HYPERTHERMIA AND THE FUNCTION OF HSP70

描述（由申请人提供）：本研究项目的长期目标是阐明Ku蛋白在细胞对应激反应中的作用，特别是对热休克和电离辐射的反应。 具体目标I侧重于Ku抑制热休克蛋白70热诱导的分子机制。我们将测试一个假设，从我们的初步研究推断，Ku 70过表达导致热休克蛋白70启动子内的CpG的超甲基化，这种超甲基化反过来抑制热休克蛋白70的热诱导。首先，将进行hsp 70启动子的亚硫酸氢盐基因组测序和基因组足迹分析，以确定CpG甲基化和蛋白结合模式如何响应Ku 70和/或Ku 80过表达而变化。第二，DNA去甲基化剂5-AzaC对热休克前后对照和Ku 70过表达细胞中hsp 70 mRNA和蛋白的热诱导，以及对hsp 70启动子CpG甲基化和蛋白结合模式的影响将被确定。这些实验的目的是建立一个坚实的联系之间的甲基化的特定CpG的热休克蛋白70启动子Ku过表达和热休克蛋白70热诱导的抑制。第三，电泳迁移率变动分析和瞬时转染实验，使用寡聚体和寡聚体连接的DNA（覆盖hsp 70启动子内的顺式元件）与特定的5-甲基胞嘧啶取代，也将进行相关的CpG甲基化，蛋白质结合，和hsp 70启动子反式激活。在特定目标II中，为了研究和剖析Ku 70和Ku 80的各个结构域在热休克和电离辐射的细胞反应中的作用，我们已经产生了稳定和组成型过表达完整或Ku 70和Ku 80的各个结构域的啮齿动物细胞系。此外，我们将利用我们最近建立的缺乏Ku 70、Ku 80或同时缺乏Ku 70和Ku 80的细胞系，单独或组合表达完整的Ku或各种Ku片段。使用这些细胞系，我们将确定哪些领域的Ku 70和Ku 80参与调节细胞对热休克和电离辐射的反应。在特定目标III中，使用表达Ku反义RNA或显性阴性Ku基因片段的重组腺病毒和腺相关病毒载体，我们将评估Ku水平和/或活性的下调是否可以在体外和体内增强肿瘤细胞的放射敏感性。所获得的信息将作为指导，在设计新的战略，使肿瘤细胞对电离辐射敏感。

DESCRIPTION (provided by applicant): The long term goal of this research project is to elucidate the roles of Ku protein in the response of cells to stress, specifically to heat shock and to ionizing radiation During the next funding period, there are three aims. Specific Aim I focuses on the molecular mechanism(s) by which Ku suppresses heat-induction of hsp70. We will test a hypothesis, inferred from our preliminary studies, that Ku70 overexpression leads to hypermethylation of CpGs within the hsp70 promoter, and that this hypermethylation in turn suppresses thermal induction of hsp70. First, bisulfite genomic sequencing and genomic footprinting of the hsp70 promoter will be carried out to determine how the CpG methylation and protein-binding patterns change in response to Ku70 and/or Ku80 overexpression. Second, the effect of DNA demethylation agent 5-AzaC on the heat-induction of hsp70 mRNA and protein, and on the CpG methylation and protein-binding patterns of hsp70 promoter in control and Ku70-overexpressing cells will be determined, both before and after heat shock. These experiments aim to establish a firm link between methylation of specific CpGs of the hsp70 promoter by Ku-overexpression and the suppression of hsp70 thermal induction. Third, electrophoretic mobility shift assays and transient transfection experiments, using oligomeric and reporter-linked DNAs (covering the cis-elements within the hsp70 promoter) with specific 5-methylcytosine substitutions, will also be performed to correlate CpG methylation, protein-binding, and hsp70 promoter transactivation. In Specific Aim II, to study and dissect the roles of various domains of Ku70 and Ku80 in cellular response to heat shock and to ionizing radiation, we have generated rodent cell lines stably and constitutively overexpresssing intact or various domains of Ku70 and Ku80. In addition, we will take advantage of the cell lines lacking Ku70, Ku80, or both Ku70 and Ku80, which we have recently established, to express intact Ku or various Ku fragments, either individually or in combinations. Using these cell lines, we will determine which domains of Ku70 and Ku80 are involved in modulating cellular responses to heat shock and ionizing radiation. In Specific Aim III, using recombinant adeno- and adeno-associated viral vectors, that express Ku antisense RNA or dominant negative Ku gene fragments, we will evaluate whether down-regulation of Ku level and/or activity can enhance the radiosensitivity of tumor cells in vitro and in vivo. The information gained will serve as a guide in the design of new strategies for sensitizing tumor cells to ionizing radiation.