Lack of internucleosomal DNA fragmentation is related to Cl(-) efflux impairment in hematopoietic cell apoptosis.
The heterodimeric DNA fragmentation factor (DFF) is responsible for DNA degradation into nucleosomal units during apoptosis. This process needs the caspase-dependent release of ICAD/DFF-45, the inhibitory subunit of DFF. Here we report that triggering apoptosis via a hyperosmotic shock in hematopoietic cells causes the appearance of mitochondrial and cytosolic alterations, activation of caspases, chromatin condensation, nuclear disruption, and DNA fragmentation. However, oligonucleosomal but not high molecular weight (50-150 kb) DNA cleavage is abolished if Cl(-) efflux is prevented by using NaCl to raise extracellular osmolarity or by Cl(-) channel blockers, even when apoptosis is initiated by other agents (staurosporine, anti-Fas antibody). In these conditions, all the apoptosis hallmarks investigated remain detectable, including the cleavage of ICAD/DFF-45. In vitro assays with lysates of cells in which Cl(-) efflux is blocked confirm the lack of internucleosomal DNA degradation. These findings establish that neither caspase activation nor ICAD/DFF-45 processing per se is sufficient to induce oligonucleosomal DNA fragmentation and that high molecular weight DNA degradation and chromatin condensation appear independently of it. Finally, they suggest that Cl(-) efflux is a necessary cofactor that intervenes specifically in the activation of the DFF endonuclease.
Rasola
A
A
Unité de Recherche en Immunologie Cellulaire et Moléculaire, Inserm U364, Faculté de Médecine, 06107 Nice Cedex 2, France.
Farahi Far
D
D
Hofman
P
P
Rossi
B
B
D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D001692:Biological Transport; D020169:Caspases / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D053938:DNA Fragmentation; D004789:Enzyme Activation; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D006801:Humans; D019169:Jurkat Cells / Q000648:ultrastructure; D008353:Mannitol / Q000494:pharmacology; D009707:Nucleosomes; D009997:Osmotic Pressure; D011506:Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology
D051017:Apoptosis Regulatory Proteins; D002712:Chlorides; D009707:Nucleosomes; D011506:Proteins; C109664:caspase-activated DNase inhibitor; D008353:Mannitol; D012965:Sodium Chloride; D020169:Caspases