EB病毒潜伏膜蛋白1多态性与鼻咽癌的关系

为了分析广东地区鼻咽癌病人和非鼻咽癌病人携带的Epstein-Barr病毒（EBV）潜伏膜蛋白1（LMP1）基因多态性,探讨LMP1基因羧基端缺失30个碱基的EBV是否与华南地区鼻咽癌高发有关。为此收集了初始的107例鼻咽癌病人和106例非鼻咽癌肿病人的漱口液,用巢式PCR扩增LMP1羧基端,观察缺失型LMP1的构成比。同时,测序分析缺失型LMP1编码区序列,了解缺失型LMP1多态性与鼻咽癌的关联度。结果：在50％的样品中可检出LMP1基因,缺失型LMP1在鼻咽癌病人和非鼻咽病人的构成比相似,约占70？％,原型和缺失型LMP1混合感染少见（0－6％）。另外,广州地区的缺失型LMP序列与上海、台湾、香港地区的缺失型LMP1基因高度同源,鼻咽癌病人是和非鼻咽癌病人携带的缺失型LMP1基因序列基本相同。此结果表明,广州地区流行的LMP1羧基端缺失30个碱基的EBV株,漱口液中检测出的缺失型与原型LMP1构成比约为7：3,在鼻咽癌病人和非鼻咽癌病人此分布情况相似。     

用鼻咽相对特异性调控区建立N-LMP1转基因小鼠

为了研究EBV LMP1在鼻咽癌发生发展中的作用 ,构建了EDL 2、PLUNC p双启动子调控鼻咽癌来源的LMP1(latentmembraneprotein 1,潜伏膜蛋白 1)的表达载体 ,采用受精卵前核显微注射法构建转基因小鼠。结果表明 ,在所获得的 5 8只转基因首建鼠中 ,4只整合阳性 ,其中的一只转基因小鼠在鼻咽、前胃、舌根等部位检测到了外源基因的表达。     

EB病毒及其癌基因LMP1对上皮细胞的转化作用

EB病毒(EBV)是一种人群感染率较高的致病性疱疹病毒,与伯基特氏淋巴瘤、鼻咽癌的关系最为明确。近年来EBV在上皮源性肿瘤发生、发展中的作用受到众多学的关注,而其癌基因LMP1在上皮源性肿瘤发病的病因学方面的作用更成为研究的热点。本综述就EBV进入上皮细胞、永生化上皮细胞的机制、LMP1的基本结构及其对上皮细胞的生物学功能作一较为详细的阐述。     

我国北方地区鼻咽癌患者EB病毒LMP1基因缺失分析

为研究我国北方地区鼻咽癌和非鼻咽癌患者Epstein-Barr病毒(EBV)潜伏膜蛋白1(LMP1)基因C端区缺失状况,探讨其在鼻咽癌发生中所起的作用.收集我国东北地区鼻咽癌石蜡组织22例,非鼻咽癌患者外周血26例,提取DNA后,采用聚合酶链反应技术(PCR)扩增LMP1基因的C末端,对其中有缺失的4例鼻咽癌进行了克隆和序列分析.结果显示:22例鼻咽癌组织标本中,有19例扩增出特异性条带,阳性率为86%.其中8例存在缺失,缺失率42%.取4例缺失样品进行序列分析,并与B95-8原型LMP1做比较,结果显示:4例鼻咽癌样品均存在30个碱基的缺失和某些位点的单点突变,并由此引起所编码的氨基酸改变.26例非鼻咽癌患者LMP1阳性扩增率为92.31%,无一例缺失.此结果表明:与广东、广西鼻咽癌高发区相比,我国北方地区鼻咽癌与非鼻咽癌患者中EB病毒LMP1基因缺失型和原型的分布有明显的地区差异.     

埃巴病毒潜伏膜蛋白的研究进展

埃巴病毒(Epstein-Barr Virus, EBV)是一种在人群中感染率高达90%的γ-疱疹病毒,其感染宿主细胞后常以潜伏形式存在并伴随终生,在一定条件诱导下,由潜伏感染转化为裂解感染,导致多种恶性肿瘤的发生。LMP1和LMP2是EBV编码的重要潜伏膜蛋白,它们锚定于细胞膜脂筏区,通过与宿主细胞内多种信号传递分子如TRAF家族蛋白、Caspase家族蛋白和STAT家族等相互作用,参与细胞内多条信号转导通路,进而影响细胞迁移与凋亡,与淋巴组织增生性疾病和恶性肿瘤的发生有着密切联系。本文阐述了LMP1和LMP2的结构特征,在宿主细胞内的基因表达调控及参与信号转导途径的机制等,旨在推进EBV发病机理研究及疫苗的研发等科研进展。     

