DNA DAMAGE AND MUTATIONS CAUSED BY ACTIVATED LEUKOCYTES

激活的多形核白细胞(PMN)已被证明能产生 会导致DNA损伤的活性氧化和自由基物种， 突变和肿瘤转化。激活的PMN是 具有生物学意义的模型，用来表征 体内由自由基和氧化剂攻击引起的DNA。我们建议 活化的人细胞DNA碱基损伤的体内特征 选择离子气相色谱-质谱法测定中性粒细胞 监测(GC-MS/SIM)以量化碱基修改，并使用 连接介导的聚合酶链式反应技术(LMPCR)定位DNA 核苷酸水平的碱基损伤。GC-MS/SIM是一种灵敏、 广谱分析技术，能够量化十种 氧自由基引起的主要碱基修饰 生理条件。我们将使用此技术来获得全局 活化的中性粒细胞诱导体内碱基损伤的评估。LMPCR是一种 测绘和量化单个核苷酸的碱基损伤的技术 通过优先在损伤部位切割DNA；产生单链 通过将它们转化为双链，连接常见的 连接子，通过聚合酶用一组适当的引物进行扩增 链式反应，聚丙烯酰胺凝胶电泳法分离条带， 印迹到膜上并进行放射性探测。每个频段对应 发生损伤的特定核苷酸；条带强度 与损坏频率相对应。在本提案中，我们将激活地图 PMN诱导的人细胞碱基损伤，对PMN的切割敏感 热哌啶；我们还将使用酶来调整技术以绘制地图 基本部位和鸟嘌呤衍生的损害，包括8-氧基瓜。基座 损伤将被定位在SupF中，这是我们突变中使用的一种附体基因 研究，以及基因组基因PGK-1；影响 活化的PMN诱导的碱基损伤在体内的分布将是 评估过了。我们还将确定In产生的突变频率 利用该突变将人AD293细胞暴露于活化的中性粒细胞 报告质粒pSL89。PSL89携带抑制tRNA基因SupF； 通过回收质粒和鉴定SupF的突变 转化细菌报告菌株MBM7070，该菌株携带 SupF-LacZ基因中可抑制的琥珀突变。个体突变 将通过标准双脱氧测序来表征，以便开发 可与LMPCR衍生的碱基进行比较的突变谱 损毁地图。这些数据将有助于阐明(S) 人体细胞暴露于一系列自由基和氧化性物质 导致突变，这是致癌的重要一步。

Activated polymorphonuclear leukocytes (PMNs) have been shown to generate reactive oxidizing and free radical species which cause DNA damage, mutation, and neoplastic transformation. Activated PMNs are a biologically significant model with which to characterize the damage to DNA caused in vivo by free radical and oxidant attack. We propose to characterize in vivo DNA base damage in human cells caused by activated PMNs using gas chromatography-mass spectrometry with selected-ion monitoring (GC-MS/SIM) to quantify base modifications, and using the ligation-mediated polymerase chain reaction technique (LMPCR) to map DNA base damage at the nucleotide level. GC-MS/SIM is a sensitive, broad-spectrum analytic technique, capable of quantifying the ten principal base modifications caused by reactive oxygen species under physiologic conditions. We will use this technique to obtain a global assessment of activated PMN-induced in vivo base damage. LMPCR is a technique which maps and quantifies base damage at individual nucleotides by cleaving DNA preferentially at damage sites; resultant single strands are visualized by converting them to double strands, ligating common linkers, amplifying with an appropriate set of primers via the polymerase chain reaction, separating bands by polyacrylamide gel electrophoresis, blotting to a membrane and radioactively probing. Each band corresponds to a particular nucleotide at which damage occurred; band intensities correspond to damage frequency. In this proposal, we will map activated PMN-induced base damage in human cells which is sensitive to cleavage by hot piperidine; we will also use enzymes to adapt the technique to map abasic sites and guanine-derived lesions, including 8-oxoGua. Base damage will be mapped in SupF, an episomal gene used in our mutation studies, and the genomic gene pgk 1; factors which influence the distribution of activated PMN-induced base damage in vivo will be assessed. We will also determine the mutation frequency generated by in vivo exposure of human Ad293 cells to activated PMNs, using the mutation reporter plasmid pSl89. pSl89 carries the suppressor tRNA gene SupF; mutations in SupF are identified by recovering the plasmid and transforming the bacterial reporter strain MBM7070, which carries a SupF-suppressible amber mutation in its lacZ gene. Individual mutations will be characterized by standard dideoxy sequencing, in order to develop a mutation spectrum which can be compared to the LMPCR-derived base damage map. These data will help elucidate the mechanism(s) by which exposure of human cells to a flux of free radical and oxidizing species causes mutation, an important step in carcinogenesis.