Butyrate is a short-chain fatty acid with significant physiological relevance for the proper functioning of the colonic epithelium. In colon cancer cell lines in vitro, butyrate consistently induces a defined maturation response, involving p21WAF1-dependent cell cycle arrest, lineage specific differentiation and apoptosis. Butyrate is a potent and direct inhibitor of histone deacetylase (HDAC) activity. Importantly, butyrate-induction of colon cell maturation is mimicked by the specific, yet structurally unrelated HDAC-inhibitor, trichostatin A, suggesting inhibition of HDAC activity is a critical component of the butyrate response. We recently used cDNA microarrays to demonstrate that butyrate-induction of colon cell maturation is driven by extensive genetic reprogramming, involving the recruitment of approximately 7 % of over 8000 sequences analyzed. Furthermore, we identified a subset of these genes which are coordinately regulated by trichostatin A, indicating a key role for HDACs in their regulation. Currently, 18 distinct HDACs have been identified in eukaryotic cells. These can be categorized into one of three distinct classes based upon their homology to their prototypical yeast HDAC. Genomewide microarray analysis in yeast has clearly demonstrated that different HDACs regulate distinct cellular processes (1, 2). Whether this is also the case in mammalian cells, and whether inhibition of specific HDACs drives different components of the butyrate response is not known. Furthermore, the link between inhibition of HDAC activity and the induction of colon cell maturation suggests a key role for HDACs in the physiological regulation of colon cell maturation in vivo. In this study, we will dissect the contribution of HDACs 1, 2 and 4, to the various components of the butyrate response.
In aim 1, we will utilize our experience with cDNA microarrays to address this question on a genomewide scale.
In aim 2, we will use the well-defined, and critical component of the butyrate response, induction of p21WAF1promoter activity, to determine the role of these same HDACs on butyrate-induction of a specific target gene.
Aim 3 will extend these observations to determine the role of HDACs in other models of colon cell maturation, and most important, in the maturation of normal colonic and small intestinal epithelial cells, in vivo. Finally, while HDAC-inhibitors induce classical anti-tumor effects in vitro their effect on intestinal tumor formation has not been tested in vivo. Therefore, in aim 4, we will capitalize on our experience with mouse models to determine the effect of two inhibitors of HDAC activity, butyrate and trichostatin A on ApcMin-initiated intestinal tumorigenesis in vivo. Collectively, these studies are designed to define the role of specific HDACs in the regulation of colon cell maturation. Understanding the critical pathways that drive normal colonic epithelial cell maturation will enhance our understanding of how colorectal tumorigenesis is initiated, which in turn, will improve our ability to prevent and treat this disease.
