. From these results, we conclude that both FXR- and p53-dependent regulatory pathways for SHP expression in BA homeostasis require the MLL3 complex; thus, the MLL3 complex is likely a master regulator of BA homeostasis. Using a common coregulator complex for multiple transcription factors, which independently control expression of the same gene, might be a prevalent theme in gene regulation and may also play critical roles in assigning a specific biological function to a coregulator complex.

  8. Dual targeting of p53 and c-Myc selectively eliminates leukaemic stem cells

    PubMed Central

    Abraham, Sheela A; Hopcroft, Lisa EM; Carrick, Emma; Drotar, Mark E; Dunn, Karen; Williamson, Andrew JK; Korfi, Koorosh; Baquero, Pablo; Park, Laura E; Scott, Mary T; Pellicano, Francesca; Pierce, Andrew; Copland, Mhairi; Nourse, Craig; Grimmond, Sean M; Vetrie, David; Whetton, Anthony D; Holyoake, Tessa L

    2016-01-01

    Summary Chronic myeloid leukaemia (CML) arises following transformation of a haemopoietic stem cell (HSC) by protein-tyrosine kinase BCR-ABL1. Direct inhibition of BCR-ABL1 kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSC), which maintain CML. LSC are independent of BCR-ABL1 for survival, providing a rationale to identify and target kinase-independent pathways. Here we show using proteomics, transcriptomics and network analyses, that in human LSC aberrantly expressed proteins, in both imatinib-responder and non-responder patients are modulated in concert with p53 and c-Myc regulation. Perturbation of both p53 and c-Myc, not BCR-ABL1 itself, leads to synergistic kill, differentiation and near elimination of transplantable human LSC in mice, whilst sparing normal HSC. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSC can be eradicated. PMID:27281222

  9. Effects of p53-knockout in vascular smooth muscle cells on atherosclerosis in mice

    PubMed Central

    Jia, Lilly; Funk, Colin D.; Jia, Zongchao; Mak, Alan S.

    2017-01-01

    In vitro and in vivo evidence has indicated that the tumor suppressor, p53, may play a significant role in the regulation of atherosclerotic plaque formation. In vivo studies using global knockout mice models, however, have generated inconclusive results that do not address the roles of p53 in various cell types involved in atherosclerosis. In this study, we have specifically ablated p53 in vascular smooth muscle cells (VSMC) in the ApoE-/- mouse model to investigate the roles of p53 in VSMC in atherosclerotic plaque formation and stability. We found that p53 deficiency in VSMC alone did not affect the overall size of atherosclerotic lesions. However, there was a significant increase in the number of p53-/- VSMC in the fibrous caps of atherosclerotic plaques in the early stages of plaque development. Loss of p53 results in migration of VSMC at a faster rate using wound healing assays and augments PDGF-induced formation of circular dorsal ruffles (CDR), known to be involved in cell migration and internalization of surface receptors. Furthermore, aortic VSMC from ApoE-/- /p53-/- mice produce significantly more podosomes and are more invasive. We conclude that p53-/- VSMC are enriched in the fibrous caps of lesions at early stages of plaque formation, which is caused in part by an increase in VSMC migration and invasion as shown by p53-/- VSMC in culture having significantly higher rates of migration and producing more CDRs and invasive podosomes. PMID:28362832

  10. AAVPG: A vigilant vector where transgene expression is induced by p53

    SciTech Connect

    Bajgelman, Marcio C.; Medrano, Ruan F.V.; Carvalho, Anna Carolina P.V.; Strauss, Bryan E.

