EBV latent membrane protein 1 effects on plakoglobin, cell growth, and migration

Latent membrane protein 1 (LMP1), the major oncoprotein of EBV, is likely responsible for many of the altered cellular growth properties in EBV-associated cancers, including nasopharyngeal carcinoma (NPC). In this study, the effects of LMP1 on cell growth and migration were studied in the context of the EBV-positive C666-1 NPC cell line. In the soft agar transformation and Transwell metastasis assays, LMP1 enhanced cell growth and migration through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappaB (NF-kappaB) signaling. Inhibitors of PI3K, Akt, and NF-kappaB signaling dramatically reduced these enhanced properties. An IkappaBalpha super-repressor also blocked these effects. However, constitutive activation of Akt alone did not alter cell growth, suggesting that both PI3K/Akt and NF-kappaB activation are required by LMP1. These enhanced effects required the full-length LMP1 encompassing both the PI3K/Akt-activating COOH-terminal activation region (CTAR) 1 and the nonredundant NF-kappaB-activating regions CTAR1 and CTAR2. LMP2A, a latent protein that is also frequently expressed in NPC, similarly activates the PI3K/Akt pathway; however, its overexpression in C666-1 cells did not affect cell growth or migration. LMP1 also decreased expression of the junctional protein plakoglobin, which was shown to be partially responsible for enhanced migration induced by LMP1. This study reveals that in epithelial cells the transforming properties of LMP1 require activation of both PI3K/Akt and NF-kappaB and shows that the loss of plakoglobin expression by LMP1 is a significant factor in the enhanced migration.     

Tumor regression by expression of high physiological levels of EBV latent membrane protein 1

Induction of apoptosis in tumor cells is one of therapeutic strategies of cancer. Previous studies indicate that LMP-1 can act as governor of cell proliferation because overexpression of latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) inhibits cell proliferation. Here we demonstrate that overexpression of the NLMP-1, isolated from an EBV strain prominent in Taiwanese population, also possess the ability to induce apoptosis of cells, and inhibit CT-26 tumor growth in mice. Mapping studies indicate that NLMP-1 transmembrane domain is required for induction of cytotoxicity. Intratumoral delivery of vectors expressing NLMP-1 or its membrane domain via electroporation induces tumor tissue damage, suppresses tumor growth in mice, and prolongs the survival of treated animals. In addition, the membrane domain of NLMP-1 alone induces effects similar to those induced by cotreatment with NLMP-1 and IL-12. Tumor-free mice at 120 days after the initial treatments were further challenged with CT-26 tumor cells. No tumor growth was observed. Thus, NLMP-1, and more specifically the transmembrane domain of NLMP-1, may be promising new therapeutic agents for control of tumor growth.     

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.     

鼻咽癌细胞系SUNE中EBV—LMP1基因对永生化人胚肾上皮细胞生长 …

研究鼻咽癌细胞系ＳＵＮＥ中ＥＢ病毒潜伏膜蛋白１基因对上皮细胞生长特性的影响、探索ＬＭＰ１在鼻咽癌发生中所起的作用。方法 用ＬＭＰ１基因真核表达质粒转染人胚肾上皮细胞,检测ＬＭＰ１的表达,观察细胞的生长状态,生长速率,在软琼脂中的集落形成能力及对裸鼠的致瘤能力。     

鼻咽癌细胞系SUNE中EBV-LMP1基因对永生化人胚肾上皮细胞生长特性的影响

目的研究鼻咽癌细胞系ＳＵＮＥ中ＥＢ病毒潜伏膜蛋白１（ＬＭＰ１）基因对上皮细胞生长特性的影响，探索ＬＭＰ１在鼻咽癌发生中所起的作用。方法用ＬＭＰ１基因真核表达质粒转染人胚肾上皮细胞，检测ＬＭＰ１的表达，观察细胞的生长状态、生长速度、在软琼脂中的集落形成能力及对裸鼠的致瘤能力。结果被ＬＭＰ１基因转染的细胞生长旺盛，丧失接触抑制，生长速度增快，在软琼脂中能够形成多个集落，并能在裸鼠体内成瘤。结论ＬＭＰ１基因能明显改变上皮细胞的生物学行为，促进细胞的生长、增殖和转化，使转染的上皮细胞获得肿瘤细胞的生长特征。     

