Reduced exosomal L-Plastin is responsible for radiation-induced bystander effect.

Radiation-induced bystander effects (RIBE) are discussed as relevant processes during radiotherapy. Irradiated cells are suggested to release growth-inhibitory/DNA-damaging factors transported to non-irradiated cells. However, the molecular nature of this phenomenon has not yet been resolved. We aimed at identifying the growth-inhibitory factor(s) transmitted to non-irradiated cells. RIBE-competent PC3 cells were used to produce conditioned medium (CM) after exposure to ionizing radiation. Indicator cells were incubated with CM and clonogenic survival as well as cell proliferation were determined as endpoints. A549 indicator cells exhibited a bystander effect upon incubation with CM from irradiated PC3 cells. This bystander effect was not due to DNA-damaging factors, but a radiation-triggered reduction of mitogenic/clonogenic activity present in CM. Several tumor cells, but not normal fibroblasts secrete this factor, whose release is reduced by irradiation. We identified L-Plastin to be responsible for the mitogenic/clonogenic activity. Removal of L-Plastin from CM by immunoprecipitation or siRNA-mediated knockdown of L-Plastin expression resulted in loss or reduction of mitogenic/clonogenic activity transmitted via CM, respectively. Exosome-transported L-Plastin was constitutively Ser5-phosphorylated, indicative of its bioactive conformation. In summary, we observed production and exosomal secretion of L-Plastin by cancer cells. Via exosome-transmitted L-Plastin, tumors induce clonogenic and mitogenic activity in cancer and normal cells of the tumor microenvironment. Irradiation inhibits L-Plastin production targeting both cancer cells and the tumor niche and may explain the high impact of radiotherapy in tumor control.

Depletion of Akt1 and Akt2 Impairs the Repair of Radiation-Induced DNA Double Strand Breaks via Homologous Recombination.

Homologous recombination repair (HRR), non-homologous end-joining (NHEJ) and alternative NHEJ are major pathways that are utilized by cells for processing DNA double strand breaks (DNA-DSBs); their function plays an important role in the radiation resistance of tumor cells. Conflicting data exist regarding the role of Akt in homologous recombination (HR), i.e., the regulation of Rad51 as a major protein of this pathway. This study was designed to investigate the specific involvement of Akt isoforms in HRR. HCT116 colon cancer cells with stable AKT-knock-out and siRNA-mediated AKT-knockdown phenotypes were used to investigate the role of Akt1 and Akt2 isoforms in HR. The results clearly demonstrated that HCT116 AKT1-KO and AKT2-KO cells have a significantly reduced Rad51 foci formation 6 h post irradiation versus parental cells. Depletion of Akt1 and Akt2 protein levels as well as inhibition of Akt kinase activity resulted in an increased number of residual-γH2AX in CENP-F positive cells mainly representing the S and G2 phase cells. Furthermore, inhibition of NHEJ and HR using DNA-PK and Rad51 antagonists resulted in stronger radiosensitivity of AKT1 and AKT2 knockout cells versus wild type cells. These data collectively show that both Akt1 and Akt2 are involved in DSBs repair through HRR.

Exposure of primary osteoblasts to combined magnetic and electric fields induced spatiotemporal endochondral ossification characteristic gene- and protein expression profiles.

