Molecular radiobiologyTGFβ-1 dependent fast stimulation of ATM and p53 phosphorylation following exposure to ionizing radiation does not involve TGFβ-receptor I signalling
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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These authors contributed equally to the work.