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Sample records for waf mdm2 p21

  1. The Expression and Clinical Significance of P21WAF1 MDM-2 in Pancreatic Carcinoma%人胰腺癌中P21WAF1、MDM-2的表达及其临床意义

    Institute of Scientific and Technical Information of China (English)

    刘振华; 冯一中; 李峰; 柴玉海

    2007-01-01

    目的 探讨P21WAF1、MDM-2在胰腺癌中的表达及其在胰腺癌发生发展中的作用.方法 应用免疫组织化学S-P法检测P21WAF1、MDM-2在10例正常胰腺组织、53例胰腺癌组织中的表达,并分析其与临床病理参数之间的关系.结果 胰腺癌组织中P21WAE1的表达显著低于正常胰腺组织(P<0.05),MDM-2的表达显著高于正常胰腺组织(P<0.05).53例胰腺癌组织中P21WAF1、MDM-2的表达随肿瘤的分化程度降低而减少,差异具有显著性(P<0.05).有淋巴结转移的胰腺癌P21WAF1的表达明显低于没有淋巴结转移者(P<0.05).P21WAF1、MDM-2的表达与患者的生存率有关.P21WAF1、MDM-2的表达与性别、年龄、肿瘤部位、肿瘤大小及临床分期无关.结论 P21WAF1、MDM-2在胰腺癌的发生、发展中起重要作用,可作为该肿瘤预后判断的理论依据.

  2. P21WAF1、MDM-2、Rb在胰腺癌中的表达及临床意义%Study on expression of P21WAF1, MDM-2 and Rb and its clinical significance in pancreatic carcinoma

    Institute of Scientific and Technical Information of China (English)

    刘振华; 冯一中; 李峰; 柴玉海; 任苏勤

    2007-01-01

    目的:探讨P21WAF1、MDM-2、Rb在胰腺癌发生过程中的作用,为预后判断提供理论依据.方法:应用免疫组织化学技术SP法检测53例胰腺癌手术患者的组织标本.结果:(1)P21WAF1在胰腺癌中的阳性表达率显著低于非肿瘤性胰腺组织(P<0.05),P21WAF1的阳性表达率与胰腺癌的病理分级、淋巴结转移呈负相关(P<0.05).(2)MDM-2在胰腺癌中的阳性表达率明显高于非肿瘤性胰腺组织(P<0.05),与胰腺癌的病理分级呈正相关(P<0.05).(3)Rb的阳性表达率在胰腺癌中明显低于非肿瘤性胰腺组织(P<0.01),与胰腺癌的病理分级呈负相关(P<0.05).(4)P21WAF1与MDM-2P21WAF1与Rb在胰腺癌中的阳性表达呈显著正相关(P<0.01).(5)生存单因素分析MDM-2及Rb均与胰腺癌的预后显著相关(P<0.01,P<0.05).结论:P21WAF1、MDM-2、Rb在胰腺癌的发生发展中均发挥一定的作用.

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

    DEFF Research Database (Denmark)

    Sørensen, Belinda Halling; Nielsen, Dorthe; Thorsteinsdottir, Unnur Arna;

    2016-01-01

    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...

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

    DEFF Research Database (Denmark)

    Sørensen, Belinda Halling; Nielsen, Dorthe; Thorsteinsdottir, Unnur Arna

    2016-01-01

    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...

  5. Differential regulation of p21 (waf1) protein half-life by DNA damage and Nutlin-3 in p53 wild-type tumors and its therapeutic implications.

    Science.gov (United States)

    Chang, Li-Ju; Eastman, Alan

    2012-09-01

    DNA damage induces the canonical p53 pathway including elevation of p21 (waf1) resulting in arrest of cell cycle progression. This can protect cells from subsequent Chk1 inhibition. Some p53 wild-type cancer cells such as HCT116 and U2OS exhibit attenuated p21 (waf1) induction upon DNA damage due to translational inhibition, and are incapable of maintaining arrest upon Chk1 inhibition. The purpose of this study was to determine whether this attenuated p21 (waf1) induction also occurred with the non-DNA damaging agent Nutlin-3 which induces p53 by disrupting binding to its negative regulator MDM2. We find that Nutlin-3 circumvented the attenuated induction of p21 (waf1) protein by increasing its half-life which led to G 1 and G 2 arrest in both cell lines. Interestingly, the p21 (waf1) protein half-life remained short on Nutlin-3 in p53 wild-type MCF10A cells; these cells achieve high p21 (waf1) levels through transcriptional upregulation. Consequently, all three p53 wild-type cells but not p53 mutant MDA-MB-231 cancer cells were protected from subsequent incubation with a combination of DNA damage plus a checkpoint inhibitor.

  6. p21WAF1/CIP1 gene DNA sequencing and its expression in human osteosarcoma

    Institute of Scientific and Technical Information of China (English)

    廖威明; 张春林; 李佛保; 曾炳芳; 曾益新

    2004-01-01

    Background Mutation and expression change of p21WAF1/CIP1 may play a role in the growth of osteosarcoma. This study was to investigate the expression of the p21WAF1/CIP1 gene in human osteosarcoma, p21WAF1/CIP1 gene DNA sequence change and their relationships with the phenotype and clinical prognosis.Methods p21WAF1/CIP1 gene in 10 normal people and the tumours of 45 osteosarcoma patients were examined using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with silver staining. The PCR product with an abnormal strand was sequenced directly. The p21WAF1/CIP1 gene mRNA and P21 protein of 45 cases of osteosarcoma were investigated by using in situ hybridization and immunohistochemistry, respectively. Results The occurrence of P21 protein in osteosarcoma was 17.78% (8/45), and p21WAF1/CIP1 mRNA expression in osteosarcoma was 42.22% (19/45). The p21WAF1/CIP1 gene DNA sequencing of amplified production showed that in p21WAF1/CIP1 gene exon 3 of 36 cases of human osteosarcoma, there were 17 cases (47.22%) with C→T at position 609; 10 normal blood samples' DNA sequence analysis yielded 8 cases (80.00%) with C→T at the same position. Conclusions Along with the increase of malignancy, the expression of p21WAF1/CIP1mRNA and P21 protein in osteosarcoma tends to decrease. It is uncommon for the p21WAF1/CIP1 gene mutation to occur in human osteosarcoma. As a result, the possible existence of tumour subtypes of p21WAF1/CIP1 gene mutation should be investigated. Our research leads to the location of p21WAF1/CIP1 gene polymorphism of Chinese osteosarcoma patients, which can provide a basis for further research.

  7. Effects of histone acetylation and DNA methylation on p21WAF1 regulation

    Institute of Scientific and Technical Information of China (English)

    Jing-Yuan Fang; You-Yong Lu

    2002-01-01

    Cell cycle progression is regulated by interactions betweencyclins and cyclin-dependent kinases (CDKs). p21wAF1 is oneof the CIP/KIP family which inhibits CDKs activity. Increasedexpression of p21WAF1 may play an important role in thegrowth arrest induced in transformed calls. Although thestability of the p21wAF1 mRNA could be altered by differentsignals, cell differentiation and numerous influencingfactors. However, recent studies suggest that two knownmechanisms of epigenesis, i. e. gene inactivation bymethylation in promoter region and changes to an inactivechromatin by histone deacetylation, seem to be the bestcandidate mechanisms for inactivation of p21WAF1. To date,almost no coding region p21wAF1 mutations have been foundin tumor cells, despite extensive screening of hundreds ofvarious tumors. Hypermethylation of the p21WAF1 promoterregion may represent an alternative mechanism by which thep21WAF1/ClPl gene can be inactivated. The reduction of cellularDNMT protein levels also induces a corresponding rapidincrease in the cell cycle regulator p21wAF1 proteindemonstrating a regulatory link between DNMT and p21WAF1which is independent of methylation of DNA. Both histonehyperacetylation and hypoacetylation appear to be importantin the carcinoma process, and induction of the p21WAF1 geneby histone hyperacetylation may be a mechanism by whichdietary fiber prevents carcinogenesis. Here, we review theinfluence of histone acetylation and DNA methylation onp21WAF1 transcription, and affection of pathways or factorsassociated such as p53, E2A, Sp1 as well as several histonedeacetylation inhibitors.

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

    Directory of Open Access Journals (Sweden)

    Dubrovsky Larisa

    2008-03-01

    Full Text Available Abstract 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.

  9. Experimental gene therapy using p21Waf1 gene for esophageal squamous cell carcinoma by gene gun technology.

    Science.gov (United States)

    Tanaka, Yuichi; Fujii, Teruhiko; Yamana, Hideaki; Kato, Seiya; Morimatsu, Minoru; Shirouzu, Kazuo

    2004-10-01

    In our previous study, the proliferation rate of esophageal squamous cell carcinoma cell lines, which poorly expressed p21Waf1, was found to be regulated by p21Waf1 gene transfection using adenovirus vector. In the present study, in order to examine the effect of p21Waf1 gene therapy in esophageal cancer, we used gene gun technology, which proved to be a powerful method to introduce the p21Waf1 gene into esophageal cancer cells. p21Waf1 transfection to KE3 and YES2 cells (weakly expressed p21Waf1 protein cells) showed a high expression of p21Waf1 protein after applying this gene gun technique. In KE3 and YES2 cells, statistical significant growth inhibition was observed after p21Waf1 transfection compared with LacZ transfection (KE3, p=0.0009; YES2, pgun technique significantly inhibited the low basal p21Waf1 expressed esophageal cancer cell growth in vitro and in vivo. Furthermore, p21Waf1 transfection strongly enhanced the effect of 5Fu suggesting that p21Waf1 may prove beneficial in chemotherapy combined with gene therapy using gene gun technology in patients with esophageal cancer who have a low level of p21Waf1 expressed tumor.

  10. P21WAF1基因与胃癌的关系

    Institute of Scientific and Technical Information of China (English)

    郭凤英; 张建中

    2006-01-01

    P21WAF1是第一个被发现的CDI 家族成员,由于发现途径不同,又名CIP1(cdk-interacting protein), WAF1(wild-type p53 activated fragment), CAP20(cdk2-associate protein), mda-6(melanoma differention associated gene).

  11. Histone acetylation regulates p21WAF1 expression in human colon cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Ying-Xuan Chen; Jing-Yuan Fang; Hong-Yin Zhu; Rong Lu; Zhong-Hua Cheng; De-Kai Qiu

    2004-01-01

    AIM: To investigate the effect of histone acetylation on regulation of p21WAF1 gene expression in human colon cancer cell lines.METHODS: Two cell lines, Colo-320 and SW1116 were treated with either trichostatin or sodium butyrate. Expressions of p21WAF1 mRNA and protein were detected by real-time RT-PCR and Western blotting, respectively. Acetylation of two regions of p21WAF1 gene-associated histones and total cellular histones were examined by chromatin immunoprecipitation assay and Western blotting. RESULTS: Trichostatin or sodium butyrate re-activated p21WAF1 transcription resulted in up-regulated p21WF1 protein level in colon cancer cell lines. Those effects were accompanied by an accumulation of acetylated histones in total cellular chromatin and p21WAF1 gene-associated region of chromatin.CONCLUSION: Histone acetylation regulates p21WAF1 expression in human colon cancer cell lines, Colo-320 and SW1116.

  12. p21WAF1/CIP1 interacts with protein kinase CK2

    DEFF Research Database (Denmark)

    Götz, C; Wagner, P; Issinger, O G

    1996-01-01

    and inhibits DNA replication. Here, we show that p21WAF1/CIP1 binds to the regulatory beta-subunit of protein kinase CK2 but not to the catalytic alpha-subunit. Binding of p21WAF1/CIP1 down regulates the kinase activity of CK2 with respect to the phosphorylation of the beta-subunit of CK2, casein and the C......p21WAF1/CIP1 which belongs to a class of regulatory proteins that interact with cyclin dependent kinases is a potent inhibitor of these kinases. The inhibition of the cyclin dependent kinases induces an arrest of cells in the G phase of the cell cycle. In addition p21WAF1/CIP1 associates with PCNA......-terminus of p53. This study demonstrates a new binding partner for the regulatory beta-subunit of protein kinase CK2 which regulates the activity of the holoenzyme....

  13. EXPRESSION AND SIGNIFICANCE OF SMAD4 AND p21WAF1 IN ENDOMETRIAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    葛秀君; 李英勇

    2003-01-01

    Objective: To investigate the expression of Smad4 and p21WAF1 in endometrial carcinoma and its clinical significance. Methods: Immunohistochemical method was used to detect Smad4 and p21WAF1 expression in 56 cases of endometrial carcinoma. Results: The positive rate of Smad4 was 80.36% in endometrial carcinoma. The Samd4 expression was significantly correlated with histological grade (P0.05). Conclusion: Smad4 may play an important role in the tumorigenesis, differentiation and progression of endometrial carcinoma. The expression of p21WAF1 was associated with the tumorigenesis of endometrial carcinoma, but the association between p21WAF1 and differentiation and progression of endometrial carcinomas needs to be further investigated.

  14. Expression and combined expression of p53, MDM-2 and p21 in 190 non-Hodgkin lymphomas%非霍奇金淋巴瘤中p53、MDM-2p21表达的意义

    Institute of Scientific and Technical Information of China (English)

    马怡晖; 朱有凯; 叶子茵; 林汉良; 林素霞; 李海刚

    2012-01-01

    目的 研究非霍奇金淋巴瘤(NHL)中p53、MDM-2p21表达及联合表达的情况,探讨它们在NHL发生发展中的作用和在临床病理诊断工作中的应用价值.方法 应用组织芯片、免疫组化及原位凋亡检测等技术对190例NHL中p53、MDM-2p21和Ki-67的表达以及凋亡进行检测并统计分析.结果 (1)p53、MDM-2p21在B-NHL中的阳性表达率分别为36.00%、8.00%和17.33%,在T/NK-NHL中的阳性表达率分别为40.00%、18.55%和24.35%;(2)MDM-2主要在侵袭性较高的NHL中表达,p53+/MDM-2+表型的NHL具有较高增殖指数,其中9例ALCL有5例为ALK阴性;(3)p53与p21阳性表达在T/NK-NHL中显著正相关(P=0.010).结论 (1)MDM-2表达可能参与恶性程度较高类型NHL的发生发展;(2)p53+/MDM-2+和p53+/p21+表型可能与侵袭性较高NHL的高度恶性、侵袭性及不良预后相关.

  15. Absence of prognostic significance of p21(WAF1/CIP1) protein expression in non-small cell lung cancer.

    Science.gov (United States)

    Dworakowska, Dorota; Jassem, Ewa; Jassem, Jacek; Boltze, Carsten; Wiedorn, Klaus Hermann; Dworakowski, Rafał; Skokowski, Jan; Jaśkiewicz, Kazimierz; Czestochowska, Eugenia

    2005-01-01

    Prognostic value of p21WAF1/CIP1 expression in non-small-cell lung cancer patients (NSCLC) remains unclear. In this study the authors investigated the clinical significance of p21WAF1/CIP1 expression in a group of 117 NSCLC patients, who underwent curative pulmonary resection. Expression of p21WAF1/CIP1 protein was assessed immunohistochemically and samples showing>5% of positive tumor cells were considered positive. Seventy-six samples (65%) showed positive nuclear p21WAF1/CIP1 protein expression. There was no relationship between the expression of p21WAF1/CIP1 protein and major clinico-pathological factors, and neither there was an impact of p21WAF1/CIP1 protein expression on disease-free and overall survival. p21WAF1/CIP1 protein occurrence was not correlated with previously determined p53 protein expression and there was also no relationship between all possible p21WAF1/CIP1/p53 phenotypes and survival. In uni- and multivariate analysis only stage of disease was independent prognostic factors. These results suggest the lack of prognostic relevance of p21WAF1/CIP1 expression (analyzed separately or jointly with p53 protein) in surgically treated NSCLC patients.

  16. POST-TRANSCRIPTIONAL REGULATION OF P21WAF1/CIP1 BY P53

    Institute of Scientific and Technical Information of China (English)

    季加孚; 张霁; 焦春雨; 顾晋; 谭立新; 张平; 李培详

    2001-01-01

    Objective: To investigate the post-transcriptional regulation of p21WAF1/CIP1 by p53. Methods: The MDA-MB-468 cells have endogenous mutant p53 and the MCF7 cells lines have wtp53. Recombinant p53 expression and p21WAF1/CIP1 induction were detected by Western blot analysis. Northern blot analysis was carried out to examine whether changes in p21WAF1/CIP1 protein levels in MCF7 cells treated with AdCMVp53 are reflected at the mRNA level. Flow cytometric analysis of MCF7 cells following overexpression of recombination. Results: The ratio of p53: p21WAF1/CIP1 was below 1 at the early stages of AdCMVp53 infection, but increased to 1.6 by day 3 and to 9.7 by day 5 post-infection. As expected, p21WAF1/CIP1 expression was not detectable in MDA-MB-468 cells despite the presence of high levels of mutant p53 protein. The G1/S ratios in untreated controls and AdCMVβgal infected MCF7 cells were 1.10 and 1.35, respectively. By Northern blot analyzing the p21WAF1/CIP1: GAPDH ratios at different time points against the ratio at time point 0, a maximum 3-fold induction of p21WAF1/CIP1 mRNA expression relative to untreated control was observed on day 1 post-infection. The flow cytometric analysis indicated that MCF7 cells infected with AdCMVp53 undergo G1 arrest at both time points studied, with G1/S ratios ranging from 5.54 at day 1 to 5.65 at day 7. The G1/S ratios in untreated controls and AdCMVβgal infected MCF7 cells were 1.10 and 1.35, respectively. Conclusion: This studydemonstrated that p53 could regulate p21WAF1/CIP1 gene expression at both the transcriptional and post-transcriptional levels in MCF7 cells. The latter mechanism may be involved in or be responsible for, the induction of cell cycle arrest by transcription-defective mutants of p53.

  17. Expression of p53, MDM2, p21, heat shock protein 70, and HPV 16/18 E6 proteins in oral verrucous carcinoma and oral verrucous hyperplasia.

    Science.gov (United States)

    Lin, Hung-Pin; Wang, Yi-Ping; Chiang, Chun-Pin

    2011-03-01

    Oral verrucous hyperplasia is a precancerous lesion of oral verrucous carcinoma. This study used immunohistochemistry to examine the expression of p53, murine double minute 2 (MDM2), p21, heat shock protein 70 (HSP 70), and human papillomavirus (HPV) 16/18 E6 proteins in 48 oral verrucous carcinoma and 30 oral verrucous hyperplasia samples. The mean labeling indices of p53, MDM2, p21, HSP 70, and HPV 16/18 E6 proteins in oral verrucous carcinoma samples were 21%, 31%, 7%, 17%, and 0.5%, respectively, and those in oral verrucous hyperplasia samples were 19%, 35%, 11%, 14%, and 0.3%, respectively. Immunohistochemistry with the above-cited 5 biomarkers could not help differentiate oral verrucous hyperplasia from oral verrucous carcinoma. The low expression of p21 may partially explain abnormal epithelial overgrowth in both verrucous lesions. The pathogenesis of both verrucous lesions may be at least partially attributed to the overexpression of MDM2 protein and moderate expression of HSP 70 protein in both lesions. Copyright © 2010 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    1998-06-01

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

  19. p21WAF1和pRb在膀胱移行细胞癌中的表达及其意义%Expression of p21WAF1 and pRb in bladder tran sitional cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    马志方; 米振国; 王东文; 刘红耀; 杨晓峰

    2001-01-01

    目的探讨p21WAF1和pRb在膀胱移行细胞癌(BTCC)中表达及相互关系和其意义.方法应用免疫组织化学SP法检测57例BTCC患者癌组织中P21WAF1和PRb的蛋白表达.结果 p21WAF1和pRb的阳性表达率分别为36.8%和45.6%,p21WAF1随病理分级升高阳性率显著下降(P<0.05),pRb的表达与BTCC的临床分期和有无转移均相关;p21WAF1和pRb的表达呈负相关(P<0.005,Kappa=-0.401),p21WAF1(-)/pRb(+)组合的发生率为38.6%, 并与BTCC的病理分级、临床分期和有无转移均相关(P<0.05).结论 p21WAF1和pBb的表达异常与BTCC的发生发展密切相关,p21WAF1的改变可能为癌变的早期事件,联合检测p21WAF1和pRb可较完整准确地评价BTCC的生物学特性,估计预后,指导治疗.

  20. Protective role of p21(Waf1/Cip1) against prostaglandin A2-mediated apoptosis of human colorectal carcinoma cells.

    Science.gov (United States)

    Gorospe, M; Wang, X; Guyton, K Z; Holbrook, N J

    1996-01-01

    Prostaglandin A2 (PGA2) suppresses tumor growth in vivo, is potently antiproliferative in vitro, and is a model drug for the study of the mammalian stress response. Our previous studies using breast carcinoma MCF-7 cells suggested that p21(Waf1/Cip1) induction enabled cells to survive PGA2 exposure. Indeed, the marked sensitivity of human colorectal carcinoma RKO cells to the cytotoxicity of PGA2 is known to be associated with a lack of a PGA2-mediated increase in p21(Waf1/Cip1) expression, inhibition of cyclin-dependent kinase activity, and growth arrest. To determine if cell death following exposure to PGA2 could be prevented by forcing the expression of p21(Waf1/Cip1) in RKO cells, we utilized an adenoviral vector-based expression system. We demonstrate that ectopic expression of p21(Waf1/Cip1) largely rescued RKO cells from PGA2-induced apoptotic cell death, directly implicating p21(Waf1/Cip1) as a determinant of the cellular outcome (survival versus death) following exposure to PGA2. To discern whether p21(Waf1/Cip1)-mediated protection operates through the implementation of cellular growth arrest, other growth-inhibitory treatments were studied for the ability to attenuate PGA2-induced cell death. Neither serum depletion nor suramin (a growth factor receptor antagonist) protected RKO cells against PGA2 cytotoxicity, and neither induced p21(Waf1/Cip1) expression. Mimosine, however, enhanced p21(Waf1/Cip1) expression, completely inhibited RKO cell proliferation, and exerted marked protection against a subsequent PGA2 challenge. Taken together, our results directly demonstrate a protective role for p21(Waf1/Cip1) during PGA2 cellular stress and provide strong evidence that the implementation of cellular growth arrest contributes to this protective influence. PMID:8943319

  1. p21(WAF1) Mediates Cell-Cycle Inhibition, Relevant to Cancer Suppression and Therapy.

    Science.gov (United States)

    El-Deiry, Wafik S

    2016-09-15

    p21 (WAF1/CIP1; CDKN1a) is a universal cell-cycle inhibitor directly controlled by p53 and p53-independent pathways. Knowledge of the regulation and function of p21 in normal and cancer cells has opened up several areas of investigation and has led to novel therapeutic strategies. The discovery in 1993 and subsequent work on p21 has illuminated basic cellular growth control, stem cell phenotypes, the physiology of differentiation, as well as how cells respond to stress. There remain open questions in the signaling networks, the ultimate role of p21 in the p53-deficiency phenotype in the context of other p53 target defects, and therapeutic strategies continue to be a work in progress. Cancer Res; 76(18); 5189-91. ©2016 AACRSee related article by El-Deiry et al., Cancer Res 1994;54:1169-74Visit the Cancer Research 75(th) Anniversary timeline.

  2. p21-Activated kinase 6 (PAK6) inhibits prostate cancer growth via phosphorylation of androgen receptor and tumorigenic E3 ligase murine double minute-2 (Mdm2).

    Science.gov (United States)

    Liu, Tong; Li, Yang; Gu, Hui; Zhu, Ge; Li, Jiabin; Cao, Liu; Li, Feng

    2013-02-01

    The androgen receptor (AR) signaling pathway plays a crucial role in the development and growth of prostate malignancies. Regulation of AR homeostasis in prostate tumorigenesis has not yet been fully characterized. In this study, we demonstrate that p21-activated kinase 6 (PAK6) inhibits prostate tumorigenesis by regulating AR homeostasis. First, we demonstrated that in normal prostate epithelium, AR co-localizes with PAK6 in the cytoplasm and translocates into the nucleus in malignant prostate. Furthermore, AR phosphorylation at Ser-578 by PAK6 promotes AR-E3 ligase murine double minute-2 (Mdm2) association, causing AR degradation upon androgen stimuli. We also showed that PAK6 phosphorylates Mdm2 on Thr-158 and Ser-186, which is critical for AR ubiquitin-mediated degradation. Moreover, we found that Thr-158 collaborates with Ser-186 for AR-Mdm2 association and AR ubiquitin-mediated degradation as it facilitates PAK6-mediated AR homeostasis. PAK6 knockdown promotes prostate tumor growth in vivo. Interestingly, we found a strong inverse correlation between PAK6 and AR expression in the cytoplasm of prostate cancer cells. These observations indicate that PAK6 may be important for the maintenance of androgen-induced AR signaling homeostasis and in prostate malignancy, as well as being a possible new therapeutic target for AR-positive and hormone-sensitive prostate cancer.

  3. p53-independent upregulation of p21WAF1 in NIH 3T3 cells malignantly transformed by mot-2

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    ot-2 protein is shown to interact with p53 and inhibit its transcriptional activation function.Mot-2 overexpressing stable clones of NIH 3T3 cells were malignantly transformed,however,they had a high level of expression of a p53 downstream gene,P21waf1.The present study was undertaken to elucidate possible molecular mechanism(s) of such upregulation.An increased level of P21waf1 expression was detected in stable transfectants although an exogenous reporter gene driven by P21waf1 promoter exhibited lower activity in these cells suggesting that some post-transcriptional mechanism contributes to upregulation.Western analyses of transient and stable clones revealed that upregulation of P21waf1 in stable NIH 3T3/mot-2 cells may be mediated by cyclin D1 and cdk-2.

  4. 肝细胞肝癌p21WAF1与p53的表达及其意义%EXPRESSION AND SIGNIFICANCE OF p21WAF1 AND p53 IN HEPATIC CELL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    戴大英; 翟为溶; 万大方; 朱腾方; 叶圣龙

    2001-01-01

    Objective To explore the expression and significance of p21WAF1 and p53 in HCC. Methods Immunohistochemical method (IHC) was used to localize and semi-quantitate the proteins of p21WAF1 and p53 and to observe the relationship between the expression of p21WAF1 and the different histopathologic characters in 38 patients of HCC and their peri-cancer tissue as well as 5 normal liver tissue. Results Of all 38 cases, both p21WAF1 and p53 expression were significantly higher in tumor than that in corresponding non-tumors liver tissue; 14 (36.8 %) of 38 cases showed p21WAF1 positive staining, 28 cases (73.7 %) were p53 positive, p21WAF1+/p53+ or p21WAF1-/p53- were observed in 18, while 20 cases showed p53+/p21WAF1- or p53-/p21WAF1+. p21WAF1+ was seen in 1 of 38 (2.6 %) corresponding non-cancerous tissue and 2 of 5 normal liver tissue. p53 protein was not detected neither in the non-tumorous tissue nor in normal liver. No significant association was found between the expressions of p21WAF1 and p53(P>0.05) in HCC. Their was no significant correlation between p21WAF1 or p53 expression and the different histopathologic characters of tumor (differentiating grades, intrahepatic metastasis and/or cancerous thrombi within portal veins). Conclusion Both p21WAF1 and p53 proteins are over expressed in HCC than that in corresponding non-tumorous liver tissue, but there is no relationship between them. Both p53-independent and p53-dependent mechanism may play a role in regulating p21WAF1 expression in HCC. p21WAF1 immunostaining cannot be used to assess the status of p53 in any given cell or tissue.%目的探讨HCC中p21WAF1与p53的表达及其意义。方法应用免疫组化法检测38例手术切取的HCC及其配对癌旁肝组织、5例正常肝组织中p21WAF1和p53的表达,并分析它们与病理形态的关系。结果 p21WAF1、p53在HCC中的表达明显高于癌旁肝组织,p21WAF1阳性14例(36.8 %),p53阳性28例(73.7 %),其中两者

  5. 乳腺癌p21(WAF1)蛋白的表达及临床病理意义%The Expression and Slgnificance of p21(WAF1) Protein in Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    滕晓东; 高翠玉; 吴德明

    2001-01-01

    目的:研究p21(WAF1)蛋白在乳腺癌的表达及临床病理意义。方法:应用微波修复免疫组化技术S-P法检测了82例乳腺癌及20例乳腺良性病变中p21(WAF1)蛋白的表达。结果:p21(WAF1)蛋白表达位于细胞核中,部分伴有浆染色;p21(WAF1)蛋白在乳腺良性病变细胞核基本不表达,部分有细胞浆着色,乳腺癌中表达率为58.54%(48/82),其阳性表达与组织高分化、ER阳性、临床分期早有关,与淋巴结有否转移、C-erbB-2、nm23蛋白表达以及是否复发及生存期长短无关。结论:细胞周期调控因子p21(WAF1)蛋白阳性诱导细胞高分化,其缺失在乳腺癌发生发展过程起重要作用。%Objectlve To study the significance of p21 (WAF1)protein expression in breast cancer.Methods The expression of p21 (WAF 1 )protein was analyzed in 82 patients with breast cancer and 20 pa-tients of benign breast diseases by microwave antigen retrieval immunohistochemical staining(S-P methods).Results The immunoreaction of p21(WAF1) protein was localized in the nuclei of cells.With the only weakcytoplasmic expression, the expression rate of p21 (WAF 1 ) in breast cancer was 58.54 % (48/82); it was corre-lated with well differentiation, positive estrogen recepter expression and early stage;but had no significant cor-relations with tumour size, lymph node status, C-erbB-2, nm 23 protein expression and prognosis of breast can-cer. Conclusions The cell cycle regulators p21(WAF1) protein expression is associated with tumour cell dif-ferentiation of breast cancer,its loss may be involved in the pathogenesis of breast cancer.

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

    Directory of Open Access Journals (Sweden)

    Zoltan Molnar

    2014-01-01

    Full Text Available 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.

  7. EBV-positive Hodgkin lymphoma is associated with suppression of p21cip1/waf1 and a worse prognosis

    Directory of Open Access Journals (Sweden)

    Hsu Su-Ming

    2010-02-01

    Full Text Available Abstract Background About 30-50% of Hodgkin lymphomas (HLs harbor the Epstein-Barr virus (EBV, but the impact of EBV infection on clinical outcomes has been unclear. EBV-encoded small RNAs (EBERs are presented in all EBV-infected cells, but their functions are still less understood. Results EBER1 was transfected into two HL cell lines, KMH2 and L428, and microarrays were used to screen for EBER1-induced changes. We found that EBER1 suppressed p21cip1/waf1 transcription in HL cell lines. In addition, positive regulators of p21cip1/waf1 transcription, such as p53, EGR1, and STAT1, were decreased. Suppression of p21cip1/waf1 in the EBER1+ HL cell lines was associated with increased resistance to histone deacetylase inhibitors or proteasome inhibitors, drugs known to cause apoptosis by increasing p21cip1/waf1 levels. On biopsy specimens, EBV+ HLs had weaker expression of both p21cip1/waf1 and active caspase 3. Clinically, suppression of p21cip1/waf1 in EBV+ HLs was associated with a worse 2-year disease-free survival rate (45% for EBV+ HLs vs. 77% for EBV- HLs, p = 0.002. Conclusion Although the underlying mechanisms are still relatively unclear, EBER1 inhibits p21cip1/waf1 transcription and prevents apoptosis through down-regulation of p53, EGR1, and STAT1. The anti-apoptotic activity of EBER1 may be important in the rescue of Reed-Sternberg cells from drug-induced apoptosis and in the clinical behaviors of EBV+ HLs.

  8. Constitutive cytoplasmic localization of p21(Waf1/Cip1) affects the apoptotic process in monocytic leukaemia

    NARCIS (Netherlands)

    Schepers, H; Geugien, M; Eggen, BJL; Vellenga, E

    2003-01-01

    In the present study, we analysed the expression and localization of p21(Waf1/Cip1) in normal and malignant haematopoietic cells. We demonstrate that in normal monocytic cells, protein kinase C (PKC)-induced p21 gene activation, which is nuclear factor-kappaB (NF-kappaB) independent, results in pred

  9. p21(WAF1/CIP1 RNA expression in highly HIV-1 exposed, uninfected individuals.

    Directory of Open Access Journals (Sweden)

    Joshua Herbeck

    Full Text Available Some individuals remain HIV-1 antibody and PCR negative after repeated exposures to the virus, and are referred to as HIV-exposed seronegatives (HESN. However, the causes of resistance to HIV-1 infection in cases other than those with a homozygous CCR5Δ32 deletion are unclear. We hypothesized that human p21WAF1/CIP1 (a cyclin-dependent kinase inhibitor could play a role in resistance to HIV-1 infection in HESN, as p21 expression has been associated with suppression of HIV-1 in elite controllers and reported to block HIV-1 integration in cell culture. We measured p21 RNA expression in PBMC from 40 HESN and 40 low exposure HIV-1 seroconverters (LESC prior to their infection using a real-time PCR assay. Comparing the 20 HESN with the highest exposure risk (median = 111 partners/2.5 years prior to the 20 LESC with the lowest exposure risk (median = 1 partner/2.5 years prior, p21 expression trended higher in HESN in only one of two experiments (P = 0.11 vs. P = 0.80. Additionally, comparison of p21 expression in the top 40 HESN (median = 73 partners/year and lowest 40 LESC (median = 2 partners/year showed no difference between the groups (P = 0.84. There was a weak linear trend between risk of infection after exposure and increasing p21 gene expression (R2 = 0.02, P = 0.12, but again only in one experiment. Hence, if p21 expression contributes to the resistance to viral infection in HESN, it likely plays a minor role evident only in those with extremely high levels of exposure to HIV-1.

  10. THE EXPRESSION AND SIGNIFICANCE OF P53 AND P21(WAF1/CIP1) IN THYROID CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    Huo Xiongwei; Ma Qingyong; Gao Yanfeng; Sun Xuejun; Liu Hao; Sheng Wei

    2005-01-01

    Objective To determine the expression of P53 and P21 (WAF1/CIP1) in thyroid carcinomas and its relationship with development and prognosis of the carcinoma. Methods 90 cases of thyroid tissues (60 thyroid carcinomas, 10 thyroid adenomas, 10 goitres and 10 normal thyroid tissues) were studied by SP immunohistochemical method. Results Positive immunoreactivity of P53 and P21(WAF1/CIP1) was found only in thyroid carcinomas. The positive rate of the P53 and P21 is 53.3% and 41.7% respectively. The positive-staining rates of P53 were higher in cases of undifferentiated carcinomas, positive metastasis lymph nodes or in stage Ⅲ, Ⅳ than those in the cases of well-differentiated, no metastasis lymph nodes, or in stage Ⅰ,Ⅱ. In addition, the positive-staining of P21(WAF1/CIP1) were lower in cases of undifferentiated carcinomas, positive metastasis lymph nodes or stage Ⅲ, Ⅳ than that in the cases of well-differentiated, no metastasis lymph nodes or in stage Ⅰ,Ⅱ. The P21 (WAF1/CIP1) expression rate in the P53 positive group was lower than that in the P53 negative group (P<0.05). Conclusion The expression of P21(WAF1/CIP1) protein in thyroid cancer is related to P53-depend pathway and P53-independent pathway, mainly the P53-depend pathway. Examination of expression of P53 and P21 (WAF1/ CIP1) proteins may be helpful to judge the thyroid cancers behavior and prognosis.

  11. Arecoline-induced growth arrest and p21WAF1 expression are dependent on p53 in rat hepatocytes.

    Science.gov (United States)

    Chou, Wen-Wen; Guh, Jinn-Yuh; Tsai, Jung-Fa; Hwang, Chi-Ching; Chen, Hung-Chun; Huang, Jau-Shyang; Yang, Yu-Lin; Hung, Wen-Chun; Chuang, Lea-Yea

    2008-01-14

    Betel-quid use is associated with the risk of liver cirrhosis and hepatocellular carcinoma and arecoline, the major alkaloid of betel-quid, is hepatotoxic in mice. Therefore, we studied the cytotoxic and genotoxic effects of arecoline in normal rat hepatocytes (Clone-9 cells). Arecoline dose-dependently (0.1-1mM) decreased cell cycle-dependent proliferation while inducing DNA damage at 24h. Moreover, arecoline (1mM)-induced apoptosis and necrosis at 24h. Arecoline dose-dependently (0.1-0.5mM) increased transforming growth factor-beta (TGF-beta) mRNA, gene transcription and bioactivity and neutralizing TGF-beta antibody attenuated arecoline (0.5mM)-inhibited cell proliferation at 24h. Arecoline (0.5mM) also increased p21(WAF1) protein expression and p21(WAF1) gene transcription. Moreover, arecoline (0.5mM) time-dependently (8-24h) increased p53 serine 15 phosphorylation. Pifithrin-alpha (p53 inhibitor) and the loss of the two p53-binding elements in the p21(WAF1) gene promoter attenuated arecoline-induced p21(WAF1) gene transcription at 24h. Pifithrin-alpha also attenuated arecoline (0.5mM)-inhibited cell proliferation at 24h. We concluded that arecoline induces cytotoxicity, DNA damage, G(0)/G(1) cell cycle arrest, TGF-beta1, p21(WAF1) and activates p53 in Clone-9 cells. Moreover, arecoline-induced p21(WAF1) is dependent on p53 while arecoline-inhibited growth is dependent on both TGF-beta and p53.

  12. The calcium channel blocker amlodipine exerts its anti-proliferative action via p21(Waf1/Cip1) gene activation.

    Science.gov (United States)

    Ziesche, Rolf; Petkov, Ventzislav; Lambers, Christopher; Erne, Paul; Block, Lutz-Henning

    2004-10-01

    Proliferation of vascular smooth muscle cells (VSMC) contributes to the progression of atherosclerotic plaques. Calcium channel blockers have been shown to reduce VSMC proliferation, but the underlying molecular mechanism remains unclear. p21(Waf1/Cip1) is a potent inhibitor of cell cycle progression. Here, we demonstrate that amlodipine (10(-6) to 10(-8) M) activates de novo synthesis of p21(Waf1/Cip1) in vitro. We show that amlodipine-dependent activation of p21(Waf1/Cip1) involves the action of the glucocorticoid receptor (GR) and C/EBP-alpha. The underlying pathway apparently involves the action of mitogen-activated protein kinase or protein kinase C, but not of extracellular signal-related kinase or changes of intracellular calcium. Amlodipine-induced p21(Waf1/Cip1) promoter activity and expression were abrogated by C/EBP-alpha antisense oligonucleotide or by the GR antagonist RU486. Amlodipine-dependent inhibition of cell proliferation was partially reversed by RU486 at 10(-8) M (58%+/-29%), antisense oligonucleotides targeting C/EBP-alpha (91%+/-26%), or antisense mRNAs targeting p21(Waf1/Cip1) (96%+/-32%, n=6); scrambled antisense oligonucleotides or those directed against C/EBP-beta were ineffective. The data suggest that the anti-proliferative action of amlodipine is achieved by induction of the p21 (Waf1/Cip1) gene, which may explain beneficial covert effects of this widely used cardiovascular therapeutic drug beyond a more limited role as a vascular relaxant.

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

    Science.gov (United States)

    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.

  14. Hispolon from Phellinus linteus has antiproliferative effects via MDM2-recruited ERK1/2 activity in breast and bladder cancer cells.

    Science.gov (United States)

    Lu, Te-Ling; Huang, Guan-Jhong; Lu, Te-Jung; Wu, Jin-Bin; Wu, Chieh-Hsi; Yang, Tung-Chuan; Iizuka, Akira; Chen, Yuh-Fung

    2009-08-01

    The MDM2 proto-oncogene is overexpressed in many human tumors. Although MDM2 inhibits tumor-suppressor function of p53, there exists a p53-independent role for MDM2 in tumorigenesis. Therefore, downregulation of MDM2 has been considered an attractive therapeutic strategy. Hispolon extracted from Phellinus species was found to induce epidermoid and gastric cancer cell apoptosis. However, the mechanisms are not fully understood. Herein, we report our findings that hispolon inhibited breast and bladder cancer cell growth, regardless of p53 status. Furthermore, p21(WAF1), a cyclin-dependent kinase inhibitor, was elevated in hispolon-treated cells. MDM2, a negative regulator of p21(WAF1), was ubiquitinated and degraded after hispolon treatment. We also found that activated ERK1/2 (extracellular signal-regulated kinase1/2) was recruited to MDM2 and involved in mediating MDM2 ubiquitination. Based on this finding, we investigated whether the sensitivity of cells to hispolon was related to ERK1/2 activity. The results indicated that cells with higher ERK1/2 activity were more sensitive to hispolon. In addition, hispolon-induced caspase-7 cleavage was inhibited by the ERK1/2 inhibitor, U0126. In conclusion, hispolon ubiquitinates and downregulates MDM2 via MDM2-recruited activated ERK1/2. Therefore, hispolon may be a potential anti-tumor agent in breast and bladder cancers.

  15. THE EXPRESSION AND CLINICAL SIGNIFICANCE OF P21 (WAF1/CIP1)AND CYCLIN D1 PROTEIN IN COLORECTAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To study the effect of P21 (WAF1/CIP1) and cyclin D1 and their relationship in colorec- tal carcinoma. Methods The expression of P21 and cyclin D1 was studied in 40 colorectal carcinoma and 10 normal tissues using S-P immunohistochemical technique. Results Decreased expression of P12 and overexpression of cyclin D1 were revealed in colorectal carcinoma. Decreased expression of P21 was related to lymph node metastasis. No cor- relation was found between cyclin D1 and clinicopathological parameters. Conclusion Decreased expression of P21 and overexpression of cyclin D1 may be involved in colorectal tumorigenesis,and were associated with poor prognosis. No correlation was found between P21 and cyclin D1 in colorectai carcinoma.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ae Jeong; Jee, Hye Jin; Song, Naree; Kim, Minjee [Department of Biochemistry, College of Medicine, Dong-A University, Busan (Korea, Republic of); Mitochondria Hub Regulation Center, College of Medicine, Dong-A University, Busan (Korea, Republic of); Jeong, Seon-Young [Mitochondria Hub Regulation Center, College of Medicine, Dong-A University, Busan (Korea, Republic of); Department of Medical Genetics, Ajou University School of Medicine (Korea, Republic of); Yun, Jeanho, E-mail: yunj@dau.ac.kr [Department of Biochemistry, College of Medicine, Dong-A University, Busan (Korea, Republic of); Mitochondria Hub Regulation Center, College of Medicine, Dong-A University, Busan (Korea, Republic of)

    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.

  17. Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis.

    Science.gov (United States)

    Ahmad, N; Feyes, D K; Agarwal, R; Mukhtar, H

    1998-06-09

    Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 --> S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.

  18. Cigarette Smoke Extract Inhibits the Proliferation of Alveolar Epithelial Cells and Augments the Expression of P21WAF1

    Institute of Scientific and Technical Information of China (English)

    Zongxian JIAO; Qilin AO; Xiaona GE; Mi XIONG

    2008-01-01

    Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke ex- tract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type Ⅱand its relationship with P21WAF1, the alveolar epithelial type Ⅱ cell line (A549) cells were chosen as surrogate cells to represent alveolar epithelial type Ⅱ cells. MTT assay was used to detect cell viability after interfered with different concentrations of cigarette smoke ex-tract. It was observed cigarette smoke extract inhibited the growth of A549 cells in a dose- and time-dependent manner. The morphological changes, involving the condensation and margination of nuclear chromatin, even karyorrhexis, were observed by both Hoechst staining and electronic mi-croscopy. Flow cytometry analysis demonstrated the increased cell percentages in G1 and subG1phases after the cells were incubated with cigarette smoke extract. The expression of p21WAF1 protein and mRNA was also significantly increased as detected by the methods of Western blot or reverse transcription-polymerase chain reaction respectively. In conclusion, cigarette smoke extract inhibits the proliferation of alveolar epithelial cell type Ⅱ and blocks them in G1/S phase. The intracellular accumulation of P21WAF1 may be one of the mechanisms which contribute to cigarette smoke ex-tract-induced inhibition of cell proliferation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hag Dong [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of); Jang, Chang-Young [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Choe, Jeong Min [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of); Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Korean Institute of Molecular Medicine and Nutrition, Seoul 136-705 (Korea, Republic of); Sohn, Jeongwon, E-mail: biojs@korea.ac.kr [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Korean Institute of Molecular Medicine and Nutrition, Seoul 136-705 (Korea, Republic of); Kim, Joon, E-mail: joonkim@korea.ac.kr [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Terrand, Jerome; Xu, Beibei; Morrissy, Steve; Dinh, Thai Nho [Department of Pharmacology,College of Medicine, University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724 (United States); Williams, Stuart [Biomedical Engineering Program, College of Medicine, University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724 (United States); Chen, Qin M., E-mail: qchen@email.arizona.edu [Department of Pharmacology,College of Medicine, University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724 (United States)

    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.

  1. The role of cyclin D2 and p21/waf1 in human T-cell leukemia virus type 1 infected cells

    Directory of Open Access Journals (Sweden)

    Pumfery Anne

    2004-04-01

    Full Text Available Abstract Background The human T-cell leukemia virus type 1 (HTLV-1 Tax protein indirectly influences transcriptional activation, signal transduction, cell cycle control, and apoptosis. The function of Tax primarily relies on protein-protein interactions. We have previously shown that Tax upregulates the cell cycle checkpoint proteins p21/waf1 and cyclin D2. Here we describe the consequences of upregulating these G1/S checkpoint regulators in HTLV-1 infected cells. Results To further decipher any physical and functional interactions between cyclin D2 and p21/waf1, we used a series of biochemical assays from HTLV-1 infected and uninfected cells. Immunoprecipitations from HTLV-1 infected cells showed p21/waf1 in a stable complex with cyclin D2/cdk4. This complex is active as it phosphorylates the Rb protein in kinase assays. Confocal fluorescent microscopy indicated that p21/waf1 and cyclin D2 colocalize in HTLV-1 infected, but not in uninfected cells. Furthermore, in vitro kinase assays using purified proteins demonstrated that the addition of p21/waf1 to cyclin D2/cdk4 increased the kinase activity of cdk4. Conclusion These data suggest that the p21/cyclin D2/cdk4 complex is not an inhibitory complex and that p21/waf1 could potentially function as an assembly factor for the cyclin D2/cdk4 complex in HTLV-1 infected cells. A by-product of this assembly with cyclin D2/cdk4 is the sequestration of p21/waf1 away from the cyclin E/cdk2 complex, allowing this active cyclin-cdk complex to phosphorylate Rb pocket proteins efficiently and push cells through the G1/S checkpoint. These two distinct functional and physical activities of p21/waf1 suggest that RNA tumor viruses manipulate the G1/S checkpoint by deregulating cyclin and cdk complexes.

  2. The role of cyclin D2 and p21/waf1 in human T-cell leukemia virus type 1 infected cells

    Science.gov (United States)

    Kehn, Kylene; Deng, Longwen; de la Fuente, Cynthia; Strouss, Katharine; Wu, Kaili; Maddukuri, Anil; Baylor, Shanese; Rufner, Robyn; Pumfery, Anne; Bottazzi, Maria Elena; Kashanchi, Fatah

    2004-01-01

    Background The human T-cell leukemia virus type 1 (HTLV-1) Tax protein indirectly influences transcriptional activation, signal transduction, cell cycle control, and apoptosis. The function of Tax primarily relies on protein-protein interactions. We have previously shown that Tax upregulates the cell cycle checkpoint proteins p21/waf1 and cyclin D2. Here we describe the consequences of upregulating these G1/S checkpoint regulators in HTLV-1 infected cells. Results To further decipher any physical and functional interactions between cyclin D2 and p21/waf1, we used a series of biochemical assays from HTLV-1 infected and uninfected cells. Immunoprecipitations from HTLV-1 infected cells showed p21/waf1 in a stable complex with cyclin D2/cdk4. This complex is active as it phosphorylates the Rb protein in kinase assays. Confocal fluorescent microscopy indicated that p21/waf1 and cyclin D2 colocalize in HTLV-1 infected, but not in uninfected cells. Furthermore, in vitro kinase assays using purified proteins demonstrated that the addition of p21/waf1 to cyclin D2/cdk4 increased the kinase activity of cdk4. Conclusion These data suggest that the p21/cyclin D2/cdk4 complex is not an inhibitory complex and that p21/waf1 could potentially function as an assembly factor for the cyclin D2/cdk4 complex in HTLV-1 infected cells. A by-product of this assembly with cyclin D2/cdk4 is the sequestration of p21/waf1 away from the cyclin E/cdk2 complex, allowing this active cyclin-cdk complex to phosphorylate Rb pocket proteins efficiently and push cells through the G1/S checkpoint. These two distinct functional and physical activities of p21/waf1 suggest that RNA tumor viruses manipulate the G1/S checkpoint by deregulating cyclin and cdk complexes. PMID:15169570

  3. Expression of p21WAF1 in Astler-Coller stage B2 colorectal cancer is associated with survival benefit from 5FU-based adjuvant chemotherapy.

    Science.gov (United States)

    Sulzyc-Bielicka, Violetta; Domagala, Pawel; Urasinska, Elzbieta; Bielicki, Dariusz; Safranow, Krzysztof; Domagala, Wenancjusz

    2011-04-01

    In several, but not all, previous studies, positive p21(WAF1) expression has been suggested as an indicator of a good prognosis in patients with stage III/IV colorectal cancer. However, it is not known whether the same is true for stage B2 patients. The purpose of this study is to assess the influence of p21(WAF1) expression in tumor cells on disease-free survival (DFS) and overall survival (OS) of Astler-Coller stage B2 and C patients with colorectal cancer who underwent 5-fluorouracil-based adjuvant chemotherapy. Nuclear p21(WAF1) was detected by immunohistochemistry in tissue microarrays from 275 colorectal cancers. The expression of p21(WAF1) was associated with DFS (p = 0.025) and OS (p = 0.008) in the subgroup of stage B2 patients that was treated with adjuvant chemotherapy. In multivariate analysis, it remained the only independent prognostic parameter in relation to DFS and OS (p = 0.035 and p = 0.02, respectively). In the subgroup of 72 stage B2 patients with positive p21(WAF1) expression but not in the subgroup of 61 stage B2 patients with negative p21(WAF1) expression, adjuvant chemotherapy was associated with better DFS (85% 5-year survival versus 65% without chemotherapy, p = 0.03) and OS (96% versus 82%, p = 0.014). In the combined stage B2 and C group of patients treated with adjuvant chemotherapy, positive p21(WAF1) expression was also associated with better DFS and OS (p = 0.03, p = 0.002, respectively). Expression of p21(WAF1) in colorectal tumor cells identifies a subgroup of Astler-Coller stage B2 patients who could benefit significantly from 5FU-based chemotherapy and may improve the selection of patients for adjuvant chemotherapy.

  4. Glycogen synthase kinase 3beta phosphorylates p21WAF1/CIP1 for proteasomal degradation after UV irradiation.

    Science.gov (United States)

    Lee, Ji Young; Yu, Su Jin; Park, Yun Gyu; Kim, Joon; Sohn, Jeongwon

    2007-04-01

    UV irradiation has been reported to induce p21(WAF1/CIP1) protein degradation through a ubiquitin-proteasome pathway, but the underlying biochemical mechanism remains to be elucidated. Here, we show that ser-114 phosphorylation of p21 protein by glycogen synthase kinase 3beta (GSK-3beta) is required for its degradation in response to UV irradiation and that GSK-3beta activation is a downstream event in the ATR signaling pathway triggered by UV. UV transiently increased GSK-3beta activity, and this increase could be blocked by caffeine or by ATR small interfering RNA, indicating ATR-dependent activation of GSK-3beta. ser-114, located within the putative GSK-3beta target sequence, was phosphorylated by GSK-3beta upon UV exposure. The nonphosphorylatable S114A mutant of p21 was protected from UV-induced destabilization. Degradation of p21 protein by UV irradiation was independent of p53 status and prevented by proteasome inhibitors. In contrast to the previous report, the proteasomal degradation of p21 appeared to be ubiquitination independent. These data show that GSK-3beta is activated by UV irradiation through the ATR signaling pathway and phosphorylates p21 at ser-114 for its degradation by the proteasome. To our knowledge, this is the first demonstration of GSK-3beta as the missing link between UV-induced ATR activation and p21 degradation.

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

    Science.gov (United States)

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

    1997-10-01

    p21WAF1/Cip1 is a recently identified gene involved in cell cycle regulation through cyclin-CDK-complex inhibition. The expression of this gene in several cell lines seems to be induced by wild-type, but not mutant, p53. p21WAF1/Cip1 expression has been studied at both mRNA and protein levels in a series of 49 normal mucosae and squamous cell carcinomas of the larynx. A significant association was found between mRNA and protein expression in tumours (P global S-phase of the carcinomas. p53 mutations (exons 5-9) were found in ten carcinomas with p21WAF1/Cip1 expression, but no p53 mutations were detected in three p21WAF1/Cip1-negative tumours. In conclusion, p21WAF1/Cip1 expression is frequently upregulated in squamous cell carcinomas of the larynx and is associated with tumour cell differentiation. p21WAF1/Cip1 expression in these tumours is independent of p53 gene mutations.

  6. In vitro effect of p21WAF-1/CIP1 gene on growth of human glioma cells mediated by EGFR targeted non-viral vector GE7 system

    Institute of Scientific and Technical Information of China (English)

    陈永新; 许秀兰; 张光霁; 王韦; 金海英; 卢亦成; 朱诚; 顾健人

    2003-01-01

    Objective: To construct the EGFR targeted non-viral vector GE7 system and explore the in vitro effect of p21WAF-1/CIP1 gene on growth of human glioma cells mediated by the GE7 system. Methods: The EGFR targeted non-viral vector GE7 gene delivery system was constructed. The malignant human glioma cell line U251MG was transfected in vitro with β-galactosidase gene(reporter gene) and p21WAF-1/CIP1 gene (therapeutic gene) using the GE7 system. By means of X-gal staining, MTS and FACS, the transfection efficiency of exogenous gene and apoptosis rate of tumor cells were examined. The expression of p21WAF-1/CIP1 gene in transfected U251MG cell was examined by immunohistochemistry staining. Results: The highest transfer rate of exogenous gene was 70%. After transfection with p21WAF-1/CIP1 gene, the expression of WAF-1 increased remarkably and steadily; the growth of U251MG cells were inhibited evidently. FACS examination showed G1 arrest. The average apoptosis rate was 25.2%. Conclusion: GE7 system has the ability to transfer exogenous gene to targeted cells efficiently, and expression of p21WAF-1/CIP1 gene can induce apoptosis of glioma cell and inhibit its growth.

  7. Misregulation of polo-like protein kinase 1, P53 and P21WAF1 in epithelial ovarian cancer suggests poor prognosis.

    Science.gov (United States)

    Zhang, Ruitao; Shi, Huirong; Ren, Fang; Liu, Huina; Zhang, Minghui; Deng, Youxing; Li, Xia

    2015-03-01

    Polo-like protein kinase 1 (PLK1), P53 and P21WAF1 are relevant to cell cycle checkpoints and cancer biology. Misregulation of PLK1, P53 and P21WAF1 has been detected in several types of malignant tumors. The present study aimed to clarify the role of PLK1, P53 and P21WAF1 in the prognosis of ovarian cancer. PLK1 and P53 shRNA lentiviral plasmids were transfected into SK-OV-3 cells, respectively. Cell proliferation, apoptosis and invasion were examined by MTT assay, flow cytometry and Matrigel assay, respectively. Survival time of the animals was observed in a xenograft model. Expression levels of PLK1, P53 and P21WAF1 were detected in different ovarian tissues by immunohistochemistry and western blot analysis. Their correlations to the clinicopathologic characteristics of the epithelial ovarian cancer (EOC) cases and their interrelationships were analyzed. Risk factors of prognosis for EOC were determined by logistic regression analysis. The survival time of EOC patients was measured by Kaplan-Meier analysis. After PLK1 or P53 knockdown, proliferation of the SK-OV-3 cells was inhibited, the apoptosis rate was increased, and cell invasion was suppressed in vitro, and the survival time was prolonged in the animals. Expression levels of P53, p-P53 (Ser15), P21WAF1, growth arrest and DNA damage‑inducible gene 45 (GADD45) and 14-3-3σ were upregulated in the SK-OV-3 cells after PLK1 knockdown, but downregulated after P53 knockdown. Higher expression levels of PLK1 and P53 were observed in patients with a higher FIGO stage and worse histological differentiation, but lower P21WAF1 was noted at a higher FIGO stage. Negative correlations were observed between expression of PLK1 and P53 and P53 and P21WAF1 in the EOC cases. PLK1, P53 and P21WAF1 could be used to assess the prognosis of EOC, respectively, but only PLK1 was found to be an independent prognostic factor. The overall survival time of subjects exhibiting PLK1-positive/P53-positive expression and PLK1-positive/P

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

    Science.gov (United States)

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

    2009-06-01

    Betel-quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53-dependent p21(WAF1) protein expression in Clone-9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3-related (ATR)-p53-p21(WAF1) and the phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline-induced p53 and p21(WAF1) protein expression in Clone-9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline-activated ATM/ATR substrate contained p-p53Ser15. Moreover, arecoline only increased the levels of the p-p53Ser6, p-p53Ser15, and p-p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline-induced p-p53Ser15 and p21(WAF1) at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30-60 min. Dominant-negative PI3K plasmids attenuated arecoline-induced p21(WAF1), but not p-p53Ser15, at 24 h. Rapamycin attenuated arecoline-induced phosphrylated p-p53Ser15, but not p21(WAF1), at 24 h. ATM shRNA, but not dominant-negative PI3K plasmids, attenuated arecoline-induced p21(WAF1) gene transcription. We conclude that arecoline activates the ATM/ATR-p53-p21(WAF1) and the PI3K/Akt-mTOR-p53 pathways in Clone-9 cells. Arecoline-induced phosphorylated p-p53Ser15 expression is dependent on ATM whereas arecoline-induced p21(WAF1) protein expression is dependent on ATM and PI3K. Moreover, p21(WAF1) gene is transcriptionally induced by arecoline-activated ATM. (c) 2009 Wiley-Liss, Inc.

  9. p21WAF1、cyclinD1和pRb在膀胱移行细胞癌中的表达及其意义%Expression of p21WAF1,cyclin D1 and pRb in bladder transitional cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    米振国; 马志方; 王东文; 刘红耀; 杨晓峰

    2002-01-01

    目的探讨p21WAF1、细胞周期蛋白D1(cyclin D1)和pRb在膀胱移行细胞癌(BTCC)中的表达及相互关系和其意义.方法应用免疫组织化学SP法检测57例BTCC患者癌组织中p21WAF1、Cyclin D1和pRb的蛋白表达.结果 p21WAF1、cyclin D1和pRb的阳性表达率分别为36.8%、49.1%和45.6%,p21WAF1随病理分级升高阳性率显著下降,cyclin D1和pRb的表达与BTCC的病理分级、临床分期和有无转移均相关,p21WAF1与pRb的表达呈负相关,cyclin D1和pRb的表达呈正相关,而p21WAF1与cyclin D1的表达无关.结论 p21WAF1/cyclin D1/pRb通路异常与BTCC的发生发展密切相关,p21WAF1的改变可能为癌变的早期事件,联合检测p21WAF1、cyclin D1和pRb可较准确地评价BTCC的生物学特性,估计预后,指导治疗.

  10. p21WAF1、Cyclin D1和PRb在膀胱移行细胞癌中的表达及其意义%Expression of p21WAF1, Cyclin D1 and PRb in Bladder Transitional Cell Carcinoma

    Institute of Scientific and Technical Information of China (English)

    米振国; 马志方; 王东文; 刘红耀; 杨晓峰

    2002-01-01

    目的:探讨p21WAF1、细胞周期蛋白D1(Cyclin D1)和PRb在膀胱移行细胞癌(BTCC)中的表达及相互关系及其意义。方法:应用免疫组织化学SP法检测57例BTCC患者癌组织中p21WAF1、Cyclin D1和PRb的蛋白表达。结果:p21WAF1、Cyclin D1和PRb的阳性表达率分别为36.8%、49.1%和45.6%,p21WAF1随病理分级升高阳性率显著下降,Cylin D1和PRb的表达与BTCC的病理分级、临床分期和有无转移均相关,p21WAF1与PRb的表达呈负相关,Cyclin D1和PRb的表达呈正相关,而p21WAF1与Cyclin D1的表达无关。结论:p21WAF1/Cyclin D1/PRb通路异常与BTCC的发生发展密切相关,p21WAF1的改变可能为癌变的早期事件,联合检测p21WAF1、Cyclin D1和PRb可较准确地评价BTCC的生物学特性,估计预后,指导治疗。

  11. Expression of p21WAF1 is related to acetylation of histone H3 in total chromatin in human colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Ying-Xuan Chen; Jing-Yuan Fang; Rong Lu; De-Kai Qiu

    2007-01-01

    AIM: To explore the relationship between acetylation of histone in total chromatin and p21WAF1 expression regulation in human colorectal carcinoma.METHODS: We analyzed the expression of tumor suppressor gene p21WAF1 mRNA by RT-PCR or realtime PCR in 33 samples of colorectal cancerous tissue,corresponding para-cancerous tissue and normal colorectal mucosa, and also examined the level of acetylated histone H3 in total chromatin using Western blotting.RESULTS: The expression level of p21WAF1 mRNA was significantly lower in colorectal cancerous tissue from 33 patients than in para-cancerous tissue and normal colorectal mucosa (2377.95 ± 865.80 vs 3216.58 ±1149.42 and 3541.61 ± 1433.17 respectively, P <0.01). In addition, when p21WAF1 mRNA expression was undectectable or at very low level (50% less than that in adjacent tissue and normal colorectal mucosa) in all tissues, the level of acetylated histone H3 in colorectal cancerous tissue was significantly lower than that in corresponding para-cancerous tissue and normal colorectal mucosa in five of seven (71.43%) cases. The transcriptional level of p21WAF1 in colorectal carcinoma might not be associated with its biological behaviors.CONCLUSION: The down-regulation of p21WAF1 transcription is involved in the tumorigenesis and development of colorectal carcinoma. The down-expression of p21WAF1 mRNA in colorectal carcinoma might be associated with histone hypoacetylation in chromatin but not with biological behaviors.

  12. [Experimental gene therapy using p21/WAF1 gene in esophageal squamous cell carcinoma--adenovirus infection and gene gun technology].

    Science.gov (United States)

    Fujii, T; Tanaka, Y; Tanaka, T; Matono, S; Sueyoshi, S; Fujita, H; Shirouzu, K; Kato, S; Yamana, H

    2001-10-01

    p21/WAF1 (p21) inhibits the activity of the cyclin/cdk complex and controls the G1 to S cell phase transition. In the present study, we used a recombinant adenoviral approach and gene gun technology to introduce p21 into esophageal cancer cells in order to assess the effect of p21 on cell growth. Infection with the p21 adenovirus (AdV) using gene gun technology resulted in inhibition of TE9 and KE3 cell growth. The levels of involucrin, which is a marker of squamous epithelium differentiation, markedly increased at 48 h and 72 h after p21 AdV infection in TE9 cells. These results indicate that p21 plays an important role in esophageal cancer cell proliferation. Overexpression of the p21 gene can inhibit cell growth and induce differentiation in esophageal cancer cells. p21 gene therapy may prove beneficial in the treatment of esophageal cancer.

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

    Directory of Open Access Journals (Sweden)

    Yu-Fang Chen

    Full Text Available 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.

  14. Expression,Purification and Spectral Characterization of p21Waf1/Cip1

    Institute of Scientific and Technical Information of China (English)

    SHI Qiao-yun; ZHENG Yong-chen; REN Jin-song; QU Xiao-gang

    2008-01-01

    p21Wafl/Cip1 ,best known as a broad-specificity inhibitor of cyclin/cyclin-dependent kinase complexes,can interact with various target proteins,and this ability relies on its structural plasticity.Therefore,studies on the structural properties of p21 are very important to understand its structure-function relationship.However,detailed studies on its secondary strcture and biophysical properties have been comparatively sparse.A human p21 gene was cloned into the temperature expression vector pBV220 and transformed into Escherichia coli strain JM109.Recombinant protein was expressed as a non-fusion protein and purified by gel filtration and anion exchange chromatography.The purified protein was verified by Western blot and the functional activity was recognized by pull-down assay.Furthermore,circular dichroisrn,fluorescence spectroscopy,and fluorescence quenching methods were used to characterize the conformational properties of the purified protein.The results indicate that it was largely unstructured under the native solution conditions,and its tryptophan residues were exposed and located in a positively charged microenvironment.This study lays a good foundation for further study of p21 binding to its different partners.

  15. The carboxy terminus of p53 mimics the polylysine effect of protein kinase CK2-catalyzed MDM2 phosphorylation

    DEFF Research Database (Denmark)

    Guerra, B; Götz, C; Wagner, P

    1997-01-01

    The oncogene product MDM2 can be phosphorylated by protein kinase CK2 in vitro 0.5-1 mol of phosphate were incorporated per mol MDM2 protein. The catalytic subunit of protein kinase CK2 (alpha-subunit) catalyzed the incorporation of twice as much phosphate into the MDM2 protein as it was obtained...... with the holoenzyme. Polylysine stimulated MDM2 phosphorylation by CK2 holoenzyme threefold in contrast to the alpha-subunit-catalyzed MDM2 phosphorylation which was reduced by about 66% when polylysine was added. Full length p53, but also a peptide representing a C-terminal fragment of the tumor suppressor gene......(264-393) was on the average close to twofold and inhibition in the case of the alpha-subunit-catalyzed MDM2 phosphorylation was about 40%. Phosphorylation of MDM2 by CK2 holoenzyme in the presence of the p21(WAF1/CIP1), known to be a potent inhibitor of cyclin-dependent protein kinases, also led to a significant reduction...

  16. Expression of p21 (Waf1/Cip1) in head and neck cancer in relation to proliferation, differentiation, p53 status and cyclin D1 expression

    NARCIS (Netherlands)

    Oijen, M.G.C.T. van; Tilanus, M.G.; Medema, R.H.; Slootweg, P.J.

    1998-01-01

    p21(Waf1/Cipl) is a critical downstream effector in the p53-dependent pathway of growth control and causes growth arrest through inhibition of cyclin-dependent kinases. In this study 67% of 43 head and neck squamous cell carcinoma (HNSCC) and 60% of 15 tumour-adjacent oral dysplasias overexpressed p

  17. Diallyl disulfide suppresses growth of HL-60 cell through increasing his-tone acetylation and p21WAF1 expression in vivo and in vitro

    Institute of Scientific and Technical Information of China (English)

    Jie ZHAO; Wei-guo HUANG; Jie HE; Hui TAN; Qian-jin LIAO; Qi SU

    2006-01-01

    Aim: To examine the differentiation induction and growth inhibition of HL-60 cells by diallyl disulfide (DADS), and its relationship with the alterations of histone acetylation and p21WAF1 expression in vitro and in vivo. Methods: Differentiation was studied by nitroblue tetrazolium (NET) reduction of HL-60 cell in vitro. HL-60 cells 5xl06 were injected into the right side of the peritoneal cavity of severe combined immunodeficiency (SCID) mice. When the peritoneal neoplasms were detected, the SCID mice were randomly divided into 3 groups and received an ip injection of vehicle alone (NS), DADS or sodium butyrate (SB). The growth inhibition of peritoneal neoplasms induced by DADS was observed by a growth curve. The cycle distribution of HL-60 cells in SCID mice was monitored by flow cytometry. The expression of acetylated histone H3, H4 and p21WAF1 were measured by Western blot. Results: After treatment with DADS for 0-72 h, the NET reduction ability of HL-60 cells increased in a time-dependent manner, compared with no treatment of HL-60 cells. In the HL-60 cells treated with DADS for 24 h, the expression of acetylated histone H3, H4, and p21WAF1 increased obviously. After treatment with DADS, tumor growth was markedly suppressed. HL-60 cells from mice treated with DADS were blocked in the G1 phase, from 25.4% to 63.4%. The tumors from the mice treated with DADS showed an increase of acetylated histone H3, H4, and p21WAF1. Conclusion: DADS could induce differentiation and inhibit the growth of HL-60 cells through increasing the expression of acetylated histone H3, H4, and p21WAF1 in vitro and in vivo.

