Mitochondrial SOD & Breast Cancer Risk-Mechanism

线粒体SOD

• 批准号: 6605783
• 负责人: JAMES Donald YAGER
• 金额: $ 8.18万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6605783
• 关键词: DNA damage; biological transport; breast neoplasm /cancer diagnosis; breast neoplasms; cancer risk; cell line; enzyme activity; enzyme mechanism; genetic polymorphism; genotype; glutathione peroxidase; hydrogen peroxide; luminescence; mitochondrial disease /disorder; oxidative stress; polymerase chain reaction; protein structure function; superoxide dismutase; superoxides; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant)The Val-9Ala polymorphism affects the secondary structure of the import sequence in mitochondrial manganese superoxide dismutase (MnSOD). The protein encoded by the Val allele exhibited decreased import in isolated rat liver mitochondria. Furthermore, Val homozygotes had an increased risk of nonfamilial cardiomyopathy in Japanese patients. In contrast, the Ala/Ala genotype was associated with increased risk of breast cancer in premenopausal women. The goals of this project are to develop preliminary results pertaining to the effects of these two MnSODs on mitochondrial and cellular levels of reactive oxygen species (ROS) that would lead to future in depth studies on how the Ala/Ala genotype may contribute to increased breast cancer risk. The Specific Aims of this project are to: 1) Determine the relationship between mitochondrial MnSOD protein levels, MnSOD activity, and superoxide and hydrogen peroxide production. Mitochondria will be isolated from several human breast cell lines with different MnSOD genotypes. The specific activity of MnSOD will be determined by determining enzyme activity and protein levels by Western blotting. The activity of mitochondrial glutathione peroxidase and GSH/GSSL levels will also be determined. 02 consumption and superoxide and hydrogen peroxide production will be determined using chemiluminescent probes during incubation under basal conditions that support mitochondrial respiration and upon treatment to induce oxidative stress. 2) Determine if the mitochondrial import of MnSOD Ala and MnSOD Val differ in mitochondria isolated from human breast cells and, through cross incubations, that the differences are solely due to the MnSOD import sequence differences as opposed to differences in mitochondria protein import capabilities. Mitochondria will be isolated from human breast cell lines, homozygous and heterozygous for the polymorphisms and import rates and levels determined using fusion proteins comprised of the Ala or Val import domains linked to a reporter. The Val and Ala constructs will each be incubated with mitochondria from Val/Val Val/Ala and Ala/Ala cells for comparison. 3) Determine the relationships among MnSOD protein levels, MnSOD activity, and markers of oxidative damage in Ala/Ala, Val/Val, and Val/Ala intact breast cells. Basal and induced levels of mitochondria superoxide, cellular hydrogen peroxide, mitochondrial DNA oxidative damage, protein oxidative damage (i.e. protein carbonyls), and activities of iron-cluster proteins (i.e. aconitase) will be compared and related to MnSOD protein level and activity. 4) From a single breast cell line, develop cell sublines expressing either MnSOD Ala or MnSOD Val to provide a cell culture model for future in depth investigation of potential mechanisms behind the increased breast cancer risk associated with the Ala/Ala genotype. Based on the results of the studies to be conducted in aims 1-3, an appropriate breast cell line(s) will be selected and a gene knock out approach used to develop the sublines referred to. The advantage of this approach is that it will provide two sublines of identical genetic backgrounds and biochemistry except for the differences in the MnSOD import sequences

描述（由申请人提供）瓦尔-9 Ala多态性影响线粒体锰超氧化物歧化酶（MnSOD）中输入序列的二级结构。由瓦尔等位基因编码的蛋白质在离体大鼠肝线粒体中表现出进口减少。此外，日本患者中，瓦尔纯合子患非家族性心肌病的风险增加。相反，Ala/Ala基因型与绝经前妇女乳腺癌风险增加相关。该项目的目标是开发有关这两种MnSOD对活性氧（ROS）的线粒体和细胞水平的影响的初步结果，这将导致未来深入研究Ala/Ala基因型如何导致乳腺癌风险增加。本研究的具体目的是：1）确定线粒体MnSOD蛋白水平、MnSOD活性与超氧化物和过氧化氢产生的关系。将从具有不同MnSOD基因型的几种人乳腺细胞系中分离线粒体。MnSOD的比活性将通过Western印迹法测定酶活性和蛋白质水平来测定。还将测定线粒体谷胱甘肽过氧化物酶活性和GSH/GSSL水平。在支持线粒体呼吸的基础条件下孵育期间和诱导氧化应激的处理后，使用荧光探针测定O2消耗以及超氧化物和过氧化氢的产生。2)确定从人乳腺细胞分离的线粒体中MnSOD Ala和MnSOD瓦尔的线粒体输入是否不同，并通过交叉孵育确定差异仅是由于MnSOD输入序列差异而不是线粒体蛋白输入能力差异。将从人乳腺细胞系中分离线粒体，对于多态性和输入速率和水平，使用由连接至报告基因的Ala或瓦尔输入结构域组成的融合蛋白测定纯合和杂合。将瓦尔和Ala构建体各自与来自瓦尔/瓦尔瓦尔/Ala和Ala/Ala细胞的线粒体一起孵育以进行比较。3)确定MnSOD蛋白水平、MnSOD活性和Ala/Ala、瓦尔/瓦尔和瓦尔/Ala完整乳腺细胞中氧化损伤标志物之间的关系。将比较线粒体超氧化物、细胞过氧化氢、线粒体DNA氧化损伤、蛋白质氧化损伤（即蛋白质羰基）和铁簇蛋白（即乌头酸酶）活性的基础和诱导水平，并将其与MnSOD蛋白水平和活性相关。4)从单个乳腺细胞系中，开发表达MnSOD Ala或MnSOD瓦尔的细胞亚系，为将来深入研究与Ala/Ala基因型相关的乳腺癌风险增加背后的潜在机制提供细胞培养模型。根据目标1-3中的研究结果，将选择适当的乳腺细胞系，并使用基因敲除方法开发所述亚系。这种方法的优点是，它将提供两个亚系相同的遗传背景和生物化学，除了在MnSOD输入序列的差异