Similarly, the mechanism thing through which butyrate and other short chain fatty acids induce the cell cycle regulatory and apoptotic effects and the mechanism by which the decision between cell death and survival is arbitrated are poorly understood. In a previous study, potential biological roles of butyrate were investigated using the established Madin Darby bovine kidney epithelial cell line. The study focused on determining whether normal bovine cells in a standard cell culture condition were sensitive to the growth inhibitory effects of butyrate. The data sug gested that sodium butyrate could induce apoptosis and cell cycle arrest in MDBK cells. Up to 38% of cells became apoptotic after 24 hours of treatment with 10 mM of butyrate. Butyrate also blocked the surviving cells at two distinct stages, G1 and M G2.

However, more studies are needed to better understand the relationship between butyrate and alterations in the expression of genes involved in cell cycle, apoptosis, and transcriptional regu lation. Recent advances in high throughput genomic tools such as microarray technology allowed us to examine the genome wide effects of sodium butyrate on MDBK cells. Results Butyrate Induces Cell Cycle Arrest and Hyperacetylation of Histone 3 in MDBK Cells We previously reported that butyrate induced cell cycle arrest in MDBK cells. Prior to microarray analysis, the butyrate induced cell cycle arrest was reconfirmed. As shown in Figure 1, after butyrate treatment for 24 h, cell population profiles changed significantly.

In the surviving cell population there was a significant increase in the number of cells in G1 whereas those in S phase were decreased. This result confirmed our prior observation that cells were arrested at the G1 S boundary and DNA replication was blocked by the butyrate treatment. We also confirmed accumulation of acetylated histone 3 due to the butyrate treatment. H3 acetylation was selected as the marker for the accumulation of acetylated histones because H3 is one of the core histones, and is highly conserved across a wide range of organisms. The antibody against the acetyl H3 is also readily available com mercially. To determine whether these biochemical attributes of butyrate were also observed in MDBK cells, two specific antibodies, the monoclonal antibody against the acetyl H3 and the monoclonal antibody against acetyl phospho H3, were used to evaluate the histone deacetylase inhibitory activity of butyrate.

Histone deacetylase inhibitors induce the hyperacetyla tion of nucleosomal histones. Evidence suggested that H3 phosphorylation is restricted to a small fraction of highly acetylated H3 histones. H3 phosphorylation is cell cycle dependent and may be associated with induced FOS and MYC oncogenes. No evidence indicates Cilengitide H3 phosphoryla tion directly results from HADC inhibitory activities. As shown in Fig. 2, butyrate treatment induced not only accumulation of hyperacetylation of H3 but also phosphor acetyl H3.