Presence of the latent membrane protein 1 gene in nasopharyngeal swabs from patients with mucosal recurrent nasopharyngeal carcinoma

BACKGROUND: Nasopharyngeal carcinoma (NPC) is the most common head and neck malignancy in southeastern China and Taiwan. Early detection of the local disease followed by timely and appropriate treatment is essential to increasing cure and survival rates. Detection of Epstein-Barr virus (EBV) genomic DNA, such as the latent membrane protein 1 gene (LMP-1), in patients postirradiation during follow-up may indicate mucosal recurrence. METHODS: Seventy-one patients with NPC underwent serial nasopharyngeal swabs for LMP-1 polymerase chain reaction assay before, during, and after irradiation. All of patients achieved a complete disease remission of the LMP-1 gene after irradiation that lasted for at least 6 months. RESULTS: The median LMP-1 disease remission time after the beginning of irradiation was 4.3 weeks. Patients with early LMP-1 disease remission ( 4 weeks) had 3-year local control rates of 93.5% and 76.9%, respectively (P = 0.0529). The LMP-1 gene was detected again (reexpression of LMP-1 [re-LMP-1]) in 10 patients after irradiation with at least 6 months of follow-up. Nine of 10 patients (90%) in the re-LMP-1 positive group and 2 of 61 patients (3.3%) in the re-LMP-1 negative group developed local recurrence. Mucosal recurrence developed in nine patients, and all displayed re-LMP-1. By detecting re-LMP-1 using nasopharyngeal swabs, mucosal recurrence was diagnosed with a sensitivity of 100% (9 of 9 patients) and a specificity of 98.4% (61 of 62 patients). The 3-year overall survival rate, the disease free survival rate for the entire group, and the estimated local mucosal control rates in the re-LMP-1 positive and re-LMP-1 negative groups were 86.5%, 76.5%, 19.4%, and 96.7%, respectively. CONCLUSIONS: Expression of EBV LMP-1 in nasopharyngeal swab specimens from patients with irradiated/treated NPC can provide a highly sensitive and specific method of forecasting mucosal recurrence. This investigation confirmed the reliability and feasibility of nasopharyngeal swabs in screening for mucosal recurrences in patients with NPC.     

EB病毒膜潜伏蛋白LMP-1筛选鼻咽癌的初步结果

Objective  

Early diagnosis of nasopharyngeal carcinoma (NPC) is an important method to improve the survival rate. However, the sensitivity and specificity of the screening protocols which was widely used in clinic now are considered to be unsatisfactory. Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP-1) is one of the proteins that have been suggested to be a classic oncogene with transformation properties. The current study set out to discuss the clinical significance of LMP-1 on the screening of NPC.     

30-bp DELETION IN LATENT MEMBRANE PROTEIN 1 （LMP-1）ONCOGENE IN LYMPHOEPITHELIAL CARCINOMA OF SALIVARY GLANDS

Objective:To investigate the 30 bp deletion in LMP-1 in lymphoepithelial carcinoma of salivary glands,and to clarify the deletion rate. Methods: 46 cases of LEC were subjected to PCR examination for the 3‘terminal region of LMP-1 gene, in order to observe the 30 bp deletion. To reduce the influence of unsuccessful DNA extraction from paraffin-embedded tissue sections,a β-actin PCR was performed at the same time.Additionally, DNA sequencing was performed on 1 case without deletion and 1 case with deletion. Results: 4 of 46 specimens were proved to contain no suitable DNA sample by β-actin gene amplification. In the remaining 42 cases, LMP-1 DNA was detected in 35/42 (83.3%)LEC cases. Two kinds of PCR products were found in these 35 cases after further DNA sequencing. 31 cases(88.6%) carried 316 bp product and 4 cases (11.4%)carried 286 bp product. Conclusion: Some LECs of salivary glands carry del-LMP-1. In our study, the deletion rate was 11.4% (4/35).     

Association of latent membrane protein 1 and matrix metalloproteinase 9 with metastasis in nasopharyngeal carcinoma

