绿茶对人胃癌细胞株中p21,p53蛋白表达的影响

应用免疫细胞化学方法检测ＳＧＣ—７９０１胃癌细胞株中ｐ２１、ｐ５３蛋白的表达,以探讨绿茶的抗癌作用机理。结果表明：绿茶提取物明显抑制ＳＧＣ—７９０１胃癌细胞株中ｐ２１ｒａｓ、ｐ５３蛋白的表达,并有剂量效应。提示绿茶对ｐ２１、ｐ５３基因突变可能有修复作用     

人MDM2基因真核表达载体的构建及其与p53的相互作用

目的:构建带Flag标签的MDM2真核表达载体,并检测MDM2与p53的相互作用。方法:从人乳腺文库中PCR扩增MDM2编码序列,将其插入pcDNA3.0-Flag载体,转染293T细胞后用Western印迹检测其在293T细胞中的表达,并通过免疫共沉淀实验检测MDM2与p53的相互作用。结果:双酶切和测序结果表明,Flag-MDM2真核表达载体构建成功,转染293T细胞后成功表达;免疫共沉淀实验证明Flag-MDM2与p53存在相互作用。结论:构建了带Flag标签的人MDM2真核表达载体,并检测了MDM2与p53之间的相互作用,为研究MDM2的功能奠定了基础。     

p53-p21通路抑制组蛋白甲基转移酶NSD2的表达

NSD2(nuclear receptor-binding SET domain 2)是一种在黑色素瘤等多种肿瘤细胞中高表达的组蛋白甲基转移酶,其在Wolf-Hirschhorn综合症(wolf-Hirschhorn syndrome,WHS)和多发性骨髓瘤(multiple myeloma,MM)疾病中表达异常的原因已经得到了较好的阐明。而NSD2在其它肿瘤中的表达为何失调还未阐明。本研究选用p53野生型的恶性黑色素瘤细胞系92-1作为细胞模型,采用DNA损伤试剂依托泊苷处理和RNA干扰技术,通过定量PCR和蛋白质免疫印迹的方法首次证实了p53-p21通路对NSD2具有抑制作用。     

核糖体蛋白参与调控p53诱导活化的分子机制

核转录因子p53是重要的肿瘤抑制因子,具有DNA损伤修复、促细胞凋亡、促细胞分化及增殖抑制等功能,并通过调控细胞周期行进和促进细胞凋亡发挥肿瘤抑制功能。原癌蛋白MDM2为p53的E3泛素化连接酶,MDM2－p53信号轴的功能异常与多种恶性肿瘤的发生发展相关。核糖体蛋白（RP）是蛋白质合成反应的关键调节蛋白,其功能失常与多种疾病相关。近年来的研究发现,RP能通过调节MDM2－p53信号轴在p53相关性肿瘤调控中发挥重要作用。我们根据目前的研究进展,对RP－MDM2－D53信号轴进行简要综述。     

p53蛋白泛素化修饰的研究进展

p53具有抑制肿瘤细胞增殖的作用,但是细胞内p53蛋白的堆积反而加速细胞衰老或凋亡,因此对p53进行严格的调控显得格外重要.泛素化、磷酸化和乙酰化是p53蛋白最主要的几种修饰形式,但近来研究表明泛素化对p53调控发挥着中心作用.MDM2是主要的负调节因子,其具有泛素连接酶的活性,早先的研究认为MDM2的作用主要是特异性结合p53并介导其在蛋白酶作用下降解,但近来的研究发现MDM2还可以介导p53的核-浆交换,这种现象在DNA损伤时尤为明显.推测MDM2介导p53的泛素化在体内可能发挥着多种调控功能.     

MDM2/MDMX异二聚体及MDMX磷酸化调控p53的研究进展

p53作为肿瘤抑制因子在维持机体内稳态和抑制肿瘤发生发展中起到关键作用。超过半数的人类肿瘤中都存在p53的突变。突变的p53具有"获得性功能",反而促进肿瘤的发生、转移和耐药。MDM2和MDMX是两个最主要的p53负调控蛋白,二者是同源蛋白,可以独自或以异二聚体的方式调控p53。在多种刺激信号下,MDM2/MDMX异二聚体对p53的负调控作用被抑制,使得p53活化进而激活下游复杂的信号网络,维持细胞内稳态。磷酸化修饰是MDMX调节的重要方式之一,对其自身的稳定性、核定位以及与MDM2、p53的相互作用均有影响。该文对以上内容进行简要综述,并对现有治疗靶标和小分子化合物进行讨论,为进一步开发新的有效的肿瘤治疗策略提供思路。     

