A Genetic Screen for Latent TGF-beta Activators

描述（由申请人提供）：TGF-β在细胞生长、基质形成、免疫系统调节和细胞凋亡中很重要。TGF-β功能的缺陷与包括肿瘤生长和转移在内的病理状况相关。由于TGF-β以非活性复合物的形式释放，其活性在细胞因子呈递的水平上受到控制，因为TGF-β从其复合物中解离是调节活性TGF-β的浓度和作用的关键步骤。控制潜在TGF-β激活的过程知之甚少。为了解决TGF-β是如何从其复合物中释放出来的，我们开发了一种用于激活剂鉴定的遗传筛选。该筛选依赖于组成性产生潜伏TGF-β的细胞。将细胞工程化以在TGF-β存在下表达绿色荧光蛋白（GFP）。用逆转录病毒感染报道细胞，所述逆转录病毒含有从激活潜伏TGF-β的细胞制备的cDNA文库。将感染的细胞分选用于GFP表达。通过回收cDNA或分离过表达的蛋白质来鉴定负责增强GFP表达的蛋白质。候选蛋白的TGF-β激活剂活性在独立的测定中得到验证。我们用已知的激活剂β 6整联蛋白进行了原理验证实验。使用来自胶质母细胞瘤细胞的激活潜伏性TGF-β的cDNA文库，我们鉴定了激活内源性潜伏性TGF-β的细胞克隆。我们建议在两个具体目标上扩展这项工作。在目标1中，我们将分析我们获得的克隆，以鉴定和表征U1240胶质母细胞瘤细胞系中的新型活化分子。在目标2中，我们将使用来自其他两种肿瘤细胞系的cDNA文库重复分离程序，所述肿瘤细胞系通过未表征的机制激活潜伏的TGF-β。这些实验将增强我们对TGF-β生物学的理解，并可能导致TGF-β激活抑制剂的开发。虽然这些研究风险很高，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.