RELATIONSHIP BETWEEN DNA DAMAGE AND CELLULAR SENESCENCE

点击翻译按钮获取中文摘要

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.