ANCHORAGE INDEPENDENT GROWTH--ROLE OF TGF-BETA

细胞锚定是几乎所有细胞类型增殖所必需的 而失去这一要求（诱导锚定独立性）， 细胞转化的标志 在分离的生长因子中， 从正常组织中，TGF-β在诱导 非锚定生长 然而，这种转变的效果， TGF-β仅限于少数成纤维细胞系（例如NRK细胞 在对TGF-β的反应中变得锚定独立，而NIH- 3 T3和正常人成纤维细胞不具有）。 或许正是因为这个原因， TGF-β生物学与锚定独立性关系 一直没有解决 我们的数据现在表明，诱导锚定独立的增长， 代表TGF-β病理学的一个重要方面。 具体地说， 我们发现（I）TGF-β不能诱导锚定非依赖性生长， 在保持其正常粘附要求以表达 细胞周期蛋白D1和（ii）这种控制的丧失--尽管在细胞周期中没有转化， 本身--使细胞容易被TGF-β转化。 因此，在本发明中， 粘附依赖性细胞周期蛋白D1表达的缺失是 转化为TGF-β。 我们现在提出四个具体目标， 鉴定TGF-β诱导的分子效应，并确定 TGF-β作用如何补充组成性细胞周期蛋白D1的作用 表达以诱导锚定独立性。 在目标1中， 检查细胞周期从G 0期到S期的进展以确定 粘附依赖性G1-cdk事件的子集，其由 诱导锚定非依赖性生长期间的TGF-β：平行 研究将确定TGF-β是否具有相同的亚细胞效应， 控制未能进行非贴壁生长的细胞。 在 目的2：异位表达TGF-β介导的细胞周期事件 以确定其哪些作用是TGF-β介导的 锚地独立性 最后，在目标3和4中，我们将 描述E2 F独立机制，调节 细胞周期蛋白A的粘附依赖性表达，并确定 TGF-β对E2 F依赖性和E2 F非依赖性细胞周期蛋白A基因的影响 在诱导锚定非依赖性生长期间表达。

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.