Seine-15 phosphorylated p53 protein as a sensor of initial DNA damage following ionizing radiation

Abstract

Purpose: Molecular data exists to suggest that focal DNA repair protein-protein interactions (ie. rad50-mre11; rad51-BRCA1) occur within the nuclei of irradiated cells at sites of DNA-dsb following IR, but whether these focal interactions occur secondary to direct signals and interactions with DNA damage checkpoint sensing protein (ie. p53, ATM) is unknown. Indeed, the wild type p53 G1-checkpoint can be activated with as little as one DNA-dsb and cause a permanent G1 arrest in lethally irradiated fibroblasts. As yet, direct evidence that the p53 protein can sense and activate DNA-dsb repair following irradiation as part of a DNA damage checkpoint response is lacking. To test the hypothesis that the p53 protein can sense DNA breaks in vivo, we have obtained data using quantitative immunofluorescence, confocal microscopy with antibodies to specific phospho-forms of p53 Materials and Methods: To avoid cell cycle bias, a number of human fibroblast cell cultures (derived from normal, AT, NBS and Li-Frauemini pateints) were irradiated in plateau-phase and the number of irradiation-induced foci were determined using confocal immunofluoresecent microscopy and image analysis at doses of 0.5 to 20 Gy and at timepoints up to 96 hours following irradiation. Primary antibodies against the ser-15/ser-20 (XRT-specific) and ser-46 (UV-specific) were used to determine subcellular localization of p53 protein after DNA damage. Results: In a dose-responsive manner, normal human fibroblasts irradiated in plateau phase (ie. GM05757) show an accumulation of discrete nuclear foci when stained with an antibody to the serine-15 phosphorylated form of p53 (ie. ser15-p53) which is a form activated by IR in an ATM-dependent manner. Dose-responsive foci can be observed within 30 minutes of IR-exposure, suggesting that p53 rapidly localizes to sites of IR-induced damage. A kinetic study of ser15-p53 accumulation in GM05757 cells suggest that despite a rapid induction of ser15-p53 following IR, a high level of residual foci remain at 24 hours which correlates to the level of rad50 foci after 10Gy. In contrast, after 2 Gy most foci have resolved at 24 hours. Rad51 foci are not dose-responsive and are invariant over the same time period. Ser15-p53 foci were not observed in ATM-/- fibroblasts (GM05823) cells, but are present in Nijmegen Breakage Syndrome fibroblasts (GM07166). Conclusion: The ser15-p53 protein is an early sensor of DNA damage which is dose-dependent. This biomarker may represent a novel predictive assay for radionsensitivity in normal and malignat cells and provide further information on the DNA repair signaling cascade.