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Sample records for p53 p21 waf

  1. G1 checkpoint is compromised in mouse ESCs due to functional uncoupling of p53-p21Waf1 signaling.

    PubMed

    Suvorova, Irina I; Grigorash, Bogdan B; Chuykin, Ilya A; Pospelova, Tatiana V; Pospelov, Valery A

    2016-01-01

    Mouse embryonic stem cells (mESCs) lack of G1 checkpoint despite that irradiation (IR) activates ATM/ATR-mediated DDR signaling pathway. The IR-induced p53 localizes in the nuclei and up-regulates p21/Waf1 transcription but that does not lead to accumulation of p21/Waf1 protein. The negative control of the p21Waf1 expression appears to occur at 2 levels of regulation. First, both p21/Waf1 gene transcription and the p21/Waf1 protein content increase in mESCs treated with histone-deacetylase inhibitors, implying its epigenetic regulation. Second, proteasome inhibitors cause the p21/Waf1 accumulation, indicating that the protein is a subject of proteasome-dependent degradation in ESСs. Then, the dynamics of IR-induced p21Waf1 protein show its accumulation at long-term time points (3 and 5 days) that coincides with an increase in the proportion of G1-phase cells, down-regulation of Oct4 and Nanog pluripotent gene transcription and activation of endoderm-specific genes sox17 and afp. In addition, nutlin-dependent stabilization of p53 in mESC was also accompanied by the accumulation of p21/Waf1 as well as restoration of G1 checkpoint and an onset of differentiation. Thus, the lack of functional p21/Waf1 is indispensable for maintaining self-renewal and pluripotency of mESCs. PMID:26636245

  2. Immunohistochemical expression of mdm2 and p21WAF1 in invasive cervical cancer: correlation with p53 protein and high risk HPV infection.

    PubMed Central

    Troncone, G; Martinez, J C; Palombini, L; De Rosa, G; Mugica, C; Rodriguez, J A; Zeppa, P; Di Vizio, D; Lucariello, A; Piris, M A

    1998-01-01

    AIM: To investigate the immunocytochemical staining pattern of mdm2 and p21WAF1 proteins in invasive cervical cancer and to determine its relation with the expression of p53 and with the high risk HPV infection. METHODS: Immunocytochemistry for p53, mdm2, and p21WAF1 was performed in 31 paraffin embedded sections of invasive cervical cancer. The results were assessed by image analysis, evaluating for each protein the optical density of the immunostained area, scored as percentage of the total nuclear area. The presence of high risk human papillomavirus (HPV) infection was detected by using the polymerase chain reaction. RESULTS: Immunostaining for both mdm2 and p21WAF1 was correlated with p53 expression; however, the correlation between p53 and mdm2 (R = 0.49; p < 0.01) was more significant than between p53 and p21WAF1 (R = 0.31; p < 0.05); the less stringent correlation between p53 and p21WAF1 might reflect the p53 independent mechanisms of p21WAF1 induction. Similar average levels of p53, mdm2, and p21WAF1 immunostaining were found in the presence or absence of high risk HPV-DNA, without significant differences between the two groups. CONCLUSIONS: These data suggest that mdm2 and p21WAF1 proteins are expressed in invasive cervical cancer and that their immunocytochemical staining pattern is not abrogated by the presence of high risk HPV genomic sequences. Images PMID:10023338

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

    SciTech Connect

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

    2011-09-01

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

  4. Transcriptional inhibition of p21WAF1/CIP1 gene (CDKN1) expression by survivin is at least partially p53-dependent: evidence for survivin acting as a transcription factor or co-factor.

    PubMed

    Tang, Lei; Ling, Xiang; Liu, Wensheng; Das, Gokul M; Li, Fengzhi

    2012-05-01

    Growing evidence suggests a role for the antiapoptotic protein survivin in promotion of cancer cell G1/S transition and proliferation. However, the underlying mechanism is unclear. Further, although upregulation of p21(WAF1/CIP1) by p53 plays an important role in p53-mediated cell G1 arrests in response to various distresses, it is unknown whether survivin plays a role in the regulation of p21(WAF1/CIP1) expression. Here, we report that exogenous expression of survivin in p53-wild type MCF-7 breast cancer cells inhibits the expression of p21(WAF1/CIP1) protein, mRNA and promoter activity, while the survivin C84A mutant and antisense failed to do so. Cotransfection experiments in the p53 mutant H1650 lung cancer cell line showed that survivin neutralizes p53-induced p21(WAF1/CIP1) expression and promoter activity. Importantly, genetically silencing of endogenous survivin using lentiviral survivin shRNA also enhances endogenous p21 in p53 wild type cancer cells, suggesting the physiological relevance of the fining. We further demonstrated that both p53 and survivin interacts on the two p53-binding sites in the p21(WAF1/CIP1) promoter (-2313 to -2212; -1452 to -1310), and survivin physically interacts with p53 in cancer cells. Together, we propose that survivin may act as a transcription factor or cofactor to interact with p53 on the p21(WAF1/CIP1) promoter leading to the inhibition of p21(WAF1/CIP1) expression at least in part by neutralizing p53-mediated transcriptional activation of the p21 gene. PMID:22503977

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

    SciTech Connect

    Tang, Lei; Ling, Xiang; Liu, Wensheng; Das, Gokul M.; Li, Fengzhi

    2012-05-04

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

  6. Knocking down p53 with siRNA does not affect the overexpression of p21WAF-1 after exposure of IMR-90 hTERT fibroblasts to a sublethal concentration of H2O2 leading to premature senescence.

    PubMed

    Zdanov, Stephanie; Debacq-Chainiaux, Florence; Toussaint, Olivier

    2007-04-01

    Premature senescence of IMR-90 human diploid fibroblasts (HDFs) expressing telomerase was induced by exposure to sublethal concentration of H(2)O(2), with appearance of several biomarkers of cellular senescence like enlarged cell shape, senescence-associated beta-galactosidase (SA ss-gal) activity, and cell cycle arrest. The induction of stress-induced premature senescence (SIPS) was associated with a transient increase in DNA-binding activity of p53 and an increased expression of p21(WAF-1). p53 small interferent RNA (siRNA) affected the basal level of p21(WAF-1) mRNA but did not affect the overexpression of p21(WAF-1) after stress. This siRNA approach confirms previous results obtained with other methods. PMID:17460194

  7. p53 can repress transcription of cell cycle genes through a p21WAF1/CIP1-dependent switch from MMB to DREAM protein complex binding at CHR promoter elements

    PubMed Central

    Quaas, Marianne; Müller, Gerd A.; Engeland, Kurt

    2012-01-01

    The tumor suppressor p53 plays an important role in cell cycle arrest by downregulating transcription. Many genes repressed by p53 code for proteins with functions in G₂/M. A large portion of these genes is controlled by cell cycle-dependent elements (CDE) and cell cycle genes homology regions (CHR) in their promoters. Cyclin B2 is an example of such a gene, with a function at the transition from G₂ to mitosis. We find that p53-dependent downregulation of cyclin B2 promoter activity is dependent on an intact CHR element. In the presence of high levels of p53 or p21WAF1/CIP1, protein binding to the CHR switches from MMB to DREAM complex by shifting MuvB core-associated proteins from B-Myb to E2F4/DP1/p130. The results suggest a model for p53-dependent transcriptional repression by which p53 directly activates p21WAF1/CIP1. The inhibitor then prevents further phosphorylation of p130 by cyclin-dependent kinases. The presence of hypophosphorylated pocket proteins shifts the equilibrium for complex formation from MMB to DREAM. In the case of promoters that do not hold CDE or E2F elements, binding of DREAM and MMB solely relies on a CHR site. Thus, p53 can repress target genes indirectly through CHR elements. PMID:23187802

  8. Correlation Among Six Biologic Factors (p53, p21{sup WAF1}, MIB-1, EGFR, HER2, and Bcl-2) and Clinical Outcomes After Curative Chemoradiation Therapy in Squamous Cell Cervical Cancer

    SciTech Connect

    Yamashita, Hideomi; Murakami, Naoya; Asari, Takao; Okuma, Kae; Ohtomo, Kuni; Nakagawa, Keiichi

    2009-07-15

    Purpose: The expressions of six cell-cycle-associated proteins were analyzed in cervical squamous cell carcinomas in correlation in a search for prognostic correlations in tumors treated with concurrent chemoradiation therapy (cCRT). Methods and Materials: The expressions of p53, p21/waf1/cip1, molecular immunology borstel-1 (MIB-1), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor type 2 (HER2), and Bcl-2 were studied using an immunohistochemical method in 57 cases of cervical squamous cell carcinoma treated with cCRT. Patients received cCRT between 1998 and 2005. The mean patient age was 61 years (range, 27-82 years). The number of patients with Stage II, III, and IVA disease was 18, 29, and 10, respectively. Results: The number of patients with tumors positive for p53, p21/waf1/cip1, MIB-1, EGFR, HER2, and Bcl-2 was 26, 24, 49, 26, 13, and 11, respectively; no significant correlation was noted. The 5-year overall survival rates of HER2-positive and -negative patients was 76% vs. 44%, which was of borderline significance (p = 0.0675). No significant correlation was noted between overall survival and expressions of p53, p21/waf1/cip1, MIB-1, EGFR, and Bcl-2. No correlation was observed between local control and expression of any of the proteins. Conclusion: Expression of HER2 protein had a weak impact of borderline significance on overall survival in squamous cell carcinoma of the uterine cervix treated with cCRT. However, no clinical associations could be established for p53, p21/waf1/cip1, MIB-1, EGFR, and Bcl-2 protein expressions.

  9. 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 p21waf1/cip1 expression

    PubMed Central

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

    2016-01-01

    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 p21waf/cip1, p27Kip1 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 p21waf/cip1 falling back moderately. Our findings suggest that attenuating dihydrofolate reductase may cause imbalanced expression of cell cycle regulators, especially up-regulation of p53-p21waf/cip1 pathway, leading to G1 cell cycle arrest, thereby inhibiting the growth of endothelial EA.hy926 cells. PMID:27013776

  10. The role of KLF4 in UVB-induced murine skin tumor development and its correlation with cyclin D1, p53, and p21(Waf1/Cip1) in epithelial tumors of the human skin.

    PubMed

    Choi, Woo Jin; Youn, Sung Hwan; Back, Jung Ho; Park, Saebomi; Park, Eun Joo; Kim, Kwang Joong; Park, Hye Rim; Kim, Arianna L; Kim, Kwang Ho

    2011-04-01

    The zinc-finger-type transcriptional factor KLF4 is expressed in a variety of tissues including skin. KLF4 can function as either a tumor suppressor or an oncogene, depending on the type of tissue in which it is expressed, by modulating the expression of various factors. To understand the role of KLF4 in human skin cancer and also to evaluate the expression of cyclin D1, p53, and p21(Waf1/Cip1) in relation to the expression of KLF4, we evaluated the pattern of KLF4 expression during UVB-induced skin tumor development in SKH-1 hairless mice and in human skin cancer. We also determined whether there are correlations between the expression of KLF4, cyclin D1, p53, and p21 and non-melanoma skin tumors. KLF4 expression was found in the basal layer of non-irradiated control murine skin. Chronic UVB irradiation caused a progressive decrease in KLF4 expression, which was substantially decreased in UVB-induced murine skin tumors. In human precancerous lesions, KLF4 expression was maintained in 64.3% of Bowen's disease samples and 90.0% of AK samples. In contrast, KLF4 expression was significantly reduced in human cancer lesions (p = 0.004). A positive correlation was found between the expression of KLF4 and p21(Waf1/Cip1) in AK, whereas there was a negative correlation between the expression of cyclin D1 and p21(Waf1/Cip1) in Bowen's disease. Thus, our results suggest that KLF4 may function as a tumor suppressor in the skin and that the deregulated expression of KLF4 in the context of p21(Waf1/Cip1) and cyclin D1 expression may be involved in skin tumorigenesis. PMID:21132436

  11. Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes*

    PubMed Central

    Choi, Won-Il; Yoon, Jae-Hyeon; Kim, Min-Young; Koh, Dong-In; Licht, Jonathan D.; Kim, Kunhong; Hur, Man-Wook

    2014-01-01

    Promyelocytic leukemia zinc finger-retinoic acid receptor α (PLZF-RARα) is an oncogene transcriptional repressor that is generated by a chromosomal translocation between the PLZF and RARα genes in acute promyelocytic leukemia (APL-type) patients. The molecular interaction between PLZF-RARα and the histone deacetylase corepressor was proposed to be important in leukemogenesis. We found that PLZF-RARα can repress transcription of the p21WAF/CDKN1A gene, which encodes the negative cell cycle regulator p21 by binding to its proximal promoter Sp1-binding GC-boxes 3, 4, 5/6, a retinoic acid response element (RARE), and distal p53-responsive elements (p53REs). PLZF-RARα also acts as a competitive transcriptional repressor of p53, RARα, and Sp1. PLZF-RARα interacts with co-repressors such as mSin3A, NCoR, and SMRT, thereby deacetylating histones Ac-H3 and Ac-H4 at the CDKN1A promoter. PLZF-RARα also interacts with the MBD3-NuRD complex, leading to epigenetic silencing of CDKN1A through DNA methylation. Furthermore, PLZF-RARα represses TP53 and increases p53 protein degradation by ubiquitination, further repressing p21 expression. Resultantly, PLZF-RARα promotes cell proliferation and significantly increases the number of cells in S-phase. PMID:24821728

  12. Expression of p53, p21(CIP1/WAF1) and eIF4E in the adjacent tissues of oral squamous cell carcinoma: establishing the molecular boundary and a cancer progression model.

    PubMed

    Li, Yi; Li, Bo; Xu, Bo; Han, Bo; Xia, Hui; Chen, Qian-Ming; Li, Long-Jiang

    2015-09-01

    The present study evaluated the expression of key molecules and the status of DNA in both oral squamous cell carcinoma (OSCC) and adjacent tissues to establish a molecular surgical boundary and provide a cancer progression model. Biopsy samples from 50 OSCC patients were divided into T (cancer), P1 (0-0.5 cm), P2 (0.5-1 cm), P3 (1-1.5 cm) and P4 (1.5-2 cm) groups based on the distances from the visible boundary of the primary focus. Twenty samples of normal mucosa were used as controls. We used immunohistochemical staining and flow cytometry to evaluate p53, p21(CIP1/WAF1), eIF4E and Ki-67 expression and to determine DNA status, respectively. Sub-mucosal invasion was present in the P1 and P2 groups as determined by haematoxylin and eosin staining. Mutant p53 expression decreased gradually from cancerous to normal mucosae, whereas p21(CIP1/WAF1) expression displayed an opposite trend. eIF4E expression decreased from cancerous to normal mucosae. Ki-67 expression, the heteroploidy ratio, S-phase fraction and proliferative index decreased gradually with the distance from the tumour centre. Based on these results, we suggest that the resection boundary in OSCC surgery should be beyond 2 cm from the tumour. Additionally, the adjacent tissues of the primary focus could be used as a model for assessing cancer progression. PMID:25835715

  13. 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

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

    2016-06-01

    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 p21(Waf1/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 p21(Waf1/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 p21(Waf1/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

  14. 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

  15. MDM2 promotes p21waf1/cip1 proteasomal turnover independently of ubiquitylation.

    PubMed

    Jin, Yetao; Lee, Hunjoo; Zeng, Shelya X; Dai, Mu-Shui; Lu, Hua

    2003-12-01

    The CDK inhibitor p21waf1/cip1 is degraded by a ubiquitin-independent proteolytic pathway. Here, we show that MDM2 mediates this degradation process. Overexpression of wild-type or ring finger-deleted, but not nuclear localization signal (NLS)-deleted, MDM2 decreased p21waf1/cip1 levels without ubiquitylating this protein and affecting its mRNA level in p53(-/-) cells. This decrease was reversed by the proteasome inhibitors MG132 and lactacystin, by p19(arf), and by small interfering RNA (siRNA) against MDM2. p21waf1/cip1 bound to MDM2 in vitro and in cells. The p21waf1/cip1-binding-defective mutant of MDM2 was unable to degrade p21waf1/cip1. MDM2 shortened the half-life of both exogenous and endogenous p21waf1/cip1 by 50% and led to the degradation of its lysine-free mutant. Consequently, MDM2 suppressed p21waf1/cip1-induced cell growth arrest of human p53(-/-) and p53(-/-)/Rb(-/-)cells. These results demonstrate that MDM2 directly inhibits p21waf1/cip1 function by reducing p21waf1/cip1 stability in a ubiquitin-independent fashion. PMID:14633995

  16. Drug 9AA reactivates p21/Waf1 and Inhibits HIV-1 progeny formation

    PubMed Central

    Wu, Weilin; Kehn-Hall, Kylene; Pedati, Caitlin; Zweier, Lynnsey; Castro, Iris; Klase, Zachary; Dowd, Cynthia S; Dubrovsky, Larisa; Bukrinsky, Michael; Kashanchi, Fatah

    2008-01-01

    It has been demonstrated that the p53 pathway plays an important role in HIV-1 infection. Previous work from our lab has established a model demonstrating how p53 could become inactivated in HIV-1 infected cells through binding to Tat. Subsequently, p53 was inactivated and lost its ability to transactivate its downstream target gene p21/waf1. P21/waf1 is a well-known cdk inhibitor (CKI) that can lead to cell cycle arrest upon DNA damage. Most recently, the p21/waf1 function was further investigated as a molecular barrier for HIV-1 infection of stem cells. Therefore, we reason that the restoration of the p53 and p21/waf1 pathways could be a possible theraputical arsenal for combating HIV-1 infection. In this current study, we show that a small chemical molecule, 9-aminoacridine (9AA) at low concentrations, could efficiently reactivate p53 pathway and thereby restoring the p21/waf1 function. Further, we show that the 9AA could significantly inhibit virus replication in activated PBMCs, likely through a mechanism of inhibiting the viral replication machinery. A mechanism study reveals that the phosphorylated p53ser15 may be dissociated from binding to HIV-1 Tat protein, thereby activating the p21/waf1 gene. Finally, we also show that the 9AA-activated p21/waf1 is recruited to HIV-1 preintegration complex, through a mechanism yet to be elucidated. PMID:18348731

  17. Absence of p21 expression is associated with abnormal p53 in human breast carcinomas.

    PubMed Central

    Ellis, P. A.; Lonning, P. E.; Borresen-Dale, A.; Aas, T.; Geisler, S.; Akslen, L. A.; Salter, I.; Smith, I. E.; Dowsett, M.

    1997-01-01

    The p53 tumour-suppressor gene is important in the regulation of cell growth and apoptosis, and loss of functional wild-type activity may be associated with tumour formation and resistance to therapy. Differentiation of functionally normal wild-type protein from mutant or abnormal protein remains difficult using either immunohistochemical assays or mutational DNA sequencing. p21(WAF1/CIP1) (p21) is induced by wild type p53 and plays an important role in promoting cell cycle arrest. To test the hypothesis that p21 protein expression may act as a downstream marker of tumours from patients with locally advanced breast cancer before treatment with doxorubicin, pretreatment p53 status had been characterized in 63 tumours by p53 protein immunostaining and DNA mutational analysis. There was a significant association between immunostaining for p53 and the presence of p53 mutations (P = 0.01). Of 56 patients available for determination of p21, 31 (55%) expressed p21 protein. Twenty-eight out of 31 patients (90%) positive for p21 had low negative p53 protein expression, whereas only 3 of 13 patients (23%) with high p53 expressed p21 (P = 0.009). No association was seen between p21 protein expression and p53 mutations (P = 0.24). The combination of p53 and p21 immunostaining results improved the specificity of the immunostaining but at a cost of significant reduction in sensitivity. Immunohistochemical assessment of p21 protein expression is inversely associated with abnormal p53 protein in human breast cancer. The detection of p21 protein expression in combination with p53 protein expression did not improve the ability of immunohistochemistry (IHC) to differentiate between normal and mutant p53 protein. Images Figure 1 PMID:9275025

  18. p21WAF1 immunohistochemical expression in breast carcinoma: correlations with clinicopathological data, oestrogen receptor status, MIB1 expression, p53 gene and protein alterations and relapse-free survival.

    PubMed Central

    Barbareschi, M.; Caffo, O.; Doglioni, C.; Fina, P.; Marchetti, A.; Buttitta, F.; Leek, R.; Morelli, L.; Leonardi, E.; Bevilacqua, G.; Dalla Palma, P.; Harris, A. L.

    1996-01-01

    p21 protein (p21) inhibitor of cyclin-dependent kinases is a critical downstream effector in the p53-specific pathway of growth control. p21 can also be induced by p53-independent pathways in relation to terminal differentiation. We investigated p21 immunoreactivity in normal breast and in 91 breast carcinomas [three in situ ductal carcinomas (DCIS) with microinfiltration and 88 infiltrating carcinomas, 17 of which with an associated DCIS; 57 node negative and 34 node positive] with long-term follow-up (median = 58 months). Seven additional breast carcinomas with known p53 gene mutations were investigated. In normal breast p21 expression was seen in the nuclei of rare luminal cells of acinar structures, and in occasional myoepithelial cells. Poorly differentiated DCIS showed high p21 expression, whereas well-differentiated DCIS tumours showed few p21-reactive cells. p21 was seen in 82 (90%) infiltrating tumours; staining was heterogeneous; the percentage of reactive nuclei ranged from 1% to 35%. High p21 expression (more than 10% of reactive cells) was seen in 24 (26%) cases, and was associated with high tumour grade (P = 0.032); no associations were seen with tumour size, metastases, oestrogen receptor status, MIB1 expression and p53 expression. p21 expression in cases with p53 gene mutations was low in six cases and high in one. High p21 expression was associated with short relapse-free survival (P = 0.003). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8688323

  19. Mutations in the TP53 gene and protein expression of p53, MDM 2 and p21/WAF-1 in primary cervical carcinomas with no or low human papillomavirus load.

    PubMed Central

    Helland, A.; Karlsen, F.; Due, E. U.; Holm, R.; Kristensen, G.; Børresen-Dale, A. l.

    1998-01-01

    Several studies have focused on the role of p53 inactivation in cervical cancer, either by inactivating mutations in the TP53 gene or by degradation of the p53 protein by human papillomavirus (HPV). In this study, primary cervical carcinomas from 365 patients were analysed for presence of HPV using both consensus primer-sets and type-specific primer-sets. Nineteen samples were determined to have no or low virus load, and were selected for further analyses: mutation screening of the TP53 gene using constant denaturant gel electrophoresis (CDGE) followed by sequencing, and protein expression of p53, MDM2 and p21 using immunohistochemistry (IHC). Mutations in the TP53 gene were found in eight samples (42%). Elevated p53 protein expression was significantly associated with presence of a mutation (P < 0.007). P21 protein expression was detected in 16 of the 19 carcinomas. No p21 expression was seen in normal cervical tissue. Two samples, both with wild-type p53, had elevated MDM2 expression. Compared with a previous study from our group, of mainly HPV-positive cervical carcinomas, in which only one sample was found to contain a TP53 mutation, a significantly higher mutation frequency (P < 0.001) was found among the carcinomas with no or low virus load. Although p53 inactivation pathways are not detected in every tumour, our study supports the hypothesis that p53 inactivation, either by binding to cellular or viral proteins or by mutation, is essential in the development of cervical carcinomas. Images Figure 1 PMID:9662253

  20. Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing.

    PubMed

    Galanos, Panagiotis; Vougas, Konstantinos; Walter, David; Polyzos, Alexander; Maya-Mendoza, Apolinar; Haagensen, Emma J; Kokkalis, Antonis; Roumelioti, Fani-Marlen; Gagos, Sarantis; Tzetis, Maria; Canovas, Begoña; Igea, Ana; Ahuja, Akshay K; Zellweger, Ralph; Havaki, Sofia; Kanavakis, Emanuel; Kletsas, Dimitris; Roninson, Igor B; Garbis, Spiros D; Lopes, Massimo; Nebreda, Angel; Thanos, Dimitris; Blow, J Julian; Townsend, Paul; Sørensen, Claus Storgaard; Bartek, Jiri; Gorgoulis, Vassilis G

    2016-07-01

    The cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4-CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery-an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs. PMID:27323328

  1. The p90 ribosomal S6 kinase (RSK) inhibitor BI-D1870 prevents gamma irradiation-induced apoptosis and mediates senescence via RSK- and p53-independent accumulation of p21WAF1/CIP1

    PubMed Central

    Neise, D; Sohn, D; Stefanski, A; Goto, H; Inagaki, M; Wesselborg, S; Budach, W; Stühler, K; Jänicke, R U

    2013-01-01

    The p90 ribosomal S6 kinase (RSK) family is a group of highly conserved Ser/Thr kinases that promote cell proliferation, growth, motility and survival. As they are almost exclusively activated downstream of extracellular signal-regulated kinases 1 and 2 (ERK1/2), therapeutic intervention by RSK inhibition is less likely to produce such severe side effects as those observed following inhibition of the upstream master regulators Raf, MEK and ERK1/2. Here, we report that BI-D1870, a potent small molecule inhibitor of RSKs, induces apoptosis, although preferentially, in a p21-deficient background. On the other hand, BI-D1870 also induces a strong transcription- and p53-independent accumulation of p21 protein and protects cells from gamma irradiation (γIR)-induced apoptosis, driving them into senescence even in the absence of γIR. Although we identified p21 in in vitro kinase assays as a novel RSK substrate that specifically becomes phosphorylated by RSK1-3 at Ser116 and Ser146, RNA-interference, overexpression and co-immunoprecipitation studies as well as the use of SL0101, another specific RSK inhibitor, revealed that BI-D1870 mediates p21 accumulation via a yet unknown pathway that, besides its off-site targets polo-like kinase-1 and AuroraB, also does also not involve RSKs. Thus, this novel off-target effect of BI-D1870 should be taken into serious consideration in future studies investigating the role of RSKs in cellular signaling and tumorigenesis. PMID:24136223

  2. Suppression of cancer cell growth by adenovirus expressing p21(WAF1/CIP1) deficient in PCNA interaction.

    PubMed

    Prabhu, N S; Blagosklonny, M V; Zeng, Y X; Wu, G S; Waldman, T; El-Deiry, W S

    1996-07-01

    p53 tumor suppression is deficient in the majority of human cancers. Efforts to understand this pathway have identified cyclin-dependent kinase (CDK) inhibitors and suggested a potential for their replacement in human cancer. In the present studies, expression of a C-terminal deletion mutant of the human p21(WAF1/CIP1) CDK inhibitor completely suppressed the growth of colon cancer cells, whereas full-length p21 only partially suppressed growth. We prepared a replication-deficient adenoviral recombinant which expresses the p21 C-terminal mutant (Ad-WAF1-341) and compared its tumor suppressive abilities with Ad-p53 and Ad-LacZ. Ad-WAF1-341- and Ad-p53-infected cancer cells, but not Ad-LacZ-infected cancer cells, expressed a nuclear protein recognized by anti-p21 antibody and were deficient in cell cycle progression. The exogenous p21 mutant interacted with CDK2 but not proliferating cell nuclear antigen following infection of p21-/- cancer cells. Ad-WAF1-341 was more potent than Ad-p53 in inhibiting DNA synthesis in human papillomavirus 16 E6-expressing cancer cells. Most importantly, the Ad-WAF1-341-infected E6-expressing cells died, whereas most of the Ad-p53-infected cells continued to proliferate. Endonucleolytic cleavage of DNA was observed in Ad-WAF1-341-infected cancer cells. These observations suggest that Ad-WAF1-341 should be evaluated in the treatment of human papillomavirus-associated neoplasia and other neoplasias resistant to p53. PMID:9816291

  3. Prognostic implications of p21 (Waf1/Cip1) immunolocalization in multiple myeloma.

    PubMed

    Ohata, Masahiko; Nakamura, Shinobu; Fujita, Hiroyuki; Isemura, Mamoru

    2005-06-01

    Protein p21 (Waf1/Cip1) plays a critical role in controlling the cell cycle especially as a check point of G1 and is intimately associated with important cellular activities including differentiation, senescence, tumorigenesis, and apoptosis. In the present study, we examined the expression of p21 in multiple myeloma (MM) cells for prognostic evaluation. The immunocytochemical localization of p21 could be categorized into nuclear and cytoplasmic types. The nuclear-type p21 localization was correlated with the severity of MM and the expression of proliferating cell nuclear antigen and p53. Patients with the nuclear-type p21 localization survived significantly shorter than those with the cytoplasmic-type localization. Thus, the present study suggests that p21-immunolocalization can be a useful prognostic marker of MM. PMID:16011301

  4. Loss of p21WAF1 compartmentalisation in sebaceous carcinoma compared with sebaceous hyperplasia and sebaceous adenoma

    PubMed Central

    McBride, S R; Leonard, N; Reynolds, N J

    2002-01-01

    Aims: Regulation of cell cycle progression is a fundamental control process, linked to cellular differentiation and apoptosis in normal tissues. p21WAF1 is a nuclear protein that regulates cell cycle progression. p21WAF1 can be transcriptionally upregulated by p53, but may be activated independently of p53—for example, during terminal differentiation. Loss of topological control of p21WAF1 expression is an early feature of malignancy in the colorectal system. Similar to the colonic mucosa, sebaceous glands contain cells that are constantly going through a process of cell division, differentiation, and cell death. This study investigated the expression of p53, p21WAF1, and the proliferation marker Ki67 in normal sebaceous glands, sebaceous adenoma, sebaceoma, and sebaceous carcinoma. Methods: Serial sections were stained with monoclonal antibodies to p21WAF1, p53, and Ki67 (MIB1) using standard immunohistochemical techniques. Results: In normal sebaceous glands, p21WAF1 positive cells were only seen within the differentiating compartment, which was spatially distinct from the cycling peripheral Ki67 positive cells. In sebaceous adenoma and sebaceoma, topological control was maintained, with the distribution of markers being similar to that seen in normal sebaceous glands. Loss of topological control of markers of cellular control was seen in sebaceous carcinoma only. This contrasts with colonic tumours, in which loss of p21 compartmentalisation is seen in adenomas at an early stage of tumour progression. Conclusion: This work confirms the hypothesis that the dysregulation of cell cycle progression is an important process in the development of malignancy within sebaceous glands, although loss of topological control was seen only in sebaceous carcinoma. PMID:12354803

  5. FOXO target gene CTDSP2 regulates cell cycle progression through Ras and p21Cip1/Waf1

    PubMed Central

    Kloet, David E.A.; Polderman, Paulien E.; Eijkelenboom, Astrid; Smits, Lydia M.; vanTriest, Miranda H.; vandenBerg, Maaike C.W.; Koerkamp, Marian J. Groot; vanLeenen, Dik; Lijnzaad, Philip; Holstege, Frank C.; Burgering, Boudewijn M.T.

    2015-01-01

    Activity of FOXO (forkhead box O) transcription factors is inhibited by growth factor–PI3K (phosphoinositide 3-kinase)–PKB (protein kinase B)/Akt signalling to control a variety of cellular processes including cell cycle progression. Through comparative analysis of a number of microarray datasets we identified a set of genes commonly regulated by FOXO proteins and PI3K–PKB/Akt, which includes CTDSP2 (C-terminal domain small phosphatase 2). We validated CTDSP2 as a genuine FOXO target gene and show that ectopic CTDSP2 can induce cell cycle arrest. We analysed transcriptional regulation after CTDSP2 expression and identified extensive regulation of genes involved in cell cycle progression, which depends on the phosphatase activity of CTDSP2. The most notably regulated gene is the CDK (cyclin-dependent kinase) inhibitor p21Cip1/Waf1 and in the present study we show that p21Cip1/Waf1 is partially responsible for the cell cycle arrest through decreasing cyclin–CDK activity. Our data suggest that CTDSP2 induces p21Cip1/Waf1 through increasing the activity of Ras. As has been described previously, Ras induces p21Cip1/Waf1 through p53-dependent and p53-independent pathways and indeed both p53 and MEK inhibition can mitigate the CTDSP2-induced p21Cip1/Waf1 mRNA up-regulation. In support of Ras activation by CTDSP2, depletion of endogenous CTDSP2 results in reduced Ras activity and thus CTDSP2 seems to be part of a larger set of genes regulated by FOXO proteins, which increase growth factor signalling upon FOXO activation. PMID:25990325

  6. Endosome traffic machinery meets the p53p21 axis

    PubMed Central

    Barroso-González, Jonathan; Thomas, Gary

    2015-01-01

    SIRT1 regulates p53 transcriptional activation in response to genotoxic insult by deacetylating key lysine residues. We recently identified the multifunctional protein PACS-2 as a SIRT1 inhibitor. After DNA damage, PACS-2 binds and inhibits SIRT1 to increase p53-dependent transactivation of the CDK inhibitor p21 (CDKN1A) and induce cell cycle arrest. PMID:25815376

  7. [Prognosis value of the immunohystochemical expresion of the p21WAF1 in the larynx cancer].

    PubMed

    García Lozano, M C; Orradre Romero, J L; Caro García, M; Abril García, A; Piris Pinilla, M A

    2005-01-01

    In this paper we carried out an immunohistochemical study of protein p21WAF1 (EA10) expression in a series of 195 patients with laryngeal carcinoma that were diagnosticated, treated and followed at the Department of Otolaryngology at "Virgen de la Salud" Hospital (Toledo, Spain). In the cases with lymph node metastasis we also studied p21WAF1 expression at this level. Furthermore we have analysed the value of protein p21WAF1 expression as a prognostic factor (tumor recurrence, deads due to cancer and survival) and we evaluate the relationship between p21WAF1 expression and other clinic and pathologic parameters. PMID:15803916

  8. ZNF307, a novel zinc finger gene suppresses p53 and p21 pathway

    SciTech Connect

    Li Jing; Wang Yuequn; Fan Xiongwei; Mo Xiaoyang; Wang Zequn; Li Yongqing; Yin Zhaochu; Deng Yun; Luo Na; Zhu Chuanbing; Liu Mingyao; Ma Qian; Ocorr, Karen Yuan Wuzhou Wu Xiushan

    2007-11-30

    We have cloned a novel KRAB-related zinc finger gene, ZNF307, encoding a protein of 545 aa. ZNF307 is conserved across species in evolution and is differentially expressed in human adult and fetal tissues. The fusion protein of EGFP-ZNF307 localizes in the nucleus. Transcriptional activity assays show ZNF307 suppresses transcriptional activity of L8G5-luciferase. Overexpressing ZNF307 in different cell lines also inhibits the transcriptional activities of p53 and p21. Moreover, ZNF307 works by reducing the p53 protein level and p53 protein reduction is achieved by increasing transcription of MDM2 and EP300. ZNF307 might suppress p53-p21 pathway through activating MDM2 and EP300 expression and inducing p53 degradation.

  9. Cristacarpin promotes ER stress-mediated ROS generation leading to premature senescence by activation of p21(waf-1).

    PubMed

    Chakraborty, Souneek; Rasool, Reyaz Ur; Kumar, Sunil; Nayak, Debasis; Rah, Bilal; Katoch, Archana; Amin, Hina; Ali, Asif; Goswami, Anindya

    2016-06-01

    Stress-induced premature senescence (SIPS) is quite similar to replicative senescence that is committed by cells exposed to various stress conditions viz. ultraviolet radiation (DNA damage), hydrogen peroxide (oxidative stress), chemotherapeutic agents (cytotoxic threat), etc. Here, we report that cristacarpin, a natural product obtained from the stem bark of Erythrina suberosa, promotes endoplasmic reticulum (ER) stress, leading to sub-lethal reactive oxygen species (ROS) generation and which eventually terminates by triggering senescence in pancreatic and breast cancer cells through blocking the cell cycle in the G1 phase. The majority of cristacarpin-treated cells responded to conventional SA-β-gal stains; showed characteristic p21(waf1) upregulation along with enlarged and flattened morphology; and increased volume, granularity, and formation of heterochromatin foci-all of these features are the hallmarks of senescence. Inhibition of ROS generation by N-acetyl-L-cysteine (NAC) significantly reduced the expression of p21(waf1), confirming that the modulation in p21(waf1) by anti-proliferative cristacarpin was ROS dependent. Further, the elevation in p21(waf1) expression in PANC-1 and MCF-7 cells was consistent with the decrease in the expression of Cdk-2 and cyclinD1. Here, we provide evidence that cristacarpin promotes senescence in a p53-independent manner. Moreover, cristacarpin treatment induced p38MAPK, indicating the ROS-dependent activation of the MAP kinase pathway, and thus abrogates the tumor growth in mouse allograft tumor model. PMID:27246693

  10. Methionine sulfoxide reductase A regulates cell growth through the p53-p21 pathway

    SciTech Connect

    Choi, Seung Hee; Kim, Hwa-Young

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Down-regulation of MsrA inhibits normal cell proliferation. Black-Right-Pointing-Pointer MsrA deficiency leads to an increase in p21 by enhanced p53 acetylation. Black-Right-Pointing-Pointer Down-regulation of MsrA causes cell cycle arrest at the G{sub 2}/M stage. Black-Right-Pointing-Pointer MsrA is a regulator of cell growth that mediates the p53-p21 pathway. -- Abstract: MsrA is an oxidoreductase that catalyzes the stereospecific reduction of methionine-S-sulfoxide to methionine. Although MsrA is well-characterized as an antioxidant and has been implicated in the aging process and cellular senescence, its roles in cell proliferation are poorly understood. Here, we report a critical role of MsrA in normal cell proliferation and describe the regulation mechanism of cell growth by this protein. Down-regulation of MsrA inhibited cell proliferation, but MsrA overexpression did not promote it. MsrA deficiency led to an increase in p21, a major cyclin-dependent kinase inhibitor, thereby causing cell cycle arrest at the G{sub 2}/M stage. While protein levels of p53 were not altered upon MsrA deficiency, its acetylation level was significantly elevated, which subsequently activated p21 transcription. The data suggest that MsrA is a regulator of cell growth that mediates the p53-p21 pathway.

  11. Cyclin kinase inhibitor p21WAF1/CIP1 in malignant melanoma: reduced expression in metastatic lesions.

    PubMed Central

    Maelandsmo, G. M.; Holm, R.; Fodstad, O.; Kerbel, R. S.; Flørenes, V. A.

    1996-01-01

    Immunohistochemical analysis of the expression of the cyclin kinase inhibitor p21WAF1/CIP1 in a panel of primary and metastatic human melanocytic tumors was performed. It was found that, independent of the p53 status, approximately 30% of the primary melanomas and 40% of the metastases completely lacked expression of this cell cycle inhibitor. Some tumors were also analyzed by Northern blotting, and in most of the cases a consistant correlation between mRNA and protein expression was observed. In four benign nevi studied, WAF1/CIP1 mRNA was expressed whereas the protein was not detected, suggesting a post-transcriptional regulation of the inhibitor in these cases. In superficial spreading melanomas, a significant correlation between protein expression and tumor thickness was found, with thin lesions showing low protein levels. Interestingly, by comparing primary and metastatic specimens obtained from the same patient, a reduction in p21WAF1/CIP1 antibody staining was observed in the latter, probably reflecting a more aggressive phenotype of the metastases. In conclusion, our results demonstrate the complexity in the relationship between p21WAF1/CIP1 expression and tumor phenotype and furthermore suggest that aberrant expression of the cyclin-dependent kinase inhibitor may be of importance in the development and progression of sporadic malignant melanoma. Images Figure 1 Figure 2 PMID:8952518

  12. Suppression of Indued Pluripotent Stem Cell Generation by the p53-p21 Pathway

    PubMed Central

    Hong, Hyenjong; Takahashi, Kazutoshi; Ichisaka, Tomoko; Aoi, Takashi; Kanagawa, Osami; Nakagawa, Masato; Okita, Keisuke; Yamanaka, Shinya

    2010-01-01

    Induced pluripotent stem (iPS) cells can be generated from somatic cells by introduction of Oct3/4, Sox2, Klf4 and c-Myc, in mouse1-4 and human5-8. Efficiency of this process, however, is low9. Pluripotency can be induced without c-Myc, but with even lower efficiency10,11. A p53 siRNA was recently shown to promote human iPS cell generation12, but specificity and mechanisms remain to be determined. Here we report that up to 10% of transduced mouse embryonic fibroblasts (MEF) lacking p53 became iPS cells, even without the Myc retrovirus. The p53 deletion also promoted induction of integration-free mouse iPS cells with plasmid transfection. Furthermore, in the p53-null background, iPS cells were generated from terminally differentiated T lymphocytes. Suppression of p53 also increased the efficiency of human iPS cell generation. DNA microarray analyses identified 34 p53-regulated genes that are common in mouse and human fibroblasts. Functional analyses of these genes demonstrate that the p53-p21 pathway serves as a safeguard not only in tumorigenicity, but also in iPS cell generation. PMID:19668191

  13. p21{sup WAF1/CIP1} deficiency induces mitochondrial dysfunction in HCT116 colon cancer cells

    SciTech Connect

    Kim, Ae Jeong; Jee, Hye Jin; Song, Naree; Kim, Minjee; Jeong, Seon-Young; Yun, Jeanho

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer p21{sup -/-} HCT116 cells exhibited an increase in mitochondrial mass. Black-Right-Pointing-Pointer The expression levels of PGC-1{alpha} and AMPK were upregulated in p21{sup -/-} HCT116 cells. Black-Right-Pointing-Pointer The proliferation of p21{sup -/-} HCT116 cells in galactose medium was significantly impaired. Black-Right-Pointing-Pointer p21 may play a role in maintaining proper mitochondrial mass and respiratory function. -- Abstract: p21{sup WAF1/CIP1} is a critical regulator of cell cycle progression. However, the role of p21 in mitochondrial function remains poorly understood. In this study, we examined the effect of p21 deficiency on mitochondrial function in HCT116 human colon cancer cells. We found that there was a significant increase in the mitochondrial mass of p21{sup -/-} HCT116 cells, as measured by 10-N-nonyl-acridine orange staining, as well as an increase in the mitochondrial DNA content. In contrast, p53{sup -/-} cells had a mitochondrial mass comparable to that of wild-type HCT116 cells. In addition, the expression levels of the mitochondrial biogenesis regulators PGC-1{alpha} and TFAM and AMPK activity were also elevated in p21{sup -/-} cells, indicating that p21 deficiency induces the rate of mitochondrial biogenesis through the AMPK-PGC-1{alpha} axis. However, the increase in mitochondrial biogenesis in p21{sup -/-} cells did not accompany an increase in the cellular steady-state level of ATP. Furthermore, p21{sup -/-} cells exhibited significant proliferation impairment in galactose medium, suggesting that p21 deficiency induces a defect in the mitochondrial respiratory chain in HCT116 cells. Taken together, our results suggest that the loss of p21 results in an aberrant increase in the mitochondrial mass and in mitochondrial dysfunction in HCT116 cells, indicating that p21 is required to maintain proper mitochondrial mass and respiratory function.

  14. The anti-leukemic activity of sodium dichloroacetate in p53mutated/null cells is mediated by a p53-independent ILF3/p21 pathway

    PubMed Central

    Agnoletto, Chiara; Brunelli, Laura; Melloni, Elisabetta; Pastorelli, Roberta; Casciano, Fabio; Rimondi, Erika; Rigolin, Gian Matteo; Cuneo, Antonio; Secchiero, Paola; Zauli, Giorgio

    2015-01-01

    B-chronic lymphocytic leukemia (B-CLL) patients harboring p53 mutations are invariably refractory to therapies based on purine analogues and have limited treatment options and poor survival. Having recently demonstrated that the mitochondria-targeting small molecule sodium dichloroacetate (DCA) exhibits anti-leukemic activity in p53wild-type B-CLL cells, the aim of this study was to evaluate the effect of DCA in p53mutated B-CLL cells and in p53mutated/null leukemic cell lines. DCA exhibited comparable cytotoxicity in p53wild-type and p53mutated B-CLL patient cell cultures, as well as in p53mutated B leukemic cell lines (MAVER, MEC-1, MEC-2). At the molecular level, DCA promoted the transcriptional induction of p21 in all leukemic cell types investigated, including p53null HL-60. By using a proteomic approach, we demonstrated that DCA up-regulated the ILF3 transcription factor, which is a known regulator of p21 expression. The role of the ILF3/p21 axis in mediating the DCA anti-leukemic activity was underscored by knocking-down experiments. Indeed, transfection with ILF3 and p21 siRNAs significantly decreased both the DCA-induced p21 expression and the DCA-mediated cytotoxicity. Taken together, our results emphasize that DCA is a small molecule that merits further evaluation as a therapeutic agent also for p53mutated leukemic cells, by acting through the induction of a p53-independent pathway. PMID:25544776

  15. 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.

  16. INMAP Overexpression Inhibits Cell Proliferation, Induces Genomic Instability and Functions through p53/p21 Pathways

    PubMed Central

    Zhu, Yan; Lei, Yan; Du, Baochen; Zheng, Yanbo; Lu, Xiangfeng; Tan, Tan; Kang, Jingting; Sun, Le; Liang, Qianjin

    2015-01-01

    INMAP is a spindle protein that plays essential role for mitosis, by ensuring spindle and centromere integrality. The aim of this study was to investigate the relevant functions of INMAP for genomic stability and its functional pathway. We overexpressed INMAP in HeLa cells, resulting in growth inhibition in monolayer cell cultures, anchorage-independent growth in soft agar and xenograft growth in nude mice. In this system caused micronuclei (MNi) formation, chromosome distortion and γH2AX expression upregulation, suggesting DNA damage induction and genomic stability impairment. As a tumour biochemical marker, lactate dehydrogenase (LDH) isoenzymes were detected to evaluate cell metabolic activity, the results confirming that total activity of LDH, as well as that of its LDH5 isoform, is significantly decreased in INMAP-overexpressing HeLa cells. The levels of p53 and p21 were upregulated, and however, that of PCNA and Bcl-2, downregulated. Indirect immunofluorescence (IIF) and coimmunoprecipitation (CoIP) analyses revealed the interaction between INMAP and p21. These results suggest that INMAP might function through p53/p21 pathways. PMID:25635878

  17. Armoring CRAds with p21/Waf-1 shRNAs: the next generation of oncolytic adenoviruses

    PubMed Central

    Höti, N; Chowdhury, WH; Mustafa, S; Ribas, J; Castanares, M; Johnson, T; Liu, M; Lupold, SE; Rodriguez, R

    2011-01-01

    Conditionally replicating adenoviruses (CRAds) represent a promising modality for the treatment of neoplastic diseases, including Prostate Cancer. Selectively replicating viruses can be generated by placing a tissue or cancer-specific promoter upstream of one or more of the viral genes required for replication (for example, E1A, E1B). We have previously reported multiple cellular processes that can attenuate viral replication, which in turn compromises viral oncolysis and tumor kill. In this study, we investigated the importance of the cyclin-dependent kinase inhibitor p21/Waf-1, on viral replication and tumor growth. To our knowledge, this is the first report describing the importance of p21/Waf-1shRNA on the induction of an androgen responsive element (ARE) based promoter driving the E1A gene. As a proof of concept, the study emphasizes the use of RNA interference technology to overcome promoter weaknesses for tissue-specific oncolytic viruses, as well as the cellular inhibitor pathways on viral life cycle. Using RNA interference against p21/Waf-1, we were able to show an increase in viral replication and viral oncolysis of prostate cancer cells. Similarly, CRAd viruses that carry p21/Waf-1 shRNA (Ad5-RV004.21) were able to prevent tumor outgrowth that resulted in a marked increase in the mean survival time of tumor-bearing mice compared with CRAd without p21/Waf-1 shRNA (Ad5-RV004). In studies combining Ad5-RV004.21 with Adriamycin, a suprar-additive effect was observed only in CRAds that harbor shRNA against p21/Waf-1. Taken together, these findings of enhanced viral replication in prostate cancer cells by using RNA interference against the cdk inhibitor p21/Waf-1 have significant implications in the development of prostate-specific CRAd therapies. PMID:20448671

  18. Loss of p21WAF1/Cip1 in Gadd45-deficient keratinocytes restores DNA repair capacity.

    PubMed

    Maeda, Tomoko; Espino, Robin A; Chomey, Eugene G; Luong, Le; Bano, Ather; Meakins, Diana; Tron, Victor A

    2005-10-01

    Ultraviolet light (UV)-induced DNA damage is repaired primarily by the nucleotide excision repair (NER) pathway. Gadd45 is a multifunctional protein that regulates NER. Gadd45-deficient keratinocytes fail to repair UV-induced DNA damage, but the mechanism by which Gadd45 stimulates repair of UV-induced DNA damage is unknown. p21WAF1/Cip1 (p21) is a well-characterized downstream target of p53 that binds to Gadd45 and proliferating cell nuclear antigen (PCNA). The role of p21 in NER is somewhat controversial, however, recent studies appear to suggest that it inhibits DNA repair by inhibiting PCNA activity. Since a physical interplay exists between p21, Gadd45 and PCNA, we hypothesized that Gadd45 promoted DNA repair via p21. Initially, we examined p21 protein expression in Gadd45-deficient and proficient mice and found a higher base level of p21 protein in Gadd45-deficient keratinocytes and in most other tissues. With these results, we next speculated on the role played by p21 in Gadd45 regulated NER, by exposing keratinocytes from wild-type, single and double knockout (Gadd45 and p21) mice to UV, and measuring the responses. We confirmed that Gadd45-deficient keratinocytes were defective in UV-induced NER, but interestingly Gadd45/p21-null keratinocytes had normal NER in response to UV. Furthermore, Gadd45/p21-null keratinocytes were more resistant to UV-induced cell death than Gadd45-deficient keratinocytes. These results support the hypothesis that Gadd45 enhances NER by negatively regulating basal p21 expression in keratinocytes. PMID:15917306

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  1. Anti-Colon Cancer Effects of 6-Shogaol Through G2/M Cell Cycle Arrest by p53/p21-cdc2/cdc25A Crosstalk.

    PubMed

    Qi, Lian-Wen; Zhang, Zhiyu; Zhang, Chun-Feng; Anderson, Samantha; Liu, Qun; Yuan, Chun-Su; Wang, Chong-Zhi

    2015-01-01

    Chemopreventive agents can be identified from botanicals. Recently, there has been strong support for the potential of 6-shogaol, a natural compound from dietary ginger (Zingiber officinale), in cancer chemoprevention. However, whether 6-shogaol inhibits the growth of colorectal tumors in vivo remains unknown, and the underlying anticancer mechanisms have not been well characterized. In this work, we observed that 6-shogaol (15 mg/kg) significantly inhibited colorectal tumor growth in a xenograft mouse model. We show that 6-shogaol inhibited HCT-116 and SW-480 cell proliferation with IC50 of 7.5 and 10 μM, respectively. Growth of HCT-116 cells was arrested at the G2/M phase of the cell cycle, primarily mediated by the up-regulation of p53, the CDK inhibitor p21(waf1/cip1) and GADD45α, and by the down-regulation of cdc2 and cdc25A. Using p53(-/-) and p53(+/+) HCT-116 cells, we confirmed that p53/p21 was the main pathway that contributed to the G2/M cell cycle arrest by 6-shogaol. 6-Shogaol induced apoptosis, mainly through the mitochondrial pathway, and the bcl-2 family might act as a key regulator. Our results demonstrated that 6-shogaol induces cancer cell death by inducing G2/M cell cycle arrest and apoptosis. 6-Shogaol could be an active natural product in colon cancer chemoprevention. PMID:26119958

  2. The p53-p21-DREAM-CDE/CHR pathway regulates G2/M cell cycle genes

    PubMed Central

    Fischer, Martin; Quaas, Marianne; Steiner, Lydia; Engeland, Kurt

    2016-01-01

    The tumor suppressor p53 functions predominantly as a transcription factor by activating and downregulating gene expression, leading to cell cycle arrest or apoptosis. p53 was shown to indirectly repress transcription of the CCNB2, KIF23 and PLK4 cell cycle genes through the recently discovered p53-p21-DREAM-CDE/CHR pathway. However, it remained unclear whether this pathway is commonly used. Here, we identify genes regulated by p53 through this pathway in a genome-wide computational approach. The bioinformatic analysis is based on genome-wide DREAM complex binding data, p53-depedent mRNA expression data and a genome-wide definition of phylogenetically conserved CHR promoter elements. We find 210 target genes that are expected to be regulated by the p53-p21-DREAM-CDE/CHR pathway. The target gene list was verified by detailed analysis of p53-dependent repression of the cell cycle genes B-MYB (MYBL2), BUB1, CCNA2, CCNB1, CHEK2, MELK, POLD1, RAD18 and RAD54L. Most of the 210 target genes are essential regulators of G2 phase and mitosis. Thus, downregulation of these genes through the p53-p21-DREAM-CDE/CHR pathway appears to be a principal mechanism for G2/M cell cycle arrest by p53. PMID:26384566

  3. The p53-p21-DREAM-CDE/CHR pathway regulates G2/M cell cycle genes.

    PubMed

    Fischer, Martin; Quaas, Marianne; Steiner, Lydia; Engeland, Kurt

    2016-01-01

    The tumor suppressor p53 functions predominantly as a transcription factor by activating and downregulating gene expression, leading to cell cycle arrest or apoptosis. p53 was shown to indirectly repress transcription of the CCNB2, KIF23 and PLK4 cell cycle genes through the recently discovered p53-p21-DREAM-CDE/CHR pathway. However, it remained unclear whether this pathway is commonly used. Here, we identify genes regulated by p53 through this pathway in a genome-wide computational approach. The bioinformatic analysis is based on genome-wide DREAM complex binding data, p53-depedent mRNA expression data and a genome-wide definition of phylogenetically conserved CHR promoter elements. We find 210 target genes that are expected to be regulated by the p53-p21-DREAM-CDE/CHR pathway. The target gene list was verified by detailed analysis of p53-dependent repression of the cell cycle genes B-MYB (MYBL2), BUB1, CCNA2, CCNB1, CHEK2, MELK, POLD1, RAD18 and RAD54L. Most of the 210 target genes are essential regulators of G2 phase and mitosis. Thus, downregulation of these genes through the p53-p21-DREAM-CDE/CHR pathway appears to be a principal mechanism for G2/M cell cycle arrest by p53. PMID:26384566

  4. Indirect p53-dependent transcriptional repression of Survivin, CDC25C, and PLK1 genes requires the cyclin-dependent kinase inhibitor p21/CDKN1A and CDE/CHR promoter sites binding the DREAM complex

    PubMed Central

    Nickel, Annina; Engeland, Kurt

    2015-01-01

    The transcription factor p53 is central to cell cycle control by downregulation of cell cycle-promoting genes upon cell stress such as DNA damage. Survivin (BIRC5), CDC25C, and PLK1 encode important cell cycle regulators that are repressed following p53 activation. Here, we provide evidence that p53-dependent repression of these genes requires activation of p21 (CDKN1A, WAF1, CIP1). Chromatin immunoprecipitation (ChIP) data indicate that promoter binding of B-MYB switches to binding of E2F4 and p130 resulting in a replacement of the MMB (Myb-MuvB) by the DREAM complex. We demonstrate that this replacement depends on p21. Furthermore, transcriptional repression by p53 requires intact DREAM binding sites in the target promoters. The CDE and CHR cell cycle promoter elements are the sites for DREAM binding. These elements as well as the p53 response of Survivin, CDC25C, and PLK1 are evolutionarily conserved. No binding of p53 to these genes is detected by ChIP and mutation of proposed p53 binding sites does not alter the p53 response. Thus, a mechanism for direct p53-dependent transcriptional repression is not supported by the data. In contrast, repression by DREAM is consistent with most previous findings and unifies models based on p21-, E2F4-, p130-, and CDE/CHR-dependent repression by p53. In conclusion, the presented data suggest that the p53-p21-DREAM-CDE/CHR pathway regulates p53-dependent repression of Survivin, CDC25C, and PLK1. PMID:26595675

  5. Analysis of p21Waf1/Cip1 expression in normal, premalignant, and malignant cells during the development of human lung adenocarcinoma.

    PubMed Central

    Hayashi, H.; Miyamoto, H.; Ito, T.; Kameda, Y.; Nakamura, N.; Kubota, Y.; Kitamura, H.

    1997-01-01

    Our studies suggested that adenocarcinoma of the peripheral lung mostly develops by several steps from atypical adenomatous hyperplasia through early adenocarcinoma to overt adenocarcinoma, and that some p53 abnormalities play an important role in this progression. In the present study, we examined by immunohistochemistry the expression of p53-inducible cyclin-dependent kinase inhibitor p21Waf1/Cip1 (p21) in the cells at various developmental stages of lung adenocarcinoma (32 lesions of adenomatous hyperplasia, 14 of early adenocarcinoma, 23 of well differentiated adenocarcinoma, and 17 of moderately or poorly differentiated adenocarcinoma) in comparison with 19 reactive proliferative lesions and analyzed the relationship between p53 and p21 expression. Bronchioalveolar cells in the normal lung expressed very little or no p21 and no p53 expression. In not only reactive but also neoplastic lesions regardless of their developmental stage, the cells expressed p21 at various frequencies. The average labeling indices ranged from 5.4 to 13.8%, and there was no significant difference between any of these categories. The expression of p21, however, tended to be relatively low in moderately and poorly differentiated adenocarcinomas (5.5%) compared to well differentiated adenocarcinomas (12.2%), and high-level p21 expressors (10% < or = positive cells) were more frequent in the latter group (1 of 17 (6%) versus 3 of 23 (35%), P < 0.05), suggesting that p21 expression is affected by the degree of differentiation of the neoplastic cells. Although the correlation was positive between the expression of p21 and p53 in reactive lesions (r = 0.88; P < 0.001), none was found in neoplastic lesions at any step or grade (-0.12 < or = r < or = 0.26). These results indicated that p21 expression depends upon p53 expression in reactive lung cells, whereas p21 expression is at least in part independent of that of p53 from the earliest to the most fully developed step of lung adenocarcinoma

  6. Evodiamine selectively targets cancer stem-like cells through the p53-p21-Rb pathway.

    PubMed

    Han, Seula; Woo, Jong Kyu; Jung, Yuchae; Jeong, Dawoon; Kang, Minsook; Yoo, Young-Ji; Lee, Hani; Oh, Seung Hyun; Ryu, Jae-Ha; Kim, Woo-Young

    2016-01-22

    In spite of the recent improvements, the resistance to chemotherapy/radiotherapy followed by relapse is the main hurdle for the successful treatment of breast cancer, a leading cause of death in women. A small population of breast cancer cells that have stem-like characteristics (cancer stem-like cells; CSLC) may contribute to this resistance and relapse. Here, we report on a component of a traditional Chinese medicine, evodiamine, which selectively targets CSLC of breast cancer cell lines MCF7 and MDAMB 231 at a concentration that does show a little or no cytotoxic effect on bulk cancer cells. While evodiamine caused the accumulation of bulk cancer cells at the G2/M phase, it did not hold CSLC in a specific cell cycle phase but instead, selectively killed CSLC. This was not due to the culture of CSLC in suspension or without FBS. A proteomic analysis and western blotting revealed that evodiamine changed the expression of cell cycle regulating molecules more efficiently in CSLC cells than in bulk cancer cells. Surprisingly, evodiamine selectively activated p53 and p21 and decreased inactive Rb, the master molecules in G1/S checkpoint. These data collectively suggest a novel mechanism involving CSLC-specific targeting by evodiamine and its possible use to the therapy of breast cancer. PMID:26713361

  7. Senescence evasion in melanoma progression: uncoupling of DNA-damage signaling from p53 activation and p21 expression.

