Involvement of caspase 3-activated DNase in internucleosomal DNA cleavage induced by diverse apoptotic stimuli

Abstract

Degradation of chromosomal DNA into nucleosomesized fragments is one of the characteristics of apoptotic cell death. Here, we examined whether caspase-activated DNase (CAD) is responsible for the DNA fragmentation that occurs upon exposure to various apoptotic stimuli. When human Jurkat cells were exposed to etoposide, or UV or g radiation, a caspase-3-like protease was activated, and nuclear DNA was fragmented. Human TF-1 cells, which are dependent on granulocyte-macrophage colony-stimulating factor (GM ± CSF), also underwent apoptosis accompanied by the activation of caspase-3-like protease and DNA fragmentation, when cultured without the cytokine. Both Jurkat and TF-1 cells expressed two forms of ICAD, ICAD-L and ICAD-S, which were cleaved upon exposure to these apoptotic stimuli. Among eight di erent caspases examined, recombinant caspases 3 and 7 speci®cally cleaved ICAD synthesized in a cell-free system. An expression plasmid containing mouse ICAD-L mutated at the caspase-3-recognition sites was then introduced into Jurkat and TF-1 cells. When the transformants were induced to undergo apoptosis (by treatment with etoposide, UV or g radiation for Jurkat cells, or factor withdrawal for TF-1 cells) they did not show DNA fragmentation, although they still died as a result of these stimuli. These results indicated that CAD, released from ICAD by caspase activation, is involved in the nuclear DNA fragmentation induced by these apoptotic stimuli.

The Contribution of Apoptosis-inducing Factor, Caspase-activated DNase, and Inhibitor of Caspase-activated DNase to the Nuclear Phenotype and DNA Degradation during Apoptosis

“…Contradictory results have been published on the relevance of ICAD processing at these Asp residues in the final nuclear apoptotic outcome. Some groups have reported that ICAD must be cleaved at both sites to lose its CAD-inhibitory activity (35). Here, we demonstrate that overexpression of the D117E ICAD mutant, but not the D224E one, is sufficient to abolish LMW DNA degradation.…”

Structure and promoter analysis of murine CAD and ICAD genes

“…ICAD-L and ICAD-S mRNAs were expressed at a similar level in various murine tissues. Equal amounts of ICAD-S and ICAD-L proteins were also detected by Western blotting in human and murine cell lines, 18,22 suggesting that the human ICAD gene also produces ICAD-S and ICAD-L mRNAs by alternative splicing. We have recently found that ICAD-L but not ICAD-S has an ability to work as a chaperone to enhance the correct folding of the CAD protein during CAD synthesis, although both ICAD-L and ICAD-S proteins efficiently inhibit the CAD' DNase activity.…”

Cellular cytotoxic response induced by highly purified multi-wall carbon nanotube in human lung cells

“…Caspase-3 activation was correlated with internucleosomal DNA fragmentation of the cells [13]. However, HTT2800 did not induce common apoptotic fragmentation of caspase-3 (Fig.…”