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a highly metastatic carcinoma whose consistent association with Epstein-Barr virus (EBV) has been established. Latent membrane protein 1 (LMP1), an EBV membrane protein expressed in latent infection, is considered to be the EBV oncoprotein. Matrix metalloproteinase 9 (MMP9), one of the MMP families, degrades Type IV collagen, a major component of extracellular matrix and is believed to be crucial for cancer invasion and metastasis. Although MMP9 is reported to be expressed in a variety of cancers, no reports concerning NPC have been published to date to the authors' knowledge. Recently, the authors have shown that LMP1 induces MMP9 in vitro cell line, which suggests the possibility of a mechanism in which LMP1 of EBV contributes to the metastasis and tumorigenesis of NPC by the induction of MMP9. METHODS: The expressions of LMP1 and MMP9 were immunohistochemically examined in 38 NPC sections, and the relation of these proteins were statistically analyzed. The authors also analyzed the associations of these proteins with clinical features. RESULTS: Both LMP1 and MMP9 proteins were predominantly immunolocalized in cancer nests. The expression of MMP9 showed a significant positive correlation with the expression of LMP1 (r = 0.75; P < 0.0001). Also, the expression of MMP9 correlated with lymph node metastasis (P = 0. 0004). CONCLUSIONS: The results suggest that the induction of MMP9 by LMP1 contributes to the metastatic potential of NPC.     

EBV-LMP1对鼻咽癌细胞系CNE1细胞凋亡的影响

背景与目的:已证实EB病毒编码的潜伏膜蛋白1(latentmembraneprotein,LMP1)具有转化致瘤作用,在鼻咽癌的发生发展中起重要作用。LMP1的转化致瘤作用可能与细胞凋亡有关,本实验目的是观察EBV-LMP1对鼻咽癌细胞系CNE1细胞凋亡的影响,探索LMP1在鼻咽癌发生发展中的可能机制。方法:用电转染的方法将带有绿色荧光蛋白GFP报道系统的EB病毒LMP1基因真核表达质粒导入CNE1,用荧光显微镜检测报道基因GFP的表达情况;用免疫组化法及Westernblot法检测目的基因LMP1的表达情况;用流式细胞仪、荧光染色及DNA片段分析等方法检测在有地塞米松磷酸钠或无血清饥饿诱导的情况下LMP1对CNE1细胞凋亡的影响。结果:CNE1G细胞(转染空载质粒PAT-GFP的CNE1细胞)及CNE1GL(转染目的基因质粒PAT-GFP-LMP1的CNE1细胞)有绿色荧光蛋白表达,CNE1细胞(未经转染的CNE1细胞)无绿色荧光蛋白表达;CNE1GL细胞LMP1呈阳性表达,而CNE1及CNE1G细胞均为阴性。CNE1、CNE1G及CNE1GL3组细胞分别用地塞米松磷酸钠或无血清1640培养液处理后均有凋亡出现,且两种诱导方法均能使CNE1GL组细胞的凋亡指数(apoptosisindex,AI)较CNE1及CNE1G组明显增高(P<0.05),而CNE1与CNE1G组的凋亡指数无明显差别。3组细胞常规培养下则无凋亡出现。结论:LMP1基因成功导入CNE1细胞     

Epstein-Barr virus-encoded latent membrane protein 1 co-expresses with epidermal growth factor receptor in nasopharyngeal carcinoma.

Latent membrane protein 1 (LMP-1) is the only Epstein-Barr virus (EBV)-encoded oncogenic protein that has been detected in nasopharyngeal carcinoma (NPC), a cancer that is closely associated with EBV. Previous in-vitro studies have demonstrated that LMP-1 can upregulate epidermal growth factor receptor (EGFR) in epithelial cells. It was not established whether this cellular effect exists in NPC. To assess the association between LMP-1 and EGFR in NPC tissues, 60 NPC specimens were examined by immunohistochemistry using anti-LMP-1 antibody (CS 1-4) and anti-EGFR antibodies (EGFR 1, EGFR 1005). The results revealed that 41 (68.3%) specimens were immunopositive for LMP-1 and 44 (73.3%) specimens over-expressed EGFR. Morphologically, the expressions of LMP-1 and EGFR were homogeneously distributed in the tumor nests. In addition, the correlation between LMP-1 and EGFR was statistically significant (P<0.001, chi2 test, d.f. = 1). To elucidate further the correlation between LMP-1 and EGFR in vivo and in situ, an indirect dual immunofluorescence assay was conducted, using secondary antibodies conjugated with fluorescein isothiocyanate (FITC) or indocarbocyanine (Cy3). The results disclosed an intimate co-expression of LMP-1 and EGFR. In summary, the data indicate that over-expression of EGFR is a common phenomenon in NPC, and that EGFR is co-expressed with LMP-1 in NPC. Thus, EBV may play a role in the tumorigenesis of NPC through the effects of LMP-1 and EGFR.     

