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.

Implication of Bcl-2 family genes in basal and d-amphetamine–induced apoptosis in preneoplastic and neoplastic rat liver lesions

“…Isolated nodules, HCCs, and small pieces of liver from each lobe were fixed for 16 hours at 0°C in paraformaldehyde and processed for embedding in paraffin, serially sectioned into 5-m thick serial slices, and stained with hematoxylin/eosin, propidium iodide, or processed for immunohistochemical analysis of glutathione-S-transferase placental form, as described. 37 To determine pulse labeling index (LI), the rats received an intraperitoneal injection of 5 mg/100 g body weight of 2-bromo-3Ј-deoxyuridine (BrdU), 2 hours before killing. BrdU incorporation into nuclei was determined immunohistochemically by the cell proliferation kit (Amersham Italia Srl).…”

Transferrin and transferrin receptor gene expression and iron uptake in hepatocellular carcinoma in the rat

“…13 However, preneoplastic and neoplastic liver lesions do not accumulate iron in a situation of iron overload. 16 Furthermore, reduced levels of iron-containing enzymes, such as cytochromes P450 and b 5 and catalase, and heme binding proteins were found in these lesions (see Eriksson and Andersson 17 ). Attempts to define the mechanisms of iron transport in preneoplastic liver lesions led to the discovery of an increased number of diferric Tf binding sites, with a normal affinity constant, with respect to normal liver.…”

“…Initiated liver cells, subjected to a selection treatment according to the ''resistant hepatocyte'' (RH) model, 4 acquire a proliferating phenotype characterized by the overexpression of some proto-oncogenes, including c-myc, c-Ha-ras, and c-Ki-ras, 5,6 as well as of ornithine decarboxylase 7 and 3-hydroxy-3-methylglutaryl CoA reductase 8 genes, which control growth-related metabolic pathways. Most of these changes appear relatively early during the carcinogenic process 6 and are maintained during the evolution of initiated cells to benign persistent nodules and hepatocellular carcinomas (HCCs).…”

Biological Effects of Polyamines on the Prevention of Aging-associated Diseases and on Lifespan Extension

“…In other studies, supplementation of methyl groups affected the methylation status and gene expression of several genes (Garcea et al, 1989;Kano et al, 2013).…”

“…Conversely, the methylation status of the region was enhanced in cells with increased polyamine concentration following spermine supplementation (upper row, right), and the amount of LFA-1 was decreased (bar in the right lower-berth compared to center bar). DFMO: DL-α-difluoromethylornithine polyamine concentrations from extracellular sources seem to activate Dnmt activities (Bestor et al, 1988;Garcea et al, 1989).…”