DNA DAMAGE AND MUTATIONS CAUSED BY ACTIVATED LEUKOCYTES

活化的多形核白细胞（PMN）已被证明可产生 导致DNA损伤的活性氧化和自由基物质， 突变和肿瘤转化。 激活的PMN是一种 具有生物学意义的模型，用于表征 DNA在体内受到自由基和氧化剂的攻击。 我们建议 表征由活化的人细胞引起的体内DNA碱基损伤 中性粒细胞的气相色谱-质谱分析 监测（GC-MS/SIM）以量化碱基修饰，并使用 连接介导的聚合酶链反应技术（LMPCR）来绘制DNA图谱 在核苷酸水平上的碱基损伤。 GC-MS/SIM是一种灵敏、 广谱分析技术，能够量化的十个 由活性氧引起的主要碱基修饰 生理条件。 我们将使用这种技术来获得一个全局 评估活化的PMN诱导的体内基底损伤。 LMPCR是一种 绘制和量化单个核苷酸碱基损伤的技术 通过优先在损伤位点切割DNA;产生单链 通过将它们转化为双链，连接共同的 接头，通过聚合酶用适当的引物组扩增 链反应，聚丙烯酰胺凝胶电泳分离条带， 印迹到膜上并进行放射性探测。 每个波段对应 对发生损伤的特定核苷酸;条带强度 对应于损坏频率。 在本提案中，我们将地图激活 PMN诱导的对切割敏感的人细胞中的碱基损伤 热哌啶;我们还将使用酶来调整该技术， 脱碱基位点和鸟嘌呤衍生的病变，包括8-oxoGua。 基地 损伤将被定位在SupF中，SupF是我们突变中使用的附加体基因 研究和基因组基因pgk 1;影响 激活的PMN诱导的基底损伤在体内的分布将是 评估。 我们还将确定在 体内暴露的人Ad 293细胞活化的中性粒细胞，使用突变 报告质粒pS189。pS189携带抑制tRNA基因SupF; 通过回收质粒鉴定SupF中的突变， 转化细菌报告菌株MBM 7070，其携带 其lacZ基因中的SupF-可抑制琥珀突变。 个体突变 将通过标准双脱氧测序进行表征，以开发 可以与LMPCR衍生碱基进行比较的突变谱 损伤图 这些数据将有助于阐明 将人体细胞暴露于自由基和氧化物质流中 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.