Department of Medical Biophysics, University of Toronto, Canada.
Radiother Oncol. 2011 Jun;99(3):307-12. doi: 10.1016/j.radonc.2011.05.039. Epub 2011 Jun 15.
The ATM kinase is activated by chromatin modification following exogenous and endogenous DSBs or cell stress, including acute anoxia. The p53 binding protein 1 (53BP1) contains multiple ATM-consensus phosphorylation sites in its N- and C-termini and may therefore be a distal read-out of ATM function. We have examined the cellular activation of these phosphorylation sites for the first time in situ following anoxic/hypoxic stress and IR-induced exogenous DSBs. We show that multiple residues of 53BP1 are phosphorylated and that these phosphoforms form discrete nuclear foci following IR or during DNA replication as exogenous or endogenous DNA double strand breaks (DSBs), respectively. Novel data pertaining to the phosphorylation of 53BP1(Ser25)in situ supports its dependency on the ATM kinase; but this occurs independently of p53 function. We show that 53BP1(Ser25) is activated specifically in S-phase cells during anoxia in an ATM-dependent manner. Exogenous DSBs form discrete IR-induced foci whereas oxygen stress induced non-localized 53BP1(Ser25) activation. Our in vitro data are supported by irradiated xenograft studies in vivo whereby 53BP1(Ser25) phosphorylation does not occur in sub-regions positive for the hypoxia marker EF5. We propose a model whereby DSBs induce chromatin modification at sites of DNA damage which are tracked by the ATM substrates γ H2AX and 53BP1(Ser25) in a mechanism distinct from p53-mediated cell cycle arrest. Together this work indicates 53BP1(Ser25), and possibly other 53BP1 phosphoforms, as a bona fide DSB-biomarkers for surveying ongoing DNA-damage related signaling in oxic and hypoxic cells during clinical radiotherapy.
ATM 激酶在染色质修饰后被激活，染色质修饰由外源和内源双链断裂（DSB）或细胞应激引起，包括急性缺氧。p53 结合蛋白 1（53BP1）在其 N 端和 C 端含有多个 ATM 一致的磷酸化位点，因此可能是 ATM 功能的远距离读出器。我们首次在体外研究了缺氧/低氧应激和 IR 诱导的外源 DSB 后这些磷酸化位点的细胞激活情况。我们表明，53BP1 的多个残基被磷酸化，并且这些磷酸化形式在 IR 或 DNA 复制过程中分别作为外源或内源 DNA 双链断裂（DSB）形成离散的核焦点。与 53BP1（Ser25）在原位磷酸化有关的新数据支持其对 ATM 激酶的依赖性；但这与 p53 功能无关。我们表明，53BP1（Ser25）在缺氧时 S 期细胞中以 ATM 依赖的方式被特异性激活。外源 DSB 形成离散的 IR 诱导焦点，而氧应激诱导非局部化的 53BP1（Ser25）激活。我们的体外数据得到了体内照射异种移植物研究的支持，即在缺氧标志物 EF5 阳性的亚区中不发生 53BP1（Ser25）磷酸化。我们提出了一个模型，即 DSB 在 DNA 损伤部位诱导染色质修饰，ATM 底物 γ H2AX 和 53BP1（Ser25）在该部位追踪 DNA 损伤，该机制与 p53 介导的细胞周期停滞不同。综上所述，这项工作表明 53BP1（Ser25），以及其他可能的 53BP1 磷酸化形式，是一种真正的 DSB 生物标志物，可用于在临床放射治疗期间监测有氧和缺氧细胞中持续的与 DNA 损伤相关的信号转导。
