Ionizing radiation induces up-regulation of functional beta1-integrin in human lung tumour cell lines in vitro

PURPOSE: Cell-matrix interactions are in part mediated through the beta1-integrin pathway regulating cell survival, proliferation, adhesion and migration. This study was performed to elucidate alterations of expression of the beta1-integrin and its co-localized protein kinase, integrin-linked kinase (ILK), after exposure to ionizing radiation in two lung carcinoma cell lines in the presence or absence of different beta1-integrin-dependent matrix proteins. MATERIALS AND METHODS: Exponentially growing A549 and SKMES1 cells grown on fibronectin, laminin, BSA or plastic were exposed to 2 Gy or 6 Gy. Besides colony formation assays (0.5-8 Gy) and immediate plating experiments, flow cytometry (for beta1-integrin) and immunoblotting (for beta1-integrin and ILK) were carried out to analyze the protein expression. The localization of both proteins plus filamentous (f-) actin was further examined by immunofluorescence staining and laser confocal scanning microscopy. Functionality of the beta1 receptor subunit after irradiation was investigated in adhesion assays. RESULTS: A549 and SKMES1 cells grown on fibronectin or laminin demonstrated a significant increase in cell survival after irradiation compared to cells grown on BSA or plastic. Immediate plating of cells after irradiation on fibronectin did not show an improved survival. Flow cytometric and Western blot data showed a dose- and matrix-dependent induction of beta1-integrin and ILK expression after irradiation within 48 h. Adhesion to fibronectin or laminin compared to BSA or plastic was increased by 10-fold after irradiation, demonstrating these specific cell surface receptors to be functional. The staining of beta1-integrin and ILK in A549 cells confirmed the radiation-induced up-regulation of both proteins. Additionally, beta1-integrin and ILK co-localized with accumulated actin fibers at the cytoplasmic face of the cell membrane at confined areas. CONCLUSIONS: Ionizing radiation strongly induced the expression of functional beta1-integrin and ILK in the two lung cancer cell lines, A549 and SKMES1, dependent on different matrices used. Additionally, the subcellular localization of both proteins was altered by irradiation, and the individual cellular radiosensitivity was reduced in the presence of an extracellular matrix. On the one hand, this may result in aggravated therapeutic approaches and on the other hand, cells could adhere more strongly in their environment by the increase in functional surface receptor density preventing metastasis. Concerning intravascular located tumour cells, beta1-integrin up-regulation might enable these cells to adhere to the endothelium, which represents a prerequisite for metastatic disease. Identification of such mechanisms will provide considerable insights into the understanding of tumorigenicity and metastatic phenotypes, possibly leading to new, optimized radiochemotherapeutic regimens.     

Integrins: signaling, disease, and therapy

BACKGROUND: Integrins are a family of transmembrane receptors that mediate cell-cell and cell-matrix adhesion. They are involved in stable cell adhesion and migration of cells. In addition, integrin-mediated interactions modulate the response to most, if not all growth factors, cytokines, and other soluble factors. PURPOSE: In this review, we briefly explain how integrins can affect the multitude of signal transduction cascades in control of survival, proliferation, and differentiation. Subsequently, we primarily focus on targeting integrins alpha5beta1 and alphanubeta3 in disease and we discuss how antagonists of these integrins, including disintegrins, RGD peptides, small molecules, and function blocking antibodies, may be of therapeutical value either alone or, especially in the treatment of cancer, in combination with existing therapeutical strategies.     

Cell Adhesion-Mediated Radioresistance (CAM-RR) Extracellular Matrix-Dependent Improvement of Cell Survival in Human Tumor and Normal Cells in Vitro

BACKGROUND: Cell-extracellular matrix (ECM) contact is thought to have great impact on cellular mechanisms resulting in increased cell survival upon exposure to ionizing radiation. Several human tumor cell lines and normal human fibroblastic cell strains of different origin, all of them expressing the wide-spread and important integrin subunit beta1, were irradiated, and clonogenic cell survival, beta 1-integrin cell surface expression, and adhesive functionality were investigated. MATERIAL AND METHODS: Human tumor cell lines A172 (glioblastoma), PATU8902 (pancreas carcinoma), SKMES1 (lung carcinoma), A549 (lung carcinoma), and IPC298 (melanoma) as well as normal human skin (HSF1) and lung fibroblasts (CCD32) and human keratinocytes (HaCaT) were irradiated with 0-8 Gy. Besides colony formation assays, beta 1-integrin cell surface expression by flow cytometry and adhesive functionality by adhesion assays were analyzed. RESULTS: All cell lines showed improved clonogenic survival after irradiation in the presence of fibronectin as compared to plastic. Irradiated cells exhibited a significant, dose-dependent increase in beta 1-integrin cell surface expression following irradiation. As a parameter of the adhesive functionality of the beta 1-integrin, a radiation-dependent elevation of cell adhesion to fibronectin in comparison with adhesion to plastic was demonstrated. CONCLUSION: The in vitro cellular radiosensitivity is highly influenced by fibronectin according to the phenomenon of cell adhesion-mediated radioresistance. Additionally, our emerging data question the results of former and current in vitro cytotoxicity studies performed in the absence of an ECM. These findings might also be important for the understanding of malignant transformation, anchorage-independent cell growth, optimization of radiotherapeutic regimes and the prevention of normal tissue side effects on the basis of experimental radiobiological data.     