Modulation of the tumor microenvironment by Epstein‐Barr virus latent membrane protein 1 in nasopharyngeal carcinoma

Latent membrane protein 1 (LMP1) is a primary oncogene encoded by the Epstein‐Barr virus, and various portions of LMP1 are detected in nasopharyngeal carcinoma (NPC) tumor cells. LMP1 has been extensively studied since the discovery of its transforming property in 1985. LMP1 promotes cancer cell growth during NPC development and facilitates the interaction of cancer cells with surrounding stromal cells for invasion, angiogenesis, and immune modulation. LMP1 is detected in 100% of pre‐invasive NPC tumors and in approximately 50% of advanced NPC tumors. Moreover, a small population of LMP1‐expressing cells in advanced NPC tumor tissue is proposed to orchestrate NPC tumor tissue maintenance and development through cancer stem cells and progenitor cells. Recent studies suggest that LMP1 activity shifts according to tumor development stage, but it still has a pivotal role during all stages of NPC development.     

Differences in the immunogenicity of latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus genomes derived from LMP1-positive and -negative nasopharyngeal carcinoma

We have previously shown that an EBV-encoded latent membrane protein 1 (LMP1) gene derived from a nude mouse-propagated nasopharyngeal carcinoma (NPC) tumor and expressed in nonimmunogenic murine mammary carcinoma S6C cells failed to convey immunogenicity (rejectability) in syngeneic mice, whereas the corresponding B-cell derived LMP1 gene made the mice highly immunogenic. This raised the question of whether LMPL-expressing NPCs have been selected for low immunogenicity at the viral gene expression level. If so, LMP1-negative tumors that carry highly methylated LMP1 regulatory sequences may not have been exposed to a similar immunoselection. In the present study, we have compared LMP1 genes derived from two LMP1-positive NPCs and two LMP1-negative NPCs. All four genes were expressed in S6C cells in parallel with the previously tested isolates from a B-cell (B95-8)-derived and a nude mouse-propagated NPC (Cao)-derived gene. As in the previous study, we have found that the B-cell-derived LMP1 isolate was highly immunogenic. LMP1-positive tumor-derived isolates were poorly immunogenic, whereas the isolates from the LMP1-negative NPC tumor had intermediate immunogenicity. Sequence data revealed that LMP1 genes from LMP1-expressing NPC had 16 amino acid substitutions, whereas LMP1 from non-LMP1-expressing NPC had only 9 amino acid changes in the coding region. Three of the changes were at shared sites, but with different modifications. The fact that the gene from non-LMP1-expressing NPC mutated at a low frequency but was more immunogenic than the LMP1 gene derived from LMP1-expressing NPC, which was highly mutated but less immunogenic, favors the idea that LMP1-positive tumors escape immunosurveillance in immunocompetent hosts by either a selective down-regulation of LMP1 expression, methylation in the LMP1 promoter sequence, or mutation of LMP1 in LMP1-expressing samples.     

Epstein-Barr Virus latent membrane protein 1 induces Snail and epithelial-mesenchymal transition in metastatic nasopharyngeal carcinoma

Background:

Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) is distinctive among head-and-neck cancers in its undifferentiated histopathology and highly metastatic character. We have recently investigated the involvement of epithelial–mesenchymal transition (EMT) in NPC. In a previous study, we found a close association of expression of LMP1, the principal EBV oncoprotein, with expression of Twist and induction of EMT.

Methods:

We analysed expression of Snail in 41 NPC tissues by immunohistochemistry. The role of Twist as well as Snail in EMT of NPC was investigated by using NP69SV40T human nasopharyngeal cells.

Results:

In NPC tissues, overexpression of Snail is associated with expression of LMP1 in carcinomatous cells. In addition, expression of Snail positively correlated with metastasis and independently correlated inversely with expression of E-cadherin. Expression of Twist had no association with expression of E-cadherin. Further, in a human nasopharyngeal cell line, LMP1 induces EMT and its associated cellular motility and invasiveness. Expression of Snail is induced by LMP1 in these cells, and small hairpin RNA (shRNA) to Snail reversed the cellular changes. By contrast, Twist did not produce EMT in these nasopharyngeal cells.

Conclusions:

This study strengthens the association of EMT with the metastatic behaviour of NPC. These results suggest that induction of Snail by the EBV oncoprotein LMP1 has a pivotal role in EMT in NPC.     

