Identification of genes epigenetically silenced by CpG methylation in human gastric carcinoma
Introduction
Globally, gastric cancer is the second most common cause of cancer death, 1 and although the incidence of gastric cancer is declining in developed countries, it remains a leading cause of cancer-related death in the majority of developing countries. Genetic and environmental factors, including Helicobacter pylori infection, are considered to contribute to the development of gastric cancer. Moreover, recent studies have shown that the silencing of tumour suppressor genes by epigenetic modification is a fundamental mechanism of the pathogenesis of human cancer.2 Gastric carcinogenesis is thought to be a multistep process that involves multiple genetic and epigenetic events, but its underlying mechanisms have yet to be clarified, although it is known that promoter hypermethylation plays an essential role in tumour suppressor gene loss of function. In terms of the molecular basis of gastric cancer, it is believed that the aberrant DNA methylation of known tumour suppressor genes is more common that their mutation.3 However, the roles played by many of the genes affected by methylation-silencing in gastric cancer remain un-revealed, and further studies are necessary to identify all the silenced genes.
The epigenetic alterations of CpG islands in the promoter region of genes by aberrant DNA methylation have been established to be a common mechanism for silencing tumour suppressor genes in cancer cells.4, 5 Aberrant DNA methylation affects important cellular pathways by silencing tumour-related genes.6 Generally, tumour cells have promoter methylation patterns that differ from those of normal cells.7 Furthermore, recently, DNA methylation has emerged as promising biomarker for cancer detection8, and a number of genes, e.g. APC, COX2, DAP-K, CDH1, p14, CDKN2A, THBS1, TIMP3, hMLH1, p15, MINT1, MINT2, MINT25, CHFR and CDH4, have been found to be aberrantly methylated in gastric cancer.9, 10, 11, 12, 13, 14 Nevertheless, many of the specific molecular events involved in the pathogenesis and progression of gastric cancer remains to be elucidated. A global approach to the identification of epigenetically silenced genes in gastric cancer cells could provide methylation signatures for early detection and prognostic stratification, identify novel targets for therapy and lead to a deeper understanding of the biology of gastric cancer.
Epigenetic silencing is not an irreversible process, for example, drugs like 5-aza-2′-deoxycytidine (5Aza-dC) cause generalised demethylation. 5Aza-dC is known to act by being incorporated into new DNA strands during replication, where it forms a covalent complex with methyltransferase active sites, thus reducing methyltransferase activity.4 On the other hand, chromatin is a DNA/histone complex, and histone deacetylation is also known to impair gene transcription.15 Furthermore, DNA methylation has been reported to promote histone deacetylation.16 Methyl-CpG binding protein 2 (MeCP2) appears to reside as a complex with histone deacetylase17, whereas DNA methyltransferase binds to histone deacetylase 2 (HDAC2) and a transcriptional co-repressor, DMAP1.18 Thus, densely methylated DNA associates with transcriptionally repressive chromatin, which is characterised by the presence of under-acetylated histones. Trichostatin A (TSA; a histone deacetylase inhibitor) has been reported to reverse the formation of transcriptionally repressive chromatin on methylated promoter templates.19 Thus, epigenetic alterations are dynamically linked, and synergy between DNA demethylation and histone deacetylase inhibition using TSA has been shown to reactivate genes silenced in carcinoma more robustly than 5Aza-dC alone.20, 21
A number of different approaches have been developed for the genome-wide identification of cancer-associated hypermethylated genes, such as, restriction landmark genome scanning, methylation-sensitive arbitrarily primed PCR and methylated CpG island amplification.22 An alternative approach is to treat cells with epigenetic modifying drugs to facilitate the expressions of hypermethylated/silenced genes and then to compare gene expression profiles by microarray analysis to identify putatively hypermethylated genes. The methylation status of such genes can then be confirmed in untreated cells by bisulphite sequencing or methylation-specific PCR analyses. Global demethylation and microarray analysis have been successfully used to identify a number of novel genes hypermethylated in colorectal and pancreatic cancer.21, 23 In the present study, we examined the global reactivations of epigenetically silenced genes using an oligonucleotide microarray in three gastric cancer cell lines. Genes that were markedly upregulated (>twofold) by 5Aza-dC treatment in these cancer cell lines were considered genes of interest. Subsequently, bisulphite sequencing and methylation-specific PCR analyses were carried out on these genes to confirm the presence of aberrantly methylated CpG dinucleotides. In the present study, we show that the methods employed preferentially selected epigenetically silenced hypermethylated genes in primary gastric carcinomas. Finally, six genes found to be hypermethylated in at least one of the three cell lines were investigated to determine whether their methylation statuses were correlated with clinicopathological parameters.
Section snippets
Cell lines and tissue samples
Three human gastric cancer cell lines, (SNU-1, -601 and -719) were obtained from the Korean Cell Line Bank (Seoul, Korea). All cell lines were grown in RPMI1640 supplemented with 10% foetal bovine serum (FBS; Hyclone, Lorgan, UT, USA) and antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin) at 37 °C in a humidified 5% CO2 incubator.
Formalin-fixed, paraffin-embedded samples of 566 gastric cancer tissue specimens, resected at Seoul National University Hospital from January 1995 to December
Identification of candidate genes reactivated by 5Aza-dC in gastric cancer cell lines
Global changes in gene expression profiles induced by 5Aza-dC in the three gastric cancer cell lines (SNU-1, -601 and -719) were determined using Affymetrix HG U133 2.0 oligonucleotide microarrays containing 22,277 transcripts representing 14,500 well-characterised human genes. A comprehensive survey for hypermethylated gene candidates associated with gastric cancer was performed by comparing mock-treated and 5Aza-dC-treated gastric cancer cells. A summary of our differential and epigenetic
Discussion
Methods of detecting DNA methylation are based on the differentiation of cytosine and 5-methylcytosine in DNA. Three strategies are currently used: (1) DNA digestion using a methylation-sensitive or -insensitive restriction endonuclease, (2) chemical modification of DNA by sodium bisulphite or metabisulphite and (3) immunoprecipitation of 5-methylcytosine to directly separate unmethylated and methylated genomic fractions. Recently, all three of these approaches have been coupled to
Conflict of interest statement
None declared.
Acknowledgement
This study was supported by a Grant (A080316) of the ’08 Good Health R&D Project, Ministry of Health & Welfare, Republic of Korea.
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