EB病毒基因BARF1在鼻咽癌细胞中的表达及意义

目的：探讨EB(Epstein—Barr)病毒新基因BARF1(BamHI A rightward open reading frame1)在人鼻咽癌组织中的表达及意义，为深入阐明EB病毒致癌机制提供实验依据。方法：提取RNA后，采用RT—PCR方法扩增标本中的EBNA1(EB virus associated nuclear antigen 1)和BARF1 mRNA，PCR产物经2％琼脂糖凝胶电泳观察并照相。结果：11例RNA合格的标本均表达EBNA1，提示病例均为EB病毒阳性病例；其中9例表达BARF1，占82％：而且9例中的7例为强阳性。结论：EB病毒新基因BARF1 mRNA在鼻咽癌细胞中高表达，这提示除了已经明确的潜伏性膜蛋白1(LMP1)以外，BARF1可能在鼻咽癌细胞恶性增殖中发挥重要作用，具体机制有待深入研究。     

A new diagnostic marker for secreted Epstein-Barr virus encoded LMP1 and BARF1 oncoproteins in the serum and saliva of patients with nasopharyngeal carcinoma.

PURPOSE: EBV has been associated with nasopharyngeal carcinomas (NPC). In North Africa, the incidence is bimodal-the first peak occurring at approximately 20 years of age and the second peak occurring at approximately 50 years. Standard diagnostic tests based on immunofluorescence using anti-IgA EBV have shown that young North African patients have a negative serology compared with older patients. We are interested in two EBV-encoded oncoproteins, LMP1 and BARF1, which have thus far not been studied in terms of their potential as diagnostic markers for NPC. These two viral oncoproteins have been detected in cell culture media, so we tested whether they could be detected in the serum and saliva of patients with NPC. EXPERIMENTAL DESIGN: LMP1 and BARF1 proteins were analyzed in the sera and saliva of young patients and adult patients with NPC from North Africa and China. We then examined whether the secreted proteins had biological activity by analyzing their mitogenic activity. RESULTS: Both LMP1 and BARF1 were present in the serum and saliva from North African and Chinese patients with NPC. All young North African patients secreted both proteins, whereas 62% and 100% of adult patients secreted LMP1 and BARF1, respectively. From animal studies, the secreted LMP1 was associated with exosome-like vesicles. These secreted EBV oncoproteins showed a powerful mitogenic activity in B cells. CONCLUSION: Both proteins will be a good diagnostic marker for NPC whereas BARF1 is a particularly promising marker for all ages of patients with NPC. Their mitogenic activity suggests their implication in the oncogenic development of NPC.     

Anti-apoptotic role of BARF1 in gastric cancer cells

Epstein-Barr virus (EBV) infection has been implicated in the carcinogenesis of several types of human cancer, including gastric cancer. In contrast to two other EBV-related malingancies, nasopharyngeal carcinoma and Hodgkins Lympomain which the latent membrane protein (LMP)-1 is often detected, in gastric cancer, BARF1, one of the early EBV genes, is frequently expressed in EBV-positive specimens. This indicates that expression of BARF1 may play a positive role in the development of gastric cancer. The aim of this study was to investigate the effect of BARF1 expression in gastric cancer cells. First, a retroviral vector containing the full length BARF1 gene was transfected into an EBV negative gastric cancer cell line, BGC823, and stable transfectants expressing ectopic BARF1 were generated. Microarray analysis was then performed and gene expression profiles were analysed and compared between the cells expressing ectopic BARF1 and the vector control. In addition, the effect of BARF1 on gastric cancer cell proliferation and apoptosis was investigated by MTT assay, DAPI staining, flow cytometry as well as Western blotting. We found that expression of BARF1 in gastric cancer cells led to significant alterations of gene expression, especially genes related to proliferation and apoptosis. In addition, the BARF1 expressing cells were more resistant to apoptosis induced by a commonly used anticancer drug, taxol. This chemo-protective effect of BARF1 was associated with increased Bcl-2 and Bax ratio and decreased expression of cleaved PARP, but not alterations in cell proliferation. Our results suggest that BARF1 expression in gastric cancer cells may provide a protective role against apoptosis through an increased Bcl-2 to Bax ratio, thus promoting cancer cell survival.     

Activation of lytic cycle of Epstein-Barr virus by suberoylanilide hydroxamic acid leads to apoptosis and tumor growth suppression of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). We reported that suberoylanilide hydroxamic acid (SAHA) induced EBV lytic cycle in EBV-positive gastric carcinoma cells and mediated enhanced cell death. However, expression of EBV lytic proteins was thought to exert antiapoptotic effect in EBV-infected cells. Here, we examined the in vitro and in vivo effects of SAHA on EBV lytic cycle induction in NPC cells and investigated the cellular consequences. Micromolar concentrations of SAHA significantly induced EBV lytic cycle in EBV-positive NPC cells. Increased apoptosis and proteolytic cleavage of poly(ADP-ribose) polymerase and caspase-3, -7 and -9 in EBV-positive versus EBV-negative NPC cells were observed. More than 85% of NPC cells expressing immediate-early (Zta), early (BMRF1) or late (gp350/220) lytic proteins coexpressed cleaved caspase-3. Tracking of expression of EBV lytic proteins and cleaved caspase-3 over time demonstrated that NPC cells proceeded to apoptosis following EBV lytic cycle induction. Inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA's induced cell death in NPC, indicating that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were also observed in NPC xenografts in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA leads to apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus-targeted therapy against EBV-positive cancer.     

