Objective: To investigate the effects of histone acetylation on the expression of p21(WAF1) and p16(INK4A) genes in two human colon cancer cell lines.
Methods: Two colon cancer cell lines (SW1116 and Colo-320) were treated with the DNA methyltransferase (DNMT) inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC) and/or the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) or sodium butyrate (NaBu). The cell cycle distribution was studied by flow cytometry (FCM). The expression of p21(WAF1) and p16(INK4A) genes mRNA was detected by real-time RT-PCR. The level of acetylated histones in chromatin associated with the p21(WAF1) and p16(INK4A) genes was examined by chromatin immunoprecipitation (ChIP) assay.
Results: TSA or NaBu blocked cells mainly in the G(1) phase, whereas 5-aza-dC treatment failed to affect cell cycle distribution. Expression of p16(INK4A) was detected slightly and p21(WAF1) was not expression in SW1116 and Colo-320 cells before treatment. In SW1116 and Colo-320 cells, the expression of p16(INK4A) gene was markedly increased after treatment of 5-aza-dC, although 5-aza-dC treatment did not activate the expression of p21(WAF1) gene. Treatment of TSA and NaBu resulted in the significant over-expression of p21(WAF1) in these two cell lines and induced an accumulation of acetylate histones H3 and H4 in chromatin associated with p21(WAF1) gene.
Conclusion: In these two human colon cancer cell lines, HDAC inhibitors stimulate the p21(WAF1) gene expression by selectively increasing the degree of acetylation of the gene-associated histones, and induce a G(1) cell cycle arrest. The expression of the p16(INK4A) gene is regulated by DNA methylation.