DIFFERENTIATION TO AN NGF-DEPENDENT STATE AND APOPTOSIS FOLLOWING NGFREMOVAL BOTH OCCUR ASYNCHRONOUSLY IN CULTURES OF PC12 CELLS

Long-term timelapse videomicroscopy was used to investigate the relati onships and transitions between mitosis, differentiation, and apoptosi s in cultures of NGF-differentiated PC12 cells. After 4 days in NGF, c ultures 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 ne uronal; 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 d ifferentiated; removal of NGF did not affect the negligible mitotic ra te 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 t he same morphology at all three differentiation states. Thus, acquisit ion of NGF-dependence in PC12 cultures seemed to be the result of a st eadily increasing percentage of cells that had each undergone a relati vely rapid transition to a postmitotic, NGF-sensitive state. These stu dies were also helpful for elucidating the timing of apoptosis. Onset of apoptosis was markedly asynchronous within a culture, but the activ e, blebbing phase, once initiated, always lasted about 45 min, regardl ess of differentiation state or time spent without NGF. Thus, the acti ve phase might represent a conserved sequence of events that every cel l must ultimately undergo before apoptotic death. (C) 1997 Academic Pr ess.