EB病毒感染与致鼻咽癌机制研究进展

Epstein-Barr病毒(EBV)的潜伏感染与鼻咽癌关系密切,在感染人体后长期潜伏,可表达多种基因,其潜伏膜蛋白(LMP)编码基因、EB病毒核抗原(EBNA)基因及EBV编码的小RNA(EBER)可以通过不同的机制致鼻咽癌。其中LMP1基因已被列为癌基因,通过激活转录核因子-κB(NF-κB)、激活蛋白1以及janus蛋白激酶-信号转导子与转录激活子信号传导通路而致瘤。     

鼻咽癌前期病变中的EB病毒感染

背景与目的：鼻咽癌中的浸润性癌细胞均感染了EB病毒（Epstein-Barr virus．EBV）。前期病变可见于早期鼻咽癌癌旁上皮。本研究旨在通过检测前期病变中的EB病毒,探讨EB病毒感染存鼻咽癌变过程中的作用,及其基因型在鼻咽癌变过程中发生的宿主内演变。方法：采用核酸原位杂交检测15例早期鼻咽癌活检组织中的EB病毒编码RNA（EBV—encoded RNA,EBER）。采用巢式PCR法检测前期病变和癌巢中的EB病毒类型和潜伏膜蛋白1（latent membrane protein 1,LMPI）EB病毒株。具有代表性的LMPI基因羧基末端PCR产物采用四色荧光终止序列技术进行DNA序列分析。结果：所有15例早期鼻咽癌中的绝大多数浸润性癌细胞均呈EBER阳性。在15例的期病变中．14例可检测到EBER阳性的异常上皮细胞和／或浸润性淋巴细胞。单个A型EB病毒可在9例癌巢（11例适用）及9例前期病变（10例通用）的DNA样本中检测到。EB病毒LMP1基因羧基末端在15例癌巢DNA样本中均可检测到,其中14例是30bp缺失型LMP1 EB病毒株,1例是野生型和30bp缺失型LMP1株的混合感染。在11例适合做EB病毒LMP1基因羧基末端扩增的前期病变的DNA样本中,5例呈野生型和30bp缺失型LMP1 EB病毒株的混合感染,4例是单个缺大型LMP1 EB病毒株感染,1例呈单个野生型LMP1 EB病毒株感染,1例呈阴性反应。野生型LMP1基因羧基末端的DNA序列与B95—8细胞的DNA序列完全一致;30bp缺大型LMP1基因羧基末端的DNA序列却其有30bp缺失（密码子：346～355）和4个错义点突变（密码子：334、335、338和366）。结论：鼻咽上皮细胞的EB病毒感染是癌变过程中侵袭前的事件;而在鼻咽癌变过程中,EB病毒基因型会产生宿主内的演变。     

EB病毒感染与致鼻咽癌机制研究进展

Epstein-Barr病毒（EBV）的潜伏感染与鼻咽癌关系密切,在感染人体后长期潜伏,可表达多种基因,其潜伏膜蛋白（LMP）编码基因、EB病毒核抗原（EBNA）基因及EBV编码的小RNA（EBER）可以通过不同的机制致鼻咽癌。其中LMPl基因已被列为癌基因,通过激活转录核因子-κB（NF-κB）、激活蛋白1以及janus蛋白激酶．信号转导子与转录激活子信号传导通路而致瘤。     

EB病毒与鼻咽癌相关性的研究进展

Epstein-Barr病毒（EBV）的潜伏感染与鼻咽癌（Nasopharyngeal carcinoma,NPC）密切相关,在感染人体后长期潜伏,可表达多种基因,其潜伏膜蛋白（LMP）编码基因、EB病毒核抗原（EBNA）基因及EBV编码的小RNA（EBER）可以通过不同的机制致鼻咽癌。本文就EBV与NPC的关系,与NPC相关的EBV的生物学特性和进展作一综述。     

