APOPTOTIC CELL-DEATH IN NEURONAL DIFFERENTIATION OF P19 EC CELLS - CELL-DEATH FOLLOWS REENTRY INTO S-PHASE

Apoptotic cell death was observed during aggregate culture of the mous e embryonal carcinoma cell line P19 exposed to all-trans retinoic acid (tRA). This finding was confirmed by genomic DNA agarose gel electrop horesis and transmission electron microscopy. Apoptosis was associated with P19 cell neuronal differentiation; alternative causes of cell de ath, i.e., cavitation-related, cytotoxicity of tRA, or spontaneous cel l death were excluded. Analysis by flow cytometry revealed that the ap optosis was likely to occur in multiplying cells is that underwent to reentering into S phase. We therefore examined 5-bromo-2'-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) ex pression and localization in the aggregates by immunofluorescent stain ing. Although the P19 cells in the aggregates exposed to tRA incorpora ted BrdU at an equivalent level to those not exposed to tRA, the cells showed diminished PCNA expression and nuclear accumulation. We propos e that P19 apoptosis during neuronal differentiation is a model system in which programmed cell death occurs simultaneously with cell divisi on leading to differentiation. (C) 1997 Wiley-Liss, Inc.