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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一，该子项目和研究者 (PI) 可能已从其他 NIH 来源获得主要资助，因此可以在其他 CRISP 机构中得到体现。列出的是该中心，该中心不一定是研究人员的机构。项目 3 正在开发量化 DNA、蛋白质和代谢物氧化修饰的方法。今年，我们的重点是在生物分子中引入 14C 同位素标记。为了表征与人类疾病相关的途径和生物标志物，这种标记是通过给细胞或动物添加标记的构件来完成的，例如用于掺入蛋白质的氨基酸或用于合成DNA的核苷。 DNA 中的 8-oxodG 是氧化应激的指示剂，与正常核苷 G、C、A 和 T 相比，它对进一步氧化更敏感。在氧化研究中，我们合成了 14C 标记的 8-oxodG 核苷。我们将 [14C]8-oxodG 化合物给予正在生长的 MCF-7 人类乳腺癌细胞，并监测这些标记细胞中新合成的 DNA 中 14C 的摄取情况。激素 17-β-雌二醇（一种天然存在的致癌物质，在乳腺细胞增殖中发挥作用）的存在导致标记的进一步氧化8-oxodG 在细胞 DNA 中形成几种新型诱变产物，这一结果表明激素诱导的乳腺癌细胞中的氧化应激可诱导新型诱变 DNA 损伤的形成，这可能在乳腺癌的发生和进展中发挥重要作用。此外，标记的 8-oxodG 中的放射性碳被掺入 RNA 中，这表明核苷酸池中的 8-oxodG 可能是由于氧化核苷酸而导致蛋白质截断或功能改变的来源。该项目的合作者包括加州大学戴维斯分校癌症中心的杰弗里·格雷格（Jeffrey Gregg）和犹他大学化学系的辛西娅·伯罗斯（Cynthia Burrows）。这项工作已提交两份手稿，其中一份正在审查中。蛋白质氧化与动脉硬化等疾病相关的假设是白细胞产生的化学物质。与 DNA、蛋白质和其他生物分子发生反应，产生大量细胞毒性和基因毒性产物。一种氧化损伤是由活化巨噬细胞中的髓过氧化物酶产生的，该酶会产生次氯酸 (HOCl)，次氯酸是漂白剂的活性成分，可氯化蛋白质中的酪氨酸残基。这种酪氨酸氯化事件可以使蛋白质和酶失活，例如 ApoA1，它是低密度脂蛋白的一个组成部分。好胆固醇?? ，含有酪氨酸残基，当氯化时，该蛋白质会完全消除与血流中胆固醇结合的能力。我们正在开发一种定量蛋白质中氯化酪氨酸的方法，其中酪氨酸残基在保留中用含 14C 的乙酸基团进行标记。色谱过程中的时间可以使标记的含氯酪氨酸的肽片段与原始母肽分离。去年，我们聘请了博士后研究员 Janna Mundt 博士来进行这些研究，她成功地概括了氯化肽的生产，并开发了一种后标记方案，该方案在源自 ApoA1 的后标记方法上得到了成功。临床样本（来自我们华盛顿大学医学中心的合作者 Jay Heinecke）和手稿将提交出版。