Disturbance of DNA methylation patterns in the early phase of hepatocarcinogenesis induced by a choline‐deficient L‐amino acid‐defined diet in rats

Abstract

The authors investigated the DNA methylation patterns of the E-cadherin, Connexin 26 (Cx26), Rassf1a and c-fos genes in the early phase of rat hepatocarcinogenesis induced by a choline-deficient L-amino acid-defined (CDAA) diet. Six-week-old F344 male rats were continuously fed with the CDAA diet, and three animals were then killed at each of 4 and 8 days and 3 weeks. Genomic DNA was extracted from livers for assessment of methylation status in the 5′ ′ ′ ′ upstream regions of E-cadherin, Cx26, Rassf1a and c-fos genes by bisulfite sequencing, compared with normal livers. The livers of rats fed the CDAA diet for 4 and 8 days and 3 weeks were methylated in E-cadherin, Cx26 and Rassf1a genes, while normal livers were all unmethylated. In contrast, normal livers were highly methylated in c-fos gene. Although the livers at 4 days were weakly methylated, those at 8 days and 3 weeks were markedly unmethylated. Methylation patterns of CpG sites in E-cadherin, Cx26 and Rassf1a were sparse and the methylation was not associated with gene repression. These results indicate that gene-specific DNA methylation patterns were found in livers of rats after short-term feeding of the CDAA diet, suggesting gene-specific hypermethylation might be involved in the early phase of rat hepatocarcinogenesis induced by the CDAA diet. (Cancer Sci 2007; 98: 1318-1322) I t is well known that unequivocal liver tumors can be induced by prolonged feeding of rats with a CD diet.(1-3) The choline deficiency causes fatty liver, cirrhosis and HCC in rats.(1-3) Possible mechanisms underlying liver carcinogenesis from the CD diet have been proposed to include the following: liver necrosis associated with subsequent regeneration; (4,5) induction of oxidative DNA damage and lipid peroxidation; (6)(7)(8)(9) and generation of genetic alterations.(10,11) It has also been considered that DNA hypomethylation might play an important role in liver carcinogenesis induced by methyl donor deficiency.(12,13) So far, hypomethylation of the c-fos, c-myc, and c-Ha-ras genes has been detected in the livers of rats fed with the CD diet. (14,15) The CDAA diet used in the present study is semi-synthetic, and provides stronger carcinogenic effects than the CD diet in rats. (16,17) The authors have previously reported the hypomethylation of c-myc in HCC resulting from the CDAA diet in rats. (18) In another study, hypermethylation of the E-cadherin and Cx26 genes was also detected in those tumors.(19) While genomewide hypomethylation occurs in several human cancer cells, sitespecific hypermethylation such as CpG islands of tumor suppressor genes, is also found.(20) It has been suggested that aberrant DNA methylation of promoter regions of genes is the major mechanism of gene silencing in the development of tumors. (21,22) In fact, aberrant DNA methylation has been found in a variety of human cancers, including liver tumors. (23)(24)(25)(26) However, it is unclear why DNA hypermethylation occurs in rat HCC induced by the CDAA diet despite methyl donor deficiency. Therefore, ...

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