SV40 Small-t Antigen and Human Cell Transformation

描述（由申请人提供）：猿猴病毒40是一种DNA肿瘤病毒，与人类间皮瘤和儿童脑肿瘤以及可能的其他人类恶性肿瘤有关。转化人类细胞需要两种SV40蛋白，即大t （LT）和小t （ST）抗原。LT是一种被充分研究的转化蛋白，它与肿瘤抑制基因如Rb和p53相互作用。我们已经证明ST对于人类细胞的转化也是必不可少的，并且两种SV40蛋白在缺乏血清的情况下合作驱动密度阻滞的人类成纤维细胞的细胞周期进程。这至少在一定程度上是通过调节细胞周期蛋白激酶抑制剂发生的，其中LT表达降低cki p21，而ST表达降低p27水平。ST与蛋白磷酸酶2A的结合对于p27的减少是必要的。我们提出的研究的第一个目的是确定p27减少的机制，以及p27减少是否足以解释ST对生长促进和转化的影响。在目的二中，我们将探讨抗凋亡激酶AKT对ST功能的意义。这方面的实验主要集中在ST激活AKT的机制上，但ST的其他潜在靶点将被研究，包括通过比较微阵列鉴定的新基因。该目标的一个重要部分是确定是否可以通过上调或下调细胞活性组合来满足人类细胞转化对ST的要求。在目标3中，将探索一个新开发的人间皮细胞转化模型。我们已经使用端粒酶和LT在培养中建立了间皮细胞，但在引入ST之前，这些细胞没有表现出诸如焦点形成或锚定独立生长等转化特性。我们将研究ST对建立的间皮细胞的影响以及特定细胞活动在这一过程中的作用。此外，表达ST的间皮细胞向更多非整倍体群体的漂移将被评估，特别是ST是否积极参与这一过程。这些方法应该为定义ST在人类系统转化中的重要作用提供关键信息，并可能作为研究其他可能是SV40肿瘤发生靶点的人类细胞类型的原型。

DESCRIPTION (provided by applicant): Simian virus 40 is a DNA tumor virus, which is associated with human mesotheliomas and pediatric brain tumors and possibly other human malignancies. Two SV40 proteins are required to transform human cells, the large-T (LT) and small-t (ST) antigens. LT is a well-studied transforming protein that interacts with tumor suppressor genes like Rb and p53. We have shown that ST is also essential for transformation of human cells and that both SV40 proteins cooperate in the absence of serum to drive cell cycle progression of density-arrested human fibroblasts. This occurs, at least in part, through regulation of cyclin-kinase inhibitors, with LT expression reducing the cki p21 while ST expression reduces p27 levels. Binding of ST to protein phosphatase 2A is necessary for this p27 reduction. The first aim of our proposed research is to determine the mechanism of p27 reduction and whether p27 reduction is sufficient to explain effects of ST on growth promotion and transformation. In aim two, we will explore the significance of the anti-apoptotic kinase, AKT, to ST function. Experiments in this aim focus primarily on mechanisms by which ST activates AKT, but other potential targets of ST will be investigated including new genes identified by comparative microarrays. An important part of this aim is to determine whether the requirement for ST in human cell transformation can be met by up- or down-regulation of a combination of cellular activities. In aim 3, will explore a newly developed model for human mesothelial cell transformation. We have established mesothelial cells in culture using telomerase and LT, but these do not exhibit transformed properties such as focus formation or anchorage independent growth until ST is introduced. We will investigate the effects of ST on established mesothelial cells and the role of specific cellular activities in this process. In addition, the drift of ST-expressing mesothelial cells toward more aneuploid populations will be evaluated with particular interest on whether ST contributes actively to this process. These approaches should provide crucial information to define the essential role of ST in transformation in human systems, and may serve as a prototype for studies of other human cell types that may be targets of SV40 tumorigenesis.