Vorce R L, Goodman J I
Department of Pharmacology and Toxicology, Michigan State University, East Lansing 48824.
Toxicol Appl Pharmacol. 1989 Sep 15;100(3):398-410. doi: 10.1016/0041-008x(89)90288-3.
The B6C3F1 mouse is a hybrid strain which exhibits a high (30%) spontaneous hepatoma incidence and sensitivity to chemical induction of liver tumors. The spontaneous hepatoma incidence of the paternal C3H/He strain is approximately 60%, while that of the maternal C57BL/6 strain is very low. The presence of activated oncogenes, primarily Ha-ras, and to a lesser extent, Ki-ras, has been reported in B6C3F1 mouse liver tumors. Because alterations in a gene's capacity for expression, as well as mutation, may be involved in oncogene activation, this investigation was directed toward an examination of a putative control point for transcription, i.e., the methylation state of a gene. Hypomethylation is believed to be necessary, but not sufficient, for transcription. It was therefore hypothesized that alterations in the methylation state of the Ha-ras and Ki-ras oncogenes may facilitate the aberrant expression of these genes in B6C3F1 mouse liver. Restriction enzyme analysis (MspI, HpaII, and HhaI) was used to assess the extent of DNA methylation. MspI digestion of B6C3F1 and C3H/He DNA revealed the absence of a 15-kb Ha-ras band present in MspI-digested C57BL/6 DNA, suggesting that the Ha-ras oncogene of B6C3F1 and C3H/He mouse liver lacks a methylated site. In other respects, the Ha-ras and Ki-ras oncogenes are methylated to a degree which suggests that these oncogenes have a low potential for expression in normal mouse liver. The methylation state of the Ha-ras and Ki-ras oncogenes was also assessed in benzidine-induced hepatomas and adjacent nontumor tissue from B6C3F1 mice. In four out of four cases, the Ha-ras oncogene was hypomethylated in tumor as compared to nontumor tissue and increased expression of the gene was detected in three out of four hepatomas; the Ki-ras oncogene was hypomethylated in two out of four cases. These results suggest that hypomethylation of oncogenes may provide an epigenetic mechanism for facilitating their aberrant expression. The lack of a methylated site observed in the Ha-ras oncogene in B6C3F1 and C3H/He mouse liver may indicate an increased potential for its expression which could, in part, account for the high propensity for hepatoma development in these two strains.
B6C3F1小鼠是一种杂交品系，具有较高（30%）的自发性肝癌发生率，且对化学诱导肝肿瘤敏感。父本C3H/He品系的自发性肝癌发生率约为60%，而母本C57BL/6品系的发生率很低。据报道，在B6C3F1小鼠肝肿瘤中存在激活的癌基因，主要是Ha-ras，其次是Ki-ras。由于基因表达能力的改变以及突变可能与癌基因激活有关，本研究旨在检查一个假定的转录控制点，即基因的甲基化状态。低甲基化被认为是转录所必需的，但并不充分。因此，推测Ha-ras和Ki-ras癌基因甲基化状态的改变可能促进这些基因在B6C3F1小鼠肝脏中的异常表达。采用限制性内切酶分析（MspI、HpaII和HhaI）来评估DNA甲基化程度。对B6C3F1和C3H/He DNA进行MspI消化后，发现MspI消化的C57BL/6 DNA中存在的15 kb Ha-ras条带缺失，这表明B6C3F1和C3H/He小鼠肝脏中的Ha-ras癌基因缺乏甲基化位点。在其他方面，Ha-ras和Ki-ras癌基因的甲基化程度表明这些癌基因在正常小鼠肝脏中的表达潜力较低。还对B6C3F1小鼠经联苯胺诱导的肝癌及相邻非肿瘤组织中Ha-ras和Ki-ras癌基因的甲基化状态进行了评估。在4例病例中，与非肿瘤组织相比，肿瘤中Ha-ras癌基因均呈低甲基化，且在4例肝癌中有3例检测到该基因表达增加；4例中有2例Ki-ras癌基因呈低甲基化。这些结果表明，癌基因的低甲基化可能为促进其异常表达提供一种表观遗传机制。在B6C3F1和C3H/He小鼠肝脏的Ha-ras癌基因中观察到缺乏甲基化位点，这可能表明其表达潜力增加，这在一定程度上可以解释这两个品系肝癌发生的高倾向性。
