Rainer Kehlbach

Radiation-induced caveolin-1 associated EGFR internalization is linked with nuclear EGFR transport and activation of DNA-PK

BackgroundTo 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.ResultsIonizing radiation resulted in src kinase stabilization, activation and subsequent src mediated caveolin-1 Y14- and EGFR Y845-phosphorylations. Both phosphorylations were radiation specific and could not be observed after treatment with EGF. Inhibition of EGFR by the antibody Erbitux resulted in a strong accumulation of caveolin/EGFR complexes within the cytoplasm, which could not be further increased by irradiation. Radiation-induced caveolin-1- and EGFR-phosphorylations were associated with nuclear EGFR transport and activation of DNA-PK, as detected by phosphorylation at T2609. Blockage of src activity by the specific inhibitor PP2, decreased nuclear transport of EGFR and inhibited caveolin-1- and DNA-PK-phosphorylation. Knockdown of src by specific siRNA blocked EGFR phosphorylation at Y845, phosphorylation of caveolin-1 at Y14 and abolished EGFR transport into the nucleus and phosphorylation of DNA-PK. Consequently, both knockdown of src by specific siRNA and also inhibition of src activity by PP2 resulted in an enhanced residual DNA-damage as quantified 24 h after irradiation and increased radiosensitivity.ConclusionSrc kinase activation following irradiation triggered caveolin-1 dependent EGFR internalization into caveolae. Subsequently EGFR shuttled into the nucleus. As a consequence, inhibition of internalization and nuclear transport of EGFR blocked radiation-induced phosphorylation of DNA-PK and hampered repair of radiation-induced double strand breaks.

Autophagy contributes to resistance of tumor cells to ionizing radiation

BACKGROUND AND PURPOSE Autophagy signaling is a novel important target to improve anticancer therapy. To study the role of autophagy on resistance of tumor cells to ionizing radiation (IR), breast cancer cell lines differing in their intrinsic radiosensitivity were used. MATERIALS AND METHODS Breast cancer cell lines MDA-MB-231 and HBL-100 were examined with respect to clonogenic cell survival and induction of autophagy after radiation exposure and pharmacological interference of the autophagic process. As marker for autophagy the appearance of LC3-I and LC3-II proteins was analyzed by SDS-PAGE and Western blotting. Formation of autophagic vacuoles was monitored by immunofluorescence staining of LC3. RESULTS LC3-I and LC3-II formation differs markedly in radioresistant MDA-MB-231 versus radiosensitive HBL-100 cells. Western blot analyses of LC3-II/LC3-I ratio indicated marked induction of autophagy by IR in radioresistant MDA-MB-231 cells, but not in radiosensitive HBL-100 cells. Indirect immunofluorescence analysis of LC3-II positive vacuoles confirmed this differential effect. Pre-treatment with 3-methyladenine (3-MA) antagonized IR-induced autophagy. Likewise, pretreatment of radioresistant MDA-231 cells with autophagy inhibitors 3-MA or chloroquine (CQ) significantly reduced clonogenic survival of irradiated cells. CONCLUSION Our data clearly indicate that radioresistant breast tumor cells show a strong post-irradiation induction of autophagy, which thus serves as a protective and pro-survival mechanism in radioresistance.

Targeting of AKT1 enhances radiation toxicity of human tumor cells by inhibiting DNA-PKcs-dependent DNA double-strand break repair

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 μmol/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-G1 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 γ-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. [Mol Cancer Ther 2008;7(7):1772–81]

Labeling of human mesenchymal stromal cells with superparamagnetic iron oxide leads to a decrease in migration capacity and colony formation ability

BACKGROUND AIMS Labeling of stem cells is crucial to allow tracking of stem cell homing and engraftment after transplantation. In this study we evaluated the influence of cell labeling procedures using clinically approved small particles of iron oxide (SPIO) with or without transfection reagents (TA) on functional parameters of human mesenchymal stem cells (MSC). METHODS The study was approved by the institutional review board of the University of Tubingen, Germany. Seven populations of bone marrow (BM)-derived human mesenchymal stem cells (MSC) were labeled with SPIO alone or in combination with various TA. Directly after labeling and two passages after labeling migration assays, quantification of colony-forming units and quantitative evaluation of the differentiation potential were performed. Quantification of the cellular total iron load (TIL), determination of the cellular viability and electron microscopy were also performed. RESULTS Labeling of mesenchymal stem cells with SPIO with or without TA did not affect cell viability and differentiation potential significantly. SPIO in combination with TA coated the cellular surface directly after labeling but was incorporated into the cells after two passages. Labeling of mesenchymal stem cells with TA led to a significant decrease of migration capacity. This effect was abolished after two passages. Labeling with and without TA led to a significant decrease in colony formation ability. This effect could also be observed after two passages. CONCLUSIONS The observed decrease of migration capacity and colony-formation ability was not associated with either TIL or localization of particles of iron oxide. SPIO labeling with and without TA had functional effects on human mesenchymal stem cells by decreasing the migration capacity and colony-formation ability of the stem cells.

