Epstein-Barr Virus: Activation of WNT

描述（由申请方提供）：本提案的目的是表征爱泼斯坦巴尔病毒（EBV）对Writ/Wingless信号通路的影响，Writ/Wingless信号通路是发育期间细胞生长和分化的关键调节因子，在癌症发展期间经常受到影响。在本申请中，我们将确定该途径是否在感染细胞系和EBV相关癌症中被激活，鉴定影响该途径调节的病毒蛋白，确定该激活的机制，并鉴定受该激活影响的细胞靶点。具体而言，我们将：1）使用细胞分级分离和免疫印迹、对β-连环蛋白应答元件的电泳迁移率变动测定（EMSA）、报告基因测定和免疫组织化学来表征EBV阳性和阴性细胞系以及EBV相关和AIDS相关癌症中的β-连环蛋白水平、定位和活性，以鉴定途径的特异性磷酸化蛋白组分并确定β-连环蛋白的细胞内定位。2)使用蛋白质和基因阵列分析确定LMP 1对writ/wingless组分的表达和定位的影响。3)确定LMP 2对Akt激活、糖原合成酶激酶β失活、B-连环蛋白定位和wnt调节的基因表达的影响。比较和对比LMP 2与LMP 2的突变形式以及PI 3-激酶和Akt的组成性激活形式。使用成纤维细胞和上皮细胞转化试验确定该途径的激活对LMP 2介导的细胞生长效应的贡献，并对表达LMP 2、LMP 2突变形式或wnt信号传导的活化组分的细胞系进行基因组表达阵列分析，以确定对基因表达的影响。4)分析表达EBV潜伏蛋白的细胞系中LEF/TCF调节的基因表达。识别转录复合物的组成部分，并确定EBV蛋白如何影响该复合物。拟议的研究代表了对可能影响EBV相关癌症的全新途径的首次研究。这些研究将确定该途径在EB病毒相关癌症发展中的贡献，并确定EB病毒蛋白对wnt/wingless信号传导调节的具体影响。详细了解这种途径的激活可能为治疗EBV相关癌症以及在免疫抑制中发展的EBV阴性的类似癌症提供新的机会。

DESCRIPTION (provided by applicant): The goal of this proposal is to characterize the effects of Epstein Barr Virus (EBV) on the Writ/Wingless signaling pathway which is a critical regulator of cell growth and differentiation during development and is frequently affected during cancer development. In this application, we will determine if this pathway is activated infected cell lines and in EBV-associated cancers, identify the viral proteins that affect regulation of this pathway, determine the mechanisms that underlie this activation, and identify the cellular targets that are affected by this activation. Specifically we will: 1) Characterize beta-catenin levels, localization, and activity in EBV positive and negative cell lines and in EBV-associated and AIDS-related cancers using cell fractionation and immunoblotting, electrophoretic mobility shift assays (EMSA) on beta-catenin responsive elements, reporter assays, and immunohistochemistry to identify specific phosphorylated protein components of the pathway and determine intracellular localization of beta-catenin. 2) Identify the effects of LMP 1 on expression and localization of components of writ/wingless using protein and gene array analyses. 3) Determine the effects of LMP2 on Akt activation, inactivation of glycogen synthase kinase beta, B-catenin localization, and wnt-regulated gene expression. Compare and contrast LMP2 with mutated forms of LMP2 and with constitutively activated forms of PI3-kinase and Akt. Determine the contribution of activation of this pathway on LMP2-mediated effects on cellular growth using fibroblast and epithelial cell transformation assays and perform genomic expression array analysis on cell lines expressing LMP2, mutant forms of LMP2, or activated components of wnt signaling to determine effects on gene expression. 4) Analyze LEF/TCF regulated gene expression in cell lines expressing EBV latent proteins. Identify components of the transcription complex and determine how EBV proteins affect this complex. The proposed studies represent the first studies on a completely new pathway likely affected in EBV-associated cancers. These studies will determine the contribution of this pathway in the development of EBV-associated cancers and identify the specific effects of EBV proteins on the regulation of wnt/wingless signaling. A detailed understanding of the activation of this pathway may provide new opportunities for treatment of EBV-associated cancers and also of similar cancers considered EBV-negative, that develop in the immunosuppressed.