Alterations in the methylation status and expression of the raf oncogene in phenobarbital-induced and spontaneous B6C3F1 mouse liver tumors

The liver tumor‐prone B6C3F1 mouse (C57BL/6 ♂ × C3H/He♀), in conjunction with the more susceptible C3H/He paternal strain and the resistant C57BL/6 maternal strain, is an excellent model for studying the mechanisms involved in carcinogenesis. The study reported here indicated that the B6C3F1 mouse inherited a maternal raf allele containing a methylated site not present in the paternal allele. Seven days after partial hepatectomy or after administration of a promoting dose of phenobarbital (PB) for 14 d; raf in B6C3F1 mouse liver was hypomethylated. The additional methylated site in the allele inherited from C57BL/6 was not maintained. The methylation status of raf in the liver of the C57BL/6 mouse was not affected by PB treatment. This indicates that the B6C3F1 mouse is less capable of maintaining methylation of raf than the C57BL/6 strain is. In both PB‐induced and spontaneous B6C3F1 liver tumors, raf was hypomethylated in a nonrandom fashion. The level of raf mRNA increased in seven of 10 PB‐induced tumors but in only one of five spontaneous tumors, whereas the level of Ha‐ras mRNA increased in nine of 10 PB‐induced tumors and in four of five spontaneous tumors. The results of our investigation (a) support the hypothesis that hypomethylation of DNA is a nongenotoxic mechanism involved in tumorigenesis, (b) support the notion that PB promotes liver tumors that develop along a pathway different from that leading to spontaneous tumors, and (c) indicate that differences in DNA methylation between C57BL/6 and B6C3F1 mice could, in part, account for the unusually high tendency of the latter strain to develop liver tumors. © 1994 Wiley‐Liss, Inc.