Aberrant DNA methylation precedes loss of heterozygosity on chromosome 16 in chronic hepatitis and liver cirrhosis
Introduction
Aberrant DNA cytosine methylation is one of the most consistent epigenetic changes in human cancers [1]. The global DNA methylation level is generally lower in cancer cells than in normal cells [2], but increased expression and activity of maintenance DNA (cytosine-5) methyltransferase (EC 2.1.1.37) are usually seen in cancer cells [3]. Some loci tend to show increased DNA methylation [4], [5], [6], [7], [8], [9], [10], whereas others are often hypomethylated [11]. DNA methylation may play a role in carcinogenesis, since DNA cytosine methylation facilitates gene mutation through deamination of 5-methylcytosine to thymine [12] and DNA methylation near the gene promoters affects the transcription of specific genes [13], [14], [15], [16], [17].
Moreover, an association between aberrant DNA methylation and chromosomal instability during carcinogenesis has been noted recently. Treatment with a DNA methyltransferase inhibitor, 5-aza-cytidine [18], and inactivating the DNA methyltransferase gene [19] each result in mitotic recombination and chromosomal deletion, probably through low global DNA methylation and chromatin configuration changes. Makos et al. suggest that DNA hypermethylation at the D17S5 locus, the location of a candidate tumor suppresser gene called HIC-1 (hypermethylated-in-cancer, [20]), predisposes the same locus to allelic loss in renal cancers [5] and neural tumors [6]. Their suggestion is based on two findings: (a) the incidence of DNA hypermethylation at the D17S5 locus exceeded that of loss of heterozygosity (LOH) at the same locus and (b) all of the cancers with LOH also showed DNA hypermethylation at the same locus [5], [6]. Most of our previous data on DNA methylation and allelic loss at the D17S5 locus in lung [8] and stomach [9] cancers and hepatocellular carcinoma (HCC; [10]) support their hypothesis.
In addition to DNA hypermethylation at the D17S5 locus, we reported previously that the DNA methyltransferase mRNA expression level is significantly higher in non-cancerous liver tissues showing chronic hepatitis and cirrhosis, which are considered to be precancerous conditions [21], [22], than in liver tissues with no remarkable histological findings [23]. The DNA methyltransferase mRNA expression level was even higher in HCC than in chronic hepatitis and cirrhosis [23]. Aberrant DNA methylation on chromosome 16, a hot spot for LOH in HCC [24], is observed frequently in chronic hepatitis and cirrhosis [7]. The degree of aberrant DNA methylation is higher in HCC than in chronic hepatitis or cirrhosis so DNA hypermethylation may precede or cause LOH on chromosome 16 [7]. Moreover, CpG methylation around the promoter region of the E-cadherin tumor suppresser gene, which is located on 16q22.1 [25] and encodes a Ca2+-dependent cell adhesion molecule [26], significantly correlates with reduced E-cadherin expression, resulting in loss of intercellular adhesiveness and destruction of tissue structure in HCC [17]. Taken together these findings suggest that aberrant DNA methylation promotes hepatocarcinogenesis, even during the precancerous stage, by predisposing some chromosomes to LOH and silencing some specific genes.
Recently, microdissection techniques and methodologies have been developed for detecting LOH in small numbers of cells from paraffin-embedded tissues. LOH has been observed in microdissected specimens from non-cancerous lesions, e.g. hyperplastic or dysplastic lesions accompanying non-small-cell lung cancer [27] and proliferative lesions accompanying breast cancer [28]. In cases of HCC, cells excised from cirrhotic foci frequently show LOH at the 8p [29], Rb [30] and p53 [31] loci. To the best of our knowledge, however, LOH on chromosome 16 has not been reported in the precancerous stage of HCC. In our previous study of DNA methylation and allelic loss on chromosome 16, LOH was not detected in non-microdissected specimens showing chronic hepatitis or cirrhosis [7].
To examine the significance of aberrant DNA methylation in hepatocarcinogenesis, we assessed the association between DNA methylation around the promoter of the E-cadherin gene and allelic loss at the D16S421 locus, which is adjacent to the E-cadherin locus, using microdissected specimens of both non-cancerous liver tissues and HCCs.
Section snippets
Patients and tissue specimens
Paired specimens of non-cancerous liver tissue and primary HCC were obtained from surgically resected materials from 38 patients who underwent therapeutic partial hepatectomy at the National Cancer Center Hospital, Tokyo, Japan. They were 31 men and 7 women with a mean age of 62±12 (mean±SD) years (range 21–78 years). Twenty-six patients were hepatitis C virus antibody positive and nine were hepatitis B virus surface antigen positive. Seven were hepatitis B and C virus negative. Histological
DNA methylation around the promoter region of the E-cadherin gene
Examples of the PCR products are shown in Fig. 1A. The DNA methylation status of the region around the promoter of the E-cadherin gene is summarized in Table 1. The incidence of DNA hypermethylation in the non-cancerous liver tissues of patients with HCCs also showing DNA hypermethylation (72%) was significantly higher than that of patients without DNA hypermethylation in their HCCs (53%, P<0.05). The incidence of DNA hypermethylation in non-cancerous tissues showing chronic hepatitis was not
Discussion
We have analyzed DNA methylation in the region around the promoter of the E-cadherin gene, which is a hot spot for aberrant DNA methylation on chromosome 16q, during hepatocarcinogenesis [17]. DNA hypermethylation in this region participates in hepatocarcinogenesis through reduced E-cadherin expression, which results in reduced intercellular adhesiveness and destruction of the tissue structure. The incidence of DNA hypermethylation in the non-cancerous liver tissues was higher in this study
Acknowledgements
This study was supported by a Grant-in-Aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control and a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan.
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