Role of 53BP1 in double strand break recognition mechanisms and cell survival after exposure to ionizing radiation : relative p53 and Chk2 dependency

889 DNA double strand break (DSB) are largely recognized as the central cellular cause of cell death after exposure to IR. In a recent study we have shown that γ-irradiation induced mitotic catastrophe occurs through a caspase-independent way in the human colorectal cancers (Zhang et al. Oncogene 2006). DSB recognition by the ATM protein is a central event during cellular response to IR leading to phosphorylation of cell cycle regulating proteins, DNA repair and apoptosis. 53BP1 is a conserved nuclear protein that is implicated in the DNA damage response, accumulates at or near sites of DNA damage, and participates in the intra S and G2/M checkpoints. The 53BP1 and NBS1 proteins are shown to be a regulators of ATM. The aim of this study was to assess the relative dependence of p53 and Chk2, two mains ATM substrates in radiation response, in the context of 53BP1 inhibition. Methods: γ-radiation was delivered by Cobalt source at a dose rate of 0.823 Gy/min. Radiosensitivity of A549 cell line and HCT116 cell lines (wild type or knocked out for Chk2 and Bax genes) with/withoutsiRNA-53BP1 or NBS1 were studied by clonogenic survival assay.The foci of53BP1, and γ-H2AX were analyzed by fluorescence immunocytochemistry. Apoptosis induction 24h after 6Gy IR with/without siRNA-53BP1 or NBS1 was determined by FACS analysis. Results: The number of foci about 53BP1 and γ-H2AX were increased 29 and 22 folds respectively in A549 as well as HCT116 WT cell lines 10 min after 2Gy IR, there is a co-localisation of 53BP1 and γ-H2AX foci. This enhancement of 53BP1 foci in response to IR was decreased 53.7% and 40.9% respectively in HCT116 p53 and Chk2-/- cells by contrast to HCT116 WT, but this decrease is not significant in Bax-/- cells . Radiation sensitivity was increased after transfection by siRNA-53BP1 in both HCT116 WT and Chk2 KO cell lines but not in p53-/- neither Bax-/- cells. SiRNA-53BP1 also enhanced the radiosensitivity in A549 cell line. This suggests that 53BP1 is involved in the recognition of DNA damage in a cell cycle check-point dependant manner. Moreover, cells exposed to 6 Gy IR manifest apoptosis signs (phosphatidyl serine exposure on the cell surface, sub-diploid DNA content) after si-53BP1 transfection by FACS analysis. Our results indicate that 53BP1 plays an important role radiation sensitivity, it is an integral component of the DNA damage-response network. The 53BP1 role in radiation response seems to be different in terms of cell survival in the absence of p53 or Chk2 suggesting a different role for these ATM substrates after DNA damage recognition by 53BP1.