Chemical Pathology of 5-aza-2'-deoxycytidine

描述（由申请方提供）：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，导致镰状细胞贫血中胎儿血红蛋白基因和人类癌症中肿瘤抑制基因的重新激活。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。我们有兴趣研究DAC的潜在化学性质，这些化学性质可能是治疗和毒性活性的原因。这项修订建议分为五个目标。在第一个目标中，我们将使用UV，NMR，GC/MS和HPLC/MS方法的电池，作为温度和pH值的函数，在水溶液中的DAC的降解进行定量研究。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，我们将使用一种新的同位素编辑NMR技术研究DAC在双链DNA中的稳定性。我们怀疑DAC在DNA中以闭环和开环形式之间的平衡存在，因为已知DAC在溶液中会发生这种平衡。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，我们将检查DAC的编码特性的寡核苷酸制备的化学合成或DAC-三磷酸掺入使用纯化的DNA聚合酶在体外测定。我们预期闭环形式将正常碱基配对。预期开环形式可直接导致坏死或作为非信息性病变。在第四个目标中，我们将使用体外测定来检查DAC的修复。将含DAC的寡核苷酸与纯化的DNA糖基化酶和全细胞提取物一起孵育。我们预期DAC的开环形式将被糖基化酶快速去除。在目标五中，我们将测量在培养物中生长的细胞中的DAC掺入和5-甲基胞嘧啶水平。这一目标应建立一个反相关性纳入DAC和甲基化胞嘧啶水平。我们进一步提出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的化学性质有重要意义，这些化学性质可能是该化合物在人体细胞中诱导的一系列生化效应的原因。

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.