Therefore it is reasonable to assume the increase detected by the anti body against phosph Acetyl H3 is mostly due to the acetylation of H3 induced by butyrate. Butyrate Induces Profound Changes in Gene Expression in MDBK Cells In our previous study no and prior to the current micro array experiment, we monitored butyrate induced cell death and cell cycle arrest in MDBK cells in a time dose dependent manner using flow cytometry and Western blotting. For the purpose of this investigation and for determining the differential gene expression induced by butyrate, we selected a single dose proven to be able to generate maximum biological impacts in cell cycle arrest. Due to research budget constraints, we selected only a sin gle time point as the initial global expression screening.

We intend to verify the genes identified with the microar ray in a time dose dependent manner using real time RT PCR in subsequent studies. We identified 450 genes sig nificantly regulated by sodium butyrate at a very stringent false discovery rate 0%. However, when relaxing the stringency threshold to FDR 10%, which should still be acceptable in most cases, there were 3662 genes significantly regulated. This percentage was con sistent with a previous report in which the authors used cDNA microarrays consisting of 8000 sequences to demonstrate that approximately 7% of sequences assayed exhibited alteration by butyrate in human colon carci noma cells. Cell Cycle Control The single largest category of genes regulated by butyrate was cell cycle control related.

Butyrate repressed the vast majority of these genes including cyc lins, cyclin dependent kinases, histone deacetylases, heli cases, chromosomal structure proteins as well as kinesins. Ubiquitin conjugating enzyme E2 C was also down regu lated. However, Max interacting protein 1 was up regulated 12. 6 fold by butyrate. Apoptosis and Extracellular Matrix Fourteen genes related to apoptosis and extracellular matrix were significantly regulated by butyrate. Generally, butyrate induced pro apoptotic genes and repressed anti apoptotic genes. Inhibin, beta A and adrenomedullin were among the induced genes while apoptosis inhibitors such as survivin and FAIM were down regulated. Interestingly, Insulin like growth factor 2 was up regulated while IGF bind ing proteins 4 and 6 were down regulated. Collagens, such as COL5A2 and COL3A1, and extracellular matrix protein Spondin 1 were repressed by butyrate while tissue inhibitor of metalloproteinase 2 was signifi cantly up regulated. Real Time RT PCR Thirty genes that represent different expression levels and functional classes were selected for real time RT PCR con firmation. The real Cilengitide time PCR data generally con firmed the microarray analysis.