Ionizing radiation induces up-regulation of functional β 1-integrin in human lung tumour cell lines in vitro

Purpose : Cell-matrix interactions are in part mediated through the β 1-integrin pathway regulating cell survival, proliferation, adhesion and migration. This study was performed to elucidate alterations of expression of the β 1-integrin and its co-localized protein kinase, integrin-linked kinase (ILK), after exposure to ionizing radiation in two lung carcinoma cell lines in the presence or absence of different β 1-integrin-dependent matrix proteins. Materials and methods : Exponentially growing A549 and SKMES1 cells grown on fibronectin, laminin, BSA or plastic were exposed to 2 Gy or 6 Gy. Besides colony formation assays (0.5-8 Gy) and immediate plating experiments, flow cytometry (for β 1-integrin) and immunoblotting (for β 1-integrin and ILK) were carried out to analyze the protein expression. The localization of both proteins plus filamentous (f-) actin was further examined by immunofluorescence staining and laser confocal scanning microscopy. Functionality of the β 1 receptor subunit after irradiation was investigated in adhesion assays. Results : A549 and SKMES1 cells grown on fibronectin or laminin demonstrated a significant increase in cell survival after irradiation compared to cells grown on BSA or plastic. Immediate plating of cells after irradiation on fibronectin did not show an improved survival. Flow cytometric and Western blot data showed a dose- and matrix-dependent induction of β 1-integrin and ILK expression after irradiation within 48 h. Adhesion to fibronectin or laminin compared to BSA or plastic was increased by 10-fold after irradiation, demonstrating these specific cell surface receptors to be functional. The staining of β 1-integrin and ILK in A549 cells confirmed the radiation-induced up-regulation of both proteins. Additionally, β 1-integrin and ILK co-localized with accumulated actin fibers at the cytoplasmic face of the cell membrane at confined areas. Conclusions : Ionizing radiation strongly induced the expression of functional β 1-integrin and ILK in the two lung cancer cell lines, A549 and SKMES1, dependent on different matrices used. Additionally, the subcellular localization of both proteins was altered by irradiation, and the individual cellular radiosensitivity was reduced in the presence of an extracellular matrix. On the one hand, this may result in aggravated therapeutic approaches and on the other hand, cells could adhere more strongly in their environment by the increase in functional surface receptor density preventing metastasis. Concerning intravascular located tumour cells, β 1-integrin up-regulation might enable these cells to adhere to the endothelium, which represents a prerequisite for metastatic disease. Identification of such mechanisms will provide considerable insights into the understanding of tumorigenicity and metastatic phenotypes, possibly leading to new, optimized radiochemotherapeutic regimens.     

Integrins: Signaling,disease, and therapy

Background: Integrins are a family of transmembrane receptors that mediate cell-cell and cell-matrix adhesion. They are involved in stable cell adhesion and migration of cells. In addition, integrin-mediated interactions modulate the response to most, if not all growth factors, cytokines, and other soluble factors.

Purpose: In this review, we briefly explain how integrins can affect the multitude of signal transduction cascades in control of survival, proliferation, and differentiation. Subsequently, we primarily focus on targeting integrins α5β1 and αvβ3 in disease and we discuss how antagonists of these integrins, including disintegrins, RGD peptides, small molecules, and function blocking antibodies, may be of therapeutical value either alone or, especially in the treatment of cancer, in combination with existing therapeutical strategies.     

