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，凋亡和坏死。然而，美国毒理学病理学家协会提出，术语胀亡（其词根含义为“肿胀”）可用作通过非凋亡途径发生的细胞死亡的替代描述符。坏死更准确地描述了肿瘤细胞死亡的后果，通常是大量细胞死亡，导致中度至重度组织损伤。胀亡是细胞死亡的一种形式，其通常响应于毒性损伤而发生，包括由化学暴露和活性氧（ROS）诱导的毒性损伤。ROS参与了多种人类疾病的发生和发展以及与化学品暴露相关的毒性。了解调节细胞对ROS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的产生与许多病理状况的发病机制有关。我们一直在使用体外模型，其中H2 O2在LLC-PK 1细胞中加入TGHQ后原位产生，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。刚性控制的功能是防止重复的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（G2 M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激活有助于LLC-PK 1细胞的凋亡细胞死亡的机制，涉及过早的染色质凝聚（PCC）和过早进入有丝分裂。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和有丝分裂灾难偶联。(2)PARP介导的组蛋白的ADP-核糖基化促进组蛋白H3磷酸化，并且这些翻译后修饰，可能与另外的修饰组合，是PCC和有丝分裂灾难所需的。(3)ROS干扰DNA损伤检查点系统的一个或多个组分，驱使细胞进入过早的有丝分裂，随后通过有丝分裂灾难死亡。(4)ROS诱导细胞周期调节因子不适当的核转位，促进过早的有丝分裂。我们的数据和其他人的数据表明，通常导致肿瘤细胞死亡（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，以产生潜在的有利（存活）组织反应。本申请中提出的实验被设计为解决这种可能性。对活性氧诱导细胞死亡机制的基本认识可能为活性氧发挥重要作用的病理学的临床干预提供策略

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