Delineation of the signaling pathways involved in glucocorticoid-induced and spontaneous apoptosis of rat thymocytes

In primary rat thymocytes, both glucocorticoids and the withdrawal of in vi vo survival factors elicit apoptosis. In this study we wanted to determine whether distinct pathways leading to apoptosis are engaged by these two sti muli. To address this question, we conducted a multiparametric analysis of cell viability, DNA fragmentation, activation of caspase-3-like activity, c ell shrinkage, the loss of mitochondrial membrane potential, and externaliz ation of phosphatidylserine in the absence and presence of protein and RNA synthesis. The role of caspase activity was also examined in both glucocort icoid- and survival factor withdrawal-induced cell death. We show that gluc ocorticoid-induced, but not spontaneous, loss of viability is dependent upo n macromolecular synthesis and caspase activity. Furthermore, glucocorticoi d-induced phosphatidylserine externalization and cell shrinkage are depende nt upon gene regulation and caspase activity, whereas these features manife st independently of gene regulation and caspase activity in spontaneous dea th. In contrast, the loss of mitochondrial membrane potential was dependent upon macromolecular synthesis only in glucocorticoid-induced death and was independent of caspases in both spontaneous and dexamethasone-induced deat h. These results suggest that thymocytes can die by a caspase-independent m echanism and that a major difference between glucocorticoid- and survival f actor deprivation-induced death is the dependence on gene expression.