DNA damage signaling in oxygen toxicity in lung cells

描述（由申请人提供）：已知高氧会产生可损伤DNA的活性氧（ROS）水平升高。细胞对DNA损伤的反应是复杂的，涉及识别DNA损伤并将信号传递给各种传感器的基因产物，这些传感器反过来抑制增殖、刺激修复或诱导凋亡。虽然已经研究了由氧化剂如过氧化氢（H2 O2）或其他过氧化物诱导的DNA损伤反应，但关于细胞如何在高氧下对DNA损伤作出反应的了解较少。阐明肺细胞对高氧DNA损伤的反应对于理解肺氧毒性至关重要。ATM（毛细血管共济失调）和ATR（ATM-Rad 3相关）是磷脂酰肌醇3-激酶相关激酶（PIKK）蛋白家族的成员，已被证明在暴露于遗传毒性物质时会抑制DNA损伤信号。本文提出的实验验证了ATR通过激活检查点蛋白p53和/或检查点激酶1（Chk 1）转导高氧介导的DNA损伤信号的假设。这导致cdc 25 C失活，其抑制cdc 2激酶活性，从而阻止细胞周期进展。首先，我们将确定ATR是否在肺细胞中的高氧中被激活。这将通过使用PIKK抑制剂，并通过涉及ATR或ATM的显性阴性构建体的遗传方法，以及使用ATM+/+或ATM-/-细胞来实现（目的1）。接下来，我们将确定高氧诱导的DNA损伤的性质以及与H2 O2或UV的不同之处（目的2）。在我们的下一个具体目标，我们将确定是否高氧特异性激活Chk 1直接或在ATR依赖的方式导致cdc 25 C失活。我们将使用Chk 1的特异性抑制剂和遗传学方法来描述Chk 1和cdc 25 C在高氧DNA损伤信号传导中的作用（目的3）。在我们的下一个具体目标（目标4）中，我们将确定cdc 2激酶活性失活的机制。我们将使用各种激酶和磷酸酶检测以及遗传和抑制剂研究来确定高氧中cdc 2抑制的机制。利用这些分子生物学方法，我们将确定DNA损伤信号转导机制，这将大大提高我们对肺细胞肺氧毒性的理解

DESCRIPTION (provided by applicant): Hyperoxia is known to produce elevated levels of reactive oxygen species (ROS) that can damage DNA. The cellular response to DNA damage is complex and involves gene-products that recognize DNA damage and transduce the signal to various sensors, which, in turn, inhibit proliferation, stimulate repair or induce apoptosis. Although the DNA damage response induced by oxidants such as hydrogen peroxide (H202) or other peroxides have been studied, less is known about how cells respond to DNA damage in hyperoxia. Elucidating how lung cells respond to hyperoxic DNA damage is critical for understanding pulmonary oxygen toxicity. ATM (ataxia telangiecea) and ATR (ATM-Rad3-related) are members of the phosphatidylinositol 3-kinase-related kinases (PIKK) family of proteins that have been shown to transduce DNA damage signals in response to exposure to genotoxic agents. The experiments proposed here test the hypothesis that ATR transduces hyperoxia-mediated DNA damage signals by activating checkpoint proteins p53 and/or checkpoint kinase1 (Chk1). This results in inactivation of cdc25C, which inhibits cdc2 kinase activity, thereby preventing cell cycle progression. First, we will determine whether ATR is activated in hyperoxia in lung cells. This will be achieved using inhibitors for PIKKs, and by genetic approaches involving dominant-negative constructs of ATR or ATM, and the use of ATM+/+ or ATM-/- cells (Aim 1). Next we will determine the nature of DNA damage induced in hyperoxia and how that differs from H202 or UV (Aim 2). In our next specific aim we will determine whether hyperoxia specifically activates Chk1 directly or in an ATR-dependent manner resulting in cdc25C inactivation. We will use specific inhibitors of Chk1 and genetic approaches to delineate the role of Chk1 and cdc25C in DNA damage signaling in hyperoxia (Aim 3). In our next specific aim (Aim 4) we will determine the mechanisms of inactivation of cdc2 kinase activity. We will use various kinase and phosphatase assays and genetic and inhibitor studies to define the mechanisms of inhibition of cdc2 in hyperoxia. Using these molecular approaches, we will define the mechanisms of DNA damage signaling n hyperoxia, which will significantly improve our understanding of pulmonary oxygen toxicity in lung cells