PROJECT 3 : QUANTITATIVE BIOMARKERS OF DNA AND PROTEIN OXIDATION

项目 3：DNA 和蛋白质氧化的定量生物标志物

• 批准号: 7358991
• 负责人: Paul Thomas Henderson
• 金额: $ 27.62万
• 依托单位: UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7358991
• 关键词: PROJECT; QUANTITATIVE; BIOMARKERS; DNA; PROTEIN

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Project 3 is developing methods to quantify oxidative modifications in DNA, proteins and metabolites. This year we have focused on introducing 14C isotope labels into biological molecules in order to characterize pathways and biomarkers of relevance to human disease. This labeling is done by either dosing cells or animals with a labeled building block such as an amino acid for incorporation into proteins or a nucleoside for synthesis of DNA. The presence of an oxidized nucleoside, 8-oxodG, in DNA acts a indicator of oxidative stress and it is more sensitive to further oxidation compared to the normal nucleosides G, C, A and T. For the DNA oxidation studies, we synthesized a 14C labeled 8-oxodG nucleoside. We dosed growing MCF-7 human breast cancer cells with the [14C]8-oxodG compound and monitored uptake of 14C into the newly synthesized DNA. Growth of these labeled cells in the presence of the hormone 17-?-estradiol (a naturally occurring carcinogen that plays a role in breast cell proliferation) caused further oxidation of the labeled 8-oxodG in the cellular DNA to form several novel mutagenic products. This result indicates that oxidative stress in breast cancer cells induced by hormones can induce the formation of novel types of mutagenic DNA damage that may play a strong role in breast cancer initiation and progression. Additionally, radiocarbon from the labeled 8-oxodG was incorporated into RNA, which indicates that 8-oxodG in the nucleotide pool may be a source for protein truncation or altered function as a result of the oxidized nucleotide interfering with transcription and possibly translation. Collaborators on this project include Jeffrey Gregg at the UC Davis Cancer Center and Cynthia Burrows at the University of Utah Department of Chemistry. This work has generated two manuscripts that were submitted for publication. One is currently under review for publication in the Nuclear Instrumentation Methods, Physics Research B and for the other, peer review is pendingfor publication in Nature. Protein oxidation is relevant to diseases such as arteriosclerosis. The hypothesis that chemical species generated by white blood cells react with DNA, protein and other biomolecules to generate a host of cytotoxic and genotoxic products. One type of oxidative damage arises from myeloperoxidase in activated macrophages that produce hypochlorous acid (HOCl), the active ingredient in bleach, which chlorinates tyrosine residues in proteins. Such tyrosine chlorination events can inactivate the proteins and enzymes. For example, ApoA1, a component of low density lipoprotein known as ¿the good cholesterol¿, contains a tyrosine residue that when chlorinated, completely abrogates the ability of the protein to bind cholesterol in the blood stream. We are developing a method to quantitate chlorinated tyrosine in proteins in which tyrosine residues are labeled with 14C-containing acetate groups. A change in retention time during chromatography can allow separation of labeled chlorotyrosine-containing peptide fragments from the pristine parent peptides. Last year we hired post-doctoral fellow Dr. Janna Mundt to carryout these studies. She has successfully recapitulated the production of chlorinated peptides and developed a postlabeling protocol that was successful for a model peptide. Once the postlabeling method has been demonstrated on ApoA1 derived from clinical samples (from our collaborator Jay Heinecke at the University of Washington Medical Center) a manuscript will be submitted for publication.

该主题项目是利用NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是针对该中心的，这不是调查人员的机构。项目3正在开发定量DNA，蛋白质和代谢物中氧化修饰的方法。今年，我们专注于将14C同位素标记引入生物学分子中，以表征与人类疾病相关的途径和生物标志物。该标记是通过用剂量细胞或具有标记的构建块的动物来完成的，例如企业中的氨基酸或用于合成DNA的核苷的氨基酸。 DNA中的氧化核侧面8-氧化物的存在作用是氧化应激的指标，并且与正常的核苷G，C，A和T相比，它对进一步的氧化更为敏感。对于DNA氧化研究，我们合成了14C标记的8-OxoDG核氧化研究。我们确实用[14C] 8-OXODG化合物生长了MCF-7人类乳腺癌细胞，并监测了14C对新合成的DNA的摄取。这些标记的细胞的生长在马酮17 - ？ - 雌二醇（一种自然存在的致癌物中，在乳腺癌细胞增殖中起作用）导致细胞DNA中标记的8-氧化物的进一步氧化，形成几种新型诱变产物。该结果表明，激素诱导的乳腺癌细胞中的氧化应激可以诱导新型诱变DNA损伤的形成，这可能在乳腺癌的启动和进展中起着强烈的作用。另外，将来自标记为8-oxoDG的放射性碳掺入RNA中，这表明核丁基池中的8-氧化物可能是蛋白质截断的来源或由于氧化的核丁基干扰转录和可能的翻译而导致的功能改变了功能。该项目的合作者包括加州大学戴维斯分校癌症中心的Jeffrey Gregg和犹他大学化学系的Cynthia Burrows。这项工作产生了两个提交出版的手稿。一个目前正在核器械方法，物理学研究B中的出版物中进行审查，另一个正在审查中，同行评审本质上正在出版。蛋白质氧化与诸如动脉粥样硬化之类的疾病有关。白细胞产生的化学物种与DNA，蛋白质和其他生物分子产生的化学物种产生了大量的细胞毒性和遗传毒性产物。一种类型的氧化损伤是由激活的巨噬细胞中的髓过氧化物酶产生的，这些巨噬细胞产生了次伐多酸（HOCL），这是漂白剂中的活性剂量，它氯化酪氨酸在蛋白质中保留。这种酪氨酸氯化事件会使蛋白质和酶失活。例如，apoA1是低密度脂蛋白的成分，称为好胆固醇，含有酪氨酸居住地，当氯化时，氯化酚含量完全消除了蛋白质在血液中结合胆固醇的能力。我们正在开发一种在蛋白质中定量氯化酪氨酸的方法，其中酪氨酸保留用14C的乙酸基团标记。色谱过程中保留时间的变化可以使标记的含毒性酪氨酸肽片段与原始父母宠物分开。去年，我们聘请了博士后研究员詹娜·蒙特（Janna Mundt）博士进行了这些研究。她已经成功地概括了氯化肽的产生，并制定了一种成功的后标签方案，该方案成功用于模型肽。一旦在临床样本中衍生出的APOA1（来自我们华盛顿大学医学中心的Jay Heinecke）的APOA1证明了后标记方法，将提交手稿供出版。