Chemical Pathology of 5-aza-2'-deoxycytidine

5-氮杂-2-脱氧胞苷的化学病理学

• 批准号: 6909684
• 负责人: Lawrence C Sowers
• 金额: $ 5.14万
• 依托单位: LOMA LINDA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6909684
• 关键词: DNA methylation; N glycosidase; antineoplastics; antisense nucleic acid; azacitidine; chemical property; chemical stability; cytotoxicity; deoxycytidine; drug metabolism; gas chromatography mass spectrometry; high performance liquid chromatography; mutagens; nuclear magnetic resonance spectroscopy; pharmacokinetics; sickling inhibitor; solubility; synthetic nucleotide; tissue /cell culture

DESCRIPTION (provided by applicant): The nucleoside analogue, 5-aza-2'-deoxycytidine (DAC) is an investigational agent for the treatment of sickle cell anemia as well as advanced human malignancies. When administered, DAC is incorporated into the DNA of replicating cells. In DNA, DAC can act as an inhibitor of cytosine methyltransferase, resulting in the reactivation of the fetal hemoglobin gene in sickle cell anemia and tumor suppressor genes in human cancer. In addition to its effects on DNA methylation, DAC also induces cellular toxicity and triggers DNA repair responses. We are interested in studying the underlying chemical properties of DAC that may account for both the therapeutic and toxic activities. This amended proposal is divided into five aims. In the first aim, we will examine quantitatively the degradation of DAC in aqueous solution as a function of both temperature and pH, using a battery of UV, NMR, GC/MS and HPLC/MS methods. In the second aim, we will build DAC into synthetic oligonucleotides using established phosphoramidite methods. We will then study the stability of DAC in duplex DNA using a novel isotope-edited NMR technique. We suspect that DAC in DNA exists as an equilibrium between ring-closed and ring-opened forms, given that this equilibrium is known to occur for DAC in solution. A solid understanding of this equilibrium is essential to determining the potential mutagenicity, cytotoxicity and mechanism of demethylation induced by DAC. In aim three, we will examine the coding properties of DAC in oligonucleotides prepared by either chemical synthesis or DAC-triphosphate incorporation using purified DNA polymerases in an in vitro assay. We expect that the ring-closed form will base pair normally. The ring-opened forms are expected to direct rniscoding or act as a non-informational lesion. In aim four, we will examine the repair of DAC using an in vitro assay. DAC-containing oligonucleotides will be incubated with purified DNA glycosylases and whole cell extracts. We anticipate that the ring-opened forms of DAC will be rapidly removed by glycosylases. In aim five, we will measure DAC incorporation and 5-methylcytosine levels in cells grown in culture. This aim should establish an inverse correlation between incorporated DAC and methylated cytosine levels. We further propose testing a novel stable-isotope mass spectrometry (GC/MS) method that might reveal additional mechanisms by which DAC could induce genome demethylation. The results of the studies proposed here should shed significant light on the chemical properties of DAC that could be responsible for the array of biochemical effects induced by this compound in human cells.

描述(申请人提供)：核苷类似物5-氮杂-2‘-脱氧胞苷(DAC)是一种用于治疗镰状细胞性贫血和晚期人类恶性肿瘤的研究药物。当给药时，DAC被整合到复制细胞的DNA中。在DNA中，DAC可以作为胞嘧啶甲基转移酶的抑制剂，导致镰状细胞性贫血中的胎儿血红蛋白基因和人类癌症中的肿瘤抑制基因重新激活。除了对DNA甲基化的影响外，DAC还可以诱导细胞毒性并触发DNA修复反应。我们有兴趣研究DAC的潜在化学性质，这可能是治疗和毒性活性的原因。这项修订后的建议分为五个目标。在第一个目标中，我们将使用一系列的UV、核磁共振、GC/MS和LC/MS方法来定量研究DAC在水溶液中的降解随温度和pH的变化。在第二个目标中，我们将使用已有的亚磷酰胺方法将DAC构建成合成的寡核苷酸。然后，我们将使用一种新的同位素编辑核磁共振技术来研究DAC在双链DNA中的稳定性。我们怀疑DNA中的DAC以环闭和环开之间的平衡形式存在，因为已知DAC在溶液中也存在这种平衡。对这一平衡的深入理解对于确定DAC的潜在致突变性、细胞毒性和去甲基化机制是至关重要的。在第三个目标中，我们将在体外实验中检测通过化学合成或DAC-三磷酸掺入方法制备的寡核苷酸中DAC的编码特性。我们预计闭环形式将正常碱基对。开环表格预计将指导rniscode或作为非信息性损害。在第四个目标中，我们将使用体外实验来检测DAC的修复。含有DAC的寡核苷酸将与纯化的DNA糖基酶和全细胞提取物孵育。我们预计开环形式的DAC将被糖基酶迅速去除。在第五个目标中，我们将测量培养细胞中的DAC掺入和5-甲基胞嘧啶水平。这一目标应该在掺入的DAC和甲基化的胞嘧啶水平之间建立负相关关系。我们进一步建议测试一种新的稳定同位素质谱学(GC/MS)方法，该方法可能揭示DAC诱导基因组去甲基化的其他机制。这里提出的研究结果应该会对DAC的化学性质提供重要的启示，这些化学性质可能是这种化合物在人类细胞中诱导的一系列生化效应的原因。