Arrest of human lung fibroblasts in G2 phase after irradiation is regulated by converging phosphatidylinositol-3 kinase and beta1-integrin signaling in vitro.
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.
Cordes
Nils
N
Institute of Radiobiology, German Armed Forces, Neuherbergstrasse 11, Munich 80937, Germany. nils_cordes@web.de
van Beuningen
Dirk
D
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
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