    PubMed

    Mackenzie Ross, Alastair D; Cook, Martin G; Chong, Heung; Hossain, Mehnaz; Pandha, Hardev S; Bennett, Dorothy C

    2013-03-01

    The best-established function of the melanoma-suppressor p16 is mediation of cell senescence, a permanent arrest following cell proliferation or certain stresses. The importance of p16 in melanoma suggests indolence of the other major senescence pathway through p53. Little or no p53 is expressed in senescent normal human melanocytes, but p16-deficient melanocytes can undergo p53-mediated senescence. As p16 expression occurs in nevi but falls with progression toward melanoma, we here investigated whether p53-dependent senescence occurs at some stage and, if not, what defects were detectable in this pathway, using immunohistochemistry. Phosphorylated checkpoint kinase 2 (CHEK2) can mediate DNA-damage signaling, and under some conditions senescence, by phosphorylating and activating p53. Remarkably, we detected no prevalent p53-mediated senescence in any of six classes of lesions. Two separate defects in p53 signaling appeared common: in nevi, lack of p53 phosphorylation by activated CHEK2, and in melanomas, defective p21 upregulation by p53 even when phosphorylated. PMID:23253087

  8. Senescence evasion in melanoma progression: uncoupling of DNA-damage signaling from p53 activation and p21 expression

    PubMed Central

    MacKenzie Ross, Alastair D; Cook, Martin G; Chong, Heung; Hossain, Mehnaz; Pandha, Hardev S; Bennett, Dorothy C

    2013-01-01

    The best-established function of the melanoma-suppressor p16 is mediation of cell senescence, a permanent arrest following cell proliferation or certain stresses. The importance of p16 in melanoma suggests indolence of the other major senescence pathway through p53. Little or no p53 is expressed in senescent normal human melanocytes, but p16-deficient melanocytes can undergo p53-mediated senescence. As p16 expression occurs in nevi but falls with progression toward melanoma, we here investigated whether p53-dependent senescence occurs at some stage and, if not, what defects were detectable in this pathway, using immunohistochemistry. Phosphorylated checkpoint kinase 2 (CHEK2) can mediate DNA-damage signaling, and under some conditions senescence, by phosphorylating and activating p53. Remarkably, we detected no prevalent p53-mediated senescence in any of six classes of lesions. Two separate defects in p53 signaling appeared common: in nevi, lack of p53 phosphorylation by activated CHEK2, and in melanomas, defective p21 upregulation by p53 even when phosphorylated. PMID:23253087

  9. Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation

    SciTech Connect

    Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

    2011-08-19

    Highlights: {yields} p21 accumulated rapidly at laser-irradiated sites via its C-terminal region. {yields} p21 colocalized with the DSB marker {gamma}-H2AX and the DSB sensor Ku80. {yields} Accumulation of p21 is dependent on PCNA, but not p53 and the NHEJ core factors. {yields} Accumulation activity of p21 was conserved among human and animal cells. {yields} p21 is a useful tool as a detection marker of DNA damaged sites. -- Abstract: The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 foci arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker {gamma}-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1-146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful

  10. Differential modulation of paclitaxel-mediated apoptosis by p21Waf1 and p27Kip1.

    PubMed

    Schmidt, M; Lu, Y; Liu, B; Fang, M; Mendelsohn, J; Fan, Z

    2000-05-11

    The impact of the cyclin dependent kinase (CDK) inhibitors p21Waf1 and p27Kip1 on paclitaxel-mediated cytotoxicity was investigated in RKO human colon adenocarcinoma cells with the ecdysone-inducible expression of p21Waf1 or p27Kip1. Ectopic expression of p27Kip1 arrested cells at G1 phase, whereas p21Waf1 expression arrested cells at G1 and G2. Expression of p21Waf1 after paclitaxel treatment produced much greater resistance to paclitaxel than did expression of p27Kip1. We attributed this difference to the additional block at G2 induced by p21Waf1, which prevented cells from entering M phase and becoming paclitaxel susceptible. Expression of p21Waf1 inhibited p34cdc2 activity and markedly reduced paclitaxel-mediated mitotic arrest, from 87.5 to 23%. In contrast, p27Kip1 expression also inhibited p34cdc2 but reduced mitotic arrest only slightly, from 87. 4 to 74.5%. We concluded that the G2 block produced by p21Waf1, but not by p27Kip1, contributed to their unequal modulation of sensitivity to paclitaxel-mediated apoptosis in RKO cells, and there is no causal relationship between paclitaxel-mediated cytotoxicity and elevation of p34cdc2 activity. PMID:10828884

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells.

    PubMed

    Cayrol, C; Knibiehler, M; Ducommun, B

    1998-01-22

    A unique feature of p21 that distinguishes it from the other cyclin-dependent kinase (CDK) inhibitors is its ability to associate with the proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerases delta and epsilon. While it is now well established that inhibition of cyclin/CDK complexes by p21 can result in G1 cell cycle arrest, the consequences of p21/PCNA interaction on cell cycle progression have not yet been determined. Here, we show, using a tetracycline-regulated system, that expression of wild-type p21 in p53-deficient DLD1 human colon cancer cells inhibits DNA synthesis and causes G1 and G2 cell cycle arrest. Similar effects are observed in cells expressing p21CDK-, a mutant impaired in the interaction with CDKs, but not in cells expressing p21PCNA-, a mutant deficient for the interaction with PCNA. Analysis of cells treated with a p21-derived PCNA-binding peptide provides additional evidence that the growth inhibitory effects of p21 and p21CDK result from their ability to bind to PCNA. Our results suggest that p21 might inhibit cell cycle progression by two independent mechanisms, inhibition of cyclin/CDK complexes, and inhibition of PCNA function resulting in both G1 and G2 arrest. PMID:9467956

  13. Zac1, an Sp1-like protein, regulates human p21{sup WAF1/Cip1} gene expression in HeLa cells

    SciTech Connect

    Liu, Pei-Yao; Hsieh, Tsai-Yuan; Liu, Shu-Ting; Chang, Yung-Lung; Lin, Wei-Shiang; Wang, Wei-Ming; Huang, Shih-Ming

    2011-12-10

    Zac1 functions as both a transcription factor and a transcriptional cofactor for p53, nuclear receptors (NRs) and NR coactivators. Zac1 might also act as a transcriptional repressor via the recruitment of histone deacetylase 1 (HDAC1). The ability of Zac1 to interact directly with GC-specific elements indicates that Zac1 possibly binds to Sp1-responsive elements. In the present study, our data show that Zac1 is able to interact directly with the Sp1-responsive element in the p21{sup WAF1/Cip1} gene promoter and enhance the transactivation activity of Sp1 through direct physical interaction. Our data further demonstrate that Zac1 might enhance Sp1-specific promoter activity by interacting with the Sp1-responsive element, affecting the transactivation activity of Sp1 via a protein-protein interaction, or competing the HDAC1 protein away from the pre-existing Sp1/HDAC1 complex. Finally, the synergistic regulation of p21{sup WAF1/Cip1} gene expression by Zac1 and Sp1 is mediated by endogenous p53 protein and p53-responsive elements in HeLa cells. Our work suggests that Zac1 might serve as an Sp1-like protein that directly interacts with the Sp1-responsive element to oligomerize with and/or to coactivate Sp1.

  14. Loss of G1/S Checkpoint in Human Immunodeficiency Virus Type 1-Infected Cells Is Associated with a Lack of Cyclin-Dependent Kinase Inhibitor p21/Waf1

    PubMed Central

    Clark, Elizabeth; Santiago, Francisco; Deng, Longwen; Chong, Siew yen; de la Fuente, Cynthia; Wang, Lai; Fu, Peng; Stein, Dana; Denny, Thomas; Lanka, Venkata; Mozafari, Fariba; Okamoto, Takashi; Kashanchi, Fatah

    2000-01-01

    Productive high-titer infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile reverse transcripts and lack of viral progeny formation. An interplay between Tat and p53 has previously been reported, where Tat inhibited the transcription of the p53 gene, which may aid in the development of AIDS-related malignancies, and p53 expression inhibited HIV-1 long terminal repeat transcription. Here, by using a well-defined and -characterized stress signal, gamma irradiation, we find that upon gamma irradiation, HIV-1-infected cells lose their G1/S checkpoints, enter the S phase inappropriately, and eventually apoptose. The loss of the G1/S checkpoint is associated with a loss of p21/Waf1 protein and increased activity of a major G1/S kinase, namely, cyclin E/cdk2. The p21/Waf1 protein, a known cyclin-dependent kinase inhibitor, interacts with the cdk2/cyclin E complex and inhibits progression of cells into S phase. We find that loss of the G1/S checkpoint in HIV-1-infected cells may in part be due to Tat's ability to bind p53 (a known activator of the p21/Waf1 promoter) and sequester its transactivation activity, as seen in both in vivo and in vitro transcription assays. The loss of p21/Waf1 in HIV-1-infected cells was specific to p21/Waf1 and did not occur with other KIP family members, such as p27 (KIP1) and p57 (KIP2). Finally, the advantage of a loss of the G1/S checkpoint for HIV-1 per se may be that it pushes the host cell into the S phase, which may then allow subsequent virus-associated processes, such as RNA splicing, transport, translation, and packaging of virion-specific genes, to occur. PMID:10799578

  15. Loss of G(1)/S checkpoint in human immunodeficiency virus type 1-infected cells is associated with a lack of cyclin-dependent kinase inhibitor p21/Waf1.

    PubMed

    Clark, E; Santiago, F; Deng, L; Chong, S; de La Fuente, C; Wang, L; Fu, P; Stein, D; Denny, T; Lanka, V; Mozafari, F; Okamoto, T; Kashanchi, F

    2000-06-01

    Productive high-titer infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile reverse transcripts and lack of viral progeny formation. An interplay between Tat and p53 has previously been reported, where Tat inhibited the transcription of the p53 gene, which may aid in the development of AIDS-related malignancies, and p53 expression inhibited HIV-1 long terminal repeat transcription. Here, by using a well-defined and -characterized stress signal, gamma irradiation, we find that upon gamma irradiation, HIV-1-infected cells lose their G(1)/S checkpoints, enter the S phase inappropriately, and eventually apoptose. The loss of the G(1)/S checkpoint is associated with a loss of p21/Waf1 protein and increased activity of a major G(1)/S kinase, namely, cyclin E/cdk2. The p21/Waf1 protein, a known cyclin-dependent kinase inhibitor, interacts with the cdk2/cyclin E complex and inhibits progression of cells into S phase. We find that loss of the G(1)/S checkpoint in HIV-1-infected cells may in part be due to Tat's ability to bind p53 (a known activator of the p21/Waf1 promoter) and sequester its transactivation activity, as seen in both in vivo and in vitro transcription assays. The loss of p21/Waf1 in HIV-1-infected cells was specific to p21/Waf1 and did not occur with other KIP family members, such as p27 (KIP1) and p57 (KIP2). Finally, the advantage of a loss of the G(1)/S checkpoint for HIV-1 per se may be that it pushes the host cell into the S phase, which may then allow subsequent virus-associated processes, such as RNA splicing, transport, translation, and packaging of virion-specific genes, to occur. PMID:10799578

  16. Increased p53 and decreased p21 accompany apoptosis induced by ultraviolet radiation in the nervous system of a crustacean.

    PubMed

    Hollmann, Gabriela; Linden, Rafael; Giangrande, Angela; Allodi, Silvana

    2016-04-01

    Ultraviolet (UV) radiation can produce biological damage, leading the cell to apoptosis by the p53 pathway. This study evaluated some molecular markers of the apoptosis pathway induced by UVA, UVB and UVA+ UVB (Solar Simulator, SIM) in environmental doses, during five consecutive days of exposure, in the brain of the crab Ucides cordatus. We evaluated the central nervous system (CNS) by immunoblotting the content of proteins p53, p21, phosphorylated AKT, BDNF, GDNF, activated caspase-3 (C3) and phosphohistone H3 (PH3); and by immunohistochemical tests of the cells labeled for PH3 and C3. After the fifth day of exposure, UVB radiation and SIM increased the protein content of p53, increasing the content of AKT and, somehow, blocking p21, increasing the content of activated caspase-3, which led the cells to apoptosis. The signs of death affected the increase in neurotrophins, such as BDNF and GDNF, stimulating the apoptotic cascade of events. Immunohistochemical assays and immunoblotting showed that apoptosis was present in the brains of all UV groups, while the number of mitotic cells in the same groups decreased. In conclusion, environmental doses of UV can cause apoptosis by increasing p53 and decreasing p21, revealing an UV-damage pathway for U. cordatus. PMID:26807499

  17. Agmatine Ameliorates High Glucose-Induced Neuronal Cell Senescence by Regulating the p21 and p53 Signaling

    PubMed Central

    Song, Juhyun; Lee, Byeori; Kang, Somang; Oh, Yumi; Kim, Eosu; Kim, Chul-Hoon; Song, Ho-Taek

    2016-01-01

    Neuronal senescence caused by diabetic neuropathy is considered a common complication of diabetes mellitus. Neuronal senescence leads to the secretion of pro-inflammatory cytokines, the production of reactive oxygen species, and the alteration of cellular homeostasis. Agmatine, which is biosynthesized by arginine decarboxylation, has been reported in previous in vitro to exert a protective effect against various stresses. In present study, agmatine attenuated the cell death and the expression of pro-inflammatory cytokines such as IL-6, TNF-alpha and CCL2 in high glucose in vitro conditions. Moreover, the senescence associated-β-galatosidase's activity in high glucose exposed neuronal cells was reduced by agmatine. Increased p21 and reduced p53 in high glucose conditioned cells were changed by agmatine. Ultimately, agmatine inhibits the neuronal cell senescence through the activation of p53 and the inhibition of p21. Here, we propose that agmatine may ameliorate neuronal cell senescence in hyperglycemia. PMID:26924930

  18. Agmatine Ameliorates High Glucose-Induced Neuronal Cell Senescence by Regulating the p21 and p53 Signaling.

    PubMed

    Song, Juhyun; Lee, Byeori; Kang, Somang; Oh, Yumi; Kim, Eosu; Kim, Chul-Hoon; Song, Ho-Taek; Lee, Jong Eun

    2016-02-01

    Neuronal senescence caused by diabetic neuropathy is considered a common complication of diabetes mellitus. Neuronal senescence leads to the secretion of pro-inflammatory cytokines, the production of reactive oxygen species, and the alteration of cellular homeostasis. Agmatine, which is biosynthesized by arginine decarboxylation, has been reported in previous in vitro to exert a protective effect against various stresses. In present study, agmatine attenuated the cell death and the expression of pro-inflammatory cytokines such as IL-6, TNF-alpha and CCL2 in high glucose in vitro conditions. Moreover, the senescence associated-β-galatosidase's activity in high glucose exposed neuronal cells was reduced by agmatine. Increased p21 and reduced p53 in high glucose conditioned cells were changed by agmatine. Ultimately, agmatine inhibits the neuronal cell senescence through the activation of p53 and the inhibition of p21. Here, we propose that agmatine may ameliorate neuronal cell senescence in hyperglycemia. PMID:26924930

  19. p21WAF1/CIP1 Expression is Differentially Regulated by Metformin and Rapamycin

    PubMed Central

    Molnar, Zoltan; Millward, Ann B.; Tse, Wai; Demaine, Andrew G.

    2014-01-01

    The mammalian target of rapamycin (mTOR) pathway plays an important role in the development of diabetic nephropathy and other age-related diseases. One of the features of DN is the elevated expression of p21WAF1/CIP1. However, the importance of the mTOR signalling pathway in p21 regulation is poorly understood. Here we investigated the effect of metformin and rapamycin on mTOR-related phenotypes in cell lines of epithelial origin. This study reports that metformin inhibits high glucose-induced p21 expression. High glucose opposed metformin in regulating cell size, proliferation, and protein synthesis. These effects were associated with reduced AMPK activation, affecting downstream mTOR signalling. However, the inhibition of the mTOR pathway by rapamycin did not have a negative effect on p21 expression, suggesting that metformin regulates p21 upstream of mTOR. These findings provide support for the hypothesis that AMPK activation may regulate p21 expression, which may have implications for diabetic nephropathy and other age-related pathologies. PMID:26464852

  20. Plant HDAC inhibitor chrysin arrest cell growth and induce p21WAF1 by altering chromatin of STAT response element in A375 cells

    PubMed Central

    2012-01-01

    Background Chrysin and its analogues, belongs to flavonoid family and possess potential anti-tumour activity. The aim of this study is to determine the molecular mechanism by which chrysin controls cell growth and induce apoptosis in A375 cells. Methods Effect of chrysin and its analogues on cell viability and cell cycle analysis was determined by MTT assay and flowcytometry. A series of Western blots was performed to determine the effect of chrysin on important cell cycle regulatory proteins (Cdk2, cyclin D1, p53, p21, p27). The fluorimetry and calorimetry based assays was conducted for characterization of chrysin as HDAC inhibitor. The changes in histone tail modification such as acetylation and methylation was studied after chrysin treatment was estimated by immuno-fluorescence and western blot analysis. The expression of Bcl-xL, survivin and caspase-3 was estimated in chrysin treated cells. The effect of chrysin on p21 promoter activity was studied by luciferase and ChIP assays. Results Chrysin cause G1 cell cycle arrest and found to inhibit HDAC-2 and HDAC-8. Chrysin treated cells have shown increase in the levels of H3acK14, H4acK12, H4acK16 and decrease in H3me2K9 methylation. The p21 induction by chrysin treatment was found to be independent of p53 status. The chromatin remodelling at p21WAF1 promoter induces p21 activity, increased STAT-1 expression and epigenetic modifications that are responsible for ultimate cell cycle arrest and apoptosis. Conclusion Chrysin shows in vitro anti-cancer activity that is correlated with induction of histone hyperacetylation and possible recruitment of STAT-1, 3, 5 proteins at STAT (−692 to −684) region of p21 promoter. Our results also support an unexpected action of chrysin on the chromatin organization of p21WAF1 promoter through histone methylation and hyper-acetylation. It proposes previously unknown sequence specific chromatin modulations in the STAT responsive elements for regulating cell cycle progression

  1. Synergistic effects of dexamethasone and genistein on the expression of Cdk inhibitor p21WAF1/CIP1 in human hepatocellular and colorectal carcinoma cells.

    PubMed

    Park, J H; Oh, E J; Choi, Y H; Kang, C D; Kang, H S; Kim, D K; Kang, K I; Yoo, M A

    2001-05-01

    Previous studies have shown that dexamethasone, a synthetic glucocorticoid, can induce a G1 arrest, however, genistein, a natural isoflavonoid phytoestrogen, induces a G2/M arrest in the cell cycle progression in various cancer cell lines. A block of cell cycle checkpoint by dexamethasone and genistein correlates with a selective induction of cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1 in a tumor suppressor p53-independent manner and abolishment of Cdk2 phosphorylation. In the present study, the effects of dexamethasone and genistein (both singly and combined) on the expression of p21 in human hepatocellular Hep G2 and colorectal Colo320 HSR carcinoma cells were evaluated. Whereas dexamethasone mildly induced the level of p21 protein, genistein strongly increased the expression of p21 protein in our experimental condition. Both compounds also activated p21 promoter reporter constructs. The combined effects of dexamethasone and genistein on the induction of p21 protein and activation of p21 promoter were synergistic in both cell lines. These findings indicate that dexamethasone and genistein act in a synergistic fashion and have potential for combination chemotherapy for the treatment of liver and colon cancer. PMID:11295047

  2. Ganglioside GM3 modulates tumor suppressor PTEN-mediated cell cycle progression--transcriptional induction of p21(WAF1) and p27(kip1) by inhibition of PI-3K/AKT pathway.

    PubMed

    Choi, Hee-Jung; Chung, Tae-Wook; Kang, Sung-Koo; Lee, Young-Choon; Ko, Jeong-Heon; Kim, Jong-Guk; Kim, Cheorl-Ho

    2006-07-01

    The simple ganglioside GM3 has been shown to have anti-proliferative effects in several in vitro and in vivo cancer models. Although the exogenous ganglioside GM3 has an inhibitory effect on cancer cell proliferation, the exact mechanism by which it prevents cell proliferation remains unclear. Previous studies showed that MDM2 is an oncoprotein that controls tumorigenesis through both p53-dependent and p53-independent mechanisms, and tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a dual-specificity phosphatase that antagonizes phosphatidylinositol 3-kinase (PI-3K)/AKT signaling, is capable of blocking MDM2 nuclear translocation and destabilizing the MDM2 protein. Results from our current study show that GM3 treatment dramatically increases cyclin-dependent kinase (CDK) inhibitor (CKI) p21(WAF1) expression through the accumulation of p53 protein by the PTEN-mediated inhibition of the PI-3K/AKT/MDM2 survival signaling in HCT116 colon cancer cells. Moreover, the data herein clearly show that ganglioside GM3 induces p53-dependent transcriptional activity of p21(WAF1), as evidenced by the p21(WAF1) promoter-driven luciferase reporter plasmid (full-length p21(WAF1) promoter and a construct lacking the p53-binding sites). Additionally, ganglioside GM3 enhances expression of CKI p27(kip1) through the PTEN-mediated inhibition of the PI-3K/AKT signaling. Furthermore, the down-regulation of the cyclin E and CDK2 was clearly observed in GM3-treated HCT116 cells, but the down-regulation of cyclin D1 and CDK4 was not. On the contrary, suppression of PTEN levels by RNA interference restores the enhanced expression of p53-dependent p21(WAF1) and p53-independent p27(kip1) through inactivating the effect of PTEN on PI-3K/AKT signaling modulated by ganglioside GM3. These results suggest that ganglioside GM3-stimulated PTEN expression modulates cell cycle regulatory proteins, thus inhibiting cell growth. We conclude that ganglioside GM3 represents a

  3. Spontaneous γH2AX Foci in Human Solid Tumor-Derived Cell Lines in Relation to p21WAF1 and WIP1 Expression

    PubMed Central

    Mirzayans, Razmik; Andrais, Bonnie; Scott, April; Wang, Ying W.; Weiss, Robert H.; Murray, David

    2015-01-01

    Phosphorylation of H2AX on Ser139 (γH2AX) after exposure to ionizing radiation produces nuclear foci that are detectable by immunofluorescence microscopy. These so-called γH2AX foci have been adopted as quantitative markers for DNA double-strand breaks. High numbers of spontaneous γH2AX foci have also been reported for some human solid tumor-derived cell lines, but the molecular mechanism(s) for this response remains elusive. Here we show that cancer cells (e.g., HCT116; MCF7) that constitutively express detectable levels of p21WAF1 (p21) exhibit low numbers of γH2AX foci (<3/nucleus), whereas p21 knockout cells (HCT116p21−/−) and constitutively low p21-expressing cells (e.g., MDA-MB-231) exhibit high numbers of foci (e.g., >50/nucleus), and that these foci are not associated with apoptosis. The majority (>95%) of cells within HCT116p21−/− and MDA-MB-231 cultures contain high levels of phosphorylated p53, which is localized in the nucleus. We further show an inverse relationship between γH2AX foci and nuclear accumulation of WIP1, an oncogenic phosphatase. Our studies suggest that: (i) p21 deficiency might provide a selective pressure for the emergence of apoptosis-resistant progeny exhibiting genomic instability, manifested as spontaneous γH2AX foci coupled with phosphorylation and nuclear accumulation of p53; and (ii) p21 might contribute to positive regulation of WIP1, resulting in dephosphorylation of γH2AX. PMID:26006237

  4. Arsenic trioxide phosphorylates c-Fos to transactivate p21{sup WAF1/CIP1} expression

    SciTech Connect

    Liu Zimiao; Huang, H.-S.

    2008-12-01

    An infamous poison, arsenic also has been used as a drug for nearly 2400 years; in recently years, arsenic has been effective in the treatment of acute promyelocytic leukemia. Increasing evidence suggests that opposite effects of arsenic trioxide (ATO) on tumors depend on its concentrations. For this reason, the mechanisms of action of the drug should be elucidated, and it should be used therapeutically only with extreme caution. Previously, we demonstrated the opposing effects of ERK1/2 and JNK on p21{sup WAF1/CIP1} (p21) expression in response to ATO in A431 cells. In addition, JNK phosphorylates c-Jun (Ser{sup 63/73}) to recruit TGIF/HDAC1 to suppress p21 gene expression. Presently, we demonstrated that a high concentration of ATO sustains ERK1/2 phosphorylation, and increases c-Fos biosynthesis and stability, which enhances p21 gene expression. Using site-directed mutagenesis, a DNA affinity precipitation assay, and functional assays, we demonstrated that phosphorylation of the C-terminus of c-Fos (Thr{sup 232}, Thr{sup 325}, Thr{sup 331}, and Ser{sup 374}) plays an important role in its binding to the p21 promoter, and in conjunction with N-terminus phosphorylation of c-Fos (Ser{sup 70}) to transactivate p21 promoter expression. In conclusion, a high concentration of ATO can sustain ERK1/2 activation to enhance c-Fos expression, then dimerize with dephosphorylated c-Jun (Ser{sup 63/73}) and recruit p300/CBP to the Sp1 sites (- 84/- 64) to activate p21 gene expression in A431 cells.

  5. Suppressor of cytokine signaling 1-dependent regulation of the expression and oncogenic functions of p21(CIP1/WAF1) in the liver.

    PubMed

    Yeganeh, M; Gui, Y; Kandhi, R; Bobbala, D; Tobelaim, W-S; Saucier, C; Yoshimura, A; Ferbeyre, G; Ramanathan, S; Ilangumaran, S

    2016-08-11

    The SOCS1 gene coding for suppressor of cytokine signaling 1 is frequently repressed in hepatocellular carcinoma (HCC), and hence SOCS1 is considered a tumor suppressor in the liver. However, the tumor-suppressor mechanisms of SOCS1 are not yet well understood. SOCS1 is known to inhibit pro-inflammatory cytokine production and signaling and to promote activation of the p53 tumor suppressor. However, we observed that SOCS1-deficient mice developed numerous and large liver tumor nodules following treatment with the hepatocarcinogen diethylnitrosamine (DEN) without showing increased interleukin-6 production or activation of p53. On the other hand, the livers of DEN-treated Socs1-null mice showed elevated levels of p21(CIP1/WAF1) protein (p21). Even though p21 generally functions as a tumor suppressor, paradoxically many cancers, including HCC, are known to express elevated levels of p21 that correlate with poor prognosis. We observed elevated p21 expression also in the regenerating livers of SOCS1-deficient mice and in cisplatin-treated Socs1-null hepatocytes, wherein the p21 protein showed increased stability. We show that SOCS1 interacts with p21 and promotes its ubiquitination and proteasomal degradation. Besides, the DEN-treated livers of Socs1-null mice showed increased nuclear and cytosolic p21 staining, and the latter was associated with growth factor-induced, phosphatidylinositol 3-kinase-dependent phosphorylation of p21 in SOCS1-deficient hepatocytes. Cytosolic p21 is often associated with malignancy and chemo-resistance in many cancers. Accordingly, SOCS1-deficient hepatocytes showed increased resistance to apoptosis that was reversed by shRNA-mediated p21 knockdown. In the regenerating livers of Socs1-null mice, increased p21 expression coincided with elevated cyclinD levels. Correspondingly, SOCS1-deficient hepatocytes showed increased proliferation to growth factor stimulation that was reversed by p21 knockdown. Overall, our findings indicate that the

  6. Loss of Notch1-dependent p21Waf1/Cip1 expression influences the Notch1 outcome in tumorigenesis

    PubMed Central

    Cialfi, Samantha; Palermo, Rocco; Manca, Sonia; De Blasio, Carlo; Vargas Romero, Paula; Checquolo, Saula; Bellavia, Diana; Uccelletti, Daniela; Saliola, Michele; D'Alessandro, Angelo; Zolla, Lello; Gulino, Alberto; Screpanti, Isabella; Talora, Claudio

    2014-01-01

    Notch signaling plays a complex role in carcinogenesis, and its signaling pathway has both tumor-suppressor and oncogenic components. In this study we investigated the effects of reactive oxygen species (ROS) on Notch1 signaling outcome in keratinocyte biology. We demonstrate that Notch1 function contributes to the arsenic-induced keratinocyte transformation. We found that acute exposure to arsenite increases oxidative stress and inhibits proliferation of keratinocyte cells by upregulation of p21waf1/Cip1. The necessity of p21waf1/Cip1 for arsenite-induced cell death was demonstrated by targeted downregulation of p21waf1/Cip1 by using RNA interference. We further demonstrated that on acute exposure to arsenite, p21waf1/Cip1 is upregulated and Notch1 downmodulated, whereas on chronic exposure to arsenite, malignant progression of arsenite-treated keratinocytes cells was accompanied by regained expression and activity of Notch1. Notch1 activity in arsenite-transformed keratinocytes inhibits arsenite-induced upregulation of p21waf1/Cip1 by sustaining c-myc expression. We further demonstrated that c-myc collaborates with Nrf2, a key regulator for the maintenance of redox homeostasis, to promote metabolic activities that support cell proliferation and cytoprotection. Therefore, Notch1-mediated repression of p21waf1/Cip1 expression results in the inhibition of cell death and keratinocytes transformation. Our results not only demonstrate that sustained Notch1 expression is at least one key event implicated in the arsenite human skin carcinogenic effect, but also may provide mechanistic insights into the molecular aspects that determine whether Notch signaling will be either oncogenic or tumor suppressive. PMID:24801890

  7. The multi-functional sorting protein PACS-2 regulates SIRT1-mediated deacetylation of p53 to modulate p21-dependent cell cycle arrest

    PubMed Central

    Atkins, Katelyn M.; Thomas, Laura L.; Barroso-González, Jonathan; Thomas, Laurel; Auclair, Sylvain; Yin, Jun; Kang, Hyeog; Chung, Jay H.; Dikeakos, Jimmy D.; Thomas, Gary

    2014-01-01

    SUMMARY SIRT1 regulates the DNA damage response by deacetylating p53, thereby repressing p53 transcriptional output. Here we demonstrate that the sorting protein PACS-2 regulates SIRT1-mediated deacetylation of p53 to modulate the DNA damage response. PACS-2 knockdown cells failed to efficiently undergo p53-induced cell cycle arrest in response to DNA damage. Accordingly, p53 acetylation was reduced both in PACS-2 knockdown cells and thymocytes from Pacs-2−/− mice, thereby blunting induction of the cyclin-dependent kinase inhibitor p21 (CDKN1A). The SIRT1 inhibitor EX-527 or SIRT1 knockdown restored p53 acetylation and p21 induction as well as p21-dependent cell cycle arrest in PACS-2 knockdown cells. Trafficking studies revealed cytoplasmic PACS-2 shuttled to the nucleus where it interacted with SIRT1 and repressed SIRT1-mediated p53 deacetylation. Correspondingly, in vitro assays demonstrated PACS-2 directly inhibited SIRT1-catalyzed p53 deacetylation. Together, these findings identify PACS-2 as an in vivo mediator of the SIRT1—p53p21 axis that modulates the DNA damage response. PMID:25159152

  8. Silibilin-induces apoptosis in breast cancer cells by modulating p53, p21, Bak and Bcl-XL pathways.

    PubMed

    Pirouzpanah, Mohammad Bagher; Sabzichi, Mehdi; Pirouzpanah, Saeed; Chavoshi, Hadi; Samadi, Nasser

    2015-01-01

    Nowadays herbal-derived medicines are attracting attention as new sources of drugs with few side effects. Silibinin is a flavonoid compound with chemotheraputic effects on different cancers such as examples in the prostate, lung, colon and breast. In the present study, the cytotoxic effects of silibinin on MCF7 breast cancer cells were investigated. Apoptosis was determined by flow cytometry and the impact of silibinin on the expression of pivotal genes including Bak, P53, P21, BRCA1, BCL-X1 and ATM was analyzed. Treatment for 24h had a significant dose-dependent inhibitory effect on cell growth (p<0.05) with dose- and time- dependent induction of apoptosis (p<0.05). In addition, there were significant increases in BRCA1, ATM, Bak and Bcl-XL gene expression at the mRNA level with different concentrations of silibinin for 24 or 48 h (p<0.05). Taken together, the results suggest that silibinin inhibits the proliferation and induces apoptosis of MCF-7 cells by down-regulating Bak, P53, P21, BRCA1, BCL-Xl and thus may be considered as an effective adjuvant drug to produce a better chemopreventive response for the cancer therapy. PMID:25773855

  9. Epigallocatechin-3-gallate prevents autoimmune-associated down- regulation of p21 in salivary gland cells through a p53-independent pathway.

    PubMed

    Dickinson, Douglas; Yu, Hongfang; Ohno, Seiji; Thomas, Cristina; Derossi, Scott; Ma, Yat-Ho; Yates, Nicole; Hahn, Emily; Bisch, Frederick; Yamamoto, Tetsuya; Hsu, Stephen

    2014-02-01

    The submandibular salivary glands of non-obese diabetic (NOD) mice, a model for Sjogren's syndrome and type-1 diabetes, show an elevated level of proliferating cell nuclear antigen (PCNA), a protein involved in cell proliferation and repair of DNA damage. We reported previously that epigallocatechin-3-gallate (EGCG), the most abundant green tea catechin, normalizes the PCNA level. PCNA's activity can be regulated by the cyclin-dependent kinase inhibitor p21, which is also important for epithelial cell differentiation. In turn, expression of p21 and PCNA are partially regulated by Rb phosphorylation levels. EGCG was found to modulate p21 expression in epithelial cells, suggesting that EGCG-induced p21 could be associated with down-regulation of PCNA in vivo. The current study examined the protein levels of p21 and p53 (which can up-regulate p21) in NOD mice fed with either water or EGCG, and the effect of EGCG on p21 and p53 in cell line models with either normal or defective Rb. In NOD mice, the p21 level was low, and EGCG normalized it. In contrast to HSG cells with functional Rb, negligible expression of p21 in NS-SVAC cells that lack Rb was not altered by EGCG treatment. Inhibition of p53 by siRNA demonstrated that p21 and p53 were induced independently in HSG cells by a physiological concentration range of EGCG, suggesting p53 could be an important but not conditional factor associated with p21 expression. In conclusion, PCNA and p21 levels are altered inversely in the NOD model for SS and in HSG cells, and warrant further study as candidate new markers for salivary dysfunction associated with xerostomia. Induction of p21 by EGCG could provide clinically useful normalization of salivary glands by promoting differentiation and reducing PCNA levels. PMID:24329914

  10. miR-30c and miR-181a synergistically modulate p53-p21 pathway in diabetes induced cardiac hypertrophy.

    PubMed

    Raut, Satish K; Singh, Gurinder B; Rastogi, Bhawna; Saikia, Uma Nahar; Mittal, Anupam; Dogra, Nilambra; Singh, Sandeep; Prasad, Rishikesh; Khullar, Madhu

    2016-06-01

    p53-p21 pathway mediates cardiomyocyte hypertrophy and apoptosis and is upregulated in diabetic cardiomyopathy (DbCM). We investigated role of microRNAs in regulating p53-p21 pathway in high glucose (HG)-induced cardiomyocyte hypertrophy and apoptosis. miR-30c and miR-181a were identified to target p53. Cardiac expression of microRNAs was measured in diabetic patients, diabetic rats, and in HG-treated cardiomyocytes. Effect of microRNAs over-expression and inhibition on HG-induced cardiomyocyte hypertrophy and apoptosis was examined. Myocardial expression of p53 and p21 genes was increased and expression of miR-30c and miR-181a was significantly decreased in diabetic patients, DbCM rats, and in HG-treated cardiomyocytes. Luciferase assay confirmed p53 as target of miR-30c and miR-181a. Over-expression of miR-30c or miR-181a decreased expression of p53, p21, ANP, cardiomyocyte cell size, and apoptosis in HG-treated cardiomyocytes. Concurrent over-expression of these microRNAs resulted in greater decrease in cardiomyocyte hypertrophy and apoptosis, suggesting a synergistic effect of these microRNAs. Our results suggest that dysregulation of miR-30c and miR-181a may be involved in upregulation of p53-p21 pathway in DbCM. PMID:27221738

  11. Gallotannin is a DNA damaging compound that induces senescence independently of p53 and p21 in human colon cancer cells.

    PubMed

    Al-Halabi, Racha; Abou Merhi, Raghida; Chakilam, Saritha; El-Baba, Chirine; Hamade, Eva; Di Fazio, Pietro; Ocker, Matthias; Schneider-Stock, Regine; Gali-Muhtasib, Hala

    2015-10-01

    The plant secondary metabolite gallotannin (GT) is the simplest hydrolyzable tannin shown to have anti-carcinogenic properties in several cell lines and to inhibit tumor development in different animal models. Here, we determined if GT induces senescence and DNA damage and investigated the involvement of p53 and p21 in this response. Using HCT116 human colon cancer cells wildtype for p53(+/+) /p21(+/+) and null for p53(+/+) /p21(-/-) or p53(-/-) /p21(+/+) , we found that GT induces senescence independently of p21 and p53. GT was found to increase the production of reactive oxygen species (ROS) by altering the redox balance in the cell, mainly by reducing the levels of glutathione and superoxide dismutase (SOD). Using the key antioxidants N-acetyl cysteine, dithiothreitol, SOD, and catalase, we showed that ROS were partially involved in the senescence response. Furthermore, GT-induced cell cycle arrest in S-phase in all HCT116 cell lines. At later time points, we noticed that p53 and p21 null cells escaped complete arrest and re-entered cell cycle provoking higher rates of multinucleation. The senescence induction by GT was irreversible and was accompanied by significant DNA damage as evidenced by p-H2AX staining. Our findings indicate that GT is an interesting anti colon cancer agent which warrants further study. PMID:24798519

  12. Negative Regulation of p21Waf1/Cip1 by Human INO80 Chromatin Remodeling Complex Is Implicated in Cell Cycle Phase G2/M Arrest and Abnormal Chromosome Stability

    PubMed Central

    Cao, Lingling; Ding, Jian; Dong, Liguo; Zhao, Jiayao; Su, Jiaming; Wang, Lingyao; Sui, Yi; Zhao, Tong; Wang, Fei; Jin, Jingji; Cai, Yong

    2015-01-01

    We previously identified an ATP-dependent human Ino80 (INO80) chromatin remodeling complex which shares a set of core subunits with yeast Ino80 complex. Although research evidence has suggested that INO80 complex functions in gene transcription and genome stability, the precise mechanism remains unclear. Herein, based on gene expression profiles from the INO80 complex-knockdown in HeLa cells, we first demonstrate that INO80 complex negatively regulates the p21Waf1/Cip1 (p21) expression in a p53-mediated mechanism. In chromatin immunoprecipitation (ChIP) and a sequential ChIP (Re-ChIP) assays, we determined that the INO80 complex and p53 can bind to the same promoter region of p21 gene (-2.2kb and -1.0kb upstream of the p21 promoter region), and p53 is required for the recruitment of the INO80 complex to the p21 promoter. RNAi knockdown strategies of INO80 not only led to prolonged progression of cell cycle phase G2/M to G1, but it also resulted in abnormal chromosome stability. Interestingly, high expression of p21 was observed in most morphologically-changed cells, suggesting that negative regulation of p21 by INO80 complex might be implicated in maintaining the cell cycle process and chromosome stability. Together, our findings will provide a theoretical basis to further elucidate the cellular mechanisms of the INO80 complex. PMID:26340092

  13. Proto-oncogene FBI-1 represses transcription of p21CIP1 by inhibition of transcription activation by p53 and Sp1.

    PubMed

    Choi, Won-Il; Jeon, Bu-Nam; Yun, Chae-Ok; Kim, Pyung-Hwan; Kim, Sung-Eun; Choi, Kang-Yell; Kim, Se Hoon; Hur, Man-Wook

    2009-05-01

    Aberrant transcriptional repression through chromatin remodeling and histone deacetylation has been postulated as the driving force for tumorigenesis. FBI-1 (formerly called Pokemon) is a member of the POK family of transcriptional repressors. Recently, FBI-1 was characterized as a critical oncogenic factor that specifically represses transcription of the tumor suppressor gene ARF, potentially leading indirectly to p53 inactivation. Our investigations on transcriptional repression of the p53 pathway revealed that FBI-1 represses transcription of ARF, Hdm2 (human analogue of mouse double minute oncogene), and p21CIP1 (hereafter indicated as p21) but not of p53. FBI-1 showed a more potent repressive effect on p21 than on p53. Our data suggested that FBI-1 is a master controller of the ARF-Hdm2-p53-p21 pathway, ultimately impinging on cell cycle arrest factor p21, by inhibiting upstream regulators at the transcriptional and protein levels. FBI-1 acted as a competitive transcriptional repressor of p53 and Sp1 and was shown to bind the proximal Sp1-3 GC-box and the distal p53-responsive elements of p21. Repression involved direct binding competition of FBI-1 with Sp1 and p53. FBI-1 also interacted with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylating Ac-H3 and Ac-H4 histones at the promoter. FBI-1 caused cellular transformation, promoted cell cycle proliferation, and significantly increased the number of cells in S phase. FBI-1 is aberrantly overexpressed in many human solid tumors, particularly in adenocarcinomas and squamous carcinomas. The role of FBI-1 as a master controller of the p53 pathway therefore makes it an attractive therapeutic target. PMID:19244234

  14. Proto-oncogene FBI-1 Represses Transcription of p21CIP1 by Inhibition of Transcription Activation by p53 and Sp1*S⃞

    PubMed Central

    Choi, Won-Il; Jeon, Bu-Nam; Yun, Chae-Ok; Kim, Pyung-Hwan; Kim, Sung-Eun; Choi, Kang-Yell; Kim, Se Hoon; Hur, Man-Wook

    2009-01-01

    Aberrant transcriptional repression through chromatin remodeling and histone deacetylation has been postulated as the driving force for tumorigenesis. FBI-1 (formerly called Pokemon) is a member of the POK family of transcriptional repressors. Recently, FBI-1 was characterized as a critical oncogenic factor that specifically represses transcription of the tumor suppressor gene ARF, potentially leading indirectly to p53 inactivation. Our investigations on transcriptional repression of the p53 pathway revealed that FBI-1 represses transcription of ARF, Hdm2 (human analogue of mouse double minute oncogene), and p21CIP1 (hereafter indicated as p21) but not of p53. FBI-1 showed a more potent repressive effect on p21 than on p53. Our data suggested that FBI-1 is a master controller of the ARF-Hdm2-p53-p21 pathway, ultimately impinging on cell cycle arrest factor p21, by inhibiting upstream regulators at the transcriptional and protein levels. FBI-1 acted as a competitive transcriptional repressor of p53 and Sp1 and was shown to bind the proximal Sp1–3 GC-box and the distal p53-responsive elements of p21. Repression involved direct binding competition of FBI-1 with Sp1 and p53. FBI-1 also interacted with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylating Ac-H3 and Ac-H4 histones at the promoter. FBI-1 caused cellular transformation, promoted cell cycle proliferation, and significantly increased the number of cells in S phase. FBI-1 is aberrantly overexpressed in many human solid tumors, particularly in adenocarcinomas and squamous carcinomas. The role of FBI-1 as a master controller of the p53 pathway therefore makes it an attractive therapeutic target. PMID:19244234

  15. Differential roles of p21(Waf1) and p27(Kip1) in modulating chemosensitivity and their possible application in drug discovery studies.

    PubMed

    Schmidt, M; Lu, Y; Parant, J M; Lozano, G; Bacher, G; Beckers, T; Fan, Z

    2001-11-01

    In this study, the differential role of the cyclin-dependent kinase (CDK) inhibitors p21(Waf1) and p27(Kip1) in cell cycle regulation was proposed for use in screening natural or synthetic compounds for cell cycle-dependent (particularly M phase-dependent) antineoplastic activity. p21(Waf1) or p27(Kip1) was ectopically expressed with an ecdysone-inducible mammalian expression system in a human colon adenocarcinoma cell line. Induction of p21(Waf1) or p27(Kip1) expression inhibited the activities of CDK2 and completely arrested cells at G(1) phase of the cell cycle by p27(Kip1) and at G(1) and G(2) phases by p21(Waf1). We examined the sensitivity of these cells to several antineoplastic agents known to be cell cycle-dependent or -independent. Substantially increased resistance to cell cycle-dependent antineoplastic agents was found in the cells when the expression of p21(Waf1) or p27(Kip1) was induced. In contrast, only a desensitization to cell cycle-independent antineoplastic agents was found in the cells arrested by p21(Waf1) or p27(Kip1). Because p21(Waf1) induces an additional block at G(2) phase that inhibits cell entry into M phase, we further examined the difference between p21(Waf1)- and p27(Kip1)-induced cells in their sensitivity to D-24851, a novel M phase-dependent compound. We found that induction of p21(Waf1) after exposure of the cells to D-24851 conferred stronger resistance than did induction of p27(Kip1). Taken together, our results suggest that the differential effect of p21(Waf1) and p27(Kip1) on cell cycle regulation may be advantageous for screening chemical libraries for novel antineoplastic candidates that are cell cycle-dependent, and M phase-dependent in particular. PMID:11641417

  16. Expression Profile of p53 and p21 in Large Bowel Mucosa as Biomarkers of Inflammatory-Related Carcinogenesis in Ulcerative Colitis

    PubMed Central

    Nichita, Luciana; Voiosu, Theodor; Bastian, Alexandra; Cioplea, Mirela; Micu, Gianina; Sticlaru, Liana; Bengus, Andreea; Voiosu, Andrei; Mateescu, Radu Bogdan

    2016-01-01

    Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease that slightly increases the risk of colorectal cancer in patients with long-standing extended disease. Overexpression of p53 and p21 in colonic epithelia is usually detected in UC patients when no dysplasia is histologically seen and it is used by pathologists as a discriminator between regenerative changes and intraepithelial neoplasia, as well as a tissue biomarker useful to predict the risk of evolution toward malignancy. We present a one-year prospective observational study including a cohort of 45 patients with UC; p53 and p21 were evaluated in epithelial cells. p53 was positive in 74 samples revealed in 5% to 90% of epithelial cells, while 63 biopsies had strong positivity for p21 in 5% to 50% of epithelial cells. Architectural distortion was significantly correlated with p53 overexpression in epithelial cells. Thus, we consider that architectural distortion is a good substitute for p53 and p21 expression. We recommend use of p53 as the most valuable tissue biomarker in surveillance of UC patients, identifying the patients with higher risk for dysplasia. Association of p21 is also recommended for a better quantification of risk and for diminishing the false-negative results. PMID:27578918

  17. Expression Profile of p53 and p21 in Large Bowel Mucosa as Biomarkers of Inflammatory-Related Carcinogenesis in Ulcerative Colitis.

    PubMed

    Popp, Cristiana; Nichita, Luciana; Voiosu, Theodor; Bastian, Alexandra; Cioplea, Mirela; Micu, Gianina; Pop, Gabriel; Sticlaru, Liana; Bengus, Andreea; Voiosu, Andrei; Mateescu, Radu Bogdan

    2016-01-01

    Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease that slightly increases the risk of colorectal cancer in patients with long-standing extended disease. Overexpression of p53 and p21 in colonic epithelia is usually detected in UC patients when no dysplasia is histologically seen and it is used by pathologists as a discriminator between regenerative changes and intraepithelial neoplasia, as well as a tissue biomarker useful to predict the risk of evolution toward malignancy. We present a one-year prospective observational study including a cohort of 45 patients with UC; p53 and p21 were evaluated in epithelial cells. p53 was positive in 74 samples revealed in 5% to 90% of epithelial cells, while 63 biopsies had strong positivity for p21 in 5% to 50% of epithelial cells. Architectural distortion was significantly correlated with p53 overexpression in epithelial cells. Thus, we consider that architectural distortion is a good substitute for p53 and p21 expression. We recommend use of p53 as the most valuable tissue biomarker in surveillance of UC patients, identifying the patients with higher risk for dysplasia. Association of p21 is also recommended for a better quantification of risk and for diminishing the false-negative results. PMID:27578918

  18. Combined loss of PUMA and p21 accelerates c-MYC-driven lymphoma development considerably less than loss of one allele of p53.

    PubMed

    Valente, L J; Grabow, S; Vandenberg, C J; Strasser, A; Janic, A

    2016-07-21

    The tumor suppressor p53 is mutated in ~50% of human cancers. P53 is activated by a range of stimuli and regulates several cellular processes, including apoptotic cell death, cell cycle arrest, senescence and DNA repair. P53 induces apoptosis via transcriptional induction of the BH3-only proteins PUMA (p53-upregulated modulator of apoptosis) and NOXA, and cell cycle arrest via p21. Induction of these processes was proposed to be critical for p53-mediated tumor suppression. It is therefore surprising that mice lacking PUMA, NOXA and p21, as well as mice bearing mutations in p53 that impair the transcriptional activation of these genes, are not tumor prone, unlike mice lacking p53 function, which spontaneously develop tumors with 100% incidence. These p53 target genes and the processes they regulate may, however, impact differently on tumor development depending on the oncogenic drivers. For example, loss of PUMA enhances c-MYC-driven lymphoma development in mice, but, interestingly, the acceleration was less impressive compared with that caused by the loss of even a single p53 allele. Different studies have reported that loss of p21 can accelerate, delay or have no impact on tumorigenesis. In an attempt to resolve this controversy, we examined whether loss of p21-mediated cell cycle arrest cooperates with PUMA deficiency in accelerating lymphoma development in Eμ-Myc mice (overexpressing c-MYC in B-lymphoid cells). We found that Eμ-Myc mice lacking both p21 and PUMA (Eμ-Myc;Puma(-/-);p21(-/-)) developed lymphoma at a rate comparable to Eμ-Myc;Puma(-/-) animals, notably with considerably longer latency than Eμ-Myc;p53(+/-)mice. Loss of p21 had no impact on the numbers, cycling or survival of pre-leukemic Eμ-Myc B-lymphoid cells, even when PUMA was lost concomitantly. These results demonstrate that even in the context of deregulated c-MYC expression, p53 must suppress tumor development by activating processes apart from, or in addition to, PUMA

  19. The cyclin-dependent kinase inhibitor butyrolactone is a potent inhibitor of p21 (WAF1/CIP1 expression).

    PubMed

    Sax, Joanna K; Dash, Bipin C; Hong, Rui; Dicker, David T; El-Deiry, Wafik S

    2002-01-01

    Butyrolactone I (BL) is a competitive inhibitor of ATP for binding and activation of cyclin-dependent kinases and is a potent inhibitor of cell cycle progression. Treatment of H460 human lung and SW480 human colon cancer cells with doses of BL that exceed the Ki for CDK inhibition but which are much lower than doses required to inhibit MAPK, PKA, PKC, or EGFR lead to a rapid significant reduction of endogenous p21 protein expression. BL-dependent inhibition of p21 expression appears to be p53-independent. BL-dependent p21 degradation was blocked by lactacystin, consistent with the hypothesis that there is accelerated p21 proteasomal degradation in the presence of BL. BL also inhibited the p53-dependent increase of p21 protein expression in cells exposed to the DNA damag-ing agent etoposide, and favored a greater G2/M arrest as compared to the non-BL exposed cells. BL accelerated the degradation of exogenously expressed p21 that was not observed with a C-terminal truncated form of p21. Degradation of exogenous p21 led to a shift to G2 accumulation in the cells exposed to BL. We conclude that BL has effects on the cell cycle beyond its role as a CDK inhibitor and can be used as a novel tool to study the mechanism of p21 degradation and the consequences towards p21- dependent checkpoints. PMID:12429914

  20. p21{sup WAF1} modulates NF-{kappa}B signaling and induces anti-apoptotic protein Bcl-2 in Tax-expressing rat fibroblast

    SciTech Connect

    Akita, Kazumasa; Kawata, Sanae; Shimotohno, Kunitada . E-mail: kshimoto@virus.kyoto-u.ac.jp

    2005-02-05

    Of the cell cycle-associated genes regulated by human T-cell leukemia virus type-1 (HTLV-1) Tax, cyclin-dependent kinase (CDK) inhibitor p21{sup WAF1} is upregulated in HTLV-1-infected cells. Previously, we reported that p21{sup WAF1} stimulated Tax-dependent NF-{kappa}B activation which influences a variety of cellular processes, including proliferation, differentiation, and apoptosis. In HTLV-1-infected cells, Tax is primarily involved in the constitutive activation of NF-{kappa}B signaling. Here, we demonstrate that p21{sup WAF1} affects Tax-dependent NF-{kappa}B signaling by inducing p100/52, an NF-{kappa}B-related protein. W4, a Tax-transformed rat fibroblast cell line, exhibits the constitutive activation of NF-{kappa}B signaling, potentially mediated by overexpression of RelB. Ectopic expression of p21{sup WAF1} in W4 cells, which lack endogenous expression due to methylation of the p21{sup WAF1} promoter, induces the expression of p100/52. Bcl-2 expression was also upregulated by ectopic p21{sup WAF1} in this cell line, suggesting that p21{sup WAF1} plays an important role in the regulation of apoptosis by modulating NF-{kappa}B signaling in Tax-expressing rat fibroblasts. We also address the expression of NF-{kappa}B-related proteins in HTLV-1-infected cells.

  1. Compound K, a Ginsenoside Metabolite, Inhibits Colon Cancer Growth via Multiple Pathways Including p53-p21 Interactions

    PubMed Central

    Zhang, Zhiyu; Du, Guang-Jian; Wang, Chong-Zhi; Wen, Xiao-Dong; Calway, Tyler; Li, Zejuan; He, Tong-Chuan; Du, Wei; Bissonnette, Marc; Musch, Mark W.; Chang, Eugene B.; Yuan, Chun-Su

    2013-01-01

    Compound K (20-O-beta-d-glucopyranosyl-20(S)-protopanaxadiol, CK), an intestinal bacterial metabolite of ginseng protopanaxadiol saponins, has been shown to inhibit cell growth in a variety of cancers. However, the mechanisms are not completely understood, especially in colorectal cancer (CRC). A xenograft tumor model was used first to examine the anti-CRC effect of CK in vivo. Then, multiple in vitro assays were applied to investigate the anticancer effects of CK including antiproliferation, apoptosis and cell cycle distribution. In addition, a qPCR array and western blot analysis were executed to screen and validate the molecules and pathways involved. We observed that CK significantly inhibited the growth of HCT-116 tumors in an athymic nude mouse xenograft model. CK significantly inhibited the proliferation of human CRC cell lines HCT-116, SW-480, and HT-29 in a dose- and time-dependent manner. We also observed that CK induced cell apoptosis and arrested the cell cycle in the G1 phase in HCT-116 cells. The processes were related to the upregulation of p53/p21, FoxO3a-p27/p15 and Smad3, and downregulation of cdc25A, CDK4/6 and cyclin D1/3. The major regulated targets of CK were cyclin dependent inhibitors, including p21, p27, and p15. These results indicate that CK inhibits transcriptional activation of multiple tumor-promoting pathways in CRC, suggesting that CK could be an active compound in the prevention or treatment of CRC. PMID:23434653

  2. Induction of p53, p21 and apoptosis by silencing the NF90/NF45 complex in human papilloma virus-transformed cervical carcinoma cells

    PubMed Central

    Shamanna, Raghavendra A.; Hoque, Mainul; Pe'ery, Tsafi; Mathews, Michael B.

    2014-01-01

    The heterodimeric nuclear factor 90/nuclear factor 45 complex (NF90/NF45) binds nucleic acids and is a multifunctional regulator of gene expression. Here we report that depletion of NF90/NF45 restores the expression of the p53 and p21 proteins in cervical carcinoma cells infected with high-risk human papillomaviruses (HPV). Knockdown of either NF90 or NF45 by RNA interference led to greatly elevated levels of p53 and p21 proteins in HPV-derived HeLa and SiHa cells, but not in other cancerous or normal cell lines. In HeLa cells, p21 mRNA increased concomitantly but the level of p53 mRNA was unaffected. RNA interference directed against p53 prevented the induction of both proteins. These results indicated that the up-regulation of p21 is due to p53-dependent transcription, whereas p53 is regulated post-transcriptionally. Proteasome-mediated turnover of p53 is accelerated by the HPV E6 and cellular E6AP proteins. We therefore examined the hypothesis that this pathway is regulated by NF90/NF45. Indeed, depletion of NF90 attenuated the expression of E6 RNA and inhibited transcription from the HPV early promoter, revealing a new role for NF90/NF45 in HPV gene expression. The transcription inhibition was largely independent of the reduction of P-TEFb levels caused by NF90 depletion. Consistent with p53 derepression, NF90/NF45-depleted HeLa cells displayed elevated PARP cleavage and susceptibility to camptothecin-induced apoptosis. We conclude that high-risk strains of HPV utilize the cellular NF90/NF45 complex for viral E6 expression in infected cervical carcinoma cell lines. Interference with NF90/NF45 function could assist in controlling cervical carcinoma. PMID:23208500

  3. Phenylbutyric acid induces the cellular senescence through an Akt/p21{sup WAF1} signaling pathway

    SciTech Connect

    Kim, Hag Dong; Jang, Chang-Young; Choe, Jeong Min; Sohn, Jeongwon; Kim, Joon

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Phenylbutyric acid induces cellular senescence. Black-Right-Pointing-Pointer Phenylbutyric acid activates Akt kinase. Black-Right-Pointing-Pointer The knockdown of PERK also can induce cellular senescence. Black-Right-Pointing-Pointer Akt/p21{sup WAF1} pathway activates in PERK knockdown induced cellular senescence. -- Abstract: It has been well known that three sentinel proteins - PERK, ATF6 and IRE1 - initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21{sup WAF1} induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21{sup WAF1} pathway by PERK inhibition.

  4. p21{sup WAF1/Cip1/Sdi1} knockout mice respond to doxorubicin with reduced cardiotoxicity

    SciTech Connect

    Terrand, Jerome; Xu, Beibei; Morrissy, Steve; Dinh, Thai Nho; Williams, Stuart; Chen, Qin M.

    2011-11-15

    Doxorubicin (Dox) is an antineoplastic agent that can cause cardiomyopathy in humans and experimental animals. As an inducer of reactive oxygen species and a DNA damaging agent, Dox causes elevated expression of p21{sup WAF1/Cip1/Sdi1} (p21) gene. Elevated levels of p21 mRNA and p21 protein have been detected in the myocardium of mice following Dox treatment. With chronic treatment of Dox, wild type (WT) animals develop cardiomyopathy evidenced by elongated nuclei, mitochondrial swelling, myofilamental disarray, reduced cardiac output, reduced ejection fraction, reduced left ventricular contractility, and elevated expression of ANF gene. In contrast, p21 knockout (p21KO) mice did not show significant changes in the same parameters in response to Dox treatment. In an effort to understand the mechanism of the resistance against Dox induced cardiomyopathy, we measured levels of antioxidant enzymes and found that p21KO mice did not contain elevated basal or inducible levels of glutathione peroxidase and catalase. Measurements of 6 circulating cytokines indicated elevation of IL-6, IL-12, IFN{gamma} and TNF{alpha} in Dox treated WT mice but not p21KO mice. Dox induced elevation of IL-6 mRNA was detected in the myocardium of WT mice but not p21KO mice. While the mechanism of the resistance against Dox induced cardiomyopathy remains unclear, lack of inflammatory response may contribute to the observed cardiac protection in p21KO mice. -- Highlights: Black-Right-Pointing-Pointer Doxorubicin induces p21 elevation in the myocardium. Black-Right-Pointing-Pointer Doxorubicin causes dilated cardiomyopathy in wild type mice. Black-Right-Pointing-Pointer p21 Knockout mice are resistant against doxorubicin induced cardiomyopathy. Black-Right-Pointing-Pointer Lack of inflammatory response correlates with the resistance in p21 knockout mice.

  5. Forkhead Box O1 (FOXO1) Protein, but Not p53, Contributes to Robust Induction of p21 Expression in Fasted Mice*

    PubMed Central

    Tinkum, Kelsey L.; White, Lynn S.; Marpegan, Luciano; Herzog, Erik; Piwnica-Worms, David; Piwnica-Worms, Helen

    2013-01-01

    Reporter mice that enable the activity of the endogenous p21 promoter to be dynamically monitored in real time in vivo and under a variety of experimental conditions revealed ubiquitous p21 expression in mouse organs including the brain. Low light bioluminescence microscopy was employed to localize p21 expression to specific regions of the brain. Interestingly, p21 expression was observed in the paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus, regions that detect nutrient levels in the blood stream and signal metabolic actions throughout the body. These results suggested a link between p21 expression and metabolic regulation. We found that short-term food deprivation (fasting) potently induced p21 expression in tissues involved in metabolic regulation including liver, pancreas and hypothalamic nuclei. Conditional reporter mice were generated that enabled hepatocyte-specific expression of p21 to be monitored in vivo. Bioluminescence imaging demonstrated that fasting induced a 7-fold increase in p21 expression in livers of reporter mice and Western blotting demonstrated an increase in protein levels as well. The ability of fasting to induce p21 expression was found to be independent of p53 but dependent on FOXO1. Finally, occupancy of the endogenous p21 promoter by FOXO1 was observed in the livers of fasted but not fed mice. Thus, fasting promotes loading of FOXO1 onto the p21 promoter to induce p21 expression in hepatocytes. PMID:23918930

  6. Forkhead box O1 (FOXO1) protein, but not p53, contributes to robust induction of p21 expression in fasted mice.

    PubMed

    Tinkum, Kelsey L; White, Lynn S; Marpegan, Luciano; Herzog, Erik; Piwnica-Worms, David; Piwnica-Worms, Helen

    2013-09-27

    Reporter mice that enable the activity of the endogenous p21 promoter to be dynamically monitored in real time in vivo and under a variety of experimental conditions revealed ubiquitous p21 expression in mouse organs including the brain. Low light bioluminescence microscopy was employed to localize p21 expression to specific regions of the brain. Interestingly, p21 expression was observed in the paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus, regions that detect nutrient levels in the blood stream and signal metabolic actions throughout the body. These results suggested a link between p21 expression and metabolic regulation. We found that short-term food deprivation (fasting) potently induced p21 expression in tissues involved in metabolic regulation including liver, pancreas and hypothalamic nuclei. Conditional reporter mice were generated that enabled hepatocyte-specific expression of p21 to be monitored in vivo. Bioluminescence imaging demonstrated that fasting induced a 7-fold increase in p21 expression in livers of reporter mice and Western blotting demonstrated an increase in protein levels as well. The ability of fasting to induce p21 expression was found to be independent of p53 but dependent on FOXO1. Finally, occupancy of the endogenous p21 promoter by FOXO1 was observed in the livers of fasted but not fed mice. Thus, fasting promotes loading of FOXO1 onto the p21 promoter to induce p21 expression in hepatocytes. PMID:23918930

  7. p21(WAF1) (/Cip1) limits senescence and acinar-to-ductal metaplasia formation during pancreatitis.

