应用蛋白质组学和RNAi技术筛选鼻咽癌细胞中p53功能相关蛋白质

为了阐明鼻咽癌中高表达的p53蛋白聚集与失活的机制,高通量地检测与p53功能相关的蛋白质,首先采用RNA干扰(RNAi)技术稳定沉默鼻咽癌细胞系CNE2的p53基因表达,然后用蛋白质组技术研究稳定沉默该基因对鼻咽癌蛋白质表达谱的影响.通过对稳定干扰p53基因后鼻咽癌细胞系CNE2的蛋白质表达谱改变的研究,用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析和电喷雾串联质谱(ESI-Q-TOF-MS)验证鉴定了22个差异表达蛋白质.在这些差异表达蛋白质中,有些是已经报道的p53功能相关蛋白质,如热休克蛋白27(HSP27)、异质性胞核核糖核蛋白K(hnRNPK)、14-3-3σ等,其他可能是新的p53功能相关蛋白质,如eIF4B、TPT1、hnRNPH3、SFRS1等.部分差异表达蛋白质如HSP27、14-3-3σ和GRP75经蛋白质印迹分析技术进行了验证,同时pcDNA3.1-FLAG-p53质粒转染CNE2细胞引起了HSP27、14-3-3σ表达下调,GRP75表达上调.在鼻咽癌细胞中鉴定的22个差异表达蛋白质大致可以分为5类,包括信号传导相关蛋白质、分子伴侣、与转录和翻译相关蛋白质、代谢相关蛋白和细胞结构相关蛋白质,涉及到细胞周期的调控、分子基因表达调控、细胞黏附、细胞代谢等众多事件,它们可能作为p53功能相关蛋白质,为阐明鼻咽癌中p53蛋白聚集及失活的机制提供了重要依据和线索.     

鼻咽癌CT 表现与p53 、p16 蛋白异常表达关系的研究

目的：研究p53、p16蛋白表达与鼻咽癌Cr表现的关系。方法：经病理证实鼻咽癌50例,全部病例做鼻咽轴位平扫,部分病例同时做冠状扫描。应用免疫组化SABC法检测所有病例中的p53、p16蛋白的表达。结果：鼻咽癌及鼻咽粘膜慢性炎症中p53蛋白表达率分别为60％和10％,p16蛋白表达率分别为32％和85％,p53、p16表达在两者间的差异有极显著意义（P〈0．005）。p53蛋白表达与鼻咽癌分化程度及Cr表现为副鼻窦受累,颅底骨质破坏相关（P〈0．05）。p16蛋白表达与鼻咽癌预后及Cr表现为颈部淋巴结转移相关（P〈0．05）。p53与p16有相关性（P〈0．005）。结论：p53、p16在鼻咽发生、发展中起重要作用,p53蛋白表达与鼻咽癌分化程度、浸润深度、颅底侵犯有一定相关性,p16蛋白表达与鼻咽癌颈部淋巴结转移和预后有一定相关性,p53,p16可作为评价鼻咽癌CT表现恶性度及预后的指标。     

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。     

人肝癌细胞瘤QGY-7703的p53基因及其表达研究

本文利用等位基因分析,PCR-SSCP,RT-PCR/序列分析,Northern印迹,免疫组织化学,Western印迹,免疫沉淀等方法对人肝癌细胞株QGY-7703的p53基因背景进行了研究,发现17号染色体短臂可能存在等位基因缺失,在p53基因的编码序列上没有发现任何突变,但发现其mRNA和蛋白表达水平很低,表明在QGY-7703细胞中p53基因的低水平表达可能同细胞的恶性程度有关。     

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—myc 3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。     

p63基因的结构与功能

p63基因是肿瘤抑制基因p53家族成员之一,与p53基因表现出高度同源性.较之p53基因,p63基因更为复杂.p63的两个不同启动子和多种内含子剪接方式,导致p63基因编码产生多种亚型P63蛋白.这些P63亚型蛋白,在不同的组织不同的发育阶段发挥不同的生物学功能.本文就p63基因结构、p63在细胞周期和凋亡中的作用,以及在表皮发育中的功能等方面的研究进展作一概述.     

