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, ...

We investigated DNA methylation patterns of E-cadherin and Connexin26 (Cx26) genes in rat hepatocellular carcinomas (HCCs) induced by a choline-deficient L-Amino Acid-defined (CDAA) diet. Six-wks-old F344 male rats were continuously fed with a CDAA diet for 75 wks, and were then killed. A total of five HCCs were obtained, and genomic DNA was extracted from each HCC for assessment of methylation status in the 5' upstream regions of E-cadherin and Cx26 genes by bisulfite sequencing, comparing to two normal liver tissues. The five HCCs showed highly methylated E-cadherin and Cx26 genes, while these genes in two normal liver tissues were all unmethylated. For analysis of gene expression, real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR) was performed. Expressions of E-cadherin and Cx26 genes were significantly reduced in the five HCCs (P < 0.0001 and P < 0.001, respectively) compared to normal liver tissues, correlating with their methylation statuses. These results suggested that hypermethylation of E-cadherin and Cx26 genes may be involved in the development of HCCs induced by a CDAA diet in rats.

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To assess the involvement of the TSLC cascade in hepatocarcinogenesis, we investigated the expression and DNA methylation patterns of the genes Tslc1 and Dal-1 in hepatocellular carcinomas (HCC) induced using N-nitrosodiethylamine (DEN) in rats. Sixweek-old male F344 rats received a single intraperitoneal injection of DEN at a dose of 10 mg/kg body weight, followed by combined treatment with partial hepatectomy and colchicine to induce cell-cycle disturbance and a selection procedure consisting of 2-acetylaminofluorene and carbon tetrachloride. Total RNA was extracted from 10 HCC, and the expression levels of Tslc1 and Dal-1 were measured using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Three of 10 HCC showed reduced expression of Tslc1, compared with normal liver tissues, but no changes in the expression level of Dal-1 were found. For DNA methylation analysis, bisulfite sequencing was performed. The 5′ ′ ′ ′ upstream region of Tslc1 was methylated in the three HCC in which its expression was reduced, but was unmethylated in normal liver tissue. Western blot analysis also revealed reduced expression of Tslc1 protein in the three HCC. These results suggest that alterations to the TSLC cascade might have a role in hepatocarcinogenesis using DEN in rats. (Cancer Sci 2007; 98: 943-948) H CC is one of the most frequent and deadly human malignancies.(1-5) Although infection with the hepatitis virus is a primary risk factor, environmental chemicals, such as aflatoxin, are also involved in the development of HCC.(6-8) Furthermore, there are various nitroso-compounds in the environment that possess hepatocarcinogenicity. To elucidate the molecular events underlying the development of HCC, animal models exposed to such nitroso-compounds are very useful. In studies of one such animal model, rats treated with DEN, the authors have so far reported the occurrence of frequent mutations in the gene encoding β-catenin,and alterations to Fhit; (10) however, no alterations have been observed in genes related to the TGF-β signaling pathway, such as those encoding Smad2, Smad4 and TGF-β receptor II. (11) TSLC1 has been identified as a tumor suppressor gene on chromosome 11q23.2 in NSCLC using functional complementation analyses.(12,13) TSLC1 encodes a membrane glycoprotein that belongs to the immunoglobulin superfamily and participates in cell adhesion. (13,14) DAL-1/4.1B was initially identified as a candidate tumor suppressor gene on 18p11.3.(15) DAL-1 belongs to the 4.1 protein family, and is an anchoring protein connecting TSLC1 to the actin cytoskeleton.(16) Recently, these TSLC cascade genes have been reported to be inactivated in several human cancers, and promoter methylation has been proposed as an important mechanism for inactivation of the genes encoding TSLC1 and DAL-1. (13,16 -23) In a recent study by the authors, reduced expression of Tslc1 due to aberrant methylation in rat lung tumors induced by nitroso-compounds was detected.(24) Therefore, to assess the involvement of the...

DAL-1 (differentially expressed in adenocarcinoma of the lung) is an actin-binding protein that has been shown to suppress growth in lung cancer cells. Recently, inactivation of the gene encoding DAL-1 due to hypermethylation has been found in several human malignancies, including lung cancers. To assess the involvement of the Dal-1 gene in rat lung carcinogenesis, we investigated the expression of Dal1 and its methylation status in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). Six-week old male Wistar rats (n = 11) were given 2,000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until they were sacrificed at 25 wk. Total RNA was extracted from 11 lung adenocarcinomas, one from each BHP treated rat, and Dal-1 gene expression was analyzed using real-time quantitative reverse transcription-polymerase chain reaction. Dal-1 expression was significantly reduced in the lung adenocarcinomas compared with three normal lung tissues (P < 0.05). For methylation analysis, bisulfite sequencing was performed using normal lung tissue and tissue from 4 tumors, all of which showed reduced expression of Dal-1. The 5' upstream region was highly methylated in all four adenocarcinomas, whereas this region was unmethylated in normal lung tissue. These results suggest that aberrant methylation of the Dal-1 gene might be involved in the development of lung adenocarcinomas induced in rats by BHP.