DNA REPAIR AND SUSCEPTIBILITY TO ENVIRONMENTAL AGENTS

描述(由申请人提供) DNA损伤被认为是肿瘤发生中的一种重要作用方式。 暴露在环境介质中可能会导致氧化和烷基化损伤 导致大量诱变的DNA加合物的形成。细胞可以做出反应 通过协调诱导DNA修复来造成DNA损伤，已经证明 即DNA损伤在不同部位的积聚和清除的差异 器官和各种物种中都存在。因此，调查人员将测试 该假说认为DNA修复的诱导程度 氧化和烷基化损伤在不同的物种和组织中有所不同，并且 这种变异有助于物种和特定部位的突变和 环境化学品的细胞毒性效应。获取与以下内容相关的数据 为了评估人类风险并探索DNA修复机制，这些实验 将使用从各种人体组织中分离出来的培养细胞进行 (例如，肝脏、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基因的基本表达水平 涉及碱基切除、核苷酸的近60个DNA修复基因簇 人类和小鼠细胞中的切除和错配修复途径。的表达 DNA修复基因将在mRNA和蛋白质水平上用多探针进行研究 核糖核酸酶保护实验和Western blotting。第二，他们 将比较DNA修复基因对氧化和氧化的转录反应 烷基化损伤。人类和小鼠细胞将暴露在电离辐射中 辐射、四氯对苯二酚、甲磺酸甲酯或N-甲基-亚硝脲。 DNA修复酶的表达将在细胞之间进行比较 利用基因芯片进行类型和种类的研究。蛋白质水平将通过以下方式进行验证 蛋白质印迹，或蛋白质组学分析。第三，调查人员将 确定碱基切除、核苷酸活性是否发生变化 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 在体外识别和修复损伤的提取物，以及一些 我们将对基本位点和单链断裂进行比较。调查人员 预计DNA修复基因的诱导程度以及 修复活性因细胞和物种的不同而有所不同， 可以确定对每种治疗的反应模式。DNA修复途径 已被证明与P53介导的细胞凋亡信号相互作用； 因此，最终目的是确定P53在组织特异性中的作用 DNA修复基因对氧化应激和蛋白质合成的转录调控 环境因素造成的烷基化破坏。在这里，调查人员将使用 野生型和P53基因缺失小鼠的细胞分离及体外处理 如上所述。 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依赖和独立 环境暴露后参与DNA修复的途径将是 已确认身份。总而言之，这些研究将汇编一个数据矩阵，说明 参与DNA修复的一组基因的表达和活性各不相同 在人类和小鼠的不同器官中。这些实验还将 为物种之间可能的差异提供关键的机械洞察力--以及 对环境因素引起的DNA损伤的组织特异性反应。

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.