PURPOSE: Molecular processes in primary osteoblasts were analyzed in response to magnetic and electric field exposure to examine its potential impact on bone healing. METHODS: Primary osteoblasts were exposed to a combination of a magnetic field and an additional electric field (EFMF) (20 Hz, 700 mV, 5 mT, continuous sinusoids) in vitro. mRNA- and protein-expressions were assessed during a time interval of 21 days and compared with expression data obtained from control osteoblasts. RESULTS: We observed an autonomous osteoblast differentiation process in vitro under the chosen cultivation conditions. The initial proliferative phase was characterized by a constitutively high mRNA expression of extracellular matrix proteins. Concurrent EFMF exposure resulted in significanly increased cell proliferation (fold change: 1.25) and reduced mRNA-expressions of matrix components (0.5-0.75). The following reorganization of the extracellular matrix is prerequisite for matrix mineralization and is characterised by increased Ca2+ deposition (1.44). On molecular level EFMF exposure led to a significant decreased thrombospondin 1 (THBS1) mRNA- (0.81) and protein- (0.54) expression, which in turn reduced the TGFß1-dependent mRNA- (0.68) and protein- (0.5) expression of transforming growth factor beta induced (ßIG-H3) significantly, an inhibitor of endochondral ossification. Consequently, EFMF exposure stimulated the expression of genes characteristic for endochondral ossification, such as collagen type 10, A1 (1.50), osteopontin (1.50) and acellular communication network factor 3 (NOV) (1.45). CONCLUSIONS: In vitro exposure of osteoblasts to EFMF supports cell differentiation and induces gene- and protein-expression patterns characteristic for endochondral ossification during bone fracture healing in vivo.

Low-dose total body irradiation facilitates antitumoral Th1 immune responses.

CD4+ T helper cells are capable of mediating long-term antitumoral immune responses. We developed a combined immunotherapy (COMBO) using tumor antigen-specific T helper 1 cells (Tag-Th1), dual PD-L1/LAG-3 immune checkpoint blockade, and a low-dose total body irradiation (TBI) of 2 Gy, that was highly efficient in controlling the tumor burden of non-immunogenic RIP1-Tag2 mice with late-stage endogenous pancreatic islet carcinomas. In this study, we aimed to explore the impact of 2 Gy TBI on the treatment efficacy and the underlying mechanisms to boost CD4+ T cell-based immunotherapies. Methods: Heavily progressed RIP1-Tag2 mice underwent COMBO treatment and their survival was compared to a cohort without 2 Gy TBI. Positron emission tomography/computed tomography (PET/CT) with radiolabeled anti-CD3 monoclonal antibodies and flow cytometry were applied to investigate 2 Gy TBI-induced alterations in the biodistribution of endogenous T cells of healthy C3H mice. Migration and homing properties of Cy5-labeled adoptive Tag-Th1 cells were monitored by optical imaging and flow cytometric analyses in C3H and tumor-bearing RIP1-Tag2 mice. Splenectomy or sham-surgery of late-stage RIP1-Tag2 mice was performed before onset of COMBO treatment to elucidate the impact of the spleen on the therapy response. Results: First, we determined a significant longer survival of RIP1-Tag2 mice and an increased CD4+ T cell tumor infiltrate when 2 Gy TBI was applied in addition to Tag-Th1 cell PD-L1/LAG-3 treatment. In non-tumor-bearing C3H mice, TBI induced a moderate host lymphodepletion and a tumor antigen-independent accumulation of Tag-Th1 cells in lymphoid and non-lymphoid organs. In RIP1-Tag2, we found increased numbers of effector memory-like Tag-Th1 and endogenous CD4+ T cells in the pancreatic tumor tissue after TBI, accompanied by a tumor-specific Th1-driven immune response. Furthermore, the spleen negatively regulated T cell effector function by upregulation PD-1/LAG-3/TIM-3 immune checkpoints, providing a further rationale for this combined treatment approach. Conclusion: Low-dose TBI represents a powerful tool to foster CD4+ T cell-based cancer immunotherapies by favoring Th1-driven antitumoral immunity. As TBI is a clinically approved and well-established technique it might be an ideal addition for adoptive cell therapy with CD4+ T cells in the clinical setting.

Nuclear EGFR as novel therapeutic target: insights into nuclear translocation and function.

