Hypomethylation of CpG Sites and c‐myc Gene Overexpression in Hepatocellular Carcinomas, but Not Hyperplastic Nodules, Induced by a Choline‐deficient L‐Amino Acid‐defined Diet in Rats

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Abstract
We have investigated aberrant methylation of CpG nucleotides (CpG sites) and gene expression of c‐myc during hepatocarcinogenesis induced by a choline‐deficient, L‐amino acid‐defined (CDAA) diet in rats. Male Fischer 344 rats, 6 weeks old, were continuously given a CDAA diet for 50 and 75 weeks and then killed. Macroscopically detectable nodules, which were histologically confirmed to be hyperplastic nodules (HNs) or well‐differentiated hepatocellular carcinomas (HCCs), were dissected free from the surrounding tissue. Normal control liver was obtained from 6‐week‐old rats. Methylation of CpG sites of the c‐myc gene was investigated in bisulfite‐treated DNA isolated from normal liver, HNs and HCCs. All 33 cytosines in the 5′‐upstream region of the c‐myc gene were fully methylated in control liver and the 4 HNs. In contrast, these cytosines were completely unmethylated in 5 HCCs. Examination of the c‐myc expression by reverse transcription‐polymerase chain reaction (RT‐PCR) analysis also showed a marked increase as compared to the low levels in normal livers and HNs. These results suggest that hypomethylation of the c‐myc gene might play a critical role in malignant transformation from HN to HCC during CDAA diet‐induced hepatocarcinogenesis in rats.