Activation of DNA-dependent protein kinase may play a role in apoptosis of human neuroblastoma cells.
Treating SH-SY5Y human neuroblastoma cells with 1 microM 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 approximately 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 microM 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.
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
B R
BR
Chakravarthy
T
T
Walker
I
I
Rasquinha
I E
IE
Hill
J P
JP
MacManus
D017209:Apoptosis / Q000502:physiology*; D001932:Brain Neoplasms / Q000201:enzymology* / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D053938:DNA Fragmentation; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D015151:Immunoblotting; D009447:Neuroblastoma / Q000201:enzymology* / Q000473:pathology; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011728:Pyridones / Q000494:pharmacology; D019311:Staurosporine / Q000037:antagonists & inhibitors / Q000633:toxicity; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
C095985:3-cyano-5-(4-pyridyl)-6-hydrazonomethyl-2-pyridone; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D006835:Hydrazones; D009687:Nuclear Proteins; D011728:Pyridones; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D020169:Caspases; D019311:Staurosporine