PATHWAYS TO ROS INDUCED CELL DEATH

描述（申请人提供）：细胞死亡的机制通常分为两种途径：细胞凋亡和坏死。然而，美国毒理学病理学家协会提出，术语“肿瘤”（其根源为“肿胀”）可用作非凋亡途径发生的细胞死亡的替代描述词。坏死更准确地描述了胶体细胞死亡的后果，通常是大量细胞死亡，导致中度至重度组织损伤。肿瘤是细胞死亡的一种形式，通常因毒性损伤而发生，包括化学暴露和活性氧 (ROS) 引起的损伤。 ROS 参与多种人类疾病和与化学品接触相关的毒性的发生和进展。了解调节细胞对 ROS 反应的因素、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 的产生与许多病理状况的发病机制有关。我们一直在使用体外模型，在 LLC-PK1 细胞中添加 TGHQ 后原位产生 H2O2，以研究肾近曲小管上皮细胞对氧化剂诱导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会导致过早进入有丝分裂。我们的初步数据表明，ROS 诱导的 ERK 激活通过涉及染色质过早凝聚 (PCC) 和过早进入有丝分裂的机制导致 LLC-PK1 细胞的胶体细胞死亡。提出了四个具体目标来检验四个相互关联的假设。 (1) ERK 激活通过下游组蛋白 H3 激酶的激活与 PCC 和有丝分裂灾难耦合。 (2) PARP 介导的组蛋白 ADP-核糖基化促进组蛋白 H3 磷酸化，并且这些翻译后修饰（可能与其他修饰相结合）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种或多种成分，促使细胞过早有丝分裂，并随后因有丝分裂灾难而死亡。 (4)ROS诱导细胞周期调节因子不适当的核转位，促进过早有丝分裂。我们和其他人的数据表明，对通常导致胶体细胞死亡（和组织坏死）的应激反应确实可以在遗传和药理学水平上进行操纵，以产生潜在有利的（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。 ROS 诱导细胞死亡机制的基础知识可能会为 ROS 发挥重要作用的病理学临床干预提供策略

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