To clarify the sequential changes in pRB and p16 during different stages of hepatocarcinogenesis such as
fibrosis,
cirrhosis,
hepatocellular adenoma (HCA), and
hepatocellular carcinoma (HCC), male Fischer 344 rats were singly injected with
diethylnitrosamine (DEN), immediately followed with
phenobarbital for 1 wk and then
thioacetamide (TAA) for 39 wk in
drinking water. Rats were killed at 9, 20, 30, and 40 wk after DEN initiation and changes of pRB level, p16 gene hypermethylation, and in vivo gankyrin expression were examined. Histologic examination showed stepwise appearances of
fibrosis,
cirrhosis, HCA, and HCC at weeks 9, 20, 30, and 40, respectively. Hypermethylation of p16 exon 1 was not found until HCA but appeared in 50% of the rats with HCC accompanied by complete loss of its
mRNA expression. The amount of
glutathione S-transferase--gankyrin bound to pRB and pRB degradation in the liver depended on the concentration of gankyrin and incubation time. Gankyrin expression preceded pRB degradation in
liver cirrhosis. In conclusion, gankyrin expression induced in
liver fibrosis accelerated the degradation of pRB during
liver cirrhosis, and inactivation of p16 exon 1 by
DNA hypermethylation occurred during the progression of
tumor cells to poorly differentiated HCC. Inactivation of pRB and/or p16 resulted in complete loss of regulation in the cell-division cycle during early and late stages, respectively, of hepatocarcinogenesis. Mol. Carcinog. 30:138--150, 2001.