The effect of Bad overexpression on apoptosis was demonstrated by a mouse Bad transgene stably expressed in NIH/3T3 cells. The cells overexpressing Bad treated with either serum starvation or ceramide showed apoptotic characteristics evident at 18 and 8 h, respectively. Whether serum deprivation and ceramide utilize a common death pathway requires further investigation. The time for the first apoptosis detection was shortened to 2 h and was prominent at 4 h, while above that time cells were maintained under serum-depleted conditions in the presence of ceramide (40 microM). Further investigation revealed that the activity of caspase-3 (CPP32) was elevated after ceramide treatment in Bad-transfected cells compared to that of the cells without Bad transfection, indicating the involvement of caspase cascade. Furthermore, the Bad-transfected cells showed reduced phosphorylation of extracellular signal-regulated kinase (ERK). Taken together, we hypothesize that Bad-overexpressing NIH/3T3 cells in the presence of ceramide undergo apoptosis by activating caspase cascade. Simultaneously, the cell survival pathway was blocked possibly by inactivation of the MAPK pathway such as the down-regulation of ERK.
Copyright 1999 Academic Press.