RELATIONSHIP BETWEEN DNA DAMAGE AND CELLULAR SENESCENCE

新研究者-研究目标：基因表达载体：开发和/或使用条件基因表达载体来调节衰老和细胞特异性基因表达。该项目的目标是开发一种载体，当在原代人培养细胞中表达时，将增加细胞衰老的速度。许多研究将DNA氧化损伤与衰老过程联系起来，并表明线粒体DNA的损伤可能是一个关键因素。然而，用氧化剂处理细胞损伤DNA会导致脂质和蛋白质氧化。我们正在开发两种反义RNA表达载体来减少线粒体或核dUTPase。在缺乏这种酶的情况下，dUTP被整合到基因组中。对细菌dUTP酶突变体的研究表明，碱基切除修复途径试图去除dUTP，导致DNA断裂和生长抑制。因此，这项工作将建立一个模型系统，其中线粒体或核基因组特异性地产生损伤，而不损害脂质或蛋白质。我们假设线粒体DNA损伤会导致线粒体功能障碍、细胞氧化应激增强和培养原代细胞复制寿命缩短。每个反义RNA特异性降低线粒体或核dUTPase的能力将首先通过与核或线粒体dUTPase-绿色荧光蛋白融合的双向启动子结合在转化细胞中瞬时表达RNA来测试。提取的细胞将被用来测量融合的表达。在确定dUTPase的下调效率后，逆转录病毒载体中的反义RNA序列将转化为逆转录病毒，用于感染原代人细胞（群体增加约15倍）。通过感染传递将确保表达载体在细胞群中以较高的频率整合到核基因组中。将检测受感染人群的线粒体功能以及核和线粒体DNA的断裂程度。这些细胞将在培养中保存，并评估与衰老相关的β -半乳糖苷酶表达和其他与衰老相关的基因表达变化，以及生长速度和细胞周期的变化。这项研究将更好地确定线粒体或核DNA损伤与复制性衰老之间的关系。

New Investigator - Research objectives: 9. Gene expression vectors: Development and/or use of conditional gene expression vectors for the regulation of aging-and cell-specific gene expression. The goal of this project is to develop a vector which when expressed in primary human cultured cells will increase the rate of cellular senescence. Many studies have correlated oxidative DNA damage with the aging process and it has been suggested that damage specifically to mitochondrial DNA may be a key factor. However, treating cells with oxidizing agents to damage DNA results in lipid and protein oxidation. We are developing two antisense RNA expression vectors to decrease either mitochondrial or nuclear dUTPase. In the absence of this enzyme, dUTP is incorporated into the genome. Studies using bacterial dUTPase mutants indicate that the base excision repair pathway attempts to remove the dUTP resulting in DNA fragmentation and inhibition of growth. This work will therefore establish a model system in which damage is specifically produced in either the mitochondrial or nuclear genome without damaging lipids or proteins. We hypothesize that mitochondrial DNA damage will result in mitochondria dysfunction, an enhanced cellular oxidative stress and a decrease in the replicative life span of primary cells in culture. The ability of each antisense RNA to specifically decrease mitochondrial or nuclear dUTPase will be first tested by transiently expressing the RNA in transformed cells from a bi-directional promoter in combination with either a nuclear or mitochondrial dUTPase-green fluorescent protein fusion. Extracts from the cells will be used to measure the expression of the fusion. Having established the efficiency of down-regulation of dUTPase, the sequence of the antisense RNA in a retroviral vector will be converted to retrovirus and used to infect primary human cells (population doubling approximately 15). Delivery by infection will ensure the expression vectors integrate into the nuclear genome at a high frequency in the cell population. The infected population will be tested for mitochondria function and the level of fragmentation of nuclear and mitochondrial DNA. The cells will be maintained in culture and assessed for senescent-associated expression of beta-galactosidase and other senescent-related changes in gene expression, as well as alteration in growth rate and cell cycle. This study will better define the relationship between mitochondrial or nuclear DNA damage and replicative senescence.