PROGRAMMED CELL-DEATH IN CORTICAL CHICK-EMBRYO ASTROCYTES IS ASSOCIATED WITH ACTIVATION OF PROTEIN-KINASE PK60 AND CERAMIDE FORMATION

Embryonic astrocytes respond readily to serine/threonine kinase regula tion in terms of cytoskeleton assembly, mitotic activity, and cell fat e. We now present evidence that these responses include apoptosis. Sta urosporine induced apoptosis in astrocyte cultures derived from chick embryo cerebral hemispheres, as assayed both by immunocytochemical det ection of new 3-hydroxy DNA ends and production of 200-bp DNA fragment laddering. Staurosporine treatment also resulted in the prolonged (>2 4 h) activation of a 60-kDa serine/threonine protein kinase (PK60), in creased ceramide formation (fourfold after 24 h), increased glutamine synthetase activity, and significant apoptosis (40%) after 24 h. PK60 was shown to be cytoskeleton associated and its activity, as measured by phosphorylation of myelin basic protein, was rapid, increased for u p to 3 h, and was stable for at least 24 h. Other protein kinase C inh ibitors, H8, sphingosine, calphostin C, or the protein kinase A inhibi tor KT5720 did not induce either PK60 activation or apoptosis. The dos e-dependent increase in [H-3] palmitate labeling of ceramide and a spe cific decrease in labeling of its precursor sphingomyelin were not blo cked by the biosynthetic inhibitor fumonisin beta 1 but were increased (in a dose-dependent manner) by the coaddition of the ceramidase inhi bitor oleoylethanolamine. Exogenous addition of C2-ceramide induced ap optosis but did not activate PK60. These results Suggest that apoptosi s in embryonic astrocytes involves pathways similar to those described in other cell types and that the activation of PK60 and formation of ceramide are early events in the pathway.