Characterization of Mutagenesis, Mutational Spectra and Mechanisms of Toxicity

项目3.一个持续的规划目标是阐明活性氮(RNS)和活性氧(ROS)增加癌症风险的机制。项目3通过测试以下假设来实现这一目标：产生NO的巨噬细胞的RNS和/或中性粒细胞的RO对DMA和其他细胞大分子的损害要么促使细胞凋亡，要么抑制凋亡并增强突变。我们使用模型来实现对DNA损伤、遗传毒性、突变性和细胞死亡的机制研究。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诱变和致命终点。炎症性肠病与结肠癌的风险之间的关系已经有了很好的记录，而且证据也清楚地表明，含有iNOS的肿瘤细胞的高频率与III期黑色素瘤患者的低存活率有关。因此，我们的第一个特定目标是阐明RNS和ROS诱导的人结肠癌细胞和人黑色素瘤细胞的剂量和剂量率、DNA损伤、突变和凋亡之间的潜在关系。P53在调节反应中的关键作用将通过对密切相关的P53突变细胞和P53缺失细胞的平行研究来评估。其次，我们将在三个环境下表征剂量和剂量率对RNS和ROS诱导的gpt报告基因突变效力和突变谱的影响：(A)在pSV2gpt转化的CHO AS52细胞中，在受控条件下暴露于NO*；(B)在AS52细胞中与激活的RAW264.7小鼠巨噬细胞和/或HL60细胞共同培养；以及(C)整合到发生炎症相关结肠腺癌的RAG 2-1-IL10-/-小鼠的基因组中。我们的第三个目标涉及DNA加合物作为致死和突变终点的生物标记物的遗传优先顺序。已经被提名为潜在的炎症性前突变损伤的加合物将首先通过插入寡核苷酸、连接到病毒基因组和在不同修复熟练程度的大肠杆菌细胞中复制来进行评估。其次，我们将使用化学-生物指纹分析这一新工具，加速DNA损伤和突变光谱与特定生物终点的损伤之间的功能联系。与RNS或ROS损伤的gpt基因突变谱特征相对应的突变性(目标#2)将从结构上进行表征(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 DMA and other cellular macromolecules by RNS from NO-producing macrophages and/or ROS from 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).