The radioprotector O-phospho-tyrosine stimulates DNA-repair via epidermal growth factor receptor- and DNA-dependent kinase phosphorylation.
OBJECTIVE
Purpose of the study was to elucidate the underlying molecular mechanism of the radioprotector O-phospho-tyrosine (P-Tyr).


METHODS
Molecular effects of P-Tyr at the level of EGFR responses were investigated in vitro with bronchial carcinoma cell line A549. Nuclear EGFR transport and DNA-PK activation were quantified after Western blotting. Residual DNA-damages were quantified by help of gammaH(2)AX focus assay.


RESULTS
As determined by dose-response curves, treatment of cells with P-Tyr for 16h before irradiation results in radioprotection. Simultaneous treatment with EGFR blocking antibody Cetuximab abolished P-Tyr associated radioprotection. At the molecular level P-Tyr mediated a general phosphorylation of EGFR and a pronounced phosphorylation of nuclear EGFR at residue Thr No. 654, also observed after treatment with ionizing radiation. This phosphorylation was associated with nuclear EGFR accumulation. Moreover, P-Tyr-triggered EGFR nuclear accumulation was associated with phosphorylation of DNA-PK at Thr 2609. This activated form of DNA-PK was not DNA associated, but after radiation, DNA binding increased, particularly after P-Tyr pre-treatment. These molecular effects of P-Tyr resulted in a reduction of residual DNA-damage after irradiation.


CONCLUSIONS
Radioprotection by P-Tyr is mediated through its stimulation of nuclear EGFR transport and concurrent, but DNA-damage independent, activation of DNA-PK. Thus, subsequent irradiation results in increased binding of DNA-PK to DNA, improved DNA-repair and increased cell survival.
Dittmann
Klaus
K
Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tübingen, Tübingen, Germany. klaus.dittmann@uni-tuebingen.de
Mayer
Claus
C
Wanner
Gabriele
G
Kehlbach
Rainer
R
Rodemann
H Peter
HP
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D015153:Blotting, Western; D002283:Carcinoma, Bronchogenic / Q000378:metabolism; D045744:Cell Line, Tumor; D000068818:Cetuximab; D004260:DNA Repair / Q000502:physiology*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000494:pharmacology / Q000502:physiology*; D011837:Radiation-Protective Agents
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D011837:Radiation-Protective Agents; D019000:Phosphotyrosine; D066246:ErbB Receptors; D051747:DNA-Activated Protein Kinase; D000068818:Cetuximab