泛素蛋白连接酶MDM2活性及稳定性调控的研究进展

泛素蛋白连接酶MDM2(Murine double minute 2)具有癌基因活性, MDM2高表达会导致抑癌基因p53失活而诱发肿瘤, 但在至少7%的肿瘤中p53基因正常而mdm2异常扩增, 表明MDM2还具有其他底物分子, 以p53不依赖的方式促进肿瘤的发生。鉴于MDM2的重要作用, 文章在基因水平、转录水平、翻译后修饰水平、相互作用分子的调节等方面系统总结了目前对MDM2调控的主要研究机制及其进展。     

增强p53、p21及抑制c-myc表达对MCF-7细胞增殖的协同抑制作用

为了探讨增强p53、p21基因表达水平和降低c-myc基因表达水平对乳腺癌细胞MCF-7增殖的协同抑制作用,以及这些基因对细胞产生效应时的相互关系,本研究中首先构建了正义的p53、p21和反义的c-myc3种真核细胞表达载体,并根据析因实验设计三种载体不同剂量组合。按照组合用质粒转染细胞,然后对转染细胞的增殖抑制率进行检测,并采用金正均Q值法、单因素方差分析中的LSD法、聚类分析法等统计学方法对结果进行统计分析。结果显示,不同量的p53、p21反义c-myc对MCF-7细胞的增殖均有抑制作用,抑制的程度各基因间存在差异。在各基因组合中,p21与反义c-myc,p53与反义c-myc联用具有协同作用,对MCF-7细胞的增殖产生更强的抑制,而p53与p21之间未显示出协同作用。对三基因协同结果进行聚类分析后,发现第一类组合协同作用最明显,第九类组合的抑制率最高。由此推测,作为抑癌基因的p53或CDK抑制基因p21高表达,同时原癌基因c-myc表达受到抑制,可相互协同显著增强对MCF-7细胞增殖的抑制作用。     

p53的两种结合蛋白：53BP1和53BP2

ｐ５３基因是一种广谱的肿瘤抑制基因,其产物ｐ５３为一多功能的转录调节因子,可以发挥调节细胞生长、细胞凋亡和ＤＮＡ修复的作用。在人类肿瘤已发现多种ｐ５３基因的点突变,但突变不是ｐ５３蛋白质丧失功能的唯一途径。胞内蛋白可能影响其活性和功能。５３ＢＰ１和５３ＢＰ２是ｐ５３在胞浆中的两种结合蛋白。本文阐述了有关这两种蛋白质的研究进展。     

甲状腺肿瘤p53mRNA及p53蛋白表达的研究

本文采用原位杂交法、免疫组织化学方法分别检测了甲状腺癌ｐ５３ｍＲＮＡ、ｐ５３蛋白的表达,结果显示：２０例甲状腺癌ｐ５３ｍＲＮＡ、ｐ５３蛋白均呈阳性反应,８例甲状腺瘤仅１例呈弱阳性反应,８例Ｇｒａｖｅｓ病全部呈阴性反应。细胞质和细胞核ｍＲＮＡ、ｐ５３蛋白灰度检测发现,甲状腺瘤细胞质、核ｐ５３ｍＲＮＡ灰度值和ｐ５３蛋白灰度值均明显高于Ｇｒａｖｅｓ病,而甲状腺癌其细胞质、核ｐ５３ｍＲＮＡ灰度值和ｐ５３蛋白灰度值又明显高于良性甲状腺瘤,提示甲状腺癌ｐ５３ｍＲＮＡ和ｐ５３蛋白的高表达可能与甲状腺肿瘤细胞分化程度有关     

Over-expression of the human MDM2 p53 binding domain by fusion to a p53 transactivation peptide

MDM2 binds to the tumor suppressor protein p53 and regulates the level of p53 in cells. Although it is possible to prepare a small amount of the region of MDM2 that binds to p53, the expression level of this fragment of MDM2 is relatively low, limiting the studies involving this protein. Here, we describe a construct for the optimized bacterial expression and purification of the MDM2 p53 binding domain. We found that the expression level of the soluble MDM2 p53 binding domain in bacteria was increased dramatically by fusing it to its interaction partner, the p53 transactivation peptide. Attachment of the p53 transactivation peptide (residues 17-29) to the N-terminus of MDM2 resulted in a more than 200-fold increase of soluble protein expression of the p53 binding domain in bacteria. To obtain the final MDM2 p53 binding domain (residues 5-109) we inserted a tobacco etch virus protease recognition site between the P53 peptide and the MDM2 p53 binding domain. To weaken the protein/peptide interaction and facilitate the separation of the protein from the complex, we introduced a point mutation of one of the key interaction residues (F19A or W23A) in the p53 peptide. The advantages of our new construct are high yield and easy purification of the MDM2 protein.     