Fibronectin and laminin increase resistance to ionizing radiation and the cytotoxic drug Ukrain in human tumour and normal cells in vitro

PURPOSE: Cell-extracellular matrix (ECM) interactions are thought to mediate drug and radiation resistance. Dependence of cell survival, beta1-integrin expression and cell cycling on the ECM proteins and beta1-integrin ligands fibronectin (FN) and laminin (LA) were examined in malignant and normal cells exposed to the cytotoxic drug Ukrain plus/minus irradiation. MATERIALS AND METHODS: Human A549 lung cancer and MDAMB231 (MDA231) breast cancer cells and normal fibroblasts (HSF1) grown on FN, LA, bovine serum albumin (BSA) or polystyrene were treated with Ukrain (1 microg ml(-1), 24 h) plus/minus irradiation (2-8 Gy) and the effects studied using colony formation assays, flow cytometry (beta1-integrin, DNA analysis) and adhesion assays. RESULTS: FN and LA reduced the cytotoxic effect of single Ukrain treatment compared with polystyrene and BSA. FN and LA also abolished Ukrain-dependent radiosensitization in A549 cells and decreased the radiosensitivity of MDA231 and HSF1 cells. Single Ukrain exposure on polystyrene significantly reduced beta1-integrin expression and promoted G2-phase accumulation of A549 cells. In contrast, Ukrain-treated MDA231 and HSF1 cells showed elevated beta1-integrin expression and no Ukrain-specific cell cycle effect. Under Ukrain-radiation exposure, irradiation, FN or LA abolished Ukrain-mediated reduction of beta1-integrin expression and G2-phase accumulation in A549 cells, whereas in MDA231 cells and fibroblasts beta1-integrin expression and cell cycle distribution were stabilized. Cell adhesion to FN or LA was significantly impaired (A549) or improved (MDA231, HSF1) upon Ukrain treatment. CONCLUSIONS: The data corroborate the findings of other groups that cell adhesion-mediated resistance to either single or combined drug and radiation exposure is tightly correlated to specific ECM proteins. By demonstrating a strong modulatory impact of FN and LA on the radiosensitivity-modifying activity of the drug Ukrain, the set findings are also highly important for the assessment of drug and radiation effects within in vitro cytotoxicity studies. The data give the first mechanistic insights into specific FN- and LA-modulated cellular resistance mechanisms as well as into the important role for beta1-integrins using the unique cytotoxic and radiosensitivity-modifying drug Ukrain.     

An essential role of integrin-linked kinase in the cellular radiosensitivity of normal fibroblasts during the process of cell adhesion and spreading

PURPOSE: In addition to focal adhesion kinase (FAK), Paxillin and p130 Crk-associated substrate (p130Cas), integrin-linked kinase (ILK) mediates signals from beta integrins for controlling, e.g., survival, adhesion and spreading. To evaluate the role of ILK in the cellular radiosensitivity at different stages of cell adhesion and spreading, ILK(floxed/floxed (fl/fl)) and ILK(-/-) mouse fibroblasts were examined. MATERIALS AND METHODS: Cells were irradiated (0 - 4 Gy, X-rays) in suspension, after varying time periods on fibronectin (FN) or after 24 h on different matrix proteins. Irradiation was combined with phosphatidylinositol-3 kinase (PI3K) inhibition using Ly294002. Clonogenic radiation survival, cell adhesion, and kinetics of protein expression and phosphorylation during FN adhesion (ILK, v-akt murine thymoma viral oncogene homolog 1 (AKT), FAK, Paxillin, p130Cas) were examined. RESULTS: In suspension and during the first hour on FN, irradiated ILK(fl/fl) cells survived significantly better than ILK(-/-) cells in a PI3K- and serum-dependent manner. 24-h cell cultures on different matrix proteins showed no difference in radiosensitivity. During FN adhesion, which was slightly impaired in ILK(-/-) cells, protein kinetics uncovered differences in AKT, FAK, Paxillin and p130Cas phosphorylation in the two cell lines. Phosphorylation of FAK, Paxillin and p130Cas was downregulated upon exposure to ionizing radiation in an ILK-independent manner. CONCLUSIONS: These findings indicate a critical function of ILK in the cellular radiosensitivity during the early stages of adhesion to and spreading on FN. On the basis of the presented data, a precise correlation of adhesion-, serum- and PI3K-mediated changes in PI3K/AKT and FAK/Paxillin/p130Cas signaling cascades was not found. However, identifying the underlying mechanisms of adhesion- and spreading-related changes in the cellular radiosensitivity might be relevant for an optimization of radiotherapeutic strategies specifically targeting cells located at the invasive edge of a malignant tumor.     

