5-AZACYTIDINE INDUCES TOXICITY IN PC12 CELLS BY APOPTOSIS

5-Azacytidine (5 Az)is a potent inhibitor of DNA methylation, and it m ay allow inactive genes to become expressed. In a previous study, we d emonstrated that 5 Az administered to the dam induced apoptosis in the brains of fetal mice. In this study, the 5 At-induced apoptosis was f urther characterized in differentiated PC 12 cells as a model for neur onal apoptosis. Cell death, determined by the activity of released lac tate dehydrogenase (LDH) into the medium, occurred from 24 to 48 hrs a fter 5 Az treatment. Toxicity for differentiated PC 12 cells was obser ved on treatment with more than 10(-1) mu g/ml of 5 Az, and it reached the maximal level at 10 mu g/ml. Cycloheximide, an inhibitor of prote in synthesis, prevented 5 Az toxicity, suggesting that this cell death required protein synthesis which could be related to the activation o f a dormant gene(s). Electrophoresis of DNA from 5 At-treated cells ev oked ladder formation, indicating the cleavage of DNA into nucleosomes . Scanning electron microscopy demonstrated bleb formation, the so-cal led apoptotic bodies on the cell surface. The biochemical and morpholo gical findings indicated that 5 At-induced cell death occurred in the form of apoptosis. 5 At-induced cell death was prevented by treatment with cAMP but not by treatment with high K+ or deoxycytidine. These re sults suggest that a cAMP-sensitive mechanism is involved in 5 Az-indu ced cell death. PC 12 cells should be of value in elucidating the mole cular mechanism of 5 At-induced neuronal apoptosis.