  18. 5-Aza-2'-deoxycytidine Activates the p53/p21waf1/Cip1 Pathway to Inhibit Cell Proliferation

    Institute of Scientific and Technical Information of China (English)

    Wei-GuoZhu; TheresaHileman; YangKe; PeichangWang; ShaoliLu; WenruiDuan; ZunyanDai; TanjunTong; MiguelA.Villalona-Calero; ChristophPlass; GregoryA.Otterson

    2005-01-01

    In addition to its demethylating function, 5-aza-2'-de- oxycytidine (5-aza-CdR) also plays an important role in inducing cell cycle arrest, differentiation, and cell death. However, the mechanism by which 5-aza-CdR in. duces antineoplastic activity is not clear. In this study, we found that 5-aza-CdR at limited concentrations(0.01-5μM) induces inhibition of cell proliferation as well as increased p53/p21waf1/Cip1 expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21wafa/Cip1 expression induced by 5-aza-CdR was not seen in A549 cells transfected with the wild-type human papilloma virus type-16 E6 gene that induces p53 degradation. Furthermore, deletion analysis and site-directed mutagenesis of the p21 promoter reveals that 5-aza-CdR induces p21wafa/Cip1 expression through two p53 binding sites in the p21 promoter. Finally, 5-aza-CdR-induced p21waf1/cip1 expression was dependent on DNA damage but not on DNA demethylation as demonstrated by comet assay and bisulfite sequencing, respectively. Our data provide useful clues for judging the therapeutic efficacy of 5-aza-CdR in the treatment of human cancer cells.

  19. Gambogic acid induces growth inhibition and differentiation via upregulation of p21waf1/cip1 expression in acute myeloid leukemia cells.

    Science.gov (United States)

    Chen, Yan; Hui, Hui; Li, Zheng; Wang, Hong-Mei; You, Qi-Dong; Lu, Na

    2014-10-01

    Gambogic acid (GA) is the major active ingredient of gamboges, a brownish to orange resin product from Garcinia hanburyi tree in Southeast Asia. This compound exhibits anti-cancer effect on solid tumors. In this study, we investigated the effects of GA on the growth and differentiation of acute myeloid leukemia cells by growth-inhibition detection, morphological changes observation, nitroblue tetrazolium reduction, and the expression of the relative cell-surface differentiation markers. The results showed that GA could inhibit cell growth and promote differentiation in U937 and HL-60 cells. In addition, GA upregulated the expression of p21waf1/cip1 in the two cell lines. Finally, downregulating the p21waf1/cip1 expression with small interfering RNA partially blocked GA-induced cell growth inhibition and differentiation. These results of this study revealed that GA may be used as one of the investigational drugs for acute myeloid leukemia.

  20. Involvement of p21Waf1/Cip1 cleavage during roscovitine-induced apoptosis in non-small cell lung cancer cells.

    Science.gov (United States)

    Zhang, Tao; Jiang, Tao; Zhang, Feng; Li, Chen; Zhou, Yong-An; Zhu, Yi-Fang; Li, Xiao-Fei

    2010-01-01

    Roscovitine, a cyclin-dependent kinases (CDKs) inhibitor, has been reported to have anti-tumor effects in some cancer cell lines by inducing apoptosis. However, the exact underlying mechanisms are not fully understood. Here, we report that roscovitine induces expression and cleavage of the universal CDK inhibitor p21Waf1/Cip1 in non-small cell lung cancer (NSCLC) A549 cells in a dose-dependent manner. Western blots of roscovitine-treated cells undergoing apoptosis consistently demonstrated a 15 kDa band that was not detected in control cultures. CDK2 activity and PCNA expression were repressed with increasing dose of roscovitine. Accompanying these molecular changes was a progressive arrest of G2 phase and decreasing of 5-bromo-2-deoxyuridine (Brdu) incorporation of S phase cells. Caspase-3 inhibitor z-DEVD-fmk almost completely abolished roscovitine-induced apoptosis, as well as the appearance of 15 kDa band, indicating that p21Waf1/Cip1 cleavage was mediated by caspase-3 activity. Furthermore, this band was predominant in the floating apoptotic cells, while weakened in the adherent cells which were vital and pre-apoptotic. We also showed that roscovitine induced an enhanced expression of gamma-H2AX, which was blocked by caspase-3 inhibition, suggesting that p21Waf1/Cip1 cleavage may interfere with DNA repair, leading to increased frequency of double strand breaks (DSBs) and enhanced apoptosis. Here we show, for the first time, that p21Waf1/Cip1 cleavage, which is mediated by caspase-3 activity, is involved in roscovitine-induced apoptosis.

  1. Expression of Survivin, CyclinD1, p21WAF1, Caspase-3 in Cervical Cancer and Its Relation with Prognosis

    Institute of Scientific and Technical Information of China (English)

    LU Shi; ZHANG Baohua; WANG Zehua

    2005-01-01

    The implications of Survivin, CyclinD1, p21WAF1, Caspase-3 in the development, progression and prognosis in cervical cancer were investigated. By using immunohistochemical SP method, the expression of Survivin, CyclinD1, p21WAF1 , Caspase-3 was detected in 41 cases of cervical cancer, 17 cases of cervical intraepithelial neoplasia (CIN) and 10 cases of normal tissues, and their relation with pathological grade, clinical stage, metastasis and survival time was analyzed.The results showed that the positive expression rate of Survivin, CyclinD1 in cervical cancer was significantly higher than in CIN group and normal control group (P<0.05). The median survival time in the patients with cervical cancer positive for Survivin and CyclinD1 was significantly shorter than in those with negative expression (P<0.05). The expression of both Survivin and CyclinD1 was not related with tumor grade, clinical stage and metastasis (P>0. 05). The positive expression rate of p21WAF1 , Caspase-3 in cervical ca rcer was significantly lower than in CIN group and normal control group (P<0.05), and had a close relation with tumor grade (P<0.05). The expression of Survivin in cervical cancer in cervical cancer was negatively associated with that of Caspase-3 (P<0.01), but positively with that of CyclinD1 (P<0.01). Cox Multivariate analysis revealed that Survivin was the independent prognostic indicator influencing the survival time of the patients with cervical cancer (P<0.05). It was suggested that the high expression of Survivin or CyclinD1, and low expression of p21WAF1 or Caspase-3 was closely correlated with the development of cervical cancer. Survivin and CyclinD1 could be used as a useful indicator to predict the prognosis of cervical cancer.

  2. Suppression of c-Myc enhances p21(WAF1/CIP1) -mediated G1 cell cycle arrest through the modulation of ERK phosphorylation by ascochlorin.

    Science.gov (United States)

    Jeong, Yun-Jeong; Hoe, Hyang-Sook; Cho, Hyun-Ji; Park, Kwan-Kyu; Kim, Dae-Dong; Kim, Cheorl-Ho; Magae, Junji; Kang, Dong Wook; Lee, Sang-Rae; Chang, Young-Chae

    2017-08-18

    Numerous anti-cancer agents inhibit cell cycle progression via a p53-dependent mechanism; however, other genes such as the proto-oncogene c-Myc are promising targets for anticancer therapy. In the present study, we provide evidence that ascochlorin, an isoprenoid antibiotic, is a non-toxic anti-cancer agent that induces G1 cell cycle arrest and p21(WAF1/CIP1) expression by downregulating of c-Myc protein expression. Ascochlorin promoted the G1 arrest, upregulated p53 and p21(WAF1/CIP1) , and downregulated c-Myc in HCT116 cells. In p53-deficient cells, ascochlorin enhanced the expression of G1 arrest-related genes except p53. Small interfering RNA (siRNA) mediated c-Myc silencing indicated that the transcriptional repression of c-Myc was related to ascochlorin-mediated modulation of p21(WAF1/CIP1) expression. Ascochlorin suppressed the stabilization of the c-Myc protein by inhibiting ERK and P70S6K/4EBP1 phosphorylation, whereas it had no effect on c-Myc degradation mediated by PI3K/Akt/GSK3β. The ERK inhibitor PD98059 and siRNA-mediated ERK silencing induced G1 arrest and p21(WAF1/CIP1) expression by downregulating c-Myc in p53-deficient cells. These results indicated that ascochlorin-induced G1 arrest is associated with the repression of ERK phosphorylation and c-Myc expression. Thus, we reveal a role for ascochlorin in inhibiting tumor growth via G1 arrest, and identify a novel regulatory mechanism for ERK /c-Myc. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  3. Expression of the cell cycle regulation proteins p53 and p21WAF1 in different types of non-dysplastic leukoplakias

    Directory of Open Access Journals (Sweden)

    Fernanda Visioli

    2012-06-01

    Full Text Available OBJECTIVES: The aim of this study was to analyze the immunolabeling of two cell cycle protein regulators, p53 and p21WAF1, in non-dysplastic leukoplakias with different epithelial alterations: acanthosis, hyperkeratosis and acanthosis combined with hyperkeratosis, and compare them with dysplastic leukoplakias. MATERIAL AND METHODS: This was a prospective cohort study involving 36 patients with oral homogeneous leukoplakias. excisional biopsies were performed and the patients remain under clinical follow-up. The leukoplakias were divided into four groups: 6 acanthosis, 9 hyperkeratosis, 10 acanthosis combined with hyperkeratosis, and 11 epithelial dysplasias. Paraffin-embebeded sections were immunostained for p53 and p21WAF1. Five hundred cells from the basal layer and 500 from the parabasal layer were counted to determine the percentage of positive cells. A qualitative analysis was also carried out to determine the presence or absence of immunohistochemical staining in the intermediate and superficial layers. Groups were compared with ANOVA (p0.05. CONCLUSIONS: Our findings failed to differentiate the non-dysplastic lesions by means of p53 and p21WAF1 immunostaining, notwithstanding similar profiles between non-dysplastic and dysplastic leukoplakias were observed.

  4. Cholesterol Retards Senescence in Bone Marrow Mesenchymal Stem Cells by Modulating Autophagy and ROS/p53/p21Cip1/Waf1 Pathway

    Directory of Open Access Journals (Sweden)

    Mingyu Zhang

    2016-01-01

    Full Text Available In the present study, we demonstrated that bone marrow mesenchymal stem cells (BMSCs of the 3rd passage displayed the senescence-associated phenotypes characterized with increased activity of SA-β-gal, altered autophagy, and increased G1 cell cycle arrest, ROS production, and expression of p53 and p21Cip1/Waf1 compared with BMSCs of the 1st passage. Cholesterol (CH reduced the number of SA-β-gal positive cells in a dose-dependent manner in aging BMSCs induced by H2O2 and the 3rd passage BMSCs. Moreover, CH inhibited the production of ROS and expression of p53 and p21Cip1/Waf1 in both cellular senescence models and decreased the percentage of BMSCs in G1 cell cycle in the 3rd passage BMSCs. CH prevented the increase in SA-β-gal positive cells induced by RITA (reactivation of p53 and induction of tumor cell apoptosis, a p53 activator or 3-MA (3-methyladenine, an autophagy inhibitor. Our results indicate that CH not only is a structural component of cell membrane but also functionally contributes to regulating cellular senescence by modulating cell cycle, autophagy, and the ROS/p53/p21Cip1/Waf1 signaling pathway.

  5. Chemosensitization by antisense oligonucleotides targeting MDM2.

    Science.gov (United States)

    Bianco, Roberto; Ciardiello, Fortunato; Tortora, Giampaolo

    2005-02-01

    The MDM2 oncogene is overexpressed in many human cancers, including sarcomas, certain hematologic malignancies, and breast, colon and prostate cancers. The p53-MDM2 interaction pathway has been suggested as a novel target for cancer therapy. To that end, several strategies have been explored, including the use of small polypeptides targeted to the MDM2-p53 binding domain, anti-MDM2 antisense oligonucleotides, and natural agents. Different generations of anti-human-MDM2 oligonucleotides have been tested in in vitro and in vivo human cancer models, revealing specific inhibition of MDM2 expression and significant antitumor activity. Use of antisense oligos potentiated the effects of growth inhibition, p53 activation and p21 induction by several chemotherapeutic agents. Increased therapeutic effectiveness of chemotherapeutic drugs in human cancer cell lines carrying p53 mutations or deletions have shown the ability of MDM2 inhibitors to act as chemosensitizers in various types of tumors through both p53-dependent and p53-independent mechanisms. Inhibiting MDM2 appears to also have a role in radiation therapy for human cancer, regardless of p53 status, providing a rationale for the development of a new class of radiosensitizers. Moreover, MDM2 antisense oligonucleotides potentiate the effect of epidermal growth factor receptor (EGFR) inhibitors by affecting in vitro and in vivo proliferation, apoptosis and protein expression in hormone-refractory and hormone-dependent human prostate cancer cells. These data support the development, among other MDM2 inhibitors, of anti-MDM2 antisense oligonucleotides as a novel class of anticancer agents, and suggest a potentially relevant role for the oligonucleotides when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.

  6. MicroRNA-17 promotes normal ovarian cancer cells to cancer stem cells development via suppression of the LKB1-p53-p21/WAF1 pathway.

    Science.gov (United States)

    Liu, Te; Qin, Wenxing; Hou, Lengchen; Huang, Yongyi

    2015-03-01

    The mechanism underlying the development of human ovarian cancer is poorly understood. The liver kinase protein, LKB1, is hypothesized to play a pivotal role in tumor cell proliferation and invasion capacity through regulation of p53 and p21/WAF1 expression. Previous studies suggest LKB1 may, in turn, be regulated by microRNA-17. Here, we examined the role of miR-17 in the expression of LKB1 and the downstream effects on proliferation and invasion capacity of normal ovarian cancer cells (OCCs) and ovarian stem cells. In this study, both the mRNA and protein expression levels of LKB1, p53, and p21 decreased in OCCs following transfection with a miR-17 expression plasmid. MiR-17 expression affected cell cycle regulation and stimulated the proliferation and invasion capacity of OCCs in vitro. ChIP assays indicated that the binding efficiency of p53 to the p21/WAF1 gene promoter was much lower in miR-17 transfected OCCs than in OCCs transfected with a mutated miR-17. Co-immunoprecipitation and western blotting showed significantly lower levels of p53 and p53 Ser15-pho in the miR-17 transfected OCCs as compared to the mutant miR-17 transfected OCCs. Xenograft experiments confirmed that suppression of tumor growth in vivo occurred in the absence of functional miR-17. These findings suggest that mature miR-17 expression may have an important role in the pathogenesis of human ovarian tumors through its interference with the LKB1-p53-p21/WAF1 pathway expression by epigenetic modification. These findings are of potential importance in the identification of novel therapeutic targets in human ovarian cancer.

  7. Evaluation of p53,p21waf1,and PCNA in the diagnosis on lung cancer%p53、p21waf1和增殖细胞核抗原诊断肺癌的评估

    Institute of Scientific and Technical Information of China (English)

    丁续红; 杨炯; 胡苏萍; 吴小军; 李清泉

    2001-01-01

    目的 评价p53、p21waf1和增殖细胞核抗原(PCNA)蛋白表达对肺癌的诊断价值。方法 采用免疫组化法检测114例肺癌和89例肺良性疾病组织标本p53、p21waf1和PCNA蛋白表达状况,计算三项指标及其组合诊断肺癌的各项参数。结果 (1)肺癌p53、p21waf1及PCNA表达阳性率分别为47.37%、75.44%和80.70%,明显高于肺良性疾病组织(P<0.001)。其OR值分别为39.15,5.75和6.76。(2)p53、p21waf1及PCNA蛋白表达与肺癌TNM分期无关。不同病理类型p53和p21waf1蛋白表达也无差异,但鳞癌PCNA表达阳性率高于小细胞癌。(3)p53诊断肺癌的阳性似然比为21.08,高于p21waf1和PCNA,但灵敏度较低。(4)PCNA诊断鳞癌的Youden指数为0.514 6,显著高于小细胞癌。但其阳性似然比为2.29,仍低于p53。(5)p53和PCNA平行试验的灵敏度为89.47%,特异度为60.67%, 其综合评价参数最高。结论 (1) 肺癌p53、p21waf1和PCNA表达均上调,三者与肺癌均有很强联系。(2)p53是一项很好的肺癌诊断单项指标。p53和PCNA平行试验诊断肺癌的综合价值较高。%Objective To evaluate the diagnostic value of p53, p21waf1 and PCNA on diagnosis of lung cancer. Methods One hundred and fourteen lung cancer patients and 89 cases with benign pulmonary diseases were studied on the expression of p53, p21waf1 and PCNA by immunohistochemical staining. The differences of p53, p21waf1 and PCNA expression status between these two groups were compared and the diagnostic value of these three markers was evaluated.Results (1) The positive rates of p53, p21waf1 and PCNA expression in lung cancer were 47.37%,75.44% and 80.70%,all significantly higher than that in benign pulmonary diseases(P<0.001), with odds ratio 39.15,5.75,6.76 respectively. (2)The positive likelihood ratio of p53 was 21.08, higher than that of p21waf1 and PCNA. Specificity and Youden index were also higher than those of p21waf1 and PCNA, but the

  8. Activation of p21(CIP1/WAF1) in mammary epithelium accelerates mammary tumorigenesis and promotes lung metastasis.

    Science.gov (United States)

    Cheng, Xiaoyun; Xia, Weiya; Yang, Jer-Yen; Hsu, Jennifer L; Chou, Chao-Kai; Sun, Hui-Lung; Wyszomierski, Shannon L; Mills, Gordon B; Muller, William J; Yu, Dihua; Hung, Mien-Chie

    2010-12-03

    While p21 is well known to inhibit cyclin-CDK activity in the nucleus and it has also been demonstrated to have oncogenic properties in different types of human cancers. In vitro studies showed that the oncogenic function of p21is closely related to its cytoplasmic localization. However, it is unclear whether cytoplasmic p21 contributes to tumorigenesis in vivo. To address this question, we generated transgenic mice expressing the Akt-phosphorylated form of p21 (p21T145D) in the mammary epithelium. The results showed that Akt-activated p21 was expressed in the cytoplasm of mammary epithelium. Overexpression of Akt-activated p21 accelerated tumor onset and promoted lung metastasis in MMTV/neu mice, providing evidence that p21, especially cytoplasmic phosphorylated p21, has an oncogenic role in promoting mammary tumorigenesis and metastasis.

  9. Lenalidomide inhibits the proliferation of CLL cells via a cereblon/p21(WAF1/Cip1)-dependent mechanism independent of functional p53.

    Science.gov (United States)

    Fecteau, Jessie-F; Corral, Laura G; Ghia, Emanuela M; Gaidarova, Svetlana; Futalan, Diahnn; Bharati, Ila Sri; Cathers, Brian; Schwaederlé, Maria; Cui, Bing; Lopez-Girona, Antonia; Messmer, Davorka; Kipps, Thomas J

    2014-09-04

    Lenalidomide has demonstrated clinical activity in patients with chronic lymphocytic leukemia (CLL), even though it is not cytotoxic for primary CLL cells in vitro. We examined the direct effect of lenalidomide on CLL-cell proliferation induced by CD154-expressing accessory cells in media containing interleukin-4 and -10. Treatment with lenalidomide significantly inhibited CLL-cell proliferation, an effect that was associated with the p53-independent upregulation of the cyclin-dependent kinase inhibitor, p21(WAF1/Cip1) (p21). Silencing p21 with small interfering RNA impaired the capacity of lenalidomide to inhibit CLL-cell proliferation. Silencing cereblon, a known molecular target of lenalidomide, impaired the capacity of lenalidomide to induce expression of p21, inhibit CD154-induced CLL-cell proliferation, or enhance the degradation of Ikaros family zinc finger proteins 1 and 3. We isolated CLL cells from the blood of patients before and after short-term treatment with low-dose lenalidomide (5 mg per day) and found the leukemia cells were also induced to express p21 in vivo. These results indicate that lenalidomide can directly inhibit proliferation of CLL cells in a cereblon/p21-dependent but p53-independent manner, at concentrations achievable in vivo, potentially contributing to the capacity of this drug to inhibit disease-progression in patients with CLL.

  10. Lenalidomide inhibits the proliferation of CLL cells via a cereblon/p21WAF1/Cip1-dependent mechanism independent of functional p53

    Science.gov (United States)

    Fecteau, Jessie-F.; Corral, Laura G.; Ghia, Emanuela M.; Gaidarova, Svetlana; Futalan, Diahnn; Bharati, Ila Sri; Cathers, Brian; Schwaederlé, Maria; Cui, Bing; Lopez-Girona, Antonia; Messmer, Davorka

    2014-01-01

    Lenalidomide has demonstrated clinical activity in patients with chronic lymphocytic leukemia (CLL), even though it is not cytotoxic for primary CLL cells in vitro. We examined the direct effect of lenalidomide on CLL-cell proliferation induced by CD154-expressing accessory cells in media containing interleukin-4 and -10. Treatment with lenalidomide significantly inhibited CLL-cell proliferation, an effect that was associated with the p53-independent upregulation of the cyclin-dependent kinase inhibitor, p21WAF1/Cip1 (p21). Silencing p21 with small interfering RNA impaired the capacity of lenalidomide to inhibit CLL-cell proliferation. Silencing cereblon, a known molecular target of lenalidomide, impaired the capacity of lenalidomide to induce expression of p21, inhibit CD154-induced CLL-cell proliferation, or enhance the degradation of Ikaros family zinc finger proteins 1 and 3. We isolated CLL cells from the blood of patients before and after short-term treatment with low-dose lenalidomide (5 mg per day) and found the leukemia cells were also induced to express p21 in vivo. These results indicate that lenalidomide can directly inhibit proliferation of CLL cells in a cereblon/p21-dependent but p53-independent manner, at concentrations achievable in vivo, potentially contributing to the capacity of this drug to inhibit disease-progression in patients with CLL. PMID:24990888

  11. Anti-cancer effects of p21WAF1/CIP1 transcriptional activation induced by dsRNAs in human hepatocellular carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Zhi-ming WU; Gang CHEN; Chun DAI; Ying HUANG; Cui-fang ZHENG; Qiong-zhu DONG; Guan WANG; Xiao-wen LI; Xiao-fei ZHANG; Bin LI

    2011-01-01

    Aim: To investigate the anti-cancer effects of p21WAF1/CIP1 transcriptional activation induced by dsRNAs in hepatocellular carcinoma (HCC) cell lines.Methods: HCC cell lines BEL7402, SMMC-7721, MHCC97L, MHCC97H, and MHCCLM3 were used. HCC ceils were treated with dsP21322 (50 nmol/L), dsControl (50 nmol/L), siP21 (50 nmol/L), or mock transfection. The expression of p21 was detected using quantitative PCR and Western blot. The effects of RNA activation on HCC cells were determined using cell viability assays, apoptosis analyses and clonogenic survival assays. Western blot was also conducted to detect the expression of Bcl-xL, survivin, cleaved caspase-3,cleaved caspase-9 and cleaved PARP.Results: At 72 to 120 h following the transfection, dsP21-322 markedly inhibited the viability of HCC cells and clone formation. At the same times, dsP21-322 caused a significant increase in HCC cell apoptosis, as demonstrated with cytometric analysis. The phenomena were correlated with decreased expression levels of the anti-apoptotic proteins Bcl-xL, surviving, and increased expression of cleaved caspase-3, cleaved caspase-9 and cleaved PARP.Conclusion: RNA-induced activation of p21 gene expression may have significant therapeutic potential for the treatment of hepatocellular carcinoma and other cancers.

  12. Platinum-(Ⅳ)-derivative satraplatin induced G2/M cell cycle perturbation via p53-p21waf1/cip1-independent pathway in human colorectal cancer cells

    Institute of Scientific and Technical Information of China (English)

    Murugan KALIMUTHO; Antonella MINUTOLO; Sandro GRELLI; Giorgio FEDERICI; Sergio BERNARDINI

    2011-01-01

    Aim:Platinum-(Ⅳ)-derivative satraplatin represents a new generation of orally available anti-cancer drugs that are under development for the treatment of several cancers.Understanding the mechanisms of cell cycle modulation and apoptosis is necessary to define the mode of action of satraplatin.In this study,we investigate the ability of satraplatin to induce cell cycle perturbation,clonogenicity loss and apoptosis in colorectal cancer (CRC) cells.Methods:CRC cells were treated with satraplatin,and the effects of satraplatin on apoptosis and the cell cycle were evaluated by flow cytometry.Western blot analysis was used to investigate the effects of satraplatin on cell cycle and apoptosis-related proteins.RTqPCR was used to evaluate p53-related mRNA modulation.Results:Satraplatin induced an accumulation of CRC cells predominantly in the G2/M phase.Increased p53 protein expression was observed in the p53 wild-type HCT116 and LoVo cells together with p21waf1/cip1 protein up-regulation.However,p21waf1/cip1 protein accumulation was not observed in the p53 mutant HCT15,HT29,and WiDr cells,even when p53 protein expression was compromised,suggesting that the cell cycle perturbation is p53-p21waf1/cip1 independent.Following a candidate approach,we found an elevated expression of 14-3-3o protein levels in CRC cells,which was independent of the status of p53,further supporting the role of satraplatin in the perturbation of the G2/M cell cycle phase.Moreover,satraplatin treatment induced apoptosis along with Bcl-2 protein down-regulation and abrogated the clonogenic formation of CRC cells in vitro.Conclusion:Collectively,our data suggest that satraplatin induces apoptosis in CRC cells,which is preceded by cell cycle arrest at G2/M due to the effect of 14-3-3σ and in a p53-p21waf1/cip1-independent manner.Taken together,these findings highlight the potential use of satraplatin for CRC treatment.

  13. Level of reactive oxygen species induced by p21Waf1/CIP1 is critical for the determination of cell fate.

    Science.gov (United States)

    Inoue, Takafumi; Kato, Kiyoko; Kato, Hidenori; Asanoma, Kazuo; Kuboyama, Ayumi; Ueoka, Yousuke; Yamaguchi, Shin-ichiro; Ohgami, Tatsuhiro; Wake, Norio

    2009-07-01

    p21(WAF(1)/)(CIP(1)) is a well-known cell cycle regulatory protein which is overexpressed in several cancer cell lines, and known to determine cell fate. We generated three recombinant adenovirus vectors that expressed either the full-length p21 (Ad-p21F), a p21 mutant with a deletion of the C-terminal proliferative cell nuclear antigen (PCNA) binding domain (Ad-p21N), or a p21 mutant with a deletion of the N-terminal cyclin-dependent kinase binding domain (Ad-p21C). We transfected these vectors into five cancer cell lines. Premature senescence was induced in all of the lines only following transfection with Ad-p21N and Ad-p21F. In addition, apoptosis was also induced in LoVo and HCT116 cells that harbored wild-type p53 and the reactive oxygen species (ROS) level was higher than in senescent cells. Finally, the induction of apoptosis was inhibited by using siRNA to downregulate p53. This observation implies that there is a feedback signaling loop involving p21/ROS/p53 in apoptotic responses. It appears to be, at least in part, driven by high levels of p21 protein. Next, we investigated the cell death effect of endogenous p21 protein on cell fate using sodium butyrate (NaB). Treatment with 1 mM NaB or 2 to 5 mM NaB induced senescence or apoptosis, respectively. The level of intracellular ROS in 5 mM NaB treated cells was 2-fold higher, compared with that in 1 mM NaB treated cells. We also demonstrated that DNA damage response signals including ataxia telangiectasia mutated, gammaH2AX, and p38 MAPK were involved in NaB-induced cell death. The magnitude of intracellular ROS levels in response to p21 elicited either senescence or apoptosis in the cancer cell lines.

  14. TATA-binding protein (TBP)-like protein is required for p53-dependent transcriptional activation of upstream promoter of p21Waf1/Cip1 gene.

    Science.gov (United States)

    Suzuki, Hidefumi; Ito, Ryo; Ikeda, Kaori; Tamura, Taka-Aki

    2012-06-01

    TATA-binding protein-like protein (TLP) is involved in development, checkpoint, and apoptosis through potentiation of gene expression. TLP-overexpressing human cells, especially p53-containing cells, exhibited a decreased growth rate and increased proportion of G(1) phase cells. TLP stimulated expression of several growth-related genes including p21 (p21(Waf1/Cip1)). TLP-mediated activation of the p21 upstream promoter in cells was shown by a promoter-luciferase reporter assay. The p53-binding sequence located in the p21 upstream promoter and p53 itself are required for TLP-mediated transcriptional activation. TLP and p53 bound to each other and synergistically enhanced activity of the upstream promoter. TLP specifically activated transcription from the endogenous upstream promoter, and p53 was required for this activation. Etoposide treatment also resulted in activation of the upstream promoter as well as nuclear accumulation of TLP and p53. Moreover, the upstream promoter was associated with endogenous p53 and TLP, and the p53 recruitment was enhanced by TLP. The results of the present study suggest that TLP mediates p53-governed transcriptional activation of the p21 upstream promoter.

  15. DDX3, a DEAD box RNA helicase with tumor growth-suppressive property and transcriptional regulation activity of the p21waf1/cip1 promoter, is a candidate tumor suppressor.

    Science.gov (United States)

    Chao, Chi-Hong; Chen, Chun-Ming; Cheng, Pei-Lin; Shih, Jing-Wen; Tsou, Ann-Ping; Lee, Yan-Hwa Wu

    2006-07-01

    DDX3 is a DEAD box RNA helicase with diverse biological functions. Using colony formation assay, our results revealed that DDX3 inhibited the colony formation ability of various tumor cells, and this inhibition might be due to a reduced growth rate caused by DDX3. Additionally, we identified p21(waf1/cip1), a cyclin-dependent kinase inhibitor, as a target gene of DDX3, and the up-regulation of p21(waf1/cip1) expression accounted for the colony-suppressing activity of DDX3. Moreover, DDX3 exerted its transactivation function on p21(waf1/cip1) promoter through an ATPase-dependent but helicase-independent mechanism, and the four Sp1 sites located within the -123 to -63 region, relative to the transcription start site of p21(waf1/cip1) promoter, were essential for the response to DDX3. Furthermore, DDX3 interacted and cooperated with Sp1 to up-regulate the promoter activity of p21(waf1/cip1). To determine the relevance of DDX3 in clinical cancers, the expression profile of DDX3 in various tumors was also examined. A declined expression of DDX3 mRNA and protein was found in approximately 58% to 73% of hepatoma specimens, which led to the reduction of p21(waf1/cip1) expression in a manner independent of p53 status. Additionally, an alteration of subcellular localization from nuclei to cytoplasm was also observed in >70% of cutaneous squamous cell carcinoma samples. Because DDX3 exhibits tumor suppressor functions, such as a growth-suppressive property and transcriptional activation of the p21(waf1/cip1) promoter, and is inactivated through down-regulation of gene expression or alteration of subcellular localization in tumor cells, all these features together suggest that DDX3 might be a candidate tumor suppressor.

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

    Science.gov (United States)

    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.

  17. Low p21(Waf1/Cip1) protein level sensitizes testicular germ cell tumor cells to Fas-mediated apoptosis

    NARCIS (Netherlands)

    Spierings, DCJ; de Vries, EGE; Stel, AJ; Rietstap, NT; Vellenga, E; de Jong, S

    2004-01-01

    In the present study, we investigated the relation between p21 expression and the sensitivity of testicular germ cell tumor (TGCT) cells to apoptotic stimuli. Despite similar cisplatin-induced wild-type p53 accumulation, the TGCT cell lines Tera and Scha expressed low p21 protein and mRNA levels in

  18. A limited role for p21(Cip1/Waf1) in maintaining normal hematopoietic stem cell functioning

    NARCIS (Netherlands)

    Van Os, Ronald; Kamminga, Leonie M.; Ausema, Albertina; Bystrykh, Leonid V.; Draijer, Deanna P.; Van Pelt, Kyrjon; Dontje, Bert; De Haan, Gerald

    2007-01-01

    Several studies have suggested that the cyclin-dependent kinase (CDK) inhibitor p21 plays a crucial role in regulating hematopoietic stem and progenitor pool size. To allow assessment of long-term stem cell functioning in vivo, we have backcrossed a p21 null allele to C57BL/6 (B6) mice, the most com

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-05-04

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

  20. p21WAF1/CIP1在兔外伤性增生性玻璃体视网膜病变发生和发展中的抑制作用%Suppressing effect of p21WAF1/CIP1 on traumatic proliferative vitreoretinopathy in rabbits

    Institute of Scientific and Technical Information of China (English)

    袁志刚; 由彩云; 韩金栋; 李海燕; 颜华

    2016-01-01

    背景 p21是一种细胞周期蛋白依赖性激酶抑制剂,能阻止细胞从G1期进入S期,抑制细胞增生,研究认为内源性p21表达的动态变化可能与细胞增生性病变有关.外伤性增生性玻璃体视网膜病变(PVR)是眼部增生性反应相关性疾病,了解PVR过程中p21表达的动态变化可能为PVR的靶向治疗提供依据.目的 检测p21WAFl/CIP1在兔外伤性PVR中的动态变化,探讨其在外伤性PVR发病机制中的作用.方法 选取青紫蓝兔54只,采用随机数字表法将实验兔随机分为正常对照组(6只)和造模后7、14、21和28 d组(每组12只),每只兔任意选取一眼作为实验眼.各模型组兔眼玻璃体腔注射人富含血小板血浆(PRP)0.4 ml,同时于鼻上方角巩膜缘后5 mm处行巩膜外冷冻约5 s,以建立外伤性PVR模型.各组兔眼行眼部B型超声检查以评估建模情况.分别于造模后7、14、21和28 d以过量麻醉法处死实验兔并制备实验眼视网膜组织切片,采用苏木精-伊红染色法检测兔眼视网膜的形态表现,分别采用免疫组织化学染色、Western blot及逆转录PCR(RT-PCR)法检测兔视网膜中p21WAFl/CIP1蛋白及其mRNA的相对表达. 结果 正常对照组兔眼眼前后节均正常,模型组兔造模后1~7 d兔眼玻璃体中增生条索逐渐变粗,可见视网膜皱褶,造模后14d兔眼出现牵引性视网膜脱离,造模后28 d兔眼漏斗状视网膜脱离.视网膜病理组织学检查显示,造模后7d兔眼视网膜表面有增生膜和炎性细胞聚集,造模后28 d可见视网膜呈花瓣形固定皱褶,视网膜结构紊乱.免疫组织化学染色显示,p21WAF1/CIP1蛋白在正常对照组兔眼视网膜神经节细胞层及内核层的细胞核内呈强阳性表达,造模后7、14、21和28 d表达强度减弱,以造模后14d表达量最低.Western blot结果显示,正常对照组和造模后7、14、21和28 d组兔眼视网膜中p21WAF1/CIP1蛋白相对表达量分别为0.74±0.08、0.60

  1. Increased levels of p21((CIP1/WAF1)) correlate with decreased chondrogenic differentiation potential in synovial membrane progenitor cells.

    Science.gov (United States)

    Masson, Anand Oliveira; Hess, Ricarda; O'Brien, Kate; Bertram, Karri L; Tailor, Pankaj; Irvine, Edward; Ren, Guomin; Krawetz, Roman J

    2015-07-01

    Cartilage injuries are a major concern in the field of orthopedics. They occur following trauma, as well as from a variety of pathological conditions including Osteoarthritis (OA). Although cartilage does not exhibit robust endogenous repair, it has been demonstrated that modulating the activity of p21 can increase the regenerative abilities of cartilage in vitro and in vivo. Since the synovial membrane is abundant with mesenchymal progenitor cells (MPCs) capable of differentiating into cartilage both in vitro and in vivo, we examined if p21 expression levels varied between MPCs derived from normal vs. OA knee joints. Analysis of p21 at the mRNA and protein levels within normal and OA MPCs demonstrated differential levels of expression between these two groups, with OA MPCs having higher p21 expression levels. The higher levels of p21 in OA MPCs are also correlated with a decreased chondrogenic differentiation capacity and synovial inflammation, however, there was no evidence of senescence in the OA cells. The results of this study suggest that cell cycle regulation in MPCs may be altered in OA and that modulation of this pathway may have therapeutic potential once the mechanism by which this regulates stem/progenitor cells is better understood.

  2. Transcriptional repression of Bmp2 by p21(Waf1/Cip1) links quiescence to neural stem cell maintenance.

    Science.gov (United States)

    Porlan, Eva; Morante-Redolat, José Manuel; Marqués-Torrejón, María Ángeles; Andreu-Agulló, Celia; Carneiro, Carmen; Gómez-Ibarlucea, Esther; Soto, Atenea; Vidal, Anxo; Ferrón, Sacri R; Fariñas, Isabel

    2013-11-01

    Relative quiescence and self renewal are defining features of adult stem cells, but their potential coordination remains unclear. Subependymal neural stem cells (NSCs) lacking cyclin-dependent kinase (CDK) inhibitor (CKI) 1a (p21) exhibit rapid expansion that is followed by their permanent loss later in life. Here we demonstrate that transcription of the gene encoding bone morphogenetic protein 2 (Bmp2) in NSCs is under the direct negative control of p21 through actions that are independent of CDK. Loss of p21 in NSCs results in increased levels of secreted BMP2, which induce premature terminal differentiation of multipotent NSCs into mature non-neurogenic astrocytes in an autocrine and/or paracrine manner. We also show that the cell-nonautonomous p21-null phenotype is modulated by the Noggin-rich environment of the subependymal niche. The dual function that we describe here provides a physiological example of combined cell-autonomous and cell-nonautonomous functions of p21 with implications in self renewal, linking the relative quiescence of adult stem cells to their longevity and potentiality.

  3. SOX9 accelerates ESC differentiation to three germ layer lineages by repressing SOX2 expression through P21 (WAF1/CIP1).

    Science.gov (United States)

    Yamamizu, Kohei; Schlessinger, David; Ko, Minoru S H

    2014-11-01

    Upon removal of culture conditions that maintain an undifferentiated state, mouse embryonic stem cells (ESCs) differentiate into various cell types. Differentiation can be facilitated by forced expression of certain transcription factors (TFs), each of which can generally specify a particular developmental lineage. We previously established 137 mouse ESC lines, each of which carried a doxycycline-controllable TF. Among them, Sox9 has unique capacity: its forced expression accelerates differentiation of mouse ESCs into cells of all three germ layers. With the additional use of specific culture conditions, overexpression of Sox9 facilitated the generation of endothelial cells, hepatocytes and neurons from ESCs. Furthermore, Sox9 action increases formation of p21 (WAF1/CIP1), which then binds to the SRR2 enhancer of pluripotency marker Sox2 and inhibits its expression. Knockdown of p21 abolishes inhibition of Sox2 and Sox9-accelerated differentiation, and reduction of Sox2 2 days after the beginning of ESC differentiation can comparably accelerate mouse ESC formation of cells of three germ layers. These data implicate the involvement of the p21-Sox2 pathway in the mechanism of accelerated ESC differentiation by Sox9 overexpression. The molecular cascade could be among the first steps to program ESC differentiation.

  4. Characterization of cells resistant to the potent histone deacetylase inhibitor spiruchostatin B (SP-B) and effect of overexpressed p21waf1/cip1 on the SP-B resistance or susceptibility of human leukemia cells.

    Science.gov (United States)

    Kanno, Syu-Ichi; Maeda, Naoyuki; Tomizawa, Ayako; Yomogida, Shin; Katoh, Tadashi; Ishikawa, Masaaki

    2012-09-01

    We previously showed that the B cell leukemia cell line NALM-6 had the highest susceptibility among a number of leukemia cell lines to spiruchostatin B (SP-B), a potent histone deacetylase (HDAC) inhibitor. We also showed that SP-B-induced cytotoxicity depended on induction of apoptosis that was mediated by p21waf1/cip1 expression. In the present study, we generated and characterized a stable, SP-B-resistant NALM-6 cell line (NALM-6/SP-B) by continuous exposure to SP-B, starting with a low SP-B concentration. NALM-6/SP-B cells were also more resistant to FK228, which has a similar chemical structure to SP-B, and were slightly more resistant to the P-gp substrates doxorubicin and vincristine than parental cells, but displayed similar susceptibility to other HDAC inhibitors and to paclitaxel as the parental cells. There was little change in the basal mRNA expression of HDAC1, p53, Bax, Bcl-2, Fas, caspase-3, c-Myc and MDR1 in NALM-6/SP-B compared to parental cells, but the mRNA expression of p21waf1/cip1 was decreased. The introduction of an exogenous p21waf1/cip1 expression vector restored SP-B induction of NALM-6/SP-B cell apoptosis. Moreover, overexpressed p21waf1/cip1 enhanced SP-B induction of the apoptosis of the human erythroleukemia leukemia cell line K562 which is less susceptible to SP-B than NALM-6 cells. These results suggest that downregulation of p21waf1/cip1, which is a characteristic feature of NALM-6/SP-B cells, was important for their resistance to SP-B, and that this SP-B resistance could be overcome by the introduction of exogenous p21waf1/cip1. Furthermore, introduction of p21waf1/cip1 to other leukemia cells such as K562 may enhance their susceptibility to SP-B. This is the first report of the characterization of SP-B-resistant cells and of the effect of overexpressed p21waf1/cip1 on the resistance or susceptibility of human leukemia cells to SP-B.

  5. Urinary bladder lesions after the chernobyl accident. Immunohistochemical assessment of p53, proliferating cell nuclear antigen, cyclin D1 and p21[sup WAF1/Cip1

    Energy Technology Data Exchange (ETDEWEB)

    Romanenko, A.; Zaparin, W.; Vinnichenko, W.; Vozianov, A. (Academy of Medical Sciences of Ukraine, Kiev (Ukraine)); Lee, C.C.R.; Yamamoto, Shinji; Hori, Taka-aki; Wanibuchi, Hideki; Fukushima, Shoji

    1999-02-01

    During the 11-year period subsequent to the Chernobyl accident, the incidence of urinary bladder cancer in Ukraine has increased from 26.2 to 36.1 per 100,000 population. Cesium-137 ([sup 137]Cs) accounts for 80-90% of the incorporated radioactivity in this population, which has been exposed to long-term, low-dose ionizing radiation, and 80% of the more labile pool of cesium is excreted via the urine. The present study was performed to evaluate the histopathological features and the immunohistochemical status of p53, p21[sup WAF1/Cip1], cyclin D1 and PCNA (proliferating cell nuclear antigen) in urinary bladder mucosa of 55 males (49-92 years old) with benign prostatic hyperplasia who underwent surgery in Kiev, Ukraine, in 1995 and 1996. Group I (28 patients) inhabiting radiocontaminated areas of the country, group II (17 patients) from Kiev city with less radiocontamination and a control group III (10 patients) living in so-called ''clean'' areas of Ukraine were compared. In groups I and II, an increase in multiple areas of moderate or severe dysplasia or carcinoma in situ was seen in 42 (93%) of 45 cases. In addition, two small transitional cell carcinomas were found in one patient in each of groups I and II. Nuclear accumulation of p53, PCNA, cyclin D1, and to a lesser extent p21[sup WAF1/Cip1], was significantly increased in both groups I and II as compared with the control group III, indicating possible transformation events or enhancement of repair activities, that may precede the defect in the regulatory pathway itself, at least in the G1 phase of the cell cycle. Our results suggest that early malignant transformation is taking place in the bladder urothelium of people in the radiocontaminated areas of Ukraine and that this could possibly lead sometime in the future to an increased incidence of urinary bladder cancer. (author)

  6. Hepatitis C virus core protein down-regulates p21(Waf1/Cip1 and inhibits curcumin-induced apoptosis through microRNA-345 targeting in human hepatoma cells.

    Directory of Open Access Journals (Sweden)

    Tzu-Yue Shiu

    Full Text Available 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 p21(Waf1/Cip1 expression. However, the mechanism of HCV core-associated p21(Waf1/Cip1 regulation remains to be further clarified. Therefore, we attempted to determine whether HCV core-modulated cellular microRNAs play an important role in regulating p21(Waf1/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 p21(Waf1/Cip1 gene expression through targeting its 3' untranslated region in human hepatoma cells. Moreover, the core protein inhibited curcumin-induced apoptosis through p21(Waf1/Cip1-targeting microRNA-345 in Huh7 cells. CONCLUSION AND SIGNIFICANCE: HCV core protein enhances the expression of microRNA-345 which then down-regulates p21(Waf1/Cip1 expression. It is the first time that HCV core protein has ever been shown to suppress p21(Waf1/Cip1 gene expression through miR-345 targeting.

  7. Inhibition of MDA-MB-231 breast cancer cell proliferation and tumor growth by apigenin through induction of G2/M arrest and histone H3 acetylation-mediated p21(WAF1/CIP1) expression.

    Science.gov (United States)

    Tseng, Tsui-Hwa; Chien, Ming-Hsien; Lin, Wea-Lung; Wen, Yu-Ching; Chow, Jyh-Ming; Chen, Chi-Kuan; Kuo, Tsang-Chih; Lee, Wei-Jiunn

    2017-02-01

    Apigenin (4',5,7-trihydroxyflavone), a flavonoid commonly found in fruits and vegetables, has anticancer properties in various malignant cancer cells. However, the molecular basis of the anticancer effect remains to be elucidated. In this study, we investigated the cellular mechanisms underlying the induction of cell cycle arrest by apigenin. Our results showed that apigenin at the nonapoptotic induction concentration inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in the MDA-MB-231 breast cancer cell line. Immunoblot analysis indicated that apigenin suppressed the expression of cyclin A, cyclin B, and cyclin-dependent kinase-1 (CDK1), which control the G2-to-M phase transition in the cell cycle. In addition, apigenin upregulated p21(WAF1/CIP1) and increased the interaction of p21(WAF1/CIP1) with proliferating cell nuclear antigen (PCNA), which inhibits cell cycle progression. Furthermore, apigenin significantly inhibited histone deacetylase (HDAC) activity and induced histone H3 acetylation. The subsequent chromatin immunoprecipitation (ChIP) assay indicated that apigenin increased acetylation of histone H3 in the p21(WAF1/CIP1) promoter region, resulting in the increase of p21(WAF1/CIP1) transcription. In a tumor xenograft model, apigenin effectively delayed tumor growth. In these apigenin-treated tumors, we also observed reductions in the levels of cyclin A and cyclin B and increases in the levels of p21(WAF1/CIP1) and acetylated histone H3. These findings demonstrate for the first time that apigenin can be used in breast cancer prevention and treatment through epigenetic regulation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 434-444, 2017.

  8. Expressions of PCNA, p53, p21WAF-1 and cell proliferation in fetal esophageal epithelia: Comparative study with adult esophagea lesions from subjects at high-incidence area for esophageal cancer in Henan, North China

    Institute of Scientific and Technical Information of China (English)

    Ying Xing; Yu Ning; Li-Qiang Ru; Li-Dong Wang

    2003-01-01

    AIM: To characterize the expression of p53, p21WAF-1 and proliferation-cell-nuclear-antigen (PCNA) in fetal esophageal epithelia and to determine the role of these genes in proliferation of fetal and adult esophageal epithelial cells.METHODS: Immunohistochemical avdin-biotin peroxidase complex (ABC) method was applied to 31 cases of fetal esophageal specimens and 194 cases of adult esophageal specimens to detect the expression of p53, p21WAF-1 and PCNA in fetal and adult esophageal epithelia.RESULTS: Both the PCNA positive immunostaining cell number and PCNA positive immunostaining rate in fetal esophageal epithelia (506±239) were significantly higher than those in adults,including normal epithelia (200±113) and epithelia with basal cell hyperplasia (BCH) (286±150) (P<0.05, ttest). However,the number of PCNA positive immunostaining cells in adult esophageal dysplasia (719±389) and squamous cell carcinoma (SCC) (1261±545) was apparently higher than that in fetal esophageal epithelia (506±239) (P<0.05, ttest). The positive immunostaining rate of P53 was 10 % (3/3L) in fetal esophageal epithelia, which was significantly lower than that in adult normal esophageal epithelia (50 %), adult epithelia with basal cell hyperplasia (62 %), dysplasia (73 %) and squamous cell carcinoma (86 %) (P<0.05, Fisher′s exact test). No p21WAF-1positive immunostaining cells were observed in fetal esophageal epithelia. However, p21WAF-1 positive immunostaining cells were observed in adult esophagus with 39 % (11/28) in normal, 38% (14/37) in BCH, 27 % (3/11) in DYS and 14 % (1/7) in SCC.CONCLUSION: PCNA could act as an indicator accurately reflecting the high proliferation status of fetal esophageal epithelium. p53 may play an important role in growth and differentiation of fetal esophageal epithelium. p21WAF-1 may have no physiological function in development of fetal esophageal epithelium.

  9. Expression of p53 and p21WAF1 Protein in Advanced Nasopharyngeal Carcinoma before and after Intratumoral Injection of RAd-p53 and Their Relationships with Short-term Prognosis%p53和p21wAF1在鼻咽癌rAd-p53瘤内注射前后的表达及其与近期预后的关系

    Institute of Scientific and Technical Information of China (English)

    兰桂萍; 司勇锋; 翁敬锦; 韦海明; 覃扬达; 何宁

    2012-01-01

    目的 探讨重组人p53腺病毒(rAd-p53)瘤内注射前后鼻咽癌(NPC)组织中p53蛋白和p21WAF1蛋白的表达情况,及其与鼻咽癌近期疗效的关系.方法 应用免疫组织化学法检测12例鼻咽慢性炎组织和63例确诊中晚期鼻咽癌组织的p53和p21WAF1蛋白表达情况.63例中晚期鼻咽癌随机分为2组:p53治疗组(32例):rAd-p53瘤内注射+同步放化疗;常规治疗组(31例):同步放化疗.分析两组治疗前及放疗至20 Gy时p53和p21 WAF1蛋白表达情况及其与预后的关系.结果 NPC组织中p53和p21 WAF1蛋白阳性表达率分别为49.21%和46.03%,和鼻咽黏膜慢性炎相比差异有统计学意义(P<0.05).NPC组织中p53和p21WAF1蛋白表达有相关性(rs=0.556,P=0.000).放疗前及放疗至20 Gy时,鼻咽癌p53治疗组和p21WAF1蛋白阳性表达率差异有统计学意义(P<0.05);两组p53蛋白和p21 WAF1蛋白阳性表达与1年无瘤生存率有关(P<0.05).结论 rAd-p53瘤内注射后p53和p21WAF1蛋白的阳性表达可能在抑制鼻咽癌复发或转移进程中起着重要作用,并预示较好的预后.%Objective To investigate the expression of p53 and p21WAF1 in the NPC cells before and after intratumoral injection of recombinant adenovirus p53(rAd-p53) and their relationships with short-term prognosis. Methods The expression of p53 and p21WAF1 protein were examined by immunochemistry in 12 cases of chronic nasopharynxitis and 63 cases with advanced stage nasopharyngeal carcinoma. Sixty-three cases of NPC were randomly divided into two groups:the p53 therapy group(32 cases) for rAd-p53 intratumoral injection plus radiotherapy and chemotherapy;and the common therapy group(31 cases) for radiotherapy and chemotherapy. The expression levels of p53 and p21WAF1 in biopsy samples from all the cases before and at the 20 Gy of radiotherapy were analyzed; And their relationship with prognosis was also analyzed. Results The expression of p53 and p21WAF1 in 63 cases of NPC was 49. 21

  10. Epstein-Barr virus nuclear antigen 3A promotes cellular proliferation by repression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1.

    Science.gov (United States)

    Tursiella, Melissa L; Bowman, Emily R; Wanzeck, Keith C; Throm, Robert E; Liao, Jason; Zhu, Junjia; Sample, Clare E

    2014-10-01

    Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14(ARF) and p16(INK4a) expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14(ARF) and p16I(NK4a). By contrast, p16(INK4a) was not detectably expressed in Wp-R BL and the low-level expression of p14(ARF) was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21(WAF1/CIP1), a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21(WAF1/CIP1) expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the

  11. Epstein-Barr virus nuclear antigen 3A promotes cellular proliferation by repression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1.

    Directory of Open Access Journals (Sweden)

    Melissa L Tursiella

    2014-10-01

    Full Text Available Latent infection by Epstein-Barr virus (EBV is highly associated with the endemic form of Burkitt lymphoma (eBL, which typically limits expression of EBV proteins to EBNA-1 (Latency I. Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R - that includes expression of the EBNA-3 proteins (3A, 3B and 3C, in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs, consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14(ARF and p16(INK4a expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14(ARF and p16I(NK4a. By contrast, p16(INK4a was not detectably expressed in Wp-R BL and the low-level expression of p14(ARF was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21(WAF1/CIP1, a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21(WAF1/CIP1 expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to

  12. EVI1 inhibits apoptosis induced by antileukemic drugs via upregulation of CDKN1A/p21/WAF in human myeloid cells.

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    Anna Rommer

    Full Text Available 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.

  13. Frequent alteration of MDM2 and p53 in the molecular progression of recurring non-Hodgkin's lymphoma

    DEFF Research Database (Denmark)

    Møller, Michael Boe; Nielsen, O; Pedersen, Niels Tinggaard

    2002-01-01

    -Hodgkin's lymphoma. METHODS AND RESULTS: We have analysed sequential biopsies from 42 non-Hodgkin's lymphoma patients immunohistochemically for p53 alterations (based on p53 and p21Waf1 expression), as well as for expression of MDM2, p27Kip1 and cyclin D3. Relapse of follicle centre lymphoma was associated with p53......-Hodgkin's lymphoma, as 2/5 (40%) diffuse large B-cell lymphomas and 3/9 (33%) T-cell non-Hodgkin's lymphomas with normal p53 at diagnosis showed p53 alterations at relapse. No indolent non-Hodgkin's lymphoma case showed MDM2 over-expression at diagnosis, whereas 4/5 (80%) transformed diffuse large B-cell lymphomas...... developed MDM2 over-expression. CONCLUSION: Our data are consistent with the notion that p53 alterations are important for the histological transformation of follicle centre lymphoma. However, the data also suggest that relapsing follicle centre lymphomas without overt transformation often have p53...

  14. KR-POK interacts with p53 and represses its ability to activate transcription of p21WAF1/CDKN1A.

    Science.gov (United States)

    Jeon, Bu-Nam; Kim, Min-Kyeong; Choi, Won-Il; Koh, Dong-In; Hong, Sung-Yi; Kim, Kyung-Sup; Kim, Minjung; Yun, Chae-Ok; Yoon, Juyong; Choi, Kang-Yell; Lee, Kyung-Ryul; Nephew, Kenneth P; Hur, Man-Wook

    2012-03-01

    Transcriptional regulation by p53 is thought to play a role in its ability to suppress tumorigenesis. However, there remain gaps in understanding about how p53 regulates transcription and how disrupting this function may promote cancer. Here we report a role in these processes for the kidney cancer-related gene KR-POK (ZBTB7C), a POZ domain and Krüppel-like zinc finger transcription factor that we found to physically interact with p53. Murine embryonic fibroblasts isolated from genetically deficient mice (Kr-pok(-/-) MEFs) exhibited a proliferative defect relative to wild-type mouse embryonic fibroblasts (MEF). The zinc finger domain of Kr-pok interacted directly with the DNA binding and oligomerization domains of p53. This interaction was essential for Kr-pok to bind the distal promoter region of the CDKN1A gene, an important p53 target gene encoding the cell-cycle regulator p21WAF1, and to inhibit p53-mediated transcriptional activation of CDKN1A. Kr-pok also interacted with the transcriptional corepressors NCoR and BCoR, acting to repress histone H3 and H4 deacetylation at the proximal promoter region of the CDKN1A gene. Importantly, Kr-pok(-/-) MEFs displayed an enhancement in CDKN1A transactivation by p53 during the DNA damage response, without any parallel changes in transcription of either the p53 or Kr-pok genes themselves. Furthermore, Kr-pok promoted cell proliferation in vitro and in vivo, and its expression was increased in more than 50% of the malignant human kidney cancer cases analyzed. Together, our findings define KR-POK as a transcriptional repressor with a pro-oncogenic role that relies upon binding to p53 and inhibition of its transactivation function.

  15. Alterations in TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIP1 expression associated with progression in B-CLL

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    Antosz Halina

    2010-04-01

    Full Text Available B-cell chronic lymphocytic leukaemia (B-CLL originates from B lymphocytes that may differ in the activationlevel, maturation state or cellular subgroups in peripheral blood. Tumour progression in CLL B cells seems to result in gradualaccumulation of the clone of resting B lymphocytes in the early phases (G0/G1 of the cell cycle. The G1 phase isimpaired in B-CLL. We investigated the gene expression of five key cell cycle regulators: TP 53, c-Myc, cyclin D2,p21WAF1/CIP1 and p27KIP1, which primarily regulate the G1 phase of the cell cycle, or S-phase entry and ultimately controlthe proliferation and cell growth as well as their role in B-CLL progression. The study was conducted in peripheral bloodCLL lymphocytes of 40 previously untreated patients. Statistical analysis of correlations of TP53, cyclin D2, c-Myc,p21WAF1/CIP1 and p27KIP1 expressions in B-CLL patients with different Rai stages demonstrated that the progression of diseasewas accompanied by increases in p53, cyclin D2 and c-Myc mRNA expression. The expression of p27KIP1 was nearlystatistically significant whereas that of p21 WAF1/CIP1 showed no such correlation. Moreover, high expression levels of TP53and c-Myc genes were found to be closely associated with more aggressive forms of the disease requiring earlier therapy.

  16. EPO gene expression promotes proliferation, migration and invasion via the p38MAPK/AP-1/MMP-9 pathway by p21WAF1 expression in vascular smooth muscle cells.

    Science.gov (United States)

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

    2015-03-01

    The use of recombinant human erythropoietin (rHuEpo) can lead to hypertrophy and hyperplasia, and has induced the proliferation of vascular smooth muscle cells (VSMCs). The effect of the EPO gene in the migration and invasion of VSMCs remains unclear. In this study, overexpression of the EPO gene increased the DNA synthesis and phosphorylation of ERK1/2 and p38MAPK in VSMCs. In addition, EPO gene expression induced the migration and invasion of VSMCs via the expression of MMP-9 by the activation of NF-κB and AP-1 binding. A blockade of p38MAPK by specific p38MAPK inhibitor SB203580 led to a suppression of the increased DNA synthesis, migration, and invasion of VSMCs that was induced by the EPO gene. SB203580 treatment blocked the increased expression of MMP-9 through the binding activity of AP-1. Transfection of the EPO gene with VSMCs was associated with the up-regulation of cyclin D1/CDK4, cyclin E/CDK2, and p21WAF1, and with the down-regulation of p27KIP1. The specific suppression of p21WAF1 expression by siRNA rescued the enhancement of DNA synthesis via the phosphorylation of p38MAPK and the increase in migration and invasion through AP-1-mediated MMP-9 expression in EPO gene transfectants. These novel findings demonstrate that p21WAF1 regulates the proliferation, migration and invasion of VSMC induced by EPO gene.

  17. P53 Mdm2 Inhibitors

    NARCIS (Netherlands)

    Khoury, Kareem; Doemling, Alex

    2012-01-01

    The protein-protein interaction (PPI) between p53 and its negative regulator MDM2 comprises one of the most important and intensely studied PPI's involved in preventing the initiation of cancer. The interaction between p53 and MDM2 is conformation-based and is tightly regulated on multiple levels. D

  18. Immunoexpression of tumor suppressor genes p53, p21WAF1/CIP1 and p27KIP1 in humam astrocystic tumors Imuno-expressão dos genes supressores tumorais p53, p21WAF1/CIP1 e p27KIP1 em tumores astrocíticos humanos

    Directory of Open Access Journals (Sweden)

    Mário Henrique Girão Faria

    2007-12-01

    Full Text Available The aim of the present study was to evaluate the tumor suppressor genes p53, p21WAF1/CIP1 and p27KIP1 expression in astrocytic tumors, correlating the findings with the histopathological grade (WHO. An immunohistochemical study of the p53, p21 and p27 proteins using the streptavidin-biotin-peroxidase method was performed in fifty-five astrocytomas (13 grade I, 14 grade II, 7 grade III and 21 grade IV and five samples of non-tumor brain tissue (negative control. p53 positive indices (PI and labeling indices (LI showed tendency to increase according to malignant progression. The nuclear expression of p27 presented similar inclination, except for the PI reduction verified in grade IV tumors. Otherwise, the cytoplasmic p27 staining was more evident between high-grade tumors (III and IV. p53 and nuclear p27 expression was correlated with the histological classification (pO presente estudo objetivou avaliar a expressão dos supressores tumorais p53, p21WAF1/CIP1 e p27KIP1 em tumores astrocíticos humanos, correlacionando os achados com a graduação histopatológica (OMS. Procedeu-se o estudo imuno-histoquímico para as proteínas p53, p21 e p27 utilizando o método da estreptavidina-biotina-peroxidase em 55 astrocitomas (13 do grau I, 14 do grau II, 7 do grau III e 21 do grau IV e 5 amostras de tecido cerebral não-tumoral (controle negativo. Os índices de positividade (PI e de marcação (LI para p53 demonstraram tendência de aumento conforme a progressão maligna. A expressão nuclear do p27 apresentou semelhante inclinação, exceto pela redução do PI verificada nos tumores do grau IV. Já a marcação citoplasmática do p27 foi mais evidente entre tumores de alto grau (III e IV. As expressões de p53 e p27 nuclear demonstraram correlação com a classificação histológica (p<0,01; teste H. Por outro lado, os índices para p21 manifestaram propensão à redução conforme a evolução maligna dos tumores astrocíticos, salvo significante

  19. 叶酸通过调节p53/p21(waf1/cip1)信号途径促进小鼠神经干细胞增殖和分化%Folic acid promote proliferation and differentiation of neural stem cells via regulation p53/p21 ( waf1/cip1 ) pathway in mice

    Institute of Scientific and Technical Information of China (English)

    王虹; 王芳; 范利军; 包金风

    2012-01-01

    目的:研究叶酸对体外培养小鼠神经干细胞(neuralstemcells,NSCs)增殖和分化的影响及作用机制.方法:采用无血清悬浮培养方法分离培养新生小鼠脑NSCs,通过MTT法检测叶酸对NSCs增殖的影响;撤除生长因子后,用含10%胎牛血清的培养基诱导分化培养6d后,采用Tuj1(神经元标记物)和GFAP(胶质细胞标记物)免疫荧光双标记法检测叶酸对NSCs分化的影响;并应用流式细胞术、RT-PCR法检测给予叶酸对NSCs细胞周期、p53和p21(waf1/cip1)mRNA水平的影响.结果:与对照组相比,MTT法测定结果显示,叶酸组NSCs增殖能力明显增强;分化后免疫荧光双标法测定显示,叶酸组Tuj1阳性细胞的比率明显增加,且差异具有显著性(P<0.01);流式细胞仪测定结果显示,叶酸组NSCs在G0/G1期细胞数量明显减少(P<0.01),而G2/M期细胞数量明显增多(P<0.01);RT-PCR结果显示,叶酸组NSCs中p53和p21mRNA表达量明显降低.结论:叶酸能促进NSCs增殖及向神经元分化;叶酸对NSCs增殖和分化的影响与调节NSCs细胞周期及p53/p21(waf1/cip1)信号转导途径相关.%Objective: To explore the effects and mechanisms of folic acid on proliferation and differentiation of neural stem cells (NSCs) in vitro in mice. Methods: NSCs were isolated from newborn mice brain and suspension cultured in serum-free medium. The effect of folic acid on proliferation of NSCs was determinated by MTT method; the differentiation of NSCs was detected by immunofluorescence double labeled with Tujl (marker of neuron) and GFAP (marker of glia) after 6 day culture in 10% fetal calf serum medium condition without growth factors. The cell cycle of NSCs was detected by flow cytometry and the mRNA levels of p53 and p21 were determinated by RT-PCR. Results: The proliferation rate of NSCs andTujl positive cells were significantly higher in folic acid group than control group(/'< 0.01); the ratio of NSCs in G0/G1 phase was decreased (P

  20. Expression of the p12 subunit of human DNA polymerase δ (Pol δ), CDK inhibitor p21(WAF1), Cdt1, cyclin A, PCNA and Ki-67 in relation to DNA replication in individual cells.

    Science.gov (United States)

    Zhao, Hong; Zhang, Sufang; Xu, Dazhong; Lee, Marietta Ywt; Zhang, Zhongtao; Lee, Ernest Yc; Darzynkiewicz, Zbigniew

    2014-01-01

    We recently reported that the p12 subunit of human DNA polymerase δ (Pol δ4) is degraded by CRL4(Cdt2) which regulates the licensing factor Cdt1 and p21(WAF1) during the G1 to S transition. Presently, we performed multiparameter laser scanning cytometric analyses of changes in levels of p12, Cdt1 and p21(WAF1), detected immunocytochemically in individual cells, vis-à-vis the initiation and completion of DNA replication. The latter was assessed by pulse-labeling A549 cells with the DNA precursor ethynyl-2'-deoxyribose (EdU). The loss of p12 preceded the initiation of DNA replication and essentially all cells incorporating EdU were p12 negative. Completion of DNA replication and transition to G2 phase coincided with the re-appearance and rapid rise of p12 levels. Similar to p12 a decline of p21(WAF1) and Cdt1 was seen at the end of G1 phase and all DNA replicating cells were p21(WAF1) and Cdt1 negative. The loss of p21(WAF1) preceded that of Cdt1 and p12 and the disappearance of the latter coincided with the onset of DNA replication. Loss of p12 leads to conversion of Pol δ4 to its trimeric form, Pol δ3, so that the results provide strong support to the notion that Pol δ3 is engaged in DNA replication during unperturbed progression through the S phase of cell cycle. Also assessed was a correlation between EdU incorporation, likely reflecting the rate of DNA replication in individual cells, and the level of expression of positive biomarkers of replication cyclin A, PCNA and Ki-67 in these cells. Of interest was the observation of stronger correlation between EdU incorporation and expression of PCNA (r = 0.73) than expression of cyclin A (r = 0.47) or Ki-67 (r = 0.47).

  1. Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometry.

