Counts J L, Kaznowski J M, McClain R M, Goodman J I
Department of Pharmacology and Toxicology, Michigan State University, East Lansing 48824, USA.
Int J Cancer. 1997 Jul 29;72(3):491-7. doi: 10.1002/(sici)1097-0215(19970729)72:3<491::aid-ijc18>3.0.co;2-f.
Modifications to DNA-5-methylcytosine (5MeC) content (i.e., alterations in the level of 5MeC) constitute epigenetic events. In general, hypomethylation of a gene is necessary but not sufficient for expression, while methylated genes typically are quiescent. Ha-ras is an oncogene commonly implicated in murine liver tumorigenesis, often, though not always, involving mutation. A PCR-based approach using pre-PCR digestion with methylation-sensitive enzymes was employed to determine the 5MeC content of the 5' flanking region of this gene in (i) B6C3F1 and C57BL/6 mouse liver from young animals (4 months old) and (ii) B6C3F1 mouse liver from aged animals (24 months old). Two segments of the 5' flanking region of Ha-ras were examined. We demonstrate the presence of 5MeC in a portion of the 5' flanking region of Ha-ras that does not share characteristics of a CpG island, while a region that shares CpG island characteristics is primarily unmethylated. Differences in methylation status in these areas of Ha-ras were not observed between B6C3F1 and C57BL/6 mouse livers. Increases in methylation status were observed with ageing in B6C3F1 mouse liver. These data provide a role for methylation in regulating Ha-ras expression in mouse liver. Ha-ras in human liver has been reported to be unmethylated. There are substantial sequence differences in a key region of the 5' flanking region of Ha-ras in mice as compared to humans. These differences in DNA methylation and sequence may, in part, provide a basis for the frequent involvement of Ha-ras in mouse liver tumors and its virtual lack of involvement in human tumors.
DNA-5-甲基胞嘧啶（5MeC）含量的改变（即5MeC水平的变化）构成表观遗传事件。一般来说，基因的低甲基化对于表达是必要的，但并不充分，而甲基化的基因通常是静止的。Ha-ras是一种癌基因，通常与小鼠肝脏肿瘤发生有关，虽然并不总是，但经常涉及突变。采用基于PCR的方法，先用甲基化敏感酶进行预PCR消化，以确定该基因5'侧翼区域在（i）幼龄动物（4个月大）的B6C3F1和C57BL/6小鼠肝脏以及（ii）老龄动物（24个月大）的B6C3F1小鼠肝脏中的5MeC含量。检测了Ha-ras 5'侧翼区域的两个片段。我们证明在Ha-ras 5'侧翼区域的一部分中存在5MeC，该部分不具有CpG岛的特征，而具有CpG岛特征的区域主要是未甲基化的。在B6C3F1和C57BL/6小鼠肝脏之间未观察到Ha-ras这些区域甲基化状态的差异。在B6C3F1小鼠肝脏中，随着年龄增长观察到甲基化状态增加。这些数据表明甲基化在调节小鼠肝脏中Ha-ras表达方面发挥作用。据报道，人类肝脏中的Ha-ras是未甲基化的。与人类相比，小鼠Ha-ras 5'侧翼区域的关键区域存在大量序列差异。DNA甲基化和序列的这些差异可能部分为Ha-ras频繁参与小鼠肝脏肿瘤而几乎不参与人类肿瘤提供了基础。
