Genes inappropriately silenced by structural chromatin changes that involve DNA methylation can be reactivated by demethylating agents, that can reverse these changes and, therefore, restore principal cellular pathways. This results in gene re-expression and reversion of some aspects of the transformed state. The demethylating agent 5-azacytidine and its deoxy derivative 5-aza-2'deoxycytidine were first synthesized in Czechoslavakia as potential chemotherapeutic agents for cancer (Cihak, 1974). These agents are incorporated into the nucleic acids of dividing cells, where they act as mechanism-based inhibitors of DNA methytransferases. They inactivate DNA cytosine C5- methyltransferases through the formation of stable complexes between the 5-aza-2'-deoxycytidine residues in DNA and the enzyme, thereby mimicking a stable transition state intermediate when bound to the methyltransferase enzyme (Sheikhnejad et al., 1999).
These powerful inhibitors of DNA methylation, can restore gene function to treated cells in culture, which has indicated that they may have potential in treating patients with malignant disease (Lubbert, 2000; Jones and Baylin, 2002).
Cells are seeded at a density of 5x10 5 /100-mm dishes, cultured for 48 hours, and treated with 0, 50, or 100 m M 5-aza-dC (Sigma Chemical Co., St. Louis, MD) (Li et al., 2001).
Forty-eight hours after treatment, cells are washed with PBS and fresh medium was added. Cells are further incubated for another 48 h before isolated total cellular RNA.
For protein studies, cells are seeded at a density of 5x10 5 /100-mm dishes, cultured for 24 h, and treated with 0, 1, 2, 3, 5, and 10 m M 5-aza-dC. After 5 days, cell supernatants are harvested and centrifuged at 1,800 rpm to pellet and remove any cell debris. Supernatants are subsequently transferred to a new tube and analyzed for protein concentrations or stored at -20 0 C .