A Genetic Screen for Latent TGF-beta Activators

描述(申请人提供)：转化生长因子-β在细胞生长、基质形成、免疫系统调节和细胞凋亡中起重要作用。转化生长因子-β功能缺陷与肿瘤生长和转移等病理条件有关。由于转化生长因子-β在非活性复合体中释放，其活性被控制在细胞因子递呈的水平，因为转化生长因子-β从其复合体中解离是调节活性转化生长因子-β的浓度和作用的关键步骤。控制潜在的转化生长因子-β激活的过程还知之甚少。为了解决转化生长因子-β是如何从其复合体中释放出来的，我们开发了一种用于激活剂识别的遗传屏幕。这种筛选依赖于结构性地产生潜在的转化生长因子-β的细胞。这些细胞被设计成在转化生长因子-β存在的情况下表达绿色荧光蛋白(GFP)。报道的细胞感染了逆转录病毒，其中含有一个从激活潜伏的转化生长因子-β的细胞中制备的cDNA库。对受感染的细胞进行GFP表达排序。通过检索或分离过表达的蛋白来鉴定负责增强GFP表达的蛋白质。在独立的实验中验证了候选蛋白的转化生长因子-β激活剂活性。我们用一种已知的激活剂--整合素进行了原理验证实验。使用激活潜伏的转化生长因子-β的胶质母细胞瘤细胞的cDNA文库，我们鉴定了激活内源性潜伏的转化生长因子-β的细胞克隆。我们建议将这项工作扩展到两个具体目标。在目标1中，我们将对我们获得的克隆进行分析，以鉴定和鉴定U1240胶质母细胞瘤细胞系中新的激活分子。在目标2中，我们将使用另外两个通过未知机制激活潜伏的转化生长因子-β的肿瘤细胞株的cDNA文库重复分离过程。这些实验将加深我们对转化生长因子-β生物学的了解，并可能导致转化生长因子-β激活抑制物的开发。虽然这些研究的风险很高，但潜在的回报是巨大的。

DESCRIPTION (provided by applicant): The TGF-betas are important in cell growth, matrix formation, immune system modulation, and apoptosis. Defects in TGF-beta function are associated with pathological conditions including tumor growth and metastasis. Because TGF-betas are released in an inactive complex, their activity is controlled at the level of cytokine presentation as the dissociation of TGF-beta from its complex is a critical step in regulating the concentration and action of active TGF-beta. The processes controlling latent TGF-beta activation are poorly understood. To address how is TGF-beta released from its complex, we developed a genetic screen for activator identification. The screen relies upon cells that constitutively produce latent TGF-beta. The cells are engineered to express green fluorescent protein (GFP) in the presence of TGF-beta. The reporter cells an infected with retroviruses containing a cDNA library prepared from cells that activate latent TGF-beta. The infected cells are sorted for GFP expression. The protein responsible for enhanced GFP expression is identified by retrieval of the cDNA or isolation of the overexpressed protein. The TGF-beta activator activity of the candidate protein is validated in independent assays. We performed proof-of-principle experiments with a known activator, beta6 integrin. Using a cDNA library from glioblastoma cells that activate latent TGF-beta, we identified cell clones that activate endogenous latent TGF-beta. We propose to extend this work in two specific aims. In Aim 1, we will analyze the clones that we have obtained to identify and characterize the novel activating molecule in the U1240 glioblastoma cell line. In Aim 2, we will repeat the isolation procedure using cDNA libraries from other two tumor cell lines that activate latent TGF-beta by uncharacterized mechanisms. These experiments will enhance our understanding of TGF-beta biology and potentially result in the development of inhibitors of TGF-beta activation. Although there is a high risk in these studies, the potential reward is great.