Characterization of Mutagenesis, Mutational Spectra and Mechanisms of Toxicity

项目3.一个持续的方案目标是阐明活性氮通过 物种(RN)和活性氧物种(ROS)会增加癌症风险。项目3涉及 这一目标是通过检验以下假设来实现的：NO-RNS对DNA和其他细胞大分子的损伤 由中性粒细胞产生巨噬细胞和/或巨噬细胞可促使细胞凋亡或抑制细胞凋亡 并增强突变。我们使用的模型能够对DNA损伤、遗传毒性、 致突变性和细胞死亡。项目3还定义了具有生物学特性的DNA损伤，解释了 细胞和生物体水平系统中的致突变和致死终点。炎症性肠病协会 疾病与结肠癌的风险是有很好的记录的，而且证据也清楚地表明 III期黑色素瘤患者存活率低的含iNOS的肿瘤细胞。因此，我们的第一个具体目标是 阐明剂量和剂量率、DNA损伤、诱变之间潜在关系的机制 RNS和RNS诱导的人结肠癌细胞和人黑色素瘤细胞的凋亡 罗斯。P53在调节反应中的关键作用将通过密切相关的平行研究进行评估 P53突变细胞和P53缺失细胞。其次，我们将表征剂量和剂量率对诱变的影响。 GPT报告基因中RNS和ROS在三种环境下的效力和突变谱：(A)在 PSV2gpt转化的CHO AS52细胞在控制条件下体外暴露于NO*；(B)AS52细胞 与激活的RAW264.7小鼠巨噬细胞和/或HL60细胞共培养；以及(C)整合到 RAG 2-1-IL10-/-发展为炎症相关结肠腺癌的小鼠的基因组。我们的第三个目标 关注DNA加合物作为致死和致突变终点的生物标记物的遗传优先顺序。加合物 已经被提名为潜在的炎症性诱变前病变将首先进行评估 插入寡脱氧核苷酸，连接到病毒基因组和在不同类型的大肠杆菌细胞中复制 修理熟练程度。其次，我们将使用化学-生物指纹这一新工具来加速 DNA损伤和突变光谱与特定生物损伤的功能联系 终端。RNS损伤的gpt基因突变谱特征对应的致突变性 或ROS(目标2)，将在结构上进行描述(与项目2合作)。

PROJECT 3. A continuing programmatic goal is elucidation of mechanisms through which reactive nitrogen species (RNS) and reactive oxygen species (ROS) contribute to increased cancer risks. Project 3 addresses this goal by testing the hypothesis that damage to DMAand other cellular macromolecules by RNS from NO- producing macrophages and/or ROSfrom neutrophils either drives cells into apoptosis or inhibits apoptosis and enhances mutation. We employ models enabling mechanistic studies of DNA damage, genotoxicity, mutagenicity and cell death. Project 3 also defines DNA lesions with biological properties that explain mutagenic and lethal endpoints in cellular- and organism-level systems. Association of inflammatory bowel disease with risk of colon cancer is well-documented, and evidence also clearly associates high frequency of iNOS-containing tumor cells with poor survival of stage III melanoma patients. Thus, our first specific aim is to elucidate mechanisms underlying relationships among dose and dose-rate, DNA damage, mutagenesis and apoptosis induced in human colon carcinoma cells and human melanoma cells by exposure to RNS and ROS. The pivotal role of p53 in modulating responses will be evaluated by parallel studies in closely related p53-mutant and p53-null cells. Second, we shall characterize effects of dose and dose-rate on mutagenic potency and mutation spectra induced by RNS and ROS in the gpt reporter gene in three settings: (a) in pSV2gpt-transformed CHO AS52 cells exposed in vitro to NO* under controlled conditions; (b) in AS52 cells co-cultivated with activated RAW264.7 mouse macrophages and/or HL60 cells; and (c) integrated into the genome of Rag 2-1- IL10-/- mice developing inflammation-related colon adenocarcinoma. Our third aim concerns genetic prioritization of DNA adducts as biomarkers of lethal and mutagenic endpoints. Adducts already nominated for potential as inflammation-derived pre-mutagenic lesions will first be evaluated by insertion into oligodeoxynucleotides, ligation into a viral genome and replication in E. coli cells of differing repair proficiency. Secondly we shall use the novel tool of chemical-biological fingerprinting to accelerate functional linkage of DNA damage and mutational spectra to lesions responsible for specific biological endpoints. Mutagenicity corresponding to features of mutational spectra in the gpt gene damaged with RNS or ROS (aim #2), will be characterized structurally (in collaboration with Project 2).