ALTERATION OF NAD METABOLISM BY CHEMICAL CARCINOGENS

化学致癌物改变 NAD 代谢

• 批准号: 3186349
• 负责人: MYRON K JACOBSON
• 金额: $ 18.88万
• 依托单位: TEXAS COLLEGE OF OSTEOPATHIC MEDICINE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 1990-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3186349
• 关键词: ADP ribosylation; DNA repair; autoradiography; chemical carcinogen; chemical carcinogenesis; enzyme inhibitors; fluorimetry; gas chromatography; gel electrophoresis; gel filtration chromatography; high performance liquid chromatography; human tissue; laboratory mouse; leukocyte activation /transformation; mass spectrometry; nicotinamide adenine dinucleotide; nitrosamines; nucleic acid metabolism; radiotracer; tissue /cell culture; tritium; tumor promoters; xanthines

The occurrence of DNA damage and cellular responses to DNA damage are involved in the initiation and perhaps the promotion phase of carcinogenesis. The proposed studies will focus on the mechanism and biological significance of the rapid alteration of poly(ADP-ribose) metabolism cause by environmentally induced DNA damage. These studies will utilize intact cultured mouse cells following treatment with N-methyl-N'-nitro-N nitrosoguanidine (MNNG). The complexity of polymers of ADP-ribose will be studied by isolation of polymers using highly selective boronate affinity resins, fractionation by HPLC molecular sieve chromatography and simultaneous determination of linear and branched internal residues and terminal residues. Such experiments will allow the determination of true polymer size and branching frequency per molecule, information necessary for evaluating the importance of non-covalent interactions of the polymer with other components of chromatin. The turnover of polymers and their distribution within chromatin fractions will be studied by combining the above methods with in vivo labeling protocols and inhibitors of poly(ADP-ribose) polymerase. Labeling methods will also be used to assess the quantitative significance of poly(ADP-ribose) metabolism in non-damaged cells. The function of poly(ADP-ribose) metabolism in cellular recovery from the cytotoxic effects of MNNG will be addressed by studies of cell cycle progression in synchronized C3H10T1/2 cells using flow cytofluorimetry, autoradiography and ADP-ribosylation inhibitors. This system will also be used to make chromatin comparisons designed to yield information concerning the stabilization or alterations of chromatin structure involving poly(ADP-ribose) and normal cell cycle progression in MNNG treated cells. The involvement of poly(ADP-ribose) metabolism in other important biological responses to DNA damage in C3H10T1/2 cells will be studied by evaluating the effect of ADP-ribosylation inhibitors and nutritional manipulation of NAD levels on mutagenesis and malignant transformation. Such studies will utilize conditions where co-cytotoxic effects will be eliminated or minimized and effects on growth during expression carefully monitored. The mechanism of poly(ADP-ribose) metabolism by hyperthermia will be studied and an assessment of a possible general role of poly(ADP-ribose) metabolism in a general cellular response to environmental stress will be made.

DNA损伤的发生和细胞对DNA损伤的反应是 参与启动阶段，或许还有推广阶段 致癌作用。 拟议的研究将重点关注机制和 聚（ADP-核糖）快速改变的生物学意义 环境引起的DNA损伤引起的新陈代谢。 这些研究将 使用处理后的完整培养小鼠细胞 N-甲基-N'-硝基-N 亚硝基胍 (MNNG)。 聚合物的复杂性 ADP-核糖将通过使用高选择性分离聚合物来研究 硼酸亲和树脂，HPLC 分子筛分级 色谱法以及直链和支链的同时测定 内部残基和末端残基。 此类实验将允许 测定真实聚合物尺寸和每个分子的支化频率， 评估非共价键重要性所需的信息 聚合物与染色质其他成分的相互作用。 这 聚合物的周转及其在染色质部分中的分布将 通过将上述方法与体内标记方案相结合进行研究 和聚（ADP-核糖）聚合酶抑制剂。 标签方法也将 用于评估聚（ADP-核糖）的定量意义 未受损细胞的新陈代谢。 聚（ADP-核糖）的功能 从 MNNG 的细胞毒性作用中恢复的细胞代谢将 通过同步 C3H10T1/2 细胞周期进展的研究来解决 使用流式细胞荧光测定法、放射自显影术和 ADP-核糖基化对细胞进行分析 抑制剂。 该系统还将用于进行染色质比较 旨在产生有关稳定或改变的信息 涉及聚（ADP-核糖）的染色质结构和正常细胞周期 MNNG 处理细胞的进展。 聚（ADP-核糖）的参与 DNA损伤的其他重要生物反应中的代谢 将通过评估效果来研究 C3H10T1/2 细胞 ADP-核糖基化抑制剂和 NAD 水平的营养控制 诱变和恶性转化。 此类研究将利用 共细胞毒性效应将被消除或最小化的条件，并且 仔细监测表达期间对生长的影响。 其机制为 将研究热疗引起的聚（ADP-核糖）代谢，并 评估聚（ADP-核糖）代谢在 一般细胞会对环境压力做出反应。