EB病毒DNA聚合酶基因的表达与纯化鼻咽癌病人诊断中的应用

以Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒（ＥＢＶ）ＤＮＡ聚合酶为抗原,建立了简便、快速、敏感和特异的鼻咽癌诊断方法。构建原表达载体ｐＲＳＥＴ－ＤＮＡ聚合酶及其亚克隆ＰＲＳＥＴ－Ａ１和ＢＬ２１（ＤＥ３）中表达的产物,经Ｗｅｓｔｅｒｎ－ｂｌｏｔ检测其抗原性并用于检测鼻咽癌（ｎａｓｏｐｈａｒｙｎｇｅａｌ ｃａｒｃｉｎｏｍａ,ＮＰＣ）病人血清中的抗体。在ＤＰＣ病人血清中存在抗ＥＢ病毒ＤＮＡ聚合酶的ＩｇＧ抗体,并证明     

鼻咽癌Epstein－Barr病毒受体（EBVR／CR＿2）基因的病毒结合区的序列测定

Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒（ＥＢＶ）进入鼻咽上皮细胞的途径,是人们研究ＥＢＶ与鼻咽癌（ＮＰＣ）的病因关系时所必须回答的一个问题。用抗ＥＢＶ受体（ＥＢＶＲ／ＣＲ＿２）单抗检测上皮细胞中该受体的表达已有报道,但对上皮细胞中ＥＢＶＲ／ＣＲ＿２的基因结构仍需研究。我们采用ＰＣＲ扩增和不对称ＰＣＲ直接测序法,首次检测了１０例ＮＰＣ及３例正常人胚鼻咽上皮（Ｈｕｍａｎｅｍｂｒｙｏｎｉｃｎａｓｏｐａｒｙｎｇｅａｌｅｐｉｔｈｅｌｉｕｍ,ＨＥＮＥ）组织样本中ＥＢＶＲ／ＣＲ＿２的ＥＢＶ结合区的编码序列。这一片段的ＤＮＡ序列测定结果显示,人ＮＰＣ细胞和正常ＨＥＮＥ细胞的ＥＢＶＲ／ＣＲ＿２的ＥＢＶ结合区的编码序列,与正常人Ｂ淋巴细胞的ＥＢＶＲ／ＣＲ＿２的相应序列完全相同,提示ＥＢＶ感染鼻咽上皮细胞可能与ＥＢＶＲ／ＣＲ＿２基因的ＥＢＶ结合区结构改变无直接关系。     

Functional expression and characterization of the Epstein-Barr virus DNA polymerase catalytic subunit.

A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 110 kDa, recognized by anti-BALF5 protein-specific polyclonal antibody. The expressed EBV DNA polymerase catalytic polypeptide was purified from the cytosolic fraction of the recombinant virus-infected insect cells. The purified protein exhibited both DNA polymerase and 3'-to-5' exonuclease activities, which were neutralized by the anti-BALF5 protein-specific antibody. These results indicate that the 3'-to-5' exonuclease activity associated with the EBV DNA polymerase (T. Tsurumi, Virology 182:376-381, 1991) is an inherent feature of the polymerase catalytic polypeptide. The DNA polymerase and the exonuclease activities of the EBV DNA polymerase catalytic subunit were sensitive to ammonium sulfate in contrast to those of the polymerase complex purified from EBV-producing lymphoblastoid cells, which were stimulated by salt. Furthermore, the template-primer preference for the polymerase catalytic subunit was different from that for the polymerase complex. These observations strongly suggest that the presence of EBV DNA polymerase accessory protein, BMRF1 gene product, does influence the enzymatic properties of EBV DNA polymerase catalytic subunit.     

用转染EB病毒基因片段的真核细胞检测鼻咽癌病人的抗EB病毒核抗原-1(EBNA-1)抗体

用转染EBV DNA Bam HI K片段后稳定表达EBNA-1的K_4细胞作为靶细胞,检测50份鼻咽癌病人和38份健康对照者血清中的IgG/EBNA-1抗体;阳性率分别为100％和92％。前者的平均几何滴度为89.4,后者为18.3,前者约为后者的5倍。同一批被检血清经SPA吸收去除IgG竞争性抑制后,鼻咽癌病人的IgA/EBNA-1抗体阳性率达78％(GMT 20.9),健康人的IgA/EBNA-1抗体阳性率仅5.3％,效价亦很低(GMT 5.2)。表明IgA/EBNA-1抗体对鼻咽癌是比较特异的,可考虑作为鼻咽癌血清学诊断的指标之一。     