The Anti-Inflammatory Effect of Low-Dose Radiation Therapy Involves a Diminished CCL20 Chemokine Expression and Granulocyte/Endothelial Cell Adhesion

BACKGROUND AND PURPOSE: Low-dose radiotherapy (LD-RT) is known to exert an anti-inflammatory effect, however, the underlying molecular mechanisms are not fully understood. The manipulation of polymorphonuclear neutrophil (PMN) function and/or recruitment may be one mechanism. Chemokines contribute to this process by creating a chemotactic gradient and by activating integrins. This study aimed to characterize the effect of LD-RT on CCL20 chemokine production and PMN/endothelial cell (EC) adhesion. MATERIAL AND METHODS: The EC line EA.hy.926 was irradiated with doses ranging from 0 to 3 Gy and was co-cultured with PMNs from healthy donors either by direct cell contact or separated by transwell membrane chambers. CXCL8, CCL18, CCL20 chemokine and tumor necrosis factor-(TNF-)alpha cytokine levels in supernatants were determined by ELISA and adhesion assays were performed. The functional impact of the cytokines transforming growth factor-(TGF-)beta(1) and TNF-alpha and of the intercellular adhesion molecule-(ICAM-)1 on CCL20 expression was analyzed by using neutralizing antibodies. RESULTS: As compared to CXCL8 and CCL18, CCL20 chemokine secretion was found to be exclusively induced by a direct cell-cell contact between PMNs and EA.hy.926 ECs in a TNF-alpha-dependent, but ICAM-1-independent manner. Furthermore, irradiation with doses between 0.5 and 1 Gy resulted in a significant reduction of CCL20 release which was dependent on TGF-beta(1) (p < 0.01). The decrease of CCL20 paralleled with a significant reduction in PMN/EA.hy.926 EC adhesion (p < 0.001). CONCLUSION: The modulation of CCL20 chemokine expression and PMN/EC adhesion adds a further facet to the plethora of mechanisms contributing to the anti-inflammatory efficacy of LD-RT.     

Funktionelle und molekulare Aspekte der antiinflammatorischen Wirkung niedrig dosierter Radiotherapie

PURPOSE: Low-dose radiotherapy (LD-RT) with single fractions between 0.1 and 1.0 Gy is known to exert an antiinflammatory effect. Although different mechanisms for the clinical efficiency were proposed, only few experimental data are still available. This paper focuses on functional and molecular aspects of LD-RT. METHODS AND RESULTS: The antiinflammatory efficiency of LD-RT in clinical studies could be confirmed in experimental models of osteoarthritis and rheumatoid arthritis. In a model of adjuvants arthritis, 5 x 1.0 Gy as well as 5 x 0.5 Gy, given at the maximum of the acute inflammation, could prevent clinically and histologically progression of the disease without affecting existing signs of inflammation. The effect of LD-RT on the adhesion of peripheral blood mononuclear cells (PBMC) and endothelial cells (EC) was analyzed in in-vitro assays. In the dose range between 0.3 and 0.7 Gy almost 4 hours after irradiation adherent cells reached a relative minimum of adhesion compared to unirradiated controls. In PBMC an discontinuous increase of apoptosis with a maximum between 0.3 and 0.5 Gy, the proteolytic shedding of L-selectin and an increased expression of the antiinflammatory cytokine interleukin 10 as well as downregulation of TNF alpha could be identified as potential mechanisms for the observed reduced adhesion. Conversely, reduced expression of E-selectin and an increased induction of transforming growth factor beta (TGF beta 1) with a maximum at 0.5 Gy could be observed in endothelial cells. Macrophages immigrating the site of inflammation are known to express inducible nitrix-oxide synthase (iNOS), which in turn mediates cytotoxic and immunmodulatory effects by producing nitric oxide (NO). LD-RT of stimulated macrophages within the dose range between 0.6 and 1.25 Gy reduced NO production and iNOS-protein expression without affecting iNOS-mRNA expression. CONCLUSION: Our experimental data have confirmed the antiinflammatory efficiency of LD-RT in vitro and in vivo, indicating effects on different cellular components and mechanisms of inflammation. The regulation of the adhesion between PBMC and endothelial cells and the effects on activated macrophages may mediate the antiinflammatory properties of LD-RT. Ongoing experiments will help to clarify the molecular mechanism.     