    PubMed

    Grabliauskaite, Kamile; Hehl, Adrian B; Seleznik, Gitta M; Saponara, Enrica; Schlesinger, Kathryn; Zuellig, Richard A; Dittmann, Anja; Bain, Martha; Reding, Theresia; Sonda, Sabrina; Graf, Rolf

    2015-02-01

    Trans-differentiation of pancreatic acinar cells into ductal-like lesions, a process defined as acinar-to-ductal metaplasia (ADM), is observed in the course of organ regeneration following pancreatitis. In addition, ADM is found in association with pre-malignant PanIN lesions and correlates with an increased risk of pancreatic adenocarcinoma (PDAC). Human PDAC samples show down-regulation of p21(WAF1) (/Cip1) , a key regulator of cell cycle and cell differentiation. Here we investigated whether p21 down-regulation is implicated in controlling the early events of acinar cell trans-differentiation and ADM formation. p21-mediated regulation of ADM formation and regression was analysed in vivo during the course of cerulein-induced pancreatitis, using wild-type (WT) and p21-deficient (p21(-/-) ) mice. Biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks of the disease and during a recovery phase. We found that p21 was strongly up-regulated in WT acinar cells during pancreatitis, while it was absent in ADM areas, suggesting that p21 down-regulation is associated with ADM formation. In support of this hypothesis, p21(-/-) mice showed a significant increase in number and size of metaplasia. In addition, p21 over-expression in acinar cells reduced ADM formation in vitro, suggesting that the protein regulates the metaplastic transition in a cell-autonomous manner. p21(-/-) mice displayed increased expression and relocalization of β-catenin both during pancreatitis and in the subsequent recovery phase. Finally, loss of p21 was accompanied by increased DNA damage and development of senescence. Our findings are consistent with a gate-keeper role of p21 in acinar cells to limit senescence activation and ADM formation during pancreatic regeneration. PMID:25212177

  8. HBP1-mediated Regulation of p21 Protein through the Mdm2/p53 and TCF4/EZH2 Pathways and Its Impact on Cell Senescence and Tumorigenesis*

    PubMed Central

    Chen, Yifan; Pan, Kewu; Wang, Pingzhang; Cao, Zhengyi; Wang, Weibin; Wang, Shuya; Hu, Ningguang; Xue, Junhui; Li, Hui; Jiang, Wei; Li, Gang; Zhang, Xiaowei

    2016-01-01

    The activity of the CDK inhibitor p21 is associated with diverse biological activities, including cell proliferation, senescence, and tumorigenesis. However, the mechanisms governing transcription of p21 need to be extensively studied. In this study, we demonstrate that the high-mobility group box-containing protein 1 (HBP1) transcription factor is a novel activator of p21 that works as part of a complex mechanism during senescence and tumorigenesis. We found that HBP1 activates the p21 gene through enhancing p53 stability by inhibiting Mdm2-mediated ubiquitination of p53, a well known positive regulator of p21. HBP1 was also found to enhance p21 transcription by inhibiting Wnt/β-catenin signaling. We identified histone methyltransferase EZH2, the catalytic subunit of polycomb repressive complex 2, as a target of Wnt/β-catenin signaling. HBP1-mediated repression of EZH2 through Wnt/β-catenin signaling decreased the level of trimethylation of histone H3 at lysine 27 of overall and specific histone on the p21 promoter, resulting in p21 transactivation. Although intricate, the reciprocal partnership of HBP1 and p21 has exceptional importance. HBP1-mediated elevation of p21 through the Mdm2/p53 and TCF4/EZH2 pathways contributes to both cellular senescence and tumor inhibition. Together, our results suggest that the HBP1 transcription factor orchestrates a complex regulation of key genes during cellular senescence and tumorigenesis with an impact on protein ubiquitination and overall histone methylation state. PMID:27129219

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

    PubMed

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

    2008-01-01

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

  10. Soluble egg antigens of Schistosoma japonicum induce senescence in activated hepatic stellate cells by activation of the STAT3/p53/p21 pathway.

    PubMed

    Chen, Jinling; Pan, Jing; Wang, Jianxin; Song, Ke; Zhu, Dandan; Huang, Caiqun; Duan, Yinong

    2016-01-01

    Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs). Recent findings suggest that senescence of activated HSCs might limit the development of liver fibrosis. Based on previously observed anti-fibrotic effects of soluble egg antigens from Schistosoma japonicum in vitro, we hypothesized that SEA might play a crucial role in alleviating liver fibrosis through promoting senescence of activated HSCs. We show here that SEA inhibited expression of α-SMA and pro-collagen I and promoted senescence of activated HSCs in vitro. In addition, SEA induced an increased expression of P-p53 and p21. Knockdown of p53 inhibited the expression of p21 and failed to induce senescence of activated-HSCs. Phosphorylated STAT3 was elevated upon SEA stimulation, while loss of STAT3 decreased the level of p53 and senescence of HSCs. Results from immunoprecipitation analysis demonstrated that SOCS3 might be involved in the SEA-induced senescence in HSCs through its interaction with p53. This study demonstrates the potential capacity of SEA in restricting liver fibrosis through promoting senescence in HSCs. Furthermore, a novel STAT3-p53-p21 pathway might participate in the observed SEA-mediated senescence of HSCs. Our results suggest that SEA might carry potential therapeutic effects of restraining liver fibrosis through promoting senescence. PMID:27489164

  11. Soluble egg antigens of Schistosoma japonicum induce senescence in activated hepatic stellate cells by activation of the STAT3/p53/p21 pathway

    PubMed Central

    Chen, Jinling; Pan, Jing; Wang, Jianxin; Song, Ke; Zhu, Dandan; Huang, Caiqun; Duan, Yinong

    2016-01-01

    Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs). Recent findings suggest that senescence of activated HSCs might limit the development of liver fibrosis. Based on previously observed anti-fibrotic effects of soluble egg antigens from Schistosoma japonicum in vitro, we hypothesized that SEA might play a crucial role in alleviating liver fibrosis through promoting senescence of activated HSCs. We show here that SEA inhibited expression of α-SMA and pro-collagen I and promoted senescence of activated HSCs in vitro. In addition, SEA induced an increased expression of P-p53 and p21. Knockdown of p53 inhibited the expression of p21 and failed to induce senescence of activated-HSCs. Phosphorylated STAT3 was elevated upon SEA stimulation, while loss of STAT3 decreased the level of p53 and senescence of HSCs. Results from immunoprecipitation analysis demonstrated that SOCS3 might be involved in the SEA-induced senescence in HSCs through its interaction with p53. This study demonstrates the potential capacity of SEA in restricting liver fibrosis through promoting senescence in HSCs. Furthermore, a novel STAT3-p53-p21 pathway might participate in the observed SEA-mediated senescence of HSCs. Our results suggest that SEA might carry potential therapeutic effects of restraining liver fibrosis through promoting senescence. PMID:27489164

  12. SEMA3B improves the survival of patients with esophageal squamous cell carcinoma by upregulating p53 and p21.

    PubMed

    Tang, Hong; Wu, Yufeng; Liu, Ming; Qin, Yanru; Wang, Haiying; Wang, Lili; Li, Shaomei; Zhu, Hui; He, Zheng; Luo, Junpeng; Wang, Hongyan; Wang, Qiming; Luo, Suxia

    2016-08-01

    As one of the most common malignancies, esophageal squamous cell carcinoma (ESCC) is ranked as the sixth leading cause of cancer-related death worldwide. In our previous study, by employing cDNA microarray analysis, semaphorin 3B (SEMA3B) was found to be significantly downregulated in ESCC. In the present study, SEMA3B downregulation at the mRNA level was found in 34 of 60 primary ESCCs (56.7%) and in 6 of 9 ESCC cell lines (66.7%) by transcription-polymerase chain reaction (RT-PCR). Moreover, immunohistochemical (IHC) staining of SEMA3B in a tissue microarray further indicated that downregulated expression of SEMA3B protein was found in 125 of 222 (56.3%) ESCC cases and downregulation of SEMA3B protein was significantly correlated with lymph node metastasis (P=0.000), advanced clinicopathological stage (P=0.001) and poor disease-specific survival (P=0.017) of ESCC patients. In addition, functional studies demonstrated that the SEMA3B gene could suppress the tumorigenic ability of ESCC cells and cell motility. Furthermore, it was found that by upregulating p53 and p21 expression and inhibiting Akt (Ser473) phosphorylation, SEMA3B could induce cell cycle arrest at G1/S phase. Taken together, our results suggest that SEMA3B may be an important tumor-suppressor gene in the malignant progression of ESCC, as well as a valuable prognostic marker for ESCC patients. PMID:27349960

  13. Msi1 promotes tumor growth and cell proliferation by targeting cell cycle checkpoint proteins p21, p27 and p53 in cervical carcinomas

    PubMed Central

    Liu, Xian; Yang, Wen-Ting; Zheng, Peng-Sheng

    2014-01-01

    Musashi RNA-binding protein1 (Msi1), a member of the RNA-binding protein family, has been reported to be a diagnostic marker and potential therapeutic target in some cancers, its function in cervical cancer remains unknown. In this study, we found Msi1 was highly expressed in cervical cancer tissues, and over-expressing Msi1 in cervical cancer cells enhanced tumor formation and cell proliferation and accelerated cells into the S phase. Whereas, down-regulating Msi1 by shRNA in cervical cancer cells inhibited tumor formation and cell proliferation and slowed cell into the S phase, suggesting that Msi1 might act as cell cycle regulator. Immunohistochemistry assay showed the negative correlation between Msi1 and p21, p27 and p53, suggesting that Msi1 might regulate these cycle regulators in cervical cancer. Moreover, the expression of the p21, p27 and p53 proteins were down-regulated in Msi1 overexpressing cervical cancer cells and up-regulated in shMsi1 cervical cancer cells. Luciferase assays and RNA-protein binding assays confirmed that Msi1 could bind to the mRNA 3′UTRs of p21, p27 and p53 and suppress the translation of these proteins. Our findings provide new evidence that Msi1 might promote cell proliferation by accelerating the cell cycle by directly targeting p21, p27 and p53. PMID:25362645

  14. Glucocorticoids induce senescence in primary human tenocytes by inhibition of sirtuin 1 and activation of the p53/p21 pathway: in vivo and in vitro evidence

    PubMed Central

    Poulsen, Raewyn C; Watts, Anna C; Murphy, Richard J; Snelling, Sarah J; Carr, Andrew J; Hulley, Philippa A

    2014-01-01

    Cellular senescence is an irreversible side effect of some pharmaceuticals which can contribute to tissue degeneration. Objective To determine whether pharmaceutical glucocorticoids induce senescence in tenocytes. Methods Features of senescence (β-galactosidase activity at pH 6 (SA-β-gal) and active mammalian/mechanistic target of rapamycin (mTOR) in cell cycle arrest) as well as the activity of the two main pathways leading to cell senescence were examined in glucocorticoid-treated primary human tenocytes. Evidence of senescence-inducing pathway induction in vivo was obtained using immunohistochemistry on tendon biopsy specimens taken before and 7 weeks after subacromial Depo-Medrone injection. Results Dexamethasone treatment of tenocytes resulted in an increased percentage of SA-βgal-positive cells. Levels of phosphorylated p70S6K did not decrease with glucocorticoid treatment indicating mTOR remained active. Increased levels of acetylated p53 as well as increased RNA levels of its pro-senescence effector p21 were evident in dexamethasone-treated tenocytes. Levels of the p53 deacetylase sirtuin 1 were lower in dexamethasone-treated cells compared with controls. Knockdown of p53 or inhibition of p53 activity prevented dexamethasone-induced senescence. Activation of sirtuin 1 either by exogenous overexpression or by treatment with resveratrol or low glucose prevented dexamethasone-induced senescence. Immunohistochemical analysis of tendon biopsies taken before and after glucocorticoid injection revealed a significant increase in the percentage of p53-positive cells (p=0.03). The percentage of p21-positive cells also tended to be higher post-injection (p=0.06) suggesting glucocorticoids activate the p53/p21 senescence-inducing pathway in vivo as well as in vitro. Conclusion As cell senescence is irreversible in vivo, glucocorticoid-induced senescence may result in long-term degenerative changes in tendon tissue. PMID:23727633

  15. Novel cis-trans interactions are involved in post-transcriptional regulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1 mRNA

    PubMed Central

    2010-01-01

    Background A variety of pathways target CDKI p21WAF1/CIP1 expression at transcriptional, post-transcriptional as well as translational levels. We previously found that cell growth suppressing retinoid CD437 enhanced expression of p21WAF1/CIP1 and DNA damage inducible GADD45 proteins in part by elevating their mRNA stability. Results Here, we investigated molecular mechanisms of CD437-dependent post-transcriptional regulation of p21WAF1/CIP1 expression. By utilizing MDA-MB-468 HBC cells expressing chimeric rabbit β-globin-p21WAF1/CIP1 transcripts we mapped multiple CD437-responsive sequences located within positions 1195 to 1795 of the 3'-untranslated region of p21WAF1/CIP1 mRNA. Several cytoplasmic proteins present in MDA-MB-468, MCF-7 HBC as well as HL-60R leukemia cells bound specifically, in vitro, with these CD437-responsive sequences. CD437 treatment of cells resulted in elevated binding of ~85 kD and ~55 kD cytoplasmic proteins with putative CD437-responsive sequences. A 12 nt RNA sequence (5'-UGUGGUGGCACA-3') present within CD437-responsive region of p21WAF1/CIP1 mRNA displayed specific and elevated binding with the above noted proteins. Treatment of cells with ActD or CHX prior to CD437 exposure did not abrogate RNA-protein interactions. However, treatment of cytoplasmic protein extracts with proteinase K or alkaline phosphatase resulted in loss of RNA-protein interactions. Conclusions CD437 regulates cell growth in part by regulating stability of p21WAF1/CIP1 mRNA that involves specific RNA-protein interactions that are phosphorylation-dependent, while not requiring nascent transcription or protein synthesis. PMID:20704727

  16. 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.

  17. Programmed cell death protein 4 suppresses CDK1/cdc2 via induction of p21(Waf1/Cip1).

    PubMed

    Göke, R; Barth, P; Schmidt, A; Samans, B; Lankat-Buttgereit, B

    2004-12-01

    We show that the recently discovered tumor suppressor pdcd4 represses the transcription of the mitosis-promoting factor cyclin-dependent kinase (CDK)1/cdc2 via upregulation of p21(Waf1/Cip1). p21(Waf1/Cip1) inhibits CDK4/6 and CDK2. Decrease of CDK4/6 and CDK2 enhances the binding of pRb to E2F/DP, which in turn together bind to and repress the cdc2 promoter. Upregulation of CDK1/cdc2 accompanied by a malignant change was previously reported in colon cancer. We show that expression of pdcd4 as an indirect suppressor of CDK1/cdc2 is lost in progressed carcinomas of lung, breast, colon, and prostate. Furthermore, it seems that localization and expression of pdcd4 directly correlate with tumor progression. Finally, the CDK1/cdc2 inhibitor roscovitine reduces the proliferation of several tumor cell lines, suggesting that inhibition of CDK1/cdc2 may be a useful strategy against malignant transformation. Therefore, pdcd4 might serve as a novel target for antineoplastic therapies. PMID:15317660

  18. 58-kDa Microspherule Protein (MSP58) Is Novel Brahma-related Gene 1 (BRG1)-associated Protein That Modulates p53/p21 Senescence Pathway*

    PubMed Central

    Hsu, Che-Chia; Lee, Yi-Chao; Yeh, Shiu-Hwa; Chen, Chang-Han; Wu, Chih-Ching; Wang, Tsui-Ying; Chen, Yu-Nong; Hung, Liang-Yi; Liu, Yao-Wen; Chen, Han-Ku; Hsiao, Yi-Ting; Wang, Wei-Sheng; Tsou, Jen-Hui; Tsou, Yi-Huan; Wu, Mei-Hsiang; Chang, Wen-Chang; Lin, Ding-Yen

    2012-01-01

    The nucleolar 58-kDa microspherule protein (MSP58) protein is a candidate oncogene implicated in modulating cellular proliferation and malignant transformation. In this study, we show that knocking down MSP58 expression caused aneuploidy and led to apoptosis, whereas ectopic expression of MSP58 regulated cell proliferation in a context-dependent manner. Specifically, ectopic expression of MSP58 in normal human IMR90 and Hs68 diploid fibroblasts, the H184B5F5/M10 mammary epithelial cell line, HT1080 fibrosarcoma cells, primary mouse embryonic fibroblasts, and immortalized NIH3T3 fibroblasts resulted in induction of premature senescence, an enlarged and flattened cellular morphology, and increased senescence-associated β-galactosidase activity. MSP58-driven senescence was strictly dependent on the presence of functional p53 as revealed by the fact that normal cells with p53 knockdown by specific shRNA or cells with a mutated or functionally impaired p53 pathway were effective in bypassing MSP58-induced senescence. At least two senescence mechanisms are induced by MSP58. First, MSP58 activates the DNA damage response and p53/p21 signaling pathways. Second, MSP58, p53, and the SWI/SNF chromatin-remodeling subunit Brahma-related gene 1 (BRG1) form a ternary complex on the p21 promoter and collaborate to activate p21. Additionally, MSP58 protein levels increased in cells undergoing replicative senescence and stress-induced senescence. Notably, the results of analyzing expression levels of MSP58 between tumors and matched normal tissues showed significant changes (both up- and down-regulation) in its expression in various types of tumors. Our findings highlight new aspects of MSP58 in modulating cellular senescence and suggest that MSP58 has both oncogenic and tumor-suppressive properties. PMID:22563078

  19. Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis; the role of p53 and p21

    PubMed Central

    Escandell, José M.; Kaler, Pawan; Recio, M. Carmen; Sasazuki, Takehiko; Shirasawa, Senji; Augenlicht, Leonard; Ríos, José-Luis; Klampfer, Lidija

    2008-01-01

    Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke3 cells. However, the presence of oncogenic k-Ras significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransfromed intestinal epithelial cells with inducible expression of k-RasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3. PMID:18561895

  20. Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis: the role of p53 and p21.

    PubMed

    Escandell, José M; Kaler, Pawan; Recio, M Carmen; Sasazuki, Takehiko; Shirasawa, Senji; Augenlicht, Leonard; Ríos, José-Luis; Klampfer, Lidija

    2008-07-15

    Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke-3 cells. However, the presence of oncogenic kRas significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransformed intestinal epithelial cells with inducible expression of kRasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3. PMID:18561895

  1. Histone deacetylase 3 represses p15{sup INK4b} and p21{sup WAF1/cip1} transcription by interacting with Sp1

    SciTech Connect

    Huang Weifeng; Tan Dapeng; Wang Xiuli; Han Songyan; Tan Jiang; Zhao Yanmei; Lu Jun . E-mail: ycsuo@nenu.edu.cn; Huang Baiqu

    2006-01-06

    Histone deacetylase 3 (HDAC3) has been implicated to play roles in governing cell proliferation. Here we demonstrated that the overexpression of HDAC3 repressed transcription of p15{sup INK4b} and p21{sup WAF1/cip1} genes in 293T cells, and that the recruitment of HDAC3 to the promoter regions of these genes was critical to this repression. We also showed that HDAC3 repressed GAL4-Sp1 transcriptional activity, and that Sp1 was co-immunoprecipitated with FLAG-tagged HDAC3. We conclude that HDAC3 can repress p15{sup INK4b} and p21{sup WAF1/cip1} transcription by interacting with Sp1. Furthermore, knockdown of HDAC3 by RNAi up-regulated the transcriptional expression of p15{sup INK4b}, but not that of p21{sup WAF1/cip1}, implicating the different roles of HDAC3 in repression of p15{sup INK4b} and p21{sup WAF1/cip1} transcription. Data from this study indicate that the inhibition of p15{sup INK4b} and p21{sup WAF1/cip1} may be one of the mechanisms by which HDAC3 participates in cell cycle regulation and oncogenesis.

  2. miR-299-5p promotes cell growth and regulates G1/S transition by targeting p21Cip1/Waf1 in acute promyelocytic leukemia

    PubMed Central

    WU, SHUN-QUAN; ZHANG, LANG-HUI; HUANG, HAO-BO; LI, YA-PING; NIU, WEN-YAN; ZHAN, RONG

    2016-01-01

    MicroRNAs (miRs) are often located in genomic breakpoint regions and are hypothesized to be important regulators involved in the regulation of critical cell processes, including cell apoptosis, proliferation and differentiation. miR-299 has been reported to be upregulated in acute promyelocytic leukemia (APL); however, the function and mechanistic role of miR-299 in APL remains unknown. The present study demonstrated mir-299 significantly induced cell growth and cell cycle progression at the G1/S transition in APL cells. Notably, the present study revealed that miR-299-5p induces these effects, whereas miR-299-3p does not. Additional studies demonstrated that in APL cells the tumor suppressor p21Cip1/Waf1 is a downstream target of miR-299; miR-299 binds directly to the 3′ untranslated region of p21Cip1/Waf1, and reduces protein, but not mRNA, levels of p21Cip1/Waf1. The present findings demonstrate that miR-299 exerts growth-promoting effects in APL cells through the suppression of p21Cip1/Waf1. Overall, the present study demonstrates that p21Cip1/Waf1 is a direct functional target of miR-299 in APL. PMID:27347210

  3. miR-24-3p Suppresses Malignant Behavior of Lacrimal Adenoid Cystic Carcinoma by Targeting PRKCH to Regulate p53/p21 Pathway

    PubMed Central

    Zhang, Hong; Tang, Hua

    2016-01-01

    MicroRNA (miRNA) may function as an oncogene or a tumor suppressor in tumorigenesis. However, the mechanism of miRNAs in adenoid cystic carcinoma (ACC) is unclear. Here, we provide evidence that miR-24-3p was downreglated and functions as a tumor suppressor in human lacrimal adenoid cystic carcinoma by suppressing proliferation and migration/invasion while promoting apoptosis. miR-24-3p down-regulated protein kinase C eta (PRKCH) by binding to its untranslated region (3’UTR). PRKCH increased the of the cell growth and migration/invasion in ACC cells and suppressed the expression of p53 and p21 in both mRNA and protein level. The overexpression of miR-24-3p decreased its malignant phenotype. Ectopic expression of PRKCH counteracted the suppression of malignancy induced by miR-24-3p, as well as ectopic expression of miR-24-3p rescued the suppression of PRKCH in the p53/p21 pathway. These results suggest that miR-24-3p promotes the p53/p21 pathway by down-regulating PRKCH expression in lacrimal adenoid cystic carcinoma cells. PMID:27351203

  4. RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC.

    PubMed

    Majumder, Mrinmoyee; House, Reniqua; Palanisamy, Nallasivam; Qie, Shuo; Day, Terrence A; Neskey, David; Diehl, J Alan; Palanisamy, Viswanathan

    2016-09-01

    RNA-binding proteins (RBP) regulate numerous aspects of co- and post-transcriptional gene expression in cancer cells. Here, we demonstrate that RBP, fragile X-related protein 1 (FXR1), plays an essential role in cellular senescence by utilizing mRNA turnover pathway. We report that overexpressed FXR1 in head and neck squamous cell carcinoma targets (G-quadruplex (G4) RNA structure within) both mRNA encoding p21 (Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A, Cip1) and the non-coding RNA Telomerase RNA Component (TERC), and regulates their turnover to avoid senescence. Silencing of FXR1 in cancer cells triggers the activation of Cyclin-Dependent Kinase Inhibitors, p53, increases DNA damage, and ultimately, cellular senescence. Overexpressed FXR1 binds and destabilizes p21 mRNA, subsequently reduces p21 protein expression in oral cancer cells. In addition, FXR1 also binds and stabilizes TERC RNA and suppresses the cellular senescence possibly through telomerase activity. Finally, we report that FXR1-regulated senescence is irreversible and FXR1-depleted cells fail to form colonies to re-enter cellular proliferation. Collectively, FXR1 displays a novel mechanism of controlling the expression of p21 through p53-dependent manner to bypass cellular senescence in oral cancer cells. PMID:27606879

  5. Tetrahydroanthraquinone Derivative (±)-4-Deoxyaustrocortilutein Induces Cell Cycle Arrest and Apoptosis in Melanoma Cells via Upregulation of p21 and p53 and Downregulation of NF-kappaB

    PubMed Central

    Genov, Miroslav; Kreiseder, Birgit; Nagl, Michael; Drucker, Elisabeth; Wiederstein, Martina; Muellauer, Barbara; Krebs, Julia; Grohmann, Teresa; Pretsch, Dagmar; Baumann, Karl; Bacher, Markus; Pretsch, Alexander; Wiesner, Christoph

    2016-01-01

    Background: Malignant melanoma is an aggressive type of skin cancer with high risk for metastasis and chemoresistance. Disruption of tightly regulated processes such as cell cycle, cell adhesion, cell differentiation and cell death are predominant in melanoma development. So far, conventional treatment options have been insufficient to treat metastatic melanoma and survival rates are poor. Anthraquinone compounds have been reported to have anti-tumorigenic potential by DNA-interaction, promotion of apoptosis and suppression of proliferation in various cancer cells. Methods: In the current study, the racemic tetrahydroanthraquinone derivative (±)-4-deoxyaustrocortilutein (4-DACL) was synthesized and the cytotoxic activity against melanoma cells and melanoma spheroids determined by CellTiter-Blue viability Assay and phase contrast microscopy. Generation of reactive oxygen species (ROS) was determined with CellROX Green and Deep Red Reagent kit and microplate-based fluorometry. Luciferase reporter gene assays for nuclear factor kappa B (NF-κB) and p53 activities and western blotting analysis were carried out to detect the expression of anti-proliferative or pro-apoptotic (p53, p21, p27, MDM2, and GADD45M) and anti-apoptotic (p65, IκB-α, IKK) proteins. Cell cycle distribution and apoptosis rate were detected by flow cytometry, the morphological changes visualized by fluorescence microscopy and the activation of different caspase cascades distinguished by Caspase Glo 3/7, 8 and 9 Assays. Results: We demonstrated that 4-DACL displayed high activity against different malignant melanoma cells and melanoma spheroids and only low toxicity to melanocytes and other primary cells. In particular, 4-DACL treatment induced mitochondrial ROS, reduced NF-κB signaling activity and increased up-regulation of the cell cycle inhibitors cyclin-dependent kinase inhibitor p21 (p21WAF1/Cip1) and the tumor suppressor protein p53 in a dose-dependent manner, which was accompanied by

  6. Doxorubicin Activates Hepatitis B Virus Replication by Elevation of p21 (Waf1/Cip1) and C/EBPα Expression

    PubMed Central

    Chen, Yu-Fang; Chong, Chin-Liew; Wu, Yi-Chieh; Wang, Yi-Ling; Tsai, Kuen-Nan; Kuo, Tzer-Min; Hong, Ming-Hsiang; Hu, Cheng-po; Chen, Mong-Liang; Chou, Yu-Chi; Chang, Chungming

    2015-01-01

    Hepatitis B virus reactivation is an important medical issue in cancer patients who undergo systemic chemotherapy. Up to half of CHB carriers receiving chemotherapy develop hepatitis and among these cases a notable proportion are associated with HBV reactivation. However, the molecular mechanism(s) through which various chemotherapeutic agents induce HBV reactivation is not yet fully understood. In this study, we investigated the role of the cell cycle regulator p21 (Waf1/Cip1) in the modulation of HBV replication when a common chemotherapeutic agent, doxorubicin, is present. We showed that p21 expression was increased by doxorubicin treatment. This elevation in p21 expression enhanced the expression of CCAAT/enhancer-binding protein α (C/EBPα); such an increase is likely to promote the binding of C/EBPα to the HBV promoter, which will contribute to the activation of HBV replication. Our current study thus reveals the mechanism underlying doxorubicin modulation of HBV replication and provides an increased understanding of HBV reactivation in CHB patients who are receiving systemic chemotherapy. PMID:26121644

  7. A Novel Interaction between FLICE-Associated Huge Protein (FLASH) and E2A Regulates Cell Proliferation and Cellular Senescence via Tumor Necrosis Factor (TNF)-Alpha-p21WAF1/CIP1 Axis

    PubMed Central

    Hirano, Takahiro; Murakami, Taichi; Ono, Hiroyuki; Sakurai, Akiko; Tominaga, Tatsuya; Takahashi, Toshikazu; Nagai, Kojiro; Doi, Toshio; Abe, Hideharu

    2015-01-01

    Dysregulation of the cell proliferation has been implicated in the pathophysiology of a number of diseases. Cellular senescence limits proliferation of cancer cells, preventing tumorigenesis and restricting tissue damage. However, the role of cellular senescence in proliferative nephritis has not been determined. The proliferative peak in experimental rat nephritis coincided with a peak in E2A expression in the glomeruli. Meanwhile, E12 (an E2A-encoded transcription factor) did not promote proliferation of Mesangial cells (MCs) by itself. We identified caspase-8-binding protein FLICE-associated huge protein (FLASH) as a novel E2A-binding partner by using a yeast two-hybrid screening. Knockdown of FLASH suppressed proliferation of MCs. This inhibitory effect was partially reversed by the knockdown of E2A. In addition, the knockdown of FLASH induced cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) expression, but did not affect p53 expression. Furthermore, overexpression of E12 and E47 induced p21, but not p53 in MCs, in the absence of FLASH. We also demonstrated that E2A and p21 expression at the peak of proliferation was followed by significant induction of FLASH in mesangial areas in rat proliferative glomerulonephritis. Moreover, we revealed that FLASH negatively regulates cellular senescence via the interaction with E12. We also demonstrated that FLASH is involved in the TNF-α-induced p21 expressions. These results suggest that the functional interaction of E2A and FLASH play an important role in cell proliferation and cellular senescence via regulation of p21 expression in experimental glomerulonephritis. PMID:26208142

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

    SciTech Connect

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

    2013-11-15

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

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

    PubMed

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

    2016-01-01

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

  10. Expression of E6, p53 and p21 proteins and physical state of HPV16 in cervical cytologies with and without low grade lesions

    PubMed Central

    Tagle, Diana K Jiménez; Sotelo, Daniel Hernández; Illades-Aguiar, Berenice; Leyva-Vazquez, Marco A; Alfaro, Eugenia Flores; Coronel, Yaneth Castro; Hernández, Oscar del Moral; Romero, Luz del Carmen Alarcón

    2014-01-01

    The aim of this study was to determine the correlation between expression of HPV16 E6, p53 and p21 proteins and the physical state of HPV16 in cervical cytologies without squamous intraepithelial lesions (Non-SIL) and with low grade squamous intraepithelial lesions (LSIL), both with HPV16 infection. 101 liquid-based cytological samples were analyzed. 50 samples were without squamous intraepithelial lesions (Non-IL) and 51 samples of low grade squamous intraepithelial lesions (LSIL), both with HPV16 infection. HPV16 infection was determined by PCR-RFLP, and the physical state of HPV16 by in situ hybridization with tyramide-amplification. The expression of E6, p53 and p21 proteins was evaluated by immunocytochemistry. The expression of HPV16 E6 protein was significantly higher in LSIL that in Non-SIL samples (p=0.006). We found a significant correlation between E6 expression and the physical state of HPV16 in Non-SIL (p=0.049). Our results suggest that high expression of E6 in LSIL is an early event of cervical carcinogenesis and perhaps can be used as an early marker. PMID:24482706

  11. Damnacanthal is a potent inducer of apoptosis with anticancer activity by stimulating p53 and p21 genes in MCF-7 breast cancer cells.

    PubMed

    Aziz, Muhammad Yusran Abdul; Omar, Abdul Rahman; Subramani, Tamilselvan; Yeap, Swee Keong; Ho, Wan Yong; Ismail, Nor Hadiani; Ahmad, Syahida; Alitheen, Noorjahan Banu

    2014-05-01

    Damnacanthal, an anthraquinone compound, is isolated from the roots of Morinda citrifolia L. (noni), which has been used for traditional therapy in several chronic diseases, including cancer. Although noni has long been consumed in Asian and Polynesian countries, the molecular mechanisms by which it exerts several benefits are starting to emerge. In the present study, the effect of damnacanthal on MCF-7 cell growth regulation was investigated. Treatment of MCF-7 cells with damnacanthal for 72 h indicated an antiproliferative activity. The MTT method confirmed that damnacanthal inhibited the growth of MCF-7 cells at the concentration of 8.2 μg/ml for 72 h. In addition, the drug was found to induce cell cycle arrest at the G1 checkpoint in MCF-7 cells by cell cycle analysis. Damnacanthal induced apoptosis, determined by Annexin V-fluorescein isothiocyanate/propidium iodide (PI) dual-labeling, acridine-orange/PI dyeing and caspase-7 expression. Furthermore, damnacanthal-mediated apoptosis involves the sustained activation of p21, leading to the transcription of p53 and the Bax gene. Overall, the present study provided significant evidence demonstrating that p53-mediated damnacanthal induced apoptosis through the activation of p21 and caspase-7. PMID:24765160

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

    PubMed Central

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

    2015-01-01

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

  13. Intracellular delivery of p53 fused to the basic domain of HIV-1 Tat.

    PubMed

    Ryu, Jiyoon; Lee, Hak Joo; Kim, Kyeong-Ae; Lee, Jae Yong; Lee, Kil Soo; Park, Jinseu; Choi, Soo Young

    2004-04-30

    p53 is a potent tumor suppressor inactivated in many cancers. In this study, the membrane permeability of the HIV-1 Tat basic domain was exploited to introduce functional p53 into cancer cells. We expressed and purified a p53 fusion protein with the HIV-1 Tat basic domain at its N terminus (Tat-p53), and examined its transduction profile and biological activity in cancer cells. Tat-p53 was efficiently delivered to both the cytoplasm and nucleus of cells, and was transcriptionally active, as judged by the level of p21/WAF1 protein and of p21 promoter activity. Transduction of cells with Tat-p53 resulted in apoptotic cell death in both p53 positive and negative human tumor cell lines. These results suggest that Tat-p53 could be useful in cancer therapy. PMID:15179054

  14. Egg antigen p40 of Schistosoma japonicum promotes senescence in activated hepatic stellate cells by activation of the STAT3/p53/p21 pathway

    PubMed Central

    Chen, Jinling; Xu, Tianhua; Zhu, Dandan; Wang, Jianxin; Huang, Caiqun; Lyu, Lei; Hu, Bin; Sun, Wei; Duan, Yinong

    2016-01-01

    Liver fibrosis is a serious disease that is characterized by the excess deposition of extracellular matrix (ECM) components. Activated hepatic stellate cells (HSCs) are a major source of ECM and serve as a key regulator in liver fibrogenesis. Inactivation of HSCs is essential for liver fibrotic regression. The present study explores the underlying mechanisms of Schistosoma japonicum egg antigen p40 (Sjp40) promoting senescence in HSCs and antifibrosis. For the first time we report that Sjp40 inhibits the activation and proliferation of an immortalized human HSC line (LX-2 cells) and promotes cellular senescence and cell cycle arrest. Sjp40 through action on the STAT3/p53/p21 pathway triggered cellular senescence, while knockdown of p53 or STAT3 partly restored cell senescence. In addition, Sjp40-induced cellular senescence caused LX-2 cells to be more sensitive to a human NK cell line (YT cells). Together these findings provide novel insights into the mechanism of antifibrosis and may have implications for the development of antifibrosis therapies. PMID:27468691

  15. Decreased PM10 Exposure Attenuates Age-Related Lung Function Decline: Genetic Variants in p53, p21, and CCND1 Modify This Effect

    PubMed Central

    Imboden, Medea; Schwartz, Joel; Schindler, Christian; Curjuric, Ivan; Berger, Wolfgang; Liu, Sally L.J.; Russi, Erich W.; Ackermann-Liebrich, Ursula; Rochat, Thierry; Probst-Hensch, Nicole M.

    2009-01-01

    Background Decreasing exposure to airborne particulates was previously associated with reduced age-related decline in lung function. However, whether the benefit from improved air quality depends on genetic background is not known. Recent evidence points to the involvement of the genes p53 and p21 and of the cell cycle control gene cyclin D1 (CCND1) in the response of bronchial cells to air pollution. Objective We determined in 4,326 participants of the Swiss Cohort Study on Air Pollution and Lung and Heart Diseases in Adults (SAPALDIA) whether four single-nucleotide polymorphisms in three genes [CCND1 (rs9344 [P242P], rs667515), p53 (rs1042522 [R72P]), and p21 (rs1801270 [S31R])] modified the previously observed attenuation of the decline in the forced expiratory flow between 25% and 75% of the forced vital capacity (FEF25–75) associated with improved air quality. Methods Subjects of the prospective population-based SAPALDIA cohort were assessed in 1991 and 2002 by spirometry, questionnaires, and biological sample collection for genotyping. We assigned spatially resolved concentrations of particulate matter with aerodynamic diameter ≤ 10 μm (PM10) to each participant’s residential history 12 months before the baseline and follow-up assessments. Results The effect of diminishing PM10 exposure on FEF25–75 decline appeared to be modified by p53 R72P, CCND1 P242P, and CCND1 rs667515. For example, a 10-μg/m3 decline in aver-age PM10 exposure over an 11-year period attenuated the average annual decline in FEF25–75 by 21.33 mL/year (95% confidence interval, 10.57–32.08) among participants homozygous for the CCND1 (P242P) GG genotype, by 13.72 mL/year (5.38–22.06) among GA genotypes, and by 6.00 mL/year (−4.54 to 16.54) among AA genotypes. Conclusions Our results suggest that cell cycle control genes may modify the degree to which improved air quality may benefit respiratory function in adults. PMID:19750108

  16. Combination treatment with proteasome inhibitors and antiestrogens has a synergistic effect mediated by p21WAF1 in estrogen receptor-positive breast cancer.

    PubMed

    Maynadier, Marie; Basile, Ilaria; Gallud, Audrey; Gary-Bobo, Magali; Garcia, Marcel

    2016-08-01

    Although antiestrogens significantly improve the survival of patients with ER-positive breast cancer, therapeutic resistance remains a major limitation. The combinatorial use of antiestrogen with other therapies was proposed to increase their efficiency and more importantly, to prevent or delay the resistance phenomenon. In the present study, we addressed their combined effects with proteasome inhibitors (PIs). The effects of antiestrogens (hydroxyl-tamoxifen, raloxifen and fulvestrant) currently used in endocrine therapy were tested in combination with PIs, bortezomib or MG132, on the growth of three ER-positive breast cancer cell lines and in two cellular models of acquired antiestrogen resistance. When compared to single treatments, these combined treatments were significantly more effective in preventing the growth of the cell lines. The regulation of key cell cycle proteins, the cyclin-dependent kinase inhibitors, p21WAF1 and p27KIP1, were also studied. Bortezomib and MG132 drastically increased p21WAF1 expression through elevation of its mRNA concentration. Notably, p27KIP1 regulation was quite different from that of p21WAF1. Furthermore, the effect of bortezomib in combination with antiestrogen was evaluated on antiestrogen-resistant cell lines. The growth of two antiestrogen-resistant cell lines appeared responsive to proteasome inhibition and was strongly decreased by a combined therapy with an antiestrogen. Collectively, these findings provide new perspectives for the use of PIs in combination with endocrine therapies for breast cancer and possibly to overcome acquired hormonal resistance. PMID:27373750

  17. EWS-FLI1 Suppresses NOTCH-Activated p53 in Ewing’s Sarcoma

    PubMed Central

    Ban, Jozef; Bennani-Baiti, Idriss M.; Kauer, Max; Schaefer, Karl-Ludwig; Poremba, Christopher; Jug, Gunhild; Schwentner, Raphaela; Smrzka, Oskar; Muehlbacher, Karin; Aryee, Dave N.T.; Kovar, Heinrich

    2016-01-01

    Although p53 is the most frequently mutated gene in cancer, half of human tumors retain wild-type p53, whereby it is unknown whether normal p53 function is compromised by other cancer-associated alterations. One example is Ewing’s sarcoma family tumors (ESFT), where 90% express wild-type p53. ESFT are characterized by EWS-FLI1 oncogene fusions. Studying 6 ESFT cell lines, silencing of EWS-FLI1 in a wild-type p53 context resulted in increased p53 and p21WAF1/CIP1 levels, causing cell cycle arrest. Using a candidate gene approach, HEY1 was linked to p53 induction. HEY1 was rarely expressed in 59 primary tumors, but consistently induced upon EWS-FLI1 knockdown in ESFT cell lines. The NOTCH signaling pathway targets HEY1, and we show NOTCH2 and NOTCH3 to be expressed in ESFT primary tumors and cell lines. Upon EWS-FLI1 silencing, NOTCH3 processing accompanied by nuclear translocation of the activated intracellular domain was observed in all but one p53-mutant cell line. In cell lines with the highest HEY1 induction, NOTCH3 activation was the consequence of JAG1 transcriptional induction. JAG1 modulation by specific siRNA, NOTCH-processing inhibition by either GSI or ectopic NUMB1, and siRNA-mediated HEY1 knockdown all inhibited p53 and p21WAF1/CIP1 induction. Conversely, forced expression of JAG1, activated NOTCH3, or HEY1 induced p53 and p21WAF1/CIP1. These results indicate that suppression of EWS-FLI1 reactivates NOTCH signaling in ESFT cells, resulting in p53-dependent cell cycle arrest. Our data link EWS-FLI1 to the NOTCH and p53 pathways and provide a plausible basis both for NOTCH tumor suppressor effects and oncogenesis of cancers that retain wild-type p53. PMID:18757425

  18. Promoter-associated endogenous and exogenous small RNAs suppress human bladder cancer cell metastasis by activating p21 (CIP1/WAF1) expression.

    PubMed

    Wang, Chenghe; Ge, Qiangqiang; Chen, Zhong; Hu, Jia; Li, Fan; Ye, Zhangqun

    2016-05-01

    Accumulating data suggest that micro RNAs (miRNAs) or double-stranded RNAs (dsRNAs) can activate gene expression by targeting promoters. The cyclin-dependent kinase inhibitor p21 (CIP1/WAF1) (p21) has also been shown to suppress epithelial-mesenchymal transition (EMT) which plays a crucial role in the early stage of tumor metastases and invasiveness. In a previous study, we have reported that miR-370-5p is low-expressed in bladder cancer (BCa) tissues and cell lines. Here, we identified that miR-370-5p and sequence homology dsRNA (dsP21-555) fully complementary to promoter hold the potent abilities to induce p21 expression. Moreover, transfection of miR-370-5p or dsP21-555 into BCa cells remarkably inverts EMT-associated genes (increases epithelial cell makers E-cadherin and β-catenin, and decreases mesenchymal cell markers ZEB1 and Vimentin) expression mainly via regulating p21 expression. Besides, through manipulating p21, both the candidates can retard BCa cell migration and invasion. In summary, our results provide evidence that both endogenous and exogenous small RNAs may function to induce p21 expression by interacting with the similar promoter region and impede BCa metastasis. PMID:26643891

  19. p21(CIP1/WAF1)-dependent inhibition of cardiac hypertrophy in response to Angiotensin II involves Akt/Myc and pRb signaling.

    PubMed

    Hauck, Ludger; Grothe, Daniela; Billia, Filio

    2016-09-01

    The cyclin-dependent kinase inhibitor p21(CIP1/WAF1) (p21) is highly expressed in the adult heart. However, in response to stress, its expression is downregulated. Therefore, we investigated the role of p21 in the regulation of cardiac hypertrophic growth. At 2 months of age, p21 knockout mice (p21KO) lack an overt cardiac phenotype. In contrast, by 10 months of age, p21KO developed age-dependent cardiac hypertrophy and heart failure. After 3 weeks of trans-aortic banding (TAB), the heart/body weight ratio in 11 week old p21KO mice increased by 57%, as compared to 42% in wild type mice indicating that p21KO have a higher susceptibility to pressure overload-induced cardiac hypertrophy. We then chronically infused 8 week old wild type mice with Angiotensin II (2.0mg/kg/min) or saline subcutaneously by osmotic pumps for 14 days. Recombinant TAT conjugated p21 protein variants (10mg/kg body weight) or saline were intraperitoneally injected once daily for 14 days into Angiotensin II and saline-infused animals. Angiotensin II treated mice developed pathological cardiac hypertrophy with an average increase of 38% in heart/body weight ratios, as compared to saline-treated controls. Reconstitution of p21 function by TAT.p21 protein transduction prevented Angiotensin II-dependent development of cardiac hypertrophy and failure. Taken together, our genetic and biochemical data show an important function of p21 in the regulation of growth-related processes in the heart. PMID:27486069

  20. Expression of TP53 Isoforms p53β or p53γ Enhances Chemosensitivity in TP53null Cell Lines

    PubMed Central

    Silden, Elisabeth; Hjelle, Sigrun M.; Wergeland, Line; Sulen, André; Andresen, Vibeke; Bourdon, Jean-Christophe; Micklem, David R.; McCormack, Emmet; Gjertsen, Bjørn Tore

    2013-01-01

    The carboxy-terminal truncated p53 alternative spliced isoforms, p53β and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53null background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53β and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53β and p53γ congenic H1299 was accompanied by increased p21(CIP1/WAF1), Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53β protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53β and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53β and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level. PMID:23409163

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

    PubMed

    Silden, Elisabeth; Hjelle, Sigrun M; Wergeland, Line; Sulen, André; Andresen, Vibeke; Bourdon, Jean-Christophe; Micklem, David R; McCormack, Emmet; Gjertsen, Bjørn Tore

    2013-01-01

    The carboxy-terminal truncated p53 alternative spliced isoforms, p53β and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53(null) background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53β and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53β and p53γ congenic H1299 was accompanied by increased p21((CIP1/WAF1)), Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53β protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53β and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53β and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level. PMID:23409163

  2. Wnt/β-Catenin Signaling Induces the Aging of Mesenchymal Stem Cells through the DNA Damage Response and the p53/p21 Pathway

    PubMed Central

    Zhang, Da-yong; Wang, Hai-jie; Tan, Yu-zhen

    2011-01-01

    Recent studies have demonstrated the importance of cellular extrinsic factors in the aging of adult stem cells. However, the effects of an aged cell–extrinsic environment on mesenchymal stem cell (MSC) aging and the factors involved remain unclear. In the current study, we examine the effects of old rat serum (ORS) on the aging of MSCs, and explore the effects and mechanisms of Wnt/β-catenin signaling on MSC aging induced by ORS treatment. Senescence-associated changes in the cells are examined with SA-β-galactosidase staining and ROS staining. The proliferation ability is detected by MTT assay. The surviving and apoptotic cells are determined using AO/EB staining. The results suggest that ORS promotes MSC senescence and reduces the proliferation and survival of cells. The immunofluorescence staining shows that the expression of β-catenin increases in MSCs of old rats. To identify the effects of Wnt/β-catenin signaling on MSC aging induced with ORS, the expression of β-catenin, GSK-3β, and c-myc are detected. The results show that the Wnt/β-catenin signaling in the cells is activated after ORS treatment. Then we examine the aging, proliferation, and survival of MSCs after modulating Wnt/β-catenin signaling. The results indicate that the senescence and dysfunction of MSCs in the medium containing ORS is reversed by the Wnt/β-catenin signaling inhibitor DKK1 or by β-catenin siRNA. Moreover, the expression of γ-H2A.X, a molecular marker of DNA damage response, p16INK4a, p53, and p21 is increased in senescent MSCs induced with ORS, and is also reversed by DKK1 or by β-catenin siRNA. In summary, our study indicates the Wnt/β-catenin signaling may play a critical role in MSC aging induced by the serum of aged animals and suggests that the DNA damage response and p53/p21 pathway may be the main mediators of MSC aging induced by excessive activation of Wnt/β-catenin signaling. PMID:21712954

  3. Stabilization of p21 (Cip1/WAF1) following Tip60-dependent acetylation is required for p21-mediated DNA damage response

    PubMed Central

    Lee, M-S; Seo, J; Choi, D Y; Lee, E-W; Ko, A; Ha, N-C; Bok Yoon, J; Lee, H-W; Pyo Kim, K; Song, J

    2013-01-01

    The molecular mechanisms controlling post-translational modifications of p21 have been pursued assiduously in recent years. Here, utilizing mass-spectrometry analysis and site-specific acetyl-p21 antibody, two lysine residues of p21, located at amino-acid sites 161 and 163, were identified as Tip60-mediated acetylation targets for the first time. Detection of adriamycin-induced p21 acetylation, which disappeared after Tip60 depletion with concomitant destabilization of p21 and disruption of G1 arrest, suggested that Tip60-mediated p21 acetylation is necessary for DNA damage-induced cell-cycle regulation. The ability of 2KQ, a mimetic of acetylated p21, to induce cell-cycle arrest and senescence was significantly enhanced in p21 null MEFs compared with those of cells expressing wild-type p21. Together, these observations demonstrate that Tip60-mediated p21 acetylation is a novel and essential regulatory process required for p21-dependent DNA damage-induced cell-cycle arrest. PMID:23238566

  4. High-Dose Estrogen and Clinical Selective Estrogen Receptor Modulators Induce Growth Arrest, p21, and p53 in Primate Ovarian Surface Epithelial Cells

    SciTech Connect

    Wright, Jay W.; Stouffer, Richard L.; Rodland, Karin D.

    2005-06-09

    Ovarian cancer is the most lethal gynecological cancer affecting women. Hormone-based therapies are variably successful in treating ovarian cancer, but the reasoning behind these therapies is paradoxical. Clinical reagents such as tamoxifen are considered to inhibit or reverse tumor growth by competitive inhibition of the estrogen receptor (ER); however high dose estrogen is as clinically effective as tamoxifen, and it is unlikely that estrogen is acting by blocking ER activity; however, it may be activating a unique function of the ER that is nonmitogenic. For poorly defined reasons, 90% of varian cancers derive from the ovarian surface epithelium (OSE). In vivo the ER-positive OSE is exposed to high estrogen levels, reaching micromolar concentrations in dominant ovarian follicles. Using cultured OSE cells in vitro, we show that these levels of estradiol (1 ug/ml; {approx}3um) block the actions of serum growth factors, activate the G1 phase retinoblastoma AQ:A checkpoint, and induce p21, an inhibitor of kinases that normally inactivate the retinoblastoma checkpoint. We also show that estradiol increases p53 levels, which may contribute to p21 induction. Supporting the hypothesis that clinical selective ER modulators activate this novel ER function, we find that micromolar doses of tamoxifen and the ''pure antiestrogen'' ICI 182,780 elicit the same effects as estradiol. We propose that, in the context of proliferation, these data clarify some paradoxical aspects of hormone-based therapy and suggest that fuller understanding of normal ER function is necessary to improve therapeutic strategies that target the ER. (J Clin Endocrinol Metab 90: 0000-0000, 2005)

  5. JKA97, a Novel Benzylidene Analog of Harmine, Exerts Anti-Cancer Effects by Inducing G1 Arrest, Apoptosis, and p53-Independent Up-Regulation of p21

    PubMed Central

    Qin, Jiang-Jiang; Wang, Ming-Hai; Sharma, Horrick; Buolamwini, John K.; Wang, Hui; Zhang, Ruiwen

    2012-01-01

    JKA97, a benzylidene analog of harmine, has been found to be a promising drug candidate for human cancer therapy, although the underlying molecular mechanisms have not been fully demonstrated. In this study, we evaluated the effects of JKA97 on human breast cancer cells in vitro and in vivo. JKA97 inhibited the growth and proliferation of MCF7 (p53 wild-type), MCF7 (p53 knockdown), and MDA-MB-468 (p53 mutant) cells in a dose-dependent manner. Treatment with JKA97 arrested breast cancer cells in G1 phase and induced apoptosis. JKA97 also significantly suppressed the growth of MCF7 and MDA-MB-468 xenograft tumors. It regulated the expression levels of G1 phase regulators, such as p21, p27, cyclinE, and cylinD1. JKA97 activated p21 transcription, independent of p53, but had little effect on p21 protein stability/degradation. In summary, our results suggest that JKA97 inhibits human breast cancer cell growth through activating p21, independent of p53, which provides a basis for developing this compound as a novel drug for human breast cancer therapy. PMID:22558087

  6. A novel tumor-promoting role for nuclear factor IA in glioblastomas is mediated through negative regulation of p53, p21, and PAI1

    PubMed Central

    Lee, Jun Sung; Xiao, Jiping; Patel, Parita; Schade, Jake; Wang, Jinhua; Deneen, Benjamin; Erdreich-Epstein, Anat; Song, Hae-Ri

    2014-01-01

    Background Nuclear factor IA (NFIA), a transcription factor and essential regulator in embryonic glial development, is highly expressed in human glioblastoma (GBM) compared with normal brain, but its contribution to GBM and cancer pathogenesis is unknown. Here we demonstrate a novel role for NFIA in promoting growth and migration of GBM and establish the molecular mechanisms mediating these functions. Methods To determine the role of NFIA in glioma, we examined the effects of NFIA in growth, proliferation, apoptosis, and migration. We used gain-of-function (overexpression) and loss-of-function (shRNA knockdown) of NFIA in primary patient-derived GBM cells and established glioma cell lines in culture and in intracranial xenografts in mouse brains. Results Knockdown of native NFIA blocked tumor growth and induced cell death and apoptosis. Complementing this, NFIA overexpression accelerated growth, proliferation, and migration of GBM in cell culture and in mouse brains. These NFIA tumor-promoting effects were mediated via transcriptional repression of p53, p21, and plasminogen activator inhibitor 1 (PAI1) through specific NFIA-recognition sequences in their promoters. Importantly, the effects of NFIA on proliferation and apoptosis were independent of TP53 mutation status, a finding especially relevant for GBM, in which TP53 is frequently mutated. Conclusion NFIA is a modulator of GBM growth and migration, and functions by distinct regulation of critical oncogenic pathways that govern the malignant behavior of GBM. PMID:24305710

  7. Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways.

    PubMed

    Huang, Wei-Ru; Chiu, Hung-Chuan; Liao, Tsai-Ling; Chuang, Kuo-Pin; Shih, Wing-Ling; Liu, Hung-Jen

    2015-01-01

    Avian reovirus (ARV) protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128) of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication. PMID:26244501

  8. Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways

    PubMed Central

    Huang, Wei-Ru; Chiu, Hung-Chuan; Liao, Tsai-Ling; Chuang, Kuo-Pin; Shih, Wing-Ling; Liu, Hung-Jen

    2015-01-01

    Avian reovirus (ARV) protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128) of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication. PMID:26244501

  9. A defect in the p53 response pathway induced by de novo purine synthesis inhibition.

    PubMed

    Bronder, Julie L; Moran, Richard G

    2003-12-01

    p53 is believed to sense cellular ribonucleotide depletion in the absence of DNA strand breaks and to respond by imposition of a p21-dependent G1 cell cycle arrest. We now report that the p53-dependent G1 checkpoint is blocked in human carcinoma cell lines after inhibition of de novo purine synthesis by folate analogs inhibitory to glycinamide ribonucleotide formyltransferase (GART). p53 accumulated in HCT116, MCF7, or A549 carcinoma cells upon GART inhibition, but, surprisingly, transcription of several p53 targets, including p21cip1/waf1, was impaired. The mechanism of this defect was examined. The p53 accumulating in these cells was nuclear but was not phosphorylated at serines 6, 15, and 20, nor was it acetylated at lysines 373 or 382. The DDATHF-stabilized p53 bound to the p21 promoter in vitro and in vivo but did not activate histone acetylation over the p53 binding sites in the p21 promoter that is an integral part of the transcriptional response mediated by the DNA damage pathway. We concluded that the robust initial response of the p53 pathway to GART inhibitors is not transcriptionally propagated to target genes due to a defect in p53 post-translational modifications and a failure to open chromatin structure despite promoter binding of this unmodified p53. PMID:14517211

  10. 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

  11. Role of Promyelocytic Leukemia Zinc Finger (PLZF) in Cell Proliferation and Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) Gene Repression*

    PubMed Central

    Choi, Won-Il; Kim, Min-Young; Jeon, Bu-Nam; Koh, Dong-In; Yun, Chae-Ok; Li, Yan; Lee, Choong-Eun; Oh, Jiyoung; Kim, Kunhong; Hur, Man-Wook

    2014-01-01

    Promyelocytic leukemia zinc finger (PLZF) is a transcription repressor that was initially isolated as a fusion protein with retinoic acid receptor α. PLZF is aberrantly overexpressed in various human solid tumors, such as clear cell renal carcinoma, glioblastoma, and seminoma. PLZF causes cellular transformation of NIH3T3 cells and increases cell proliferation in several cell types. PLZF also increases tumor growth in the mouse xenograft tumor model. PLZF may stimulate cell proliferation by controlling expression of the genes of the p53 pathway (ARF, TP53, and CDKN1A). We found that PLZF can directly repress transcription of CDKN1A encoding p21, a negative regulator of cell cycle progression. PLZF binds to the proximal Sp1-binding GC-box 5/6 and the distal p53-responsive elements of the CDKN1A promoter to repress transcription. Interestingly, PLZF interacts with Sp1 or p53 and competes with Sp1 or p53. PLZF interacts with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylates Ac-H3 and Ac-H4 histones at the CDKN1A promoter, which indicated the involvement of the corepressor·HDACs complex in transcription repression by PLZF. Also, PLZF represses transcription of TP53 and also decreases p53 protein stability by ubiquitination. PLZF may act as a potential proto-oncoprotein in various cell types. PMID:24821727

  12. FGFR1 signaling stimulates proliferation of human mesenchymal stem cells by inhibiting the cyclin-dependent kinase inhibitors p21(Waf1) and p27(Kip1).

    PubMed

    Dombrowski, Christian; Helledie, Torben; Ling, Ling; Grünert, Martin; Canning, Claire A; Jones, C Michael; Hui, James H; Nurcombe, Victor; van Wijnen, Andre J; Cool, Simon M

    2013-12-01

    Signaling through fibroblast growth factor receptor one (FGFR1) is a known inducer of proliferation in both embryonic and human adult mesenchymal stem cells (hMSCs) and positively regulates maintenance of stem cell viability. Leveraging the mitogenic potential of FGF2/FGFR1 signaling in stem cells for therapeutic applications necessitates a mechanistic understanding of how this receptor stimulates cell cycle progression. Using small interfering RNA (siRNA) depletion, antibody-inhibition, and small molecule inhibition, we establish that FGFR1 activity is rate limiting for self-renewal of hMSCs. We show that FGFR1 promotes stem cell proliferation through multiple mechanisms that unite to antagonize cyclin-dependent kinase (CDK) inhibitors. FGFR1 not only stimulates c-Myc to suppress transcription of the CDK inhibitors p21(Waf1) and p27(Kip1), thus promoting cell cycle progression but also increases the activity of protein kinase B (AKT) and the level of S-phase kinase-associated protein 2 (Skp2), resulting in the nuclear exclusion and reduction of p21(Waf1). The in vivo importance of FGFR1 signaling for the control of proliferation in mesenchymal progenitor populations is underscored by defects in ventral mesoderm formation during development upon inhibition of its signaling. Collectively, these studies demonstrate that FGFR1 signaling mediates the continuation of MSC growth and establishes a receptor target for enhancing the expansion of mesenchymal progenitors while maintaining their multilineage potential. PMID:23939995

  13. The role of p21 Waf1/Cip1 in large airway epithelium in smokers with and without COPD.

    PubMed

    Chiappara, Giuseppina; Gjomarkaj, Mark; Virzì, Alessia; Sciarrino, Serafina; Ferraro, Maria; Bruno, Andreina; Montalbano, Angela Marina; Vitulo, Patrizio; Minervini, Marta Ida; Pipitone, Loredana; Pace, Elisabetta

    2013-10-01

    Airway epithelium alterations, including squamous cell metaplasia, characterize smokers with and without chronic obstructive pulmonary disease (COPD). The p21 regulates cell apoptosis and differentiation and its role in COPD is largely unknown. Molecules regulating apoptosis (cytoplasmic p21, caspase-3), cell cycle (nuclear p21), proliferation (Ki67/PCNA), and metaplasia (survivin) in central airways from smokers (S), smokers-COPD (s-COPD) and non-smokers (Controls) were studied. The role of cigarette smoke extracts (CSE) in p21, survivin, apoptosis (caspase-3 and annexin-V binding) and proliferation was assessed in a bronchial epithelial cell line (16HBE). Immunohistochemistry, image analysis in surgical samples and flow-cytometry and carboxyfluorescein succinimidyl ester proliferative assay in 16HBE with/without CSE were applied. Cytoplasmic and nuclear p21, survivin, and Ki67 expression significantly increased in large airway epithelium in S and in s-COPD in comparison to Controls. Caspase-3 was similar in all the studied groups. p21 correlated with epithelial metaplasia, PCNA, and Ki67 expression. CSE increased cytoplasmic p21 and survivin expression but not apoptosis and inhibited the cell proliferation in 16HBE. In large airway epithelium of smokers with and without COPD, the cytoplasmic p21 inhibits cell apoptosis, promotes cell proliferation and correlates with squamous cell metaplasia thus representing a potential pre-oncogenic hallmark. PMID:23639631

  14. p53 protein oxidation in cultured cells in response to pyrrolidine dithiocarbamate: a novel method for relating the amount of p53 oxidation in vivo to the regulation of p53-responsive genes.

