ALTERATION OF NAD METABOLISM BY CHEMICAL CARCINOGENS

化学致癌物改变 NAD 代谢

• 批准号: 6469170
• 负责人: MYRON K JACOBSON
• 金额: $ 23.59万
• 依托单位: UNIVERSITY OF ARIZONA
• 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 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6469170
• 关键词: ADP ribosylation; DNA damage; DNA repair; N methyl N' nitro N nitrosoguanidine; NAD nucleosidase; adenine phosphoribosyltransferase; animal genetic material tag; carcinogen testing; chemical carcinogenesis; enzyme activity; glycation; histones; laboratory mouse; laboratory rabbit; mutagen testing; mutagens; neoplasm /cancer genetics; nicotinamide adenine dinucleotide; nucleotide metabolism; radiation carcinogen; radiation carcinogenesis

Cancer results when cells survive DNA damage and progressively lose genomic integrity. Compelling evidence now demonstrates that poly(ADP- ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are part of a complex network of proteins that function to oppose the loss of genomic integrity by detecting DNA damage and mediating cellular responses, which can range from DNA repair leading to recovery of normal cell function to the elimination of damaged cells by apoptosis or necrosis. The hypothesis to be tested in this proposal is that the coordinated activities of PARP and PARG are essential for the maintenance of genomic integrity. Specific aim 1 is to determine specific functions of PARG. This will be accomplished by the controlled depletion of the cellular content of PARG, using inducible expression of a stably transfected antisense clone of PARG cDNA in 3T3L1 cells. The objective is to approach a PARG null phenotype. Specific aim 2 is to determine if an optimal ratio of PARP to PARG is required for modulation of cellular responses to DNA damage. The cellular content of PARG will be progressively elevated by the inducible overexpression of a sense clone of PARG in 3T3L1 cells with a normal content of PARP and in cells derived from animals containing a disrupted PARP gene. In both specific aims 1 and 2, biochemical and biological functions affected by the genetic modulation of PARG will be assessed. In the absence of genotoxic stress, cell viability and inherent genomic stability will be determined by colony formation, rates of growth, and SCE frequency. Following genotoxic stress, the effects of modulation on the frequency of SCE, chromosomal aberrations, DNA base excision repair, p53 function, and cytotoxic responses involving apoptosis and necrosis will be determined. Analysis of alterations of the kinetics of NAD and ADP-ribose polymer metabolism will be used to evaluate the function of PARG in these cellular responses. Specific aim 3 is to determine specific functions of PARG structures revealed by cDNA cloning, including a putative regulatory domain and a nuclear location signal. Antibodies specific to the regulatory domain will be produced to follow its cellular fate and genetic modulation of this domain will be used to search for its function. Understanding how PARG functions is essential to the long term objectives of this proposal, which are to enhance protective cellular responses that maintain genomic integrity and to design therapies that target this pathway to facilitate the destruction of cancer cells.

当细胞生存DNA损伤并逐渐失去时，癌症会产生 基因组完整性。 令人信服的证据现在表明poly（adp- 核糖）聚合酶（PARP）和聚（ADP-核糖）糖脂酶（PARG）为 复杂的蛋白质网络的一部分，该网络起作用以反对损失 通过检测DNA损伤和介导细胞来构成基因组完整性 反应，范围从DNA修复范围导致正常的恢复 细胞功能通过细胞凋亡消除受损细胞或 坏死。在此提议中要检验的假设是 PARP和PARG的协调活动对于 维持基因组完整性。 具体目标1是确定 PARG的特定功能。 这将由受控的 使用诱导表达的PARG的细胞含量的耗竭 在3T3L1细胞中PARG cDNA的稳定转染的反义克隆 目的是接近PARG NULL表型。 具体目标2是 确定是否需要PARP与PARG的最佳比率 调节细胞对DNA损伤的反应。 细胞含量 PARG的诱导过表达将逐步提高 在3T3L1细胞中具有正常含量PARP的PARG的感觉克隆 在源自包含pARP基因的动物的细胞中。 在 具体目标1和2，生化和生物学功能 将评估受PARG遗传调节的影响。在 缺乏遗传毒性应激，细胞活力和固有基因组 稳定性将由菌落形成，增长率和 SCE频率。 遵循遗传毒性应激，调节的影响 关于SCE的频率，染色体畸变，DNA碱基切除 维修，p53功能和涉及细胞凋亡和的细胞毒性反应 将确定坏死。 分析动力学的改变 NAD和ADP-核糖聚合物代谢将用于评估 PARG在这些细胞反应中的功能。 具体目标3是 确定cDNA揭示的PARG结构的特定功能 克隆，包括推定的监管域和核位置 信号。 将产生针对调节域的特定抗体 遵循其细胞命运和该域的遗传调制将 用于搜索其功能。 了解Parg如何功能 对于本提案的长期目标至关重要，这是 增强维持基因组完整性的保护性细胞反应 并设计针对这种途径以促进的疗法 癌细胞的破坏。