Emerging evidence suggests the existence of a new mode of epidermal growth factor receptor (EGFR) signaling in which activated EGFR undergoes nuclear translocation following treatment with ionizing radiation. The authors provide evidence that the nuclear EGFR transport is a stress-specific cellular reaction, which is linked to src-dependent EGFR internalization into caveolae. These flask-shaped pits can fuse with endoplasmic reticulum and the EGFR is sorted into a perinuclear localization. This compartment may serve as a reservoir for nuclear EGFR transport which is regulated by PKCepsilon (protein kinase Cepsilon). Nuclear EGFR is able to induce transcription of genes essential for cell proliferation and cell-cycle regulation. Moreover, nuclear EGFR has physical contact with compounds of the DNA repair machinery and is involved in removal of DNA damage. Anti-EGFR strategies target radiation-associated EGFR nuclear translocation in different manners. EGFR-inhibitory antibodies, i.e., cetuximab (Erbitux((R))), can block nuclear translocation by EGFR immobilization within the cytosol in responder cell lines, whereas tyrosine kinase inhibitors rather target nuclear kinase activity of EGFR linked with cytosolic or nuclear functions. However, both strategies can inhibit DNA repair following irradiation.

PI3K-Akt signaling regulates basal, but MAP-kinase signaling regulates radiation-induced XRCC1 expression in human tumor cells in vitro.

As demonstrated recently, ionizing radiation (IR) can mediate phosphorylation of DNA-PKcs in human tumor cells through stimulation of the PI3K/Akt pathway. It is also known that DNA-PKcs directly interacts the X-ray repair cross-complementing group 1 protein (XRCC1) involved in base excision repair (BER). Therefore, in the present study we investigated the role of PI3K/Akt activity and DNA-PKcs on XRCC1 expression/stabilization. In contrast to the DNA-PKcs-deficient glioblastoma cell line MO59J, the DNA-PKcs-proficient counterpart MO59K as well as human lung adenocarcinoma A549 cells presented a high basal level of XRCC1 expression. Radiation doses of 3-12Gy did not stimulate a further enhanced expression of XRCC1 in DNA-PKcs-proficient cells (MO59K and A549) within 180min post-irradiation. However, a marked induction of XRCC1 expression was apparent in DNA-PKcs-deficient MO59J cells. Targeting of DNA-PKcs as well as PI3K/Akt pathway by specific kinase inhibitors and/or siRNA reduced basal XRCC1 expression in un-irradiated DNA-PKcs-proficient cells to the level observed in DNA-PKcs-deficient cells. Reduction of basal expression of XRCC1 by XRCC1-siRNA, AKT-siRNA as well as DNA-PKcs inhibitor facilitated IR-induced XRCC1 expression. XRCC1 expression induced by irradiation, however, was independent of PI3K/Akt signaling, but dependent of MAPK-ERK1/2. By immuno-precipitation experiments and confocal microscopy a complex formation of XRCC1 and DNA-PKcs was shown. Applying gamma-H2AX foci analysis it was shown that basal expression of XRCC1 is important for the repair of IR-induced DNA-double strand breaks (DNA-DSBs). These data indicate that IR-induced XRCC1 expression is dependent on the expression level of DNA-PKcs and basal activity status of PI3K/Akt signaling. Likewise, potential of IR-induced XRCC1 expression depends on its basal expression level.

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

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. RESULTS: Ionizing 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. CONCLUSION: Src 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.

Celecoxib induced tumor cell radiosensitization by inhibiting radiation induced nuclear EGFR transport and DNA-repair: a COX-2 independent mechanism.