    Science.gov (United States)

    Darzynkiewicz, Zbigniew; Zhao, Hong; Zhang, Sufang; Lee, Marietta Y W T; Lee, Ernest Y C; Zhang, Zhongtao

    2015-05-20

    During our recent studies on mechanism of the regulation of human DNA polymerase δ in preparation for DNA replication or repair, multiparameter imaging cytometry as exemplified by laser scanning cytometry (LSC) has been used to assess changes in expression of the following nuclear proteins associated with initiation of DNA replication: cyclin A, PCNA, Ki-67, p21(WAF1), DNA replication factor Cdt1 and the smallest subunit of DNA polymerase δ, p12. In the present review, rather than focusing on Pol δ, we emphasize the application of LSC in these studies and outline possibilities offered by the concurrent differential analysis of DNA replication in conjunction with expression of the nuclear proteins. A more extensive analysis of the data on a correlation between rates of EdU incorporation, likely reporting DNA replication, and expression of these proteins, is presently provided. New data, specifically on the expression of cyclin D1 and cyclin E with respect to EdU incorporation as well as on a relationship between expression of cyclin A vs. p21(WAF1) and Ki-67 vs. Cdt1, are also reported. Of particular interest is the observation that this approach makes it possible to assess the temporal sequence of degradation of cyclin D1, p21(WAF1), Cdt1 and p12, each with respect to initiation of DNA replication and with respect to each other. Also the sequence or reappearance of these proteins in G2 after termination of DNA replication is assessed. The reviewed data provide a more comprehensive presentation of potential markers, whose presence or absence marks the DNA replicating cells. Discussed is also usefulness of these markers as indicators of proliferative activity in cancer tissues that may bear information on tumor progression and have a prognostic value.

  2. P21Cip/WAF1及P27Kip1与增殖细胞核抗原在人骨髓间充质干细胞向肝细胞分化过程中的表达%Expression of P21Cip/WAF1,P27Kip1 and proliferating cell nuclear antigen during differentiation of human bone marrow mesenchymal stem cells into hepatocytes

    Institute of Scientific and Technical Information of China (English)

    汤志华; 闵军; 陈积圣; 叶林; 余杰雄

    2009-01-01

    背景:P21CIp/WAF1、P27Kip1是两种重要的细胞周期的负调控因子,增殖细胞核抗原是反映干细胞增殖的指标.目的:观察体外诱导人骨髓间充质干细胞向肝细胞分化过程中P21Cip/WAF1,P27KIp1及增殖细胞核抗原的表达变化.设计、时间及地点:细胞学基因水平观察,于2006-12/2007-06在中山大学附属第二医院林百欣医学研究中心实验室完成.材料:骨髓来源于中山大学附属第二医院行骨髓穿刺的健康供者.方法:全骨髓贴壁筛选法体外分离培养人骨髓间充质干细胞,传至第5代后,加入含20 μg/L肝细胞生长因子、10 μg/L成纤维细胞生长因子4的opti-MEM I低血清培养基向肝细胞诱导分化.主要观察指标:诱导后RT-PCR检测细胞内P21Cip/WAF1,P27 KIp1及增殖细胞核抗原基因mRNA的表达.结果:骨髓间充质干细胞向肝细胞定向诱导后0,1,3,5,7 d,细胞内P21CIp/WAF1 mRNA表达的相对量逐渐升高(P<0.01),P27Kip1 mRNA表达的相对量无明显差异(P>0.05),增殖细胞核抗原mRNA表达的相对量除诱导后0,1 d无明显差异(P>0.05)外,其余各时间点均逐渐降低(P<0.01).结论:P21 CIp/WAF1高表达抑制了骨髓间充质干细胞增殖,同时在其向肝细胞分化过程中起一定调控作用,P27KIp1可能并不起作用,增殖细胞核抗原的表达则与P21Cip/WAF1相反,呈逐步减少趋势.

  3. Nuclear translocation of p21{sup WAF1/CIP1} protein prior to its cytosolic degradation by UV enhances DNA repair and survival

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Young; Kim, Hee Suk; Kim, Joo Young [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Sohn, Jeongwon, E-mail: biojs@korea.ac.kr [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of)

    2009-12-25

    We previously reported that UV induced rapid proteasomal degradation of p21 protein in an ubiquitination-independent manner. Here, UV-induced p21 proteolysis was found to occur in the cytosol. Before cytosolic degradation, however, p21 protein translocated to and transiently accumulated in the nucleus. Nuclear translocation of p21 was not required for its degradation, but rather promoted DNA repair and cell survival. Overexpression of the wild type p21, but not the one with defective nuclear localization signal (NLS), reduced UV-induced DNA damage and cell death. Some of p21 protein translocated to the nucleus were associated with chromatin-bound PCNA and saved from UV-induced proteolysis. These data together show that p21 translocates to the nucleus to participate in DNA repair, while the rest is rapidly degraded in the cytosol. We propose that our findings reflect a mechanism to facilitate removal of damaged cells, enhancing DNA repair at the same time.

  4. Estudo de p27, p21, p16 em epitélio escamoso normal, papiloma escamoso e carcinoma de células escamosas da cavidade oral Comparative analysis of the immunohistochemistry expression of p27, p21WAF/Cip1, and p16INK4a in oral normal epithelium, squamous papilloma and squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Ana Beatriz Piazza Queiroz

    2009-12-01

    Full Text Available INTRODUÇÃO E OBJETIVO: O tipo de câncer oral mais frequente é o carcinoma de células escamosas, que corresponde a 95% dos casos(9. O papiloma escamoso oral é uma neoplasia benigna normalmente associada à infecção pelo papilomavírus humano (HPV(21. A análise da literatura mostra alterações nos genes reguladores do ciclo celular p27, p21WAF/Cip1 e p16INK4a, porém sem uma definição de seus papéis na carcinogênese oral. O objetivo foi caracterizar imuno-histoquimicamente p27, p21WAF/Cip1 e p16NK4a em epitélio escamoso normal, papilomas escamosos e carcinomas de células escamosas da cavidade oral. MÉTODOS: Imuno-histoquímica para p27, p21WAF/Cip1 e p16NK4a em 32 casos de epitélio escamoso normal, 30 casos de papiloma escamoso e 34 de carcinoma de células escamosas da cavidade oral. RESULTADOS: p27: 97,06% dos casos de carcinoma de células escamosas apresentaram imunopositividade focal. O grupo papiloma escamoso apresentou 33,33% e o grupo controle, 18,75%. p21WAF/Cip1: 100% de imunopositividade focal tanto no grupo controle como no grupo carcinoma de células escamosas, e 90% no grupo papiloma escamoso. p16INK4a: 100% de imunopositividade focal para os grupos controle e papiloma escamoso, e 94% para o grupo carcinoma de células escamosas. CONCLUSÃO: Imuno-histoquimicamente demonstrou-se diferença significativa para p27 quando feita comparação dos grupos controle e papiloma escamoso com o grupo carcinoma de células escamosas. O p21WAF/Cip1 não demonstrou poder de diferenciar os grupos analisados. O p16INK4a apresentou imunopositividade difusa em uma minoria dos casos do grupo carcinoma de células escamosas. O grupo papiloma escamoso se comportou de maneira similar ao grupo controle em relação aos três marcadores.INTRODUCTION: The most frequent type of oral cancer is the squamous cell carcinoma, which corresponds to 95% of the cases(9.The oral squamous papilloma is a benign neoplasia, commonly associated with

  5. Inhibition of the growth of human hepatoma cell line both in vitro and in vivo by transducing CKI gene p21WAF-1 with GE7 targeting gene delivery system

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous gene pCEP-p21WAF-1 into human hepatocellular carcinoma cell both in vitro and in vivo. After in vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. The in vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untransfected control. The average tumor weight of the experiment group was (0.083 ± 0.043) g, while that of the control group was (0.281± 0.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous gene pCEP-p21WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growth with high efficacy both in vivo and in vitro.

  6. Inhibition of the growth of human hepatoma cell line both in vitro and in vivo by transducing CKI gene p21WAF-1 with GE7 targeting gene delivery system

    Institute of Scientific and Technical Information of China (English)

    韩峻松; 田培坤; 柳湘; 姚明; 顾健人

    2000-01-01

    The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous gene pCEP-p21WAF-1 into human hepatocellular carcinoma cell both in vitro and in vivo. After in vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. The in vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untrans-fected control. The average tumor weight of the experiment group was (0.083 ?0.043) g, while that of the control group was (0.28110.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous gene pCEP-p21WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growt

  7. {sup 123}I-labeled HIV-1 tat peptide radioimmunoconjugates are imported into the nucleus of human breast cancer cells and functionally interact in vitro and in vivo with the cyclin-dependent kinase inhibitor, p21{sup WAF-1/Cip-1}

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Meiduo [University Health Network, Division of Nuclear Medicine, Toronto, ON (Canada); University of Toronto, Department of Pharmaceutical Sciences, Toronto, ON (Canada); Chen, Paul; Wang, Judy; Scollard, Deborah A. [University Health Network, Division of Nuclear Medicine, Toronto, ON (Canada); Vallis, Katherine A. [University Health Network, Department of Radiation Oncology, Toronto, ON (Canada); University of Toronto, Department of Medical Biophysics, Toronto, ON (Canada); Reilly, Raymond M. [University Health Network, Division of Nuclear Medicine, Toronto, ON (Canada); University of Toronto, Department of Pharmaceutical Sciences, Toronto, ON (Canada); University of Toronto, Department of Medical Imaging, Toronto, ON (Canada); University of Toronto, Leslie Dan Faculty of Pharmacy, Toronto, ON (Canada)

    2007-03-15

    To evaluate the internalization and nuclear translocation of {sup 123}I-tat-peptide radioimmunoconjugates in MDA-MB-468 breast cancer cells and their ability to interact with the cyclin-dependent kinase inhibitor, p21{sup WAF-1/Cip-1}. Peptides [GRKKRRQRRRPPQGYGC] harboring the nuclear-localizing sequence from HIV tat domain were conjugated to anti-p21{sup WAF-1/Cip-1} antibodies. Immunoreactivity was assessed by Western blot using lysate from MDA-MB-468 cells exposed to EGF to induce p21{sup WAF-1/Cip-1}. Internalization and nuclear translocation were measured. The ability of tat-anti-p21{sup WAF-1/Cip-1} to block G{sub 1}-S phase arrest in MDA-MB-468 cells caused by EGF-induced p21{sup WAF-1/Cip-1} was evaluated. Tumor and normal tissue uptake were determined at 48 h p.i. in athymic mice implanted s.c. with MDA-MB-468 xenografts injected intratumorally with EGF. There was 13.4{+-}0.2% of radioactivity internalized by MDA-MB-468 cells incubated with {sup 123}I-tat-anti-p21{sup WAF-1/Cip-1} and 34.6{+-}3.1% imported into the nucleus. Tat-anti-p21{sup WAF-1/Cip-1}(8 {mu}M) decreased the proportion of EGF-treated cells in G{sub 1} phase from 81.9{+-}0.7% to 46.1{+-}0.7% (p<0.001), almost restoring the G{sub 1} phase fraction to that of unexposed cells (25.8{+-}0.2%). Non-specific tat-mouse IgG did not block EGF-induced G{sub 1}-S phase arrest. Tumor uptake of radioactivity was higher in mice injected with EGF to induce p21{sup WAF-1/Cip-1} than in mice not receiving EGF (3.1{+-}0.4% versus 1.8{+-}0.2% ID/g; p=0.04). Western blot analysis of tumors revealed a threefold increase in the p21{sup WAF-1/Cip-1}/{beta}-actin ratio. We conclude that intracellular and nuclear epitopes in cancer cells can be functionally targeted with tat-radioimmunoconjugates to exploit many more epitopes for imaging and radiotherapeutic applications than have previously been accessible. (orig.)

  8. MST kinases monitor actin cytoskeletal integrity and signal via c-Jun N-terminal kinase stress-activated kinase to regulate p21Waf1/Cip1 stability.

    Science.gov (United States)

    Densham, Ruth M; O'Neill, Eric; Munro, June; König, Ireen; Anderson, Kurt; Kolch, Walter; Olson, Michael F

    2009-12-01

    As well as providing a structural framework, the actin cytoskeleton plays integral roles in cell death, survival, and proliferation. The disruption of the actin cytoskeleton results in the activation of the c-Jun N-terminal kinase (JNK) stress-activated protein kinase (SAPK) pathway; however, the sensor of actin integrity that couples to the JNK pathway has not been characterized in mammalian cells. We now report that the mammalian Ste20-like (MST) kinases mediate the activation of the JNK pathway in response to the disruption of the actin cytoskeleton. One consequence of actin disruption is the JNK-mediated stabilization of p21(Waf1/Cip1) (p21) via the phosphorylation of Thr57. The expression of MST1 or MST2 was sufficient to stabilize p21 in a JNK- and Thr57-dependent manner, while the stabilization of p21 by actin disruption required MST activity. These data indicate that, in addition to being components of the Salvador-Warts-Hippo tumor suppressor network and binding partners of c-Raf and the RASSF1A tumor suppressor, MST kinases serve to monitor cytoskeletal integrity and couple via the JNK SAPK pathway to the regulation of a key cell cycle regulatory protein.

  9. TReP-132 Controls Cell Proliferation by Regulating the Expression of the Cyclin-Dependent Kinase Inhibitors p21WAF1/Cip1 and p27Kip1

    Science.gov (United States)

    Gizard, Florence; Robillard, Romain; Barbier, Olivier; Quatannens, Brigitte; Faucompré, Anne; Révillion, Françoise; Peyrat, Jean-Philippe; Staels, Bart; Hum, Dean W.

    2005-01-01

    The transcriptional regulating protein of 132 kDa (TReP-132) has been identified in steroidogenic tissues, where it acts as a coactivator of steroidogenic factor 1 (SF-1). We show here that TReP-132 plays a role in the control of cell proliferation. In human HeLa cells, TReP-132 knockdown by using small interfering RNA resulted in increased G1→S cell cycle progression. The growth-inhibitory effects of TReP-132 was further shown to be mediated by induction of G1 cyclin-dependent kinase inhibitors p21WAF1 (p21) and p27KIP1 (p27) expression levels. As a consequence, G1 cyclin/cyclin-dependent kinase activities and pRB phosphorylation were markedly reduced, and cell cycle progression was blocked in the G1 phase. The stimulatory effect of TReP-132 on p21 and p27 gene transcription involved interaction of TReP-132 with the transcription factor Sp1 at proximal Sp1-binding sites in their promoters. Moreover, in different breast tumor cell lines, endogenous TReP-132 expression was positively related with a lower proliferation rate. In addition, TReP-132 knockdown resulted in enhanced cell proliferation and lowered p21 and p27 mRNA levels in the steroid-responsive and nonresponsive T-47D and MDA-MB-231 cell lines, respectively. Finally, a statistic profiling of human breast tumor samples highlighted that expression of TReP-132 is correlated with p21 and p27 levels and is associated with lower tumor incidence and aggressiveness. Together, these results identify TReP-132 as a basal cell cycle regulatory protein acting, at least in part, by interacting with Sp1 to activate the p21 and p27 gene promoters. PMID:15899840

  10. Expression of p21(Cip1/Waf1/Sdi1) and p27(Kip1) cyclin-dependent kinase inhibitors during human hematopoiesis.

    Science.gov (United States)

    Taniguchi, T; Endo, H; Chikatsu, N; Uchimaru, K; Asano, S; Fujita, T; Nakahata, T; Motokura, T

    1999-06-15

    Expression of p21 and p27 cyclin-dependent kinase inhibitors is associated with induced differentiation and cell-cycle arrest in some hematopoietic cell lines. However, it is not clear how these inhibitors are expressed during normal hematopoiesis. We examined various human hematopoietic colonies derived from cord blood CD34(+) cells, bone marrow, and peripheral blood cells using a quantitative reverse transcription-polymerase chain reaction assay, immunochemistry, and/or Western blot analysis. p21 mRNA was expressed increasingly over time in all of the colonies examined (granulocytes, macrophages, megakaryocytes, and erythroblasts), whereas p27 mRNA levels remained low, except for erythroid bursts. Erythroid bursts expressed both p21 and p27 mRNAs with differentiation but expressed neither protein, whereas both proteins were expressed in megakaryocytes and peripheral blood monocytes. In bone marrow, p21 was immunostained almost exclusively in a subset of megakaryocytes and p27 protein was present in megakaryocytes, plasma cells, and endothelial cells. In megakaryocytes, reciprocal expression of p27 to Ki-67 was evident and an inverse relationship between p21 and Ki-67 positivities was also present, albeit less obvious. These observations suggest that a complex lineage-specific regulation is involved in p21 and p27 expression and that these inhibitors are involved in cell-cycle exit in megakaryocytes.

  11. 硒对乳腺癌细胞株p21WAF1/CIP1启动子的调控作用%Regulatingrole of p21WAF1/CIP1 promoter by selenium on breast cancer cell

    Institute of Scientific and Technical Information of China (English)

    申社林; 李兵; 李朝争; 李建广; 郭靠山

    2009-01-01

    目的:研究硒对p21的转录调控及其调控位点.方法:通过向转染了重组质粒pGL3- p21p的乳腺癌细胞株MCF7先后加入不同的p21因子启动子的负调节因子和乳酸硒,对比分析荧光素酶表达活性,以确定硒对p21的转录调控及调控位点,并验证硒对癌细胞的生长的负调控作用.结果:perifosine、depsipeptide、apicidin、butyrate与硒共同诱导荧光素酶,酶活性表达无显著差异;而C-Myc与醋酸硒先后诱导酶活性表达差异显著.结论:硒对癌细胞具有诱导凋亡的作用,转录调节位点在p21启动子的sp1结合位点.

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

    Directory of Open Access Journals (Sweden)

    Hilary V Clegg

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

  13. MicroRNAs regulate p21(Waf1/Cip1) protein expression and the DNA damage response in human embryonic stem cells.

    Science.gov (United States)

    Dolezalova, Dasa; Mraz, Marek; Barta, Tomas; Plevova, Karla; Vinarsky, Vladimir; Holubcova, Zuzana; Jaros, Josef; Dvorak, Petr; Pospisilova, Sarka; Hampl, Ales

    2012-07-01

    Studies of human embryonic stem cells (hESCs) commonly describe the nonfunctional p53-p21 axis of the G1/S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs, but p21 protein is not detectable. In this article, we provide evidence that expression of p21 protein is directly regulated by the microRNA (miRNA) pathway under standard culture conditions and after DNA damage. The DDR in hESCs leads to upregulation of tens of miRNAs, including hESC-specific miRNAs such as those of the miR-302 family, miR-371-372 family, or C19MC miRNA cluster. Most importantly, we show that the hESC-enriched miRNA family miR-302 (miR-302a, miR-302b, miR-302c, and miR-302d) directly contributes to regulation of p21 expression in hESCs and, thus, demonstrate a novel function for miR-302s in hESCS. The described mechanism elucidates the role of miRNAs in regulation of important molecular pathway governing the G1/S transition checkpoint before as well as after DNA damage.

  14. Nucleostemin knocking-down causes cell cycle arrest and apoptosis in human T-cell acute lymphoblastic leukemia MOLT-4 cells via p53 and p21Waf1/Cip1 up-regulation.

    Science.gov (United States)

    Rahmati, Marveh; Moosavi, Mohammad Amin; Zarghami, Nosratollah

    2014-12-01

    Nucleostemin (NS), a recently discovered nucleolar protein, is essential for maintaining self-renewal and proliferation of embryonic and adult stem cells as well as cancerous cells. The aim of this study was to determine biological function of NS in MOLT-4 cells as a human T-cell acute lymphocytic leukemia (T-ALL) model. Efficacy of a specific small interference RNA on NS depletion was studied by quantitative polymerase chain reaction and western blotting. The growth rate and viability were analyzed by trypan blue exclusion test. Fluorescent microscopy was used for detecting apoptosis. Cell cycle and apoptosis were mechanistically studied by flow cytometry and western blotting. Knockdown of NS inhibited proliferation, arrested the cell cycle, and induced apoptosis through p53 and p21(Waf1/Cip1) pathways in MOLT-4 cells. These findings demonstrate critical roles of NS in MOLT-4 cells and may implicate on its therapeutic potential in this human T-ALL model.

  15. Gene expression of the p16(INK4a)-Rb and p19(Arf)-p53-p21(Cip/Waf1) signaling pathways in the regulation of hematopoietic stem cell aging by ginsenoside Rg1.

    Science.gov (United States)

    Yue, Z; Rong, J; Ping, W; Bing, Y; Xin, Y; Feng, L D; Yaping, W

    2014-12-04

    The elucidation of the molecular mechanisms underlying the effects of traditional Chinese medicines in clinical practice is a key step toward their worldwide application, and this topic is currently a subject of intense research interest. Rg1, a component of ginsenoside, has recently been shown to perform several pharmacological functions; however, the underlying mechanisms of these effects remain unclear. In the present study, we investigated whether Rg1 has an anti-senescence effect on hematopoietic stem cells (HSCs) and the possible molecular mechanisms driving any effects. The results showed that Rg1 could effectively delay tert-butyl hydroperoxide (t-BHP)-induced senescence and inhibit gene expression in the p16(INK4a)-Rb and p19(Arf)-p53-p21(Cip/Waf1) signaling pathways in HSCs. Our study suggested that these two signaling pathways might be potential targets for elucidating the molecular mechanisms of the Rg1 anti-senescence effect.

  16. 8-60hIPP5(m)-induced G2/M cell cycle arrest involves activation of ATM/p53/p21(cip1/waf1) pathways and delayed cyclin B1 nuclear translocation.

    Science.gov (United States)

    Zeng, Qi-Yan; Zeng, Lin-Jie; Huang, Yu; Huang, Yong-Qi; Zhu, Qi-Fang; Liao, Zhi-Hong

    2014-01-01

    Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression and cell cycle progression. The active mutant IPP5 (8-60hIPP5(m)), the latest member of the inhibitory molecules for PP1, has been shown to inhibit the growth of human cervix carcinoma cells (HeLa). In order to elucidate the underlying mechanisms, the present study assessed overexpression of 8-60hIPP5(m) in HeLa cells. Flow cytometric and biochemical analyses showed that overexpression of 8-60hIPP5(m) induced G2/M-phase arrest, which was accompanied by the upregulation of cyclin B1 and phosphorylation of G2/M-phase proteins ATM, p53, p21(cip1/waf1) and Cdc2, suggesting that 8-60hIPP5(m) induces G2/M arrest through activation of the ATM/p53/p21(cip1/waf1)/Cdc2/ cyclin B1 pathways. We further showed that overexpression of 8-60hIPP5(m) led to delayed nuclear translocation of cyclin B1. 8-60hIPP5(m) also could translocate to the nucleus in G2/M phase and interact with pp1α and Cdc2 as demonstrated by co-precipitation assay. Taken together, our data demonstrate a novel role for 8-60hIPP5(m) in regulation of cell cycle in HeLa cells, possibly contributing to the development of new therapeutic strategies for cervix carcinoma.

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

    Science.gov (United States)

    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.

  18. Influence of p53 and p21Waf1 expression on G2/M phase arrest of colorectal carcinoma HCT116 cells to proteasome inhibitors.

    Science.gov (United States)

    Kim, On Hee; Lim, Jun Hee; Woo, Kyung Jin; Kim, Young-Ho; Jin, Ing-Nyol; Han, Sang Tae; Park, Jong-Wook; Kwon, Taeg Kyu

    2004-04-01

    Ubiquitin-mediated protein degradation in vertebrates has been implicated in cell cycle control. In this report we explored the effects of proteasome inhibitors (MG132, lactacystin and ALLN) on cell cycle distribution. Colorectal carcinoma HCT116 cells were treated with proteasome inhibitor MG132. The results showed that MG132 inhibited cell proliferation in a dose-dependent manner. MG132 arrested HCT116 cells at G2/M phase, which was associated with drug-induced blockade of p53 degradation and/or induction of p53-related gene expression along with the accumulation of cyclin B, cyclin A and p21. MG132 treated HCT116 (wild-type) had a similar cell cycle distribution as the MG132 treated HCT116 (p53-/-) and HCT116 (p21-/-) cells, suggesting that p53 and p21 may not be essential for MG132-induced G2/M phase arrest. The release experiments from nocodazole-induced mitotic phase cells indicated that MG132 inhibits the proliferation of HCT116 cells via arrest in the G2 phase. In addition, when HCT116 cells were exposed to combination of sodium butyrate and MG132 enhanced cell growth inhibition and induction of apoptosis were observed.

  19. Immunohistochemical expression of cyclin D1, p16Ink4a, p21WAF1, and Ki-67 correlates with the severity of cervical neoplasia.

    Science.gov (United States)

    Portari, Elyzabeth A; Russomano, Fábio B; de Camargo, Maria J; Machado Gayer, Carlos R; da Rocha Guillobel, Heloísa C; Santos-Rebouças, Cíntia B; Brito Macedo, Jacyara M

    2013-09-01

    High-risk human papillomaviruses are closely associated with cervical cancer and its precursor lesions through interactions between the E6 and E7 oncoproteins and the cell-cycle regulatory proteins, such as p53 and pRb, respectively. As other molecules involved in the cell-cycle control seem to be important for human papillomavirus (HPV)-mediated cervical carcinogenesis, we have analyzed the expression of p53, p21, p16, cyclin D1, and Ki-67 and the presence of HPV (HPV pool and HPV-16) by immunohistochemical studies using tissue microarray in low squamous intraepithelial lesions (n=50), high squamous intraepithelial lesions (n=98), and cervical carcinoma (n=18). We have found a significant increase in the expression of p16 and p21 (Pcancer. In contrast, cyclin D1 expression showed a significant decrease in more severe lesions (PKi-67, p21, and p53 positivity increased with the cell-layer level and the lesion severity, with stronger correlations being observed for p16 and Ki-67. High positivity for HPV pool (96.3%) and HPV-16 (77.5%) immunostaining was detected in all cases, with an association between p16 and cyclin D1 expression and HPV-16 infection. Our tissue microarray results corroborate the usefulness of the immunohistochemical assessment of cell-cycle biomarkers in distinguishing different groups of precursor lesions of the cervix and cervical carcinoma.

  20. HCC和HepG2细胞表达HCV C蛋白、p14ARF、p21WAF1的意义探讨%The expression of HCV core protein, p14ARF and p21WAF1 in HCC and HepG2

    Institute of Scientific and Technical Information of China (English)

    王英禹; 朗红娟; 张志培; 张瑞

    2006-01-01

    目的 检测HCV C蛋白、p14、p21在HCC和表达野生p53 HepG2中的表达,初步探讨C蛋白在HCC和HepG2中对p14-p53-p21凋亡通路的作用.方法 收集42例HCC石蜡组织,采用免疫组织化学EnVision法检测HCC组织中核心蛋白、p14和p21的表达,用统计学方法及临床联系分析它们之间的关系;用细胞化学EnVision法和免疫荧光法检测核心蛋白、p53、p14和p24在HepG2细胞中的表达.结果 C蛋白、p14和p21的阳性表达主要定位于细胞核膜和细胞核中;HCC组织中C蛋白、p14和p21阳性率分别为40.5%、45.24%、19.05%;3组间的Kruskal-Wallis检验P=0.03,差异显著;C蛋白与p14、p21间及p14与P21间蛋白阳性强度相关性分析显示,P值分别为0.000、0.43、-0.34,相关系数rs分别为0.64、-0.29、-0.33.HepG2细胞有较高的C蛋白和P53表达及少量的p14、p21蛋白表达.结论 在C蛋白阳性的HCC中p14的表达与C蛋白有关,HCC中p21表达缺陷是十分常见的;C蛋白在HCC中可能影响p53通路,下调p21的表达,阻止其凋亡作用;HepG2细胞永生化特性可能与HCV或HCV C蛋白有关.

  1. Oncogene Mdm2 takes part in hepatocarcinogenesis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective:To investigate the role of Mdm2 expression in hepatocellular carcinoma.Methods: Streptavidin-peroxidase conjugation method (SP)was used to observe the expression of Mdm2 and p53 proteins in 61 cases of hepatocellular carcinoma(HCC)and 59 cases of corresponding paracancerous tissues,among which p53 mutations in exons 5~8 were detected in 21 cases by PCR-SSCP.Results:Positive nuclear P53 and Mdm2 immunostains were demonstrated in 57.38% and 26.23% of HCC,1.69% and 3.39% of corresponding paracancerous tissues respectively.The expression of p53 and Mdm2 proteins in hepatocellular carcinoma was significantly higher than that in paracancerous tissues(P<0.01).The expression of P53 and Mdm2 was not significantly correlated.42.86% of hepatocellualr carcinomas showed mutations in exon 7 of p53 gene,and no mutation was found in exons 5,6,8 and paracancerous tissues. 66.67% of mutational cases had P53 overexpression and 11.11%(1/9)showed overexpression of both P53 and Mdm2,Mdm2 overexpression also appeared in 25% of cases without mutations.Conclusions:Mdm2-induced p53 inactivation and p53 gene mutation play an important role in carcinogenesis of hepatocellular carcinoma.Tumorigenic property of Mdm2 itself,together with p53 mutation,may take part in hepatocarcinogenesis.

  2. Stress-induced alternative splice forms of MDM2 and MDMX modulate the p53-pathway in distinct ways.

    Directory of Open Access Journals (Sweden)

    Aishwarya G Jacob

    Full Text Available MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway and the nature of its interaction with MDMX remain unclear. Also, the role of the architecturally similar MDMX-ALT2 and its influence of the MDM2-MDMX-p53 axis are yet to be elucidated. We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2. Additionally, we demonstrate that MDMX-ALT2 is able to dimerize with both full-length MDMX and MDM2 and that the expression of MDM2-ALT1 and MDMX-ALT2 leads to the upregulation of p53 protein, and also of its downstream target p21. Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21. Finally we present evidence that MDM2-ALT1 and MDMX-ALT2 expression can activate subtly distinct subsets of p53-transcriptional targets implying that these splice variants can modulate the p53 tumor suppressor pathway in unique ways. In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

  3. Effect of human papillomavirus on cell cycle-related proteins p16INK4A, p21waf1/cip1, p53 and cyclin D1 in sinonasal inverted papilloma and laryngeal carcinoma. An in situ hybridization study

    Directory of Open Access Journals (Sweden)

    Marian Danilewicz

    2011-04-01

    Full Text Available Human papillomavirus (HPV infection is implicated as an important risk factor in the development of head and neck cancers. Many studies focusing on the relationships between HPV infection and cell cycle proteins immunoexpression in laryngeal lesions have provided contradictory results. The aim of this study was to evaluate the relationships between HPV DNA presence and p16INK4a, p21waf1/cip1, p53 and cyclin D1 immunoexpression in heterogenous HPV-positive and HPV-negative groups of laryngeal cancers and inverted papillomas. The HPV DNA expression was detected using an in situ hybridization method and immunoexpression of p16INK4a, p21waf1/cip1, p53 and cyclin D1 using immunohistochemistry. The immunoexpression of p21waf1/ /cip1 and p53 proteins was lower in the HPV-positive group compared to the HPV-negative group, although only the difference of p53 staining was statistically significant. The immunoexpression of p16INK4a and cyclin D1 was significantly increased in the HPV-positive group compared to the HPV-negative group. The increased immunoexpression of p16INK4a and cyclin D1, and the lower immunoexpression of p21waf1/cip1 and p53 in the HPV-positive group compared to the HPV-negative group, supports the hypothesis that HPV may play an important role in cell cycle dysregulation. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 1, pp. 34–40

  4. Induction of cyclin-dependent kinase inhibitor, p21WAF1, by treatment with 3,4-dihydro-6-[4-(3,4)-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinoline (vesnarinone) in a human salivary cancer cell line with mutant p53 gene.

    Science.gov (United States)

    Sato, M; Kawamata, H; Harada, K; Nakashiro, K; Ikeda, Y; Gohda, H; Yoshida, H; Nishida, T; Ono, K; Kinoshita, M; Adachi, M

    1997-01-30

    It has been found by PCR-SSCP analysis and direct DNA sequencing that a human salivary adenosquamous carcinoma-forming cell line, TYS, has a mutant p53 gene at codon 281Asp-->His. When TYS cells were treated with a differentiation-inducing agent, vesnarinone, cellular proliferation was significantly inhibited on the basis of MTT assay. In addition, it has been found by Northern blotting and/or immunoblotting that expression of p21WAF1 and transforming growth factor-beta (TGF-beta) is up-regulated by treating TYS cells with vesnarinone. TGF-beta 1 alone also induced p21WAF1 expression in TYS cells. Moreover, it has been shown by ELISA that the treatment of TYS cells with vesnarinone results in the enhanced generation of latent TGF-beta 1. The expression of TGF-beta receptor (T beta R), including T beta R-I, T beta R-II and T beta R-III, on TYS cells was detected by affinity cross-linking using 125I-TGF-beta 1 and addition of active TGF-beta 1 into serum-free culture medium inhibited the growth of TYS cells in a concentration-dependent manner. These findings suggest that vesnarinone might directly induce expression of p21WAF1 gene in TYS cells, the product of which may be associated with the inhibition of cell growth and induce differentiation.

  5. Trichostatin A Inhibits Proliferation and Induces Expression of p21WAF and p27 in Human Brain Tumor Cell Lines%曲古抑菌素A抑制人脑肿瘤细胞增殖及提高p21和p27蛋白表达

    Institute of Scientific and Technical Information of China (English)

    王之敏; 胡锦; 等

    2002-01-01

    . Expression patterns of accumulation of highly acetylated histone H3,H4; p53 and cell cycle-associated p21waf,p27 which were induced by TSA were determined by using Western blot analysis. Results: TSA inhibited the proliferation of brain tumor cell lines at nanomolar concentrations and induced accumulation of highly acetylased histone moleculars. Treatment with TSA at 0.33μ M for 24h significantly induced cell apoptosis.In addition to the suppression of cell growth, the up regulation of p21waf and p27 expression was observed within 48h after the treatment.p21 protein levels were increased at early time points and reached maximal levels at 8h, while p27 protein levels were increased after 8h. However, there was no significant changes of acetylased p53 and endogenous p53 protein levels were observed. Conclusion:TSA may inhibit brain tumor cell growth in vitro, which is otherwise particularly resistant to chemotherapy. TSA acts as an anti-tumor agent could be through co-operation between p21 and p27 in growth inhibition, irrespective of endogenous p53 status.

  6. Mdm2 ligase dead mutants did not act in a dominant negative manner to re-activate p53, but promoted tumor cell growth.

    Science.gov (United States)

    Swaroop, Manju; Sun, Yi

    2003-01-01

    Mdm2 (murine double minute 2) is an oncogene, first identified in BALB/c 3T3 cells. Over-expression and gene amplification of Mdm2 were found in a variety of human cancers. Recently, Mdm2 was found to be an E3 ubiquitin ligase that promotes degradation of p53, which contributes significantly to its oncogenic activity. In this study, we test a hypothesis that Mdm2 ligase dead mutants, which retained p53 binding activity but lost degradation activity, would act in a dominant negative manner to re-activate p53, especially upon stressed conditions. Five Mdm2 constructs expressing wild-type and E3 ligase-dead Mdm2 proteins were generated in a Tet-Off system and transfected into MCF-7 breast cancer cells (p53+/+ with Mdm2 overexpression) as well as MCF10A immortalized breast cells (p53+/+ without Mdm2 overexpression) as a normal control. We found that expression of Mdm2 mutants were tightly regulated by doxycycline. Withdrawal of doxycycline in culture medium triggered overexpression of Mdm2 mutants. However, expression of ligase dead mutants in MCF7 and MCF10A cells did not reactivate p53 as shown by a luciferase-reporter transcription assay and Western blot of p53 and its downstream target p21 under either unstressed condition or after exposure to DNA damaging agents. Biologically, over-expression of Mdm2 mutants had no effect on p53-induced apoptosis following DNA damage. Interestingly, over-expression of Mdm2 mutants promoted growth of MCF7 tumor cells probably via a p53-independent mechanism. Over-expression of Mdm2 mutants, however, had no effect on the growth of normal MCF10A cells and did not cause their transformation. Thus, ligase dead mutants of Mdm2 did not act in a dominant negative manner to reactivate p53 and they are not oncogenes in MCF10A cells.

  7. Effects of Radix notoginseng extracts drug-containing serum on expressions of bcl-2, Bax and p21WAF1 proteins in MNNG transformed GES-1 cells%三七提取物含药血清对MNNG转化后GES-1细胞凋亡相关基因蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    李军祥; 王志斌; 朱陵群; 牛福玲; 崔巍

    2008-01-01

    Objective: To investigate the effects of Radix notoginseng extracts drug-containing serum on the expressions of apoptosis-regulating proteins including Bax, bcl-2 and p21WAF1 in precancerous gastric cells. Methods: The N-methyI-N'-nitro-N-nitroso-guanidine (MNNG) transformed eternalized human gastric mucosa epithelium GES-I cell line (MC cell) was used in vitro as a model of gastric precancerous lesion. The medicated canine serum was prepared by feeding to the adult Beagle dog with Radix notoginseng extracts and obtaining the serum after 2-hour medication. MC cells were cultured with medicated canine serum (medicated serum group) or non-medicated canine serum (normal control group) for 72 hours. Expressions of Bax, bcl-2 and p21WAF1 proteins were detected by immunocytochemical assay and the average optical density of the cells was determined by an image analysis system. Results. Compared with those of the normal control group, Bax and p21WAF1 expressions in medicated serum group were significantly enhanced (P<0.01), while the expression of bcl-2 was significantly reduced (P 001). Conclusion. Radix notoginseng extracts may inhibit the proliferation and promote the apoptosis of precancerous gastric cells through altering expressions of the bcl-2, Bax and p21WAF1 genes.%目的:研究三七提取物犬药物血清作用于胃癌前细胞后,其凋亡相关基因蛋白表达的变化.方法:采用被N-甲基-N-硝基-N-亚硝基胍(N-methyl-N-nitroso-guanidine,MNNG)转化后的永生化人胃黏膜上皮细胞系GES-1细胞(简称MC细胞)作为胃癌前病变细胞的体外研究模型,用三七提取物一次性灌胃彼格犬,取给药后2 h的血清作为实验药物血清.以免疫组织化学法检测药物血清对MC细胞作用72 h后bcl-2、Bax和p21WAF13种凋亡相关基因蛋白表达情况,并与正常培养的MC细胞相比较.结果:药物血清作用后的MC细胞中Bax和p21WAF1的表达较正常培养的MC细胞升高(P<0.01);Bc1-2表达较

  8. Homozygous mdm2 SNP309 cancer cells with compromised transcriptional elongation at p53 target genes are sensitive to induction of p53-independent cell death.

    Science.gov (United States)

    Rosso, Melissa; Polotskaia, Alla; Bargonetti, Jill

    2015-10-27

    A single nucleotide polymorphism (T to G) in the mdm2 P2 promoter, mdm2 SNP309, leads to MDM2 overexpression promoting chemotherapy resistant cancers. Two mdm2 G/G SNP309 cancer cell lines, MANCA and A875, have compromised wild-type p53 that co-localizes with MDM2 on chromatin. We hypothesized that MDM2 in these cells inhibited transcription initiation at the p53 target genes p21 and puma. Surprisingly, following etoposide treatment transcription initiation occurred at the compromised target genes in MANCA and A875 cells similar to the T/T ML-1 cell line. In all cell lines tested there was equally robust recruitment of total and initiated RNA polymerase II (Pol II). We found that knockdown of MDM2 in G/G cells moderately increased expression of subsets of p53 target genes without increasing p53 stability. Importantly, etoposide and actinomycin D treatments increased histone H3K36 trimethylation in T/T, but not G/G cells, suggesting a G/G correlated inhibition of transcription elongation. We therefore tested a chemotherapeutic agent (8-amino-adenosine) that induces p53-independent cell death for higher clinically relevant cytotoxicity. We demonstrated that T/T and G/G mdm2 SNP309 cells were equally sensitive to 8-amino-adenosine induced cell death. In conclusion for cancer cells overexpressing MDM2, targeting MDM2 may be less effective than inducing p53-independent cell death.

  9. Mir-660 is downregulated in lung cancer patients and its replacement inhibits lung tumorigenesis by targeting MDM2-p53 interaction.

    Science.gov (United States)

    Fortunato, O; Boeri, M; Moro, M; Verri, C; Mensah, M; Conte, D; Caleca, L; Roz, L; Pastorino, U; Sozzi, G

    2014-12-11

    Lung cancer represents the leading cause of cancer-related death in developed countries. Despite the advances in diagnostic and therapeutic techniques, the 5-year survival rate remains low. The research for novel therapies directed to biological targets has modified the therapeutic approach, but the frequent engagement of resistance mechanisms and the substantial costs, limit the ability to reduce lung cancer mortality. MicroRNAs (miRNAs) are small noncoding RNAs with known regulatory functions in cancer initiation and progression. In this study we found that mir-660 expression is downregulated in lung tumors compared with adjacent normal tissues and in plasma samples of lung cancer patients with poor prognosis, suggesting a potential functional role of this miRNA in lung tumorigenesis. Transient and stable overexpression of mir-660 using miRNA mimics reduced migration, invasion, and proliferation properties and increased apoptosis in p53 wild-type lung cancer cells (NCI-H460, LT73, and A549). Furthermore, stable overexpression using lentiviral vectors in NCI-H460 and A549 cells inhibited tumor xenograft growth in immunodeficient mice (95 and 50% reduction compared with control, respectively), whereas the effects of mir-660 overexpression were absent in H1299, a lung cancer cell line lacking p53 locus, both in in vitro and in vivo assays. We identified and validated mouse double minute 2 (MDM2) gene, a key regulator of the expression and function of p53, as a new direct target of mir-660. In addition, mir-660 expression reduced both mRNA and protein expression of MDM2 in all cell lines and stabilized p53 protein levels resulting in an upregulation of p21(WAF1/CIP1) in p53 wild-type cells. Our finding supports that mir-660 acts as a tumor suppressor miRNA and we suggest the replacement of mir-660 as a new therapeutic approach for p53 wild-type lung cancer treatment.

  10. Specific inhibition of Mdm2-mediated neddylation by Tip60

    DEFF Research Database (Denmark)

    Dohmesen, Christoph; Koeppel, Max; Dobbelstein, Matthias

    2007-01-01

    Tip60 is a histone acetyl transferase (HAT) and a cofactor of transcription, but also an interaction partner of the Mdm2 oncoprotein. The functional consequences of this interaction are only partially understood and were further explored in this study. We found that Tip60 is capable of selectively...... inhibiting the Mdm2- mediated conjugation of Nedd8 to p53, whereas it did not affect p53 ubiquitination. In contrast, the known Mdm2 antagonist p14arf preferentially blocked Ubiquitin conjugation by Mdm2. To identify underlying mechanisms, we studied the intracellular localization of Tip60 and Mdm2. Both...... proteins relocalized each other to the PML nuclear bodies, but a similar localization pattern was observed even in the absence of PML. Analysis of Tip60 deletion mutants revealed that some mutants, while still interacting with Mdm2, failed to relocalize it and to inhibit Mdm2-mediated neddylation...

  11. Stable low-level expression of p21WAF1/CIP1 in A549 human bronchogenic carcinoma cell line-derived clones down-regulates E2F1 mRNA and restores cell proliferation control

    Directory of Open Access Journals (Sweden)

    Crawford Erin L

    2006-01-01

    Full Text Available Abstract Background Deregulated cell cycle progression and loss of proliferation control are key properties of malignant cells. In previous studies, an interactive transcript abundance index (ITAI comprising three cell cycle control genes, [MYC × E2F1]/p21 accurately distinguished normal from malignant bronchial epithelial cells (BEC, using a cut-off threshold of 7,000. This cut-off is represented by a line with a slope of 7,000 on a bivariate plot of p21 versus [MYC × E2F1], with malignant BEC above the line and normal BEC below the line. This study was an effort to better quantify, at the transcript abundance level, the difference between normal and malignant BEC. The hypothesis was tested that experimental elevation of p21 in a malignant BEC line would decrease the value of the [MYC × E2F1]/p21 ITAI to a level below this line, resulting in loss of immortality and limited cell population doubling capacity. In order to test the hypothesis, a p21 expression vector was transfected into the A549 human bronchogenic carcinoma cell line, which has low constitutive p21 TA expression relative to normal BEC. Results Following transfection of p21, four A549/p21 clones with stable two-fold up-regulated p21 expression were isolated and expanded. For each clone, the increase in p21 transcript abundance (TA was associated with increased total p21 protein level, more than 5-fold reduction in E2F1 TA, and 10-fold reduction in the [MYC × E2F1]/p21 ITAI to a value below the cut-off threshold. These changes in regulation of cell cycle control genes were associated with restoration of cell proliferation control. Specifically, each transfectant was capable of only 15 population doublings compared with unlimited population doublings for parental A549. This change was associated with an approximate 2-fold increase in population doubling time to 38.4 hours (from 22.3 hrs, resumption of contact-inhibition, and reduced dividing cell fraction as measured by flow

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

    Science.gov (United States)

    With prostate being the highest zinc-accumulating tissue before the onset of cancer, the effects of physiologic levels of zinc on Akt-Mdm2-p53 and Akt-p21 signaling axes in human normal prostate epithelial cells (PrEC) and malignant prostate LNCaP cells were examined. Cells were cultured for 6 d in...

  13. 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

    Energy Technology Data Exchange (ETDEWEB)

    Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Das, Dipon; Siddharth, Sumit [Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024 (India); Choudhuri, Tathagata [Institute of Life Sciences, Nalco Square, Bhubaneswar, Orissa 751023 (India); Department of Biotechnology, Visva Bharati University, Santiniketan, West Bengal (India); Kundu, Chanakya Nath, E-mail: cnkundu@gmail.com [Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024 (India)

    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

  14. Targeting RING domains of Mdm2-MdmX E3 complex activates apoptotic arm of the p53 pathway in leukemia/lymphoma cells.

    Science.gov (United States)

    Wu, W; Xu, C; Ling, X; Fan, C; Buckley, B P; Chernov, M V; Ellis, L; Li, F; Muñoz, I G; Wang, X

    2015-12-31

    Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2-p53 interface or MdmX ((MDM4), mouse double minute 4)-p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2-MdmX really interesting new gene (RING)-RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2-MdmX RING-RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2-MdmX RING domain inhibitors)) that specifically inhibit Mdm2-MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2-MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2-MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.

  15. 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.

    Science.gov (United States)

    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.

  16. Novel Insights into the Molecular Mechanisms Governing Mdm2 Ubiquitination and Destruction

    OpenAIRE

    Fukushima, Hidefumi; Inuzuka, Hiroyuki; Shaik, Shavali; Wei, Wenyi

    2010-01-01

    The Mdm2/p53 pathway is compromised in more than 50% of all human cancers, therefore it is an intensive area of research to understand the upstream regulatory pathways governing Mdm2/p53 activity. Mdm2 is frequently overexpressed in human cancers while the molecular mechanisms underlying the timely destruction of Mdm2 remain unclear. We recently reported that Casein Kinase I phosphorylates Mdm2 at multiple sites to trigger Mdm2 interaction with, and subsequent ubiquitination and destruction b...

  17. Potential Landscape and Flux of p53-Mdm2 Oscillator Mediated by Mdm2 Degradation Rate

    Science.gov (United States)

    Bi, Yuanhong; Yang, Zhuoqin

    The dynamics of the tumor suppressor p53 can play a crucial role in deciding cell fate after DNA damage. In this paper, we explore the dynamics and stability of p53 mediated by Mdm2 degradation rate in p53-Mdm2 oscillator through bifurcation, the potential landscape and flux. Based on the investigation of the bifurcation, we find that p53 can exhibit rich dynamics including monostability, bistability of two stable steady states and oscillation behaviors as well as bistability between a stable steady state and an oscillatory state. The stability of these states are further validated by the potential landscape. In addition, oscillatory behaviors of p53 are explored by means of the negative gradient of the potential landscape and the probability flux. It is shown that the negative gradient of the potential landscape can attract the system towards the oscillatory path and the flux can drive oscillation along the path. Moreover, the quicker the flux runs, the smaller the period is. Besides, stability and sensitivity of the system are explored by the barrier height and the entropy production rate in a single cell level, and we further compare the potential landscapes at single and population cell levels. Our results may be useful for understanding the regulation of p53 signaling pathways in response to DNA damage.

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

    Indian Academy of Sciences (India)

    Indrani Bose; Bhaswar Ghosh

    2007-08-01

    The p53 protein is well-known for its tumour suppressor function. The p53-MDM2 negative feedback loop constitutes the core module of a network of regulatory interactions activated under cellular stress. In normal cells, the level of p53 proteins is kept low by MDM2, i.e. MDM2 negatively regulates the activity of p53. In the case of DNA damage, the p53-mediated pathways are activated leading to cell cycle arrest and repair of the DNA. If repair is not possible due to excessive damage, the p53-mediated apoptotic pathway is activated bringing about cell death. In this paper, we give an overview of our studies on the p53-MDM2 module and the associated pathways from a systems biology perspective. We discuss a number of key predictions, related to some specific aspects of cell cycle arrest and cell death, which could be tested in experiments.

  19. Mdm2 Splice isoforms regulate the p53/Mdm2/Mdm4 regulatory circuit via RING domain-mediated ubiquitination of p53 and Mdm4.

    Science.gov (United States)

    Fan, Chuandong; Wang, Xinjiang

    2017-02-06

    p53 is regulated by heterodimer E3 ligase Mdm2-Mdm4 via RING domain interaction. Mdm2 transcripts undergo alternative splicing, and Mdm2 splice isoforms are increased in cancer and induced by DNA damage. Although two major Mdm2 splice isoforms that do not bind to p53 were reported to impact the p53 pathway, the underlying biochemical mechanisms were not understood. Here, we show that these Mdm2 splice isoforms ubiquitinate Mdm2 and Mdm4 in vitro and regulate the activity of Mdm2-Mdm4 E3 complex in cells. The Mdm2 isoforms are capable of promoting p53 ubiquitination in the absence of Mdm2 or Mdm4. The two isoforms stimulate Mdm2 or Mdm4 activity for p53 ubiquitination in vitro and promote degradation of p53 and Mdm4 in cells. However, the Mdm2 isoforms have opposing effects on the steady-state p53 levels depending on the stoichiometric ratios of Mdm2, Mdm4 and the isoforms, causing either decreased or increased p53 levels in cells. Our data indicate that the Mdm2 splice isoforms can act as independent E3 ligases for p53 when Mdm2 and Mdm4 are absent, form potent heterodimer E3 ligases with either Mdm2 or Mdm4 for targeting p53 degradation, or act as inhibitory regulators of Mdm2-Mdm4 E3 ligase activity by downregulating Mdm4. These findings suggest that Mdm2 splice isoforms may play critical roles in the regulatory loop of p53/Mdm2-Mdm4 via a RING domain-mediated biochemical mechanism.

  20. mRNA display selection of an optimized MDM2-binding peptide that potently inhibits MDM2-p53 interaction.

    Directory of Open Access Journals (Sweden)

    Hirokazu Shiheido

    Full Text Available p53 is a tumor suppressor protein that prevents tumorigenesis through cell cycle arrest or apoptosis of cells in response to cellular stress such as DNA damage. Because the oncoprotein MDM2 interacts with p53 and inhibits its activity, MDM2-p53 interaction has been a major target for the development of anticancer drugs. While previous studies have used phage display to identify peptides (such as DI that inhibit the MDM2-p53 interaction, these peptides were not sufficiently optimized because the size of the phage-displayed random peptide libraries did not cover all of the possible sequences. In this study, we performed selection of MDM2-binding peptides from large random peptide libraries in two stages using mRNA display. We identified an optimal peptide named MIP that inhibited the MDM2-p53 and MDMX-p53 interactions 29- and 13-fold more effectively than DI, respectively. Expression of MIP fused to the thioredoxin scaffold protein in living cells by adenovirus caused stabilization of p53 through its interaction with MDM2, resulting in activation of the p53 pathway. Furthermore, expression of MIP also inhibited tumor cell proliferation in a p53-dependent manner more potently than DI. These results show that two-stage, mRNA-displayed peptide selection is useful for the rapid identification of potent peptides that target oncoproteins.

  1. p53 and MDM2 protein expression in actinic cheilitis.

    Science.gov (United States)

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

    2008-01-01

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

  2. p53 and MDM2 protein expression in actinic cheilitis

    Directory of Open Access Journals (Sweden)

    Maria da Conceição Andrade de Freitas

    2008-12-01

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

  3. A dynamic P53-MDM2 model with time delay

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-11-15

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

  4. Oncoprotein MDM2 Overexpression is Associated with Poor Prognosis in Distinct Non-Hodgkin's Lymphoma Entities

    DEFF Research Database (Denmark)

    Møller, Michael Boe; Nielsen, O; Pedersen, Niels Tinggaard

    1999-01-01

    MDM2 is an oncoprotein involved in the regulation of p53. MDM2 exerts its tumorigenic potential through p53-dependent and -independent mechanisms. It is frequently overexpressed in various malignancies. Little is known about the prognostic value of MDM2 expression in non-Hodgkin's lymphomas (NHL......). We analyzed MDM2 expression immunohistochemically in 188 NHL cases from a prospective population-based NHL registry. The aim was to identify MDM2 expression profiles in various histological NHL subtypes and analyze whether MDM2 expression correlated with clinical variables and p53 status. MDM2...... overexpression was present in 42 (22%) of 188 cases. The frequency was highest in aggressive/very aggressive NHL (P MDM2 overexpression was associated with higher-grade disease (P = .008). MDM2 overexpression was not related to a phenotype indicating...

  5. The p53-MDM2/MDMX axis - A chemotype perspective

    NARCIS (Netherlands)

    Khoury, Kareem; Popowicz, Grzegorz M.; Holak, Tad A.; Doemling, Alexander

    The protein-protein interaction (PPI) of the tumor suppressor p53 and its negative regulator MDM2 consists of the most intense studied PPI with a group of small molecular weight antagonists described and many more disclosed in patent literature. Due to the A-level structural insight into p53

  6. Immunohistochemical detection of P53 and Mdm2 in vitiligo

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2014-04-01

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

  8. Therapeutic considerations for Mdm2: not just a one trick pony

    Science.gov (United States)

    Lehman, Jason A.; Eitel, Jacob A.; Batuello, Christopher N.; Mayo, Lindsey D.

    2008-01-01

    Background The mdm2 proto-oncogene is elevated in numerous late stage cancers. The Mdm2 protein manifests its oncogenic properties in part through inactivation of the tumor suppressor protein p53. Recent efforts in anti-cancer drug design have focused on the identification of small molecules that disrupt the Mdm2-p53 interaction, in hopes of re-engaging the p53 pathway. Objective In addition to binding p53, Mdm2 complexes with numerous proteins involved in DNA repair, translation, metabolic activities, tumor growth and apoptosis. Additional biochemical analysis is required to understand how Mdm2 integrates into all of these cellular processes. Post-translational modifications to Mdm2 can alter its ability to associate with numerous proteins. Changes in protein structure may also affect the ability of small molecule inhibitors to effectively antagonize Mdm2. Conclusion The complexity of Mdm2 modification has been largely neglected during the development of previous Mdm2 inhibitors. Future high-throughput or in silico screening efforts will need to recognize the importance of post-translational modifications to Mdm2. Furthermore, the identification of molecules that target other domains in Mdm2 may provide a tool to prevent other pivotal p53-independent functions of Mdm2. These aims provide a useful roadmap for the discovery of new Mdm2 binding compounds with therapeutic potency that may exceed its predecessors. PMID:19738896

  9. Mdm2’s Dilemma: To Degrade or To Translate p53?

    OpenAIRE

    2012-01-01

    In this issue of Cancer Cell, Gajjar et al. provide insight into how Mdm2 can both inhibit and enhance p53 activity. In the basal setting, Mdm2 binds p53 and promotes p53 degradation. Under stress conditions, ATM-dependent phosphorylation of Mdm2 results in its recruitment to p53 mRNA, thereby stimulating p53 translation.

  10. Inhibition of p21-mediated ROS accumulation can rescue p21-induced senescence

    OpenAIRE

    2002-01-01

    The cyclin-dependent kinase (CDK) inhibitor p21Waf1/Cip1/Sdi1 was identified initially as a gene induced in senescent cells and itself has been shown to cause permanent growth arrest/senescence. Reactive oxygen species (ROS), a byproduct of oxidative processes, can also induce an irreversible growth arrest similar to senescence. Here we show that p21 increased intracellular levels of ROS both in normal fibroblasts and in p53-negative cancer cells. N-acetyl-l-cysteine, an ROS inhibitor, rescue...

  11. Novel targeted therapeutics: inhibitors of MDM2, ALK and PARP

    Directory of Open Access Journals (Sweden)

    Hsueh Chung-Tsen

    2011-04-01

    Full Text Available Abstract We reviewed preclinical data and clinical development of MDM2 (murine double minute 2, ALK (anaplastic lymphoma kinase and PARP (poly [ADP-ribose] polymerase inhibitors. MDM2 binds to p53, and promotes degradation of p53 through ubiquitin-proteasome degradation. JNJ-26854165 and RO5045337 are 2 small-molecule inhibitors of MDM2 in clinical development. ALK is a transmembrane protein and a member of the insulin receptor tyrosine kinases. EML4-ALK fusion gene is identified in approximately 3-13% of non-small cell lung cancer (NSCLC. Early-phase clinical studies with Crizotinib, an ALK inhibitor, in NSCLC harboring EML4-ALK have demonstrated promising activity with high response rate and prolonged progression-free survival. PARPs are a family of nuclear enzymes that regulates the repair of DNA single-strand breaks through the base excision repair pathway. Randomized phase II study has shown adding PARP-1 inhibitor BSI-201 to cytotoxic chemotherapy improves clinical outcome in patients with triple-negative breast cancer. Olaparib, another oral small-molecule PARP inhibitor, demonstrated encouraging single-agent activity in patients with advanced breast or ovarian cancer. There are 5 other PARP inhibitors currently under active clinical investigation.

  12. Structure of a stapled peptide antagonist bound to nutlin-resistant Mdm2.

    Directory of Open Access Journals (Sweden)

    Sharon Min Qi Chee

    Full Text Available As key negative regulator of the p53 tumour suppressor, Mdm2 is an attractive therapeutic target. Small molecules such as Nutlin have been developed to antagonise Mdm2, resulting in p53-dependent death of tumour cells. We have recently described a mutation in Mdm2 (M62A, which precludes binding of Nutlin, but not p53. This Nutlin-resistant variant is not, however, refractory to binding and inhibition by stapled peptide antagonists targeting the same region of Mdm2. A detailed understanding of how stapled peptides are recalcitrant to Mdm2 mutations conferring Nutlin-resistance will aid in the further development of potent Mdm2 antagonists. Here, we report the 2.00 Å crystal structure of a stapled peptide antagonist bound to Nutlin resistant Mdm2. The stapled peptide relies on an extended network of interactions along the hydrophobic binding cleft of Mdm2 for high affinity binding. Additionally, as seen in other stapled peptide structures, the hydrocarbon staple itself contributes to binding through favourable interactions with Mdm2. The structure highlights the intrinsic plasticity present in both Mdm2 and the hydrocarbon staple moiety, and can be used to guide future iterations of both small molecules and stapled peptides for improved antagonists of Mdm2.

  13. mdm-2 gene amplification in 3T3-L1 preadipocytes.

    Science.gov (United States)

    Berberich, S J; Litteral, V; Mayo, L D; Tabesh, D; Morris, D

    1999-05-01

    In this study the regulation of the murine double minute-2 (mdm-2) gene was examined in NIH 3T3-L1 preadipocytes. The 3T3-L1 cell line, under proper conditions, has the capacity to differentiate from fibroblasts into adipocytes [15]. A recent report demonstrated that mdm-2 overexpression could block myogenesis [12]. While examining the regulation of the mdm-2 gene during adipogenesis, it was discovered that 3T3-L1 cells possess a 36-fold elevation of mdm-2 mRNA relative to A31 cells, another immortalized Balb/c 3T3 fibroblast cell line that lacks the capacity to differentiate. Based on Southern blot analysis, the increase in mdm-2 mRNA was the result of a mdm-2 gene amplification. The level of Mdm-2 protein in undifferentiated 3T3-L1 cells was elevated relative to A31 fibroblasts and resulted from translation of mRNA transcripts initiating from the p53-independent P1 promoter. We also examined how mdm-2 and p53 levels changed as undifferentiated fibroblasts converted to adipocytes. While mdm-2 mRNA levels remained elevated, p53 mRNA, protein, and DNA-binding activity decreased. These results suggest that adipogenesis is unaffected by elevated Mdm-2 levels and that the overexpression of mdm-2 mRNA is predominantly p53 independent.

  14. Structural Basis of Competitive Recognition of p53 and MDM2 by HAUSP/USP7: Implications for the Regulation of the p53-MDM2 Pathway.

    Directory of Open Access Journals (Sweden)

    2006-01-01

    Full Text Available Herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7, a deubiquitylating enzyme of the ubiquitin-specific processing protease family, specifically deubiquitylates both p53 and MDM2, hence playing an important yet enigmatic role in the p53-MDM2 pathway. Here we demonstrate that both p53 and MDM2 specifically recognize the N-terminal tumor necrosis factor-receptor associated factor (TRAF-like domain of HAUSP in a mutually exclusive manner. HAUSP preferentially forms a stable HAUSP-MDM2 complex even in the presence of excess p53. The HAUSP-binding elements were mapped to a peptide fragment in the carboxy-terminus of p53 and to a short-peptide region preceding the acidic domain of MDM2. The crystal structures of the HAUSP TRAF-like domain in complex with p53 and MDM2 peptides, determined at 2.3-A and 1.7-A resolutions, respectively, reveal that the MDM2 peptide recognizes the same surface groove in HAUSP as that recognized by p53 but mediates more extensive interactions. Structural comparison led to the identification of a consensus peptide-recognition sequence by HAUSP. These results, together with the structure of a combined substrate-binding-and-deubiquitylation domain of HAUSP, provide important insights into regulation of the p53-MDM2 pathway by HAUSP.

  15. 睾丸肿瘤组织中mdm-2基因表达及临床意义%Mdm-2 Gene Expression in Human Testicular cancer

    Institute of Scientific and Technical Information of China (English)

    吴先旺; 徐元虎

    2003-01-01

    探讨mdm-2基因与睾丸肿瘤发生及预后的关系.应用免疫组化方法检测27 例睾丸肿瘤中mdm-2蛋白表达水平.发现有12 例标本mdm-2蛋白阳性表达,并且与睾丸肿瘤的病理分级和临床分期有关,提示睾丸肿瘤恶性程度与mdm-2蛋白表达具有明显相关性.mdm-2蛋白表达可能成为睾丸肿瘤预后的指标.

  16. A dynamic p53-mdm2 model with distributed delay

    Science.gov (United States)

    Horhat, Raluca; Horhat, Raul Florin

    2014-12-01

    Specific activator and repressor transcription factors which bind to specific regulator DNA sequences, play an important role in gene activity control. Interactions between genes coding such transcripion factors should explain the different stable or sometimes oscillatory gene activities characteristic for different tissues. In this paper, the dynamic P53-Mdm2 interaction model with distributed delays is investigated. Both weak and Dirac kernels are taken into consideration. For Dirac case, the Hopf bifurcation is investigated. Some numerical examples are finally given for justifying the theoretical results.

  17. Ribosomal protein S7 is both a regulator and a substrate of MDM2.

    Science.gov (United States)

    Zhu, Yan; Poyurovsky, Masha V; Li, Yingchun; Biderman, Lynn; Stahl, Joachim; Jacq, Xavier; Prives, Carol

    2009-08-14

    MDM2 associates with ribosomal protein S7, and this interaction is required to inhibit MDM2's E3 ligase activity, leading to stabilization of MDM2 and p53. Notably, the MDM2 homolog MDMX facilitates the inhibition of MDM2 E3 ligase activity by S7. Further, ablation of S7 inhibits MDM2 and p53 accumulation induced by different stress signals in some cell types. Thus, ribosomal/nucleolar stress is likely a key integrating event in DNA damage signaling to p53. Interestingly, S7 is itself a substrate for MDM2 E3 ligase activity both in vitro and in vivo. An S7-ubiquitin fusion protein (S7-Ub) selectively inhibits MDM2 degradation of p53 and is unaffected by MDMX. S7-Ub promotes apoptosis to a greater extent than S7 alone. This indicates that MDM2 ubiquitination of S7 is involved in sustaining the p53 response. Thus, S7 functions as both effector and affector of MDM2 to ensure a proper cellular response to different stress signals.

  18. Escape, or Vanish: Control the Fate of p53 through MDM2-Mediated Ubiquitination.

    Science.gov (United States)

    Wei, Jinlian; Yang, Yingrui; Lu, Mengchen; Xu, Lili; Liu, Fang; Yuan, Zhenwei; Bao, Qichao; Jiang, Zhengyu; Xu, Xiaoli; Guo, Xiaoke; Zhang, Xiaojin; You, Qidong; Sun, Haopeng

    2015-01-01

    p53 protein is a prominent tumor suppressor to induce cell cycle arrest, apoptosis and senescence, which attracts significant interest to cancer treatment. Therefore, it would be particularly important to restore the wild-type p53 that retains latent functions in the approximately 50% of tumors. MDM2 (murine double minute 2), the principal cellular antagonist of p53, has long been believed to suppress p53 activity through two main mechanisms: promoting degradation via its E3 ligase activity and masking p53 transcriptional activation by direct binding. Targeting MDM2 E3 ligase activity is becoming a potential antitumor strategy resulting from MDM2's decisive role in controlling the fate of p53: p53 is going to degradation when entrapped into MDM2-mediated ubiquitination, where p53 can escape by abrogating MDM2 E3 ligase activity using regulators. The intensive focus on regulating MDM2 ubiquitin E3 ligase activity has led to the rapid progress of its inhibitors, which may be possible to help p53 escape from degradation and restore its function to control tumor growth. This review summarizes the current inhibitors of MDM2 E3 ligase in cancer therapy based on the understanding the regulation of MDM2 E3 ubiquitin ligase activity, including post-translational modification, interactions between MDM2 and its cofactors, and regulation of MDM2 stability.

  19. Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Kasim, Vivi, E-mail: vivikasim78@gmail.com [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Huang, Can; Zhang, Jing; Jia, Huizhen; Wang, Yunxia [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Yang, Li [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Miyagishi, Makoto [Molecular Composite Medicine Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566 (Japan); Wu, Shourong, E-mail: shourongwu@hotmail.com [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044 (China)

    2014-07-04

    Highlights: • MDM2 is a novel positive regulator of TAp73 transcriptional activity. • MDM2 colocalizes together and physically interacts with E2F1. • Synergistic cooperation of MDM2 and E2F1 is crucial for TAp73 transcription. • MDM2 regulates TAp73 transcriptional activity in a p53-independent manner. - Abstract: TAp73, a structural homologue of p53, plays an important role in tumorigenesis. E2F1 had been reported as a transcriptional regulator of TAp73, however, the detailed mechanism remains to be elucidated. Here we reported that MDM2-silencing reduced the activities of the TAp73 promoters and the endogenous TAp73 expression level significantly; while MDM2 overexpression upregulated them. We further revealed that the regulation of TAp73 transcriptional activity occurs as a synergistic effect of MDM2 and E2F1, most probably through their physical interaction in the nuclei. Furthermore, we also suggested that MDM2 might be involved in DNA damage-induced TAp73 transcriptional activity. Finally, we elucidated that MDM2-silencing reduced the proliferation rate of colon carcinoma cells regardless of the p53 status. Our data show a synergistic effect of MDM2 and E2F1 on TAp73 transcriptional activity, suggesting a novel regulation pathway of TAp73.

