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

https://doi.org/10.1016/j.radonc.2007.05.013Get rights and content

Abstract

Background and purpose

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

Materials and methods

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

Results

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

Conclusions

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

Section snippets

Cell culture

The study was performed using a human bronchial carcinoma cell line A549 (ATCC, USA). Cells were cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (BioWhittaker, USA) and standard amounts of antibiotics (penicillin 100 U/ml; streptomycin 100 μg/ml). With the exception of cell cycle analyses, 72 h serum-starved cells were used for all experimental conditions applied.

TGFβ-1 antisense A549 subclones

A549 cells were stably transfected with the pTETon- and the empty pTRE2-Hygro- or

Clonogenic survival

As demonstrated in Fig. 1a, radiation sensitivity of A549 wild type cells was not influenced by treatment with doxycyclin. In contrast doxycycline induction (dox+) of the anti-TGFβ-1 construct resulted in a significantly better clonogenic cell survival as compared to the not induced condition (without doxycycline, dox), i.e., survival fraction of dox cells at 3 Gy (SF3) was 0.4025 ± 0.0204 whereas SF3 in dox+ cells was 0.5710 ± 0.0246 (Fig. 1b).

Flow cytometry

Cell cycle analysis of A549 cells untreated or

Discussion

TGFβ-1 is an important tumour suppressor [8] and a potent inducer of growth inhibition and differentiation in several cell types i.e., epithelial cells and fibroblasts [5], [16], [19], [26], [28] and plays an important role in the induction and manifestations of radiation induced fibrosis [1], [4], [17], [25]. As shown for normal fibroblasts and several tumour cell lines ionizing radiation effectively induces expression and activation of TGFβ-1 [2], [16], [23], [33], suggesting a role for

Conflict of interest statement

The authors do not have any financial or personal relationship with other people or organisations that inappropriately influence their work.

Acknowledgements

This work was supported by grants from the BMVg (FV M SAB1 2 A002) and the BMBF-IZKF Program (01KS9601) awarded to HPR and from the Dutch Cancer Society awarded to EMW.

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