Irradiation and various cytotoxic drugs enhance tyrosine phosphorylation and beta(1)-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.

Development and evaluation of a skin organ model for the analysis of radiation effects.

BACKGROUND AND PURPOSE: The reaction of tissues to ionizing radiation involves alterations in cell-cell and cell-matrix interactions mediated by cellular adhesion molecules. The aim of this study was to develop and evaluate an artificial skin organ model for the analysis of radiation effects. MATERIAL AND METHODS: A human co-culture system consisting of the spontaneously immortalized keratinocyte cell line HaCaT and primary HDFa fibroblasts embedded into a collagen sponge was established. This skin organ model has been characterized and evaluated for its suitability for radiobiological investigations. For that purpose, expression of beta(1)-integrin following irradiation was compared in the skin organ model and in HaCaT monolayer cells (FACScan and immunohistochemistry). Furthermore, the influence of ionizing radiation on DNA fragmentation was investigated in the skin organ model (TUNEL assay). RESULTS: The novel skin organ model showed characteristics of human skin as demonstrated by cytokeratin and Ki-67 immunoreactivity and by electron microscopy. A single dose of 5 Gy X-irradiation induced an upregulation of beta(1)-integrin expression both in the skin organ model and in HaCaT cells. Following irradiation, beta(1)-integrin immunoreactivity was intensified in the upper layers of the epidermis equivalent whereas it was almost absent in the deeper layers. Additionally, irradiation of the skin organ model also caused a marked increase of DNA fragmentation. CONCLUSION: These results demonstrate that the novel skin organ model is suitable to investigate cellular radiation effects under three-dimensional conditions. This allows to investigate radiation effects which cannot be demonstrated in monolayer cell cultures.

Arrest of human lung fibroblasts in G2 phase after irradiation is regulated by converging phosphatidylinositol-3 kinase and beta1-integrin signaling in vitro.

PURPOSE: Cell-matrix interactions might confer cellular radioresistance in vitro. As a function of radiation, the impact of fibronectin (FN) and phosphatidylinositol-3 kinase (PI3K)-related signaling on survival, the cell cycle, and the beta1-integrin signaling kinases integrin-linked kinase (ILK), protein kinase Balpha/Akt (PKBalpha/Akt), and glycogen synthase kinase-3beta (GSK-3beta) was examined in normal lung fibroblasts in vitro. METHODS AND MATERIALS: Normal human CCD32 lung fibroblasts grown on polystyrene, FN, or poly-L-lysine were irradiated with 0-8 Gy. Colony forming assays, flow cytometric DNA analysis, immunoblotting (Chk1, Chk2, Cdc25C, Cdk1, 14-3-3, p53, p21), and protein kinase assays (ILK, PKBalpha/Akt, GSK-3beta) were performed with or without PI3K inhibition using LY294002 or wortmannin. RESULTS: FN significantly elevated clonogenic survival of CCD32 cells after irradiation compared with polystyrene or poly-L-lysine. FN improved accumulation of irradiated cells in G(2)/M (60%) compared with polystyrene (43%). LY294002 prevented radiation-dependent G(2) blockage on polystyrene; on FN, G(2) arrest was only slightly reduced. Radiation- and PI3K inhibition-related changes in expression and phosphorylation of the various cell cycle proteins tested correlated with the cell cycle data acquired. The kinase activities of ILK, PKBalpha/Akt, and GSK-3beta were strongly induced by irradiation on polystyrene, but not on FN, which was a result of a FN-mediated increase of basal kinase activities. In contrast to polystyrene, FN enabled radiation-dependent induction of ILK and GSK-3beta in a PI3K-independent manner. CONCLUSION: The data indicate a tight convergence of cell-matrix and cell-growth factor interactions that seem to optimize the cellular responsiveness to ionizing radiation in terms of survival and G(2) arrest. ILK, PKBalpha/Akt, and GSK-3beta involved in integrin signaling were uncovered as new molecular factors within the cellular radiation response. Our findings might also provide insight into normal tissue effects and cellular radioresistance.

Comparison of endogenous TP53 genomic status with clonogenicity and different modes of cell death after X irradiation.

Although extensive data indicate that the tumor suppressor TP53 modifies the radiation responses of human and rodent cells, the exact relationship between TP53 and radiation responsiveness remains controversial. To elucidate the relevance of endogenous TP53 genomic status to radiosensitivity in a cell-type-independent manner, different cells of 10 human tumor cell lines with different tissues of origin were examined for TP53 status. The TP53 status was compared with radiation-related cell survival parameters (D(q), D(0), SF2) and with the mode of cell death. Different modes of cell death were examined by measuring radiation-induced micronucleation, apoptosis and abnormal cells. Alterations of the TP53 gene were detected in eight cell lines. No splicing mutation was found. Five cell lines showed codon 68 polymorphism. Codon 72 alterations were found in four cell lines. "Hot spot" alterations were detected in only two of 10 cell lines. Although the cells differed widely in survival parameters (D(q), D(0), SF2) and modes of cell death (micronucleation/apoptosis/abnormal cells) after irradiation, significant cell-type-independent correlations were obtained between the multiple cell death parameter micronucleation/apoptosis/abnormal cells and SF2 (P < 0.001) and D(q) (P = 0.003). Moreover, cells with a wild-type TP53 gene were more resistant to X rays than cells with a mutated TP53 gene or cells that were TP53-deficient. The alterations within exons 5-10 of the TP53 correlated with a enhanced radiosensitivity. For the first time, we demonstrated a correlation between endogenous genetic alterations within exons 5-10 of TP53 and radiation-related cell survival and cell death. This indicates a new molecular relevance of TP53 status to intrinsic cellular radiosensitivity.