PURPOSE: The purpose of the study was to elucidate the molecular mechanisms mediating radiosensitization of human tumor cells by the selective cyclooxygenase (COX)-2 inhibitor celecoxib. METHODS AND MATERIALS: Experiments were performed using bronchial carcinoma cells A549, transformed fibroblasts HH4dd, the FaDu head-and-neck tumor cells, the colon carcinoma cells HCT116, and normal fibroblasts HSF7. Effects of celecoxib treatment were assessed by clonogenic cell survival, Western analysis, and quantification of residual DNA damage by gammaH(2)AX foci assay. RESULTS: Celecoxib treatment resulted in a pronounced radiosensitization of A549, HCT116, and HSF7 cells, whereas FaDu and HH4dd cells were not radiosensitized. The observed radiosensitization could neither be correlated with basal COX-2 expression pattern nor with basal production of prostaglandin E2, but was depended on the ability of celecoxib to inhibit basal and radiation-induced nuclear transport of epidermal growth factor receptor (EGFR). The nuclear EGFR transport was strongly inhibited in A549-, HSF7-, and COX-2-deficient HCT116 cells, which were radiosensitized, but not in FaDu and HH4dd cells, which resisted celecoxib-induced radiosensitization. Celecoxib inhibited radiation-induced DNA-PK activation in A549, HSF7, and HCT116 cells, but not in FaDu and HH4dd cells. Consequentially, celecoxib increased residual gammaH2AX foci after irradiation, demonstrating that inhibition of DNA repair has occurred in responsive A549, HCT116, and HSF7 cells only. CONCLUSIONS: Celecoxib enhanced radiosensitivity by inhibition of EGFR-mediated mechanisms of radioresistance, a signaling that was independent of COX-2 activity. This novel observation may have therapeutic implications such that COX-2 inhibitors may improve therapeutic efficacy of radiation even in patients whose tumor radioresistance is not dependent on COX-2.

The radioprotector Bowman-Birk proteinase inhibitor stimulates DNA repair via epidermal growth factor receptor phosphorylation 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 gammaH(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.

Activation of protein kinase Cepsilon stimulates DNA-repair via epidermal growth factor receptor nuclear accumulation.

PURPOSE: To elucidate the interaction between radioprotector O-phospho-l-tyrosine (P-Tyr) with epidermal growth factor receptor (EGFR). METHODS: Molecular effects of P-Tyr at the level of EGFR responses were investigated in vitro with TP53-wildtype bronchial carcinoma cell line A549, which is radio-protected by P-Tyr treatment. Nuclear EGFR accumulation was followed by confocal microscopy and Western blotting. PKCepsilon protein expression was impaired by specific siRNA. Residual DNA-damage was quantified with gammaH(2)AX foci analysis. RESULTS: P-Tyr mediated radio-protection was associated with nuclear EGFR accumulation. Radiation-induced nuclear EGFR presented increased phosphorylation at residue No. T654. We identified PKCepsilon as responsible for T654-phosphorylation. Knockdown of PKCepsilon by siRNA blocked both radiation- and P-Tyr-triggered nuclear EGFR accumulation. Furthermore, nuclear accumulation of EGFR was associated with increased phosphorylation of DNA-dependent protein kinase (DNA-PK) at residue No. T2609, essential for DNA-repair. Consequently P-Tyr mediated effects upon DNA-PK resulted in a significant reduction of radiation-induced residual gammaH(2)AX-foci. Knockdown of PKCepsilon increased radiation-induced residual damage and abolished the P-Tyr associated radioprotection. In addition, P-Tyr mediated radioprotection was completely absent in colony formation assay. CONCLUSION: The data presented herein suggest that P-Tyr-treatment mediates activation of PKCepsilon, which triggers nuclear EGFR accumulation. Nuclear EGFR is involved in phosphorylation of DNA-PK at Thr2609, which has a significant impact upon DNA-DSB repair.

Glucose starvation impairs DNA repair in tumour cells selectively by blocking histone acetylation.

