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

BACKGROUND AND PURPOSE: It has been proposed that radiation induced stimulation of ATM and downstream components involves activation of TGFbeta-1 and that this may be due to TGFbeta-1-receptor I-Smad signalling. Therefore, the aim of this study was to clarify the distinct role of TGFbeta-1-receptor I-Smad signalling in mediating ATM activity following radiation exposure.

MATERIALS AND

METHODS

A549 cells were stably transfected with a conditionally regulatable TGFbeta-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 TGFbeta-1. Furthermore Western and immunoblot analyses were performed on serum-starved cells to investigate the dependence of ATM- and p53-stimulation on TGFbeta-1-receptor I-Smad signalling by applying a specific TGFbeta-1-receptor I inhibitor.

RESULTS

Knock down of TGFbeta-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 TGFbeta-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 TGFbeta-1-receptor I-Smad signalling was not involved in this fast activation of ATM and p53.

CONCLUSIONS

We confirm that TGFbeta-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 TGFbeta-1-receptor I-Smad signalling as recently proposed.