The ATM kinase has previously been shown to respond to the DNA damage induced by reoxygenation following hypoxia by initiating a Chk 2-dependent cell cycle arrest in the G 2 phase. Here we show that ATM is both phosphorylated and active during exposure to hypoxia in the absence of DNA damage, detectable by either comet assay or 53BP1 focus formation. Hypoxia-induced activation of ATM correlates with oxygen concentrations low enough to cause a replication arrest and is entirely independent of hypoxia-inducible factor 1 status. In contrast to damage-activated ATM, hypoxia-activated ATM does not form nuclear foci but is instead diffuse throughout the nucleus. The hypoxia-induced activity of both ATM and the related kinase ATR is independent of NBS1 and MRE11, indicating that the MRN complex does not mediate the DNA damage response to hypoxia. However, the mediator MDC1 is required for efficient activation of Kap1 by hypoxiainduced ATM, indicating that similarly to the DNA damage response, there is a requirement for MDC1 to amplify the ATM response to hypoxia. However, under hypoxic conditions, MDC1 does not recruit BRCA1/ 53BP1 or RNF8 activity. Our findings clearly demonstrate that there are alternate mechanisms for activating ATM that are both stress-specific and independent of the presence of DNA breaks.Sensing and responding to DNA damage is crucial for maintaining cellular homeostasis and preventing the development of cancer. The cellular response to DNA damage can be divided into three parts: sensing the type of damage, activating DNA damage signaling pathways, and repairing the damage. The proteins involved in these three processes act as sensors, transducers, and effectors of the DNA damage response (41, 54). The ATM kinase is one of the key transducers of the DNA double-stranded break response. ATM has been identified as the product mutated or inactivated in ataxia telangiectasia (AT) patients and belongs to the phosphatidyl inositol-3-kinase-like kinase (PIKK) family, together with its family members ATR, DNA-PK, and mTOR. Elegant studies have demonstrated that ATM is present as an inactive dimer that undergoes rapid autophosphorylation on serine 1981 after DNA damage (4) and is recruited to sites of DNA strand breaks (2). Various proteins, such as the MRE11-Rad50-NBS1 (MRN) complex (49) and MDC1 (20, 43), have been described to be essential for the efficient response of ATM to DNA damage. Activated ATM phosphorylates downstream targets, such as p53, Chk2, and BRCA 1 and 2, that are involved in DNA repair, cell cycle control, and apoptosis.Low-oxygen tension or hypoxia is a common feature in all solid tumors (15). It is strongly associated with tumor development, malignant progression, metastatic outgrowth, and resistance to therapy and is considered an independent prognostic indicator for poor patient prognosis in various tumor types. Tumor hypoxia results from an imbalance between the cellular oxygen consumption rate of cells and the delivery of oxygen to cells (50). Interestingly, the level of tumor hy...

Questions exist concerning the effects of acute versus chronic hypoxic conditions on DNA replication and genomic stability that may influence tumorigenesis. Severe hypoxia causes replication arrest independent of S-phase checkpoint, DNA damage response, or transformation status. Arrests occur during both the initiation and elongation phases of DNA replication, correlated with a rapid decrease in available deoxynucleotide triphosphates. With fluctuating oxygen tensions in tumors, arrested hypoxic cells may undergo rapid reperfusion and reoxygenation that leads to reoxygenation-induced DNA damage. In cells subjected to chronic hypoxia, we found that replicative restart was inhibited along with numerous replication factors, including MCM6 and RPA, the latter of which limits the hypoxia-induced DNA damage response. In contrast, in cells where replicative restart occurred, it was accompanied by extensive reoxygenation-induced DNA damage and compromised DNA repair. We found that cells reoxygenated after acute hypoxia underwent rapid p53-dependent apoptosis. Our findings suggest that cells lacking functional p53 are more susceptible to genomic instability and potentially tumorigenesis if they experience reoxygenation after acute exposure to hypoxia. Cancer Res; 70(3); 925-35. ©2010 AACR.