ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATM in response to reoxygenation.
The ATR kinase phosphorylates both p53 and Chk1 in response to extreme hypoxia (oxygen concentrations of less than 0.02%). In contrast to ATR, loss of ATM does not affect the phosphorylation of these or other targets in response to hypoxia. However, hypoxia within tumors is often transient and is inevitably followed by reoxygenation. We hypothesized that ATR activity is induced under hypoxic conditions because of growth arrest and ATM activity increases in response to the oxidative stress of reoxygenation. Using the comet assay to detect DNA damage, we find that reoxygenation induced significant amounts of DNA damage. Two ATR/ATM targets, p53 serine 15 and histone H2AX, were both phosphorylated in response to hypoxia in an ATR-dependent manner. These phosphorylations were then maintained in response to reoxygenation-induced DNA damage in an ATM-dependent manner. The reoxygenation-induced p53 serine 15 phosphorylation was inhibited by the addition of N-acetyl-l-cysteine (NAC), indicating that free radical-induced DNA damage was mediated by reactive oxygen species. Taken together these data implicate both ATR and ATM as critical roles in the response of hypoxia and reperfusion in solid tumors.
Hammond
Ester M
EM
Center for Clinical Sciences Research, Department of Radiation Oncology, Stanford University, Stanford, California 94303-5152, USA.
Dorie
Mary Jo
MJ
Giaccia
Amato J
AJ
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D004249:DNA Damage / Q000502:physiology; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D009369:Neoplasms / Q000378:metabolism; D009687:Nuclear Proteins; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D010100:Oxygen / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D012694:Serine; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D010100:Oxygen