Functional investigations on human mesenchymal stem cells exposed to magnetic fields and labeled with clinically approved iron nanoparticles.

BackgroundFor clinical applications of mesenchymal stem cells (MSCs), labeling and tracking is crucial to evaluate cell distribution and homing. Magnetic resonance imaging (MRI) has been successfully established detecting MSCs labeled with superparamagnetic particles of iron oxide (SPIO). Despite initial reports that labeling of MSCs with SPIO is safe without affecting the MSCs biology, recent studies report on influences of SPIO-labeling on metabolism and function of MSCs. Exposition of cells and tissues to high magnetic fields is the functional principle of MRI. In this study we established innovative labeling protocols for human MSCs using clinically established SPIO in combination with magnetic fields and investigated on functional effects (migration assays, quantification of colony forming units, analyses of gene and protein expression and analyses on the proliferation capacity, the viability and the differentiation potential) of magnetic fields on unlabeled and labeled human MSCs. To evaluate the imaging properties, quantification of the total iron load per cell (TIL), electron microscopy, and MRI at 3.0 T were performed.ResultsHuman MSCs labeled with SPIO permanently exposed to magnetic fields arranged and grew according to the magnetic flux lines. Exposure of MSCs to magnetic fields after labeling with SPIO significantly enhanced the TIL compared to SPIO labeled MSCs without exposure to magnetic fields resulting in optimized imaging properties (detection limit: 1,000 MSCs). Concerning the TIL and the imaging properties, immediate exposition to magnetic fields after labeling was superior to exposition after 24 h. On functional level, exposition to magnetic fields inhibited the ability of colony formation of labeled MSCs and led to an enhanced expression of lipoprotein lipase and peroxisome proliferator-activated receptor-γ in labeled MSCs under adipogenic differentiation, and to a reduced expression of alkaline phosphatase in unlabeled MSCs under osteogenic differentiation as detected by qRT-PCR. Moreover, microarray analyses revealed that exposition of labeled MSCs to magnetic fields led to an up regulation of CD93 mRNA and cadherin 7 mRNA and to a down regulation of Zinc finger FYVE domain mRNA. Exposition of unlabeled MSCs to magnetic fields led to an up regulation of CD93 mRNA, lipocalin 6 mRNA, sialic acid acetylesterase mRNA, and olfactory receptor mRNA and to a down regulation of ubiquilin 1 mRNA. No influence of the exposition to magnetic fields could be observed on the migration capacity, the viability, the proliferation rate and the chondrogenic differentiation capacity of labeled or unlabeled MSCs.ConclusionsIn our study an innovative labeling protocol for tracking MSCs by MRI using SPIO in combination with magnetic fields was established. Both, SPIO and the static magnetic field were identified as independent factors which affect the functional biology of human MSCs. Further in vivo investigations are needed to elucidate the molecular mechanisms of the interaction of magnetic fields with stem cell biology.

Radiation-induced lipid peroxidation activates src kinase and triggers nuclear EGFR transport

PURPOSE Elucidation of the molecular mechanism of radiation-induced activation of src kinase, which initiates EGFR internalization and nuclear transport. MATERIAL AND METHODS Radiation-induced src activation was investigated in the bronchial carcinoma cell line A549. Proteins were Western blotted and quantified by the help of specific antibodies. Residual DNA-damage was quantified with gammaH(2)AX-foci analysis. Radiation-induced lipid peroxidation was prevented by acetyl-cysteine. RESULTS The radiation-induced src activation and EGFR stabilization could be mimicked by addition of hydroxy-nonenal (HNE), one of the major lipid peroxidation products. Radiation-generated HNE is bound to EGFR and src and correlated with complex formation between both following radiation. Treatment with HNE activated src and stimulated radiation-associated EGFR and caveolin 1 phosphorylations resulting in increased nuclear transport of EGFR. Consequently, radiation-induced phosphorylation and activation of DNA-PK were increased. This phosphorylation was associated with improved removal of residual damage 24h after irradiation. Inhibition of radiation-induced HNE generation by acetyl-cysteine blocked radiation-induced src activation and EGFR phosphorylation. CONCLUSIONS HNE generated in response to radiation exposure activates src kinase and is involved in regulation of radiation-stimulated DNA-repair processes.

