Differentiation to an NGF-dependent state and apoptosis following NGF removal both occur asynchronously in cultures of PC12 cells

Long-term timelapse videomicroscopy was used to investigate the relationships and transitions between mitosis, differentiation, and apoptosis in cultures of NGF-differentiated PC12 cells. After 4 days in NGF, cultures were at an early stage of neuronal differentiation. Removal of NGF led to an appreciable increase in apoptosis with no effect on the relatively high mitotic rate. After 7 days in NGF, cells were more neuronal; NGF withdrawal again resulted in no change in the low mitotic rate but an even greater increase in apoptosis, eventually leading to considerable net loss of cells. After 10 days, cells were terminally differentiated; removal of NGF did not affect the negligible mitotic rate but induced a dramatic increase in apoptosis resulting in death of most of the cells. Apoptosis in the fraction of cells that had become NGF-dependent followed a similar timecourse and was characterized by the same morphology at all three differentiation states. Thus, acquisition of NGF-dependence in PC12 cultures seemed to be the result of a steadily increasing percentage of cells that had each undergone a relatively rapid transition to a postmitotic, NGF- sensitive state. These studies were also helpful for elucidating the timing of apoptosis. Onset of apoptosis was markedly asynchronous within a culture, but the active, blebbing phase, once initiated, always lasted about 45 min, regardless of differentiation state or time spent without NGF. Thus, the active phase might represent a conserved sequence of events that every cell must ultimately undergo before apoptotic death.