Novel mechanisms of cell cycle checkpoint activation and maintenance in hyperoxia

Cells are continuously challenged by endogenously produced metabolic by-products including reactive oxygen species (ROS) which damage DNA and other macromolecules. Exposure to elevated levels of atmospheric oxygen (hyperoxia) accelerates endogenous ROS production resulting in chronic oxidative stress and progressive DNA damage. Since oxidative lung injury limits the clinical utility of supplemental oxygen treatment, further examination of the cellular response to hyperoxia is needed. As with other forms of DNA damage, hyperoxia stimulates the activation of cell cycle checkpoints resulting in p53-dependent expression of p21 which inhibits proliferation and protects against cell death. While checkpoints are activated transiently in response to acute DNA damage, the function of checkpoints in response to chronic damage as seen with hyperoxia is not well understood. In these studies, we test the hypothesis that unique mechanisms function in activation and maintenance of checkpoints in response to chronic damage caused by hyperoxia. Previous results from our laboratory established a role for the PI 3-kinase related kinase (PIKK) ATM in activating the p53/p21 pathway in hyperoxia. The results presented here demonstrate that ATM regulates p53 at later exposures in hyperoxia, but is dispensable for early activation. Instead, the recently recognized PIKK hSMG-1 is rapidly activated and mediates signaling at early times in hyperoxia. Examination of checkpoint function revealed that sustained p21 expression over time in hyperoxia lowers abundance of PCNA, a DNA polymerase co-factor, thus allowing p21 to inhibit proliferation and maintain checkpoint integrity over prolonged periods of damage. Additionally, chronic hyperoxia decreased p21 stability through a pathway involving PIKK signaling perhaps to regulate PCNA-dependent replication or repair. Together, these finding provide significant insight into the cellular response to hyperoxia and help elucidate checkpoint function under chronic damaging conditions. A clear picture of how cell cycle checkpoints operate in hyperoxia may promote the development of novel interventions to mitigate the deleterious effects of prolonged oxygen treatment and also clarify the role of these pathways in other disease states associated with chronic oxidant production.