INTERFERON ACTION ON CELL STRUCTURE AND PROLIFERATION

干扰素(IFN)是一种细胞因子，已记录在案的疗效 治疗毛细胞白血病，并显示出令人振奋的结果 在治疗肾细胞癌中，慢性粒细胞 白血病、肝炎和获得性免疫缺陷综合征。这个 提出的研究计划的目标是定义分子 干扰素抗增殖作用的基础，重点是 干扰素α受体的特性及干扰素的拮抗作用 α-生长因子在信号转导水平上的作用 和干扰素α诱导的生长因子下调 受体表达。关于建立科学发展观基础的研究 干扰素的抗增殖作用关键是通过比较 干扰素对金黄色葡萄球菌敏感和耐药的影响分析 肾癌细胞株均表达干扰素受体。 这样的细胞系允许进行关键实验来识别特定的 干扰素α对细胞的抑制作用及其机制 扩散。使用这样的细胞系，我们将定义 干扰素α诱导的受体二聚化、高亲和力和低亲和力 结合组分和干扰素α的糖基化部分 干扰素诱导的干扰素刺激基因表达的受体 以及抗增殖和抗病毒活性的诱导。 根据我们最近的发现，干扰素α刺激GTP结合 抑制细胞内花生四烯酸(AA)的释放 敏感但不在耐药细胞中，我们假设这些可能 作为抗增殖剂的早期措施，或许是必须采取的措施 干扰素α的作用。GTP结合蛋白在干扰素中的作用 将调查激活ISG的行动和 干扰素的抗增殖作用。我们认为干扰素可以阻断信号 相关生长因子(EGF， PDGF、胰岛素等)。我们将直接检测干扰素的作用 对花生四烯酸(AA)释放、蛋白激酶C活性和 添加前后膜GTP酶活性的变化 增长因素。干扰素对细胞功能的一些重要作用 只有在几个小时后才会显现出来。基于我们的 干扰素下调生长因子受体表达的研究 阿尔法治疗减弱了细胞对这种情况的反应能力 因素，我们将确定干扰素是否调节生长因子 受体在转录和转录后的表达 转录水平。我们将定义转录后基因 测定干扰素的磷酸化和循环对干扰素的影响 胰岛素、转铁蛋白和EGF受体的表达，以及 这些受体的mRNA。我们计划定义分子基础 通过确定是否存在 5‘区上游的特异性干扰素反应元件 EGF受体基因的起始点。这将使用以下工具完成 克隆了该基因的片段并进行了瞬时表达分析。我们 也将决定是否存在转录因子 绑定这样的元素。

Interferons (IFNs) are cytokines that have documented efficacy in the treatment of hairy cell leukemia, and show promising results in the treatment of renal cell carcinoma, chronic myelocytic leukemia, hepatitis and acquired immune deficiency syndrome. The goal of the proposed research program is to define the molecular basis of the antiproliferative action of IFNs, focussing on characterization of the IFN alpha receptor; the antagonism of IFN alpha with growth factors at the level of signal transduction pathways; and IFN alpha-induced down-regulation of growth factor receptor expression. Studies of the basis for the antiproliferative action of IFN are critically aided by comparative analysis of the effects of IFN on sensitive and resistant Daudi and renal carcinoma cell lines all of which express IFN receptors. Such cell lines permit critical experiments to identify specific mechanisms underlying the inhibitory action of IFN alpha on cell proliferation. Using such cell lines, we will define the roles of IFN alpha-induced receptor dimerization, the high and low affinity binding components, and the glycosylated portion of the IFN alpha receptor in IFN-induced expression of IFN-stimulated genes (ISGs) and the induction of antiproliferative and antiviral activities. Based on our recent findings that IFN alpha stimulates GTP binding to membranes and depresses arachidonic acid (AA) release in IFN- sensitive but not in resistant cells, we hypothesize that these may be early and perhaps obligatory steps in the antiproliferative action of IFN alpha. The role of GTP-binding proteins in IFN action will be investigated in the activation of ISGs and the antiproliferative action of IFN. We propose that IFN blocks signal transduction pathways activated by relevant growth factors (EGF, PDGF, insulin, etc.). We will directly examine the effects of IFN on arachidonic acid (AA) release, protein kinase C activity, and membrane GTPase activity in the presence and absence of added growth factors. Some important effects of IFN on cell function become manifest only after a period of several hours. Based on our findings that down-regulation of growth factor receptors by IFN alpha treatment attenuates the ability of cells to respond to such factors, we will determine whether IFN modulates growth factor receptor expression at both transcriptional and post- transcriptional levels. We will define post-transcriptional effects of IFN alpha by measuring the phosphorylation and recycling of insulin, transferrin and EGF receptors, and the stability of the mRNA for these receptors. We plan to define the molecular basis of transcriptional regulation by determining whether there are specific IFN-responsive elements in the 5' region upstream from the start site of the EGF receptor gene. This will be done using cloned segments of this gene and transient expression assays. We will also determine whether there are transcription factors that bind such elements.