  20. Serdemetan antagonizes the Mdm2-HIF1α axis leading to decreased levels of glycolytic enzymes.

    Directory of Open Access Journals (Sweden)

    Jason A Lehman

    Full Text Available Serdemetan (JNJ-26854165, an antagonist to Mdm2, was anticipated to promote the activation of p53. While regulation of p53 by Mdm2 is important, Mdm2 also regulates numerous proteins involved in diverse cellular functions. We investigated if Serdemetan would alter the Mdm2-HIF1α axis and affect cell survival in human glioblastoma cells independently of p53. Treatment of cells with Serdemetan under hypoxia resulted in a decrease in HIF1α levels. HIF1α downstream targets, VEGF and the glycolytic enzymes (enolase, phosphoglycerate kinase1/2, and glucose transporter 1, were all decreased in response to Serdemetan. The involvement of Mdm2 in regulating gene expression of glycolytic enzymes raises the possibility of side effects associated with therapeutically targeting Mdm2.

  1. Clinicopathological significance of p53 and mdm2 protein expression in human pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Ming Dong; Gang Ma; Wei Tu; Ke-Jian Guo; Yu-Lin Tian; Yu-Ting Dong

    2005-01-01

    AIM: To study the clinicopathological significance of p53 and mdm2 protein expression in human pancreatic cancer. METHODS: To investigate the expression of p53 and mdm2 in pancreatic cancer by immunohistochemistry, and the relationships between the p53 and mdm2 protein expression and clinicopathological parameters in pancreatic cancer.RESULTS: The positive expression of p53 protein was found in 40 of 59 patients (67.8%) and that of mdm2 protein in 17 of 59 patients (28.8%). No obvious relationships were found between p53 as well as mdm2 expression and sex, tumor site, TNM staging and histological differentiation. p53 expression was increased in patients younger than 65 years old, while mdm2 had no relationship with age. The survival time of the patients with the positive expression of p53 and mdm2 proteins was obviously shorter than the other groups. CONCLUSION: Both p53 and mdm2 presented relatively high expression in human pancreatic cancer. The overexpression of p53 and mdm2 might reflect the malignant proliferation of pancreatic cancer and their co-expression might be helpful to evaluate the prognosis of the patients with pancreatic cancer.

  2. Mdm2 controls CREB-dependent transactivation and initiation of adipocyte differentiation

    DEFF Research Database (Denmark)

    Hallenborg, Philip; Feddersen, Søren; Francoz, S.

    2012-01-01

    The role of the E3 ubiquitin ligase murine double minute 2 (Mdm2) in regulating the stability of the p53 tumor suppressor is well documented. By contrast, relatively little is known about p53-independent activities of Mdm2 and the role of Mdm2 in cellular differentiation. Here we report a novel r...... in the myoblast cell line C2C12, it is conceivable that Mdm2 acts as a switch in cell fate determination. Cell Death and Differentiation (2012) 19, 1381-1389; doi:10.1038/cdd.2012.15; published online 2 March 2012...

  3. Mouse models of Mdm2 and Mdm4 and their clinical implications

    Institute of Scientific and Technical Information of China (English)

    Shunbin Xiong

    2013-01-01

    Mdm2 and Mdm4 are two key negative regulators of the tumor suppressor p53.Deletion of either Mdm2 or Mdm4 induces p53-dependent early embryonic lethality in knockout mouse models.The tissuespecific deletion of Mdm2 induces p53-dependent apoptosis,whereas the deletion of Mdm4 induces both p53-dependent apoptosis and cell cycle arrest.Compared to Mdm4 deletion,Mdm2 deletion causes more severe phenotypic defects.Disrupting the Mdm2 and Mdm4 interaction using knockin mice models causes embryonic lethality that can be completely rescued by the concomitant loss of p53,suggesting that Mdm2 and Mdm4 heterodimerization is critical to inhibit p53 activity during embryogenesis.Overexpression of Mdm2 and Mdm4 in mice induces spontaneous tumorigenesis,which clearly indicates that Mdm2 and Mdm4 are bona fide oncogenes.Studies from these mouse models strongly suggest that blocking Mdm2-and Mdm4-mediated p53 inhibition is an appealing therapeutic strategy for cancer patients with wild-type p53 alleles.

  4. Primary extraskeletal osteosarcoma: a clinicopathological study of 18 cases focusing on MDM2 amplification status.

    Science.gov (United States)

    Yamashita, Kyoko; Kohashi, Kenichi; Yamada, Yuichi; Nishida, Yoshihiro; Urakawa, Hiroshi; Oda, Yoshinao; Toyokuni, Shinya

    2017-05-01

    Extraskeletal osteosarcoma (ESOS) is an uncommon malignant neoplasm. Most ESOSs are high grade, although some low-grade cases have been reported. A few cases of ESOS with MDM2 amplification have also been reported, suggesting some similarity to skeletal low-grade osteosarcoma such as parosteal osteosarcoma, where MDM2 is often amplified. However, the frequency of low-grade cases and cases with MDM2 amplification among ESOSs remains unknown, and their relationship is unclear. To clarify this, we examined 18 primary ESOS cases clinically, pathologically, and genetically, focusing on their MDM2 amplification status. Our cases comprised 10 men and 8 women whose mean age was 58.6 years; the most common site of the lesion was the thigh and buttock. There were one histologically low-grade case evaluated by biopsy specimen with an aggressive course and 2 relatively low-grade cases whose lesions were of low grade for the most part. MDM2 amplification status was revealed by fluorescence in situ hybridization in all 18 cases; 2 patients-histologically intermediate- and high-grade cases-were found to have MDM2 amplification. In conclusion, this study indicates that histologically low-grade and relatively low-grade cases of ESOS are not always associated with MDM2 amplification. The ESOS case with MDM2 amplification could be high grade, although MDM2-amplified dedifferentiated liposarcoma with osteogenic differentiation should be ruled out in making the diagnosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3

    DEFF Research Database (Denmark)

    Hallenborg, P.; Siersbæk, M.; Barrio-Hernandez, I.

    2016-01-01

    The ubiquitin ligase MDM2 is best known for balancing the activity of the tumor suppressor p53. We have previously shown that MDM2 is vital for adipocyte conversion through controlling Cebpd expression in a p53-independent manner. Here, we show that the proadipogenic effect of MDM2 relies...... on activation of the STAT family of transcription factors. Their activation was required for the cAMP-mediated induction of target genes. Interestingly, rather than influencing all cAMP-stimulated genes, inhibition of the kinases directly responsible for STAT activation, namely JAKs, or ablation of MDM2, each...

  6. Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function.

    Science.gov (United States)

    Chen, D; Zhang, Z; Li, M; Wang, W; Li, Y; Rayburn, E R; Hill, D L; Wang, H; Zhang, R

    2007-08-01

    As a major negative regulator of p53, the MDM2 oncogene plays an important role in carcinogenesis and tumor progression. MDM2 promotes p53 proteasomal degradation and negatively regulates p53 function. The mechanisms by which the MDM2-p53 interaction is regulated are not fully understood, although several MDM2-interacting molecules have recently been identified. To search for novel MDM2-binding partners, we screened a human prostate cDNA library by the yeast two-hybrid assay using full-length MDM2 protein as the bait. Among the candidate proteins, ribosomal protein S7 was identified and confirmed as a novel MDM2-interacting protein. Herein, we demonstrate that S7 binds to MDM2, in vitro and in vivo, and that the interaction between MDM2 and S7 leads to modulation of MDM2-p53 binding by forming a ternary complex among MDM2, p53 and S7. This results in the stabilization of p53 protein through abrogation of MDM2-mediated p53 ubiquitination. Consequently, S7 overexpression increases p53 transactivational activities, induces apoptosis, and inhibits cell proliferation. The identification of S7 as a novel MDM2-interacting partner contributes to elucidation of the complex regulation of the MDM2-p53 interaction and has implications in cancer prevention and therapy.

  7. Mice with a Mutation in the Mdm2 Gene That Interferes with MDM2/Ribosomal Protein Binding Develop a Defect in Erythropoiesis

    Science.gov (United States)

    Kamio, Takuya; Gu, Bai-wei; Olson, Timothy S.; Zhang, Yanping; Mason, Philip J.; Bessler, Monica

    2016-01-01

    MDM2, an E3 ubiquitin ligase, is an important negative regulator of tumor suppressor p53. In turn the Mdm2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited when certain ribosomal proteins, including RPL5 and RPL11, bind to MDM2. This inhibition, and the resulting increase in p53 levels has been proposed to be responsible for the red cell aplasia seen in Diamond-Blackfan anemia (DBA) and in 5q- myelodysplastic syndrome (MDS). DBA and 5q- MDS are associated with inherited (DBA) or acquired (5q- MDS) haploinsufficiency of ribosomal proteins. A mutation in Mdm2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation (Mdm2C305F), retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we noticed that Mdm2C305F homozygous mice had altered hematopoiesis. These mice developed a mild macrocytic anemia with reticulocytosis. In the bone marrow (BM), these mice showed a significant decrease in Ter119hi cells compared to wild type (WT) littermates, while no decrease in the number of mature erythroid cells (Ter119hiCD71low) was found in the spleen, which showed compensated bone marrow hematopoiesis. In methylcellulose cultures, BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls (p < 0.01). This erythropoietic defect was abrogated by concomitant p53 deficiency (Trp53ko/ko). Further investigation revealed that in Mdm2C305F animals, there was a decrease in Lin-Sca-1+c-Kit+ (LSK) cells, accompanied by significant decreases in multipotent progenitor (MPP) cells (p < 0.01). Competitive BM repopulation experiments showed

  8. Mice with a Mutation in the Mdm2 Gene That Interferes with MDM2/Ribosomal Protein Binding Develop a Defect in Erythropoiesis.

    Directory of Open Access Journals (Sweden)

    Takuya Kamio

    Full Text Available MDM2, an E3 ubiquitin ligase, is an important negative regulator of tumor suppressor p53. In turn the Mdm2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited when certain ribosomal proteins, including RPL5 and RPL11, bind to MDM2. This inhibition, and the resulting increase in p53 levels has been proposed to be responsible for the red cell aplasia seen in Diamond-Blackfan anemia (DBA and in 5q- myelodysplastic syndrome (MDS. DBA and 5q- MDS are associated with inherited (DBA or acquired (5q- MDS haploinsufficiency of ribosomal proteins. A mutation in Mdm2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation (Mdm2C305F, retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we noticed that Mdm2C305F homozygous mice had altered hematopoiesis. These mice developed a mild macrocytic anemia with reticulocytosis. In the bone marrow (BM, these mice showed a significant decrease in Ter119hi cells compared to wild type (WT littermates, while no decrease in the number of mature erythroid cells (Ter119hiCD71low was found in the spleen, which showed compensated bone marrow hematopoiesis. In methylcellulose cultures, BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls (p < 0.01. This erythropoietic defect was abrogated by concomitant p53 deficiency (Trp53ko/ko. Further investigation revealed that in Mdm2C305F animals, there was a decrease in Lin-Sca-1+c-Kit+ (LSK cells, accompanied by significant decreases in multipotent progenitor (MPP cells (p < 0.01. Competitive BM repopulation experiments

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

    Directory of Open Access Journals (Sweden)

    Chin-Yu Lai

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

  10. Association between MDM2-SNP309 and hepatocellular carcinoma in Taiwanese population

    Institute of Scientific and Technical Information of China (English)

    Jyh-Der Leu; I-Feng Lin; Ying-Fang Sun; Su-Mei Chen; Chih-Chao Liu; Yi-Jang Lee

    2009-01-01

    AIM: To investigate the risk association and compare the onset age of hepatocellular carcinoma (HCC)patients in Taiwan with different genotypes of MDM2-SNP309.METHODS: We analyzed MDM2-SNP309 genotypes from 58 patients with HCC and 138 cancer-free healthy controls consecutively. Genotyping of MDM2-SNP309 was conducted by restriction fragment length polymorphism assay.RESULTS: The proportion of homozygous MDM2-SNP309 genotype (G/G) in cases and cancer-free healthy controls was similar (17.2% vs 16.7%). Multivariate analysis showed that the risk of G/G genotype of MDM2-SNP309 vs wild-type T/T genotype in patients with HCC was not significant (OR = 1.265, 95%CI = 0.074-21.77) after adjustment for sex, hepatitis B or C virus infection, age, and cardiovascular disease/diabetes. Nevertheless, there was a trend that GG genotype of MDM2-SNP309 might increase the risk in HCC patients infected with hepatitis virus (OR = 2.568,95% CI = 0.054-121.69). Besides, the homozygous MDM2-SNP309 genotype did not exhibit a significantly earlier age of onset for HCC.CONCLUSION: Current data suggest that the association between MDM2-SNP309 GG genotype and HCC is not significant, while the risk may be enhanced in patients infected by hepatitis virus in Taiwan.

  11. Scaffold hopping via ANCHOR.QUERY : beta-lactams as potent p53-MDM2 antagonists

    NARCIS (Netherlands)

    Shaabani, S.; Neochoritis, C. G.; Twarda-Clapa, Aleksandra; Musielak, Bogdan; Holak, Tad A.; Domling, A.

    2017-01-01

    Using the pharmacophore-based virtual screening platform ANCHOR.QUERY, we morphed our recently described Ugi-4CR scaffold towards a beta-lactam scaffold with potent p53-MDM2 antagonizing activities. 2D-HSQC and FP measurements confirm potent MDM2 binding. Molecular modeling studies were used to unde

  12. MDM2 antagonist Nutlin-3a potentiates antitumour activity of cytotoxic drugs in sarcoma cell lines

    Directory of Open Access Journals (Sweden)

    Lothe Ragnhild A

    2011-05-01

    Full Text Available Abstract Background Frequent failure and severe side effects of current sarcoma therapy warrants new therapeutic approaches. The small-molecule MDM2 antagonist Nutlin-3a activates the p53 pathway and efficiently induces apoptosis in tumours with amplified MDM2 gene and overexpression of MDM2 protein. However, the majority of human sarcomas have normal level of MDM2 and the therapeutic potential of MDM2 antagonists in this group is still unclear. We have investigated if Nutlin-3a could be employed to augment the response to traditional therapy and/or reduce the genotoxic burden of chemotherapy. Methods A panel of sarcoma cell lines with different TP53 and MDM2 status were treated with Nutlin-3a combined with Doxorubicin, Methotrexate or Cisplatin, and their combination index determined. Results Clear synergism was observed when Doxorubicin and Nutlin-3a were combined in cell lines with wild-type TP53 and amplified MDM2, or with Methotrexate in both MDM2 normal and amplified sarcoma cell lines, allowing for up to tenfold reduction of cytotoxic drug dose. Interestingly, Nutlin-3a seemed to potentiate the effect of classical drugs as Doxorubicin and Cisplatin in cell lines with mutated TP53, but inhibited the effect of Methotrexate. Conclusion The use of Nutlin in combination with classical sarcoma chemotherapy shows promising preclinical potential, but since clear biomarkers are still lacking, clinical trials should be followed up with detailed tumour profiling.

  13. Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity*

    Science.gov (United States)

    Ranaweera, Ruchira S.; Yang, Xiaolu

    2013-01-01

    The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes. PMID:23671280

  14. Mouse modeling of the MDM2/MDMX-p53 signaling axis.

    Science.gov (United States)

    Tackmann, Nicole R; Zhang, Yanping

    2017-01-17

    It is evident that p53 activity is critical for tumor prevention and stress response through its transcriptional activation of genes affecting cellular senescence, apoptosis, cellular metabolism, and DNA repair. The regulation of p53 is highly complex, and MDM2 and MDMX are thought to be critical for deciding the fate of p53, both through inhibitory binding and posttranslational modification. Many mouse models have been generated to study the regulation of p53 in vivo, and they have altered our interpretations of how p53 is regulated by MDM2 and MDMX. Although MDM2 is absolutely required for p53 regulation, certain functions are dispensable under unstressed conditions, including the ability of MDM2 to degrade p53. MDMX, on the other hand, may only be required in select situations, like embryogenesis. These models have also clarified how cellular stress signals modify the p53-inhibiting activities of MDM2 and MDMX in vivo It is clear that more work will need to be performed to further understand the contexts for each of these signals and the requirements of various MDM2 and MDMX functions. Here, we will discuss what we have learned from mouse modeling of MDM2 and MDMX and underscore the ways in which these models could inform future therapies.

  15. Systems biology analysis reveals role of MDM2 in diabetic nephropathy

    Science.gov (United States)

    Saito, Rintaro; Rocanin-Arjo, Anaïs; You, Young-Hyun; Darshi, Manjula; Van Espen, Benjamin; Miyamoto, Satoshi; Pham, Jessica; Pu, Minya; Romoli, Simone; Natarajan, Loki; Ju, Wenjun; Kretzler, Matthias; Nelson, Robert; Ono, Keiichiro; Thomasova, Dana; Mulay, Shrikant R.; Ideker, Trey; D’Agati, Vivette; Beyret, Ergin; Belmonte, Juan Carlos Izpisua; Anders, Hans Joachim

    2016-01-01

    To derive new insights in diabetic complications, we integrated publicly available human protein-protein interaction (PPI) networks with global metabolic networks using metabolomic data from patients with diabetic nephropathy. We focused on the participating proteins in the network that were computationally predicted to connect the urine metabolites. MDM2 had the highest significant number of PPI connections. As validation, significant downregulation of MDM2 gene expression was found in both glomerular and tubulointerstitial compartments of kidney biopsy tissue from 2 independent cohorts of patients with diabetic nephropathy. In diabetic mice, chemical inhibition of MDM2 with Nutlin-3a led to reduction in the number of podocytes, increased blood urea nitrogen, and increased mortality. Addition of Nutlin-3a decreased WT1+ cells in embryonic kidneys. Both podocyte- and tubule-specific MDM2-knockout mice exhibited severe glomerular and tubular dysfunction, respectively. Interestingly, the only 2 metabolites that were reduced in both podocyte and tubule-specific MDM2-knockout mice were 3-methylcrotonylglycine and uracil, both of which were also reduced in human diabetic kidney disease. Thus, our bioinformatics tool combined with multi-omics studies identified an important functional role for MDM2 in glomeruli and tubules of the diabetic nephropathic kidney and links MDM2 to a reduction in 2 key metabolite biomarkers. PMID:27777973

  16. Platycodin D, a metabolite of Platycodin grandiflorum, inhibits highly metastatic MDA-MB-231 breast cancer growth in vitro and in vivo by targeting the MDM2 oncogene.

    Science.gov (United States)

    Kong, Ya; Lu, Zong-Liang; Wang, Jia-Jia; Zhou, Rui; Guo, Jing; Liu, Jie; Sun, Hai-Lan; Wang, He; Song, Wei; Yang, Jian; Xu, Hong-Xia

    2016-09-01

    The objective of the present study was to explore the in vitro and in vivo anticancer effects of Platycodin D (PD), derived from Platycodin grandiflorum, on highly metastatic MDA-MB-231 breast cancer cells. Using the MTT assay, we found that PD inhibited MDA-MB-231 cell growth in a concentration-dependent manner, with an IC50 value of 7.77±1.86 µM. Further studies showed that PD had anti-proliferative effects and induced cell cycle arrest in the G0/G1 phase. To explore the detailed mechanism(s) by which PD suppressed MDA-MB-231 cell growth, western blot analyses were used to detect the expression levels of proteins related to cell proliferation and survival. The data showed that PD decreased the expression of proteins related to the G0/G1 phases, downregulated the protein expression of MDM2, MDMX, and mutant p53, and increased the expression levels of p21 and p27 in vitro. We verified the effects of PD on the expression of MDM2, MDMX, mutant p53, p21 and p27 using a pcDNA3-Flag-MDM2 plasmid and MDM2 siRNA transfection, and found that PD inhibited MDA-MB-231 cell viability by targeting MDM2 and mutant p53. Compared with the corresponding parental cells, the cells with siRNA-MDM2 transfection had a greater decrease in cell viability and proliferation, while those with pcDNA3-MDM2 plasmid transfection did not show any increase in the effects of PD. We also established a MDA-MB-231 xenograft model in BALB/c nude mice, and found that PD significantly inhibited the growth of MDA-MB-231 xenograft tumors in these mice. The expression levels of various proteins in the tumor tissue exhibited changes similar to those observed in vitro. These findings indicate that PD exerted in vitro and in vivo anticancer effects against MDA-MB-231 breast cancer cells, that PD is a potential MDM2/MDMX inhibitor, and that the anticancer effects of PD were likely associated with its inhibition of these proteins. Our observations help to identify a mechanism by which PD functions as

  17. Novel small molecule inhibitors of the p53-MDM2 /MDM4 interaction for induction of apoptosis in AML

    NARCIS (Netherlands)

    Köhler, L.M.; Beck, B.; Huang, H.; Holak, T.; Dömling, A.; Subklewe, M.

    2012-01-01

    A promising new approach in cancer treatment is the inhibition of murine double minute 2 and 4 (MDM2/MDM4) which is a negative regulator of p53. In neoplasia with unmutated p53 the inactivation of MDM2/4 promotes apoptosis and growth arrest. A derivative of the first described p53/MDM2 antagonist Nu

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

    Directory of Open Access Journals (Sweden)

    Reem Malek

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

  19. MdmX Protects p53 from Mdm2-Mediated Degradation

    OpenAIRE

    2000-01-01

    The p53 tumor suppressor protein is stabilized in response to cellular stress, resulting in activation of genes responsible for either cell cycle arrest or apoptosis. The cellular pathway for releasing normal cells from p53-dependent cell cycle arrest involves the Mdm2 protein. Recently, a p53-binding protein with homology to Mdm2 was identified and called MdmX. Like Mdm2, MdmX is able to bind p53 and inhibit p53 transactivation; however, the ability of MdmX to degrade p53 has yet to be exami...

  20. Regulation of transcription functions of the p53 tumor suppressor by the mdm-2 oncogene.

    OpenAIRE

    1995-01-01

    BACKGROUND: Mdm-2, a zinc finger protein, negatively regulates the p53 tumor suppressor gene product by binding to it and preventing transcriptional activation (16). MATERIALS AND METHODS: Assays for p53 mediated transcription, repression and activation by mutant and wild-type p53 proteins were used to measure the ability of mdm-2 to block each activity. RESULTS: Mdm-2 was able to inhibit all three functions of the wild-type and mutant p53 activities; transcriptional activation by the wild-ty...

  1. Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation

    DEFF Research Database (Denmark)

    Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid

    2004-01-01

    and induction of cell death. We have used the osteosarcoma cell line U2OS cells provided with E7 and the cdk2 inhibitor p21 (cip1/waf1) under inducible control, as a model system for the analysis of E7-mediated apoptosis. Our data shows that simultaneous expression of E7 and p21 proteins induces cell death...

  2. Polymorphism of MDM2 promoter 309 (rs 2279744) and the risk of PCOS.

    Science.gov (United States)

    Chan, Ying; Jiang, Hongguo; Yang, Xiaoling; Li, Dongya; Ma, Lan; Luo, Ying; Tang, Wenru

    2016-01-01

    This study aimed at evaluating possible association between MDM2 SNP309 polymorphism (rs 2279744) and polycystic ovary syndrome (PCOS). One hundred and twenty-five women with PCOS and two hundred and fifty women without PCOS were collected from the department of reproductive medicine of college hospital in this case-control study. Peripheral blood samples were collected from all participants and DNA was extracted, MDM2 SNP309 polymorphism (rs 2279744) was determined from the 125 cases and 250 controls. Women were grouped into PCOS (n = 125) group and control group (n = 250). Odds ratios (OR) and 95% confidence intervals (CI) were used to evaluate the association between MDM2 SNP309 polymorphism (rs 2279744) and PCOS. The distribution of T allele was significant higher in PCOS cases than controls. MDM2 SNP 309 T allele is associated with PCOS.

  3. Mdm2 Phosphorylation Regulates Its Stability and Has Contrasting Effects on Oncogene and Radiation-Induced Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Michael I. Carr

    2016-09-01

    Full Text Available ATM phosphorylation of Mdm2-S394 is required for robust p53 stabilization and activation in DNA-damaged cells. We have now utilized Mdm2S394A knockin mice to determine that phosphorylation of Mdm2-S394 regulates p53 activity and the DNA damage response in lymphatic tissues in vivo by modulating Mdm2 stability. Mdm2-S394 phosphorylation delays lymphomagenesis in Eμ-myc transgenic mice, and preventing Mdm2-S394 phosphorylation obviates the need for p53 mutation in Myc-driven tumorigenesis. However, irradiated Mdm2S394A mice also have increased hematopoietic stem and progenitor cell functions, and we observed decreased lymphomagenesis in sub-lethally irradiated Mdm2S394A mice. These findings document contrasting effects of ATM-Mdm2 signaling on p53 tumor suppression and reveal that destabilizing Mdm2 by promoting its phosphorylation by ATM would be effective in treating oncogene-induced malignancies, while inhibiting Mdm2-S394 phosphorylation during radiation exposure or chemotherapy would ameliorate bone marrow failure and prevent the development of secondary hematological malignancies.

  4. Identification of functional DNA variants in the constitutive promoter region of MDM2.

    Science.gov (United States)

    Lalonde, Marie-Eve; Ouimet, Manon; Larivière, Mathieu; Kritikou, Ekaterini A; Sinnett, Daniel

    2012-09-01

    Although mutations in the oncoprotein murine double minute 2 (MDM2) are rare, MDM2 gene overexpression has been observed in several human tumors. Given that even modest changes in MDM2 levels might influence the p53 tumor suppressor signaling pathway, we postulated that sequence variation in the promoter region of MDM2 could lead to disregulated expression and variation in gene dosage. Two promoters have been reported for MDM2; an internal promoter (P2), which is located near the end of intron 1 and is p53-responsive, and an upstream constitutive promoter (P1), which is p53-independent. Both promoter regions contain DNA variants that could influence the expression levels of MDM2, including the well-studied single nucleotide polymorphism (SNP) SNP309, which is located in the promoter P2; i.e., upstream of exon 2. In this report, we screened the promoter P1 for DNA variants and assessed the functional impact of the corresponding SNPs. Using the dbSNP database and genotyping validation in individuals of European descent, we identified three common SNPs (-1494 G > A; indel 40 bp; and -182 C > G). Three major promoter haplotypes were inferred by using these three promoter SNPs together with rs2279744 (SNP309). Following subcloning into a gene reporter system, we found that two of the haplotypes significantly influenced MDM2 promoter activity in a haplotype-specific manner. Site-directed mutagenesis experiments indicated that the 40 bp insertion/deletion variation is causing the observed allelic promoter activity. This study suggests that part of the variability in the MDM2 expression levels could be explained by allelic p53-independent P1 promoter activity.

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

    Science.gov (United States)

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

    2014-02-01

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

  6. Low Prevalence of TP53 Mutations and MDM2 Amplifications in Pediatric Rhabdomyosarcoma

    Directory of Open Access Journals (Sweden)

    Simona Ognjanovic

    2012-01-01

    Full Text Available The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer. The reported prevalence of mutations in rhabdomyosarcoma (RMS varies widely, with recent larger studies suggesting that TP53 mutations in pediatric RMS may be extremely rare. Overexpression of MDM2 also attenuates p53 function. We have performed TP53 mutation/MDM2 amplification analyses in the largest series analyzed thus far, including DNA isolated from 37 alveolar and 38 embryonal RMS tumor samples obtained from the Cooperative Human Tissue Network (CHTN. Available samples were frozen tumor tissues (N=48 and histopathology slides. TP53 mutations in exons 4–9 were analyzed by direct sequencing in all samples, and MDM2 amplification analysis was performed by differential PCR on a subset of 22 samples. We found only one sample (1/75, 1.3% carrying a TP53 mutation at codon 259 (p.D259Y and no MDM2 amplification. Two SNPs in the TP53 pathway, associated with accelerated tumor onset in germline TP53 mutation carriers, (TP53 SNP72 (rs no. 1042522 and MDM2 SNP309 (rs no. 2279744, were not found to confer earlier tumor onset. In conclusion, we confirm the extremely low prevalence of TP53 mutations/MDM2 amplifications in pediatric RMS (1.33% and 0%, respectively. The possible inactivation of p53 function by other mechanisms thus remains to be elucidated.

  7. MDM2 is a novel E3 ligase for HIV-1 Vif

    Directory of Open Access Journals (Sweden)

    Tomonaga Mitsunori

    2009-01-01

    Full Text Available Abstract The human immunodeficiency virus type 1 (HIV-1 Vif plays a crucial role in the viral life cycle by antagonizing a host restriction factor APOBEC3G (A3G. Vif interacts with A3G and induces its polyubiquitination and subsequent degradation via the formation of active ubiquitin ligase (E3 complex with Cullin5-ElonginB/C. Although Vif itself is also ubiquitinated and degraded rapidly in infected cells, precise roles and mechanisms of Vif ubiquitination are largely unknown. Here we report that MDM2, known as an E3 ligase for p53, is a novel E3 ligase for Vif and induces polyubiquitination and degradation of Vif. We also show the mechanisms by which MDM2 only targets Vif, but not A3G that binds to Vif. MDM2 reduces cellular Vif levels and reversely increases A3G levels, because the interaction between MDM2 and Vif precludes A3G from binding to Vif. Furthermore, we demonstrate that MDM2 negatively regulates HIV-1 replication in non-permissive target cells through Vif degradation. These data suggest that MDM2 is a regulator of HIV-1 replication and might be a novel therapeutic target for anti-HIV-1 drug.

  8. MDM2-MDM4 molecular interaction investigated by atomic force spectroscopy and surface plasmon resonance.

    Science.gov (United States)

    Moscetti, Ilaria; Teveroni, Emanuela; Moretti, Fabiola; Bizzarri, Anna Rita; Cannistraro, Salvatore

    Murine double minute 2 (MDM2) and 4 (MDM4) are known as the main negative regulators of p53, a tumor suppressor. They are able to form heterodimers that are much more effective in the downregulation of p53. Therefore, the MDM2-MDM4 complex could be a target for promising therapeutic restoration of p53 function. To this aim, a deeper understanding of the molecular mechanisms underlining the heterodimerization is needed. The kinetic and thermodynamic characterization of the MDM2-MDM4 complex was performed with two complementary approaches: atomic force spectroscopy and surface plasmon resonance. Both techniques revealed an equilibrium dissociation constant (KD ) in the micromolar range for the MDM2-MDM4 heterodimer, similar to related complexes involved in the p53 network. Furthermore, the MDM2-MDM4 complex is characterized by a relatively high free energy, through a single energy barrier, and by a lifetime in the order of tens of seconds. New insights into the MDM2-MDM4 interaction could be highly important for developing innovative anticancer drugs focused on p53 reactivation.

  9. Recombinant human MDM2 oncoprotein shows sequence composition selectivity for binding to both RNA and DNA.

    Science.gov (United States)

    Challen, Christine; Anderson, John J; Chrzanowska-Lightowlers, Zofia M A; Lightowlers, Robert N; Lunec, John

    2012-03-01

    MDM2 is a 90 kDa nucleo-phosphoprotein that binds p53 and other proteins contributing to its oncogenic properties. Its structure includes an amino proximal p53 binding site, a central acidic domain and a carboxy region which incorporates Zinc and Ring Finger domains suggestive of nucleic acid binding or transcription factor function. It has previously been reported that a bacculovirus expressed MDM2 protein binds RNA in a sequence-specific manner through the Ring Finger domain, however, its ability to bind DNA has yet to be examined. We report here that a bacterially expressed human MDM2 protein binds both DNA as well as the previously defined RNA consensus sequence. DNA binding appears selective and involves the carboxy-terminal domain of the molecule. RNA binding is inhibited by an MDM2 specific antibody, which recognises an epitope within the carboxy region of the protein. Selection cloning and sequence analysis of MDM2 DNA binding sequences, unlike RNA binding sequences, revealed no obvious DNA binding consensus sequence, but preferential binding to oligopurine:pyrimidine-rich stretches. Our results suggest that the observed preferential DNA binding may occur through the Zinc Finger or in a charge-charge interaction through the Ring Finger, thereby implying potentially different mechanisms for DNA and RNA MDM2 binding.

  10. World Area Forecast System (WAFS)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The World Area Forecast System (WAFS) is a worldwide system by which world area forecast centers provide aeronautical meteorological en-route forecasts in uniform...

  11. Expression and significance of P53 protein and MDM-2 protein in human gliomas

    Institute of Scientific and Technical Information of China (English)

    WANG An-liu; LIU Zhao-xia; LI Guang; ZHANG Li-wei

    2011-01-01

    Background P53 is one of the most studied tumor suppressors in the cancer research, and over 50% of human tumors carry P53 mutations. MDM-2 is amplified and/or overexpressed in a variety of human tumors of diverse tissue origin. The aim of this study was to examine the expression of P53 protein and MDM-2 protein in gliomas, and to investigate the relationship between the expression of the two proteins and the histopathological grades of glioma. The relationship between MDM-2 protein expression and P53 protein expression was also analyzed.Methods The expression of P53 protein and MDM-2 protein was immunohistochemically detected using monoclonal antibodies in 242 paraffin embedded tissues, including 30 normal brain tissues from patients with craniocerebral injury and 212 tissues from patients with primary glioma (grade Ⅰ-Ⅱ group: 5 cases of grade Ⅰ, 119 cases of grade Ⅱ; and grade Ⅲ-Ⅳ group: 53 cases of grade Ⅲ, and 35 cases of grade Ⅳ).Results The P53 positive rate was significantly higher in the glioma groups than in the control group (P <0.0001). The P53 positive rate was significantly higher in glioma tissues of grade Ⅲ-V than in glioma tissues of grade Ⅰ-Ⅱ group (P=0.001). The MDM-2 positive rate was significantly higher in glioma groups than in the control group (P <0.0001).There was no significant difference in the MDM-2 positive rate between the two glioma groups (P=0.936). The expression of P53 protein was not related to expression of MDM-2 protein (P=0.069)Conclusions Overexpression of P53 protein might be related to the occurrence and progression of glioma.Overexpression of MDM-2 protein may play an important role in glioma tumorigenesis, but may not be involved in glioma progression. The overexpression of MDM-2 protein was an early event in malignant transformation of glioma. MDM-2 may be a key player in glioma in its own right.

  12. The oncoprotein HBXIP modulates the feedback loop of MDM2/p53 to enhance the growth of breast cancer.

    Science.gov (United States)

    Li, Hang; Liu, Qian; Wang, Zhen; Fang, Runping; Shen, Yu; Cai, Xiaoli; Gao, Yuen; Li, Yinghui; Zhang, Xiaodong; Ye, Lihong

    2015-09-11

    MDM2 and p53 form a negative feedback loop, in which p53 as a transcription factor positively regulates MDM2 and MDM2 negatively regulates tumor suppressor p53 through promoting its degradation. However, the mechanism of the feedback loop is poorly understood in cancers. We had reported previously that the oncoprotein hepatitis B X-interacting protein (HBXIP) is a key oncoprotein in the development of cancer. Thus, we supposed that HBXIP might be involved in the event. Here, we observed that the expression levels of HBXIP were positively correlated to those of MDM2 in clinical breast cancer tissues. Interestingly, HBXIP was able to up-regulate MDM2 at the levels of mRNA and protein in MCF-7 breast cancer cells. Mechanically, HBXIP increased the promoter activities of MDM2 through directly binding to p53 in the P2 promoter of MDM2. Strikingly, we identified that the acetyltransferase p300 was recruited by HBXIP to p53 in the promoter of MDM2. Moreover, we validated that HBXIP enhanced the p53 degradation mediated by MDM2. Functionally, the knockdown of HBXIP or/and p300 inhibited the proliferation of breast cancer cells in vitro, and the depletion of MDM2 or overexpression of p53 significantly blocked the HBXIP-promoted growth of breast cancer in vitro and in vivo. Thus, we concluded that highly expressed HBXIP accelerates the MDM2-mediated degradation of p53 in breast cancer through modulating the feedback loop of MDM2/p53, resulting in the fast growth of breast cancer cells. Our findings provide new insights into the mechanism of the acceleration of the MDM2/p53 feedback loop in the development of cancer.

  13. p53 expression and relationship with MDM2 amplification in breast carcinomas.

    Science.gov (United States)

    Buyukpinarbasili, Nur; Gucin, Zuhal; Ersoy, Yeliz Emine; İlbak, Ayca; Kadioglu, Huseyin; Muslumanoglu, Mahmut

    2016-04-01

    Carcinoma of the breast, like other malignancies, is a genetic disease with multiple genetic events leading to the malignant phenotype. p53 mutations are the most common genetic events in human cancer. Inactivation of p53 can be a result of mutation in gene sequence. One of the main structures that regulate p53 stabilization is MDM2. It suppresses p53 transcriptional activation by recognizing transactivation domain of p53. The loss of MDM2 function on p53 regulation results in deprivation of p53 tumor suppressor ability. Single nucleotide polymorphisms (SNP309 T->G exchange) or MDM2 amplification has been proposed to play a role in this issue. In the present study, our aim is to analyze p53 and MDM2 status and investigate their interactions in human sporadic breast carcinoma. The study groups were separated according to their molecular classifications. In each group, histologic type of the tumor, conventional prognostic parameters, p53, and MDM2 interactions were compared statistically. Tumors are divided into 4 subtypes due to estrogen and progesterone receptor status, HER-2, and Ki-67 proliferation index results. According to this classification, 23 cases are in the luminal A, 32 cases are in the luminal B, 15 cases are in the HER-2 positive, and 22 cases are in the triple-negative group, with a total of 92 cases. p53 expression is low in luminal breast carcinomas than HER-2 and triple-negative subtypes. MDM2 amplification frequency was found to be 5.4% in total. MDM2 gene amplification does not have a significant role in breast carcinogenesis, but other possible mechanisms may play a role in its inactivation.

  14. A new subtype of high-grade mandibular osteosarcoma with RASAL1/MDM2 amplification.

    Science.gov (United States)

    Guérin, Maxime; Thariat, Juliette; Ouali, Mounia; Bouvier, Corinne; Decouvelaere, Anne-Valérie; Cassagnau, Elisabeth; Aubert, Sébastien; Lepreux, Sébastien; Coindre, Jean-Michel; Valmary-Degano, Séverine; Larousserie, Frédérique; Meilleroux, Julie; Projetti, Fabrice; Stock, Nathalie; Galant, Christine; Marie, Béatrice; Peyrottes, Isabelle; de Pinieux, Gonzague; Gomez-Brouchet, Anne

    2016-04-01

    In contrast to long bone osteosarcoma, mandibular osteosarcoma is highly heterogeneous and morphologically overlaps with benign tumors, obscuring diagnosis and treatment selection. Molecular characterization is difficult due to the paucity of available specimens of this rare disease. We aimed to characterize the spectrum of mandibular osteosarcoma using immunohistochemistry and molecular techniques (quantitative polymerase chain reaction and sequencing) and compare them with benign fibro-osseous lesions. Forty-nine paraffin-embedded mandible osteosarcoma tissue samples were collected retrospectively and compared with 10 fibrous dysplasia and 15 ossifying fibroma cases. These were analyzed for molecular markers thought to differ between the different diseases and subtypes: MDM2 (murine double-minute type 2) overexpression, GNAS (guanine nucleotide-binding protein/α subunit) mutations, and amplification of MDM2 and/or RASAL1 (RAS protein activator like 1). Five fibroblastic high-grade osteosarcoma subtypes showed MDM2 amplification, including 2 with a microscopic appearance of high-grade osteosarcoma with part low-grade osteosarcoma (differentiated/dedifferentiated osteosarcoma) and MDM2 overexpression. The other 3 contained a coamplification of MDM2 and RASAL1, a signature also described for juvenile ossifying fibroma, with no overexpression of MDM2. These were of the giant cell-rich high-grade osteosarcoma, with areas mimicking juvenile ossifying fibroma (ossifying fibroma-like osteosarcoma). Our results show that some diagnosed high-grade osteosarcomas are differentiated/dedifferentiated osteosarcomas and harbor an overexpression and amplification of MDM2. In addition, juvenile ossifying fibromas can potentially evolve into giant cell-rich high-grade osteosarcomas and are characterized by a RASAL1 amplification (osteosarcoma with juvenile ossifying fibroma-like genotype). Thus, the presence of a RASAL1 amplification in ossifying fibroma may indicate a requirement

  15. Small-molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: implications for therapy.

    Science.gov (United States)

    Tovar, Christian; Rosinski, James; Filipovic, Zoran; Higgins, Brian; Kolinsky, Kenneth; Hilton, Holly; Zhao, Xiaolan; Vu, Binh T; Qing, Weiguo; Packman, Kathryn; Myklebost, Ola; Heimbrook, David C; Vassilev, Lyubomir T

    2006-02-07

    The p53 tumor suppressor retains its wild-type conformation and transcriptional activity in half of all human tumors, and its activation may offer a therapeutic benefit. However, p53 function could be compromised by defective signaling in the p53 pathway. Using a small-molecule MDM2 antagonist, nutlin-3, to probe downstream p53 signaling we find that the cell-cycle arrest function of the p53 pathway is preserved in multiple tumor-derived cell lines expressing wild-type p53, but many have a reduced ability to undergo p53-dependent apoptosis. Gene array analysis revealed attenuated expression of multiple apoptosis-related genes. Cancer cells with mdm2 gene amplification were most sensitive to nutlin-3 in vitro and in vivo, suggesting that MDM2 overexpression may be the only abnormality in the p53 pathway of these cells. Nutlin-3 also showed good efficacy against tumors with normal MDM2 expression, suggesting that many of the patients with wild-type p53 tumors may benefit from antagonists of the p53-MDM2 interaction.

  16. Immunohistochemical detection of p53 and MDM2 expressions in liposarcoma with World health organization classification

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    A Arici

    2013-01-01

    Full Text Available Background: Liposarcomas are among the most common soft tissue sarcomas in adulthood. Aim: The purpose of the study is to perform a histopathologic typing according to World Health Organization (WHO classification of cases diagnosed with liposarcoma and to examine the difference of p53 and MDM2 expressions. Materials and Methods: The haematoxylin-eosin stained sections of 48 subjects enrolled in the study have been evaluated on the basis of the WHO classification for liposarcoma and sections stained using p53 and MDM2. Statistical Analysis Used: Chi-Square test was applied. Results: 20 subjects were diagnosed with well-differentiated liposarcoma (WLS, 16 myxoid liposarcoma (ML, 7 pleomorphic liposarcoma (PL, and 5 de-differentiated liposarcoma (DLS. The number of cases stained positive with MDM2 and p53 were positive correlated in all subjects (P = 0.02. p53 and MDM2 positivity increased in high grade tumors (P = 0.01. Conclusion: p53 and MDM2 immuno-reactivity was found to be potentially useful in liposarcoma diagnosis but a definitive implication would be rather unhealthy due to the small number of cases in our study.

  17. MDM2 inhibitor Nutlin-3a suppresses proliferation and promotes apoptosis in osteosarcoma cells

    Institute of Scientific and Technical Information of China (English)

    Bo Wang; Liming Fang; Hui Zhao; Tong Xiang; Dechun Wang

    2012-01-01

    Restoring p53 activity by inhibiting the interaction between p53 and the mouse double minutes clone 2 (MDM2) offers an attractive approach to cancer therapy.Nutlin-3a is a small-molecule inhibitor that inhibits MDM2 binding to p53 and subsequent p53-dependent DNA damage signaling.In this study,we determined the efficacy of Nutlin-3a in inducing p53-mediated cell death in osteosarcoma (OS) cell lines both in vivo and in vitro.Targeted disruption of the p53-MDM2 interaction by Nutlin-3a stabilizes p53 and selectively activates the p53 pathway only in OS cells with wild-type p53,resulting ina pronounced anti-proliferative and cytotoxic effect due to G1 cell cycle arrest and apoptosis both in vitro and in vivo.p53 dependence of these alternative outcomes of Nutlin-3a treatment was shown by the abrogation of these effects when p53 was knocked-down by small interfering RNA.These data suggest that the disruption of p53-MDM2 interaction by Nutlin-3a might be beneficial for OS patients with MDM2 amplification and wt p53 status.

  18. MDM2 E3 ubiquitin ligase mediates UT-A1 urea transporter ubiquitination and degradation.

    Science.gov (United States)

    Chen, Guangping; Huang, Haidong; Fröhlich, Otto; Yang, Yuan; Klein, Janet D; Price, S Russ; Sands, Jeff M

    2008-11-01

    UT-A1 is the primary urea transporter in the apical plasma membrane responsible for urea reabsorption in the inner medullary collecting duct. Although the physiological function of UT-A1 has been well established, the molecular mechanisms that regulate its activity are less well understood. Analysis of the UT-A1 amino acid sequence revealed a potential MDM2 E3 ubiquitin ligase-binding motif in the large intracellular loop of UT-A1, suggesting that UT-A1 urea transporter protein may be regulated by the ubiquitin-proteasome pathway. Here, we report that UT-A1 is ubiquitinated and degraded by the proteasome but not the lysosome proteolytic pathway. Inhibition of proteasome activity causes UT-A1 cell surface accumulation and concomitantly increases urea transport activity. UT-A1 interacts directly with MDM2; the binding site is located in the NH2-terminal p53-binding region of MDM2. MDM2 mediates UT-A1 ubiquitination both in vivo and in vitro. Overexpression of MDM2 promotes UT-A1 degradation. The mechanism is likely to be physiologically important as UT-A1 ubiquitination was identified in kidney inner medullary tissue. The ubiquitin-proteasome degradation pathway provides an important novel mechanism for UT-A1 regulation.

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

    Directory of Open Access Journals (Sweden)

    Richard Murdoch Montgomery

    2015-07-01

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

  20. Mechanism of p53 downstream effectors p21 and Gadd45 in DNA damage surveillance

    Institute of Scientific and Technical Information of China (English)

    孟祥兵; 董燕; 孙志贤

    1999-01-01

    Both p21WAF1/CIP1) and Gadd45 were activated in a p53-dependent manner in MCF-7 cells after being exposed to ionizing radiation. In order to investigate their roles in DNA damage surveillance, p21as/MCF-7 cells stably transfected by p21 antisense expression plasmid pC-WAF1-AS and Gadd45as/MCF-7 stably transfected by Gadd45 antisense expression plasmid pCMVas45 were established. It was observed that G1 arrest induced by radiation was significantly reduced in Gadd45as/MCF-7 cells as well as in p21as/MCF-7 cells. Repair of radiation damaged report gene greatly reduced in Gadd45as/MCF-7 and p21as/MCF-7 cells. Apoptosis significantly increased in p21as/MCF-7 after exposure to radiation. These results suggest that both p21 and Gadd45 support cellular survival by taking roles in G1 arrest and DNA repair, furthermore, p21 protects cells from death by inhibiting apoptosis after exposure to ionizing radiation.

  1. Expression and significance of p53 and mdm2 in patients with leukoplakia cancer

    Institute of Scientific and Technical Information of China (English)

    Juan-Juan Cui; Xiao-Lan Han; Wei-Min Wang

    2013-01-01

    Objective:To study the relationship of the expressions of p53 and mdm2 in leukoplakia cancer. Methods:RT-PCR was used to detect the mRNA of p53, mdm2 in patients with leukoplakia cancer.The frequencies of p53, mdm2 in peripheral blood were detected by flow cytometric analysis.Results:The expression of p53mRNA in normal oral mucosa, simple oral leukoplakia, no-simple oral leukoplakia and leukoplakia cancer were7.7%,27.3%,33.3%,56.8%, respectively. The frequencies of p53 in normal oral mucosa, simple oral leukoplakia, no-simple oral leukoplakia and leukoplakia cancer were(0.3±0.1)%,(1.6±0.9)%,(1.9±1.1)%,(3.4±1.8)%.The expression of mdm2 mRNA in normal oral mucosa, simple oral leukoplakia, no-simple oral leukoplakia and leukoplakia cancer were0.0%,6.8%,11.1%,37.8%, respectively.The frequencies ofmdm2 in normal oral mucosa, simple oral leukoplakia, no-simple oral leukoplakia and leukoplakia cancer were(0.1±0.1)%,(0.8±0.6)%,(1.2±0.8)%,(1.2±0.8)%.There was a positively correlation between p53 mRNA and mdm2 mRNA.Conclusions:The positive rate of p53 and mdm2 cells in the peripheral blood increases in patients with leukoplakia cancer tissue and has positive correlation with the severity of leukoplakia cancer.

  2. MDM2/MDMX: Master negative regulators for p53 and RB.

    Science.gov (United States)

    Hu, Linshan; Zhang, Haibo; Bergholz, Johann; Sun, Shengnan; Xiao, Zhi-Xiong Jim

    2016-03-01

    MDM2 (mouse double minute 2 homolog) and MDMX (double minute X human homolog, also known as MDM4) are critical negative regulators of tumor protein p53. Our recent work shows that MDMX binds to and promotes degradation of retinoblastoma protein (RB) in an MDM2-dependent manner. In a xenograft tumor growth mouse model, silencing of MDMX results in inhibition of p53-deficient tumor growth, which can be effectively reversed by concomitant RB silencing. Thus, MDMX exerts its oncogenic activity via suppression of RB.

  3. Quantitative assessment of the p53-Mdm2 feedback loop using protein lysate microarrays.

    Science.gov (United States)

    Ramalingam, Sundhar; Honkanen, Peter; Young, Lynn; Shimura, Tsutomu; Austin, John; Steeg, Patricia S; Nishizuka, Satoshi

    2007-07-01

    Mathematical simulations of the p53-Mdm2 feedback loop suggest that both proteins will exhibit impulsive expression characteristics in response to high cellular stress levels. However, little quantitative experimental evaluation has been done, particularly of the phosphorylated forms. To evaluate the mathematical models experimentally, we used lysate microarrays from an isogenic pair of gamma-ray-irradiated cell lysates from HCT116 (p53(+/+) and p53(-/-)). Both p53 and Mdm2 proteins showed expected pulses in the wild type, whereas no pulses were seen in the knockout. Based on experimental observations, we determined model parameters and generated an in silico "knockout," reflecting the experimental data, including phosphorylated proteins.

  4. Ling Zhi-8 mediates p53-dependent growth arrest of lung cancer cells proliferation via the ribosomal protein S7-MDM2-p53 pathway.

    Science.gov (United States)

    Wu, Chien-Ting; Lin, Tung-Yi; Hsu, Hsien-Yeh; Sheu, Fuu; Ho, Chau-Mei; Chen, Edmund I-T

    2011-12-01

    Ling Zhi-8 (LZ-8), an immunomodulatory protein, is derived from and has been cloned from the medicinal mushroom Ganoderma lucidum (Reishi or Ling Zhi); this protein exhibits immunomodulating and antitumor properties. We investigated the effects of recombinant LZ-8 protein (rLZ-8) on the proliferation of A549 human lung cancer cells. Here, we showed that rLZ-8 inhibits cell growth and that this is correlated with increased G(1) arrest. The treatment of A549 cells with rLZ-8 activated p53 and p21 expression, and both the G(1) arrest and the antigrowth effect were found to be p53 dependent. It was further demonstrated that rLZ-8 inhibited tumor growth in mice transplanted with Lewis lung carcinoma cells. Interestingly, rLZ-8 treatment was found to lead to nucleolar stress (or ribosomal stress) as evidenced by inhibition of precursor ribosomal RNA synthesis and reduced polysome formation in A549 cells. These changes resulted in an increasing binding of ribosomal protein S7 to MDM2 and a decreased interaction between MDM2 and p53. Taking these results together, we have identified a novel rLZ-8 antitumor function that positively modulates p53 via ribosomal stress and inhibits lung cancer cell growth in vitro and in vivo. Our current results suggest that rLZ-8 may have potential as a therapeutic intervention for the treatment of cancers that contain wild-type p53 and high expression of MDM2.

  5. Core Binding Factor β Protects HIV, Type 1 Accessory Protein Viral Infectivity Factor from MDM2-mediated Degradation.

    Science.gov (United States)

    Matsui, Yusuke; Shindo, Keisuke; Nagata, Kayoko; Yoshinaga, Noriyoshi; Shirakawa, Kotaro; Kobayashi, Masayuki; Takaori-Kondo, Akifumi

    2016-11-25

    HIV, type 1 overcomes host restriction factor apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins by organizing an E3 ubiquitin ligase complex together with viral infectivity factor (Vif) and a host transcription cofactor core binding factor β (CBFβ). CBFβ is essential for Vif to counteract APOBEC3 by enabling the recruitment of cullin 5 to the complex and increasing the steady-state level of Vif protein; however, the mechanisms by which CBFβ up-regulates Vif protein remains unclear. Because we have reported previously that mouse double minute 2 homolog (MDM2) is an E3 ligase for Vif, we hypothesized that CBFβ might protect Vif from MDM2-mediated degradation. Co-immunoprecipitation analyses showed that Vif mutants that do not bind to CBFβ preferentially interact with MDM2 and that overexpression of CBFβ disrupts the interaction between MDM2 and Vif. Knockdown of CBFβ reduced the steady-state level of Vif in MDM2-proficient cells but not in MDM2-null cells. Cycloheximide chase analyses revealed that Vif E88A/W89A, which does not interact with CBFβ, degraded faster than wild-type Vif in MDM2-proficient cells but not in MDM2-null cells, suggesting that Vif stabilization by CBFβ is mainly caused by impairing MDM2-mediated degradation. We identified Vif R93E as a Vif variant that does not bind to MDM2, and the virus with this substitution mutation was more resistant to APOBEC3G than the parental virus. Combinatory substitution of Vif residues required for CBFβ binding and MDM2 binding showed full recovery of Vif steady-state levels, supporting our hypothesis. Our data provide new insights into the mechanism of Vif augmentation by CBFβ.

  6. The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells*

    Science.gov (United States)

    Duan, Yajian; Ma, Gaoen; Huang, Xionggao; D'Amore, Patricia A.; Zhang, Feng; Lei, Hetian

    2016-01-01

    The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR. PMID:27246850

  7. The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells.

    Science.gov (United States)

    Duan, Yajian; Ma, Gaoen; Huang, Xionggao; D'Amore, Patricia A; Zhang, Feng; Lei, Hetian

    2016-07-29

    The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. MDM2/p53通路与心血管疾病的研究进展%The Research Progress of Mdm2/p53 Pathway and Cardiovascular Disease

    Institute of Scientific and Technical Information of China (English)

    羊暑艳

    2011-01-01

    鼠双微基因2(MDM2)是一原癌基因,因mRNA的不同剪切可形成多种不同相对分子质量的变异体,如p57、p64、p85、p90等.在正常细胞中,MDM2和野生型p53有着精细的平衡,其相互调节形成负反馈回路:p53诱导MDM2表达,MDM2与p53结合形成p53-MDM2复合物,使p53泛素化而被蛋白酶降解.MDM2-p53反馈体系与其他信号转导途径一起形成调控网络,参与细胞生长抑制、凋亡、细胞周期调控等各种生物进程.现对MDM2/p53途径在心血管疾病中的研究进展进行综述.%Murine double minute 2( Mdm2 )gene is an oncogene,due to the different cuttings of mRNA, a variety of variants with different molecular weight can be formed, such as p57, p64, p85, p90 and so on. In normal cells, Mdm2 and wild - type p53 has a delicate balance. The Interaction between them form a negative feedback loop:p53 induces the expression of Mdm2,the combination of Mdm2 and p53 forms a compound named p53-Mdm2. It makes p53 to be ubiquitinationed and then degraded by protease. Regulatory networks formed by Mdm2-p53 feedback system and other signaling pathways involved in cell growth inhibition,apop-tosis, cell cycle regulation and other biological processes. Here is to review the progress of the Mdm2/p53 pathways in cardiovascular disease research.

  9. mdm-2扩-增与胃癌及淋巴转移的关系%The relationship between mdm-2 amplification and gastric cancer,its lymph node metastasis

    Institute of Scientific and Technical Information of China (English)

    时军; 杨书文; 陈道达; 吕有勇

    2000-01-01

    目的探索mdm-2扩增与胃癌及其淋巴转移的关系.方法用DC-PCR技术定量分析32例胃癌及转移灶中mdm-2扩增.结果 mdm-2在转移淋巴结中的扩增频率(57.1%)高于胃原发癌中的扩增频率(37.5%),3例淋巴管内有瘤栓、淋巴结转移阴性的原发癌灶中发现mdm-2扩增.结论 mdm-2扩增与胃癌的淋巴转移关系较密切,它可能成为监测胃癌淋巴转移的潜在性分子标志物.

  10. MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3.

    Science.gov (United States)

    Hallenborg, P; Siersbæk, M; Barrio-Hernandez, I; Nielsen, R; Kristiansen, K; Mandrup, S; Grøntved, L; Blagoev, B

    2016-06-30

    The ubiquitin ligase MDM2 is best known for balancing the activity of the tumor suppressor p53. We have previously shown that MDM2 is vital for adipocyte conversion through controlling Cebpd expression in a p53-independent manner. Here, we show that the proadipogenic effect of MDM2 relies on activation of the STAT family of transcription factors. Their activation was required for the cAMP-mediated induction of target genes. Interestingly, rather than influencing all cAMP-stimulated genes, inhibition of the kinases directly responsible for STAT activation, namely JAKs, or ablation of MDM2, each resulted in abolished induction of a subset of cAMP-stimulated genes, with Cebpd being among the most affected. Moreover, STATs were able to interact with the transcriptional cofactors CRTC2 and CRTC3, hitherto only reported to associate with the cAMP-responsive transcription factor CREB. Last but not least, the binding of CRTC2 to a transcriptional enhancer that interacts with the Cebpd promoter was dramatically decreased upon JAK inhibition. Our data reveal the existence of an unusual functional interplay between STATs and CREB at the onset of adipogenesis through shared CRTC cofactors.

  11. Regulation of kidney development by the Mdm2/Mdm4-p53 axis.

    Science.gov (United States)

    El-Dahr, Samir; Hilliard, Sylvia; Saifudeen, Zubaida

    2017-01-17

    While p53 activity is required for tumor suppression, unconstrained p53 activity on the other hand is detrimental to the organism, resulting in inappropriate cellular death or proliferation defects. Unimpeded p53 activity is lethal in the developing embryo, underlining the need for maintaining a tight control on p53 activity during this period. The critical role of the negative regulators of p53, Mdm2 and Mdm4, in vertebrate development came to light by fatal disruption of embryogenesis that was observed with Mdm2 and Mdm4 gene deletions in mice. Embryonic lethality was rescued only by superimposing p53 removal. Here we summarize the contribution of the Mdm2/Mdm4-p53 axis that occurs at multiple steps of kidney development. Conditional, cell type-specific deletions reveal distinct functions of these proteins in renal morphogenesis. The severe impact on the renal phenotype from targeted gene deletions underscores the critical role played by the Mdm2/Mdm4-p53 nexus on nephrogenesis, and emphasizes the need to monitor patients with aberrations in this pathway for kidney function defects and associated cardiovascular dysfunction.

  12. Outside the p53 RING: Transcription Regulation by Chromatin-Bound MDM2.

    Science.gov (United States)

    Jain, Abhinav K; Barton, Michelle C

    2016-06-16

    Evidence mounts, via two studies published in Molecular Cell (Riscal et al., 2016; Wienken et al., 2016), that chromatin-bound MDM2 impacts pluripotency and metabolism to promote survival and proliferation of cancer cells, independently of p53 degradation.

  13. Overexpression of SKI oncoprotein leads to p53 degradation through regulation of MDM2 protein sumoylation.

    Science.gov (United States)

    Ding, Boxiao; Sun, Yin; Huang, Jiaoti

    2012-04-27

    Protooncogene Ski was identified based on its ability to transform avian fibroblasts in vitro. In support of its oncogenic activity, SKI was found to be overexpressed in a variety of human cancers, although the exact molecular mechanism(s) responsible for its oncogenic activity is not fully understood. We found that SKI can negatively regulate p53 by decreasing its level through up-regulation of MDM2 activity, which is mediated by the ability of SKI to enhance sumoylation of MDM2. This stimulation of MDM2 sumoylation is accomplished through a direct interaction of SKI with SUMO-conjugating enzyme E2, Ubc9, resulting in enhanced thioester bond formation and mono-sumoylation of Ubc9. A mutant SKI defective in transformation fails to increase p53 ubiquitination and is unable to increase MDM2 levels and to increase mono-sumoylation of Ubc9, suggesting that the ability of SKI to enhance Ubc9 activity is essential for its transforming function. These results established a detailed molecular mechanism that underlies the ability of SKI to cause cellular transformation while unraveling a novel connection between sumoylation and tumorigenesis, providing potential new therapeutic targets for cancer.

  14. Influence of MDM2 polymorphisms on squamous cell carcinoma susceptibility: a meta-analysis

    Science.gov (United States)

    Yu, Huanxin; Li, Haiyan; Zhang, Jinling; Liu, Gang

    2016-01-01

    Purpose Controversial associations between single-nucleotide polymorphisms (rs2279744, rs937283, rs3730485) of the MDM2 gene and the etiology of squamous cell carcinomas (SCCs) have been reported. This merits further comprehensive assessment. Materials and methods We systematically reviewed the available data and conducted an updated meta-analysis to evaluate the genetic effect of MDM2 polymorphisms in SCC susceptibility, using Stata/SE 12.0 software. Results After screening, 7,987 SCC cases and 12,954 controls from 26 eligible case–control studies were enrolled. Overall, compared with the control group, a significantly increased SCC risk was observed for the MDM2 rs2279744 polymorphism in the Asian population (test of association: odds ratio [OR] 1.12, P=0.027 for G vs T; OR 1.26, P=0.016 for GG vs TT; OR 1.25, P0.05). Conclusion Our results highlight a positive association between the GG genotype of MDM2 rs2279744 polymorphism and an increased risk of esophageal SCC in the Asian population, which needs to be clarified by more large-scale studies.

  15. TP53 and MDM2 gene polymorphisms and risk of hepatocellular carcinoma among Italian patients

    Directory of Open Access Journals (Sweden)

    Buonaguro Franco M

    2011-08-01

    Full Text Available Abstract Background Single-nucleotide polymorphisms within TP53 gene (codon 72 exon 4, rs1042522, encoding either arginine or proline and MDM2 promoter (SNP309; rs2279744, have been independently associated with increased risk of several cancer types. Few studies have analysed the role of these polymorphisms in the development of hepatocellular carcinoma. Methods Genotype distribution of TP53 codon 72 and MDM2 SNP309 in 61 viral hepatitis-related hepatocellular carcinoma cases and 122 blood samples (healthy controls from Italian subjects were determined by PCR and restriction fragment length polymorphism (RFLP. Results Frequencies of TP53 codon 72 alleles were not significantly different between cases and controls. A significant increase of MDM2 SNP309 G/G and T/G genotypes were observed among hepatocellular carcinoma cases (Odds Ratio, OR = 3.56, 95% Confidence Limits, 95% CI = 1.3-9.7; and OR = 2.82, 95% CI = 1.3-6.4, respectively. Conclusions These results highlight a significant role of MDM2 SNP309 G allele as a susceptibility gene for the development of viral hepatitis-related hepatocellular carcinoma among Italian subjects.

  16. A fluorescent probe for imaging p53-MDM2 protein-protein interaction.

    Science.gov (United States)

    Liu, Zhenzhen; Miao, Zhenyuan; Li, Jin; Fang, Kun; Zhuang, Chunlin; Du, Lupei; Sheng, Chunquan; Li, Minyong

    2015-04-01

    In this article, we describe a no-wash small-molecule fluorescent probe for detecting and imaging p53-MDM2 protein-protein interaction based on an environment-sensitive fluorescent turn-on mechanism. After extensive biological examination, this probe L1 exhibited practical activity and selectivity in vitro and in cellulo.

  17. Combined targeting of MDM2 and CDK4 is synergistic in dedifferentiated liposarcomas.

    Science.gov (United States)

    Laroche-Clary, Audrey; Chaire, Vanessa; Algeo, Marie-Paule; Derieppe, Marie-Alix; Loarer, François L; Italiano, Antoine

    2017-06-19

    MDM2 and CDK4 are frequently co-amplified in well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS). We aimed to determine whether combined MDM2/CDK4 targeting is associated with higher antitumour activity than a single agent in preclinical models of DDLPS. DDLPS cells were exposed to RG7388 (MDM2 antagonist) and palbociclib (CDK4 inhibitor), and apoptosis and signalling/survival pathway perturbations were monitored by flow cytometry and Western blotting. Xenograft mouse models were used to assess tumour growth and survival. Treatment efficacy was assessed by Western blotting, histopathology and tumour volume. RG7388 and palbociclib together exerted a greater antitumour effect than either drug alone, with significant differences in cell viability after a 72-h treatment with RG7388 and/or palbociclib. The combination treatment significantly increased apoptosis compared to the single agents. We then analysed the in vivo antitumour activity of RG7388 and palbociclib in a xenograft model of DDLPS. The combination regimen reduced the tumour growth rate compared with a single agent alone and significantly increased the median progression-free survival. Our results provide a strong rationale for evaluating the therapeutic potential of CDK4 inhibitors as potentiators of MDM2 antagonists in DDLPS and justify clinical trials in this setting.

  18. Study of MDM2 and SUMO-1 expression in actinic cheilitis and lip cancer.

    Science.gov (United States)

    Oliveira Alves, Mônica Ghislaine; da Mota Delgado, Adriana; Balducci, Ivan; Carvalho, Yasmin Rodarte; Cavalcante, Ana Sueli Rodrigues; Almeida, Janete Dias

    2014-11-01

    Actinic cheilitis exhibits a potential of malignant transformation in 10-20 % of cases. The objective of this study was to compare the expression of MDM2 and SUMO-1 proteins between actinic cheilitis (AC) and squamous cell carcinoma (SCC) of the lip. The sample consisted of lower lip mucosa specimens obtained from cases with a clinical and histopathological diagnosis of AC (n = 26) and SCC (n = 25) and specimens of labial semi-mucosa (n = 15) without clinical alterations or inflammation. The tissue samples were stained with hematoxylin-eosin and anti-MDM2 and anti-SUMO-1 antibodies. Data were analyzed by the Kruskal-Wallis and Dunn's tests (5 %). The median expression of MDM2 (kW = 36.8565; df = 3-1 = 2; p = 0.0001) and SUMO-1 (kW = 32.7080; df = 3-1 = 2; p = 0.0001) was similar in cases of AC and SCC of the lip, but differed significantly from that observed for normal labial semi-mucosa. Despite the limitations of the present study, immunohistochemistry demonstrated the overexpression of important proteins (MDM2 and SUMO-1) related to regulatory mechanisms of apoptosis in AC and SCC of the lip, but further studies are needed.