    2013-12-15

    Using p53 to drive transgene expression from viral vectors may provide on demand expression in response to physiologic stress, such as hypoxia or DNA damage. Here we introduce AAVPG, an adeno-associated viral (AAV) vector where a p53-responsive promoter, termed PG, is used to control transgene expression. In vitro assays show that expression from the AAVPG-luc vector was induced specifically in the presence of functional p53 (1038±202 fold increase, p<0.001). The AAVPG-luc vector was an effective biosensor of p53 activation in response to hypoxia (4.48±0.6 fold increase in the presence of 250 µM CoCl{sub 2}, p<0.001) and biomechanical stress (2.53±0.4 fold increase with stretching, p<0.05). In vivo, the vigilant nature of the AAVPG-luc vector was revealed after treatment of tumor-bearing mice with doxorubicin (pre-treatment, 3.4×10{sup 5}±0.43×10{sup 5} photons/s; post-treatment, 6.6×10{sup 5}±2.1×10{sup 5} photons/s, p<0.05). These results indicate that the AAVPG vector is an interesting option for detecting p53 activity both in vitro and in vivo. - Highlights: • AAV vector where transgene expression is controlled by the tumor suppressor p53. • The new vector, AAVPG, shown to function as a biosensor of p53 activity, in vitro and in vivo. • The p53 activity monitored by the AAVPG vector is relevant to cancer and other diseases. • AAVPG reporter gene expression was activated upon DNA damage, hypoxia and mechanical stress.

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

    PubMed Central

    Zhao, M; Zimmermann, A

    1997-01-01

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

  12. Induced p53 loss in mouse luminal cells causes clonal expansion and development of mammary tumours

    PubMed Central

    Tao, Luwei; Xiang, Dongxi; Xie, Ying; Bronson, Roderick T.; Li, Zhe

    2017-01-01

    Most breast cancers may have a luminal origin. TP53 is one of the most frequently mutated genes in breast cancers. However, how p53 deficiency contributes to breast tumorigenesis from luminal cells remains elusive. Here we report that induced p53 loss in Krt8+ mammary luminal cells leads to their clonal expansion without directly affecting their luminal identity. All induced mice develop mammary tumours with 9qA1 (Yap1) and/or 6qA2 (Met) amplification(s). These tumours exhibit a mammary stem cell (MaSC)-like expression signature and most closely resemble claudin-low breast cancer. Thus, although p53 does not directly control the luminal fate, its loss facilitates acquisition of MaSC-like properties by luminal cells and predisposes them to development of mammary tumours with loss of luminal identity. Our data also suggest that claudin-low breast cancer can develop from luminal cells, possibly via a basal-like intermediate state, although further study using a different luminal promoter is needed to fully support this conclusion. PMID:28194015

  13. Proliferating cell nuclear antigen, p53 and micro vessel density: Grade II vs. Grade III astrocytoma.

    PubMed

    Malhan, Priya; Husain, Nuzhat; Bhalla, Shalini; Gupta, Rakesh K; Husain, Mazhar

    2010-01-01

    Histological classification and grading are prime procedures in the management of patients with astrocytoma, providing vital data for therapeutic decision making and prognostication. However, it has limitations in assessing biological tumor behavior. This can be overcome by using newer immunohistochemical techniques. This study was carried out to compare proliferative indices using proliferating cell nuclear antigen (PCNA), extent of p53 expression and micro vessel morphometric parameters in patients with low grade and anaplastic astrocytoma. Twenty-five patients, each of grade II and grade III astrocytoma were evaluated using monoclonal antibodies to PCNA, p53 protein and factor VIII related antigen. PCNA, p53-labeling indices were calculated along with micro vessel morphometric analysis using Biovis Image plus Software. Patients with grade III astrocytoma had higher PCNA and p53 labeling indices as compared with grade II astrocytoma (29.14 plus/minus 9.87% vs. 16.84 plus/minus 6.57%, p 0.001; 18.18 plus/minus 6.14% vs. 6.14 plus/minus 7.23%, p 0.001, respectively). Micro vessel percentage area of patients with grade III astrocytoma was also (4.26 plus/minus 3.70 vs. 1.05 plus/minus 0.56, p 0.001), higher along with other micro vessel morphometric parameters. Discordance between histology and one or more IHC parameters was seen in 5/25 (20%) of patients with grade III astrocytoma and 9/25 (36%) of patients with grade II disease. PCNA and p53 labeling indices were positively correlated with Pearson's correlation, p less than 0.001 for both). Increased proliferative fraction, genetic alterations and neovascularization mark biological aggressiveness in astrocytoma. Immunohistochemical evaluation scores over meet the challenge of accurate prognostication of this potentially fatal malignancy.