    PubMed Central

    Wu, H H; Thomas, J A; Momand, J

    2000-01-01

    A novel method was developed to determine the oxidation status of proteins in cultured cells. Methoxy-polyethylene glycol-maleimide MW 2000 (MAL-PEG) was used to covalently tag p53 protein that was oxidized at cysteine residues in cultured cells. Treatment of MCF7 breast cancer cells with pyrrolidine dithiocarbamate (PDTC), a metal chelator, resulted in a minimum of 25% oxidation of p53. The oxidized p53 had an average of one cysteine residue oxidized per p53 protein molecule. The effect of PDTC treatment on downstream components of the p53 signal-transduction pathway was tested. PDTC treatment prevented actinomycin D-mediated up-regulation of two p53 effector gene products, murine double minute clone 2 oncoprotein and p21(WAF1/CIP1) (where WAF1 corresponds to wild-type p53-activated fragment 1 and CIP1 corresponds to cyclin-dependent kinase-interacting protein 1). Actinomycin D treatment led to accumulation of p53 protein in the nucleus. However, when cells were simultaneously treated with PDTC and actinomycin D, p53 accumulated in both the nucleus and the cytoplasm. The data indicate that an average of one cysteine residue per p53 protein molecule is highly sensitive to oxidation and that p53 can be efficiently oxidized by PDTC in cultured cells. PDTC-mediated oxidation of p53 correlates with altered p53 subcellular localization and reduced activation of p53 downstream effector genes. The novel method for detecting protein oxidation detailed in the present study may be used to determine the oxidation status of specific proteins in cells. PMID:10998350

  15. Nitric oxide-induced cellular stress and p53 activation in chronic inflammation

    PubMed Central

    Hofseth, Lorne J.; Saito, Shin'ichi; Hussain, S. Perwez; Espey, Michael G.; Miranda, Katrina M.; Araki, Yuzuru; Jhappan, Chamelli; Higashimoto, Yuichiro; He, Peijun; Linke, Steven P.; Quezado, Martha M.; Zurer, Irit; Rotter, Varda; Wink, David A.; Appella, Ettore; Harris, Curtis C.

    2003-01-01

    Free radical-induced cellular stress contributes to cancer during chronic inflammation. Here, we investigated mechanisms of p53 activation by the free radical, NO. NO from donor drugs induced both ataxia-telangiectasia mutated (ATM)- and ataxia-telangiectasia mutated and Rad3-related-dependent p53 posttranslational modifications, leading to an increase in p53 transcriptional targets and a G2/M cell cycle checkpoint. Such modifications were also identified in cells cocultured with NO-releasing macrophages. In noncancerous colon tissues from patients with ulcerative colitis (a cancer-prone chronic inflammatory disease), inducible NO synthase protein levels were positively correlated with p53 serine 15 phosphorylation levels. Immunostaining of HDM-2 and p21WAF1 was consistent with transcriptionally active p53. Our study highlights a pivotal role of NO in the induction of cellular stress and the activation of a p53 response pathway during chronic inflammation. PMID:12518062

  16. Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1

    PubMed Central

    Su, Liang-Cheng; Jiang, Shih Sheng; Chan, Tzu-Min; Chang, Chung-Ho; Chen, Li-Tzong; Kung, Hsing-Jien; Wang, Horng-Dar; Chuu, Chih-Pin

    2015-01-01

    Prostate cancer (PCa) patients receiving the androgen ablation therapy ultimately develop recurrent castration-resistant prostate cancer (CRPC) within 1–3 years. Treatment with caffeic acid phenethyl ester (CAPE) suppressed cell survival and proliferation via induction of G1 or G2/M cell cycle arrest in LNCaP 104-R1, DU-145, 22Rv1, and C4–2 CRPC cells. CAPE treatment also inhibited soft agar colony formation and retarded nude mice xenograft growth of LNCaP 104-R1 cells. We identified that CAPE treatment significantly reduced protein abundance of Skp2, Cdk2, Cdk4, Cdk7, Rb, phospho-Rb S807/811, cyclin A, cyclin D1, cyclin H, E2F1, c-Myc, SGK, phospho-p70S6kinase T421/S424, phospho-mTOR Ser2481, phospho-GSK3α Ser21, but induced p21Cip1, p27Kip1, ATF4, cyclin E, p53, TRIB3, phospho-p53 (Ser6, Ser33, Ser46, Ser392), phospho-p38 MAPK Thr180/Tyr182, Chk1, Chk2, phospho-ATM S1981, phospho-ATR S428, and phospho-p90RSK Ser380. CAPE treatment decreased Skp2 and Akt1 protein expression in LNCaP 104-R1 tumors as compared to control group. Overexpression of Skp2, or siRNA knockdown of p21Cip1, p27Kip1, or p53 blocked suppressive effect of CAPE treatment. Co-treatment of CAPE with PI3K inhibitor LY294002 or Bcl-2 inhibitor ABT737 showed synergistic suppressive effects. Our finding suggested that CAPE treatment induced cell cycle arrest and growth inhibition in CRPC cells via regulation of Skp2, p53, p21Cip1, and p27Kip1. PMID:25788262

  17. Germ cell tumors of the testis overexpress wild-type p53.

    PubMed Central

    Guillou, L.; Estreicher, A.; Chaubert, P.; Hurlimann, J.; Kurt, A. M.; Metthez, G.; Iggo, R.; Gray, A. C.; Jichlinski, P.; Leisinger, H. J.; Benhattar, J.

    1996-01-01

    Several recent studies have suggested that testicular germ cell tumors express high levels of wild-type p53 protein. To clarify and confirm this unexpected result, we have investigated seminomatous and nonseminomatous germ cell tumors at the genomic, mRNA, and protein levels. Thirty-five tumors were examined for p53 overexpression using antibodies directed against the p53 (PAb1801, PAb240, and CM1), mdm2 (IF2), and p21Waf1/Clp1 (EA10) proteins. Thirty-two tumors were screened for p53 mutations by single-strand conformation polymorphism analysis. Eighteen tumors were screened with a functional assay that tests the transcriptional competence of human p53 protein expressed in yeast. On frozen sections, 100, 65, 35, 73, and 0% of tumors reacted with the CM1, PAb240, PAb1801, IF2, and EA10 antibodies, respectively. No p53 mutations were detected by single-strand conformation polymorphism or by functional assay. The fact that many tumors overexpress wild-type p53 but not mdm2 rules out mdm2 overexpression as a general explanation for the presence of wild-type p53 in these tumors. The absence of p21 overexpression suggests that p53 may be unable to activate transcription of critical target genes, which may explain why the presence of wild-type p53 is tolerated in this tumor type, although the mechanism for this transcriptional inactivity remains to be established. Images Figure 1 Figure 2 PMID:8863671

  18. Histone acetylation may suppress human glioma cell proliferation when p21 WAF/Cip1 and gelsolin are induced.

    PubMed Central

    Kamitani, Hideki; Taniura, Seijiro; Watanabe, Kenji; Sakamoto, Makoto; Watanabe, Takashi; Eling, Thomas

    2002-01-01

    Histone deacetylase inhibitors that increase histone acetylation on transformed cells are being investigated as unique anticancer drugs. The aim of this investigation was to evaluate an antiproliferative activity of the histone deacetylase inhibitors sodium butyrate (NaBT) and trichostatin A on 5 glioma cell lines, T98G, A172, U-87 MG, U-118 MG, and U-373 MG, with the examination of the altered expressions in p21 and gelsolin genes. Treatment with 5-mM NaBT and 40 ng/ml trichostatin A for 48 h caused more than a 50% growth inhibition in 5 cell lines as measured by cell proliferation assays. An increase in histone acetylation was confirmed in each cell line. After treatment with 5 mM NaBT, T98G, A172, and U118 cells undergo apoptosis as indicated by DNA ladder formation. Treatment with NaBT and trichostatin A also decreased DNA synthesis as examined by the fluorescence-activated cell sorting analysis in T98G and U87 cells. In addition to the suppression of cell growth, the up regulation of p21 and gelsolin expression was observed after treatment with NaBT, especially in T98G cells. Maximum expression of p21 and gelsolin was observed within 24 h after treatment. Results from our in vitro studies indicate that the treatment of human glioma cells with one of the histone deacetylase inhibitors suppresses cell growth with decreasing DNA synthesis and stimulates apoptosis, and that associated molecular mechanisms responsible for these effects include increased histone acetylation as well as enhanced expression of p21 and gelsolin. PMID:11916500

  19. Accumulation of soluble and nucleolar-associated p53 proteins following cellular stress.

    PubMed

    Klibanov, S A; O'Hagan, H M; Ljungman, M

    2001-05-01

    The tumor suppressor p53 is a nucleocytoplasmic shuttling protein that accumulates in the nucleus of cells exposed to various cellular stresses. One important role of nuclear p53 is to mobilize a stress response by transactivating target genes such as the p21(Waf1) gene. In this study, we investigated more closely the localization of p53 in cells following various stresses. Immunocytochemistry of fixed human fibroblasts treated with either UV light, the kinase and transcription inhibitor DRB or the proteasome inhibitor MG132 revealed abundant p53 localized to the nucleus. When cells treated with UV or DRB were permeabilized prior to fixation to allow soluble proteins to diffuse, the nuclear p53 signal was abolished. However, in cells treated with MG132, residual p53 localized to distinct large foci. Furthermore, nucleolin co-localized with p53 to these foci, suggesting that these foci were nucleolar structures. Interestingly, the MDM2 protein was found to co-localize with p53 to nucleolar structures following proteasome inhibition. Our results suggest that the p53 proteins accumulating in the nucleus following UV-irradiation or blockage of transcription are freely soluble and, thus, should be able to roam the nucleus to ensure high occupancy of p53 binding sites. However, inhibition of proteasome activity may be a unique stress in that it leads to the sequestering of p53 proteins to the nucleolus, thereby blunting the p53-mediated transactivation of target genes. PMID:11329373

  20. Hepatitis C Virus Core Protein Down-Regulates p21Waf1/Cip1 and Inhibits Curcumin-Induced Apoptosis through MicroRNA-345 Targeting in Human Hepatoma Cells

    PubMed Central

    Shiu, Tzu-Yue; Huang, Shih-Ming; Shih, Yu-Lueng; Chu, Heng-Cheng; Chang, Wei-Kuo; Hsieh, Tsai-Yuan

    2013-01-01

    Background Hepatitis C virus (HCV) has been reported to regulate cellular microRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma, but HCV core-modulated cellular microRNAs are unknown. The HCV core protein regulates p21Waf1/Cip1 expression. However, the mechanism of HCV core-associated p21Waf1/Cip1 regulation remains to be further clarified. Therefore, we attempted to determine whether HCV core-modulated cellular microRNAs play an important role in regulating p21Waf1/Cip1 expression in human hepatoma cells. Methods Cellular microRNA profiling was investigated in core-overexpressing hepatoma cells using TaqMan low density array. Array data were further confirmed by TaqMan real-time qPCR for single microRNA in core-overexpressing and full-length HCV replicon-expressing cells. The target gene of microRNA was examined by reporter assay. The gene expression was determined by real-time qPCR and Western blotting. Apoptosis was examined by annexin V-FITC apoptosis assay. Cell cycle analysis was performed by propidium iodide staining. Cell proliferation was analyzed by MTT assay. Results HCV core protein up- or down-regulated some cellular microRNAs in Huh7 cells. HCV core-induced microRNA-345 suppressed p21Waf1/Cip1 gene expression through targeting its 3′ untranslated region in human hepatoma cells. Moreover, the core protein inhibited curcumin-induced apoptosis through p21Waf1/Cip1-targeting microRNA-345 in Huh7 cells. Conclusion and Significance HCV core protein enhances the expression of microRNA-345 which then down-regulates p21Waf1/Cip1 expression. It is the first time that HCV core protein has ever been shown to suppress p21Waf1/Cip1 gene expression through miR-345 targeting. PMID:23577194

  1. miR-30e controls DNA damage-induced stress responses by modulating expression of the CDK inhibitor p21WAF1/CIP1 and caspase-3

    PubMed Central

    Sohn, Dennis; Peters, Dominik; Piekorz, Roland P.; Budach, Wilfried; Jänicke, Reiner U.

    2016-01-01

    MicroRNAs (miRNAs), a class of small non-coding RNAs that usually cause gene silencing by translational repression or degradation of mRNAs, are implicated in DNA damage-induced stress responses. To identify senescence-associated miRNAs, we performed microarray analyses using wild-type and p53-deficient HCT116 colon carcinoma cells that following gamma-irradiation (γIR) are driven into senescence and apoptosis, respectively. Several miRNAs including miR-30e were found upregulated in a p53-dependent manner specifically in senescent cells, but not in apoptotic cells. Overexpression of miR-30e in HCT116 cells not only inhibited γIR-, etoposide- or miR-34a-induced caspase-3-like DEVDase activities and cell death, but greatly accelerated and augmented their senescent phenotype. Consistently, procaspase-3 protein, but not mRNA decreased in the presence of miR-30e, whereas expression of the cyclin-dependent kinase inhibitor p21 increased both at the mRNA and protein level. Performing luciferase reporter gene assays, we identified the 3′-UTR of the caspase-3 mRNA as a direct miR-30e target. In contrast, although miR-30e was unable to bind to the p21 mRNA, it increased expression of a luciferase construct containing the p21 promoter, suggesting that the miR-30e-mediated upregulation of p21 occurs indirectly at the transcriptional level. Interestingly, despite suppressing procaspase-3 expression, miR-30e was unable to protect RKO colon carcinoma cells from DNA damage-induced death or to induce senescence, as miR-30e completely fails to upregulate p21 in these cells. These data suggest that miR-30e functions in a cell type-dependent manner as an important molecular switch for DNA damage-induced stress responses and may thus represent a target of therapeutic value. PMID:26895377

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

    SciTech Connect

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

    2008-02-15

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

  3. Retardation of cell growth by avian reovirus p17 through the activation of p53 pathway

    SciTech Connect

    Liu, H.-J.; Lin, P.-Y.; Lee, J.-W.; Hsu, H.-Y.; Shih, W.-L. . E-mail: shihwl@mail.tcu.edu.tw

    2005-10-21

    The second open reading frame of avian reovirus S1 gene segment encodes a 17 kDa non-structural protein, named p17. The biological role of p17 is fully unknown so far. Using trypan blue dye exclusion and MTT assay, we demonstrated that the ectopic expression of p17 results in the reduction of viable cell number and cell proliferation rate of Vero, BHK, 293, and HeLa cells. Measurement of LDH activity and DNA fragmentation analysis revealed that p17 expression did not cause cell death or apoptosis. These data indicated that the p17 possessed the growth retardation function. Semi-quantitative RT-PCR and Western blotting revealed that p17-expressing cells induced the expression of CDK inhibitor p21{sup cip1/waf1} in a time- and dose-dependent manner, but the transcripts of CDK inhibitor p15{sup INK4b}, p16{sup INK4a}, or p27{sup kip} were not altered. In the presence of p17, the p53 protein level and p53-driven reporter activity were elevated significantly. Dominant negative p53 alleviated the p21 accumulation, p53 activation, and growth inhibition effect induced by p17. Taken together, these studies revealed a possible intrinsic function of p17 in growth regulation through the activation of p53 and p21{sup cip1/waf1}.

  4. Effect of Boschniakia rossica on expression of GST-P, p53 and p21(ras)proteins in early stage of chemical hepatocarcinogenesis and its anti-inflammatory activities in rats.

    PubMed

    Yin, Zong-Zhu; Jin, Hai-Ling; Yin, Xue-Zhe; Li, Tian-Zhu; Quan, Ji-Shu; Jin, Zeng-Nan

    2000-12-01

    AIM:To investigate the effect of Boschniakia rossica (BR) extract on expression of GST-P, p53 and p21(ras) proteins in early stage of chemical hepatocarcinogenesis in rats and its anti-inflammatory activities.METHODS:The expression of tumor marker-placental form glutathione S-transferase (GST-P), p53 and p21(ras) proteins were investigated by immunohisto-chemical techniques and ABC method. Anti-inflammatory activities of BR were studied by xylene and croton oil-induced mouse ear edema, carrageenin, histamine and hot scald-induced rat pow edema, adjuvant-induced rat arthritis and cotton pellet induced mouse granuloma formation methods.RESULTS:The 500mg/kg of BR-H2O extract frac-tionated from BR-Methanol extract had inhibitory effect on the formation of DEN-induced GST-P-positive foci in rat liver (GST-P staining was 78% positive in DEN+AAF group vs 20% positive in DEN+AAF+BR group, P<0.05) and the expression of mutant p53 and p21(ras) protein was lower than that of hepatic preneoplastic lesions (33% and 22% positive respectively in DEN+AAF group vs negative in DEN+AAF+BR group). Both CH(2)Cl(2) and H(2)O extracts from BR had anti-inflamatory effect in xylene and crotonoil induced mouse ear edema (inhibitory rates were 26%-29% and 35%-59%, respectively). BR H(2)O extract exhibited inhibitory effect in carrageenin, histamine and hot scald-induced hind paw edema and adjuvant-induced arthritis in rats and cotton pellet-induced granuloma formation in mice.CONCLUSION:BR extract exhibited inhibitory effect on formation of preneoplastic hepatic foci in early stage of rat chemical hepato-carcinogenesis.Both CH(2)Cl(2) and H(2)O extracts from BR exerted anti-inflammatory effect in rats and mice. PMID:11819701

  5. Enhanced sensitivity to irinotecan by Cdk1 inhibition in the p53-deficient HT29 human colon cancer cell line.

    PubMed

    Abal, Miguel; Bras-Goncalves, Rui; Judde, Jean-Gabriel; Fsihi, Hafida; De Cremoux, Patricia; Louvard, Daniel; Magdelenat, Henri; Robine, Sylvie; Poupon, Marie-France

    2004-03-01

    Mutations in the tumor-suppressor gene p53 have been associated with advanced colorectal cancer (CRC). Irinotecan (CPT-11), a DNA topoisomerase 1 inhibitor, has been recently incorporated to the adjuvant therapy. Since the DNA-damage checkpoint depends on p53 activation, the status of p53 might critically influence the response to CPT-11. We analysed the sensitivity to CPT-11 in the human colon cancer cell line HT29 (mut p53) and its wild-type (wt)-p53 stably transfected subclone HT29-A4. Cell-cycle analysis in synchronised cells demonstrated the activation of transfected wt-p53 and a p21(WAF1/CIP1)-dependent cell-cycle blockage in the S phase. Activated wt-p53 increased apoptosis and enhanced sensitivity to CPT-11. In p53-deficient cells, cDNA-macroarray analysis and western blotting showed an accumulation of the cyclin-dependent kinase (cdk)1/cyclin B complex. Subsequent p53-independent activation of the cdk-inhibitor (cdk-I) p21(WAF1/CIP1) prevented cell-cycle progression. Cdk1 induction was exploited in vivo to improve the sensitivity to CPT-11 by additional treatment with the cdk-I CYC-202. We demonstrate a gain of sensitivity to CPT-11 in a p53-mutated colon cancer model either by restoring wild-type p53 function or by sequential treatment with cdk-Is. Considering that mutations in p53 are among the most common genetic alterations in CRC, a therapeutic approach specifically targeting p53-deficient tumors could greatly improve the treatment outcomes. PMID:15001986

  6. EVI1 Inhibits Apoptosis Induced by Antileukemic Drugs via Upregulation of CDKN1A/p21/WAF in Human Myeloid Cells

    PubMed Central

    Rommer, Anna; Steinmetz, Birgit; Herbst, Friederike; Hackl, Hubert; Heffeter, Petra; Heilos, Daniela; Filipits, Martin; Steinleitner, Katarina; Hemmati, Shayda; Herbacek, Irene; Schwarzinger, Ilse; Hartl, Katharina; Rondou, Pieter; Glimm, Hanno; Karakaya, Kadin; Krämer, Alwin; Berger, Walter; Wieser, Rotraud

    2013-01-01

    Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in acute myeloid leukemia (AML). Despite of its clinical importance, little is known about the mechanism through which EVI1 confers resistance to antileukemic drugs. Here, we show that a human myeloid cell line constitutively overexpressing EVI1 after infection with a retroviral vector (U937_EVI1) was partially resistant to etoposide and daunorubicin as compared to empty vector infected control cells (U937_vec). Similarly, inducible expression of EVI1 in HL-60 cells decreased their sensitivity to daunorubicin. Gene expression microarray analyses of U937_EVI1 and U937_vec cells cultured in the absence or presence of etoposide showed that 77 and 419 genes were regulated by EVI1 and etoposide, respectively. Notably, mRNA levels of 26 of these genes were altered by both stimuli, indicating that EVI1 regulated genes were strongly enriched among etoposide regulated genes and vice versa. One of the genes that were induced by both EVI1 and etoposide was CDKN1A/p21/WAF, which in addition to its function as a cell cycle regulator plays an important role in conferring chemotherapy resistance in various tumor types. Indeed, overexpression of CDKN1A in U937 cells mimicked the phenotype of EVI1 overexpression, similarly conferring partial resistance to antileukemic drugs. PMID:23457546

  7. A p53 growth arrest protects fibroblasts from anticancer agents.

    PubMed

    McCormack, E S; Bruskin, A M; Borzillo, G V

    1997-01-01

    Reversible inhibitors of the cell cycle such as the TGF-betas have been exploited to protect dividing cells from exposure to anticancer drugs and radiation. Here, rat embryo fibroblast (REF) lines expressing different p53 mutations were used to test whether the p53 growth arrest could also chemoprotect cells from high doses of anticancer drugs. Whereas the doubling times of the different REF lines at 37 degrees C were similar, cells bearing temperature-sensitive mutations (mouse 135V or human 143A) were growth arrested at 31 degrees C. Temperature-dependent p53 activity was associated with increased levels of MDM2 and p21/WAF1, and the induction of an integrated p53-responsive luciferase gene. The REF lines exhibited similar sensitivities to common anticancer drugs when grown at 37 degrees C. However, when exposed to the same agents following transient incubation at 31 degrees C, the p53-arrested cells exhibited a marked survival advantage as shown by colony-forming assays. Chemoprotection was not universal, in that colony formation was not enhanced significantly after treatment with cisplatin or 5-fluorouracil, two drugs which can cause cellular damage throughout the cell cycle. Like other negative growth regulators, an activated p53 checkpoint may mediate the survival of cells exposed to drugs that target DNA synthesis or mitosis. PMID:9351895

  8. 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.

  9. IL-1-induced ERK1/2 activation up-regulates p21{sup Waf1/Cip1} protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375

    SciTech Connect

    Arakawa, Tomohiro; Hayashi, Hidetoshi; Itoh, Saotomo; Takii, Takemasa; Onozaki, Kikuo

    2010-02-12

    IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21{sup Waf1/Cip1} (p21) protein. Here, we demonstrate that IL-1 induces the stabilization of p21 protein via ERK1/2 pathway. The degradation of p21 was inhibited by IL-1, however the ubiquitination level of p21 was not affected. In addition, the degradation of non-ubiquitinated form of lysine less mutant p21-K6R was also inhibited by IL-1, suggesting that IL-1 stabilized p21 protein via ubiquitin-independent pathway. Furthermore, the inhibition of p21 protein degradation was prevented by a selective inhibitor of ERK1/2 pathway, PD98059. These results suggest that IL-1-induced ERK1/2 activation leads to the up-regulation of p21 by inhibiting degradation via ubiquitin-independent pathway in human melanoma cells A375.

  10. Cadmium Impairs p53 Activity in HepG2 Cells.

    PubMed

    Urani, C; Melchioretto, P; Fabbri, M; Bowe, G; Maserati, E; Gribaldo, L

    2014-01-01

    Cadmium and cadmium compounds are contaminants of the environment, food, and drinking water and are important constituents of cigarette smoke. Cd exposure has also been associated with airborne particulate CdO and with Cd-containing quantum dots in medical therapy. Adverse cadmium effects reported in the literature have stimulated during recent years an ongoing discussion to better elucidate cadmium outcomes at cell and molecular level. The present work is designed to gain an insight into the mechanism of p53 impairment at gene and protein level to understand Cd-induced resistance to apoptosis. We used a hepatoma cell line (HepG2) derived from liver, known to be metal responsive. At genotoxic cadmium concentrations no cell cycle arrest was observed. The p53 at gene and protein level was not regulated. Fluorescence images showed that p53 was correctly translocated into the nucleus but that the p21(Cip1/WAF-1), a downstream protein of p53 network involved in cell cycle regulation, was not activated at the highest cadmium concentrations used. The miRNAs analysis revealed an upregulation of mir-372, an miRNA able to affect p21(Cip1/WAF-1) expression and promote cell cycle progression and proliferation. The role of metallothioneins and possible conformational changes of p53 are discussed. PMID:25101185

  11. Differential targeting of the cyclin-dependent kinase inhibitor, p21CIP1/WAF1, by chelators with anti-proliferative activity in a range of tumor cell-types

    PubMed Central

    Moussa, Rayan S.; Kovacevic, Zaklina; Richardson, Des R.

    2015-01-01

    Chelators such as 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (311) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) target tumor cell iron pools and inhibit proliferation. These agents also modulate multiple targets, one of which is the cyclin-dependent kinase inhibitor, p21. Hence, this investigation examined the mechanism of action of these compounds in targeting p21. All the chelators up-regulated p21 mRNA in the five tumor cell-types assessed. In contrast, examining their effect on total p21 protein levels, these agents induced either: (1) down-regulation in MCF-7 cells; (2) up-regulation in SK-MEL-28 and CFPAC-1 cells; or (3) had no effect in LNCaP and SK-N-MC cells. The nuclear localization of p21 was also differentially affected by the ligands depending upon the cell-type, with it being decreased in MCF-7 cells, but increased in SK-MEL-28 and CFPAC-1 cells. Further studies assessing the mechanisms responsible for these effects demonstrated that p21 expression was not correlated with p53 status, suggesting a p53-independent mechanism. Considering this, we examined proteins that modulate p21 independently of p53, namely NDRG1, MDM2 and ΔNp63. These studies demonstrated that a dominant negative MDM2 isoform (p75MDM2) closely resembled p21 expression in response to chelation in three cell lines. These data suggest MDM2 may be involved in the regulation of p21 by chelators. PMID:26335183

  12. UV irradiation leads to transient changes in phosphorylation and stability of tumor suppressor protein p53.

    PubMed

    Scheidtmann, K; Landsberg, G

    1996-12-01

    Tumor suppressor protein p53 is thought to play a crucial role in maintaining the integrity of the genome. DNA damage caused by genotoxic drugs, UV or gamma-irradiation leads to accumulation of p53 and activation of its DNA binding and transcriptional activities and subsequently to cell cycle arrest or apoptosis. We investigated whether the apparent activation of p53 might be due to post-translational modification. The rat fibroblast cell lines REF52, 208F, and rat1 were irradiated with W-A and the synthesis, stability and phosphorylation state of p53 were investigated by pulse chase experiments, SDS-PAGE and two-dimensional phosphopeptide mapping. The three cell lines exhibited different sensitivities and biological responses to UV irradiation, REF52 cells responded with a growth arrest whereas 208F and rat1 cells underwent apoptosis. The fate of p53 was similar in all cases. Both the stability of p53 and its phosphorylation increased instantaneously but transiently. However, the amount of p53 that accumulated after UV treatment was much higher in 208F and rat1 than in REF52 cells. Interestingly, p53 that was synthesized early after irradiation was stable for more than 14 h whereas molecules synthesized 8 or more hours post irradiation were increasingly susceptible to degradation. Moreover, between 14 and 20 h after treatment, the rate of synthesis of p53 decreased to a level lower than in untreated cells suggesting negative feed back control. The expression of different p53-responsive genes, waf1/cip1, Gadd45, and bax was investigated by protein analyses. Surprisingly, p21(waf1) was expressed only in REF52 cells but not in the others. Furthermore, UV irradiation led only to a moderate increase of p21(waf1) expression. Expression of Gadd45 and box was detectable in both cell types but its expression did not change significantly upon UV treatment. Our results suggest i) that both cell types share a common pathway which upon UV irradiation results in enhanced

  13. Cucurbitacin E Induces G(2)/M Phase Arrest through STAT3/p53/p21 Signaling and Provokes Apoptosis via Fas/CD95 and Mitochondria-Dependent Pathways in Human Bladder Cancer T24 Cells.

    PubMed

    Huang, Wen-Wen; Yang, Jai-Sing; Lin, Meng-Wei; Chen, Po-Yuan; Chiou, Shang-Ming; Chueh, Fu-Shin; Lan, Yu-Hsuan; Pai, Shu-Jen; Tsuzuki, Minoru; Ho, Wai-Jane; Chung, Jing-Gung

    2012-01-01

    Cucurbitacin E, a tetracyclic triterpenes compound extracted from cucurbitaceous plants, has been shown to exhibit anticancer and anti-inflammatory activities. The purpose of this study was to elucidate whether cucurbitacin E promotes cell cycle arrest and induces apoptosis in T24 cells and further to explore the underlying molecular mechanisms. The effects of cucurbitacin E on T24 cell's growth and accompanied morphological changes were examined by MTT assay and a phase-contrast microscope. DNA content, mitochondrial membrane potential (ΔΨ(m)) and annexin V/PI staining were determined by flow cytometry. The protein levels were measured by Western blotting. Our results demonstrated that cucurbitacin E-induced G(2)/M arrest was associated with a marked increase in the levels of p53, p21 and a decrease in phospho-signal transducer and activator of transcription 3 (STAT3), cyclin-dependent kinase 1 (CDK1) and cyclin B. Cucurbitacin E-triggered apoptosis was accompanied with up-regulation of Fas/CD95, truncated BID (t-BID) and a loss of ΔΨ(m), resulting in the releases of cytochrome c, apoptotic protease activating factor 1 (Apaf-1) and apoptosis-inducing factor (AIF), and sequential activation of caspase-8, caspase-9, and caspase-3. Our findings provided the first evidence that STAT3/p53/p21 signaling, Fas/CD95 and mitochondria-dependent pathways play critical roles in cucurbitacin E-induced G(2)/M phase arrest and apoptosis of T24 cells. PMID:22272214

  14. Cucurbitacin E Induces G2/M Phase Arrest through STAT3/p53/p21 Signaling and Provokes Apoptosis via Fas/CD95 and Mitochondria-Dependent Pathways in Human Bladder Cancer T24 Cells

    PubMed Central

    Huang, Wen-Wen; Yang, Jai-Sing; Lin, Meng-Wei; Chen, Po-Yuan; Chiou, Shang-Ming; Chueh, Fu-Shin; Lan, Yu-Hsuan; Pai, Shu-Jen; Tsuzuki, Minoru; Ho, Wai-Jane; Chung, Jing-Gung

    2012-01-01

    Cucurbitacin E, a tetracyclic triterpenes compound extracted from cucurbitaceous plants, has been shown to exhibit anticancer and anti-inflammatory activities. The purpose of this study was to elucidate whether cucurbitacin E promotes cell cycle arrest and induces apoptosis in T24 cells and further to explore the underlying molecular mechanisms. The effects of cucurbitacin E on T24 cell's growth and accompanied morphological changes were examined by MTT assay and a phase-contrast microscope. DNA content, mitochondrial membrane potential (ΔΨm) and annexin V/PI staining were determined by flow cytometry. The protein levels were measured by Western blotting. Our results demonstrated that cucurbitacin E-induced G2/M arrest was associated with a marked increase in the levels of p53, p21 and a decrease in phospho-signal transducer and activator of transcription 3 (STAT3), cyclin-dependent kinase 1 (CDK1) and cyclin B. Cucurbitacin E-triggered apoptosis was accompanied with up-regulation of Fas/CD95, truncated BID (t-BID) and a loss of ΔΨm, resulting in the releases of cytochrome c, apoptotic protease activating factor 1 (Apaf-1) and apoptosis-inducing factor (AIF), and sequential activation of caspase-8, caspase-9, and caspase-3. Our findings provided the first evidence that STAT3/p53/p21 signaling, Fas/CD95 and mitochondria-dependent pathways play critical roles in cucurbitacin E-induced G2/M phase arrest and apoptosis of T24 cells. PMID:22272214

  15. miR-29c-3p promotes senescence of human mesenchymal stem cells by targeting CNOT6 through p53-p21 and p16-pRB pathways.

    PubMed

    Shang, Jin; Yao, Yuan; Fan, Xin; Shangguan, Lei; Li, Jie; Liu, Huan; Zhou, Yue

    2016-04-01

    Mesenchymal stem cells (MSCs) are important seed cells for tissue engineering and are promising targets for cell-based therapies. However, the replicative senescence of MSCs during in vitro culture limits their research and clinical applications. The molecular mechanisms underlying the replicative senescence of MSCs are not fully understood. Evidence suggests that miRNAs play important roles in replicative senescence. A microarray analysis found that the miR-29c-3p level was significantly increased during the MSC senescence process. In our study, we investigated the roles of miR-29c-3p in senescence of MSCs. We cultured MSCs for long periods of time, up and down-regulated the miR-29c-3p expression in MSCs, and examined the senescent phenotype changes. The over-expression of miR-29c-3p led to enhanced senescence-associated-β-galactosidase (SA-β-gal) staining, senescence associated secretory phenotype (SASP), senescence associated heterochromatic foci (SAHF), reduced proliferation ability, retarded osteogenic differentiation and corresponding changes in senescence markers, whereas the miR-29c-3p down-regulation had the opposite results. Dual-luciferase reporter assays demonstrated that CNOT6 is the target gene of miR-29c-3p. Knockdown of CNOT6 confirmed its inhibitory effects on the senescence of MSCs. In addition, Western blot results showed that both the p53-p21 and the p16-pRB pathways were activated during the miR-29c-3p-induced senescence of MSCs. In conclusion, our results demonstrate that miR-29c-3p promotes the senescence of MSCs by targeting CNOT6 through p53-p21 and p16-pRB pathways and highlight the contribution of post-transcriptional regulation to stem cell senescence. PMID:26792405

  16. Cordyceps militaris (L.) Link Fruiting Body Reduces the Growth of a Non-Small Cell Lung Cancer Cell Line by Increasing Cellular Levels of p53 and p21.

    PubMed

    Bizarro, Ana; Ferreira, Isabel C F R; Soković, Marina; van Griensven, Leo J L D; Sousa, Diana; Vasconcelos, M Helena; Lima, Raquel T

    2015-01-01

    Cordyceps militaris (L.) Link, an edible entomopathogenic fungus widely used in traditional Chinese medicine, has numerous potential medicinal properties including antitumor activity. The methanolic extract of C. militaris fruiting body was recently shown to have tumor cell growth inhibitory activity in several human tumor cell lines. Nonetheless, the mechanism of action involved is still not known. This work aimed at further studying the effect of the methanolic extract of C. militaris regarding its antitumor mechanism of action, using the non-small cell lung cancer cell line (NCI-H460) as a model. Results showed that treatment with the extract decreased cellular proliferation, induced cell cycle arrest at G0/G1 and increased apoptosis. In addition, the extract increased the levels of p53 and p21. Moreover, an increase in p-H2A.X and 53BP1 levels, together with an increase in the number of 53BP1 foci/cell (all indicative of DNA damage), were also observed after treatment with the extract. This work suggests that this extract affected NCI-H460 cellular viability through a mechanism involving DNA damage and p53 activation. This further supports the potential of this extract as a source of bioactive compounds, which may be used in anticancer strategies. PMID:26263965

  17. Beta-escin inhibits colonic aberrant crypt foci formation in rats and regulates the cell cycle growth by inducing p21(waf1/cip1) in colon cancer cells.

    PubMed

    Patlolla, Jagan M R; Raju, Jayadev; Swamy, Malisetty V; Rao, Chinthalapally V

    2006-06-01

    Extracts of Aesculus hippocastanum (horse chestnut) seed have been used in the treatment of chronic venous insufficiency, edema, and hemorrhoids. Most of the beneficial effects of horse chestnut are attributed to its principal component beta-escin or aescin. Recent studies suggest that beta-escin may possess anti-inflammatory, anti-hyaluronidase, and anti-histamine properties. We have evaluated the chemopreventive efficacy of dietary beta-escin on azoxymethane-induced colonic aberrant crypt foci (ACF). In addition, we analyzed the cell growth inhibitory effects and the induction of apoptosis in HT-29 human colon cancer cell line. To evaluate the inhibitory properties of beta-escin on colonic ACF, 7-week-old male F344 rats were fed experimental diets containing 0%, 0.025%, or 0.05% beta-escin. After 1 week, the rats received s.c. injections of azoxymethane (15 mg/kg body weight, once weekly for 2 weeks) or an equal volume of normal saline (vehicle). Rats were continued on respective experimental diets and sacrificed 8 weeks after the azoxymethane treatment. Colons were evaluated histopathologically for ACF. Administration of dietary 0.025% and 0.05% beta-escin significantly suppressed total colonic ACF formation up to approximately 40% (P < 0.001) and approximately 50% (P < 0.0001), respectively, when compared with control diet group. Importantly, rats fed beta-escin showed dose-dependent inhibition (approximately 49% to 65%, P < 0.0001) of foci containing four or more aberrant crypts. To understand the growth inhibitory effects, HT-29 human colon carcinoma cell lines were treated with various concentrations of beta-escin and analyzed by flow cytometry for apoptosis and cell cycle progression. Beta-escin treatment in HT-29 cells induced growth arrest at the G1-S phase, which was associated with the induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1), and this correlated with reduced phosphorylation of retinoblastoma protein. Results also indicate that

  18. Resveratrol and curcumin synergistically induces apoptosis in cigarette smoke condensate transformed breast epithelial cells through a p21(Waf1/Cip1) mediated inhibition of Hh-Gli signaling.

    PubMed

    Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Siddharth, Sumit; Das, Dipon; Nayak, Anmada; Kundu, Chanakya Nath

    2015-09-01

    Combination therapy using two or more small molecule inhibitors of aberrant signaling cascade in aggressive breast cancers is a promising therapeutic strategy over traditional monotherapeutic approaches. Here, we have studied the synergistic mechanism of resveratrol and curcumin induced apoptosis using in vitro (cigarette smoke condensate mediated transformed breast epithelial cell, MCF-10A-Tr) and in vivo (tumor xenograft mice) model system. Resveratrol exposure increased the intracellular uptake of curcumin in a dose dependent manner and caused apoptosis in MCF-10A-Tr cells. Approximately, ten fold lower IC50 value was noted in cells treated with the combination of resveratrol (3μM) and curcumin (3μM) in comparison to 30μM of resveratrol or curcumin alone. Resveratrol+curcumin combination caused apoptosis by increasing Bax/Bcl-xL ratio, Cytochrome C release, cleaved product of PARP and caspase 3 in cells. Interestingly, this combination unaltered the protein expressions of WNT-TCF and Notch signaling components, β-catenin and cleaved notch-1 val1744, respectively. Furthermore, the combination also significantly decreased the intermediates of Hedgehog-Gli cascade including SMO, SHH, Gli-1, c-MYC, Cyclin-D1, etc. and increased the level of p21(Waf/Cip1) in vitro and in vivo. A significant reduction of Gli- promoter activity was noted in combinational drug treated cells in comparison to individual drug treatment. Un-alteration of the expressions of the above proteins and Gli1 promoter activity in p21(Waf/Cip1) knockout cells suggests this combination caused apoptosis through p21(Waf/Cip1). Thus, our findings revealed resveratrol and curcumin synergistically caused apoptosis in cigarette smoke induced breast cancer cells through p2(Waf/Cip1) mediated inhibition of Hedgehog-Gli cascade. PMID:26212257

  19. A Temperature Sensitive Variant of p53 Drives p53-Dependent MicroRNA Expression without Evidence of Widespread Post-Transcriptional Gene Silencing

    PubMed Central

    Cabrita, Miguel A.; Vanzyl, Erin J.; Hamill, Jeff D.; Pan, Elysia; Marcellus, Kristen A.; Tolls, Victoria J.; Alonzi, Rhea C.; Pastic, Alyssa; Rambo, Teeghan M. E.; Sayed, Hadil; McKay, Bruce C.

    2016-01-01

    The p53 tumour suppressor is a transcription factor that can regulate the expression of numerous genes including many encoding proteins and microRNAs (miRNAs). The predominant outcomes of a typical p53 response are the initiation of apoptotic cascades and the activation of cell cycle checkpoints. HT29-tsp53 cells express a temperature sensitive variant of p53 and in the absence of exogenous DNA damage, these cells preferentially undergo G1 phase cell cycle arrest at the permissive temperature that correlates with increased expression of the cyclin-dependent kinase inhibitor p21WAF1. Recent evidence also suggests that a variety of miRNAs can induce G1 arrest by inhibiting the expression of proteins like CDK4 and CDK6. Here we used oligonucleotide microarrays to identify p53-regulated miRNAs that are induced in these cells undergoing G1 arrest. At the permissive temperature, the expression of several miRNAs was increased through a combination of either transcriptional or post-transcriptional regulation. In particular, miR-34a-5p, miR-143-3p and miR-145-5p were strongly induced and they reached levels comparable to that of reference miRNAs (miR-191 and miR-103). Importantly, miR-34a-5p and miR-145-5p are known to silence the Cdk4 and/or Cdk6 G1 cyclin-dependent kinases (cdks). Surprisingly, there was no p53-dependent decrease in the expression of either of these G1 cdks. To search for other potential targets of p53-regulated miRNAs, p53-downregulated mRNAs were identified through parallel microarray analysis of mRNA expression. Once again, there was no clear effect of p53 on the repression of mRNAs under these conditions despite a remarkable increase in p53-induced mRNA expression. Therefore, despite a strong p53 transcriptional response, there was no clear evidence that p53-responsive miRNA contributed to gene silencing. Taken together, the changes in cell cycle distribution in this cell line at the permissive temperature is likely attributable to transcriptional

  20. Characterization of coordinated immediate responses by p16INK4A and p53 pathways in UVB-irradiated human skin cells.

    PubMed

    Abd Elmageed, Zakaria Y; Gaur, Rajiv L; Williams, Mandy; Abdraboh, Mohamed E; Rao, Prakash N; Raj, Madhwa H G; Ismail, Fathi M; Ouhtit, Allal

    2009-01-01

    While the precise mechanisms of melanoma development are unknown, recent in vivo studies have revealed that the p16(Ink4a)/Rb pathway is disrupted in melanomagenesis. Here, we characterize the role of p16/Rb in coordinating the early events in UVB-irradiated skin. Foreskins and melanoma cell cultures were irradiated with low and high acute UVB doses and examined for cell-cycle- and apoptosis-associated genes. In melanoma cells, low UVB dose upregulated p16, p53, and p21 expression levels in Malme-3M, and high UVB dose accentuated the expression of p53 and p21(Cip1/Waf1), in particular; however, in SkMel-28 cells only p16 expression was upregulated in response to UV irradiation. In HaCaT cells, high UVB dose caused dramatic increase in p53 expression followed by upregulation of p21(Cip1/Waf1) and Bax, and downregulation of Bcl-2 leading to apoptosis. In HaCaT cells, reinstatement of p16 pathway restored cell-cycle arrest in response to low dose. Foreskin organ culture experiments confirmed our in vitro cell results. These data indicate that the p53 and p16 pathways respond independently to UVB insult. The p16 pathway is favored at low doses and results in cell-cycle arrest; the p53 pathway is more responsive to higher doses and induces apoptosis depending on p53 mutation status. PMID:18719612

  1. XAF1 directs apoptotic switch of p53 signaling through activation of HIPK2 and ZNF313.

    PubMed

    Lee, Min-Goo; Han, Jikhyon; Jeong, Seong-In; Her, Nam-Gu; Lee, Jin-Hee; Ha, Tae-Kyu; Kang, Min-Ju; Ryu, Byung-Kyu; Chi, Sung-Gil

    2014-10-28

    X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a tumor suppressor that is frequently inactivated in many human cancers. However, the molecular mechanism underlying its growth-inhibitory function remains largely unknown. Here, we report that XAF1 forms a positive feedback loop with p53 and acts as a molecular switch in p53-mediated cell-fate decisions favoring apoptosis over cell-cycle arrest. XAF1 binds directly to the N-terminal proline-rich domain of p53 and thus interferes with E3 ubiquitin ligase MDM2 binding and ubiquitination of p53. XAF1 stimulates homeodomain-interacting protein kinase 2 (HIPK2)-mediated Ser-46 phosphorylation of p53 by blocking E3 ubiquitin ligase Siah2 interaction with and ubiquitination of HIPK2. XAF1 also steps up the termination of p53-mediated cell-cycle arrest by activating zinc finger protein 313 (ZNF313), a p21(WAF1)-targeting ubiquitin E3 ligase. XAF1 interacts with p53, Siah2, and ZNF313 through the zinc finger domains 5, 6, and 7, respectively, and truncated XAF1 isoforms preferentially expressed in cancer cells fail to form a feedback loop with p53. Together, this study uncovers a novel role for XAF1 in p53 stress response, adding a new layer of complexity to the mechanisms by which p53 determines cell-fate decisions. PMID:25313037

  2. Reactivation of p53 by Novel MDM2 Inhibitors: Implications for Pancreatic Cancer Therapy

    PubMed Central

    Azmi, A.S.; Philip, P.A.; Wang, Z.; Banerjee, S.; Zafar, S.F.; Goustin, A.-S.; Almhanna, K.; Yang, D.; Wang, S.; Sarkar, F.H.; Mohammad, R.M.

    2013-01-01

    The present study is the first to show in pancreatic cancer (PC) the growth inhibition and apoptosis by novel MDM2 inhibitors (MI-319 & 219) through reactivation of p53 pathway. Our results highlight two new secondary targets of MDM2 inhibitor ‘SIRT1’ and Ku70. SIRT1 has a role in ageing and cancer and is known to regulate p53 signaling through acetylation. Ku70 is a key component of non-homologous end joining machinery in the DNA damage pathway and is known to regulate apoptosis by blocking Bax entry into mitochondria. Given the growth inhibition and apoptosis by MI-219, MI-319 was accompanied by increase in levels of p53 along with p21WAF1 and the proapoptotic Puma. SiRNA against p21WAF1 abrogated the growth inhibition of PC cells confirming p21WAF1 as a key player downstream of activated p53. Immunoprecipitation-western blot analysis revealed reduced association of MDM2-p53 interaction in drug exposed PC cells. In combination studies, the inhibitors synergistically augmented anti-tumor effects of therapeutic drug gemcitabine both in terms of cell growth inhibition as well as apoptosis. Surface plasmon resonance studies confirmed strong binding between MI-319 and Ku70 (KD 170 nM). Western blot revealed suppression of SIRT1 and Ku70 with simultaneous upregulation of acetyl-p53 (Lys379) and Bax. Co-Immunoprecipitation studies confirmed that MI-319 could disrupt Ku70-Bax and SIRT1-Bax interaction. Further, using wt-p53 xenograft of Capan-2, we found that oral administration of MI-319 at 300 mg/kg for 14 days resulted in significant tumor growth inhibition without any observed toxicity to the animals. No tumor inhibition was found in mut-p53 BxPC-3 xenografts. In light of our results, the inhibitors of MDM2 warrant clinical investigation as new agents for PC treatment. PMID:20370686

  3. Momilactone B induces apoptosis and G1 arrest of the cell cycle in human monocytic leukemia U937 cells through downregulation of pRB phosphorylation and induction of the cyclin-dependent kinase inhibitor p21Waf1/Cip1.

    PubMed

    Park, Cheol; Jeong, Na Young; Kim, Gi-Young; Han, Min Ho; Chung, Ill-Min; Kim, Wun-Jae; Yoo, Young Hyun; Choi, Yung Hyun

    2014-04-01

    Momilactone B, a terpenoid phytoalexin present in rice bran, has been shown to exhibit several biological activities. The present study was conducted using cultured human leukemia U937 cells to elucidate the possible mechanisms by which momilactone B exerts its anticancer activity, which to date has remained poorly understood. Momilactone B treatment of U937 cells resulted in a dose-dependent inhibition of cell growth and induced apoptotic cell death as detected by chromatin condensation, DNA fragmentation, the cleavage of poly(ADP-ribose) polymerase and Annexin V-FITC staining. Flow cytometric analysis revealed that momilactone B resulted in G1 arrest in cell cycle progression, which was associated with the dephosphorylation of retinoblastoma protein (pRB) and enhanced binding of pRB with the E2F transcription factor family proteins. Treatment with momilactone B also increased the expression of cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1 in a p53-independent manner, without any noticeable changes in G1 cyclins and cyclin-dependent kinases (Cdks), except a slight decrease in cyclin E. Moreover, in vitro kinase assay indicated that momilactone B significantly decreased Cdk4- and Cdk6-associated kinase activities through a notably increased binding of p21 to Cdk4 and Cdk6. Our results demonstrated that momilactone B caused G1 cell cycle arrest and apoptosis in U937 cells through the induction of p21 expression, inhibition of Cdk/cyclin-associated kinase activities, and reduced phosphorylation of pRB, which may be related to anticancer activity. PMID:24503697

  4. TSPYL5 is involved in cell growth and the resistance to radiation in A549 cells via the regulation of p21{sup WAF1/Cip1} and PTEN/AKT pathway

    SciTech Connect

    Kim, Eun Jin; Lee, So Yong; Kim, Tae Rim; Choi, Soo Im; Cho, Eun Wie; Kim, Kug Chan; Kim, In Gyu

    2010-02-12

    TSPYL5, encoding testis-specific Y-like protein, has been postulated to be a tumor suppressor gene, and its hypermethylation is often associated with human disease, especially cancer. In this study, we report that the TSPYL5 gene was less methylated (30%) in A549 lung adenocarcinoma cells, which are relatively resistant to {gamma}-radiation, than in H460 lung cancer cells, in which the TSPYL5 gene was hypermethylated (95%); thus, the expression level of TSPYL5 is much higher in A549 cells than in H460 cells. We showed that TSPYL5 suppression with silencing RNA in A549 cells up-regulated cellular PTEN, followed by down-regulation of AKT activation. Therefore, blockage of TSPYL5 sensitized A549 cells to cytotoxic agents such as {gamma}-radiation. In addition, TSPYL5 suppression also showed an increased level of p21{sup WAF1/Cip1} and subsequently induced inhibition of cell growth in A549 cells. The overexpression of TSPYL5 in H460 cells showed the opposite effects. This study provides the first demonstration that TSPYL5 modulates cell growth and sensitization of cells to the detrimental effects of damaging agents via regulation of p21{sup WAF1/Cip1} and PTEN/AKT pathway.

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

    SciTech Connect

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

    2007-07-06

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

  6. Heterogeneity of p53-pathway Protein Expression in Chemosensitive Chronic Lymphocytic Leukemia: A Pilot Study

    PubMed Central

    Groves, Michael J; MacCallum, Stephanie F; Boylan, Michael T; Haydock, Sally; Cunningham, Joan; Gelly, Keith; Gowans, Duncan; Kerr, Ron; Coates, Philip J; Tauro, Sudhir

    2012-01-01

    The presence of p53-pathway dysfunction in chronic lymphocytic leukemia (CLL) can be used to identify patients with chemotherapy-refractory disease. Therapeutic responses are known to vary between patients with chemosensitive CLL and may relate to differences in p53-pathway activity. We hypothesized that the magnitude or type of p53-pathway protein expression is heterogeneous in patients with chemosensitive disease and could associate with white cell responses. In this pilot study, changes in p53 and its transcriptional targets, p21/waf1 and MDM2 were analyzed by immunoblotting and densitometry in CLL cells from 10 patients immediately prior to the start of chemotherapy, and after culture for 24 hours (h) with fludarabine (n=7) or chlorambucil (n=3). The in vitro response was also compared to that in vivo in circulating cells pre-treatment, and at 24h and 96h of chemotherapy. Disease responses were evident in all patients after the first treatment-cycle. Significant p53 induction was observed in CLL cells treated in vitro and in vivo. Greater heterogeneity in the expression-intensity was observed in vivo (σ2=45.15) than in vitro (σ2=1.33) and the results failed to correlate (r2=0.18, p=0.22). p21/waf1 and MDM2 expression-profiles were also dissimilar in vitro and in vivo. Higher in vivo (but not in vitro) responses associated with changes in white cell count (p=0.026). Thus, heterogeneity of p53-pathway activity exists in chemosensitive CLL; in unselected patients, in vivo changes do not correlate with those in vitro, but may associate with post-treatment white cell responses. PMID:22962562

  7. Modulation of p53-mediated transcriptional repression and apoptosis by the adenovirus E1B 19K protein.

    PubMed Central

    Sabbatini, P; Chiou, S K; Rao, L; White, E

    1995-01-01

    BRK cell lines that stably express adenovirus E1A and a murine temperature-sensitive p53 undergo apoptosis when p53 assumes the wild-type conformation. Expression of the E1B 19,000-molecular-weight (19K) protein rescues cells from this p53-mediated apoptosis and diverts cells to a growth-arrested state. As p53 likely functions as a tumor suppressor by regulating transcription, the ability of the E1B 19K protein to regulate p53-mediated transactivation and transcriptional repression was investigated. In promoter-reporter assays the E1B 19K did not block p53-mediated transactivation but did alleviate p53-mediated transcriptional repression. E1B 19K expression permitted efficient transcriptional activation of the p21/WAF-1/cip-1 mRNA by p53, consistent with maintenance of the growth arrest function of p53. The E1B 19K protein is thereby unique among DNA virus-transforming proteins that target p53 for inactivation in that it selectively modulates the transcriptional properties of p53. The E1B 19K protein also rescued cells from apoptosis induced by inhibitors of transcription and protein synthesis. This suggests that cell death may result from the inhibition of expression of survival factors which function to maintain cell viability. p53 may induce apoptosis through generalized transcriptional repression. In turn, the E1B 19K protein may prevent p53-mediated apoptosis by alleviating p53-mediated transcriptional repression. PMID:7823921

  8. Ligand-dependent interaction of the glucocorticoid receptor with p53 enhances their degradation by Hdm2

    PubMed Central

    Sengupta, Sagar; Wasylyk, Bohdan

    2001-01-01

    The glucocorticoid receptor (GR) and the tumor supressor p53 mediate different stress responses. We have studied the mechanism of their mutual inhibition in normal endothelial cells (HUVEC) in response to hypoxia, a physiological stress, and mitomycin C, which damages DNA. Dexamethasone (Dex) stimulates the degradation of endogenous GR and p53 by the proteasome pathway in HUVEC under hypoxia and mitomycin C treatments, and also in hepatoma cells (HepG2) under normoxia. Dex inhibits the functions of p53 (apoptosis, Bax, and p21WAF1/CIP1 expression) and GR (PEPCK and G-6-Pase expression). Endogenous p53 and GR form a ligand-dependent trimeric complex with Hdm2 in the cytoplasm. Disruption of the p53–HDM2 interaction prevents Dex-induced ubiquitylation of GR and p53. The ubiquitylation of GR requires p53, the interaction of p53 with Hdm2, and E3 ligase activity of Hdm2. These results provide a mechanistic basis for GR and p53 acting as opposing forces in the decision between cell death and survival. PMID:11562347

  9. Synergistic efficacy of sorafenib and genistein in growth inhibition by down regulating angiogenic and survival factors and increasing apoptosis through upregulation of p53 and p21 in malignant neuroblastoma cells having N-Myc amplification or non-amplification.

    PubMed

    Roy Choudhury, Subhasree; Karmakar, Surajit; Banik, Naren L; Ray, Swapan K

    2010-12-01

    Neuroblastoma is an extracranial, solid, and heterogeneous malignancy in children. The conventional therapeutic modalities are mostly ineffective and thus new therapeutic strategies for malignant neuroblastoma are urgently warranted. We examined the synergistic efficacy of combination of sorafenib (SF) and genistein (GST) in human malignant neuroblastoma SK-N-DZ (N-Myc amplified) and SH-SY5Y (N-Myc non-amplified) cell lines. MTT assay showed dose-dependent decrease in cell viability and the combination therapy more prominently inhibited the cell proliferation in both cell lines than either treatment alone. Apoptosis was confirmed morphologically by Wright staining. Flow cytometric analysis of cell cycle phase distribution and Annexin V-FITC/PI staining showed increase in subG1 DNA content and early apoptosis, respectively, after treatment with the combination of drugs. Apoptosis was further confirmed by scanning electron microscopy. Combination therapy showed activation of caspase-8, cleavage of Bid to tBid, increase in p53 and p21 expression, down regulation of anti-apoptotic Mcl-1, and increase in Bax:Bcl-2 ratio to trigger apoptosis. Down regulation of MDR, hTERT, N-Myc, VEGF, FGF-2, NF-κB, p-Akt, and c-IAP2 indicated suppression of angiogenic and survival pathways. Mitochondrial release of cytochrome c and Smac into cytosol indicated involvement of mitochondia in apoptosis. Increases in proteolytic activities of calpain and caspase-3 were also confirmed. Our results suggested that combination of SF and GST inhibited angiogenic and survival factors and increased apoptosis via receptor and mitochondria mediated pathways in both neuroblastoma SK-N-DZ and SH-SY5Y cell lines. Thus, this combination of drugs could be a potential therapeutic strategy against human malignant neuroblastoma cells having N-Myc amplification or non-amplification. PMID:19777160

  10. Tangeretin, a citrus pentamethoxyflavone, exerts cytostatic effect via p53/p21 up-regulation and suppresses metastasis in 7,12-dimethylbenz(α)anthracene-induced rat mammary carcinoma.

    PubMed

    Arivazhagan, Lakshmi; Sorimuthu Pillai, Subramanian

    2014-11-01

    Breast cancer is the most commonly diagnosed cancer among women worldwide, which is characterized by unregulated cell growth and metastasis. Many bioactive compounds of plant origin such as tangeretin have been shown to possess potent antioxidant and anticancerous properties. In the present study we have investigated the chemotherapeutic effect of tangeretin against 7,12-dimethylbenz(α)anthracene (DMBA)-induced rat mammary carcinogenesis and studied its underlying mechanism of action. Breast cancer was induced by "air pouch technique" with a single dose of 25mg/kg of DMBA. Tangeretin (50 mg/kg) was administered orally for four weeks. Remarkably, tangeretin treatment controlled the growth of cancer cells which was clearly evidenced by morphological and histological analysis. Also, serum levels of estradiol, progesterone and prolactin; lipid bound sialic acid and total sialic acid and the tissue levels of nitric oxide and protein carbonyls of cancer induced animals were decreased upon tangeretin treatment. Staining of breast tissues for nucleolar organizer regions, mast cells, glycoproteins, lipids and collagen showed that tangeretin treatment to breast cancer induced rats significantly reduced tumorigenesis. Oral tangeretin treatment also effectively reduced the tumor cell proliferation markers such as PCNA, COX-2 and Ki-67. Further, tangeretin treatment arrested the cancer cell division at the G1/S phase via p53/p21 up-regulation and inhibited metastasis by suppressing matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor. Taken together, the data provides new evidence on the mechanism of action of tangeretin in breast cancer and hence extends the hypothesis supporting its potential use in chemotherapy. PMID:25151216

  11. Transforming growth factor beta 1 increases the stability of p21/WAF1/CIP1 protein and inhibits CDK2 kinase activity in human colon carcinoma FET cells.

    PubMed

    Gong, JianGen; Ammanamanchi, Sudhakar; Ko, Tien C; Brattain, Michael G

    2003-06-15

    We examined transforming growth factor-beta 1 (TGF-beta 1) effects on cell cycle progression of human colon carcinoma FET cells. TGF-beta 1 inhibited DNA synthesis and cyclin-dependent kinase (CDK) activity after release from growth arrest in association with induction of the p21 CDK inhibitor, whereas cyclins, CDKs, and p27 protein levels remained relatively unchanged. The decrease in CDK2 kinase activity was the result of increased p21 association with cyclin A-CDK2 and cyclin E-CDK2. TGF-beta 1 treatment in late G(1) showed reduced induction of p21 protein levels in association with increased DNA synthesis. Consequently, p21 induction in early G(1) is critical for TGF-beta 1 inhibition of CDK2 kinase activity. Although TGF-beta 1 treatments in late G(1) failed to induce p21 protein, p21 mRNA induction was observed in late G(1) and in S phase. Further analysis showed that TGF-beta 1 treatment in early G(1) increases p21 protein stability throughout the G(1) and S phases of the cell cycle. Our results demonstrate that TGF-beta 1 stimulation of p21 is regulated at the posttranscriptional and transcriptional levels. This is a novel mechanism of TGF-beta 1 inhibition requiring early G(1) induction and stabilization of p21 protein, which binds to and inhibits cyclin E-CDK2 and cyclin A-CDK2 kinase activity rather than direct modulation of cyclin or CDK protein levels as seen in other systems. PMID:12810668

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

    PubMed Central

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

    2008-01-01

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

  13. Polymorphisms in the 5′ upstream regulatory region of p21WAF1/CIP1 and susceptibility to oesophageal squamous cell carcinoma

    PubMed Central

    Yang, Wenjun; Li, Yong; Ning, Tao; Cai, Hong; Chen, Zhiqiang; Dong, Ying; Ke, Yang

    2016-01-01

    This study aims to scan the 5′-upstream regulatory region of the p21 gene to identify all putative functional single nucleotide polymorphisms (SNPs) and to evaluate the contribution of p21 variants to oesophageal squamous cell carcinoma (ESCC) in the Chinese Han population. Common SNPs were identified, and both locus-based and haplotype-based association tests were used to evaluate the potential risk of these p21 gene polymorphisms for ESCC. Immunohistochemistry assay was further performed to detect the P21 protein expression in ESCC specimens. Twenty three SNPs were identified and seven Tagging SNPs were chosen to represent all 23 SNPs. Univariate analysis indicated that the rs3829963 C and the rs2395655 G alleles increased susceptibility to ESCC (OR = 1.606 and OR = 1.572, respectively). The rs3829963 C and rs2395655 G alleles, combined with cigarette smoking, could further increase the risk for ESCC (OR = 2.657 and OR = 2.828, respectively). Additionally, the rs2395655 G allele appeared to elevate the positive rate of P21 expression in ESCC tissues, as compared to the A allele. This report demonstrates for the first time that rs3829963 and rs2395655, in the promoter of the p21 gene are potentially functional, modulating susceptibility to ESCC among the high-risk cigarette-smoking Chinese population. PMID:26932598

  14. Polymorphisms in the 5' upstream regulatory region of p21(WAF1/CIP1) and susceptibility to oesophageal squamous cell carcinoma.

