Shaw R J, Liloglou T, Rogers S N, Brown J S, Vaughan E D, Lowe D, Field J K, Risk J M
Molecular Genetics & Oncology Group, School of Dental Sciences, University of Liverpool, Liverpool L69 3GN, UK.
Br J Cancer. 2006 Feb 27;94(4):561-8. doi: 10.1038/sj.bjc.6602972.
Methylation profiling of cancer tissues has identified this mechanism as an important component of carcinogenesis. Epigenetic silencing of tumour suppressor genes through promoter methylation has been investigated by a variety of means, the most recent of which is pyrosequencing. We have investigated quantitative methylation status in oral squamous cell carcinoma patients. Fresh tumour tissue and normal control tissue from resection margin was obtained from 79 consecutive patients undergoing resection of oral squamous cell carcinoma. DNA was extracted and bisulphite treated. PCR primers were designed to amplify 75-200 bp regions of the CpG rich gene promoters of p16, RARbeta, E-cadherin, cytoglobin and cyclinA1. Methylation status of 4-5 CpG sites per gene was determined by pyrosequencing. Significant CpG methylation of gene promoters within tumour specimens was found in 28% for p16, 73% for RARbeta, 42% for E-cadherin, 65% for cytoglobin and 53% for cyclinA1. Promoter methylation was significantly elevated in tumours compared to normal tissue for p16 (P = 0.048), cytoglobin (P = 0.002) and cyclin A1 (P = 0.001) but not in RARbeta (P = 0.088) or E-cadherin (P = 0.347). Concordant methylation was demonstrated in this tumour series (P = 0.03). Significant differences in degree of methylation of individual CpG sites were noted for all genes except RARbeta and these differences were in a characteristic pattern that was reproduced between tumour samples. Cyclin A1 promoter methylation showed an inverse trend with histological grade. Promoter methylation analysis using pyrosequencing reveals valuable quantitative data from several CpG sites. In contrast to qualitative data generated from methylation specific PCR, our data demonstrated p16 promoter methylation in a highly tumour specific pattern. Significant tumour specific methylation of cyclin A1 promoter was also seen. Cytoglobin is a novel candidate tumour suppressor gene highly methylated in upper aero-digestive tract squamous cancer.
癌症组织的甲基化谱分析已将这一机制确定为致癌作用的一个重要组成部分。通过多种方法对肿瘤抑制基因通过启动子甲基化导致的表观遗传沉默进行了研究，其中最新的方法是焦磷酸测序。我们对口腔鳞状细胞癌患者的甲基化定量状态进行了研究。从79例连续接受口腔鳞状细胞癌切除术的患者中获取新鲜肿瘤组织和手术切缘的正常对照组织。提取DNA并进行亚硫酸氢盐处理。设计PCR引物以扩增p16、RARβ、E-钙黏蛋白、细胞珠蛋白和细胞周期蛋白A1富含CpG的基因启动子的75-200 bp区域。通过焦磷酸测序确定每个基因4-5个CpG位点的甲基化状态。在肿瘤标本中发现，p16基因启动子的显著CpG甲基化率为28%，RARβ为73%，E-钙黏蛋白为42%，细胞珠蛋白为65%，细胞周期蛋白A1为53%。与正常组织相比，肿瘤中p16（P = 0.048）、细胞珠蛋白（P = 0.002）和细胞周期蛋白A1（P = 0.001）的启动子甲基化显著升高，但RARβ（P = 0.088）或E-钙黏蛋白（P = 0.347）则不然。在这个肿瘤系列中证实了一致性甲基化（P = 0.03）。除RARβ外，所有基因的单个CpG位点甲基化程度均存在显著差异，且这些差异呈特征性模式，在肿瘤样本之间可重复出现。细胞周期蛋白A1启动子甲基化与组织学分级呈相反趋势。使用焦磷酸测序进行的启动子甲基化分析揭示了来自几个CpG位点的有价值的定量数据。与甲基化特异性PCR产生的定性数据相反，我们的数据显示p16启动子甲基化具有高度肿瘤特异性模式。细胞周期蛋白A1启动子也存在显著的肿瘤特异性甲基化。细胞珠蛋白是一种新的候选肿瘤抑制基因，在上呼吸道消化道鳞状癌中高度甲基化。
