Chemical Pathology of 5-aza-2'-deoxycytidine

5-氮杂-2-脱氧胞苷的化学病理学

基本信息

批准号：
6761772
负责人：
Lawrence C Sowers
金额：
$ 29.19万
依托单位：
LOMA LINDA UNIVERSITY
依托单位国家：
美国
项目类别：
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

项目摘要

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以及晚期人类恶性肿瘤的研究药物。施用后，DAC 会掺入复制细胞的 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中的胎儿血红蛋白基因和人类癌症中的肿瘤抑制基因的重新激活。除了对 DNA 甲基化产生影响外，DAC 还会诱导细胞毒性并触发 DNA 修复反应。我们有兴趣研究 DAC 的潜在化学特性，这些特性可能解释其治疗和毒性活性。该修改后的提案分为五个目标。第一个目标是，我们将使用一系列 UV、NMR、GC/MS 和 HPLC/MS 方法，定量检测 DAC 在水溶液中的降解随温度和 pH 的变化。在第二个目标中，我们将使用已建立的亚磷酰胺方法将 DAC 构建到合成寡核苷酸中。然后，我们将使用新型同位素编辑 NMR 技术研究双链 DNA 中 DAC 的稳定性。我们怀疑 DNA 中的 DAC 以闭环和开环形式之间的平衡形式存在，因为已知溶液中的 DAC 会发生这种平衡。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。在目标三中，我们将在体外测定中使用纯化的 DNA 聚合酶检查通过化学合成或 DAC-三磷酸掺入制备的寡核苷酸中 DAC 的编码特性。我们预计闭环形式将正常碱基配对。开环形式预计将直接编码或充当非信息损伤。在目标四中，我们将使用体外测定检查 DAC 的修复情况。含有 DAC 的寡核苷酸将与纯化的 DNA 糖基化酶和全细胞提取物一起孵育。我们预计 DAC 的开环形式将被糖基酶快速去除。在目标五中，我们将测量培养细胞中的 DAC 掺入和 5-甲基胞嘧啶水平。这一目标应在掺入的 DAC 和甲基化胞嘧啶水平之间建立负相关。我们进一步建议测试一种新型稳定同位素质谱 (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，DAC 的化学性质可能是该化合物在人体细胞中引起一系列生化效应的原因。