Previous studies have demonstrated that programmed cell death takes place at different stages during the development of the CNS in vivo. Our purpose in this study was to detect early programmed cell death associated with the induction of differentiation by retinoic acid (RA) in the NT2 cell line. By using the annexin V labeling as a marker of apoptosis, a significant apoptotic cell death was quantified during the third and the fourth days of the RA treatment. Double-labeling studies using the staining of the genomic DNA strand breaks with the terminal deoxyribosyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay and either nestin or microtubule-associated protein 2 (MAP2) showed that 1) the early apoptotic cell death affected mostly nestin-positive cells and 2) after 8 days of differentiation, although cells with neuronal phenotypes are present, no colabeled TUNEL/MAP2 cells were detected. With regard to the neuronal protein MAP2, we observed discrete immunolabeling of a few NT2 cells as early as day 3 of the differentiation and a significant emergence of MAP2-immunopositive cells at days 6-8. Thus, our results show that, when as a whole the differentiating NT2 cell population is considered, 1) the apoptotic cell death observed during the third day of differentiation occurs mostly in undifferentiated cells, 2) this process coincides with the first detection of the neuronal phenotype in NT2 cell cultures, and 3) the end of the cell death period in NT2 cell cultures is marked by both the accumulation of MAP2-positive cells and the beginning of expression of the Bcl-2 protein in the cultures.
Copyright 2002 Wiley-Liss, Inc.