实时定量PCR方法检测鼻咽癌病人全血EB病毒拷贝数的实验研究

EBV is detected in more and more tumors, and is relative to carcinogenesis. We studied the copies of EBV DNA in whole blood of NPC patients and healthy controls by real-time quantitative PCR. In the 73 NPC patients and 83 controls, the positive rate of EBV in blood of NPC is 46.6%, while 13.3% in control. The mean copy number is 3.9 x 10(4) copys/microgram DNA in controls, which is much higher than NPC patients (which is 1.7 x 10(5) copies/microgram DNA). EBV infection is relative with NPC, while the lytic form of EBV maybe more important than its latent form. These results suggest that whole blood EBV DNA may be a valuable tool for molecular diagnosis of NPC.     

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     

Detection of Epstein-Barr virus in salivas and throat washings in healthy adults and children

It is well known that Epstein-Barr virus (EBV) is excreted from oral regions in the patients with infectious mononucleosis. We analyzed the prevalence of EBV in saliva and throat washings from healthy people in Japan by the polymerase chain reaction assay. EBV DNA was detected in 43 (90%) of the 48 throat washings from healthy adults (21 to 57 years old) and in 35 (38%) of the 93 salivas from healthy children (0 to 6 years old). The percentages of the EBV DNA-positive ratio in salivas increased in proportion relative to the increase of the children's ages. EBV type 1 was predominant and was detected in 86 and 94% of adults and children, respectively. Umbilical cord lymphocytes were transformed by some throat washings from EBV seropositive donors. EBV DNA was detected in throat washings from two healthy adults whose EBV antibody was not detected. In both cases, higher amounts of EBV DNA were detected in their peripheral blood mononuclear cells than in those of other, EBV antibody-positive donors. These results demonstrated the incidence of EBV excretion in oral regions of healthy individuals in Japan and defined a novel type of EBV infection in healthy adults.     

RPA3 is a potential marker of prognosis and radioresistance for nasopharyngeal carcinoma

Radioresistance‐induced residual and recurrent tumours are the main cause of treatment failure in nasopharyngeal carcinoma (NPC). Thus, the mechanisms of NPC radioresistance and predictive markers of NPC prognosis and radioresistance need to be investigated and identified. In this study, we identified RPA3 as a candidate radioresistance marker using RNA‐seq of NPC samples. In vitro studies further confirmed that RPA3 affected the radiosensitivity of NPC cells. Specifically, the overexpression of RPA3 enhanced radioresistance and the capacity for DNA repair of NPC cells, whereas inhibiting RPA3 expression sensitized NPC cells to irradiation and decreased the DNA repair capacity. Furthermore, the overexpression of RPA3 enhanced RAD51 foci formation in NPC cells after irradiation. Immunohistochemical assays in 104 NPC specimens and 21 normal epithelium specimens indicated that RPA3 was significantly up‐regulated in NPC tissues, and a log‐rank test suggested that in patients with NPC, high RPA3 expression was associated with shorter overall survival (OS) and a higher recurrence rate compared with low expression (5‐year OS rates: 67.2% versus 86.2%; 5‐year recurrence rates: 14.8% versus 2.3%). Moreover, TCGA data also indicated that high RPA3 expression correlated with poor OS and a high recurrence rate in patients with head and neck squamous cell carcinoma (HNSC) after radiotherapy. Taken together, the results of our study demonstrated that RPA3 regulated the radiosensitivity and DNA repair capacity of NPC cells. Thus, RPA3 may serve as a new predictive biomarker for NPC prognosis and radioresistance to help guide the diagnosis and individualized treatment of patients with NPC.     

EB病毒白介素-10基因在广东地区人群中的变异特征及与鼻咽癌的相关性

EB病毒（Epstein-Barr virus,EBV）基因变异与EBV相关疾病发生的关系尚无明确结论,本研究旨在了解广东地区人群中EBV白介素-10基因（viral interleukin-10,vIL-10）基因变异特征,探讨其变异与鼻咽癌（nasopharyngeal carcinoma,NPC）发生的关系。采用PCR和DNA测序检测NPC组织和健康成年人咽漱液中vIL-10序列。50例NPC和70例健康人群完成序列测定,其中42例（84.0%）NPC和60例（85.7%）健康人群与EBV标准株B95-8氨基酸序列一致,被分类为B95-8型;剩余8例（16.0%）NPC和10例（14.3%）健康人群在信号肽区域出现氨基酸突变,被分类为SPM变异型。2种型别及4处共有突变在2种人群中的分布无统计学意义（P>0.05）。结果表明,广东地区NPC和健康人群中EBV vIL-10高度保守,均以B95-8型为主,而SPM变异型的地域性分布及与鼻咽癌的确切关系有待进一步研究。     

