Growth inhibition induced by Ro 31-8220 and calphostin C in human glioblastoma cell lines is associated with apoptosis and inhibition of CDC2 kinase

Protein kinase C (PKC) is a central component in signal transduction and gr owth control and might be an appropriate target for the chemotherapy of hum an brain tumors. This study demonstrates that the staurosporine derivative Po 31-8220, a potent PKC inhibitor, inhibited the growth of 7 human brain t umor cell lines with an IC50 of about 2 mu M. Calphostin C, a structurally unrelated PKC inhibitor, inhibited the growth of two of these cell lines wi th an IC50 of about 100 to 300 nM Drug withdrawal and clonogenicity assays indicated that the growth inhibition by both of these compounds was irrever sible. Morphologic studies, DNA fragmentation studies and flow cytometric a ssays showed that the treated glioblastoma cells underwent apoptosis Treatm ent of glioblastoma cells with Po 31-8220 lead to a rapid decline in the le vel of the anti-apoptosis protein bcl-2. At least thr ee of the glioblastom a cell lines carried mutant p53 alleles with missense mutations in the DNA binding domain of p53. Therefore, the induction of apoptosis in these cell lines occurred through a p53-independent mechanism. Furthermore treatment o f these glioblastoma cell lines with Po 31-8220 or calphostin C led to art increase of cells in the G(2)-M phase of the cell cycle. This correlated wi th a decrease in CDC2-associated histone HI kinase activity, as well as a d ecrease in the level of the CDC2 protein as shown by immunoblotting. When a dded to subcellular assays Po 31-8220 markedly inhibited CDC2 histone HI ki nase activity with an IC50 of 100 nM, but calphostin C directly inhibited t his kinase activity only at very high concentrations (above 100 mu M). Thus these compounds inhibit the growth of glioblastoma cells through novel mec hanisms. Po 31-8220 in particular might be a useful agent for the treatment of human brain tumors.