HYPERTHERMIA AND THE FUNCTION OF HSP70

这项研究项目的长期目标是阐明Ku蛋白在细胞对 压力，特别是对热休克和电离辐射的压力，在下一个供资期间，有三个 目标 具体目的I重点研究Ku抑制热休克蛋白70热诱导的分子机制(S 我们将检验从我们的初步研究中推断的一个假设，即Ku70的过度表达会导致高... HSP70启动子内CPGS的甲基化，这种超甲基化反过来抑制热 首先是HSP70的诱导、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甲基化和蛋白质结合模式如何变化对Ku70的响应 和/或KuS0的过表达第二，DNA去甲基化试剂5-azaC对热诱导的影响 HSP70基因和蛋白以及HSP70启动子CpG甲基化和蛋白结合模式的研究 对照和Ku70过表达的细胞将被确定，在热休克之前和之后 实验的目的是在HSP70启动子的特定CPGS甲基化之间建立牢固的联系。 热诱导HSP70的过表达和抑制第三，凝胶迁移率改变分析 以及使用寡聚体和报告分子连接的DNA(覆盖顺式元件)的瞬时转染实验 在HSP70启动子内)与特定的5-甲基胞嘧啶取代也将被执行以关联 CpG甲基化、蛋白结合和HSP70启动子反式激活 在特定目的II中，研究和剖析Ku70和Ku80的各个结构域在细胞中的作用 对热休克和电离辐射的响应，我们已经稳定和稳定地产生了啮齿动物细胞系。 过表达完整或不同结构域的Ku70和Ku80此外，我们还将利用细胞 缺少Ku70、Ku80或同时缺少KuT0和Ku80的品系，这是我们最近建立的，用来表达完整的Ku或 不同的Ku片段，无论是单独的还是使用这些细胞系的组合，我们将确定哪些 Ku70和Ku80结构域参与调节细胞对热休克和电离辐射的反应 在特定目的III中，使用腺相关和腺相关病毒载体，表达Ku 反义RNA或显性负性Ku基因片段，我们将评估Ku是否下调 水平和/或活性可增强肿瘤细胞的体外和体内放射敏感性 将在设计使肿瘤细胞对电离辐射敏感的新策略方面起到指导作用。

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 hyper- methylation 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 KuS0 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 KuT0 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.