DNA REPAIR AND SUSCEPTIBILITY TO ENVIRONMENTAL AGENTS

描述（由申请人提供） DNA损伤被认为是致癌过程中的一种重要作用方式。 暴露于环境因子可能导致氧化和烷基化损伤 导致形成许多致突变DNA加合物。细胞可以做出反应 通过协调诱导DNA修复来修复DNA损伤， 不同组织中DNA损伤的积累和去除的差异 在不同的物种中存在。因此，调查人员将测试 这一假说认为，DNA修复的诱导程度， 氧化和烷基化损伤在不同物种和组织中不同， 这种变异有助于种属和位点特异性诱变， 环境化学物质的细胞毒性作用。获取与以下内容相关的数据 人类风险评估和探索DNA修复的机制，实验 将使用从各种人体组织中分离的培养细胞进行 (e.g.,肝、脑、皮肤、骨髓）和来自小鼠的匹配组织。 首先，研究人员将评估一种新的基因的基础表达水平。 近60个DNA修复基因簇参与碱基切除，核苷酸 切除和错配修复途径。表达 DNA修复基因将利用多探针技术在mRNA和蛋白质水平上进行研究 RNA酶保护试验和蛋白质印迹法。二是 将比较DNA修复基因的转录反应， 烷基化损伤人类和小鼠细胞将暴露于电离 辐射、四氯氢醌、甲磺酸甲酯或N-甲基-N亚硝基脲。 DNA修复酶的表达将在细胞间进行比较。 使用cDNA阵列的类型和物种。蛋白质水平将由以下人员验证： 蛋白质印迹法或蛋白质组学分析。第三，调查人员将 确定碱基切除、核苷酸 由环境因素引起的切除和错配修复与 DNA修复基因的诱导程度。在这里，无细胞的效率 提取物来识别和修复体外病变，以及一些 将比较碱性位点和单链断裂。调查人员 预期DNA修复基因的诱导程度以及 修复的活性在细胞和物种之间会有所不同， 可以识别对每种治疗的反应模式。DNA修复途径 已显示与p53介导的凋亡信号相互作用; 因此，最终的目的是确定p53在组织特异性 调节DNA修复基因对氧化和 环境因素造成的烷基化损害。在这里，调查人员将使用 从野生型和p53缺失小鼠中分离的细胞，并在体外将它们作为 上面详述。 DNA损伤的积累和DNA修复基因表达的变化将 被研究。研究人员预计，p53依赖性和独立性 环境暴露后参与DNA修复的途径将 鉴定总的来说，这些研究将汇编一个数据矩阵， 参与DNA修复的一组基因的表达和活性 在人类和老鼠的不同器官中。这些实验也将 为物种间可能的差异提供了至关重要的机制性见解， 组织特异性反应的DNA损伤诱导的环境因素。

DESCRIPTION (provided by applicant) DNA damage is recognized as an important mode of action in carcinogenesis. Exposure to environmental agents may result in oxidative and alkylation damage leading to formation of a number of mutagenic DNA adducts. Cells can respond to DNA damage by coordinated induction of DNA repair and it has been shown that differences in the accumulation and removal of DNA lesions in different organs and in various species exist. Therefore, the investigators will test the hypothesis that the extent of induction of DNA repair in response to oxidative and alkylation damage varies in different species and tissues, and that such variation contributes to species and site-specific mutagenic and cytotoxic effects of environmental chemicals. To obtain data relevant for human risk assessment and to explore mechanisms of DNA repair, the experiments will be carried out using cultured cells isolated from various human tissues (e.g., liver, brain, skin, bone marrow) and matching tissues from mouse. First, the investigators will evaluate the basal levels of expression of a cluster of nearly 60 DNA repair genes involved in base excision, nucleotide excision and mismatch repair pathways in human and mouse cells. Expression of DNA repair genes will be studied on the mRNA and protein level by using multiprobe RNase protection assay and Western blotting, respectively. Second, they will compare the transcriptional response of DNA repair genes to oxidative and alkylation damage. Human and mouse cells will be exposed to ionizing radiation, tetrachlorohydroquinone, methyl methanesulfonate, or N-methyl-Nnitrosourea. Expression of DNA repair enzymes will be compared between cell types and species using cDNA arrays. Protein levels will be verified by Western blotting, or by proteomics analysis. Third, the investigators will determine whether changes in the activity of base excision, nucleotide excision and mismatch repair caused by environmental agents correlate with a degree of induction of DNA repair genes. Here, the efficiency of cell-free extracts to recognize and repair lesions in vitro, as well as a number of basic sites and single strand breaks will be compared. The investigators expect that the degree of induction of DNA repair genes as well as the activity of repair will differ between cells and species and that unique patterns of response to each treatment can be identified. DNA repair pathways have been shown to interact with p53-mediated signaling to apoptosis; therefore, the final aim is to determine the role of p53 in tissue-specific regulation of transcriptional responses of DNA repair genes to oxidative and alkylation damage by environmental agents. Here, the investigators will use cells isolated from wild type and p53-null mice and treat them in vitro as detailed above. Accumulation of DNA lesions and changes in expression of DNA repair genes will be studied. The investigators anticipate that p53-dependent and independent pathways involved in DNA repair following environmental exposure will be identified. Collectively, these studies will compile a matrix of data on how expression and activity of a cluster of genes involved in DNA repair varies among different organs from both humans and mice. These experiments will also provide crucial mechanistic insights into possible differences in species- and tissue-specific responses to DNA damage induced by environmental agents.