Characterization of CPP32-like protease activity following apoptotic challenge in SH-SY5Y neuroblastoma cells.
We characterized the activation of interleukin-1beta-converting enzyme (ICE)-like proteases (caspases) in human neuroblastoma cells (SH-SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti-poly(ADP-ribose) polymerase (anti-PARP) and anti-CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl-DEVD-7-amido-4-methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32-like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH-SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH-SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.
Posmantur
R
R
Department of Immunopathology, Parke-Davis Pharmaceutical Research, Warner-Lambert Company, Ann Arbor, Michigan 48105, U.S.A.
McGinnis
K
K
Nadimpalli
R
R
Gilbertsen
R B
RB
Wang
K K
KK
D000918:Antibody Specificity; D017209:Apoptosis / Q000502:physiology*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D020170:Caspase 1; D053148:Caspase 3; D020169:Caspases; D003374:Coumarins; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006801:Humans; D006868:Hydrolysis; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology / Q000201:enzymology; D009842:Oligopeptides; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000201:enzymology
D003374:Coumarins; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D005456:Fluorescent Dyes; D009842:Oligopeptides; D011480:Protease Inhibitors; C102580:acetyl-aspartyl-glutamyl-valyl-aspartyl-amino-4-methylcoumarin; C093260:benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene; D001224:Aspartic Acid; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D020170:Caspase 1; D019311:Staurosporine