DNAJB1 stabilizes MDM2 and contributes to cancer cell proliferation in a p53-dependent manner

Both MDM2 and MDMX regulate p53, but these proteins play different roles in this process. To clarify the difference, we performed a yeast 2 hybrid (Y2H) screen using the MDM2 acidic domain as bait. DNAJB1 was found to specifically bind to MDM2, but not MDMX, in vitro and in vivo. Further investigation revealed that DNAJB1 stabilizes MDM2 at the post-translational level. The C-terminus of DNAJB1 is essential for its interaction with MDM2 and for MDM2 accumulation. MDM2 was degraded faster by a ubiquitin-mediated pathway when DNAJB1 was depleted. DNAJB1 inhibited the MDM2-mediated ubiquitination and degradation of p53 and contributed to p53 activation in cancer cells. Depletion of DNAJB1 in cancer cells inhibited activity of the p53 pathway, enhanced the activity of the Rb/E2F pathway, and promoted cancer cell growth in vitro and in vivo. This function was p53 dependent, and either human papillomavirus (HPV) E6 protein or siRNA against p53 was able to block the contribution caused by DNAJB1 depletion. In this study, we discovered a new MDM2 interacting protein, DNAJB1, and provided evidence to support its p53-dependent tumor suppressor function.     

RYBP stabilizes p53 by modulating MDM2

The mouse double minute 2 (MDM2)–p53 interaction regulates the activity of p53 and is a potential target for human cancer therapy. Here, we report that RYBP (RING1‐ and YY1‐binding protein), a member of the polycomb group (PcG), interacts with MDM2 and decreases MDM2‐mediated p53 ubiquitination, leading to stabilization of p53 and an increase in p53 activity. RYBP induces cell‐cycle arrest and is involved in the p53 response to DNA damage. Expression of RYBP is decreased in human cancer tissues compared with adjacent normal tissues. These results show that RYBP is a new regulator of the MDM2–p53 loop and that it has tumour suppressor activity.     

E2F1 inhibits MDM2 expression in a p53-dependent manner

MDM2 expression is down-regulated upon E2F1 over-expression, but the mechanism is not well defined. In the current study, we found that E2F1 inhibits MDM2 expression by suppressing its promoter activity. Although E2F1 binds to the MDM2 promoter, the inhibitory effect of E2F1 on the MDM2 promoter does not require the direct binding. We demonstrate that E2F1 inhibits MDM2 promoter activity in a p53-dependent manner. Knockdown of p53 in U2OS cells impairs the inhibitory effect of E2F1 on the MDM2 promoter. Consistent with this observation, E2F1 does not inhibit MDM2 promoter activity in p53-deficient H1299 cells, and the inhibition is restored when p53 is expressed exogenously. Both E2F1 and p53 are up-regulated after DNA damage stimulation. We show that such stimulation induces E2F1 to inhibit MDM2 promoter activity and promote p53 accumulation. Furthermore, inhibition of MDM2 by E2F1 promotes E2F1 induced apoptosis. These data suggest that E2F1 regulates the MDM2-p53 pathway by inhibiting p53 induced up-regulation of MDM2.     

erbB-2表达抑制与EGF对p21、p53基因转录活性的促进

原癌基因ｅｒｂＢ－２的异常表达存在于人类多种肿瘤中,与肿瘤的发生、发展密切相关［１］．我们曾构建了反义ｅｒｂＢ－２逆转录病毒重组载体,将其转染存在该基因异常表达的人胃癌细胞系ＢＧＣ－８２３,达到了特异抑制ｅｒｂＢ－２表达、抑制瘤细胞恶性增殖并部分阻断...     

CREB represses p53-dependent transactivation of MDM2 through the complex formation with p53 and contributes to p53-mediated apoptosis in response to glucose deprivation

Recently, we have described that CREB (cAMP-responsive element-binding protein) has the ability to transactivate tumor suppressor p53 gene in response to glucose deprivation. In this study, we have found that CREB forms a complex with p53 and represses p53-mediated transactivation of MDM2 but not of p21^WAF1. Immunoprecipitation analysis revealed that CREB interacts with p53 in response to glucose deprivation. Forced expression of CREB significantly attenuated the up-regulation of the endogenous MDM2 in response to p53. By contrast, the mutant form of CREB lacking DNA-binding domain (CREBΔ) had an undetectable effect on the expression level of the endogenous MDM2. During the glucose deprivation-mediated apoptosis, there existed an inverse relationship between the expression levels of MDM2 and p53/CREB. Additionally, p53/CREB complex was dissociated from MDM2 promoter in response to glucose deprivation. Collectively, our present results suggest that CREB preferentially down-regulates MDM2 and thereby contributing to p53-mediated apoptosis in response to glucose deprivation.