    PubMed

    Yang, Wenjun; Li, Yong; Ning, Tao; Cai, Hong; Chen, Zhiqiang; Dong, Ying; Ke, Yang

    2016-01-01

    This study aims to scan the 5'-upstream regulatory region of the p21 gene to identify all putative functional single nucleotide polymorphisms (SNPs) and to evaluate the contribution of p21 variants to oesophageal squamous cell carcinoma (ESCC) in the Chinese Han population. Common SNPs were identified, and both locus-based and haplotype-based association tests were used to evaluate the potential risk of these p21 gene polymorphisms for ESCC. Immunohistochemistry assay was further performed to detect the P21 protein expression in ESCC specimens. Twenty three SNPs were identified and seven Tagging SNPs were chosen to represent all 23 SNPs. Univariate analysis indicated that the rs3829963 C and the rs2395655 G alleles increased susceptibility to ESCC (OR = 1.606 and OR = 1.572, respectively). The rs3829963 C and rs2395655 G alleles, combined with cigarette smoking, could further increase the risk for ESCC (OR = 2.657 and OR = 2.828, respectively). Additionally, the rs2395655 G allele appeared to elevate the positive rate of P21 expression in ESCC tissues, as compared to the A allele. This report demonstrates for the first time that rs3829963 and rs2395655, in the promoter of the p21 gene are potentially functional, modulating susceptibility to ESCC among the high-risk cigarette-smoking Chinese population. PMID:26932598

  15. Nucleolin inhibits Hdm2 by multiple pathways leading to p53 stabilization.

    PubMed

    Saxena, A; Rorie, C J; Dimitrova, D; Daniely, Y; Borowiec, J A

    2006-11-23

    Nucleolin is a c-Myc-induced gene product with defined roles in ribosomal RNA processing and the inhibition of chromosomal DNA replication following stress. Here we find that changes in nucleolin protein levels in unstressed cells cause parallel changes in the amount of p53 protein. Alterations in p53 levels arise from nucleolin binding to the p53 antagonist Hdm2, resulting in the inhibition of both p53 ubiquitination and Hdm2 auto-ubiquitination. Nucleolin does not alter p53 ubiquitination by human papillomavirus E6, indicating that the effect is specific for Hdm2. Although the inhibition of ligase activity would be expected to stabilize Hdm2, we instead find that nucleolin also reduces Hdm2 protein levels, demonstrating that nucleolin inhibits Hdm2 using multiple mechanisms. Increases in nucleolin levels in unstressed cells led to higher expression of p21(cip1/waf1), a reduced rate of cellular proliferation, and an increase in apoptosis. Thus, nucleolin has a number of properties in common with the tumor suppressor ARF (alternate reading frame). We propose that nucleolin, like ARF, responds to hyperproliferative signals by upregulation of p53 through Hdm2 inhibition. PMID:16751805

  16. EPO gene expression induces the proliferation, migration and invasion of bladder cancer cells through the p21WAF1‑mediated ERK1/2/NF-κB/MMP-9 pathway.

    PubMed

    Park, Sung Lyea; Won, Se Yeon; Song, Jun-Hui; Kim, Wun-Jae; Moon, Sung-Kwon

    2014-11-01

    Erythropoietin (EPO) is a cytokine that modulates the production of red blood cells. Previous studies have contradicted the assumed role of EPO in tumor cell proliferation. In the present study, we investigated the effect of EPO in the proliferation, migration and invasion that is involved in the signaling pathways and cell-cycle regulation of bladder cancer 5637 cells. The results showed that an overexpression of the EPO gene has a potent stimulatory effect on DNA synthesis, migration and invasion. EPO gene expression increased the expression of matrix metalloproteinase (MMP)-9 via the binding activity of NF-κB, AP-1 and Sp-1 in 5637 cells. The transfection of 5637 cells with the EPO gene induced the phosphorylation of ERK1/2. Treatment with ERK1/2 inhibitor U0126 significantly inhibited the increased proliferation, migration and invasion of EPO gene-transfected cells. U0126 treatment suppressed the induction of MMP-9 expression through NF-κB binding activity in EPO gene transfectants. In addition, EPO gene expression was correlated with the upregulation of cyclins/CDKs and the upregulation of the CDK inhibitor p21WAF1 expression. Finally, the inhibition of p21WAF1 function by siRNA blocked the proliferation, migration, invasion and phosphorylation of ERK1/2 signaling, as well as MMP-9 expression and activation of NF-κB in EPO gene-transfected cells. These novel findings suggest that the molecular mechanisms of EPO contribute to the progression and development of bladder tumors. PMID:25175278

  17. Bioluminescence Imaging Captures the Expression and Dynamics of Endogenous p21 Promoter Activity in Living Mice and Intact Cells▿

    PubMed Central

    Tinkum, Kelsey L.; Marpegan, Luciano; White, Lynn S.; Sun, Jinwu; Herzog, Erik D.; Piwnica-Worms, David; Piwnica-Worms, Helen

    2011-01-01

    To interrogate endogenous p21WAF1/CIP1 (p21) promoter activity under basal conditions and in response to various forms of stress, knock-in imaging reporter mice in which expression of firefly luciferase (FLuc) was placed under the control of the endogenous p21 promoter within the Cdkn1a gene locus were generated. Bioluminescence imaging (BLI) of p21 promoter activity was performed noninvasively and repetitively in mice and in cells derived from these mice. We demonstrated that expression of FLuc accurately reported endogenous p21 expression at baseline and under conditions of genotoxic stress and that photon flux correlated with mRNA abundance and, therefore, bioluminescence provided a direct readout of p21 promoter activity in vivo. BLI confirmed that p53 was required for activation of the p21 promoter in vivo in response to ionizing radiation. Interestingly, imaging of reporter cells demonstrated that p53 prevents the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway from activating p21 expression when quiescent cells are stimulated with serum to reenter the cell cycle. In addition, low-light BLI identified p21 expression in specific regions of individual organs that had not been observed previously. This inducible p21FLuc knock-in reporter strain will facilitate imaging studies of p53-dependent and -independent stress responses within the physiological context of the whole animal. PMID:21791610

  18. Bioluminescence imaging captures the expression and dynamics of endogenous p21 promoter activity in living mice and intact cells.

    PubMed

    Tinkum, Kelsey L; Marpegan, Luciano; White, Lynn S; Sun, Jinwu; Herzog, Erik D; Piwnica-Worms, David; Piwnica-Worms, Helen

    2011-09-01

    To interrogate endogenous p21(WAF1/CIP1) (p21) promoter activity under basal conditions and in response to various forms of stress, knock-in imaging reporter mice in which expression of firefly luciferase (FLuc) was placed under the control of the endogenous p21 promoter within the Cdkn1a gene locus were generated. Bioluminescence imaging (BLI) of p21 promoter activity was performed noninvasively and repetitively in mice and in cells derived from these mice. We demonstrated that expression of FLuc accurately reported endogenous p21 expression at baseline and under conditions of genotoxic stress and that photon flux correlated with mRNA abundance and, therefore, bioluminescence provided a direct readout of p21 promoter activity in vivo. BLI confirmed that p53 was required for activation of the p21 promoter in vivo in response to ionizing radiation. Interestingly, imaging of reporter cells demonstrated that p53 prevents the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway from activating p21 expression when quiescent cells are stimulated with serum to reenter the cell cycle. In addition, low-light BLI identified p21 expression in specific regions of individual organs that had not been observed previously. This inducible p21(FLuc) knock-in reporter strain will facilitate imaging studies of p53-dependent and -independent stress responses within the physiological context of the whole animal. PMID:21791610

  19. Runt-related Transcription Factor 1 (RUNX1) Stimulates Tumor Suppressor p53 Protein in Response to DNA Damage through Complex Formation and Acetylation*

    PubMed Central

    Wu, Dan; Ozaki, Toshinori; Yoshihara, Yukari; Kubo, Natsumi; Nakagawara, Akira

    2013-01-01

    Representative tumor suppressor p53 plays a critical role in the regulation of proper DNA damage response. In this study, we have found for the first time that Runt-related transcription factor 1 (RUNX1) contributes to p53-dependent DNA damage response. Upon adriamycin (ADR) exposure, p53 as well as RUNX1 were strongly induced in p53-proficient HCT116 and U2OS cells, which were closely associated with significant transactivation of p53 target genes, such as p21WAF1, BAX, NOXA, and PUMA. RUNX1 was exclusively expressed in the cell nucleus and formed a complex with p53 in response to ADR. Chromatin immunoprecipitation assay demonstrated that p53 together with RUNX1 are efficiently recruited onto p53 target gene promoters following ADR exposure, indicating that RUNX1 is involved in p53-mediated transcriptional regulation. Indeed, forced expression of RUNX1 stimulated the transcriptional activity of p53 in response to ADR. Consistent with these observations, knockdown of RUNX1 attenuated ADR-mediated induction of p53 target genes and suppressed ADR-dependent apoptosis. Furthermore, RUNX1 was associated with p300 histone acetyltransferase, and ADR-dependent acetylation of p53 at Lys-373/382 was markedly inhibited in RUNX1 knockdown cells. In addition, knockdown of RUNX1 resulted in a significant decrease in the amount of p53-p300 complex following ADR exposure. Taken together, our present results strongly suggest that RUNX1 is required for the stimulation of p53 in response to DNA damage and also provide novel insight into understanding the molecular mechanisms behind p53-dependent DNA damage response. PMID:23148227

  20. Multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after DNA damage.

    PubMed

    Karimian, Ansar; Ahmadi, Yasin; Yousefi, Bahman

    2016-06-01

    An appropriate control over cell cycle progression depends on many factors. Cyclin-dependent kinase (CDK) inhibitor p21 (also known as p21(WAF1/Cip1)) is one of these factors that promote cell cycle arrest in response to a variety of stimuli. The inhibitory effect of P21 on cell cycle progression correlates with its nuclear localization. P21 can be induced by both p53-dependent and p53-independent mechanisms. Some other important functions attributed to p21 include transcriptional regulation, modulation or inhibition of apoptosis. These functions are largely dependent on direct p21/protein interactions and also on p21 subcellular localizations. In addition, p21 can play a role in DNA repair by interacting with proliferating cell nuclear antigen (PCNA). In this review, we will focus on the multiple functions of p21 in cell cycle regulation, apoptosis and gene transcription after DNA damage and briefly discuss the pathways and factors that have critical roles in p21 expression and activity. PMID:27156098

  1. MicroRNA-128-2 targets the transcriptional repressor E2F5 enhancing mutant p53 gain of function.

    PubMed

    Donzelli, S; Fontemaggi, G; Fazi, F; Di Agostino, S; Padula, F; Biagioni, F; Muti, P; Strano, S; Blandino, G

    2012-06-01

    p53 mutations have profound effects on non-small-cell lung cancer (NSCLC) resistance to chemotherapeutic treatments. Mutant p53 proteins are usually expressed at high levels in tumors, where they exert oncogenic functions. Here we show that p53R175H, a hotspot p53 mutant, induces microRNA (miRNA)-128-2 expression. Mutant p53 binds to the putative promoter of miR128-2 host gene, ARPP21, determining a concomitant induction of ARPP21 mRNA and miR-128-2. miR-128-2 expression in lung cancer cells inhibits apoptosis and confers increased resistance to cisplatin, doxorubicin and 5-fluorouracyl treatments. At the molecular level, miR-128-2 post-transcriptionally targets E2F5 and leads to the abrogation of its repressive activity on p21(waf1) transcription. p21(waf1) protein localizes to the cytoplasmic compartment, where it exerts an anti-apoptotic effect by preventing pro-caspase-3 cleavage. This study emphasizes miRNA-128-2 role as a master regulator in NSCLC chemoresistance. PMID:22193543

  2. MicroRNA-128-2 targets the transcriptional repressor E2F5 enhancing mutant p53 gain of function

    PubMed Central

    Donzelli, S; Fontemaggi, G; Fazi, F; Di Agostino, S; Padula, F; Biagioni, F; Muti, P; Strano, S; Blandino, G

    2012-01-01

    p53 mutations have profound effects on non-small-cell lung cancer (NSCLC) resistance to chemotherapeutic treatments. Mutant p53 proteins are usually expressed at high levels in tumors, where they exert oncogenic functions. Here we show that p53R175H, a hotspot p53 mutant, induces microRNA (miRNA)-128-2 expression. Mutant p53 binds to the putative promoter of miR128-2 host gene, ARPP21, determining a concomitant induction of ARPP21 mRNA and miR-128-2. miR-128-2 expression in lung cancer cells inhibits apoptosis and confers increased resistance to cisplatin, doxorubicin and 5-fluorouracyl treatments. At the molecular level, miR-128-2 post-transcriptionally targets E2F5 and leads to the abrogation of its repressive activity on p21waf1 transcription. p21waf1 protein localizes to the cytoplasmic compartment, where it exerts an anti-apoptotic effect by preventing pro-caspase-3 cleavage. This study emphasizes miRNA-128-2 role as a master regulator in NSCLC chemoresistance. PMID:22193543

  3. Activation of p53 with Ilimaquinone and Ethylsmenoquinone, Marine Sponge Metabolites, Induces Apoptosis and Autophagy in Colon Cancer Cells

    PubMed Central

    Lee, Hyun-Young; Chung, Kyu Jin; Hwang, In Hyun; Gwak, Jungsuk; Park, Seoyoung; Ju, Bong Gun; Yun, Eunju; Kim, Dong-Eun; Chung, Young-Hwa; Na, MinKyun; Song, Gyu-Yong; Oh, Sangtaek

    2015-01-01

    The tumor suppressor, p53, plays an essential role in the cellular response to stress through regulating the expression of genes involved in cell cycle arrest, apoptosis and autophagy. Here, we used a cell-based reporter system for the detection of p53 response transcription to identify the marine sponge metabolites, ilimaquinone and ethylsmenoquinone, as activators of the p53 pathway. We demonstrated that ilimaquinone and ethylsmenoquinone efficiently stabilize the p53 protein through promotion of p53 phosphorylation at Ser15 in both HCT116 and RKO colon cancer cells. Moreover, both compounds upregulate the expression of p21WAF1/CIP1, a p53-dependent gene, and suppress proliferation of colon cancer cells. In addition, ilimaquinone and ethylsmenoquinone induced G2/M cell cycle arrest and increased caspase-3 cleavage and the population of cells that positively stained with Annexin V-FITC, both of which are typical biochemical markers of apoptosis. Furthermore, autophagy was elicited by both compounds, as indicated by microtubule-associated protein 1 light chain 3 (LC3) puncta formations and LC3-II turnover in HCT116 cells. Our findings suggest that ilimaquinone and ethylsmenoquinone exert their anti-cancer activity by activation of the p53 pathway and may have significant potential as chemo-preventive and therapeutic agents for human colon cancer. PMID:25603347

  4. FHL2 mediates p53-induced transcriptional activation through a direct association with HIPK2

    SciTech Connect

    Lee, Sang-Wang . E-mail: umsj@sejong.ac.kr

    2006-01-27

    To understand the molecular mechanism underlying HIPK2 regulation of the transcriptional activation by p53, we sought to identify the protein that interacts with HIPK2. From our yeast two-hybrid screen, we found that four and a half LIM domains 2 (FHL2) could bind to the C-terminal half of HIPK2. Further assays in yeast mapped the minimal interaction domain to amino acids 812-907 in HIPK2. The interaction was confirmed using a GST pull-down assay in vitro, and an immunoprecipitation (IP) assay and fluorescence microscopy in vivo. FHL2 alone spread throughout both the cytoplasm and nucleus but was redistributed to dot-like structures in the nucleus when HIPK2 was coexpressed in HEK293 cells. When tethered to the Gal4-responsive promoter through the Gal4 DBD fusion, FHL2 showed autonomous transcriptional activity that was enhanced by wild-type HIPK2, but not by the kinase-defective mutant. In addition, FHL2 increased the p53-dependent transcriptional activation and had an additive effect on the activation when coexpressed with HIPK2, which was again not observed with the kinase-defective mutant of HIPK2. Finally, we found a ternary complex of p53, HIPK2, and FHL2 using IP, and their recruitment to the p53-responsive p21Waf1 promoter in chromatin IP assays. Overall, our findings indicate that FHL2 can also regulate p53 via a direct association with HIPK2.

  5. Proliferation arrest in G1 phase of a non-small cell lung cancer cell line (NSCLC-N6) treated by an original compound methyl-4-methoxy-3-(3-methyl-2-butanoyl) benzoate (VT1) independently of the p53/p21 cascade.

    PubMed

    Jacquot, Catherine; Moreau, Dimitri; Tomasoni, Christophe; Juge, Marcel; Coiffard, Laurence; Roussis, Vasilios; Roussakis, Christos

    2003-08-01

    Non-small cell lung cancers remain difficult to treat and have a high death rate and poor 5-year survival. New therapeutic strategies are urgently needed, which should be more specific for the cancer cell and less toxic for normal cells. In this respect, induction of the terminal differentiation of tumor cells appears to be a particularly suitable approach, which can only be achieved after proliferation arrest in G1 phase. This study describes the activity of a chemical compound with an original structure, namely methyl-4-methoxy-3-(3-methyl-2-butanoyl) benzoate (VT1), which induced irreversible proliferation arrest in G1 phase of two lung cancer lines, NSCLC-N6 and NSCLC-derived A549 cells. The p53 gene is now unanimously regarded as a key gene for cell cycle arrest in G1 phase, and A549 cells possess a wild-type p53 gene. The similarity of effects obtained on both lines led us to consider whether the p53/p21 cascade was activated in NSCLC-N6 cells during VT1 treatment. The mutational status of p53 gene was first established in the NSCLC-N6 line using a PCR SSCP technique, and a reporter gene was then used to assess the functionality of P53 protein. PMID:12851701

  6. Role of p14ARF-HDM2-p53 axis in SOX6-mediated tumor suppression.

    PubMed

    Wang, J; Ding, S; Duan, Z; Xie, Q; Zhang, T; Zhang, X; Wang, Y; Chen, X; Zhuang, H; Lu, F

    2016-03-31

    Sex-determining region Y box 6 (SOX6) has been described as a tumor-suppressor gene in several cancers. Our previous work has suggested that SOX6 upregulated p21(Waf1/Cip1)(p21) expression in a p53-dependent manner; however, the underlying mechanism has remained elusive. In this study, we confirmed that SOX6 can suppress cell proliferation in vitro and in vivo by stabilizing p53 protein and subsequently upregulating p21. Co-immunoprecipitation and immunocytofluorescence assays demonstrated that SOX6 can promote formation of the p14ARF-HDM2-p53 ternary complex by promoting translocation of p14ARF (p14 alternate reading frame tumor suppressor) to the nucleoplasm, thereby inhibiting HDM2-mediated p53 nuclear export and degradation. Chromatin immunoprecipitation combined with PCR assay proved that SOX6 can bind to a potential binding site in the regulatory region of the c-Myc gene. Furthermore, we confirmed that SOX6 can downregulate the expression of c-Myc, as well as its direct target gene nucleophosmin 1 (NPM1), and that the SOX6-induced downregulation of NPM1 is linked to translocation of p14ARF to the nucleoplasm. Finally, we showed that the highly conserved high-mobility group (HMG) domain of SOX6 is required for SOX6-mediated p53 stabilization and tumor inhibitory activity. Collectively, these results reveal a new mechanism of SOX6-mediated tumor suppression involving p21 upregulation via the p14ARF-HDM2-p53 axis in an HMG domain-dependent manner. PMID:26119940

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

    PubMed Central

    2014-01-01

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

  8. Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type p53 function.

    PubMed

    Thiery, Jérôme; Dorothée, Guillaume; Haddada, Hedi; Echchakir, Hamid; Richon, Catherine; Stancou, Rodica; Vergnon, Isabelle; Benard, Jean; Mami-Chouaib, Fathia; Chouaib, Salem

    2003-06-15

    Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21(waf/CIP1). Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We demonstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway. PMID:12794118

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

    PubMed Central

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

    2010-01-01

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

  10. Survivin safeguards chromosome numbers and protects from aneuploidy independently from p53

    PubMed Central

    2014-01-01

    Background Survivin, a member of the inhibitor of apoptosis (IAP) gene family, has a dual role in mitosis and in apoptosis. It is abundantly expressed in every human tumor, compared with normal tissues. During mitosis Survivin assembles with the chromosomal passenger complex and regulates chromosomal segregation. Here, we aim to explore whether interference with the mitotic function of Survivin is linked to p53-mediated G1 cell cycle arrest and affects chromosomal stability. Methods In this study, we used HCT116, SBC-2, and U87-MG and generated corresponding isogenic p53-deficient cells. Retroviral vectors were used to stably knockdown Survivin. The resulting phenotype, in particular the mechanisms of cell cycle arrest and of initiation of aneuploidy, were investigated by Western Blot analysis, confocal laser scan microscopy, proliferation assays, spectral karyotyping and RNAi. Results In all cell lines Survivin-RNAi did not induce instant apoptosis but caused polyplodization irrespective of p53 status. Strikingly, polyploidization after knockdown of Survivin resulted in merotelic kinetochore spindle assemblies, γH2AX-foci, and DNA damage response (DDR), which was accompanied by a transient p53-mediated G1-arrest. That p53 wild type cells specifically arrest due to DNA damage was shown by simultaneous inhibition of ATM and DNA-PK, which abolished induction of p21waf/cip. Cytogenetic analysis revealed chromosomal aberrations indicative for DNA double strand break repair by the mechanism of non-homologous end joining (NHEJ), only in Survivin-depleted cells. Conclusion Our findings suggest that Survivin plays an essential role in proper amphitelic kinetochore-spindle assembly and that constraining Survivin’s mitotic function results in polyploidy and aneuploidy which cannot be controlled by p53. Therefore, Survivin critically safeguards chromosomal stability independently from p53. PMID:24886358

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

    SciTech Connect

    Komissarova, Elena V.; Rossman, Toby G.

    2010-03-15

    Arsenite is an environmental pollutant. Exposure to inorganic arsenic in drinking water is associated with elevated cancer risk, especially in skin. Arsenite alone does not cause skin cancer in animals, but arsenite can enhance the carcinogenicity of solar UV. Arsenite is not a significant mutagen at non-toxic concentrations, but it enhances the mutagenicity of other carcinogens. The tumor suppressor protein P53 and nuclear enzyme PARP-1 are both key players in DNA damage response. This laboratory demonstrated earlier that in cells treated with arsenite, the P53-dependent increase in p21{sup WAF1/CIP1} expression, normally a block to cell cycle progression after DNA damage, is deficient. Here we show that although long-term exposure of human keratinocytes (HaCaT) to a nontoxic concentration (0.1 muM) of arsenite decreases the level of global protein poly(ADP-ribosyl)ation, it increases poly(ADP-ribosyl)ation of P53 protein and PARP-1 protein abundance. We also demonstrate that exposure to 0.1 muM arsenite depresses the constitutive expression of p21 mRNA and P21 protein in HaCaT cells. Poly(ADP-ribosyl)ation of P53 is reported to block its activation, DNA binding and its functioning as a transcription factor. Our results suggest that arsenite's interference with activation of P53 via poly(ADP-ribosyl)ation may play a role in the comutagenic and cocarcinogenic effects of arsenite.

  12. Reinitiation of DNA Synthesis and Cell Division in Senescent Human Fibroblasts by Microinjection of Anti-p53 Antibodies

    PubMed Central

    Gire, Veronique; Wynford-Thomas, David

    1998-01-01

    In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the “young” morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four- to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function. PMID:9488478

  13. FAK and p53 protein interactions.

    PubMed

    Golubovskaya, Vita M; Cance, William G

    2011-09-01

    Focal Adhesion Kinase plays a major role in cell adhesion, motility, survival, proliferation, metastasis, angiogenesis and lymphangiogenesis. In 2004, we have cloned the promoter sequence of FAK and found that p53 inhibits its activity (BBA, v. 1678, 2004). In 2005, we were the first group to show that FAK and p53 proteins directly interact in the cells (JBC, v. 280, 2005). We have shown that FAK and p53 proteins interact in the cytoplasm and in the nucleus by immunoprecipitation, pull-down and confocal microscopy assays. We have shown that FAK inhibited activity of p53 with the transcriptional targets: p21, Bax and Mdm-2 through protein-protein interactions. We identified the 7 amino-acid site in p53 that is involved in interaction with FAK protein. The present review will discuss the interaction of FAK and p53 proteins and discuss the mechanism of FAK-p53 loop regulation: inhibition of FAK promoter activity by p53 protein and also inhibition of p53 transcriptional activity by FAK protein. PMID:21355845

  14. Estrogens decrease {gamma}-ray-induced senescence and maintain cell cycle progression in breast cancer cells independently of p53

    SciTech Connect

    Toillon, Robert-Alain . E-mail: robert.toillon@univ-lille1.fr; Magne, Nicolas; Laios, Ioanna; Castadot, Pierre; Kinnaert, Eric; Van Houtte, Paul; Desmedt, Christine B.Sc.; Leclercq, Guy; Lacroix, Marc

    2007-03-15

    Purpose: Sequential administration of radiotherapy and endocrine therapy is considered to be a standard adjuvant treatment of breast cancer. Recent clinical reports suggest that radiotherapy could be more efficient in association with endocrine therapy. The aim of this study was to evaluate the estrogen effects on irradiated breast cancer cells (IR-cells). Methods and Materials: Using functional genomic analysis, we examined the effects of 17-{beta}-estradiol (E{sub 2}, a natural estrogen) on MCF-7 breast cancer cells. Results: Our results showed that E{sub 2} sustained the growth of IR-cells. Specifically, estrogens prevented cell cycle blockade induced by {gamma}-rays, and no modification of apoptotic rate was detected. In IR-cells we observed the induction of genes involved in premature senescence and cell cycle progression and investigated the effects of E{sub 2} on the p53/p21{sup waf1/cip1}/Rb pathways. We found that E{sub 2} did not affect p53 activation but it decreased cyclin E binding to p21{sup waf1/cip1} and sustained downstream Rb hyperphosphorylation by functional inactivation of p21{sup waf1/cip1}. We suggest that Rb inactivation could decrease senescence and allow cell cycle progression in IR-cells. Conclusion: These results may help to elucidate the molecular mechanism underlying the maintenance of breast cancer cell growth by E{sub 2} after irradiation-induced damage. They also offer clinicians a rational basis for the sequential administration of ionizing radiation and endocrine therapies.

  15. Karyopherin α2 induces apoptosis in tongue squamous cell carcinoma CAL-27 cells through the p53 pathway.

    PubMed

    Gao, Li; Yu, Lei; Li, Chun-Ming; Li, Ying; Jia, Bao-Lin; Zhang, Bin

    2016-06-01

    Tumor onset and progression are associated with dysfunction of the nuclear transport machinery at the level of import and export receptors. However, the role of Karyopherin α2 (KPNA2) in human tongue squamous cell carcinoma (TSCC) remains unknown. We assessed the proliferation, apoptosis and migration of TSCC CAL-27 cells using wound healing, Transwell and MTT assays, western blotting, electron microscopy and acridine orange/ethidium bromide staining following knockdown of KPNA2. The results revealed the antiproliferative, proapoptotic and anti-migratory effects of KPNA2 silencing on the TSCC CAL-27 cells. Moreover, the knockdown of KPNA2 proved to be accompanied by the upregulation of active caspase-3, cytochrome c, Bax, Bad and decreased expression of Bcl-2, p-Bad and XIAP. KPNA2 activated the caspase-dependent pathway in the CAL-27 cells with upregulation of p53, p21Cip1/Waf1 and p16INK4a. Thus, the present study demonstrated that p53/p21Cip1/Waf1/p16INK4a may be an important pathway involved in the function of KPNA2 in TSCC CAL-27 cells. PMID:27109484

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

    SciTech Connect

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

    2007-01-15

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

  17. p21 controls patterning but not homologous recombination in RPE development.

    PubMed

    Bishop, A J R; Kosaras, B; Hollander, M C; Fornace, A; Sidman, R L; Schiestl, R H

    2006-01-01

    p21/WAF1/CIP1/MDA6 is a key cell cycle regulator. Cell cycle regulation is an important part of development, differentiation, DNA repair and apoptosis. Following DNA damage, p53 dependent expression of p21 results in a rapid cell cycle arrest. p21 also appears to be important for the development of melanocytes, promoting their differentiation and melanogenesis. Here, we examine the effect of p21 deficiency on the development of another pigmented tissue, the retinal pigment epithelium. The murine mutation pink-eyed unstable (p(un)) spontaneously reverts to a wild-type allele by homologous recombination. In a retinal pigment epithelium cell this results in pigmentation, which can be observed in the adult eye. The clonal expansion of such cells during development has provided insight into the pattern of retinal pigment epithelium development. In contrast to previous results with Atm, p53 and Gadd45, p(un) reversion events in p21 deficient mice did not show any significant change. These results suggest that p21 does not play any role in maintaining overall genomic stability by regulating homologous recombination frequencies during development. However, the absence of p21 caused a distinct change in the positions of the reversion events within the retinal pigment epithelium. Those events that would normally arrest to produce single cell events continued to proliferate uncovering a cell cycle dysregulation phenotype. It is likely that p21 is involved in controlling the developmental pattern of the retinal pigment. We also found a C57BL/6J specific p21 dependent ocular defect in retinal folding, similar to those reported in the absence of p53. PMID:16202662

  18. Regulated recruitment of tumor suppressor BRCA1 to the p21 gene by coactivator methylation

    PubMed Central

    Lee, Young-Ho; Bedford, Mark T.; Stallcup, Michael R.

    2011-01-01

    Tumor suppression by p53 and BRCA1 involves regulation of cell cycle, apoptosis, and DNA repair and is influenced by transcriptional coactivators and post-translational modifications. Here we show that coactivator-associated arginine methyltransferase 1 (CARM1) methylates Arg 754 in the KIX region of coactivator p300. Methylated p300 and p300 protein fragments are preferentially recognized by BRCT domains of BRCA1, identifying the BRCT domain as a novel methylarginine-binding module. CARM1 and p300 cooperate with BRCA1 and p53 to induce expression of the critical cell cycle and proliferation regulator p21WAF1/CIP1 in response to DNA damage. This induction was severely attenuated by elimination of CARM1 or its methyltransferase activity, or by mutation of Arg 754 of p300. Absence of CARM1 methyltransferase activity led to failure of cells to arrest in the G1 phase of the cell cycle in response to DNA damage. CARM1 methyltransferase activity was required for induction of some p53 target genes (p21 and Gadd45) but not others (Bax) by DNA damage. Recruitment of BRCA1 to the p53-binding region of the p21 promoter in response to DNA damage required methylation of Arg 754 of p300 by CARM1. Thus, coactivator methylation may be crucial for fine-tuning the tumor suppressor function of BRCA1 and other BRCT domain proteins. PMID:21245169

  19. HER-2, p53, p21 and hormonal receptors proteins expression as predictive factors of response and prognosis in locally advanced breast cancer treated with neoadjuvant docetaxel plus epirubicin combination

    PubMed Central

    Tiezzi, Daniel G; Andrade, Jurandyr M; Ribeiro-Silva, Alfredo; Zola, Fábio E; Marana, Heitor RC; Tiezzi, Marcelo G

    2007-01-01

    Background Neoadjuvant chemotherapy has been considered the standard care in locally advanced breast cancer. However, about 20% of the patients do not benefit from this clinical treatment and, predictive factors of response were not defined yet. This study was designed to evaluate the importance of biological markers to predict response and prognosis in stage II and III breast cancer patients treated with taxane and anthracycline combination as neoadjuvant setting. Methods Sixty patients received preoperative docetaxel (75 mg/m2) in combination with epirubicin (50 mg/m2) in i.v. infusion in D1 every 3 weeks after incisional biopsy. They received adjuvant chemotherapy with CMF or FEC, attaining axillary status following definitive breast surgery. Clinical and pathologic response rates were measured after preoperative therapy. We evaluated the response rate to neoadjuvant chemotherapy and the prognostic significance of clinicopathological and immunohistochemical parameters (ER, PR, p51, p21 and HER-2 protein expression). The median patient age was 50.5 years with a median follow up time 48 months after the time of diagnosis. Results Preoperative treatment achieved clinical response in 76.6% of patients and complete pathologic response in 5%. The clinical, pathological and immunohistochemical parameters were not able to predict response to therapy and, only HER2 protein overexpression was associated with a decrease in disease free and overall survival (P = 0.0007 and P = 0.003) as shown by multivariate analysis. Conclusion Immunohistochemical phenotypes were not able to predict response to neoadjuvant chemotherapy. Clinical response is inversely correlated with a risk of death in patients submitted to neoadjuvant chemotherapy and HER2 overexpression is the major prognostic factor in stage II and III breast cancer patients treated with a neoadjuvant docetaxel and epirubicin combination. PMID:17324279

  20. Anti-lung cancer potential of pure esteric-glycoside condurangogenin A against nonsmall-cell lung cancer cells in vitro via p21/p53 mediated cell cycle modulation and DNA damage-induced apoptosis

    PubMed Central

    Sikdar, Sourav; Mukherjee, Avinaba; Khuda-Bukhsh, Anisur Rahman

    2015-01-01

    Background: Marsdenia condurango (condurango) is a tropical woody vine native to South America. Our earlier study was limited to evaluation of anti-cancer potentials of crude condurango extract and its glycoside-rich components in vitro on lung cancer. Objective: This study aims at evaluating the effect of the single isolated active ingredient condurangogenin A (ConA; C32H42O7) on A549, H522 and H460-nonsmall-cell lung cancer cells. Materials and Methods: ConA was isolated by column chromatography and analyzed by mass spectroscopy, Fourier transform infrared spectroscopy and proton-nuclear magnetic resonance. diphenyltetrazolium bromide assays were conducted on three cell-types using 6%-alcohol as control. Critical studies on cellular morphology, cell-cycle regulation, reactive oxygen species, mitochondrial membrane potential, and DNA-damage were made, and expressions of related signaling markers studied. Results: As IC50 doses of ConA proved to be too high and toxic to both A549 and H522 cells, all experimental studies were carried out on H460 cells with the IC50 dose (32 μg/ml − 24 h). Cellular morphology revealed typical apoptotic features after ConA treatment. At early treatment hours (2 h-12 h), maximum cells were arrested at G0/G1 phase that could be correlated with reduced level of cyclin D1-CDK with p21 up-regulation. At 18 h − 24 h, sub G0/G1 cell population was increased gradually, as revealed from cytochrome-c release and caspase-3 activation, further confirming the apoptosis-inducing ability of ConA at later phases. Gradual increase of TUNEL-positive cells with significant modulation of mitochondria-dependent apoptotic markers at longer time-points would establish apoptosis-induction property of ConA, indicating its potential as a strong candidate for anti-cancer drug formulation. Conclusion: Further studies are warranted against other types of cancer cells and animal models before its possible human use. PMID:26109778

  1. p53 and Cell Cycle Dependent Transcription of kinesin family member 23 (KIF23) Is Controlled Via a CHR Promoter Element Bound by DREAM and MMB Complexes

    PubMed Central

    Quaas, Marianne; Hoffmann, Saskia; Knörck, Arne; Gumhold, Catalina; Rother, Karen

    2013-01-01

    The microtubule-dependent molecular motor KIF23 (Kinesin family member 23) is one of two components of the centralspindlin complex assembled during late stages of mitosis. Formation of this complex is known as an essential step for cytokinesis. Here, we identified KIF23 as a new transcriptional target gene of the tumor suppressor protein p53. We showed that p53 reduces expression of KIF23 on the mRNA as well as the protein level in different cell types. Promoter reporter assays revealed that this repression results from downregulation of KIF23 promoter activity. CDK inhibitor p21WAF1/CIP1 was shown to be necessary to mediate p53-dependent repression. Furthermore, we identified the highly conserved cell cycle genes homology region (CHR) in the KIF23 promoter to be strictly required for p53-dependent repression as well as for cell cycle-dependent expression of KIF23. Cell cycle- and p53-dependent regulation of KIF23 appeared to be controlled by differential binding of DREAM and MMB complexes to the CHR element. With this study, we describe a new mechanism for transcriptional regulation of KIF23. Considering the strongly supporting function of KIF23 in cytokinesis, its p53-dependent repression may contribute to the prevention of uncontrolled cell growth. PMID:23650552

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

    PubMed

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

    2010-11-15

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

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

    PubMed

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

    2016-08-18

    Genome-wide chromatin studies identified the tumor suppressor p53 as both a promoter and an enhancer-binding transcription factor. As an enhancer factor, p53 can induce local production of enhancer RNAs, as well as transcriptional activation of distal neighboring genes. Beyond the regulation of protein-coding genes, p53 has the capacity to regulate long intergenic noncoding RNA molecules (lincRNAs); however, their importance to the p53 tumor suppressive function remains poorly characterized. Here, we identified and characterized a novel p53-bound intronic enhancer that controls the expression of its host, the lincRNA00475 (linc-475). We demonstrate the requirement of linc-475 for the proper induction of a p53-dependent cell cycle inhibitory response. We further confirm the functional importance of linc-475 in the maintenance of CDKN1A/p21 levels, a cell cycle inhibitor and a major p53 target gene, following p53 activation. Interestingly, loss of linc-475 reduced the binding of both p53 and RNA polymerase II (RNAPII) to the promoter of p21, attenuating its transcription rate following p53 activation. Altogether, our data suggest a direct role of p53-bound enhancer domains in the activation of lincRNAs required for an efficient p53 transcriptional response. PMID:26776159

  4. Mucin1 shifts Smad3 signaling from the tumor-suppressive pSmad3C/p21(WAF1) pathway to the oncogenic pSmad3L/c-Myc pathway by activating JNK in human hepatocellular carcinoma cells.

    PubMed

    Li, Qiongshu; Liu, Guomu; Yuan, Hongyan; Wang, Juan; Guo, Yingying; Chen, Tanxiu; Zhai, Ruiping; Shao, Dan; Ni, Weihua; Tai, Guixiang

    2015-02-28

    Mucin1 (MUC1) is a transmembrane glycoprotein that acts as an oncogene in human hepatic tumorigenesis. Hepatocellular carcinoma (HCC) cells often gain advantage by reducing the tumor-suppressive activity of transforming growth factor beta (TGF-β) together with stimulation of its oncogenic activity as in MUC1 expressing HCC cells; however, molecular mechanisms remain largely unknown. Type I TGF-β receptor (TβRI) and c-Jun NH2-terminal kinase (JNK) differentially phosphorylate Smad3 mediator to create 2 phosphorylated forms: COOH-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Here, we report that MUC1 overexpression in HCC cell lines suppresses TβRI-mediated pSmad3C signaling which involves growth inhibition by up-regulating p21(WAF1). Instead, MUC1 directly activates JNK to stimulate oncogenic pSmad3L signaling, which fosters cell proliferation by up-regulating c-Myc. Conversely, MUC1 gene silencing in MUC1 expressing HCC cells results in preserved tumor-suppressive function via pSmad3C, while eliminating pSmad3L-mediated oncogenic activity both in vitro and in vivo. In addition, high correlation between MUC1 and pSmad3L/c-Myc but not pSmad3C/p21(WAF1) expression was observed in HCC tissues from patients. Collectively, these results indicate that MUC1 shifts Smad3 signaling from a tumor-suppressive pSmad3C/p21(WAF1) to an oncogenic pSmad3L/c-Myc pathway by directly activating JNK in HCC cells, suggesting that MUC1 is an important target for HCC therapy. PMID:25714018

  5. Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

    PubMed Central

    Geng, Deyu; Zhang, Zhixia; Guo, Huarong

    2012-01-01

    p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl) phthalate (DEHP), a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner. PMID:25585933

  6. Cancer-associated S100P protein binds and inactivates p53, permits therapy-induced senescence and supports chemoresistance

    PubMed Central

    Gibadulinova, Adriana; Pastorek, Michal; Filipcik, Pavel; Radvak, Peter; Csaderova, Lucia; Vojtesek, Borivoj; Pastorekova, Silvia

    2016-01-01

    S100P belongs to the S100 family of calcium-binding proteins regulating diverse cellular processes. Certain S100 family members (S100A4 and S100B) are associated with cancer and used as biomarkers of metastatic phenotype. Also S100P is abnormally expressed in tumors and implicated in migration-invasion, survival, and response to therapy. Here we show that S100P binds the tumor suppressor protein p53 as well as its negative regulator HDM2, and that this interaction perturbs the p53-HDM2 binding and increases the p53 level. Paradoxically, the S100P-induced p53 is unable to activate its transcriptional targets hdm2, p21WAF, and bax following the DNA damage. This appears to be related to reduced phosphorylation of serine residues in both N-terminal and C-terminal regions of the p53 molecule. Furthermore, the S100P expression results in lower levels of pro-apoptotic proteins, in reduced cell death response to cytotoxic treatments, followed by stimulation of therapy-induced senescence and increased clonogenic survival. Conversely, the S100P silencing suppresses the ability of cancer cells to survive the DNA damage and form colonies. Thus, we propose that the oncogenic role of S100P involves binding and inactivation of p53, which leads to aberrant DNA damage responses linked with senescence and escape to proliferation. Thereby, the S100P protein may contribute to the outgrowth of aggressive tumor cells resistant to cytotoxic therapy and promote cancer progression. PMID:26967060

  7. Targeting the p53 pathway.

    PubMed

    Golubovskaya, Vita M; Cance, William G

    2013-10-01

    This article summarizes data on translational studies to target the p53 pathway in cancer. It describes the functions of the p53 and Mdm-2 signaling pathways, and discusses current therapeutic approaches to target p53 pathways, including reactivation of p53. In addition, direct interaction and colocalization of the p53 and focal adhesion kinase proteins in cancer cells have been demonstrated, and different approaches to target this interaction are reviewed. This is a broad review of p53 function as it relates to the diagnosis and treatment of a wide range of cancers. PMID:24012397

  8. p53-related apoptosis resistance and tumor suppression activity in UVB-induced premature senescent human skin fibroblasts.

    PubMed

    Chen, Wenqi; Kang, Jian; Xia, Jiping; Li, Yanhua; Yang, Bo; Chen, Bin; Sun, Weiling; Song, Xiuzu; Xiang, Wenzhong; Wang, Xiaoyong; Wang, Fei; Wan, Yinsheng; Bi, Zhigang

    2008-05-01

    Chronic exposure to solar UV irradiation leads to photoaging, immunosuppression, and ultimately carcinogenesis. Cellular senescence is thought to play an important role in tumor suppression and apoptosis resistance. However, the relationships among stress-induced premature senescence (SIPS), tumorigenesis and apoptosis induced by UVB remain unknown. We developed a model of UVB-induced premature senescence in human skin fibroblasts (HSFs). After five repeated subcytotoxic UVB exposures at a dose of 10 mJ/cm2, the following biomarkers of senescence were markedly present: senescence-associated beta-galactosidase (SA beta-gal) activity, growth arrest, and the overexpression of senescence-associated genes. Firstly, there was an increase in the proportion of cells positive for SA beta-gal activity. Secondly, there was a loss of replicative potential as assessed by MTT assay. FACS analysis showed that UVB-stressed HSFs were blocked mostly in the G1 phase of the cell cycle, and replicative senescence, and protein expression of p53, p21(WAF-1) and p16(INK-4a) increased significantly. Thirdly, the mRNA levels of three senescence-associated genes, fibronectin, osteonectin and SM22, also increased. A real time PCR array to investigate the mRNA expression of p53-related genes involved in growth arrest, apoptosis and tumorigenesis indicated that p53, p21, p19, Hdm2, and Bax were up-regulated, and bcl, HIF-1alpha and VEGF were down-regulated. Collectively, our data suggest that UVB-induced SIPS plays an important role in p53-related apoptosis resistance and tumor suppression activity. PMID:18425358

  9. YEATS4 is a novel oncogene amplified in non-small cell lung cancer that regulates the p53 pathway

    PubMed Central

    Pikor, Larissa A.; Lockwood, William W.; Thu, Kelsie L.; Vucic, Emily A.; Chari, Raj; Gazdar, Adi F.; Lam, Stephen; Lam, Wan L.

    2013-01-01

    Genetic analyses of lung cancer have helped found new treatments in this disease. We conducted an integrative analysis of gene expression and copy number in 261 non-small cell lung cancers (NSCLC) relative to matched normal tissues to define novel candidate oncogenes, identifying 12q13-15 and more specifically the YEATS4 gene as amplified and overexpressed in ~20% of the NSCLC cases examined. Overexpression of YEATS4 abrogated senescence in human bronchial epithelial cells (HBECs). Conversely, RNAi-mediated attenuation of YEATS4 in human lung cancer cells reduced their proliferation and tumor growth, impairing colony formation and inducing cellular senescence. These effects were associated with increased levels of p21WAF1 and p53 and cleavage of PARP, implicating YEATS4 as a negative regulator of the p21-p53 pathway. We also found that YEATS4 expression affected cellular responses to cisplastin, with increased levels associated with resistance and decreased levels with sensitivity. Taken together, our findings reveal YEATS4 as a candidate oncogene amplified in NSCLC, and a novel mechanism contributing to NSCLC pathogenesis. PMID:24170126

  10. Interleukin-20 Promotes Migration of Bladder Cancer Cells through Extracellular Signal-regulated Kinase (ERK)-mediated MMP-9 Protein Expression Leading to Nuclear Factor (NF-κB) Activation by Inducing the Up-regulation of p21WAF1 Protein Expression*

    PubMed Central

    Lee, Se-Jung; Cho, Seok-Cheol; Lee, Eo-Jin; Kim, Sangtae; Lee, Soo-Bok; Lim, Jung-Hyurk; Choi, Yung Hyun; Kim, Wun-Jae; Moon, Sung-Kwon

    2013-01-01

    The role of inflammatory cytokine interleukin-20 (IL-20) has not yet been studied in cancer biology. Here, we demonstrated up-regulation of both IL-20 and IL-20R1 in muscle-invasive bladder cancer patients. The expressions of IL-20 and IL-20R1 were observed in bladder cancer 5637 and T-24 cells. We found that IL-20 significantly increased the expression of matrix metalloproteinase (MMP)-9 via binding activity of NF-κB and AP-1 in bladder cancer cells and stimulated the activation of ERK1/2, JNK, p38 MAPK, and JAK-STAT signaling. Among the pathways examined, only ERK1/2 inhibitor U0126 significantly inhibited IL-20-induced migration and invasion. Moreover, siRNA knockdown of IL-20R1 suppressed migration, invasion, ERK1/2 activation, and NF-κB-mediated MMP-9 expression induced by IL-20. Unexpectedly, the cell cycle inhibitor p21WAF1 was induced by IL-20 treatment without altering cell cycle progression. Blockade of p21WAF1 function by siRNA reversed migration, invasion, activation of ERK signaling, MMP-9 expression, and activation of NF-κB in IL-20-treated cells. In addition, IL-20 induced the activation of IκB kinase, the degradation and phosphorylation of IκBα, and NF-κB p65 nuclear translocation, which was regulated by ERK1/2. IL-20 stimulated the recruitment of p65 to the MMP-9 promoter region. Finally, the IL-20-induced migration and invasion of cells was confirmed by IL-20 gene transfection and by addition of anti-IL-20 antibody. This is the first report that p21WAF1 is involved in ERK1/2-mediated MMP-9 expression via increased binding activity of NF-κB, which resulted in the induction of migration in IL-20/IL-20R1 dyad-induced bladder cancer cells. These unexpected results might provide a critical new target for the treatment of bladder cancer. PMID:23271730

  11. Regulation of the Cyclin-dependent Kinase Inhibitor 1A Gene (CDKN1A) by the Repressor BOZF1 through Inhibition of p53 Acetylation and Transcription Factor Sp1 Binding*

    PubMed Central

    Kim, Min-Kyeong; Jeon, Bu-Nam; Koh, Dong-In; Kim, Kyung-Sup; Park, So-Yoon; Yun, Chae-Ok; Hur, Man-Wook

    2013-01-01

    The human POZ domain and Krüppel-like zinc finger (POK) family proteins play important roles in the regulation of apoptosis, cell proliferation, differentiation, development, oncogenesis, and tumor suppression. A novel POK family transcription factor, BTB/POZ and zinc finger domains factor on chromosome 1 (BOZF-1; also called ZBTB8A), contains a POZ domain and two C2H2-type Krüppel-like zinc fingers and is localized at nuclear speckles. Compared with paired normal tissues, BOZF1 expression is increased in cancer tissues of the prostate, breast, and cervix. BOZF1 repressed the transcription of p21WAF/CDKN1A by acting on the proximal promoter concentrated with Sp1-binding GC boxes. BOZF1 competed with Sp1 in binding to GC boxes 1–5/6 of the CDKN1A proximal promoter. In addition, BOZF1 interacted with p53 and decreased the acetylation of p53 by p300, which reduced the DNA binding activity of p53 at the far distal p53-binding element. BOZF1 blocked the two major molecular events that are important in both constitutive and inducible transcription activation of CDKN1A. BOZF1 is unique in that it bound to all the proximal GC boxes to repress transcription, and it inhibited p53 acetylation without affecting p53 stability. BOZF1 might be a novel proto-oncoprotein that stimulates cell proliferation. PMID:23329847

  12. Resveratrol mediated cell death in cigarette smoke transformed breast epithelial cells is through induction of p21Waf1/Cip1 and inhibition of long patch base excision repair pathway

    SciTech Connect

    Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Das, Dipon; Siddharth, Sumit; Choudhuri, Tathagata; Kundu, Chanakya Nath

    2014-03-15

    Cigarette smoking is a key factor for the development and progression of different cancers including mammary tumor in women. Resveratrol (Res) is a promising natural chemotherapeutic agent that regulates many cellular targets including p21, a cip/kip family of cyclin kinase inhibitors involved in DNA damage-induced cell cycle arrest and blocking of DNA replication and repair. We have recently shown that cigarette smoke condensate (CSC) prepared from commercially available Indian cigarette can cause neoplastic transformation of normal breast epithelial MCF-10A cell. Here we studied the mechanism of Res mediated apoptosis in CSC transformed (MCF-10A-Tr) cells in vitro and in vivo. Res mediated apoptosis in MCF-10A-Tr cells was a p21 dependent event. It increased the p21 protein expression in MCF-10A-Tr cells and MCF-10A-Tr cells-mediated tumors in xenograft mice. Res treatment reduced the tumor size(s) and expression of anti-apoptotic proteins (e.g. PI3K, AKT, NFκB) in solid tumor. The expressions of cell cycle regulatory (Cyclins, CDC-2, CDC-6, etc.), BER associated (Pol-β, Pol-δ, Pol-ε, Pol-η, RPA, Fen-1, DNA-Ligase-I, etc.) proteins and LP-BER activity decreased in MCF-10A-Tr cells but remain significantly unaltered in isogenic p21 null MCF-10A-Tr cells after Res treatment. Interestingly, no significant changes were noted in SP-BER activity in both the cell lines after Res exposure. Finally, it was observed that increased p21 blocks the LP-BER in MCF-10A-Tr cells by increasing its interaction with PCNA via competing with Fen-1 after Res treatment. Thus, Res caused apoptosis in CSC-induced cancer cells by reduction of LP-BER activity and this phenomenon largely depends on p21. - Highlights: • Resveratrol (Res) caused reduction of MCF-10A-Tr cell growth by inducing apoptosis. • Res caused cell cycle arrest and DNA damage in p21 dependent manner. • Res mediated LP-BER reduction in MCF-10A-Tr cells was a p21 dependent phenomenon. • Res inhibits BER and PI

  13. Kaposi's sarcoma-associated herpesvirus K8beta is derived from a spliced intermediate of K8 pre-mRNA and antagonizes K8alpha (K-bZIP) to induce p21 and p53 and blocks K8alpha-CDK2 interaction.

    PubMed

    Yamanegi, Koji; Tang, Shuang; Zheng, Zhi-Ming

    2005-11-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a lymphotropic DNA tumor virus that induces Kaposi's sarcoma and AIDS-related primary effusion lymphoma. KSHV open reading frame 50 and K8 genes in early viral lytic infection express, respectively, a tricistronic and a bicistronic pre-mRNA, which undergo alternative splicing to create two major spliced mRNA isoforms, alpha and beta, by inclusion (beta) or exclusion (alpha) of an intron at nucleotides 75563 to 75645. This intron contains some suboptimal features, which cause the intron 5' splice site (ss) to interact weakly with U1 snRNA and the 3' ss to bind a U2 auxiliary factor, U2AF, with low affinity. Optimization of this intron in K8 (K8 intron 2) promoted the interaction of the 5' ss with U1 and the 3' ss with U2AF, resulting in a substantial increase in intron splicing. Splicing of K8 intron 2 has also been shown to be stimulated by the splicing of a downstream intron. This was confirmed by the insertion of a human beta-globin intron into the K8beta exon 3-exon 4 splice junction, which promoted splicing of K8beta intron 2 and conversion of the K8beta mRNA to the K8alpha mRNA that encodes a K-bZIP protein. Intron 2 contains a premature termination codon, yet the K8beta mRNA is insensitive to nonsense-mediated mRNA decay, suggesting that the truncated K8beta protein may have a biological function. Indeed, although the truncated K8beta protein is missing only a C-terminal leucine zipper domain from the K-bZIP, its expression antagonizes the ability of the K-bZIP to induce p53 and p21 and blocks K-bZIP-CDK2 interaction through interfering K8alpha mRNA production. PMID:16254356

  14. Disruption of focal adhesion kinase and p53 interaction with small molecule compound R2 reactivated p53 and blocked tumor growth

    PubMed Central

    2013-01-01

    Background Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor kinase that plays a major role in cancer cell survival and metastasis. Methods We performed computer modeling of the p53 peptide containing the site of interaction with FAK, predicted the peptide structure and docked it into the three-dimensional structure of the N-terminal domain of FAK involved in the complex with p53. We screened small molecule compounds that targeted the site of the FAK-p53 interaction and identified compounds (called Roslins, or R compounds) docked in silico to this site. Results By different assays in isogenic HCT116p53+/+ and HCT116 p53-/- cells we identified a small molecule compound called Roslin 2 (R2) that bound FAK, disrupted the binding of FAK and p53 and decreased cancer cell viability and clonogenicity in a p53-dependent manner. In addition, dual-luciferase assays demonstrated that the R2 compound increased p53 transcriptional activity that was inhibited by FAK using p21, Mdm-2, and Bax-promoter targets. R2 also caused increased expression of p53 targets: p21, Mdm-2 and Bax proteins. Furthermore, R2 significantly decreased tumor growth, disrupted the complex of FAK and p53, and up-regulated p21 in HCT116 p53+/+ but not in HCT116 p53-/- xenografts in vivo. In addition, R2 sensitized HCT116p53+/+ cells to doxorubicin and 5-fluorouracil. Conclusions Thus, disruption of the FAK and p53 interaction with a novel small molecule reactivated p53 in cancer cells in vitro and in vivo and can be effectively used for development of FAK-p53 targeted cancer therapy approaches. PMID:23841915

  15. The mitochondrial p53 pathway

    PubMed Central

    Vaseva, Angelina V.; Moll, Ute M.

    2010-01-01

    p53 is one of the most mutated tumor suppressors in human cancers and as such has been intensively studied for a long time. p53 is a major orchestrator of the cellular response to a broad array of stress types by regulating apoptosis, cell cycle arrest, senescence, DNA repair and genetic stability. For a long time it was thought that these functions of p53 solely rely on its function as a transcription factor, and numerous p53 target genes have been identified [1]. In the last 8 years however, a novel transcription-independent proapoptotic function mediated by the cytoplasmic pool of p53 has been revealed. p53 participates directly in the intrinsic apoptosis pathway by interacting with the multidomain members of the Bcl-2 family to induce mitochondrial outer membrane permeabilization. Our review will discuss these studies, focusing on recent advances in the field. PMID:19007744

  16. Regulation of rheumatoid synoviocyte proliferation by endogenous p53 induction

    PubMed Central

    Migita, K; Tanaka, F; Yamasaki, S; Shibatomi, K; Ida, H; Kawakami, A; Aoyagi, T; Kawabe, Y; Eguchi, K

    2001-01-01

    The p53 tumour suppressor protein protects cells from tumorigenic alterations by inducing either cell growth arrest or apoptosis. In the present study, we investigated the role of endogenous p53 expressed in rheumatoid arthritis synovial fibroblasts which show transformed-appearing phenotypes. Type B synovial cells (fibroblast-like synovial cells) were exposed to a proteasome inhibitor, carbobenzoxyl-leucinyl-leucinyl-leucinal (MG-132). During this process, the expressions of p53 and p21 were examined by Western blot. Cell cycle analysis of the synovial cells was determined by DNA staining using propidium iodide (PI). Inhibition of proteasome resulted in the accumulation of p53 which was followed by an increase in the amount of a cyclin-dependent kinase (CDK)-inhibitor, p21. As a consequence, the retinoblastoma gene product, Rb, remained in the hypophosphorylated state, thus preventing PDGF-stimulated synovial cells from progressing into S-phase. This study shows that endogenous p53, which is inducible in rheumatoid synovial cells, is functionally active based on the findings that its expression blocks the G1/S transition by inhibiting the CDK-mediated phosphorylation of Rb via p21 induction. Thus the induction of p53 using proteasome inhibitor may provide a new approach in the treatment of RA. PMID:11703379

  17. Estradiol induces functional inactivation of p53 by intracellular redistribution.

    PubMed

    Molinari, A M; Bontempo, P; Schiavone, E M; Tortora, V; Verdicchio, M A; Napolitano, M; Nola, E; Moncharmont, B; Medici, N; Nigro, V; Armetta, I; Abbondanza, C; Puca, G A

    2000-05-15

    Estrogen treatment of MCF-7 cells grown in serum-free medium induced a modification of the intracellular distribution of p53 protein. Western blot analysis and immunofluorescence staining showed that p53 was localized in the nucleus of untreated cell and that after 48 h of hormone treatment, it was mostly localized in the cytoplasm. This effect was blocked by the antiestrogen ICI182,780. Intracellular redistribution of p53 was correlated to a reduced expression of the WAF1/CIP1 gene product and to the presence of degradation fragments of p53 in the cytosol. Estradiol treatment prevented the growth inhibition induced by oligonucleotide transfection, simulating DNA damage. This observation indicated that the wild-type p53 gene product present in the MCF-7 cell could be inactivated by estradiol through nuclear exclusion to permit the cyclin-dependent phosphorylation events leading to the G1-S transition. In addition, the estradiol-induced inactivation of p53 could be involved in the tumorigenesis of estrogen-dependent neoplasm. PMID:10825127

  18. The p53 circuit board

    PubMed Central

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

    2012-01-01

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

  19. Modulation of p53β and p53γ expression by regulating the alternative splicing of TP53 gene modifies cellular response.

