Role of Prdx 1 in Natural Killer Cells and Tumorigenesis

Prdx 1 在自然杀伤细胞和肿瘤发生中的作用

• 批准号: 6780310
• 负责人: CAROLA ANKE NEUMANN
• 金额: $ 10.8万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-02 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6780310
• 关键词: aging; antioxidants; athymic mouse; biological signal transduction; calcium flux; chromophore; endocytosis; enzyme linked immunosorbent assay; erythrocytes; fibroblasts; flow cytometry; free radical oxygen; guanine nucleotide binding protein; hydrogen peroxide; immunofluorescence technique; laboratory mouse; leukocyte activation /transformation; natural killer cells; neoplasm /cancer immunology; neoplastic transformation; oxidation reduction reaction; p53 gene /protein; peroxidases; protein localization; tissue /cell culture; transfection /expression vector; tumor necrosis factor alpha; western blottings

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.

描述（由申请人提供） 该提议将有助于了解抗氧化蛋白过氧化物氧还蛋白1（Prdx 1）在自然杀伤细胞功能和肿瘤易感性中的作用。活性氧（ROS）是衰老和许多疾病如动脉粥样硬化、自身免疫性疾病、神经元变性和癌症的原因之一。抗氧化剂的一个作用是平衡氧化还原稳态，以避免破坏ROS的过度水平。Prdx1也被认为是来自红细胞（RBC）的自然杀伤（NK）细胞激活剂。小鼠Prdx 1的缺失会导致溶血性贫血和癌症导致过早死亡。Prdx 1缺陷小鼠表现出NK细胞活性降低和细胞损伤增加，如小鼠胚胎成纤维细胞（MEF）中的蛋白质氧化和DNA损伤。Prdx 1缺陷小鼠为研究ROS在疾病发展中的影响提供了一个很好的工具。 目标1.目的：明确Prdx1在自然杀伤细胞激活中的作用。目前还不完全清楚外源性Prdx 1如何激活NK细胞。因此，将证明Prdx 1可能由RBC分泌并与NK细胞膜或胞质溶胶共定位。将通过使用荧光激活细胞分选仪（FACS）分析、免疫荧光或亚细胞分级分离进行定位研究。将通过使用荧光染料检查NK细胞的ROS含量、pH和钙（Ca2+）含量。将通过蛋白质印迹法研究NK细胞的信号传导。红细胞和NK细胞需要Prdx 1来表达肿瘤坏死因子α，我将研究这种产生是否依赖于Prdx 1的抗氧化活性。所有研究将包括来自Prdx 1野生型和无效小鼠以及重组Prdx 1的RBC和NK细胞。 目的2将研究Prdx1和氧化应激作为细胞转化的特异性调节因子。ROS是否在细胞转化中发挥特定作用尚不完全清楚。将来自Prdx 1野生型和缺失小鼠的MEF用H2O2长期应激并用致癌Ras转化以测试ROS是否可以增强Ras诱导的转化。已知肿瘤抑制基因p53是氧化还原敏感蛋白。为了研究p53是否受到ROS的影响以及这是否可以导致Ras的转化敏感性增强，来自p53和Prdx 1缺陷小鼠的原代MEF将在用H2O2和致癌Ras慢性应激处理后转化。