Irradiation and Various Cytotoxic Drugs Enhance Tyrosine Phosphorylation and β1-Integrin Clustering in Human A549 Lung Cancer Cells in a Substratum-Dependent Manner in Vitro

BACKGROUND AND PURPOSE: Interactions of cells with a substratum, especially extracellular matrix proteins, initiate clustering of integrin receptors in the cell membrane. This process represents the initial step for the activation of signaling pathways regulating survival, proliferation, differentiation, adhesion, and migration, and could, furthermore, be important for cellular resistance mediating mechanisms against radiation or cytotoxic drugs. The lack of data elucidating the impact of irradiation or cytotoxic drugs on this important phenomenon led to this study on human A549 lung cancer cells in vitro. MATERIAL AND METHODS: The human lung carcinoma cell line A549 grown on polystyrene or fibronectin (FN) was irradiated with 0-8 Gy or treated with cisplatin (0.1-50 microM), paclitaxel (0.1-50 nM), or mitomycin (0.1-50 microM). Colony formation assays, immunofluorescence staining in combination with activation of integrin clustering using anti-beta(1)-integrin antibodies (K20), and Western blotting for tyrosine phosphorylation under treatment of cells with the IC(50) for irradiation (2 Gy; IC(50) = 2.2 Gy), cisplatin (2 microM), paclitaxel (5 nM), or mitomycin (7 microM) were performed. RESULTS: Attachment of cells to FN resulted in a significantly reduced radio- and chemosensitivity compared to polystyrene. The clustering of beta(1)-integrins examined by immunofluorescence staining was only stimulated by irradiation, cisplatin, paclitaxel, or mitomycin in case of cell attachment to FN. By contrast, tyrosine phosphorylation, as one of the major events following beta(1)-integrin clustering, showed a 3.7-fold, FN-related enhancement, and treatment of cells with the IC(50) of radiation, cisplatin, paclitaxel, or mitomycin showed a substratum-dependent induction. CONCLUSION: For the first time, a strong influence of irradiation and a variety of cytotoxic drugs on the clustering of beta(1)-integrins could be shown. This event is a prerequisite for tyrosine phosphorylation and, thus, the activation of cellular mechanisms regulating survival, proliferation, and adhesion. These data are not only important for the understanding of cellular resistance against cytotoxic agents but, furthermore, for tumor progression, anchorage-independent cell growth, and, possibly, the optimization of radiochemotherapeutic strategies.     

Mononuclear cell adhesion and cell adhesion molecule liberation after X-irradiation of activated endothelial cells in vitro

PURPOSE: Increased expression of cell adhesion molecules on endothelial cells is an important early event in inflammation. Low-dose radiotherapy is very effective anti-inflammatory treatment. The hypothesis that it may act by modulation of cell adhesion molecule expression in activated endothelial cells and the subsequent adhesion of mononuclear cells onto the activated endothelial cells was tested. MATERIALS AND METHODS: EA.hy.926 endothelial cells were irradiated with 0.3-10 Gy X-rays at different times before or after stimulation with TNFalpha. ICAM-1 or E-selectin expression was measured by ELISA and FACS. Isolated peripheral blood mononuclear cells were incubated with an activated and irradiated confluent monolayer of endothelial cells 4 h, 12 h or 24 h after stimulation, and adhesion was determined in dynamic and static adhesion assays. RESULTS: In the static adhesion assay, where integrin-mediated adhesion dominates, radiation doses of 0.3-0.6 Gy reduced the adhesion of mononuclear cells onto EA.hy.926-EC in vitro by 25-40% and 15-25% of the control level 4 h and 24 h after stimulation, respectively, but increased adhesion 12 h after stimulation. In the dynamic adhesion assay, where selectin-mediated adhesion dominates, radiation doses of 0.3-0.6 Gy reduced the adhesion events by 40-50% and 30-40% of the control level 4 h and 24 h after stimulation, respectively, and again increased adhesion 12h after stimulation. X-ray doses of < or =5 Gy did not induce ICAM-1 expression, or modulate TNFalpha-induced ICAM-1 expression. E-selectin expression was, however, increased in a dose-dependent way 6 h after irradiation. In contrast, X-irradiation 2-5 h before stimulation decreased the characteristic transient expression of E-selectin after TNFalpha stimulation. CONCLUSIONS: Modulation of E-selectin liberation on activated endothelial cells may be one mechanism to decrease leukocyte adhesion after low-dose irradiation in vitro, and could be involved in the therapeutic action of anti-inflammatory radiotherapy.     