    PubMed

    Marcel, V; Fernandes, K; Terrier, O; Lane, D P; Bourdon, J-C

    2014-09-01

    In addition to the tumor suppressor p53 protein, also termed p53α, the TP53 gene produces p53β and p53γ through alternative splicing of exons 9β and 9γ located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53β and p53γ at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9β/9γ. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and α variant, supporting our experimental data. Using siRNA specifically targeting exons 9β/9γ, we demonstrate that cell growth can be driven by modulating p53β and p53γ expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53β and p53γ promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53β enhanced p53α transcriptional activity on the p21 and Bax promoters, while p53γ increased p53α transcriptional activity on the Bax promoter only. Moreover, p53β and p53γ co-immunoprecipitate with p53α only in the presence of p53-responsive promoter. Interestingly, although p53β and p53γ promote apoptosis in MCF7 cells, p53β and p53γ maintain cell growth in response to TG003 in a p53α-dependent manner. The dual activities of p53β and p53γ isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53β and p53γ regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers. PMID:24926616

  20. Dihydroptychantol A, a macrocyclic bisbibenzyl derivative, induces autophagy and following apoptosis associated with p53 pathway in human osteosarcoma U2OS cells

    SciTech Connect

    Li Xia; Wu, William K.K.; Sun Bin; Cui Min; Liu Shanshan; Gao Jian; Lou Hongxiang

    2011-03-01

    Dihydroptychantol A (DHA), a novel macrocyclic bisbibenzyl compound extracted from liverwort Asterella angusta, has antifungal and multi-drug resistance reversal properties. Here, the chemically synthesized DHA was employed to test its anti-cancer activities in human osteosarcoma U2OS cells. Our results demonstrated that DHA induced autophagy followed by apoptotic cell death accompanied with G{sub 2}/M-phase cell cycle arrest in U2OS cells. DHA-induced autophagy was morphologically characterized by the formation of double membrane-bound autophagic vacuoles recognizable at the ultrastructural level. DHA also increased the levels of LC3-II, a marker of autophagy. Surprisingly, DHA-mediated apoptotic cell death was potentiated by the autophagy inhibitor 3-methyladenine, suggesting that autophagy may play a protective role that impedes the eventual cell death. Furthermore, p53 was shown to be involved in DHA-meditated autophagy and apoptosis. In this connection, DHA increased nuclear expression of p53, induced p53 phosphorylation, and upregulated p53 target gene p21{sup Waf1/Cip1}. In contrast, cytoplasmic p53 was reduced by DHA, which contributed to the stimulation of autophagy. In relation to the cell cycle, DHA decreased the expression of cyclin B{sub 1}, a cyclin required for progression through the G{sub 2}/M phase. Taken together, DHA induces G{sub 2}/M-phase cell cycle arrest and apoptosis in U2OS cells. DHA-induced apoptosis was preceded by the induction of protective autophagy. DHA-mediated autophagy and apoptosis are associated with the cytoplasmic and nuclear functions of p53.

  1. A vitamin D3 analog induces a G1-phase arrest in CaCo-2 cells by inhibiting cdk2 and cdk6: roles of cyclin E, p21Waf1, and p27Kip1.

    PubMed

    Scaglione-Sewell, B A; Bissonnette, M; Skarosi, S; Abraham, C; Brasitus, T A

    2000-11-01

    Previous studies by our laboratory have shown that a noncalcemic fluorinated analog of 1alpha,25-dihydroxyvitamin D3, 1alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholcal ciferol (F6-D3), significantly reduced the frequency of colonic adenomas and completely abolished the development of colonic adenocarcinomas in rats treated with azoxymethane. The mechanisms involved in this analog's chemopreventive actions, however, remain unclear. In the present study, we now show that although both 1alpha,25-dihydroxyvitamin D3 and F6-D3 inhibited the proliferation of CaCo-2 cells, a human colonic adenocarcinoma cell line, by increasing their doubling times, only F6-D3 caused an arrest of these cells in the G1 phase of their cell cycle. This arrest was accompanied by an increase in the expression of the cyclin-dependent kinase (cdk) inhibitor proteins, p2Waf1 and p27Kip1, which served to decrease the activity of cyclin-dependent kinase 2 and cyclin-dependent kinase 6, whereas the expression and phosphorylation of pRB were unchanged. In contrast to the increased expression of these cdk inhibitors, the expression of cyclin E was decreased, which further inhibited the activity of cyclin-dependent kinase 2. Collectively, the inhibition of these cyclin-dependent kinases served to arrest the CaCo-2 cells, independent of changes in pRB. Furthermore, antibody neutralization studies suggest that transforming growth factor-beta may mediate the coassociations between cdk2 and p27Kip1 and cyclin E induced by F6-D3. These data indicate that cell cycle arrest may, at least in part, underlie the chemopreventive actions of F6-D3 observed in the azoxymethane model of colon cancer. Furthermore, if the antiproliferative action observed in CaCo-2 cells also occurs in human colonic epithelium, F6-D3 may have chemopreventive potential against human colon cancer, as well. PMID:11089522

  2. p53 Prevents Entry into Mitosis with Uncapped Telomeres

    PubMed Central

    Thanasoula, Maria; Escandell, Jose Miguel; Martinez, Paula; Badie, Sophie; Muñoz, Purificacion; Blasco, María A.; Tarsounas, Madalena

    2016-01-01

    Summary Telomeres are protected by capping structures consisting of core protein complexes that bind with sequence specificity to telomeric DNA (reviewed in [1]). In their absence, telomeres trigger a DNA damage response, materialized in accumulation at the telomere of damage response proteins, e.g., phosphorylated histone H2AX (γH2AX), into telomere-dysfunction-induced foci [2, 3]. Telomere uncapping occurs transiently in every cell cycle in G2 [4], following DNA replication, but little is known about how protective structures are reassembled or whether this process is controlled by the cell-cycle surveillance machinery. Here, we report that telomere capping is monitored at the G2/M transition by the p53/p21 damage response pathway. Unlike their wild-type counterparts, human and mouse cells lacking p53 or p21 progress into mitosis prematurely with persisting uncapped telomeres. Furthermore, artificially uncapped telomeres delay mitotic entry in a p53- and p21-dependent manner. Uncapped telomeres that persist in mitotic p53-deficient cells are shorter than average and religate to generate end-to-end fusions. These results suggest that a p53-dependent pathway monitors telomere capping after DNA replication and delays G2/M progression in the presence of unprotected telomeres. This mechanism maintains a cell-cycle stage conducive for capping reactions and prevents progression into stages during which uncapped telomeres are prone to deleterious end fusions. PMID:20226664

  3. Inhibition of p53 transcriptional activity by human cytomegalovirus UL44.

    PubMed

    Kwon, Yejin; Kim, Mi-Na; Young Choi, Eun; Heon Kim, Jung; Hwang, Eung-Soo; Cha, Chang-Yong

    2012-05-01

    Human cytomegalovirus (HCMV) stimulates cellular synthesis of DNA and proteins and induces transition of the cell cycle from G(1) to S and G(2) /M phase, in spite of increased amounts of p53 in the infected cells. The immediate early protein IE2-86  kDa (IE86) tethers a transcriptional repression domain to p53; however, its repression of p53 function is not enough to abrogate the G(1) checkpoint function of p53. Other HCMV proteins that suppress the activity of p53 were investigated in this study. Of the HCMV proteins that bind to p53 when assessed by immunoprecipitation and immunoblot analysis, HCMV UL44 was chosen as a candidate protein. It was found that reporter gene containing p53 consensus sequence was activated by transfection with wild type p53, but when plasmids of p53 with IE86 or UL44 were co-transfected, p53 transcriptional activity was decreased to 3-7% of the p53 control in a dose-dependent manner. When the deletion mutant of UL44 was co-transected with p53, the carboxyl one-third portion of UL44 had little effect on inhibition of p53 transcriptional activity. The amount of mRNA p21 was measured in H1299 by real time PCR after transfection of the combination of p53 and UL44 vectors and it was found that p21 transcription by p53 was inhibited dose-dependently by UL44. Increased G0/G1 and decreased S phases in p53 wild type-transfected H1299 cells were recovered to the level of p53 mutant type-transfected ones by the additional transfection of UL44 in a dose-dependent manner. In conclusion, the transcriptional activity of p53 is suppressed by UL44 as well as by IE86. PMID:22376288

  4. p53-Dependent apoptosis induced in human bronchial epithelial (16-HBE) cells by PM(2.5) sampled from air in Guangzhou, China.

    PubMed

    Zhou, Bo; Liang, Guiqiang; Qin, Huiyan; Peng, Xiaowu; Huang, Jiongli; Li, Qin; Qing, Li; Zhang, Li'e; Chen, Li; Ye, Li; Niu, Piye; Zou, Yunfeng

    2014-12-01

    Epidemiological studies have shown that air pollution particulate matter (PM) is associated with increased respiratory morbidity and mortality. However, the mechanisms are not fully understood. Oxidative stress-mediated apoptosis plays an important role in the occurrence of respiratory diseases. In this study, human bronchial epithelial (16-HBE) cells were exposed to different concentrations (16-128 µg/ml) of PM(2.5) for 24 h to investigate the apoptosis induced by PM(2.5). The results showed that PM(2.5) exposure significantly induced apoptosis, DNA strand breaks, and oxidative damage in a dose-dependent manner in 16-HBE cells. The expression of p53 and p73 increased significantly along with the dose of PM(2.5) in 16-HBE cells, whereas the expression of p21(Cip1/WAF1) decreased; the expression of mdm2 increased and then decreased, but not significantly. Taken together, these observations indicate that PM(2.5) may lead to oxidative damage and induce apoptosis through the p53-dependent pathway in 16-HBE cells. p53-Dependent apoptosis mediated by DNA strand breaks may be an important mechanism of PM(2.5)-induced apoptosis in 16-HBE cells. PMID:25133668

  5. The novel partnership of L-GILZ and p53: a new affair in cancer?

    PubMed Central

    Ayroldi, Emira; Marchetti, Cristina; Riccardi, Carlo

    2015-01-01

    A recent report from our laboratory reveals how long glucocorticoid-induced leucine zipper (L-Gilz) protein binds to p53 and mouse double minute 2 homolog (Mdm2), thus dissociating the p53/Mdm2 complex and activating p53 with subsequent activation of downstream genes p21 and p53 upregulated modulator of apoptosis (Puma). p53 activation appears to be the mechanism by which both basal and glucocorticoid (GC)-induced L-Gilz inhibits proliferation and induces antioncogenic activity in human cancer. PMID:27308427

  6. p53: Guardian of Ploidy

    PubMed Central

    Aylon, Yael; Oren, Moshe

    2011-01-01

    Aneuploidy, often preceded by tetraploidy, is one of the hallmarks of solid tumors. Indeed, both aneuploidy and tetraploidy are oncogenic occurrences that are sufficient to drive neoplastic transformation and cancer progression. True to form, the tumor suppressor p53 obstructs propagation of these dangerous chromosomal events by either instigating irreversible cell cycle arrest or apoptosis. The tumor suppressor Lats2, along with other tumor inhibitory proteins such as BRCA1/2 and BubR1, are central to p53-dependent elimination of tetraploid cells. Not surprisingly, these proteins are frequently inactivated or downregulated in tumors, synergizing with p53 inactivation to establish an atmosphere of “tolerance” for a nondiploid state. PMID:21852209

  7. 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.

  8. MOZ increases p53 acetylation and premature senescence through its complex formation with PML.

    PubMed

    Rokudai, Susumu; Laptenko, Oleg; Arnal, Suzzette M; Taya, Yoichi; Kitabayashi, Issay; Prives, Carol

    2013-03-01

    Monocytic leukemia zinc finger (MOZ)/KAT6A is a MOZ, Ybf2/Sas3, Sas2, Tip60 (MYST)-type histone acetyltransferase that functions as a coactivator for acute myeloid leukemia 1 protein (AML1)- and Ets family transcription factor PU.1-dependent transcription. We previously reported that MOZ directly interacts with p53 and is essential for p53-dependent selective regulation of p21 expression. We show here that MOZ is an acetyltransferase of p53 at K120 and K382 and colocalizes with p53 in promyelocytic leukemia (PML) nuclear bodies following cellular stress. The MOZ-PML-p53 interaction enhances MOZ-mediated acetylation of p53, and this ternary complex enhances p53-dependent p21 expression. Moreover, we identified an Akt/protein kinase B recognition sequence in the PML-binding domain of MOZ protein. Akt-mediated phosphorylation of MOZ at T369 has a negative effect on complex formation between PML and MOZ. As a result of PML-mediated suppression of Akt, the increased PML-MOZ interaction enhances p21 expression and induces p53-dependent premature senescence upon forced PML expression. Our research demonstrates that MOZ controls p53 acetylation and transcriptional activity via association with PML. PMID:23431171

  9. Lysines in the tetramerization domain of p53 selectively modulate G1 arrest.

    PubMed

    Beckerman, Rachel; Yoh, Kathryn; Mattia-Sansobrino, Melissa; Zupnick, Andrew; Laptenko, Oleg; Karni-Schmidt, Orit; Ahn, Jinwoo; Byeon, In-Ja; Keezer, Susan; Prives, Carol

    2016-06-01

    Functional in a tetrameric state, the protein product of the p53 tumor suppressor gene confers its tumor-suppressive activity by transactivating genes which promote cell-cycle arrest, senescence, or programmed cell death. How p53 distinguishes between these divergent outcomes is still a matter of considerable interest. Here we discuss the impact of 2 mutations in the tetramerization domain that confer unique properties onto p53. By changing lysines 351 and 357 to arginine, thereby blocking all post-translational modifications of these residues, DNA binding and transcriptional regulation by p53 remain virtually unchanged. On the other hand, by changing these lysines to glutamine (2KQ-p53), thereby neutralizing their positive charge and potentially mimicking acetylation, p53 is impaired in the induction of cell cycle arrest and yet can still effectively induce cell death. Surprisingly, when 2KQ-p53 is expressed at high levels in H1299 cells, it can bind to and transactivate numerous p53 target genes including p21, but not others such as miR-34a and cyclin G1 to the same extent as wild-type p53. Our findings show that strong induction of p21 is not sufficient to block H1299 cells in G1, and imply that modification of one or both of the lysines within the tetramerization domain may serve as a mechanism to shunt p53 from inducing cell cycle arrest. PMID:27210019

  10. p53 and ATF4 mediate distinct and additive pathways to skeletal muscle atrophy during limb immobilization

    PubMed Central

    Fox, Daniel K.; Ebert, Scott M.; Bongers, Kale S.; Dyle, Michael C.; Bullard, Steven A.; Dierdorff, Jason M.; Kunkel, Steven D.

    2014-01-01

    Immobilization causes skeletal muscle atrophy via complex signaling pathways that are not well understood. To better understand these pathways, we investigated the roles of p53 and ATF4, two transcription factors that mediate adaptations to a variety of cellular stresses. Using mouse models, we demonstrate that 3 days of muscle immobilization induces muscle atrophy and increases expression of p53 and ATF4. Furthermore, muscle fibers lacking p53 or ATF4 are partially resistant to immobilization-induced muscle atrophy, and forced expression of p53 or ATF4 induces muscle fiber atrophy in the absence of immobilization. Importantly, however, p53 and ATF4 do not require each other to promote atrophy, and coexpression of p53 and ATF4 induces more atrophy than either transcription factor alone. Moreover, muscle fibers lacking both p53 and ATF4 are more resistant to immobilization-induced atrophy than fibers lacking only p53 or ATF4. Interestingly, the independent and additive nature of the p53 and ATF4 pathways allows for combinatorial control of at least one downstream effector, p21. Using genome-wide mRNA expression arrays, we identified p21 mRNA as a skeletal muscle transcript that is highly induced in immobilized muscle via the combined actions of p53 and ATF4. Additionally, in mouse muscle, p21 induces atrophy in a manner that does not require immobilization, p53 or ATF4, and p21 is required for atrophy induced by immobilization, p53, and ATF4. Collectively, these results identify p53 and ATF4 as essential and complementary mediators of immobilization-induced muscle atrophy and discover p21 as a critical downstream effector of the p53 and ATF4 pathways. PMID:24895282

  11. Novel Pactamycin Analogs Induce p53 Dependent Cell-Cycle Arrest at S-Phase in Human Head and Neck Squamous Cell Carcinoma (HNSCC) Cells

    PubMed Central

    Guha, Gunjan; Liang, Xiaobo; Kulesz-Martin, Molly F.; Mahmud, Taifo; Indra, Arup Kumar; Ganguli-Indra, Gitali

    2015-01-01

    Pactamycin, although putatively touted as a potent antitumor agent, has never been used as an anticancer drug due to its high cytotoxicity. In this study, we characterized the effects of two novel biosynthetically engineered analogs of pactamycin, de-6MSA-7-demethyl-7-deoxypactamycin (TM-025) and 7-demethyl-7-deoxypactamycin (TM-026), in head and neck squamous cell carcinoma (HNSCC) cell lines SCC25 and SCC104. Both TM-025 and TM-026 exert growth inhibitory effects on HNSCC cells by inhibiting cell proliferation. Interestingly, unlike their parent compound pactamycin, the analogs do not inhibit synthesis of nascent protein in a cell-based assay. Furthermore, they do not induce apoptosis or autophagy in a dose- or a time-dependent manner, but induce mild senescence in the tested cell lines. Cell cycle analysis demonstrated that both analogs significantly induce cell cycle arrest of the HNSCC cells at S-phase resulting in reduced accumulation of G2/M-phase cells. The pactamycin analogs induce expression of cell cycle regulatory proteins including master regulator p53, its downstream target p21Cip1/WAF1, p27kip21, p19, cyclin E, total and phospho Cdc2 (Tyr15) and Cdc25C. Besides, the analogs mildly reduce cyclin D1 expression without affecting expression of cyclin B, Cdk2 and Cdk4. Specific inhibition of p53 by pifithrin-α reduces the percentage of cells accumulated in S-phase, suggesting contribution of p53 to S-phase increase. Altogether, our results demonstrate that Pactamycin analogs TM-025 and TM-026 induce senescence and inhibit proliferation of HNSCC cells via accumulation in S-phase through possible contribution of p53. The two PCT analogs can be widely used as research tools for cell cycle inhibition studies in proliferating cancer cells with specific mechanisms of action. PMID:25938491

  12. Bisnaphthalimidopropyl diaminodicyclohexylmethane induces DNA damage and repair instability in triple negative breast cancer cells via p21 expression.

    PubMed

    Barron, Gemma A; Goua, Marie; Kuraoka, Isao; Bermano, Giovanna; Iwai, Shigenori; Kong Thoo Lin, Paul

    2015-12-01

    Bisnaphthalimidopropyl diaminodicyclohexylmethane (BNIPDaCHM) bisintercalates to DNA and is a potential anti-cancer therapeutic. In an attempt to elucidate the mechanism(s) underlying the potential of BNIPDaCHM; earlier work was extended to investigate its effect on DNA damage and repair as well as cell cycle modulation, in a triple negative breast cancer (TNBC) cell line in vitro. BNIPDaCHM significantly decreased cell viability in a concentration (≥ 5 μM) and time (≥ 24 h) dependent manner. The mechanism of this growth inhibition involved alterations to cell cycle progression, an increase in the sub-G1 population and changes to plasma membrane integrity/permeability observed by flow cytometry and fluorescence microscopy with acridine orange/ethidium bromide staining. Using single cell gel electrophoresis (Comet assay) and fluorescence microscopy to detect γ-H2AX-foci expression; it was found that after 4 h, ≥ 0.1 μM BNIPDaCHM treatment-induced significant DNA double strand breaks (DSBs). Moreover, exposure to a non-genotoxic concentration of BNIPDaCHM induced a significant decrease in the repair of oxidative DNA strand breaks induced by hydrogen peroxide. Also, BNIPDaCHM-treatment induced a significant time dependent increase in p21(Waf/Cip1) mRNA expression but, did not alter p53 mRNA expression. In conclusion, BNIPDaCHM treatment in MDA-MB-231 cells was associated with a significant induction of DNA DSBs and inhibition of DNA repair at non-genotoxic concentrations via p53-independent expression of p21(Waf1/Cip1). The latter may be a consequence of novel interactions between BNIPDaCHM and MDA-MB-231 cells which adds to the spectrum of therapeutically relevant activities that may be exploited in the future design and development of naphthalimide-based therapeutics. PMID:26499071

  13. p53 negatively regulates Aurora A via both transcriptional and posttranslational regulation

    PubMed Central

    Wu, Chun-Chi; Yang, Tsung-Ying; Yu, Chang-Tze Ricky; Phan, Liem; Ivan, Cristina; Sood, Anil K.; Hsu, Shih-Lan; Lee, Mong-Hong

    2012-01-01

    p53 plays an important role in mitotic checkpoint, but what its role is remains enigmatic. Aurora A is a Ser/Thr kinase involved in correcting progression of mitosis. Here, we show that p53 is a negative regulator for Aurora A. We found that p53 deficiency leads to Aurora A elevation. Ectopic expression of p53 or DNA damage-induced expression of p53 can suppress the expression of Aurora A. Mechanistic studies show that p53 is a negative regulator for Aurora A expression through both transcriptional and posttranslational regulation. p53 knockdown in cancer cells reduces the level of p21, which, in turn, increases the activity of CDK2 followed by induction of Rb1 hyperphosphorylation and its dissociation with transcriptional factor E2F3. E2F3 can bind to Aurora A gene promoter, potentiating Aurora A gene expression and p53 deficiency, enhancing the binding of E2F3 on Aurora A promoter. Also, p53 deficiency leads to decelerating Aurora A’s turnover rate, due to the fact that p53 deficiency causes the downregulation of Fbw7α, a component of E3 ligase of Aurora A. Consistently, p53 knockdown-mediated Aurora A elevation is mitigated when Fbw7α is ectopically expressed. Thus, p53-mediated Aurora A degradation requires Fbw7α expression. Significantly, inverse correlation between p53 and Aurora A elevation is translated into the deregulation of centrosome amplification. p53 knockdown leads to high percentages of cells with abnormal amplification of centrosome. These data suggest that p53 is an important negative regulator of Aurora A, and that loss of p53 in many types of cancer could lead to abnormal elevation of Aurora A and dysregulated mitosis, which provides a growth advantage for cancer cells. PMID:22894933

  14. Pathologies Associated with the p53 Response

    PubMed Central

    Gudkov, Andrei V.; Komarova, Elena A.

    2010-01-01

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

  15. Regulation of Mutant p53 Protein Expression

    PubMed Central

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

    2015-01-01

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

  16. Wild-type p53 and p73 negatively regulate expression of proliferation related genes.

    PubMed

    Scian, M J; Carchman, E H; Mohanraj, L; Stagliano, K E R; Anderson, M A E; Deb, D; Crane, B M; Kiyono, T; Windle, B; Deb, S P; Deb, S

    2008-04-17

    When normal cells come under stress, the wild-type (WT) p53 level increases resulting in the regulation of gene expression responsible for growth arrest or apoptosis. Here we show that elevated levels of WT p53 or its homologue, p73, inhibit expression of a number of cell cycle regulatory and growth promoting genes. Our analysis also identified a group of genes whose expression is differentially regulated by WT p53 and p73. We have infected p53-null H1299 human lung carcinoma cells with recombinant adenoviruses expressing WT p53, p73 or beta-galactosidase, and have undertaken microarray hybridization analyses to identify genes whose expression profile is altered by p53 or p73. Quantitative real-time PCR verified the repression of E2F-5, centromere protein A and E, minichromosome maintenance proteins (MCM)-2, -3, -5, -6 and -7 and human CDC25B after p53 expression. 5-Fluorouracil treatment of colon carcinoma HCT116 cells expressing WT p53 results in a reduction of the cyclin B2 protein level suggesting that DNA damage may indeed cause repression of these genes. Transient transcriptional assays verified that WT p53 repressed promoters of a number of these genes. Interestingly, a gain-of-function p53 mutant instead upregulated a number of these promoters in transient transfection. Using promoter deletion mutants of MCM-7 we have found that WT p53-mediated repression needs a minimal promoter that contains a single E2F site and surrounding sequences. However, a single E2F site cannot be significantly repressed by WT p53. Many of the genes identified are also repressed by p21. Thus, our work shows that WT p53 and p73 repress a number of growth-related genes and that in many instances this repression may be through the induction of p21. PMID:17982488

  17. 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells.

    PubMed

    Bauer, Matthias R; Joerger, Andreas C; Fersht, Alan R

    2016-09-01

    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-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. PMID:27551077

  18. Reversible induction of translational isoforms of p53 in glucose deprivation

    PubMed Central

    Khan, D; Katoch, A; Das, A; Sharathchandra, A; Lal, R; Roy, P; Das, S; Chattopadhyay, S; Das, S

    2015-01-01

    Tumor suppressor protein p53 is a master transcription regulator, indispensable for controlling several cellular pathways. Earlier work in our laboratory led to the identification of dual internal ribosome entry site (IRES) structure of p53 mRNA that regulates translation of full-length p53 and Δ40p53. IRES-mediated translation of both isoforms is enhanced under different stress conditions that induce DNA damage, ionizing radiation and endoplasmic reticulum stress, oncogene-induced senescence and cancer. In this study, we addressed nutrient-mediated translational regulation of p53 mRNA using glucose depletion. In cell lines, this nutrient-depletion stress relatively induced p53 IRES activities from bicistronic reporter constructs with concomitant increase in levels of p53 isoforms. Surprisingly, we found scaffold/matrix attachment region-binding protein 1 (SMAR1), a predominantly nuclear protein is abundant in the cytoplasm under glucose deprivation. Importantly under these conditions polypyrimidine-tract-binding protein, an established p53 ITAF did not show nuclear-cytoplasmic relocalization highlighting the novelty of SMAR1-mediated control in stress. In vivo studies in mice revealed starvation-induced increase in SMAR1, p53 and Δ40p53 levels that was reversible on dietary replenishment. SMAR1 associated with p53 IRES sequences ex vivo, with an increase in interaction on glucose starvation. RNAi-mediated-transient SMAR1 knockdown decreased p53 IRES activities in normal conditions and under glucose deprivation, this being reflected in changes in mRNAs in the p53 and Δ40p53 target genes involved in cell-cycle arrest, metabolism and apoptosis such as p21, TIGAR and Bax. This study provides a new physiological insight into the regulation of this critical tumor suppressor in nutrient starvation, also suggesting important functions of the p53 isoforms in these conditions as evident from the downstream transcriptional target activation. PMID:25721046

  19. Allele Specific p53 Mutant Reactivation

    PubMed Central

    Yu, Xin; Vazquez, Alexei; Levine, Arnold J.; Carpizo, Darren R.

    2012-01-01

    Summary Rescuing the function of mutant p53 protein is an attractive cancer therapeutic strategy. Using the NCI anticancer drug screen data, we identified two compounds from the thiosemicarbazone family that manifest increased growth inhibitory activity in mutant p53 cells, particularly for the p53R175 mutant. Mechanistic studies reveal that NSC319726 restores WT structure and function to the p53R175 mutant. This compound kills p53R172H knock-in mice with extensive apoptosis and inhibits xenograft tumor growth in a 175-allele specific mutant p53 dependent manner. This activity depends upon the zinc ion chelating properties of the compound as well as redox changes. These data identify NSC319726 as a p53R175 mutant reactivator and as a lead compound for p53 targeted drug development. PMID:22624712

  20. Prospective therapeutic applications of p53 inhibitors

    SciTech Connect

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

    2005-06-10

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

  1. Exogenous p53 and ASPP2 expression enhances rAdV-TK/GCV-induced death in hepatocellular carcinoma cells lacking functional p53

    PubMed Central

    Guo, Xianghua; Wei, Feili; Yin, Jiming; Zang, Yunjin; Li, Ning; Chen, Dexi

    2016-01-01

    Suicide gene therapy using herpes simplex virus-1 thymidine kinase (HSV-TK) in combination with ganciclovir (GCV) has emerged as a potential new method for treating cancer. We hypothesize that the efficacy of HSV-TK/GCV therapy is at least partially dependent on p53 status in hepatocellular carcinoma (HCC) patients. Using recombinant adenoviral vectors (rAdV), TK, p53, and ASPP2 were overexpressed individually and in combination in Hep3B (p53 null) and HepG2 (p53 wild-type) cell lines and in primary HCC tumor cells. p53 overexpression induced death in Hep3B cells, but not HepG2 cells. ASPP2 overexpression increased rAdV-TK/GCV-induced HepG2 cell death by interacting with endogenous p53. Similarly, ASPP2 reduced survival in rAdV-TK/GCV-treated primary HCC cells expressing p53 wild-type but not a p53 R249S mutant. Mutated p53 was unable to bind to ASPP2, suggesting that the increase in rAdV-TK/GCV-induced cell death resulting from ASPP2 overexpression was dependent on its interaction with p53. Additionally, γ-H2AX foci, ATM phosphorylation, Bax, and p21 expression increased in rAdV-TK/GCV-treated HepG2 cells as compared to Hep3B cells. This suggests that the combined use of HSV-TK, GCV, rAdV-p53 and rAdV-ASPP2 may improve therapeutic efficacy in HCC patients lacking functional p53. PMID:26934443

  2. The therapeutic potential of p53 reactivation by nutlin-3a in ALK+ anaplastic large cell lymphoma with wild-type or mutated p53.

    PubMed

    Drakos, E; Atsaves, V; Schlette, E; Li, J; Papanastasi, I; Rassidakis, G Z; Medeiros, L J

    2009-12-01

    p53 is expressed frequently, but is rarely mutated in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) tumours. Nutlin-3a is a recently developed small molecule that targets Mdm2, a critical negative regulator of p53, and disrupts the p53-Mdm2 interaction resulting in p53 stabilization and activation. We show that nutlin-3a activates p53 in ALK+ ALCL cells carrying a wild type (wt) or mutated but partially functional p53 gene resulting in p53-dependent cell-cycle arrest and apoptosis. Cell-cycle arrest was associated with upregulation of the cyclin-dependent kinase inhibitor p21. Nutlin-3a-induced apoptotic cell death was accompanied by Bax and Puma upregulation, downregulation of Bcl-xl, survivin, and caspase-3 cleavage, and this was reduced when p53-dependent transactivation activity was inhibited by pifithrin-alpha, or when pifithrin-mu was used to inhibit direct p53 targeting of mitochondria. Nutlin-3a sensitized the activation of the extrinsic apoptotic pathway in wt-p53 ALK+ ALCL cells, in part, through upregulation of DR-5 and downregulation of c-Flip(S/L), and was synergistic with TRAIL in cell death induction. In addition, nutlin-3a treatment enhanced doxorubicin cytotoxicity against ALK+ ALCL cells harbouring mt p53, and this was associated with p73 upregulation. These data suggest that disruption of the p53-mdm2 interaction by nutlin-3a offers a novel therapeutic approach for ALK+ ALCL patients. PMID:19741726

  3. ZBTB2, a Novel Master Regulator of the p53 Pathway*

    PubMed Central

    Jeon, Bu-Nam; Choi, Won-Il; Yu, Mi-Young; Yoon, A-Rum; Kim, Myung-Hwa; Yun, Chae-Ok; Hur, Man-Wook

    2009-01-01

    We found that ZBTB2, a POK family transcription factor, is a potent repressor of the ARF-HDM2-p53-p21 pathway important in cell cycle regulation. ZBTB2 repressed transcription of the ARF, p53, and p21 genes, but activated the HDM2 gene. In particular, ZBTB2 repressed transcription of the p21 gene by acting on the two distal p53 binding elements and the proximal Sp1 binding GC-box 5/6 elements. ZBTB2 directly interacted with Sp1 via its POZ domain and zinc fingers, which was important in the repression of transcription activation by Sp1. ZBTB2 and Sp1 competed with each other in binding to the GC-box 5/6 elements and the two p53 binding elements. ZBTB2 directly interacted with p53 via its zinc fingers, inhibiting p53 binding and repressing transcription activation by p53. The POZ domain, required for transcription repression, interacted with corepressors such as BCoR, NCoR, and SMRT. The interactions deacetylated histones Ac-H3 and -H4 at the proximal promoter. Although ectopic ZBTB2 stimulated cell proliferation, knock-down of ZBTB2 expression decreased cell proliferation and DNA synthesis. Overall, our data suggest that ZBTB2 is a potential proto-oncogenic master control gene of the p53 pathway and, in particular, is a potent transcription repressor of the cell cycle arrest gene p21 by inhibiting p53 and Sp1. PMID:19380588

  4. Sodium orthovanadate inhibits p53-mediated apoptosis.

    PubMed

    Morita, Akinori; Yamamoto, Shinichi; Wang, Bing; Tanaka, Kaoru; Suzuki, Norio; Aoki, Shin; Ito, Azusa; Nanao, Tomohisa; Ohya, Soichiro; Yoshino, Minako; Zhu, Jin; Enomoto, Atsushi; Matsumoto, Yoshihisa; Funatsu, Osamu; Hosoi, Yoshio; Ikekita, Masahiko

    2010-01-01

    Sodium orthovanadate (vanadate) inhibits the DNA-binding activity of p53, but its precise effects on p53 function have not been examined. Here, we show that vanadate exerts a potent antiapoptotic activity through both transcription-dependent and transcription-independent mechanisms relative to other p53 inhibitors, including pifithrin (PFT) alpha. We compared the effects of vanadate to PFTalpha and PFTmicro, an inhibitor of transcription-independent apoptosis by p53. Vanadate suppressed p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. Similarly, vanadate suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of SaOS-2 cells. In radioprotection assays, which rely on p53, vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Together, our findings indicated that vanadate effectively suppresses p53-mediated apoptosis by both transcription-dependent and transcription-independent pathways, and suggested that both pathways must be inhibited to completely block p53-mediated apoptosis. PMID:20048077

  5. Resveratrol contributes to chemosensitivity of malignant mesothelioma cells with activation of p53.

    PubMed

    Lee, Yoon-Jin; Park, Ihl-Sung; Lee, Yong-Jin; Shim, Jung-Hyun; Cho, Moon-Kyun; Nam, Hae-Seon; Park, Ji Woong; Oh, Myung-Ho; Lee, Sang-Han

    2014-01-01

    Resveratrol is a naturally occurring polyphenolic phytoalexin with chemopreventive properties. We previously reported a synergistic anti-proliferative effect of resveratrol and clofarabine against malignant mesothelioma (MM) cells. Here, we further investigated molecular mechanisms involved in the synergistic interaction of these compounds in MM MSTO-211H cells. Resveratrol, in combination with clofarabine, time-dependently induced a strong cytotoxic effect with the nuclear accumulation of phospho-p53 (p-p53) in MSTO-211H cells, but not in normal mesothelial MeT-5A cells. Combination treatment up-regulated the levels of p-p53, cleaved caspase-3, and cleaved PARP proteins. Gene silencing with p53-targeting siRNA attenuated the sensitivity of cells to the combined treatment of two compounds. Analyses of p53 DNA binding assay, p53 reporter gene assay, and RTP-CR toward p53-regulated genes, including Bax, PUMA, Noxa and p21, demonstrated that induced p-p53 is transcriptionally active. These results were further confirmed by the siRNA-mediated knockdown of p53 gene. Combination treatment significantly caused the accumulation of cells at G1 phase with the increases in the sub-G0/G1 peak, DNA ladder, nuclear fragmentation, and caspase-3/7 activity. Taken together, these results demonstrate that resveratrol and clofarabine synergistically elicit apoptotic signal via a p53-dependent pathway, and provide a scientific rationale for clinical evaluation of resveratrol as a promising chemopotentiator in MM. PMID:24239893

  6. 2-Phenylethynesulfonamide (PES) uncovers a necrotic process regulated by oxidative stress and p53.

    PubMed

    Mattiolo, Paolo; Barbero-Farran, Ares; Yuste, Víctor J; Boix, Jacint; Ribas, Judit

    2014-10-01

    2-Phenylethynesulfonamide (PES) or pifithrin-μ is a promising anticancer agent with preferential toxicity for cancer cells. The type of cell death and the molecular cascades activated by this compound are controversial. Here, we demonstrate PES elicits a caspase- and BAX/BAK-independent non-necroptotic necrotic cell death, since it is not inhibited by necrostatin-1. This process is characterized by an early generation of reactive oxygen species (ROS) resulting in p53 up-regulation. Accordingly, thiolic antioxidants protect cells from PES-induced death. Furthermore, inhibiting the natural sources of glutathione with l-buthionine-sulfoximine (BSO) strongly cooperates with PES in triggering cytotoxicity. Genetically modified p53-null or p53 knocked-down cells show resistance to PES-driven necrosis. The predominant localization of p53 in chromatin-enriched fractions added to the up-regulation of the p53-responsive gene p21, strongly suggest the involvement of a transcription-dependent p53 program. On the other hand, we report an augmented production of ROS in p53-positive cells that, added to the increased p53 content in response to PES-elicited ROS, suggests that p53 and ROS are mutually regulated in response to PES. In sum, p53 up-regulation by ROS triggers a positive feedback loop responsible of further increasing ROS production and reinforcing PES-driven non-necroptotic necrosis. PMID:25139326

  7. 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. PMID:26596838

  8. Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation.

    PubMed

    Wang, Xinwei; Wei, Liang; Cramer, Julie M; Leibowitz, Brian J; Judge, Colleen; Epperly, Michael; Greenberger, Joel; Wang, Fengchao; Li, Linheng; Stelzner, Matthias G; Dunn, James C Y; Martin, Martin G; Lagasse, Eric; Zhang, Lin; Yu, Jian

    2015-01-01

    Exposure to high levels of ionizing radiation (IR) leads to debilitating and dose-limiting gastrointestinal (GI) toxicity. Using three-dimensional mouse crypt culture, we demonstrated that p53 target PUMA mediates radiation-induced apoptosis via a cell-intrinsic mechanism, and identified the GSK-3 inhibitor CHIR99021 as a potent radioprotector. CHIR99021 treatment improved Lgr5+ cell survival and crypt regeneration after radiation in culture and mice. CHIR99021 treatment specifically blocked apoptosis and PUMA induction and K120 acetylation of p53 mediated by acetyl-transferase Tip60, while it had no effect on p53 stabilization, phosphorylation or p21 induction. CHIR99021 also protected human intestinal cultures from radiation by PUMA but not p21 suppression. These results demonstrate that p53 posttranslational modifications play a key role in the pathological and apoptotic response of the intestinal stem cells to radiation and can be targeted pharmacologically. PMID:25858503

  9. Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation

    PubMed Central

    Wang, Xinwei; Wei, Liang; Cramer, Julie M.; Leibowitz, Brian J.; Judge, Colleen; Epperly, Michael; Greenberger, Joel; Wang, Fengchao; Li, Linheng; Stelzner, Matthias G.; Dunn, James C. Y.; Martin, Martin G.; Lagasse, Eric; Zhang, Lin; Yu, Jian

    2015-01-01

    Exposure to high levels of ionizing radiation (IR) leads to debilitating and dose-limiting gastrointestinal (GI) toxicity. Using three-dimensional mouse crypt culture, we demonstrated that p53 target PUMA mediates radiation-induced apoptosis via a cell-intrinsic mechanism, and identified the GSK-3 inhibitor CHIR99021 as a potent radioprotector. CHIR99021 treatment improved Lgr5+ cell survival and crypt regeneration after radiation in culture and mice. CHIR99021 treatment specifically blocked apoptosis and PUMA induction and K120 acetylation of p53 mediated by acetyl-transferase Tip60, while it had no effect on p53 stabilization, phosphorylation or p21 induction. CHIR99021 also protected human intestinal cultures from radiation by PUMA but not p21 suppression. These results demonstrate that p53 posttranslational modifications play a key role in the pathological and apoptotic response of the intestinal stem cells to radiation and can be targeted pharmacologically. PMID:25858503

  10. DNA-mediated oxidation of p53.

    PubMed

    Schaefer, Kathryn N; Barton, Jacqueline K

    2014-06-01

    Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs over long distances through the π-stacked bases and leads to the oxidative dissociation of p53. The extent of protein dissociation depends upon the redox potential of the DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using oligonucleotides containing both synthetic and human p53 consensus sequences with an appended photooxidant, anthraquinone. Greater p53 dissociation is observed from sequences containing low-redox potential purine regions, particularly guanine triplets. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites corresponding to sites of preferred electron hole localization were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets. Oxidative DNA damage is inhibited in the presence of p53, but only at sites in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress as well as possible biological implications have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell. PMID:24853816

  11. Atypical role of sprouty in p21 dependent inhibition of cell proliferation in colorectal cancer.

    PubMed

    Zhang, Qiong; Shim, Katherine; Wright, Kevin; Jurkevich, Alexander; Khare, Sharad

    2016-09-01

    Sprouty (SPRY) appears to act as a tumor suppressor in cancer, whereas we reported that SPRY2 functions as a putative oncogene in colorectal cancer (CRC) [Oncogene, 2010, 29: 5241-5253]. In general, various studies established inhibition of cell proliferation by SPRY in cancer. The mechanisms by which SPRY regulates cell proliferation in CRC are investigated. We demonstrate, for the first time, suppression of SPRY2 augmented EGF-dependent oncogenic signaling, however, surprisingly decreased cell proliferation in colon cancer cells. Our data suggest that cell cycle inhibitor p21(WAF1/CIP1) transcriptional activity being regulated by SPRY2. Indeed, suppression of SPRY2 significantly increased p21(WAF1/CIP1) mRNA and protein expression as well as p21(WAF1/CIP1) promoter activity. Conversely, overexpressing SPRY2 triggered a decrease in p21(WAF1/CIP1) promoter activity. Concurrent down-regulation of both SPRY1 and SPRY2 also increased p21(WAF1/CIP1) expression in colon cancer cells. Increased nuclear localization of p21(WAF1/CIP1) in SPRY2 downregulated colon cancer cells may explain the inhibition of cell proliferation in colon cancer cells. Underscoring the biological relevance of these findings in SPRY1 and SPRY2 mutant mouse, recombination of floxed SPRY1 and SPRY2 alleles in mouse embryonic fibroblasts (MEFs) resulted in increased expression and nuclear localization of p21(WAF1/CIP1) and decreased cell proliferation. In CRC, the relationship of SPRY with p21 may provide unique strategies for cancer prevention and treatment. © 2015 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc. PMID:26293890

  12. p53 mutation heterogeneity in cancer

    SciTech Connect

    Soussi, T. . E-mail: thierry.soussi@free.fr; Lozano, G.

    2005-06-10

    The p53 gene is inactivated in about 50% of human cancers and the p53 protein is an essential component of the cell response induced by genotoxic stresses such as those generated by radiotherapy or chemotherapy. It is therefore highly likely that these alterations are an important component in tumor resistance to therapy. The particular characteristics of these alterations, 80% of which are missense mutations leading to functionally heterogeneous proteins, make p53 a unique gene in the class of tumor suppressor genes. A considerable number of mutant p53 proteins probably have an oncogenic activity per se and therefore actively participate in cell transformation. The fact that the apoptotic and antiproliferative functions of p53 can be dissociated in certain mutants also suggests another level of complexity in the relationships between p53 inactivation and neoplasia.

  13. Mutant p53: one name, many proteins

    PubMed Central

    Freed-Pastor, William A.; Prives, Carol

    2012-01-01

    There is now strong evidence that mutation not only abrogates p53 tumor-suppressive functions, but in some instances can also endow mutant proteins with novel activities. Such neomorphic p53 proteins are capable of dramatically altering tumor cell behavior, primarily through their interactions with other cellular proteins and regulation of cancer cell transcriptional programs. Different missense mutations in p53 may confer unique activities and thereby offer insight into the mutagenic events that drive tumor progression. Here we review mechanisms by which mutant p53 exerts its cellular effects, with a particular focus on the burgeoning mutant p53 transcriptome, and discuss the biological and clinical consequences of mutant p53 gain of function. PMID:22713868

  14. 4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells

    PubMed Central

    Sharma, Abha; Sharma, Rajendra; Chaudhary, Pankaj; Vatsyayan, Rit; Pearce, Virginia; Jeyabal, Prince V.S.; Zimniak, Piotr; Awasthi, Sanjay; Awasthi, Yogesh C.

    2009-01-01

    4-Hydroxynonenal (4-HNE) has been suggested to be involved in stress-induced signaling for apoptosis. In present studies, we have examined the effects of 4-HNE on the intrinsic apoptotic pathway associated with p53 in human retinal pigment epithelial (RPE and ARPE-19) cells. Our results show that 4-HNE causes induction, phosphorylation, and nuclear accumulation of p53 which is accompanied with down regulation of MDM2, activation of the pro-apoptotic p53 target genes viz. p21 and Bax, JNK, caspase3, and onset of apoptosis in treated RPE cells. Reduced expression of p53 by an efficient silencing of the p53 gene resulted in a significant resistance of these cells to 4-HNE-induced cell death. The effects of 4-HNE on the expression and functions of p53 are blocked in GSTA4-4 over expressing cells indicating that 4-HNE-induced, p53-mediated signaling for apoptosis is regulated by GSTs. Our results also show that the induction of p53 in tissues of mGsta4 (-/-) mice correlate with elevated levels of 4-HNE due to its impaired metabolism. Together, these studies suggest that 4-HNE is involved in p53-mediated signaling in in vitro cell cultures as well as in vivo that can be regulated by GSTs. PMID:18930016

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

    NASA Technical Reports Server (NTRS)

    Kohli, M.; Jorgensen, T. J.

    1999-01-01

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

  16. The p53-dependent radioadaptive response

    NASA Astrophysics Data System (ADS)

    Ohnishi, Takeo

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

  17. Mitochondrial death functions of p53

    PubMed Central

    Marchenko, N D; Moll, U M

    2014-01-01

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

  18. Lysosomal destabilization in p53-induced apoptosis

    PubMed Central

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

    2002-01-01

    The tumor suppressor wild-type p53 can induce apoptosis. M1-t-p53 myeloid leukemic cells have a temperature-sensitive p53 protein that changes its conformation to wild-type p53 after transfer from 37°C to 32°C. We have now found that these cells showed an early lysosomal rupture after transfer to 32°C. Mitochondrial damage, including decreased membrane potential and release of cytochrome c, and the appearance of apoptotic cells occurred later. Lysosomal rupture, mitochondrial damage, and apoptosis were all inhibited by the cytokine IL-6. Some other compounds can also inhibit apoptosis induced by p53. The protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone inhibited the decrease in mitochondrial membrane potential and cytochrome c release, the Ca2+-ATPase inhibitor thapsigargin inhibited only cytochrome c release, and the antioxidant butylated hydroxyanisole inhibited only the decrease in mitochondrial membrane potential. In contrast to IL-6, these other compounds that inhibited some of the later occurring mitochondrial damage did not inhibit the earlier p53-induced lysosomal damage. The results indicate that apoptosis is induced by p53 through a lysosomal-mitochondrial pathway that is initiated by lysosomal destabilization, and that this pathway can be dissected by using different apoptosis inhibitors. These findings on the induction of p53-induced lysosomal destabilization can also help to formulate new therapies for diseases with apoptotic disorders. PMID:11959917

  19. Regulation of P53 stability in p53 mutated human and mouse hepatoma cells.

    PubMed

    Hailfinger, Stephan; Jaworski, Maike; Marx-Stoelting, Philip; Wanke, Ines; Schwarz, Michael

    2007-04-01

    The tumor suppressor p53 is frequently mutated in cancer. We have investigated the regulation of P53 in p53 wild type mouse hepatoma cells (line 55.1c), in p53 heterozygeously mutated cells (56.1b) and in p53 defective cells (lines 56.1d, 70.4 and HUH7) under various experimental settings. The basal levels of P53 were low in 55.1c cells, but nuclear accumulation occurred upon UV-irradiation. Similarly, UV-exposure induced stabilization of P53 in the heterozygeously p53 mutated 56.1b hepatoma cells. By contrast, the 3 hepatoma lines, which lack transcriptionally active P53, demonstrated high basal nuclear concentrations of P53 protein and, unexpectedly, showed loss of P53 upon UV-irradiation. Expression of p53 mRNA was also decreased in p53 defective cells after 24 hr post UV-irradiation, which may be linked to induction of apoptosis of the irradiated cells under these conditions. Other stressors like H2O2 also mediated a decrease in P53 concentration in p53 defective cells. This effect occurred at very low concentrations and was already detectable 1-2 hr after exposure of cells. There were no signs of apoptosis of H2O2-exposed cells at this time point and no significant changes in p53 mRNA or MDM2 level. These unexpected findings indicate a new aspect related to regulation of P53 stability in cells with a defect in the tumor suppressor protein. PMID:17205518

  20. Microbial Regulation of p53 Tumor Suppressor.

    PubMed

    Zaika, Alexander I; Wei, Jinxiong; Noto, Jennifer M; Peek, Richard M

    2015-09-01

    p53 tumor suppressor has been identified as a protein interacting with the large T antigen produced by simian vacuolating virus 40 (SV40). Subsequent research on p53 inhibition by SV40 and other tumor viruses has not only helped to gain a better understanding of viral biology, but also shaped our knowledge of human tumorigenesis. Recent studies have found, however, that inhibition of p53 is not strictly in the realm of viruses. Some bacterial pathogens also actively inhibit p53 protein and induce its degradation, resulting in alteration of cellular stress responses. This phenomenon was initially characterized in gastric epithelial cells infected with Helicobacter pylori, a bacterial pathogen that commonly infects the human stomach and is strongly linked to gastric cancer. Besides H. pylori, a number of other bacterial species were recently discovered to inhibit p53. These findings provide novel insights into host-bacteria interactions and tumorigenesis associated with bacterial infections. PMID:26379246

  1. Microbial Regulation of p53 Tumor Suppressor

    PubMed Central

    Zaika, Alexander I.; Wei, Jinxiong; Noto, Jennifer M.; Peek, Richard M.

    2015-01-01

    p53 tumor suppressor has been identified as a protein interacting with the large T antigen produced by simian vacuolating virus 40 (SV40). Subsequent research on p53 inhibition by SV40 and other tumor viruses has not only helped to gain a better understanding of viral biology, but also shaped our knowledge of human tumorigenesis. Recent studies have found, however, that inhibition of p53 is not strictly in the realm of viruses. Some bacterial pathogens also actively inhibit p53 protein and induce its degradation, resulting in alteration of cellular stress responses. This phenomenon was initially characterized in gastric epithelial cells infected with Helicobacter pylori, a bacterial pathogen that commonly infects the human stomach and is strongly linked to gastric cancer. Besides H. pylori, a number of other bacterial species were recently discovered to inhibit p53. These findings provide novel insights into host–bacteria interactions and tumorigenesis associated with bacterial infections. PMID:26379246

  2. 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

  3. The heme-p53 interaction: Linking iron metabolism to p53 signaling and tumorigenesis.

    PubMed

    Shen, Jia; Sheng, Xiangpeng; Chang, ZeNan; Wu, Qian; Xie, Dong; Wang, Fudi; Hu, Ronggui

    2016-01-01

    Recently, we reported that heme binds to tumor suppressor p53 protein (TP53, best known as p53) and promotes its nuclear export and cytosolic degradation, whereas iron chelation stabilizes p53 protein and suppresses tumors in a p53-dependent manner. This not only provides mechanistic insights into tumorigenesis associated with iron excess, but also helps guide the administration of chemotherapy based on iron deprivation in the clinic. PMID:27308524

  4. The heme–p53 interaction: Linking iron metabolism to p53 signaling and tumorigenesis

    PubMed Central

    Shen, Jia; Sheng, Xiangpeng; Chang, ZeNan; Wu, Qian; Xie, Dong; Wang, Fudi; Hu, Ronggui

    2016-01-01

    Recently, we reported that heme binds to tumor suppressor p53 protein (TP53, best known as p53) and promotes its nuclear export and cytosolic degradation, whereas iron chelation stabilizes p53 protein and suppresses tumors in a p53-dependent manner. This not only provides mechanistic insights into tumorigenesis associated with iron excess, but also helps guide the administration of chemotherapy based on iron deprivation in the clinic. PMID:27308524

  5. Tumor Protein 53-Induced Nuclear Protein 1 Enhances p53 Function and Represses Tumorigenesis.

    PubMed

    Shahbazi, Jeyran; Lock, Richard; Liu, Tao

    2013-01-01

    Tumor protein 53-induced nuclear protein 1 (TP53INP1) is a stress-induced p53-target gene whose expression is modulated by transcription factors such as p53, p73, and E2F1. TP53INP1 gene encodes two isoforms of TP53INP1 proteins, TP53INP1α and TP53INP1β, both of which appear to be key elements in p53 function. In association with homeodomain-interacting protein kinase-2 (HIPK2), TP53INP1 phosphorylates p53 protein at Serine-46. This enhances p53 protein stability and its transcriptional activity, leading to transcriptional activation of p53-target genes such as p21 and PIG3, cell growth arrest and apoptosis upon DNA damage stress. The anti-proliferative and pro-apoptotic activities of TP53INP1 indicate that TP53INP1 has an important role in cellular homeostasis and DNA damage response. Deficiency in TP53INP1 expression results in increased tumorigenesis, whereas TP53INP1 expression is repressed during early stages of cancer by factors such as miR-155. This review aims to summarize the roles of TP53INP1 in blocking tumor progression through p53-dependant and p53-independent pathways, as well as the elements which repress TP53INP1 expression, hence highlighting its potential as a therapeutic target in cancer treatment. PMID:23717325

  6. 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

  7. Expression of p53β and Δ133p53 isoforms in different gastric tissues

    PubMed Central

    Ji, Wansheng; Zhang, Na; Zhang, Hongmei; Ma, Jingrong; Zhong, Hua; Jiao, Jianxin; Gao, Zhixing

    2015-01-01

    This study aims to detect the mRNA of p53β and Δ133p53 isoforms in three gastric carcinoma cell lines and tissues of superficial gastritis, atrophic gastritis, gastric carcinoma, or paracancerous area. Nested reverse transcription PCR was used to detect the mRNA of p53β and Δ133p53 isoforms in tissues of superficial gastritis, chronic atrophic gastritis, gastric cancer cell lines (SGC-7901, MKN45, KATO III), gastric adenocarcinoma, and paracancerous lesion. The amplified products were shown by agarose gel electrophoresis. The expression difference among various tissues was analyzed by x2 tests. The positive rates of ∆133p53 mRNA were 73.3% (11/15) in gastric adenocarcinoma and 20% (3/15) in paracancerous tissue, whereas the positive rates of p53β mRNA were 20% (3/15) in gastric adenocarcinoma and 66.7% (10/15) in paracancerous tissue. The difference between adenocarcinoma and paracancerous tissues was significant (P<0.05). The positive rates of ∆133p53 mRNA were 25% (5/20), 50% (15/30), and 75% (15/20), respectively, in superficial gastritis, atrophic gastritis, and gastric adenocarcinoma; the positive rates of p53β mRNA were 65% (13/20), 33.3% (10/30), and 25% (5/20), respectively, in superficial gastritis, atrophic gastritis, and gastric adenocarcinoma. The difference between adenocarcinoma and superficial gastritis samples was significant (P<0.05). Both p53β and ∆133p53 mRNAs were positive in MKN45; only p53β mRNA was detected in SGC7901; neither p53β nor ∆133p53 mRNA was detected in KATO III. ∆133p53 and p53β, which are possible indicators for the diagnosis and biological therapy of gastric carcinoma, were expressed differentially in different gastric tissues. PMID:26617756

  8. p53 and Mitochondrial Function in Neurons

    PubMed Central

    Wang, David B.; Kinoshita, Chizuru; Kinoshita, Yoshito; Morrison, Richard S.

    2014-01-01

    The p53 tumor suppressor plays a central role in dictating cell survival and death as a cellular sensor for a myriad of stresses including DNA damage, oxidative and nutritional stress, ischemia and disruption of nucleolar function. Activation of p53-dependent apoptosis leads to mitochondrial apoptotic changes via the intrinsic and extrinsic pathways triggering cell death execution most notably by release of cytochrome c and activation of the caspase cascade. Although it was previously believed that p53 induces apoptotic mitochondrial changes exclusively through transcription-dependent mechanisms, recent studies suggest that p53 also regulates apoptosis via a transcription-independent action at the mitochondria. Recent evidence further suggests that p53 can regulate necrotic cell death and autophagic activity including mitophagy. An increasing number of cytosolic and mitochondrial proteins involved in mitochondrial metabolism and respiration are regulated by p53, which influences mitochondrial ROS production as well. Cellular redox homeostasis is also directly regulated by p53 through modified expression of pro- and anti-oxidant proteins. Proper regulation of mitochondrial size and shape through fission and fusion assures optimal mitochondrial bioenergetic function while enabling adequate mitochondrial transport to accommodate local energy demands unique to neuronal architecture. Abnormal regulation of mitochondrial dynamics has been increasingly implicated in neurodegeneration, where elevated levels of p53 may have a direct contribution as the expression of some fission/fusion proteins are directly regulated by p53. Thus, p53 may have a much wider influence on mitochondrial integrity and function than one would expect from its well-established ability to transcriptionally induce mitochondrial apoptosis. However, much of the evidence demonstrating that p53 can influence mitochondria through nuclear, cytosolic or intra-mitochondrial sites of action has yet to be

  9. 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.

  10. Inactivation of p53 Is Sufficient to Induce Development of Pulmonary Hypertension in Rats

    PubMed Central

    Jacquin, S.; Rincheval, V.; Mignotte, B.; Richard, S.; Humbert, M.; Mercier, O.; Londoño-Vallejo, A.; Fadel, E.; Eddahibi, S.

    2015-01-01

    Objective Pulmonary artery smooth muscle cells (PA-SMCs) in pulmonary arterial hypertension (PAH) show similarities to cancer cells. Due to the growth-suppressive and pro-apoptotic effects of p53 and its inactivation in cancer, we hypothesized that the p53 pathway could be altered in PAH. We therefore explored the involvement of p53 in the monocrotaline (MCT) rat model of pulmonary hypertension (PH) and the pathophysiological consequences of p53 inactivation in response to animal treatment with pifithrin-α (PFT, an inhibitor of p53 activity). Methods and Results PH development was assessed by pulmonary arterial pressure, right ventricular hypertrophy and arterial wall thickness. The effect of MCT and PFT on lung p53 pathway expression was evaluated by western blot. Fourteen days of daily PFT treatment (2.2 mg/kg/day), similar to a single injection of MCT (60 mg/kg), induced PH and aggravated MCT-induced PH. In the first week after MCT administration and prior to PH development, p53, p21 and MDM2 protein levels were significantly reduced; whereas PFT administration effectively altered the protein level of p53 targets. Anti-apoptotic and pro-proliferative effects of PFT were revealed by TUNEL and MTT assays on cultured human PA-SMCs treated with 50 μM PFT. Conclusions Pharmacological inactivation of p53 is sufficient to induce PH with a chronic treatment by PFT, an effect related to its anti-apoptotic and pro-proliferative properties. The p53 pathway was down-regulated during the first week in the rat MCT model. These in vivo experiments implicate the p53 pathway at the initiation stages of PH pathogenesis. PMID:26121334

  11. Simian virus 40 T antigen can regulate p53-mediated transcription independent of binding p53.

    PubMed Central

    Rushton, J J; Jiang, D; Srinivasan, A; Pipas, J M; Robbins, P D

    1997-01-01

    A simian virus 40 (SV40) T-antigen mutant containing only the N-terminal 136 amino acids, able to bind to Rb and p300 but not p53, partially inhibited p53-mediated transcription without affecting the ability of p53 to bind DNA. These results suggest that SV40 T antigen can regulate p53-mediated transcription either directly through protein-protein association or indirectly through interaction with factors which may function to confer p53-mediated transcription. PMID:9188637

  12. The novel tryptamine derivative JNJ-26854165 induces wild-type p53- and E2F1-mediated apoptosis in acute myeloid and lymphoid leukemias

    PubMed Central

    Kojima, Kensuke; Burks, Jared K.; Arts, Janine; Andreeff, Michael

    2010-01-01

    Development of small-molecule activators of p53 is currently focused on malignancies containing a wild-type p53 genotype, which is present in most leukemias. JNJ-26854165 is one of such p53-activating agents, but its mechanism of action remains to be elucidated. Here, we report the effects of JNJ-26854165 in acute leukemias. JNJ-26854165 treatment induced p53-mediated apoptosis in acute leukemia cells with wild-type p53, in which p53 rapidly drives transcription-independent apoptosis followed by activation of a transcription-dependent pathway. JNJ-26854165 accelerated the proteasome-mediated degradation of p21, and antagonized the transcriptional induction of p21 by p53. Interestingly, JNJ-26854165 induced S-phase delay and upregulated E2F1 expression in p53 mutant cells, resulting in apoptosis preferentially of S-phase cells. E2F1 knockdown blocked apoptosis induced by JNJ-26854165 in p53 mutant cells. Apoptotic activity of JNJ-26854165 against primary acute leukemia cells was maintained in leukemia/stroma cocultures, unlike doxorubicin, which has reduced cytrotoxicity in coculture systems. JNJ-26854165 synergizes with AraC or doxorubicin to induce p53-mediated apoptosis. Our data suggest that JNJ-26854165 may provide a novel therapeutic approach for the treatment of acute leukemias. The presence of p53-independent apoptotic activity in addition to p53-mediated apoptosis induction, if operational in vivo, may prevent the selection of p53 mutant subclones during therapy. PMID:20736344

  13. Indoleamine 2,3-dioxygenase increases p53 levels in alloreactive human T cells, and both indoleamine 2,3-dioxygenase and p53 suppress glucose uptake, glycolysis and proliferation.

