OXIDATIVE DNA DAMAGE & OXYSTEROL INDUCED APOPTOSIS

这个项目的主要目的是研究线粒体在氧固醇诱导的过程中的作用。 人淋巴细胞性白血病细胞的凋亡。氧化甾醇是一类在不同细胞类型，特别是白血病淋巴样细胞中诱导细胞凋亡的强效诱导剂。氧化甾醇也是涉及胆固醇生物合成、细胞生长和细胞凋亡的基因的强大转录调节因子。尽管氧合甾醇诱导淋巴样细胞凋亡的分子基础尚不清楚，但已有研究表明，氧合甾醇诱导的细胞凋亡是通过产生活性氧来实现的。前一个资助周期的研究结果表明，氧化甾醇处理CEM细胞会导致线粒体ATP水平下降和线粒体膜电位丧失。这个项目将验证这样的假设，即ROS损伤，特别是对线粒体DNA的损伤，在氧固醇诱导的人类白血病细胞凋亡中起到了作用。我们的假设预测，线粒体DNA的氧化损伤可以导致线粒体功能障碍和诱导细胞凋亡。为了验证这一假说，我们将：1)确定氧化甾醇敏感的人淋巴样细胞在暴露于氧化甾醇后，对核和线粒体DNA的氧化损伤的形成和修复是否高于氧化甾醇抵抗细胞；2)确定氧化甾醇敏感和耐药的人淋巴细胞在氧化甾醇暴露后的线粒体功能和功能障碍；以及3)确定氧化应激、DNA修复和凋亡相关基因在氧化甾醇诱导的淋巴细胞凋亡过程中的表达谱。该项目将有助于为一项旨在了解线粒体在细胞凋亡过程中的作用的研究项目奠定基础。

The major objective of this project is to study the role of mitochondria in oxysterol-induced apoptosis of human lymphoblastic leukemia cells. Oxysterols represent a class of potent inducers of apoptosis in various cell types, particularly leukemic lymphoid cells. Oxysterols also act as powerful transcriptional regulators of genes involved in cholesterol biosynthesis, cell growth and apoptosis. Although the molecular basis of oxysterol-induced apoptosis of lymphoid cells remains unknown, some studies have suggested that oxysterol induction of apoptosis is mediated by reactive oxygen species generation. Results obtained in the previous funding cycle show that oxysterol treatment of CEM cells induces a decrease in ATP levels and loss of mitochondrial membrane potential. This project will test the hypothesis that ROS damage particularly to mitochondrial DNA plays a role in oxysterol-induced apoptosis of human leukemia cells. Our hypothesis predicts that oxidative damage to mitochondrial DNA can lead to mitochondrial dysfunction and induction of apoptosis. To test this hypothesis we will: 1) determine whether the formation and repair of oxidative damage to nuclear and mitochondrial DNA are higher in oxysterol sensitive human lymphoid cells than in oxysterol-resistant cells after exposure to oxysterols, 2) determine mitochondrial function and dysfunction in oxysterol-sensitive and resistant human lymphoid cells after exposure to oxysterols, and 3) determine the expression profile of genes involved in oxidative stress, DNA repair and apoptosis during oxysterol-induced apoptosis of lymphoid cells. This project will assist in laying the foundation for a research project directed towards the understanding of the role of mitochondria in the process of apoptosis.