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p53-dependent induction of p21(Cip1/WAF1/Sdi1) protects against oxygen-induced toxicity
C E Helt1, R C Rancourt, R J Staversky
1Departments of Pediatrics and Environmental Medicine, The University of Rochester, Rochester, New York 14642, USA.
Abstract:
The beneficial effects of supplemental oxygen delivered to patients suffering from acute respiratory distress is offset by its reduction to genotoxic reactive oxygen species (ROS) that inhibit proliferation and kill pulmonary cells. Cells respond to oxygen-induced damage by expressing the tumor suppressor p53 and the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1) (p21), which limits proliferation by blocking entry into S phase. Since preventing DNA synthesis during genotoxic stress may enhance survival, the current study examines whether hyperoxia induces p21 through a p53-dependent pathway and whether p21 protects cells from the toxic effects of oxygen. HCT116 colon carcinoma cells and clonal lines lacking p53 or p21were used in this study because they allow direct cytotoxic comparisons between isogenic cells, without complications arising from unknown genetic differences between nonhomologous cell lines. Hyperoxia (95% O2, 5% CO2) increased p53 abundance, phosphorylation of p53 on serine 15, and p21 mRNA and protein in parental HCT116 cells that ceased proliferation. In contrast, p21 was not detected in either p53- or p21-deficient HCT116 cells, which exited the G1 compartment and were arrested in S and G2/M phases during hyperoxia. Trypan blue-dye exclusion revealed that induction of p21 markedly enhanced survival during exposure and colony survival assays showed that p21 enhanced the ability to resume proliferation during recovery in room air. The observation that p53-dependent induction of p21 prevents exit from G1 and promotes survival during hyperoxia is consistent with the importance of limiting DNA replication during genotoxic stress caused by oxygen exposure.
Insights
Supplemental oxygen can be toxic, causing cell damage via reactive oxygen species (ROS). This study shows that p53-dependent p21 induction protects cells from oxygen toxicity by limiting DNA replication and enhancing survival.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Supplemental oxygen, while beneficial for acute respiratory distress, generates genotoxic reactive oxygen species (ROS).
- ROS can inhibit cell proliferation and cause cell death, particularly in pulmonary cells.
- Cells possess defense mechanisms, including the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, to counteract oxygen-induced damage.
Purpose of the Study:
- To investigate if hyperoxia induces p21 via a p53-dependent pathway.
- To determine if p21 confers protection against the toxic effects of oxygen.
- To understand the role of p21 in cell cycle regulation during genotoxic stress.
Main Methods:
- Utilized HCT116 colon carcinoma cells and isogenic variants lacking p53 or p21.
- Exposed cells to hyperoxia (95% O2, 5% CO2) to induce oxidative stress.
- Assessed p53 and p21 expression, cell cycle progression, and cell viability using techniques like Western blotting, RT-PCR, and trypan blue exclusion assays.
Main Results:
- Hyperoxia increased p53 and p21 expression in parental HCT116 cells, leading to cell cycle arrest in the G1 phase.
- p53- and p21-deficient cells failed to induce p21, exited G1, and arrested in S and G2/M phases.
- p21 induction significantly enhanced cell survival during hyperoxia exposure and improved recovery of proliferation.
Conclusions:
- Hyperoxia induces p21 in a p53-dependent manner.
- p21 plays a critical role in protecting cells from oxygen-induced genotoxicity by preventing DNA replication.
- Limiting DNA synthesis via p21 is a key survival mechanism during oxidative stress.