Epstein-Barr virus in the pathogenesis of NPC

Epstein-Barr virus (EBV) is consistently detected in nasopharyngeal carcinoma (NPC) from regions of high and low incidence. EBV DNA within the tumor is homogeneous with regard to the number of terminal repeats. The detection of a single form of viral DNA suggests that the tumors are clonal proliferations of a single cell that was initially infected with EBV. Specific EBV genes are consistently expressed within the NPC tumors and in early, dysplastic lesions. The viral proteins, latent membrane protein 1 and 2, have profound effects on cellular gene expression and cellular growth, resulting in the highly invasive, malignant growth of NPC tumors. In addition to potential genetic changes, the establishment of a latent, transforming infection in epithelial cells is likely to be a major contributing factor to the development of this tumor.     

Expression of tumor necrosis factor receptor-associated factor 1 in nasopharyngeal carcinoma: possible upregulation by Epstein-Barr virus latent membrane protein 1

EBV infection is associated with virtually all cases of undifferentiated NPC, and the EBV-encoded LMP1 is expressed in a proportion of cases. LMP1 has transforming functions similar to members of the TNF receptor family and activates intracellular signaling cascades through interaction with TRAFs. In B cells, expression of TRAF1 is in turn upregulated by LMP1. LMP1 signaling in epithelial cells may be affected by the presence or absence of TRAF1. By immunohistochemistry, we detected TRAF1 expression in 17 of 42 (40%) EBV+ undifferentiated NPCs. All 7 LMP1+ NPC biopsies were also TRAF1+. Using an RNAse protection assay, high-level TRAF1 expression was detected in an LMP1-expressing NPC-derived cell line (C15) and expression was weaker in 2 LMP1- cell lines (C17, C19). Finally, LMP1 upregulated TRAF1 expression in an EBV- keratinocyte cell line. Our results demonstrate that TRAF1 is expressed in NPC tumor cells in vivo and suggest that TRAF1 expression may be upregulated by LMP1 in NPC. An antiapoptotic function has been proposed for TRAF1, and this may be relevant for the pathogenesis of NPC.     

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.     

Mitogenic activity of Epstein-Barr virus-encoded BARF1 protein

We previously reported that BARF1 gene has either an immortalizing effect, when expressed in primary primate epithelial cells, or a malignant transforming activity, when expressed in established and nontumoral rodent fibroblast or human B-cell lines. As predicted from sequence analysis, we found that BARF1 coded protein can be secreted from different cell lines, among them BARF1-transfected Balb/c3T3 rodent fibroblasts. Thus, as an initial step to clarify BARF1 oncogenic functions, we investigated whether the secreted form of BARF1 protein can activate the cell cycle as a growth factor. Since efficient BARF1 expression could be obtained from 293-tTA cells infected with a tetracycline-regulatable recombinant adenovirus, secreted BARF1 product could be purified from the culture medium of such cells by ammonium sulfate precipitation, ion exchange chromotography and sucrose gradient sedimentation. We describe in this paper that addition of a purified product of secreted BARF1 protein to serum-free culture medium of Balb/c3T3 rodent fibroblasts, human Louckes B-cell line and primary monkey kidney epithelial cells resulted in a cell cycle activation that was inhibited by affinity-purified anti-BARF1 antibody. Our demonstration of a specific stimulation of cell cycle in vitro by BARF1 secreted product suggests that this EBV-encoded BARF1 protein could act as a growth factor in vivo.     

鼻咽癌组织中 EBV-BARF0基因序列的测定

目的： EB病毒 (Epstein-Barr virus,EBV)存在的变异影响到其生物学功能（如 LMP1基因）。 BamHI A区右向框 0（ BamHI A rightward fram0, BARF0）是在鼻咽癌 (Nasopharyngeal Carcinoma,NPC)病人中检出率很高的一个 EB病毒基因,对其变异性的研究尚未见报道,本研究是为了明确广东鼻咽癌组织中 EBV-BARF0基因的序列及其变异。方法：应用 PCR技术从 20例鼻咽癌组织中,扩增 EBV基因 BARF0,并对它们的序列进行测定。结果：与标准株 B95-8相比,鼻咽癌组织中 EBV-BARF0均有 4个位点发生突变： 160473（ G→ T）、 160545（ C→ T）、 160701（ C→ A）、 160707（ G→ C）,并导致相应的氨基酸的改变： 2（ Ala→ Ser）、 26（ Leu→ Phe）、 78（ Arg→ Ser）、 80（ Ala→ Pro）。结论：由于 EBV的 BARF0基因在鼻咽癌细胞株及活检组织中 100％表达,在淋巴瘤组织及淋巴瘤细胞株中表达较低或不表达,本研究首次报道鼻咽癌组织中 EBV-BARF0的序列分析,与 B95-8相比存在变异,并导致了蛋白质的一级结构的改变,推测该基因很可能在鼻咽癌变过程中起着重要的作用。