Role of Prdx 1 in Natural Killer Cells and Tumorigenesis

描述(由申请人提供) 这一建议将有助于理解抗氧化蛋白Peroxiredox1(Prdx1)在自然杀伤细胞功能和肿瘤易感性中的作用。活性氧(ROS)是导致衰老和许多疾病的原因之一，如动脉粥样硬化、自身免疫性疾病、神经元变性和癌症。抗氧化剂的作用之一是平衡氧化还原动态平衡，以避免破坏过量的ROS。Prdx1也是已知的来自红细胞(RBC)的自然杀伤(NK)细胞激活剂。小鼠中Prdx1的缺失会导致溶血性贫血和癌症导致的过早死亡。Prdx1基因缺陷小鼠在胚胎成纤维细胞(MEF)中表现出NK细胞活性降低和细胞损伤，如蛋白质氧化和DNA损伤。Prdx1基因缺陷小鼠为研究ROS在疾病发展中的作用提供了一个很好的工具。 目的1.明确Prdx1在自然杀伤细胞激活中的作用。目前尚不完全清楚外源性Prdx1是如何激活NK细胞的。因此，Prdx1可能由红细胞分泌，并与NK细胞膜或胞浆共定位。定位研究将通过使用荧光激活细胞分类器(FACS)分析、免疫荧光或亚细胞分级进行。用荧光染料检测NK细胞的ROS含量、pH和钙(Ca~(2+))含量。NK细胞的信号转导将通过Western blotting进行研究。红细胞和NK细胞需要Prdx1来表达肿瘤坏死因子α，我将研究这种产生是否依赖于Prdx1的抗氧化活性。所有研究将包括来自Prdx1野生型和缺失小鼠的红细胞和NK细胞以及重组Prdx1。 目的2将研究Prdx1和氧化应激源作为细胞转化的特定调节因素。ROS是否在细胞转化中起着特定的作用，目前还不完全清楚。将Prdx1野生型和空白小鼠的MEF用H202慢性应激，并用致癌RAS转化，以检测ROS是否能增强RAS诱导的转化。肿瘤抑制基因P53是已知的氧化还原敏感蛋白。为了研究ROS是否影响P53，以及这是否会导致RAS对转化敏感性的增强，将P53和Prdx1基因缺陷小鼠的原代MEF经H202和致癌RAS慢性应激处理后进行转化。

DESCRIPTION (provided by applicant) This proposal will help to understand the role of the antioxidant protein peroxiredoxin 1 (Prdx1) in natural killer cell function and tumor susceptibility. Reactive oxygen species (ROS) are one cause of aging and many diseases such as atherosclerosis, autoimmune disorders, neuronal degeneration and cancer. One role of antioxidants is to balance the redox homeostasis to avoid damaging excessive levels of ROS. Prdx1 is also known to be a natural killer (NK) cell activator from red blood cells (RBCs). Loss of Prdx1 in mice leads to premature death due to hemolytic anemia and cancer. Prdx1 deficient mice show decreased NK cell activity and increased cellular damage such as protein oxidation and DNA damage in murine embryonic fibroblasts (MEFs). The Prdx1 deficient mice provide an excellent tool to study the impact of ROS in disease development. Aim 1. To define the role of Prdx1 in natural killer cell activation. It is not fully understood how exogenous Prdx1 activates NK cells. Therefore, it will be demonstrated that Prdx1 is probably secreted by RBCs and co-localizes with the NK cell membrane or cytosol. Localization studies will be done by using fluorescence activated cell sorter (FACS) analysis, immunofluorescence or subcellular fractionation. NK cells will be examined for their ROS content, pH and calcium (Ca2+) content by using fluorescence dyes. Signaling of NK cells will be studied by Western blotting. RBCs and NK cells need Prdx1 to express Tumor Necrosis Factor alpha, I will study whether this production is dependent on Prdx1 antioxidant activity. All studies will include RBCs and NK cells from Prdx1 wildtype and null mice and recombinant Prdx1. Aim 2 will investigate Prdx1 and oxidative stressors as specific regulators of cellular transformation. It is not fully understood whether ROS play a specific role in cellular transformation. MEFs from Prdx1 wildtype and null mice will be chronically stressed with H202 and transformed with oncogenic Ras to test whether ROS can enhance Ras induced-transformation. The tumor suppressor gene p53 is known to be a redox sensitive protein. To study whether p53 is affected by ROS and whether this can lead to enhanced transformation susceptibility by Ras, primary MEFs from p53 and Prdx1 deficient mice will be transformed after chronic stress treatment with H202 and oncogenic Ras.