P21 regulation of cell survival and death in response to hyperoxic stress

Reactive oxygen species (ROS) generated from endogenous or exogenous sources can cause oxidative damage to macromolecular targets such as DNA, proteins, lipids and carbohydrates. DNA damage caused by oxidative stress may result in activation of growth arrest, DNA repair and/or cell death. The cyclin-dependent kinase inhibitor, p21Cip1/Waf1/sdi1, is responsible for initiating growth arrest during the DNA damage response. Hyperoxic damage is mediated by ROS, resulting in up-regulation of p21 and activation of G 1 growth arrest. P21 also plays a protective role during the damage response since mice lacking P21 had increased hyperoxic lung injury and mortality even though the lung is a highly senescent tissue. This evidence led to the hypothesis that p21 has distinct pro-survival functions during hyperoxic stress which are separate from its growth suppressive properties. The objective of this thesis was to investigate the protective role of p21 during hyperoxic stress. Studies using H1299 lung adenocarcinoma cells with conditional over-expression of full length p21 or p21 domains capable of cell cycle inhibition show that p21-mediated cytoprotection against hyperoxia is uncoupled from its ability to inhibit proliferation. Low levels of p21 inhibited cell proliferation while higher levels were required for protection. Mutation of the nuclear localization sequence of p21 ablated its growth suppressive activities, but the cytoprotective function of p21 remained intact thereby indicating that cytoplasmic localization of p21 was involved in the protective mechanism. The anti-apoptotic protein, Bcl-XL, was identified as an inhibitor of hyperoxic death whose expression declined during hyperoxia, but was maintained by p21 expression. Pro-apoptotic Bax and Bak activation both contributed to hyperoxic cell death but only Bax was inhibited by Bcl-X L. Taken together, the studies in this thesis clearly implicate p21 as a major regulator in determining cell survival during oxidative damage. The mechanism of p21 survival is uncoupled from its ability to regulate growth and is mediated through Bcl-XL inhibition of Bax-dependent death.