Differentiation-associated apoptosis of neural stem cells is effected by Bcl-2 overexpression: impact on cell lineage determination

Apoptosis is an integral part of neural development. To elucidate the impor tance of programmed cell death on cell lineage determination we utilized mu rine PCC7-Mz1 cells, a model system for neural differentiation. Treatment o f pluripotent PCC7-Mz1 stem cells with 0.1 muM all-trans retinoic acid (RA) causes a cease of proliferation and an initiation of differentiation into neurons, glial cells and fibroblasts. Simultaneously, a fraction of the cel l culture (ca. 25%) dies within 24 h by apoptosis. We transfected PCC7-Mz1 cells with the human bcl-2 cDNA and generated PCC7-Mz-Bcl-2 cell lines expr essing two- to tenfold higher levels of Bcl-2 than parental cells. Overexpr ession of Bcl-2 resulted in hypophosphorylation of the retinoblastoma (Rh) protein and consequently prolonged the doubling time of the culture from 18 h to 23 h. RA-induced apoptosis was drastically reduced to 3 to 15% depend ing on the level of Bcl-2 expression. RA-induced caspase activation, cytoch rome c release from the mitochondria to the cytosol and DNA fragmentation w as completely blocked. Furthermore, treating Bcl-2 cultures with ceramide ( 10 muM), a second messenger mediating the RA-initiated death signal in pare ntal cells, no longer caused DNA laddering. Bcl-2 overexpression did not in terfere with the potential of PCC7-Mz cells to develop into neurons, glial cells and fibroblasts. However, the relative distribution of cell types in the culture was shifted such that the fraction of neurons was reduced to ha lf (from 60 to 30%) with a concomitant increase in the number of glial and fibroblastoid cells. Furthermore, Bcl-2-overexpressing neurons, but not neu rons of parental or mock-transfected PCC7-Mz1 cultures, were able to grow a s single cells.