Hammond Ester M, Green Susannah L, Giaccia Amato J
Center for Clinical Sciences Research, Department of Radiation Oncology, Stanford University, Stanford, CA 94303-5152, USA.
Mutat Res. 2003 Nov 27;532(1-2):205-13. doi: 10.1016/j.mrfmmm.2003.08.017.
Severe levels of hypoxia (oxygen concentrations of less that 0.02%) have been shown to induce a rapid S-phase arrest. The mechanism behind hypoxia-induced S-phase arrest is unclear, we show here that it was not mediated by a shortage of nucleosides and was not dependent on p53, p21 or Hif 1alpha status. The drugs aphidicolin and hydroxyurea both induce rapid replication arrest and have been used throughout the literature to study the ATR-mediated response to stalled replication. We have shown previously that hypoxia induces ATR-dependent phosphorylation of p53, Chk1 and histone H2AX. Using comet-assays to detect DNA-damage we found that both aphidicolin and hydroxyurea induced significant levels of DNA-damage while hypoxia did not. Here we show that like aphidicolin and hydroxyurea, hypoxia induces phosphorylation of Nbs1 at serine 343 and Rad17 serine 645. Hypoxia-dependent phosphorylation of Nbs1 and Rad17 was ATM-independent and therefore likely to be a result of the ATR kinase activity. In contrast, p53 was phosphorylated differentially in response to the three treatments considered here. p53 was phosphorylated at serine 15 in response to all three treatments but was only phosphorylated at serine 20 in response to the drug treatments. We propose that treatment with either aphidicolin or hydroxyurea leads to not only replication arrest but also DNA-damage and therefore both ATM and ATR-mediated signaling. In contrast replication arrest induced by severe hypoxia is sensed exclusively through ATR, with ATM only having a role to play after re-oxygenation.
已表明严重程度的缺氧（氧气浓度低于0.02%）会诱导快速的S期停滞。缺氧诱导S期停滞背后的机制尚不清楚，我们在此表明它不是由核苷短缺介导的，也不依赖于p53、p21或Hif 1α的状态。药物阿非迪霉素和羟基脲均能诱导快速的复制停滞，并且在整个文献中都被用于研究ATR介导的对停滞复制的反应。我们之前已经表明缺氧会诱导p53、Chk1和组蛋白H2AX的ATR依赖性磷酸化。使用彗星试验检测DNA损伤，我们发现阿非迪霉素和羟基脲均诱导了显著水平的DNA损伤，而缺氧则没有。在此我们表明，与阿非迪霉素和羟基脲一样，缺氧会诱导Nbs1在丝氨酸343位点和Rad17在丝氨酸645位点的磷酸化。Nbs1和Rad17的缺氧依赖性磷酸化不依赖于ATM，因此很可能是ATR激酶活性的结果。相比之下，p53对这里考虑的三种处理的磷酸化反应有所不同。p53在丝氨酸15位点对所有三种处理都有磷酸化反应，但仅在丝氨酸20位点对药物处理有磷酸化反应。我们提出，用阿非迪霉素或羟基脲处理不仅会导致复制停滞，还会导致DNA损伤，因此会引发ATM和ATR介导的信号传导。相比之下，严重缺氧诱导的复制停滞仅通过ATR被感知，ATM仅在再给氧后才发挥作用。
