Protooncogene methylation and expression in regenerating liver and preneoplastic liver nodules induced in the rat by diethylnitrosamine: effects of variations of S-adenosylmethionine: S-adenosylhomocysteine ratio

Abstract

S-adenosylmethionine:S-adenosylhomocysteine (SAM/SAH) ratio, 5-methylcytosine (5mC) DNA content, and methylation and expression of c-myc, c-Ha-ras and c-Ki-ras have been studied in liver nodules, induced by diethylnitrosamine according to the 'resistant hepatocyte' model, and in regenerating liver (RL) between 0.5 and 72 h after partial hepatectomy (PH). Nodules, 11, 13 and 21 weeks after initiation, grew actively, showed a low tendency to remodel (persistent nodules), and did not exhibit carcinomatous changes. They underwent extensive remodeling after a 1-week SAM treatment (64 mumol/kg/day), and decreased in size and number after a 3-11-week treatment. A low SAM/SAH ratio was coupled, in nodules, with a high labeling index (LI), 2-fold fall in 5mC DNA content, increase in c-myc, c-Ha-ras and c-Ki-ras expression and hypomethylation of CCGG sequences in the DNA hybridizing with the three protooncogenes. In RL a low SAM/SAH ratio, overall DNA hypomethylation and enhanced c-myc expression were first observed 0.5 h after PH, reached a peak at 5 h and progressively returned to pre-PH levels later on. Maximum expression of c-Ha-ras and c-Ki-ras occurred 24-30 h after PH, roughly coincident with the LI peak. However, no great modifications of the methylation pattern of protooncogene CCGG sequence occurred at any time after PH, indicating the presence of hypomethylated genes and/or DNA sequences different from those investigated in this paper. SAM injection to nodule-bearing rats, for 1-11 weeks before killing, and to hepatectomized rats, 2 days before PH and then up to killing, largely prevented decrease in the SAM/SAH ratio and overall DNA methylation and inhibited LI and protooncogene expression. In nodules these effects were proportional to the treatment length and coupled with methylation of CpG residues in the CCGG sequence of the three protooncogenes studied. SAM treatment left the methylation pattern of these genes unchanged in RL. Kinetics of increase in protooncogene expression suggest a role in the regulation of cell cycle in RL. However, decrease in the SAM/SAH ratio, protooncogene hypomethylation and enhanced expression are apparently stable in nodules 11-21 weeks after initiation and could be implicated in continuous nodule growth and progression. Control of DNA methylation and gene expression by exogenous SAM could be a mechanism of the SAM anti-progression effect.