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损伤并将信号转导到各种传感器的基因产物，这些传感器反过来抑制增殖，刺激修复或诱导细胞凋亡。虽然过氧化氢（H202）或其他过氧化物等氧化剂诱导的DNA损伤反应已经被研究过，但对高氧条件下细胞如何对DNA损伤作出反应尚不清楚。阐明肺细胞对高氧DNA损伤的反应是理解肺氧毒性的关键。ATM （ataxia telangiecea）和ATR （ATM- rad3相关）是磷脂酰肌醇3激酶相关激酶（PIKK）蛋白家族的成员，已被证明在暴露于基因毒性物质时可转导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 （Chk1）来转导高氧介导的DNA损伤信号的假设。这导致cdc25C失活，从而抑制cdc2激酶活性，从而阻止细胞周期的进展。首先，我们将确定ATR是否在肺细胞高氧状态下被激活。这将通过使用PIKKs抑制剂，通过涉及ATR或ATM的显性阴性结构的遗传方法以及使用ATM+/+或ATM-/-细胞来实现（Aim 1）。接下来，我们将确定高氧诱导的DNA损伤的性质，以及它与H202或紫外线的区别（目的2）。在我们的下一个具体目标中，我们将确定高氧是直接特异性激活Chk1还是以atr依赖的方式导致cdc25C失活。我们将使用Chk1的特异性抑制剂和遗传学方法来描述Chk1和cdc25C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cdc2激酶活性失活的机制。我们将使用各种激酶和磷酸酶测定以及遗传和抑制剂研究来确定高氧条件下cdc2抑制的机制。利用这些分子方法，我们将定义DNA损伤信号在高氧下的机制，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