PROJECT 3 : QUANTITATIVE BIOMARKERS OF DNA AND PROTEIN OXIDATION

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。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，我们的重点是将14C同位素标签引入生物分子，以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建块来完成的，例如用于结合到蛋白质中的氨基酸或用于合成DNA的核苷。DNA中氧化核苷8-oxodG的存在是氧化应激的一个指标，与正常核苷G、C、a和t相比，它对进一步氧化更敏感。对于DNA氧化研究，我们合成了一个14C标记的8-oxodG核苷。我们给生长中的MCF-7人乳腺癌细胞注射了[14C]8-氧化g化合物，并监测14C进入新合成的DNA的吸收情况。这些标记细胞在激素17-？雌二醇（一种在乳腺细胞增殖中起作用的天然致癌物）引起细胞DNA中标记的8-oxodG进一步氧化，形成几种新的致突变产物。这一结果表明，激素诱导的乳腺癌细胞氧化应激可诱导新型诱变DNA损伤的形成，这种损伤可能在乳腺癌的发生和发展中起重要作用。此外，来自标记的8-oxodG的放射性碳被结合到RNA中，这表明核苷酸池中的8-oxodG可能是蛋白质截断或功能改变的来源，因为氧化的核苷酸干扰转录和可能的翻译。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。这项工作产生了两份手稿，已提交出版。其中一篇论文目前正在接受《核仪器方法，物理研究B》杂志的评审，另一篇正在接受《自然》杂志的同行评审。蛋白质氧化与动脉硬化等疾病有关。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的假说。一种氧化损伤是由活化的巨噬细胞中的髓过氧化物酶引起的，它产生次氯酸（HOCl），这是漂白剂的活性成分，它使蛋白质中的酪氨酸残基氯化。酪氨酸氯化反应可使蛋白质和酶失活。例如，ApoA1是低密度脂蛋白的一种成分，被称为“好胆固醇”，它含有酪氨酸残留物，当氯化时，它完全剥夺了蛋白质在血液中结合胆固醇的能力。我们正在开发一种定量蛋白质中氯化酪氨酸的方法，其中酪氨酸残基用含14c的醋酸基团标记。色谱过程中保留时间的改变可以使标记的含氯酪氨酸肽片段从原始亲本肽中分离出来。去年，我们聘请了博士后Janna Mundt博士来进行这些研究。她成功地概括了氯化肽的生产，并开发了一种成功用于模型肽的后标记方案。一旦对临床样本（来自华盛顿大学医学中心的合作者Jay Heinecke）衍生的ApoA1证明了后标记方法，将提交手稿发表。

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.