ALTERNATE SIGNALING PATHWAYS FOR INTERFERON ALPHA

干扰素诱导不同基因家族的转录，这些基因 蛋白质产物被认为介导了对细胞生长的抑制 和病毒复制以及细胞分化和免疫的调节 功能。一种保守的顺式干扰素刺激反应元件 (ISRE)，已在许多干扰素的5‘侧翼区被发现 刺激基因(ISG)启动子。干扰素-α通过以下途径向细胞发出信号 酪氨酸激酶介导的转录因子的磷酸化 绑定到ISRE的复合体(ISGF3)。然而，生化和遗传 有证据表明存在多种细胞内信号 干扰素-α的途径。我们的主要目标是了解基本的 干扰素-α信号转导机制的研究进展 小路。这些信息应该被证明对设计新的 使用干扰素和其他细胞因子的治疗方法。我们已经提供了 替代信号转导存在的直接证据 使用生化、病毒抑制剂和突变体的干扰素-α途径 细胞系接近了。我们建议定义一组基因，利用 生物化学和分子生物学中的交替途径及其特征 水平，这些途径的不同组成部分。 为了实现我们的目标，我们提出了以下具体目标： 1)确定和表征干扰素-α的替代途径 转导我们将使用ISGF3有明确突变的细胞系 依赖于信号。我们将描述干扰素-α反应的特征 依赖ISGF3的信号转导受腺病毒5E1a抑制的细胞 制作。我们将研究干扰素对U2a中IRF-1基因的诱导作用 在依赖ISGF3的信号转导中有缺陷并使用聚合酶链式反应的细胞 鉴定和克隆U2A、293诱导的其他mRNAs的方法 和表达E1a的HeLa细胞对干扰素-α的反应。要隔离和 鉴定对干扰素-α无反应的突变细胞系，我们 将通过选择干扰素介导的方式产生新的突变细胞系 转录自ISGF3-的药物选择标志物的表达 独立的IRF-1启动子。 2)在生物化学水平上定义独特成分的作用 可能同时参与ISGF3依赖和非依赖的信号转导 将确定可能与干扰素-α受体相互作用的成分 利用亲和层析和免疫共沉淀法提取提取物 从正常和突变细胞系制备。定义行动模式的步骤 对于ISRE和IR元件结合蛋白IBF-1，我们将使用 转染法、无细胞转录反应及分离 基因抑制因子。

Interferons induce transcription of a diverse family of genes whose protein products are believed to mediate the inhibition of cell growth and viral replication, and regulation of cell differentiation and immune functions. A well conserved cis-acting IFN stimulated response element (ISRE), has been identified in the 5' flanking region of many interferon stimulated gene (ISG) promoters. IFN-alpha signals cells through tyrosine kinase mediated phosphorylation of a transcription factor complex (ISGF3) that binds to the ISRE. However, biochemical and genetic evidence suggests the existence of multiple intracellular signaling pathways for IFN-alpha. Our major goal is to understand the basic mechanisms underlying IFN-alpha signal transduction through alternate pathways. Such information should prove useful in designing new therapeutic approaches using IFNs and other cytokines. We have provided direct evidence for the existence of alternate signal transduction pathways for IFN-alpha using biochemical, viral inhibitor, and mutant cell line approaches. We propose to define the sets of genes utilizing the alternate pathways and characterizing at a biochemical and molecular level, the different components of these pathways. To achieve our goal, we propose the following specific aims: 1) To identify and characterize alternate pathways for IFN-alpha transduction we will use cell lines with defined mutations in ISGF3 dependent signaling. We will characterize the IFN-alpha response in cells where ISGF3-dependent signaling is inhibited by adenovirus 5 E1A production. We will study the IFN-induction of the IRF-1 gene in U2A cells which are defective in ISGF3-dependent signaling and use a PCR approach to identify and clone other mRNAs which are induced in U2A, 293 and E1A-expressing HeLa cells in response to IFN-alpha. To isolate and characterize mutant cell lines which are unresponsive to IFN-alpha, we will generate new mutant cell lines by selecting for IFN mediated expression of drug selectable markers transcribed from the ISGF3- independent IRF-1 promoter. 2) To define at a biochemical level the action of unique components which may be involved in both ISGF3 -dependent and -independent signaling we will identify components that may interact with the IFN-alpha receptor using affinity chromatography and co-immunoprecipitation of extracts prepared from normal and mutant cell lines. To define the mode of action of the ISRE and IR element binding protein, IBF-1, we will use transfection assays, cell-free transcription reactions and the isolation of genetic suppressor elements.