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...

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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We have already reported that epidermal growth factor receptor/phosphatidylinositol 3-kinase/AKT signaling is an important pathway in regulating radiation sensitivity and DNA double-strand break (DNA-dsb) repair of human tumor cells. In the present study, we investigated the effect of AKT1 on DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and DNA-dsb repair in irradiated non-small cell lung cancer cell lines A549 and H460. Treatment of cells with the specific AKT pathway inhibitor API-59CJ-OH (API; 1-5 Mmol/L) reduced clonogenic survival between 40% and 85% and enhanced radiation sensitivity of both cell lines significantly. As indicated by fluorescence-activated cell sorting analysis (sub-G 1 cells) and poly(ADP-ribose) polymerase cleavage, API treatment or transfection with AKT1-small interfering RNA (siRNA) induced apoptosis of H460 but not of A549 cells. However, in either apoptosis-resistant A549 or apoptosis-sensitive H460 cells, API and/or AKT1-siRNA did not enhance poly(ADP-ribose) polymerase cleavage and apoptosis following irradiation. Pretreatment of cells with API or transfection with AKT1-siRNA strongly inhibited radiation-induced phosphorylation of DNA-PKcs at T2609 and S2056 as well as repair of DNA-dsb as measured by the ;-H2AX foci assay. Coimmunoprecipitation experiments showed a complex formation of activated AKT and DNA-PKcs, supporting the assumption that AKT plays an important regulatory role in the activation of DNA-PKcs in irradiated cells. Thus, targeting of AKT enhances radiation sensitivity of lung cancer cell lines A549 and H460 most likely through specific inhibition of DNA-PKcs-dependent DNA-dsb repair but not through enhancement of radiation-induced apoptosis.

SPIO or USPIO labeling without TAs has an influence on gene expression of MSCs upregulating transferrin receptor. Furthermore, SPIO labeling with a TA will coat the cellular surface.

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IntroductionExpression of Y-box binding protein-1 (YB-1) is associated with tumor progression and drug resistance. Phosphorylation of YB-1 at serine residue 102 (S102) in response to growth factors is required for its transcriptional activity and is thought to be regulated by cytoplasmic signaling phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. These pathways can be activated by growth factors and by exposure to ionizing radiation (IR). So far, however, no studies have been conducted on IR-induced YB-1 phosphorylation.MethodsIR-induced YB-1 phosphorylation in K-RAS wild-type (K-RASwt) and K-RAS-mutated (K-RASmt) breast cancer cell lines was investigated. Using pharmacological inhibitors, small interfering RNA (siRNA) and plasmid-based overexpression approaches, we analyzed pathways involved in YB-1 phosphorylation by IR. Using γ-H2AX foci and standard colony formation assays, we investigated the function of YB-1 in repair of IR-induced DNA double-stranded breaks (DNA-DSB) and postirradiation survival was investigated.ResultsThe average level of phosphorylation of YB-1 in the breast cancer cell lines SKBr3, MCF-7, HBL100 and MDA-MB-231 was significantly higher than that in normal cells. Exposure to IR and stimulation with erbB1 ligands resulted in phosphorylation of YB-1 in K-RASwt SKBr3, MCF-7 and HBL100 cells, which was shown to be K-Ras-independent. In contrast, lack of YB-1 phosphorylation after stimulation with either IR or erbB1 ligands was observed in K-RASmt MDA-MB-231 cells. Similarly to MDA-MB-231 cells, YB-1 became constitutively phosphorylated in K-RASwt cells following the overexpression of mutated K-RAS, and its phosphorylation was not further enhanced by IR. Phosphorylation of YB-1 as a result of irradiation or K-RAS mutation was dependent on erbB1 and its downstream pathways, PI3K and MAPK/ERK. In K-RASmt cells K-RAS siRNA as well as YB-1 siRNA blocked repair of DNA-DSB. Likewise, YB-1 siRNA increased radiation sensitivity.ConclusionsIR induces YB-1 phosphorylation. YB-1 phosphorylation induced by oncogenic K-Ras or IR enhances repair of DNA-DSB and postirradiation survival via erbB1 downstream PI3K/Akt and MAPK/ERK signaling pathways.

a b s t r a c tNuclear localisation of EGFR is associated with treatment resistance of tumor cells. The aim of this study was to identify molecular targets to block nuclear shuttling of EGFR. Mutation of Thr654, located within the putative EGFR NLS demonstrated that phosphorylation of this residue is essential for nuclear EGFR shuttling following irradiation. Deletion of Thr654 blocked nuclear transport of EGFR, whereas mutation to Glu increased shuttling. Treatment with a peptide, corresponding to the phosphorylated NLS, abolished nuclear EGFR transport and reduced radiation-induced activation of DNA-PK, essential for DNA-repair. In accordance with that, lack of nuclear EGFR increased residual DNA damage in tumor cells and reduced cellular survival following irradiation. Blockage of nuclear EGFR shuttling may be a new strategy to fight treatment resistance.