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 importance of programmed cell death on cell lineage determination we utilized murine PCC7-Mzl cells, a model system for neural differentiation. Treatment of pluripotent PCC7-Mzl stem cells with 0.1 microM 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 cell culture (ca. 25%) dies within 24 h by apoptosis. We transfected PCC7-Mzl cells with the human bcl-2 cDNA and generated PCC7-Mz-Bcl-2 cell lines expressing two- to tenfold higher levels of Bcl-2 than parental cells. Overexpression of Bcl-2 resulted in hypophosphorylation of the retinoblastoma (Rb) 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% depending on the level of Bcl-2 expression. RA-induced caspase activation, cytochrome c release from the mitochondria to the cytosol and DNA fragmentation was completely blocked. Furthermore, treating Bcl-2 cultures with ceramide (10 microM), a second messenger mediating the RA-initiated death signal in parental cells, no longer caused DNA laddering. Bcl-2 overexpression did not interfere 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 half (from 60 to 30%) with a concomitant increase in the number of glial and fibroblastoid cells. Furthermore, Bcl-2-overexpressing neurons, but not neurons of parental or mock-transfected PCC7-Mzl cultures, were able to grow as single cells.