  19. Chlamydia infection depends on a functional MDM2-p53 axis.

    Science.gov (United States)

    González, Erik; Rother, Marion; Kerr, Markus C; Al-Zeer, Munir A; Abu-Lubad, Mohammad; Kessler, Mirjana; Brinkmann, Volker; Loewer, Alexander; Meyer, Thomas F

    2014-11-13

    Chlamydia, a major human bacterial pathogen, assumes effective strategies to protect infected cells against death-inducing stimuli, thereby ensuring completion of its developmental cycle. Paired with its capacity to cause extensive host DNA damage, this poses a potential risk of malignant transformation, consistent with circumstantial epidemiological evidence. Here we reveal a dramatic depletion of p53, a tumor suppressor deregulated in many cancers, during Chlamydia infection. Using biochemical approaches and live imaging of individual cells, we demonstrate that p53 diminution requires phosphorylation of Murine Double Minute 2 (MDM2; a ubiquitin ligase) and subsequent interaction of phospho-MDM2 with p53 before induced proteasomal degradation. Strikingly, inhibition of the p53-MDM2 interaction is sufficient to disrupt intracellular development of Chlamydia and interferes with the pathogen's anti-apoptotic effect on host cells. This highlights the dependency of the pathogen on a functional MDM2-p53 axis and lends support to a potentially pro-carcinogenic effect of chlamydial infection.

  20. The p53 Tumor Suppressor Protein Does Not Regulate Expression of Its Own Inhibitor, MDM2, Except under Conditions of Stress

    OpenAIRE

    2000-01-01

    MDM2 is an important regulator of the p53 tumor suppressor protein. MDM2 inhibits p53 by binding to it, physically blocking its ability to transactivate gene expression, and stimulating its degradation. In cultured cells, mdm2 expression can be regulated by p53. Hence, mdm2 and p53 can interact to form an autoregulatory loop in which p53 activates expression of its own inhibitor. The p53/MDM2 autoregulatory loop has been elucidated within cultured cells; however, regulation of mdm2 expression...

  1. Rational design and synthesis of 1,5-disubstituted tetrazoles as potent inhibitors of the MDM2-p53 interaction

    NARCIS (Netherlands)

    Surmiak, Ewa; Neochoritis, Constantinos G; Musielak, Bogdan; Twarda-Clapa, Aleksandra; Kurpiewska, Katarzyna; Dubin, Grzegorz; Camacho, Carlos; Holak, Tad A; Dömling, Alexander

    2016-01-01

    Using the computational pharmacophore-based ANCHOR.QUERY platform a new scaffold was discovered. Potent compounds evolved inhibiting the protein-protein interaction p53-MDM2. An extensive SAR study was performed based on our four-point pharmacophore model, yielding derivatives with affinity to MDM2

  2. Rational design and synthesis of 1,5-disubstituted tetrazoles as potent inhibitors of the MDM2-p53 interaction

    NARCIS (Netherlands)

    Surmiak, Ewa; Neochoritis, Constantinos G; Musielak, Bogdan; Twarda-Clapa, Aleksandra; Kurpiewska, Katarzyna; Dubin, Grzegorz; Camacho, Carlos; Holak, Tad A; Dömling, Alexander

    2016-01-01

    Using the computational pharmacophore-based ANCHOR.QUERY platform a new scaffold was discovered. Potent compounds evolved inhibiting the protein-protein interaction p53-MDM2. An extensive SAR study was performed based on our four-point pharmacophore model, yielding derivatives with affinity to MDM2

  3. Overexpression of MDM2 protein in ameloblastomas as compared to adenomatoid odontogenic tumor

    Directory of Open Access Journals (Sweden)

    A Krishna

    2012-01-01

    Full Text Available Background: Recent studies on odontogenic tumors have identified various molecular alterations responsible for their development, and determination of epithelial proliferation is a useful means of investigating the differences in biologic behavior of these tumors. One such specific marker to identify proliferative activity and tumor aggressiveness by immunohistochemistry (IHC is MDM2, 90-95kDa protein. Objective: This immunohistochemical study using MDM2 expression was undertaken to understand better the diverse biological activity of two groups of odontogenic tumors namely ameloblastoma and adenomatoid odontogenic tumor (AOT based on their cell proliferation activity. Materials and Methods: A total of 50 cases, comprising of 36 ameloblastoma samples and 14 AOT samples, were subjected to heat-induced antigen retrieval method using citrate buffer in a pressure cooker. Consequently, the sections were stained with MDM2 monoclonal antibody and visualized using an LSAB+ kit. Results: In ameloblastomas, statistically significant association was seen between plexiform ameloblastomas, follicular ameloblastomas with granular cell changes, desmoplastic and unicystic variants. The predominant nuclear staining by MDM2 revealed overexpression in ameloblastomas as compared to AOT. Conclusion: The MDM2 overexpression noticed in plexiform ameloblastoma, follicular ameloblastoma with granular cell changes and acanthomatous ameloblastoma when compared to simple unicystic and desmoplastic ameloblastoma suggest a relatively enhanced proliferative phenotype of these solid multicystic variants of ameloblastomas. On overall comparison, higher expression was noted in ameloblastomas when compared to AOT. This indicates differences in the aggressive nature between these two groups of odontogenic tumors favoring the perception of a greater aggressive nature of ameloblastomas.

  4. MDM2 gene amplification: a new independent factor of adverse prognosis in non-small cell lung cancer (NSCLC).

    Science.gov (United States)

    Dworakowska, Dorota; Jassem, Ewa; Jassem, Jacek; Peters, Brigitte; Dziadziuszko, Rafał; Zylicz, Maciej; Jakóbkiewicz-Banecka, Joanna; Kobierska-Gulida, Grazyna; Szymanowska, Amelia; Skokowski, Jan; Roessner, Albert; Schneider-Stock, Regine

    2004-03-01

    The prognostic impact of MDM2 amplification in non-small cell lung cancer (NSCLC) remains unknown. In this study, we investigated the occurrence of MDM2 amplification in surgically treated NSCLC patients. Molecular data were correlated with clinicopathological factors and evaluated for their prognostic value. The study group included 116 NSCLC patients who underwent pulmonary resection between 1996 and 1999. MDM2 amplification was assessed by real-time PCR using hybridization probe format on a LightCycler (Roche). The calculated ratio was a MDM2 value normalized to the amplification of the housekeeping gene phenylalaninhydroxylase (PAH). Survival curves were drawn according to the Kaplan-Meier method and compared with the use of the log-rank test. Multivariate analysis was based on Cox regression analysis. MDM2 amplification was found in 24 patients (21%). There was no relationship between MDM2 amplification and clinicopathological factors, such as sex, age and stage of disease, pT, pN, histology and tumor differentiation. Median disease-free survival (DFS) in patients with and without MDM2 amplification was 3 and 31 months, and 5-year DFS 24 and 33%, respectively (log-rank, P = 0.02). Likewise, median overall survival (OS) in patients with and without MDM2 amplification was 9 and 33 months, respectively, and 5-year OS 24 and 39%, respectively (log-rank, P = 0.01). The strong prognostic relevance of MDM2 amplification for both DFS and OS was confirmed in multivariate analysis (P < 0.01 for both comparisons). Our results suggest that MDM2 gene amplification analysis provides additional prognostic information in surgically treated NSCLC patients.

  5. MAGE-A Cancer/Testis Antigens Inhibit MDM2 Ubiquitylation Function and Promote Increased Levels of MDM4.

    Directory of Open Access Journals (Sweden)

    Lynnette Marcar

    Full Text Available Melanoma antigen A (MAGE-A proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i uncouple the ubiquitin ligase and degradation functions of MDM2; (ii act as potent inhibitors of E3 ligase function; and (iii regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically.

  6. MAGE-A Cancer/Testis Antigens Inhibit MDM2 Ubiquitylation Function and Promote Increased Levels of MDM4

    Science.gov (United States)

    Marcar, Lynnette; Ihrig, Bianca; Hourihan, John; Bray, Susan E.; Quinlan, Philip R.; Jordan, Lee B.; Thompson, Alastair M.; Hupp, Ted R.; Meek, David W.

    2015-01-01

    Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i) uncouple the ubiquitin ligase and degradation functions of MDM2; (ii) act as potent inhibitors of E3 ligase function; and (iii) regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically. PMID:26001071

  7. p21Cip1 Protection against Hyperoxia Requires Bcl-XL and Is Uncoupled from Its Ability to Suppress Growth

    OpenAIRE

    2006-01-01

    The cyclin-dependent kinase inhibitor p21Cip1/Waf1/Sdi1 protects the lung against hyperoxia, but the mechanism of protection remains unclear because loss of p21 does not lead to aberrant cell proliferation. Because some members of the Bcl-2 gene family have been implicated in hyperoxia-induced cell death, the current study investigated their expression as well as p21-dependent growth suppression and cytoprotection. Conditional overexpression of full-length p21, its amino-terminal cyclin-bindi...

  8. Mechanisms of MEOX1 and MEOX2 regulation of the cyclin dependent kinase inhibitors p21 and p16 in vascular endothelial cells.

    Science.gov (United States)

    Douville, Josette M; Cheung, David Y C; Herbert, Krista L; Moffatt, Teri; Wigle, Jeffrey T

    2011-01-01

    Senescence, the state of permanent cell cycle arrest, has been associated with endothelial cell dysfunction and atherosclerosis. The cyclin dependent kinase inhibitors p21(CIP1/WAF1) and p16(INK4a) govern the G(1)/S cell cycle checkpoint and are essential for determining whether a cell enters into an arrested state. The homeodomain transcription factor MEOX2 is an important regulator of vascular cell proliferation and is a direct transcriptional activator of both p21(CIP1/WAF1) and p16(INK4a). MEOX1 and MEOX2 have been shown to be partially functionally redundant during development, suggesting that they regulate similar target genes in vivo. We compared the ability of MEOX1 and MEOX2 to activate p21(CIP1/WAF1) and p16(INK4a) expression and induce endothelial cell cycle arrest. Our results demonstrate for the first time that MEOX1 regulates the MEOX2 target genes p21(CIP1/WAF1) and p16(INK4a). In addition, increased expression of either of the MEOX homeodomain transcription factors leads to cell cycle arrest and endothelial cell senescence. Furthermore, we show that the mechanism of transcriptional activation of these cyclin dependent kinase inhibitor genes by MEOX1 and MEOX2 is distinct. MEOX1 and MEOX2 activate p16(INK4a) in a DNA binding dependent manner, whereas they induce p21(CIP1/WAF1) in a DNA binding independent manner.

  9. Mechanisms of MEOX1 and MEOX2 regulation of the cyclin dependent kinase inhibitors p21 and p16 in vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Josette M Douville

    Full Text Available Senescence, the state of permanent cell cycle arrest, has been associated with endothelial cell dysfunction and atherosclerosis. The cyclin dependent kinase inhibitors p21(CIP1/WAF1 and p16(INK4a govern the G(1/S cell cycle checkpoint and are essential for determining whether a cell enters into an arrested state. The homeodomain transcription factor MEOX2 is an important regulator of vascular cell proliferation and is a direct transcriptional activator of both p21(CIP1/WAF1 and p16(INK4a. MEOX1 and MEOX2 have been shown to be partially functionally redundant during development, suggesting that they regulate similar target genes in vivo. We compared the ability of MEOX1 and MEOX2 to activate p21(CIP1/WAF1 and p16(INK4a expression and induce endothelial cell cycle arrest. Our results demonstrate for the first time that MEOX1 regulates the MEOX2 target genes p21(CIP1/WAF1 and p16(INK4a. In addition, increased expression of either of the MEOX homeodomain transcription factors leads to cell cycle arrest and endothelial cell senescence. Furthermore, we show that the mechanism of transcriptional activation of these cyclin dependent kinase inhibitor genes by MEOX1 and MEOX2 is distinct. MEOX1 and MEOX2 activate p16(INK4a in a DNA binding dependent manner, whereas they induce p21(CIP1/WAF1 in a DNA binding independent manner.

  10. p53蛋白和MDM 2蛋白在食管鳞状细胞癌中的表达及意义%Expression of p53 and MDM 2 Protein and their Significance in Esophageal Carcinomas

    Institute of Scientific and Technical Information of China (English)

    赵仲生; 张梅; 徐文娟

    1998-01-01

    为了探讨肿瘤抑制基因p53和癌基因MDM 2在食管鳞状细胞癌中的表达及其意义,采用免疫组织化学链菌素亲生物素蛋白过氧化物酶连接法(SP法),检测了68例食管鳞状细胞癌中p53蛋白和MDM 2蛋白的表达,结果显示p53蛋白和MDM 2蛋白阳性率分别为60.3%(41/68)和42.6%(29/68),p53蛋白阳性率与食管鳞状细胞癌分级呈显著正相关(P<0.01),MDM 2蛋白阳性率与食管鳞状细胞癌分级呈显著负相关(P<0.01),并与食管鳞状细胞癌临床病理分期呈显著负相关(P<0.05).在68例食管鳞状细胞癌中,p53蛋白阳性表达者41例,其中p53蛋白和MDM 2蛋白表达均阳性者12例,MDM 2蛋白表达阴性者29例;在68例食管癌中,MDM 2蛋白阳性表达者29例,其中MDM2与p53蛋白均阳性者12例,p53蛋白表达阴性者17例,两者呈显著负相关(P<0.01),p53蛋白和MDM 2蛋白表达均阴性者10例.可以认为p53蛋白和MDM 2蛋白表达可作为食管鳞状细胞癌病理分级参考指标之一,MDM 2蛋白表达还可作为食管鳞状细胞癌临床病理分期的参考指标之一,并间接证明MDM 2蛋白对p53蛋白表达具有负性调节作用.

  11. LC-MS/MS-based targeted proteomics quantitatively detects the interaction between p53 and MDM2 in breast cancer.

    Science.gov (United States)

    Zhang, Wen; Zhong, Ting; Chen, Yun

    2017-01-30

    In breast cancer, p53 could be functionally compromised by interaction with several proteins. Among those proteins, MDM2 serves as a pivotal negative regulator and counteracts p53 activation. Thus, the ability to quantitatively and accurately monitor the changes in level of p53-MDM2 interaction with disease state can enable an improved understanding of this protein-protein interaction (PPI), provide a better insight into cancer development and allow the emergence of advanced treatments. However, rare studies have evaluated the quantitative extent of PPI including p53-MDM2 interaction so far. In this study, a LC-MS/MS-based targeted proteomics assay was developed and coupled with co-immunoprecipitation (Co-IP) for the quantification of p53-MDM2 complex. A p53 antibody with the epitope residing at 156-214 residues achieved the greatest IP efficiency. 321KPLDGEYFTLQIR333 (p53) and 327ENWLPEDK334 (MDM2) were selected as surrogate peptides in the targeted analysis. Stable isotope-labeled synthetic peptides were used as internal standards. An LOQ (limit of quantification) of 2ng/mL was obtained. Then, the assay was applied to quantitatively detect total p53, total MDM2 and p53-MDM2 in breast cells and tissue samples. Western blotting was performed for a comparison. Finally, a quantitative time-course analysis in MCF-7 cells with the treatment of nutlin-3 as a PPI inhibitor was also monitored.

  12. Protein-peptide molecular docking with large-scale conformational changes: the p53-MDM2 interaction

    Science.gov (United States)

    Ciemny, Maciej Pawel; Debinski, Aleksander; Paczkowska, Marta; Kolinski, Andrzej; Kurcinski, Mateusz; Kmiecik, Sebastian

    2016-12-01

    Protein-peptide interactions are often associated with large-scale conformational changes that are difficult to study either by classical molecular modeling or by experiment. Recently, we have developed the CABS-dock method for flexible protein-peptide docking that enables large-scale rearrangements of the protein chain. In this study, we use CABS-dock to investigate the binding of the p53-MDM2 complex, an element of the cell cycle regulation system crucial for anti-cancer drug design. Experimental data suggest that p53-MDM2 binding is affected by significant rearrangements of a lid region - the N-terminal highly flexible MDM2 fragment; however, the details are not clear. The large size of the highly flexible MDM2 fragments makes p53-MDM2 intractable for exhaustive binding dynamics studies using atomistic models. We performed extensive dynamics simulations using the CABS-dock method, including large-scale structural rearrangements of MDM2 flexible regions. Without a priori knowledge of the p53 peptide structure or its binding site, we obtained near-native models of the p53-MDM2 complex. The simulation results match well the experimental data and provide new insights into the possible role of the lid fragment in p53 binding. The presented case study demonstrates that CABS-dock methodology opens up new opportunities for protein-peptide docking with large-scale changes of the protein receptor structure.

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

    Science.gov (United States)

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

    2017-02-24

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

  14. A systems wide mass spectrometric based linear motif screen to identify dominant in-vivo interacting proteins for the ubiquitin ligase MDM2.

    Science.gov (United States)

    Nicholson, Judith; Scherl, Alex; Way, Luke; Blackburn, Elizabeth A; Walkinshaw, Malcolm D; Ball, Kathryn L; Hupp, Ted R

    2014-06-01

    Linear motifs mediate protein-protein interactions (PPI) that allow expansion of a target protein interactome at a systems level. This study uses a proteomics approach and linear motif sub-stratifications to expand on PPIs of MDM2. MDM2 is a multi-functional protein with over one hundred known binding partners not stratified by hierarchy or function. A new linear motif based on a MDM2 interaction consensus is used to select novel MDM2 interactors based on Nutlin-3 responsiveness in a cell-based proteomics screen. MDM2 binds a subset of peptide motifs corresponding to real proteins with a range of allosteric responses to MDM2 ligands. We validate cyclophilin B as a novel protein with a consensus MDM2 binding motif that is stabilised by Nutlin-3 in vivo, thus identifying one of the few known interactors of MDM2 that is stabilised by Nutlin-3. These data invoke two modes of peptide binding at the MDM2 N-terminus that rely on a consensus core motif to control the equilibrium between MDM2 binding proteins. This approach stratifies MDM2 interacting proteins based on the linear motif feature and provides a new biomarker assay to define clinically relevant Nutlin-3 responsive MDM2 interactors.

  15. Mdm2 is required for survival of hematopoietic stem cells/progenitors via dampening of ROS-induced p53 activity

    Science.gov (United States)

    Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic st...

  16. Novel small molecule inhibitors of MDM2/4-p53 interaction, YH264 and its ethyl ester YH263: Preclinical evaluation

    NARCIS (Netherlands)

    Christner, Susan M.; Clausen, Dana M.; Beumer, Jan H.; Parise, Robert A.; Huang, Yi; Dömling, Alexander S.; Eiseman, Julie L.

    2012-01-01

    Introduction: In p53+/+ cells, expression of MDM2/4 leads to turnover of p53 and inhibition of downstream gene transcription decreasing cell cycle arrest or apoptosis. Prevention of MDM2/4-p53 interaction is a promising therapeutic strategy. Two in-house developed small molecule inhibitors of MDM2/4

  17. MDM2 SNP309, gene-gene interaction, and tumor susceptibility: an updated meta-analysis

    Directory of Open Access Journals (Sweden)

    Wu Wei

    2011-05-01

    Full Text Available Abstract Background The tumor suppressor gene p53 is involved in multiple cellular pathways including apoptosis, transcriptional control, and cell cycle regulation. In the last decade it has been demonstrated that the single nucleotide polymorphism (SNP at codon 72 of the p53 gene is associated with the risk for development of various neoplasms. MDM2 SNP309 is a single nucleotide T to G polymorphism located in the MDM2 gene promoter. From the time that this well-characterized functional polymorphism was identified, a variety of case-control studies have been published that investigate the possible association between MDM2 SNP309 and cancer risk. However, the results of the published studies, as well as the subsequent meta-analyses, remain contradictory. Methods To investigate whether currently published epidemiological studies can clarify the potential interaction between MDM2 SNP309 and the functional genetic variant in p53 codon72 (Arg72Pro and p53 mutation status, we performed a meta-analysis of the risk estimate on 27,813 cases with various tumor types and 30,295 controls. Results The data we reviewed indicated that variant homozygote 309GG and heterozygote 309TG were associated with a significant increased risk of all tumor types (homozygote comparison: odds ratio (OR = 1.25, 95% confidence interval (CI = 1.13-1.37; heterozygote comparison: OR = 1.10, 95% CI = 1.03-1.17. We also found that the combination of GG and Pro/Pro, TG and Pro/Pro, GG and Arg/Arg significantly increased the risk of cancer (OR = 3.38, 95% CI = 1.77-6.47; OR = 1.88, 95% CI = 1.26-2.81; OR = 1.96, 95% CI = 1.01-3.78, respectively. In a stratified analysis by tumor location, we also found a significant increased risk in brain, liver, stomach and uterus cancer (OR = 1.47, 95% CI = 1.06-2.03; OR = 2.24, 95%CI = 1.57-3.18; OR = 1.54, 95%CI = 1.04-2.29; OR = 1.34, 95%CI = 1.07-1.29, respectively. However, no association was seen between MDM2 SNP309 and tumor susceptibility

  18. Rare Aggressive Behavior of MDM2-Amplified Retroperitoneal Dedifferentiated Liposarcoma, with Brain, Lung and Subcutaneous Metastases

    Science.gov (United States)

    Ben Salha, Imen; Zaidi, Shane; Noujaim, Jonathan; Miah, Aisha B.; Fisher, Cyril; Jones, Robin L.; Thway, Khin

    2016-01-01

    Dedifferentiated liposarcoma (DDL) is a histologically pleomorphic sarcoma, traditionally defined as well-differentiated liposarcoma with abrupt transition to high grade, non-lipogenic sarcoma. It can occur as part of recurrent well-differentiated liposarcoma, or may arise de novo. DDL most frequently occurs within the retroperitoneum, and while it is prone to local recurrence, it usually has a lower rate of metastasis than other pleomorphic sarcomas. We describe a case of retroperitoneal dedifferentiated liposarcoma in a 63-year-old male, who showed MDM2 amplification with fluorescence in situ hybridization, which displayed unusually aggressive behavior, with brain, lung and subcutaneous soft tissue metastases. As previous reports of metastatic liposarcoma have largely grouped DDL in with other (genetically and clinically distinct) liposarcoma subtypes, we highlight and discuss the rare occurrence of brain metastasis in MDM2-amplified retroperitoneal liposarcoma.

  19. MDM2 SNP309 contributes to tumor susceptibility: A meta-analysis

    Institute of Scientific and Technical Information of China (English)

    Xiaoman Wo; Dong Han; Haiming Sun; Yang Liu; Xiangning Meng; Jing Bai; Feng Chen

    2011-01-01

    The potentially functional polymorphism,SNP309,in the promoter region of MDM2 gene has been implicated in cancer risk,but individual published studies showed inconclusive results.To obtain a more precise estimate of the association between MDM2 SNP309 and risk of cancer,we performed a meta-analysis of 70 individual studies in 59 publications that included 26,160 cases with different types of tumors and 33,046 controls.Summary odds ratios (OR) and corresponding 95% confidence intervals (CIs) were estimated using fixed- and random-effects models when appropriate.Overall,the variant genotypes were associated with a significantly increased cancer risk for all cancer types in different genetic models (GG vs.TT:OR,1.123; 95% CI,1.056-1.193; GG/GT vs.TT:OR,1.028; 95% CI,1.006-1.050).In the stratified analyses,the increased risk remained for the studies of most types of cancers,Asian populations,and hospital-/population-based studies in different genetic models,whereas significantly decreased risk was found in prostate cancer (GG vs.TT:OR,0.606; 95% CI,0.407-0.903; GG/GT vs.TT:OR,0.748; 95% CI,0.579-0.968).In conclusion,the data of meta-analysis suggests that MDM2 SNP309 is a potential biomarker for cancer risk.

  20. Genetic variants in TP53 and MDM2 associated with male infertility in Chinese population

    Institute of Scientific and Technical Information of China (English)

    Cong Huang; Wei Liu; Gui-Xiang Ji; Ai-Hua Gu; Jian-Hua Qu; Ling Song; Xin-Ru Wang

    2012-01-01

    The TP53,a transcriptional regulator and tumor suppressor,is functionally important in spermatogenesis.MDM2 is a key regulator of the p53 pathway and modulates p53 activity.Both proteins have been functionally linked to germ cell apoptosis,which may affect human infertility,but very little is known on how common polymorphisms in these genes may influence germ cell apoptosis and the risk of male infertility.Thus,this study was designed to test whether three previously described polymorphisms 72Arg>Pro (rs1042522) and the Ex2+ 19C>T (rs2287498) in TP53,and the 5' untranslated region (5' UTR) 309T>G (rs937283) in MDM2,are associated with idiopathic male infertility in a Chinese population.The three polymorphisms were genotyped using OpenArray assay in a hospital-based case-control study,including 580 infertile patients and 580 fertile controls.Our analyses revealed that TP53 Ex2+ 19C>T and MDM2309T>G polymorphisms are associated with mate infertility.Furthermore,we detected a nearly statistically significant additive interaction between TP53 rs2287498 and MDM2 rs937283 for the development of male.infertility (Pinteraction=0.055).In summary,this study found preliminary evidence,demonstrating that genetic variants in genes of the TP53 pathway are risk factors for male infertility.

  1. C-terminal substitution of MDM2 interacting peptides modulates binding affinity by distinctive mechanisms.

    Directory of Open Access Journals (Sweden)

    Christopher J Brown

    Full Text Available The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19-26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD and an equivalent phage optimized peptide (12/1 were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design.

  2. Bcl-2/MDM2 Dual Inhibitors Based on Universal Pyramid-Like α-Helical Mimetics.

    Science.gov (United States)

    Wang, Ziqian; Song, Ting; Feng, Yingang; Guo, Zongwei; Fan, Yudan; Xu, Wenjie; Liu, Lu; Wang, Anhui; Zhang, Zhichao

    2016-04-14

    No α-helical mimetic that exhibits Bcl-2/MDM2 dual inhibition has been rationally designed due to the different helicities of the α-helixes at their binding interfaces. Herein, we extracted a one-turn α-helix-mimicking ortho-triarene unit from o-phenylene foldamers. Linking benzamide substrates with a rotatable C-N bond, we constructed a novel semirigid pyramid-like scaffold that could support its two-turn α-helix mimicry without aromatic stacking interactions and could adopt the different dihedral angles of the key residues of p53 and BH3-only peptides. On the basis of this universal scaffold, a series of substituent groups were installed to capture the key residues of both p53TAD and BimBH3 and balance the differences of the bulks between them. Identified by FP, ITC, and NMR spectroscopy, a compound 6e (zq-1) that directly binds to Mcl-1, Bcl-2, and MDM2 with balanced submicromolar affinities was obtained. Cell-based experiments demonstrated its antitumor ability through Bcl-2/MDM2 dual inhibition simultaneously.

  3. MDM2 Amplification and PI3KCA Mutation in a Case of Sclerosing Rhabdomyosarcoma.

    Science.gov (United States)

    Kikuchi, Ken; Wettach, George R; Ryan, Christopher W; Hung, Arthur; Hooper, Jody E; Beadling, Carol; Warrick, Andrea; Corless, Christopher L; Olson, Susan B; Keller, Charles; Mansoor, Atiya

    2013-01-01

    A rare sclerosing variant of rhabdomyosarcoma characterized by prominent hyalinization and pseudovascular pattern has recently been described as a subtype biologically distinct from embryonal, alveolar, and pleomorphic forms. We present cytogenetic and molecular findings as well as experimental studies of an unusual case of sclerosing rhabdomyosarcoma. The primary lesion arose within the plantar subcutaneous tissue of the left foot of an otherwise healthy 23-year-old male who eventually developed pulmonary nodules despite systemic chemotherapy. Two genetic abnormalities identified in surgical and/or autopsy samples of the tumor were introduced into 10T1/2 murine fibroblasts to determine whether these genetic changes cooperatively facilitated transformation and growth. Cytogenetic analysis revealed a complex abnormal hyperdiploid clone, and MDM2 gene amplification was confirmed by fluorescence in situ hybridization. Cancer gene mutation screening using a combination of multiplexed PCR and mass spectroscopy revealed a PIK3CA exon 20 H1047R mutation in the primary tumor, lung metastasis, and liver metastasis. However, this mutation was not cooperative with MDM2 overexpression in experimental assays for transformation or growth. Nevertheless, MDM2 and PIK3CA are genes worthy of further investigation in patients with sclerosing rhabdomyosarcoma and might be considered in the enrollment of these patients into clinical trials of targeted therapeutics.

  4. MDM2 Amplification and PI3KCA Mutation in a Case of Sclerosing Rhabdomyosarcoma

    Directory of Open Access Journals (Sweden)

    Ken Kikuchi

    2013-01-01

    Full Text Available A rare sclerosing variant of rhabdomyosarcoma characterized by prominent hyalinization and pseudovascular pattern has recently been described as a subtype biologically distinct from embryonal, alveolar, and pleomorphic forms. We present cytogenetic and molecular findings as well as experimental studies of an unusual case of sclerosing rhabdomyosarcoma. The primary lesion arose within the plantar subcutaneous tissue of the left foot of an otherwise healthy 23-year-old male who eventually developed pulmonary nodules despite systemic chemotherapy. Two genetic abnormalities identified in surgical and/or autopsy samples of the tumor were introduced into 10T1/2 murine fibroblasts to determine whether these genetic changes cooperatively facilitated transformation and growth. Cytogenetic analysis revealed a complex abnormal hyperdiploid clone, and MDM2 gene amplification was confirmed by fluorescence in situ hybridization. Cancer gene mutation screening using a combination of multiplexed PCR and mass spectroscopy revealed a PIK3CA exon 20 H1047R mutation in the primary tumor, lung metastasis, and liver metastasis. However, this mutation was not cooperative with MDM2 overexpression in experimental assays for transformation or growth. Nevertheless, MDM2 and PIK3CA are genes worthy of further investigation in patients with sclerosing rhabdomyosarcoma and might be considered in the enrollment of these patients into clinical trials of targeted therapeutics.

  5. 睾丸肿瘤组织中mdm-2基因表达及临床意义%Mdm-2 Gene Expression in Human Testicular Cancer

    Institute of Scientific and Technical Information of China (English)

    徐元虎; 张培华

    2001-01-01

    为了探讨mdm-2基因与睾丸肿瘤发生及预后的关系,应用免疫组化方法检测27例睾丸肿瘤中mdm-2蛋白表达水平,发现有12例为mdm-2蛋白阳性表达,并且与睾丸肿瘤的病理分级和临床分期有关.提示睾丸肿瘤恶性程度与mdm-2蛋白表达具有明显相关性.mdm-2蛋白表达可能成为睾丸肿瘤预后的指标.

  6. MDM-2、bcl-2 和Ki-67在子宫颈癌中的表达及其意义%Expression and Evaluation of MDM-2, bcl-2 and Ki-67 in Cervical Carcinoma

    Institute of Scientific and Technical Information of China (English)

    李宏艳; 张建中

    2008-01-01

    目的 探讨MDM-2、bcl-2和Ki-67在子宫颈癌的发生、发展过程中的作用.方法 采用免疫组化法检测MDM-2、bcl-2和Ki-67在31例子宫颈癌、13例子宫颈非典型性增生、15例正常子宫颈组织中的表达水平.结果 在31例子宫颈癌组织中MDM-2表达阳性11例,bcl-2阳性13例,Ki-67阳性15例.bcl-2、MDM-2和Ki-67三者共同阳性9例(29.03%).对31例子宫颈癌组织中MDM-2与bcl-2共同阳性者、MDM-2与Ki-67共同阳性者及Ki-67与bcl-2共同阳性者进行配对资料χ2检验,P<0.05.子宫颈癌组织中bcl-2、MDM-2和Ki-6阳性表达率彼此间存在着明显的正性相关关系.结论 联合检测MDM-2、bcl-2和Ki-67在子宫颈癌中的表达,显示三者具有明显相关性,与子宫颈癌的发生、发展有关.

  7. Convergent solid-phase and solution approaches in the synthesis of the cysteine-rich Mdm2 RING finger domain.

    Science.gov (United States)

    Vasileiou, Zoe; Barlos, Kostas; Gatos, Dimitrios

    2009-12-01

    The RING finger domain of the Mdm2, located at the C-terminus of the protein, is necessary for regulation of p53, a tumor suppressor protein. The 48-residues long Mdm2 peptide is an important target for studying its interaction with small anticancer drug candidates. For the chemical synthesis of the Mdm2 RING finger domain, the fragment condensation on solid-phase and the fragment condensation in solution were studied. The latter method was performed using either protected or free peptides at the C-terminus as the amino component. Best results were achieved using solution condensation where the N-component was applied with the C-terminal carboxyl group left unprotected. The developed method is well suited for large-scale synthesis of Mdm2 RING finger domain, combining the advantages of both solid-phase and solution synthesis.

  8. P53 and Murine Double Mimute 2 (MDM2) Expression Changes and Significance in Different Types of Endometrial Lesions

    Science.gov (United States)

    Jiang, Zhongyong; Xu, Wanqing; Dan, Gang; Liu, Yuan; Xiong, Jie

    2016-01-01

    Background Endometrial lesions are common in obstetrics and gynecology, including endometrial polyps, uterine adenomyosis, and malignant endometrial adenocarcinoma. Endometrial lesions seriously affect women’s health, fertility, quality of life, and life safety. As a pro-apoptosis gene, p53 is considered to be closely related with human tumors. Murine double mimute 2 (MDM2) is an oncogene that can promote tumor occurrence and development. P53 and MDM2 expression and significance in different types of endometrial lesions have not been fully elucidated. Material/Methods Normal endometrium, endometrial polyps, uterine adenomyosis, and endometrial adenocarcinoma tissue samples were collected. Real-time PCR was used to detect p53 and MDM2 mRNA expression. Immunohistochemical staining and Western blot analysis were applied to test p53 and MDM2 protein expression. Their correlation with clinical staging of endometrial adenocarcinoma was analyzed. Results P53 and MDM2 mRNA and protein expression were significantly elevated in the endometrial polyps group and the endometrial adenocarcinoma group compared with the normal control group (Pendometrial adenocarcinoma compared with endometrial polyps (P0.05). P53 and MDM2 mRNA and protein level showed a positive correlation. Significantly higher expression of p53 or MDM2 was observed in patients with stage III compared to those in patients with stage II. Higher expression was also observed in patients with stage II than in patients with stage I. Conclusions P53 and MDM2 mRNA and protein were elevated in endometrial polyps and endometrial adenocarcinoma and their expressions were correlated with clinical staging of endometrial adenocarcinoma. They can promote cancer occurrence and development, and can be treated to assist diagnosis and provide a reference for treatment. PMID:27924072

  9. MDM2 turnover and expression of ATRX determine the choice between quiescence and senescence in response to CDK4 inhibition.

    Science.gov (United States)

    Kovatcheva, Marta; Liu, David D; Dickson, Mark A; Klein, Mary E; O'Connor, Rachael; Wilder, Fatima O; Socci, Nicholas D; Tap, William D; Schwartz, Gary K; Singer, Samuel; Crago, Aimee M; Koff, Andrew

    2015-04-10

    CDK4 inhibitors (CDK4i) earned Breakthrough Therapy Designation from the FDA last year and are entering phase III clinical trials in several cancers. However, not all tumors respond favorably to these drugs. CDK4 activity is critical for progression through G1 phase and into the mitotic cell cycle. Inhibiting this kinase induces Rb-positive cells to exit the cell cycle into either a quiescent or senescent state. In this report, using well-differentiated and dedifferentiated liposarcoma (WD/DDLS) cell lines, we show that the proteolytic turnover of MDM2 is required for CDK4i-induced senescence. Failure to reduce MDM2 does not prevent CDK4i-induced withdrawal from the cell cycle but the cells remain in a reversible quiescent state. Reducing MDM2 in these cells drives them into the more stable senescent state. CDK4i-induced senescence associated with loss of MDM2 is also observed in some breast cancer, lung cancer and glioma cell lines indicating that this is not limited to WD/DDLS cells in which MDM2 is overexpressed or in cells that contain wild type p53. MDM2 turnover depends on its E3 ligase activity and expression of ATRX. Interestingly, in seven patients the changes in MDM2 expression were correlated with outcome. These insights identify MDM2 and ATRX as new regulators controlling geroconversion, the process by which quiescent cells become senescent, and this insight may be exploited to improve the activity of CDK4i in cancer therapy.

  10. MDM2 Associates with Polycomb Repressor Complex 2 and Enhances Stemness-Promoting Chromatin Modifications Independent of p53

    DEFF Research Database (Denmark)

    Wienken, Magdalena; Dickmanns, Antje; Nemajerova, Alice

    2016-01-01

    The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion...... in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically...... associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell...

  11. HAUSP-nucleolin interaction is regulated by p53-Mdm2 complex in response to DNA damage response.

    Science.gov (United States)

    Lim, Key-Hwan; Park, Jang-Joon; Gu, Bon-Hee; Kim, Jin-Ock; Park, Sang Gyu; Baek, Kwang-Hyun

    2015-08-04

    HAUSP (herpes virus-associated ubiquitin specific protease, known as ubiquitin specific protease 7), one of DUBs, regulates the dynamics of the p53 and Mdm2 network in response to DNA damage by deubiquitinating both p53 and its E3 ubiquitin ligase, Mdm2. Its concerted action increases the level of functional p53 by preventing proteasome-dependent degradation of p53. However, the protein substrates that are targeted by HAUSP to mediate DNA damage responses in the context of the HAUSP-p53-Mdm2 complex are not fully identified. Here, we identified nucleolin as a new substrate for HAUSP by proteomic analysis. Nucleolin has two HAUSP binding sites in its N- and C-terminal regions, and the mutation of HAUSP interacting peptides on nucleolin disrupts their interaction and it leads to the increased level of nucleolin ubiquitination. In addition, HAUSP regulates the stability of nucleolin by removing ubiquitin from nucleolin. Nucleolin exists as a component of the HAUSP-p53-Mdm2 complex, and both Mdm2 and p53 are required for the interaction between HAUSP and nucleolin. Importantly, the irradiation increases the HAUSP-nucleolin interaction, leading to nucleolin stabilization significantly. Taken together, this study reveals a new component of the HAUSP-p53-Mdm2 complex that governs dynamic cellular responses to DNA damage.

  12. Phosphorylation of the Mdm2 oncoprotein by the c-Abl tyrosine kinase regulates p53 tumor suppression and the radiosensitivity of mice.

    Science.gov (United States)

    Carr, Michael I; Roderick, Justine E; Zhang, Hong; Woda, Bruce A; Kelliher, Michelle A; Jones, Stephen N

    2016-12-27

    The p53 tumor suppressor acts as a guardian of the genome by preventing the propagation of DNA damage-induced breaks and mutations to subsequent generations of cells. We have previously shown that phosphorylation of the Mdm2 oncoprotein at Ser394 by the ATM kinase is required for robust p53 stabilization and activation in cells treated with ionizing radiation, and that loss of Mdm2 Ser394 phosphorylation leads to spontaneous tumorigenesis and radioresistance in Mdm2(S394A) mice. Previous in vitro data indicate that the c-Abl kinase phosphorylates Mdm2 at the neighboring residue (Tyr393) in response to DNA damage to regulate p53-dependent apoptosis. In this present study, we have generated an Mdm2 mutant mouse (Mdm2(Y393F)) to determine whether c-Abl phosphorylation of Mdm2 regulates the p53-mediated DNA damage response or p53 tumor suppression in vivo. The Mdm2(Y393F) mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Although apoptosis is unaltered in these mice, they recover more rapidly from radiation-induced bone marrow ablation and are more resistant to whole-body radiation-induced lethality. These data reveal an in vivo role for c-Abl phosphorylation of Mdm2 in regulation of p53 tumor suppression and bone marrow failure. However, c-Abl phosphorylation of Mdm2 Tyr393 appears to play a lesser role in governing Mdm2-p53 signaling than ATM phosphorylation of Mdm2 Ser394. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2(Y393F/S394A) mice and Mdm2(S394A) mice display similar phenotypes.

  13. Expression and clinical significance of Pokemon and mdm2 proteins in breast cancer%Pokemon、mdm2蛋白在乳腺癌中的表达及临床意义

    Institute of Scientific and Technical Information of China (English)

    刘海峰; 曲宏岩

    2008-01-01

    目的 探讨Pokemon和mdm2蛋白与乳腺癌发生、发展的关系及其临床意义.方法 采用免疫组织化学S-P法检测54例乳腺癌及18例正常乳腺组织中Pokemon和mdm2蛋白的表达水平,分析其与乳腺癌临床病理学特征的关系,以及Pokemon与mdm2蛋白的相关性.结果 在乳腺癌和正常乳腺组织中,Pokemon蛋白的阳性表达率分别为74.1%(40/54)、16.7%(3/18);mdm2蛋白的阳性表达率分别为81.5%(44/54)、22.2%(4/18).两组中Pokemon与mdm2蛋白的阳性表达率均有统计学意义(P0.05 ).Pokemon蛋白表达与mdm2蛋白表达呈正相关(rs=0.588 ,P<0.05).结论 Pokemon与mdm2蛋白可能共同参与了乳腺癌的发生、发展和转移.Pokemon有望成为乳腺癌治疗的一个有效的靶基因,从而为乳腺癌基因治疗开辟新的途径.

  14. Aberrant splicing of the DMP1-ARF-MDM2-p53 pathway in cancer.

    Science.gov (United States)

    Inoue, Kazushi; Fry, Elizabeth A

    2016-07-01

    Alternative splicing (AS) of mRNA precursors is a ubiquitous mechanism for generating numerous transcripts with different activities from one genomic locus in mammalian cells. The gene products from a single locus can thus have similar, dominant-negative or even opposing functions. Aberrant AS has been found in cancer to express proteins that promote cell growth, local invasion and metastasis. This review will focus on the aberrant splicing of tumor suppressor/oncogenes that belong to the DMP1-ARF-MDM2-p53 pathway. Our recent study shows that the DMP1 locus generates both tumor-suppressive DMP1α (p53-dependent) and oncogenic DMP1β (p53-independent) splice variants, and the DMP1β/α ratio increases with neoplastic transformation of breast epithelial cells. This process is associated with high DMP1β protein expression and shorter survival of breast cancer (BC) patients. Accumulating pieces of evidence show that ARF is frequently inactivated by aberrant splicing in human cancers, demonstrating its involvement in human malignancies. Splice variants from the MDM2 locus promote cell growth in culture and accelerate tumorigenesis in vivo. Human cancers expressing these splice variants are associated with advanced stage/metastasis, and thus have negative clinical impacts. Although they lack most of the p53-binding domain, their activities are mostly dependent on p53 since they bind to wild-type MDM2. The p53 locus produces splice isoforms that have either favorable (β/γ at the C-terminus) or negative impact (Δ40, Δ133 at the N-terminus) on patients' survival. As the oncogenic AS products from these loci are expressed only in cancer cells, they may eventually become targets for molecular therapies.

  15. The regulation of the p53/MDM2 feedback loop by microRNAs

    OpenAIRE

    2015-01-01

    Tumor suppressor p53 and its signaling pathway play a central role in tumor prevention. The E3 ubiquitin ligase MDM2, which is a direct p53 transcriptional target and also the most critical negative regulator of p53, forms an autoregulatory negative feedback loop with p53 in the cell to tightly regulate the levels and activity of p53. MicroRNAs (miRNAs) are endogenously expressed small non-coding RNAs that play a critical role in the post-translational regulation of gene expression. Recent st...

  16. MDM2 promoter del1518 polymorphism and cancer risk: evidence from 22,931 subjects

    Directory of Open Access Journals (Sweden)

    Hua WF

    2017-07-01

    Full Text Available Wenfeng Hua,1,* Anqi Zhang,2,* Ping Duan,2,* Jinhong Zhu,3 Yuan Zhao,2 Jing He,4 Zhi Zhang1 1Department of Laboratory Medicine and Central Laboratories, Guangdong Second Provincial General Hospital, Guangzhou, Guangdong, 2Department of Obstetrics and Gynecology, The Second Affiliated Hospital & Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 3Molecular Epidemiology Laboratory and Department of Laboratory Medicine, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, 4Department of Pediatric Surgery, Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, Guangdong, People’s Republic of China *These authors contributed equally to this work Abstract: Studies have shown that single-nucleotide polymorphisms in MDM2 gene may play important roles in the development of malignant tumor. The association of del1518 polymorphism (rs3730485 in the MDM2 promoter with cancer susceptibility has been extensively studied; however, the results are contradictory. To quantify the association between this polymorphism and overall cancer risk, we conducted a meta-analysis with 12,905 cases and 10,026 controls from 16 eligible studies retrieved from PubMed, Embase, and Chinese Biomedical (CBM databases. We assessed the strength of the connection using odds ratios (ORs and 95% confidence intervals (CIs. In summary, no significant associations were discovered between the del1518 polymorphism and overall cancer risk (Del/Del vs Ins/Ins: OR =1.01, 95% CI =0.90–1.14; Ins/Del vs Ins/Ins: OR =1.03, 95% CI =0.96–1.12; recessive model: OR =0.98, 95% CI =0.90–1.07; dominant model: OR =1.03, 95% CI =0.94–1.12; and Del vs Ins: OR =1.01, 95% CI =0.94–1.07. In the stratified analysis by source of control, quality score, cancer type, and ethnicity, no significant associations were found. Despite some limitations, the current meta-analysis provides solid

  17. MDM2–MDM4 molecular interaction investigated by atomic force spectroscopy and surface plasmon resonance

    Directory of Open Access Journals (Sweden)

    Moscetti I

    2016-08-01

    Full Text Available Ilaria Moscetti,1 Emanuela Teveroni,2,3 Fabiola Moretti,3 Anna Rita Bizzarri,1 Salvatore Cannistraro1 1Biophysics and Nanoscience Centre, Department DEB, Università della Tuscia, Viterbo, Italy; 2Department of Endocrinology and Metabolism, Università Cattolica di Roma, Roma, Italy; 3Institute of Cell Biology and Neurobiology, Consiglio Nazionale delle Ricerche (CNR, Roma, Italy Abstract: Murine double minute 2 (MDM2 and 4 (MDM4 are known as the main negative regulators of p53, a tumor suppressor. They are able to form heterodimers that are much more effective in the downregulation of p53. Therefore, the MDM2–MDM4 complex could be a target for promising therapeutic restoration of p53 function. To this aim, a deeper understanding of the molecular mechanisms underlining the heterodimerization is needed. The kinetic and thermodynamic characterization of the MDM2–MDM4 complex was performed with two complementary approaches: atomic force spectroscopy and surface plasmon resonance. Both techniques revealed an equilibrium dissociation constant (KD in the micromolar range for the MDM2–MDM4 heterodimer, similar to related complexes involved in the p53 network. Furthermore, the MDM2–MDM4 complex is characterized by a relatively high free energy, through a single energy barrier, and by a lifetime in the order of tens of seconds. New insights into the MDM2–MDM4 interaction could be highly important for developing innovative anticancer drugs focused on p53 reactivation. Keywords: MDM2, MDM4, atomic force spectroscopy, surface plasmon resonance

  18. Expression of p53 and MDM2 in the retinas of three RP mice during the development of disease%p53和MDM2在3种RP小鼠视网膜中的表达

    Institute of Scientific and Technical Information of China (English)

    申煌煊; 张清炯; 肖学珊; 黎仕强; 贾小云; 郭向明

    2002-01-01

    目的了解凋亡相关基因p53和MDM2与视网膜色素变性(retinitis pigmentosa, RP)发病的关系.方法以RP模式动物rds小鼠、rd小鼠、C3H小鼠及正常对照C3B小鼠为材料,提取不同发病时间(出生后7、12、17、23、29、37和50d)视网膜的总RNA,以β-actin和L19 核糖体蛋白基因为内对照,RT-PCR分析小鼠发病过程中视网膜p53和MDM2的表达.结果在C3B小鼠视网膜中,p53和MDM2的表达完全同步.出生后7d,高表达;7~12d期间,表达量迅速下降;12~50d期间,表达水平低,但基本稳定.p53和MDM2在rds小鼠的表达水平低,但基本上是同步、稳定表达.在rd小鼠中,p53的表达变化不大,但MDM2的表达则与p53不同步,类似于C3B小鼠MDM2的表达.在C3H小鼠的视网膜,p53和MDM2的表达也不同步.7~23d期间,p53的表达基本稳定;自23d后逐步下降,其中23~29d期间,p53的表达下降最快.而MDM2的表达总体变化不大.结论在这3种RP模式动物中,虽然凋亡相关基因p53和MDM2存在着表达差异,但其发病过程中视网膜细胞的变性死亡可能是通过p53非依赖型途径进行的.

  19. Interplay among p53, MDM2, and MDMX and Progress in Related Studies%p53与MDM2及MDMX 的相互作用和研究进展

    Institute of Scientific and Technical Information of China (English)

    李小龙

    2012-01-01

    p53是重要的抑癌基因,在细胞周期阻滞、DNA损伤修复及细胞凋亡等生物过程中发挥重要作用,并已成为潜在的肿瘤治疗靶点.MDM2(Mdm2 p53 binding protein homolog)及MDMX(Mdm4 p53 binding protein homolog)是p53的主要抑制因子,两者相互协同并通过不同的信号途径抑制p53的活性.MDM2是p53的E3连接酶,介导p53的泛素化从而降低p53的稳定性.MDMX则主要通过与p53的转录活性区结合,抑制p53对其下游基因的转录活性,但并不介导p53的降解.MDM2与MDMX通过不同机制协同对p53产生抑制作用,其具体分子过程及作用机制繁多且复杂.本文就p53、MDM2及MDMX的相互作用及各蛋白的功能进行综述.%P53 is one of the most important tumor suppressors. The resukts from functional analysis revealed that p53 is a potent stress responder that regulates cell-cycle arrest, DNA repair, cell senescence, and apoptosis. P53 is also recognized as a potential therapy target. MDM2 and MDMX are two major p53 suppressors, which can both inhibit p53 activity via different mechanisms. MDM2 is an E3 ligase of p53, which can promote p53 ubiquitination and can regulate p53 degradation and stabilization. MDMX does not have E3 ligase activity, but can directly bind to the N terminus of p53 and can inhibit p53 transactivation. Studies have shown that MDM2 and MDMX could work together to regulate p53 activity in a more complicated mechanism. The present work reviews the interplay among p53, MDM2, and MDMX.

  20. Expressions of P16, P53 and MDM2 protein in retinoblastoma%视网膜母细胞瘤中MDM2、P16和P53的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    雷晓华; 朱润庆; 胡学斌

    2006-01-01

    目的 研究P16、P53和癌基因MDM2在视网膜母细胞瘤的表达作用.方法 对已明确诊断的42例视网膜母细胞瘤标本采用免疫组织化学方法检测P16、P53和MDM2表达.结果 42例标本中P16、P53和MDM2阳性检出率分别是35.7%、69.1%和38.1%,P53和MDM2的共表达率为23.8%.结论 P16、P53和MDM2在视网膜母细胞瘤的产生和发展过程中起重要作用.

  1. Pharmacologic ATM but not ATR kinase inhibition abrogates p21-dependent G1 arrest and promotes gastrointestinal syndrome after total body irradiation

    Science.gov (United States)

    Vendetti, Frank P.; Leibowitz, Brian J.; Barnes, Jennifer; Schamus, Sandy; Kiesel, Brian F.; Abberbock, Shira; Conrads, Thomas; Clump, David Andy; Cadogan, Elaine; O’Connor, Mark J.; Yu, Jian; Beumer, Jan H.; Bakkenist, Christopher J.

    2017-01-01

    We show that ATM kinase inhibition using AZ31 prior to 9 or 9.25 Gy total body irradiation (TBI) reduced median time to moribund in mice to 8 days. ATR kinase inhibition using AZD6738 prior to TBI did not reduce median time to moribund. The striking finding associated with ATM inhibition prior to TBI was increased crypt loss within the intestine epithelium. ATM inhibition reduced upregulation of p21, an inhibitor of cyclin-dependent kinases, and blocked G1 arrest after TBI thereby increasing the number of S phase cells in crypts in wild-type but not Cdkn1a(p21CIP/WAF1)−/− mice. In contrast, ATR inhibition increased upregulation of p21 after TBI. Thus, ATM activity is essential for p21-dependent arrest while ATR inhibition may potentiate arrest in crypt cells after TBI. Nevertheless, ATM inhibition reduced median time to moribund in Cdkn1a(p21CIP/WAF1)−/− mice after TBI. ATM inhibition also increased cell death in crypts at 4 h in Cdkn1a(p21CIP/WAF1)−/−, earlier than at 24 h in wild-type mice after TBI. In contrast, ATR inhibition decreased cell death in crypts in Cdkn1a(p21CIP/WAF1)−/− mice at 4 h after TBI. We conclude that ATM activity is essential for p21-dependent and p21-independent mechanisms that radioprotect intestinal crypts and that ATM inhibition promotes GI syndrome after TBI. PMID:28145510

  2. Pharmacologic ATM but not ATR kinase inhibition abrogates p21-dependent G1 arrest and promotes gastrointestinal syndrome after total body irradiation.

    Science.gov (United States)

    Vendetti, Frank P; Leibowitz, Brian J; Barnes, Jennifer; Schamus, Sandy; Kiesel, Brian F; Abberbock, Shira; Conrads, Thomas; Clump, David Andy; Cadogan, Elaine; O'Connor, Mark J; Yu, Jian; Beumer, Jan H; Bakkenist, Christopher J

    2017-02-01

    We show that ATM kinase inhibition using AZ31 prior to 9 or 9.25 Gy total body irradiation (TBI) reduced median time to moribund in mice to 8 days. ATR kinase inhibition using AZD6738 prior to TBI did not reduce median time to moribund. The striking finding associated with ATM inhibition prior to TBI was increased crypt loss within the intestine epithelium. ATM inhibition reduced upregulation of p21, an inhibitor of cyclin-dependent kinases, and blocked G1 arrest after TBI thereby increasing the number of S phase cells in crypts in wild-type but not Cdkn1a(p21(CIP/WAF1))-/- mice. In contrast, ATR inhibition increased upregulation of p21 after TBI. Thus, ATM activity is essential for p21-dependent arrest while ATR inhibition may potentiate arrest in crypt cells after TBI. Nevertheless, ATM inhibition reduced median time to moribund in Cdkn1a(p21(CIP/WAF1))-/- mice after TBI. ATM inhibition also increased cell death in crypts at 4 h in Cdkn1a(p21(CIP/WAF1))-/-, earlier than at 24 h in wild-type mice after TBI. In contrast, ATR inhibition decreased cell death in crypts in Cdkn1a(p21(CIP/WAF1))-/- mice at 4 h after TBI. We conclude that ATM activity is essential for p21-dependent and p21-independent mechanisms that radioprotect intestinal crypts and that ATM inhibition promotes GI syndrome after TBI.

  3. p21 is dispensable for AID-mediated class switch recombination and mutagenesis of immunoglobulin genes during somatic hypermutation.

    Science.gov (United States)

    Shansab, Maryam; Selsing, Erik

    2011-03-01

    In B cells, activation-induced cytidine deaminase (AID) induces somatic hypermutation (SHM) at rearranged immunoglobulin (Ig) variable (V) regions. Previous studies have shown that both monoubiquitination of proliferating cell nuclear antigen (PCNA) and translesional DNA polymerase activity are important for inducing mutagenesis during SHM. Regulation of PCNA ubiquitination by p21, also known as Cdkn1a and p21(Cip1/Waf1), is an important mechanism that controls mutation loads in mammalian cells. In this study, we have assessed whether p21 has an in vivo function in regulating mutagenesis in B cells by analyzing SHM frequency in p21-deficient mice. Our results show that p21 is dispensable for SHM. This suggests that, during SHM of Ig genes, p21 does not act to regulate mutagenesis load. We also show that p21 transcript levels are the same in both wildtype and AID-deficient B cells during B cell activation, and that AID-mediated class switch recombination (CSR) is not affected by p21 deficiency; thereby indicating that p21 regulation in B cells is not altered by AID-induced DNA damage and that p21 has no affect on AID-dependent Ig gene diversification. Our results suggest that regulation of p21 in activated B cells is probably more important for maintaining proper cell cycle progression as opposed to promoting SHM of Ig genes.

  4. The dynamics of stress p53-Mdm2 network regulated by p300 and HDAC1.

    Science.gov (United States)

    Arora, Akshit; Gera, Saurav; Maheshwari, Tanuj; Raghav, Dhwani; Alam, Md Jahoor; Singh, R K Brojen; Agarwal, Subhash M

    2013-01-01

    We construct a stress p53-Mdm2-p300-HDAC1 regulatory network that is activated and stabilised by two regulatory proteins, p300 and HDAC1. Different activation levels of [Formula: see text] observed due to these regulators during stress condition have been investigated using a deterministic as well as a stochastic approach to understand how the cell responds during stress conditions. We found that these regulators help in adjusting p53 to different conditions as identified by various oscillatory states, namely fixed point oscillations, damped oscillations and sustain oscillations. On assessing the impact of p300 on p53-Mdm2 network we identified three states: first stabilised or normal condition where the impact of p300 is negligible, second an interim region where p53 is activated due to interaction between p53 and p300, and finally the third regime where excess of p300 leads to cell stress condition. Similarly evaluation of HDAC1 on our model led to identification of the above three distinct states. Also we observe that noise in stochastic cellular system helps to reach each oscillatory state quicker than those in deterministic case. The constructed model validated different experimental findings qualitatively.

  5. The dynamics of stress p53-Mdm2 network regulated by p300 and HDAC1.

    Directory of Open Access Journals (Sweden)

    Akshit Arora

    Full Text Available We construct a stress p53-Mdm2-p300-HDAC1 regulatory network that is activated and stabilised by two regulatory proteins, p300 and HDAC1. Different activation levels of [Formula: see text] observed due to these regulators during stress condition have been investigated using a deterministic as well as a stochastic approach to understand how the cell responds during stress conditions. We found that these regulators help in adjusting p53 to different conditions as identified by various oscillatory states, namely fixed point oscillations, damped oscillations and sustain oscillations. On assessing the impact of p300 on p53-Mdm2 network we identified three states: first stabilised or normal condition where the impact of p300 is negligible, second an interim region where p53 is activated due to interaction between p53 and p300, and finally the third regime where excess of p300 leads to cell stress condition. Similarly evaluation of HDAC1 on our model led to identification of the above three distinct states. Also we observe that noise in stochastic cellular system helps to reach each oscillatory state quicker than those in deterministic case. The constructed model validated different experimental findings qualitatively.

  6. Stress-specific response of the p53-Mdm2 feedback loop

    Directory of Open Access Journals (Sweden)

    Jensen Mogens H

    2010-07-01

    Full Text Available Abstract Background The p53 signalling pathway has hundreds of inputs and outputs. It can trigger cellular senescence, cell-cycle arrest and apoptosis in response to diverse stress conditions, including DNA damage, hypoxia and nutrient deprivation. Signals from all these inputs are channeled through a single node, the transcription factor p53. Yet, the pathway is flexible enough to produce different downstream gene expression patterns in response to different stresses. Results We construct a mathematical model of the negative feedback loop involving p53 and its inhibitor, Mdm2, at the core of this pathway, and use it to examine the effect of different stresses that trigger p53. In response to DNA damage, hypoxia, etc., the model exhibits a wide variety of specific output behaviour - steady states with low or high levels of p53 and Mdm2, as well as spiky oscillations with low or high average p53 levels. Conclusions We show that even a simple negative feedback loop is capable of exhibiting the kind of flexible stress-specific response observed in the p53 system. Further, our model provides a framework for predicting the differences in p53 response to different stresses and single nucleotide polymorphisms.

  7. Characterizing the Free-Energy Landscape of MDM2 Protein-Ligand Interactions by Steered Molecular Dynamics Simulations.

    Science.gov (United States)

    Hu, Guodong; Xu, Shicai; Wang, Jihua

    2015-12-01

    Inhibition of p53-MDM2 interaction by small molecules is considered to be a promising approach to re-activate wild-type p53 for tumor suppression. Several inhibitors of the MDM2-p53 interaction were designed and studied by the experimental methods and the molecular dynamics simulation. However, the unbinding mechanism was still unclear. The steered molecular dynamics simulations combined with Brownian dynamics fluctuation-dissipation theorem were employed to obtain the free-energy landscape of unbinding between MDM2 and their four ligands. It was shown that compounds 4 and 8 dissociate faster than compounds 5 and 7. The absolute binding free energies for these four ligands are in close agreement with experimental results. The open movement of helix II and helix IV in the MDM2 protein-binding pocket upon unbinding is also consistent with experimental MDM2-unbound conformation. We further found that different binding mechanisms among different ligands are associated with H-bond with Lys51 and Glu25. These mechanistic results may be useful for improving ligand design.

  8. The cholesterol metabolite 27-hydroxycholesterol regulates p53 activity and increases cell proliferation via MDM2 in breast cancer cells.

    Science.gov (United States)

    Raza, Shaneabbas; Ohm, Joyce E; Dhasarathy, Archana; Schommer, Jared; Roche, Conor; Hammer, Kimberly D P; Ghribi, Othman

    2015-12-01

    Estrogen is synthesized from cholesterol and high cholesterol levels are suggested to be associated with increased risk of estrogen receptor(ER)-positive breast cancer. The cholesterol metabolite 27-hydroxycholesterol (27-OHC) was recently identified as a selective estrogen receptor modulator (SERM) and may therefore impact breast cancer progression. However, the mechanisms by which 27-OHC may contribute to breast cancer are not all known. We determined the extent to which 27-OHC regulates cell proliferation in MCF7 ER-positive breast cancer cell line involving the tumor suppressor protein p53. We found that treatment of MCF7 cells with 27-OHC resulted reduced p53 transcriptional activity. Conversely, treatment of the ER-negative MDA-MB 231 cells with 27-OHC induced no significant change in p53 activity. Exposure of MCF7 cells to 27-OHC was also associated with increased protein levels of the E3 ubiquitin protein ligase MDM2 and decreased levels of p53. Moreover, 27-OHC also enhanced physical interaction between p53 and MDM2. Furthermore, 27-OHC-induced proliferation was attenuated using either the p53 activator Tenovin-1 or the MDM2 inhibitor Nutlin-3 and Mdm2 siRNA. Taken together, our results indicate that 27-OHC may contribute to ER-positive breast cancer progression by disrupting constitutive p53 signaling in an MDM2-dependent manner.

  9. PTCH-1 and MDM2 expression in ameloblastoma from a West African sub-population: implication for chemotherapeutics.

    Science.gov (United States)

    Udeabor, Samuel Ebele; Adisa, Akinyele Olumuyiwa; Lawal, Ahmed Oluwatoyin; Barbeck, Mike; Booms, Patrick; Sader, Robert Alexander; Ghanaati, Shahram

    2015-01-01

    Ameloblastoma is a slow growing, painless odontogenic swelling which can attain sizes that result in severe deformities of the craniofacial complex. It is the most commonly encountered odontogenic tumor in Nigeria. Surgical intervention is currently the method of treatment; however identification of altered molecular pathways may inform chemotherapeutic potential. The Protein Patched homolog 1 (PTCH-1) is overexpressed in ameloblastoma. Also, mutation in the MDM2 gene can reduce the tumor suppressor function of p53 and promote ameloblastoma growth. No study however has characterized the molecular profile of African cases of ameloblastoma with a view to developing chemotherapeutic alternatives. The objective was to characterize the PTCH-1 genetic profile of Ameloblastoma in Nigerian patients as a first step in investigating its potential for chemotherapeutic intervention. Twenty-eight FFPE blocks of ameloblastoma cases from Nigerian patients were prepared for antibody processing to PTCH-1 (Polyclonal Anti-PTCH antibody ab39266) and MDM2 (Monoclonal Anti-MDM2 antibody (2A10) ab16895). Cytoplasmic brown staining was considered as positive for PTCH while nuclear staining was positive for MDM2. Moderate and strong expressions for PTCH in ameloblast and stellate reticulum were 78.6% and 60.7% respectively. Only 3 (10.7%) cases expressed MDM2. The importance of our study is that it supports, in theory, anti-PTCH/SHH chemotherapeutics for Nigerian ameloblastoma cases and also infers the possible additional use of anti-p53 agents.

  10. 荧光定量PCR法检测MDM2T309G位点多态性%Novel real-time PCR methods for clinical detection of MDM2 T309G polymorphism

    Institute of Scientific and Technical Information of China (English)

    陈红; 谢丽; 胡文静; 禹立霞; 钱晓萍; 刘宝瑞

    2011-01-01

    Objective To establish a simple, convenient method for genotyping of MDM2 SNP309. Methods Using SYBR GREEN PCR with Tm-shift primers and genomic DNA purified with TIANamp DNA blood kit, we established a new method to detect genotyping of MDM2 SNP309. And we also genotyped MDM2 SNP3O9 by SYBR GREEN PCR with Tm-shift primers using whole blood DNA extracted by boiling method. Furthermore, genomic DNA were purified using boiling method from different blood samples kept at room temperature for different time or treated with multiple freezing and thawing as well as hematoiogic abnormal blood samples. Quantitative PCR were carried out to observe the amplification efficiency of the obtained DNA templates to assess the DNA extraction method. Results We established a novel, SYBR GREEN PCR method for MDM2 SNP309 genotyping. DNA purified by both methods mentioned before were suitable for MDM2 SNP309 SYBR GREEN PCR. DNA extracted using boiling method could be used for poly-merase chain reaction even when the blood sample were disposed in different ways. Conclusion In the present research, we established methods for MDM2 SNP309 genotype. Boiling method for DNA extraction is proved to be a simpler and quicker way of processing whole-blood samples compared with the DNA templates, and is effective for both PCR-RFLP and SYBR GREEN PCR.%目的 建立一种简便易行的MDM2基因单核苷酸多态性(SNP)309位点的检测方法.方法 用DNA提取试剂盒提取外周血DNA,建立MDM2 SNP309位点荧光定量PCR熔解曲线检测法.应用煮沸法处理血样取得粗制DNA,行荧光定量PCR检测,建立位点特异引物法检测MDM2 SNP309位点基因型.比较静置不同时间、反复冻融等处理以及存在血液学指标异常的血样经煮沸法提取DNA对荧光定量PCR扩增反应的影响,通过观察PCR扩增情况,评估不同处理方法对PCR扩增反应的影响.结果 荧光定量建立MDM2 SNP309位点PCR检测方法.煮沸法提取的DNA可用于普通PCR

  11. Expression of Brn-3a and MDM-2 in Cervical Neoplasia%Brn-3a和MDM-2在宫颈癌及癌前病变中的表达

    Institute of Scientific and Technical Information of China (English)

    黄雅; 冯玉昆; 李建军; 于璐

    2009-01-01

    目的:探讨Brn-3a和MDM-2在宫颈癌和癌前病变中的表达及其作为宫颈癌和癌前病变生物标志物的可行性和临床意义.方法:利用SP免疫组化法检测110例蜡块标本中Brn-3a和MDM-2的表达.结果:宫颈癌及癌前病变各组的Brn-3a表达阳性率均高于对照组,差异有统计学意义(P<0.05);SCC组及CINⅢ组的MDM-2表达阳性率均高于对照组,差异有统计学意义(P<0.05),SCC组与CIN Ⅰ组比较,差异有统计学意义(P<0.05).Brn-3a NtMDM-2在各临床分期及组织学分级中的阳性表达无统计学差异(P>0.05)Brn-3a与MDM-2蛋白阳性表达之间无相关关系(P>0.05).结论:Brn-3a和MDM-2可作为宫颈癌前病变的生物标志物.

