The radioprotector Bowman-Birk proteinase inhibitor stimulates DNA repair via epidermal growth factor receptor phosphorylation and nuclear transport.
OBJECTIVE
The purpose of the study was to elucidate the underlying molecular mechanism of the radioprotector, Bowman-Birk proteinase inhibitor (BBI), and its interaction with EGFR nuclear transport.


METHODS
Molecular effects of BBI at the level of EGFR responses were investigated in vitro with wt. TP53 bronchial carcinoma cell line A549 and the transformed fibroblast cell line HH4dd characterized by a mt. TP53. EGFR and associated protein expression were quantified by Western blotting and confocal microscopy in the cytoplasmic and nuclear cell fraction. Residual DNA double strand breaks were quantified by means of a gammaH(2)AX focus assay.


RESULTS
Both irradiation and BBI-treatment stimulated EGFR internalization into the cytoplasm. This process involved src kinase activation, EGFR phosphorylation at Y845, and caveolin 1 phosphorylation at Y14. EGFR internalization correlated with nuclear EGFR transport and was associated with phosphorylation of EGFR at T654. Nuclear EGFR was linked with DNA-PK complex formation and activation. Furthermore, nuclear EGFR was found in complex with TP53, phosphorylated at S15, and with MDC1, following irradiation and BBI treatment. It is noteworthy that MDC1 was strongly decreased in the nuclear EGFR complex in cells with mt. TP53 and failed to be increased by either BBI treatment or irradiation. Interestingly, in cells with mt. TP53 the BBI mediated stimulation of double strand break repair was hampered significantly.


CONCLUSIONS
These data indicate that BBI stimulates complex formation between EGFR, TP53 and MDC1 protein in wt. TP53 cells only. Since MDC1 is essential for recruitment of DNA repair foci, this observation may explain how BBI selectively stimulated repair of DNA double strand breaks in wt. TP53 cells.
Dittmann
Klaus
K
Department of Radiation Oncology, University of Tübingen, Tübingen, Germany. klaus.dittmann@uni-tuebingen.de
Mayer
Claus
C
Kehlbach
Rainer
R
Rodemann
H Peter
HP
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000187:drug effects*; D066246:ErbB Receptors / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D015534:Trans-Activators / Q000378:metabolism; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C413163:MDC1 protein, human; D009687:Nuclear Proteins; D015842:Serine Proteinase Inhibitors; C495901:TP53 protein, human; D015534:Trans-Activators; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D016159:Tumor Suppressor Protein p53; D066246:ErbB Receptors