Chemical Pathology of 5-aza-2'-deoxycytidine

5-氮杂-2-脱氧胞苷的化学病理学

• 批准号: 7077939
• 负责人: Lawrence C Sowers
• 金额: $ 5.27万
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7077939
• 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（由申请人提供）：核苷类似物5-aza-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。给药后，DAC被纳入复制细胞的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胎儿血红蛋白基因和人类癌症肿瘤抑制基因的再激活。除了对DNA甲基化的影响外，DAC还诱导细胞毒性并引发DNA修复反应。我们感兴趣的是研究DAC的潜在化学性质，它可以解释治疗和毒性活性。修订后的建议分为五个目标。在第一个目标中，我们将使用UV， NMR， GC/MS和HPLC/MS方法，定量地研究DAC在水溶液中的降解作为温度和pH值的函数。在第二个目标中，我们将使用已建立的酰胺磷方法将DAC构建为合成寡核苷酸。然后，我们将使用一种新的同位素编辑核磁共振技术研究DAC在双链DNA中的稳定性。我们怀疑DNA中的DAC存在于环闭合和环打开形式之间的平衡，因为已知这种平衡发生在溶液中的DAC中。深入了解这种平衡对于确定DAC诱导的潜在致突变性、细胞毒性和去甲基化机制至关重要。在第三个目标中，我们将在体外实验中研究通过化学合成或使用纯化DNA聚合酶将DAC-三磷酸结合制备的寡核苷酸中DAC的编码特性。我们期望环封闭形式的碱基对是正常的。开环的形式预计会引导rna编码或作为非信息病变。在目标四中，我们将使用体外实验检查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一起孵育。我们预计开环形式的DAC将被糖基酶迅速去除。在目标五中，我们将测量DAC掺入和5-甲基胞嘧啶水平在培养细胞中生长。目的是建立掺入DAC和甲基化胞嘧啶水平之间的负相关关系。我们进一步建议测试一种新的稳定同位素质谱（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对DAC的化学性质有重要的启示，这些化学性质可能是该化合物在人类细胞中诱导的一系列生化效应的原因。