Clinical and immunological effect of intravesical interleukin-2 on superficial bladder cancer

TheBamHI Z EBV replication activator (ZEBRA) protein is involved in the switch from latency to productive cycle of Epstein-Barr virus. A recombinant ZEBRA protein was synthesized and assessed in enzymelinked immunosorbent assay (ELISA) for serum IgG response in nasopharyngeal carcinoma (NPC) patients. In 100 NPC serum samples that were positive for IgA to the EBV viral capsid antigen (VCA), 75% had IgG anti-ZEBRA antibodies. In contrast, only 3/83 (3.6%) serum samples from healthy donors and 2/50 (4%) from other cancers were positive for IgG to ZEBRA. Interestingly, in a selected group of 100 NPC sera negative for IgA to VCA, 25% contained IgG anti-ZEBRA antibodies. This suggests that the ELISA for IgG anti-ZEBRA may also identify earlier cases of NPC not detected by the conventional immunofluorescence test for IgA to VCA.     

Immunophenotyping at the time of diagnosis distinguishes two groups of nasopharyngeal carcinoma patients: implications for adoptive immunotherapy

Background: Adoptive immunotherapy with EBV-specific CTLs (EBV-CTL) has been used to treat EBV-associated nasopharyngeal carcinoma (NPC) but only a fraction of the patients shows noticeable clinical response.Patients and Methods: Sixty-seven newly diagnosed NPC patients from 2005 to 2007 and 21 healthy donors were collected. Immunological parameters and immune function of PBMCs and EBV-CTL were analyzed by flow cytometer analysis (FACS) and ⁵¹Cr releasing experiment; Molecular characteristics on NPC tumor cells were investigated by immunochemical staining and statistic analysis.Results: NPC patients can be classified into two groups based on the percentage of CD3⁺ T cells in peripheral blood before accepted any treatment, (>52.6%, mean-2SE from healthy controls, NPC Group 1; <52.6%, NPC Group 2). The patients in Group 2 showed a significant decrease of CD3⁺CD8⁺ T-cells, CD3⁺CD4⁺ T-cells and CD3⁺CD45RO⁺ memory T cells, and increase of CD3^-CD16⁺ NK cells compared to Group 1 patients and healthy controls (P<0.001). EBV-specific T cell responses, were weaker in this group of patients and their tumor cells expressed lower levels of the EBV encoded latent membrane protein (LMP)-1 and HLA class II protein compared with the patients of NPC Group 1 (P<0.05) .Conclusion: These findings demonstrate that NPC patients could be distinguished on the basis of their immune status which will affect the efficacy of EBV-CTL immunotherapy.     

Patients with systemic lupus erythematosus have abnormally elevated Epstein–Barr virus load in blood

Various genetic and environmental factors appear to be involved in systemic lupus erythematosus (SLE). Epstein–Barr virus (EBV) is among the environmental factors that are suspected of predisposing to SLE, based on the characteristics of EBV itself and on sequence homologies between autoantigens and EBV antigens. In addition, higher titers of anti-EBV antibodies and increased EBV seroconversion rates have been observed in SLE patients as compared with healthy control individuals. Serologic responses do not directly reflect EBV status within the body. Clarification of the precise status of EBV infection in SLE patients would help to improve our understanding of the role played by EBV in this disease. In the present study we determined EBV types in SLE patients (n = 66) and normal control individual (n = 63) by direct PCR analysis of mouthwash samples. We also compared EBV load in blood between SLE patients (n = 24) and healthy control individuals (n = 29) using semiquantitative PCR assay. The number of infections and EBV type distribution were similar between adult SLE patients and healthy control individuals (98.5% versus 94%). Interestingly, the EBV burden in peripheral blood mononuclear cells (PBMCs) was over 15-fold greater in SLE patients than in healthy control individuals (mean ± standard deviation: 463 ± 570 EBV genome copies/3 μg PBMC DNA versus 30 ± 29 EBV genome copies/3 μg PBMC DNA; P = 0.001), suggesting that EBV infection is abnormally regulated in SLE. The abnormally increased proportion of EBV-infected B cells in the SLE patients may contribute to enhanced autoantibody production in this disease.     

Specific immune serum to the Epstein-Barr virus DNA polymerase.

