Nucleolar activation during artificial demethylation of DNA in cultured pig embryonic kidney cells.

The level of nucleolar activity after incubation of pig embryonic kidney cells with an inhibitor of enzymatic methylation of DNA, 5-azacytidine, which decreased DNA methylation from (3.0 +/- 0.2) to (1.0 +/- 0.3) mol% of 5-methylcytosine in total cytosine, was studied. Addition of 5-azacytidine to the culture medium for 12 or 24 h followed by incubation of the cells in fresh 5-azacytidine-free culture medium for 48-72 h led to the activation of rRNA synthesis. This was evident from the increased level of incorporation of [3H]uridine recorded by biochemical techniques and also from the increase in the intensity with which the nucleoli were stained with silver nitrate and from the changes in their ultrastructure. When the cells were maintained in the presence of sodium butyrate, an inhibitor of histone deacetylases, rRNA synthesis was activated in stationary phase cultures, but the inhibitor had no marked effect on the nucleolar activity of cells which had been preincubated with 5-azacytidine. The results obtained are discussed from the standpoint of the mechanisms associated with the regulation of the transcription of ribosomal genes.