Analysis of functional properties of the Acute Promylocytic Leukemia (APL) oncogene PML-RARalpha using a novel ex-vivo assay

使用新型离体测定法分析急性早幼粒细胞白血病 (APL) 癌基因 PML-RARalpha 的功能特性

• 批准号: 194977922
• 负责人: Dr. Thomas Martin Sternsdorf
• 金额: --
• 依托单位: Forschungsinstitut Kinderkrebs-Zentrum Hamburg gGmbH
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/194977922?language=en
• 关键词: Analysis; functional; properties; Acute; Promylocytic

This proposal aims at analyzing functional properties of the leukemia-inducing PML-RARα fusion protein. Our previous data suggest, that both the RARα part, and also the PML part provide unique features, which prime them to become leukemia-inducing through their fusion in APL. We will use an in-vitro immortalization assay, which I have established, based on the observation, that expression of a fusion protein of murine PML and human RARα (mPR) by retroviral transduction can immortalize primary murine bone marrow cells, providing a rapid in vitro-assay (mPR assay). We want to use this assay to investigate the contribution of the different structural domains of mPML-RARα for this phenomenon, by performing domain swapping and mutagenesis experiments. Aim 1: determination of relevant features on the RARα side: Analysis of DNA binding and interaction with the cofactors RXR and SMRT. Aim 2: determination of relevant features on the PML side: Analysis of RING finger, B Boxes and coiled coil structural domains. Aim 3: Experiments correlating the immortalisation results with leukemogenesis, using classical transduction/ transplantation experiments in the mouse. If successful, our method will provide a fast way to determine effects of specific changes in the oncogene, saving time and significantly reducing necessary animal experiments.

本研究旨在分析白血病诱导PML-RARα融合蛋白的功能特性。我们以前的数据表明，RARα部分和PML部分都提供了独特的特征，这些特征使它们通过与APL融合而成为白血病诱导因子。我们将使用一种体外永生化试验，我已经建立了该试验，基于观察结果，即通过逆转录病毒转导表达鼠PML和人RARα（mPR）的融合蛋白可以使原代鼠骨髓细胞永生化，从而提供了一种快速的体外试验（mPR试验）。我们希望通过进行结构域交换和诱变实验，使用该试验研究mPML-RARα的不同结构域对该现象的贡献。目的1：确定RARα侧的相关特征：分析DNA结合以及与辅因子RXR和SMRT的相互作用。目的2：确定PML侧的相关特征：分析RING指、B盒和卷曲螺旋结构域。目标三：使用小鼠中的经典转导/移植实验将永生化结果与白血病发生相关联的实验。如果成功的话，我们的方法将提供一种快速的方法来确定癌基因中特定变化的影响，节省时间并显着减少必要的动物实验。