ALTERNATE SIGNALING PATHWAYS FOR INTERFERON ALPHA

干扰素诱导多种基因家族的转录， 蛋白质产物被认为介导细胞生长的抑制 和病毒复制，以及调节细胞分化和免疫 功能协调发展的 一个高度保守的顺式作用干扰素刺激反应元件 （ISRE），已在许多干扰素的5 '侧翼区中鉴定 受激基因（ISG）启动子。 IFN-α信号细胞通过 酪氨酸激酶介导的转录因子磷酸化 复合物（ISGF3）结合到ISRE。 然而，生物化学和遗传学 有证据表明存在多种细胞内信号 IFN-α的通路。 我们的主要目标是了解 IFN-α信号转导的机制 途径。 这些信息应证明有助于设计新的 使用IFN和其他细胞因子的治疗方法。 我们提供了 交替信号转导存在的直接证据 使用生物化学、病毒抑制剂和突变体的IFN-α途径 细胞系接近。 我们建议定义的基因集利用 在生物化学和分子生物学上， 这些途径的不同组成部分。 为了实现我们的目标，我们提出以下具体目标： 1)识别和表征IFN-α的替代途径 我们将使用在ISGF3中具有确定突变的细胞系 依赖信号 我们将描述IFN-α的反应， 腺病毒5 E1A抑制ISGF3依赖性信号传导的细胞 生产 我们将在U2A中研究IRF-1基因的IFN诱导 在ISGF3依赖性信号传导中有缺陷并使用PCR的细胞 鉴定和克隆U2A中诱导的其他mRNA的方法，293 和表达E1A的HeLa细胞对IFN-α的应答。 分离和 表征对IFN-α无应答的突变细胞系，我们 将通过选择IFN介导的突变细胞系来产生新的突变细胞系。 从ISGF3 - 1转录的药物选择性标记的表达 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， 可能参与ISGF3依赖和非依赖信号传导， 将鉴定可能与IFN-α受体相互作用的成分 使用亲和层析和提取物的免疫共沉淀 由正常和突变细胞系制备。 定义作用模式 的ISRE和IR元件结合蛋白，IBF-1，我们将使用 转染测定、无细胞转录反应和分离 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.