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蛋白在细胞应激反应中的作用，特别是对热休克和电离辐射的反应。在下一个资助期内，有三个目标。重点研究Ku抑制hsp70热诱导的分子机制。我们将验证从我们的初步研究中推断出的一个假设，即Ku70过表达导致hsp70启动子内CpGs的超甲基化，而这种超甲基化反过来又抑制hsp70的热诱导。首先，亚硫酸盐基因组测序和hsp70启动子的基因组足迹将进行，以确定CpG甲基化和蛋白质结合模式如何响应Ku70和/或Ku80过表达。其次，在热休克前后，研究DNA去甲基化剂5-AzaC对热诱导hsp70 mRNA和蛋白的影响，以及对对照组和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。这些实验旨在通过ku -过表达hsp70启动子的特定CpGs甲基化与抑制hsp70热诱导之间建立牢固的联系。第三，电泳迁移率转移分析和瞬时转染实验，使用低聚和报告子连接的dna（覆盖hsp70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CpG甲基化，蛋白质结合和hsp70启动子反式激活的关联。在Specific Aim II中，为了研究和解剖Ku70和Ku80的各个结构域在细胞对热休克和电离辐射的反应中的作用，我们产生了稳定且组成性过表达Ku70和Ku80的完整或多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Ku70， Ku80或Ku70和Ku80的细胞系来表达完整的Ku或各种Ku片段，无论是单独的还是组合的。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Specific Aim III中，我们将使用表达Ku反义RNA或显性负Ku基因片段的重组腺病毒和腺相关病毒载体，评估Ku水平和/或活性的下调是否可以增强肿瘤细胞在体外和体内的放射敏感性。所获得的信息将作为指导设计新的策略，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.