  14. Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway.

    PubMed Central

    Chowdary, D R; Dermody, J J; Jha, K K; Ozer, H L

    1994-01-01

    The wild-type p53 gene product plays an important role in the control of cell proliferation, differentiation, and survival. Altered function is frequently associated with changes in p53 stability. We have studied the role of the ubiquitination pathway in the degradation of p53, utilizing a temperature-sensitive mutant, ts20, derived from the mouse cell line BALB/c 3T3. We found that wild-type p53 accumulates markedly because of decreased breakdown when cells are shifted to the restrictive temperature. Introduction of sequences encoding the human ubiquitin-activating enzyme E1 corrects the temperature sensitivity defect in ts20 and prevents accumulation of p53. The data therefore strongly indicate that wild-type p53 is degraded intracellularly by the ubiquitin-mediated proteolytic pathway. Images PMID:8114731

  15. p53 induction and cell viability modulation by genotoxic individual chemicals and mixtures.

    PubMed

    Di Paolo, Carolina; Müller, Yvonne; Thalmann, Beat; Hollert, Henner; Seiler, Thomas-Benjamin

    2017-03-16

    The binding of the p53 tumor suppression protein to DNA response elements after genotoxic stress can be quantified by cell-based reporter gene assays as a DNA damage endpoint. Currently, bioassay evaluation of environmental samples requires further knowledge on p53 induction by chemical mixtures and on cytotoxicity interference with p53 induction analysis for proper interpretation of results. We investigated the effects of genotoxic pharmaceuticals (actinomycin D, cyclophosphamide) and nitroaromatic compounds (4-nitroquinoline 1-oxide, 3-nitrobenzanthrone) on p53 induction and cell viability using a reporter gene and a colorimetric assay, respectively. Individual exposures were conducted in the absence or presence of metabolic activation system, while binary and tertiary mixtures were tested in its absence only. Cell viability reduction tended to present direct correlation with p53 induction, and induction peaks occurred mainly at chemical concentrations causing cell viability below 80%. Mixtures presented in general good agreement between predicted and measured p53 induction factors at lower concentrations, while higher chemical concentrations gave lower values than expected. Cytotoxicity evaluation supported the selection of concentration ranges for the p53 assay and the interpretation of its results. The often used 80% viability threshold as a basis to select the maximum test concentration for cell-based assays was not adequate for p53 induction assessment. Instead, concentrations causing up to 50% cell viability reduction should be evaluated in order to identify the lowest observed effect concentration and peak values following meaningful p53 induction.

  16. The oncogenic effects of p53-inducible gene 3 (PIG3) in colon cancer cells

    PubMed Central

    Park, Seon-Joo; Kim, Hong Beum; Kim, Jeeho

    2017-01-01

    The p53-inducible gene 3 (PIG3), initially identified as a gene downstream of p53, plays an important role in the apoptotic process triggered by p53-mediated reactive oxygen species (ROS) production. Recently, several studies have suggested that PIG3 may play a role in various types of cancer. However, the functional significance of PIG3 in cancer remains unclear. Here, we found that PIG3 was highly expressed in human colon cancer cell lines compared to normal colonderived fibroblasts. Therefore, we attempted to elucidate the functional role of PIG3 in colon cancer. PIG3 overexpression increases the colony formation, migration and invasion ability of HCT116 colon cancer cells. Conversely, these tumorigenic abilities were significantly decreased in in vitro studies with PIG3 knockdown HCT116 cells. PIG3 knockdown also attenuated the growth of mouse xenograft tumors. These results demonstrate that PIG3 is associated with the tumorigenic potential of cancer cells, both in vitro and in vivo, and could play a key oncogenic role in colon cancer. PMID:28280421

  17. Sensitization of osteosarcoma cells to apoptosis by oncostatin M depends on STAT5 and p53.

    PubMed

    Chipoy, C; Brounais, B; Trichet, V; Battaglia, S; Berreur, M; Oliver, L; Juin, P; Rédini, F; Heymann, D; Blanchard, F

