title: Apoptotic DNA degradation into oligonucleosomal fragments, but not apoptotic nuclear morphology, relies on a cytosolic pool of DFF40/CAD endonuclease.
abstract: 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.
authors.affiliation: Cell Death, Senescence, and Survival Group, Departament de Bioquimica i Biologia Molecular and Institut de Neurociencies, Facultat de Medicina, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
authors.forename: Victoria
authors.initials: V
authors.lastname: Iglesias-Guimarais
authors.forename: Estel
authors.initials: E
authors.lastname: Gil-Guiñon
authors.forename: Gisela
authors.initials: G
authors.lastname: Gabernet
authors.forename: Mercè
authors.initials: M
authors.lastname: García-Belinchón
authors.forename: María
authors.initials: M
authors.lastname: Sánchez-Osuna
authors.forename: Elisenda
authors.initials: E
authors.lastname: Casanelles
authors.forename: Joan X
authors.initials: JX
authors.lastname: Comella
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Yuste
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009154:Mutation; D000075223:Poly-ADP-Ribose Binding Proteins; D019311:Staurosporine / Q000494:pharmacology
chemical_list: D051017:Apoptosis Regulatory Proteins; D004791:Enzyme Inhibitors; D000075223:Poly-ADP-Ribose Binding Proteins; C109664:caspase-activated DNase inhibitor; C113696:DFFB protein, human; D003851:Deoxyribonucleases; D053148:Caspase 3; D019311:Staurosporine