Infidelity of Cytosine Methylation and Human Cancer

描述(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学改变。启动子高甲基化(表观遗传学)导致多个肿瘤抑制基因沉默。人们对甲基化模式改变的机制知之甚少。在之前的资助阶段，我们专注于炎症介导的DNA损伤，因为炎症和癌症的发展之间存在着长期的联系。通过使用体外和模型系统的一系列研究，我们确定了由激活的中性粒细胞和嗜酸性粒细胞产生的反应分子可以产生一系列DNA加合物。这些产品中有5-氯胞嘧啶(5ClC)和5-溴胞嘧啶(5BrC)。我们证实，含有甲基结合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损伤产物可能会作为欺骗性的表观遗传信号，导致局部超甲基化。其他几种加合物被证明干扰DNA-蛋白质的相互作用，潜在地导致甲基化的丧失。尽管多项研究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存在5ClC和5BrC。这里讨论了阻碍这些测量的几个方法学问题。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，免疫细胞产生的活性分子必须穿过细胞膜进入细胞核，才能与DNA发生反应。初步数据支持这样的假设，即氯胺或溴胺的形成可能促进反应分子向核的输送，一些叔胺，包括尼古丁，可能催化卤胺向胞嘧啶的转移。这一假设将在目标2中使用目标1中开发的方法进行验证。启动子区域中5-卤代胞嘧啶的存在可能同时起到沉默转录和进一步甲基化的作用。为了开始检验这一假设，我们提出了 目标3中的新方法，既可以在培养的人类细胞DNA中选择性地产生5-卤胞嘧啶，又可以在DNA序列水平上进行检测。在目标4中，我们建议使用在先前目标中开发的方法来直接测量正常组织和肿瘤组织DNA中5-卤代胞嘧啶的存在。我们首次报道了人体外科组织中5ClC和5BrC的测定。完成这里提出的目标将使我们能够深入研究炎症、DNA损伤和癌症病因之间的联系。 公共卫生相关性：我们的实验室一直在研究炎症如何导致DNA损伤，因为众所周知，炎症和癌症之间存在相关性。我们研究了炎症过程中产生的反应分子引起的DNA损伤的类型，并确定了特定形式的DNA损伤，包括我们认为可能导致肿瘤细胞异常甲基化导致恶性转化的5-溴胞嘧啶(5BrC)和5-氯胞嘧啶(5ClC)。这些研究的结果可能会导致合理的方法来降低癌症发病率。

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. PUBLIC HEALTH RELEVANCE: Our laboratory has been studying how inflammation can result in DNA damage because of the well known correlations between inflammation and cancer. We have studied the types of DNA damage induced by reactive molecules generated during inflammation, and we have identified specific forms of DNA damage including 5-bromocytosine (5BrC) and 5-chlorocytosine (5ClC) that we propose could contribute to aberrant methylation in tumor cells leading to malignant transformation. Results of these studies could lead to rational approaches to reduce cancer incidence.