SV40 Small-t Antigen and Human Cell Transformation

描述（由申请方提供）：猿猴病毒40是一种DNA肿瘤病毒，与人类间皮瘤和小儿脑肿瘤以及可能的其他人类恶性肿瘤相关。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，与肿瘤抑制基因如Rb和p53相互作用。我们已经表明，ST也是人类细胞的转化所必需的，并且两种SV40蛋白在不存在血清的情况下合作驱动密度停滞的人成纤维细胞的细胞周期进展。这至少部分地通过调节细胞周期蛋白激酶抑制剂而发生，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的结合是p27减少所必需的。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。目的二：探讨抗凋亡激酶AKT对ST功能的影响。在这个目标的实验主要集中在ST激活AKT的机制，但ST的其他潜在目标将进行调查，包括比较微阵列识别的新基因。这一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的组合来满足人细胞转化中对ST的需求。在目标3中，将探索一种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，但是这些细胞在ST引入之前没有表现出转化的特性，例如焦点形成或锚定独立生长。我们将研究ST对已建立的间皮细胞的影响以及在此过程中特定细胞活动的作用。此外，ST表达间皮细胞向更多非整倍体群体的漂移将进行评估，特别关注ST是否积极参与这一过程。这些方法应该提供关键的信息，以确定ST在人类系统中的转化的重要作用，并可能作为其他人类细胞类型的研究，可能是SV40肿瘤发生的目标原型。

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.