Bachman Ammie N, Curtin Geoffrey M, Doolittle David J, Goodman Jay I
Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824, USA.
Toxicol Sci. 2006 Jun;91(2):406-18. doi: 10.1093/toxsci/kfj179. Epub 2006 Mar 28.
Altered DNA methylation, an epigenetic mechanism, likely contributes to tumorigenesis, with an inverse relationship existing between methylation in a promoter region and transcription. Using the SENCAR two-stage mouse skin tumorigenesis model, altered methylation was characterized in precancerous tissue and in tumor tissue. Mouse skin was initiated with 7,12-dimethylbenz[a]anthracene and promoted three times a week with 3, 9, 18, or 27 mg cigarette smoke condensate (CSC) for 4, 8, or 29 weeks; tumors were collected at 29 weeks. In addition, reversibility of changes in methylation was assessed following cessation of the promoting stimulus. DNA was isolated, and GC-rich methylation was assessed quantitatively via methylation-sensitive restriction digestion, arbitrarily primed PCR, and electrophoretic separation of PCR products. Analysis focused on regions of altered methylation (RAMs), which persisted from 4 to 8 weeks and from 8 weeks to tumor tissue. Persistent RAMs (i.e., seen in precancerous tissue and carried forward to tumors) are likely to play a key role in tumorigenesis. Twenty-two CpG sites in the upstream region of the Ha-ras promoter were unmethylated in control skin, 27 mg CSC, and tumor tissue. At two CpG sites closer to the transcriptional start site the incidence of hypomethylation increased with the dose of CSC. Hypomethylation was detected in all tumor samples. Expression of Ha-ras increased with 18 and 27 mg CSC promotion and more so in tumor tissue. These data support our hypothesis that tumor promotion involves instability of the epigenome, providing an environment where changes in the methylation status of specific regions of the genome accumulate progressively and contribute to the clonal expansion of initiated cells that leads to tumor formation.
DNA甲基化改变作为一种表观遗传机制，可能在肿瘤发生过程中发挥作用，启动子区域的甲基化与转录之间存在负相关关系。利用SENCAR两阶段小鼠皮肤肿瘤发生模型，对癌前组织和肿瘤组织中的甲基化改变进行了表征。用7,12-二甲基苯并[a]蒽引发小鼠皮肤，并每周用3、9、18或27mg香烟烟雾浓缩物（CSC）进行三次促癌处理，持续4、8或29周；在29周时收集肿瘤。此外，在停止促癌刺激后评估甲基化变化的可逆性。分离DNA，并通过甲基化敏感限制性消化、任意引物PCR和PCR产物的电泳分离对富含GC的甲基化进行定量评估。分析集中在甲基化改变区域（RAMs），这些区域在4至8周以及从8周持续到肿瘤组织中存在。持续存在的RAMs（即在癌前组织中出现并延续至肿瘤）可能在肿瘤发生中起关键作用。在对照皮肤、27mg CSC和肿瘤组织中，Ha-ras启动子上游区域的22个CpG位点未发生甲基化。在更靠近转录起始位点的两个CpG位点，低甲基化的发生率随CSC剂量增加而升高。在所有肿瘤样本中均检测到低甲基化。Ha-ras的表达在18mg和27mg CSC促癌处理时增加，在肿瘤组织中增加更为明显。这些数据支持了我们的假设，即肿瘤促进涉及表观基因组的不稳定性，提供了一个基因组特定区域甲基化状态变化逐渐积累并促进起始细胞克隆扩增从而导致肿瘤形成的环境。
