PATHWAYS TO ROS INDUCED CELL DEATH

描述(由申请人提供)：细胞死亡的机制通常分为两种途径，即凋亡和坏死。然而，美国毒理病理学家学会建议将其词根为“肿胀”的术语“肿胀”用作非凋亡途径引起的细胞死亡的替代描述。坏死更准确地描述了癌性细胞死亡的后果，通常是大量细胞的死亡，导致中度到严重的组织损伤。浮游症是细胞死亡的一种形式，通常发生在对毒性损伤的反应中，包括由化学物质暴露和活性氧(ROS)引起的损伤。ROS参与了与化学物质接触相关的各种人类疾病和毒性的发生和发展。了解调节细胞对ROS的反应的因素，以及它们与细胞成分相互作用的分子机制，以及这种相互作用的后果，是生物医学研究的重要基本目标。ROS的产生与许多病理疾病的发病机制有关。我们一直使用体外模型，在LLC-PK1细胞中加入TGHQ后原位产生H202，以研究肾近端小管上皮细胞对氧化剂诱导的损伤的细胞和分子反应。严格控制的作用是防止重复的DNA复制(S期停滞)，而不干预有丝分裂，或在DNA复制完成之前启动有丝分裂(G2M停滞)(“有丝分裂灾难”)。DNA损伤后这些细胞周期检查点的丢失可能允许过早进入有丝分裂。我们的初步数据表明，ROS诱导的ERK激活通过一种涉及过早染色质凝结(PCC)和过早进入有丝分裂的机制导致LLC-PK1细胞的死亡。为了检验四个相互关联的假说，本文提出了四个具体目标。(1)ERK激活通过激活下游的组蛋白H3激酶与PCC和有丝分裂灾难偶联。(2)PARP介导的组蛋白ADP-核糖化促进了组蛋白H3的磷酸化，这些翻译后修饰可能与额外的修饰结合在一起，是PCC和有丝分裂灾难所必需的。(3)ROS干扰DNA损伤检查点系统的一个或多个组件，促使细胞过早有丝分裂，随后因有丝分裂灾难而死亡。(4)ROS诱导细胞周期调节因子的核不适当移位，促进早熟有丝分裂。我们和其他人的数据表明，对通常导致肿瘤性细胞死亡(和组织坏死)的应激反应确实可以在遗传和药物水平上进行操纵，以产生潜在的有利(可存活的)组织反应。本申请中提出的实验旨在解决这种可能性。对ROS诱导细胞死亡机制的基础知识可能为临床干预ROS发挥重要作用的病理学提供策略

DESCRIPTION (provided by applicant): Mechanisms of cell death are usually classified into two pathways, apoptosis and necrosis. However, it has been proposed by the American Society of Toxicologic Pathologists that the term oncosis, with its root meaning of "swelling" be used as the alternate descriptor of cell death occurring by non-apoptotic pathways. Necrosis more accurately describes the consequences of oncotic cell death, usually the death of a large number of cells which results in moderate to severe tissue injury. Oncosis is a form of cell death that typically occurs in response to toxic injury, including that induced by chemical exposure and reactive oxygen species (ROS). ROS are involved in the initiation and progression of a variety of human diseases and toxicities associated with chemical exposure. An understanding of the factors that regulate the cellular response to ROS and of the molecular mechanisms by which they interact with cellular constituents, and the consequences of such interactions, are important fundamental goals of biomedical research. The generation of ROS has been implicated in the pathogenesis of many pathological conditions. We have been using an in vitro model, in which H202 is generated in situ following the addition of TGHQ to LLC-PK1 cells, to investigate the cellular and molecular response of renal proximal tubule epithelial cells to oxidant-induced injury. Rigid controls function to prevent repeated rounds of DNA replication (S-phase arrest) without intervening mitoses, or the initiation of mitosis (G2M arrest) before DNA replication is complete ("mitotic catastrophe"). Loss of these cell cycle checkpoints after DNA damage may permit premature entry into mitosis. Our Preliminary data indicate that ROS-induced ERK activation contributes to oncotic cell death of LLC-PK1 cells by a mechanism that involves premature chromatin condensatior (PCC) and premature entry into mitosis. Four Specific Aims are proposed to test four inter-related hypotheses. (1) ERK activation is coupled to PCC and mitotic catastrophe via the activation of downstream histone H3 kinases. (2) PARP mediated ADP-ribosylation of histones facilitates histone H3 phosphorylation, and these post-translational modifications, perhaps in combination with additional modifications, are required for PCC and mitotic catastrophe. (3) ROS interfere with one or more components of the DNA damage check point system, driving the cells into premature mitosis, and subsequently death by mitotic catastrophe. (4) ROS induce the inappropriate nuclear translocation of cell cycle regulators, promoting premature mitosis. Our data, and that of others, indicate that responses to stress that usually result in oncotic cell death (and tissue necrosis) can indeed be manipulated, at the genetic and pharmacological level, to produce a potentially favorable (survivable) tissue response. Experiments proposed in the present application are designed to address this possibility. Basic knowledge of the mechanisms by which ROS induce cell death may yield strategies for clinical interventions in pathologies in which ROS play a prominent role