Preapoptotic chromatin condensation upstream of the mitochondrial checkpoint.
When added for a short period (2-4 h) to cells, the kinase inhibitor staurosporine (STS), can trigger double strand breaks, the formation of nuclear foci containing phosphorylated H2AX, Chk2, and p53, a decrease in transcription, and a minor degree of peripheral chromatin condensation. This "preapoptotic chromatin condensation" (PACC) occurs before mitochondrial membrane permeabilization (MMP) and caspase activation become detectable and is not inhibited by Z-VAD-fmk or Bcl-2. PACC is followed by classical apoptosis, when cells are cultured overnight, even when STS is removed from the system. After overnight incubation, STS-pretreated cells manifest mitochondrial cytochrome c release, caspase activation, phosphatidylserine exposure, and apoptotic DNA fragmentation. Caspase or MMP inhibitors did not influence the advent of PACC yet did suppress the evolution of PACC toward apoptosis. Importantly, two unrelated MMP inhibitors (viral mitochondrial inhibitor of apoptosis (vMIA) from cytomegalovirus and mitochondrion-targeted Bcl-2) had a larger range of effects than the pan-caspase inhibitor Z-VAD-fmk. Caspase inhibition simply prevented the transition from PACC to apoptosis yet did not reverse PACC and did not restore transcription. In contrast, Bcl-2 and vMIA allowed for the repair of the DNA lesions, correlating with the reestablishment of active transcription. PACC could also be induced by a gross perturbation of RNA synthesis or primary DNA damage. Again, inhibition of MMP (but not that of caspases) reversed PACC induced by these stimuli. In synthesis, our data reveal the unexpected capacity of STS to induce DNA lesions and suggest qualitative differences in the cytoprotective and DNA repair-inducing potential of different apoptosis inhibitors.
Andreau
Karine
K
CNRS-UMR8125, Institut Gustave Roussy, Pavillon de Recherche 1, 39 rue Camille-Desmoulins, F-94805 Villejuif, France.
Castedo
Maria
M
Perfettini
Jean-Luc
JL
Roumier
Thomas
T
Pichart
Evelyne
E
Souquere
Sylvie
S
Vivet
Sonia
S
Larochette
Nathanael
N
Kroemer
Guido
G
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D053938:DNA Fragmentation; D004260:DNA Repair; D003609:Dactinomycin / Q000494:pharmacology; D004789:Enzyme Activation; D005434:Flow Cytometry; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry; D006801:Humans; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008564:Membrane Potentials; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D010718:Phosphatidylserines / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology*; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D002843:Chromatin; C468783:H2AX protein, human; D006657:Histones; D010718:Phosphatidylserines; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D003609:Dactinomycin; D045304:Cytochromes c; D004247:DNA; D020169:Caspases; D020782:Matrix Metalloproteinases; D019311:Staurosporine; D002945:Cisplatin