Ionizing radiation, but not stimulation with epidermal growth factor (EGF), triggers EGF receptor (EGFR) import into the nucleus in a probably karyopherin ␣-linked manner. An increase in nuclear EGFR is also observed after treatment with H 2 O 2 , heat, or cisplatin. During this process, the proteins Ku70/80 and the protein phosphatase 1 are transported into the nucleus. As a consequence, an increase in the nuclear kinase activity of DNA-dependent kinase (DNA-PK) and increased formation of the DNA end-binding protein complexes containing DNA-PK, essential for repair of DNA-strand breaks, occurred. Blockade of EGFR import by the anti-EGFR monoclonal antibody C225 abolished EGFR import into the nucleus and radiation-induced activation of DNA-PK, inhibited DNA repair, and increased radiosensitivity of treated cells. Our data implicate a novel function of the EGFR during DNA repair processes.The epidermal growth factor receptor (EGFR) 1 is essential for mediation of both proliferative and survival signals to cells (1). At least five mitogenic growth factors bind to and activate EGFR. In addition to EGF these factors include transforming growth factor-␣, amphiregulin, heparin binding EGF (2), and epiregulin (3). Moreover, in recent years it became apparent that in addition to ligand binding-induced activation of the EGFR, ligand-independent receptor activating processes also exist (4). Activation of the EGFR signaling pathway by ligands has been linked with increased cell proliferation, angiogenesis, and metastasis and decreased apoptosis (5). As a consequence it is assumed that increased EGFR signaling plays an important role in tumorigenesis (6, 7). The mechanisms of ligand-independent EGFR activation and also the importance for the cell fate are not understood so far. But it is noteworthy that the ligand-independent activation always is linked to exposure to genotoxic stress (4,8,9). However, the differences of the observed cell response after ligand-induced EGFR activation, e.g. cell proliferation, and ligandindependent activation, e.g. cell cycle arrest, argue rather for a link to DNA repair processes for ligand-independent activation. This idea was strengthened by the observation that many tumor cells show an increased radiosensitivity after inhibition of EGFR signaling (10). The most detrimental DNA damages after treatment with ionizing radiation are double-strand strands (11), which are preferentially repaired in mammalians by nonhomologous end-joining (12). Looking for a connection between radiation-induced EGFR activation and DNA repair, we found a report stating that there is a physical interaction of EGFR and DNAdependent kinase (DNA-PK) (13) after cell treatment with the EGFR blocking antibody C225. As a consequence, the nuclear DNA-PK protein and activity was reduced, providing an explanation for the radiosensitizing effect of the EGFR blockage. However, this paper did not answer the question of the functional role of the physical interaction between EGFR and DNA-PK. Therefore, we addressed herein the...

Secondary mitotic human skin fibroblast populations in vitro underwent 53 ± 6 cumulative population doublings (CPD) in 302 ± 27 days. When the growth capacity of the mitotic fibroblasts is exhausted, and if appropriate methods are applied, the fibroblasts differentiate spontaneously into postmitotic fibroblast populations, which were kept in stationary culture for up to 305 ± 41 additional days. Mitotic and postmitotic fibroblast populations are heterogeneous populations with reproducible changes in the proportions of mitotic

Background: To elucidate the role of src kinase in caveolin-1 driven internalization and nuclear transport of EGFR linked to regulation of DNA-repair in irradiated cells.

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Purpose: It is known that blockage of epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K) activity enhances radiation sensitivity of human tumor cells presenting a K-RAS mutation. In the present study, we investigated whether impaired repair of DNA doublestrand breaks (DSB) is responsible for the radiosensitizing effect of EGFR and PI3K inhibition in K-RAS mutated (K-RAS mt ) cells. Experimental Design: The effect of the EGFR tyrosine kinase inhibitor BIBX1382BS (BIBX) on cellular radiosensitivity was determined in K-RAS mt (A549) and K-RAS wt (FaDu) cell lines by clonogenic survival assay. Radiation-induced phosphorylation of H2AX (Ser 139 ), ATM (Ser 1981 ), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs; Thr 2609 ) was analyzed by immunoblotting. Twenty-four hours after irradiation, residual DSBs were quantified by identification of gH2AX foci and frequency of micronuclei. Results: BIBX reduced clonogenic survival of K-RAS mt -A549 cells, but not of K-RAS wt -FaDu cells, after single-dose irradiation. Analysis of the radiation-induced H2AX phosphorylation revealed that BIBX, as well as the PI3K inhibitor LY294002, leads to a marked reduction of P-H2AX in K-RAS mt -A549 and MDA-MB-231 cells, but not in K-RAS wt -FaDu and HH4ded cells. Likewise, radiation-induced autophosphorylation of DNA-PKcs at Thr 2609 was only blocked in A549 cells by these two inhibitors and AKT1 small interfering RNA transfection. However, neither in K-RAS mt nor in K-RAS wt cells the inhibitors did affect radiation-induced ATM phosphorylation. As a consequence of inhibitor treatment, a significant enhancement of both residual DSBs and frequency of micronuclei was apparent only in A549 but not in FaDu cells following radiation. Conclusion: Targeting of the EGFR-dependent PI3K-AKT pathway in K-RAS-mutated A549 cells significantly affects postradiation survival by affecting the activation of DNA-PKcs, resulting in a decreased DSB repair capacity.

The novel findings on radiation-induced EGFR-signaling and its involvement in regulating DNA-double strand break repair need further investigations of the detailed mechanisms involved. The results to be obtained may not only improve our knowledge on basic mechanisms of radiation sensitivity/resistance but also will promote translational approaches to test new strategies for clinically applicable molecular targeting.

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