Nuclear EGFR shuttling induced by ionizing radiation is regulated by phosphorylation at residue Thr654

MINT‐7987956: Karyopherin alpha (uniprotkb:P52294) physically interacts (MI:0915) with EGFR (uniprotkb:P00533) by anti bait coimmunoprecipitation (MI:0006)

Selection of radioresistant tumor cells and presence of ALDH1 activity in vitro.

BACKGROUND Tumor resistance to radiotherapy has been hypothesized to be mediated by a tumor subpopulation, called cancer stem cells (CSCs). Based on the proposed function of CSCs in radioresistance, we explored the cancer stem cell properties of cells selected for radioresistance phenotype. MATERIALS AND METHODS A549 and SK-BR-3 cells were radioselected with four single doses of 4 or 3 Gy in intervals of 10-12 days and used for colony formation assay and γ-H2AX foci formation assay. Expression of putative stem cell markers, i.e. Sox2, Oct4, ALDH1, and CD133 were analyzed using Western blotting. A549 and SK-BR-3 cells sorted based on their ALDH1 activity were analyzed in clonogenic survival assays. RESULTS Radioselected A549 and SK-BR-3 cells (A549-R, SK-BR-3-R) showed increased radioresistance and A549-R cells presented enhanced repair of DNA-double strand breaks. PI3K inhibition significantly reduced radioresistance of A549-R cells. Cell line specific differences in the expression of the putative CSC markers Sox2 and Oct4 were observed when parental and radioselected cells were compared but could not be directly correlated to the radioresistant phenotype. However, enzyme activity of the putative stem cell marker ALDH1 showed a correlation to radioresistance. CONCLUSIONS Subpopulations of pooled radioresistant colonies, selected by various radiation exposures were analyzed for the presence of putative stem cell markers. Although the pattern of Sox2, Oct4, and CD133 expression was not generally associated with radioresistance, presence of ALDH1 seems to be indicative for subpopulations with increased radioresistance.

Nuclear epidermal growth factor receptor modulates cellular radio-sensitivity by regulation of chromatin access

PURPOSE Nuclear EGFR is involved in cellular stress management and regulation of cellular radio-sensitivity. The aim of this study was to elucidate the molecular mode of nuclear EGFR action. METHODS Radiation induced nuclear EGFR-shuttling and EGFR-foci formation was analyzed with immunohistochemistry and confocal microscopy. Composition of γH(2)AX-protein complexes was analyzed by western-blotting after immuno-precipitation. Functional relevance of nuclear EGFR was analyzed after siRNA mediated depletion of EGFR with respect to activation of ATM, histone H3 acetylation, residual DNA-damage and cell survival after irradiation. RESULTS Following radiation nuclear EGFR was localized in foci similar to γH(2)AX. EGFR co-localized in a sub-fraction of γH(2)AX-foci. Analysis of composition of γH(2)AX-complexes revealed presence of EGFR, ATM, promyelocytic leukemia protein (PML), histone H3 and hetero-chromatin binding protein (HP1) in response to radiation. Depletion of EGFR protein inhibited ATM activation due to inhibition of acetylase TIP60 activity following irradiation. Consequently, histone H3 acetylation and phosphorylation was blocked and chromatin could not be opened for repair. Thus, residual DNA-damage was increased 24 h after irradiation and cells were radio-sensitized. Comparable results were obtained when cells were treated with EGFR-NLS-peptide, which blocks EGFR nuclear shuttling specifically. CONCLUSIONS Nuclear EGFR is part of DNA-damage repair complex and is involved in regulation of TIP60-acetylase activity. TIP60 is essential for ATM activation and chromatin relaxation which is a prerequisite for DNA-repair in heterochromatic DNA. Thus interventional EGFR strategies during tumor treatment may also interact with DNA-repair by blocking access to damaged DNA.

Long‐term effects of repetitive exposure to a static magnetic field (1.5 T) on proliferation of human fetal lung fibroblasts

The aim of the study was to assess the effects of repetitive exposures to a static magnetic field (1.5 T) on human fetal lung fibroblast (HFL) proliferation. HFL were exposed three times a week for 1 hr to a static magnetic field for 3 weeks. Cells were subcultured every week. Population doublings (PD) and cumulative population doublings (CPD) were calculated weekly. Colony formation assays, bromodeoxyuridine enzyme‐linked immunosorbent assay, and cell cycle analysis were performed weekly. After the third week, proliferation kinetics were assessed. Over a period of 3 weeks no statistically significant differences between the PD and CPD of exposed and control cells could be detected. Clonogenic activity, DNA synthesis, cell cycle, and proliferation kinetics were not altered by magnetic field exposure. The data do not provide evidence that repetitive exposures to a static magnetic field (1.5 T) exert effects on HFL proliferation. Magn Reson Med 41:464–468, 1999.