ANCHORAGE INDEPENDENT GROWTH--ROLE OF TGF-BETA

几乎所有类型的细胞的增殖都需要细胞锚定 而失去这一要求(导致锚地独立)是 这是细胞转化的标志。在分离出的增长因素中 从正常组织中，转化生长因子-β在诱导 锚地--独立增长。然而，这一转变效应 转化生长因子-β仅限于少数成纤维细胞系(如NRK细胞 在对转化生长因子-β的反应中，变得不依赖锚定，而NIH- 3T3和正常人成纤维细胞不表达)。或许正是因为这个原因， 转化生长因子-β生物学与支抗独立性的关系 一直没有得到解决。 我们的数据现在表明，锚定非依赖生长的诱导 代表了转化生长因子-β病理学的一个重要方面。具体来说， 我们发现：(1)转化生长因子-β不能诱导非锚定生长 在保持正常黏附要求的细胞中表达 细胞周期蛋白D1和(Ii)失去这种控制--尽管不转化为 本身--使细胞容易受到转化生长因子-β的影响。因此， 失去黏附依赖的细胞周期蛋白D1的表达是 转化生长因子-β转化。我们现在提出四个具体目标： 确定转化生长因子-β诱导的分子效应(S)并测定 转化生长因子-β作用如何补充结构性细胞周期蛋白D1的作用 诱导锚定独立性的表达。在目标1中，我们将 检测细胞周期从G0期到S期的进程以确定 黏附依赖的G1-CDK事件的子集，由 锚定非依赖性生长诱导过程中的转化生长因子-β：平行 研究将确定转化生长因子-β是否具有相同的亚细胞效应(S) 不能独立于锚定生长的对照细胞。在……里面 目的2、异位表达转化生长因子-β介导的细胞周期事件 确定哪些影响是转化生长因子-β介导的原因 安克雷奇--独立。最后，在目标3和4中，我们将 描述E2F独立的机制，该机制调节 细胞周期蛋白A的黏附依赖性表达及其作用机制的研究 转化生长因子β对E2F依赖和非E2F依赖的细胞周期蛋白A基因的影响 在非锚定生长诱导过程中的表达。

Cell anchorage is required for the proliferation of almost all cell types and loss of this requirement (induction of anchorage-independence) is a hallmark of cell transformation. Among the growth factors isolated from normal tissue, TGF-beta is outstanding in its ability to induce anchorage-independent growth. However, this transforming effect of TGF-beta is restricted to a few fibroblastic cell lines (e.g. NRK cells become anchorage-independent in response to TGF-beta whereas NIH- 3T3 and normal human fibroblasts do not). Perhaps for this reason, the relationship between TGF-beta biology and anchorage-independence has never been resolved. Our data now indicate that induction of anchorage-independent growth represents an important aspect of TGF-beta pathology. Specifically, we find that (I) TGF-beta fails to induce anchorage-independent growth in cells that retain their normal adhesion requirement for expression of cyclin D1 and (ii) loss of this control--although non-transforming in itself-- renders cells susceptible to transformation by TGF-beta. Thus, loss of adhesion-dependent cyclin D1 expression is a prerequisite for transformation by TGF-beta. We now propose four specific aims to identify the molecular effect(s) induced by TGF-beta and determine how TGF-beta action complements the effect of constitutive cyclin D1 expression to induce anchorage-independence. In aim 1, we will examine cell cycle progression from G0 to S phase to identify the subset of adhesion-dependent G1-cdk events that are stimulated by TGF-beta during induction of anchorage-independent growth: parallel studies will determine if TGF-beta has the same subcellular effect(s) on control cells that fail to undergo anchorage-independent growth. In aim 2, we will ectopically express TGF-beta mediated cell cycle events to determine which of its effects are causal for TGF-beta mediated anchorage-independence. Finally, in aims 3 and 4, we will characterize the E2F-independent mechanism that regulates the adhesion-dependent expression of cyclin A and determine the effects of TGF-beta on E2F-dependent and E2F-independent cyclin A gene expression during induction of anchorage-independent growth.