    2007-10-11

    Oncostatin M (OSM), a cytokine of the interleukin-6 family, induces growth arrest and differentiation of osteoblastic cells into glial-like/osteocytic cells. Here, we asked whether OSM regulates apoptosis of normal or transformed (osteosarcoma) osteoblasts. We show that OSM sensitizes cells to apoptosis induced by various death inducers such as staurosporine, ultraviolet or tumor necrosis factor-alpha. Apoptosis is mediated by the mitochondrial pathway, with release of cytochrome c from the mitochondria to the cytosol and activation of caspases-9 and -3. DNA micro-arrays revealed that OSM modulates the expression of Bax, Bad, Bnip3, Bcl-2 and Mcl-1. Pharmacological inhibitors, dominant-negative signal transducer and activator of transcriptions (STATs), stable RNA interference and knockout cells indicated that the transcription factors p53 and STAT5, which are activated by OSM, are implicated in the sensitization to apoptosis, being responsible for Bax induction and Bcl-2 reduction, respectively. These results indicate that, in addition to growth arrest and induced differentiation, OSM also sensitizes normal and transformed osteoblasts to apoptosis by a mechanism implicating (i) activation and nuclear translocation of STAT5 and p53 and (ii) an increased Bax/Bcl-2 ratio. Therefore, association of OSM with kinase inhibitors such as Sts represents new therapeutic opportunities for wild-type p53 osteosarcoma.

  18. Enigma negatively regulates p53 through MDM2 and promotes tumor cell survival in mice.

    PubMed

    Jung, Cho-Rok; Lim, Jung Hwa; Choi, Yoonjung; Kim, Dae-Ghon; Kang, Koo Jeong; Noh, Seung-Moo; Im, Dong-Soo

    2010-12-01

    The human E3 ubiquitin ligase murine double minute 2 (MDM2) targets the tumor suppressor p53 for ubiquitination and degradation but also promotes its own ubiquitination and subsequent degradation. As the balance between MDM2 and p53 levels plays a crucial role in regulating cell proliferation and apoptosis, we sought to identify factors selectively inhibiting MDM2 self-ubiquitination. Here we have shown that the LIM domain protein Enigma directly interacts with MDM2 to form a ternary complex with p53 in vitro and in human hepatoma and colon carcinoma cell lines and mouse embryonic fibroblasts. We found that Enigma elicited p53 degradation by inhibiting MDM2 self-ubiquitination and increasing its ubiquitin ligase activity toward p53 in cells. Moreover, mitogenic stimuli such as serum, FGF, and HGF increased Enigma transcription via induction of serum response factor (SRF), leading to MDM2 stabilization and subsequent p53 degradation. We observed similar results in the livers of mice treated with HGF. In humans, we found SRF and Enigma coexpressed with MDM2 but not p53 in several liver and stomach tumors. Finally, we showed that Enigma promoted cell survival and chemoresistance by suppressing p53-mediated apoptosis in both cell lines and a mouse xenograft model. Our findings suggest a role for Enigma in tumorigenesis and uncover a mechanism whereby mitogens attenuate p53 antiproliferative activity through an SRF/Enigma/MDM2 pathway.

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

    DOE PAGES

    Botcheva, Krassimira; McCorkle, Sean R.

    2014-11-21

    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 reportmore » 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). In conclusion, 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.« less

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

    SciTech Connect

    Botcheva, Krassimira; McCorkle, Sean R.

    2014-11-21

    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). In conclusion, 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.

  1. Cell context dependent p53 genome-wide binding patterns and enrichment at repeats.

    PubMed

    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.