Mononuclear cell adhesion and cell adhesion molecule liberation after X-irradiation of activated endothelial cells in vitro

Purpose : Increased expression of cell adhesion molecules on endothelial cells is an important early event in inflammation. Low-dose radiotherapy is very effective anti-inflammatory treatment. The hypothesis that it may act by modulation of cell adhesion molecule expression in activated endothelial cells and the subsequent adhesion of mononuclear cells onto the activated endothelial cells was tested. Materials and methods : EA.hy.926 endothelial cells were irradiated with 0.3-10 Gy X-rays at different times before or after stimulation with TNFα. ICAM-1 or E-selectin expression was measured by ELISA and FACS. Isolated peripheral blood mononuclear cells were incubated with an activated and irradiated confluent monolayer of endothelial cells 4 h, 12 h or 24 h after stimulation, and adhesion was determined in dynamic and static adhesion assays. Results : In the static adhesion assay, where integrin-mediated adhesion dominates, radiation doses of 0.3-0.6 Gy reduced the adhesion of mononuclear cells onto EA.hy.926-EC in vitro by 25-40% and 15-25% of the control level 4 h and 24 h after stimulation, respectively, but increased adhesion 12 h after stimulation. In the dynamic adhesion assay, where selectin-mediated adhesion dominates, radiation doses of 0.3-0.6 Gy reduced the adhesion events by 40-50% and 30-40% of the control level 4 h and 24 h after stimulation, respectively, and again increased adhesion 12h after stimulation. X-ray doses of ≤5 Gy did not induce ICAM-1 expression, or modulate TNF α -induced ICAM-1 expression. E-selectin expression was, however, increased in a dose-dependent way 6 h after irradiation. In contrast, X-irradiation 2-5 h before stimulation decreased the characteristic transient expression of E-selectin after TNF α stimulation. Conclusions : Modulation of E-selectin liberation on activated endothelial cells may be one mechanism to decrease leukocyte adhesion after low-dose irradiation in vitro, and could be involved in the therapeutic action of anti-inflammatory radiotherapy.     

The impact of conventional and heavy ion irradiation on tumor cell migration in vitro

PURPOSE: The influence of X-ray and (12)C heavy ion irradiation on tumor cell migration and of beta(3) and beta(1) integrin expression was investigated. MATERIAL AND METHODS: Two different tumor cell lines (U87 glioma and HCT116 colon carcinoma cells) were irradiated with 1, 3, or 10 Gy X-rays or (12)C heavy ions. 24 h after irradiation a standardized Boyden Chamber assay for migration analysis was performed and cells were lysed for Western blotting. RESULTS: Radiation-induced influences were cell line- and radiation type-dependent. X-rays decreased HCT116 migration at higher doses and appear to increase U87 migration after 3 Gy. Heavy ions decreased migration of both cell lines dose-dependently. A trend of increased beta(3) and beta(1) integrin expression in U87 cells after both radiation types was observed. beta(1) integrin expression in HCT116 cells was increased after X-rays but decreased after heavy ion irradiation. CONCLUSIONS: Results suggest that irradiation of tumor cells can modulate their migratory behavior. An increased migration, as shown with U87, leaves a higher probability of metastatic induction after irradiation of solid tumors in vivo, whereas an invariably reduced tumor cell migration, as shown after heavy ion treatment, could diminish the hazard of radiation-induced metastasis. As integrin expression and migration were only partially correlated, other migration-related surface molecules may be more relevant for radiation effects on tumor cell motility.     

Anti-inflammatory effect of low-dose X-irradiation and the involvement of a TGF-beta1-induced down-regulation of leukocyte/endothelial cell adhesion

PURPOSE: Low-dose radiotherapy (LD-RT) is known to exert an anti-inflammatory effect, but the underlying radiobiological and immunological mechanisms remain elusive. In recent studies, we observed a reduced adhesion of peripheral blood mononuclear cells (PBMC) to endothelial cells (EC) after LD-RT (0.3-0.7 Gy). This shows that this treatment affects the initial steps of the inflammatory response. To explore the role of inflammatory mediators in this process, we investigated the expression of Transforming growth factor beta(1) (TGF-beta(1)) and Interleukin 6 (IL-6) after LD-RT. MATERIALS AND METHODS: EC were grown to subconfluence and irradiated with single-dose LD-RT. Twenty-hours after irradiation, EC were treated with IL-1beta for 4 h and then incubated with peripheral blood mononuclear cells (PBMC). Adherent PBMC were counted when using light microscopy. Expression of the cytokines TGF-beta(1) and IL-6 was measured by ELISA, and mRNA levels were analysed by the RNAse-protection assay (RPA). Surface expression of E-selectin was quantified by flow cytometry. RESULTS: A relative minimum of adhesion was observed after LD-RT between 0.3 and 0.7 Gy. This was paralleled by an expression maximum of TGF-beta(1) and IL-6, as shown by protein and mRNA levels, respectively. Neutralization of TGF-beta(1) by monoclonal antibodies, but not of IL-6, increased PBMC adhesion to EC nearly to control levels. In addition, fluorescence activated cell sorter (FACS) analysis of irradiated EC demonstrated a down-regulation of E-selectin in the same dose range. CONCLUSION: Low-dose X-irradiation between 0.3 and 0.7 Gy induced a relative maximum of TGF-beta(1) production by stimulated EC. This results in a down-regulation of leukocyte/PBMC adhesion and may contribute to the anti-inflammatory effect of LD-RT.     

