Dirk van Beuningen

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 α and β subunits was quantified: β1 (CD29), α2 (CD49b), α5 (CD49e) and α6 (CD49f). The expression of α1, α2, α5, and α6 changes as a function of time after irradiation (5 Gy). For β1 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 β1-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.Hintergrund: Die Adhäsion zwischen Tumorzellen und Endothelzellen sowie der extrazellulären Matrix ist ein entscheidender Schritt in der Initialphase einer Metastasierung. Da eine Bestrahlung von Tumoren mittels Radiotherapie Änderungen der Zelloberfläche bewirken kann, wurde der Effekt von ionisierender Strahlung auf die Expression von Integrinen in der kolorektalen Tumorzelllinie COLO-320 sowie die Modulationder Adhäsionsfähigkeit der bestrahlten Zellen an Kollagen und Fibronektin untersucht. Material und Methode: Die Zelloberflächenexpression einer Reihe von Integrinen wurde durchflusszytometrisch an COLO-320-Zellen bis zu 144 Stunden nach Bestrahlung bestimmt. Die funktionelle Bedeutung einer gesteigerten Adhäsionsmolekülexpression wurde mittels Zell-Matrix-Adhäsionsversuchen und Rezeptorblockaden untersucht. Ergebnisse: Die Zelloberflächenexpression der folgenden α- und β-Integrinuntereinheiten wurde quantifiziert: β1 (CD29), α2 (CD49b), α5 (CD49e) und α6 (CD49f). Die Expression von α1, α2, α5 und α6 änderte sich zeitabhängig nach Bestrahlung (5 Gy). Für β1 konnte auch eine Dosisunabhängigkeit zwischen 1 und 5 Gy gezeigt werden. Adhäsionsversuche bestätigten einen zeitabhängigen Anstieg sowohl der Adhäsion zu Kollagen als auch zu Fibronektin. Der strahleninduzierte Anstieg der Adhäsion wurde signifiaktn durch einen CD29-Antikörper inhibiert. Schlussfolgerungen: Ionisierende Strahlung moduliert die Zelloberflächenexpression von Integrinen und Zell-Matrix-Interaktionen. Die β1-Integrinuntereinheit spielt eine wichtige Rolle bei der strahleninduzierten Adhäsion sowohl an Kollagen als auch an Fibronektin. Mögliche Konsequenzen dieser In-vitro-Ergebnisse für eine Radiotherapie kolorektaler Tumoren in vivo werden diskutiert.

