TRANSFORMATION BY EBV LATENT MEMBRANE PROTEINS 1 AND 2

EBV 潜伏膜蛋白 1 和 2 的转化

• 批准号: 6930190
• 负责人: NANCY JOAN RAAB-TRAUB
• 金额: $ 22.51万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6930190
• 关键词: B cell lymphoma; Epstein Barr virus; Herpesviridae disease; carcinogens; cell line; fibroblasts; gene expression; genetically modified animals; host organism interaction; immunoglobulin genes; laboratory mouse; latent virus infection; microarray technology; molecular oncology; neoplasm /cancer genetics; neoplastic transformation; nuclear factor kappa beta; oncogenic virus; viral carcinogenesis; virus cytopathogenic effect; virus protein

The viral proteins, latent membrane protein 1 (LMP1) and latent membrane protein 2 (LMP2), are expressed in many of the malignancies associated with EBV. We have previously produced and characterized transgenic mice that express LMP1 in B-lymphocytes using the immunoglobulin heavy chain promoter/enhancer (Ig-LMPl). These mice develop clonal B-cell lymphomas that express LMP1 at high levels. The clonal development of these cancers indicates that additional genetic changes must occur that complement the cellular pathways activated by LMP1. During this last period of funding, we have identified remarkable oncogenic synergy between the loss of the p16INK4/p19ARF locus and expression of LMP1 in B-lymphocytes. To evaluate the effects of LMP1 on epithelial cell growth, we have produced transgenic mice that express LMP1 under the control of the keratin 14 (K14) promoter. In these mice using classical tumor initiation and promotion analyses, LMP1 functions largely as a tumor promoter with a possible role in tumor progression. Transgenic mice that express LMP2 in B-lymphocytes using the Ig promoter and in epithelial cells from the K14 promoter have been obtained from Dr. Richard Longnecker. The K14-LMP2 transgenic mice will be tested to determine if LMP2 affects the oncogenic process through initiation, promotion, or progression in skin tests. Possible synergistic effects of LMP1 and LMP2 expression on B-cell and epithelial cell growth will be analyzed in dually transgenic mice and in transformation assays in vitro. Our specific aims are: 1) The transgenic LMP1+ lymphocytes, LMP1+ lymphomas, LMP1+ p16 null lymphomas, and LMP1+ p53 heterozygous lymphomas will be further characterized to identify activated signaling pathways and effects on cellular gene expression. The growth properties of the LMP1+ lymphocytes and lymphoma cells will be analyzed in vitro. 2) LMP1 and LMP2 affect and activate distinct cellular signaling pathways. To test the hypothesis that coordinate expression of LMP1 and LMP2 activates complementing pathways that synergistically alter growth regulation, Ig-LMP1/LMP2 transgenic mice will be produced by cross-breeding. The time to tumor development and the growth and biochemical properties of the cells in vitro will be determined. 3) To determine the effects of expression of LMP1 and LMP2 in normal epithelial cells, K14 promoter transgenic mice that express LMP1 and/or LMP2 in epithelial cells will be tested in classic skin carcinogenesis assays in combination with exposure to carcinogens and tumor promoters. 4) Characterize LMP1 transformation of rodent fibroblasts. The essential domains of LMP1 will be identified and the signaling pathways that are activated will be determined. The effects on rodent fibroblast growth properties of LMP1 alone and in the absence of the p16 and p19 tumor suppressor genes will be determined.

潜伏膜蛋白1(LMP1)和潜伏膜蛋白2(LMP2)在许多与EBV相关的恶性肿瘤中都有表达。我们之前已经利用免疫球蛋白重链启动子/增强子(Ig-LMP1)培育并鉴定了在B淋巴细胞中表达LMP1的转基因小鼠。这些小鼠患上克隆性B细胞淋巴瘤 高水平表达LMP1的细胞。这些癌症的克隆性发展表明，必须发生额外的基因变化，以补充LMP1激活的细胞通路。在这最后的资助期间，我们发现p16INK4/p19ARF基因缺失和B淋巴细胞中LMP1的表达之间存在显著的致癌协同作用。为了评估LMP1对上皮细胞生长的影响，我们培育了在角蛋白14(K14)启动子控制下表达LMP1的转基因小鼠。在这些使用经典肿瘤启动和促进分析的小鼠中，LMP1在很大程度上作为肿瘤促进剂发挥作用，可能在肿瘤进展中发挥作用。理查德·朗纳克博士已经获得了在使用Ig启动子的B淋巴细胞和使用K14启动子的上皮细胞中表达LMP2的转基因小鼠。将对K14-LMP2转基因小鼠进行测试，以确定LMP2是否通过皮肤测试的启动、促进或进展影响肿瘤形成过程。LMP1和LMP2表达对B细胞和上皮细胞生长的可能的协同作用将在双转基因小鼠和体外转化试验中进行分析。我们的具体目标是：1)转基因LMP1+淋巴细胞、LMP1+淋巴瘤、LMP1+p16缺失淋巴瘤和 LMP1+P53杂合子淋巴瘤将进一步确定激活的信号通路和对细胞基因表达的影响。LMP1+淋巴细胞和淋巴瘤细胞的生长特性将在体外进行分析。2)LMP1和LMP2影响和激活不同的细胞信号通路。为了验证LMP1和LMP2协同表达激活互补途径从而协同改变生长调节的假设，将通过杂交产生Ig-LMP1/LMP2转基因小鼠。肿瘤发展的时间和细胞的生长和生化特性将在体外确定。3)为了确定LMP1和LMP2在正常皮肤上皮细胞中的表达情况，对在正常皮肤上皮细胞中表达LMP1和/或LMP2的K14启动子转基因小鼠进行了经典的皮肤致癌实验，并联合使用致癌物和促癌剂。4)研究啮齿动物成纤维细胞对LMP1的转化。LMP1的基本结构域将被确定，激活的信号通路将被确定。LMP1单独和在缺乏p16和p19抑癌基因的情况下对啮齿动物成纤维细胞生长特性的影响将被确定。