The relationship between potentially lethal damage repair and intrinsic radiosensitivity of human cells

The intrinsic radiosensitivity of exponentially growing cells (exp) was compared to that of immediately plated plateau phase cells (ip) using published data on 60 human cell lines (27 fibroblast lines and 33 tumour cell lines). The values for alpha, D and S2 are not significantly different for the two groups; beta is significantly higher in ip cells. This produces a smaller alpha/beta ratio in ip cells than in exp cells. The influence of potentially lethal damage (PLD) repair was assessed by comparing the radiosensitivities of ip cells and plateau phase cells with delayed plating (dp). The published data for 81 human cell lines (48 fibroblasts and 33 tumour lines) were used. PLD repair was found to lead to a decrease in alpha and an increase in D and S2, whereas neither beta nor the alpha/beta ratio changed significantly. The relationship between PLD repair and intrinsic radiosensitivity was assessed by repair capacity and the repair ratio. The fitted relationship is a bell-shaped curve with a maximum at 2.2 Gy for repair capacity. The fitted curve predicts that repair capacity is zero at a D up of 0.28 Gy and at 4 Gy. Thus, PLD repair is a reasonable reflection of intrinsic radiosensitivity up to 2.2 Gy. Above 2.2 Gy, the relationship is reversed: the greater the radioresistance, the lower the PLD repair.     

Interaction of vascular smooth muscle cells with collagen-impregnated embolization coils studied with a novel quantitative in vitro model

BACKGROUND AND PURPOSE: Modifications of aneurysm occlusion devices and other biologically active molecules may reduce the risk of recanalization by promoting vascular cell migration, adhesion, and proliferation. Our purpose was to apply in vitro methods in the qualitative and quantitative analysis of vascular smooth muscle cell (VSMC) interactions with collagen-impregnated microcoils. METHODS: The adhesion of rat aortic VSMCs to collagen fiber bundles (CFBs), nitinol coils, and collagen-impregnated nitinol coils (CINCs) was examined by using an assay consisting of monopulse exposure to increasing concentrations of rat aortic VSMCs. Exposed devices were washed and examined by using confocal fluorescence microscopy. Adhesion coefficients, which quantitatively express the cell-binding quality of a surface, were determined by using a mathematical model for cell-device interactions. RESULTS: VSMCs, attached to devices, spread out and extended cytoplasmic projections over the contact surface. Cell distribution was random on CFBs and within interloop troughs on nitinol coils. On collagen-impregnated coils, VSMCs were selectively concentrated on the collagen between coil loops. The average adhesion coefficient was 25.0 for CFBs, 8.5 for CINCs (250-microm pitch), and 6.5 for nitinol coils. Adhesion coefficient differences for the three devices were significant (P =.044). CONCLUSION: The monopulse exposure assay is a simple and reproducible in vitro test that provides qualitative information about the morphology and topography of cell-device contacts and permits quantitative measurement of the intrinsic cell-binding quality of the test device. VSMCs exposed to collagen-impregnated microcoils selectively attach to collagen. Collagen enhances the rate of VSMC adhesion to embolic devices, and the degree of enhancement correlates with the surface area constituted by collagen.     

Adhesion molecules,catecholamines and leucocyte redistribution during and following exercise

The circulating blood normally contains no more than 1-2% of the body's population of leucocytes. The numbers and phenotypes of circulating leucocyte subsets can change dramatically during and immediately following exercise. The surface expression of adhesion molecules makes an important contribution to such responses by changing patterns of cell trafficking. Alterations in the surface expression of adhesion molecules could reflect a shedding of molecules, selective apoptosis or differential trafficking of cells with a particular phenotype, effects from mechanical deformation of the cytoplasm, active biochemical processes involving cytokines, catecholamines, glucocorticoids or other hormones, or changes in the induction of adhesion molecules. The expression of adhesion molecules changes with maturation and activation of leucocytes. Typically, mature cells express lower densities of L-selectin (CD62L), the homing receptor for secondary lymphoid organs, and higher densities of LFA-1 (CD11a), the molecule associated with trafficking to non-lymphoid reservoir sites. The neutrophils and natural killer cells that are mobilised during exercise also express high levels of Mac-1 (CD11b), a marker associated with cellular activation. Possibly, exercise demarginates older cells that are awaiting destruction in the spleen. Plasma concentrations of catecholamines rise dramatically with exercise, and there is growing evidence that catecholamines, acting through a cyclic adenosine monophosphate second messenger system, play an important role in modifying the surface expression of adhesion molecules. Analogous changes can be induced by other forms of stress that release catecholamines or by catecholamine infusion, and responses are blocked by beta(2)-blocking agents. Catecholamines also modify adherence and expression of adhesion molecules in vitro. Cell trafficking is modified by genetic deficiencies in the expression of adhesion molecules, but leucocyte responses to exercise and catecholamines are generally unaffected by splenectomy. A number of clinical conditions including atherogenesis and metaplasia are marked by an altered expression of adhesion molecules. The effects of exercise on these molecules could thus have important health implications.     