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 resistancemediating 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 µM), paclitaxel (0.1–50 nM), or mitomycin (0.1–50 µM). Colony formation assays, immunofluorescence staining in combination with activation of integrin clustering using anti-β1-integrin antibodies (K20), and Western blotting for tyrosine phosphorylation under treatment of cells with the IC50 for irradiation (2 Gy; IC50 = 2.2 Gy), cisplatin (2 µM), paclitaxel (5 nM), or mitomycin (7 µM) were performed.Results:Attachment of cells to FN resulted in a significantly reduced radio- and chemosensitivity compared to polystyrene. The clustering of β1-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 β1-integrin clustering, showed a 3.7-fold, FN-related enhancement, and treatment of cells with the IC50 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 β1-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.Hintergrund und Ziel:Interaktionen von Zellen mit einem Substrat, insbesondere spezifischen Proteinen der extrazellulären Matrix, initiieren ein Zusammenballen (Clustering) von Integrinrezeptoren in der Zellmembran. Dieser Prozess repräsentiert den Initialschritt für die Aktivierung von Signalwegen, die Überleben, Proliferation, Differenzierung, Adhäsion und Migration regulieren. Aufgrund fehlender Daten, die den Einfluss von Bestrahlung oder Zytostatika auf dieses evtl. auch für Resistenzmechanismen wichtige Phänomen näher beschreiben, wurde die vorliegende In-vitro-Studie an humanen A549-Bronchialkarzinomzellen durchgeführt.Material und Methodik:Humane, auf Polystyrol oder Fibronectin (FN) wachsende A549-Bronchialkarzinomzellen wurden mit aufsteigenden Dosen von 0–8 Gy bestrahlt oder mit Cisplatin (0,1–50 µM), Paclitaxel (0,1–50 nM) oder Mitomycin (0,1–50 µM) behandelt. Koloniebildungsassays, Immunfluoreszenzfärbungen in Kombination mit der Aktivierung des Integrinclusterings sowie Western-Blotting zum Nachweis der Tyrosinphosphorylierung wurden unter Behandlung mit den IC50 für die Bestrahlungsdosis und die Zytostatikakonzentrationen durchgeführt.Ergebnisse:Die Adhäsion von Zellen an FN resultierte in einer signifikant reduzierten Radio- und Chemosensibilität im Gegensatz zu Polystyrol. Das Clustering von β1-Integrinen, mittels Immunfluoreszenzfärbung untersucht, wurde durch Bestrahlung, Cisplatin, Paclitaxel und Mitomycin lediglich in Zellen, die auf FN wuchsen, stimuliert. Im Gegensatz hierzu zeigte die Untersuchung der Tyrosinphosphorylierung, als eines der Hauptereignisse im Anschluss an ein β1-Integrin-Clustering, eine 3,7fache, FN-bedingte Verstärkung. Behandlung der Zellen mit den IC50 für Bestrahlung, Cisplatin, Paclitaxel oder Mitomycin führte zu einer substratabhängigen Induktion der Tyrosinphosphorylierung.Schlussfolgerung:Erstmals wird in dieser Studie ein starker Einfluss von Bestrahlung und einer Reihe von Zytostatika auf das β1-Integrin-Clustering gezeigt. Dieses Ereignis ist eine Grundvoraussetzung für die Tyrosinphosphorylierung und folglich die Aktivierung zellulärer Regulationsmechanismen für Überleben, Proliferation und Adhäsion. Diese Daten sind wichtig zum Verständnis von adhäsionsunabhängigem Zellwachstum und Tumorwachstum, aber insbesondere auch von zellulären Resistenzmechanismen gegenüber zytotoxischen Agenzien oder Bestrahlung. Langfristig bietet dies möglicherweise eine Chance zur Optimierung radiochemotherapeutischer Behandlungsstrategien in der Klinik.

Tissue engineering with HaCaT cells and a fibroblast cell line

Abstract Most skin models consist of primary cells. Our aim was to develop a highly reproducible skin model consisting only of cell lines to investigate irradiation effects. The spontaneously immortalized human keratinocyte line HaCaT is known for its capacity for epidermal differentiation. As an organotypic coculture, HaCaT cells were grown air-exposed on top of a dermis equivalent consisting of a murine fibroblast cell line (L929) in collagen. The technique for the preparation of this coculture system is described. After 3 weeks a multilayered epithelium with signs of differentiation developed. The expression of several markers for differentiation and basal membrane formation were compared with those of healthy human skin by immunohistochemical staining. In the epithelium of the skin model several cytokeratins, especially keratin 10, and involucrin were expressed comparable to normal skin. Laminin expression was found along the basal zone of the epithelium. BrdU labeling indicating proliferation was mainly found in the basal parts of the epithelium. Differentiated cells showing DNA fragmentation were detected in the upper parts of the epithelium by the TUNEL assay. Fluorescence in situ hybridization was used to discriminate between HaCaT and L929 cells in the coculture. Some L929 cells growing on top of the epithelium could be detected. This might have been due to an invasion of highly proliferating L929 cells and might be one of the limits of tissue engineering with cell lines. In conclusion, the organotypic coculture used as a skin model is a promising additional tool for addressing specific research questions.

