title: Arrest of human lung fibroblasts in G2 phase after irradiation is regulated by converging phosphatidylinositol-3 kinase and beta1-integrin signaling in vitro.
abstract: OBJECTIVE
  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
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Radiobiology, German Armed Forces, Neuherbergstrasse 11, Munich 80937, Germany. nils_cordes@web.de
authors.forename: Nils
authors.initials: N
authors.lastname: Cordes
authors.forename: Dirk
authors.initials: D
authors.lastname: van Beuningen
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects*; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D002867:Chromones / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004790:Enzyme Induction; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects*; D005353:Fibronectins / Q000494:pharmacology*; D016195:G2 Phase; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D066298:In Vitro Techniques; D019012:Integrin beta1 / Q000378:metabolism; D008168:Lung / Q000166:cytology; D009025:Morpholines / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011137:Polystyrenes; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction; D000077191:Wortmannin; D000099200:Scaffold Protein ILK
chemical_list: D000730:Androstadienes; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D002867:Chromones; D016895:Culture Media, Serum-Free; D005353:Fibronectins; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D019012:Integrin beta1; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011137:Polystyrenes; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin; D000099200:Scaffold Protein ILK; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494918:AKT1 protein, human; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; C000605506:GSK3B protein, human