Stage-specific expression of DNaseγ during B-cell development and its role in B-cell receptor-mediated apoptosis in WEHI-231 cells

D Shiokawa, Y Shika, S Araki, S Sunaga, R Mizuta, D Kitamura, S Tanuma
2007 Cell Death and Differentiation  
Here, we describe the non-redundant roles of caspase-activated DNase (CAD) and DNasec during apoptosis in the immature B-cell line WEHI-231. These cells induce DNA-ladder formation and nuclear fragmentation by activating CAD during cytotoxic drug-induced apoptosis. Moreover, these apoptotic manifestations are accompanied by inhibitor of CAD (ICAD) cleavage and are abrogated by the constitutive expression of a caspase-resistant ICAD mutant. No such nuclear changes occur during oxidative
more » ... oxidative stress-induced necrosis, indicating that neither CAD nor DNasec functions under necrotic conditions. Interestingly, the DNAladder formation and nuclear fragmentation induced by B-cell receptor ligation occur in the absence of ICAD cleavage and are not significantly affected by the ICAD mutant. Both types of nuclear changes are preceded by the upregulation of DNasec expression and are strongly suppressed by 4-(4,6-dichloro-[1, 3, 5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid (DR396), which is a specific inhibitor of DNasec. Our results suggest that DNasec provides an alternative mechanism for inducing nuclear changes when the working apoptotic cascade is unsuitable for CAD activation. During B-lymphocyte development in the bone marrow, somatic gene rearrangements occur at the immunoglobulin (Ig) loci through V(D)J recombination. 1 This produces a diverse repertoire of B cells, and allows the immune system to recognize a wide range of foreign antigens. However, creating such variety carries the risk of generating self-reactive B cells. To avoid autoimmune manifestations, self-reactive B cells can be rendered inactive (anergy), reconstituted in their antigen receptor (receptor editing) or eliminated by apoptosis (clonal deletion) at an immature stage of development. 2 The clonal deletion of self-reactive B cells depends upon the sensitivity of their B-cell receptors (BCRs) to self-antigens. The strong engagement of surface IgM causes immature B cells to exit from the cell cycle and undergo apoptosis. 3,4 This cellular suicide program allows potentially harmful or redundant cells to be individually eliminated, in order to maintain healthy homeostasis in multicellular organisms. 5 It is distinguished from necrosis by its specific morphologies (such as balloon-cell formation, chromatin condensation, and nuclear fragmentation) and biochemical markers (including the cleavage of death substrates by caspases and the degradation of genomic DNA into nucleosomal fragments). 6,7 The occurrence of nucleosomal DNA fragmentation is an important feature of apoptosis, and the presence of a ladder configuration in agarose gel electrophoresis provides reliable evidence of this process. 7 Apoptotic signals are initiated by various physiological and pathological stimuli, and are transmitted by the successive activation of several protease cascades, including those of calpains, cathepsins, and caspases. 8, 9 Although the utilization of apical proteases differs depending on the type of apoptotic stimulus, they subsequently promote the activation of downstream effector caspases (caspase-3, caspase-6, and caspase-7), which catalyze the proteolysis of death substrates (such as poly (ADP-ribose) polymerase-1 (PARP-1) and DNA-fragmentation factor 45 (DFF45)/inhibitor of caspase-activated DNase (ICAD)). 8 DFF is a heterodimeric complex of DFF40 and DFF45, which is purified from the S100 fraction of HeLa cells on the basis of its activity to induce DNA-ladder formation in a cell-free apoptosis system. 10 The murine homologs of DFF40 and DFF45 -caspase-activated DNase (CAD) and ICAD, respectively -have been isolated and characterized. 11 CAD exists in living cells as an inactive complex with its natural inhibitor ICAD. Upon apoptosis induction, CAD is released from the complex by the caspase-3-mediated cleavage of ICAD, and catalyzes nucleosomal DNA fragmentation in dying cells. 11 Thus, CAD is considered to have a major role in apoptosis accompanied by caspase-3 activation. The existence of a nuclear apoptotic Ca 2 þ /Mg 2 þ -dependent DNase has long been suspected. We previously identified and
doi:10.1038/sj.cdd.4402086 pmid:17218958 fatcat:fadetls7qzf4lfu53srv3nbrae