ATM protein is required for radiation-induced apoptosis and acts before mitochondrial collapse.
OBJECTIVE
To define the role of the ataxia telangiectasia (A-T) mutated gene (ATM) in activation and progress of apoptosis.


METHODS
Three normal and three A-T EBV-transformed cell lines were studied. Following irradiation (IR), Fas activation or ceramide exposure, viability and apoptosis were measured by trypan blue dye exclusion assay and as sub-G1 cell fraction by flow cytometric analysis of propidium iodide stained cultures, respectively. Activation of caspase-3 was evaluated by immunoblot and by an in vitro activity assay on cytosolic cell extracts. To assess changes in mitochondrial potential and reactive oxygen species, cells were stained by 3,3'-dihexyloxacarbocynine iodide or hydroethidine, respectively, and scored by flow cytometry.


RESULTS
The observations establish that A-T cells are equipped with a proficient apoptotic machinery, as demonstrated by their ability to undergo mitochondrial collapse and caspase-3 activation after Fas activation or ceramide treatment. Both treatments have a similar cytotoxic effect on normal and A-T cells. In contrast, in spite of the stronger cytotoxicity induced by IR exposure, irradiated A-T cells are unable to undergo mitochondrial collapse and caspase-3 activation.


CONCLUSIONS
The data indicate that ATM is necessary in the initiation of molecular pathway(s) leading to IR-induced apoptosis, and suggest that increased radiosensitivity of A-T cells is more likely a direct consequence of necrotic cell death.
Vit
J P
JP
UMR 218 du Centre National de la Recherche Scientifique, LRC n 1 du Commissariat à l'Energie Atomique, Institut Curie-Recherche, Paris, France.
Moustacchi
E
E
Rosselli
F
F
D017209:Apoptosis / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects*; D001260:Ataxia Telangiectasia / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D064007:Ataxia Telangiectasia Mutated Proteins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D018797:Cell Cycle Proteins; D002460:Cell Line; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins; D004789:Enzyme Activation / Q000528:radiation effects; D006801:Humans; D008928:Mitochondria / Q000378:metabolism* / Q000528:radiation effects*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D011065:Poly(ADP-ribose) Polymerases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases