MOLECULAR EVENTS IN THE PATHOGENESIS OF KAPOSI'S SARCOMA

KS是一种多克隆恶性肿瘤，高发于 艾滋病毒感染者和一些非艾滋病毒感染者的亚群 受感染的人群。将尝试作为确定感染的辅助因素 KS细胞一直没有结果，流行病学数据表明这种共同的 因素。 我们已经确定KS细胞与正常细胞有显著的不同 内皮细胞对诱导VCAM-1的药物的反应性， 尤其是它们对双链RNA的分子反应性。这 很可能是由于双链RNA的差异激活所致 活化蛋白激酶，PKR。这是一种重要的激酶，在 宿主抗病毒反应，许多病毒已经进化出机制来 颠覆PKR的抗病毒活性。PKR是一种干扰素反应基因 最近发现这是一种肿瘤抑制基因。我们 假设PKR的功能改变，可能是由于病毒 抑制物在KS细胞中的表达，在KS的发病机制中起重要作用。我们 假设临床对干扰素的反应可能源于较高的水平 PKR蛋白克服PKR对肿瘤的部分功能抑制作用 抑制器功能。我们已经证明了微血管内皮细胞 转染有PKR功能的病毒抑制物或 功能失活野生型PKR的突变体PKR发展成KS样蛋白 表型，支持PKR功能改变是 在KS的发展中至关重要。 在这项提案中，我们将充分描述PKR的诱导和激活 KS细胞与正常内皮细胞比较。我们将研究以下方面的变化 在翻译因子eIF的磷酸化中，自动磷酸化- 2α，与正常内皮细胞相比，KS细胞中NF-kappaB的活化。 然后我们将确定KS细胞中的大分子成分 对职能上的差异负责。 我们还将识别在KS细胞中差异表达的基因 与正常和激活的微血管内皮细胞相比。Cdna 将从培养的KS细胞和培养的皮肤创建文库 微血管内皮细胞在相同条件下生长。 将对克隆的基因进行差异筛选，以确定 与正常内皮细胞相比，KS细胞中特异表达的基因 并与细胞因子激活的内皮细胞进行比较。功能性 这些基因的分析将通过表达选定的序列在 培养的微血管内皮细胞是否产生KS 表型。将进行原位杂交和免疫组织化学 定位这些基因在KS中的表达，而不是非受累组织。 这些基因与PKR活性改变的关系也将是 探索过了。

KS is a polyclonal malignancy that occurs at high frequency in subpopulations of HIV-infected individuals as well as in some non-HIV infected populations. Attempts as identifying an infectious co-factor in KS cells have been unfruitful, epidemiologic data suggesting such a co- factor. We have determined that KS cells differ dramatically from normal endothelial cells in their responsiveness to agents that induce VCAM-1, especially in their molecular responsiveness to double stranded RNA. This is most likely due to differential activation of the double stranded RNA activated protein kinase, PKR. This is a kinase that is important in the host anti-viral response, and many viruses have evolved mechanisms to subvert the antiviral activities of PKR. PKR is an IFN-responsive gene that has recently been shown to be a tumor suppressor gene. We hypothesize that functional alterations of PKR, possibly due to a viral inhibitor in the KS cells, are important in the pathogenesis of KS. We hypothesize that clinical responses to IFN might result from higher levels of PKR protein overcoming some of the functional inhibition of PKRs tumor suppressor function. We have demonstrated that microvascular endothelial cells transfected with either a viral inhibitor of PKR function or a mutant PKR that functionally inactivates wild type PKR develop a KS-like phenotype, supporting the hypothesis that alteration of PKR function is critical in the development of KS. In this proposal, we will fully characterize PKR induction and activation in KS cells compared to normal ECs. We will examine changes in autophosphorylation, in phosphorylation of the translation factor eIF- 2alpha, and in activation of NF-kappaB in KS cells compared to normal ECs. We will then identify the macromolecular components in KS cells that are responsible for the functional differences. We will also identify genes that are differentially expressed in KS cells compared to normal and activated microvascular endothelial cells. cDNA libraries will be created from cultured KS cells and from cultured skin microvascular endothelial cells grown under identical conditions. Differential screening of cloned genes will be undertaken to identify genes expressed exclusively in the KS cells compared to normal endothelial cells and compared to cytokine activated endothelial cells. Functional assays of these genes will be done by expressing selected sequences in cultured microvascular endothelial cells to see if they generate a KS phenotype. In situ hybridization and immunohistochemistry will be done to localize the expression of these genes in KS versus uninvolved tissue. The relationship of these genes to altered PKR activity will also be explored.