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 β1-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 β1-integrin expression both in the skin organ model and in HaCaT cells. Following irradiation, β1-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.Hintergrund und Ziel:Die Reaktion von Gewebe auf ionisierende Strahlung bewirkt Änderungen in Zell-Zell- und Zell-Matrix-Interaktionen und wird durch zelluläre Adhäsionsmoleküle vermittelt. Das Ziel dieser Studie bestand darin, ein geeignetes Modell zur Untersuchung kutaner Strahlenreaktionen zu entwickeln und zu evaluieren.Material und Methodik:Es wurde ein humanes Kokultursystem etabliert, das aus der spontan immortalisierten Keratinozytenzelllinie HaCaT und primären Fibroblasten (HDFa) besteht, welche in einen speziellen Kollagenschwamm eingebettet sind. Dieses Hautorganmodell wurde charakterisiert und hinsichtlich seiner Eignung für strahlenbiologische Untersuchungen evaluiert. Hierzu wurde die Expression von β1-Integrin nach ionisierender Bestrahlung vergleichend im Organmodell und an HaCaT-Zellen untersucht (FACScan und Immunhistochemie). Des Weiteren wurde der Einfluss von ionisierender Strahlung auf die DNA-Fragmentierung im Organmodell analysiert (TUNEL-Assay).Ergebnisse:Das neue Hautorganmodell zeigte Charakteristika menschlicher Haut, wie durch immunhistochemische Färbungen von Zytokeratin und Ki-67 sowie durch elektronenmikroskopische Untersuchungen demonstriert werden konnte. Sowohl im Hautorganmodell als auch in HaCaT-Zellen wurde die Expression von β1-Integrin durch ionisierende Strahlung (5 Gy) heraufreguliert. Im Hautorganmodell fanden sich darüber hinaus nach Bestrahlung Umverteilungsphänomene des β1-Integrin-Signals in die oberen Schichten des Epidermisäquivalents. Des Weiteren konnte gezeigt werden, dass die Bestrahlung einen deutlichen Anstieg der DNA-Fragmentierung im Hautorganmodell bewirkte.Schlussfolgerung:Die vorliegenden Ergebnisse zeigen, dass das neue Hautorganmodell gut geeignet ist, um zelluläre Strahleneffekte unter dreidimensionalen Bedingungen zu analysieren. Es können Effekte untersucht werden, die sich in Monolayerzellkulturen nicht darstellen lassen.

Fas/Fas ligand system and apoptosis induction in testicular carcinoma

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).

APOPTOSIS IN HUMAN EMBRYONAL CELL CARCINOMA: PRELIMINARY RESULTS

Abstract Disorders in the regulation of apoptotic cell death may contribute to cancer. Furthermore, lymphocytes are supposed to play a role in counteracting tumorigenesis by inducing apoptosis in different human tumors. In this study, for the first time, tumor cell and lymphocyte apoptosis were investigated systematically in human embryonal cell carcinoma. DNA fragmentation and DNA condensation were measured simultaneously on double-fluorescence-labeled testis tumor sections using immunofluorescence microscopy. Different apoptotic indices (AIs), based either on biochemical (DNA fragmentation) or morphological criteria (DNA condensation) alone or on a combination of both, were determined in different histological regions in and around the tumor. Using morphological criteria alone, 40–75% of all apoptotic cells were not detected. Based on previous observations this finding might be related to subsets of apoptotic cells which induce the process of DNA condensation without activation of processes responsible for DNA fragmentation. Moreover, the AIs of tumor cells and lymphocytes were highest in the tumor region, compared with regions around the tumor and distant from it; these findings are discussed in the context of the Fas/FasL system.

Comparison of Endogenous TP53 Genomic Status with Clonogenicity and Different Modes of Cell Death after X Irradiation

Abstract Lu-Hesselmann, J., Abend, M. and van Beuningen, D. Comparison of Endogenous TP53 Genomic Status with Clonogenicity and Different Modes of Cell Death after X Irradiation. Radiat. Res. 161, 39–47 (2004). 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 (Dq, D0, 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 (Dq, D0, 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 Dq (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.