Caspase-activated DNase is necessary and sufficient for oligonucleosomal DNA breakdown, but not for chromatin disassembly during caspase-dependent apoptosis of LN-18 glioblastoma cells.
Caspase-dependent apoptosis is a controlled type of cell death characterized by oligonucleosomal DNA breakdown and major nuclear morphological alterations. Other kinds of cell death do not share these highly distinctive traits because caspase-activated DNase (DFF40/CAD) remains inactive. Here, we report that human glioblastoma multiforme-derived LN-18 cells do not hydrolyze DNA into oligonucleosomal fragments after apoptotic insult. Furthermore, their chromatin remains packaged into a single mass, with no signs of nuclear fragmentation. However, ultrastructural analysis reveals that nuclear disassembly occurs, although compacted chromatin does not localize into apoptotic nuclear bodies. Caspases become properly activated, and ICAD, the inhibitor of DFF40/CAD, is correctly processed. Using cell-free in vitro assays, we show that chromatin from isolated nuclei of LN-18 cells is suitable for hydrolysis into oligonuclesomal fragments by staurosporine-pretreated SH-SY5Y cytoplasms. However, staurosporine-pretreated LN-18 cytoplasms do not induce DNA laddering in isolated nuclei from either LN-18 or SH-SY5Y cells because LN-18 cells express lower amounts of DFF40/CAD. DFF40/CAD overexpression makes LN-18 cells fully competent to degrade their DNA into oligonucleosome-sized fragments, and yet they remain unable to arrange their chromatin into nuclear clumps after apoptotic insult. Indeed, isolated nuclei from LN-18 cells were resistant to undergoing apoptotic nuclear morphology in vitro. The use of LN-18 cells has uncovered a previously unsuspected cellular model, whereby a caspase-dependent chromatin package is DFF40/CAD-independent, and DFF40/CAD-mediated double-strand DNA fragmentation does not warrant the distribution of the chromatin into apoptotic nuclear bodies. The studies highlight a not-yet reported DFF40/CAD-independent mechanism driving conformational nuclear changes during caspase-dependent cell death.
From the Cell Death, Senescence, and Survival Group, Departament de Bioquímica i Biologia Molecular and Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallés, and the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 08193 Cerdanyola del Vallés, Barcelona, Spain.
María
M
Sánchez-Osuna
From the Cell Death, Senescence, and Survival Group, Departament de Bioquímica i Biologia Molecular and Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallés, and the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 08193 Cerdanyola del Vallés, Barcelona, Spain.
Mercè
M
Garcia-Belinchón
From the Cell Death, Senescence, and Survival Group, Departament de Bioquímica i Biologia Molecular and Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallés, and the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 08193 Cerdanyola del Vallés, Barcelona, Spain.
Victoria
V
Iglesias-Guimarais
From the Cell Death, Senescence, and Survival Group, Departament de Bioquímica i Biologia Molecular and Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallés, and the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 08193 Cerdanyola del Vallés, Barcelona, Spain.
Estel
E
Gil-Guiñón
From the Cell Death, Senescence, and Survival Group, Departament de Bioquímica i Biologia Molecular and Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallés, and the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 08193 Cerdanyola del Vallés, Barcelona, Spain.
Elisenda
E
Casanelles
From the Cell Death, Senescence, and Survival Group, Departament de Bioquímica i Biologia Molecular and Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallés, and the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 08193 Cerdanyola del Vallés, Barcelona, Spain victor.yuste@uab.cat.
Victor J
VJ
Yuste
D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000378:metabolism*; D045744:Cell Line, Tumor; D042002:Chromatin Assembly and Disassembly / Q000187:drug effects; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D003851:Deoxyribonucleases / Q000378:metabolism*; D005909:Glioblastoma / Q000235:genetics / Q000473:pathology*; D006801:Humans; D008970:Molecular Weight; D009707:Nucleosomes / Q000187:drug effects / Q000235:genetics*; D019311:Staurosporine / Q000494:pharmacology
D009707:Nucleosomes; D004247:DNA; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease; D020169:Caspases; D019311:Staurosporine
Apoptosis; Apoptotic Nuclear Morphology; Caspase; Chromatin Disassembly; DFF40/CAD; DNA; Deoxyribonuclease (DNase); Nucleus; Oligonucleosomal DNA Fragmentation