Effect of 5-azacytidine on the methylation aspects of NMDA receptor NR2B gene in the cultured cortical neurons of mice.

Our previous study revealed that the exposure of the drug 5-Azacytidine and ethanol to the cultured cortical neurons of mice causes demethylation of cytosine residues in the CpG island of the NMDA receptor NR2B gene (Marutha Ravindran and Ticku, Mol Brain Res 121:19-27, 2004). In the present study, we further analyzed methylation in the CpG island with various concentration frame and time frame of exposure of the cultured cortical neurons with 5-azacytidine to identify whether methylation in the NR2B gene is site specific or region specific. Methylation was studied by digesting the genomic DNA with methylation sensitive HpaII, MspI, AciI or HhaI enzyme following the exposure of cultured cortical neurons of mice with 5-azacytidine by performing PCR and Southern hybridization. We observed demethylation of DNA at 1, 3 and 5 muM concentrations of 5-azacytidine in the regions (5982-6155), (6743-7466) and at 3 and 5 muM concentrations of 5-azacytidine used in the region (6477-6763). Similarly in the time frame study with 5-azacytidine, demethylation of DNA was observed at 24 h and 36 h of incubation with 5-azacytidine in the regions (5982-6155), (6743-7466) and at 36 h of incubation with 5-azacytidine used in the region (6477-6763). Our experimental results demonstrate that the methylation in the CpG islands of the NR2B gene may not be site specific or region specific in the cultured cortical neurons of mice.