  12. The Expression of MDM-2 and Rb in Pancreatic Carcinoma and Its Clinical Significance%MDM-2、Rb在胰腺癌中的表达及其临床意义

    Institute of Scientific and Technical Information of China (English)

    刘振华; 冯一中; 柴玉海; 李峰; 任苏勤

    2007-01-01

    目的 探讨MDM-2、Rb在胰腺癌生长、转移过程中的作用,为患者预后判断提供理论依据.方法 应用免疫组织化学S-P法,检测MDM.2、Rb在53例胰腺癌、10非肿瘤性胰腺组织中的表达,并分析它们与胰腺癌临床病理参数及患者生存率的关系.结果 (1)MDM-2在胰腺癌中的阳性表达率明显高于非肿瘤性胰腺组织(P<0.05),与胰腺癌的病理分级成正相关(P<0.05).(2)Rb的阳性表达率在胰腺癌中显著低于非肿瘤性胰腺组织(P<0.01),与胰腺癌的病理分级成负相关(p<0.05).(3)MDM-2与Rb在胰腺癌中的阳性表达无显著相关.(4)MDM-2及Rb的阳性表达经生存单因素分析显示与患者的预后显著相关(P<0.01,P<0.05).结论 MDM-2、Rb在胰腺癌的发生发展中均发挥一定的作用,它们在胰腺癌的发生中可能起协同作用.

  13. Cytoplasmic p21 is a potential predictor for cisplatin sensitivity in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Liu Ronghua

    2011-09-01

    Full Text Available Abstract Background P21(WAF1/Cip1 binds to cyclin-dependent kinase complexes and inhibits their activities. It was originally described as an inhibitor of cancer cell proliferation. However, many recent studies have shown that p21 promotes tumor progression when accumulated in the cell cytoplasm. So far, little is known about the correlation between cytoplasmic p21 and drug resistance. This study was aimed to investigate the role of p21 in the cisplatin resistance of ovarian cancer. Methods RT-PCR, western blot and immunofluorescence were used to detect p21 expression and location in cisplatin-resistant ovarian cancer cell line C13* and its parental line OV2008. Regulation of cytoplasmic p21 was performed through transfection of p21 siRNA, Akt2 shRNA and Akt2 constitutively active vector in the two cell lines; their effects on cisplatin-induced apoptosis were evaluated by flow cytometry. Tumor tissue sections of clinical samples were analyzed by immunohistochemistry. Results p21 predominantly localizes to the cytoplasm in C13* compared to OV2008. Persistent exposure to low dose cisplatin in OV2008 leads to p21 translocation from nuclear to cytoplasm, while it had not impact on p21 localization in C13*. Knockdown of cytoplasmic p21 by p21 siRNA transfection in C13* notably increased cisplatin-induced apoptosis through activation of caspase 3. Inhibition of p21 translocation into the cytoplasm by transfection of Akt2 shRNA into C13* cells significantly increased cisplatin-induced apoptosis, while induction of p21 translocation into the cytoplasm by transfection of constitutively active Akt2 in OV2008 enhanced the resistance to cisplatin. Immunohistochemical analysis of clinical ovarian tumor tissues demonstrated that cytoplasmic p21 was negatively correlated with the response to cisplatin based treatment. Conclusions Cytoplasmic p21 is a novel biomarker of cisplatin resistance and it may represent a potential therapeutic target for ovarian tumors

  14. A re-examination of the MDM2/p53 interaction leads to revised design criteria for novel inhibitors.

    Science.gov (United States)

    Vasilevich, Natalya I; Afanasyev, Ilya I; Kovalskiy, Dmitry A; Genis, Dmitry V; Kochubey, Valery S

    2014-11-01

    The general model of epitope-type MDM2 inhibitor was developed based on the structural information on the complexes between MDM2 and various low molecular weight ligands found in the PDB database. Application of this model to our in-house library has led us to a new scaffold capable of interrupting protein-protein interactions. A synthetic library based on this and related scaffolds resulted in new classes of compounds that possess biochemical and cellular activity and good pharmacokinetic properties. We assume that such general approach to PPI inhibitors design may be useful for the development of inhibitors of various PPI types, including Bcl/XL.

  15. Design, synthesis and biological evaluation of sulfamide and triazole benzodiazepines as novel p53-MDM2 inhibitors.

    Science.gov (United States)

    Yu, Zhiliang; Zhuang, Chunlin; Wu, Yuelin; Guo, Zizhao; Li, Jin; Dong, Guoqiang; Yao, Jianzhong; Sheng, Chunquan; Miao, Zhenyuan; Zhang, Wannian

    2014-09-05

    A series of sulfamide and triazole benzodiazepines were obtained with the principle of bioisosterism. The p53-murine double minute 2 (MDM2) inhibitory activity and in vitro antitumor activity were evaluated. Most of the novel benzodiazepines exhibited moderate protein binding inhibitory activity. Particularly, triazole benzodiazepines showed good inhibitory activity and antitumor potency. Compound 16 had promising antitumor activity against the U-2 OS human osteosarcoma cell line with an IC50 value of 4.17 μM, which was much better than that of nutlin-3. The molecular docking model also successfully predicted that this class of compounds mimicked the three critical residues of p53 binding to MDM2.

  16. Expression and clinical significance of Pokemon and mdm2 proteins in gallbladder cancer%Pokemon及mdm2蛋白在胆囊癌组织中的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    李达; 张水军; 赵永福; 吴阳; 唐哲

    2012-01-01

    目的 探讨Pokemon及mdm2蛋白与胆囊癌的发生、发展的关系及其临床意义.方法 采用免疫组织化学S-P法检测40例胆囊癌、12例胆囊腺瘤、20例胆囊息肉及20例胆囊炎组织中Pokemon和mdm2蛋白的表达水平,分析其与胆囊癌临床病理学特征的关系,以及Pokemon和mdm2蛋白在组织中表达的相关性.结果 在检测的胆囊癌、胆囊腺瘤、胆囊息肉及胆囊炎组织中Pokemon蛋白的阳性表达率分别为60.0%、25.0%、30.0%、25.0%,mdm2 蛋白的阳性表达率分别为57.5%、16.7%、35.0%、30.0%,胆囊癌组织中Pokemon及mdm2蛋白的阳性表达率高于其他组,差异有统计学意义(P< 0.05);Pokemon及mdm2蛋白的表达与肿瘤的病理学分级及Nevin分期相关(P<0.05),与患者性别、年龄、肿瘤组织学类型及是否合并结石无关(P>0.05).Poketnon蛋白的表达与mdm2蛋白的表达呈正相关(r=0.434,p<0.05).结论 Pokemon蛋白和mdm2蛋白可能共同参与了胆囊癌的发生、发展和转移的过程,Pokemon基因有望成为胆囊癌治疗的一个有效的靶基因,从而为胆囊癌的治疗开辟新的途径.%Objective To investigate the relationship between POK erythroid myeloid ontogenic factor (Pokemon) and mdm2 proteins expressions and gallbladder cancer and its clinical significance.Methods The Pokemon and mdm2 proteins were analyzed in 40 gallbladder cancer patients,12 gallbladder adenoma patients,20 gallbladder polyps patients and 20 chronic cholecystitis patients.The relationship between the expressions of Pokemon and mdm2 and clinic-pathological features of gallbladder cancer were analyzed.The correlation of Pokemon expression with mdm2 expression in tissues was analyzed as well.Results In the tissues of gallbladder cancer,gallbladder adenoma,gallbladder polyps and chronic cholecystitis,the positive expression rates of Pokemon protein were 60.0%,25.0%,30.0%,25.0%,the positive expression rates of mdm2 were 57

  17. 1,25(OH)2D3 inhibits the progression of hepatocellular carcinoma via downregulating HDAC2 and upregulating P21(WAFI/CIP1).

    Science.gov (United States)

    Huang, Jian; Yang, Guozhen; Huang, Yunzhu; Kong, Weiying; Zhang, Shu

    2016-02-01

    Vitamin D, termed 1,25(OH)2D3 in it's active form, activity is associated with a reduced risk of hepatocellular carcinoma (HCC) and is an important immune regulator. However, the detail molecular mechanisms underlying the effects of 1,25(OH)2D3 on the progression of HCC are widely unknown. Histone deacetwylase 2 (HDAC2) is usually expressed at high levels in tumors, and its downregulation leads to high expression levels of cell cycle components, including p21(WAF1/Cip1), a well-characterized modulator, which is critical in cell senescence and apoptosis. The present study investigated whether vitamin D inhibits HCC via the regulation of HDAC2 and p21(WAF1/Cip1). Firstly, the toxic concentrations of 1,25(OH)2D3 were determined, according to trypan blue and [(3)H]thymidine incorporation assays. Secondly, HCC cells lines were treated with different concentrations of 1,25(OH)2D3. The expression of HDAC2 was either silenced via short hairpin (sh)RNA or induced by transfection of plasmids expressing the HDAC2 gene in certain HCC cells. Finally the mRNA and protein levels of HDAC2 and p21(WAF1/Cip1) were measured using reverse transcription-quantitative polymerase chain reaction and western blot analyses. The results revealed that 1,25(OH)2D3 treatment reduced the expression of HDAC2 and increased the expression of p21(WAF1/Cip1), in a dose-dependent manner, resulting in the reduction of HCC growth. Elevated levels of HDAC2 reduced the expression of p21(WAF1/Cip1), resulting in an increase in HCC growth. HDAC2 shRNA increased the expression of p21(WAF1/Cip1), resulting in reduction in HCC growth. Thus, 1,25(OH)2D3 exerted antitumorigenic effects through decreasing the expression levels of HDAC2 and increasing the expression of p21(WAF1/Cip1), which inhibited the development of HCC and may indicate the possible underlying mechanism. These results suggest that vitamin D3 may be developed as a potential drug for effective therapy in the treatment of HCC.

  18. The nucleolar SUMO-specific protease SMT3IP1/SENP3 attenuates Mdm2-mediated p53 ubiquitination and degradation

    Energy Technology Data Exchange (ETDEWEB)

    Nishida, Tamotsu, E-mail: nishida@gene.mie-u.ac.jp [Department of Human Functional Genomics, Life Science Research Center, Mie University, 1577 Kurima-machiya, Tsu 514-8507 (Japan); Yamada, Yoshiji [Department of Human Functional Genomics, Life Science Research Center, Mie University, 1577 Kurima-machiya, Tsu 514-8507 (Japan)

    2011-03-11

    Research highlights: {yields} SMT3IP1 interacts with p53 and Mdm2, and desumoylates both proteins. {yields} SMT3IP1 competes with p53 for binding to the central acidic domain of Mdm2. {yields} SMT3IP1 binding to Mdm2 inhibits Mdm2-mediated p53 ubiquitination and degradation. {yields} We postulate that SMT3IP1 acts as a new regulator of the p53-Mdm2 pathway. -- Abstract: SUMO (small ubiquitin-like modifier) modification plays multiple roles in several cellular processes. Sumoylation is reversibly regulated by SUMO-specific proteases. SUMO-specific proteases have recently been implicated in cell proliferation and early embryogenesis, but the underlying mechanisms remain unknown. Here, we show that a nucleolar SUMO-specific protease, SMT3IP1/SENP3, controls the p53-Mdm2 pathway. We found that SMT3IP1 interacts with p53 and Mdm2, and desumoylates both proteins. Overexpression of SMT3IP1 in cells resulted in the accumulation of Mdm2 in the nucleolus and increased stability of the p53 protein. In addition, SMT3IP1 bound to the acidic domain of Mdm2, which also mediates the p53 interaction, and competed with p53 for binding. Increasing expression of SMT3IP1 suppressed Mdm2-mediated p53 ubiquitination and subsequent proteasomal degradation. Interestingly, the desumoylation activity of SMT3IP1 was not necessary for p53 stabilization. These results suggest that SMT3IP1 is a new regulator of the p53-Mdm2 pathway.

  19. 核糖体蛋白家族成员与 MDM 2-p53通路调控的研究进展%Research progress of the relationship between ribosomal protein and MDM 2-p 5 3 pathway

    Institute of Scientific and Technical Information of China (English)

    刘弘扬; 孙冬琳; 孙海明; 金焰

    2016-01-01

    肿瘤是危害人类生命健康的最主要的疾病之一。 p53是目前为止研究最多的抑癌基因,p53的功能紊乱是肿瘤发生最主要的原因。核糖体蛋白发挥核糖体外功能抑制鼠双微体基因2( murine double minute 2, MDM 2)并激活 P 53是近十年的研究热点。本文就核糖体蛋白与 MDM 2-p 53通路调控关系及其与肿瘤治疗的研究进展进行综述。%Cancer is one of the most life threatening diseases .The dysfunction of the p 5 3 , as one of the most studied tumor-suppressor genes so far , is the main reason for tumorigenesis .Many studies over the past decades reveal that the ribosome-free ribosomal protein inhibits the MDM 2 and actives the P 5 3 .This review focuseon the progress in studying on the relationship between the ribosomal protein on the MDM 2-p 5 3 regulatory pathway and its roles incancer therapy .

  20. Galectin-3 regulates p21 stability in human prostate cancer cells

    Science.gov (United States)

    Wang, Yi; Balan, Vitaly; Kho, Dhonghyo; Hogan, Victor; Nangia-Makker, Pratima; Raz, Avraham

    2014-01-01

    Galectin-3 (Gal-3) is a multifunctional protein involved in cancer through regulation of cell adhesion, cell growth, apoptosis, and metastasis, while p21 (Cip1/WAF1) is a negative regulator of the cell cycle, involved in apoptosis, transcription, DNA repair and metastasis. The results presented here demonstrate for the first time that the level of Gal-3 protein is associated with the level of p21 protein expression in human prostate cancer cells and the effects of Gal-3 on cell growth and apoptosis were reversed by modulating p21 expression level. Furthermore, Gal-3 regulates p21 expression at the post-translational level by stabilizing p21 protein via the carbohydrate-recognition domain (CRD). This is the first report suggesting a molecular function not yet described for Gal-3 as the regulator of p21 protein stability. This study provides a unique insight into the relationship of these two molecules during prostate cancer progression, and may provide a novel therapeutic target. PMID:23160381

  1. Association between polymorphism of MDM2 and susceptibility of primary hepatic carcinoma:A Meta analysis%MDM2基因多态与肝癌遗传易感性的Meta分析

    Institute of Scientific and Technical Information of China (English)

    万艳; 任仰武; 周宝森

    2011-01-01

    目的:根据已发表的相关文献,综合评估鼠双微体同源基因2(MDM2)多态与肝癌遗传易感性之间的关系.方法:按照统一的检索策略在相关中英文数据库中全面检索相关文献,对文献进行筛选、评价后获得相关研究的结果数据,然后应用Stata 10软件中Meta分析的方法,计算合并OR值及95%CI,并进行敏感性分析和发表偏倚的估计.结果:共有国内外5篇合格文献纳入本研究,累计病例和对照数分别为738和1 062例.合并结果显示,携带GG等位基因型者患肝癌的危险性是携带TT等位基因型的2.39倍(95%CI=1.81~3.15,P<0.001),携带TG等位基因型者患肝癌的危险性是携带TT等位基因型的1.65倍(95%CI=1.31~2.08,P<0.001).发表偏倚评估未发现明显的偏倚.结论:MDM2SNP309多态中GG、TG等位基因型可能与肝癌的易感性升高有关.%OBJECTIVE: To explore whether the polymorphism of murine double minute 2 (MDM2) contribute to the genetic susceptibility to primary hepatic carcinoma (PHC) based on the published studies. METHODS: All studies related to MDM2 and primary hepatic carcinoma were published in English and Chinese language and the allele frequencies of MDM2 and outcomes were abstracted after assessed under the same criteria. The pooled Ors with their 95%Cis were calculated using Meta methods in Stata 10 software. Publication bias and sensitivity were evaluated at the same time. RESULTS: Five studies with 738 cases and 1062 controls were included based on the selection criteria. The pooled results indicated MDM2 309GG genotype could increase the susceptibility to PHC with OR 2. 39(95%CI = 1. 81 - 3. 15, P<0. 001) compared to the wild allele MDM2 309TT, and MDM2 309TG genotype could increase the susceptibility to PHC with OR 1. 65 (95%CI=1.31-2. 08, P<0. 001) compared to the wild allele MDM2 309TT. The publication bias was not significant. CONCLUSION: MDM2 309GG, 309TG genotype seems to be involved in the risk of

  2. Positive effect of Mdm2 on p53 expression explains excitability of p53 in response to DNA damage.

    Science.gov (United States)

    Eliaš, Ján

    2017-04-07

    Most of the existing biological models consider Mdm2 as a dominant negative regulator of p53 appearing in several negative feedback loops. However, in addition to targeting p53 for degradation, Mdm2 in tight cooperation with MdmX can control expression levels of p53 through enhanced induction of p53 synthesis in response to DNA damage. Whilst ATM-dependent phosphorylation of p53 is not observed to be important in this enhanced synthesis, ATM-dependent phosphorylation of Mdm2 (as well as MdmX) is essential for its dual role, which is accompanied with widely oscillating p53. In the light of these new observations we formulate a novel molecular mechanism which, in silico, is capable of triggering p53 oscillations. The mechanism that is based on Mdm2's dual regulation of p53 can provide mechanistic insights into an excitability of the p53 network, thus it contributes to understanding of variability of p53 dynamics in response to single and double strand breaks.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    P53 is rarely mutated in cutaneous T-cell lymphoma (CTCL) and is therefore a promising target for innovative therapeutic approaches. Nutlin-3a is an inhibitor of MDM2 (human homolog of murine double minute 2), which disrupts its interaction with p53, leading to the stabilization and activation of p...

  4. Next-generation repeat-free FISH probes for DNA amplification in glioblastoma in vivo: Improving patient selection to MDM2-targeted inhibitors.

    Science.gov (United States)

    Brunelli, Matteo; Eccher, Albino; Cima, Luca; Trippini, Tobia; Pedron, Serena; Chilosi, Marco; Barbareschi, Mattia; Scarpa, Aldo; Pinna, Giampietro; Cabrini, Giulio; Pilotto, Sara; Carbognin, Luisa; Bria, Emilio; Tortora, Giampaolo; Fioravanzo, Adele; Schiavo, Nicola; Meglio, Mario; Sava, Teodoro; Belli, Laura; Martignoni, Guido; Ghimenton, Claudio

    2017-01-01

    A next-generation FISH probe mapping to the MDM2 locus-specific region has recently been designed. The level of MDM2 gene amplification (high versus low) may allow selection of patients for cancer treatment with MDM2 inhibitors and may predict their responsiveness. We investigated the spectrum of MDM2 gene alterations using the new probes in vivo after visualizing single neoplastic cells in situ from a series of glioblastomas. Signals from next-generation repeat-free FISH interphase probes were identified in tissue microarrays that included 3 spots for each of the 48 cases. The murine double minutes (MDM2)-specific DNA probe and the satellite enumeration probe for chromosome 12 were used. Three cases (6%) showed more than 25 signals (high gene amplification), and 7 (15%) showed 3-10 signals (gains); among these, 4 cases (8%) had an equal number of MDM2 and centromeric signals on chromosome 12 (polyploidy). Genomic heterogeneity was observed only in 3 cases with low gene amplification. In our series, 6% of glioblastomas exhibited high MDM2 amplification (in vivo) with a pattern related to the known double minutes/chromothripsis phenomenon (in situ), and only cases with low amplification showed genomic heterogeneity. We concluded that the rate of MDM2 gene amplification can be a useful predictive biomarker to improve patient selection.

  5. MDM2 phenotypic and genotypic profiling, respective to TP53 genetic status, in diffuse large B-cell lymphoma patients treated with rituximab-CHOP immunochemotherapy

    DEFF Research Database (Denmark)

    Xu-Monette, Zijun Y; Møller, Michael; Tzankov, Alexander

    2013-01-01

    MDM2 is a key negative regulator of the tumor suppressor p53, however, the prognostic significance of MDM2 overexpression in diffuse large B-cell lymphoma (DLBCL) has not been defined convincingly. In a p53-genetically defined large cohort of de novo DLBCL patients treated with R-CHOP chemotherap...

  6. Structural Exploration and Conformational Transitions in MDM2 upon DHFR Interaction from Homo sapiens: A Computational Outlook for Malignancy via Epigenetic Disruption.

    Science.gov (United States)

    Banerjee, Arundhati; Ray, Sujay

    2016-01-01

    Structural basis for exploration into MDM2 and MDM2-DHFR interaction plays a vital role in analyzing the obstruction in folate metabolism, nonsynthesis of purines, and further epigenetic regulation in Homo sapiens. Therefore, it leads to suppression of normal cellular behavior and malignancy. This has been earlier documented via yeast two-hybrid assays. So, with a novel outlook, this study explores the molecular level demonstration of the best satisfactory MDM2 model selection after performing manifold modeling techniques. Z-scores and other stereochemical features were estimated for comparison. Further, protein-protein docking was executed with MDM2 and the experimentally validated X-ray crystallographic DHFR. Residual disclosure from the best suited simulated protein complex disclosed 18 side chain and 3 ionic interactions to strongly accommodate MDM2 protein into the pocket-like zone in DHFR due to the positive environment by charged residues. Lysine residues from MDM2 played a predominant role. Moreover, evaluation from varied energy calculations, folding rate, and net area for solvent accessibility implied the active participation of MDM2 with DHFR. Fascinatingly, conformational transitions from coils to helices and β-sheets after interaction with DHFR affirm the conformational strength and firmer interaction of human MDM2-DHFR. Therefore, this probe instigates near-future clinical research and interactive computational investigations with mutations.

  7. Structural Exploration and Conformational Transitions in MDM2 upon DHFR Interaction from Homo sapiens: A Computational Outlook for Malignancy via Epigenetic Disruption

    Science.gov (United States)

    Banerjee, Arundhati; Ray, Sujay

    2016-01-01

    Structural basis for exploration into MDM2 and MDM2-DHFR interaction plays a vital role in analyzing the obstruction in folate metabolism, nonsynthesis of purines, and further epigenetic regulation in Homo sapiens. Therefore, it leads to suppression of normal cellular behavior and malignancy. This has been earlier documented via yeast two-hybrid assays. So, with a novel outlook, this study explores the molecular level demonstration of the best satisfactory MDM2 model selection after performing manifold modeling techniques. Z-scores and other stereochemical features were estimated for comparison. Further, protein-protein docking was executed with MDM2 and the experimentally validated X-ray crystallographic DHFR. Residual disclosure from the best suited simulated protein complex disclosed 18 side chain and 3 ionic interactions to strongly accommodate MDM2 protein into the pocket-like zone in DHFR due to the positive environment by charged residues. Lysine residues from MDM2 played a predominant role. Moreover, evaluation from varied energy calculations, folding rate, and net area for solvent accessibility implied the active participation of MDM2 with DHFR. Fascinatingly, conformational transitions from coils to helices and β-sheets after interaction with DHFR affirm the conformational strength and firmer interaction of human MDM2-DHFR. Therefore, this probe instigates near-future clinical research and interactive computational investigations with mutations. PMID:27213086

  8. In vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and metabolism of pyrazole-based small molecule inhibitors of Mdm2/4-p53 interaction

    NARCIS (Netherlands)

    Christner, Susan M; Clausen, Dana M; Beumer, Jan H; Parise, Robert A; Guo, Jianxia; Huang, Yijun; Dömling, Alexander S; Eiseman, Julie L

    2015-01-01

    PURPOSE: The interaction of p53 with its negative regulators Mdm2/4 has been widely studied (Khoury and Domling in Curr Pharm Des 18(30):4668-4678, 2012). In p53(+/+) cells, expression of Mdm2/4 leads to p53 turnover, inhibition of downstream transcription, decreasing cell cycle arrest, or apoptosis

  9. P53 Gene Mutation and Expression of MDM2, P53, P16 Protein and their Relationship in Human Glioma

    Institute of Scientific and Technical Information of China (English)

    CUI Wen; WU Renliang; CAO Huiling; GAO Jifa; WANG Xu; REN Qiwei

    2005-01-01

    To investigate the effect of P53 protein accumulation and p53 gene mutation in the pathogenesis of glioma and to study the role of MDM2, P53 and P16 protein in glioma formation and progression and their relationship with each other, LSAB immunohistochemical staining method and non-isotopic PCR-SSCP techniques were used to detect the expression of MDM2, P53 and P16 pro tein and p53 gene mutation in 48 cases of gliomas. The results showed that the positive expression rate of MDM2, P53 and the negative rate of P16 was 22.9 %, 41.7 % and 60.4 %, respectively.The latter two in high grade (grade Ⅲ , Ⅳ) gliomas had a significantly higher rate than in the low grade (grade Ⅱ ) gliomas. Moreover, the co-expression of MDM2 and P53 protein was confirmed in only 1 of 48 cases. No significant difference was found in the rate of the expression of MDM2 between high grade and low grade gliomas (P>0.1) . PCR SSCP results showed that mutation of 5-8 exons of p53 gene was detected in 17 out of 48 cases (35.42 %) . Mutation was detected in 16of 20 cases of positive p53 expression, and another one was detected in 28 cases of negative expression cases. The correlation between p53 mutation and p53 immunopositivity was observed in 89.6% of the cases. P53 gene mutation and the level of MDM2, P53 and P16 protein were not related to age, gender of the patients, tumor location and size. It is concluded that the mutation of p53 and deletion of p16 might play important roles in the tumorigenesis of gliomas and it was significantly associated with the grade of tumor differentiation. P53 protein accumulation can indirectly reflect p53 mutation. MDM2 amplification and overexpression might be an early event in the growth of human gliomas.

  10. Regulation of p21ras activity

    DEFF Research Database (Denmark)

    Lowy, D R; Zhang, K; DeClue, J E

    1992-01-01

    The ras genes encode GTP/GDP-binding proteins that participate in mediating mitogenic signals from membrane tyrosine kinases to downstream targets. The activity of p21ras is determined by the concentration of GTP-p21ras, which is tightly regulated by a complex array of positive and negative control...... mechanisms. GAP and NF1 can negatively regulate p21ras activity by stimulating hydrolysis of GTP bound to p21ras. Other cellular factors can positively regulate p21ras by stimulating GDP/GTP exchange....

  11. Influence of the MDM2 single nucleotide polymorphism SNP309 on tumour development in BRCA1 mutation carriers

    Directory of Open Access Journals (Sweden)

    Johnson Peter W

    2006-03-01

    Full Text Available Abstract Background The MDM2 gene encodes a negative regulator of the p53 tumour suppressor protein. A single nucleotide polymorphism (SNP in the MDM2 promoter (a T to G exchange at nucleotide 309 has been reported to produce accelerated tumour formation in individuals with inherited p53 mutations. We have investigated the effect of the MDM2 SNP309 on clinical outcome in a cohort of patients with germline mutations of BRCA1. Methods Genomic DNA was obtained for 102 healthy controls and 116 patients with established pathogenic mutations of BRCA1 and Pyrosequencing technology™ was used to determine the genotype at the MDM2 SNP309 locus. Results The polymorphism was present in 52.9% of the controls (G/T in 37.3% and G/G in 15.6% and 58.6% of the BRCA1 mutation carriers (47.4% G/T and 11.2% G/G. Incidence of malignancy in female BRCA1 carriers was not significantly higher in SNP309 carriers than in wildtype (T/T individuals (72.7% vs. 75.6%, p = 1.00. Mean age of diagnosis of first breast cancer was 41.2 years in the SNP309 G/G genotype carriers, 38.6 years in those with the SNP309 G/T genotype and 39.0 years in wildtype subjects (p = 0.80. Conclusion We found no evidence that the MDM2 SNP309 accelerates tumour development in carriers of known pathogenic germline mutations of BRCA1.

  12. HIF-1α, MDM2, CDK4, and p16 expression in ischemic fasciitis, focusing on its ischemic condition.

    Science.gov (United States)

    Yamada, Yuichi; Kinoshita, Izumi; Kohashi, Kenichi; Yamamoto, Hidetaka; Kuma, Yuki; Ito, Takamichi; Koda, Kenji; Kisanuki, Atsushi; Kurosawa, Manabu; Yoshimura, Michiko; Furue, Masutaka; Oda, Yoshinao

    2017-07-01

    Ischemic fasciitis is a benign myofibroblastic lesion, occurring in the sacral region or proximal thigh of elderly or bedridden individuals. The pathogenesis of ischemic fasciitis is thought to be based on ischemic condition; however, it has never been demonstrated. In this study, we examined the expression of ischemia-associated proteins in ischemic fasciitis by immunohistochemical and genetic methods. Specifically, this study aimed to reveal the expression of HIF-1α, MDM2, CDK4, p16, and gene amplification of MDM2 gene. Seven cases of ischemic fasciitis from among the soft-tissue tumors registered at our institution were retrieved. Histopathological findings were as follows: poorly demarcated nodular masses, a proliferation of spindle-shaped fibroblastic or myofibroblastic cells with oval nuclei and eosinophilic or pale cytoplasm, zonal fibrinous deposition, pseudocystic degeneration, granulation-like proliferation of capillary vessels, ganglion-like cells, myxoid or hyalinized stroma, and chronic inflammatory infiltration. Immunohistochemically, the spindle cells were positive for HIF-1α (7/7 cases), MDM2 (4/7 cases), CDK4 (4/7 cases), p16 (7/7 cases), p53 (2/7 case), cyclin D1 (7/7 cases), and alpha-smooth muscle actin (6/7 cases). Neither MDM2 gene amplification nor USP6 gene split signal was detected in any case. Overexpression of the above proteins may be associated with the pathogenic mechanism of ischemic fasciitis. It is noted that the immunohistochemical positivity of MDM2, CDK4, and p16 do not necessarily indicate malignant neoplasm such as dedifferentiated liposarcoma.

  13. The role of NPM, p14arf and MDM2 in precursors of bronchial squamous cell carcinoma.

    Science.gov (United States)

    Mascaux, C; Bex, F; Martin, B; Burny, A; Haller, A; Paesmans, M; Willard-Gallo, K; Ninane, V; Sculier, J-P

    2008-09-01

    Murine double minute clone 2 (MDM2), p14 alternate reading frame (p14arf), and nucleophosmin (NPM) regulate p53 activity. A total of 200 biopsies, including normal bronchial, pre-invasive and invasive tissues, were examined for changes in NPM, p14arf, MDM2 and p53 expression patterns by immunohistochemistry and immunofluorescence with confocal microscopy. NPM and p14arf displayed a diffuse nuclear staining in most normal bronchial tissue. The fraction of biopsies displaying an increased MDM2 staining or a nucleolar relocalisation of NPM increased at mild and moderate dysplasia, respectively. Two different modifications occurred in p14arf expression, i.e. its loss or its nucleolar relocalisation, both increasing at severe dysplasia and both being associated with high MDM2 expression. In addition, the nucleolar relocalisation of p14arf was associated with that of NPM. Immunofluorescence staining indicated that NPM and p14arf either co-localised in the nucleoplasm or in the nucleoli, before and as a result of severe dysplasia, respectively. MDM2 was not detected in the nucleoli. Thus, changes occur in murine double minute clone 2, p14 alternate reading frame and nucleophosmin level of expression and/or cellular distribution during early steps of lung carcinogenesis. Their relative localisation as determined by immunofluorescence, supports the hypothesis that p14 alternate reading frame nucleolar relocalisation impairs p14 alternate reading frame-murine double minute clone 2 complex formation and that nucleophosmin might sequester p14 alternate reading frame. The demonstration of this hypothesis requires further functional studies.

  14. The MDM-2 Antagonist Nutlin-3 Promotes the Maturation of Acute Myeloid Leukemic Blasts

    Directory of Open Access Journals (Sweden)

    Paola Secchiero

    2007-10-01

    Full Text Available The small-molecule inhibitor of murine double minute (MDM-2, Nutlin-3, induced variable apoptosis in primary acute myeloid leukemia (AML blasts, promoted myeloid maturation of surviving cells, as demonstrated by analysis of CD11 b, CD14 surface antigens, by morphologic examination. Although the best-characterized activity of Nutlin-3 is activation of the p53 pathway, Nutlin-3 induced maturation also in one AML sample characterized by p53 deletion, as well as in the p53-/- human myeloblastic HL-60 cell line. At the molecular level, the maturational activity of Nutlin-3 in HL-60 cells was accompanied by the induction of E2F1 transcription factor, it was significantly counteracted by specific gene knockdown with small interfering RNA for E2F1. Moreover, Nutlin-3, as well as tumor necrosis factor (TNF α, potentiated the maturational activity of recombinant TNF-related apoptosis-inducing lig, (TRAIL in HL-60 cells. However, although TNF-α significantly counteracted the proapoptotic activity of TRAIL, Nutlin-3 did not interfere with the proapoptotic activity of TRAIL. Taken together, these data disclose a novel, potentially relevant therapeutic role for Nutlin-3 in the treatment of both p53 wild-type, p53-/- AML, possibly in association with recombinant TRAIL.

  15. MOXA推出支持MDM 2.0的Rcore平台

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Moxa在所有嵌入式计算机中都预装了操作系统,Moxa同时提供中间件、范例程序和开发工具,为编程人员带来了友善的应用软件开发环境,减少系统集成所需的开发时间和人力。Moxa Device Manager 2.0(MDM 2.0)作为简单易用的远程管理工具,可通过浏览器管理Moxa立即可用系列嵌八式计算机。Moxa的嵌入式计算机为现场数据采集和工业级控制应用提供了优越的前端控制主机;但这些前端主机通常位于远程场所。MDM就是为了让系统管理者更简单且快速地管理他们的远程嵌入式计算机而设计。

  16. 结肠腺癌患者癌组织MDM2、TBX2和Pokemon表达及相关性

    Institute of Scientific and Technical Information of China (English)

    崔婷; 魏强

    2014-01-01

    目的:分析结肠腺癌患者癌组织TBX2、MDM2和Pokemon表达及相关性。方法结肠腺癌患者119例为观察组,留取术后新鲜组织。收集距肿瘤边缘>5 cm的正常结肠黏膜新鲜组织78例为对照组。应用流式细胞术检测 TBX2、MDM2和 Pokemon表达。结果观察组 TBX2、MDM2和Pokemon表达量明显高于对照组(P<0.0001),观察组TBX2、MDM2和Pokemon表达与肿瘤分化程度、浸润深度、临床分期和Ki67表达密切相关。观察组TBX2和MDM2表达正相关,而TBX2和Pokemon、MDM2和Pokemon未见明显相关性。结论结肠腺癌患者术后组织TBX2、MDM2和Pokemon异常表达对肿瘤性病变的发生和进展有重要促进作用。

  17. Disruption of the 5S RNP-Mdm2 interaction significantly improves the erythroid defect in a mouse model for Diamond-Blackfan anemia.

    Science.gov (United States)

    Jaako, P; Debnath, S; Olsson, K; Zhang, Y; Flygare, J; Lindström, M S; Bryder, D; Karlsson, S

    2015-11-01

    Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2(C305F) knock-in mice that have a disrupted 5S RNP-Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2(C305F) reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP-Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP-Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

  18. Ribosomal protein S7 regulates arsenite-induced GADD45α expression by attenuating MDM2-mediated GADD45α ubiquitination and degradation.

    Science.gov (United States)

    Gao, Ming; Li, Xiaoguang; Dong, Wen; Jin, Rui; Ma, Hanghang; Yang, Pingxun; Hu, Meiru; Li, Yi; Hao, Yi; Yuan, Shengtao; Huang, Junjian; Song, Lun

    2013-05-01

    The stress-responding protein, GADD45α, plays important roles in cell cycle checkpoint, DNA repair and apoptosis. In our recent study, we demonstrate that GADD45α undergoes a dynamic ubiquitination and degradation in vivo, which process can be blocked by the cytotoxic reagent, arsenite, resulting in GADD45α accumulation to activate JNKs cell death pathway, thereby revealing a novel mechanism for the cellular GADD45α functional regulation. But the factors involved in GADD45α stability modulations are unidentified. Here, we demonstrated that MDM2 was an E3 ubiquitin ligase for GADD45α. One of MDM2-binding partner, ribosomal protein S7, interacted with and stabilized GADD45α through preventing the ubiquitination and degradation of GADD45α mediated by MDM2. This novel function of S7 is unrelated to p53 but seems to depend on S7/MDM2 interaction, for the S7 mutant lacking MDM2-binding ability lost its function to stabilize GADD45α. Further investigations indicated that arsenite treatment enhanced S7-MDM2 interaction, resulting in attenuation of MDM2-dependent GADD45α ubiquitination and degradation, thereby leading to GADD45α-dependent cell death pathway activation. Silencing S7 expression suppressed GADD45α-dependent cytotoxicity induced by arsenite. Our findings thus identify a novel function of S7 in control of GADD45α stabilization under both basal and stress conditions and its significance in mediating arsenite-induced cellular stress.

  19. NF-kappaB inhibits T-cell activation-induced, p73-dependent cell death by induction of MDM2.

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    Busuttil, Valere; Droin, Nathalie; McCormick, Laura; Bernassola, Francesca; Candi, Eleonora; Melino, Gerry; Green, Douglas R

    2010-10-19

    NF-κB is a key transcription factor involved in the regulation of T-cell activation and proliferation upon engagement of the T-cell receptor (TCR). T cells that lack the IκB kinase (IKKβ) are unable to activate NF-κB, and rapidly undergo apoptosis upon activation. NF-κB activation following T-cell receptor engagement induces the expression of Mdm2 through interaction with NF-κB sites in its P1 promoter, and enforced expression of Mdm2 protected T cells deficient for NF-κB activation from activation-induced cell death. In T cells with intact NF-κB signaling, ablation or pharmacologic inhibition of Mdm2 resulted in activation-induced apoptosis. Mdm2 coprecipitates with p73 in activated T cells, and apoptosis induced by inhibition of Mdm2 was p73-dependent. Further, Bim was identified as a p73 target gene required for cell death induced by Mdm2 inhibition, and a p73-responsive element in intron 1 of Bim was characterized. Our results demonstrate a pathway for survival of activated T cells through NF-κB-induced Mdm2, which blocks Bim-dependent apoptosis through binding and inhibition of p73.

  20. Non-degradative ubiquitination of the Notch1 receptor by the E3 ligase MDM2 activates the Notch signalling pathway.

    Science.gov (United States)

    Pettersson, Susanne; Sczaniecka, Matylda; McLaren, Lorna; Russell, Fiona; Gladstone, Karen; Hupp, Ted; Wallace, Maura

    2013-03-15

    The Notch receptor is necessary for modulating cell fate decisions throughout development, and aberrant activation of Notch signalling has been associated with many diseases, including tumorigenesis. The E3 ligase MDM2 (murine double minute 2) plays a role in regulating the Notch signalling pathway through its interaction with NUMB. In the present study we report that MDM2 can also exert its oncogenic effects on the Notch signalling pathway by directly interacting with the Notch 1 receptor through dual-site binding. This involves both the N-terminal and acidic domains of MDM2 and the RAM [RBP-Jκ (recombination signal-binding protein 1 for Jκ)-associated molecule] and ANK (ankyrin) domains of Notch 1. Although the interaction between Notch1 and MDM2 results in ubiquitination of Notch1, this does not result in degradation of Notch1, but instead leads to activation of the intracellular domain of Notch1. Furthermore, MDM2 can synergize with Notch1 to inhibit apoptosis and promote proliferation. This highlights yet another target for MDM2-mediated ubiquitination that results in activation of the protein rather than degradation and makes MDM2 an attractive target for drug discovery for both the p53 and Notch signalling pathways.

  1. Expression of MDM2 mRNA, MDM2, P53 and P16 Proteins in Urothelial Lesions in the View of the WHO 4th Edition Guidelines as A Molecular Insight towards Personalized Medicine

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    Olfat Hammam

    2017-08-01

    Full Text Available AIM: Here we imposed a multimarker molecular panel composed of P53, MDM2 protein & mRNA & P16 with the identification of sensitive and specific cut offs among the Egyptian urothelial carcinomas bilharzial or not emphasize the pathological and molecular classifications, pathways and prognosis as a privilege for adjuvant therapy. METHODS: Three hundred and ten urothelial lesions were pathologically evaluated and grouped as follows: 50 chronic cystitis as benign, 240 urothelial carcinomas and 20 normal bladder tissue as a control. Immunohistochemistry for MDM Protein, P16 & p53 and In Situ Hybridization for MDM2mRNA were done. RESULTS: MDM2mRNA overexpression correlated with low grade low stage non invasive tumors, while P53 > 40% & p16 40% & P16 10% from high grade, high stage invasive urothelial carcinomas (with p53 > 40, p16 40 & p16 < 10%, together with the histopathological features can distinguish in situ urothelial lesions from dysplastic and atypical lesions.

  2. Differential diagnosis of atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma: utility of p16 in combination with MDM2 and CDK4 immunohistochemistry.

    Science.gov (United States)

    Kammerer-Jacquet, Solène-Florence; Thierry, Sixte; Cabillic, Florian; Lannes, Morgane; Burtin, Florence; Henno, Sébastien; Dugay, Frédéric; Bouzillé, Guillaume; Rioux-Leclercq, Nathalie; Belaud-Rotureau, Marc-Antoine; Stock, Nathalie

    2017-01-01

    The differential diagnosis between atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDLPS) and dedifferentiated liposarcoma (DDLPS) from their morphologic counterparts is challenging. Currently, the diagnosis is guided by MDM2 and CDK4 immunohistochemistry (IHC) and is confirmed by the amplification of the corresponding genes. Recently, p16 IHC has been proposed as a useful diagnostic biomarker. The objective was to assess the utility of p16 IHC in the differential diagnosis of ALT/WDLPS and DDLPS. Our series included 101 tumors that were previously analyzed using fluorescence in situ hybridization for MDM2 and CDK4 amplification. We compared sensitivity and specificity of p16 IHC to MDM2 and CDK4 IHC in the differential diagnosis of ALT-WDLPS (n=19) versus benign adipocytic tumors (n=44) and DDLPS (n=18) versus mimicking sarcomas (n=20). In the differential diagnosis of ALT-WDLPS, p16 had a sensitivity of 89.5% but a specificity of 68.2%, which was impaired by false-positive lipomas with secondary changes, especially in biopsies. Likewise, in the differential diagnosis of DDLPS, p16 had a sensitivity of 94.4% and a specificity of 70%, which hampered its use as a single marker. However, adding p16 to MDM2 and/or CDK4 increased diagnostic specificity. Indeed, MDM2+/p16+ tumors were all ALT-WDLPS, and MDM2-/p16- tumors were all benign adipocytic tumors. Moreover, all MDM2+/CDK4+/p16+ tumors were DDLPS, and the MDM2-/CDK4-/p16- tumor was an undifferentiated sarcoma. Although the use of p16 as a single immunohistochemical marker is limited by its specificity, its combination with MDM2 and CDK4 IHC may help discriminate ALT-WDLPS/DDLPS.

  3. MDM2 Inhibitor, Nutlin 3a, Induces p53 Dependent Autophagy in Acute Leukemia by AMP Kinase Activation.

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    Gautam Borthakur

    Full Text Available MDM2 (mouse double minute 2 inhibitors that activate p53 and induce apoptosis in a non-genotoxic manner are in clinical development for treatment of leukemias. P53 can modulate other programmed cell death pathways including autophagy both transcriptionally and non-transcriptionally. We investigated autophagy induction in acute leukemia by Nutlin 3a, a first-in-class MDM2 inhibitor. Nutlin 3a induced autophagy in a p53 dependent manner and transcriptional activation of AMP kinase (AMPK is critical, as this effect is abrogated in AMPK -/- mouse embryonic fibroblasts. Nutlin 3a induced autophagy appears to be pro-apoptotic as pharmacological (bafilomycin or genetic inhibition (BECLIN1 knockdown of autophagy impairs apoptosis induced by Nutlin 3a.

  4. Design, Synthesis and Biological Evaluation of Sulfamide and Triazole Benzodiazepines as Novel p53-MDM2 Inhibitors

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    Zhiliang Yu

    2014-09-01

    Full Text Available A series of sulfamide and triazole benzodiazepines were obtained with the principle of bioisosterism. The p53-murine double minute 2 (MDM2 inhibitory activity and in vitro antitumor activity were evaluated. Most of the novel benzodiazepines exhibited moderate protein binding inhibitory activity. Particularly, triazole benzodiazepines showed good inhibitory activity and antitumor potency. Compound 16 had promising antitumor activity against the U-2 OS human osteosarcoma cell line with an IC50 value of 4.17 μM, which was much better than that of nutlin-3. The molecular docking model also successfully predicted that this class of compounds mimicked the three critical residues of p53 binding to MDM2.

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

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    Jayoung Kim

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

  6. A p53-independent role for the MDM2 antagonist Nutlin-3 in DNA damage response initiation

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    Kumar Sonia

    2011-02-01

    Full Text Available Abstract Background The mammalian DNA-damage response (DDR has evolved to protect genome stability and maximize cell survival following DNA-damage. One of the key regulators of the DDR is p53, itself tightly regulated by MDM2. Following double-strand DNA breaks (DSBs, mediators including ATM are recruited to the site of DNA-damage. Subsequent phosphorylation of p53 by ATM and ATM-induced CHK2 results in p53 stabilization, ultimately intensifying transcription of p53-responsive genes involved in DNA repair, cell-cycle checkpoint control and apoptosis. Methods In the current study, we investigated the stabilization and activation of p53 and associated DDR proteins in response to treatment of human colorectal cancer cells (HCT116p53+/+ with the MDM2 antagonist, Nutlin-3. Results Using immunoblotting, Nutlin-3 was observed to stabilize p53, and activate p53 target proteins. Unexpectedly, Nutlin-3 also mediated phosphorylation of p53 at key DNA-damage-specific serine residues (Ser15, 20 and 37. Furthermore, Nutlin-3 induced activation of CHK2 and ATM - proteins required for DNA-damage-dependent phosphorylation and activation of p53, and the phosphorylation of BRCA1 and H2AX - proteins known to be activated specifically in response to DNA damage. Indeed, using immunofluorescent labeling, Nutlin-3 was seen to induce formation of γH2AX foci, an early hallmark of the DDR. Moreover, Nutlin-3 induced phosphorylation of key DDR proteins, initiated cell cycle arrest and led to formation of γH2AX foci in cells lacking p53, whilst γH2AX foci were also noted in MDM2-deficient cells. Conclusion To our knowledge, this is the first solid evidence showing a secondary role for Nutlin-3 as a DDR triggering agent, independent of p53 status, and unrelated to its role as an MDM2 antagonist.

  7. Polymorphisms at p53, p73, and MDM2 loci modulate the risk of tobacco associated leukoplakia and oral cancer.

    Science.gov (United States)

    Misra, Chaitali; Majumder, Mousumi; Bajaj, Swati; Ghosh, Saurabh; Roy, Bidyut; Roychoudhury, Susanta

    2009-09-01

    Polymorphisms at loci controlling cellular processes such as cell cycle, DNA repair, and apoptosis may modulate the risk of cancer. We examined the association of two linked polymorphisms (G4C14-A4T14) at p73 and one polymorphism (309G > T) at MDM2 promoter with the risk of leukoplakia and oral cancer. The p73 and MDM2 genotypes were determined in 197 leukoplakia patients, 310 oral cancer patients and in 348 healthy control subjects. The p73 GC/AT genotype increased the risk of leukoplakia (OR = 1.6, 95% CI = 1.1-2.3) and oral cancer (OR = 2.4, 95% CI = 1.7-3.3) but the 309G > T MDM2 polymorphism independently could not modify the risk of any of the diseases. Stratification of the study population into subgroups with different tobacco habits showed that the risk of the oral cancer is not modified further for the individuals carrying p73 risk genotype. However, leukoplakia patients with smokeless tobacco habit showed increased risk with combined GC/AT and AT/AT (OR = 3.0, 95% CI = 1.3-7.0) genotypes. A combined analysis was done with our previous published data on p53 codon 72 pro/arg polymorphism. Analysis of pair wise genotype combinations revealed increase in risk for specific p73-MDM2 and p73-p53 genotype combinations. Finally, the combined three loci analyses revealed that the presence of at least one risk allele at all three loci increases the risk of both leukoplakia and oral cancer.

  8. MDM2 antagonists synergize with PI3K/mTOR inhibition in well-differentiated/dedifferentiated liposarcomas.

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    Laroche, Audrey; Chaire, Vanessa; Algeo, Marie-Paule; Karanian, Marie; Fourneaux, Benjamin; Italiano, Antoine

    2017-08-15

    Well-differentiated/dedifferentiated liposarcoma (WDLPS/DDLPS) are characterized by a consistent amplification of the MDM2 gene. The PI3K/AKT/mTOR pathway has been suggested to play also an important role in their tumorigenesis. Our goal was to determine whether combined MDM2 and PI3K/AKT/mTOR targeting is associated with higher anti-tumor activity than single agent alone in preclinical models of WDLPS/DDLPS. WDLPS/DDLPS cells were exposed to RG7388 (MDM2 antagonist) and BEZ235 (PI3K/mTOR dual inhibitor) after which apoptosis and signaling/survival pathway perturbations were monitored by flow cytometry and Western blot analysis. Xenograft mouse models were used to assess tumor growth and animal survival. Western blotting, histopathology, and tumor volume evolution were used for the assessment of treatment efficacy. The PI3K/AKT/mTOR was upregulated in up to 81% of the human WDLPS/DDLPS samples analysed. Treatment with RG7388 and BEZ235 resulted in a greater tumor activity than either drug alone with a significant difference in terms of cell viability after 72h of treatment with RG-73888 alone, BEZ235 alone and a combination of both agents. Consistent with these observations, we found a significant increase in apoptosis with the combination versus the single agent treatment alone. We then analysed the in vivo antitumor activity of RG7388 and BEZ235 in a xenograft model of DDLPS. The combination regimen significantly reduced tumor growth rate in comparison with single agent alone. Our results represent the first in vivo evidence of synergy between MDM2 and PI3K/AKT/mTOR antagonists and represent a strong rationale to evaluate the therapeutic potential of such a combination in WDLPS/DDLPS.

  9. The pharmacodynamics of the p53-Mdm2 targeting drug Nutlin: the role of gene-switching noise.

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    Krzysztof Puszynski

    2014-12-01

    Full Text Available In this work we investigate, by means of a computational stochastic model, how tumor cells with wild-type p53 gene respond to the drug Nutlin, an agent that interferes with the Mdm2-mediated p53 regulation. In particular, we show how the stochastic gene-switching controlled by p53 can explain experimental dose-response curves, i.e., the observed inter-cell variability of the cell viability under Nutlin action. The proposed model describes in some detail the regulation network of p53, including the negative feedback loop mediated by Mdm2 and the positive loop mediated by PTEN, as well as the reversible inhibition of Mdm2 caused by Nutlin binding. The fate of the individual cell is assumed to be decided by the rising of nuclear-phosphorylated p53 over a certain threshold. We also performed in silico experiments to evaluate the dose-response curve after a single drug dose delivered in mice, or after its fractionated administration. Our results suggest that dose-splitting may be ineffective at low doses and effective at high doses. This complex behavior can be due to the interplay among the existence of a threshold on the p53 level for its cell activity, the nonlinearity of the relationship between the bolus dose and the peak of active p53, and the relatively fast elimination of the drug.

  10. Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Eysturskard, Jonhard; Nielsen, Peter E

    2010-01-01

    ABSTRACT: BACKGROUND: Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human ca...... method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs.......ABSTRACT: BACKGROUND: Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human...

  11. Monitoring p53 by MDM2 and MDMX is required for endocrine pancreas development and function in a spatio-temporal manner.

    Science.gov (United States)

    Zhang, Yiwei; Zeng, Shelya X; Hao, Qian; Lu, Hua

    2017-03-01

    Although p53 is not essential for normal embryonic development, it plays a pivotal role in many biological and pathological processes, including cell fate determination-dependent and independent events and diseases. The expression and activity of p53 largely depend on its two biological inhibitors, MDM2 and MDMX, which have been shown to form a complex in order to tightly control p53 to an undetectable level during early stages of embryonic development. However, more delicate studies using conditional gene-modification mouse models show that MDM2 and MDMX may function separately or synergistically on p53 regulation during later stages of embryonic development and adulthood in a cell and tissue-specific manner. Here, we report the role of the MDM2/MDMX-p53 pathway in pancreatic islet morphogenesis and functional maintenance, using mouse lines with specific deletion of MDM2 or MDMX in pancreatic endocrine progenitor cells. Interestingly, deletion of MDM2 results in defects of embryonic endocrine pancreas development, followed by neonatal hyperglycemia and lethality, by inducing pancreatic progenitor cell apoptosis and inhibiting cell proliferation. However, unlike MDM2-knockout animals, mice lacking MDMX in endocrine progenitor cells develop normally. But, surprisingly, the survival rate of adult MDMX-knockout mice drastically declines compared to control mice, as blockage of neonatal development of endocrine pancreas by inhibition of cell proliferation and subsequent islet dysfunction and hyperglycemia eventually lead to type 1 diabetes-like disease with advanced diabetic nephropathy. As expected, both MDM2 and MDMX deletion-caused pancreatic defects are completely rescued by loss of p53, verifying the crucial role of the MDM2 and/or MDMX in regulating p53 in a spatio-temporal manner during the development, functional maintenance, and related disease progress of endocrine pancreas. Also, our study suggests a possible mouse model of advanced diabetic nephropathy

  12. Predictive and prognostic impact of TP53 mutations and MDM2 promoter genotype in primary breast cancer patients treated with epirubicin or paclitaxel.

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    Ranjan Chrisanthar

    Full Text Available BACKGROUND: TP53 mutations have been associated with resistance to anthracyclines but not to taxanes in breast cancer patients. The MDM2 promoter single nucleotide polymorphism (SNP T309G increases MDM2 activity and may reduce wild-type p53 protein activity. Here, we explored the predictive and prognostic value of TP53 and CHEK2 mutation status together with MDM2 SNP309 genotype in stage III breast cancer patients receiving paclitaxel or epirubicin monotherapy. EXPERIMENTAL DESIGN: Each patient was randomly assigned to treatment with epirubicin 90 mg/m(2 (n = 109 or paclitaxel 200 mg/m(2 (n = 114 every 3rd week as monotherapy for 4-6 cycles. Patients obtaining a suboptimal response on first-line treatment requiring further chemotherapy received the opposite regimen. Time from last patient inclusion to follow-up censoring was 69 months. Each patient had snap-frozen tumor tissue specimens collected prior to commencing chemotherapy. PRINCIPAL FINDINGS: While TP53 and CHEK2 mutations predicted resistance to epirubicin, MDM2 status did not. Neither TP53/CHEK2 mutations nor MDM2 status was associated with paclitaxel response. Remarkably, TP53 mutations (p = 0.007 but also MDM2 309TG/GG genotype status (p = 0.012 were associated with a poor disease-specific survival among patients having paclitaxel but not patients having epirubicin first-line. The effect of MDM2 status was observed among individuals harbouring wild-type TP53 (p = 0.039 but not among individuals with TP53 mutated tumors (p>0.5. CONCLUSION: TP53 and CHEK2 mutations were associated with lack of response to epirubicin monotherapy. In contrast, TP53 mutations and MDM2 309G allele status conferred poor disease-specific survival among patients treated with primary paclitaxel but not epirubicin monotherapy.

  13. Induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway delays the initiation but fails to eradicate established murine acute myeloid leukemia.

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    Jaako, P; Ugale, A; Wahlestedt, M; Velasco-Hernandez, T; Cammenga, J; Lindström, M S; Bryder, D

    2017-01-01

    Mutations resulting in constitutive activation of signaling pathways that regulate ribosome biogenesis are among the most common genetic events in acute myeloid leukemia (AML). However, whether ribosome biogenesis presents as a therapeutic target to treat AML remains unexplored. Perturbations in ribosome biogenesis trigger the 5S ribonucleoprotein particle (RNP)-Mdm2-p53 ribosomal stress pathway, and induction of this pathway has been shown to have therapeutic efficacy in Myc-driven lymphoma. In the current study we address the physiological and therapeutic role of the 5S RNP-Mdm2-p53 pathway in AML. By utilizing mice that have defective ribosome biogenesis due to downregulation of ribosomal protein S19 (Rps19), we demonstrate that induction of the 5S RNP-Mdm2-p53 pathway significantly delays the initiation of AML. However, even a severe Rps19 deficiency that normally results in acute bone marrow failure has no consistent efficacy on already established disease. Finally, by using mice that harbor a mutation in the Mdm2 gene disrupting its binding to 5S RNP, we show that loss of the 5S RNP-Mdm2-p53 pathway is dispensable for development of AML. Our study suggests that induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway holds limited potential as a single-agent therapy in the treatment of AML.

  14. MDM2 promoter polymorphism and p53 codon 72 polymorphism in chronic myeloid leukemia: the association between MDM2 promoter genotype and disease susceptibility, age of onset, and blast-free survival in chronic phase patients receiving imatinib.

    Science.gov (United States)

    Liu, Yi-Chang; Hsiao, Hui-Hua; Yang, Wen-Chi; Liu, Ta-Chih; Chang, Chao-Sung; Yang, Ming-Yu; Lin, Pai-Mei; Hsu, Jui-Feng; Lee, Ching-Ping; Lin, Sheng-Fung

    2014-12-01

    The genetic or functional inactivation of the p53 pathway plays an important role with regards to disease progression from the chronic phase (CP) to blast phase (BP) and imatinib treatment response in chronic myeloid leukemia (CML). Two functional single nucleotide polymorphisms (SNPs), p53 R72P and MDM2 SNP309, are associated with alternation of p53 activity, however the association regarding CML susceptibility and BP transformation under imatinib treatment is unclear. The MDM2 SNP309 genotype was determined by polymerase chain reaction-restriction fragment length polymorphism and confirmed by direct sequencing from 116 CML patients, including 104 in the CP at diagnosis, and 162 healthy Taiwanese controls. The p53 R72P polymorphism was examined in all CML patients. The SNP309 G/G genotype was associated with an increased risk of CML susceptibility (OR: 1.82, 95% CI: 1.03-3.22, P = 0.037), and an earlier age of disease onset (log-rank P = 0.005) compared with the T/T + T/G genotypes. Higher MDM2 mRNA expression was found in G/G genotype compared with T/T (P = 0.034) and T/T + T/G (P = 0.056) genotypes. No associations were found between the p53 R72P genotypes and clinical parameters and survival outcomes. Among 62 CP patients receiving imatinib as first-line therapy, the G/G genotype was associated with a shorter blast-free survival (log-rank P = 0.048) and more clonal evolution compared with the T/T + T/G genotypes. In patients with advanced diseases at diagnosis, the G/G genotype was associated with a poor overall survival (log-rank P = 0.006). Closely monitoring CML patients harboring the G/G genotype and further large-scale studies are warranted.

  15. Effects of the single nucleotide polymorphism at MDM2 309 on breast cancer patients with/without BRCA1/2 mutations

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    Sharon Nir

    2009-02-01

    Full Text Available Abstract Background A germ line single nucleotide polymorphism (SNP in the first intron of the gene encoding MDM2 at position 309, an important modulator of p53, has been described. BRCA1/2 mutation have been associated with increased rates of breast cancers with mutated P53. It was shown that the presence of MDM2 309 SNP correlated with younger cancer onset age in individuals with a p53 mutations. The differential effects of this SNP were also linked to estrogen receptor activation. Here we report on our study of 453 Ashkenazi breast cancer patients of whom 180 were positive for the known Ashkenazi BRCA1/2 mutations Methods DNA from breast cancer patients was obtained for analysis of one of the three common BRCA1/2 mutations and MDM2 SNP309. Data regarding cancer onset and death ages was obtained from our database and Statistical analysis was performed using the SPSS® statistical package (SPCC Inc., Chicago, IL, and JMP® software (SAS Institute, Cary, NC. Results The percentage of MDM2 SNP309 in control and BRCA 1/2 population which is similar to that reported for other Jewish Ashkenazi populations at 52.2% for the heterozygotes and 25.0% for MDM2SNP309G/G and 22.8% for MDM2SNP309T/T. There was not a statistical significant difference in median age of disease onset in the different MDM2 SNP309 subgroups of the BRCA1/2 carriers. When we further divided the group into under and above 51 years old ( presumed menopause age in the BRCA1 positive subset we found that there were less patients of the MDM2SNP309 G/G versus the MDM2SNP309 T/T in the over 51 patient group (p = 0.049. This result has been obtained in a relatively small subgroup and is of borderline statistical significance. Interestingly, in the BRCA1/2 mutation carriers, we found a survival advantage for patients harboring the SNP309 G/G genotype (p = 0.0086 but not for the 272 patients not harbouring this mutations. Conclusion MDM2SNP309G/G main effect on BRCA1/2 positive mutation

  16. HMG-CoA reductase inhibitors, statins, induce phosphorylation of Mdm2 and attenuate the p53 response to DNA damage.

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    Pääjärvi, Gerd; Roudier, Emilie; Crisby, Milita; Högberg, Johan; Stenius, Ulla

    2005-03-01

    3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, statins, are widely used cholesterol-lowering drugs and have been shown to have anticancer effects in many models. We have investigated the effect of statins on Mdm2, a p53-specific ubiquitin ligase. It was found that pravastatin induced Mdm2 phosphorylation at Ser166 and at 2A10 antibody-specific epitopes in HepG2 cells, while mRNA levels were unchanged. Furthermore, pravastatin was found to induce phosphorylation of mTOR at Ser2448. Ser166 phosphorylation of Mdm2 was abrogated by an inhibitor of mTOR, rapamycin, but not by the PI3-kinase inhibitors LY294002 and wortmannin. Ser166 phosphorylation of Mdm2 has been associated to active Mdm2 and has been shown to increase its ubiquitin ligase activity and lead to increased p53 degradation. Our data show that statins attenuated the p53 response to DNA damage. Thus, in HepG2 cells pravastatin and simvastatin pretreatment attenuated the p53 response to DNA damage induced by 5-fluorouracil and benzo(a)pyrene. Similar attenuation was induced when p53 stabilization was induced by the inhibitor of nuclear export, leptomycin B. Furthermore, in the DNA-damaged cells, half-lives of Mdm2 and p53 were decreased by statins, indicating a more rapid formation of p53/Mdm2 complexes and facilitated p53 degradation. The induction of p53 responsive genes and apoptosis was attenuated. Mdm2 and p53 were also studied in vivo in rat liver employing immunohistochemistry, and it was found that constitutive Mdm2 expression was changed in livers of pravastatin-treated rats. We also show that the p53 response to a challenging dose of diethylnitrosamine was attenuated in hepatocytes in situ and in primary cultures of hepatocytes by pravastatin pretreatment. Taken together, these data indicate that statins induce an mTOR-dependent Ser166 phosphorylation of Mdm2, and this effect may attenuate the duration and intensity of the p53 response to DNA damage in hepatocytes.

  17. MDM2 SNP309 promoter polymorphism and p53 mutations in urinary bladder carcinoma stage T1

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    Olsson Hans

    2013-01-01

    Full Text Available Abstract Background Urinary bladder carcinoma stage T1 is an unpredictable disease that in some cases has a good prognosis with only local or no recurrence, but in others can appear as a more aggressive tumor with progression to more advanced stages. The aim here was to investigate stage T1 tumors regarding MDM2 promoter SNP309 polymorphism, mutations in the p53 gene, and expression of p53 and p16 measured by immunohistochemistry, and subsequently relate these changes to tumor recurrence and progression. We examined a cohort of patients with primary stage T1 urothelial carcinoma of the bladder and their tumors. Methods After re-evaluation of the original slides and exclusions, the study population comprised 141 patients, all with primary stage T1 urothelial carcinoma of the bladder. The hospital records were screened for clinical parameters and information concerning presence of histologically proven recurrence and progression. The paraffin-embedded tumor material was evaluated by immunohistochemistry. Any mutations found in the p53 gene were studied by single-strand conformation analysis and Sanger sequencing. The MDM2 SNP309 polymorphism was investigated by pyrosequencing. Multivariate analyses concerning association with prognosis were performed, and Kaplan-Meier analysis was conducted for a combination of changes and time to progression. Results Of the 141 patients, 82 had at least one MDM2 SNP309 G allele, and 53 had a mutation in the p53 gene, but neither of those anomalies was associated with a worse prognosis. A mutation in the p53 gene was associated with immunohistochemically visualized p53 protein expression at a cut-off value of 50%. In the group with p53 mutation Kaplan-Meier analysis showed higher rate of progression and shorter time to progression in patients with immunohistochemically abnormal p16 expression compared to them with normal p16 expression (p = 0.038. Conclusions MDM2 SNP309 promoter polymorphism and mutations in

  18. d-Amino acid mutation of PMI as potent dual peptide inhibitors of p53-MDM2/MDMX interactions.

    Science.gov (United States)

    Li, Xiang; Liu, Chao; Chen, Si; Hu, Honggang; Su, Jiacan; Zou, Yan

    2017-09-07

    According to the previously reported potent dual l-peptide PMI of p53-MDM2/MDMX interactions, a series of d-amino acid mutational PMI analogues, PMI-1-4, with enhanced proteolytic resistence and in vitro tumor cell inhibitory activities were reported, of which Liposome-PMI-1 showed a stronger inhibitory activity against the U87 cell lines than Nutlin-3. This d-amino acid mutation strategy may give a hand for enhancing the potential of peptide drugs. Copyright © 2017. Published by Elsevier Ltd.

  19. Immediate-early gene product ICP22 inhibits the trans-transcription activating function of P53-mdm-2

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    As a product of HSVI immediate-early gene, ICP22 is capable of interacting with various cellular tran-scriptive and regulatory molecules during viral infection so as to impact the normal cellular molecular mechanism. ICP22 expressed in transfected cells can push the cells’ entering into S phase with binding to mdm-1 promoter region and impact its trans-transcription activating effect by P53. Consequently, the MDM-2 binds to P53, and the degradation effects by the ubiquitous pathway are decreased, improving indirectly the P53 levels in cells and making the cells progress into the S phase.

  20. Immediate-early gene product ICP22 inhibits the trans-transcription activating function of P53-mdm-2

    Institute of Scientific and Technical Information of China (English)

    GUO HongXiong; CUN Wei; LIU LongDing; WANG LiChun; ZHAO HongLing; DONG ChengHong; LI QiHan

    2007-01-01

    As a product of HSVI immediate-early gene, ICP22 is capable of interacting with various cellular transcriptive and regulatory molecules during viral infection so as to impact the normal cellular molecular mechanism. ICP22 expressed in transfected cells can push the cells' entering into S phase with binding to mdm-1 promoter region and impact its trans-transcription activating effect by P53. Consequently, the MDM-2 binds to P53, and the degradation effects by the ubiquitous pathway are decreased, improving indirectly the P53 levels in cells and making the cells progress into the S phase.