EB 病毒LMP1 在肿瘤研究中的进展

EB病毒(Epstein-Barr virus,EBV)是具有致瘤潜能的疱疹病毒,与多种恶性肿瘤的发生相关。EB病毒编码的潜伏性膜蛋白1(Latent membrane protein-1,LMP1)作为其主要的致瘤蛋白,能通过细胞内多种信号传导通路,调节和控制细胞的生长、增殖、分化、迁移与凋亡,从而在癌变的发生和发展过程中发挥重要的作用。本文主要就LMP1的结构、生物学功能、介导的信号通路及其与肿瘤关系的相关进展做一阐述。     

携带绿色荧光报告基因的EBV-LMP1真核表达载体构建与鼻咽癌细胞中的表达

潜伏膜蛋白1(LMP1)是由EB病毒编码的致瘤蛋白,众多研究表明LMP1蛋白可通过NF-κB、p38 MAPK、c-JNK等多条重要信号通路引起鼻咽癌细胞的生物学行为改变。我们从EB病毒阳性的B95-8狨猴淋巴瘤细胞中克隆EB病毒LMP1 c DNA,构建携带绿色荧光基因的真核表达质粒p IRES2-Zs-Green1-LMP1,通过脂质体转染的方法将质粒导入鼻咽癌细胞株CNE1、CNE2中,利用质粒所携带的绿色荧光蛋白表达粗略计算转染的效率,通过免疫细胞化学(ICC)、RT-PCR、Western-Blot检测该质粒的表达。本实验室所构建的p IRES2-Zs-Green1-LMP1表达质粒能在鼻咽癌细胞内表达LMP1蛋白,为后续的实验研究奠定基础。     

表达Rta的杆状病毒载体和更昔洛韦联用治疗鼻咽癌的实验研究

目的 联合使用一种新的表达Epstein Barr病毒（EBV）裂解性复制极早期基因Rta的杆状病毒载体及抗病毒药物更昔洛韦,试验其对激活鼻咽癌细胞中潜伏性EBV的复制和杀伤肿瘤细胞的作用。方法 用携带EBV的复制起点OriP和CMV启动子表达Rta基因的重组杆状病毒处理EBV潜伏感染的鼻咽癌细胞Hone1-EBV,和由该细胞系建立的裸鼠肿瘤模型,同时联合使用更昔洛韦,研究细胞和肿瘤生长的情况。结果 重组杆状病毒和更昔洛韦联合处理肿瘤细胞后,细胞生长活性仅为对照的51%,裸鼠活体实验中肿瘤生长也受到明显的抑制,10 d后瘤体积仅为对照的20%,瘤重仅为对照的30%。组织化学分析证实大量肿瘤细胞坏死。结论 杆状病毒载体与更昔洛韦联合可以用于EBV相关的鼻咽癌的治疗。     

EB病毒编码的潜伏膜蛋白1参与鼻咽癌恶性演进的转录调控实验研究

探讨EB病毒潜伏膜蛋白1(LMP1)激活激活蛋白1(AP1),和核转录因子(NF-κB)在鼻咽癌细胞SUNE-1及亚细胞株恶性演进中的作用.运用报告基因法和凝胶电泳迁移率法(EMSA)分析AP1和NF-κB反式激活活性和DNA结合活性,蛋白质印迹检测蛋白质表达;裸鼠致瘤实验结合组织制片研究瘤细胞的成瘤和转移能力. 结果显示恶性程度不同的SUNE-1亚细胞株的反式激活活性、DNA结合活性、LMP1蛋白表达及c-Jun氨基端激酶(JNK)活性均存在明显差异,且与细胞恶性程度正相关.这些结果提示LMP1活化AP1和NF-κB的信号通路参与了鼻咽癌细胞SUNE-1的恶性演进过程.     

