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） 该提案将有助于了解抗氧化蛋白过氧化还原蛋白1（Prdx1）在自然杀伤细胞功能和肿瘤易感性中的作用。活性氧（ROS）是衰老和许多疾病的原因之一，例如动脉粥样硬化、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稳态，以避免破坏过量的活性氧。 Prdx1 还被认为是红细胞 (RBC) 中的自然杀伤 (NK) 细胞激活剂。小鼠失去 Prdx1 会导致因溶血性贫血和癌症而过早死亡。 Prdx1 缺陷小鼠表现出 NK 细胞活性降低和细胞损伤增加，例如小鼠胚胎成纤维细胞 (MEF) 中的蛋白质氧化和 DNA 损伤。 Prdx1 缺陷小鼠为研究 ROS 在疾病发展中的影响提供了一个极好的工具。 目标 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 如何激活 NK 细胞。因此，将证明Prdx1可能由红细胞分泌并与NK细胞膜或细胞质共定位。定位研究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来完成。将使用荧光染料检查 NK 细胞的 ROS 含量、pH 值和钙 (Ca2+) 含量。 NK 细胞的信号传导将通过蛋白质印迹法进行研究。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抗氧化活性。所有研究将包括来自 Prdx1 野生型和无效小鼠以及重组 Prdx1 的红细胞和 NK 细胞。 目标 2 将研究 Prdx1 和氧化应激源作为细胞转化的特定调节因子。 ROS 是否在细胞转化中发挥特定作用尚不完全清楚。来自 Prdx1 野生型和无效小鼠的 MEF 将受到 H2O2 的长期应激，并用致癌 Ras 进行转化，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是一种氧化还原敏感蛋白。为了研究 p53 是否受到 ROS 的影响以及这是否会导致 Ras 转化敏感性增强，来自 p53 和 Prdx1 缺陷小鼠的原代 MEF 在用 H2O2 和致癌 Ras 进行慢性应激处理后将发生转化。