Infidelity of Cytosine Methylation and Human Cancer

描述（由申请人提供）：人类癌症的发展需要一系列的基因变化，包括突变和表观遗传改变。启动子超甲基化（表观遗传）导致多种肿瘤抑制基因沉默。我们对甲基化模式改变的机制知之甚少。在之前的资助期间，我们专注于炎症介导的DNA损伤，因为炎症与癌症的发展之间存在长期联系。通过体外和模型系统的一系列研究，我们确定由活化的中性粒细胞和嗜酸性粒细胞产生的反应性分子可以产生一系列DNA加合物。这些产物包括5-氯胞嘧啶（5ClC）和5-溴胞嘧啶（5BrC）。我们确定含有甲基结合结构域的蛋白质，以及人类维持甲基转移酶DNMT1，不能将这些加合物与5-甲基胞嘧啶区分开来。因此，这些炎症产生的DNA损伤产物可能作为欺诈性的表观遗传信号，导致局部高甲基化。其他几种加合物被证明会干扰dna -蛋白质的相互作用，可能导致甲基化的丧失。尽管多项研究已经测量了与人类疾病相关的氯化和溴化氨基酸，但文献基本上没有提到人体组织中5ClC和5BrC的存在。这里讨论了妨碍这些测量的几个方法问题。如目标1所述，我们拥有开发所需分析方法所需的试剂和专业知识。在人体组织中，免疫细胞产生的反应性分子必须穿过细胞膜进入细胞核才能与DNA发生反应。初步数据支持以下假设：氯胺或溴胺的形成可能促进活性分子向细胞核的传递，一些叔胺，包括尼古丁，可能催化卤素从卤胺向胞嘧啶的转移。这一假设将在Aim 2中使用Aim 1中开发的方法进行测试。启动子区域中5-盐胞嘧啶的存在可以沉默转录并“播种”进一步的甲基化。为了验证这个假设，我们提出

DESCRIPTION (provided by applicant): The development of cancer in man requires a series of genetic changes including both mutations and epigenetic alterations. Promoter hypermethylation (epigenetic) leads to the silencing of multiple tumor suppressor genes. Very little is known about mechanisms by which methylation patterns are altered. In the previous period of funding, we focused on inflammation-mediated DNA damage due to the long-standing association between inflammation and the development of cancer. Through a series of studies using in vitro and model systems, we established that reactive molecules generated by activated neutrophils and eosinophils can generated an array of DNA adducts. Among these products are 5-chlorocytosine (5ClC) and 5-bromocytosine (5BrC). We established that proteins containing methyl-binding domains, as well as the human maintenance methyltransferase DNMT1, do not distinguish these adducts from 5-methylcytosine. Therefore, these inflammation- generated DNA damage products could act as fraudulent epigenetic signals resulting in local hypermethylation. Several other adducts were shown to interfere with DNA-protein interactions, potentially leading to loss of methylation. Although multiple studies have measured chlorinated and brominated amino acids associated with human disease, the literature is essentially silent on the presence of 5ClC and 5BrC in human tissues. Several methodological issues are discussed here that have hampered these measurements. As described in Aim 1, we have the reagents and expertise needed to develop the required analytical methods. In human tissues, reactive molecules generated by immune cells must cross the cell membrane and enter the nucleus in order to react with the DNA. Preliminary data support the hypothesis that the formation of chloramines or bromamines might facilitate the delivery of reactive molecules to the nucleus, and that some tertiary amines, including nicotine, might catalyze halogen transfer from the haloamines to cytosine. This hypothesis will be tested in Aim 2 using methods developed in Aim 1. The presence of the 5-halocytosines within promoter regions could serve to both silence transcription and "seed" further methylation. In order to begin testing this hypothesis, we present new methods in Aim 3 that both selectively generate 5-halocytosine in the DNA of human cells in culture and allow its detection at the DNA sequence level. In Aim 4, we propose to use methods developed in the previous aims to directly measure the presence of 5- halocytosines in the DNA of normal and tumor tissues. We present for the first time, the measurement of 5ClC and 5BrC in human surgical tissues. Completion of the aims proposed here will allow an in- depth examination of the connection between inflammation, DNA damage and cancer etiology.