BACKGROUND AND PURPOSE: Tumour cells are characterized by aerobic glycolysis and thus have high glucose consumption. Because repairing radiation-induced DNA damage is an energy-demanding process, we hypothesized that glucose starvation combined with radiotherapy could be an effective strategy to selectively target tumour cells. MATERIAL AND METHODS: We glucose-starved tumour cells (A549, FaDu) in vitro and analysed their radiation-induced cell responses compared to normal fibroblasts (HSF7). RESULTS: Irradiation depleted intracellular ATP levels preferentially in cancer cells. Consequently, glucose starvation impaired DNA double-strand break (DSB) repair and radiosensitized confluent tumour cells but not normal fibroblasts. In proliferating tumour cells glucose starvation resulted in a reduction of proliferation, but failed to radiosensitize cells. Glucose supply was indispensable during the late DSB repair in confluent tumour cells starting approximately 13 h after irradiation, and glucose starvation inhibited radiation-induced histone acetylation, which is essential for chromatin relaxation. Sirtinol - an inhibitor of histone deacetylases - reverted the effects of glucose depletion on histone acetylation and DNA DSB repair in tumour cells. Furthermore, a glucose concentration of 2.8 mmol/L was sufficient to impair DSB repair in tumour cells and reduced their clonogenic survival under a fractionated irradiation regimen. CONCLUSIONS: In resting tumour cells, glucose starvation combined with irradiation resulted in the impairment of late DSB repair and the reduction of clonogenic survival, which was associated with disrupted radiation-induced histone acetylation. However, in normal cells, DNA repair and radiosensitivity were not affected by glucose depletion.

The radioprotector O-phospho-tyrosine stimulates DNA-repair via epidermal growth factor receptor- and DNA-dependent kinase phosphorylation.

BACKGROUND AND PURPOSE: Purpose of the study was to elucidate the underlying molecular mechanism of the radioprotector O-phospho-tyrosine (P-Tyr). METHODS: Molecular effects of P-Tyr at the level of EGFR responses were investigated in vitro with bronchial carcinoma cell line A549. Nuclear EGFR transport and DNA-PK activation were quantified after Western blotting. Residual DNA-damages were quantified by help of gammaH(2)AX focus assay. RESULTS: As determined by dose-response curves, treatment of cells with P-Tyr for 16h before irradiation results in radioprotection. Simultaneous treatment with EGFR blocking antibody Cetuximab abolished P-Tyr associated radioprotection. At the molecular level P-Tyr mediated a general phosphorylation of EGFR and a pronounced phosphorylation of nuclear EGFR at residue Thr No. 654, also observed after treatment with ionizing radiation. This phosphorylation was associated with nuclear EGFR accumulation. Moreover, P-Tyr-triggered EGFR nuclear accumulation was associated with phosphorylation of DNA-PK at Thr 2609. This activated form of DNA-PK was not DNA associated, but after radiation, DNA binding increased, particularly after P-Tyr pre-treatment. These molecular effects of P-Tyr resulted in a reduction of residual DNA-damage after irradiation. CONCLUSIONS: Radioprotection by P-Tyr is mediated through its stimulation of nuclear EGFR transport and concurrent, but DNA-damage independent, activation of DNA-PK. Thus, subsequent irradiation results in increased binding of DNA-PK to DNA, improved DNA-repair and increased cell survival.

Inhibition of cyclooxygenase-2 activity by celecoxib does not lead to radiosensitization of human prostate cancer cells in vitro.

PURPOSE: To evaluate the potential radiosensitizing effect of the specific COX-2 inhibitor celecoxib (Celebrex) on prostate carcinoma cells in vitro. MATERIALS AND METHODS: The influence of celecoxib (concentration range 5 to 75 microM) on radiation-induced cellular and clonogenic survival was investigated in prostate carcinoma cell lines PC-3, DU145, LNCaP and normal prostate epithelial cells (PrEC). Western blot analysis and ELISA were used to determine the impact of radiation alone or radiation combined with celecoxib treatment on COX-2 expression and prostaglandin E2 synthesis. To evaluate induction of celecoxib-induced apoptosis cell cycle analysis has been performed. RESULTS: Celecoxib (5, 10 and 25 microM) in combination with single-dose irradiation of 2 Gy induced a significant radiosensitization in normal prostate epithelial cells which could not be observed for any of the prostate carcinoma cell lines investigated. Increased COX-2 protein expression in PC-3 cells was obvious only after IR with 15 Gy, while PGE2 production was elevated following irradiation (2-15 Gy) in a dose-dependent manner. Treatment with celecoxib alone or in combination with IR led to a dose-dependent increase in COX-2 protein expression. Nevertheless pre-treatment with celecoxib caused a marked reduction of radiation-induced enzyme activity as tested at the level of PGE2 production, both in PC-3 and DU145 cells. Following fractionated irradiation with single doses of 2 Gy, elevated COX-2 protein expression as well as enhanced PGE2 production was observed already after the second fraction in PC-3 cells. Pre-treatment with celecoxib reduced the amount of PGE(2) significantly, but not of COX-2 protein. CONCLUSIONS: Our data obtained for the human prostate cancer cell lines do not indicate that a marked inhibition of prostaglandin E2 synthesis by celecoxib leads to enhanced radiosensitization. Thus, in terms of radiosensitization the analysed prostate cancer cells can be classified as non-responders to celecoxib treatment.

EGFR-targeted anti-cancer drugs in radiotherapy: preclinical evaluation of mechanisms.

Preclinical and clinical results indicate that the EGFR can mediate radioresistance in different solid human tumours. Combination of radiotherapy and EGFR inhibitors can improve local tumour control compared to irradiation alone and has been introduced into clinical radiotherapy practice. So far several mechanisms have been identified in preclinical studies to contribute to improved local tumour control after radiation combined with EGFR inhibitors. These include direct kill of cancer stem cells by EGFR inhibitors, cellular radiosensitization through modified signal transduction, inhibition of repair of DNA damage, reduced repopulation and improved reoxygenation during fractionated radiotherapy. Effects and mechanisms may differ for different classes of EGFR inhibitors, for different tumours and for normal tissues. The mechanisms underlying this heterogeneity are currently poorly understood, and predictive assays are not available yet. Importantly, mechanisms and predictors for the combined effects of radiation with EGFR inhibitors appear to be considerably different to those for application of EGFR inhibitors alone or in combination with chemotherapy. Therefore to further evaluate the efficacy and mechanisms of EGFR-inhibition in combined treatments, radiotherapy-specific preclinical research strategies, which include in vivo experiments using local tumour control as an endpoint, as well as animal studies on normal tissue toxicity are needed.

Radiation-induced EGFR-signaling and control of DNA-damage repair.

PURPOSE: Over the last decade evidence has accumulated indicating that cell membrane-bound growth factor receptor of the erbB family and especially the epidermal growth factor receptor EGFR (erbB1) mediates resistance of tumor cells to both chemo- and radiotherapy when mutated or overexpressed. More recently a novel link between EGFR signaling pathways and DNA repair mechanisms, especially non-homologous end joining (NHEJ) repair could be demonstrated. The following review summarizes the current knowledge on the role of EGFR and its downstream signaling pathways in the regulation of cellular radiation response and DNA repair. CONCLUSION: 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.

Blockage of epidermal growth factor receptor-phosphatidylinositol 3-kinase-AKT signaling increases radiosensitivity of K-RAS mutated human tumor cells in vitro by affecting DNA repair.

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 double-strand 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 (Ser139), ATM (Ser1981), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs; Thr2609) was analyzed by immunoblotting. Twenty-four hours after irradiation, residual DSBs were quantified by identification of gammaH2AX 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 Thr2609 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.

New roles for nuclear EGFR in regulating the stability and translation of mRNAs associated with VEGF signaling.

Cell membrane-associated epidermal growth factor receptor (EGFR) translocates into a perinuclear/nuclear location upon stimulation, where it complexes with mRNAs. Treatment with radiation and cisplatin decreases the amounts of mRNAs present within this complex. Gene array analyses of mRNAs in complex with immunoprecipitated nEGFR revealed significant enrichment of different mRNA species compared to the control immunoprecipitation. Functional annotation with help of DAVID Gene Ontology Analysis identified under other terms the HIF-1A/VEGF signaling pathway as one of the top scoring KEGG pathways. RT-PCR and western blots revealed the radiation-induced expression of mRNAs and proteins involved in HIF-1A/VEGF signaling. Simultaneously, the levels of the corresponding validated miRNAs within the complex containing nEGFR and mRNAs were decreased. This finding argues that an mRNA/miRNA/nEGFR complex regulates protein expression. Indeed, we detected the GW182, AGO2, PABPC1 and cNOT1 proteins, which belong to the deadenylase complex, in a complex with nuclear EGFR. Erlotinib-mediated inhibition of EGFR kinase reduced the radiation-induced increase in mRNA expression. In this context, erlotinib reduced AGO2 phosphorylation by the EGFR kinase at residue Y393, which was associated with increased cNOT1 deadenylase activity and reduced mRNA stability. To prove the roles of miRNAs in this context, we transfected cells with an inhibitor of Hsa-mir-1180p5, which targets the NFATC4 mRNA, an mRNA associated with VEGF signaling, or pretreated cells with erlotinib. Indeed, Hsa-mir-1180p5 knockdown increased and the erlotinib treatment decreased the expression of the NFATC4 protein. The expression of the NFATC4 protein controlled the cloning efficiency and radiosensitivity of A549 and FaDu tumor cells. Thus, this study is the first to show that a membrane-located tyrosine kinase receptor, such as EGFR, is internalized to a nuclear/perinuclear location upon exposure to stress and modulates the stability and translation of miRNA-selected mRNAs. This mechanism enables cells to directly express proteins in response to EGFR activation and may contribute to treatment resistance in EGFR-overexpressing tumors.

The radioprotector ortho-phospho-L-tyrosine (pTyr) attenuates the side effects of fractionated irradiation in retinoblastoma mouse models but also decreases the local tumour control.

BACKGROUND: Radiotherapy (RT) is used to treat retinoblastoma (Rb), the most frequent ocular tumour in children. Besides eradicating the tumour, RT can cause severe side effects including secondary malignancies. This study aimed to define whether the radioprotector ortho-phospho-L-tyrosine (pTyr) prevents RT-induced side effects and affects local tumour control in a xenograft and a genetic orthotopic Rb mouse model. METHODS: B6;129-Rb1tm3Tyj/J (Rb+/-) and Y79-Rb cell-xenografted nude mice were fractionated external beam irradiated (15 fractions of 5Gy 6MV photons during 3weeks) with or without pTyr pre-treatment (100mg/kg BW, 16h prior to each irradiation). One, three, six and nine months after RT, tumour control and RT toxicity were evaluated using in vivo imaging and histology. We also analysed pTyr dependant post irradiation cell survival and p53 activity in vitro. RESULTS: In vitro pTyr pre-treatment showed no radioprotection on Y79 cells, but led to p53 stabilisation in unirradiated Y79 cells and to a facilitation of radiation-induced p21 up-regulation, confirming a modulation of p53 activity by pTyr. In both mouse models, secondary tumours were undetectable. In Rb+/- mice, pTyr significantly lowered RT-induced greying of the fur, retinal thickness reduction and photoreceptor loss. However, in the xenografted Rb model, pTyr considerably decreased RT-mediated tumour control, which was observed in 16 out of 22 control eyes but in none of the 24 pTyr treated eyes. CONCLUSIONS: In Rb+/- mice pTyr significantly prevents RT-induced greying of the fur as well as retinal degeneration. However, since non-irradiated control mice were not used in our study, a formal possibility exists that the effect shown in the retina of Rb+/- mice may be due to ageing of the animals and/or actions of pTyr alone. Unfortunately, as tested in a xenograft model, pTyr treatment reduced the control of Rb tumours.

Inhibition of DNA repair as a mechanism of enhanced radioresponse of head and neck carcinoma cells by a selective cyclooxygenase-2 inhibitor, celecoxib.

PURPOSE: Previously, we reported that inhibitors of cyclooxygenase-2 (COX-2) enzyme enhanced murine and human tumor cell response to radiation in vitro and in vivo. However, the molecular mechanisms mediating the effects of COX-2 inhibitors are not clear. The present study was designed to investigate the ability of celecoxib, a selective COX-2 inhibitor, to sensitize human head-and-neck cancer cell line, HN5, to radiation, and examine its effects on DNA repair, which may be a potential mechanism of radiosensitization. METHODS AND MATERIALS: Cells were assessed for the effect of celecoxib (5-50 microM), by 3-[4,5-dimethylthiozol-2-yl]-2,5-diphenyltetrazolium bromide assay for growth inhibition and by clonogenic cell survival assay for the radiosensitizing effect. Kinase assay and Western analysis were conducted to assess the effect of celecoxib on DNA-dependent protein kinase catalytic subunit (PKcs) and Ku proteins. Electrophoretic mobility shift assays (EMSA) were performed to determine the DNA-binding activity of Ku/DNA-PKcs protein complex and nuclear factor kappa B (NFkappaB). RESULTS: Celecoxib (10 and 50 microM, for 2 days) inhibited the HN5 cell growth and significantly enhanced the cell radiosensitivity in a dose-dependent manner. It also reduced the shoulder region on the radiation-survival curve, suggesting that inhibition of DNA repair processes may have occurred. Western blot analysis demonstrated that celecoxib downregulated the expression of Ku70 protein and inhibited the kinase activity of DNA-PKcs, which are involved in the double-stranded DNA-break repair machinery. By EMSA, it was further shown that celecoxib reduced DNA-binding activity of Ku/DNA-PKcs protein complex. In addition, celecoxib inhibited the constitutively active NFkappaB and the radiation-induced NFkappaB in HN5 cells, suggesting that NFkappaB may play a role in mediating the effects of celecoxib. CONCLUSIONS: Celecoxib strongly enhanced the sensitivity of HN5 carcinoma cells to radiation, which, mechanistically, can be attributed to the inhibition of DNA repair processes in radiation-damaged cells.

Inhibition of radiation-induced EGFR nuclear import by C225 (Cetuximab) suppresses DNA-PK activity.

BACKGROUND AND PURPOSE: Inhibition of EGFR-function can induce radiosensitization in tumor cells. Purpose of our investigation was to identify the possible molecular mechanism of radiosensitization following treatment with anti-EGFR-antibody C225 (Cetuximab). MATERIALS AND METHODS: The effect of C225 on radiation response was determined in human cell lines of bronchial carcinoma (A549) and breast adenoma cells (MDA MB 231). The molecular effects of C225 on EGFR-function after irradiation were analyzed applying western blotting, immune-precipitation and kinase assays. Effects on DNA-repair were detected by quantification of gamma-H2AX positive foci 24h after irradiation. RESULTS: The EGFR specific antibody C225 induced radiosensitization in A549 and also in MDA MB 231 cells. Radiosensitization in A549 was associated with blockage of radiation-induced EGFR transport into the nucleus, and immobilized the complex of EGFR with DNA-dependent protein kinase (DNA-PK) in the cytoplasm. As a consequence radiation-induced DNA-PK activation was abolished, a process that is essential for DNA-repair after radiation exposure. Likewise C225 treatment increased the residual amount of gamma-H2AX-positive foci 24h after irradiation in A549 and in MDA MB 231 cells. CONCLUSIONS: Our results suggest that irradiation induced DNA-PK activation-essential for DNA repair-may be hampered specifically by use of the anti-EGFR-antibody C225. This process is associated with radiosensitization.