ATM protein is required for radiation-induced apoptosis and acts before mitochondrial collapse
J P Vit1, E Moustacchi, F Rosselli
1UMR 218 du Centre National de la Recherche Scientifique, LRC n 1 du Commissariat à l'Energie Atomique, Institut Curie-Recherche, Paris, France.
Purpose:
To define the role of the ataxia telangiectasia (A-T) mutated gene (ATM) in activation and progress of apoptosis.
Material And 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.
Insights
The ataxia telangiectasia mutated (ATM) gene is crucial for initiating apoptosis after irradiation. A-T cells exhibit radiosensitivity due to necrotic cell death, not impaired apoptosis machinery.
Area of Science:
- Cell Biology
- Molecular Genetics
- Cancer Research
Background:
- Ataxia telangiectasia (A-T) is a rare genetic disorder.
- The ataxia telangiectasia mutated (ATM) gene plays a role in DNA damage response.
- Apoptosis, or programmed cell death, is a critical cellular process.
Purpose of the Study:
- To elucidate the role of the ATM gene in initiating and progressing apoptosis.
- To investigate the mechanisms underlying the radiosensitivity observed in A-T cells.
Main Methods:
- Utilized normal and A-T Epstein-Barr virus-transformed cell lines.
- Assessed cell viability and apoptosis via trypan blue exclusion and flow cytometry.
- Measured caspase-3 activation, mitochondrial potential, and reactive oxygen species.
Main Results:
- A-T cells possess functional apoptosis machinery, activating caspase-3 and undergoing mitochondrial collapse upon Fas or ceramide treatment.
- Irradiated A-T cells failed to activate caspase-3 or exhibit mitochondrial collapse, despite increased cytotoxicity.
- Normal and A-T cells showed similar responses to Fas activation and ceramide exposure.
Conclusions:
- ATM is essential for initiating the apoptotic pathway following ionizing radiation (IR).
- The radiosensitivity of A-T cells likely results from a failure in IR-induced apoptosis, leading to necrotic cell death.
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