Epstein-Barr virus (EBV) DNA polymerase was released from phorbol ester-treated tamarin (Saguinus oedipus) cells (B95-8) and prepared for use as an antigen by sequential column chromatography with DEAE-Sephadex A-25, DEAE-cellulose, phosphocellulose, and single-stranded DNA cellulose. Proteins from single-stranded DNA cellulose with DNA polymerase activity in 100 mM ammonium sulfate were mixed with complete Freund adjuvant and injected intradermally into rats and rabbits. Immune sera that were screened for specific antibody by indirect immunofluorescence procedures reacted with approximately 3% of the cells in EBV-producer cultures (B95-8 and P3HR-1) but not with EBV genome-negative cells (BJAB). In functional enzyme assays, immune sera or the immunoglobulin fraction inhibited the activity of purified EBV DNA polymerase 90%. Inhibition of enzyme activity was not affected by absorption of immune sera with insoluble matrices of proteins prepared with tamarin and human cells which lacked the EBV genome. Cellular DNA polymerase alpha was not inhibited by immune sera to the EBV enzyme.     

Effect of SPLUNC1 protein on the Pseudomonas aeruginosa and Epstein-Barr virus

Short palate, lung and nasal epithelium clone 1 (SPLUNC1) gene coded a secreted protein found at the surface of nasopharyngeal epithelium, which may be an innate immunity defensive molecular and a risk factor for nasopharyngeal carcinoma (NPC). Here, we observed the effects of SPLUNC1 on the Gram negative bacteria Pseudomonas aeruginosa, evaluated the ability of SPLUNC1 protein binding to lipopolysaccharide. To observe the effect of SPLUNC1 protein on Epstein-Barr virus (EBV), we raised three EBV-transformed B-lymphocyte lines and treated the cells by SPLUNC1 protein; cellular disruption, apoptosis, EBV DNA content, and viral oncogene expression were analyzed. We found that SPLUNC1 protein can bind to bacterial lipopolysaccharide, inhibit the growth of P. aeruginosa, enhance the disruption and apoptosis of EBV-infected B-lymphocytes, downregulate protein expression of EBV latent membrane protein 1, while upregulate protein expression of EBV envelope glycoprotein gp350/220. The total EBV DNA in the culture medium was decreased significantly after 7 days of treatment by SPLUNC1. This study shows that SPLUNC1 not only has the role of antibacteria and antivirus, but also inhibits the potential oncogenicity of EBV in respiratory epithelium. Hou-De Zhou and Xiao-Ling Li contributed equally to this work.     

采用原位杂交及电镜检测CR2转染小鼠细胞的EB病毒感染

EB病毒不能感染小鼠是因为小鼠CR2受体构像与人的不同,通过对小鼠CR2受体进行定点空变,然后将野生型和突变型小鼠CR2／1（MCR2／1）及人CR2（hCR2）用基因转移技术导入小鼠鼻咽上皮细胞系（TMNE）进行表达,观察转染阳性细胞是否具有结合EB病毒的能力,EBER－1杂交结果显示,只有转染hCR2和空变型MCR2（mtMCR2)的TMNE细胞可以感染EB病毒,但是前感染EB病毒的阳性率比后高的高得多。电镜结果也进一步证实EB病毒可以感染这两种细胞,这为进一步研究EB病毒进入细胞的机制及建立EB病毒相关的鼻咽癌动物模型奠定了良好的基础。     

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.     

Isolation and characteristics of endonuclease tightly bound to alpha-polymerase from the rat liver

Three major polypeptides are found in purified DNA polymerase alpha from rat liver: 160, 77 and 58 kDa. The electrophoretic analysis has identified polypeptide 160 kDa as the catalytically active subunit of DNA polymerase alpha. The other two polypeptides showed no DNA polymerase activity. Individual polypeptide p77 kDa purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to produce antibodies in rabbits. Immunoblot analysis indicated that the complex DNA polymerase alpha-3'-5'-exonuclease contained polypeptide p77 kDa. To elucidate the function of the p77 kDa protein we have prepared an immunoabsorbent column with antibodies against the p77 kDa polypeptide. The antibody column purified p77 kDa protein was homogeneous according to sodium dodecyl sulfate gel electrophoresis. The activity of alpha-polymerase was increased approximately 10-fold as a result of purification of DNA polymerase alpha from the p77 kDa protein. The in vitro experiments showed the identity of the p77 kDa polypeptide to endonuclease. It cleaved both single-stranded and double-stranded DNA. The function of endonuclease p77 kDA in complex with DNA polymerase alpha remains obscure.