    PubMed

    Eleftheriadis, Theodoros; Pissas, Georgios; Antoniadi, Georgia; Spanoulis, Aginor; Liakopoulos, Vassilios; Stefanidis, Ioannis

    2014-12-01

    Indoleamine 2,3-dioxygenase (IDO) suppresses adaptive immunity by inhibiting T-cell proliferation and altering glucose metabolism. The tumor suppressor p53 also alters these cellular processes with similar results. The effect of IDO on p53 and on glucose metabolism was evaluated in alloreactive T cells. Mixed-lymphocyte reactions (MLRs) were performed in the presence or not of the IDO inhibitor, 1-dl-methyl-tryptophan (1-MT) and/or the p53 inhibitor, pifithrin-α (PFT). Cell proliferation, glucose consumption and lactate production were assessed. 1-MT increased cell proliferation, glucose influx and lactate production, whereas PFT enhanced cell proliferation and glucose influx, leaving lactate production unaffected. In MLR-derived T cells, protein analysis revealed that IDO activated general control non-derepressible 2 kinase and induced p53, p-p53 (p53 phosphorylated at serine 15) and p21. In addition, both IDO and p53 decreased glucose transporter 1 and TP53-induced glycolysis and apoptosis regulator and increased synthesis of cytochrome c oxidase 2. IDO also reduced lactate dehydrogenase-A and glutaminase 2 levels, whereas p53 left them unaffected. Neither 1-MT nor PFT affected glucose-6-phosphate dehydrogenase. In conclusion, in alloreactive T cells, IDO increases p53 levels, and both IDO and p53 inhibit cell proliferation, glucose consumption and glycolysis. Lactate production and glutaminolysis are also suppressed by IDO, but not by p53. PMID:25064493

  14. NuMA Is Required for the Selective Induction of p53 Target Genes

    PubMed Central

    Ohata, Hirokazu; Miyazaki, Makoto; Otomo, Ryo; Matsushima-Hibiya, Yuko; Otsubo, Chihiro; Nagase, Takahiro; Arakawa, Hirofumi; Yokota, Jun; Nakagama, Hitoshi

    2013-01-01

    The p53 tumor suppressor protein is a transcription factor controlling various outcomes, such as growth arrest and apoptosis, through the regulation of different sets of target genes. The nuclear mitotic apparatus protein (NuMA) plays important roles in spindle pole organization during mitosis and in chromatin regulation in the nucleus during interphase. Although NuMA has been shown to colocalize with several nuclear proteins, including high-mobility-group proteins I and Y and GAS41, the role of NuMA during interphase remains unclear. Here we report that NuMA binds to p53 to modulate p53-mediated transcription. Acute and partial ablation of NuMA attenuates the induction of the proarrested p21 gene following DNA damage, subsequently causing impaired cell cycle arrest. Interestingly, NuMA knockdown had little effect on the induction of the p53-dependent proapoptotic PUMA gene. Furthermore, NuMA is required for the recruitment of cyclin-dependent kinase 8 (Cdk8), a component of the Mediator complex and a promoter of p53-mediated p21 gene function. These data demonstrate that NuMA is critical for the target selectivity of p53-mediated transcription. PMID:23589328

  15. Protective role of p53 in skin cancer: Carcinogenesis studies in mice lacking epidermal p53.

    PubMed

    Page, Angustias; Navarro, Manuel; Suarez-Cabrera, Cristian; Alameda, Josefa P; Casanova, M Llanos; Paramio, Jesús M; Bravo, Ana; Ramirez, Angel

    2016-04-12

    p53 is a protein that causes cell cycle arrest, apoptosis or senescence, being crucial in the process of tumor suppression in several cell types. Different in vitro and animal models have been designed for the study of p53 role in skin cancer. These models have revealed opposing results, as in some experimental settings it appears that p53 protects against skin cancer, but in others, the opposite conclusion emerges. We have generated cohorts of mice with efficient p53 deletion restricted to stratified epithelia and control littermates expressing wild type p53 and studied their sensitivity to both chemically-induced and spontaneous tumoral transformation, as well as the tumor types originated in each experimental group. Our results indicate that the absence of p53 in stratified epithelia leads to the appearance, in two-stage skin carcinogenesis experiments, of a higher number of tumors that grow faster and become malignant more frequently than tumors arisen in mice with wild type p53 genotype. In addition, the histological diversity of the tumor type is greater in mice with epidermal p53 loss, indicating the tumor suppressive role of p53 in different epidermal cell types. Aging mice with p53 inactivation in stratified epithelia developed spontaneous carcinomas in skin and other epithelia. Overall, these results highlight the truly protective nature of p53 functions in the development of cancer in skin and in other stratified epithelia. PMID:26959115

  16. Protective role of p53 in skin cancer: Carcinogenesis studies in mice lacking epidermal p53

    PubMed Central

    Page, Angustias; Navarro, Manuel; Suarez-Cabrera, Cristian; Alameda, Josefa P.; Casanova, M. Llanos; Paramio, Jesús M.; Bravo, Ana; Ramirez, Angel

    2016-01-01

    p53 is a protein that causes cell cycle arrest, apoptosis or senescence, being crucial in the process of tumor suppression in several cell types. Different in vitro and animal models have been designed for the study of p53 role in skin cancer. These models have revealed opposing results, as in some experimental settings it appears that p53 protects against skin cancer, but in others, the opposite conclusion emerges. We have generated cohorts of mice with efficient p53 deletion restricted to stratified epithelia and control littermates expressing wild type p53 and studied their sensitivity to both chemically-induced and spontaneous tumoral transformation, as well as the tumor types originated in each experimental group. Our results indicate that the absence of p53 in stratified epithelia leads to the appearance, in two-stage skin carcinogenesis experiments, of a higher number of tumors that grow faster and become malignant more frequently than tumors arisen in mice with wild type p53 genotype. In addition, the histological diversity of the tumor type is greater in mice with epidermal p53 loss, indicating the tumor suppressive role of p53 in different epidermal cell types. Aging mice with p53 inactivation in stratified epithelia developed spontaneous carcinomas in skin and other epithelia. Overall, these results highlight the truly protective nature of p53 functions in the development of cancer in skin and in other stratified epithelia. PMID:26959115

  17. AMPKα1 deficiency promotes cellular proliferation and DNA damage via p21 reduction in mouse embryonic fibroblasts

    PubMed Central

    Xu, Hairong; Zhou, Yanhong; Coughlan, Kathleen A.; Ding, Ye; Wang, Shaobin; Wu, Yue; Song, Ping; Zou, Ming-Hui

    2014-01-01

    Emerging evidence suggests that activation of adenosine monophosphate-activated protein kinase (AMPK), an energy gauge and redox sensor, controls the cell cycle and protects against DNA damage. However, the molecular mechanisms by which AMPKα isoform regulates DNA damage remain largely unknown. The aim of this study was to determine if AMPKα deletion contributes to cellular hyperproliferation by reducing p21WAF1/Cip1 (p21) expression thereby leading to accumulated DNA damage. The markers for DNA damage, cell cycle proteins, and apoptosis were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J), AMPKα1, or AMPKα2 homozygous deficient (AMPKα1−/−, AMPKα2−/−) mice by Western blot, flow cytometry, and cellular immunofluorescence staining. Deletion of AMPKα1, the predominant AMPKα isoform, but not AMPKα2 in immortalized MEFs led to spontaneous DNA double-strand breaks (DSB) which corresponded to repair protein p53-binding protein1 (53BP1) foci formation and subsequent apoptosis. Furthermore, AMPKα1 localizes to chromatin and AMPKα1 deletion down-regulates cyclin-dependent kinase inhibitor, p21, an important protein that plays a role in decreasing the incidence of spontaneous DSB via inhibition of cell proliferation. In addition, AMPKα1 null cells exhibited enhanced cell proliferation. Finally, p21 overexpression partially blocked the cellular hyperproliferation of AMPKα1-deleted MEFs via the inhibition of cyclin-dependent kinase 2 (CDK2). Taken together, our results suggest that AMPKα1 plays a fundamental role in controlling the cell cycle thereby affecting DNA damage and cellular apoptosis. PMID:25307521

  18. Expression of p53 in endometrial polyps with special reference to the p53 signature.

    PubMed

    Sho, Tomoko; Hachisuga, Toru; Kawagoe, Toshinori; Urabe, Rie; Kurita, Tomoko; Kagami, Seiji; Shimajiri, Shohei; Fujino, Yoshihisa

    2016-07-01

    We herein examined the significance of the p53 expression in endometrial polyps (EMPs). A total of 133 EMPs, including 62 premenopausal and 71 postmenopausal women with EMP, were immunohistochemically studied for the expression of estrogen receptor (ER)-alpha, Ki-67 and p53. Apoptotic cells were identified using a TUNEL assay. A DNA sequence analysis of TP53 exons 5 to 9 was performed. Among the premenopausal EMPs, a multivariate analysis showed the labeling index (LI) for Ki-67 to correlate significantly with that for p53 (P<0.001), but not that for apoptosis. On the contrary, among the postmenopausal EMPs, the LI for Ki-67 correlated significantly with that for apoptosis (P<0.001). The p53 signature (p53S) was defined by endometrial epithelial cells, which are morphologically benign in appearance but display 12 or more consecutive epithelial cell nuclei with strong p53 immunostaining. The p53S was found in nine (12.7%) postmenopausal EMPs (mean age: 70.2 years). The median Ki-67 index for the p53S was 7%, with no significant difference from that of the glands of the postmenopausal EMPs without the p53S (P=0.058). The median apoptotic index for the p53S was 0%, which was significantly lower than that of the postmenopausal EMPs without the p53S (P=0.002). Two of four p53Ss showed TP53 mutations according to the DNA sequence analysis. The presence of the p53S is not rare in postmenopausal EMPs with an advanced age. Among postmenopausal EMPs, the LI of Ki-67 significantly correlates with that of apoptosis. However, such a positive correlation between the LI of Ki-67 and apoptosis is not observed in p53S. PMID:26727623

  19. p53 is required for metformin-induced growth inhibition, senescence and apoptosis in breast cancer cells.

    PubMed

    Li, Puyu; Zhao, Ming; Parris, Amanda B; Feng, Xiaoshan; Yang, Xiaohe

    2015-09-01

    The p53 tumor repressor gene is commonly mutated in human cancers. The tumor inhibitory effect of metformin on p53-mutated breast cancer cells remains unclear. Data from the present study demonstrated that p53 knockdown or mutation has a negative effect on metformin or phenformin-induced growth inhibition, senescence and apoptosis in breast cancer cells. We also found that p53 reactivating agent nutlin-3α and CP/31398 promoted metformin-induced growth inhibition, senescence and apoptosis in MCF-7 (wt p53) and MDA-MB-231 (mt p53) cells, respectively. Treatment of MCF-7 cells with metformin or phenformin induced increase in p53 protein levels and the transcription of its downstream target genes, Bax and p21, in a dose-dependent manner. Moreover, we demonstrated that AMPK-mTOR signaling played a role in metformin-induced p53 up-regulation. The present study showed that p53 is required for metformin or phenformin-induced growth inhibition, senescence and apoptosis in breast cancer cells. The combination of metformin with p53 reactivating agents, like nutlin-3α and CP/31398, is a promising strategy for improving metformin-mediated anti-cancer therapy, especially for tumors with p53 mutations. PMID:26225749

  20. p53, Stem Cells, and Reprogramming

    PubMed Central

    Spike, Benjamin T.; Wahl, Geoffrey M.

    2011-01-01

    p53 is well recognized as a potent tumor suppressor. In its classic role, p53 responds to genotoxic insults by inducing cell cycle exit or programmed cell death to limit the propagation of cells with corrupted genomes. p53 is also implicated in a variety of other cellular processes in which its involvement is less well understood including self-renewal, differentiation, and reprogramming. These activities represent an emerging area of intense interest for cancer biologists, as they provide potential mechanistic links between p53 loss and the stem cell–like cellular plasticity that has been suggested to contribute to tumor cell heterogeneity and to drive tumor progression. Despite accumulating evidence linking p53 loss to stem-like phenotypes in cancer, it is not yet understood how p53 contributes to acquisition of “stemness” at the molecular level. Whether and how stem-like cells confer survival advantages to propagate the tumor also remain to be resolved. Furthermore, although it seems reasonable that the combination of p53 deficiency and the stem-like state could contribute to the genesis of cancers that are refractory to treatment, direct linkages and mechanistic underpinnings remain under investigation. Here, we discuss recent findings supporting the connection between p53 loss and the emergence of tumor cells bearing functional and molecular similarities to stem cells. We address several potential molecular and cellular mechanisms that may contribute to this link, and we discuss implications of these findings for the way we think about cancer progression. PMID:21779509

  1. Nucleolar stress with and without p53.

    PubMed

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

    2014-01-01

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

  2. Nucleolar stress with and without p53

    PubMed Central

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

    2014-01-01

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

  3. p53 Acts as a Co-Repressor to Regulate Keratin 14 Expression during Epidermal Cell Differentiation

    PubMed Central

    Chao, Chung-Faye; Lu, Mei-Hua; Lin, Hwang-Chi; Chiou, Shih-Hwa; Tao, Pao-Luh; Chen, Jang-Yi

    2012-01-01

    During epidermal cell differentiation, keratin 14 (K14) expression is down-regulated, p53 expression varies, and the expression of the p53 target genes, p21 and 14-3-3σ, increases. These trends suggest that the relative transcriptional activity of p53 is increased during epidermal cell differentiation. To determine the relationship between K14 and p53, we constructed K14 promoters of various sizes and found that wild-type p53 could repress the promoter activity of all of the K14 promoter constructs in H1299 cells. K14-p160 contains an SP1 binding site mutation that prevents p53 from repressing K14 expression. Using a DNA affinity precipitation assay, we confirmed that p53 forms a complex with SP1 at the SP1 binding site between nucleotides -48 and -43 on the K14 promoter. Thus, our data indicate that p53 acts as a co-repressor to down-regulate K14 expression by binding to SP1. Next, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal cell differentiation model to examine the inhibition of K14 expression caused by increased p53 activity. Human ovarian teratocarcinoma C9 cells were treated with TPA to induce differentiation. Over-expression of the dominant negative p53 mutant ΔTAp53, which inhibits p53 activity, prevented the TPA-induced K14 down-regulation in C9 cells. Furthermore, treatment of normal primary human foreskin keratinocytes (PHFK) with the p53 inhibitor pifithrin-α (PFT-α) showed that the inhibition of p53 activity relieves K14 repression during epidermal cell differentiation. Finally, we found that TPA induces the phosphorylation of p53 at residue 378, which enhances the affinity of p53 to bind to Sp1 and repress K14 expression. PMID:22911849

  4. SMG7 is a critical regulator of p53 stability and function in DNA damage stress response

    PubMed Central

    Luo, Hongwei; Cowen, Lauren; Yu, Guowu; Jiang, Wenguo; Tang, Yi

    2016-01-01

    The p53 tumor suppressor functions as a transcription factor and plays a pivotal role in regulation of cellular response to DNA damage by activating various genes including those involved in cell cycle arrest. p53 stability is essential for its function during stress response; however, the molecular mechanism for DNA damage-induced stabilization of p53 is not fully understood. In our present study, we have identified SMG7 (suppressor with morphological defects in genitalia 7), also known as EST1C, as a novel p53-binding protein. SMG7 is an mRNA surveillance factor implicated in degradation of p53 mRNA-containing nonsense mutations, yet it is completely unknown whether SMG7 regulates p53 function. Here, we show that SMG7 has a crucial role in p53-mediated response to genotoxic stress by regulating p53 stability. Using somatic gene knockout, we found that deletion of SMG7 abrogates DNA damage-induced p53 stabilization, although it exhibits minimal effect on the basal levels of p53. Importantly, loss of SMG7 impairs p53-mediated activation of p21 and cell cycle arrest following DNA damage. Pharmacological inhibition of Mdm2, a major E3 ubiquitin ligase for p53, restored p53 stability in gamma-irradiated SMG7-deficient cells. Furthermore, SMG7 physically interacts with Mdm2 and promotes ATM-mediated inhibitory phosphorylation of Mdm2 following ionizing radiation. Therefore, our present data demonstrate that SMG7 is critical for p53 function in DNA damage response, and reveal the SMG7-mediated phosphorylation of Mdm2 as a previously unknown mechanism for p53 regulation.

  5. SMG7 is a critical regulator of p53 stability and function in DNA damage stress response.

    PubMed

    Luo, Hongwei; Cowen, Lauren; Yu, Guowu; Jiang, Wenguo; Tang, Yi

    2016-01-01

    The p53 tumor suppressor functions as a transcription factor and plays a pivotal role in regulation of cellular response to DNA damage by activating various genes including those involved in cell cycle arrest. p53 stability is essential for its function during stress response; however, the molecular mechanism for DNA damage-induced stabilization of p53 is not fully understood. In our present study, we have identified SMG7 (suppressor with morphological defects in genitalia 7), also known as EST1C, as a novel p53-binding protein. SMG7 is an mRNA surveillance factor implicated in degradation of p53 mRNA-containing nonsense mutations, yet it is completely unknown whether SMG7 regulates p53 function. Here, we show that SMG7 has a crucial role in p53-mediated response to genotoxic stress by regulating p53 stability. Using somatic gene knockout, we found that deletion of SMG7 abrogates DNA damage-induced p53 stabilization, although it exhibits minimal effect on the basal levels of p53. Importantly, loss of SMG7 impairs p53-mediated activation of p21 and cell cycle arrest following DNA damage. Pharmacological inhibition of Mdm2, a major E3 ubiquitin ligase for p53, restored p53 stability in gamma-irradiated SMG7-deficient cells. Furthermore, SMG7 physically interacts with Mdm2 and promotes ATM-mediated inhibitory phosphorylation of Mdm2 following ionizing radiation. Therefore, our present data demonstrate that SMG7 is critical for p53 function in DNA damage response, and reveal the SMG7-mediated phosphorylation of Mdm2 as a previously unknown mechanism for p53 regulation. PMID:27462439

  6. Lupeol induces p53 and cyclin-B-mediated G2/M arrest and targets apoptosis through activation of caspase in mouse skin

    SciTech Connect

    Nigam, Nidhi Prasad, Sahdeo; George, Jasmine; Shukla, Yogeshwer

    2009-04-03

    Lupeol, present in fruits and medicinal plants, is a biologically active compound that has been shown to have various pharmacological properties in experimental studies. In the present study, we demonstrated the modulatory effect of lupeol on 7,12-dimethylbenz[a]anthracene (DMBA)-induced alterations on cell proliferation in the skin of Swiss albino mice. Lupeol treatment showed significant (p < 0.05) preventive effects with marked inhibition at 48, 72, and 96 h against DMBA-mediated neoplastic events. Cell-cycle analysis showed that lupeol-induced G2/M-phase arrest (16-37%) until 72 h, and these inhibitory effects were mediated through inhibition of the cyclin-B-regulated signaling pathway involving p53, p21/WAF1, cdc25C, cdc2, and cyclin-B gene expression. Further lupeol-induced apoptosis was observed, as shown by an increased sub-G1 peak (28%) at 96 h, with upregulation of bax and caspase-3 genes and downregulation of anti-apoptotic bcl-2 and survivin genes. Thus, our results indicate that lupeol has novel anti-proliferative and apoptotic potential that may be helpful in designing strategies to fight skin cancer.

  7. Intestinal Dysplasia Induced by Simian Virus 40 T Antigen Is Independent of p53

    PubMed Central

    Markovics, Jennifer A.; Carroll, Patrick A.; Robles, M. Teresa Sáenz; Pope, Hannah; Coopersmith, Craig M.; Pipas, James M.

    2005-01-01

    Transgenic mice expressing simian virus 40 large T antigen in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. Hyperplasia is dependent on T antigen binding to the retinoblastoma (pRb) family of tumor suppressor proteins. Mice expressing a truncated T antigen that inactivates the pRb-family, but is defective for binding p53, exhibit hyperplasia but do not progress to dysplasia. We hypothesized that the inhibition of the pRb family leads to entry of enterocytes into the cell cycle, resulting in hyperplasia, while inactivation of p53 is required for progression to dysplasia. Therefore, we examined T antigen/p53 complexes from the intestines of transgenic mice. We found that T antigen did not induce p53 stabilization, and we could not detect T antigen/p53 complexes in villus enterocytes. In contrast, T antigen expression led to a large increase in the levels of the cyclin-dependent kinase inhibitor p21. Furthermore, mice in which pRb was inactivated by a truncated T antigen in a p53 null background exhibited intestinal hyperplasia but no progression to dysplasia. These data indicate that loss of p53 function does not play a role in T antigen-induced dysplasia in the intestine. Rather, some unknown function of T antigen is essential for progression beyond hyperplasia. PMID:15919904

  8. Neocarzinostatin induces an effective p53-dependent response in human papillomavirus-positive cervical cancer cells.

    PubMed

    Bañuelos, Adriana; Reyes, Elba; Ocadiz, Rodolfo; Alvarez, Elizabeth; Moreno, Martha; Monroy, Alberto; Gariglio, Patricio

    2003-08-01

    Human papillomavirus (HPV) E6 viral oncoprotein plays an important role during cervical carcinogenesis. This oncoprotein binds the tumor suppressor protein p53, leading to its degradation via the ubiquitin-proteasome pathway. Therefore, it is generally assumed that in HPV-positive cancer cells p53 function is completely abolished. Nevertheless, recent findings suggest that p53 activity can be recovered in cells expressing endogenous E6 protein. To investigate whether p53-dependent functions controlling genome integrity, cell proliferation, and apoptosis can be reactivated in cervical cancer cells, we examined the capacity of HeLa, INBL, CaSki, C33A, and ViBo cell lines to respond to neocarzinostatin (NCS), a natural product which induces single- and double-strand breaks in DNA. We found that NCS treatment inhibits cellular proliferation through G2 cell cycle arrest and apoptosis induction. This effect was preceded by nuclear accumulation of p53 protein and by an increase of p21 transcripts. Although apoptosis was blocked in ViBo cells (HPV-negative), nuclear accumulation of transcriptionally active p53 and inhibition of cell proliferation are observed after NCS treatment. These results suggest that HPV-positive cervical cancer cells are capable of responding efficiently to DNA damage provoked by NCS treatment through a p53-dependent pathway in spite of the presence of E6 protein. PMID:12750435

  9. The p53 Codon 72 Pro/Pro Genotype Identifies Poor-Prognosis Neuroblastoma Patients: Correlation with Reduced Apoptosis and Enhanced Senescence by the p53-72P Isoform12

    PubMed Central

    Cattelani, Sara; Ferrari-Amorotti, Giovanna; Galavotti, Sara; Defferrari, Raffaella; Tanno, Barbara; Cialfi, Samantha; Vergalli, Jenny; Fragliasso, Valentina; Guerzoni, Clara; Manzotti, Gloria; Soliera, Angela Rachele; Menin, Chiara; Bertorelle, Roberta; McDowell, Heather P; Inserra, Alessandro; Belli, Maria Luisa; Varesio, Luigi; Tweddle, Deborah; Tonini, Gian Paolo; Altavista, Pierluigi; Dominici, Carlo; Raschellà, Giuseppe; Calabretta, Bruno

    2012-01-01

    The p53 gene is rarely mutated in neuroblastoma, but codon 72 polymorphism that modulates its proapoptotic activity might influence cancer risk and clinical outcome. We investigated whether this polymorphism affects neuroblastoma risk and disease outcome and assessed the biologic effects of the p53-72R and p53-72P isoforms in p53-null cells. Comparison of 288 healthy subjects and 286 neuroblastoma patients revealed that the p53-72 polymorphism had no significant impact on the risk of developing neuroblastoma; however, patients with the Pro/Pro genotype had a shorter survival than those with the Arg/Arg or the Arg/Pro genotypes even in the stage 3 and 4 subgroup without MYCN amplification. By Cox regression analysis, the p53 Pro/Pro genotype seems to be an independent marker of poor prognosis (hazard ratio = 2.74; 95% confidence interval = 1.14–6.55, P = .014) together with clinical stage, MYCN status, and age at diagnosis. In vitro, p53-72P was less effective than p53-72R in inducing apoptosis and inhibiting survival of p53-null LAN-1 cells treated with etoposide, topotecan, or ionizing radiation but not taxol. By contrast, p53-72P was more effective in promoting p21-dependent accelerated senescence, alone or in the presence of etoposide. Thus, the p53-72 Pro/Pro genotype might be a marker of poor outcome independent of MYCN amplification, possibly improving risk stratification. Moreover, the lower apoptosis and the enhanced accelerated senescence by the p53-72P isoform in response to DNA damage suggest that patients with neuroblastoma with the p53-72 Pro/Pro genotype may benefit from therapeutic protocols that do not rely only on cytotoxic drugs that function, in part, through p53 activation. PMID:22904680

  10. Gold (I) N-heterocyclic carbene complex inhibits mouse melanoma growth by p53 upregulation

    PubMed Central

    2014-01-01

    Background Cancer treatment using gold (I) complexes is becoming popular. In this study, a gold (I) N-heterocyclic complex designated as complex 3 was synthesized, its cytotoxicity was examined, and its anti-melanoma activity was evaluated in vitro and in vivo. Methods Viability of cancer cells was determined by MTT assay upon treatment with various concentrations of a gold (I) N-heterocyclic carbene complex (complex 3) in a dose and time dependent manner. Mouse melanoma cells B16F10 were selected for further apoptotic studies, including flowcytometric analysis of annexin binding, cell cycle arrest, intracellular ROS generation and loss in the mitochondrial membrane potential. ELISA based assays were done for caspase activities and western blots for determining the expression of various survival and apoptotic proteins. Immunocytology was performed to visualize the translocation of p53 to the nucleus. B16F10 cells were inoculated into mice and post tumor formation, complex 3 was administered. Immunohistology was performed to determine the expressions of p53, p21, NF-κB (p65 and p50), MMP-9 and VEGF. Student’s t test was used for determining statistical significance. The survival rate data were analyzed by Kaplan-Meier plots. Results Complex 3 markedly inhibited the growth of HCT 116, HepG2, and A549, and induced apoptosis in B16F10 cells with nuclear condensation, DNA fragmentation, externalization of phosphatidylserine, activation of caspase 3 and caspase 9, PARP cleavage, downregulation of Bcl-2, upregulation of Bax, cytosolic cytochrome c elevation, ROS generation, and mitochondrial membrane potential loss indicating the involvement of an intrinsic mitochondrial death pathway. Further, upregulation of p53, p-p53 (ser 15) and p21 indicated the role of p53 in complex 3 mediated apoptosis. The complex reduced tumor size, and caused upregulation of p53 and p21 along with downregulation of NF-κB (p65 and p50), VEGF and MMP-9. These results suggest that it induced

  11. LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner

    PubMed Central

    Kramer, Holly B.; Lai, Chun-Fui; Patel, Hetal; Periyasamy, Manikandan; Lin, Meng-Lay; Feller, Stephan M.; Fuller-Pace, Frances V.; Meek, David W.; Ali, Simak; Buluwela, Laki

    2016-01-01

    Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53. PMID:26400164

  12. LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner.

    PubMed

    Kramer, Holly B; Lai, Chun-Fui; Patel, Hetal; Periyasamy, Manikandan; Lin, Meng-Lay; Feller, Stephan M; Fuller-Pace, Frances V; Meek, David W; Ali, Simak; Buluwela, Laki

    2016-01-29

    Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53. PMID:26400164

  13. p53-directed translational control can shape and expand the universe of p53 target genes

    PubMed Central

    Zaccara, S; Tebaldi, T; Pederiva, C; Ciribilli, Y; Bisio, A; Inga, A

    2014-01-01

    The increasing number of genome-wide transcriptome analyses focusing on p53-induced cellular responses in many cellular contexts keeps adding to the already numerous p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses, we performed a translatome analysis through polysomal profiling on MCF7 cells upon 16 hours of doxorubicin or nutlin-3a treatment. The comparison between the transcriptome and the translatome revealed a considerable level of uncoupling, characterized by genes whose transcription variations did not correlate with translation variations. Interestingly, uncoupled genes were associated with apoptosis, DNA and RNA metabolism and cell cycle functions, suggesting that post-transcriptional control can modulate classical p53-regulated responses. Furthermore, even for well-established p53 targets that were differentially expressed both at the transcriptional and translational levels, quantitative differences between the transcriptome, subpolysomal and polysomal RNAs were evident. As we searched mechanisms underlying gene expression uncoupling, we identified the p53-dependent modulation of six RNA-binding proteins, where hnRNPD (AUF1) and CPEB4 are direct p53 transcriptional targets, whereas SRSF1, DDX17, YBX1 and TARDBP are indirect targets (genes modulated preferentially in the subpolysomal or polysomal mRNA level) modulated at the translational level in a p53-dependent manner. In particular, YBX1 translation appeared to be reduced by p53 via two different mechanisms, one related to mTOR inhibition and the other to miR-34a expression. Overall, we established p53 as a master regulator of translational control and identified new p53-regulated genes affecting translation that can contribute to p53-dependent cellular responses. PMID:24926617

  14. p53-directed translational control can shape and expand the universe of p53 target genes.

    PubMed

    Zaccara, S; Tebaldi, T; Pederiva, C; Ciribilli, Y; Bisio, A; Inga, A

    2014-10-01

    The increasing number of genome-wide transcriptome analyses focusing on p53-induced cellular responses in many cellular contexts keeps adding to the already numerous p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses, we performed a translatome analysis through polysomal profiling on MCF7 cells upon 16 hours of doxorubicin or nutlin-3a treatment. The comparison between the transcriptome and the translatome revealed a considerable level of uncoupling, characterized by genes whose transcription variations did not correlate with translation variations. Interestingly, uncoupled genes were associated with apoptosis, DNA and RNA metabolism and cell cycle functions, suggesting that post-transcriptional control can modulate classical p53-regulated responses. Furthermore, even for well-established p53 targets that were differentially expressed both at the transcriptional and translational levels, quantitative differences between the transcriptome, subpolysomal and polysomal RNAs were evident. As we searched mechanisms underlying gene expression uncoupling, we identified the p53-dependent modulation of six RNA-binding proteins, where hnRNPD (AUF1) and CPEB4 are direct p53 transcriptional targets, whereas SRSF1, DDX17, YBX1 and TARDBP are indirect targets (genes modulated preferentially in the subpolysomal or polysomal mRNA level) modulated at the translational level in a p53-dependent manner. In particular, YBX1 translation appeared to be reduced by p53 via two different mechanisms, one related to mTOR inhibition and the other to miR-34a expression. Overall, we established p53 as a master regulator of translational control and identified new p53-regulated genes affecting translation that can contribute to p53-dependent cellular responses. PMID:24926617

  15. The role of p53 in ribosomopathies.

    PubMed

    Fumagalli, Stefano; Thomas, George

    2011-04-01

    Impaired ribosome biogenesis is the underlying cause of the pathological conditions collectively known as ribosomopathies. Several hypotheses have been advanced to explain the mechanisms by which deficiencies in ribosome biogenesis interfere with developmental processes leading eventually to the emergence of these diseases. In recent years it has become clear that perturbation of this process triggers a cell-cycle checkpoint that, through activation of the tumor-suppressor p53, leads to cell-cycle arrest and apoptosis. Indeed, evidence is accumulating from studies in animal models that the unscheduled activation of p53 is responsible for perturbations in tissue homeostasis that cause the development of ribosomopathies such as Treacher-Collins syndrome (TCS) and 5q(-) syndrome. These findings imply that inhibition of p53, or better, of mechanisms that specifically lead to p53 activation in response to inhibition of ribosome biogenesis, could be targeted in the treatment of ribosomopathies where activation of p53 is shown to play a pathogenic role. PMID:21435506

  16. The induction of polyploidy or apoptosis by the Aurora A kinase inhibitor MK8745 is p53-dependent

    PubMed Central

    Ho, Alan L; Schwartz, Gary K

    2012-01-01

    Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small-molecule inhibitors of Aurora kinases are currently undergoing clinical trials. Aurora A kinase is essential for successful mitotic transition. MK8745 is a novel and selective small-molecule inhibitor of Aurora A kinase. MK8745 induced apoptotic cell death in a p53-dependent manner when tested in vitro in cell lines of multiple lineages. Cells expressing wild-type p53 showed a short delay in mitosis followed by cytokinesis, resulting in 2N cells along with apoptosis. However, cells lacking or with mutant p53 resulted in a prolonged arrest in mitosis followed by endoreduplication and polyploidy. Cytokinesis was completely inhibited in p53-deficient cells, as observed by the absence of 2N cell population. The induction of apoptosis in p53-proficient cells was associated with activation of caspase 3 and release of cytochrome c but was independent of p21. Exposure of p53 wild-type cells to MK8745 resulted in the induction of p53 phosphorylation (ser15) and an increase in p53 protein expression. p53-dependent apoptosis by MK8745 was further confirmed in HCT 116 p53−/− cells transfected with wild-type p53. Transient knockdown of Aurora A by specific siRNA recapitulated these p53-dependent effects, with greater percent induction of apoptosis in p53 wild-type cells. In conclusion, our studies show p53 as a determining factor for induction of apoptosis vs. polyploidy upon inhibition of Aurora A. PMID:22293494

  17. TRIM32 is a novel negative regulator of p53.

    PubMed

    Liu, Juan; Zhu, Yu; Hu, Wenwei; Feng, Zhaohui

    2015-01-01

    To ensure proper function, the tumor suppressor p53 is tightly regulated through different post-translational modifications, particularly ubiquitination. Recently, TRIM32 was identified as a p53-regulated gene and an E3 ubiquitin ligase of p53. Thus, TRIM32 and p53 form a novel auto-regulatory negative feedback loop for p53 regulation in cells. PMID:27308422

  18. TRIM32 is a novel negative regulator of p53

    PubMed Central

    Liu, Juan; Zhu, Yu; Hu, Wenwei; Feng, Zhaohui

    2015-01-01

    To ensure proper function, the tumor suppressor p53 is tightly regulated through different post-translational modifications, particularly ubiquitination. Recently, TRIM32 was identified as a p53-regulated gene and an E3 ubiquitin ligase of p53. Thus, TRIM32 and p53 form a novel auto-regulatory negative feedback loop for p53 regulation in cells. PMID:27308422

  19. p53 and rapamycin are additive

    PubMed Central

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

    2015-01-01

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

  20. p53 Suppresses Tetraploid Development in Mice

    PubMed Central

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

    2015-01-01

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

  1. TFIIS.h, a new target of p53, regulates transcription efficiency of pro-apoptotic bax gene

    PubMed Central

    Liao, Jun-Ming; Cao, Bo; Deng, Jun; Zhou, Xiang; Strong, Michael; Zeng, Shelya; Xiong, Jianping; Flemington, Erik; Lu, Hua

    2016-01-01

    Tumor suppressor p53 transcriptionally regulates hundreds of genes involved in various cellular functions. However, the detailed mechanisms underlying the selection of p53 targets in response to different stresses are still elusive. Here, we identify TFIIS.h, a transcription elongation factor, as a new transcriptional target of p53, and also show that it can enhance the efficiency of transcription elongation of apoptosis-associated bax gene, but not cell cycle-associated p21 (CDKN1A) gene. TFIIS.h is revealed as a p53 target through microarray analysis of RNAs extracted from cells treated with or without inauhzin (INZ), a p53 activator, and further confirmed by RT-q-PCR, western blot, luciferase reporter, and ChIP assays. Interestingly, knocking down TFIIS.h impairs, but overexpressing TFIIS.h promotes, induction of bax, but not other p53 targets including p21, by p53 activation. In addition, overexpression of TFIIS.h induces cell death in a bax- dependent fashion. These findings reveal a mechanism by which p53 utilizes TFIIS.h to selectively promote the transcriptional elongation of the bax gene, upsurging cell death in response to severe DNA damage. PMID:27005522

  2. Dux4 induces cell cycle arrest at G1 phase through upregulation of p21 expression

    SciTech Connect

    Xu, Hongliang; Wang, Zhaoxia; Jin, Suqin; Hao, Hongjun; Zheng, Lemin; Zhou, Boda; Zhang, Wei; Lv, He; Yuan, Yun

    2014-03-28

    Highlights: • Dux4 induced TE671 cell proliferation defect and G1 phase arrest. • Dux4 upregulated p21 expression without activating p53. • Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. • Sp1 binding site was required for Dux4-induced p21 promoter activation. - Abstract: It has been implicated that Dux4 plays crucial roles in development of facioscapulohumeral dystrophy. But the underlying myopathic mechanisms and related down-stream events of this retrogene were far from clear. Here, we reported that overexpression of Dux4 in a cell model TE671 reduced cell proliferation rate, and increased G1 phase accumulation. We also determined the impact of Dux4 on p53/p21 signal pathway, which controls the checkpoint in cell cycle progression. Overexpression of Dux4 increased p21 mRNA and protein level, while expression of p53, phospho-p53 remained unchanged. Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. Furthermore, we demonstrated that enhanced Dux4 expression increased p21 promoter activity and elevated expression of Sp1 transcription factor. Mutation of Sp1 binding site decreased dux4 induced p21 promoter activation. Chromatin immunoprecipitation (ChIP) assays confirmed the Dux4-induced binding of Sp1 to p21 promoter in vivo. These results suggest that Dux4 might induce proliferation inhibition and G1 phase arrest through upregulation of p21.

  3. KAP1 dictates p53 response induced by chemotherapeutic agents via Mdm2 interaction

    SciTech Connect

    Okamoto, Koji . E-mail: kojokamo@gan2.res.ncc.go.jp; Kitabayashi, Issay; Taya, Yoichi . E-mail: ytaya@gan2.res.ncc.go.jp

    2006-12-08

    KAP1 recruits many proteins involved in gene silencing and functions as an integral part of co-repressor complex. KAP1 was identified as Mdm2-binding protein and shown to form a complex with Mdm2 and p53 in vivo. We examined the role of KAP1 in p53 activation after the treatment of cells with different types of external stresses. KAP1 reduction markedly enhanced the induction of p21, a product of the p53 target gene, after treatment with actinomycin D or {gamma}-irradiation, but not with camptothecin. Treatment with actinomycin D, but not with camptothecin, augmented the interaction of p53 with Mdm2 and KAP1. Further, KAP1 reduction in actinomycin D-treated cells facilitated cell cycle arrest and negatively affected clonal cell growth. Thus, the reduction of KAP1 levels promotes p53-dependent p21 induction and inhibits cell proliferation in actinomycin D-treated cells. KAP1 may serve as a therapeutic target against cancer in combination with actinomycin D.

  4. Δ113p53/Δ133p53 converts P53 from a repressor to a promoter of DNA double-stand break repair

    PubMed Central

    Gong, Lu; Chen, Jun

    2016-01-01

    ABSTRACT In response to DNA damage, p53 (TP53, best known as p53) is quickly activated leading to cell cycle arrest or apoptosis to ensure genomic integrity; however, this represses DNA double-strand break (DSB) repair. Our recent work revealed that Δ113p53/Δ133p53 protein is accumulated at a later stage upon DNA DSB stress to switch p53 signaling from repression to promotion of DNA DSB repair. PMID:27308550

  5. Delayed cell cycle progression in selenoprotein W depleted cells is regulated by a mitogen-activated protein kinase kinase 4–p38–p53 pathway

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a p53- and p21Cip1-dependent G1-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser33 in p53, which is associated with decreased p53...

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

    PubMed Central

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

    2012-01-01

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

  7. Autoantibody recognition mechanisms of p53 epitopes

    NASA Astrophysics Data System (ADS)

    Phillips, J. C.

    2016-06-01

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

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

    SciTech Connect

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

    2007-09-01

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

  9. Activation of p53-Dependent Growth Suppression in Human Cells by Mutations in PTEN or PIK3CA▿

    PubMed Central

    Kim, Jung-Sik; Lee, Carolyn; Bonifant, Challice L.; Ressom, Habtom; Waldman, Todd

    2007-01-01

    In an effort to identify genes whose expression is regulated by activated phosphatidylinositol 3-kinase (PI3K) signaling, we performed microarray analysis and subsequent quantitative reverse transcription-PCR on an isogenic set of PTEN gene-targeted human cancer cells. Numerous p53 effectors were upregulated following PTEN deletion, including p21, GDF15, PIG3, NOXA, and PLK2. Stable depletion of p53 led to reversion of the gene expression program. Western blots revealed that p53 was stabilized in HCT116 PTEN−/− cells via an Akt1-dependent and p14ARF-independent mechanism. Stable depletion of PTEN in untransformed human fibroblasts and epithelial cells also led to upregulation of p53 and senescence-like growth arrest. Simultaneous depletion of p53 rescued this phenotype, enabling PTEN-depleted cells to continue proliferating. Next, we tested whether oncogenic PIK3CA, like inactivated PTEN, could activate p53. Retroviral expression of oncogenic human PIK3CA in MCF10A cells led to activation of p53 and upregulation of p53-regulated genes. Stable depletion of p53 reversed these PIK3CA-induced expression changes and synergized with oncogenic PIK3CA in inducing anchorage-independent growth. Finally, targeted deletion of an endogenous allele of oncogenic, but not wild-type, PIK3CA in a human cancer cell line led to a reduction in p53 levels and a decrease in the expression of p53-regulated genes. These studies demonstrate that activation of PI3K signaling by mutations in PTEN or PIK3CA can lead to activation of p53-mediated growth suppression in human cells, indicating that p53 can function as a brake on phosphatidylinositol (3,4,5)-triphosphate-induced mitogenesis during human cancer pathogenesis. PMID:17060456

  10. Glycogen Synthase Kinase-3β (GSK3β) Binds to and Promotes the Actions of p53*

    PubMed Central

    Watcharasit, Piyajit; Bijur, Gautam N.; Song, Ling; Zhu, Jianhui; Chen, Xinbin; Jope, Richard S.

    2006-01-01

    The recent discovery of direct interactions between two important regulators of cell fate, the tumor suppressor p53 and glycogen synthase kinase-3β (GSK3β), led us to examine the mechanism and outcomes of this interaction. Two regions of p53 were identified that regulate its binding to GSK3β. Deletion of the p53 activation domain-1 (AD1), but not mutations that prevent MDM2 binding through the AD1 domain, enhanced GSK3β binding to p53, indicating that the AD1 domain interferes with p53 binding to GSK3β. Deletion of the p53 basic domain (BD) abrogated GSK3β binding, and a ten amino acid region within the C-terminal BD domain was identified as necessary for binding to GSK3β. GSK3β activity was not required for p53 binding, but inhibition of GSK3β stabilized the association, suggesting a transient interaction during which active GSK3β promotes actions of p53. This regulatory role of GSK3β was demonstrated by large reductions of p53-induced increases in the levels of MDM2, p21, and Bax when GSK3β was inhibited. Besides promoting p53-mediated transcription, GSK3β also contributed to mitochondrial p53 apoptotic signaling. After DNA damage, mitochondrial GSK3β co-immunoprecipitated with p53 and was activated, and inhibition of GSK3β blocked cytochrome c release and caspase-3 activation. Thus, GSK3β interacts with p53 in both the nucleus and mitochondria and promotes its actions at both sites. PMID:14523002

  11. Endothelial p53 Deletion Improves Angiogenesis and Prevents Cardiac Fibrosis and Heart Failure Induced by Pressure Overload in Mice

    PubMed Central

    Gogiraju, Rajinikanth; Xu, Xingbo; Bochenek, Magdalena L.; Steinbrecher, Julia H.; Lehnart, Stephan E.; Wenzel, Philip; Kessel, Michael; Zeisberg, Elisabeth M.; Dobbelstein, Matthias; Schäfer, Katrin

    2015-01-01

    Background Cardiac dysfunction developing in response to chronic pressure overload is associated with apoptotic cell death and myocardial vessel rarefaction. We examined whether deletion of tumor suppressor p53 in endothelial cells may prevent the transition from cardiac hypertrophy to heart failure. Methods and Results Mice with endothelial‐specific deletion of p53 (End.p53‐KO) were generated by crossing p53fl/fl mice with mice expressing Cre recombinase under control of an inducible Tie2 promoter. Cardiac hypertrophy was induced by transverse aortic constriction. Serial echocardiography measurements revealed improved cardiac function in End.p53‐KO mice that also exhibited better survival. Cardiac hypertrophy was associated with increased p53 levels in End.p53‐WT controls, whereas banded hearts of End.p53‐KO mice exhibited lower numbers of apoptotic endothelial and non‐endothelial cells and altered mRNA levels of genes regulating cell cycle progression (p21), apoptosis (Puma), or proliferation (Pcna). A higher cardiac capillary density and improved myocardial perfusion was observed, and pharmacological inhibition or genetic deletion of p53 also promoted endothelial sprouting in vitro and new vessel formation following hindlimb ischemia in vivo. Hearts of End.p53‐KO mice exhibited markedly less fibrosis compared with End.p53‐WT controls, and lower mRNA levels of p53‐regulated genes involved in extracellular matrix production and turnover (eg, Bmp‐7, Ctgf, or Pai‐1), or of transcription factors involved in controlling mesenchymal differentiation were observed. Conclusions Our analyses reveal that accumulation of p53 in endothelial cells contributes to blood vessel rarefaction and fibrosis during chronic cardiac pressure overload and suggest that endothelial cells may be a therapeutic target for preserving cardiac function during hypertrophy. PMID:25713289

  12. Molecular mechanisms of nutlin-induced apoptosis in multiple myeloma: evidence for p53-transcription-dependent and -independent pathways.

    PubMed

    Saha, Manujendra N; Jiang, Hua; Chang, Hong

    2010-09-15

    Multiple myeloma (MM) is an incurable plasma cell malignancy in which p53 is rarely mutated. Thus, activation of the p53 pathway by a small molecule inhibitor of the p53-MDM2 interaction, nutlin, in MM cells retaining wild type p53 is an attractive therapeutic strategy. Recently we reported that nutlin plus velcade (a proteasome inhibitor) displayed a synergistic response in MM. However, the mechanism of the p53-mediated apoptosis in MM has not been fully understood. Our data show that nutlin-induced apoptosis correlated with reduction in cell viability, upregulation of p53, p21 and MDM2 protein levels with a simultaneous increase in pro-apoptotic targets PUMA, Bax and Bak and downregulation of anti-apoptotic targets Bcl2 and survivin and activation of caspase in MM cells harboring wild type p53. Nutlin-induced apoptosis was inhibited when activation of caspase was blocked by the caspase inhibitor. Nutlin caused mitochondrial translocation of p53 where it binds with Bcl2, leading to cytochrome C release. Moreover, blocking the transcriptional arm of p53 by the p53-specific transcriptional inhibitor, pifithrin-α, not only inhibited nutlin-induced upregulation of p53-transcriptional targets but also augmented apoptosis in MM cells, suggesting an association of transcription-independent pathway of apoptosis. However, inhibitor of mitochondrial translocation of p53, PFT-μ, did not prevent nutlin-induced apoptosis, suggesting that the p53 transcription-dependent pathway was also operational in nutlin-induced apoptosis in MM. Our study provides the evidence that nutlin-induced apoptosis in MM cells is mediated by transcription-dependent and -independent pathways and supports further clinical evaluation of nutlin as a novel therapeutic agent in MM. PMID:20595817

  13. Gelsolin negatively regulates the activity of tumor suppressor p53 through their physical interaction in hepatocarcinoma HepG2 cells

    SciTech Connect

    An, Joo-Hee; Kim, Jung-Woong; Jang, Sang-Min; Kim, Chul-Hong; Kang, Eun-Jin; Choi, Kyung-Hee

    2011-08-19

    Highlights: {yields} The actin binding protein Gelsolin (GSN) interacts with transcription factor p53. {yields} GSN interacts with transactivation- and DNA binding domains of p53. {yields} GSN represses transactivity of p53 via inhibition of nuclear translocation of p53. {yields} GSN inhibits the p53-mediated apoptosis in hepatocarcinoma HepG2 cells. -- Abstract: As a transcription factor, p53 modulates several cellular responses including cell-cycle control, apoptosis, and differentiation. In this study, we have shown that an actin regulatory protein, gelsolin (GSN), can physically interact with p53. The nuclear localization of p53 is inhibited by GSN overexpression in hepatocarcinoma HepG2 cells. Additionally, we demonstrate that GSN negatively regulates p53-dependent transcriptional activity of a reporter construct, driven by the p21-promoter. Furthermore, p53-mediated apoptosis was repressed in GSN-transfected HepG2 cells. Taken together, these results suggest that GSN binds to p53 and this interaction leads to the inhibition of p53-induced apoptosis by anchoring of p53 in the cytoplasm in HepG2 cells.

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

    PubMed

    2003-01-01

    Introgen's adenoviral p53 gene therapy [INGN 201, ADVEXIN] is in clinical development for the treatment of various cancers. The p53 tumour suppressor gene is deleted or mutated in many tumour cells and is one of the most frequently mutated genes in human tumours. INGN 201 has been shown to kill cancer cells directly. In August 2002, Introgen announced plans to file an application for INGN 201 with the European Agency for the Evaluation of Medicinal Products (EMEA) for the treatment of head and neck cancer; the European filing will be submitted simultaneously with the previously scheduled (planned for 2004) submission of a Biologics License Application (BLA) for ADVEXIN to the US FDA. On 20 February 2003, INGN 201 received orphan drug designation from the US FDA for head and neck cancer. INGN 201 is available for licensing although Introgen favours retaining partial or full rights to the therapy in the US. Introgen Therapeutics and its collaborative partner for the p53 programme, Aventis Gencell, have been developing p53 gene therapy products. The agreement was originally signed by Rhône-Poulenc Rorer's Gencell division, which became Aventis Gencell after Rhône-Poulenc Rorer merged with Hoechst Marion Roussel to form Aventis Pharma. According to the original agreement, Introgen was responsible for phase I and preclinical development in North America, while Aventis Gencell was responsible for clinical trials conducted in Europe and for clinical trials in North America beyond phase I. In April 2001, Aventis Gencell and Introgen restructured their existing collaboration agreement for p53 gene therapy products. Aventis Gencell indicated that p53 research had suffered from internal competition for resources and was pulling back from its development agreement with Introgen for p53 gene therapy products. Introgen will assume responsibility for worldwide development of all p53 programmes and will obtain exclusive worldwide commercial rights to p53-based gene therapy

  15. Titanium dioxide nanoparticles trigger p53-mediated damage response in peripheral blood lymphocytes.

    PubMed

    Kang, Su Jin; Kim, Byeong Mo; Lee, Young Joon; Chung, Hai Won

    2008-06-01

    Titanium dioxide nanoparticles (nano-TiO2) are widely used as a photocatalyst in air and water remediation. These nanoparticles are known to induce toxicity; however, their cytotoxic mechanism is not fully understood. In this study, we investigated the underlying mechanism of nano-TiO2-induced cytotoxicity in peripheral blood lymphocytes. We examined the genotoxic effects of nano-TiO2 in lymphocytes using alkaline single-cell gel electrophoresis (Comet) and cytokinesis-block micronucleus (CBMN) assays. Lymphocytes treated with nano-TiO2 showed significantly increased micronucleus formation and DNA breakage. Western-blot analysis to identify proteins involved in the p53-mediated response to DNA damage revealed the accumulation of p53 and activation of DNA damage checkpoint kinases in nano-TiO2-treated lymphocytes. However, p21 and bax, downstream targets of p53, were not affected, indicating that nano-TiO2 does not stimulate transactivational activity of p53. The generation of reactive oxygen species (ROS) in nano-TiO2-treated cells was also observed, andN-acetylcysteine (NAC) supplementation inhibited the level of nano-TiO2-induced DNA damage. Given that ROS-induced DNA damage leads to p53 activation in the DNA damage response, our results suggest that nano-TiO2 induces ROS generation in lymphocytes, thereby activating p53-mediated DNA damage checkpoint signals. PMID:18418868

  16. 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.

  17. Acetylation of the p53 DNA binding domain regulates apoptosis induction.

    PubMed Central

    Sykes, Stephen M.; Mellert, Hestia S.; Holbert, Marc A.; Li, Keqin; Marmorstein, Ronen; Lane, William S.; McMahon, Steven B.

    2007-01-01

    SUMMARY The ability of p53 to induce apoptosis plays an important role in tumor suppression. Here we describe a previously unknown post-translational modification of the DNA-binding domain of p53. This modification, acetylation of lysine 120, occurs rapidly after DNA damage and is catalyzed by the MYST family acetyltransferases hMOF and TIP60. Mutation of lysine 120 to arginine, as occurs in human cancer, debilitates K120 acetylation and diminishes p53-mediated apoptosis without affecting cell-cycle arrest. The K120R mutation selectively blocks the transcription of pro-apoptotic target genes such as BAX and PUMA while the non-apoptotic targets p21 and hMDM2 remain unaffected. Consistent with this, depletion of hMOF and/or TIP60 inhibits the ability of p53 to activate BAX and PUMA transcription. Furthermore, the acetyl-lysine 120 form of p53 specifically accumulates at pro-apoptotic target genes. These data suggest that K120 acetylation may help distinguish the cell cycle arrest and apoptotic functions of p53. PMID:17189187

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

    PubMed Central

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

    2015-01-01

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

  19. Oxidative stress-induced p53 activity is enhanced by a redox-sensitive TP53INP1 SUMOylation

    PubMed Central

    Peuget, S; Bonacci, T; Soubeyran, P; Iovanna, J; Dusetti, N J

    2014-01-01

    Tumor Protein p53-Induced Nuclear Protein 1 (TP53INP1) is a tumor suppressor that modulates the p53 response to stress. TP53INP1 is one of the key mediators of p53 antioxidant function by promoting the p53 transcriptional activity on its target genes. TP53INP1 expression is deregulated in many types of cancers including pancreatic ductal adenocarcinoma in which its decrease occurs early during the preneoplastic development. In this work, we report that redox-dependent induction of p53 transcriptional activity is enhanced by the oxidative stress-induced SUMOylation of TP53INP1 at lysine 113. This SUMOylation is mediated by PIAS3 and CBX4, two SUMO ligases especially related to the p53 activation upon DNA damage. Importantly, this modification is reversed by three SUMO1-specific proteases SENP1, 2 and 6. Moreover, TP53INP1 SUMOylation induces its binding to p53 in the nucleus under oxidative stress conditions. TP53INP1 mutation at lysine 113 prevents the pro-apoptotic, antiproliferative and antioxidant effects of TP53INP1 by impairing the p53 response on its target genes p21, Bax and PUMA. We conclude that TP53INP1 SUMOylation is essential for the regulation of p53 activity induced by oxidative stress. PMID:24608790

  20. LincRNA-p21 acts as a mediator of ING1b-induced apoptosis

    PubMed Central

    Tran, U M; Rajarajacholan, U; Soh, J; Kim, T-s; Thalappilly, S; Sensen, C W; Riabowol, K

    2015-01-01

    ING1b is a tumor suppressor that affects transcription, cell cycle control and apoptosis. ING1b is deregulated in disease, and its activity is closely linked to that of p53. In addition to regulating protein-coding genes, we found that ING1b also influences the expression of large intergenic non-coding RNAs (lincRNAs). In particular, lincRNA-p21 was significantly induced after DNA-damage stress or by ING1b overexpression. Furthermore, lincRNA-p21 expression in response to DNA damage was significantly attenuated in cells lacking ING1b. LincRNA-p21 is also a target of p53 and can trigger apoptosis in mouse cell models. We found that this function of lincRNA-p21 is conserved in human cell models. Moreover, ING1b and p53 could function independently to influence lincRNA-p21 expression. However, their effects become more additive under conditions of stress. In particular, ING1b regulates lincRNA-p21 levels by binding to its promoter and is required for induction of lincRNA-p21 by p53. The ability of ING1b to cause apoptosis is also impaired in the absence of lincRNA-p21. Surprisingly, deletion of the ING1b plant homeodomain, which allows it to bind histones and regulate chromatin structure, did not alter regulation of lincRNA-p21. Our findings suggest that ING1b induces lincRNA-p21 expression independently of histone 3 lysine 4 trimethylation mark recognition and that lincRNA-p21 functions downstream of ING1b. Thus, regulation at the level of lincRNA-p21 may represent the point at which ING1b and p53 pathways converge to induce apoptosis under specific stress conditions. PMID:25741593

  1. Exercise Activates p53 and Negatively Regulates IGF-1 Pathway in Epidermis within a Skin Cancer Model.

    PubMed

    Yu, Miao; King, Brenee; Ewert, Emily; Su, Xiaoyu; Mardiyati, Nur; Zhao, Zhihui; Wang, Weiqun

    2016-01-01

    Exercise has been previously reported to lower cancer risk through reducing circulating IGF-1 and IGF-1-dependent signaling in a mouse skin cancer model. This study aims to investigate the underlying mechanisms by which exercise may down-regulate the IGF-1 pathway via p53 and p53-related regulators in the skin epidermis. Female SENCAR mice were pair-fed an AIN-93 diet with or without 10-week treadmill exercise at 20 m/min, 60 min/day and 5 days/week. Animals were topically treated with TPA 2 hours before sacrifice and the target proteins in the epidermis were analyzed by both immunohistochemistry and Western blot. Under TPA or vehicle treatment, MDM2 expression was significantly reduced in exercised mice when compared with sedentary control. Meanwhile, p53 was significantly elevated. In addition, p53-transcriptioned proteins, i.e., p21, IGFBP-3, and PTEN, increased in response to exercise. There was a synergy effect between exercise and TPA on the decreased MDM2 and increased p53, but not p53-transcripted proteins. Taken together, exercise appeared to activate p53, resulting in enhanced expression of p21, IGFBP-3, and PTEN that might induce a negative regulation of IGF-1 pathway and thus contribute to the observed cancer prevention by exercise in this skin cancer model. PMID:27509024

  2. Exercise Activates p53 and Negatively Regulates IGF-1 Pathway in Epidermis within a Skin Cancer Model

    PubMed Central

    Yu, Miao; King, Brenee; Ewert, Emily; Su, Xiaoyu; Mardiyati, Nur; Zhao, Zhihui; Wang, Weiqun

    2016-01-01

    Exercise has been previously reported to lower cancer risk through reducing circulating IGF-1 and IGF-1-dependent signaling in a mouse skin cancer model. This study aims to investigate the underlying mechanisms by which exercise may down-regulate the IGF-1 pathway via p53 and p53-related regulators in the skin epidermis. Female SENCAR mice were pair-fed an AIN-93 diet with or without 10-week treadmill exercise at 20 m/min, 60 min/day and 5 days/week. Animals were topically treated with TPA 2 hours before sacrifice and the target proteins in the epidermis were analyzed by both immunohistochemistry and Western blot. Under TPA or vehicle treatment, MDM2 expression was significantly reduced in exercised mice when compared with sedentary control. Meanwhile, p53 was significantly elevated. In addition, p53-transcriptioned proteins, i.e., p21, IGFBP-3, and PTEN, increased in response to exercise. There was a synergy effect between exercise and TPA on the decreased MDM2 and increased p53, but not p53-transcripted proteins. Taken together, exercise appeared to activate p53, resulting in enhanced expression of p21, IGFBP-3, and PTEN that might induce a negative regulation of IGF-1 pathway and thus contribute to the observed cancer prevention by exercise in this skin cancer model. PMID:27509024

  3. Gradual reduction in rRNA transcription triggers p53 acetylation and apoptosis via MYBBP1A

    PubMed Central

    Kumazawa, Takuya; Nishimura, Kazuho; Katagiri, Naohiro; Hashimoto, Sayaka; Hayashi, Yuki; Kimura, Keiji

    2015-01-01

    The nucleolus, whose primary function is ribosome biogenesis, plays an essential role in p53 activation. Ribosome biogenesis is inhibited in response to cellular stress and several nucleolar proteins translocate from the nucleolus to the nucleoplasm, where they activate p53. In this study, we analysed precisely how impaired ribosome biogenesis regulates the activation of p53 by depleting nucleolar factors involved in rRNA transcription or rRNA processing. Nucleolar RNA content decreased when rRNA transcription was inhibited. In parallel with the reduced levels of nucleolar RNA content, the nucleolar protein Myb-binding protein 1 A (MYBBP1A) translocated to the nucleoplasm and increased p53 acetylation. The acetylated p53 enhanced p21 and BAX expression and induced apoptosis. In contrast, when rRNA processing was inhibited, MYBBP1A remained in the nucleolus and nonacetylated p53 accumulated, causing cell cycle arrest at the G1 phase by inducing p21 but not BAX. We propose that the nucleolus functions as a stress sensor to modulate p53 protein levels and its acetylation status, determining cell fate between cell cycle arrest and apoptosis by regulating MYBBP1A translocation. PMID:26044764

  4. DNA damage-induced regulatory interplay between DAXX, p53, ATM kinase and Wip1 phosphatase

    PubMed Central

    Brazina, Jan; Svadlenka, Jan; Macurek, Libor; Andera, Ladislav; Hodny, Zdenek; Bartek, Jiri; Hanzlikova, Hana

    2015-01-01

    Death domain-associated protein 6 (DAXX) is a histone chaperone, putative regulator of apoptosis and transcription, and candidate modulator of p53-mediated gene expression following DNA damage. DAXX becomes phosphorylated upon DNA damage, however regulation of this modification, and its relationship to p53 remain unclear. Here we show that in human cells exposed to ionizing radiation or genotoxic drugs etoposide and neocarzinostatin, DAXX became rapidly phosphorylated in an ATM kinase-dependent manner. Our deletion and site-directed mutagenesis experiments identified Serine 564 (S564) as the dominant ATM-targeted site of DAXX, and immunofluorescence experiments revealed localization of S564-phosphorylated DAXX to PML nuclear bodies. Furthermore, using a panel of human cell types, we identified the p53-regulated Wip1 protein phosphatase as a key negative regulator of DAXX phosphorylation at S564, both in vitro and in cells. Consistent with the emerging oncogenic role of Wip1, its DAXX-dephosphorylating impact was most apparent in cancer cell lines harboring gain-of-function mutant and/or overexpressed Wip1. Unexpectedly, while Wip1 depletion increased DAXX phosphorylation both before and after DNA damage and increased p53 stability and transcriptional activity, knock-down of DAXX impacted neither p53 stabilization nor p53-mediated expression of Gadd45a, Noxa, Mdm2, p21, Puma, Sesn2, Tigar or Wip1. Consistently, analyses of cells with genetic, TALEN-mediated DAXX deletion corroborated the notion that neither phosphorylated nor non-phosphorylated DAXX is required for p53-mediated gene expression upon DNA damage. Overall, we identify ATM kinase and Wip1 phosphatase as opposing regulators of DAXX-S564 phosphorylation, and propose that the role of DAXX phosphorylation and DAXX itself are independent of p53-mediated gene expression. PMID:25659035

