Three independent mechanisms for arrest in G₂ after 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 0-2hours after IR (0.5 or 4Gy). 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 (≥6hours after IR) of cells that were in G₂/S/G₁ at the time of irradiation (4Gy) was inactivated by caffeine. This late G₂-arrest could not be explained by down-regulation of genes with functions in G₂/mitosis (e.g. PLK1, CCNB1/2), since the down-regulation was transient and not accompanied by reduced protein levels. However, the persistent phosphorylation of CHEK1 after 4Gy 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 G₂-arrest, but mediated an intermediate arrest (2-10hours after IR) independently of ATM and CHEK1. In conclusion, the IR-induced arrest in G₂ is mediated by ATM immediately after irradiation, with TP53 for independent and transient back-up, while CHEK1 is necessary for the late arrest.