  1. PKCeta enhances cell cycle progression, the expression of G1 cyclins and p21 in MCF-7 cells.

    Science.gov (United States)

    Fima, E; Shtutman, M; Libros, P; Missel, A; Shahaf, G; Kahana, G; Livneh, E

    2001-10-11

    Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. However, not much is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that the expression of PKCeta in MCF-7 cells, under the control of a tetracycline-responsive inducible promoter, enhanced cell growth and affected the cell cycle at several points. The induced expression of another PKC isoform, PKCdelta, in MCF-7 cells had opposite effects and inhibited their growth. PKCeta expression activated cellular pathways in these cells that resulted in the increased expression of the G1 phase cyclins, cyclin D and cyclin E. Expression of the cyclin-dependent kinase inhibitor p21(WAF1) was also specifically elevated in PKCeta expressing cells, but its overall effects were not inhibitory. Although, the protein levels of the cyclin-dependent kinase inhibitor p27(KIP1) were not altered by the induced expression of PKCeta, the cyclin E associated Cdk2 kinase activity was in correlation with the p27(KIP1) bound to the cyclin E complex and not by p21(WAF1) binding. PKCeta expression enhanced the removal of p27(KIP1) from this complex, and its re-association with the cyclin D/Cdk4 complex. Reduced binding of p27(KIP1) to the cyclin D/Cdk4 complex at early time points of the cell cycle also enhanced the activity of this complex, while at later time points the decrease in bound p21(WAF1) correlated with its increased activity in PKCeta-expressing cells. Thus, PKCeta induces altered expression of several cell cycle functions, which may contribute to its ability to affect cell growth.

  2. Neisseria meningitidis causes cell cycle arrest of human brain microvascular endothelial cells at S phase via p21 and cyclin G2.

    Science.gov (United States)

    Oosthuysen, Wilhelm F; Mueller, Tobias; Dittrich, Marcus T; Schubert-Unkmeir, Alexandra

    2016-01-01

    Microbial pathogens have developed several mechanisms to modulate and interfere with host cell cycle progression. In this study, we analysed the effect of the human pathogen Neisseria meningitidis on cell cycle in a brain endothelial cell line as well as in primary brain endothelial cells. We found that N.  Meningitidis causes an accumulation of cells in the S phase early at 3 and at 24 h post-infection that was paralleled by a decrease of cells in G2/M phase. Importantly, the outer membrane proteins of the colony opacity-associated (Opa) protein family as well as the Opc protein proved to trigger the accumulation of cells in the S phase. A focused cell cycle reverse transcription quantitative polymerase chain reaction-based array and integrated network analysis revealed changes in the abundance of several cell cycle regulatory mRNAs, including the cell cycle inhibitors p21(WAF1/CIP1) and cyclin G2. These alterations were reflected in changes in protein expression levels and/or relocalization in N. meningitidis-infected cells. Moreover, an increase in p21(WAF1/CIP1) expression was found to be p53 independent. Genetic ablation of p21(WAF1/CIP1) and cyclin G2 abrogated N. meningitidis-induced S phase accumulation. Finally, by measuring the levels of the biomarker 8-hydroxydeoxyguanosine and phosphorylation of the histone variant H2AX, we provide evidence that N. meningitidis induces oxidative DNA damage in infected cells.

  3. Expressions of MDM2, Livin and Caspase-3 protein and mRNA in endometrial adenocarcinomas%学位论文摘要

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Objective To investigate the relationship of the expression of MDM2,Livin and Caspase-3 protein and mRNA in the development of endometrioid adenocarcinoma (EA). Methods The expression levels of MDM2, Livin and Caspase-3 proteins and mRNA in EA tissues (n = 72), endometrial hyperplasia tissues (n = 60) and normal tissues ( n = 30) were examined by tissue microarray technique, immunohistochemistry( SP method) and in situ hybridization method. Results The positive expression rates of MDM2, Livin and Caspase-3 protein and mRNA in EA were respectively 80. 6% ( 58/72 ), 80. 6% ( 58/72 ), 33.3% ( 24/72 ) and 73.6% ( 53/72 ), 75.0% ( 54/72 ),27.8% (20/72). The positive rates of both MDM2 and Livin protein and mRNA in EA were higher than that in normal endometrium and endometrial hyperplasia( P < 0. 01 ). However, the positive rate of Caspase-3 in EA was lower than that in normal endometrium and endometrial hyperplasia( P < 0. 01 ). The positive expressions of MDM2 protein and mRNA were not related to the histological grade, FIGO stage, depth of invasion and lymph node metastasis. The positive expressions of Livin and Caspase-3 protein and mRNA were related to histological grade (P <0. 01 ,P <0.05 ), but they were not related to FIGO stage, depth of invasion and the lymph node metastasis. The expressions of MDM2, Livin and Caspase-3 protein were positively correlated with their mRNA. The expression of Livin was negatively correlated Caspase-3. Conclusion The expressions of MDM2, Livin and Caspase-3 protein and mRNA correlate with the dedvelopment and progression of EA, which may be valuable biomarkers to detect the early carcinogenesis and prognosis of EA.

  4. Effect of sodium arsenite on the gene and protein expression of p53, mdm2 and Kras in islet cells%亚砷酸钠对胰岛细胞中 p53、mdm2和Kras基因及蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    姚晓峰; 孙睿; 姜丽平; 耿成燕; 仲来福; 郑白璐; 杨光; 刘爽; 孙鲜策

    2014-01-01

    Objective To investigate the effect of sodium arsenite on p53, mdm2 and Kras expressions in islet βcells (INS-1) of rat.Methods The levels of wild type p53 (Tp53), mdm2 and Kras in sodium arsenite-treated rat islet βcells were detected by real-time PCR.The expression of mutant p53 and mdm2 protein were detected by western blot. Results After treatment with sodium arsenite, the level of Tp53 decreased, but those of mdm2、Kras increased.The protein expressions of mutant p53 and mdm2 increased.Conclusion Sodium arsenite could induce the transformation of Tp53 to mutant p53, and increase the level of mdm2 and Kras in INS-1 cells.%目的:探讨砷对大鼠胰岛β细胞(INS-1)p53、mdm2和Kras基因和蛋白表达的影响。方法荧光实时定量PCR法检测亚砷酸钠对大鼠胰岛β细胞中野生型p53(Tp53)、mdm2和Kras基因表达的影响,Western blot检测亚砷酸钠对大鼠胰岛β细胞突变型p53和mdm2蛋白表达的影响。结果亚砷酸钠作用于INS-1细胞后,Tp53基因水平降低,mdm2、Kras基因水平升高;突变型p53和mdm2蛋白表达增加。结论亚砷酸钠可使INS-1细胞中抑癌基因Tp53向突变型p53转变,癌基因mdm2和Kras的水平升高。

  5. Pitx2 expression promotes p21 expression and cell cycle exit in neural stem cells.

    Science.gov (United States)

    Heldring, Nina; Joseph, Bertrand; Hermanson, Ola; Kioussi, Chrissa

    2012-11-01

    Cortical development is a complex process that involves many events including proliferation, cell cycle exit and differentiation that need to be appropriately synchronized. Neural stem cells (NSCs) isolated from embryonic cortex are characterized by their ability of self-renewal under continued maintenance of multipotency. Cell cycle progression and arrest during development is regulated by numerous factors, including cyclins, cyclin dependent kinases and their inhibitors. In this study, we exogenously expressed the homeodomain transcription factor Pitx2, usually expressed in postmitotic progenitors and neurons of the embryonic cortex, in NSCs with low expression of endogenous Pitx2. We found that Pitx2 expression induced a rapid decrease in proliferation associated with an accumulation of NSCs in G1 phase. A search for potential cell cycle inhibitors responsible for such cell cycle exit of NSCs revealed that Pitx2 expression caused a rapid and dramatic (≉20-fold) increase in expression of the cell cycle inhibitor p21 (WAF1/Cip1). In addition, Pitx2 bound directly to the p21 promoter as assessed by chromatin immunoprecipitation (ChIP) in NSCs. Surprisingly, Pitx2 expression was not associated with an increase in differentiation markers, but instead the expression of nestin, associated with undifferentiated NSCs, was maintained. Our results suggest that Pitx2 promotes p21 expression and induces cell cycle exit in neural progenitors.

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

    Directory of Open Access Journals (Sweden)

    Mariell Pettersson

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

  7. A stapled peptide antagonist of MDM2 carried by polymeric micelles sensitizes glioblastoma to temozolomide treatment through p53 activation.

    Science.gov (United States)

    Chen, Xishan; Tai, Lingyu; Gao, Jie; Qian, Jianchang; Zhang, Mingfei; Li, Beibei; Xie, Cao; Lu, Linwei; Lu, Wuyuan; Lu, Weiyue

    2015-11-28

    Antagonizing MDM2 and MDMX to activate the tumor suppressor protein p53 is an attractive therapeutic paradigm for the treatment of glioblastoma multiforme (GBM). However, challenges remain with respect to the poor ability of p53 activators to efficiently cross the blood-brain barrier and/or blood-brain tumor barrier and to specifically target tumor cells. To circumvent these problems, we developed a cyclic RGD peptide-conjugated poly(ethylene glycol)-co-poly(lactic acid) polymeric micelle (RGD-M) that carried a stapled peptide antagonist of both MDM2 and MDMX (sPMI). The peptide-carrying micelle RGD-M/sPMI was prepared via film-hydration method with high encapsulation efficiency and loading capacity as well as ideal size distribution. Micelle encapsulation dramatically increased the solubility of sPMI, thus alleviating its serum sequestration. In vitro studies showed that RGD-M/sPMI efficiently inhibited the proliferation of glioma cells in the presence of serum by activating the p53 signaling pathway. Further, RGD-M/sPMI exerted potent tumor growth inhibitory activity against human glioblastoma in nude mouse xenograft models. Importantly, the combination of RGD-M/sPMI and temozolomide--a standard chemotherapy drug for GBM increased antitumor efficacy against glioblastoma in experimental animals. Our results validate a combination therapy using p53 activators with temozolomide as a more effective treatment for GBM.

  8. Natural product ginsenoside 25-OCH3-PPD inhibits breast cancer growth and metastasis through down-regulating MDM2.

    Directory of Open Access Journals (Sweden)

    Wei Wang

    Full Text Available Although ginseng and related herbs have a long history of utility for various health benefits, their application in cancer therapy and underlying mechanisms of action are not fully understood. Our recent work has shown that 20(S-25-methoxyl-dammarane-3β, 12β, 20-triol (25-OCH(3-PPD, a newly identified ginsenoside from Panax notoginseng, exerts activities against a variety of cancer cells in vitro and in vivo. This study was designed to investigate its anti-breast cancer activity and the underlying mechanisms of action. We observed that 25-OCH(3-PPD decreased the survival of breast cancer cells by induction of apoptosis and G1 phase arrest and inhibited the growth of breast cancer xenografts in vivo. We further demonstrated that, in a dose- and time-dependent manner, 25-OCH(3-PPD inhibited MDM2 expression at both transcriptional and post-translational levels in human breast cancer cells with various p53 statuses (wild type and mutant. Moreover, 25-OCH(3-PPD inhibited in vitro cell migration, reduced the expression of epithelial-to-mesenchymal transition (EMT markers, and prevented in vivo metastasis of breast cancer. In summary, 25-OCH(3-PPD is a potential therapeutic and anti-metastatic agent for human breast cancer through down-regulating MDM2. Further preclinical and clinical development of this agent is warranted.

  9. MDM-2和bcl-2在子宫颈癌中的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    张巍

    2009-01-01

    宫颈癌是全球妇女恶性肿瘤中仅次于乳腺癌的第2个最常见恶性肿瘤。每年全世界新增病例大约500000例。在发展中国家妇女中发病率居第1位。子宫颈癌的早期诊断是子宫颈癌防治的重要策略之一。子宫颈癌的发生涉及多因素、多基因、多环节。本研究采用免疫组化法,检测了MDM-2和bcl-2在宫颈癌中的表达,旨在探讨MDM-2和bcl-2在宫颈癌的发生、发展过程中的相互关系。

  10. Npas4 Regulates Mdm2 and thus Dcx in Experience-Dependent Dendritic Spine Development of Newborn Olfactory Bulb Interneurons

    Directory of Open Access Journals (Sweden)

    Sei-ichi Yoshihara

    2014-08-01

    Full Text Available Sensory experience regulates the development of various brain structures, including the cortex, hippocampus, and olfactory bulb (OB. Little is known about how sensory experience regulates the dendritic spine development of OB interneurons, such as granule cells (GCs, although it is well studied in mitral/tufted cells. Here, we identify a transcription factor, Npas4, which is expressed in OB GCs immediately after sensory input and is required for dendritic spine formation. Npas4 overexpression in OB GCs increases dendritic spine density, even under sensory deprivation, and rescues reduction of dendrite spine density in the Npas4 knockout OB. Furthermore, loss of Npas4 upregulates expression of the E3-ubiquitin ligase Mdm2, which ubiquitinates a microtubule-associated protein Dcx. This leads to reduction in the dendritic spine density of OB GCs. Together, these findings suggest that Npas4 regulates Mdm2 expression to ubiquitinate and degrade Dcx during dendritic spine development in newborn OB GCs after sensory experience.

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

    Science.gov (United States)

    Pettersson, Mariell; Quant, Maria; Min, Jaeki; Iconaru, Luigi; Kriwacki, Richard W; Waddell, M Brett; Guy, R Kiplin; Luthman, Kristina; Grøtli, Morten

    2015-01-01

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

  12. Induction of apoptosis in Ehrlich ascites tumour cells via p53 activation by a novel small-molecule MDM2 inhibitor - LQFM030.

    Science.gov (United States)

    da Mota, Mariana F; Cortez, Alane P; Benfica, Polyana L; Rodrigues, Bruna Dos S; Castro, Thalyta F; Macedo, Larissa M; Castro, Carlos H; Lião, Luciano M; de Carvalho, Flávio S; Romeiro, Luiz A S; Menegatti, Ricardo; Verli, Hugo; Villavicencio, Bianca; Valadares, Marize C

    2016-09-01

    The activation of the p53 pathway through the inhibition of MDM2 has been proposed as a novel therapeutic strategy against tumours. A series of cis-imidazoline analogues, termed nutlins, were reported to displace the recombinant p53 protein from its complex with MDM2 by binding to MDM2 in the p53 pocket, and exhibited an antitumour activity both in vitro and in vivo. Thus, the purpose of this study was to evaluate the antitumour properties of LQFM030 (2), a nutlin analogue created by employing the strategy of molecular simplification. LQFM030 (2) cytotoxicity was evaluated in Ehrlich ascites tumour (EAT) cells, p53 wild type, by the trypan blue exclusion test, and the mechanisms involved in EAT cell death were investigated by light and fluorescence microscopy, flow cytometry, real-time PCR and Western blotting. Our results demonstrate that LQFM030 has dose-dependent antiproliferative activity and cytotoxic activity on EAT cells, induces the accumulation of p53 protein and promotes cell cycle arrest and apoptosis. p53 gene transcription was unaffected by LQFM030 (2); however, MDM2 mRNA increased and MDM2 protein decreased. These results suggest that the small-molecule p53 activator LQFM030 (2) has the potential for further development as a novel cancer therapeutic agent. © 2016 Royal Pharmaceutical Society.

  13. Computational Studies of Difference in Binding Modes of Peptide and Non-Peptide Inhibitors to MDM2/MDMX Based on Molecular Dynamics Simulations

    Directory of Open Access Journals (Sweden)

    Yuxin Zhang

    2012-02-01

    Full Text Available Inhibition of p53-MDM2/MDMX interaction is considered to be a promising strategy for anticancer drug design to activate wild-type p53 in tumors. We carry out molecular dynamics (MD simulations to study the binding mechanisms of peptide and non-peptide inhibitors to MDM2/MDMX. The rank of binding free energies calculated by molecular mechanics generalized Born surface area (MM-GBSA method agrees with one of the experimental values. The results suggest that van der Waals energy drives two kinds of inhibitors to MDM2/MDMX. We also find that the peptide inhibitors can produce more interaction contacts with MDM2/MDMX than the non-peptide inhibitors. Binding mode predictions based on the inhibitor-residue interactions show that the π–π, CH–π and CH–CH interactions dominated by shape complimentarity, govern the binding of the inhibitors in the hydrophobic cleft of MDM2/MDMX. Our studies confirm the residue Tyr99 in MDMX can generate a steric clash with the inhibitors due to energy and structure. This finding may theoretically provide help to develop potent dual-specific or MDMX inhibitors.

  14. 树舌多糖GF对HepA瘤细胞MDM-2基因表达的影响研究

    Institute of Scientific and Technical Information of China (English)

    于赫; 于英君

    2005-01-01

    应用免疫组化技术对HepA瘤细胞中MDM-2基因的表达量进行分析,以探讨树舌多糖抗肿瘤的分子机制.目的:研究树舌多糖GF对HepA瘤细胞MDM-2表达的影响.方法:应用SP免疫组化染色技术和多媒体彩色病理图文分析系统测定HepA瘤细胞中MDM-2的表达.结果:树舌多糖组中MDM-2的表达量均显著低于模型组(P<0.01),树舌多糖组与猪苓多糖组无显著性差异.结论:初步认为树舌多糖GF可通过降低MDM-2的表达而抑制HepA瘤细胞的增殖.

  15. 膀胱癌中mdm2、p53蛋白和粘着斑激酶表达的病理意义

    Institute of Scientific and Technical Information of China (English)

    桂津; 罗金芳; 李如昌; 林梅绥; 许祖德

    2001-01-01

    目的探讨mdm2、p53蛋白和粘着斑激酶(FAK)在膀胱移行细胞癌(TCC)中的表达与肿瘤生物行为的关系.方法采用免疫组织化学方法,测定mdm2、p53蛋白和FAK在81例TCC中的表达情况.结果mdm2、p53蛋白和FAK在TCC组织中的表达明显高于癌旁粘膜,mdm2在p53阳性TCC组中的表达明显高于阴性组.结论mdm2和p53在TCC发展过程中可能具有协同作用,联合检测mdm2、p53蛋白和FAK可作为TCC有价值的诊断指标.

  16. Hydrophobic Interactions Are a Key to MDM2 Inhibition by Polyphenols as Revealed by Molecular Dynamics Simulations and MM/PBSA Free Energy Calculations.

    Science.gov (United States)

    Verma, Sharad; Grover, Sonam; Tyagi, Chetna; Goyal, Sukriti; Jamal, Salma; Singh, Aditi; Grover, Abhinav

    2016-01-01

    p53, a tumor suppressor protein, has been proven to regulate the cell cycle, apoptosis, and DNA repair to prevent malignant transformation. MDM2 regulates activity of p53 and inhibits its binding to DNA. In the present study, we elucidated the MDM2 inhibition potential of polyphenols (Apigenin, Fisetin, Galangin and Luteolin) by MD simulation and MM/PBSA free energy calculations. All polyphenols bind to hydrophobic groove of MDM2 and the binding was found to be stable throughout MD simulation. Luteolin showed the highest negative binding free energy value of -173.80 kJ/mol followed by Fisetin with value of -172.25 kJ/mol. It was found by free energy calculations, that hydrophobic interactions (vdW energy) have major contribution in binding free energy.

  17. Hydrophobic Interactions Are a Key to MDM2 Inhibition by Polyphenols as Revealed by Molecular Dynamics Simulations and MM/PBSA Free Energy Calculations.

    Directory of Open Access Journals (Sweden)

    Sharad Verma

    Full Text Available p53, a tumor suppressor protein, has been proven to regulate the cell cycle, apoptosis, and DNA repair to prevent malignant transformation. MDM2 regulates activity of p53 and inhibits its binding to DNA. In the present study, we elucidated the MDM2 inhibition potential of polyphenols (Apigenin, Fisetin, Galangin and Luteolin by MD simulation and MM/PBSA free energy calculations. All polyphenols bind to hydrophobic groove of MDM2 and the binding was found to be stable throughout MD simulation. Luteolin showed the highest negative binding free energy value of -173.80 kJ/mol followed by Fisetin with value of -172.25 kJ/mol. It was found by free energy calculations, that hydrophobic interactions (vdW energy have major contribution in binding free energy.

  18. The Heparanase Inhibitor PG545 Attenuates Colon Cancer Initiation and Growth, Associating with Increased p21 Expression

    Directory of Open Access Journals (Sweden)

    Preeti Singh

    2017-03-01

    Full Text Available Heparanase activity is highly implicated in cellular invasion and tumor metastasis, a consequence of cleavage of heparan sulfate and remodeling of the extracellular matrix underlying epithelial and endothelial cells. Heparanase expression is rare in normal epithelia, but is often induced in tumors, associated with increased tumor metastasis and poor prognosis. In addition, heparanase induction promotes tumor growth, but the molecular mechanism that underlines tumor expansion by heparanase is still incompletely understood. Here, we provide evidence that heparanase down regulates the expression of p21 (WAF1/CIP1, a cyclin-dependent kinase inhibitor that attenuates the cell cycle. Notably, a reciprocal effect was noted for PG545, a potent heparanase inhibitor. This compound efficiently reduced cell proliferation, colony formation, and tumor xenograft growth, associating with a marked increase in p21 expression. Utilizing the APC Min+/− mouse model, we show that heparanase expression and activity are increased in small bowel polyps, whereas polyp initiation and growth were significantly inhibited by PG545, again accompanied by a prominent induction of p21 levels. Down-regulation of p21 expression adds a novel feature for the emerging pro-tumorigenic properties of heparanase, while the potent p21 induction and anti-tumor effect of PG545 lends optimism that it would prove an efficacious therapeutic in colon carcinoma patients.

  19. Influences of MDM2 on epithelial mesenchymal transition of human breast cancer cells and its molecular mechanism%MDM2对人乳腺癌细胞上皮间质转化的影响及其机制研究

    Institute of Scientific and Technical Information of China (English)

    闫彩云; 仇金荣; 卢建磊; 殷咏梅

    2014-01-01

    目的:探讨鼠双微粒体2( MDM2)在上皮间质转化( EMT )过程中的作用及其分子机制。方法采用Western blotting检测人乳腺癌 MCF-7、MDA-MB-231和 MDA-MB-435细胞株中 MDM2及 EMT相关标记分子( E-cadherin、N-cadherin及Vimentin)水平;分别于MCF-7细胞中瞬时转染MDM2空质粒pcmv、MDM2过表达质粒pcmv-MDM2,MDA-MB-231细胞中瞬时转染针对MDM2三个不同靶点的干扰质粒36h后观察细胞形态,并采用Western blotting和免疫荧光检测EMT相关标记分子及MDM2的表达情况;采用Western blotting及实时定量PCR检测在MCF-7、MDA-MB-231细胞过表达MDM2后对促EMT转录因子Snail1、Twist 蛋白和mRNA水平的影响。结果 MCF-7细胞过表达MDM2后形态由鹅卵石形变为纺锤形, E-cadherin蛋白随MDM2表达水平的上调表达降低;MDA-MB-231细胞敲低MDM2后形态由长梭形变为卵圆形,Vimentin、N-cadherin蛋白随MDM2表达水平的下调表达依次降低;MCF-7及MDA-MB-231细胞过表达MDM2后Snail1、Twist的蛋白及mRNA水平均升高。结论在乳腺癌细胞株中MDM2可促进 EMT发生,其可能通过上调转录因子 Snail1和 Twist表达来实现。%Objective To explore the influence of murine double minute 2 ( MDM2) on epithelial mesenchymal transition ( EMT) of human breast cancer cells and its molecular mechanism. Methods Western blotting method was performed to examine the protein expressions of MDM2 and EMT-related proteins ( E-cadherin, N-cadherin and Vimentin) in human breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-435. The optical microscope was used to observe the morphological changes at 36th hour after transiently transfection of MCF-7 cells with empty plasmid ( pcmv) or MDM2 overexpression plasmid ( pcmv-MDM2) and MDA-MB-231 cells with three different interference plasmids. Meanwhile, the expressions of MDM2 and EMT related molecular markers were measured by Western blotting and immunofluorescence assays

  20. p16INK4a hypermethylation and p53, p16 and MDM2 protein expression in Esophageal Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Memar Bahram

    2010-04-01

    Full Text Available Abstract Background Tumor suppressor genes p53 and p16INK4a and the proto-oncogene MDM2 are considered to be essential G1 cell cycle regulatory genes whose loss of function is associated with ESCC carcinogenesis. We assessed the aberrant methylation of the p16 gene and its impact on p16INK4a protein expression and correlations with p53 and MDM2 protein expressions in patients with ESCC in the Golestan province of northeastern Iran in which ESCC has the highest incidence of cancer, well above the world average. Methods Cancerous tissues and the adjacent normal tissue obtained from 50 ESCC patients were assessed with Methylation-Specific-PCR to examine the methylation status of p16. The expression of p16, p53 and MDM2 proteins was detected by immunohistochemical staining. Results Abnormal expression of p16 and p53, but not MDM2, was significantly higher in the tumoral tissue. p53 was concomitantly accumulated in ESCC tumor along with MDM2 overexpression and p16 negative expression. Aberrant methylation of the p16INK4a gene was detected in 31/50 (62% of esophageal tumor samples, while two of the adjacent normal mucosa were methylated (P p16INK4a aberrant methylation was significantly associated with decreased p16 protein expression (P = 0.033, as well as the overexpression of p53 (P = 0.020. Conclusions p16 hypermethylation is the principal mechanism of p16 protein underexpression and plays an important role in ESCC development. It is associated with p53 protein overexpression and may influence the accumulation of abnormally expressed proteins in p53-MDM2 and p16-Rb pathways, suggesting a possible cross-talk of the involved pathways in ESCC development.

  1. p16INK4a hypermethylation and p53, p16 and MDM2 protein expression in esophageal squamous cell carcinoma.

    Science.gov (United States)

    Taghavi, Noushin; Biramijamal, Firouzeh; Sotoudeh, Masoud; Khademi, Hooman; Malekzadeh, Reza; Moaven, Omeed; Memar, Bahram; A'rabi, Azadeh; Abbaszadegan, Mohammad Reza

    2010-04-13

    Tumor suppressor genes p53 and p16INK4a and the proto-oncogene MDM2 are considered to be essential G1 cell cycle regulatory genes whose loss of function is associated with ESCC carcinogenesis. We assessed the aberrant methylation of the p16 gene and its impact on p16INK4a protein expression and correlations with p53 and MDM2 protein expressions in patients with ESCC in the Golestan province of northeastern Iran in which ESCC has the highest incidence of cancer, well above the world average. Cancerous tissues and the adjacent normal tissue obtained from 50 ESCC patients were assessed with Methylation-Specific-PCR to examine the methylation status of p16. The expression of p16, p53 and MDM2 proteins was detected by immunohistochemical staining. Abnormal expression of p16 and p53, but not MDM2, was significantly higher in the tumoral tissue. p53 was concomitantly accumulated in ESCC tumor along with MDM2 overexpression and p16 negative expression. Aberrant methylation of the p16INK4a gene was detected in 31/50 (62%) of esophageal tumor samples, while two of the adjacent normal mucosa were methylated (P p16 protein expression (P = 0.033), as well as the overexpression of p53 (P = 0.020). p16 hypermethylation is the principal mechanism of p16 protein underexpression and plays an important role in ESCC development. It is associated with p53 protein overexpression and may influence the accumulation of abnormally expressed proteins in p53-MDM2 and p16-Rb pathways, suggesting a possible cross-talk of the involved pathways in ESCC development.

  2. Fluorescence In Situ Hybridization for MDM2 Amplification as a Routine Ancillary Diagnostic Tool for Suspected Well-Differentiated and Dedifferentiated Liposarcomas: Experience at a Tertiary Center

    Directory of Open Access Journals (Sweden)

    Khin Thway

    2015-01-01

    Full Text Available Background. The assessment of MDM2 gene amplification by fluorescence in situ hybridization (FISH has become a routine ancillary tool for diagnosing atypical lipomatous tumor (ALT/well-differentiated liposarcoma and dedifferentiated liposarcoma (WDL/DDL in specialist sarcoma units. We describe our experience of its utility at our tertiary institute. Methods. All routine histology samples in which MDM2 amplification was assessed with FISH over a 2-year period were included, and FISH results were correlated with clinical and histologic findings. Results. 365 samples from 347 patients had FISH for MDM2 gene amplification. 170 were positive (i.e., showed MDM2 gene amplification, 192 were negative, and 3 were technically unsatisfactory. There were 122 histologically benign cases showing a histology:FISH concordance rate of 92.6%, 142 WDL/DDL (concordance 96.5%, and 34 cases histologically equivocal for WDL (concordance 50%. Of 64 spindle cell/pleomorphic neoplasms (in which DDL was a differential diagnosis, 21.9% showed MDM2 amplification. Of the cases with discrepant histology and FISH, all but 3 had diagnoses amended following FISH results. For discrepancies of benign histology but positive FISH, lesions were on average larger, more frequently in “classical” (intra-abdominal or inguinal sites for WDL/DDL and more frequently core biopsies. Discrepancies of malignant histology but negative FISH were smaller, less frequently in “classical” sites but again more frequently core biopsies. Conclusions. FISH has a high correlation rate with histology for cases with firm histologic diagnoses of lipoma or WDL/DDL. It is a useful ancillary diagnostic tool in histologically equivocal cases, particularly in WDL lacking significant histologic atypia or DDL without corresponding WDL component, especially in larger tumors, those from intra-abdominal or inguinal sites or core biopsies. There is a significant group of well-differentiated adipocytic neoplasms

  3. Single-nucleotide polymorphisms p53 G72C and Mdm2 T309G in patients with psoriasis, psoriatic arthritis, and SAPHO syndrome.

    Science.gov (United States)

    Assmann, Gunter; Wagner, Annette D; Monika, Mueller; Pfoehler, Claudia; Pfreundschuh, Michael; Tilgen, Wolfgang; Roemer, Klaus

    2010-08-01

    Psoriasis (Ps), psoriatic arthritis (PsA), and SAPHO syndrome are diseases of unknown etiology that share common clinical features; however, family studies support the hypothesis of a genetic background for each of these diseases. To study the two common single-nucleotide polymorphisms (SNP) in the murine-double-minute-2-(Mdm2) and p53 genes in patients with Ps, PsA, and SAPHO syndrome. Genomic DNA was obtained from 187 patients with Ps, 50 with PsA, and 36 with SAPHO as well as 478 healthy controls. Mdm2-gene SNP T309G and p53-gene SNP G72C genotypes were determined by the polymerase chain reaction. Genotype and allele frequencies were analyzed with chi(2)-tests. Among the patients with Ps and PsA, no differences in allele or genotype frequencies of the p53-gene SNP G72C and Mdm2-gene SNP T309G were detected. However, in the SAPHO patients group, the frequencies of the Mdm2 SNP309 G allele and the genotype SNP 309 GG were significantly increased compared with the controls (G allele: 51.4 vs. 38.7%, P = 0.034; genotype GG: 36.1 vs. 14.2%, P = 0.002). In addition, the frequencies of the p53 SNP72 C allele and the genotype SNP 72 CC were also increased in the SAPHO patients cohort (C allele: 36.1 vs. 25.6%, P = 0.05; genotype CC: 16.7 vs. 6.3%, P = 0.05). SAPHO syndrome may be linked to an imbalance between MDM2 and p53 regulation with a "weak" p53-response associated with the Mdm2 SNP 309 G allele. In contrast, the p53 network does not seem to play a major role in pathogenesis of Ps or PsA.

  4. Clinical significance of mdm2 and p53 expression in orbital rhabdomyosarcoma%原癌基因mdm2及p53基因与眼眶横纹肌肉瘤生物学特性研究

    Institute of Scientific and Technical Information of China (English)

    顼晓琳; 李彬; 孙宪丽; 李辽青

    2004-01-01

    目的探讨原癌基因mdm2、p53基因在眼眶横纹肌肉瘤(ORMS)发病机制中的可能作用,初步论证mdm2、p53作为ORMS生物学特性标记物和预后指标的可行性.方法应用免疫组化法检测31例ORMS标本中mdm2、p53、Ki-67蛋白的表达,Ki-67蛋白表达作为增殖指标反映肿瘤细胞的增殖状态.应用原位杂交方法检测31例ORMS标本中mdm2、p53基因在mRNA水平的表达.分析mdm2、p53蛋白表达与年龄、性别、病理类型、分化程度、增殖程度等临床组织病理学指标之间的关系.结果 31例ORMS中,mdm2、p53蛋白水平表达阳性率分别为77.4%(24/31)和71.0%(22/31),mdm2与p53蛋白共同表达阳性率是61.3%.mdm2、p53在蛋白水平表达与其各自的mRNA水平表达之间具有较好一致性.(1)按肿瘤细胞分化程度分为高、中、低3个组:中、低分化组mdm2蛋白阳性率和mdm2与p53蛋白共同表达阳性率均较高分化组高,差异有显著意义(P=0.007、0.009).(2)按Ki-67表达与否分为高、低增殖组:高增殖组p53蛋白表达阳性率较低增殖组高,差异有显著意义(P=0.044).mdm2、p53蛋白表达与其他临床病理学指标之间无显著相关性(P>0.05).结论 mdm2和p53与ORMS的分化和增殖密切相关,mdm2与p53共同表达提示mdm2可能与p53相结合而调节其活性.mdm2和p53的异常表达可能导致细胞异型性和过度增殖,在ORMS的发生和发展中起重要作用.mdm2和p53在一定程度上能够反映肿瘤的分化和增殖特性,有可能成为判断该肿瘤预后的指标之一.

  5. The MDM2-p53-pyruvate carboxylase signalling axis couples mitochondrial metabolism to glucose-stimulated insulin secretion in pancreatic β-cells

    DEFF Research Database (Denmark)

    Li, Xiaomu; Cheng, Kenneth K. Y.; Liu, Zhuohao

    2016-01-01

    Mitochondrial metabolism is pivotal for glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. However, little is known about the molecular machinery that controls the homeostasis of intermediary metabolites in mitochondria. Here we show that the activation of p53 in β-cells, by genetic...... oxaloacetate and NADPH, and impaired oxygen consumption. The defective GSIS and mitochondrial metabolism in MDM2-null islets can be rescued by restoring PC expression. Under diabetogenic conditions, MDM2 and p53 are upregulated, whereas PC is reduced in mouse β-cells. Pharmacological inhibition of p53...

  6. Nondenaturing polyacrylamide gel electrophoresis to study the dissociation of the p53·MDM2/X complex by potentially anticancer compounds.

    Science.gov (United States)

    Sgammato, Roberta; Desiderio, Doriana; Lamberti, Anna; Raimo, Gennaro; Novellino, Ettore; Carotenuto, Alfonso; Masullo, Mariorosario

    2015-12-01

    A new analytical method to study the dissociation of the complexes between the oncosuppressor p53 and its negative modulators murine double-minute protein 2 (MDM2) or MDMX, is proposed. This technique is reliable to determine the dissociative power exerted by small molecules on the complex taking advantage of the appearance of migrating MDM2 or MDMX in a native polyacrylamide gel, when inhibitors are added to the complex mixture. Therefore, we propose this new approach to easily screen library of compounds, with potential pharmacological anticancer activity. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Analysis of MDM2 and MDM4 single nucleotide polymorphisms, mRNA splicing and protein expression in retinoblastoma.

    Directory of Open Access Journals (Sweden)

    Justina McEvoy

    Full Text Available Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic RB1 gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191 was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.

  8. WAF Dance etendab "Väikest printsi" / Karin Klaus

    Index Scriptorium Estoniae

    Klaus, Karin

    2006-01-01

    Pärnu tantsukool WAF Dance toob 17. ja 18. juunil Endla teatris lavale Saint-Exupery "Väikese printsi". See on moodsa tantsuteatri koolitus- ja etendusprojekt. Lavastaja ja koreograaf on Kate Pringle Londonist

  9. WAF Dance etendab "Väikest printsi" / Karin Klaus

    Index Scriptorium Estoniae

    Klaus, Karin

    2006-01-01

    Pärnu tantsukool WAF Dance toob 17. ja 18. juunil Endla teatris lavale Saint-Exupery "Väikese printsi". See on moodsa tantsuteatri koolitus- ja etendusprojekt. Lavastaja ja koreograaf on Kate Pringle Londonist

  10. Development of cell-penetrating peptide-based drug leads to inhibit MDMX:p53 and MDM2:p53 interactions.

    Science.gov (United States)

    Philippe, Grégoire; Huang, Yen-Hua; Cheneval, Olivier; Lawrence, Nicole; Zhang, Zhen; Fairlie, David P; Craik, David J; de Araujo, Aline Dantas; Henriques, Sónia Troeira

    2016-11-01

    The transcription factor p53 has a tumor suppressor role in leading damaged cells to apoptosis. Its activity is regulated/inhibited in healthy cells by the proteins MDM2 and MDMX. Overexpression of MDM2 and/or MDMX in cancer cells inactivates p53, facilitating tumor development. A 12-mer dual inhibitor peptide (pDI) was previously reported to be able to target and inhibit MDMX:p53 and MDM2:p53 interactions with nanomolar potency in vitro. With the aim of improving its cellular inhibitory activity, we produced a series of constrained pDI analogs featuring lactam staples that stabilize the bioactive helical conformation and fused them with a cell-penetrating peptide to increase cytosol delivery. We compared pDI and its analogs on their inhibitory potency, toxicity, and ability to enter cancer cells. Overall, the results show that these analogs keep their nanomolar affinity for MDM2 and MDMX and are highly active against cancer cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 853-863, 2016.

  11. The MDM2–p53–pyruvate carboxylase signalling axis couples mitochondrial metabolism to glucose-stimulated insulin secretion in pancreatic β-cells

    Science.gov (United States)

    Li, Xiaomu; Cheng, Kenneth K. Y.; Liu, Zhuohao; Yang, Jin-Kui; Wang, Baile; Jiang, Xue; Zhou, Yawen; Hallenborg, Philip; Hoo, Ruby L. C.; Lam, Karen S. L.; Ikeda, Yasuhiro; Gao, Xin; Xu, Aimin

    2016-01-01

    Mitochondrial metabolism is pivotal for glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. However, little is known about the molecular machinery that controls the homeostasis of intermediary metabolites in mitochondria. Here we show that the activation of p53 in β-cells, by genetic deletion or pharmacological inhibition of its negative regulator MDM2, impairs GSIS, leading to glucose intolerance in mice. Mechanistically, p53 activation represses the expression of the mitochondrial enzyme pyruvate carboxylase (PC), resulting in diminished production of the TCA cycle intermediates oxaloacetate and NADPH, and impaired oxygen consumption. The defective GSIS and mitochondrial metabolism in MDM2-null islets can be rescued by restoring PC expression. Under diabetogenic conditions, MDM2 and p53 are upregulated, whereas PC is reduced in mouse β-cells. Pharmacological inhibition of p53 alleviates defective GSIS in diabetic islets by restoring PC expression. Thus, the MDM2–p53–PC signalling axis links mitochondrial metabolism to insulin secretion and glucose homeostasis, and could represent a therapeutic target in diabetes. PMID:27265727

  12. Non-linear feedback control of the p53 protein-mdm2 inhibitor system using the derivative-free non-linear Kalman filter.

    Science.gov (United States)

    Rigatos, Gerasimos G

    2016-06-01

    It is proven that the model of the p53-mdm2 protein synthesis loop is a differentially flat one and using a diffeomorphism (change of state variables) that is proposed by differential flatness theory it is shown that the protein synthesis model can be transformed into the canonical (Brunovsky) form. This enables the design of a feedback control law that maintains the concentration of the p53 protein at the desirable levels. To estimate the non-measurable elements of the state vector describing the p53-mdm2 system dynamics, the derivative-free non-linear Kalman filter is used. Moreover, to compensate for modelling uncertainties and external disturbances that affect the p53-mdm2 system, the derivative-free non-linear Kalman filter is re-designed as a disturbance observer. The derivative-free non-linear Kalman filter consists of the Kalman filter recursion applied on the linearised equivalent of the protein synthesis model together with an inverse transformation based on differential flatness theory that enables to retrieve estimates for the state variables of the initial non-linear model. The proposed non-linear feedback control and perturbations compensation method for the p53-mdm2 system can result in more efficient chemotherapy schemes where the infusion of medication will be better administered.

  13. In squamous cell carcinoma of the vulva, overexpression of p53 is a late event and neither p53 nor mdm2 expression is a useful marker to predict lymph node metastases

    NARCIS (Netherlands)

    Emanuels, AG; Koudstaal, J; Burger, MPM; Hollema, H

    1999-01-01

    To offer more tailored treatment to individual patients with squamous cell carcinoma of the vulval more accurate prediction of lymph node metastases is required. As p53 and mdm2 are genes known to be involved in the development of other tumours, we studied expression of p53 and mdm2 in carcinogenesi

  14. In squamous cell carcinoma of the vulva, overexpression of p53 is a late event and neither p53 nor mdm2 expression is a useful marker to predict lymph node metastases

    NARCIS (Netherlands)

    Emanuels, AG; Koudstaal, J; Burger, MPM; Hollema, H

    1999-01-01

    To offer more tailored treatment to individual patients with squamous cell carcinoma of the vulval more accurate prediction of lymph node metastases is required. As p53 and mdm2 are genes known to be involved in the development of other tumours, we studied expression of p53 and mdm2 in carcinogenesi

  15. Association between MDM2 SNP309 T>G polymorphism and the risk of bladder cancer: new data in a Chinese population and an updated meta-analysis

    Directory of Open Access Journals (Sweden)

    Xie LG

    2015-12-01

    Full Text Available Linguo Xie,1,2,* Yan Sun,2,* Tao Chen,1,2,* Dawei Tian,1,2 Yujuan Li,3 Yu Zhang,1,2 Na Ding,2 Zhonghua Shen,1,2 Hao Xu,1,2 Xuewu Nian,4 Nan Sha,1,2 Ruifa Han,1,2 Hailong Hu,1,2 Changli Wu1,2 Objective: Human murine double minute 2 protein (MDM2 is mainly a negative regulator of p53 tumor suppressor pathway. We aimed to investigate the association between MDM2 SNP309 polymorphism and bladder cancer risk. Methods: A total of 535 bladder cancer patients and 649 health controls were recruited for our study. MDM2 SNP309 T>G polymorphism was genotyped by polymerase chain reaction-ligase detection reaction method. Logistic regression was used to analyze the relationship between the genotype and susceptibility of bladder cancer. Kaplan–Meier estimates and log-rank test were obtained to analyze the association between the genotype and risk of recrudesce in nonmuscle-invasive bladder cancer patients. A multivariable Cox proportional hazards model was fitted to identify independent prognostic factors. To further investigate the association, we conducted a meta-analysis including six studies. Results: The frequency of the MDM2 SNP309 T>G polymorphism showed no significant difference between cases and controls (all P>0.05. In the stratification analysis, the results showed that G allele carriers were prone to have a significant decrease in risk of low-grade bladder cancer (adjusted odds ratio: 0.613, 95% confidence interval: 0.427–0.881, and G variant was associated with a significantly reduced risk of recurrence in nonmuscle-invasive bladder cancer patients with or without chemotherapy (P<0.05. The results of the meta-analysis showed that G allele and GG genotype of MDM2 SNP309 polymorphism were significantly associated with increased risk of bladder cancer in Caucasians (both P<0.05, and no association was observed in total populations and Asians (P>0.05. Conclusion: MDM2 SNP309 T>G polymorphism has no influence on bladder cancer risk in Asians, but

  16. Study on expressions of MDM2 and SPIN1mRNA in cancer of larynx%MDM2和SPIN1mRNA 在喉癌组织中的表达及其相关性研究

    Institute of Scientific and Technical Information of China (English)

    马伟军; 任晓勇; 许珉

    2014-01-01

    Objective:To observe the expressions of MDM 2 and SPIN1 mRNA in tissue of cancer of lar-ynx and investigate their correlations and explore their roles in tumor genesis .Method :The expressions of MDM2 and SPIN1mRNA in 32 cancer of larynx tissues and adjacent tissues detected by RT-PCR .Result :There were signif-icant differences in the expressions of MDM2 of 32 samples ,MDM2 mRNA presented higher level expression in cancer of larynx tissues (P< 0 .01) ,while SPIN1mRNA expressions of two tissues show no significant difference . MDM2 mRNA expression increased along with the increased tumor differentiation degree of cancer of larynx (P<0 . 01) .There were no correlation between the expressions of MDM 2 and SPIN1 in cancer of larynx .Conclusion :MDM2 may play an important role in tumor genesis of cancer of larynx ,while SPIN1 may play an unimportant role in tumor genesis of cancer of larynx .Their relationship will be researched in further study .%目的:研究喉癌组织中MDM2和SPIN1mRNA的表达,探讨其相关性及在肿瘤发生发展中的作用。方法:应用real-time PCR对32例喉癌及癌旁组织标本的MDM2、SPIN1在mR-NA水平进行检测。结果:MDM2在喉癌组织中的表达明显高于癌旁正常组织,差异具有显著性( P<0.01);SPIN1在喉癌组织和癌旁正常组织中表达无显著性差异( P>0.05)。MDM2在喉癌中的表达与肿瘤的病理分化呈负相关(P<0.01)。SPIN1的表达和肿瘤分化无关( P >0.05)。喉癌组织中MDM2和SPIN1表达无显著相关性( P >0.05)。结论:MDM2基因在喉癌的发生发展中可能发挥重要作用,而SPIN1基因在喉癌的发生发展过程中可能不发挥主要作用,两者之间的相互作用机制有待进一步研究。

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

    Science.gov (United States)

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

    2017-03-10

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

  18. Effects of MDM2, MDM4 and TP53 codon 72 polymorphisms on cancer risk in a cohort study of carriers of TP53 germline mutations.

    Directory of Open Access Journals (Sweden)

    Shenying Fang

    Full Text Available BACKGROUND: Previous studies have shown that MDM2 SNP309 and p53 codon 72 have modifier effects on germline P53 mutations, but those studies relied on case-only studies with small sample sizes. The impact of MDM4 polymorphism on tumor onset in germline mutation carriers has not previously been studied. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed 213 p53 germline mutation carriers including 168(78.9% affected with cancer and 174 who had genotypic data. We analyzed time to first cancer using Kaplan-Meier and Cox proportional hazards methods, comparing risks according to polymorphism genotypes. For MDM2 SNP309, a significant difference of 9.0 years in the average age of cancer diagnosis was observed between GG/GT and TT carriers (18.6 versus 27.6 years, P = 0.0087. The hazards ratio was 1.58 (P = 0.03 comparing risks among individuals with GG/GT to risk among TT, but this effect was only significant in females (HR = 1.60, P = 0.02. Compared to other genotypes, P53 codon 72 PP homozygotes had a 2.24 times (P = 0.03 higher rate for time to develop cancer. We observed a multiplicative joint effect of MDM2 and p53 codon72 polymorphism on risk. The MDM4 polymorphism had no significant effects. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the MDM2 SNP309 G allele is associated with cancer risk in p53 germline mutation carriers and accelerates time to cancer onset with a pronounced effect in females. A multiplicative joint effect exists between the MDM2 SNP309 G allele and the p53 codon 72 G allele in the risk of cancer development. Our results further define cancer risk in carriers of germline p53 mutations.

  19. Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf(-/-) mouse model.

    Science.gov (United States)

    Chapeau, Emilie A; Gembarska, Agnieszka; Durand, Eric Y; Mandon, Emeline; Estadieu, Claire; Romanet, Vincent; Wiesmann, Marion; Tiedt, Ralph; Lehar, Joseph; de Weck, Antoine; Rad, Roland; Barys, Louise; Jeay, Sebastien; Ferretti, Stephane; Kauffmann, Audrey; Sutter, Esther; Grevot, Armelle; Moulin, Pierre; Murakami, Masato; Sellers, William R; Hofmann, Francesco; Jensen, Michael Rugaard

    2017-03-21

    Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF, activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201. Constitutive PB mutagenesis in Arf(-/-) mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess the pharmacologic effects of HDM201. Sixteen out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. A comparison of tumors with acquired resistance to HDM201 and untreated tumors identified 87 genes that were differentially and significantly targeted by the PB transposon. Resistant tumors displayed a complex clonality pattern suggesting the emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in transformation-related protein 53 (Trp53) in 54% of tumors and transposon-mediated gain-of-function alterations in B-cell lymphoma-extra large (Bcl-xL), Mdm4, and two TP53 family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 protein expression was confirmed in resistant tumors, as well as in HDM201-resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro. Collectively, our findings identify several potential mechanisms by which TP53 wild-type tumors may escape MDM2-targeted therapy.

  20. Rapid detection of SNP (c.309T>G in the MDM2 gene by the Duplex SmartAmp method.

    Directory of Open Access Journals (Sweden)

    Yasuaki Enokida

    Full Text Available BACKGROUND: Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans. METHODOLOGY: In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named "Duplex SmartAmp," which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms. RESULTS AND CONCLUSIONS: By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.

  1. MDM2 and HIF1alpha expression levels in different histologic subtypes of malignant pleural mesothelioma: correlation with pathological and clinical data.

    Science.gov (United States)

    Pasello, Giulia; Urso, Loredana; Mencoboni, Manlio; Grosso, Federica; Ceresoli, Giovanni Luca; Lunardi, Francesca; Vuljan, Stefania Edith; Bertorelle, Roberta; Sacchetto, Valeria; Ciminale, Vincenzo; Rea, Federico; Favaretto, Adolfo; Conte, PierFranco; Calabrese, Fiorella

    2015-12-08

    Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited treatment options. Sarcomatoid/biphasic mesotheliomas are characterized by more aggressive behaviour and a poorer prognosis compared with the epithelioid subtype. To date prognostic and tailored therapeutic biomarkers are lacking. The present study analyzed the expression levels of MDM2 and HIF1alpha in different histologic subtypes from chemonaive MPM patients. Diagnostic biopsies of MPM patients from four Italian cancer centers were centrally collected and analyzed. MDM2 and HIF1alpha expression levels were investigated through immunohistochemistry and RT-qPCR. Pathological assessment of necrosis, inflammation and proliferation index was also performed. Molecular markers, pathological features and clinical characteristics were correlated to overall survival (OS) and progression free survival (PFS). Sixty MPM patients were included in the study (32 epithelioid and 28 non-epithelioid). Higher levels of MDM2 (p sarcomatoid/biphasic subtypes. Higher levels of inflammation were significantly associated with epithelioid subtype (p = 0.044). MDM2 expression levels were correlated with HIF1alpha levels (p = 0.0001), necrosis (p = 0.008) and proliferation index (p = 0.009). Univariate analysis showed a significant correlation of non-epithelioid histology (p = 0.04), high levels of necrosis (p = 0.037) and proliferation index (p = 0.0002) with shorter PFS. Sarcomatoid/biphasic and epithelioid mesotheliomas showed different MDM2 and HIF1alpha expression levels and were characterized by different levels of necrosis, proliferation and inflammation. Further studies are warranted to confirm a prognostic and predictive role of such markers and features.

  2. Potential genotype-specific single nucleotide polymorphism interaction of common variation in p53 and its negative regulator mdm2 in cholangiocarcinoma susceptibility.

    Science.gov (United States)

    Zimmer, Vincent; Höblinger, Aksana; Mihalache, Florentina; Assmann, Gunter; Acalovschi, Monica; Lammert, Frank

    2012-07-01

    Aberrant cell cycle control and apoptosis deregulation are involved in biliary carcinogenesis. The tumor suppressor gene p53 and its key negative regulator murine double minute 2 (mdm2) cooperate in modulating these basic cell functions and germline p53 alteration promotes cholangiocarcinoma (CCA) formation in animal models. The potential association between common functional genetic variation in p53 (SNP72 G/C) and mdm2 (SNP309 T/G) and susceptibility to bile duct cancer, however, has not been studied. p53/SNP72 G/C (rs1042522) and mdm2/SNP309 T/G (rs2279744) were genotyped in 182 Caucasian CCA patients and 350 controls using TaqMan assays. Allelic and genotypic differences, including exploratory data analyses (according to gender, tumor localization, early onset and genotypic interactions) were compared in contingency tables using the χ(2) and Fisher's exact tests. The overall comparison of allele and genotype frequencies yielded no significant association between either SNP and CCA susceptibility. Similarly, gender- and localization-specific analyses did not reveal deviations in allelic or genotypic distributions. In carriers of the low-apoptotic p53 genotype CC, the mdm2 SNP309 T allele conferred borderline significant CCA risk [P=0.049; odds ratio (OR), 4.36; 95% CI, 0.92-20.77]. Power analysis confirmed adequate statistical power to exclude major SNP effects (each >97% for OR 1.7). Collectively, the results we obtained from the largest European CCA cohort do not support the hypothesis of a prominent role of common p53 and mdm2 variation in the genetic susceptibility to bile duct cancer. However, epistatic effects may modulate genetic CCA risk in individual subsets.

  3. MDM2 and HIF1alpha expression levels in different histologic subtypes of malignant pleural mesothelioma: correlation with pathological and clinical data

    Science.gov (United States)

    Mencoboni, Manlio; Grosso, Federica; Ceresoli, Giovanni Luca; Lunardi, Francesca; Vuljan, Stefania Edith; Bertorelle, Roberta; Sacchetto, Valeria; Ciminale, Vincenzo; Rea, Federico; Favaretto, Adolfo; Conte, PierFranco; Calabrese, Fiorella

    2015-01-01

    Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited treatment options. Sarcomatoid/biphasic mesotheliomas are characterized by more aggressive behaviour and a poorer prognosis compared with the epithelioid subtype. To date prognostic and tailored therapeutic biomarkers are lacking. The present study analyzed the expression levels of MDM2 and HIF1alpha in different histologic subtypes from chemonaive MPM patients. Diagnostic biopsies of MPM patients from four Italian cancer centers were centrally collected and analyzed. MDM2 and HIF1alpha expression levels were investigated through immunohistochemistry and RT-qPCR. Pathological assessment of necrosis, inflammation and proliferation index was also performed. Molecular markers, pathological features and clinical characteristics were correlated to overall survival (OS) and progression free survival (PFS). Sixty MPM patients were included in the study (32 epithelioid and 28 non-epithelioid). Higher levels of MDM2 (p < 0.001), HIF1alpha (p = 0.013), necrosis (p = 0.013) and proliferation index (p < 0.001) were seen mainly in sarcomatoid/biphasic subtypes. Higher levels of inflammation were significantly associated with epithelioid subtype (p = 0.044). MDM2 expression levels were correlated with HIF1alpha levels (p = 0.0001), necrosis (p = 0.008) and proliferation index (p = 0.009). Univariate analysis showed a significant correlation of non-epithelioid histology (p = 0.04), high levels of necrosis (p = 0.037) and proliferation index (p = 0.0002) with shorter PFS. Sarcomatoid/biphasic and epithelioid mesotheliomas showed different MDM2 and HIF1alpha expression levels and were characterized by different levels of necrosis, proliferation and inflammation. Further studies are warranted to confirm a prognostic and predictive role of such markers and features. PMID:26544728

  4. The importance of ribosome production, and the 5S RNP-MDM2 pathway, in health and disease.

    Science.gov (United States)

    Pelava, Andria; Schneider, Claudia; Watkins, Nicholas J

    2016-08-15

    Ribosomes are abundant, large RNA-protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease. © 2016 The Author(s).

  5. miR-339-5p regulates the p53 tumor-suppressor pathway by targeting MDM2

    DEFF Research Database (Denmark)

    Jansson, M D; Djodji Damas, Nkerorema; Lees, M

    2014-01-01

    MicroRNAs (miRNAs) regulate many key cancer-relevant pathways and may themselves possess oncogenic or tumor-suppressor functions. Consequently, miRNA dysregulation has been shown to be a prominent feature in many human cancers. The p53 tumor suppressor acts as a negative regulator of cell...... proliferation in response to stress and represents the most commonly lost and mutated gene in human cancers. The function of p53 is inhibited by the MDM2 oncoprotein. Using a high-throughput screening approach, we identified miR-339-5p as a regulator of the p53 pathway. We demonstrate that this regulation...... inhibition of miR-339-5p function perturbs the p53 response in cancer cells, allowing an increased proliferation rate. In addition, miR-339-5p expression is downregulated in tumors harboring wild-type TP53, suggesting that reduction of miR-339-5p level helps to suppress the p53 response in p53-competent...

  6. Evidence that proteasome-dependent degradation of the retinoblastoma protein in cells lacking A-type lamins occurs independently of gankyrin and MDM2.

    Directory of Open Access Journals (Sweden)

    Ryan T Nitta

    Full Text Available BACKGROUND: A-type lamins, predominantly lamins A and C, are nuclear intermediate filaments believed to act as scaffolds for assembly of transcription factors. Lamin A/C is necessary for the retinoblastoma protein (pRB stabilization through unknown mechanism(s. Two oncoproteins, gankyrin and MDM2, are known to promote pRB degradation in other contexts. Consequently, we tested the hypothesis that gankyrin and/or MDM2 are required for enhanced pRB degradation in Lmna-/- fibroblasts. Principal Findings. To determine if gankyrin promotes pRB destabilization in the absence of lamin A/C, we first analyzed its protein levels in Lmna-/- fibroblasts. Both gankyrin mRNA levels and protein levels are increased in these cells, leading us to further investigate its role in pRB degradation. Consistent with prior reports, overexpression of gankyrin in Lmna+/+ cells destabilizes pRB. This decrease is functionally significant, since gankyrin overexpressing cells are resistant to p16(ink4a-mediated cell cycle arrest. These findings suggest that lamin A-mediated degradation of pRB would be gankyrin-dependent. However, effective RNAi-enforced reduction of gankyrin expression in Lmna-/- cells was insufficient to restore pRB stability. To test the importance of MDM2, we disrupted the MDM2-pRB interaction by transfecting Lmna-/- cells with p14(arf. p14(arf expression was also insufficient to stabilize pRB or confer cell cycle arrest, suggesting that MDM2 also does not mediate pRB degradation in Lmna-/- cells. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that pRB degradation in Lmna-/- cells occurs by gankyrin and MDM2-independent mechanisms, leading us to propose the existence of a third proteasome-dependent pathway for pRB degradation. Two findings from this study also increase the likelihood that lamin A/C functions as a tumor suppressor. First, protein levels of the oncoprotein gankyrin are elevated in Lmna-/- fibroblasts. Second, Lmna-/- cells are refractory to

  7. 胃黏膜病变中突变型p53、Mdm2和PCNA蛋白表达及与幽门螺杆菌感染的关系%Expressions of mutant p53, Mdm2 and PCNA protein in gastric mucosal lesions and their relationship with Helicobacter pylori infection

    Institute of Scientific and Technical Information of China (English)

    齐淑文; 舒徐; 陈江; 吕农华

    2009-01-01

    目的 探讨胃黏膜病变中突变型p53、Mdm2、增殖细胞核抗原(PCNA)蛋白的表达及其与幽门螺杆菌(Hp)感染的关系.方法 应用免疫组化PV-9000法染色及Giemsa染色检测胃黏膜病变中突变型p53、Mdm2、PCNA蛋白的表达和Hp感染情况.结果 在慢性浅表性胃炎(CSG)、慢性萎缩性胃炎伴肠上皮化生(CAG伴IM)、不典型增生(Dys)及胃癌(GC)中,突变型p53、Mdm2、PCNA阳性表达随病变发展而逐渐升高,突变型p53在CSG组高于GC组,GC组高于CAG伴IM组(P均<0.05).不同胃黏膜病变之间Mdm2比较均有统计学差异(P<0.05).在同一病变中Hp阳性者突变型p53、Mdm2、PCNA阳性表达一般高于Hp阴性者,突变型p53在CAG伴IM组Hp阳性与阴性者间有统计学差异(P<0.05),PCNA和Mdm2在Dys组Hp阳性与阴性者有统计学差异(P<0.05).GC组中突变型p53、Mdm2、PCNA蛋白表达无相关关系.结论 突变型p53、Mdm2和PCNA蛋白的表达及Hp感染共同参与胃黏膜病变的发生、发展.

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

    Directory of Open Access Journals (Sweden)

    Huarong Guo

    2012-09-01

    Full Text Available 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.

  9. Inhibitors of p21-activated kinases (PAKs).

    Science.gov (United States)

    Rudolph, Joachim; Crawford, James J; Hoeflich, Klaus P; Wang, Weiru

    2015-01-08

    The p21-activated kinase (PAK) family of serine/threonine protein kinases plays important roles in cytoskeletal organization, cellular morphogenesis, and survival, and members of this family have been implicated in many diseases including cancer, infectious diseases, and neurological disorders. Owing to their large and flexible ATP binding cleft, PAKs, particularly group I PAKs (PAK1, -2, and -3), are difficult to drug; hence, few PAK inhibitors with satisfactory kinase selectivity and druglike properties have been reported to date. Examples are a recently discovered group II PAK (PAK4, -5, -6) selective inhibitor series based on a benzimidazole core, a group I PAK selective series based on a pyrido[2,3-d]pyrimidine-7-one core, and an allosteric dibenzodiazepine PAK1 inhibitor series. Only one compound, an aminopyrazole based pan-PAK inhibitor, entered clinical trials but did not progress beyond phase I trials. Clinical proof of concept for pan-group I, pan-group II, or PAK isoform selective inhibition has yet to be demonstrated.

  10. Expression signature based on TP53 target genes doesn't predict response to TP53-MDM2 inhibitor in wild type TP53 tumors.

    Science.gov (United States)

    Sonkin, Dmitriy

    2015-10-22

    A number of TP53-MDM2 inhibitors are currently under investigation as therapeutic agents in a variety of clinical trials in patients with TP53 wild type tumors. Not all wild type TP53 tumors are sensitive to such inhibitors. In an attempt to improve selection of patients with TP53 wild type tumors, an mRNA expression signature based on 13 TP53 transcriptional target genes was recently developed (Jeay et al. 2015). Careful reanalysis of TP53 status in the study validation data set of cancer cell lines considered to be TP53 wild type detected TP53 inactivating alterations in 23% of cell lines. The subsequent reanalysis of the remaining TP53 wild type cell lines clearly demonstrated that unfortunately the 13-gene signature cannot predict response to TP53-MDM2 inhibitor in TP53 wild type tumors.

  11. Downregulation of extracellular signal-regulated kinase 1/2 activity by calmodulin KII modulates p21Cip1 levels and survival of immortalized lymphocytes from Alzheimer's disease patients.

    Science.gov (United States)

    Esteras, Noemí; Alquézar, Carolina; Bermejo-Pareja, Félix; Bialopiotrowicz, Emilia; Wojda, Urszula; Martín-Requero, Angeles

    2013-04-01

    Previously, we reported a Ca(2+)/calmodulin (CaM)-dependent impairment of apoptosis induced by serum deprivation in Alzheimer's disease (AD) lymphoblasts. These cell lines showed downregulation of extracellular signal-regulated kinase (ERK)1/2 activity and elevated content of p21 compared with control cells. The aim of this study was to delineate the molecular mechanism underlying the distinct regulation of p21 content in AD cells. Quantitative reverse transcription polymerase chain reaction analysis demonstrated increased p21 messenger RNA (mRNA) levels in AD cells. The ERK1/2 inhibitor, PD98059, prevented death of control cells and enhanced p21 mRNA and protein levels. The CaM antagonist, calmidazolium, and the CaMKII inhibitor, KN-62, normalized the survival pattern of AD lymphoblasts by augmenting ERK1/2 activation and reducing p21 mRNA and protein levels. Upregulation of p21 transcription in AD cells appears to be the consequence of increased activity of forkhead box O3a (FOXO3a) as the result of diminished ERK1/2-mediated phosphorylation of this transcription factor, which in turn facilitates its nuclear accumulation. Murine double minute 2 (MDM2) protein levels were decreased in AD cells relative to control lymphoblasts, suggesting an impairment of FOXO3a degradation.

  12. Markov models of the apo-MDM2 lid region reveal diffuse yet two-state binding dynamics and receptor poses for computational docking

    Science.gov (United States)

    Mukherjee, Sudipto; Pantelopulos, George A.; Voelz, Vincent A.

    2016-08-01

    MDM2 is a negative regulator of p53 activity and an important target for cancer therapeutics. The N-terminal lid region of MDM2 modulates interactions with p53 via competition for its binding cleft, exchanging slowly between docked and undocked conformations in the absence of p53. To better understand these dynamics, we constructed Markov State Models (MSMs) from large collections of unbiased simulation trajectories of apo-MDM2, and find strong evidence for diffuse, yet two-state folding and binding of the N-terminal region to the p53 receptor site. The MSM also identifies holo-like receptor conformations highly suitable for computational docking, despite initiating trajectories from closed-cleft receptor structures unsuitable for docking. Fixed-anchor docking studies using a test set of high-affinity small molecules and peptides show simulated receptor ensembles achieve docking successes comparable to cross-docking studies using crystal structures of receptors bound by alternative ligands. For p53, the best-scoring receptor structures have the N-terminal region lid region bound in a helical conformation mimicking the bound structure of p53, suggesting lid region association induces receptor conformations suitable for binding. These results suggest that MD + MSM approaches can sample binding-competent receptor conformations suitable for computational peptidomimetic design, and that inclusion of disordered regions may be essential to capturing the correct receptor dynamics.

  13. Stapled α-helical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy.

    Science.gov (United States)

    Chang, Yong S; Graves, Bradford; Guerlavais, Vincent; Tovar, Christian; Packman, Kathryn; To, Kwong-Him; Olson, Karen A; Kesavan, Kamala; Gangurde, Pranoti; Mukherjee, Aditi; Baker, Theresa; Darlak, Krzysztof; Elkin, Carl; Filipovic, Zoran; Qureshi, Farooq Z; Cai, Hongliang; Berry, Pamela; Feyfant, Eric; Shi, Xiangguo E; Horstick, James; Annis, D Allen; Manning, Anthony M; Fotouhi, Nader; Nash, Huw; Vassilev, Lyubomir T; Sawyer, Tomi K

    2013-09-03

    Stapled α-helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy.

  14. Common polymorphisms in TP53 and MDM2 and the relationship to TP53 mutations and clinical outcomes in women with ovarian and peritoneal carcinomas.

    Science.gov (United States)

    Galic, Vijaya; Willner, Julia; Wollan, Melissa; Garg, Ruchi; Garcia, Rochelle; Goff, Barbara A; Gray, Heidi J; Swisher, Elizabeth M

    2007-03-01

    The importance of somatic TP53 mutations and germline TP53 codon 72 genotype in the survival of women with epithelial ovarian cancer is controversial. Recent data suggest that a promoter polymorphism in the MDM2 gene may influence age of cancer onset in a gender-specific fashion. We sought to determine the relationship between somatic TP53 mutations, germline genotypes at TP53 codon 72 and MDM2 SNP309, and overall survival and response to chemotherapy in a large series of patients with ovarian and peritoneal carcinomas. Of the 188 cancers, 103 (54.8%) had a TP53 mutation, of which 71% were missense mutations and 29% were null mutations. TP53 mutation status and mutation type (null vs. missense) did not influence response to therapy or overall survival. Women with the codon 72 Pro/Pro had a decreased overall survival (median, 29 months) compared with women with one or two arginine alleles (median, 49 months; P=0.04). Somatic mutation or deletion was equally common for either codon 72 allele. Age of diagnosis was not influenced by codon 72 but showed a trend for younger age in women with somatic TP53 mutations and the MDM2 G/G genotype. Copyright (c) 2006 Wiley-Liss, Inc.