Specific latent membrane protein 1 gene sequences in type 1 and type 2 Epstein-Barr virus from nasopharyngeal carcinoma in Hong Kong

We reported previously that a characteristic Epstein-Barr virus latent membrane protein 1 (EBV-LMP1) gene was associated with nasopharyngeal carcinoma (NPC) in Hong Kong. It showed a 30 bp deletion at the carboxyl terminus with specific amino acid substitution Asp at codon 335 with reference to Gly in B95-8 LMP1. This deletion variant Asp335 was present in over 90% of NPC biopsy specimens. The present study attempted to determine the whole encoding sequence of the LMP1 gene in different EBV isolates from NPC, and its relation with EBV types. We found that 92% (34/37) of primary NPC tumours harboured EBV-1 and possessed the LMP1 deletion variant, of which 86% were Asp335 and 6% were Gly335. EBV-2 was present in 8% (3/37) of tumours and all contained the retention variant of the LMP1 gene. Sequencing of the whole encoding region of the LMP1 gene revealed that the deletion variant Asp335 and deletion variant Gly335 carried similar sequences. They showed 43 common nucleotide substitutions in 41 codons with reference to B95-8. The retention variant showed 52 base changes in 46 codons compared with B95-8. The amino acid alterations in both the deletion and retention variants were mostly clustered at the transmembrane domain of the protein. Furthermore, half of the substitutions were common to both variants, suggesting a common evolutionary selection pressure. Nonetheless, the 2 LMP1 variants showed differences in nucleotide alterations and were associated with different EBV types, suggesting the presence of 2 distinct EBV strains in Hong Kong NPC. Int. J. Cancer 76:399–406, 1998.© 1998 Wiley-Liss, Inc.     

EBV-LMP1对鼻咽癌细胞系CNE1细胞转移相关因素的影响

背景与目的:已证实EB病毒(Epstein-Barrvirus,EBV)编码的潜伏膜蛋白1(latentmembraneprotein1,LMP1)能够诱导鼻咽癌细胞中基质金属蛋白酶9(matrixmetalloproteinase-9,MMP-9)的表达。本实验的目的是观察EB病毒潜伏膜蛋白1(EBV-LMP1)对鼻咽癌细胞系CNE1细胞转移相关因素的影响,探讨LMP1在鼻咽癌侵袭、转移过程中的作用。方法:用免疫组化法及Westernblot法检测CNE1-GL(转染LMP1基因的CNE1细胞)和CNE1细胞中MMP-9的表达情况;用细胞-基质粘附实验、细胞运动实验和肿瘤细胞重组基底膜侵袭实验检测LMP1对CNE1细胞粘附、运动及侵袭能力的影响。结果:免疫组化法及Westernblot法结果均显示CNE1-GL细胞中MMP-9的表达明显高于CNE1细胞(P<0.05);肿瘤细胞-基质粘附实验结果显示,CNE1-GL的粘附能力(平均吸光度值为1.2508±0.0711),高于CNE1细胞(平均吸光度值为0.9519±0.068),两者相比差异有显著性(P<0.01)。运动实验及重组基底膜侵袭实验结果均显示,穿过游离的聚乙烯吡咯烷酮膜(polyvinylpyrroli-done-free,PVP-F)的CNE1-GL细胞数明显高于CNE1细胞(P<0.01)。结论:LMP1能够诱导CNE1细胞中MMP-9的表达,且增强CNE1细胞与基底膜的粘附能力、运动能力及侵袭能力。     

EB病毒LMP1对人鼻咽癌细胞转移能力的影响

背景与目的:EB病毒编码的潜伏膜蛋白1(latent membrane protein 1,LMP1)在鼻咽癌的浸润和转移过程中起重要的作用。本实验目的在于研究EB病毒LMP1对人鼻咽癌细胞转移能力的影响和探讨其中的相关机制。方法:应用免疫细胞化学RT-PCR法和Western blot检测LMP1、E-cadherin和intercellular adhesion molecule-1 (ICAM-1)在人高分化鼻咽癌细胞系CNE1和转染了LMP1基因的CNE1-GL细胞中的表达。应用细胞-细胞粘附实验、细胞-基质粘附实验、划痕实验和细胞运动实验观察LMP1对鼻咽癌细胞粘附和运动能力的影响。结果:免疫细胞化学结果显示:LMP1在CNE1和CNE1-GL细胞中的阳性率分别为0和(96.60±3.03)%(P<0.01);E-cadherin蛋白的阳性率分别为(37.47±1.50)%和(19.53±1.92)%(P<0.01);ICAM-1蛋白的阳性率分别为(5.27±1.45)%和(93.33±4.23)%(P<0.01)。RT-PCR和Westernblot结果表明,与CNE1细胞相比较,CNE1-GL细胞中E-cadherin的mRNA和蛋白表达明显降低(P<0.01),而ICAM-1的mRNA和蛋白表达则明显升高(P<0.01)。肿瘤细胞同质粘附实验显示,CNE1-GL细胞的同质粘附能力较CNE1细胞弱(P<0.05)。肿瘤细胞-基质粘附实验得出CNE1-GL细胞的异质粘附能力(A值=0.60±0.03)较CNE1细胞(A值=0.46±0.01)强(P<0.01)。肿瘤细胞运动实验显示,穿过PVPF膜的CNE1-GL细胞数多于CNE1细胞(119.3±6.0 vs 46.3±7.0,P<0.05)。结论:LMP1通过抑制E-cadherin表达、促进ICAM-1表达从而抑制肿瘤细胞的同质粘附能力、增强其异质粘附能力和运动能力来参与鼻咽癌的侵袭和转移。     

