HYPERTHERMIA AND THE FUNCTION OF HSP70

描述(申请人提供)：这项研究项目的长期目标是阐明Ku蛋白在细胞对应激，特别是对热休克和电离辐射的反应中的作用，在下一个资助期，有三个目标。具体目的一重点研究Ku抑制热休克蛋白70热诱导的分子机制(S)。我们将检验一个假说，从我们的初步研究推断，Ku70的过度表达导致HSP70启动子内CPGS的超甲基化，而这种超甲基化反过来抑制HSP70的热诱导。首先，将进行亚硫酸氢盐基因组测序和HSP70启动子的基因组足迹，以确定CpG甲基化和蛋白结合模式如何随着Ku70和/或Ku80的过度表达而改变。其次，研究DNA去甲基化试剂5-azaC对热诱导HSP70基因和蛋白表达的影响，以及对对照细胞和Ku70过表达细胞中HSP70启动子CpG甲基化和蛋白结合模式的影响。这些实验旨在建立Ku过表达引起的HSP70启动子特定CPGS的甲基化与HSP70热诱导抑制之间的牢固联系。第三，还将使用具有特定5-甲基胞嘧啶取代的寡聚和报告连接的DNA(覆盖HSP70启动子内的顺式元件)，进行电泳迁移率改变分析和瞬时转染实验，以关联CpG甲基化、蛋白质结合和HSP70启动子的反式激活。为了研究和剖析Ku70和Ku80不同结构域在细胞对热休克和电离辐射反应中的作用，我们建立了稳定和结构性过表达完整或不同结构域的啮齿动物细胞系。此外，我们将利用最近建立的缺失Ku70和Ku80或同时缺失Ku70和Ku80的细胞系，单独或组合表达完整的Ku或各种Ku片段。利用这些细胞系，我们将确定Ku70和Ku80的哪些结构域参与调节细胞对热休克和电离辐射的反应。在特定的目的III中，我们将利用表达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，评估下调Ku水平和/或活性是否可以在体内外提高肿瘤细胞的放射敏感性。所获得的信息将在设计使肿瘤细胞对电离辐射敏感的新策略时起到指导作用。

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.