PARENT SESSION
3:45 PM to 5:15 PM
Tuesday, April 23, 2002
Mini-Symposium 12
Cell Cycle and Apoptosis
Room: Nevada 4-5
, Co-Chair: Muschel, Ruth1; Haimovitz-Friedman, Adriana21University of Pennsylvania, Philadelphia, PA2Memorial Sloan-Kettering Cancer Center, New York, NY
(MS12-2) Ser15-phosphorylated p53: a Sensor of DNA damage.
Al Rashid, Shahnaz*,1,3, Jalali, Farid1,3, Lilge, Lothar1,3, Benchimol, Sam1,3, Bristow, Robert1,2,3, 1 Ontario Cancer Institute-Princess Margaret Hospital (UHN), Toronto, Ontario3 Department of Medical Biophysics, University of Toronto, Toronto, Ontario2 Department of Radiation Oncology, University of Toronto, Toronto, Ontario
ABSTRACT-
Exposure to DNA damage results in delays in the cell cycle progression of mammalian cells, presumably to allow time for DNA repair and thereby maintain genomic stability. Focal DNA repair protein-protein interactions (eg. Rad50/Mre11; Rad51/BRCA1) occur within the nuclei of irradiated cells at sites of DNA double-strand breaks (dsbs), but whether these focal interactions occur secondary to direct signals and interactions with DNA damage checkpoint sensing proteins (ie. p53, ATM) is unknown. Recently, a specific ionizing radiation (IR)-induced, serine15-phosphorylated form of p53 (ie. Ser15-p53), was shown to be nuclear-bound following DNA damage and resistant to ongoing mdm-2 ubiquitination, suggesting a novel role for this sub-pool of p53 in the DNA damage response cascade. However, its exact function remains unclear. Using quantitative immunofluorescence confocal microscopy of normal human fibroblasts irradiated in plateau phase (ie. GM05757) and stained with Ser15-p53-specific antibodies, a discrete nuclear sub-pool of Ser15-p53 foci was observed to accumulate within 30 minutes post-IR in a dose-responsive manner. The formation of Ser15-p53 foci is ATM-dependent and wortmannin-sensitive (ie. PI3-kinase dependent), but can form in the absence of the Rad50/Mre11/Nbs1 repair complex. Furthermore, the residual number of Ser15-p53 foci at 24 hours appears to be consistent with the estimated number of residual DNA-dsbs based on DNA-dsb rejoining assays (ie. CFGE) in the same cell line. In subsequent experiments, Ser15-p53 foci were maximally induced by agents that caused DNA-dsbs and base damage (ie. IR, bleomycin and MMS), but rarely by other types of DNA damage (ie. MMC, hydroxyurea, UV radiation and H202), suggesting an association with DNA-dsb and base excision repair pathways. Consistent with this hypothesis, a proportion of Ser15-p53 foci do co-localize with Rad50 and Mre11 proteins. Our results suggest a novel biology for the Ser15-p53 phospho-form as a direct sensor of DNA damage and provides a link between cell cycle checkpoint control and DNA repair. Ongoing experiments include scoring residual Ser15-p53 foci at later time points to address whether they correlate with final clonogenic radiation survival and may be a useful surrogate as a predictive assay of normal tissue radiosensitivity.
KEYWORDS: p53, DNA-dsbs, fibroblasts, cell-cycle