Apoptotic DNA degradation into oligonucleosomal fragments, but not apoptotic nuclear morphology, relies on a cytosolic pool of DFF40/CAD endonuclease
Iglesias Guimarais, Victoria (Universitat Autònoma de Barcelona. Institut de Neurociències)
Gil Guiñon, Estel (Universitat Autònoma de Barcelona. Institut de Neurociències)
Gabernet, Gisela (Universitat Autònoma de Barcelona. Institut de Neurociències)
Garcia i Belinchón, Maria Mercè (Universitat Autònoma de Barcelona. Institut de Neurociències)
Sánchez-Osuna, María (Universitat Autònoma de Barcelona. Institut de Neurociències)
Casanelles Abella, Elisenda (Universitat Autònoma de Barcelona. Institut de Neurociències)
Comella i Carnicé, Joan Xavier, 1963- (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Yuste, Victor J.. (Víctor José) (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)

Data:	2012
Resum:	Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation.
Ajuts:	Ministerio de Ciencia e Innovación FEDER/BFU2008-01328 Ministerio de Ciencia e Innovación FEDER/TRA2009-0185 Ministerio de Ciencia e Innovación FEDER/SAF2010-19953 Instituto de Salud Carlos III CIBERNED/CB06/05/0042 Agència de Gestió d'Ajuts Universitaris i de Recerca 2009/SGR-346
Drets:	Tots els drets reservats.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Apoptosis ; Caspase ; Cell death ; Neuroblastoma ; Subcellular fractionation ; DFF40/CAD ; Oligonucleosomal DNA degradation
Publicat a:	Journal of biological chemistry, Vol. 287, Num. 10 (Mar 2012) , p. 7766-7779, ISSN 1083-351X

DOI: 10.1074/jbc.M111.290718
PMID: 22253444

18 p, 6.6 MB

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