BCL-2 INHIBITS RETINOIC ACID-INDUCED APOPTOSIS DURING THE NEURAL DIFFERENTIATION OF EMBRYONAL STEM-CELLS

We report here that all trans-retinoic acid (RA), a classical morphoge n, induces apoptosis during the neural differentiation of the embryoni c stem cell line P19. The apoptotic cells showed, in addition to DNA c leavage, typical morpholo,oical changes including chromatin condensati on, nuclear fragmentation, and cytoplasmic vacuolation. These apoptoti c changes became obvious by 12 h after the addition of RA. The endogen ous expression of bcl-2 in surviving cells was down-regulated during t his process, and the compelled expression of bcl-2 by retroviral vecto rs reduced the number of apoptotic cells. Apoptosis was partially inhi bited by adding antisense oligonucleotides against RA receptors (RARs) simultaneously or by transfecting a plasmid vector flanked with a RA- responsive element. Antisense oligonucleotides against retinoid X rece ptors (RXRs), the receptors for 9 cis-RA, did not inhibit apoptosis in duced by all trans-RA. Cycloheximide and actinomycin D, inhibitors of protein and RNA syntheses, respectively, suppressed apoptosis. No chan ges were seen in the expression of tumor necrosis factors, their recep tors, Fas, FasL, p53, or c-myc, molecules which have been suggested to participate in the apoptotic process. Addition of neurotrophins to th e culture medium did not affect apoptosis. These findings suggest that the signals mediated by RARs, the differentiation signals themselves, promote expression of molecules essential for apoptosis. Furthermore, we observed that RA induced apoptosis of cerebral neurons from murine embryos in primary culture, which suggests that RA might participate in cell death which occurs during neural development.