SV40 Small-t Antigen and Human Cell Transformation

描述(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瘤有关，可能还与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。它是一种经过充分研究的转化蛋白，与Rb和P53等肿瘤抑制基因相互作用。我们已经证明，ST对人类细胞的转化也是必不可少的，并且这两种SV40蛋白在没有血清的情况下协同作用，推动密度受阻的人成纤维细胞的细胞周期进展。这至少部分是通过调节细胞周期蛋白激酶抑制物来实现的，其中LT的表达降低了CKI p21，而ST的表达降低了p27的水平。ST与蛋白磷酸酶2A的结合是这种p27还原所必需的。我们提出的研究的第一个目的是确定p27降低的机制，以及p27降低是否足以解释ST对生长促进和转化的影响。在第二个目标中，我们将探讨抗凋亡激酶AKT在ST功能中的意义。这一目标的实验主要集中在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确定的新基因。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一系列细胞活动来满足人类细胞转化中对ST的要求。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建立了培养的间皮细胞，但在引入ST之前，这些细胞不会表现出转化的特性，如病灶形成或锚定无关生长。我们将研究ST对已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，ST表达的间皮细胞向更多非整倍体群体的漂移将被特别感兴趣地评估ST是否在这一过程中起积极作用。这些方法应该提供关键的信息，以确定ST在人类系统转化中的重要作用，并可能作为研究其他人类细胞类型的原型，这些细胞类型可能是SV40肿瘤发生的靶点。

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.