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

    PubMed Central

    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

  3. p53-Mediated Cellular Response to DNA Damage in Cells with Replicative Hepatitis B Virus

    NASA Astrophysics Data System (ADS)

    Puisieux, Alain; Ji, Jingwei; Guillot, Celine; Legros, Yann; Soussi, Thierry; Isselbacher, Kurt; Ozturk, Mehmet

    1995-02-01

    Wild-type p53 acts as a tumor suppressor gene by protecting cells from deleterious effects of genotoxic agents through the induction of a G_1/S arrest or apoptosis as a response to DNA damage. Transforming proteins of several oncogenic DNA viruses inactivate tumor suppressor activity of p53 by blocking this cellular response. To test whether hepatitis B virus displays a similar effect, we studied the p53-mediated cellular response to DNA damage in 2215 hepatoma cells with replicative hepatitis B virus. We demonstrate that hepatitis B virus replication does not interfere with known cellular functions of p53 protein.

  4. Progesterone facilitates chromosome instability (aneuploidy) in p53 null normal mammary epithelial cells

    NASA Technical Reports Server (NTRS)

    Goepfert, T. M.; McCarthy, M.; Kittrell, F. S.; Stephens, C.; Ullrich, R. L.; Brinkley, B. R.; Medina, D.

    2000-01-01

    Mammary epithelial cells from p53 null mice have been shown recently to exhibit an increased risk for tumor development. Hormonal stimulation markedly increased tumor development in p53 null mammary cells. Here we demonstrate that mammary tumors arising in p53 null mammary cells are highly aneuploid, with greater than 70% of the tumor cells containing altered chromosome number and a mean chromosome number of 56. Normal mammary cells of p53 null genotype and aged less than 14 wk do not exhibit aneuploidy in primary cell culture. Significantly, the hormone progesterone, but not estrogen, increases the incidence of aneuploidy in morphologically normal p53 null mammary epithelial cells. Such cells exhibited 40% aneuploidy and a mean chromosome number of 54. The increase in aneuploidy measured in p53 null tumor cells or hormonally stimulated normal p53 null cells was not accompanied by centrosome amplification. These results suggest that normal levels of progesterone can facilitate chromosomal instability in the absence of the tumor suppressor gene, p53. The results support the emerging hypothesis based both on human epidemiological and animal model studies that progesterone markedly enhances mammary tumorigenesis.

  5. Photodynamic injury of isolated crayfish neuron and surrounding glial cells: the role of p53

    NASA Astrophysics Data System (ADS)

    Sharifulina, S. A.; Uzdensky, A. B.

    2015-03-01

    The pro-apoptotic transcription factor p53 is involved in cell responses to injurious impacts. Using its inhibitor pifithrin- α and activators tenovin-1, RITA and WR-1065, we studied its potential participation in inactivation and death of isolated crayfish mechanoreceptor neuron and satellite glial cells induced by photodynamic treatment, a strong inducer of oxidative stress. In dark, p53 activation by tenovin-1 or WR-1065 shortened activity of isolated neurons. Tenovin-1 and WR-1065 induced apoptosis of glial cells, whereas pifithrin-α was anti-apoptotic. Therefore, p53 mediated glial apoptosis and suppression of neuronal activity after axotomy. Tenovin-1 but not other p53 modulators induced necrosis of axotomized neurons and surrounding glia, possibly, through p53-independent pathway. Under photodynamic treatment, p53 activators tenovin-1 and RITA enhanced glial apoptosis indicating the pro-apoptotic activity of p53. Photoinduced necrosis of neurons and glia was suppressed by tenovin-1 and, paradoxically, by pifithrin-α. Modulation of photoinduced changes in the neuronal activity and necrosis of neurons and glia was possibly p53-independent. The different effects of p53 modulators on neuronal and glial responses to axotomy and photodynamic impact were apparently associated with different signaling pathways in neurons and glial cells.

  6. Distinct patterns of cleavage and translocation of cell cycle control proteins in CD95-induced and p53-induced apoptosis.