MMP-9、LMP-1蛋白在鼻咽癌中的表达及其与远处转移的相关性研究

目的：研究基质金属蛋白酶-9（MMP-9）、潜伏膜蛋白-1（LMP-1）在鼻咽癌中的表达及其对鼻咽癌远处转移的影响。方法：收集83例鼻咽癌患者的临床资料及鼻咽部瘤体活检组织蜡块。采用免疫组化s—P法检测标本中MMP-9、LMP-1的表达。结果：MMP-9阳性表达率为65％（54／83）,其表达与颈淋巴结转移、远处转移关系密切（P〈0．05）。LMP-1阳性表达率为57％（47／83）,其表达与颈淋巴结转移、远处转移关系密切（P〈0．05）;且与MMP-9表达呈显著正相关（rJ=0．327,P〈0．05）。鼻咽癌远处转移的危险因素是临床分期（Ⅲ、Ⅳ期）、颈淋巴结N,阳性、MMP-9阳性表达、LMP-1阳性表达（P〈0．05）。MMP-9、LMP-1均为阳性表达者远处转移风险更高（P〈0．05）。结论：MMP-9、LMP-1阳性表达与远处转移明显相关,可作为鼻咽癌远处转移的预测指标。     

Interleukins,laminin and epstein - barr virus latent membrane protein 1 (EBV LMP1) Promote metastatic phenotype in nasopharyngeal carcinoma

Background  

Nasopharyngeal carcinoma (NPC) is a type of neoplasm that is highly prevalent in East Asia and Africa with Epstein-Barr virus (EBV), genetic, and dietary factors implicated as possible aetiologic factors. Previous studies suggested the association of certain cytokines with the invasion and metastatic properties of NPC. The present study examined the roles of EBV latent membrane protein-1 (LMP1), interleukin-6 (IL-6), interleukin-10 (IL-10), transforming growth factor-beta 1 (TGF-β1) and laminin in the regulation of matrix-metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) in NPC. The effects of these factors on bmi-1, an oncogene, and ngx6, a tumour suppressor gene, were also investigated.     

EBV-LMP1上调Ezrin表达对鼻咽癌细胞转移潜能的影响

背景与目的:细胞骨架相关蛋白Ezrin的异常表达与肿瘤侵袭转移密切相关,既往研究已证实EB病毒潜伏膜蛋白1(Epstein-Barr virus latent membrane protein1,EBV-LMP1)可促进鼻咽癌细胞的转移能力。本研究旨在进一步探讨EBV-LMP1是否通过改变Ezrin的表达来影响鼻咽癌细胞的转移能力。方法:采用免疫细胞化学和Western blot检测两种鼻咽癌细胞株-CNE1细胞(EBV阴性)和CNE1-GL细胞(稳定转染EBV-LMP1)中LMP1和Ezrin的表达;细胞-基质粘附实验检测CNE1、CNE1-GL和经Ezrin抗体预处理的CNE1-GL细胞(AntiEzrin-CNE1-GL)对细胞外基质的粘附力;Transwell小室法检测上述3种细胞的运动和对重组基底膜侵袭能力。结果:CNE1细胞中Ezrin阴性表达,而CNE1-GL细胞中Ezrin强阳性表达。CNE1-GL细胞对细胞外基质的粘附率[(89.38±6.12)%]强于CNE1细胞[(49.42±5.37)%](P<0.001),而AntiEzrin-CNE1-GL细胞对基质的粘附率[(56.94±4.08)%]明显下降,与CNE1-GL相比,差异有统计学意义(P<0.05)。运动实验和侵袭实验均提示CNE1-GL细胞运动、侵袭能力(107±11和179±25)强于CNE1细胞(27±3和46±6),差异均有统计学意义(P<0.001),而AntiEzrin-CNE1-GL细胞的运动、侵袭能力(38±4和51±5)较CNE1-GL细胞显均显著下降(P<0.001)。结论:CNE1细胞中EBV-LMP1可通过上调Ezrin表达来促进细胞转移。