Epigenetic regulation of p16INK4a in human gastric cancer.

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Epigenetic regulation of p16INK4a in human gastric cancer. Epigenetic regulation of p16INK4a in human gastric cancer. A, MSP to identify changes in the methylation status at p16INK4a promoter regions in human gastric cancer cell lines. Primers were designed to study DNA methylation patterns in CpG islands of the p16INK4a promoter start site (genomic position: +167, genomic position is the location of the 5′ nucleotide of the sense primer in relation to the major transcriptional start site defined in the Genbank accession numbers: X94154). In addition, the effect of treatment of human gastric cancer cell lines with 5-aza-dC was investigated. The presence of a PCR product in lanes labeled U indicates an unmethylated status for the p16INK4a promoter region, whereas the presence of a PCR product in lanes labeled M indicates a methylated promoter status. B, p16INK4a levels are evaluated in HDAC2 knockdown or 5-aza-dC treated cells to ascertain the effects of HDAC2 knockdown or 5-aza-dC treatment on p16INK4a protein expression in gastric cancer cell lines. C, cell proliferation of MKN-1, SNU-216, SNU-484, and SNU-719 human gastric cancer cell lines treated with 5-aza-dC (2 μmol/L). Cells were grown in 24-well plates in medium supplemented with 5-aza-dC for up to 96 hours. Cell proliferation was determined by the MTT assay. Data are expressed as means ± SDs. *, P < 0.01. All measurements were conducted in triplicate, and each experiment was repeated at least twice. D, Western blotting of HDAC2 and p16INK4a expressions in human normal gastric mucosa and cancer tissues. The blot shown is typical of at least 3 individual experiments. E, analysis of the methylation status of the p16INK4a promoter by MSP in paired-gastric cancer tissues of the same patient. N, normal gastric mucosa; T, gastric cancer tissue. F, chromatin-IP to evidence HDAC2 or acetylated histone H4 DNA binding. A marked increased in acetylated histone H4 DNA binding is noted in HDAC2 gene silenced MKN-1 human gastric cancer cells expressing short hairpin RNAs. This coincides with recovered gene expression of p16INK4a. IgG was used as a negative control, whereas total input DNA (Bottom) was used as a positive PCR control for the target promoter. Depicted is the proximal p16INK4a promoter regions, that is, nucleotide −177 to +135, and the result shown is representative of 3 conducted experiments. Jeong Kyu Kim et al. Mol Cancer Res 2013;11:62-73 ©2013 by American Association for Cancer Research