EB病毒LMP1促鼻咽癌细胞生长与CD23、bcl-2表达和凋亡的关系

目的：探讨ＥＢ病毒ＬＭＰＩ表达促鼻咽细胞系生长作用与ＣＤ２３、ｂｃｌ－２表达和细胞凋亡的关系。方法：用免疫组化ＬＳＡＢ法检测ＬＭＰＩ、ＣＤ２３和ｂｃｌ－２蛋白的表达;用ＭＴＴ法测定鼻咽癌细胞系的生长能力;用ＤＮＡＪ电泳法、流式细胞法和ＴＵＮＥＬ法检测癌细胞凋亡;用ＣＤ２３单抗阻断和ＭＴＴ法观察ＣＤ２３单抗对鼻咽产纱生长的影响。结果：表达ＬＭＰＩ的鼻咽癌细胞系（Ｌ－ＣＮＥＩ）的生长能力明显增强,并有     

蛋白激酶CK2β在食管癌组织中的表达及临床意义

目的探讨蛋白激酶CK2β在食管癌组织中的表达及其临床意义。方法选取2010年2月至2013年1月经手术病理证实的原发性食管癌患者58例,采用免疫组织化学法分别检测食管癌及癌旁正常食管黏膜组织芯片中CK2β的表达水平。结果CK2β在食管癌组织细胞中的细胞核和细胞浆均有表达,呈黄色或棕褐色颗粒状分布。其中,58例食管癌组织有3例（5．2％）阴性,2例（3．5％）弱阳性,6例（10．3％）中阳性,47例（81．0％）强阳性;CK2β在食管癌旁正常黏膜组织中多呈阴性表达,仅有3例（5．2％）呈弱阳性。两者阳性表达率差异有统计学意义（P〈0．01）。结论蛋白激酶CK2β在食管癌组织中呈明显高阳性表达,可以作为食管癌早期诊断和治疗的敏感性及特异性指标,具有重要的临床应用及研究意义。     

Survivin在食管鳞癌组织中的表达及其意义

目的：探讨Survivin在食管鳞癌中的表达及其临床意义。方法：采用免疫组织化学检测47例食管鳞癌组织和10例正常食管黏膜的Survivin蛋白的表达，探讨其与食管鳞癌临床病理特征及预后之间的关系。结果：Survivin阳性表达定位于肿瘤细胞胞浆内。47例食管鳞癌组织Survivin阳性表达率为93．6％。在正常食管黏膜无阳性表达。Survivin表达水平与组织学分化程度有相关关系，高分化鳞癌中低表达者居多，而中分化鳞癌中高表达者居多，二者呈负相关（P〈0．05）。总体生存率单因素分析显示Survivin高表达组生存预后明显差于低表达组，有显著性意义（P〈0．05）。Logistic多因素回归分析显示Survivin表达水平为独立预后因素（P〈0．05）。Survivin表达水平与食管癌治疗失败（手术后3年内复发和转移死亡）有明显相关关系，高表达组发生治疗失败的病例明显增多（P〈0．01）。结论：Survivin表达水平与组织学分化程度有相关关系，为食管癌的独立预后因素；Survivin高表达预示食管癌治疗失败的机会增大。     

食管鳞状细胞癌组织中核干细胞因子蛋白和mRNA的半定量表达

目的 探讨食管鳞状细胞癌组织中核干细胞因子(NS)蛋白和mRNA的半定量表达情况.方法 应用免疫组化SABC法和逆转录聚合酶链反应(RT-PCR)法,检测62例食管鳞状细胞癌组织及其相对应的31例癌旁不典型增生组织和62例正常食管黏膜组织中NS蛋白和mRNA的半定量表达情况.结果 正常食管黏膜组织、癌旁不典型增生组织和食管鳞状细胞癌组织中,NS蛋白阳性表达率分别为17.7%(11/62)、41.9%(13/31)和69.4%(43/62),组间比较,差异有统计学意义(χ2=33.676,P＜0.01).食管鳞状细胞癌组织中,NS蛋白阳性表达率与其组织学分级、侵袭程度及淋巴结转移密切相关(均为P＜0.05),而与年龄、性别和病理分型无关(均为P＞0.05).食管鳞状细胞癌组织中,NS mRNA的相对含量(0.971±0.121)高于癌旁不典型增生组织(0.913±0.085)和正常食管黏膜组织(0.866±0.103),组间比较,差异有统计学意义(F=14.829,P＜0.01).不同组织学分级、不同侵袭程度及有无淋巴结转移的食管鳞状细胞癌组织之间,NS mRNA的相对含量差异均有统计学意义(均为P＜0.05),NSmRNA的相对含量与年龄、性别和病理分型无关(均为P＞0.05).结论 食管鳞状细胞癌组织中,NS mRNA的表达升高,其高表达与食管鳞状细胞癌的发生发展有关.     

