Cell adhesion to the extracellular matrix protein fibronectin modulates radiation-dependent G2 phase arrest involving integrin-linked kinase (ILK) and glycogen synthase kinase-3beta (GSK-3beta) in vitro.
Cell adhesion to extracellular matrix (ECM) is thought to confer resistance against cell-damaging agents, that is, drugs and radiation, in tumour and normal cells in vitro. The dependence of cell survival on beta1-integrin-linked kinase (ILK), protein kinase Balpha/Akt (PKBalpha/Akt) and glycogen synthase kinase-3beta (GSK-3beta) activity, which participate in beta1-integrin signalling and cell cycle progression was investigated as a function of radiation exposure. Colony-formation assays on polystyrene, fibronectin (FN), laminin (LA), bovine serum albumin (BSA) or poly-L-lysine (poly-L) (0-8 Gy), kinase assays, flow cytometric DNA and annexin-V analysis and immunoblotting were performed in nonirradiated and irradiated (2 or 6 Gy) A549 human lung cancer cells and CCD32 normal human lung fibroblasts. Cell contact to FN in contrast to polystyrene elevated basal ILK, PKBalpha/Akt and GSK-3beta kinase activities in A549 and CCD32 cells, as well as the basal amount of A549 G2 phase cells. Irradiation on FN or LA as compared to polystyrene, BSA or poly-L significantly improved cell survival. Following irradiation, kinase activities were stimulated strongly on polystyrene but showed to be less prominent on FN, which was because of the FN-related basal induction. Following irradiation, FN compared to polystyrene enlarged and prolonged G2 arrest in both the cell lines. For the analysis of phosphatidylinositol-3 kinase (PI3-K) dependence of protein kinases and cell cycle transition, the PI3-K inhibitors LY294002 and wortmannin were used showing decreased kinase activities, antiproliferative and radiation-dependent G2 accumulation-abrogating effects accompanied by downregulation of cyclin D1 and phospho-pRb in cells attached to polystyrene. Fibronectin partly abrogated these effects PI3-K-independently. These findings suggest a novel pathway that makes direct phosphorylation of GSK-3beta by ILK feasible after irradiation. Conclusively, the data indicate that ILK, PKBalpha/Akt and GSK-3beta are involved in modulations of the cell cycle after irradiation. These interactions are strictly dependent on ECM components in a cell line-specific manner. Our findings provide molecular insights into mechanisms likely to be important for ECM-dependent cell survival and cellular radioresistance as well as tumour growth.
Cordes
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Institute of Radiobiology, German Armed Forces Medical Academy, Neuherbergstrasse 11, 80937 Munich, Germany. nils_cordes@web.de
van Beuningen
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D
D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000502:physiology*; D005353:Fibronectins / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D005434:Flow Cytometry; D016195:G2 Phase / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007700:Kinetics; D008168:Lung; D008175:Lung Neoplasms; D009025:Morpholines / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay; D014965:X-Rays; D000099200:Scaffold Protein ILK
D017304:Annexin A5; D002867:Chromones; D004791:Enzyme Inhibitors; D005353:Fibronectins; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D009025:Morpholines; D017346:Protein Serine-Threonine Kinases; D000099200:Scaffold Protein ILK; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C000605506:GSK3B protein, human