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Endothelial adhesion of synchronized gastric tumor cells changes during cell cycle transit and correlates with the expression level of CD44 splice variants

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World Journal of Gastroenterology 2005年第40期11卷 6243-6248页

作者： Anton Oertl Jens Castein Tobias Engl Wolf-Dietrich Beecken dietger jonas Richard Melamed Roman A. Blaheta Zentrum der Chirurgie

AIM To study adhesion capacity and CD44 expression of human gastric adenocarcinoma MKN45 cells at different stages of a first cell cycle. METHODS MKN45 cells were synchronized by aphidicolin and assayed for adhesion to an endothelial cell （HUVEC） monolayer. Surface expression of CD44 and CD44 splice variants on MKN45 cells was evaluated by flow cytometry. Functional relevance of CD44 adhesion receptors was investigated by blocldng studies using anti CD44 monodonal antibodies or by hyaluronan digestion. RESULTS： Adhesion of MKN45 to HUVEC was increased during G2/M transit, after which adhesion returned to baseline levels with cell cycle completion. In parallel, CD44 splice vadants CD44v4, CD44v5, and CD44v7 were all upregulated on MKN45 during cell cycle progression with a maximum effect in G2/M. The function of CD44 surface receptors was assessed with specific receptor blocking monoclonal antibodies or removal of hyaluronan by digestion with hyaluronidase. Both strategies inhibited tumor cell adhesion to HUVEC by nearly 50%, which indicates that MKN45-HUVEC-interaction is CD44 dependent. CONCLUSION CD44 expression level is linked to the cell cycle in gastrointestinal tumor cells, which in turn leads to cell cycle dependent alterations of their adhesion behaviour to endothelium.

关键词： Cell cycle CD44 Adhesion Gastrointestinal tumors

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Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique

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World Journal of Gastroenterology 2005年第14期11卷 2080-2087页

作者： Marcus K.H. Auth Kim A. Boost Kerstin Leckel Wolf-Dietrich Beecken Tobias Engl dietger jonas Elsie Oppermann Philip Hilgard Bernd H. Markus Wolf-Otto Bechstein Roman A. Blaheta Universit(a|¨)ts-Kinderklinik Essen Abteilung für Allgemeine P(a|¨)diatrie Germany Zentrum der An(a|¨)sthesiologie und Wiederbelebung Johann Wolfgang Goethe-Universit(a|¨)t Frankfurt am MainGermany Klinik für Allgemein-und Gef(a|¨)βchirurgie Germany Klinik für Urologie und Kinderurologie Zentrum der Chirurgie Johann Wolfgang Goethe-Universit(a|¨)tFrankfurt am MainGermany Klinik für Allgemein- und Gef(a|¨)βchirurgie Germany Universit(a|¨)tsklinikum Essen Abteilung für Gastroenterologie und Hepatologie Germany Klinik für Allgemein- und Viszeralchirurgie St(a|¨)dt. Klinikum Kemperhof KoblenzGermany

AIM:Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GMCSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome dedifferentiation, which occurs during continuous stimulation by means of growth factors.

关键词： Human hepatocytes Differentiation Hepatocyte growth factor Epidermal growth factor HGF receptor EGF receptor Cytokeratin Collagen gel

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