Wiegman Erwin M, Blaese Marcel A, Loeffler Heidi, Coppes Rob P, Rodemann H Peter
Department of Radiation Oncology, Eberhard-Karls-University Tuebingen, Germany.
Radiother Oncol. 2007 Jun;83(3):289-95. doi: 10.1016/j.radonc.2007.05.013. Epub 2007 Jun 8.
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.
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.
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.
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.
有人提出，辐射诱导的ATM及下游成分的激活涉及TGFβ-1的活化，这可能是由于TGFβ-1受体I-Smad信号传导所致。因此，本研究的目的是阐明TGFβ-1受体I-Smad信号传导在辐射暴露后介导ATM活性中的独特作用。
用条件可调节的TGFβ-1反义构建体（Tet-on系统）稳定转染A549细胞，以检测照射后的克隆形成活性。通过免疫印迹法在非循环、血清饥饿的细胞中测定ATM、p53和chk2的磷酸化谱。同样，使用A549野生型细胞来确定细胞周期分布与照射的关系，照射前有无用CMK预处理，CMK是一种参与潜伏TGFβ-1激活的弗林蛋白酶的特异性抑制剂。此外，对血清饥饿的细胞进行蛋白质免疫印迹分析，通过应用特异性TGFβ-1受体I抑制剂来研究ATM和p53刺激对TGFβ-1受体I-Smad信号传导的依赖性。
用反义构建体敲低TGFβ-1可显著提高电离辐射后克隆形成细胞的存活率。同样，CMK处理减少了辐射诱导的A549细胞G1期阻滞。此外，TGFβ-1敲低以及CMK处理均抑制了辐射后ATM、p53和chk2的快速磷酸化。然而，使用特异性抑制剂表明，TGFβ-1受体I-Smad信号传导不参与ATM和p53的这种快速激活。
我们证实TGFβ-1在照射细胞中对ATM和p53信号传导的刺激中起关键作用。然而，这种快速刺激似乎不依赖于最近提出的TGFβ-1受体I-Smad信号传导的激活。
