Cell adhesion to the extracellular matrix protein fibronectin modulates radiation-dependent G2 phase arrest involving integrin-linked kinase (ILK) and glycogen synthase kinase-3β (GSK-3β) in vitro

Abstract

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 b1-integrin-linked kinase (ILK), protein kinase Ba/Akt (PKBa/Akt) and glycogen synthase kinase-3b (GSK-3b) activity, which participate in b1-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, PKBa/Akt and GSK-3b 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-Kindependently. These findings suggest a novel pathway that makes direct phosphorylation of GSK-3b by ILK feasible after irradiation. Conclusively, the data indicate that ILK, PKBa/Akt and GSK-3b 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.

Integrin‐dependent amplification of the G2 arrest induced by ionizing radiation

“…Cell division is a highly regulated process that is modulated in response to adverse conditions including ionizing radiation [40,41]. Signaling via adhesion of integrins to laminin 5 is known to be involved in progression through the G1 phase of the cell cycle [32,33,42] and other integrin/ligand interactions in the G2/M transition [35,43]. Our data support adhesion-mediated regulation of the G2 phase through the integrin ligand laminin 5.…”

“…G1 phase and S phase [34] are influenced by integrins and it has been recently shown that adhesion of cells to fibronectin through integrin receptors plays a role in regulating the G2 phase and entry into mitosis [35,36]. Signaling events through cell adhesion to laminin 5 through the α6β4 integrin may affect cell cycle regulation after DNA damage.…”

Knockdown of peroxisome proliferator-activated receptor-β induces less differentiation and enhances cell–fibronectin adhesion of colon cancer cells

“…ILK is the binding partner of integrin, associating with the cytoplasmic domain of integrin b1 and mediating integrin-dependent cell attachment (Hannigan et al, 1996). Inhibition of ILK activity or expression results in the inhibition of cell adhesion by its downstream signaling to protein kinase B/Akt and glycogen synthase kinase-3b (Cordes and van Beuningen, 2003;Hannigan et al, 2005)…”

Integrin-linked kinase interacts with caspase-9 and -8 in an adhesion-dependent manner for promoting radiation-induced apoptosis in human leukemia cells

“…The nonradioactive Akt kinase assay kit purchased from New England Biolabs GmbH (Frankfurt a.M., Germany) was modified to detect ILK kinase activity as described previously (Cordes and van Beuningen, 2003).…”

“…In great similarity to adherent growing cells (Zhang et al, 2002;Cordes and van Beuningen, 2003;Seidler et al, 2005), these events confer resistance to genotoxic agents such as ionizing radiation or drugs like bleomycin, etoposide or fludarabine.…”