EB病毒潜伏膜蛋白1调节EGF受体核移位

EB病毒编码的病毒潜伏膜蛋白1(LMP1)是重要的致瘤蛋白, 一直以来是EB病毒致瘤机制的研究核心. 传统的受体学说认为, 细胞膜受体作为第一信使, 在细胞膜上与其配体结合发挥生物学效应. 但近年来, 对EGFR家族成员受体核移位在基因表达调控中意义的研究, 拓宽了人们对细胞膜受体生物学功能的认识. 利用我们建立的Tet-on系统调控LMP1表达的细胞系, 采用Western blot和激光共聚焦显微镜等技术证实, LMP1可调控EGFR的核移位, 并呈一定的剂量效应. 通过GFP与EGFR及不同突变体的融合蛋白在细胞内的表达发现, EGFR的核定位信号在其核移位过程中起着一定的作用, 并初步发现LMP1调控EGFR核移位为配体非依赖性.     

Variable expression of latent membrane protein in nasopharyngeal carcinoma can be related to methylation status of the Epstein-Barr virus BNLF-1 5''-flanking region.

Seven virus-coded proteins, the nuclear proteins EBNA-1 to EBNA-6 and the latent membrane protein (LMP), are regularly expressed in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines. In nasopharyngeal carcinoma (NPC), only EBNA-1 is regularly expressed; LMP is detected in about 65% of the tumors. In Burkitt's lymphoma tumors only EBNA-1 is expressed. We have recently shown that the methylation patterns of the EBV genome varied between these cell types. In virally transformed lymphoblastoid cell lines of normal origin, the EBV DNA is completely unmethylated. In contrast, in the Burkitt's lymphoma-derived cell line Rael and in a nude mouse-passaged NPC tumor, C15, there was an extensive methylation of CpG pairs. The methylation extended into the coding regions of the two expressed genes, EBNA-1 (in both tumor types) and LMP (in C15). Two presumptive control regions were exempted from this overall methylation: the oriP that contains both an origin of DNA replication and an EBNA-1-dependent enhancer and the 5'-flanking region of the BNLF-1 open reading frame that codes for LMP. The latter was only exempted in the LMP expressing NPC. We have now investigated the relation between expression of LMP and methylation of DNA in the 5'-flanking 1 kb region of BNLF-1, coding for LMP. LMP was methylated in 3 of 12 NPC biopsies that did not express LMP but was partially or totally unmethylated in the remaining 9 that expressed the protein. The three BNLF-1 exons were highly methylated in all the tumors. The oriP region was unmethylated in all the tumors, as in the previously studied Rael cell line and nude mouse-passaged NPC. Also, the BamHI W enhancer region involved in the expression of EBNA nuclear proteins was methylated. None of the biopsies expressed EBNA-2. Our data show that the EBV genomes are highly methylated in NPC tumors. The strong reverse correlation between the methylation of the putative control region of the LMP gene and the expression of LMP suggests that methylation has a role in the regulation of this gene.     

Tet调控的EB病毒LMP1基因导入鼻咽癌细胞系表达的研究

本文利用Ｔｅｔ－ｏｎ基因表达系统建立了一株可诱导ＥＢ病毒ＬＭＰ１基因表达的鼻咽癌细胞株。首先将Ｔｅｔ－ｏｎ基因调控系统的调节质粒ｐＴｅｔ－ｏｎ导入鼻咽癌细胞系ＨＮＥ２中,用ｒｔＴＡ反应性芝光素酶报道基因ｐＴＲＥ－ｌｕｃ挑选高诱导、低背景的克隆,第二轮转染将构建的反应质粒ｐＴＲＥ－ＬＭＰ１导入得到稳定转染的阳性克隆,对各克隆用Ｗｅｓｔｅｒｎ印迹方法进行强力霉素诱导效应检测,其中Ｌ７对Ｄｏｘ具有良好的     

Expression of the BNLF-1 oncogene of Epstein-Barr virus in the skin of transgenic mice induces hyperplasia and aberrant expression of keratin 6.

The BNLF-1 gene of Epstein-Barr virus (EBV) encodes the latent membrane protein (LMP), one of the putative oncogene products of the virus. This gene has been expressed from two different enhancer-promoter constructs in transgenic mice, to determine its biological activity and possible contribution to oncogenesis. While transgenic mice expressing LMP in many tissues demonstrated poor viability, expression of LMP specifically in the epidermis induces a phenotype of hyperplastic dermatosis. Concomitant with the expression of LMP in this tissue (and in the esophagus) is an induction of the expression of a hyperproliferative keratin, K6, at aberrant locations within the epidermis. The epithelial hyperplastic phenotype caused by the LMP-encoding transgenes implies that the LMP plays a role in the acanthotic condition of the tongue epithelium in the human EBV- and HIV-associated syndrome oral hairy leukoplakia, as well as possibly predisposing the nasopharyngeal epithelium to carcinogenesis.     