EB病毒LMP1与E-cadherin、ICAM-1在鼻咽癌组织中的表达及其相关性

 目的研究EB病毒潜伏膜蛋白1(LMP1)对鼻咽癌细胞黏附分子E-cadherin和ICAM-1表达的影响,探讨LMP1在鼻咽癌转移中的作用及其相关的机制。方法应用免疫组织化学(SP法)分别检测LMP1、E-cadherin和ICAM-1蛋白在31例鼻咽粘膜慢性炎、32例鼻咽癌和24例鼻咽癌颈部淋巴结转移灶中的表达。结果LMP1和ICAM-1蛋白在鼻咽癌和鼻咽癌淋巴结转移灶中的阳性表达率显著高于在鼻咽粘膜慢性炎组织中的阳性表达率,E-cadherin蛋白在鼻咽癌和鼻咽癌淋巴结转移灶中的阳性表达率则显著低于在鼻咽粘膜慢性炎组织中的阳性表达率。Spearman相关分析显示,在鼻咽癌组织和鼻咽癌淋巴结转移灶中,LMP1与E-cadherin的表达呈负相关,而与ICAM-1的表达呈正相关。结论在鼻咽癌和鼻咽癌颈部淋巴结转移灶中LMP1可能通过抑制E-cadherin的表达和促进ICAM-1的表达进而影响肿瘤细胞的黏附性,促进鼻咽癌的侵袭和转移。     

EB病毒LMP1与E-cadherin、ICAM-1在鼻咽癌组织中的表达及其相关性北大核心

目的研究EB病毒潜伏膜蛋白1(LMP1)对鼻咽癌细胞黏附分子E-cadherin和ICAM-1表达的影响,探讨LMP1在鼻咽癌转移中的作用及其相关的机制。方法应用免疫组织化学(SP法)分别检测LMP1、E-cadherin和ICAM-1蛋白在31例鼻咽粘膜慢性炎、32例鼻咽癌和24例鼻咽癌颈部淋巴结转移灶中的表达。结果LMP1和ICAM-1蛋白在鼻咽癌和鼻咽癌淋巴结转移灶中的阳性表达率显著高于在鼻咽粘膜慢性炎组织中的阳性表达率,E-cadherin蛋白在鼻咽癌和鼻咽癌淋巴结转移灶中的阳性表达率则显著低于在鼻咽粘膜慢性炎组织中的阳性表达率。Spearman相关分析显示,在鼻咽癌组织和鼻咽癌淋巴结转移灶中,LMP1与E-cadherin的表达呈负相关,而与ICAM-1的表达呈正相关。结论在鼻咽癌和鼻咽癌颈部淋巴结转移灶中LMP1可能通过抑制E-cadherin的表达和促进ICAM-1的表达进而影响肿瘤细胞的黏附性,促进鼻咽癌的侵袭和转移。     

EBV-LMP2多肽所激活的特异性CTL对鼻咽癌细胞杀伤活性的研究

[目的]研究EB病毒编码的LMP2多肽片段所激活的肿瘤特异性CTL对鼻咽癌细胞的杀伤作用,探讨利用LMP2多肽对鼻咽癌进行免疫治疗.[方法]通过细胞免疫方法鉴定鼻咽癌细胞(CNE2)中的LMP2蛋白是否表达;外周血分离树突状细胞(DC)体外培养,以LMP2多肽片段负载DC激活产生特异性CTL;通过流式细胞仪、MTT法检测特异性CTL对CNE2细胞杀伤活性.[结果]在CNE2中表达LMP2蛋白,LMP2多肽所激活的肿瘤特异性CTL对鼻咽癌细胞具有杀伤活性.[结论]LMP2多肽片段所激活的肿瘤特异性CTL在体外对鼻咽癌细胞具有杀伤作用.     

EB病毒编码小RNA多态性与鼻咽癌的关系

目的 探讨EB病毒编码小RNA（EBERs)多态性与鼻咽癌发病趋势的联系。方法 巢式PCR扩增EBER－1和EBER－2序列，直接测序，比较鼻咽癌高发区广东地区鼻咽癌组织、正常人鼻咽黏膜组织和鼻咽癌低发区哈尔滨地区正常人漱口液感染的EB病毒EBERs序列差异。结果 广东地区鼻咽癌组织感染的EBERs具备1型病毒的特征。与原型株B95．8相比，广东地区鼻咽癌组织来源的EBER－2基因存在8处碱基替换和2处碱基缺失。但是，广东正常人鼻咽黏膜组织和哈尔滨地区正常人漱口液来源的EBERs也有相同的碱基替换和缺失。结论 鼻咽癌高发区和低发区流行的EB病毒EBERs序列呈现高度一致性。     

