ALTERATION OF NAD METABOLISM BY CHEMICAL CARCINOGENS

DNA损伤的发生和细胞对DNA损伤的反应是 参与启动和可能的推广阶段 致癌。拟议的研究将集中在机制和 聚腺苷二磷酸核糖快速变化的生物学意义 由环境引起的DNA损伤引起的新陈代谢。这些研究将 利用原代培养的小鼠细胞 N-甲基-N‘-硝基-N亚硝胺(MNNG)。聚合体的复杂性 ADP-核糖的研究将通过高度选择性地分离聚合物来进行 高效液相分子筛分级法制备硼酸亲和树脂 直链和支链的层析及同时测定 内部残基和末端残基。这样的实验将允许 测定每个分子的真实聚合物尺寸和支化频率， 评估非共价的重要性所需的信息 该聚合物与染色质的其他成分的相互作用。这个 聚合物的周转及其在染色质组分中的分布将 通过将上述方法与体内标记方案相结合进行研究 和聚腺苷二磷酸核糖聚合酶的抑制剂。标签方法也将 用于评价聚(ADP-核糖)的定量意义 未受损细胞的新陈代谢。聚腺苷二磷酸核糖的功能 新陈代谢在细胞中从MNNG的细胞毒效应中恢复 通过对同步化C3H10T1/2的细胞周期进程的研究来解决 流式细胞荧光法、放射自显影和ADP核糖化 抑制剂。这个系统也将被用来进行染色质比较 旨在提供关于稳定化或改变的信息 染色质结构涉及多(ADP-核糖)和正常细胞周期 MNNG处理细胞的研究进展。聚腺苷二磷酸核糖的参与 对DNA损伤的其他重要生物反应中的代谢 C3H10T1/2细胞的研究将通过评估 腺苷二磷酸核糖基化抑制剂和营养调控NAD水平的研究 诱变和恶变。这样的研究将利用 消除或最小化共细胞毒性效应的条件以及 在表达过程中对生长的影响被仔细监测。它的作用机制 将研究高温下聚(ADP-核糖)的代谢，并对其进行分析 多聚(ADP-核糖)代谢在急性胰腺炎中可能的一般作用的评估 我们将对环境压力做出一般的细胞反应。

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.