The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor.

The Epstein-Barr virus (EBV)-encoded LMP1 protein is an important component of the process of transformation by EBV. LMP1 is essential for transformation of B lymphocytes, most likely because of its profound effects on cellular gene expression. Although LMP1 is expressed in the majority of nasopharyngeal carcinoma (NPC) tumors, the effect of LMP1 on cellular gene expression and its contribution to the development of malignancy in epithelial cells is largely unknown. In this study the effects of LMP1 on the expression and tyrosine kinase activity of the epidermal growth factor receptor (EGFR) were investigated in C33A human epithelial cells. Stable or transient expression of LMP1 in C33A cells increased expression of the EGFR at both the protein and mRNA levels. In contrast, expression of the EGFR was not induced by LMP1 in EBV-infected B lymphocytes. Stimulation of LMP1-expressing C33A cells with epidermal growth factor (EGF) caused rapid tyrosine phosphorylation of the EGFR (pp170) as well as several other proteins, including pp120, pp85, pp75, and pp55, indicating that the EGFR induced by LMP1 is functional. LMP1 also induced expression of the A20 gene in C33A epithelial cells. In C33A cells, LMP1 expression increased the proliferative response to EGF, as LMP1-expressing C33A cells continued to increase in number when plated in serum-free media supplemented with EGF, while the neo control cells exhibited very low levels of viability and did not proliferate. Immunoblot analysis of protein extracts from nude mouse-passaged NPC tumors also demonstrated that the EGFR is overexpressed in primary NPC tumors as well as those passaged in nude mice. This study suggests that the alteration in the growth patterns of C33A cells expressing LMP1 is a result of increased proliferative signals due to enhanced EGFR expression, as well as protection from cell death due to LMP1-induced A20 expression. The induction of EGFR and A20 by LMP1 may be an important component of EBV infection in epithelial cells and could contribute to the development of epithelial malignancies such as NPC.     

Epstein-Barr-virus-encoded LMP2A induces primary epithelial cell migration and invasion: possible role in nasopharyngeal carcinoma metastasis

Nonkeratinizing nasopharyngeal carcinomas (NPC) are >95% associated with the expression of the Epstein-Barr virus (EBV) LMP2A latent protein. However, the role of EBV, in particular, LMP2A, in tumor progression is not well understood. Using Affymetrix chips and a pattern-matching computational technique (neighborhood analysis), we show that the level of LMP2A expression in NPC biopsy samples correlates with that of a cellular protein, integrin-alpha-6 (ITGalpha6), that is associated with cellular migration in vitro and metastasis in vivo. We have recently developed a primary epithelial model from tonsil tissue to study EBV infection in epithelial cells. Here we report that LMP2A expression in primary tonsil epithelial cells causes them to become migratory and invasive, that ITGalpha6 RNA levels are up-regulated in epithelial cells expressing LMP2, and that ITGalpha6 protein levels are increased in the migrating cells. Blocking antibodies against ITGalpha6 abrogated LMP2-induced invasion through Matrigel by primary epithelial cells. Our results provide a link between LMP2A expression, ITGalpha6 expression, epithelial cell migration, and NPC metastasis and suggest that EBV infection may contribute to the high incidence of metastasis in NPC progression.     

Epstein-Barr virus-encoded latent membrane protein 1 impairs G2 checkpoint in human nasopharyngeal epithelial cells through defective Chk1 activation

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, particularly in southern regions of China. EBV infection is closely associated with NPC and has long been postulated to play an etiological role in the development of NPC. However, the role of EBV in malignant transformation of nasopharyngeal epithelial cells remains enigmatic. The current hypothesis of NPC development is that premalignant nasopharyngeal epithelial cells harboring genetic alterations support EBV infection and expression of EBV genes induces further genomic instability to facilitate the development of NPC. The latent membrane protein 1 (LMP1) is a well-documented EBV-encoded oncogene. The involvement of LMP1 in human epithelial malignancies has been implicated, but the mechanisms of oncogenic actions of LMP1, particularly in nasopharyngeal cells, are unclear. Here we observed that LMP1 expression in nasopharyngeal epithelial cells impaired G2 checkpoint, leading to formation of unrepaired chromatid breaks in metaphases after γ-ray irradiation. We further found that defective Chk1 activation was involved in the induction of G2 checkpoint defect in LMP1-expressing nasopharyngeal epithelial cells. Impairment of G2 checkpoint could result in loss of the acentrically broken chromatids and propagation of broken centric chromatids in daughter cells exiting mitosis, which facilitates chromosome instability. Our findings suggest that LMP1 expression facilitates genomic instability in cells under genotoxic stress. Elucidation of the mechanisms involved in LMP1-induced genomic instability in nasopharyngeal epithelial cells will shed lights on the understanding of role of EBV infection in NPC development.     