鼻咽癌组织中EB病毒感染与癌基因表达的关系

目的 观察EB病毒（epstein-barr virus,EBV)RNA、潜伏膜蛋白（LMP－1）、细胞周期素D1（cyclin D1)及c-fos在鼻咽癌组织中的表达，并分析其相互关系。方法 采用原位分子杂交技术检测36例鼻咽癌组织中EBV编码的小分子RAN（encoded small RNAs,EBERs)，采用免疫组化技术检测LMP－1及cyclin D1、c-fos的表达。结果 36例鼻咽癌组织中EBV RNAs阳性率为100．0％（36／36），LMP－1阳性率为44．4％（16／36），cyclin D1阳性率为52．8％（19／36），c-fos阳性率为61．1％（22／36）。LMP－1表达与cyclin D1、c-fos表达有明显的关系。结论 鼻咽癌发生是多步骤、多基因改变的过程。鼻咽活检组织中有EBERs及LMP－1的检出，对鼻咽癌的诊断具有一定的价值。     

鼻咽癌中EB病毒LMP1基因N端Xho I酶切位点的丢失

背景与目的：众所周知,EB病毒LMP1基因在鼻咽癌变过程起着一定的作用。本研究通过检测广东地区鼻咽癌组织EB病毒LMP1基因N-末端区Xho I酶切位点的丢失,探讨LMP1基因变异在鼻咽癌发生发展中的作用。方法：收集中山大学肿瘤防治中心鼻咽癌患者鼻咽新鲜活检标本63例。收集EB病毒健康携带者外周血单个核细胞(PBMCs)10例作为对照。采用QIAamp DNA Mini Kit和QIAamp DNA Blood Mini Kit分别抽取组织和外周血单个核细胞的DNA,应用巢式PCR扩增EB病毒LMP1基因的N-末端区,并用Xho I对扩增产物进行酶切。采用四色荧光末端终止法对扩增产物进行序列分析。结果：10例健康携带者外周血单个核细胞的EB病毒LMP1基因N-末端区均未见Xho I酶切位点的丢失。63例鼻咽癌组织中有50例(79．37％)出现Xho I酶切位点的丢失(Xho I—loss),还有4例(6．34％,)为Xho I酶切位点部分丢失,只有9例(14．29％)未见Xho I酶切位点的丢失(wt-Xho I)。除了Xho I酶切位点的丢失(nt：169423～169428;GAGCTC→GA□TCTC)外,还发现四个错义点突变。结论：本研究所检测的广东地区EB病毒健康携带者外周血单个核细胞所携带的：EB病毒LMP1基因为wt—Xho I,而在鼻咽癌组织中主要为Xho I-loss。因此,我们认为EB病毒LMP1基因N-末端区Xho I酶切位点的丢失和其他的错义点突变可能是在鼻咽癌的发生发展过程中产生的。     

Epstein-barr virus genotypes in NPC biopsies from North Africa

The genotypes of Epstein-Barr virus (EBV) were investigated in North African nasopharyngeal carcinoma (NPC) biopsies, nasopharyngeal chronic inflammation (NCI) biopsies, and saliva of healthy individuals from Algeria and Tunisia where there is an intermediate incidence of NPC. The prevalence of A-type virus in NPC, NCI biopsies and saliva of healthy individuals was found in these regions by means of a PCR assay. Restriction enzyme polymorphism analysis by Southern blotting revealed that all North African EBV variants have a conserved restriction site on BamHI W'-1′ and XhoI LMP gene. No additional BamHI enzyme site on the BamHI-F fragment was observed; however, the presence of an extra BamHI site on the BamHI-H fragment giving 2 H1 and H2 fragment-like EBV M-ABA strains was found. All EBV strains present in NPC or NCI biopsies at all ages were homogeneous in these polymorphisms and no correlation was observed between the EBV genotypes from NPC patients and clinical stages of the cancer. These characteristics revealed a significant difference between the EBV variants common in Chinese NPC and those in North African NPC.     

Identification and prevalence of CD8(+) T-cell responses directed against Epstein-Barr virus-encoded latent membrane protein 1 and latent membrane protein 2

Epstein-Barr virus (EBV) is associated with several human malignancies that each show different viral gene expression profiles. In malignancies such as Hodgkin's disease and nasopharyngeal carcinoma only Epstein-Barr nuclear antigen 1 (EBNA1) and varying levels of latent membrane proteins 1 and 2 (LMP1 and -2) are expressed. Since endogenously expressed EBNA1 is protected from CTL recognition, LMP1 and LMP2 are the most likely target antigens for anti-tumor immunotherapy. Therefore, we sought to identify in a systematic way CD8(+) T-cell responses directed against eptitopes derived from LMP1 and LMP2. Using IFNgamma-ELISPOT assays of interferon-gamma release, peripheral blood mononuclear cells (PBMC) of healthy donors were screened with peptide panels (15 mer overlapping by 10) spanning the LMP1 and LMP2 sequences of the prototype EBV strain B95.8. When positive responses were found, CD4(+) or CD8(+) T cells were depleted from donor PBMC to determine the origin of the responder population. We detected CD8(+) T-cell responses to LMP1 in 9/50(18%) donors and to LMP2 in 15/28 (54%) donors. In addition to the already described epitopes, 3 new LMP1- and 5 new LMP2-derived CD8(+) epitopes were identified. In most donors LMP1- and LMP2-specific CD8(+) precursor frequencies were low compared with precursors against immunodominant EBV epitopes from latent (EBNA3A, -3B and -3C) and lytic cycle antigens. These results demonstrate that CD8(+) memory T cell responses to LMP1 and especially to LMP2 do exist in Caucasians, albeit at low levels and could potentially be exploited for therapeutic use.     

