MOLECULAR EVENTS IN THE PATHOGENESIS OF KAPOSIS SARCOMA

KS是一种多克隆恶性肿瘤，发生率高， 艾滋病毒感染者的亚群以及一些非艾滋病毒感染者 感染人群。 试图鉴定一种感染性辅因子， KS细胞一直没有结果，流行病学数据表明这种共同作用， 因子 我们已经确定KS细胞与正常的KS细胞有很大的不同， 内皮细胞对诱导VCAM-1的试剂的反应性， 特别是在它们对双链RNA的分子响应性方面。 这 很可能是由于双链RNA的差异激活 活化蛋白激酶。 这是一种激酶， 宿主的抗病毒反应，许多病毒已经进化出机制， 破坏PKR的抗病毒活性。 PKR是一个IFN应答基因 最近被证明是一种肿瘤抑制基因。 我们 假设PKR功能改变，可能是由于病毒 KS细胞中的抑制剂，在KS的发病机制中是重要的。 我们 假设对IFN的临床反应可能是由于较高的水平 PKR蛋白克服PKR肿瘤的一些功能抑制 抑制功能 我们已经证明微血管内皮细胞 用PKR功能的病毒抑制剂或 使野生型PKR功能失活的突变型PKR发展出KS样 表型，支持PKR功能改变是 这对KS的发展至关重要。 在这个建议中，我们将充分表征PKR的诱导和激活 KS细胞与正常EC相比。 我们将研究 自磷酸化，在翻译因子eIF的磷酸化中- 2 α，并与正常EC相比，在KS细胞中激活NF-κ B。 然后，我们将确定KS细胞中的大分子成分， 对功能差异负责。 我们还将鉴定KS细胞中差异表达的基因 与正常和活化的微血管内皮细胞相比。 cDNA 将从培养的KS细胞和培养的皮肤中创建文库 微血管内皮细胞在相同条件下生长。 将对克隆基因进行差异筛选， 与正常内皮细胞相比， 细胞并与细胞因子活化的内皮细胞进行比较。 功能 这些基因的测定将通过在大肠杆菌中表达选定的序列来进行。 培养微血管内皮细胞，观察它们是否产生KS， 表型 将进行原位杂交和免疫组织化学， 将这些基因的表达定位在KS与未受累组织中。 这些基因与PKR活性改变的关系也将被研究。 探讨了

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.