    PubMed Central

    Park, Weon Seo; Jung, Kyeong Cheon; Chung, Doo Hyun; Nam, Woo-Dong; Choi, Won Jin; Bae, Youngmee

    2003-01-01

    Apoptotic cell death induced by p53 occurs at a late G1 cell cycle checkpoint termed the restriction (R) point, and it has been proposed that p53-induced apoptosis causes upregulation of CD95. However, as cells with defective in CD95 signaling pathway are still sensitive to p53-induced apoptosis, CD95 cannot be the sole factor resulting in apoptosis. In addition, unlike p53-induced apoptosis, the relationship between CD95-mediated apoptosis and the cell cycle is not clearly understood. It would therefore be worth investigating whether CD95-mediated cell death is pertinent with p53-induced apoptosis in view of cell cycle related molecules. In this report, biochemical analysis showed that etoposide-induced apoptosis caused the induction and the nuclear translocation of effector molecules involved in G1 cell cycle checkpoint. However, there was no such translocation in the case of CD95-mediated death. Thus, although both types of apoptosis involved caspase activation, the cell cycle related proteins responded differently. This argues against the idea that p53-induced apoptosis occurs through the induction of CD95/CD95L expression. PMID:12923319

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

    SciTech Connect

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

    2006-10-15

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

  8. Prognostic impact of c-Rel nuclear expression and REL amplification and crosstalk between c-Rel and the p53 pathway in diffuse large B-cell lymphoma.

    PubMed

    Li, Ling; Xu-Monette, Zijun Y; Ok, Chi Young; Tzankov, Alexandar; Manyam, Ganiraju C; Sun, Ruifang; Visco, Carlo; Zhang, Mingzhi; Montes-Moreno, Santiago; Dybkaer, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Richards, Kristy L; Hsi, Eric D; Choi, William W L; van Krieken, J Han; Huh, Jooryung; Ponzoni, Maurilio; Ferreri, Andrés J M; Møller, Michael B; Wang, Jinfen; Parsons, Ben M; Winter, Jane N; Piris, Miguel A; Pham, Lan V; Medeiros, L Jeffrey; Young, Ken H

    2015-09-15

    Dysregulated NF-κB signaling is critical for lymphomagenesis. The regulation, function, and clinical relevance of c-Rel/NF-κB activation in diffuse large B-cell lymphoma (DLBCL) have not been well studied. In this study we analyzed the prognostic significance and gene-expression signature of c-Rel nuclear expression as surrogate of c-Rel activation in 460 patients with de novo DLBCL. Nuclear c-Rel expression, observed in 137 (26.3%) DLBCL patients frequently associated with extranoal origin, did not show significantly prognostic impact in the overall- or germinal center B-like-DLBCL cohort, likely due to decreased pAKT and Myc levels, up-regulation of FOXP3, FOXO3, MEG3 and other tumor suppressors coincided with c-Rel nuclear expression, as well as the complicated relationships between NF-κB members and their overlapping function. However, c-Rel nuclear expression correlated with significantly poorer survival in p63+ and BCL-2- activated B-cell-like-DLBCL, and in DLBCL patients with TP53 mutations. Multivariate analysis indicated that after adjusting clinical parameters, c-Rel positivity was a significantly adverse prognostic factor in DLBCL patients with wild type TP53. Gene expression profiling suggested dysregulations of cell cycle, metabolism, adhesion, and migration associated with c-Rel activation. In contrast, REL amplification did not correlate with c-Rel nuclear expression and patient survival, likely due to co-amplification of genes that negatively regulate NF-κB activation. These insights into the expression, prognostic impact, regulation and function of c-Rel as well as its crosstalk with the p53 pathway underscore the importance of c-Rel and have significant therapeutic implications.

  9. TopBP1 contributes to the chemoresistance in non-small cell lung cancer through upregulation of p53

    PubMed Central

    Lv, Yinxiang; Huo, Yanan; Yu, Xican; Liu, Rongrong; Zhang, Shufen; Zheng, Xiaoxiao; Zhang, Xianning