The 30-bp deletion variant: a polymorphism of latent membrane protein 1 prevalent in endemic and non-endemic areas of nasopharyngeal carcinomas in China.

Development of nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. However, NPC occurs with a marked geographic and racial distribution, whereas EBV infection is ubiquitous in the world. This leads to a question whether certain subtypes of EBV have a greater potential to induce cell transformation. Latent membrane protein 1 (LMP1) is an EBV-encoded oncogenic protein and its 30-bp deleted variant (del-LMP1) has been reported to be predominant in biopsies of NPC. We have assessed the polymorphism of LMP1 in 47 biopsies of NPC, 107 cases of throat washings (TWs) from NPC patients, and 106 cases of TWs from non-NPC patients in Guangzhou, an endemic area of NPC in southern China, as well as 103 cases of TWs from healthy donors in Haerbin, a non-endemic area of NPC in northern China. Our results found a similar extent of the LMP1 polymorphism between NPC patients and non-NPC patients in Guangzhou, with the del-LMP1 being predominant in both Guangzhou and Haerbin. Sequence analyses showed identical substitutions in other coding regions of the del-LMP1 isolated from Guangzhou and Haerbin. These results indicate that del-LMP1 represents a geographic or race-associated polymorphism rather than an NPC disease phenotype-associated polymorphism.     

Establishment and characterization of a novel nasopharyngeal carcinoma cell line (SUNE2) from a Cantonese patient

The undifferentiated form of nasopharyngeal carcinoma (NPC) is the most common malignant head and neck cancer in South China, especially in Cantonese populations. However, few NPC cell lines have been established from the patients in this region. In this study, we established a new NPC cell line, termed SUNE2, from a Cantonese patient with undifferentiated NPC. This cell line had extremely low concentrations of Epstein-Barr virus (EBV) DNA in long-term culture and expressed low levels of latent membrane protein 1 (LMP1), latent membrane protein 2A (LMP2A), BamH1-A right frame 1 (BARF1), EBV-encoded RNA-1 (EBER1), and EBV-encoded RNA-2 (EBER2) in early passages. SUNE2 cells also showed much stronger transforming ability than 5-8F cells in colony formation assays and anchorage- independent growth assays in soft agar, and they only need 2 weeks to form tumors in nude mice. In summary, the SUNE2 cell line is a new in vitro model that can be used for further research on the mechanisms underlying the occurrence and development of NPC.     

Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma

Expression of the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNA 1 to 6) and membrane-associated protein (LMP) was investigated by immunoblotting in 83 nasopharyngeal carcinoma (NPC) biopsies and 25 other tumor and normal tissue specimens from the head and neck region. Fifty-eight of the 83 NPC biopsies were large enough to yield parallel data on virus DNA and viral expression. All 16 cases of clinically diagnosed and histologically confirmed NPCs from North Africa contained EBV DNA and expressed EBNA-1. Of 31 clinically diagnosed NPCs from China, 29 contained EBV DNA and 25 of these expressed EBNA-1. One control tissue biopsy from the oropharynx of NPC patients contained EBV DNA, but none expressed EBNA-1. The latent membrane protein (LMP) was detected in 22/31 of the Chinese and in 10/16 of the North African NPC biopsies. None of the NPC biopsies or control tissues expressed detectable amounts of EBNA 2 or any of the other 4 nuclear antigens which are invariably expressed in EBV-transformed B cells. A smaller number of tumors from Malaysia and East Africa exhibited a similar pattern of expression. EBV was rescued from a nude-mouse-passaged North African NPC tumor by co-cultivation of the tumor cells with umbilical cord blood lymphocytes. The tumor expressed EBNA 1 and LMP, but not EBNA 2 or the other 4 EBNAs. The resulting LCLs expressed all 6 nuclear antigens, EBNA 1 to 6 and LMP. Our data suggest that expression of the EBV genome is regulated in a tissue-specific fashion.     

