Characterization of Mutational Spectra, Mechanisms of Toxicity and Homologous Rec

突变谱的表征、毒性机制和同源重组

• 批准号: 6990331
• 负责人: GERALD N WOGAN
• 金额: $ 27.15万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-13 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6990331
• 关键词: DNA damage; apoptosis; cytotoxicity; free radical oxygen; gene mutation; genetic recombination; loss of heterozygosity; macrophage; model design /development; nitric oxide; nitrogen metabolism; p53 gene /protein; peroxynitrites; tissue /cell culture

The goal of this project is to characterize mechanisms of mutagenicity, apoptosis and homologous recombination resulting from DNA damage induced by NO, with particular emphasis on how specific types of damage may contribute to tumor initiation and development. The ability of macrophages and neutrophils to overproduce NO and reactive oxygen species is well-established, as is the ability of these reactive species to induce lethal and mutagenic DNA damage. Work in Dr. Tennebaum's Project has established that the chemistry of cellular damage induced by NO, although complex, may result from only two fundamental processes: (1) reaction with oxygen to form N203, a nitrosating agent; and (2) reaction with superoxide to form peroxynitrite (ONOO-). This project is designed to test the hypothesis that mutations, alterations in apoptotic signaling and homologous recombination result from DNA damage produced by NO and ONOO- in chemically-defined systems as well as by activated phagocytes. Our first specific aim will be to determine effects of dose and dose-rate on mutagenicity and mutation spectra induced by NO and ONOO- in the supF gene of pSP189 replicated in human cells. Second, we shall determine relationships among NO and ONOO- dose and dose-rate, DNA damage, mutagenesis and apoptosis in cultured human cells. Cells to be used include human lymphoblastoid TK6 cells and human colon adenocarcinoma HCT116 cells; the pivotal role of p53 in modulating responses will be evaluated by parallel studies in isogenic p53 knockout cells. Our third line of investigation will focus on relative contributions of NO and reactive oxygen species generated by activated macrophages to mutagenesis of endogenous and exogenous genes in co-cultivated target cells. In the fourth specific aim, we propose to identify the DNA lesion(s) giving rise to mutations observed in NO-treated cells. Oligonucleotides will be synthesized containing modified bases formed by NO, inserted into the genome of M 13 bacteriophage and replicated in E. coil. The type and amount of genetic change, if any, will be characterized, and genetic requirements for mutagenesis by specific lesions will be defined. Preliminary studies revealed that homologous recombination was a critical determinant of NO-induced lethality in E. coll. In the final specific aim, we therefore propose to determine relationships between NO-induced DNA lesions and homologous recombination in mammalian cells, inasmuch as LOH, an important contributor to tumorigenesis can result from aberrant recombination events.

该项目的目标是表征由NO诱导的DNA损伤引起的致突变性、凋亡和同源重组的机制，特别强调特定类型的损伤如何有助于肿瘤的发生和发展。巨噬细胞和嗜中性粒细胞过度产生NO和活性氧的能力是公认的，这些活性物质诱导致死性和致突变性DNA损伤的能力也是如此。Tennebaum博士的项目中的工作已经确定，由NO诱导的细胞损伤的化学虽然复杂，但可能仅由两个基本过程引起：（1）与氧反应以形成N2 O3，一种亚硝化剂;以及（2）与超氧化物反应以形成过氧亚硝酸盐（ONOO-）。 该项目的目的是测试的假设，突变，改变凋亡信号和同源重组的结果从DNA损伤产生的NO和ONOO-在化学定义的系统，以及激活的吞噬细胞。我们的第一个具体目标将是确定剂量和剂量率对致突变性和突变谱诱导的NO和ONOO-在人细胞中复制的pSP 189的supF基因。其次，我们将确定NO和ONOO-剂量和剂量率，DNA损伤，诱变和细胞凋亡在培养的人细胞之间的关系。待使用的细胞包括人淋巴母细胞样TK 6细胞和人结肠腺癌HCT 116细胞; p53在调节应答中的关键作用将被阐明。 通过在同基因p53敲除细胞中的平行研究进行评价。我们的第三条调查线将集中在NO和活性氧物种产生的活化巨噬细胞在共培养的靶细胞的内源性和外源性基因的诱变的相对贡献。在第四个具体目标中，我们提出鉴定在NO处理的细胞中观察到的引起突变的DNA损伤。将合成的含有NO形成的修饰碱基的寡核苷酸插入到M13噬菌体的基因组中，并在E. 线圈将对遗传变化的类型和数量（如有）进行表征，并定义特定病变致突变的遗传要求。初步研究表明，同源重组是NO诱导大肠杆菌致死的关键决定因素。因此，在最后的具体目标中，我们建议确定哺乳动物细胞中NO诱导的DNA损伤和同源重组之间的关系，因为洛是异常重组事件可能导致肿瘤发生的重要因素。