Time‐ and dose‐dependent changes of intracellular cytokine and cytokine receptor profile of Ewing tumour subpopulations under the influence of ionizing radiation

Purpose: Cytokines and their corresponding cell surface receptors are involved in intercellular signalling pathways and in the radioresistance of normal and malignant cells. The aim was the characterization of the expression of intracellular cytokines, their receptors and apoptosis‐associated markers under the influence of radiation.

Materials and methods: Two Ewing tumours were characterized in vitro before and 4, 24 and 72?h after radiation with 5 and 10?Gy, and in vivo 4, 6 and 15 days after radiation with 5 and 30?Gy by five parameter flow cytometry. Direct fluorescence‐conjugated antibodies directed against intracellular cytokines (interferon‐gamma, tumour necrosis factor [TNF]‐alpha, interleukin 1) and their receptors (CD119, CD120a, CD121a) were used. Annexin V and 7‐amino‐actinomycin D were used to identify radiation‐induced apoptosis.

Results: Inter‐ and intra‐individual heterogeneities were identified by the expression of cytokine receptors and the intracellular cytokine profile before radiation. Time‐ and dose‐dependent up‐regulation of the cytokines TNF‐alpha and interleukin 1 were found in vitro. In vivo, an up‐regulation of CD120a and CD121a was detectable on tumour cell subpopulations. For interferon‐gamma and CD119, no changes were seen.

Conclusions: The observed radiation‐induced changes of cytokine and receptor profile are an indication for complex intercellular interactions in view of radioresistance‐associated mechanisms between cell populations within one individual tumour. The observed heterogeneous response on radiation might have therapeutic implications for an individualized therapy based on combined radiation and cytokine modulation, defined by flow cytometric characterization of markers potentially informative for radioresistance.     

An essential role of integrin-linked kinase in the cellular radiosensitivity of normal fibroblasts during the process of cell adhesion and spreading

Purpose: In addition to focal adhesion kinase (FAK), Paxillin and p130 Crk-associated substrate (p130Cas), integrin-linked kinase (ILK) mediates signals from β integrins for controlling, e.g., survival, adhesion and spreading. To evaluate the role of ILK in the cellular radiosensitivity at different stages of cell adhesion and spreading, ILK^{floxed/floxed (fl/fl)} and ILK^?/? mouse fibroblasts were examined.

Materials and methods: Cells were irradiated (0 – 4 Gy, X-rays) in suspension, after varying time periods on fibronectin (FN) or after 24 h on different matrix proteins. Irradiation was combined with phosphatidylinositol-3 kinase (PI3K) inhibition using Ly294002. Clonogenic radiation survival, cell adhesion, and kinetics of protein expression and phosphorylation during FN adhesion (ILK, v-akt murine thymoma viral oncogene homolog 1 (AKT), FAK, Paxillin, p130Cas) were examined.

Results: In suspension and during the first hour on FN, irradiated ILK^fl/fl cells survived significantly better than ILK^?/? cells in a PI3K- and serum-dependent manner. 24-h cell cultures on different matrix proteins showed no difference in radiosensitivity. During FN adhesion, which was slightly impaired in ILK^?/? cells, protein kinetics uncovered differences in AKT, FAK, Paxillin and p130Cas phosphorylation in the two cell lines. Phosphorylation of FAK, Paxillin and p130Cas was downregulated upon exposure to ionizing radiation in an ILK-independent manner.

Conclusions: These findings indicate a critical function of ILK in the cellular radiosensitivity during the early stages of adhesion to and spreading on FN. On the basis of the presented data, a precise correlation of adhesion-, serum- and PI3K-mediated changes in PI3K/AKT and FAK/Paxillin/p130Cas signaling cascades was not found. However, identifying the underlying mechanisms of adhesion- and spreading-related changes in the cellular radiosensitivity might be relevant for an optimization of radiotherapeutic strategies specifically targeting cells located at the invasive edge of a malignant tumor.