    2016-01-01

    Resistance to chemotherapeutic drugs is a major obstacle in non-small cell lung cancer (NSCLC) therapy. The molecular determinants of NSCLC resistance to doxorubicin are unknown. In the present study, we investigated whether topoisomerase IIβ binding protein 1 (TopBP1) was involved in the chemoresistance to doxorubicin in NSCLC cancer. We found that p53-deficient lung cancer cells (NCI-H1299) displayed the greatest resistance to doxorubicin compared with NCI-H358, A549, and HCC827 cells with p53 expression. The expression of TopBP1 was significantly higher in NCI-H1299 cells than the other three tumor cell lines. In addition, TopBP1 knockdown with specific small interfering RNA in NCI-H1299 cells enhanced the doxorubicin chemosensitivity and decreased the expression of p53 in the presence of doxorubicin. After doxorubicin administration, co-immunoprecipitation assay showed that TopBP1 promoted the expression of p53 in NCI-H1299 cells. These results for the first time demonstrated that TopBP1 plays an important role in NSCLC chemoresistance via upregulation of p53. Therefore, inhibition of TopBP1, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant NSCLC. PMID:27729767

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

    SciTech Connect

    Leão, Mariana; Gomes, Sara; Bessa, Cláudia; Soares, Joana; Raimundo, Liliana; Monti, Paola; Fronza, Gilberto; Pereira, Clara; Saraiva, Lucília

    2015-01-01

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

  11. p53 protein expression independently predicts outcome in patients with lower-risk myelodysplastic syndromes with del(5q)

    PubMed Central

    Saft, Leonie; Karimi, Mohsen; Ghaderi, Mehran; Matolcsy, András; Mufti, Ghulam J.; Kulasekararaj, Austin; Göhring, Gudrun; Giagounidis, Aristoteles; Selleslag, Dominik; Muus, Petra; Sanz, Guillermo; Mittelman, Moshe; Bowen, David; Porwit, Anna; Fu, Tommy; Backstrom, Jay; Fenaux, Pierre; MacBeth, Kyle J.; Hellström-Lindberg, Eva

    2014-01-01

    Del(5q) myelodysplastic syndromes defined by the International Prognostic Scoring System as low- or intermediate-1-risk (lower-risk) are considered to have an indolent course; however, recent data have identified a subgroup of these patients with more aggressive disease and poorer outcomes. Using deep sequencing technology, we previously demonstrated that 18% of patients with lower-risk del(5q) myelodysplastic syndromes carry TP53 mutated subclones rendering them at higher risk of progression. In this study, bone marrow biopsies from 85 patients treated with lenalidomide in the MDS-004 clinical trial were retrospectively assessed for p53 expression by immunohistochemistry in association with outcome. Strong p53 expression in ≥1% of bone marrow progenitor cells, observed in 35% (30 of 85) of patients, was significantly associated with higher acute myeloid leukemia risk (P=0.0006), shorter overall survival (P=0.0175), and a lower cytogenetic response rate (P=0.009), but not with achievement or duration of 26-week transfusion independence response. In a multivariate analysis, p53-positive immunohistochemistry was the strongest independent predictor of transformation to acute myeloid leukemia (P=0.0035). Pyrosequencing analysis of laser-microdissected cells with strong p53 expression confirmed the TP53 mutation, whereas cells with moderate expression predominantly had wild-type p53. This study validates p53 immunohistochemistry as a strong and clinically useful predictive tool in patients with lower-risk del(5q) myelodysplastic syndromes. This study was based on data from the MDS 004 trial (clinicaltrials.gov identifier: NCT00179621). PMID:24682512

  12. Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction.

    PubMed

    Tran, T Q; Lowman, X H; Reid, M A; Mendez-Dorantes, C; Pan, M; Yang, Y; Kong, M

    2017-04-06

    Cancer cells depend on glutamine to sustain their increased proliferation and manage oxidative stress, yet glutamine is often depleted at tumor sites owing to excessive cellular consumption and poor vascularization. We have previously reported that p53 protein, although a well-known tumor suppressor, can contribute to cancer cell survival and adaptation to low-glutamine conditions. However, the TP53 gene is frequently mutated in tumors, and the role of mutant p53 (mutp53) in response to metabolic stress remains unclear. Here, we demonstrate that tumor-associated mutp53 promotes cancer cell survival upon glutamine deprivation both in vitro and in vivo. Interestingly, cancer cells expressing