Autocrine regulation of γ-irradiation-induced DNA damage response via extracellular nucleotides-mediated activation of P2Y6 and P2Y12 receptors.
A key component of the response to DNA damage caused by ionizing radiation is DNA repair. Release of extracellular nucleotides, such as ATP, from cells plays a role in signaling via P2 receptors. We show here that release of ATP, followed by activation of P2Y receptors, is involved in the response to γ-irradiation-induced DNA damage. Formation of phosphorylated histone variant H2AX (γH2AX) foci, which are induced in nuclei by DNA damage and contribute to accumulation of DNA-repair factors, was increased at 1-3h after γ-ray irradiation (2.0Gy) of human lung cancer A549 cells. Focus formation was suppressed by pre-treatment with the ecto-nucleotidase apyrase. Pre-treatment with ecto-nucleotidase inhibitor ARL67156 or post-treatment with ATP or UTP facilitated induction of γH2AX, indicating that extracellular nucleotides play a role in induction of γH2AX foci. Next, we examined the effect of P2 receptor inhibitors on activation of ataxia telangiectasia mutated (ATM; a protein kinase) and accumulation of 53BP1 (a DNA repair factor), both of which are important for DNA repair, at DNA damage sites. P2Y6 receptor antagonist MRS2578, P2Y12 receptor antagonist clopidogrel, and P2X7 receptor antagonists A438079 and oxATP significantly inhibited these processes. Release of ATP was detected within 2.5min after irradiation, but was blocked by A438079. Activation of ATM and accumulation of 53BP1 were decreased in P2Y6 or P2Y12 receptor-knockdown cells. We conclude that autocrine/paracrine signaling through P2X7-dependent ATP release and activation of P2Y6 and P2Y12 receptors serves to amplify the cellular response to DNA damage caused by γ-irradiation.
Department of Radiation Biosciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-0022, Japan.
Naoko
N
Nishimaki
Mitsutoshi
M
Tsukimoto
Akihiro
A
Kitami
Shuji
S
Kojima
D000255:Adenosine Triphosphate / Q000378:metabolism; D001081:Apyrase / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D019898:Autocrine Communication / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D000077144:Clopidogrel; D004249:DNA Damage / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005720:Gamma Rays / Q000009:adverse effects*; D006657:Histones / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D017879:Isothiocyanates / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7 / Q000187:drug effects / Q000378:metabolism; D058925:Receptors, Purinergic P2Y12 / Q000187:drug effects / Q000378:metabolism*; D013777:Tetrazoles / Q000494:pharmacology; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology; D013988:Ticlopidine / Q000031:analogs & derivatives / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D000071857:Tumor Suppressor p53-Binding Protein 1
C523668:3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methylpyridine; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D017879:Isothiocyanates; C541384:N,N''-1,4-butanediylbis(N'-(3-isothiocyanatophenyl))thiourea; D011725:Pyridines; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D058925:Receptors, Purinergic P2Y12; C498301:TP53BP1 protein, human; D013777:Tetrazoles; D025521:Tumor Suppressor Proteins; D000071857:Tumor Suppressor p53-Binding Protein 1; C099845:purinoceptor P2Y6; D000255:Adenosine Triphosphate; D000077144:Clopidogrel; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D001081:Apyrase; D013890:Thiourea; D013988:Ticlopidine