Apoptosis of human glioma cells in response to calphostin C, a specific protein kinase C inhibitor.

Calphostin C acts at the regulatory domain as a highly selective inhibitor of protein kinase C (PKC), and staurosporine acts at the catalytic domain as a nonspecific PKC inhibitor. The authors investigated the capacity of calphostin C and staurosporine to promote apoptotic fragmentation of DNA in four human glioma cell lines. The exposure of glioma cell lines to 100 nM calphostin C for 2 to 8 hours induced a decrease in particulate PKC activities and exposure for 16 to 24 hours produced a concentration-dependent increase in internucleosomal DNA cleavage on agarose gel electrophoresis. In addition, the human glioma cells showed the classic morphological features of apoptosis: cell shrinkage, nuclear condensation, and the formation of apoptotic bodies. A 24-hour exposure to staurosporine failed to induce internucleosomal DNA fragmentation at concentrations generally used to achieve maximum inhibition of enzyme activity (50 nM) but promoted fragmentation at considerably higher concentration (more than 200 nM). Deoxyribonucleic acid fragments obtained from cells exposed to 100 nM calphostin C for 16 to 24 hours possessed predominantly 5'-phosphate termini, consistent with the action of a Ca++/Mg(++)-dependent endonuclease. Northern and Western blot analyses revealed that the exposure to 100 nM calphostin C for 4 hours failed to alter bcl-2 transcript and protein, but exposure for more than 8 hours decreased the amount of bcl-2 transcript and protein. Together, these observations suggest that calphostin C is capable of inducing apoptotic DNA fragmentation and cell death in a highly concentration dependent manner in human glioma cells and that the apoptosis is closely associated with the decrease in transcription and translation of bcl-2.