Modulation of X-ray-induced apoptosis in human keratinocytes (HaCaT) by 1,25-dihydroxyvitamin D3.

Possible effects of 1,25-dihydoxyvitamin D3 (vitamin D) on ionizing radiation-induced cell damage have been unknown until now. The task of the present study was to analyze, in a human keratinocyte cell line (HaCaT), the effects of a preincubation with vitamin D on the X-ray-induced mRNA expression of different genes related to apoptosis (gene array). The first results show that ionizing radiation leads to a down-regulation of various apoptosis-relevant genes in HaCaT cells pretreated with vitamin D. Therefore it can be speculated that vitamin D could prove to be a promising radioprotective substance.

Medical management principles for radiation accidents.

The medical management of radiation accidents requires intensive planning and action. This article looks at the medical management of recent radiation accidents to derive principles for structuring and organizing the treatment of patients who may have radiation-induced health impairments. Although the radiation accidents in Tokai-mura, Japan and Lilo, Georgia were small-scale accidents, they illustrate important and characteristic symptoms and clinical developments. There are lessons to be learned and conclusions to be drawn for the military medical officers concerned with problems of medical management after radiation accidents.

Ionizing radiation modulates cell surface integrin expression and adhesion of COLO-320 cells to collagen and fibronectin in vitro.

PURPOSE: Adhesion of tumor cells to endothelial cells and to the extracellular matrix is a key step in the initial phase of metastasis. Since radiotherapy of tumors can induce alterations of the cell surface, we investigated the effect of ionizing radiation on the expression of integrins in the colorectal tumor cell line COLO-320 and the modulation of adhesion capacity of irradiated cells to collagen and fibronectin. MATERIAL AND METHODS: The cell surface expression of a broad range of integrins on COLO-320 cells was determined by flow cytometry during 144 hours after X-irradiation. The functional significance of increased adhesion molecule expression was assessed by cell-matrix adhesion and receptor blocking experiments. RESULTS: Cell surface expression of the following integrin alpha and beta subunits was quantified: beta1 (CD29), alpha 2 (CD49b), alpha 5 (CD49e) and alpha 6 (CD49f). The expression of alpha 1, alpha 2, alpha 5, and alpha 6 changes as a function of time after irradiation (5 Gy). For beta1 even a function of dose (1-5 Gy) could be shown. Adhesion experiments confirmed a time dependent increase in adhesion to both collagen and fibronectin. Radiation-induced increase in adhesion was inhibited significantly by using a CD29 antibody. CONCLUSIONS: Ionizing radiation modulates cell surface expression of integrins and cell-matrix interactions. The beta1-integrin subunit plays an important role in radiation-induced adhesion to both collagen and fibronectin. Possible consequences of these in-vitro results for radiotherapy of colorectal tumors in vivo are discussed.

Fas/Fas ligand system and apoptosis induction in testicular carcinoma.

BACKGROUND: Tumor-infiltrating, Fas ligand (FasL)-expressing lymphocytes are able to eliminate Fas-bearing tumor cells by apoptosis induction. Activated cytotoxic T-cells that express Fas may enter apoptosis in the presence of FasL tumor cells. To date, no studies of patients with testicular carcinoma have correlated the differential expression of Fas and FasL in both cell types with the corresponding apoptotic index (AI). METHODS: Fas and FasL were investigated immunohistochemically in paraffin embedded tissue sections from 25 patients with nonseminomatous testicular tumors. The percentages of positive cells and the ratios of Fas cells to FasL cells were correlated with the AI of tumor cells and lymphocytes, respectively, using Spearman correlations. RESULTS: No association was found between the rate of FasL positive cells and AI of the other cell type or between the rate of Fas positive cells and the AI of the same cell type. Ratios between Fas positive cells and FasL positive cells were not correlated with the AI; however, a significant positive correlation was found between the AI of tumor cells and the AI of lymphocytes. CONCLUSIONS: It seems unlikely that the Fas/FasL system is responsible for immune escape of the tumor in testicular carcinoma. Rather, the significant positive correlation between the AIs of tumor cells and lymphocytes implicate a previously unknown mechanism of apoptosis induction in both cell types.