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.