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Pàgina inicial > Articles > Articles publicats > Apoptotic DNA degradation into oligonucleosomal fragments, but not apoptotic nuclear morphology, relies on a cytosolic pool of DFF40/CAD endonuclease |
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 |
18 p, 6.6 MB |