ALTERATION OF NAD METABOLISM BY CHEMICAL CARCINOGENS

当细胞在DNA损伤后存活并逐渐丧失时，就会产生癌症 基因组的完整性。令人信服的证据表明，聚(ADP- 核糖聚合酶(PARP)和聚(ADP-核糖)糖水解酶(PARG)是 一个复杂的蛋白质网络的一部分，起到对抗损失的作用 通过检测DNA损伤和介导细胞 反应范围从DNA修复到恢复正常 通过细胞凋亡或细胞死亡来消除受损细胞的功能 坏死。这项提议中要检验的假设是 PARP和PARG的协调活动对于 保持基因组的完整性。具体目标1是确定 PARG的具体功能。这将由受控者完成 利用诱导性表达技术耗尽PARG的细胞内容 在3T3L1细胞中获得稳定表达的PARG反义克隆。 目的是探讨PARG缺失的表型。具体目标2是 确定是否需要PARP与PARG的最佳比例 调节细胞对DNA损伤的反应。蜂窝内容 通过可诱导的过度表达将逐渐提高PARG的 PARP含量正常的3T3L1细胞中PARG正义克隆的构建 以及来自含有被破坏的PARP基因的动物的细胞。在……里面 具体目标1和2，生化和生物功能 受PARG基因调控的影响将被评估。在 没有遗传毒性应激、细胞活力和固有基因组 稳定性将取决于菌落形成、生长速度和 姐妹染色单体交换频率。在基因毒性应激后，调制的影响 姐妹染色单体交换频率、染色体畸变率、DNA碱基切除 修复、P53功能和细胞毒反应，涉及细胞凋亡和 将确定是否有坏死。动力学变化的分析 NAD和ADP-核糖聚合物代谢的变化将被用来评估 PARG在这些细胞反应中的作用。具体目标3是 确定c DNA揭示的PARG结构的特定功能 克隆，包括假定的调控结构域和核位置 信号。将产生针对受调控结构域的抗体 为了追随它的细胞命运和这个领域的遗传调控， 用于搜索其功能。了解PARG的工作原理 对这项提案的长期目标至关重要，即 增强保护性细胞反应，保持基因组完整性 并设计针对这一途径的疗法，以促进 破坏癌细胞。

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.