Chk2 tumor suppressor protein regulates the damage response to hypoxia and reoxygenation

The dynamic nature of the tumor microenvironment lends itself to the formation of oxygen gradients. As a result, regions with in the tumor can become chronically or acutely hypoxic. The efficacy of both chemotherapy and radiotherapy is dramatically reduced under these conditions. Additional complications arise when acute hypoxia is followed by reoxygenation, the combination of which acts as a selective pressure, resulting in the expansion of populations that are resistant to apoptosis and other growth controlling mechanisms, contributing to tumor formation. Under hypoxia, cells undergo G1 and replication arrest. Our studies indicate that replication arrest is independent of Hif-1alpha, protein Rb, protein 107, and protein 130. This arrest is also intact in Rb protein triple knockouts (TKO). During reoxygenation, the cell undergoes a damage response that leads to arrest in the G2 phase of the cell cycle. We found that this arrest was dependent on the protein kinase Chk2, which was phosphorylated in an ATM dependent manner during both hypoxia and reoxygenation. Loss of Chk2 resulted in attenuated G2 arrest, increased apoptosis and increased sensitivity to reoxygenation as measured by colony forming efficiency. The importance of Chk2 is beginning to be recognized as an increasing number of tumors with mutations or post-translational changes in this protein are being identified. Reoxygenation stress also has an impact on other pathologies. Hypoxia and reoxygenation occurs during stroke, myocardial infarction, organ transplant and other ischemic injuries. Understanding the molecular mechanisms involved in the reoxygenation damage response will benefit not only cancer biology, but other fields of study as well. In the first chapter, an introduction to tumor biology and early studies on hypoxia are summarized. The second chapter of this thesis presents studies designed to investigate the mechanisms regulating cell cycle response to hypoxia, as well as early reoxygenation studies done with a former graduate student in the lab, Dr. Susanna Green. Chapter three presents a characterization of the DNA damage response to hypoxia and reoxygenation. The fourth chapter describes the mechanism of G2 arrest observed following hypoxia and reoxygenation, and discusses future directions of the project.