Epigenetics are involved in the regulation of the cell cycle and expression of tumor suppressor genes in human colon cancer cells

Abstract

OBJECTIVE: To investigate the effects of DNA methylation and histone acetylation on the cell cycle progression and expression of tumor suppressor genes in human colon cancer (HCC) cell lines.METHODS: Three HCC cell lines (HT‐29, SW1116 and Colo‐320) were treated with the DNA methyl­ation inhibitor, 5‐aza‐2'‐deoxycytidine (5‐aza‐dC) or/and histone deacetylase (HDAC) inhibitors, tricho­statin A (TSA) or sodium butyrate. The methylation status of the promoter of the p16INK4A gene was assayed by methylation‐specific PCR (MSP). The expression of p16INK4A and p21WAF1 was analyzed by RT‐PCR. The cell cycle distribution was determined by flow cytometry.RESULTS: Before treatment, p16INK4A expression was slightly detected in the three cell lines (HT‐29, SW1116 and Colo‐320) and p21WAF1 expression was not detected in SW1116 and Colo‐320 cells. The methylation level of the p16INK4A gene promoter significantly decreased and mRNA expression markedly increased in HT‐29 cells after treatment with 1 µmol/L, but not 10 µmol/L, of 5‐aza‐dC for 24 h. In the SW1116 and Colo‐320 cells, the expression of p16INK4A was markedly enhanced at 10 µmol/L or 5 µmol/L of 5‐aza‐dC for 24 h. However, p21WAF1 gene expression was not detected. Interestingly, after treatment with TSA or sodium butyrate, the transcription of p21WAF1 was significantly upregulated in these two cell lines. Furthermore, 5‐aza‐dC did not affect cell cycle distribution, but TSA or sodium butyrate blocked the cell cycle, mainly in the G1 phase.CONCLUSIONS: The expression of the p16INK4A gene is regulated by DNA methylation in three HCC cell lines. The expression of p21WAF1 gene is regulated by histone acetylation in SW1116 and Colo‐320. In these two cell lines, histone hyperacetylation causes a G1 cell cycle arrest.