鼻咽癌的CT 显像与VEGF 和p53 蛋白表达的相关性研究

目的:探讨鼻咽癌患者CT表现与血管内皮细胞生长因子以及p53蛋白表达的相关性.方法:选择经病理确诊的鼻咽癌患者36例,行螺旋CT扫描并采用免疫组化法检测肿瘤组织中VEGF和p53蛋白的表达,分析影像学特征与VEGF及p53蛋白表达的相关性.结果:鼻咽癌患者肿瘤组织中VEGF的表达与CT征象中的侵及后鼻孔、颅底骨质破坏及颈部淋巴结转移正相关,p53蛋白的表达与颈部淋巴结转移正相关.结论:肿瘤组织中VEGF和p53蛋白表达与鼻咽癌的部分CT影像学特征相关,可作为评价鼻咽癌CT表现恶性度及预后的指标.     

p53过表达抑制同源盒基因NKX3.1启动子活性

NKX3.1是前列腺特异表达的同源盒基因,在前列腺癌的发生发展中起重要作用,而在前列腺癌进展中常会发生p53的基因突变.为研究两者之间的关系,构建NKX-3.1启动子(1 040bp)-荧光素酶报告基因重组质粒（pGL3-1040）及其缺失突变体,瞬时转染前列腺癌细胞LNCaP.通过荧光素酶表达活性分析,检测p53过表达对NKX3.1启动子活性的影响.结果表明：p53在LNCaP细胞中过表达可明显抑制NKX3.1启动子活性;RT-PCR及Western印迹检测p53过表达对NKX3.1表达的影响.结果表明,p53过表达可以明显抑制同源盒基因NKX3.1的表达.通过TRANSFAC软件分析,在NKX3.1基因上游-526至-507区存在一个p53反应元件的5′核心序列.缺失pGL3-1040中的p53反应元件核心序列并不能消除p53对NKX3.1启动子的抑制作用,表明p53不是通过p53反应元件直接抑制NKX3.1启动子活性.进一步通过5′缺失突变分析,发现NKX3.1启动子-140～+8 bp区仍受p53负调控.此148 bp区域中含有一个Sp1和一个CREB元件,瞬时共转染Sp1表达载体或CREB表达载体的结果表明,p53并不是通过与Sp1或CREB相互作用对NKX3.1启动子发挥抑制作用的.上述结果表明,p53过表达可以抑制同源盒基因NKX3.1启动子活性,下调NKX3.1基因的转录,其调控机制有待进一步研究.     

p21和p27基因在眼睑皮脂腺腺癌中的表达及意义

目的探讨p21和p27基因在眼睑皮脂腺腺癌中的表达差异。方法收集武汉大学人民医院和武汉大学中南医院病理科2000-2008年手术切除及活检的眼睑皮脂腺腺癌(eyelid sebaceous gland grandular cancer,ESGGC)标本共20例,另取癌周围组织5例作对照。采用免疫组织化学方法观察各组组织内p21和p27基因的表达。并利用HPIAS-2000图像分析系统测定p21和p27基因在以上各组中表达的平均光密度和平均阳性面积率。对各组组织中p21和p27基因表达的阳性面积率作双变量相关分析。结果 1.p21和p27基因在眼睑皮脂腺腺癌组织中呈低表达;p21和p27基因在癌旁组织中呈高表达。经单因素方差分析,组间有显著性差异(P0.05?。经q检验,眼睑皮脂腺腺癌与癌旁组织之间,p21和p27基因表达的平均光密度及阳性面积率有显著性差异(P0.05?。2.p21和p27基因之间的表达呈显著正相关。结论1.p21和p27基因在眼睑皮脂腺腺癌组织中异常表达,对眼睑皮脂腺腺癌的发生和发展起了重要作用;2.p21和p27基因之间的表达呈显著正相关,它们在皮脂腺腺癌的发生和发展过程中可能起协同作用。     

