Three independent mechanisms for arrest in G2after ionizing radiation

Abstract

Cell cycle checkpoints ensure that eukaryotic cells do not enter mitosis after ionizing irradiation (IR). The G2-arrest after IR is the result of activation of multiple signalling pathways, the contributions of which vary with time after irradiation. We have studied the time evolution of the IR-induced G2-arrest in human B-lymphocyte cancer cell lines, as well as the molecular mechanisms responsible for the arrest. Cells that were in G2 phase at the time of irradiation experienced a transient arrest that blocked entry into mitosis at 0-2 hours after IR (0.5 or 4 Gy). Activation of ATM and CHEK2 occurred at the same time as this early arrest and was, like the arrest, abrogated by the ATM-inhibitor KU-55933. A late, permanent and AtM-independent arrest (≥6 hours after IR) of cells that were in G 2/S/G1 at the time of irradiation (4 Gy) was inactivated by caffeine. This late G2-arrest could not be explained by downregulation of genes with functions in G2/mitosis (e.g., PLK1, CCNB1/2), since the downregulation was transient and not accompanied by reduced protein levels. However, the persistent phosphorylation of CHEK1 after 4 Gy suggested a role for CHEK1 in the late arrest, consistent with the abrogation of the arrest in CHEK1-depleted cells. tp53 was not necessary for the late G 2-arrest, but mediated an intermediate arrest (2-10 hours after IR) independently of ATM and CHEK1. In conclusion, the IR-induced arrest in G 2 is mediated by ATM immediately after irradiation, with TP53 for independent and transient back-up, while CHEK1 is necessary for the late arrest. © 2011 Landes Bioscience.