Epstein-Barr Virus: Activation of WNT

描述(申请人提供)：这项建议的目标是表征EB病毒(EBV)对WRIT/Wingless信号通路的影响，该信号通路是细胞发育过程中生长和分化的关键调节因子，经常在癌症发展过程中受到影响。在这项应用中，我们将确定这一途径是否被激活，在感染细胞系和EBV相关癌症中，识别影响这一途径调控的病毒蛋白，确定这种激活的机制，并确定受这种激活影响的细胞靶点。具体地说，我们将：1)通过细胞分级和免疫印迹、对β-连环蛋白反应元件的电泳迁移率改变分析(EMSA)、报告分析和免疫组织化学来鉴定该通路的特定磷酸化蛋白成分，并确定该通路的特定磷酸化蛋白成分，从而表征EBV阳性和阴性细胞系以及EBV相关和艾滋病相关癌症中的β-连环蛋白水平、定位和活性。2)利用蛋白质和基因芯片分析鉴定LMP-1对WRIT/WING基因表达和定位的影响。3)研究LMP2对Akt激活、糖原合成酶激酶β失活、B-连环蛋白定位以及WNT调控基因表达的影响。比较和对比LMP2与LMP2的突变形式以及PI3-激酶和Akt的结构性激活形式。通过成纤维细胞和上皮细胞转化实验，确定该通路的激活在LMP2介导的细胞生长效应中的作用，并对表达LMP2、LMP2的突变形式或WNT信号的激活成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LeF/TCF在表达EBV潜伏蛋白的细胞系中的调控基因表达。确定转录复合体的成分，并确定EBV蛋白如何影响该复合体。这项拟议的研究是对EBV相关癌症中可能受到影响的全新途径的首次研究。这些研究将确定这一途径在EBV相关癌症的发展中的作用，并确定EBV蛋白在Wnt/Wingless信号调节中的特定作用。对这一途径激活的详细了解可能会为治疗EBV相关癌症和被认为是EBV阴性的类似癌症提供新的机会，这些癌症发生在免疫抑制的人群中。

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.