med.ufl.edu/biochem/keithr/research.html
DNA Methylation as a Chemoprevention Target.
Research Interests-Keith D. Robertson
My research interests are in the area of epigenetics, specifically DNA methylation, and its role in cancer and development. Most of our work centers on the enzymes that carry out the DNA methylation reaction in mammalian cells, the DNA methyltransferases (or DNMTs). We want to understand how cells determine which sequences will be methylated and which will not, and how this regulation goes awry in cancer cells as well as how it is involved in cell differentiation. Specific research interests fall into four general areas: (1) The DNMTs are expressed in recombinant form and their properties studied using in vitro and in vivo enzyme assays. (2) Their protein-interaction partners in vivo are identified using biochemical methods and yeast two-hybrid assays and the functional consequences of the interactions are studied in cells and in reconstituted in vitro systems. (3) We also use high-throughput gene expression techniques, coupled with pharmacologic manipulation of DNA methylation levels, to uncover genes aberrantly silenced by DNA methylation in cancer. (4) Lastly, we are interested in understanding the epigenetic make-up of pluripotent cells and how DNA methylation is involved in regulating their differentiation.
Unlike tumor suppressor genes that have been inactivated by genetic mutations, genes silenced by DNA methylation are intact and can be reactivated by small molecule inhibitors of the DNMTs (Kopelovich et al. 2003). Therapies targeting DNA methylation and the DNA methyltransferases have therefore attracted significant interest as a means for cancer prevention. Inhibitors of DNA methylation, such as 5-aza-2’-deoxycytidine (5-azadC), zebularine (Cheng et al. 2004), antisense oligonucleotides to DNMT1 (MacLeod et al. 1995), and procainamide (Lin et al. 2001), are capable of reversing aberrant promoter hypermethylation, resulting in gene reactivation and restoration of cell growth control, apoptosis, and DNA repair capacity (Murakami et al. 1995; Bender et al. 1998; Herman et al. 1998; Zhu et al. 2001). 5-azadC administered to ApcMin/+ mice, a model for human colon cancer, resulted in dramatically reduced rates of intestinal tumor formation (Laird et al. 1995). In other studies, 5-azadC treatment diminished the formation of aberrant crypt foci in the colons of selenium-deficient rats that had been treated with carcinogens (Davis et al. 2002). Administration of 5-azadC to mice also prevented lung tumor development in animals treated with a carcinogen found in tobacco smoke (Belinsky et al. 2003). Thus there is ample evidence that targeting epigenetic changes in ‘normal’ tissues with small molecule DNMT inhibitors results in dramatic reductions in certain cancers in model systems. Although existing demethylating agents such as 5-azadC may not be ideal for use in a prevention strategy (requiring long-term administration) due to safety concerns, other agents such as procainamide, zebularine, and valproic acid (Detich et al. 2003) may be safer for long-term use. In addition, there is now significant interest in the pharmaceutical industry in developing new and more specific inhibitors of epigenetic modifications; therefore new drugs better suited for a chemoprevention strategy will likely be available within the next few years. DNA methylation changes are very early events directly contributing to many cancers, they can be readily detected with non-invasive assays, and DNMT inhibitors dramatically reduce the incidence of colon cancer in high-risk model systems. These aspects make promoter hypermethylation an excellent early detection biomarker and target for cancer chemoprevention.