  5. Cholesterol Secosterol Aldehydes Induce Amyloidogenesis and Dysfunction of Wild Type Tumor Protein p53

    PubMed Central

    Nieva, Jorge; Song, Byeong-Doo; Rogel, Joseph K.; Kujawara, David; Altobel, Lawrence; Izharrudin, Alicia; Boldt, Grant E.; Grover, Rajesh K.; Wentworth, Anita D.; Wentworth, Paul

    2011-01-01

    SUMMARY Epidemiologic and clinical evidence points to an increased risk of cancer when coupled with chronic inflammation. However, the molecular mechanisms that underpin this interrelationship remain largely unresolved. Herein we show that the inflammation-derived cholesterol 5,6-secosterol aldehydes, atheronal-A (KA) and –B (ALD), but not the PUFA-derived aldehydes 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE), induce misfolding of wild-type p53 into an amyloidogenic form that binds thioflavin T and Congo Red dyes but cannot bind to a consensus DNA sequence. Treatment of lung carcinoma cells with KA and ALD leads to a loss of function of extracted p53, as determined by analysis of extracted nuclear protein and in activation of p21. Our results uncover a plausible chemical link between inflammation and cancer and expands the already pivotal role of p53 dysfunction and cancer risk. PMID:21802012

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

    PubMed Central

    Hoang, Kimson; Ankney, John A.; Nguyen, Stephanie T.; Rushing, Amanda W.; Polakowski, Nicholas; Miotto, Benoit; Lemasson, Isabelle

    2016-01-01

    Adult T-cell leukemia (ATL) is an often fatal malignancy caused by infection with the complex retrovirus, human T-cell Leukemia Virus, type 1 (HTLV-1). In ATL patient samples, the tumor suppressor, p53, is infrequently mutated; however, it has been shown to be inactivated by the viral protein, Tax. Here, we show that another HTLV-1 protein, HBZ, represses p53 activity. In HCT116 p53+/+ cells treated with the DNA-damaging agent, etoposide, HBZ reduced p53-mediated activation of p21/CDKN1A and GADD45A expression, which was associated with a delay in G2 phase-arrest. These effects were attributed to direct inhibition of the histone acetyltransferase (HAT) activity of p300/CBP by HBZ, causing a reduction in p53 acetylation, which has be linked to decreased p53 activity. In addition, HBZ bound to, and inhibited the HAT activity of HBO1. Although HBO1 did not acetylate p53, it acted as a coactivator for p53 at the p21/CDKN1A promoter. Therefore, through interactions with two separate HAT proteins, HBZ impairs the ability of p53 to activate transcription. This mechanism may explain how p53 activity is restricted in ATL cells that do not express Tax due to modifications of the HTLV-1 provirus, which accounts for a majority of patient samples. PMID:26625199

  7. IGF-I enhances cellular senescence via the reactive oxygen species-p53 pathway.

    PubMed

    Handayaningsih, Anastasia-Evi; Takahashi, Michiko; Fukuoka, Hidenori; Iguchi, Genzo; Nishizawa, Hitoshi; Yamamoto, Masaaki; Suda, Kentaro; Takahashi, Yutaka

    2012-08-24

    Cellular senescence is characterized by growth arrest, enlarged and flattened cell morphology, the expression of senescence-associated β-galactosidase (SA-β-gal), and by activation of tumor suppressor networks. Insulin-like growth factor-I (IGF-I) plays a critical role in cellular growth, proliferation, tumorigenesis, and regulation of aging. In the present study, we show that IGF-I enhances cellular senescence in mouse, rat, and human primary cells in the confluent state. IGF-I induced expression of a DNA damage marker, γH2AX, the increased levels of p53 and p21 proteins, and activated SA-β-gal. In the confluent state, an altered downstream signaling of IGF-I receptor was observed. Treatment with a reactive oxygen species (ROS) scavenger, N-acetylcystein (NAC) significantly suppressed induction of these markers, indicating that ROS are involved in the induction of cellular senescence by IGF-I. In p53-null mouse embryonic fibroblasts, the IGF-I-induced augmentation of SA-β-gal and p21 was inhibited, demonstrating that p53 is required for cellular senescence induced by IGF-I. Thus, these data reveal a novel pathway whereby IGF-I enhances cellular senescence in the ROS and p53-dependent manner and may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging. PMID:22877754

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

    PubMed Central

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

    2016-01-01

    ABSTRACT Recent evidence indicates that canonical functions of p53 (i.e., apoptosis and growth arrest) are dispensable for p53-mediated tumor suppression. We have uncovered a novel function of p53 that contributes to tumor suppression through regulation of cystine metabolism, reactive oxygen species responses, and ferroptosis. The p53-mediated ferroptotic response via SLC7A11 denotes an extra layer of defense against tumorigenesis in conjunction with other p53 functions. PMID:27314071

  9. Pharmacological Activation of p53 in Cancer Cells

    PubMed Central

    Athar, Mohammad; Elmets, Craig A.; Kopelovich, Levy

    2013-01-01

    Tumor suppressor p53 is a transcription factor that regulates a large number of genes and guards against genomic instability. Under multiple cellular stress conditions, p53 functions to block cell cycle progression transiently unless proper DNA repair occurs. Failure of DNA repair mechanisms leads to p53-mediated induction of cell death programs. p53 also induces permanent cell cycle arrest known as cellular senescence. During neoplastic progression, p53 is often mutated and fails to efficiently perform these functions. It has been observed that cancers carrying a wild-type p53 may also have interrupted downstream p53 regulatory signaling leading to disruption in p53 functions. Therefore, strategies to reactivate p53 provide an attractive approach for blocking tumor pathogenesis and its progression. p53 activation may also lead to regression of existing early neoplastic lesions and therefore may be important in developing cancer chemoprevention protocols. A large number of small molecules capable of reactivating p53 have been developed and some are progressing through clinical trials for prospective human applications. However, several questions remain to be answered at this stage. For example, it is not certain if pharmacological activation of p53 will restore all of its multifaceted biological responses, assuming that the targeted cell is not killed following p53 activation. It remains to be demonstrated whether the distinct biological effects regulated by specific post-transnationally modified p53 can effectively be restored by refolding mutant p53. Mutant p53 can be classified as a loss of function or gain of function protein depending on the type of mutation. It is also unclear whether reactivation of mutant p53 has similar consequences in cells carrying gain-of-function and loss-of-function p53 mutants. This review provides a description of various pharmacological approaches tested to activate p53 (both wild-type and mutant) and to assess the effects of

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

    PubMed

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

    2016-05-01

    Recent evidence indicates that canonical functions of p53 (i.e., apoptosis and growth arrest) are dispensable for p53-mediated tumor suppression. We have uncovered a novel function of p53 that contributes to tumor suppression through regulation of cystine metabolism, reactive oxygen species responses, and ferroptosis. The p53-mediated ferroptotic response via SLC7A11 denotes an extra layer of defense against tumorigenesis in conjunction with other p53 functions. PMID:27314071

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

    PubMed

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

    2009-11-01

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

  12. Targeting Oncogenic Mutant p53 for Cancer Therapy

    PubMed Central

    Parrales, Alejandro; Iwakuma, Tomoo

    2015-01-01

    Among genetic alterations in human cancers, mutations in the tumor suppressor p53 gene are the most common, occurring in over 50% of human cancers. The majority of p53 mutations are missense mutations and result in the accumulation of dysfunctional p53 protein in tumors. These mutants frequently have oncogenic gain-of-function activities and exacerbate malignant properties of cancer cells, such as metastasis and drug resistance. Increasing evidence reveals that stabilization of mutant p53 in tumors is crucial for its oncogenic activities, while depletion of mutant p53 attenuates malignant properties of cancer cells. Thus, mutant p53 is an attractive druggable target for cancer therapy. Different approaches have been taken to develop small-molecule compounds that specifically target mutant p53. These include compounds that restore wild-type conformation and transcriptional activity of mutant p53, induce depletion of mutant p53, inhibit downstream pathways of oncogenic mutant p53, and induce synthetic lethality to mutant p53. In this review article, we comprehensively discuss the current strategies targeting oncogenic mutant p53 in cancers, with special focus on compounds that restore wild-type p53 transcriptional activity of mutant p53 and those reducing mutant p53 levels. PMID:26732534

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

    PubMed Central

    Vilborg, Anna; Glahder, Jacob A.; Wilhelm, Margareta T.; Bersani, Cinzia; Corcoran, Martin; Mahmoudi, Salah; Rosenstierne, Maiken; Grandér, Dan; Farnebo, Marianne; Norrild, Bodil; Wiman, Klas G.

    2009-01-01

    The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3′ UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link between the tumor suppressor p53 and posttranscriptional gene regulation via AREs in mRNA. PMID:19805223

  14. Transcriptional Activation of p53 during Cold Induced Torpor in the 13-Lined Ground Squirrel Ictidomys tridecemlineatus

    PubMed Central

    Hefler, Joshua; Wu, Cheng-Wei; Storey, Kenneth B.

    2015-01-01

    The transcription factor p53 is located at the centre of multiple pathways relating the cellular response to stress. Commonly known as a tumor suppressor, it is responsible for initiating diverse actions to protect the integrity of the genome, ranging from cell cycle arrest to apoptosis. This study investigated the regulation of p53 protein in hibernating 13-lined ground squirrel Ictidomys tridecemlineatus during multiple stages of the torpor-arousal cycle. Transcript and protein levels of p53 were both elevated in the skeletal muscle during early and late torpor stages of the hibernation cycle. Nuclear localization of p53 was also increased during late torpor, and this is associated with an increase in its DNA binding activity and expression of p53 transcriptional targets p21CIP, gadd45α, and 14-3-3σ. The increase in p53 transcriptional activity appears to be independent of its phosphorylation at Ser-15, Ser-46, and Ser-392, consistent with an absence of checkpoint kinase activation during torpor. Sequence analysis revealed unique amino acid substitutions in the ground squirrel p53 protein, which may contribute to an increase in protein stability compared to nonhibernators. Overall, the study results provided evidences for a potential role of p53 in the protection of the skeletal muscle during torpor. PMID:26843984

  15. Transcriptional Activation of p53 during Cold Induced Torpor in the 13-Lined Ground Squirrel Ictidomys tridecemlineatus.

    PubMed

    Hefler, Joshua; Wu, Cheng-Wei; Storey, Kenneth B

    2015-01-01

    The transcription factor p53 is located at the centre of multiple pathways relating the cellular response to stress. Commonly known as a tumor suppressor, it is responsible for initiating diverse actions to protect the integrity of the genome, ranging from cell cycle arrest to apoptosis. This study investigated the regulation of p53 protein in hibernating 13-lined ground squirrel Ictidomys tridecemlineatus during multiple stages of the torpor-arousal cycle. Transcript and protein levels of p53 were both elevated in the skeletal muscle during early and late torpor stages of the hibernation cycle. Nuclear localization of p53 was also increased during late torpor, and this is associated with an increase in its DNA binding activity and expression of p53 transcriptional targets p21CIP, gadd45α, and 14-3-3σ. The increase in p53 transcriptional activity appears to be independent of its phosphorylation at Ser-15, Ser-46, and Ser-392, consistent with an absence of checkpoint kinase activation during torpor. Sequence analysis revealed unique amino acid substitutions in the ground squirrel p53 protein, which may contribute to an increase in protein stability compared to nonhibernators. Overall, the study results provided evidences for a potential role of p53 in the protection of the skeletal muscle during torpor. PMID:26843984

  16. Iron Metabolism Regulates p53 Signaling through Direct Heme-p53 Interaction and Modulation of p53 Localization, Stability, and Function

    PubMed Central

    Shen, Jia; Sheng, Xiangpeng; Chang, ZeNan; Wu, Qian; Wang, Sheng; Xuan, Zongliang; Li, Dan; Wu, Yalan; Shang, Yongjia; Kong, Xiangtao; Yu, Long; Li, Lin; Ruan, Kangchen; Hu, Hongyu; Huang, Ying; Hui, Lijian; Xie, Dong; Wang, Fudi; Hu, Ronggui

    2014-01-01

    SUMMARY Iron excess is closely associated with tumorigenesis in multiple types of human cancers, with underlying mechanisms yet unclear. Recently, iron deprivation has emerged as a major strategy for chemotherapy, but it exerts tumor suppression only on select human malignancies. Here, we report that the tumor suppressor protein p53 is downregulated during iron excess. Strikingly, the iron polyporphyrin heme binds to p53 protein, interferes with p53-DNA interactions, and triggers both nuclear export and cytosolic degradation of p53. Moreover, in a tumorigenicity assay, iron deprivation suppressed wild-type p53-dependent tumor growth, suggesting that upregulation of wild-type p53 signaling underlies the selective efficacy of iron deprivation. Our findings thus identify a direct link between iron/heme homeostasis and the regulation of p53 signaling, which not only provides mechanistic insights into iron-excess-associated tumorigenesis but may also help predict and improve outcomes in iron-deprivation-based chemotherapy. PMID:24685134

  17. The dichotomy of p53 regulation by noncoding RNAs.

    PubMed

    Deng, Qipan; Becker, Lindsey; Ma, Xiaodong; Zhong, Xiaoming; Young, Ken; Ramos, Kenneth; Li, Yong

    2014-06-01

    The p53 tumor suppressor gene is the most frequently mutated gene in cancer. Significant progress has been made to discern the importance of p53 in coordinating cellular responses to DNA damage, oncogene activation, and other stresses. Noncoding RNAs are RNA molecules functioning without being translated into proteins. In this work, we discuss the dichotomy of p53 regulation by noncoding RNAs with four unconventional questions. First, is overexpression of microRNAs responsible for p53 inactivation in the absence of p53 mutation? Second, are there somatic mutations in the noncoding regions of the p53 gene? Third, is there a germline mutant in the noncoding regions of the p53 gene that predisposes carriers to cancer? Fourth, can p53 activation mediated by a noncoding RNA mutation cause cancer? This work highlights the prominence of noncoding RNAs in p53 dysregulation and tumorigenesis. PMID:24706938

  18. P53 licensed to kill? Operating the assassin.

    PubMed

    Haupt, Susan; Louria-Hayon, Igal; Haupt, Ygal

    2003-01-01

    The p53 protein is a key player in the cellular response to stress. Proper regulation of p53 is imperative for the suppression of tumor development. This regulation is largely governed by its master inhibitor, Mdm2, which both blocks p53 activities and promotes its destabilization. This tight regulation of p53 by Mdm2 must be interrupted under stress conditions in order for p53 to be stabilized in an active form. A combined action of partner proteins and modifying enzymes is essential for the relief of p53 from Mdm2. The recent revelation of p53 association with the PML-nuclear bodies provides one explanation of how this regulatory network is coordinated within the nucleus in response to certain stress conditions. Thus, it is not only the nature of the p53 regulatory complex but also the spatial and temporal context of this association that governs the output inhibitory signals mediated by p53. PMID:12461776

  19. 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

  20. Distinct phosphatases antagonize the p53 response in different phases of the cell cycle.

    PubMed

    Shaltiel, Indra A; Aprelia, Melinda; Saurin, Adrian T; Chowdhury, Dipanjan; Kops, Geert J P L; Voest, Emile E; Medema, René H

    2014-05-20

    The basic machinery that detects DNA damage is the same throughout the cell cycle. Here, we show, in contrast, that reversal of DNA damage responses (DDRs) and recovery are fundamentally different in G1 and G2 phases of the cell cycle. We find that distinct phosphatases are required to counteract the checkpoint response in G1 vs. G2. Whereas WT p53-induced phosphatase 1 (Wip1) promotes recovery in G2-arrested cells by antagonizing p53, it is dispensable for recovery from a G1 arrest. Instead, we identify phosphoprotein phosphatase 4 catalytic subunit (PP4) to be specifically required for cell cycle restart after DNA damage in G1. PP4 dephosphorylates Krüppel-associated box domain-associated protein 1-S473 to repress p53-dependent transcriptional activation of p21 when the DDR is silenced. Taken together, our results show that PP4 and Wip1 are differentially required to counteract the p53-dependent cell cycle arrest in G1 and G2, by antagonizing early or late p53-mediated responses, respectively. PMID:24711418

  1. 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

  2. Discovery of new low-molecular-weight p53-Mdmx disruptors and their anti-cancer activities.

    PubMed

    Uesato, Shinichi; Matsuura, Yoshihiro; Matsue, Saki; Sumiyoshi, Takaaki; Hirata, Yoshiyuki; Takemoto, Suzuho; Kawaratani, Yasuyuki; Yamai, Yusuke; Ishida, Kyoji; Sasaki, Tsutomu; Enari, Masato

    2016-04-15

    Although several p53-Mdm2-binding disruptors have been identified to date, few studies have been published on p53-Mdmx-interaction inhibitors. In the present study, we demonstrated that o-aminothiophenol derivatives with molecular weights of 200-300 selectively inhibited the p53-Mdmx interaction. S-2-Isobutyramidophenyl 2-methylpropanethioate (K-178) (1c) activated p53, up-regulated the expression of its downstream genes such as p21 and Mdm2, and preferentially inhibited the growth of cancer cells with wild-type p53 over those with mutant p53. Furthermore, we found that the S-isobutyryl-deprotected forms 1b and 3b of 1c and S-2-benzamidophenyl 2-methylpropanethioate (K-181) (3c) preferentially inhibited the p53-Mdmx interaction over the p53-Mdm2 interaction, respectively, by using a Flag-p53 and glutathione S-transferase (GST)-fused protein complex (Mdm2, Mdmx, DAPK1, or PPID). In addition, the interaction of p53 with Mdmx was lost by replacing a sulfur atom with an oxygen atom in 1b and 1c. These results suggest that sulfides such as 1b, 3b, 4b, and 5b interfere with the binding of p53-Mdmx, resulting in the dissociation of the two proteins. Furthermore, the results of oral administration experiments using xenografts in nude mice indicated that 1c reduced the volume of tumor masses to 49.0% and 36.6% that of the control at 100mg/kg and 150mg/kg, respectively, in 40days. PMID:27010502

  3. p53 independent G0/G1 arrest and apoptosis induced by a novel retinoid in human breast cancer cells.

    PubMed

    Shao, Z M; Dawson, M I; Li, X S; Rishi, A K; Sheikh, M S; Han, Q X; Ordonez, J V; Shroot, B; Fontana, J A

    1995-08-01

    The biological activity of a novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) was investigated in human breast carcinoma (HBC) cells. Although capable of selective binding to the RAR gamma nuclear receptor, AHPN inhibited the growth of a number of HBC cell lines via RAR- or RXR-independent pathways. AHPN also inhibited the growth of the human leukemia cell line HL-60R which does not possess functional RARs. RA significantly inhibited AP-1 mediated gene activation in MCF-7 cells while AHPN displayed no such anti-AP-1 activity. Retinoids normally are cytostatic in their inhibition of breast carcinoma growth and permit cell proliferation upon their removal, wher as AHPN induced G0/G1 arrest within 6h followed by apoptosis. In MCF-7 cells that harbor wild type p53, AHPN-induced G0/G1 arrest and apoptosis was accompanied by p53-independent regulation of WAF1/CIP1 as well as bax mRNA levels while bcl-2 mRNA levels were decreased. In MDA-MB-231 cells which possess a mutant p53, AHPN-mediated G0/G1 arrest and apoptosis was also associated with a concomitant up regulation of WAF1/CIP1 mRNA while these cells did not express bax or bcl-2 messages. Thus AHPN represents a novel retinoid that induces G0/G1 arrest and apoptosis via a unique pathway which appears to involve activation of known downstream effectors of p53 in a p53-independent manner. PMID:7630633

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

    SciTech Connect

    Dai, Jiawen; Itahana, Koji; Baskar, Rajamanickam

    2015-02-27

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

  5. Daxx cooperates with the Axin/HIPK2/p53 complex to induce cell death.

    PubMed

    Li, Qinxi; Wang, Xuan; Wu, Xiaoling; Rui, Yanning; Liu, Wei; Wang, Jifeng; Wang, Xinghao; Liou, Yih-Cherng; Ye, Zhiyun; Lin, Sheng-Cai

    2007-01-01

    Daxx, a death domain-associated protein, has been implicated in proapoptosis, antiapoptosis, and transcriptional regulation. Many factors known to play critically important roles in controlling apoptosis and gene transcription have been shown to associate with Daxx, including the Ser/Thr protein kinase HIPK2, promyelocytic leukemia protein, histone deacetylases, and the chromatin remodeling protein ATRX. Although it is clear that Daxx may exert multiple functions, the underlying mechanisms remain far from clear. Here, we show that Axin, originally identified for its scaffolding role to control beta-catenin levels in Wnt signaling, strongly associates with Daxx at endogenous levels. The Daxx/Axin complex formation is enhanced by UV irradiation. Axin tethers Daxx to the tumor suppressor p53, and cooperates with Daxx, but not DaxxDeltaAxin, which is unable to interact with Axin, to stimulate HIPK2-mediated Ser(46) phosphorylation and transcriptional activity of p53. Interestingly, Axin and Daxx seem to selectively activate p53 target genes, with strong activation of PUMA, but not p21 or Bax. Daxx-stimulated p53 transcriptional activity was significantly diminished by small interfering RNA against Axin; Daxx fails to inhibit colony formation in Axin(-/-) cells. Moreover, UV-induced cell death was attenuated by the knockdown of Axin and Daxx. All these results show that Daxx cooperates with Axin to stimulate p53, and implicate a direct role for Axin, HIPK2, and p53 in the proapoptotic function of Daxx. We have hence unraveled a novel aspect of p53 activation and shed new light on the ultimate understanding of the Daxx protein, perhaps most pertinently, in relation to stress-induced cell death. PMID:17210684

  6. The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis

    SciTech Connect

    Tsujita, Maristela; Batista, Wagner L.; Ogata, Fernando T.; Monteiro, Hugo P. Arai, Roberto J.

    2008-05-16

    p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras{sup C118S}) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG.

  7. The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis.

    PubMed

    Tsujita, Maristela; Batista, Wagner L; Ogata, Fernando T; Stern, Arnold; Monteiro, Hugo P; Arai, Roberto J

    2008-05-16

    p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras(C118S)) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG. PMID:18325324

  8. mTORC1 and p53

    PubMed Central

    Hasty, Paul; Sharp, Zelton Dave; Curiel, Tyler J.; Campisi, Judith

    2013-01-01

    A balance must be struck between cell growth and stress responses to ensure that cells proliferate without accumulating damaged DNA. This balance means that optimal cell proliferation requires the integration of pro-growth and stress-response pathways. mTOR (mechanistic target of rapamycin) is a pleiotropic kinase found in complex 1 (mTORC1). The mTORC1 pathway governs a response to mitogenic signals with high energy levels to promote protein synthesis and cell growth. In contrast, the p53 DNA damage response pathway is the arbiter of cell proliferation, restraining mTORC1 under conditions of genotoxic stress. Recent studies suggest a complicated integration of these pathways to ensure successful cell growth and proliferation without compromising genome maintenance. Deciphering this integration could be key to understanding the potential clinical usefulness of mTORC1 inhibitors like rapamycin. Here we discuss how these p53-mTORC1 interactions might play a role in the suppression of cancer and perhaps the development of cellular senescence and organismal aging. PMID:23255104

  9. High levels of p53 protein expression do not correlate with p53 gene mutations in anaplastic large cell lymphoma.

    PubMed Central

    Cesarman, E.; Inghirami, G.; Chadburn, A.; Knowles, D. M.

    1993-01-01

    Strong immunohistochemical reactivity for p53 tumor suppressor gene product has been reported in a variety of different human malignancies including CD30- (Ki-1) positive anaplastic large cell lymphoma (ALCL). Although high levels of p53 protein have been interpreted as abnormal, rapidly proliferating benign and neoplastic lymphoid cells may have increased p53 expression in the absence of structural alterations. On the other hand, mutations in the p53 gene can lead to a lack of p53 protein production. Structural alterations of the p53 gene have not been documented in cases of ALCL and the mechanism for an abnormal pattern of p53 expression in these lymphomas has not been elucidated. Therefore, to determine whether an altered pattern of p53 expression correlates with mutations in the p53 locus in ALCL, we analyzed the expression of p53 protein immunohistochemically, compared it with the proliferation index using monoclonal antibody Ki-67, and assessed the presence of mutations in exons 5 though 9 of the p53 gene using a single-strand conformation polymorphism assay in a panel of 17 ALCLs. Furthermore, we studied the presence of allelic deletions of chromosome 17p by restriction fragment length polymorphism analysis. We found significant levels of p53 protein expression in 12 of the 15 cases studied, but identified mutations in only one of 17 cases. An allelic deletion in chromosome 17p was identified only in the one case containing a mutated p53 gene. Whereas the case containing structural alterations in the p53 gene did have strong p53 immunoreactivity, 11 cases that lacked p53 mutations in the regions examined also had significant levels of p53. Thus, our studies indicate that strong immunohistochemical reactivity for p53 is not a reliable indicator of the presence of structural alterations of p53 gene exons 5 through 9 in ALCL. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8103295

  10. Inhibition of cyclin-dependent kinases by p21.

    PubMed Central

    Harper, J W; Elledge, S J; Keyomarsi, K; Dynlacht, B; Tsai, L H; Zhang, P; Dobrowolski, S; Bai, C; Connell-Crowley, L; Swindell, E

    1995-01-01

    p21Cip1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. We have explored the interaction of p21 with the currently known Cdks. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5-15 nM) but is much less effective toward Cdc2/cyclin B (Ki approximately 400 nM) and Cdk5/p35 (Ki > 2 microM), and does not associate with Cdk7/cyclin H. Overexpression of P21 arrests cells in G1. Thus, p21 is not a universal inhibitor of Cdks but displays selectivity for G1/S Cdk/cyclin complexes. Association of p21 with Cdks is greatly enhanced by cyclin binding. This property is shared by the structurally related inhibitor p27, suggesting a common biochemical mechanism for inhibition. With respect to Cdk2 and Cdk4 complexes, p27 shares the inhibitory potency of p21 but has slightly different kinase specificities. In normal diploid fibroblasts, the vast majority of active Cdk2 is associated with p21, but this active kinase can be fully inhibited by addition of exogenous p21. Reconstruction experiments using purified components indicate that multiple molecules of p21 can associate with Cdk/cyclin complexes and inactive complexes contain more than one molecule of p21. Together, these data suggest a model whereby p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression. Images PMID:7626805

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2016-01-01

    p53 function is frequently inhibited in cancer either through mutations or by increased degradation via MDM2 and/or E6AP E3-ubiquitin ligases. Most agents that restore p53 expression act by binding MDM2 or E6AP to prevent p53 degradation. However, fewer compounds directly bind to and activate p53. Here, we identified compounds that shared a core structure that bound p53, caused nuclear localization of p53 and caused cell death. To identify these compounds, we developed a novel cell-based screen to redirect p53 degradation to the Skip-Cullin-F-box (SCF) ubiquitin ligase complex in cells expressing high levels of p53. In a multiplexed assay, we coupled p53 targeted degradation with Rb1 targeted degradation in order to identify compounds that prevented p53 degradation while not inhibiting degradation through the SCF complex or other proteolytic machinery. High-throughput screening identified several leads that shared a common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that stabilized p53. Surface plasmon resonance analysis indicated that these compounds bound p53 with a KD of 200 ± 52 nM. Furthermore, these compounds increased p53 nuclear localization and transcription of the p53 target genes PUMA, BAX, p21 and FAS in cancer cells. Although p53-null cells had a 2.5±0.5-fold greater viability compared to p53 wild type cells after treatment with core compounds, loss of p53 did not completely rescue cell viability suggesting that compounds may target both p53-dependent and p53-independent pathways to inhibit cell proliferation. Thus, we present a novel, cell-based high-throughput screen to identify a 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that bound to p53 and increased p53 activity in cancer cells. These compounds may serve as anti-neoplastic agents in part by targeting p53 as well as other potential pathways. PMID:27124407

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

    PubMed Central

    Bechill, John; Zhong, Rong; Zhang, Chen; Solomaha, Elena

    2016-01-01

    p53 function is frequently inhibited in cancer either through mutations or by increased degradation via MDM2 and/or E6AP E3-ubiquitin ligases. Most agents that restore p53 expression act by binding MDM2 or E6AP to prevent p53 degradation. However, fewer compounds directly bind to and activate p53. Here, we identified compounds that shared a core structure that bound p53, caused nuclear localization of p53 and caused cell death. To identify these compounds, we developed a novel cell-based screen to redirect p53 degradation to the Skip-Cullin-F-box (SCF) ubiquitin ligase complex in cells expressing high levels of p53. In a multiplexed assay, we coupled p53 targeted degradation with Rb1 targeted degradation in order to identify compounds that prevented p53 degradation while not inhibiting degradation through the SCF complex or other proteolytic machinery. High-throughput screening identified several leads that shared a common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that stabilized p53. Surface plasmon resonance analysis indicated that these compounds bound p53 with a KD of 200 ± 52 nM. Furthermore, these compounds increased p53 nuclear localization and transcription of the p53 target genes PUMA, BAX, p21 and FAS in cancer cells. Although p53-null cells had a 2.5±0.5-fold greater viability compared to p53 wild type cells after treatment with core compounds, loss of p53 did not completely rescue cell viability suggesting that compounds may target both p53-dependent and p53-independent pathways to inhibit cell proliferation. Thus, we present a novel, cell-based high-throughput screen to identify a 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that bound to p53 and increased p53 activity in cancer cells. These compounds may serve as anti-neoplastic agents in part by targeting p53 as well as other potential pathways. PMID:27124407

  14. Implication of K-ras and p53 in colorectal cancer carcinogenesis in Tunisian population cohort.

    PubMed

    Ines, Chaar; Donia, Ounissi; Rahma, Boughriba; Ben Ammar, Azza; Sameh, Amara; Khalfallah, Taher; Abdelmajid, Ben Hmida; Sabeh, Mzabi; Saadia, Bouraoui

    2014-07-01

    According to the multistep route of genetic alterations in the colorectal adenoma-carcinoma sequence, the complex K-ras/p53 mutation is one of the first alterations to occur and represent an important genetic event in colorectal cancer (CRC). An evaluation of the mutation spectra in K-ras and p53 gene was effected in 167 Tunisian patients with sporadic CRC to determine whether our populations have similar pattern of genetic alteration as in Maghrebin's population. Mutation patterns of codon 12-13 of K-ras and exon 5-8 of p53 were analyzed by immunohistochemistry and PCR-SSCP and confirmed by sequencing. Mutations in the K-ras gene were detected in 31.13 % and affect the women more than the men (p = 0.008). Immunostaining showed that expression of p21 ras was correlated with the advanced age (p = 0.004), whereas loss of signal was associated with mucinous histotype (p = 0.003). Kaplan-Meier survival curve found that patients with the K-ras mutation had a shorter survival compared with patients without mutation (p = 0.005). Alteration in p53 was seen in 17.4 % of patients and affects three hot spot codons such as 175, 245, and 248. Overexpression of p53 was seen in 34.1 % and correlated with tumor node metastasis (TNM) advanced stage (p = 0.037) and mucinous histotype (p = 0.001). A high concordance between p53 expression and alteration (p<0.005) was shown. Concomitant mutations in K-ras and p53 gene were detected in only 4 % of tumors. K-ras and p53 undergo separate pathways in colorectal tumorogenesis. Interestingly, mutations in the K-ras gene might be considered a valuable prognostic factor correlated to poor outcome. p53 gene alterations were rather low in our set, and methylation pattern of p53 is required to elucidate the molecular basis of this protein in CRC. PMID:24763823

  15. IGF-I enhances cellular senescence via the reactive oxygen species-p53 pathway

    SciTech Connect

    Handayaningsih, Anastasia-Evi; Takahashi, Michiko; Fukuoka, Hidenori; Iguchi, Genzo; Nishizawa, Hitoshi; Yamamoto, Masaaki; Suda, Kentaro; Takahashi, Yutaka

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Cellular senescence plays an important role in tumorigenesis and aging process. Black-Right-Pointing-Pointer We demonstrated IGF-I enhanced cellular senescence in primary confluent cells. Black-Right-Pointing-Pointer IGF-I enhanced cellular senescence in the ROS and p53-dependent manner. Black-Right-Pointing-Pointer These results may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging. -- Abstract: Cellular senescence is characterized by growth arrest, enlarged and flattened cell morphology, the expression of senescence-associated {beta}-galactosidase (SA-{beta}-gal), and by activation of tumor suppressor networks. Insulin-like growth factor-I (IGF-I) plays a critical role in cellular growth, proliferation, tumorigenesis, and regulation of aging. In the present study, we show that IGF-I enhances cellular senescence in mouse, rat, and human primary cells in the confluent state. IGF-I induced expression of a DNA damage marker, {gamma}H2AX, the increased levels of p53 and p21 proteins, and activated SA-{beta}-gal. In the confluent state, an altered downstream signaling of IGF-I receptor was observed. Treatment with a reactive oxygen species (ROS) scavenger, N-acetylcystein (NAC) significantly suppressed induction of these markers, indicating that ROS are involved in the induction of cellular senescence by IGF-I. In p53-null mouse embryonic fibroblasts, the IGF-I-induced augmentation of SA-{beta}-gal and p21 was inhibited, demonstrating that p53 is required for cellular senescence induced by IGF-I. Thus, these data reveal a novel pathway whereby IGF-I enhances cellular senescence in the ROS and p53-dependent manner and may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging.

  16. Activation and activities of the p53 tumour suppressor protein

    PubMed Central

    Bálint, É; Vousden, K H

    2001-01-01

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

  17. Watching the watcher: regulation of p53 by mitochondria

    PubMed Central

    Holley, Aaron K; St Clair, Daret K

    2009-01-01

    p53 has been referred to as the ‘guardian of the genome’ because of its role in protecting the cell from DNA damage. p53 performs its duties by regulating cell-cycle progression and DNA repair and, in cases of irreparable DNA damage, by executing programmed cell death. Mitochondria are an important target of transcription-dependent and -independent actions of p53 to carry out the apoptotic function. However, increasing evidence suggests that p53 activity is regulated by mitochondria. Cellular insults that alter mitochondrial function can have important consequences on p53 activity. In light of these new findings, the following review focuses on p53/mitochondria connections, in particular how reactive oxygen species generated at mitochondria regulate p53 activity. A better understanding of the mechanisms by which mitochondria regulate p53 may have an impact on our understanding of the development and progression of many diseases, especially cancer. PMID:19243304

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

    PubMed Central

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

    2015-01-01

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

  19. Wild type p53 reactivation: from lab bench to clinic.

    PubMed

    Selivanova, Galina

    2014-08-19

    The p53 tumor suppressor is the most frequently inactivated gene in cancer. Several mouse models have demonstrated that the reconstitution of the p53 function suppresses the growth of established tumors. These facts, taken together, promote the idea of p53 reactivation as a strategy to combat cancer. This review will focus on recent advances in the development of small molecules which restore the function of wild type p53 by blocking its inhibitors Mdm2 and MdmX or their upstream regulators and discuss the impact of different p53 functions for tumor prevention and tumor eradication. Finally, the recent progress in p53 research will be analyzed concerning the role of p53 cofactors and cellular environment in the biological response upon p53 reactivation and how this can be applied in clinic. PMID:24726725

  20. In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene.

    PubMed

    Wolf, D; Laver-Rudich, Z; Rotter, V

    1985-08-01

    The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53-specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription-translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-H7 and the cloned p53 gene indicated the presence of seven major exons. PMID:3018534

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  3. Silencing of protein kinase D2 induces glioma cell senescence via p53-dependent and -independent pathways

    PubMed Central

    Bernhart, Eva; Damm, Sabine; Heffeter, Petra; Wintersperger, Andrea; Asslaber, Martin; Frank, Saša; Hammer, Astrid; Strohmaier, Heimo; DeVaney, Trevor; Mrfka, Manuel; Eder, Hans; Windpassinger, Christian; Ireson, Christopher R.; Mischel, Paul S.; Berger, Walter; Sattler, Wolfgang

    2014-01-01

    Background Glioblastoma multiforme (GBM) is a highly aggressive tumor of the central nervous system with a dismal prognosis for affected patients. Aberrant protein kinase C (PKC) signaling has been implicated in gliomagenesis, and a member of the PKC-activated protein kinase D (PRKD) family, PRKD2, was identified as mediator of GBM growth in vitro and in vivo. Methods The outcome of PRKD2 silencing and pharmacological inhibition on glioma cell proliferation was established with different glioma cell lines. Western blotting, senescence assays, co-immunoprecipitation, fluorescence activated cell sorting, quantitative PCR, and immunofluorescence microscopy were utilized to analyze downstream signaling. Results RNA-interference (21-mer siRNA) and pharmacological inhibition (CRT0066101) of PRKD2 profoundly inhibited proliferation of p53wt (U87MG, A172, and primary GBM2), and p53mut (GM133, T98G, U251, and primary Gli25) glioma cells. In a xenograft experiment, PRKD2 silencing significantly delayed tumor growth of U87MG cells. PRKD2 silencing in p53wt and p53mut cells was associated with typical hallmarks of senescence and cell cycle arrest in G1. Attenuated AKT/PKB phosphorylation in response to PRKD2 silencing was a common observation made in p53wt and p53mut GBM cells. PRKD2 knockdown in p53wt cells induced upregulation of p53, p21, and p27 expression, decreased phosphorylation of CDK2 and/or CDK4, hypophosphorylation of retinoblastoma protein (pRb), and reduced transcription of E2F1. In p53mut GM133 and primary Gli25 cells, PRKD2 silencing increased p27 and p15 and reduced E2F1 transcription but did not affect pRb phosphorylation. Conclusions PRKD2 silencing induces glioma cell senescence via p53-dependent and -independent pathways. PMID:24463355

  4. Identification of p53-target genes in Danio rerio

    PubMed Central

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

    2016-01-01

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

  5. Identification of p53-target genes in Danio rerio.

    PubMed

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

    2016-01-01

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

  6. Repression of p53-target gene Bbc3/PUMA by MYSM1 is essential for the survival of hematopoietic multipotent progenitors and contributes to stem cell maintenance.

    PubMed

    Belle, J I; Petrov, J C; Langlais, D; Robert, F; Cencic, R; Shen, S; Pelletier, J; Gros, P; Nijnik, A

    2016-05-01

    p53 is a central mediator of cellular stress responses, and its precise regulation is essential for the normal progression of hematopoiesis. MYSM1 is an epigenetic regulator essential for the maintenance of hematopoietic stem cell (HSC) function, hematopoietic progenitor survival, and lymphocyte development. We recently demonstrated that all developmental and hematopoietic phenotypes of Mysm1 deficiency are p53-mediated and rescued in the Mysm1(-/-)p53(-/-) mouse model. However, the mechanisms triggering p53 activation in Mysm1(-/-) HSPCs, and the pathways downstream of p53 driving different aspects of the Mysm1(-/-) phenotype remain unknown. Here we show the transcriptional activation of p53 stress responses in Mysm1(-/-) HSPCs. Mechanistically, we find that the MYSM1 protein associates with p53 and colocalizes to promoters of classical p53-target genes Bbc3/PUMA (p53 upregulated modulator of apoptosis) and Cdkn1a/p21. Furthermore, it antagonizes their p53-driven expression by modulating local histone modifications (H3K27ac and H3K4me3) and p53 recruitment. Using double-knockout mouse models, we establish that PUMA, but not p21, is an important mediator of p53-driven Mysm1(-/-) hematopoietic dysfunction. Specifically, Mysm1(-/-)Puma(-/-) mice show full rescue of multipotent progenitor (MPP) viability, partial rescue of HSC quiescence and function, but persistent lymphopenia. Through transcriptome analysis of Mysm1(-/-)Puma(-/-) MPPs, we demonstrate strong upregulation of other p53-induced mediators of apoptosis and cell-cycle arrest. The full viability of Mysm1(-/-)Puma(-/-) MPPs, despite strong upregulation of many other pro-apoptotic mediators, establishes PUMA as the essential non-redundant effector of p53-induced MPP apoptosis. Furthermore, we identify potential mediators of p53-dependent but PUMA-independent Mysm1(-/-)hematopoietic deficiency phenotypes. Overall, our study provides novel insight into the cell-type-specific roles of p53 and its downstream

  7. Repression of p53-target gene Bbc3/PUMA by MYSM1 is essential for the survival of hematopoietic multipotent progenitors and contributes to stem cell maintenance

    PubMed Central

    Belle, J I; Petrov, J C; Langlais, D; Robert, F; Cencic, R; Shen, S; Pelletier, J; Gros, P; Nijnik, A

    2016-01-01

    p53 is a central mediator of cellular stress responses, and its precise regulation is essential for the normal progression of hematopoiesis. MYSM1 is an epigenetic regulator essential for the maintenance of hematopoietic stem cell (HSC) function, hematopoietic progenitor survival, and lymphocyte development. We recently demonstrated that all developmental and hematopoietic phenotypes of Mysm1 deficiency are p53-mediated and rescued in the Mysm1−/−p53−/− mouse model. However, the mechanisms triggering p53 activation in Mysm1−/− HSPCs, and the pathways downstream of p53 driving different aspects of the Mysm1−/− phenotype remain unknown. Here we show the transcriptional activation of p53 stress responses in Mysm1−/− HSPCs. Mechanistically, we find that the MYSM1 protein associates with p53 and colocalizes to promoters of classical p53-target genes Bbc3/PUMA (p53 upregulated modulator of apoptosis) and Cdkn1a/p21. Furthermore, it antagonizes their p53-driven expression by modulating local histone modifications (H3K27ac and H3K4me3) and p53 recruitment. Using double-knockout mouse models, we establish that PUMA, but not p21, is an important mediator of p53-driven Mysm1−/− hematopoietic dysfunction. Specifically, Mysm1−/−Puma−/− mice show full rescue of multipotent progenitor (MPP) viability, partial rescue of HSC quiescence and function, but persistent lymphopenia. Through transcriptome analysis of Mysm1−/−Puma−/− MPPs, we demonstrate strong upregulation of other p53-induced mediators of apoptosis and cell-cycle arrest. The full viability of Mysm1−/−Puma−/− MPPs, despite strong upregulation of many other pro-apoptotic mediators, establishes PUMA as the essential non-redundant effector of p53-induced MPP apoptosis. Furthermore, we identify potential mediators of p53-dependent but PUMA-independent Mysm1−/−hematopoietic deficiency phenotypes. Overall, our study provides novel insight into the cell-type-specific roles of p

  8. Computational identification of a transiently open L1/S3 pocket for reactivation of mutant p53

    PubMed Central

    Wassman, Christopher D.; Baronio, Roberta; Demir, Özlem; Wallentine, Brad D.; Chen, Chiung-Kuang; Hall, Linda V.; Salehi, Faezeh; Lin, Da-Wei; Chung, Benjamin P.; Wesley Hatfield, G.; Richard Chamberlin, A.; Luecke, Hartmut; Lathrop, Richard H.; Kaiser, Peter; Amaro, Rommie E.

    2013-01-01

    The tumour suppressor p53 is the most frequently mutated gene in human cancer. Reactivation of mutant p53 by small molecules is an exciting potential cancer therapy. Although several compounds restore wild-type function to mutant p53, their binding sites and mechanisms of action are elusive. Here computational methods identify a transiently open binding pocket between loop L1 and sheet S3 of the p53 core domain. Mutation of residue Cys124, located at the centre of the pocket, abolishes p53 reactivation of mutant R175H by PRIMA-1, a known reactivation compound. Ensemble-based virtual screening against this newly revealed pocket selects stictic acid as a potential p53 reactivation compound. In human osteosarcoma cells, stictic acid exhibits dose-dependent reactivation of p21 expression for mutant R175H more strongly than does PRIMA-1. These results indicate the L1/S3 pocket as a target for pharmaceutical reactivation of p53 mutants. PMID:23360998

  9. p53 Tumor Suppressor Protein Stability and Transcriptional Activity Are Targeted by Kaposi's Sarcoma-Associated Herpesvirus-Encoded Viral Interferon Regulatory Factor 3

    PubMed Central

    Baresova, Petra; Musilova, Jana; Pitha, Paula M.

    2014-01-01

    Viruses have developed numerous strategies to counteract the host cell defense. Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus linked to the development of Kaposi's sarcoma, Castleman's disease, and primary effusion lymphoma (PEL). The virus-encoded viral interferon regulatory factor 3 (vIRF-3) gene is a latent gene which is involved in the regulation of apoptosis, cell cycle, antiviral immunity, and tumorigenesis. vIRF-3 was shown to interact with p53 and inhibit p53-mediated apoptosis. However, the molecular mechanism underlying this phenomenon has not been established. Here, we show that vIRF-3 associates with the DNA-binding domain of p53, inhibits p53 phosphorylation on serine residues S15 and S20, and antagonizes p53 oligomerization and the DNA-binding affinity. Furthermore, vIRF-3 destabilizes p53 protein by increasing the levels of p53 polyubiquitination and targeting p53 for proteasome-mediated degradation. Consequently, vIRF-3 attenuates p53-mediated transcription of the growth-regulatory p21 gene. These effects of vIRF-3 are of biological relevance since the knockdown of vIRF-3 expression in KSHV-positive BC-3 cells, derived from PEL, leads to an increase in p53 phosphorylation, enhancement of p53 stability, and activation of p21 gene transcription. Collectively, these data suggest that KSHV evolved an efficient mechanism to downregulate p53 function and thus facilitate uncontrolled cell proliferation and tumor growth. PMID:24248600

  10. Cellular adaptation to hypoxia and p53 transcription regulation.

    PubMed

    Zhao, Yang; Chen, Xue-qun; Du, Ji-zeng

    2009-05-01

    Tumor suppressor p53 is the most frequently mutated gene in human tumors. Meanwhile, under stress conditions, p53 also acts as a transcription factor, regulating the expression of a series of target genes to maintain the integrity of genome. The target genes of p53 can be classified into genes regulating cell cycle arrest, genes involved in apoptosis, and genes inhibiting angiogenesis. p53 protein contains a transactivation domain, a sequence-specific DNA binding domain, a tetramerization domain, a non-specific DNA binding domain that recognizes damaged DNA, and a later identified proline-rich domain. Under stress, p53 proteins accumulate and are activated through two mechanisms. One, involving ataxia telangiectasia-mutated protein (ATM), is that the interaction between p53 and its down-regulation factor murine double minute 2 (MDM2) decreases, leading to p53 phosphorylation on Ser15, as determined by the post-translational mechanism; the other holds that p53 increases and is activated through the binding of ribosomal protein L26 (RPL26) or nucleolin to p53 mRNA 5( untranslated region (UTR), regulating p53 translation. Under hypoxia, p53 decreases transactivation and increases transrepression. The mutations outside the DNA binding domain of p53 also contribute to tumor progress, so further studies on p53 should also be focused on this direction. The subterranean blind mole rat Spalax in Israel is a good model for hypoxia-adaptation. The p53 of Spalax mutated in residue 172 and residue 207 from arginine to lysine, conferring it the ability to survive hypoxic conditions. This model indicates that p53 acts as a master gene of diversity formation during evolution. PMID:19434769

  11. p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression

    PubMed Central

    Xia, Peng; Sun, Yifu; Zheng, Changjun; Hou, Tingting; Kang, Mingyang; Yang, Xiaoyu

    2015-01-01

    Purpose: The aim of this study was to investigate the association between osteosarcoma (OS) and Fanconi anemia (FA) related pathways and the molecular mechanisms. Methods: siRNA for Fanconi anemia complementation group D2 (FANCD2) was constructed and transfected into the osteosarcoma cell line MG-63 cells. Expression of TP53INP1, p53, p21, caspase-9, and caspase-3 mRNA in MG-63 cells were examined by real-time fluorescence quantitative PCR, and the protein levels were also determined by western blot. Results: After silence of the FANCD2 gene in MG-63 cells, cell proliferation was inhibited, cell cycle was arrested and cell apoptosis was induced. The apoptosis was mediated by the p53 signaling pathway. After FANCD2 expression was inhibited, TP53INP1 gene expression was up-regulated, phosphorylation of p53 was promoted and the p21 protein was activated, leading to cell cycle arrested in G1, finally resulted in caspase-dependent cell apoptosis. Conclusions: Inhibition of FANCD2 gene expression can induce apoptosis of osteosarcoma cells, which indicated that FANCD2 played an important role in the development of osteosarcoma and it might be a potential target for treatment of osteosarcoma. PMID:26379910

  12. Transcriptional control of human p53-regulated genes.

    PubMed

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

    2008-05-01

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

  13. [Bcl-2 inhibits p53-induced apoptosis after genotoxic damage by inhibitors of nuclear import of p53].

    PubMed

    Beham, A; Schumacher, G; McDonnell, T J; Marin, M C; Jauch, K W

    1998-01-01

    The tumor suppressor gene p53 in overexpressed in 50% of colorectal carcinomas and is an interesting target for gene therapeutic approaches. Furthermore the protooncogen bcl-2 is known to inhibit p53 induced apoptosis and is expressed in some colorectal carcinomas. In this study mechanism of bcl-2 cell death inhibition after p53 induction were evaluated. The human colon carcinoma cell line RKO posses wild-type p53 and also expresses bcl-2 protein. RKO cells were treated with liposomal bcl-2 antisense oligonucleotides (AS), control oligonucleotides (CO) and empty liposomes (EL) resulting in decreased bcl-2 expression. After induction of p53 with gamma-irradiation p53 protein expression was induced in AS, CO and EL pretreated cells. Microscopy and immunoblotting was used to characterize subcellular localization of p53 protein. Further p53 subcellular localisation was examined after p53 transfer of wt p53 cDNA in three bcl-2 expressing cell lines. Most of the p53 protein remained localized in the cytosol and apoptosis was decreased in bcl-2 expressing cells assessed by flow cytometric analysis (Ao). Our data suggests that bcl-2 is able to modulate transmembrane trafficking of p53. This resulted in inhibition of cell death implicating that bcl-2 function is involved in regulation of transmembrane gradients. PMID:14518224

  14. 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.

  15. Loss of DDB1 Leads to Transcriptional p53 Pathway Activation in Proliferating Cells, Cell Cycle Deregulation, and Apoptosis in Zebrafish Embryos

    PubMed Central

    Hu, Zhilian; Holzschuh, Jochen; Driever, Wolfgang

    2015-01-01

    DNA damage-binding protein 1 (DDB1) is a large subunit of the heterodimeric DDB complex that recognizes DNA lesions and initiates the nucleotide excision repair process. DDB1 is also a component of the CUL4 E3 ligase complex involved in a broad spectrum of cellular processes by targeted ubiquitination of key regulators. Functions of DDB1 in development have been addressed in several model organisms, however, are not fully understood so far. Here we report an ENU induced mutant ddb1 allele (ddb1m863) identified in zebrafish (Danio rerio), and analyze its effects on development. Zebrafish ddb1 is expressed broadly, both maternally and zygotically, with enhanced expression in proliferation zones. The (ddb1m863 mutant allele affects the splice acceptor site of exon 20, causing a splicing defect that results in truncation of the 1140 amino acid protein after residue 800, lacking part of the β-propeller domain BPC and the C-terminal helical domain CTD. ddb1m863 zygotic mutant embryos have a pleiotropic phenotype, including smaller and abnormally shaped brain, head skeleton, eyes, jaw, and branchial arches, as well as reduced dopaminergic neuron groups. However, early forming tissues develop normally in zygotic ddb1m863 mutant embryos, which may be due to maternal rescue. In ddb1m863 mutant embryos, pcna-expressing proliferating cell populations were reduced, concurrent with increased apoptosis. We also observed a concomitant strong up-regulation of transcripts of the tumor suppressor p53 (tp53) and the cell cycle inhibitor cdkn1a (p21a/bCIP1/WAF1) in proliferating tissues. In addition, transcription of cyclin genes ccna2 and ccnd1 was deregulated in ddb1m863 mutants. Reduction of p53 activity by anti-sense morpholinos alleviated the apoptotic phenotype in ddb1m863 mutants. These results imply that Ddb1 may be involved in maintaining proper cell cycle progression and viability of dividing cells during development through transcriptional mechanisms regulating genes

  16. p53 genes function to restrain mobile elements

    PubMed Central

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

    2016-01-01

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

  17. p53 genes function to restrain mobile elements.

    PubMed

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

    2016-01-01

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

  18. A novel p53-binding domain in CUL7.

    PubMed

    Kasper, Jocelyn S; Arai, Takehiro; DeCaprio, James A

    2006-09-15

    CUL7 is a member of the cullin RING ligase family and forms an SCF-like complex with SKP1 and FBXW8. CUL7 is required for normal mouse embryonic development and cellular proliferation, and is highly homologous to PARC, a p53-associated, parkin-like cytoplasmic protein. We determined that CUL7, in a manner similar to PARC, can bind directly to p53 but does not affect p53 expression. We identified a discrete, co-linear domain in CUL7 that is conserved in PARC and HERC2, and is necessary and sufficient for p53-binding. The presence of p53 stabilized expression of this domain and we demonstrate that this p53-binding domain of CUL7 contributes to the cytoplasmic localization of CUL7. The results support the model that p53 plays a role in regulation of CUL7 activity. PMID:16875676

  19. Chemical Variations on the p53 Reactivation Theme

    PubMed Central

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

    2016-01-01

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

  20. A novel p53-binding domain in CUL7

    SciTech Connect

    Kasper, Jocelyn S.; Arai, Takehiro; De Caprio, James A. . E-mail: james_decaprio@dfci.harvard.edu

    2006-09-15

    CUL7 is a member of the cullin RING ligase family and forms an SCF-like complex with SKP1 and FBXW8. CUL7 is required for normal mouse embryonic development and cellular proliferation, and is highly homologous to PARC, a p53-associated, parkin-like cytoplasmic protein. We determined that CUL7, in a manner similar to PARC, can bind directly to p53 but does not affect p53 expression. We identified a discrete, co-linear domain in CUL7 that is conserved in PARC and HERC2, and is necessary and sufficient for p53-binding. The presence of p53 stabilized expression of this domain and we demonstrate that this p53-binding domain of CUL7 contributes to the cytoplasmic localization of CUL7. The results support the model that p53 plays a role in regulation of CUL7 activity.

  1. 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.

  2. Overexpression of p53 mRNA in colorectal cancer and its relationship to p53 gene mutation.

    PubMed Central

    el-Mahdani, N.; Vaillant, J. C.; Guiguet, M.; Prévot, S.; Bertrand, V.; Bernard, C.; Parc, R.; Béréziat, G.; Hermelin, B.

    1997-01-01

    We analysed the frequency of p53 mRNA overexpression in a series of 109 primary colorectal carcinomas and its association with p53 gene mutation, which has been correlated with short survival. Sixty-nine of the 109 cases (63%) demonstrated p53 mRNA overexpression, without any correlation with stage or site of disease. Comparison with p53 gene mutation indicated that, besides cases in which p53 gene mutation and p53 mRNA overexpression were either both present (40 cases) or both absent (36 cases), there were also cases in which p53 mRNA was overexpressed in the absence of any mutation (29 cases) and those with a mutant gene in which the mRNA was not overexpressed (four cases). Moreover, the mutant p53 tumours exhibited an increase of p53 mRNA expression, which was significantly higher in tumours expressing the mutated allele alone than in tumours expressing both wild- and mutated-type alleles. These data (1) show that p53 mRNA overexpression is a frequent event in colorectal tumours and is not predictive of the status of the gene, i.e. whether or not a mutation is present; (2) provide further evidence that p53 protein overexpression does not only result from an increase in the half-life of mutated p53 and suggest that inactivation of the p53 function in colorectal cancers involves at least two distinct mechanisms, including p53 overexpression and/or mutation; and (3) suggest that p53 mRNA overexpression is an early event, since it is not correlated with Dukes stage. PMID:9052405

  3. Regulation of p53 during senescence in normal human keratinocytes

    PubMed Central

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

    2015-01-01

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

  4. p53 Loss Increases the Osteogenic Differentiation of BMSCs

    PubMed Central

    He, Yunlong; de Castro, Luis F; Shin, Min Hwa; Dubois, Wendy; Yang, Howard H.; Jiang, Shunlin; Mishra, Pravin J.; Ren, Ling; Gou, Hongfeng; Lal, Ashish; Khanna, Chand; Merlino, Glenn; Lee, Maxwell; Robey, Pamela G.; Huang, Jing

    2014-01-01

    The tumor suppressor, p53, plays a critical role in suppressing osteosarcoma. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested to give rise to osteosarcomas. However, the role of p53 in BMSCs has not been extensively explored. Here, we report that p53 regulates the lineage choice of mouse BMSCs (mBMSCs). Compared to mBMSCs with wild type p53, mBMSCs deficient in p53 have enhanced osteogenic differentiation, but with similar adipogenic and chondrogenic differentiation. The role of p53 in inhibiting osteogenic lineage differentiation is mainly through the action of Runx2, a master transcription factor required for the osteogenic differentiation of mBMSCs. We find that p53 indirectly represses the expression of Runx2 by activating the microRNA-34 family, which suppresses the translation of Runx2. Since osteosarcoma may derive from BMSCs, we examined whether p53 has a role in the osteogenic differentiation of osteosarcoma cells and found that osteosarcoma cells with p53 deletion have higher levels of Runx2 and faster osteogenic differentiation than those with wild type p53. A systems biology approach reveals that p53-deficient mBMSCs are more closely related to human osteosarcoma while mBMSCs with wild type p53 are similar to normal human BMSCs. In summary, our results indicate that p53 activity can influence cell fate specification of mBMSCs, and provide molecular and cellular insights into the observation that p53 loss is associated with increased osteosarcoma incidence. PMID:25524638

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

    SciTech Connect

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

    2013-06-15

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

  6. Drug resistance to inhibitors of the human double minute-2 E3 ligase is mediated by point mutations of p53, but can be overcome with the p53 targeting agent RITA.

    PubMed

    Jones, Richard J; Bjorklund, Chad C; Baladandayuthapani, Veerabhadran; Kuhn, Deborah J; Orlowski, Robert Z

    2012-10-01

    The human double minute (HDM)-2 E3 ubiquitin ligase plays a key role in p53 turnover and has been validated preclinically as a target in multiple myeloma (MM) and mantle cell lymphoma (MCL). HDM-2 inhibitors are entering clinical trials, and we therefore sought to understand potential mechanisms of resistance in lymphoid models. Wild-type p53 H929 MM and Granta-519 MCL cells resistant to MI-63 or Nutlin were generated by exposing them to increasing drug concentrations. MI-63-resistant H929 and Granta-519 cells were resistant to Nutlin, whereas Nutlin-resistant cells displayed cross-resistance to MI-63. These cells also showed cross-resistance to bortezomib, doxorubicin, cisplatin, and melphalan, but remained sensitive to the small molecule inhibitor RITA (reactivation of p53 and induction of tumor cell apoptosis). HDM-2 inhibitor-resistant cells harbored increased p53 levels, but neither genotoxic nor nongenotoxic approaches to activate p53 induced HDM-2 or p21. Resequencing revealed wild-type HDM-2, but mutations were found in the p53 DNA binding and dimerization domains. In resistant cells, RITA induced a G(2)-M arrest, upregulation of p53 targets HDM-2, PUMA, and NOXA, and PARP cleavage. Combination regimens with RITA and MI-63 resulted in enhanced cell death compared with RITA alone. These findings support the possibility that p53 mutation could be a primary mechanism of acquired resistance to HDM-2 inhibitors in MCL and MM. Furthermore, they suggest that simultaneous restoration of p53 function and HDM-2 inhibition is a rational strategy for clinical translation. PMID:22933706

  7. A minimally invasive assay for individual assessment of the ATM/CHEK2/p53 pathway activity

    PubMed Central

    Kabacik, Sylwia; Ortega-Molina, Ana; Efeyan, Alejo; Finnon, Paul; Bouffler, Simon; Serrano, Manuel

    2011-01-01

    Ionizing radiation induces DNA Double-Strand Breaks (DSBs), which activate the ATM/CHEK2/p53 pathway leading to cell cycle arrest and apoptosis through transcription of genes including CDKN1A (p21) and BBC3 (PUMA). This pathway prevents genomic instability and tumorigenesis as demonstrated in heritable syndromes [e.g., Ataxia Telangiectasia (AT); Li-Fraumeni syndrome (LFS