Sánchez-Osuna María, Garcia-Belinchón Mercè, Iglesias-Guimarais Victoria, Gil-Guiñón Estel, Casanelles Elisenda, Yuste Victor J
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.
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
J Biol Chem. 2014 Jul 4;289(27):18752-69. doi: 10.1074/jbc.M114.550020. Epub 2014 May 17.
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.
半胱天冬酶依赖性凋亡是一种受调控的细胞死亡类型，其特征在于寡核小体DNA断裂和主要的核形态改变。其他类型的细胞死亡不具备这些高度独特的特征，因为半胱天冬酶激活的脱氧核糖核酸酶（DFF40/CAD）仍处于无活性状态。在此，我们报告多形性胶质母细胞瘤来源的LN-18细胞在遭受凋亡刺激后不会将DNA水解成寡核小体片段。此外，它们的染色质仍包裹成单一团块，没有核碎片化的迹象。然而，超微结构分析显示核解体发生了，尽管致密的染色质没有定位到凋亡核小体中。半胱天冬酶被正确激活，并且DFF40/CAD的抑制剂ICAD被正确加工。使用无细胞体外试验，我们表明来自LN-18细胞分离核的染色质适合被星形孢菌素预处理的SH-SY5Y细胞质水解成寡核小体片段。然而，星形孢菌素预处理的LN-18细胞质不会在来自LN-18或SH-SY5Y细胞的分离核中诱导DNA梯状条带形成，因为LN-18细胞表达较低量的DFF40/CAD。DFF40/CAD的过表达使LN-18细胞完全有能力将其DNA降解成寡核小体大小的片段，但它们在遭受凋亡刺激后仍无法将染色质排列成核团块。实际上，来自LN-18细胞的分离核在体外对经历凋亡核形态具有抗性。LN-18细胞的使用揭示了一种先前未被怀疑的细胞模型，即半胱天冬酶依赖性染色质包装是不依赖DFF40/CAD的，并且DFF40/CAD介导的双链DNA断裂并不保证染色质分布到凋亡核小体中。这些研究突出了一种尚未报道的不依赖DFF40/CAD的机制，该机制在半胱天冬酶依赖性细胞死亡过程中驱动核构象变化。
