Poly (ADP-ribose) polymerase induction is an early signal of apoptosis in human neuroblastoma.
Poly (ADP-ribose) polymerase (PARP) is an abundant chromatin associated protein important in DNA repair, maintenance of chromosomal stability and programmed cell death. Here we report that an increase in caspase 3-activity and cleavage of PARP serves as an early execution phase signal in human neuroblastoma. Human neuroblastoma SK-N-SH cells were exposed to a protein kinase inhibitor, staurosporine, or a topoisomerase II inhibitor, etoposide, at various concentrations and time points. Cells exposed to staurosporine (0.1 microM) for 30 min showed an increase in caspase 3-activity and by 1 h an increase in PARP 116-kDa band and an 85-kDa cleavage product, which further increased in density with time after treatment. Quantitative analysis for condensed chromatin material using bisbenzimide, and DNA fragmentation enzyme immunoassays showed a significant increase in apoptosis 5 h after staurosporine treatment. This was further confirmed with a Klenow fragment of DNA polymerase I assay which primarily detects single-stranded DNA breaks. A significant decrease in mitochondrial metabolism occurred within 8-12 h after treatment. Studies using Trypan Blue exclusion, and lactic dehydrogenase (LDH) release revealed a significant increase in membrane permeability 8 h after staurosporine (0.1 microM) or etoposide (10 microM) treatments. Cleavage of lamin B1, a protein important in maintaining the nuclear envelope integrity was observed 12 h after staurosporine treatment. Our results show that activation of caspase 3 followed by PARP cleavage occur at much earlier time point than any other morphological or biochemical parameters of apoptosis or cytotoxicity.
Bursztajn
S
S
Department of Neurosurgery, Biomedical Research Institute F6-21, Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130, USA. sbursz@lsumc.edu
Feng
J J
JJ
Berman
S A
SA
Nanda
A
A
D017209:Apoptosis; D015415:Biomarkers; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002843:Chromatin / Q000187:drug effects; D004256:DNA Polymerase I; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D009447:Neuroblastoma / Q000201:enzymology / Q000473:pathology*; D011065:Poly(ADP-ribose) Polymerases / Q000096:biosynthesis*; D019311:Staurosporine / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D015415:Biomarkers; D002843:Chromatin; D004791:Enzyme Inhibitors; D005047:Etoposide; D007770:L-Lactate Dehydrogenase; D011065:Poly(ADP-ribose) Polymerases; D004256:DNA Polymerase I; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine