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.

Nanog Signaling Mediates Radioresistance in ALDH-Positive Breast Cancer Cells.

Recently, cancer stem cells (CSCs) have been identified as the major cause of both chemotherapy and radiotherapy resistance. Evidence from experimental studies applying both in vitro and in vivo preclinical models suggests that CSCs survive after conventional therapy protocols. Several mechanisms are proposed to be involved in CSC resistance to radiotherapy. Among them, stimulated DNA double-strand break (DSB) repair capacity in association with aldehyde dehydrogenase (ALDH) activity seems to be the most prominent mechanism. However, thus far, the pathway through which ALDH activity stimulates DSB repair is not known. Therefore, in the present study, we investigated the underlying signaling pathway by which ALDH activity stimulates DSB repair and can lead to radioresistance of breast cancer cell lines in vitro. When compared with ALDH-negative cells, ALDH-positive cells presented significantly enhanced cell survival after radiation exposure. This enhanced cell survival was associated with stimulated Nanog, BMI1 and Notch1 protein expression, as well as stimulated Akt activity. By applying overexpression and knockdown approaches, we clearly demonstrated that Nanog expression is associated with enhanced ALDH activity and cellular radioresistance, as well as stimulated DSB repair. Akt and Notch1 targeting abrogated the Nanog-mediated radioresistance and stimulated ALDH activity. Overall, we demonstrate that Nanog signaling induces tumor cell radioresistance and stimulates ALDH activity, most likely through activation of the Notch1 and Akt pathways.

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.

Stress-Induced Phosphorylation of Nuclear YB-1 Depends on Nuclear Trafficking of p90 Ribosomal S6 Kinase.

Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1) phosphorylation at Ser-102 in KRAS wild-type (KRASwt) cells, whereas in KRAS mutated (KRASmut) cells, YB-1 is constitutively phosphorylated, independent of IR or EGF. YB-1 activity stimulates the repair of IR-induced DNA double-strand breaks (DSBs) in the nucleus. Thus far, the YB-1 nuclear translocation pattern after cell exposure to various cellular stressors is not clear. In the present study, we investigated the pattern of YB-1 phosphorylation and its possible translocation to the nucleus in KRASwt cells after exposure to IR, EGF treatment, and conditional expression of mutated KRAS(G12V). IR, EGF, and conditional KRAS(G12V) expression induced YB-1 phosphorylation in both the cytoplasmic and nuclear fractions of KRASwt cells. None of the stimuli induced YB-1 nuclear translocation, while p90 ribosomal s6 kinase (RSK) translocation was enhanced in KRASwt cells after any of the stimuli. EGF-induced RSK translocation to the nucleus and nuclear YB-1 phosphorylation were completely blocked by the EGF receptor kinase inhibitor erlotinib. Likewise, RSK inhibition blocked RSK nuclear translocation and nuclear YB-1 phosphorylation after irradiation and KRAS(G12V) overexpression. In summary, acute stimulation of YB-1 phosphorylation does not lead to YB-1 translocation from the cytoplasm to the nucleus. Rather, irradiation, EGF treatment, or KRAS(G12V) overexpression induces RSK activation, leading to its translocation to the nucleus, where it activates already-existing nuclear YB-1. Our novel finding illuminates the signaling pathways involved in nuclear YB-1 phosphorylation and provides a rationale for designing appropriate targeting strategies to block YB-1 in oncology as well as in radiation oncology.

Akt1 Stimulates Homologous Recombination Repair of DNA Double-Strand Breaks in a Rad51-Dependent Manner.

Akt1 is known to promote non-homologous end-joining (NHEJ)-mediated DNA double-strand break (DSB) repair by stimulation of DNA-PKcs. In the present study, we investigated the effect of Akt1 on homologous recombination (HR)-dependent repair of radiation-induced DSBs in non-small cell lung cancer (NSCLC) cells A549 and H460. Akt1-knockdown (Akt1-KD) significantly reduced Rad51 protein level, Rad51 foci formation and its colocalization with γH2AX foci after irradiation. Moreover, Akt1-KD decreased clonogenicity after treatment with Mitomycin C and HR repair, as tested by an HR-reporter assay. Double knockdown of Akt1 and Rad51 did not lead to a further decrease in HR compared to the single knockdown of Rad51. Consequently, Akt1-KD significantly increased the number of residual DSBs after irradiation partially independent of the kinase activity of DNA-PKcs. Likewise, the number of residual BRCA1 foci, indicating unsuccessful HR events, also significantly increased in the irradiated cells after Akt1-KD. Together, the results of the study indicate that Akt1 seems to be a regulatory component in the HR repair of DSBs in a Rad51-dependent manner. Thus, based on this novel role of Akt1 in HR and the previously described role of Akt1 in NHEJ, we propose that targeting Akt1 could be an effective approach to selectively improve the killing of tumor cells by DSB-inducing cytotoxic agents, such as ionizing radiation.

Phosphatidylinositol 3-kinase/Akt signaling as a key mediator of tumor cell responsiveness to radiation.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is a key cascade downstream of several protein kinases, especially membrane-bound receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) family members. Hyperactivation of the PI3K/Akt pathway is correlated with tumor development, progression, poor prognosis, and resistance to cancer therapies, such as radiotherapy, in human solid tumors. Akt/PKB (Protein Kinase B) members are the major kinases that act downstream of PI3K, and these are involved in a variety of cellular functions, including growth, proliferation, glucose metabolism, invasion, metastasis, angiogenesis, and survival. Accumulating evidence indicates that activated Akt is one of the major predictive markers for solid tumor responsiveness to chemo/radiotherapy. DNA double-strand breaks (DNA-DSB), are the prime cause of cell death induced by ionizing radiation. Preclinical in vitro and in vivo studies have shown that constitutive activation of Akt and stress-induced activation of the PI3K/Akt pathway accelerate the repair of DNA-DSB and, consequently, lead to therapy resistance. Analyzing dysregulations of Akt, such as point mutations, gene amplification or overexpression, which results in the constitutive activation of Akt, might be of special importance in the context of radiotherapy outcomes. Such studies, as well as studies of the mechanism(s) by which activated Akt1 regulates repair of DNA-DSB, might help to identify combinations using the appropriate molecular targeting strategies with conventional radiotherapy to overcome radioresistance in solid tumors. In this review, we discuss the dysregulation of the components of upstream regulators of Akt as well as specific modifications of Akt isoforms that enhance Akt activity. Likewise, the mechanisms by which Akt interferes with repair of DNA after exposure to ionizing radiation, will be reviewed. Finally, the current status of Akt targeting in combination with radiotherapy will be discussed.

Potential roles of the NFκB and glutathione pathways in mature human erythrocytes.

Anucleated erythrocytes were long considered as oxygen-transporting cells with limited regulatory functions. Components of different nuclear signaling pathways have not been investigated in those cells, yet. Surprisingly, we repeatedly found significant amounts of transcription factors in purified erythrocyte preparations, i.e. nuclear factor κB (NFκB), and major components of the canonical NFκB signaling pathway. To investigate the functional role of NFκB signaling, the effects of the preclinical compounds Bay 11-7082 and parthenolide on the survival of highly purified erythrocytes were investigated. Interestingly, both inhibitors of the NFκB pathway triggered erythrocyte programmed cell death as demonstrated by enhanced phospholipid scrambling (phosphatidylserine exposure) and cell shrinkage. Anucleated erythrocytes are an ideal cellular model allowing the study of nongenomic mechanisms contributing to suicidal cell death. As NFκB inhibitors might also interfere with the anti-oxidative defense systems of the cell, we measured the levels of reduced glutathione (GSH) after challenge with the inhibitors. Indeed, incubation of erythrocytes with Bay 11-7082 clearly decreased erythrocyte GSH levels. In conclusion, the pharmacological inhibitors of the NFκB pathway Bay 11-7082 and parthenolide interfere with the survival of erythrocytes involving mechanisms other than disruption of NFκB-dependent gene expression. Besides affecting erythrocyte survival, NFκB inhibition and induction of erythrocyte phosphatidylserine exposure may influence blood clotting. Future studies will be aimed at discriminating between NFκB-dependent and NFκB-independent GSH-mediated effects of Bay 11-7082 and parthenolide on erythrocyte death.

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.

Impact of oncogenic K-RAS on YB-1 phosphorylation induced by ionizing radiation.

INTRODUCTION: Expression 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. METHODS: IR-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. RESULTS: The 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. CONCLUSIONS: IR 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.

The NFĸB pathway inhibitors Bay 11-7082 and parthenolide induce programmed cell death in anucleated Erythrocytes.

The preclinical compounds Bay 11-7082 and parthenolide trigger apoptosis, an effect contributing to their antiinflammatory action. The substances interfere with the activation and nuclear translocation of nuclear factor NFκB, by inhibiting NFκB directly (parthenolide) or by interfering with the inactivation of the NFκB inhibitory protein IκB-α (Bay 11-7082). Beyond that, the substances may be effective in part by nongenomic effects. Similar to apoptosis of nucleated cells, erythrocytes may undergo apoptosis-like cell death (eryptosis) characterized by cell membrane scrambling with phosphatidylserine exposure, and cell shrinkage. Thus, erythrocytes allow the study of nongenomic mechanisms contributing to suicidal cell death, e.g. Ca(2+) leakage or glutathione depletion. The present study utilized Western blotting to search for NFκB and IκB-α expression in erythrocytes, FACS analysis to determine cytosolic Ca(2+) (Fluo3 fluorescence), phosphatidylserine exposure (annexin V binding), and cell volume (forward scatter), as well as an enzymatic method to determine glutathione levels. As a result, both NFκB and IκB-α are expressed in erythrocytes. Targeting the NFκB pathway by Bay 11-7082 (IC(50) ≈ 10 µM) and parthenolide (IC(50) ≈ 30 µM) triggered suicidal erythrocyte death as shown by annexin V binding and decrease of forward scatter. Bay 11-7082 treatment further increased intracellular Ca(2+) and led to depletion of reduced glutathione. The effects of Bay 11-7082 and parthenolide on annexin V binding could be fully reversed by the antioxidant N-acetylcysteine. In conclusion, the pharmacological inhibitors of NFκB, Bay 11-7082 and parthenolide, interfere with the survival of erythrocytes involving mechanisms other than disruption of NFκB-dependent gene expression.

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.

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-59 CJ-OH (API; 1-5 micromol/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 gamma-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.

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.

Stimulated PI3K-AKT signaling mediated through ligand or radiation-induced EGFR depends indirectly, but not directly, on constitutive K-Ras activity.

Previous results showed an inducible radiation sensitivity selectively observable for K-RAS-mutated cell lines as a function of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor blockade of phosphatidylinositol 3-kinase (PI3K)-AKT signaling. Therefore, the role of K-Ras activity for a direct (i.e., through activation of PI3K by K-Ras) or an indirect stimulation of PI3K-AKT signaling (through K-Ras activity-dependent EGFR ligand production) was investigated by means of small interfering RNA and inhibitor approaches as well as ELISA measurements of EGFR ligand production. K-RASmt tumor cells presented a constitutively activated extracellular signal-regulated kinase-1/2 signaling, resulting in enhanced production and secretion of the EGFR ligand amphiregulin (AREG). Medium supernatants conditioned by K-RASmt tumor cells equally efficiently stimulated EGFR signaling into the PI3K-AKT and mitogen-activated protein kinase pathways. Knocking down K-Ras expression by specific small interfering RNA markedly affected autocrine production of AREG, but not PI3K-AKT signaling, after treatment of K-RAS-mutated or wild-type cells with EGFR ligands or exposure to ionizing radiation. These results indicate that PI3K-mediated activation of AKT in K-RASmt human tumor cells as a function of EGFR ligand or radiation stimulus is independent of a direct function of K-Ras enzyme activity but depends on a K-Ras-mediated enhanced production of EGFR ligands (i.e., most likely AREG) through up-regulated extracellular signal-regulated kinase-1/2 signaling. The data provide new differential insight into the importance of K-RAS mutation in the context of PI3K-AKT-mediated radioresistance of EGFR-overexpressing or EGFR-mutated tumors.

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.

Diversity of fibroblasts--a review on implications for skin tissue engineering.

Enormous advances in the development of skin substitutes have occurred in the past 3 decades. Major obstacles yet to be overcome in the quest for an optimal skin substitute include controlling scar formation, contraction and the loss of adnexal structures. Mesenchyme-derived signals are essential for epithelial proliferation, skin morphogenesis, homeostasis and differentiation. Having previously shown that fibroblasts differentiate along a lineage from highly proliferative progenitor fibroblasts with characteristic spindle-shaped appearance to differentiated postmitotic polygonal fibrocytes, we have now established that the different subsets of fibroblasts exert significantly different patterns of cytokine release and that the highest levels of keratinocyte growth factor and transforming growth factor-beta1 expression result from differentiated fibroblasts. Coculture studies with keratinocytes reveal that postmitotic fibroblasts stimulate keratinocyte proliferation to a greater extent than progenitor fibroblasts. Acellular and fibroblast-seeded dermal substitutes have been shown to improve scarring and contraction in animal studies, the latter substitutes yielding the most favorable results. Fibroblasts from different body sites display different functional properties which may affect their suitability for dermal substitutes. Future in vivo human studies in tissue-engineered dermal substitutes will likely focus on fibroblast-seeded lattices and the impact of fibroblast subpopulations and bone marrow-derived mesenchymal stem cells on dermal regeneration.

Responses of normal cells to ionizing radiation.

Radiation-induced alterations in cellular tissue homeostasis triggered by various molecular responses at the level of inter- and intracellular signaling processes cause both acute and late effects in normal tissue after radiation therapy. Some of the underlying molecular and cellular response pathways leading to radiation-induced tissue remodeling will be discussed, with special emphasis on vascular and parenchymal tissues.