In vitro induction of apoptosis of developing brain cells by 5-azacytidine.

We had found that 5-azacytidine (5Az), a cytidine analogue, could produce apoptosis of fetal developing neuronal cells on the day after injection of the agent into dams by the i.p. route at 11 days of gestation. To make a further understanding of the phenomenon by comparing the results between the in-vivo and in-vitro system, this study was carried out. Entire cephalic parts of the fetuses were collected aseptically at days 11 of gestation and a mixed culture, consisting of neuronal and mesenchymal cells, was established after one week of incubation. The silver-staining revealed pyknotic nuclei and loss of dendrites of neuronal cells in the lower (5 micrograms/ml) dose of the 5 Az-added group. SEM showed shrinkage of the cell body and blebbing formation on the cell surface. TEM evoked margination, segmentation and complete condensation of the nuclear chromatin. Scattered positive signals identical to the apoptotic cells and aggregated fragmented DNA were detected by the TUNEL method. Treatment of higher doses of 5Az (50 and 500 micrograms/ml), however, induced necrosis of both neuronal and mesenchymal cells, light- and electron-microscopically. On the contrary, the control group (0 microgram/ml) showed normal development of neuronal cells and very few positive signals of physiological apoptosis. It was concluded that 5Az could induce apoptosis of developing neuronal cells at lower doses, but necrosis of a wider cell population at higher doses. Involvement of hypomethylation, an important biochemical function of 5Az, in apoptosis was also speculated.