Activation of DNA‐Dependent Protein Kinase May Play a Role in Apoptosis of Human Neuroblastoma Cells

Abstract

Abstract : Treating SH‐SY5Y human neuroblastoma cells with 1 μM staurosporine resulted in a three‐ to fourfold higher DNA‐dependent protein kinase (DNA‐PK) activity compared with untreated cells. Time course studies revealed a biphasic effect of staurosporine on DNA‐PK activity : an initial increase that peaked by 4 h and a rapid decline that reached ~5‐10% that of untreated cells by 24 h of treatment. Staurosporine induced apoptosis in these cells as determined by the appearance of internucleosomal DNA fragmentation and punctate nuclear morphology. The maximal stimulation of DNA‐PK activity preceded significant morphological changes that occurred between 4 and 8 h (40% of total number of cells) and increased with time, reaching 70% by 48 h. Staurosporine had no effect on caspase‐1 activity but stimulated caspase‐3 activity by 10‐15‐fold in a time‐dependent manner, similar to morphological changes. Similar time‐dependent changes in DNA‐PK activity, morphology, and DNA fragmentation occurred when the cells were exposed to either 100 μM ceramide or UV radiation. In all these cases the increase in DNA‐PK activity preceded the appearance of apoptotic markers, whereas the loss in activity was coincident with cell death. A cell‐permeable inhibitor of DNA‐PK, OK‐1035, significantly reduced staurosporine‐induced punctate nuclear morphology and DNA fragmentation. Collectively, these results suggest an intriguing possibility that activation of DNA‐PK may be involved with the induction of apoptotic cell death.