Epstein—Barr病毒基因组在鼻咽癌组织中转录的特征

对ＥＢ病毒基因在鼻咽癌活检组织细胞内的转录进行了较系统的探测。实验结果表明,ＥＢ病毒基因组在鼻咽癌活检组织中以附加体（Ｅｐｉｓｏｍｅ）形式存在,而其基因转录有如下特征：（１）ＥＢ病毒在所有鼻咽癌组织细胞中都表达ＥＢＮＡ－１,并且此基因转录产物由一个在ＢａｍＨＩ－Ｆ区的启动子（Ｆｐ）驱动;（２）潜伏感染膜蛋白（Ｌａｔｅｎｔ ｍｅｍｂｒａｎｅ ｐｒｏｔｅｉｎ,ＬＭＰ）和末端蛋白（Ｔｅｒｍｉｎａｌ ｐｒ     

Epstein-Barr Virus_Encoded LMP1 Upregulates MicroRNA-21 to Promote the Resistance of Nasopharyngeal Carcinoma Cells to Cisplatin-Induced Apoptosis by Suppressing PDCD4 and Fas-L

Approximately 30% of patients with Epstein-Barr virus (EBV)-positive advanced nasopharyngeal carcinoma (NPC) display chemoresistance to cisplatin-based regimens, but the underlying mechanisms are unclear. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, contributes substantially to the oncogenic potential of EBV through the activation of multiple signaling pathways, and it is closely associated with a poorer prognosis for NPC. Recent studies show that EBV infection can induce the expression of many cellular miRNAs, including microRNA-21, a biomarker for chemoresistance. However, neither a link between LMP1 expression and miR-21 upregulation nor their cross talk in affecting chemoresistance to cisplatin have been reported. Here, we observed that stable LMP1-transformed NPC cells were less sensitive to cisplatin treatment based on their proliferation, colony formation, the IC₅₀ value of cisplatin and the apoptosis index. Higher levels of miR-21 were found in EBV-carrying and LMP1-positive cell lines, suggesting that LMP1 may be linked to miR-21 upregulation. These data were confirmed by our results that exogenous LMP1 increased miR-21 in both transiently and stably LMP1-transfected cells, and the knock down of miR-21 substantially reversed the resistance of the NPC cells to cisplatin treatment. Moreover, the proapoptotic factors programmed cell death 4 (PDCD4) and Fas ligand (Fas-L), which were negatively regulated by miR-21, were found to play an important role in the program of LMP1-dependent cisplatin resistance. Finally, we demonstrated that LMP1 induced miR-21 expression primarily by modulating the PI3K/AKT/FOXO3a signaling pathway. Taken together, we revealed for the first time that viral LMP1 triggers the PI3K/Akt/FOXO3a pathway to induce human miR-21 expression, which subsequently decreases the expression of PDCD4 and Fas-L, and results in chemoresistance in NPC cells.     

In vitro anti-tumor immune response induced by dendritic cells transfected with EBV-LMP2 recombinant adenovirus

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is a high-incidence tumor in southern China. Latent membrane proteins 2 (LMP2) is a subdominant antigen of EBV. The present study was to develop a dendritic cells (DCs)-based cancer vaccine (rAd-LMP2-DC) and to study its biological characteristics and its immune functions. Our results showed that LMP2 gene transfer did not alter the typical morphology of mature DC, and the representative phenotypes of mature DC (CD80, CD83, and CD86) were highly expressed in rAd-LMP2-DCs. The expression of LMP2 in rAd-LPM2-DCs was about 84.54%, which suggested efficient gene transfer. Transfected DCs markedly increased antigen-specific T-cell proliferation. The specific cytotoxicity against NPC cell was significantly higher than that in controls (p < 0.05), and enhanced with increased stimulations by transfected DCs. In addition, phenotypic analysis demonstrated that the LMP2-specific CTLs consisted of both CD4(+) and CD8(+) T cells. These results showed that development of DC-based vaccine by transfection with malignancy-associated virus antigens could elicit potent CTL response and provide a potential strategy of immunotherapy for EBV-associated NPC.