Arrest of human lung fibroblasts in G 2 phase after irradiation is regulated by converging phosphatidylinositol-3 kinase and β1-integrin signaling in vitro

Abstract

Purpose 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 β1-integrin signaling kinases integrin-linked kinase (ILK), protein kinase Bα/Akt (PKBα/Akt), and glycogen synthase kinase-3β (GSK-3β) was examined in normal lung fibroblasts in vitro. Methods and materials 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, PKBα/Akt, GSK-3β) 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, PKBα/Akt, and GSK-3β 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-3β in a PI3K-independent manner. Conclusion 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, PKBα/Akt, and GSK-3β 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.