载体介导的小干扰RNA诱导p53基因的沉默

目的:筛选有效抑制p53基因表达的靶位点,以研究其在哺乳动物细胞中的功能.方法:用Ambion公司的RNA干扰(RNAi)设计软件设计了3段针对野生型p53基因的长度为19 nt的反向重复序列,在人工退火条件下形成双链,将其构建到pSilencer3.1-H1 neo载体上,与pcDNA3.1-HA-p53(野生型)载体共转染Cos7及293T细胞,用Western blot检测HA-p53蛋白质的表达;将pCMV-GFP-p53与p53-RNAi或空载体共转入Cos7细胞,于激光扫描共聚焦显微镜下比较GFP-p53的表达;将p53-RNAi和空载体分别转入BEL-7405-p53-RE-luc 细胞,在不同剂量化疗药物阿霉素的作用下,通过活体成像仪观察内源p53的表达;利用G418(neo)筛选获得稳定整合p53-RNAi的H460细胞(p53野生型)株,用流式细胞仪分析正常及p53基因沉默细胞的周期变化.结果:筛选到一个有效的p53基因沉默位点,并在H460细胞中得到p53基因沉默的稳定细胞株.结论:获得了有效的p53基因RNAi位点,为进一步研究p53基因在细胞中的功能提供了有力的工具.     

Erp1p and Erp2p, Partners for Emp24p and Erv25p in a Yeast p24 Complex

Six new members of the yeast p24 family have been identified and characterized. These six genes, named ERP1-ERP6 (for Emp24p- and Erv25p-related proteins) are not essential, but deletion of ERP1 or ERP2 causes defects in the transport of Gas1p, in the retention of BiP, and deletion of ERP1 results in the suppression of a temperature-sensitive mutation in SEC13 encoding a COPII vesicle coat protein. These phenotypes are similar to those caused by deletion of EMP24 or ERV25, two previously identified genes that encode related p24 proteins. Genetic and biochemical studies demonstrate that Erp1p and Erp2p function in a heteromeric complex with Emp24p and Erv25p.     

Viral Oncoproteins Discriminate between p53 and the p53 Homolog p73

p73 is a recently identified member of the p53 family. Previously it was shown that p73 can, when overproduced in p53-defective tumor cells, activate p53-responsive promoters and induce apoptosis. In this report we describe the generation of anti-p73 monoclonal antibodies and confirm that two previously described p73 isoforms are produced in mammalian cells. Furthermore, we show that these two isoforms can bind to canonical p53 DNA-binding sites in electrophoretic mobility shift assays. Despite the high degree of similarity between p53 and p73, we found that adenovirus E1B 55K, simian virus 40 T, and human papillomavirus E6 do not physically interact with p73. The observation that viral oncoproteins discriminate between p53 and p73 suggests that the functions of these two proteins may differ under physiological conditions. Furthermore, they suggest that inactivation of p73 may not be required for transformation.     

Baculovirus p33 Binds Human p53 and Enhances p53-Mediated Apoptosis

In vertebrates, p53 participates in numerous biological processes including cell cycle regulation, apoptosis, differentiation, and oncogenic transformation. When insect SF-21 cells were infected with a recombinant of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) overexpressing human p53, p53 formed a stable complex with the product of the AcMNPV orf92, a novel protein p33. The interaction between p53 and p33 was further confirmed by immunoprecipitation studies. When individually expressed in SF-21 cells, human p53 localized mainly in the nucleus whereas baculovirus p33 displayed diffuse cytoplasmic staining and punctuate nuclear staining. However, coexpression of p33 with p53 resulted in exclusive nuclear localization of p33. In both SF-21 and TN-368 cells, p53 expression induced typical features of apoptosis including nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Coexpression of p53 with a baculovirus inhibitor of apoptosis, p35, OpIAP, or CpIAP, blocked apoptosis, whereas coexpression with p33 enhanced p53-mediated apoptosis approximately twofold. Expression of p53 in SF-21 cells stably expressing OpIAP inhibited cell growth in the presence or absence of p33. Thus, human p53 can influence both insect cell growth and death and baculovirus p33 can modulate the death-inducing effects of p53.     