Absence of p53 gene mutations in primary nasopharyngeal carcinomas.

Alterations in the p53 tumor suppressor gene and Epstein-Barr virus status were investigated in 15 nasopharyngeal carcinoma (NPC) biopsies, 4 xenografts, and 2 cell lines from the Cantonese region of southern China. One other established NPC cell line obtained from a northern Chinese patient was also studied. Restriction fragment length polymorphism analysis revealed a loss of heterozygosity for chromosome 17p, where the p53 gene resides, in only one of 15 NPC biopsies. Polymerase chain reaction-single-stranded conformational polymorphism analysis and direct sequencing failed to detect sequence alterations in exons 5 through 8 of the p53 gene in the 15 tumors and in the 4 NPC xenografts, all of which tested positive for Epstein-Barr virus. In contrast, the 3 NPC cell lines were all negative for Epstein-Barr virus and contained G----C transversions in the p53 gene, with cell lines CNE-1 and CNE-2 harboring identical AGA (arginine) to ACA (threonine) changes at codon 280. These results suggest that p53 inactivation is not a necessary component of nasopharyngeal carcinogenesis in Cantonese but may be important in the establishment of cell lines derived from these tumors.     

In vivo expansion of LMP 1- and 2-specific T-cells in a patient who received donor-derived EBV-specific T-cells after allogeneic stem cell transplantation

Immunotherapy approaches with antigen-specific cytotoxic T lymphocytes (CTLs) have provided safe and effective prophylaxis and treatment of Epstein-Barr virus (EBV)-associated lymphomas arising after bone marrow transplantation. EBV is also associated with other malignancies including ∼40% of cases of Hodgkin's disease, making this tumor another potential target for EBV-targeted immunotherapy. This study describes a patient with multiple relapsed EBV positive Hodgkin's Disease who received both autologous and allogeneic EBV CTL lines. After multiple chemotherapeutic and radiotherapy regimens including two autologous stem cell transplants, he received two doses of gene-marked autologous EBV-specific CTL which resulted in disease stabilization for 6 months. The gene-marked EBV-CTL persisted for 12 months in the peripheral blood after which he proceeded to unrelated donor stem cell transplant followed by immunotherapy with donor-derived EBV-specific CTL. Despite low levels of donor chimerism, the patient remains in complete remission 5 years post-allogeneic SCT. Comparison of the autologous and the donor-derived CTL lines showed that the donor line had specificity for two tumor-associated EBV antigens, latent membrane protein (LMP)1 and 2 compared to the autologous line, which only had specificity for LMP2 epitopes. Following infusion of the donor-derived CTL, functional analyses showed that T-cells reactive with both LMP1 and LMP2 epitopes expanded in the peripheral blood, suggesting that strategies to increase their frequency may result in a broader cytotoxic response against EBV+ Hodgkin tumors.     

V-val subtype of Epstein-Barr virus nuclear antigen 1 preferentially exists in biopsies of nasopharyngeal carcinoma

Epstein-Barr virus (EBV) has been suggested to be involved in pathogenesis of nasopharyngeal carcinoma (NPC). However, EBV infection is ubiquitous, whereas NPC occurs with strong geographic and racial distribution. Whether a substrain of EBV contributes to this phenomenon remains uncertain. Epstein-Barr virus nuclear antigen 1 (EBNA-1) is one of the most frequently detected EBV proteins in NPC tissues. Based on the polymorphism of amino acids at position 487, EBNA-1 is classified into five subtypes: P-ala, P-thr, V-val, V-leu and V-pro. To examine the relationship between subtypes of EBNA-1 and NPC, we determined the subtypes of EBNA-1 in biopsies of NPC, peripheral blood lymphocytes (PBL), and throat washings (TWs) obtained in endemic and non-endemic areas of NPC within China. The results revealed that V-val was the only subtype detected in NPC tissue, whereas three subtypes of EBNA-1, V-val, P-ala, and P-thr, were detected in PBL and TWs irrespective of origin, and mixed infection of V-val and P-ala was also observed. In addition, the variations of V-val derived from biopsies of NPC were identical to those derived from PBL and TWs in the context of N-terminus and C-terminus of EBNA-1. These facts indicate that a substrain of EBV with V-val subtype of EBNA-1 infects NPC preferentially and a susceptibility to a particular EBV isolate in the nasopharynx may exist during development of NPC.