  15. Phosphorylation of murine double minute clone 2 (MDM2) protein at serine-267 by protein kinase CK2 in vitro and in cultured cells

    DEFF Research Database (Denmark)

    Hjerrild, M; Milne, D; Dumaz, N

    2001-01-01

    -site phosphorylation, may itself be a target for stress signalling (SUMO is small ubiquitin-related modifier-1). In the present study we show that, like p53, the MDM2 protein is a substrate for phosphorylation by the protein kinase CK2 (CK2) in vitro. CK2 phosphorylates a single major site, Ser(267), which lies within...... the central acidic domain of MDM2. Fractionation of cellular extracts revealed the presence of a single Ser(267) protein kinase which co-purified with CK2 on ion-exchange chromatography and, like CK2, was subject to inhibition by micromolar concentrations of the CK2-specific inhibitor 5......,6-dichlororibofuranosylbenzimidazole. Radiolabelling of cells expressing tagged recombinant wild-type MDM2 or a S267A (Ser(267)-->Ala) mutant, followed by phosphopeptide analysis, confirmed that Ser(267) is a cellular target for phosphorylation. Ser(267) mutants are still able to direct the degradation of p53, but in a slightly...

  16. Expression of Bcl- 2 and MDM- 2, and HPV- 16 Infection Rate in Esophageal Carcinoma and in Its Pre - cancerous Lesion%食管癌及其癌前病变中Bcl-2和MDM-2表达以及HPV-16感染率的研究

    Institute of Scientific and Technical Information of China (English)

    吕怀盛; 张建中; 景丽; 秦璟

    2004-01-01

    目的:对食管癌及其癌前病变中Bcl-2、MDM-2和HPV-16进行分析,以探讨食管癌发生原因及其机制.方法:采用免疫组化方法,对正常食管上皮、食管癌及其癌前病变中Bcl-2、MDM-2蛋白的表达和HPV-16进行对比观察.结果:9例正常食管鳞状上皮中有1例Bcl-2染色阳性(11.11%),MDM-2和HPV-16全部阴性;39例食管癌中Bcl-2、MDM-2、HPV-16染色分别有33例(84.61%)、25例(64.10%)和13例(33.33%)染色阳性;31例非典型增生中Bcl-2、MDM-2、HPV-16染色分别有7例(22.58%)、11例(35.48%)和3例(9.68%)染色阳性.三项指标在三组间比较均具有显著性差异(P<0.05);食管癌HPV-16阳性(33.33%)与非典型增生(9.68%)有显著性差异(χ2=4.14,P<0.05);食管癌MDM-2与Bcl-2染色阳性率间具有明显的相关关系(χ2=7.689,P<0.01).结论:食管癌常伴有HPV-16感染,食管癌的发生与Bcl-2和MDM-2基因异常表达有密切关系.

  17. Study on expression changes of MDM2 gene in SD rats' blood after chronic 137Cs γ ray irradiation%137 Cs γ射线慢性照射诱导大鼠血MDM2基因表达改变研究

    Institute of Scientific and Technical Information of China (English)

    王超; 刘建功; 党旭红; 刘红艳; 左雅慧; 段志凯; 刘占旗

    2013-01-01

    目的 探讨长期慢性137Cs γ射线照射对大鼠血中MDM2基因表达改变的影响.方法 无特定病原体级雄性SD大鼠120只,随机分成1个对照组,11个剂量组,每组均为10只,各剂量组137Cs γ射线慢性累积照射分别为0.05、0.10、0.20、0.40、0.60、0.80、1.00、2.00、3.00、4.00、5.00 Gy,对照组不予照射.照射完成后采腹主动脉血,利用实时荧光定量聚合酶链反应法检测各组血中MDM2基因表达的剂量-效应关系.结果 各剂量组和对照组比较,血中MDM2基因的表达量均发生变化,从整体水平看随受照剂量的增加,基因表达量先上升后下降.其中,0.05、0.10Gy剂量组中的基因表达量升高,其后0.20~1.00 Gy剂量范围MDM2基因的相对表达量出现一定程度回落,以0.20 Gy剂量组中的基因表达量降至最低点;0.20~2.00 Gy剂量范围随着照射剂量增大,MDM2基因的相对表达量呈线性关系升高,2.00 Gy剂量组的MDM2基因表达量最高;其后随着剂量的增加,MDM2基因的相对表达量再次出现下降趋势.拟合建立了0.20~2.00 Gy剂量范围内MDM2基因表达量与照射剂量之间的回归方程为多=1.918 9x+0.386 0,决定系数=0.99,P<0.01.结论 慢性累积照射可诱导大鼠血中MDM2基因表达发生改变,其总体表达趋势是先上升后下降,其中0.20~2.00 Gy剂量内MDM2基因表达量与照射剂量有较好的剂量-效应关系.

  18. Association of MDM-2 SNP309 polymorphism and age at onset and risk of hepatocellular carcinoma%鼠MDM-2 SNP309位点与肝细胞癌发病年龄与风险关系的分析

    Institute of Scientific and Technical Information of China (English)

    张艳艳; 毛良勤; 曾小云; 谢志春; 仇小强; 余红平

    2012-01-01

    目的:探讨广西地区人群鼠双微粒体-2基因(MDM-2)启动子区309位点单核苷酸多态性(SNP)与肝细胞癌(hepatocellular carcinoma,HCC)发病年龄和发病风险的关系.方法:运用聚合酶链反应-限制性片段长度多态性方法,对985例HCC病例和992例非肿瘤对照者的MDM-2 SNP309位点(T>G,rs2279744)基因型进行检测,并分析该SNP 与HCC发病年龄和发病风险的关系.结果:经年龄、性别、民族、吸烟、饮酒、HBV及HCV感染等因素校正后,MDM-2SNP309位点与HCC发病风险之间无统计学关联(TG vs TT∶ OR=1.19,95%CI∶ 0.86~1.65; GG vs TT∶ OR=1.28,95%CI∶0.89~1.85;TG+GG vs TT∶ OR=1.22,95%CI:0.90~1.66).在女性HCC患者中,与携带MDM-2SNP309位点TG+GG基因型的女性HCC患者相比(44.8岁),携带TT基因型的女性患者HCC发病年龄提前4.6岁(49.4岁),Log-rank检验:x2=7.372,P=0.007.在男性患者中未发现此类似结果.结论:MDM-2 SNP309位点多态性可能对HCC的发病风险无单独效应作用,但其TT基因型可能与女性HCC的发病年龄提前有关联.本研究结果需要大样本量的研究进一步验证.%OBJECTIVE: To explore associations between MDM-2 single nucleotide polymorphism (SNP) 309 (T> G, rs2279744) and the age at onset and risk of hepatocellular carcinoma (HCC) in Guangxi population. METHODS: A hospital-based case-control study was conducted in 985 cases with HCC and 992 cancer-free controls. SNP309 genotypes were detected using a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay, and associations of MDM-2 SNP309 with risk and age of onset of HCC were assessed. RESULTS: The variant genotypes of MDM-2 SNP309 were not significantly associated with risk of HCC (TG vs TT: OR=1. 19, 95%CI: 0. 86-1. 65; GG vs TT: OR=1. 28, 95%CI: 0.89-1.85; TG+GG vsTT: OR=1. 22, 95%CI: 0. 90-1. 66). It was found that female patients carrying the TT genotype (44. 8 year-old) showed a 4. 6-year earlier age at

  19. Correlation of biological behaviors with mdm-2 and p53 gene changes in primary gastric cancer and metastasis%mdm-2和p53基因变异与胃癌及淋巴转移生物学行为的关系

    Institute of Scientific and Technical Information of China (English)

    时军; 陈道达; 崔建涛; 吕有勇

    2000-01-01

    目的:分析癌基因mdm-2和抑癌基因p53在胃癌及其转移灶中的变异及相互关系,探讨胃癌及其转移的分子机制.方法:采用DC-PCR、PCR-SSCP及DNA测序技术检测32例胃癌及其转移灶中的mdm-2扩增和p53突变.结果:mdm-2在转移淋巴结中的扩增频率(12/21,57.1%)高于胃原发癌灶(12/32,37.5%),3例淋巴微转移灶中出现mdm 2的扩增.在7例肠型胃癌的原发癌或转移癌灶中同时检测出mdm-2扩增和p53突变.肝转移癌灶中p53突变位点和突变方式与胃原发癌之间有差异.结论:mdm-2扩增可能与胃癌细胞的淋巴高转移潜能有关.p53突变和mdm-2扩增多并存于肠型胃癌中.在胃原发癌和肝转移癌中癌细胞的基因改变存在着异质性.

  20. Polyomavirus-associated Trichodysplasia spinulosa involves hyperproliferation, pRB phosphorylation and upregulation of p16 and p21.

    Directory of Open Access Journals (Sweden)

    Siamaque Kazem

    Full Text Available Trichodysplasia spinulosa (TS is a proliferative skin disease observed in severely immunocompromized patients. It is characterized by papule and trichohyalin-rich spicule formation, epidermal acanthosis and distention of dysmorphic hair follicles overpopulated by inner root sheath cells (IRS. TS probably results from active infection with the TS-associated polyomavirus (TSPyV, as indicated by high viral-load, virus protein expression and particle formation. The underlying pathogenic mechanism imposed by TSPyV infection has not been solved yet. By analogy with other polyomaviruses, such as the Merkel cell polyomavirus associated with Merkel cell carcinoma, we hypothesized that TSPyV T-antigen promotes proliferation of infected IRS cells. Therefore, we analyzed TS biopsy sections for markers of cell proliferation (Ki-67 and cell cycle regulation (p16ink4a, p21waf, pRB, phosphorylated pRB, and the putatively transforming TSPyV early large tumor (LT antigen. Intense Ki-67 staining was detected especially in the margins of TS hair follicles, which colocalized with TSPyV LT-antigen detection. In this area, staining was also noted for pRB and particularly phosphorylated pRB, as well as p16ink4a and p21waf. Healthy control hair follicles did not or hardly stained for these markers. Trichohyalin was particularly detected in the center of TS follicles that stained negative for Ki-67 and TSPyV LT-antigen. In summary, we provide evidence for clustering of TSPyV LT-antigen-expressing and proliferating cells in the follicle margins that overproduce negative cell cycle regulatory proteins. These data are compatible with a scenario of TSPyV T-antigen-mediated cell cycle progression, potentially creating a pool of proliferating cells that enable viral DNA replication and drive papule and spicule formation.

  1. Polyomavirus-associated Trichodysplasia spinulosa involves hyperproliferation, pRB phosphorylation and upregulation of p16 and p21.

    Science.gov (United States)

    Kazem, Siamaque; van der Meijden, Els; Wang, Richard C; Rosenberg, Arlene S; Pope, Elena; Benoit, Taylor; Fleckman, Philip; Feltkamp, Mariet C W

    2014-01-01

    Trichodysplasia spinulosa (TS) is a proliferative skin disease observed in severely immunocompromized patients. It is characterized by papule and trichohyalin-rich spicule formation, epidermal acanthosis and distention of dysmorphic hair follicles overpopulated by inner root sheath cells (IRS). TS probably results from active infection with the TS-associated polyomavirus (TSPyV), as indicated by high viral-load, virus protein expression and particle formation. The underlying pathogenic mechanism imposed by TSPyV infection has not been solved yet. By analogy with other polyomaviruses, such as the Merkel cell polyomavirus associated with Merkel cell carcinoma, we hypothesized that TSPyV T-antigen promotes proliferation of infected IRS cells. Therefore, we analyzed TS biopsy sections for markers of cell proliferation (Ki-67) and cell cycle regulation (p16ink4a, p21waf, pRB, phosphorylated pRB), and the putatively transforming TSPyV early large tumor (LT) antigen. Intense Ki-67 staining was detected especially in the margins of TS hair follicles, which colocalized with TSPyV LT-antigen detection. In this area, staining was also noted for pRB and particularly phosphorylated pRB, as well as p16ink4a and p21waf. Healthy control hair follicles did not or hardly stained for these markers. Trichohyalin was particularly detected in the center of TS follicles that stained negative for Ki-67 and TSPyV LT-antigen. In summary, we provide evidence for clustering of TSPyV LT-antigen-expressing and proliferating cells in the follicle margins that overproduce negative cell cycle regulatory proteins. These data are compatible with a scenario of TSPyV T-antigen-mediated cell cycle progression, potentially creating a pool of proliferating cells that enable viral DNA replication and drive papule and spicule formation.

  2. 急性淋巴细胞性白血病患儿细胞中MDM-2蛋白表达与化疗反应的关系

    Institute of Scientific and Technical Information of China (English)

    邵秀梅; 杨献华

    2001-01-01

    @@ 1998年1 2月至2000年3月,我们对25例住院的急性淋巴细胞白血病(ALL)患儿细胞中MDM-2蛋白的表达水平及其对化疗效果的影响进行了研究,以探讨MDM-2蛋白异常表达与化疗反应的关系.

  3. A functional polymorphism T309G in MDM2 gene promoter, intensified by Helicobacter pylori lipopolysaccharide, is associated with both an increased susceptibility and poor prognosis of gastric carcinoma in Chinese patients.

    Science.gov (United States)

    Pan, Xiaolin; Li, Yuqin; Feng, Jin; Wang, Xiaoyong; Hao, Bo; Shi, Ruihua; Zhang, Guoxin

    2013-03-18

    Studies on the association between MDM2 SNP309 (T > G) and gastric cancer have reported conflicting results. Thus, the aim of this study was to investigate whether MDM2 SNP309 is associated with susceptibility and prognosis of gastric carcinoma in Chinese patients. Total of 574 gastric carcinoma cases and 574 age- and sex-matched healthy controls were included. MDM2 polymorphism was detected by PCR- RFLP and infection of Helicobacter pylori (H. pylori) by a validated serology test. The functionality of MDM2 SNP309, with or without H. pylori lipopolysaccharide (LPS), was examined by dual-luciferase assay. Kaplan-Meier survival curves were used to evaluate survival. Additional, a meta-analysis was conducted to verity the findings. MDM2 SNP309G/G genotype was associated with an increased risk of gastric carcinoma when compared with T/T genotype or T carriers (both P gastric carcinoma risk. SNP309G/G was identified as an independent marker of poor overall survival of carcinoma. In vitro, the luciferase assay further showed an increased transcriptional activity of SNP309G allele compared with SNP309T allele, and the function of polymorphism T309G in MDM2 gene promoter was intensified by H. pylori LPS. Pooled results from the meta-analysis confirmed that SNP309G/G genotype had a significantly increased risk of gastric carcinoma compared with T/T genotype or T carriers, consistent with the case-control findings. MDM2 SNP309G allele is associated with an increased risk and poor prognosis of gastric carcinoma in Chinese patients. Additional, there is a joint effect of MDM2 SNP309G/G allele and H. pylori infection on gastric carcinoma development, which may attribute to H. pylori LPS.

  4. Targeting p53-MDM2 negative feedback loops for treatment of cancer%针对p53-鼠双微体基因负反馈环的肿瘤治疗

    Institute of Scientific and Technical Information of China (English)

    吴学元; 马巍

    2009-01-01

    MDM2 controls p53 function through inhibiting p53-mediated transcriptional activity and promoting p53 degradation.Over-expression of MDM2 inactivates p53 and induces wide-type p53 cells malignant transformation.So inhibition of the p53-MDM2 interaction with synthetic molecules should therefore lead to both the nuclear accumulation and the activation of p53 followed by the death of the tumor cells from apoptosis.Inhibitors of the p53-MDM2 interaction might be attractive new anticancer agents that could be used to activate wild-type p53 in tumors.%鼠双微体基因(MDM2)通过抑制p53介导的转录活性和促进p53降解来控制p53的功能.MDM2过度表达引起p53失活,导致p53野生细胞恶性转化.人工合成抑制p53-MDM2蛋白复合体相互作用的小分子可促使p53核内积聚、活化p53,从而诱导肿瘤细胞的死亡.针对p53-MDM2相互作用的抑制剂可成为治疗p53野生型肿瘤系的新型抗癌药物.

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

    Science.gov (United States)

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

    2014-01-01

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

  6. P21 deficiency delays regeneration of skeletal muscular tissue.

    Directory of Open Access Journals (Sweden)

    Nobuaki Chinzei

    Full Text Available The potential relationship between cell cycle checkpoint control and tissue regeneration has been indicated. Despite considerable research being focused on the relationship between p21 and myogenesis, p21 function in skeletal muscle regeneration remains unclear. To clarify this, muscle injury model was recreated by intramuscular injection of bupivacaine hydrochloride in the soleus of p21 knockout (KO mice and wild type (WT mice. The mice were sacrificed at 3, 14, and 28 days post-operation. The results of hematoxylin-eosin staining and immunofluorescence of muscle membrane indicated that muscle regeneration was delayed in p21 KO mice. Cyclin D1 mRNA expression and both Ki-67 and PCNA immunohistochemistry suggested that p21 deficiency increased cell cycle and muscle cell proliferation. F4/80 immunohistochemistry also suggested the increase of immune response in p21 KO mice. On the other hand, both the mRNA expression and western blot analysis of MyoD, myogenin, and Pax7 indicated that muscular differentiation was delayed in p21KO mice. Considering these results, we confirmed that muscle injury causes an increase in cell proliferation. However, muscle differentiation in p21 KO mice was inhibited due to the low expression of muscular synthesis genes, leading to a delay in the muscular regeneration. Thus, we conclude that p21 plays an important role in the in vivo healing process in muscular injury.

  7. EGCG经AKT1-Mdm-2-p53途径抑制卵巢癌HO-891O细胞的增殖%Epigallocatechin-3-gallate Inhibits Proliferation of Ovarian Cancer HO-8910 Cells by AKT 1-Mdm-2-p53 Pathway

    Institute of Scientific and Technical Information of China (English)

    李维; 罗瞳; 罗招阳

    2011-01-01

    Objective: To investigate mechanism of Epigallocatechin-3-gallate (EGCG) inhibited the proliferation of ovarian cancer HO-8910 cells. Methods: EGCG inhibited HO-8910 cellular proliferation was observed by the drawing cellular growth curve, plate colony formation and soft agar colony formation. The expressions of AKT1, Mdm-2 and p53 proteins were detected by Western-blotting. Results: 1. The results of cellular growth curve, plate colony formation and soft agar colony formation showed that EGCG active depressed the proliferation of HO-8910 cells with treatment time extended (n=3, P<0.05). 2. The western-blotting results exhibited that the expressions of AKT1 and Mdm-2 proteins in HO-8910 cells obviously decreased, but the expression of p53 protein increased after EGCG dealing with it (P<0.05). Conclusion: EGCG depressed the expressions of AKT1, Mdm-2 proteins in HO-8910 cells and increased the expression of p53 protein to inhibit the cellular proliferation.%初步探讨EGCG对卵巢癌HO-8910细胞增殖的抑制作用及其机制.方法:通过绘制细胞生长曲线、平皿克隆和软琼脂集落形成实验观察EGCG对HO-8910细胞增殖的抑制作用;Western-blotting检测AKT1、Mdm-2与p53蛋白的表达.结果:(1)细胞生长曲线、平皿克隆和软琼脂集落形成实验结果显示,EGCG可有效抑制HO-8910细胞的增殖(n=3,P<0.05).(2)Westemblotting检测结果显示,EGCG处理后AKT1与Mdm-2蛋白表达均降低,而p53蛋白表达升高(P<0.05).结论:EGCG通过抑制HO-8910细胞中AKT1与Mdm-2蛋白表达,促使p53蛋白表达而发挥其对细胞增殖的抑制作用.

  8. An MDM2 antagonist (MI-319 restores p53 functions and increases the life span of orally treated follicular lymphoma bearing animals

    Directory of Open Access Journals (Sweden)

    Yang Dajun

    2009-12-01

    Full Text Available Abstract Background MI-319 is a synthetic small molecule designed to target the MDM2-P53 interaction. It is closely related to MDM2 antagonists MI-219 and Nutlin-3 in terms of the expected working mechanisms. The purpose of this study was to evaluate anti-lymphoma activity of MI-319 in WSU-FSCCL, a B-cell follicular lymphoma line. For comparison purpose, MI-319, MI-219 and Nutlin-3 were assessed side by side against FSCCL and three other B-cell hematological tumor cell lines in growth inhibition and gene expression profiling experiments. Results MI-319 was shown to bind to MDM2 protein with an affinity slightly higher than that of MI-219 and Nutlin-3. Nevertheless, cell growth inhibition and gene expression profiling experiments revealed that the three compounds have quite similar potency against the tumor cell lines tested in this study. In vitro, MI-319 exhibited the strongest anti-proliferation activity against FSCCL and four patient cells, which all have wild-type p53. Data obtained from Western blotting, cell cycle and apoptosis analysis experiments indicated that FSCCL exhibited strong cell cycle arrest and significant apoptotic cell death; cells with mutant p53 did not show significant apoptotic cell death with drug concentrations up to 10 μM, but displayed weaker and differential cell cycle responses. In our systemic mouse model for FSCCL, MI-319 was tolerated well by the animals, displayed effectiveness against FSCCL-lymphoma cells in blood, brain and bone marrow, and achieved significant therapeutic impact (p 28% (%ILS, 14.4 days increase in median survival days. Conclusion Overall, MI-319 probably has an anti-lymphoma potency equal to that of MI-219 and Nutlin-3. It is a potent agent against FSCCL in vitro and in vivo and holds the promises to be developed further for the treatment of follicular lymphoma that retains wild-type p53.

  9. Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2017-09-01

    The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

  10. P53、MDM2在鼻咽癌组织中的表达及其与鼻咽癌生物学行为、预后关系的分析研究%Expression of p53 and MDM2 in Nasopharyngeal Carcinoma and their Correlation with the Biological Behavior and Prognosis of Nasopharyngeal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    张鹏; 吴松珂; 徐鹏; 冯梅; 范子煊; 李厨荣; 付彬玉; 王卫东; 郎锦义

    2012-01-01

    目的:探讨P53、MDM2在鼻咽癌组织中的表达及与鼻咽癌生物学行为和生存率之间的关系.方法:回顾分析我院2005年~2009年行根治性放化疗治疗的96例鼻咽癌患者,所有患者治疗前行鼻咽部活检病理证实,均采用6MV-X线调强放疗联合以顺铂为主的方案同步化疗,随访3年以上.通过免疫组化SP法检测96例鼻咽癌标本中P53、MDM2蛋白的表达水平,并分析其与临床参数、预后之间关系.结果:P53、MDM2在96例鼻咽癌中阳性表达率分别为65.6%(63/96)、79.17%(76/96).P53的阳性表达率与不同T分期有关(P=0.000);MDM2在不同N分期的鼻咽癌组织中的表达的差异性具有统计学意义(P=0.001);P53的阳性表达在不同临床分期中的差异性具有统计学意义(P=0.037).P53与MDM2的表达呈负相关(r=-3.24,P<0.05).3年无瘤生存率(DFS)及总生存率(OS)分别为73.9%、84.4%.结论:P53、MDMZ的表达与鼻咽癌的生物学行为有关;P53与MDM2的表达呈负相关;P53表达是影响总生存率的预后因素,可以作为评价预后的指标.%Objective; To determine the expression of p53 and MDM2 in nasopharyngeal carcinoma and to investigate the relationship between them and biological behavior , survival rate of nasopharyngeal carcinoma. Methods: Clinical data of 96 nasopharyngeal carcinoma patients accepted radical radiotherapy and chemotherapy in our hospital from 2005 - 2009 were retrospective analyzed. All patients were diagnosed by biopsy of the nasopharynx before treatment. Patients accepted the 6-MV X-ray intensity-modulated radiotherapy combined with cisplatin-based chemotherapy regimen and were followed up for more than 3 years. The expression levels of P53, MDM2 protein in 96 cases of nasopharyngeal specimens were detected by immunohistochemical SP method and their relationship with the clinical parameters, prognosis were analyzed. Results; The positive expression rate of P53 and MDM2 was 65. 6% (63/96) , 79. 17% (76

  11. Neferine, an alkaloid ingredient in lotus seed embryo, inhibits proliferation of human osteosarcoma cells by promoting p38 MAPK-mediated p21 stabilization.

    Science.gov (United States)

    Zhang, Xiyu; Liu, Zhaojian; Xu, Bing; Sun, Zhaoliang; Gong, Yaoqin; Shao, Changshun

    2012-02-29

    Identification of natural products that have antitumor activity is invaluable to the chemoprevention and therapy of cancer. The embryos of lotus (Nelumbo nucifera) seeds are consumed in beverage in some parts of the world for their presumed health-benefiting effects. In this report we studied the effects of neferine, a major alkaloid component in lotus embryos, on human osteosarcoma cells and the underlying mechanisms. We found that neferine possessed a potent growth-inhibitory effect on human osteosarcoma cells, but not on non-neoplastic human osteoblast cells. The inhibitory effect of neferine on human osteosarcoma cells was largely attributed to cell cycle arrest at G1. The induction of G1 arrest was p21(WAF1/CIP1)-dependent, but was independent of p53 or RB (retinoblastoma-associated protein). The up-regulation of p21 by neferine was due to an increase in the half-life of p21 protein. We examined four kinases that are known to affect the stabilization of p21, and found that p38 MAPK and JNK were activated by neferine. However, only SB203580 (an inhibitor of p38), but not SP600125 (the inhibitor of JNK), can attenuate the up-regulation of p21 in response to neferine. Furthermore, the p21-stabilizing effect of neferine was abolished when p38 was silenced by RNA interference. Finally, we showed that neferine treatment led to an increased phosphorylation of p21 at Ser130 that was dependent on p38. Our results for the first time showed a direct antitumor effect of neferine, suggesting that consumption of neferine may have cancer-preventive and cancer-therapeutic benefit.

  12. Synthesis of spiro[isoindole-1,5’-isoxazolidin]-3(2H)-ones as potential inhibitors of the MDM2-p53 interaction

    Science.gov (United States)

    Cirmi, Santa; Mancuso, Raffaella; Nicolò, Francesco; Lanza, Giuseppe; Legnani, Laura; Campisi, Agata; Chiacchio, Maria A; Navarra, Michele; Gabriele, Bartolo

    2016-01-01

    A series of spiro[isoindole-1,5-isoxazolidin]-3(2H)-ones has been synthesized by 1,3-dipolar cycloaddition of N-benzylnitrone with isoindolin-3-methylene-1-ones. The regio- and stereoselectivity of the process have been rationalized by computational methods. The obtained compounds show cytotoxic properties and antiproliferative activity in the range of 9–22 μM. Biological tests suggest that the antitumor activity could be linked to the inhibition of the protein–protein p53-MDM2 interaction. Docking measurements support the biological data. PMID:28144352

  13. Synthesis of spiro[isoindole-1,5’-isoxazolidin]-3(2H-ones as potential inhibitors of the MDM2-p53 interaction

    Directory of Open Access Journals (Sweden)

    Salvatore V. Giofrè

    2016-12-01

    Full Text Available A series of spiro[isoindole-1,5-isoxazolidin]-3(2H-ones has been synthesized by 1,3-dipolar cycloaddition of N-benzylnitrone with isoindolin-3-methylene-1-ones. The regio- and stereoselectivity of the process have been rationalized by computational methods. The obtained compounds show cytotoxic properties and antiproliferative activity in the range of 9–22 μM. Biological tests suggest that the antitumor activity could be linked to the inhibition of the protein–protein p53-MDM2 interaction. Docking measurements support the biological data.

  14. The isolation, total synthesis and structure elucidation of chlorofusin, a natural product inhibitor of the p53-MDM2 protein-protein interaction

    Science.gov (United States)

    Clark, Ryan C.; Lee, Sang Yeul; Searcey, Mark; Boger, Dale L.

    2009-01-01

    Inhibitors of key protein-protein interactions are emerging as exciting therapeutic targets for the treatment of cancer. One such interaction between MDM2 (HDM2) and p53, that silences the tumour suppression activities of p53, was found to be inhibited by the recently isolated natural product chlorofusin. Synthetic studies on this complex natural product summarized herein have served to reassign its chromophore relative stereochemistry, assign its absolute stereochemistry, and provided access to a series of key analogues and partial structures for biological evaluation. PMID:19642417

  15. 肺小细胞癌p53基因分析与mdm-2基因蛋白表达的关系%Relationship of the analyse of p53 gene and the expression of mdm-2 gene protein in small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    杨廷桐; 张俊; 郑杰; 吴秉铨

    1999-01-01

    目的:探讨肺小细胞癌(SCLC)的p53基因改变与mdm-2基因蛋白表达的关系.方法:应用免疫组化LSAB法和聚合酶链反应-单链构象多态性(PCR-SSCP)分析的方法,对14例SCLC的石蜡切片组织进行研究.结果:免疫组化染色p53蛋白阳性9例,阳性率为64.3%(9/14);mdm-2蛋白阳性4例,阳性率为28.6%(4/14).p53基因第5、6、7、8外显子,突变率分别为21.4%(3/14);14.3(2/14);14.3%(2/14);7.1%(1/14),总突变率为57.1%(8/14).结论:SCLC中存在较高的p53基因突变率和mdm-2基因蛋白的明显表达.

  16. Prevalence of p21 immunohistochemical expression in esophageal adenocarcinoma Prevalência da expressão imunoistoquímica da proteína p21 em adenocarcinoma do esôfago

    Directory of Open Access Journals (Sweden)

    Maitê de Mello Villwock

    2006-09-01

    Full Text Available BACKGROUND: In western societies, the prevalence of adenocarcinoma of the gastroesophageal junction has increased in recent years. It is commonly accepted today that esophageal adenocarcinoma develops from a premalignant lesion: Barrett's esophagus. This type of carcinoma is hardly diagnosed at early stages, which results in significant mortality. Molecular biology studies have shown that most malignant tumors originate from the interaction between inherited characteristics and external factors, which may cause genetic changes that interfere with the control over the differentiation and growth of cells in susceptible individuals. p21 (WAF1/CIP1 has a key role in the regulation of the cell cycle, and its immunohistochemical expression has been investigated in several tumors, showing that it influences the prognosis of various neoplasms. AIM: To check the prevalence of p21 protein expression in patients with esophageal adenocarcinoma diagnosed in the last 5 years by the Group for Surgeries of the Esophagus and Stomach of "Hospital de Clínicas de Porto Alegre", RS, Brazil. METHODS: The study population consisted of 42 patients with esophageal adenocarcinoma diagnosed by the Group for Surgeries of the Esophagus and Stomach between January 1998 and December 2002. The expression of p21 protein was determined by immunohistochemistry using primary antibody, p21, clone SX118, code M7202 (Dako, and assessed according to the immunoreactive scoring system. RESULTS: Of 42 analyzed patients, 83.3% were male and older than 40 years. Among these, 56.2% were submitted to curative resection: total gastrectomy and transhiatal esophagogastrectomy. The remaining patients were submitted to palliative surgery or did not undergo any surgical treatment. Only five patients received adjuvant chemotherapy and radiation therapy, either alone or combined. Advanced disease (stages III and IV was detected in 78.6% of the patients. Only nine patients were positive for p21

  17. Pre-clinical evaluation of the MDM2-p53 antagonist RG7388 alone and in combination with chemotherapy in neuroblastoma.

    Science.gov (United States)

    Chen, Lindi; Rousseau, Raphaël F; Middleton, Steven A; Nichols, Gwen L; Newell, David R; Lunec, John; Tweddle, Deborah A

    2015-04-30

    Neuroblastoma is a predominantly p53 wild-type (wt) tumour and MDM2-p53 antagonists offer a novel therapeutic strategy for neuroblastoma patients. RG7388 (Roche) is currently undergoing early phase clinical evaluation in adults. This study assessed the efficacy of RG7388 as a single-agent and in combination with chemotherapies currently used to treat neuroblastoma in a panel of neuroblastoma cell lines. RG7388 GI50 concentrations were determined in 21 p53-wt and mutant neuroblastoma cell lines of varying MYCN, MDM2 and p14(ARF) status, together with MYCN-regulatable Tet21N cells. The primary determinant of response was the presence of wt p53, and overall there was a >200-fold difference in RG7388 GI50 concentrations for p53-wt versus mutant cell lines. Tet21N MYCN+ cells were significantly more sensitive to RG7388 compared with MYCN- cells. Using median-effect analysis in 5 p53-wt neuroblastoma cell lines, selected combinations of RG7388 with cisplatin, doxorubicin, topotecan, temozolomide and busulfan were synergistic. Furthermore, combination treatments led to increased apoptosis, as evident by higher caspase-3/7 activity compared to either agent alone. These data show that RG7388 is highly potent against p53-wt neuroblastoma cells, and strongly supports its further evaluation as a novel therapy for patients with high-risk neuroblastoma and wt p53 to potentially improve survival and/or reduce toxicity.

  18. The silence of p66(Shc) in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway.

    Science.gov (United States)

    Zhang, Ling; Zhu, Shengtao; Shi, Xuesen; Sha, Weihong

    2015-01-01

    Colon cancer is the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. The previous studies demonstrated that p66(Shc) protein, a member of Shc family, is highly expressed in colon cancer cells, but the role of p66(Shc) in the progress of colon cancer still unknown. In this study, we found that p66(Shc) highly expressed in colon cancer tissue and colon cancer cell line SW620 cells, HCT8 cells, HCT116 cells and CaCO2 cells. The silence of p66(Shc) in HCT8 cells reduced the proliferation and accelerated the apoptosis, in addition, the expression of pro-apoptotic proteins caspase-3, caspase-9, Bax was enhanced and the expression of anti-apoptotic protein Bcl-2 was declined. Moreover, the cell cycle arrest in G0/G1 phase after HCT8 cells treated with p66(Shc) siRNA. Furthermore, after HCT8 cells treated with p66(Shc) siRNA, the phosphorylation of PI3K and AKT was significantly suppressed, and the expression of Mdm-2, a downstream of AKT, was obviously prohibited, while the expression of p53 was enhanced. These results indicate that the silence of p66(Shc) in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway, it may provide a promising approach to prevent the progress of colon cancer cell.

  19. The silence of p66Shc in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway

    Science.gov (United States)

    Zhang, Ling; Zhu, Shengtao; Shi, Xuesen; Sha, Weihong

    2015-01-01

    Colon cancer is the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. The previous studies demonstrated that p66Shc protein, a member of Shc family, is highly expressed in colon cancer cells, but the role of p66Shc in the progress of colon cancer still unknown. In this study, we found that p66Shc highly expressed in colon cancer tissue and colon cancer cell line SW620 cells, HCT8 cells, HCT116 cells and CaCO2 cells. The silence of p66Shc in HCT8 cells reduced the proliferation and accelerated the apoptosis, in addition, the expression of pro-apoptotic proteins caspase-3, caspase-9, Bax was enhanced and the expression of anti-apoptotic protein Bcl-2 was declined. Moreover, the cell cycle arrest in G0/G1 phase after HCT8 cells treated with p66Shc siRNA. Furthermore, after HCT8 cells treated with p66Shc siRNA, the phosphorylation of PI3K and AKT was significantly suppressed, and the expression of Mdm-2, a downstream of AKT, was obviously prohibited, while the expression of p53 was enhanced. These results indicate that the silence of p66Shc in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway, it may provide a promising approach to prevent the progress of colon cancer cell. PMID:26464652

  20. Elucidating the digital control mechanism for DNA damage repair with the p53-Mdm2 system: single cell data analysis and ensemble modelling.

    Science.gov (United States)

    Ogunnaike, Babatunde A

    2006-02-22

    Recent experimental evidence about DNA damage response using the p53-Mdm2 system has raised some fundamental questions about the control mechanism employed. In response to DNA damage, an ensemble of cells shows a damped oscillation in p53 expression whose amplitude increases with increased DNA damage--consistent with 'analogue' control. Recent experimental results, however, show that the single cell response is a series of discrete pulses in p53; and with increase in DNA damage, neither the height nor the duration of the pulses change, but the mean number of pulses increase--consistent with 'digital' control. Here we present a system engineering model that uses published data to elucidate this mechanism and resolve the dilemma of how digital behaviour at the single cell level can manifest as analogue ensemble behaviour. First, we develop a dynamic model of the p53-Mdm2 system that produces non-oscillatory responses to a stress signal. Second, we develop a probability model of the distribution of pulses in a cell population, and combine the two with the simplest digital control algorithm to show how oscillatory responses whose amplitudes grow with DNA damage can arise from single cell behaviour in which each single pulse response is independent of the extent of DNA damage. A stochastic simulation of the hypothesized control mechanism reproduces experimental observations remarkably well.

  1. Aberrations of the p53 pathway components p53, MDM2 and CDKN2A appear independent in diffuse large B cell lymphoma

    DEFF Research Database (Denmark)

    Møller, Michael Boe; Ino, Y; Gerdes, A M

    1999-01-01

    The two gene products of the CDKN2A gene, p16 and p19ARF, have recently been linked to each of two major tumour suppressor pathways in human carcinogenesis, the RB1 pathway and the p53 pathway. p16 inhibits the phosphorylation of the retinoblastoma gene product by cyclin D-dependent kinases...... of aberrations of CDKN2A, MDM2 and p53, since this has not been analysed previously. We investigated 37 DLCL for aberrations of p15, p16, p19ARF, MDM2, and p53 at the epigenetic, genetic and/or protein levels. Homozygous deletion of CDKN2A was detected in seven (19%) of 37 tumours, and another three cases were...... hypermethylated at the 5' CpG island of p16. No point mutations were found in CDKN2B or CDKN2A. Immunohistochemical staining of formalin-fixed, paraffin-embedded tissue for p16 confirmed these results, as all tumours with alterations of CDKN2A were p16 immunonegative. We found p53 mutations in eight (22%) cases...

  2. Long non-coding RNA ENST00462717 suppresses the proliferation, survival, and migration by inhibiting MDM2/MAPK pathway in glioma.

    Science.gov (United States)

    Wang, Aiqin; Meng, Mingzhu; Zhao, Xiuhe; Kong, Lina

    2017-04-01

    Gliomas are the most common and aggressive primary malignant tumor in the central nervous system, and requires new biomarkers and therapeutic methods. Long noncoding RNAs (lncRNAs) are important factors in numerous human diseases, including cancer. But studies on lncRNAs and gliomas are limited. In this study, we investigated the expression patterns of lncRNAs in 3 pairs of glioma samples and adjacent non-tumor tissues via microarray and selected the most down-regulated lnc00462717 to further verify its roles in glioma. We observed that decreased lnc00462717 expression was associated with the malignant status in glioma. In vitro experiment demonstrated that lnc00462717 overexpression suppressed glioma cell proliferation, survival and migration while knockdown of lnc00462717 had an opposite result. Moreover, we identified MDM2 as a direct target of lnc00462717 and lnc00462717 played a role by partially regulating the MDM2/MAPK pathway. In conclusion, lnc00462717 may function in suppressing glioma cell proliferation, survival, migration and may potentially serve as a novel biomarker and therapeutic target for glioma.

  3. Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

    Science.gov (United States)

    Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

    2015-02-01

    In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on

  4. Expression of P14ARF, MDM2 and mutant type P53 in skin tissue of coal-burning-type of endemic arseniasis patients%燃煤污染型地方性砷中毒患者皮肤组织P14ARF和MDM2及突变型P53蛋白表达

    Institute of Scientific and Technical Information of China (English)

    夏玉洁; 张爱华; 韩雪; 黄晓欣

    2012-01-01

    目的 观察燃煤污染型地方性砷中毒患者皮肤组织突变型P53( P53mt)蛋白及其下游基因P14ARG、MDM2蛋白的表达水平,为揭示砷致皮肤损害的分子机制提供依据.方法 以自愿手术治疗的60例燃煤污染型地方性砷中毒患者作为观察对象,依据皮肤病理学诊断分为一般皮肤病变组(35例)、癌前病变组(19例)、癌变组(6例);以某医院经病理学诊断无异常的非肿瘤手术患者的9例皮肤组织为对照,采用免疫组织化学EnVision二步法检测皮肤组织中P14ARF、MDM2和P53mt蛋白的表达情况.结果 4组皮肤组织P14ARF蛋白表达阳性率比较,差异有统计学意义(x2=9.39,P<0.05);其中癌前病变组、癌变组[46.1%(6/19)、33.3%(2/6)]明显低于对照组[88.9%(8/9),P均<0.05];且随着皮肤病变程度的加重,P14ARF蛋白表达呈降低趋势(P<0.05).4组皮肤组织MDM2、P53mt蛋白表达阳性率比较,差异有统计学意义(x2值分别为6.21、20.64,P均< 0.05);其中一般病变组、癌前病变组和癌变组[54.2%( 19/35)、63.2%(10/19)、66.7%(4/6)和25.7%(9/35)、73.7%(14/19)、83.3%(5/6)]明显高于对照组(0、0,P均<0.05);随着皮肤病变程度的加重,MDM2和P53mt蛋白表达呈增强趋势(P均<0.05).结论 燃煤污染型地方性砷中毒患者皮肤组织P53mt蛋白表达阳性率升高,且P14ARF和MDM2蛋白表达异常,后者所致的细胞周期和凋亡调控失常可能是砷致皮肤病损发生发展的原因之一.%Objective To determine the protein expression of P14ARF,MDM2 and mutant type P53 (P53mt) in skin specimens of coal-burning-type of endemic arseniasis patients and to reveal the molecular mechanism of the disease.Methods Sixty skin specimens from 60 endemic arseniasis patients including 35 of skin lesions patients,19 of precancerous lesion and 6 of skin cancer and 9 normal skin specimens from non-cancer patients were studied.Expression of P14~,MDM2 and P53mt was evaluated

  5. EXPRESSION OF △Np63 AND MDM2 PROTEINS IN TRANSITIONAL CELL CARCINOMA OF URINARY BLADDER%膀胱移行细胞癌组织△Np63和MDM2蛋白表达及意义

    Institute of Scientific and Technical Information of China (English)

    张鲁伟; 周荣祥

    2007-01-01

    目的 探讨缺乏酸性N端反式激活区的截短型p63(△Np63)和MDM2蛋白在膀胱移行细胞癌(TCC)组织中的表达及其临床意义.方法 用免疫组织化学法,检测35例石蜡包埋TCC组织标本中△Np63和MDM2的表达,以38例正常膀胱黏膜作为对照.结果 膀胱癌组30例(83.3%)△Np63蛋白呈阳性表达,膀胱黏膜组7例(18.4%)呈阳性表达,两组比较差异有显著性(uc=5.75,P<0.01).膀胱癌组15例(42.9%)MDM2蛋白呈阳性表达,膀胱黏膜组3例(7.9%)呈阳性表达,两组差异有显著性(x2=11.99,P<0.01).在不同病理分级、临床分期TCC中,△Np63表达差异有显著性(H=21.09、14.99,P<0.01).随着TCC病理分级、临床分期升高,△Np63表达明显增强,呈显著正相关(r=0.64、0.51,P<0.01).在不同病理分级、临床分期TCC中,MDM2表达差异有显著性(x2=16.63,P<0.01;x2=6.72,P<0.05).30例△Np63蛋白阳性表达TCC组织中MDM2蛋白阳性表达10例(33.3%),两者呈显著相关性(x2=7.78,P<0.05).△Np63阳性表达主要集中在低分化、浸润性TCC中,而MDM2则主要表达在高分化、浅表TCC中.结论 △Np63和MDM2与膀胱肿瘤的发生发展密切相关,可作为膀胱肿瘤早期诊断和判断恶性程度的参考指标.

  6. DDX3 loss by p53 inactivation promotes tumor malignancy via the MDM2/Slug/E-cadherin pathway and poor patient outcome in non-small-cell lung cancer.

    Science.gov (United States)

    Wu, D-W; Lee, M-C; Wang, J; Chen, C-Y; Cheng, Y-W; Lee, H

    2014-03-20

    P53 inactivation by p53 mutation and E6 oncoprotein has a crucial role in human carcinogenesis. DDX3 has been shown to be a target of p53. In this study, we hypothesized that DDX3 loss by p53 inactivation may promote tumor malignancy and poor patients' outcome. Mechanically, DDX3 loss by p53 knockdown and E6 overexpression was observed in A549 lung cancer cells. Conversely, DDX3 expression was markedly elevated by wild-type (WT) p53 ectopic expression in p53-null H1299 cells, E6-knockdown TL-1 lung cancer and SiHa cervical cancer cells. Interestingly, DDX3 loss promotes soft-agar growth and invasive capability; however, both capabilities were suppressed by DDX3 overexpression. We next expected that DDX3 loss might result in Slug-suppressed E-cadherin expression via decreased MDM2-mediated Slug degradation. As expected, MDM2 transcription is suppressed by DDX3 loss via decreased SP1 binding activity to the MDM2 promoter. Consequently, Slug expression was elevated by the reduction of MDM2 because of DDX3 loss, and E-cadherin expression was suppressed by Slug. Consistent observations in the correlation of DDX3 loss with MDM2, Slug and E-cadherin were seen in lung tumors from lung cancer patients. In addition, patients with low-DDX3 tumors had poorer survival and relapse than patients with high-DDX3 tumors. In conclusion, we suggest that DDX3 loss by p53 inactivation via MDM2/Slug/E-cadherin pathway promotes tumor malignancy and poor patient outcome.

  7. Evaluation of TP53 Pro72Arg and MDM2 SNP285-SNP309 polymorphisms in an Italian cohort of LFS suggestive patients lacking identifiable TP53 germline mutations.

    Science.gov (United States)

    Ponti, Francesca; Corsini, Serena; Gnoli, Maria; Pedrini, Elena; Mordenti, Marina; Sangiorgi, Luca

    2016-10-01

    Li-Fraumeni syndrome (LFS) is a rare genetic cancer predisposition disease, partly determined by the presence of a TP53 germline mutation; lacking thereof, in presence of a typical LFS phenotype, defines a wide group of 'LFS Suggestive' patients. Alternative LFS susceptibility genes have been investigated without promising results, thus suggesting other genetic determinants involvement in cancer predisposition. Hence, this study explores the single and combined effects of cancer risk, age of onset and cancer type of three single nucleotide polymorphisms (SNPs)-TP53 Pro72Arg, MDM2 SNP285 and SNP309-already described as modifiers on TP53 mutation carriers but not properly investigated in LFS Suggestive patients. This case-control study examines 34 Italian LFS Suggestive lacking of germline TP53 mutations and 95 tumour-free subjects. A significant prevalence of homozygous MDM2 SNP309 G in the LFS Suggestive group (p TP53 positive families. Conversely its anticipating role on tumour onset has not been confirmed, as in our results it was associated with the SNP309 T allele. A strong combined outcome with a 'dosage' effect has also been reported for TP53 P72 and MDM2 SNP309 G allele on cancer susceptibility (p < 0.0005). Whereas the MDM2 SNP285 C allele neutralizing effect on MDM2 SNP309 G variant is not evident in our population. Although it needs further evaluations, obtained results strengthen the role of MDM2 SNP309 as a genetic factor in hereditary predisposition to cancer, so improving LFS Suggestive patients management.

  8. Determination of mRNA, and protein levels of p53, MDM2 and protein kinase CK2 subunits in F9 cells after treatment with the apoptosis-inducing drugs cisplatin and carboplatin

    DEFF Research Database (Denmark)

    Siemer, S; Ornskov, D; Guerra, B

    1999-01-01

    Protein kinase CK2 is a pleiotropic serine/threonine kinase which has been shown to phosphorylate numerous substrates. Evidence is accumulating that CK2 may exist complexed to a variety of cellular proteins, e.g. p53, MDM2, and A-Raf. Here, we explored the effects of the chemotherapeutic drugs...... cisplatin and carboplatin on the mRNA and protein levels of p53, MDM2 and CK2 in a murine teratocarcinoma cell line F9. Northern and Western blot analyses were performed and the CK2 activity was determined. The degree of apoptosis after drug treatment was assessed using the TUNEL test. Six hours after...

  9. An Integrative Analysis Reveals a Central Role of P53 Activation via MDM2 in Zika Virus Infection Induced Cell Death

    Directory of Open Access Journals (Sweden)

    Yue Teng

    2017-07-01

    Full Text Available Zika virus (ZIKV infection is an emerging global threat that is suspected to be associated with fetal microcephaly. However, the molecular mechanisms underlying ZIKV disease pathogenesis in humans remain elusive. Here, we investigated the human protein interaction network associated with ZIKV infection using a systemic virology approach, and reconstructed the transcriptional regulatory network to analyze the mechanisms underlying ZIKV-elicited microcephaly pathogenesis. The bioinformatics findings in this study show that P53 is the hub of the genetic regulatory network for ZIKV-related and microcephaly-associated proteins. Importantly, these results imply that the ZIKV capsid protein interacts with mouse double-minute-2 homolog (MDM2, which is involved in the P53-mediated apoptosis pathway, activating the death of infected neural cells. We also found that synthetic mimics of the ZIKV capsid protein induced cell death in vitro and in vivo. This study provides important insight into the relationship between ZIKV infection and brain diseases.

  10. The suppression of prostate LNCaP cancer cells growth by Selenium nanoparticles through Akt/Mdm2/AR controlled apoptosis.

    Science.gov (United States)

    Kong, Ling; Yuan, Qing; Zhu, Huarui; Li, Ying; Guo, Quanyi; Wang, Qin; Bi, Xiaolin; Gao, Xueyun

    2011-09-01

    The trace element Selenium is suggested having cancer prevention activity and used as food supplement. Previous results had shown Selenium nanoparticles are safer compared with other Selenium compounds like selenomethionine, sodium selenite and monomethylated Selenium, however, its anticancer activity and intrinsic mechanisms are still elusive. Here, we prepared Selenium nanoparticles and investigated its inherent anticancer mechanisms. We found Selenium nanoparticles inhibit growth of prostate LNCaP cancer cells partially through caspases mediated apoptosis. Selenium nanoparticles suppress transcriptional activity of androgen receptor via down-regulating its mRNA and protein expression. Moreover, Selenium nanoparticles activate Akt kinase by increasing its phosphorylation, promote Akt-dependent androgen receptor phosphorylation and Mdm2 regulated degradation through proteasome pathway. We suggest Selenium nanoparticles suppress prostate cancer cells growth by disrupting androgen receptor, implicating a potential application in cancer treatment.

  11. Lipid Synthetic Transcription Factor SREBP-1a Activates p21WAF1/CIP1, a Universal Cyclin-Dependent Kinase Inhibitor

    OpenAIRE

    INOUE, Noriyuki; Shimano, Hitoshi; Nakakuki, Masanori; Matsuzaka, Takashi; Nakagawa, Yoshimi; Yamamoto, Takashi; Sato, Ryuichiro; Takahashi, Akimitsu; Sone, Hirohito; Yahagi, Naoya; Suzuki, Hiroaki; Toyoshima, Hideo; Yamada, Nobuhiro

    2005-01-01

    Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that regulate lipid synthetic genes. In contrast to SREBP-2, which regulates cellular cholesterol level in normal cells, SREBP-1a is highly expressed in actively growing cells and activates entire programs of genes involved in lipid synthesis such as cholesterol, fatty acids, triglycerides, and phospholipids. Previously, the physiological relevance of this potent activity of SREBP-1a has been thought ...

  12. Giant cell rich osteosarcoma revisited-diagnostic criteria and histopathologic patterns, Ki67, CDK4, and MDM2 expression, changes in response to bisphosphonate and denosumab treatment.

    Science.gov (United States)

    Chow, Louis Tsun Cheung

    2016-06-01

    Defining giant cell-rich osteosarcoma (GCRO) as "an osteosarcoma in which more than 50% of the tumor consists of numerous uniformly distributed osteoclastic giant cells amidst oval or spindle mononuclear cells embedded in a fibrovascular stroma," eight such cases identified among 265 cases of osteosarcoma were analysed. Their age ranges from 11 to 33 years, with peak incidence in the second decade and equal sex distribution. Seventy-five percent presented with pain, commonest in the knee, affecting the metaphysis. Most appeared radiologically as well-circumscribed expansile multiloculated osteolytic lesions, and many are displayed periosteal reaction. They showed several distinct histologic patterns: the stromal and giant cell, fibrohistiocytic, aneurysmal-cystic, osteoblastoma-like, and parosteal and fibrous dysplasia-like patterns. Focal subtle lacelike osteoid deposition, permeative infiltration into adjacent native bony trabeculae and over 30 % Ki67 proliferative index were characteristic. There was no CDK4 and MDM2 amplification. In those having bisphosphonate and denosumab treatment, there was limited focal necrosis with reduction in the number of giant cells and broad trabecular woven bone formation but no giant osteoclast was seen. Two patients with initial diagnosis of giant cell tumor treated by curettage and local resection pursued aggressive clinical courses, died after 14 and 21 months. The others survived 12 to 110 months. GCRO accounts for about 3 % of all osteosarcomas and apart from its more frequent diaphyseal location and associated normal bone-specific alkaline phosphate levels; it shares with conventional high-grade osteosarcoma the same patient demographics, sites of occurrence, absence of CDK4 and MDM2 amplification, and probably clinical course.

  13. Biochemical Characterization of Complexes with p21, a CDK Inhibitor

    Science.gov (United States)

    1998-08-01

    additional experiments to further characterize p28 and p40 , two potentially novel proteins that co-fractionated with p21 on glycerol gradients, sizing...well as with amino- and carboxy-terminal fragments of p21. Neither p28 nor p40 was captured in preliminary binding experiments, suggesting that these...an additional step of 1.0 HMGNB (25 mM Z 75 HEPES [pH 7.6], 1 M NaCI, 10% glycerol, 0.1% Nonidet P-40 [NP-40], 5 mM U P-mercaptoethanol, and 0.2 mM

  14. Cytoplasmic translocation of p21 mediates NUPR1-induced chemoresistance: NUPR1 and p21 in chemoresistance.

    Science.gov (United States)

    Vincent, Andrew J; Ren, Suping; Harris, Lillianne G; Devine, Daniel J; Samant, Rajeev S; Fodstad, Oystein; Shevde, Lalita A

    2012-09-21

    The expression of Nuclear Protein 1 (NUPR1) is associated with chemoresistance in multiple malignancies. We previously reported that NUPR1 functions as a transcriptional cofactor for the p300-p53 complex and transcriptionally regulates p21 expression. In the present study we investigated the activity of NUPR1 in p53-deficient, triple-negative, inflammatory SUM159 breast cancer cells. Our studies reveal that NUPR1 confers growth benefit and chemoresistance by causing Akt-mediated phosphorylation and subsequent cytoplasmic re-localization of p21 and activation of the anti-apoptotic Bcl-xL protein. Our findings elucidate a NUPR1-PI-3-K/Akt-phospho-p21 axis that functions in p53-negative, inflammatory breast cancer cells to enhance chemoresistance in breast cancer.

  15. Bacterial cyclomodulin Cif blocks the host cell cycle by stabilizing the cyclin-dependent kinase inhibitors p21 and p27.

    Science.gov (United States)

    Samba-Louaka, Ascel; Nougayrède, Jean-Philippe; Watrin, Claude; Jubelin, Grégory; Oswald, Eric; Taieb, Frédéric

    2008-12-01

    The cycle inhibiting factor (Cif) is a cyclomodulin produced by enteropathogenic and enterohemorrhagic Escherichia coli. Upon injection into the host cell by the bacterial type III secretion system, Cif inhibits the G2/M transition via sustained inhibition of the mitosis inducer CDK1 independently of the DNA damage response. In this study, we show that Cif induces not only G2, but also G1 cell cycle arrest depending on the stage of cells in the cell cycle during the infection. In various cell lines including differentiated and untransformed enterocytes, the cell cycle arrests are correlated with the accumulation of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). Cif-induced cyclin-dependent kinase inhibitor accumulation is independent of the p53 pathway but occurs through inhibition of their proteasome-mediated degradation. Our results provide a direct link between the mode of action of Cif and the host cell cycle control.

  16. NOXA、PUMA等基因在乳腺癌中的表达及其意义%The Expression Characteristics of MDM2, PCNA, NOXA and PUMA in Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    王尚文; 佟钧; 诸晨; 平妮娜; 喻箴; 崔明; 何越峰

    2016-01-01

    目的 比较癌组织与正常组织中MDM2、PCNA、NOXA、PUMA表达的差异,了解乳腺癌与表达的关系.方法 通过SYBR Green实时荧光定量PCR方法检测乳腺癌组织和正常乳腺组织中MDM2、PCNA、NOXA、PUMA 4个基因的含量.结果 (1) NOXA、和PUMA在乳腺癌组织中表达升高(P<0.05);(2) PCNA和NOXA的表达存在正相关关系(r=0.82,P<0.05));PCNA和MDM2的表达存在正相关关系(r=0.80,P<0.05);PUMA和MDM2的表达存在正相关关系(r=0.82,P<0.05).结论 NOXA、PUMA在乳腺癌组织中处于高表达状态,4个基因表达与乳腺癌有关.

  17. Markers aiding the diagnosis of chondroid tumors: an immunohistochemical study including osteonectin, bcl-2, cox-2, actin, calponin, D2-40 (podoplanin), mdm-2, CD117 (c-kit), and YKL-40

    DEFF Research Database (Denmark)

    Daugaard, Søren; Christensen, Lise H; Høgdall, Estrid

    2009-01-01

    (s) for the different subgroups. Archival material from three extraskeletal myxoid chondrosarcomas, five chordomas, five chondromyxoid fibromas, five chondroblastomas and 25 chondrosarcomas was stained with antibodies against osteonectin, bcl-2, cox-2, actin, calponin, D2-40 (podoplanin), mdm-2, CD117 (c-kit) and YKL......) and extraskeletal myxoid chondrosarcomas (n=3) were positive for bcl-2. In contrast to all other tumors, two of three extraskeletal myxoid chondrosarcomas were also positive for CD17 and negative for osteonectin, cox-2, mdm-2 and actin. All five chordomas were negative for D2-40 and positive for mdm-2 and YKL-40....... The diagnosis of chondrosarcoma may be aided by its positivity for D2-40 and YKL-40 and its lack of reactivity for actin and CD117. This should be seen in the light of no reaction for D2-40 in chordomas and a corresponding lack of reaction for osteonectin, cox-2, mdm-2 and actin in extraskeletal myxoid...

  18. Evaluation of MDM2 and CDK4 amplification by real-time PCR on paraffin wax-embedded material: a potential tool for the diagnosis of atypical lipomatous tumours/well-differentiated liposarcomas.

    Science.gov (United States)

    Hostein, I; Pelmus, M; Aurias, A; Pedeutour, F; Mathoulin-Pélissier, S; Coindre, J M

    2004-01-01

    Atypical lipomatous tumours/well-differentiated liposarcomas and dedifferentiated liposarcomas are characterized by 12q13-15 region amplification. In contrast, this molecular event has not been reported in benign lipomas. Within the 12q13-15 chromosomal region, the MDM2, SAS, HMGA2, and CDK4 genes are the most frequent targets of amplification. A series of lipomas (36 cases) and liposarcomas (48 cases) was analysed for MDM2 and CDK4 gene amplification by real-time PCR. MDM2 and CDK4 gene amplification was detected in 2.8% and 5.6% of lipomas and 98.2% and 82.4% of liposarcomas, respectively. Moreover, co-amplification of the two genes as well as a higher-level amplification was observed more frequently in dedifferentiated liposarcomas than in atypical lipomatous tumours/well-differentiated liposarcomas. Real-time PCR proved to be a fast and reliable method to characterize lipomas and liposarcomas by quantification of MDM2 and CDK4 gene amplification. It is applicable to paraffin wax-embedded tissues and could be useful when histological diagnosis is difficult.

  19. Molecular mechanisms of MYCN-dependent apoptosis and the MDM2-p53 pathway: an Achille’s heel to be exploited for the therapy of MYCN amplified neuroblastoma

    Directory of Open Access Journals (Sweden)

    Marialaura ePetroni

    2012-10-01

    Full Text Available The p53 oncosuppressor is very seldom mutated in neuroblastoma, but several mechanisms cooperate to its functional inactivation in this tumor. Increased MDM2 levels, due to genetic amplification or constitutive inhibition of p14ARF, significantly contribute to this event highlighting p53 reactivation as an attractive perspective for neuroblastoma treatment.In addition to its role in tumorigenesis, MYCN sensitizes untransformed and cancer cells to apoptosis. This is associated to a fine modulation of the MDM2-p53 pathway. Indeed MYCN induces p53 and MDM2 transcription, and, by evoking a DNA damage response (DDR, it stabilizes p53 and its proapoptotic kinase HIPK2. Through the regulation of the HIPK2-p53 inhibitor HMGA1 and the homeobox proteins BMI-1 and TWIST-1, MYCN establishes a delicate balance between pro- and anti-apoptotic molecules that might be easily perturbed by a variety of insults, leading to cell death. MDM2-p53 antagonists, such as Nutlin-3, are strikingly prone to inducing death in MYCN-amplified neuroblastoma, by further pushing on HIPK2 accumulation. Here we discuss implications and caveats of exploiting this pathway and its connections to MYCN-induced DDR for a tailored therapy of MYCN-amplified neuroblastoma.

  20. p21-activated kinase family: promising new drug targets

    Directory of Open Access Journals (Sweden)

    Huynh N

    2015-05-01

    Full Text Available Nhi Huynh, Hong He Department of Surgery, University of Melbourne, Austin Health, Melbourne, VIC, Australia Abstract: The p21-activated kinase (PAK family of serine/threonine protein kinases are downstream effectors of the Rho family of GTPases. PAKs are frequently upregulated in human diseases, including various cancers, and their overexpression correlates with disease progression. Current research findings have validated important roles for PAKs in cell proliferation, survival, gene transcription, transformation, and cytoskeletal remodeling. PAKs are shown to act as a converging node for many signaling pathways that regulate these cellular processes. Therefore, PAKs have emerged as attractive targets for treatment of disease. This review discusses the physiological and pathological roles of PAKs, validation of PAKs as new promising drug targets, and current challenges and advances in the development of PAK-targeted anticancer therapy, with a focus on PAKs and human cancers. Keywords: p21-activated kinase, cancer, inhibitor

  1. The NEDD8 inhibitor MLN4924 increases the size of the nucleolus and activates p53 through the ribosomal-Mdm2 pathway.

    Science.gov (United States)

    Bailly, A; Perrin, A; Bou Malhab, L J; Pion, E; Larance, M; Nagala, M; Smith, P; O'Donohue, M-F; Gleizes, P-E; Zomerdijk, J; Lamond, A I; Xirodimas, D P

    2016-01-28

    The ubiquitin-like molecule NEDD8 is essential for viability, growth and development, and is a potential target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human and germline cells of Caenorhabditis elegans in the absence of nucleolar fragmentation. SILAC proteomics and monitoring of rRNA production, processing and ribosome profiling shows that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of inhibitors of NEDDylation and provides a mechanism for p53 activation upon NEDD8 inhibition. It also indicates that targeting the nucleolar proteome without affecting nucleolar transcription initiates the required signalling events for the control of cell cycle regulators.

  2. E2/ER β Enhances Calcineurin Protein Degradation and PI3K/Akt/MDM2 Signal Transduction to Inhibit ISO-Induced Myocardial Cell Apoptosis

    Directory of Open Access Journals (Sweden)

    Kuan-Ho Lin

    2017-04-01

    Full Text Available Secretion of multifunctional estrogen and its receptor has been widely considered as the reason for markedly higher frequency of heart disease in men than in women. 17β-Estradiol (E2, for instance, has been reported to prevent development of cardiac apoptosis via activation of estrogen receptors (ERs. In addition, protein phosphatase such as protein phosphatase 1 (PP1 and calcineurin (PP2B are also involved in cardiac hypertrophy and cell apoptosis signaling. However, the mechanism by which E2/ERβ suppresses apoptosis is not fully understood, and the role of protein phosphatase in E2/ERβ action also needs further investigation. In this study, we observed that E2/ERβ inhibited isoproterenol (ISO-induced myocardial cell apoptosis, cytochrome c release and downstream apoptotic markers. Moreover, we found that E2/ERβ blocks ISO-induced apoptosis in H9c2 cells through the enhancement of calcineurin protein degradation through PI3K/Akt/MDM2 signaling pathway. Our results suggest that supplementation with estrogen and/or overexpression of estrogen receptor β gene may prove to be effective means to treat stress-induced myocardial damage.

  3. Kõlar Hi-Waf / Ell-Maaja Randküla

    Index Scriptorium Estoniae

    Randküla, Ell-Maaja, 1939-2016

    2005-01-01

    Disain: Galina Burnakova. Tootja: AS Sarkop. Kõlar Hi-Waf tehti Brüsselis galeriis Creneau International toimuvale näitusele viimiseks. Galina Burnakovast, tema erialasest tegevusest. Ill.: kõlari vaade, foto sisearhtektist

  4. Kõlar Hi-Waf / Ell-Maaja Randküla

    Index Scriptorium Estoniae

    Randküla, Ell-Maaja, 1939-2016

    2005-01-01

    Disain: Galina Burnakova. Tootja: AS Sarkop. Kõlar Hi-Waf tehti Brüsselis galeriis Creneau International toimuvale näitusele viimiseks. Galina Burnakovast, tema erialasest tegevusest. Ill.: kõlari vaade, foto sisearhtektist

  5. 鼠双微粒体-2基因沉默对人肝癌HepG2细胞移植瘤生长的抑制作用%shRNA-mediated silencing of MDM2 inhibits growth of HepG2 hepatocellular carcinoma cells xenografted in nude mice

    Institute of Scientific and Technical Information of China (English)

    赵燕颖; 李亚刚; 孙远杰; 刘海鹏; 杨泽成; 张舵舵; 赵春燕

    2013-01-01

    目的 构建鼠双微粒体-2基因(MDM2)靶向小分子干扰RNA (siRNA)质粒,研究其对人肝癌HepG2细胞裸鼠皮下移植瘤生长的抑制作用,探讨其在肝癌基因治疗中的可行性.方法 构建MDM2的siRNA真核表达载体pSilencer-siRNA-MDM2 (siMDM2),建立裸鼠荷瘤模型,分别给予阴性对照质粒和siMDM2质粒处理.监测肿瘤生长变化,RT-PCR、Western blot方法检测MDM2及p53 mRNA和蛋白的表达变化.正态分布数据的组间比较用单因素方差分析及LSD检验.结果 裸鼠体内实验结果显示,转染siMDM2-1和siMDM2-2组与对照组相比,肿瘤增长受到明显抑制,抑瘤率分别为60.6%和54.6%.RT-PCR和Westem blot结果示MDM2的mRNA和蛋白质表达下调,而p53的mRNA和蛋白质表达上调.与空白组比较,转染siMDM2-1和siMDM2-2组的MDM2mRNA表达分别下调62.8%和61.5%,MDM2蛋白表达分别下调60.7%和59.5%;p53 mRNA表达分别上调47.1%和45.6%,p53蛋白表达分别上调45.9%和44.3%,差异均具有统计学意义(F值分别为75.099、47.860、17.676和235.770,P值均<0.01).结论 siRNA沉默MDM2基因能抑制人肝癌HepG2细胞裸鼠皮下移植瘤的形成,可能与其在体内有效下调MDM2和上调p53的mRNA及蛋白质表达有关.%Objecive To construct a short hairpin (sh)RNA targeting the gene encoding the MDM2 oncoprotein in order to investigate its role in human hepatocellular carcinoma (HCC) and its potential for use as a gene therapy strategy to inhibit HCC growth in vivo.Methods Small interfering (si)RNAs were designed targeting the