Cooperation between p27 and p107 during endochondral ossification suggests a genetic pathway controlled by p27 and p130

Pocket proteins and cyclin-dependent kinase (CDK) inhibitors negatively regulate cell proliferation and can promote differentiation. However, which members of these gene families, which cell type they interact in, and what they do to promote differentiation in that cell type during mouse development are largely unknown. To identify the cell types in which p107 and p27 interact, we generated compound mutant mice. These mice were null for p107 and had a deletion in p27 that prevented its binding to cyclin-CDK complexes. Although a fraction of these animals survived into adulthood and looked similar to single p27 mutant mice, a larger number of animals died at birth or within a few weeks thereafter. These animals displayed defects in chondrocyte maturation and endochondral bone formation. Proliferation of chondrocytes was increased, and ectopic ossification was observed. Uncommitted mouse embryo fibroblasts could be induced into the chondrocytic lineage ex vivo, but these cells failed to mature normally. These results demonstrate that p27 carries out overlapping functions with p107 in controlling cell cycle exit during chondrocyte maturation. The phenotypic similarities between p107(-/-) p27(D51/D51) and p107(-/-) p130(-/-) mice and the cells derived from them suggest that p27 and p130 act in an analogous pathway during chondrocyte maturation.     

p53, p63 and p73--solos, alliances and feuds among family members

p53 controls crucial stress responses that play a major role in preventing malignant transformation. Hence, inactivation of p53 is the single most common genetic defect in human cancer. With the recent discovery of two close structural homologs, p63 en p73, we are getting a broader view of a fascinating gene family that links developmental biology with tumor biology. While unique roles are apparent for each of these genes, intimate biochemical cross-talk among family members suggests a functional network that might influence many different aspects of individual gene action. The most interesting part of this family network derives from the fact that the p63 and p73 genes are based on the "two-genes-in-one" idea, encoding both agonist and antagonist in the same open reading frame. In this review, we attempt to present an overview of the current status of this fast moving field.     

Pil1p and Lsp1p negatively regulate the 3-phosphoinositide-dependent protein kinase-like kinase Pkh1p and downstream signaling pathways Pkc1p and Ypk1p

The Saccharomyces cerevisiae homologs, Pkh1/2p, of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1) regulate the Pkc1-MAP kinase cascade and the partially parallel Ypk1/2p pathway(s) that control growth and cell integrity. Mammalian PDK1 is regulated by 3-phosphoinositides, whereas Pkh1/2p are regulated by sphingolipid long-chain bases (LCBs). Recently Pkh1/2p were found to complex with two related proteins, Pil1p (Ygr086) and Lsp1p (Ypl004). Because these two proteins are not related to any known protein we sought to characterize their functions. We show that Pkh1p phosphorylates both proteins in vitro in a reaction that is only weakly regulated by LCBs. In contrast, LCBs inhibit phosphorylation of Pil1p by Pkh2p, whereas LCBs stimulate phosphorylation of Lsp1p by Pkh2p. We find that Pil1p and Lsp1p down-regulate resistance to heat stress and, specifically, that they down-regulate the activity of the Pkc1p-MAP and Ypk1p pathways during heat stress. Pil1p and Lsp1p are thus the first proteins identified as regulators of Pkh1/2p. An unexpected finding was that the level of Ypk1p is greatly reduced in pkc1Delta cells, indicating that Pkc1p controls the level of Ypk1p. Homologs of Pil1p and Lsp1p are widespread in nature, and our results suggest that they may be negative regulators of PDK-like protein kinases and their downstream cellular pathways that control cell growth and survival.     

p53 Family members p63 and p73 are SAM domain-containing proteins.

Homologs of the tumor suppressor p53, called p63 and p73, have been identified. The p63 and p73 family members possess a domain structure similar to p53, but contain variable C-terminal extensions. We find that some of the C-terminal extensions contain Sterile Alpha Motif (SAM) domains. SAM domains are protein modules that are involved in protein-protein interactions. Consistent with this role, the C-terminal SAM domains of the p63 and p73 may regulate function by recruiting other protein effectors.     

p73 and p63: Estranged relatives?

Comment on: Holembowski L, et al. Cell Cycle 2011; 10:680-9.