Induction of receptor for advanced glycation end products by EBV latent membrane protein 1 and its correlation with angiogenesis and cervical lymph node metastasis in nasopharyngeal carcinoma

PURPOSE: The EBV oncoprotein, latent membrane protein 1 (LMP1), contributes to the metastasis of nasopharyngeal carcinoma (NPC) by inducing factors to promote tumor invasion and angiogenesis. The receptor for advanced glycation end products (RAGE) is associated with abnormal angiogenesis in diabetic microangiopathies. Moreover, some papers have suggested the association of RAGE overexpression with tumor metastasis; thus, the associations of RAGE with LMP1 and angiogenesis in NPC were examined. EXPERIMENTAL DESIGN: Forty-two patients with NPC were evaluated for expressions of LMP1, RAGE, and S100 proteins and for microvessel counts by immunohistochemistry. Then, the RAGE induction by LMP1 was examined with Western blotting and luciferase reporter assay. RESULTS: The microvessel counts were significantly higher in patients with high LMP1 expression or high RAGE expression compared with cases with low expressions (P=0.0049 and P<0.0001), respectively. Patients with advanced N classification were also significantly increased in these groups (P=0.0484 and P=0.0005). The expressions of LMP1 and RAGE proteins were clearly correlated in NPC tissues (P=0.0093). Transient transfection with LMP1 expression plasmid induced RAGE protein in Ad-AH cells. The expression of LMP1 transactivated the RAGE promoter as shown by luciferase reporter assay. Mutation of the reporter at nuclear factor-kappaB binding site (-671 to -663) abolished transactivation of the RAGE promoter by LMP1. CONCLUSION: These results suggest that LMP1-induced RAGE enhances lymph node metastasis through the induction of angiogenesis in NPC. Nuclear factor-kappaB binding site (-671 to -663) is essential for transactivation of the RAGE promoter by LMP1.     

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.     

TSLC1在食管鳞癌中的表达及临床意义

目的：研究食管鳞状细胞癌组织中肺癌肿瘤抑制因子1（TSLC1）基因的表达及临床病理意义,探讨其在食管鳞状细胞癌发生、发展过程中的作用。方法：应用免疫组化（SP法）检测50例食管鳞状细胞癌标本及癌旁正常食管组织中TSLC1基因的表达情况,分析其与食管鳞状细胞癌患者临床病理学参数之间的关系。结果：50例食管鳞状细胞癌组织中TSLC1基因表达阳性率为48.0%（24/50）,其表达与癌组织浸润深度、区域淋巴结转移及TNM分期有关（P〈0.05）。与性别、年龄、肿瘤大体分型、肿瘤大小、位置、分化程度无关（P〉0.05）。50例癌旁正常食管组织中TSLC1基因高表达,阳性率为94.0%（47/50）。食管鳞状细胞癌组织中的阳性表达率显著低于癌旁正常食管组织,两者相比差异有统计学意义（P〈0.01）。结论：食管鳞状细胞癌组织中有明显的TSLC1基因的表达缺失,TSLC1蛋白表达缺失与食管鳞状细胞癌的发生、发展有关。     

