Establishment of a novel clonal murine bone marrow stromal cell line for assessment of p53 responses to genotoxic stress.
The p53 protein is widely regarded as an important sensor of genotoxic damage in cells, and mutations in p53 are the most frequent observed in human cancers. Rapid assays for evaluating the potential of a chemical or physical agent to alter the transcriptional regulatory role of p53 may therefore serve as useful tools in toxicological research. In this study, the use of enhanced green fluorescent protein (EGFP) as a live cell reporter to assess the transactivation response of p53 to chemical and physical agents was evaluated. A stable murine bone marrow stromal cell line (D2XRIIGFP24) expressing EGFP under control of p53 response elements was established. D2XRIIGFP24 cells displayed low constitutive background fluorescence which was significantly enhanced in response to exposure to agents that induced p53 protein levels. Increases in EGFP fluorescence in response to oxidative and nitrosative stress as well as UVC irradiation were dose-dependent, detectable within 3 h of exposure and correlated closely with the amount of p53 protein accumulated within the cell. The results demonstrate the potential for rapid and early detection of p53 transactivation using the EGFP reporter approach and indicate this approach is adaptable to a variety of fluorescent assay techniques and a useful cell model for molecular toxicology research.
Gorbunov
Nikolai V
NV
Pacific Northwest National Laboratory, Richland, WA, USA. nikolai.gorbunov@na.amedd.army.mil
Morris
James E
JE
Greenberger
Joel S
JS
Thrall
Brian D
BD
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002460:Cell Line; D002999:Clone Cells; D020552:Comet Assay; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide / Q000633:toxicity; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D009152:Mutagenicity Tests / Q000379:methods*; D009569:Nitric Oxide / Q000633:toxicity; D018384:Oxidative Stress; D013312:Stress, Physiological / Q000235:genetics*; D017154:Stromal Cells; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays / Q000009:adverse effects
D008164:Luminescent Proteins; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins; D009569:Nitric Oxide; D006861:Hydrogen Peroxide