The radioprotector Bowman-Birk proteinase inhibitor stimulates DNA repair via epidermal growth factor receptor phosphorytation and nuclear transport

Background and purpose: 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. Materials and 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 gamma H(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. Conclusion: 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. (c) 2008 Elsevier Ireland Ltd. All rights reserved.