EHD2蛋白在食管鳞状细胞癌组织中的表达及意义

目的:探讨EHD2蛋白在食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)组织中的表达情况及意义。方法:采用Western blot技术和免疫组织化学法(SP法)检测ESCC和相应癌旁正常组织EHD2蛋白的表达水平,分析其表达与患者临床病理学参数的关系。结果:在8对新鲜食管鳞癌组织中,EHD2蛋白在食管鳞癌组织中的表达明显低于癌旁正常组织(P<0.01);在44例石蜡标本中,EHD2蛋白在食管鳞癌组织的阳性表达率明显低于癌旁正常组织(P<0.05),且EHD2蛋白的表达明显与有无淋巴结转移(P=0.006)和组织分化程度相关(P=0.023),与患者的年龄、性别、肿瘤大小和TNM分期无相关性(P>0.05)。结论:EHD2蛋白在食管鳞癌组织中低表达,提示EHD2蛋白可能是新的食管鳞癌抑癌因子,并有望为食管鳞癌的预后判断和治疗提供新的思路。     

Expression of ras oncogene p21 protein in esophageal squamous cell carcinoma

In order to investigate the value of ras oncogene expression as a prognostic indicator in esophageal squamous cell carcinoma, we evaluated the level of ras oncogene protein product (p21) in 52 specimens resected between 1977 and 1986. All patients were followed until death or for at least 2 years. Pathology slides and archival paraffin blocks were retrieved for confirmation of the original diagnosis, study of histopathologic features, and measurement of p21 content. P21 titers were obtained using the RAP-5 monoclonal antibody in a semiquantitative immunohistochemical assay. Titer was expressed as the highest dilution of antibody giving definitive staining using the avidin-biotin peroxidase method. Ras oncogene was expressed in 88.5% of the specimens. We did not find a significant correlation between ras expression and any of a variety of clinical and histopathologic prognostic parameters. Although patients' median survival after resection of specimens with ras oncogene expression was less than half the median survival after removal of tumors without such expression, this difference was not statistically significant. Further prospective investigations are needed to assess the role of ras oncogene evaluation in clinical practice.     

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.     

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.     

S期激酶相关蛋白2在食管癌中的表达及其意义

目的:探讨S期激酶相关蛋白2(S-phase kinase-interacing protein 2,SKP2)在食管癌组织中的扩增、表达状态及其临床意义.方法:应用荧光原位杂交、RT-PCR、Western印迹和免疫组织化学方法检测SKP2在食管癌组织中的扩增和表达水平,结合临床病理资料进行统计学分析.结果:食管癌组织中SKP2的扩增频率为46.0%,SKP2扩增与肿瘤淋巴结转移和临床分期具有显著相关性(P<0.05).在SKP2扩增的食管癌组织中SKP2 mRNA和蛋白的表达水平均明显升高.免疫组织化学检测结果表明,食管癌组织中SKP2蛋白的过表达与肿瘤淋巴结转移和肿瘤分期有显著相关性(P<0.05),与预后无相关性(P>0.05).结论:SKP2可能具有癌基因的潜能,在食管癌的发生、发展和淋巴结转移中发挥一定作用.     

P16和CyclinE蛋白在食管鳞状细胞癌中的表达及意义

目的探讨P16和cyclinE蛋白在食管鳞状上皮、增生上皮和癌变上皮中表达状况及其与鳞状细胞癌发生、发展和转移的相关性。方法采用SP免疫组织化学方法,检测食管鳞癌72例,粘膜上皮增生21例,正常对照13例和淋巴结转移癌15例中P16和cyclinE基因蛋白的表达。结果P16在正常上皮组无阳性表达,增生组为23.81%,鳞癌组为38.89%;高分化鳞癌组阳性表达率(41.67%)显著高于低分化鳞癌组(27.27%)(P<0.05);有淋巴结转移组(16.67%)和无淋巴结转移组(42.85%)比较具有显著性差异(P<0.05);原发癌组阳性率显著高于淋巴结转移癌组(P<0.05)。cyclinE在正常上皮组无阳性表达,增生上皮组为33.3%,鳞癌组为51.4%;高分化鳞癌组阳性表达率(44.44%)显著低于低分化鳞癌组(81.81%)(P<0.05);淋巴结转移组cyclinE阳性表达率(79.17%)和淋巴结无转移组(48.57%)比较有显著性差异(P<0.05)。P16和cyclinE表达与肿瘤部位和浸润深度相关性不明显。结论在食管癌中P16和cyclinE基因蛋白的表达与病理分化程度有关;检测P16和cyclinE对食管癌早期诊断有指导作用。