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


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.
Wiegman
Erwin M
EM
Department of Radiation Oncology, Eberhard-Karls-University Tuebingen, Germany.
Blaese
Marcel A
MA
Loeffler
Heidi
H
Coppes
Rob P
RP
Rodemann
H Peter
HP
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D011839:Radiation, Ionizing; D018125:Receptors, Transforming Growth Factor beta / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects* / Q000528:radiation effects*; D051899:Smad2 Protein / Q000378:metabolism / Q000528:radiation effects; D013997:Time Factors; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D015854:Up-Regulation
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D018125:Receptors, Transforming Growth Factor beta; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D053773:Transforming Growth Factor beta1; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases