Poly (ADP-ribose) polymerase induction is an early signal of apoptosis in human neuroblastoma

Poly (ADP-ribose) polymerase (PARP) is an abundant chromatin associated pro tein important in DNA repair, maintenance of chromosomal stability and prog rammed cell death. Here we report that an increase in caspase S-activity an d cleavage of PARP serves as an early execution phase signal in human neuro blastoma. Human neuroblastoma SK-N-SH cells were exposed to a protein kinas e inhibitor, staurosporine, or a topoisomerase II inhibitor, etoposide, at various concentrations and time points. Cells exposed to staurosporine (0.1 mu M) for 30 min showed an increase in caspase 3-activity and by 1 h an in crease in PARP 116-kDa band and an 85-kDa cleavage product, which further i ncreased in density with time after treatment. Quantitative analysis for co ndensed chromatin material using bisbenzimide, and DNA fragmentation enzyme immunoassays showed a significant increase in apoptosis 5 h after staurosp orine treatment. This was further confirmed with a Klenow fragment of DNA p olymerase I assay which primarily detects single-stranded DNA breaks. A sig nificant decrease in mitochondrial metabolism occurred within 8-12 h after treatment. Studies using Trypan Blue exclusion, and lactic dehydrogenase (L DH) release revealed a significant increase in membrane permeability 8 h af ter staurosporine (0.1 mu M) or etoposide (10 mu M) treatments. Cleavage of lamin B1, a protein important in maintaining the nuclear envelope integrit y was observed 12 h after staurosporine treatment. Our results show that ac tivation of caspase 3 followed by PARP cleavage occur at much earlier time point than any other morphological or biochemical parameters of apoptosis o r cytotoxicity. (C) 2000 Elsevier Science B.V. All rights reserved.