Dittmann Klaus, Mayer Claus, Kehlbach Rainer, Rodemann H Peter
Department of Radiation Oncology, University of Tübingen, Tübingen, Germany.
Radiother Oncol. 2008 Mar;86(3):375-82. doi: 10.1016/j.radonc.2008.01.007. Epub 2008 Jan 30.
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.
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.
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.
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.
本研究旨在阐明辐射防护剂鲍曼-伯克蛋白酶抑制剂（BBI）的潜在分子机制及其与表皮生长因子受体（EGFR）核转运的相互作用。
在体外，利用野生型TP53支气管癌细胞系A549和以突变型TP53为特征的转化成纤维细胞系HH4dd，研究BBI在EGFR反应水平的分子效应。通过蛋白质免疫印迹法和共聚焦显微镜对细胞胞质和细胞核部分的EGFR及相关蛋白表达进行定量分析。利用γH(2)AX焦点分析法对残留的DNA双链断裂进行定量分析。
辐射和BBI处理均刺激EGFR内化进入细胞质。该过程涉及src激酶激活、Y845位点的EGFR磷酸化以及Y14位点的小窝蛋白1磷酸化。EGFR内化与核EGFR转运相关，并与T654位点的EGFR磷酸化有关。核EGFR与DNA-PK复合物的形成和激活有关。此外，在辐射和BBI处理后，发现核EGFR与S15位点磷酸化的TP53以及MDC1形成复合物。值得注意的是，在具有突变型TP53的细胞中，核EGFR复合物中的MDC1显著减少，并且BBI处理或辐射均未能使其增加。有趣的是，在具有突变型TP53的细胞中，BBI介导的双链断裂修复刺激作用受到显著阻碍。
这些数据表明，BBI仅在野生型TP53细胞中刺激EGFR、TP53和MDC1蛋白之间的复合物形成。由于MDC1对于DNA修复位点的募集至关重要，这一观察结果可能解释了BBI如何选择性地刺激野生型TP53细胞中DNA双链断裂的修复。
