Inflammation Induced Cytosine Deamination and the Initiation of Cancer

项目总结/摘要 癌症仍然是美国和全世界的主要死亡原因之一， 每年有100万人死于癌症。为了减少这种公共卫生负担，调查最 通常观察到的取代突变，即胞嘧啶到胸腺嘧啶的转变，是至关重要的。这方面的一个例子 在炎症驱动的结肠炎相关结肠直肠癌（CAC）中优势明显。发展 CAC涉及早期关键的p53突变，其由5'胞嘧啶、3'胞嘧啶和5 '胞嘧啶-胸腺嘧啶转变主导。 鸟嘌呤（CpG）二核苷酸。这些突变是由5-甲基胞嘧啶脱氨成胸腺嘧啶， T：G错配中间体。随后的复制将突变传播到相反的链，形成 a T：碱基对。虽然在CAC诊断时已经证实了p53突变，但很少有研究表明， 已经做了检测低频突变和错配中间体的确切位置， 非癌组织也不知道突变在癌症诊断之前存在多久或如何存在 炎性病症可促进胞嘧啶向胸腺嘧啶的转变。目前的测序方法 涉及标准PCR掩盖了罕见但潜在重要突变的存在，因为扩增 以指数方式增加主导序列并仅报告该序列。解决这些 对于尚未解答的问题，我们的目标是机械地确定炎症如何增加这些疾病的发生率 关键突变，并开发新的方法来检测低频率的错配中间体和突变。 鉴于炎症在CAC病因学中的关键作用，我们假设炎症增加了CAC的发病率。 通过为竞争性DNA修复途径提供底物， 环境将大大增加突变和错配中间体的数量，从而导致癌症。 发展我们将通过首先证明我们的竞争DNA的机械假说来解决这个假设 修复，然后开发方法，以确定在p53 突变热点，通过三个特定目的描述：1）确定一种潜在的机制， 炎症介质可以诱导特征性胞嘧啶到胸腺嘧啶的转换; 2）设计一种新的 糖基化酶-质谱分析，以测量错配中间体的总体水平，并确定 炎症影响错配的中间产物; 3）开发一种新的方法来扫描p53外显子7， 胞嘧啶脱氨突变和错配中间体。通过测量低频错配 在肿瘤形成之前很久，我们将能够估计肿瘤中的细胞数量。 包含关键突变的群体，并预测其先前的DNA损伤历史。机械研究将 阐明炎症在肿瘤发生的开始阶段的作用，并共同，这一结果 该提案可能导致CAC或其他炎症的新治疗靶点或诊断方法 条件

PROJECT SUMMARY/ABSTRACT Cancer remains one of the leading causes of death in the United States and worldwide with more than 8.8 million cancer related deaths each year. To reduce this public health burden, investigation of the most commonly observed substitution mutation, a cytosine to thymine transition, is crucial. An example of this predominance is evident in inflammation driven colitis associated colorectal cancer (CAC). Development of CAC involves an early critical p53 mutation dominated by cytosine to thymine transitions at 5’ cytosine, 3’ guanine (CpG) dinucleotides. These mutations arise from 5-methylcytosine deamination into thymine, forming a T:G mispaired intermediate. Subsequent replication propagates the mutation to the opposing strand, forming a T:A base pair. Although mutations in p53 have been demonstrated at the time of CAC diagnosis, little has been done to detect the exact location of low frequency mutations and mispaired intermediates in noncancerous tissues. It is also unknown how long a mutation is present before cancer diagnosis or how inflammatory conditions can promote cytosine to thymine transitions. Current sequencing methodologies involving standard PCR mask the presence of rare but potentially important mutations because amplification exponentially increases the predominant sequence and reports only that sequence. To solve these unanswered questions, we aim to mechanistically determine how inflammation increases the rate of these critical mutations and develop novel methods to detect low frequency mispaired intermediates and mutations. Given the critical role of inflammation in the etiology of CAC, we hypothesize that inflammation increases the number of mutations by providing substrates for competing DNA repair pathways and that an inflammatory environment will greatly increase the number of mutations and mispaired intermediates which leads to cancer development. We will address this hypothesis by first proving our mechanistic hypothesis of competing DNA repair and then developing methods to determine the number of global mispaired intermediates at a p53 mutation hotspot, described through three Specific Aims: 1) determine a potential mechanism by which inflammatory mediators can induce characteristic cytosine to thymine transitions; 2) design a novel glycosylase-mass spectrometry assay to measure global levels of mispaired intermediates and determine how inflammation affects mispaired intermediate production; and 3) develop a novel method to scan p53 exon 7 for cytosine deamination mutations and mispaired intermediates. By measuring low frequency mispaired intermediates and mutations long before tumor formation, we will be able to estimate the number of cells in a population that contains critical mutation and predict its prior DNA damage history. The mechanistic studies will elucidate the role of inflammation on the beginning stages of tumorigenesis, and together, the results of this proposal could lead to novel therapeutic targets or diagnostic methods for CAC or other inflammatory conditions.