Altered methylation of ras oncogenes in benzidine-induced B6C3F1 mouse liver tumors

Abstract

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 presen 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.