TGF beta-1 dependent fast stimulation of ATM and p53 phosphorylation following exposure to ionizing radiation does not involve TGF beta-receptor I signalling

Background and purpose: It has been proposed that radiation induced stimulation of ATM and downstream components involves activation of TGF beta-1 and that this may be due to TGF beta-1-receptor I-Smad signalling. Therefore, the aim of this study was to clarify the distinct role of TGF beta-1-receptor I-Smad signalling in mediating ATM activity following radiation exposure. Materials and methods: A549 cells were stably transfected with a conditionally regulatable TGF beta-1 antisense construct (Tet-on-system) to test clonogenic activity following irradiation. Phosphorylation profile of ATM, p53, and chk2 was determined in non-cycling, serum-starved cells by immunoblotting. Likewise, A549 wild type cells were used to identify cell cycle distribution as a function of irradiation with or without pretreatment with CMK, a specific inhibitor of furin protease involved in activation of latent TGF beta-1. Furthermore Western and immunoblot analyses were performed on serum-starved cells to investigate the dependence of ATM- and p53-stimulation on TGF beta-1 -receptor I-Smad signalling by applying a specific TGF beta-I-receptor I inhibitor. Results: Knock down of TGF beta-1 by an antisense construct significantly increased clonogenic cell survival following exposure to ionizing radiation. Likewise, CMK treatment diminished the radiation induced G1 arrest of A549 cells. Moreover, both TGF beta-1-knock down as well as CMK treatment inhibited the fast post-radiation phosphorylation of ATM, p53, and chk2. However, as shown by the use of a specific inhibitor TGF beta-l-receptor I-Smad signalling was not involved in this fast activation of ATM and p53. Conclusions: We confirm that TGF beta-1 plays a critical role in the stimulation of ATM- and p53 signalling in irradiated cells. However, this fast stimulation seems not to be dependent on activation of TGF beta-1-receptor I-Smad signalling as recently proposed. (C) 2007 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 83 (2007) 289-295.