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. 2003 May 6;88(9):1470-9.
doi: 10.1038/sj.bjc.6600912.

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

N Cordes  1 , D van Beuningen
Affiliations

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

N Cordes et al. Br J Cancer. .

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 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.

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Figures

Figure 1
Figure 1
Analysis of substrate-dependent clonogenic cell survival was accomplished by plating human lung carcinoma cells A549 and normal human lung fibroblasts CCD32 24 h prior to irradiation onto polystyrene (•) or a specific protein (▴: FN, LA, BSA, poly-L). P<0.05 was found for radiation doses ⩾4 Gy for FN- or LA-attached A549 cells and ⩾2 Gy for FN- or LA-attached CCD32 cells, compared to polystyrene, BSA or poly-L. Each data point represents the mean±s.d. of three independent experiments (n=18).
Figure 2
Figure 2
Protein kinase activities of ILK (A) and PKBα/Akt (B) and GSK-3β phosphorylation at the amino-acid residue Ser9 (C) were examined in A549 and CCD32 cells attached to polystyrene (P) or FN at 5 or 60 min after irradiation with 6 Gy (right panels). Basal kinase activities were strongly stimulated by FN in both tumour and normal cells (left panels). Radiation-dependent increases of ILK and PKBα/Akt activity and GSK-3β phosphorylation demonstrated to be pronounced on polystyrene and less prominent on FN. Additionally, ILK, PKBα/Akt and GSK-3β protein were detected to exclude changes in total protein amounts and β-actin served as loading control (data not shown). Each data point shown represents the fold induction (means±s.d.) of protein kinase activity or Ser9 phosphorylation of GSK-3β from the densitometric protein band analysis of three independent experiments in relation to untreated controls (mock). Inset, photographic demonstration of one exemplary protein kinase assay used for densitometric analysis. To examine the dependence of ILK, PKBα/Akt activity and GSK-3β phosphorylation on the PI3-K pathway, cells attached to FN or polystyrene were incubated with the PI3-K-specific inhibitors LY294002 (50 μM) (D) or wortmannin (100 nM) (E) in serum-free medium and then irradiated with 6 Gy (IR). The data uncovered PI3-K-dependent decrease of basal and radiation-induced kinase activities (ILK and PKBα/Akt at 5 min; GSK-3β at 60 min) in cells grown on FN or polystyrene. However, irradiation on FN was able to stimulate ILK and GSK-3β independent of PI3-K indicating the involvement of yet unknown signalling pathways.
Figure 3
Figure 3
(A–F) An 18-h inhibition of PI3-K by LY294002 using concentrations from 0.1 to 100 μM in combination with serum starvation resulted in a nonlinear and polystyrene (P)- or FN-independent dose–response relation for A549 (A) and CCD32 cells (D). Determination of apoptosis of A549 (B) or CCD32 cells (E) induced by LY294002 (LY) plus serum starvation (SD) on polystyrene or FN in comparison with untreated controls (nt) was performed by annexin-V staining and flow cytometric analysis. Cell viability was >90% in all the cases. The radiation-dependent clonogenic cell survival of A549 (C) and CCD32 cells (F) grown on FN or polystyrene was tested after an 18-h incubation with the PI3-K inhibitor LY294002 (50 μM) in serum-free medium. The data indicate no significant change of the clonogenic survival of irradiated cells on FN, whereas on polystyrene the survival was further reduced by LY294002 at 2 and 6 Gy in A549 cells and at 6 Gy in normal lung fibroblasts significantly (P<0.05).
Figure 4
Figure 4
G2 phase distribution of nonirradiated or irradiated (2 or 6 Gy) A549 (•) and CCD32 cells (○) grown on polystyrene or FN is shown. Each data point represents the mean ± s.d. of three independent experiments. A549 cells, but not CCD32 cells, indicated an elevated basal amount of G2 cells at FN presence. Fibronectin prolonged and increased the radiation-induced G2 phase blockage in both the cell lines in a dose-dependent manner.
Figure 5
Figure 5
Changes in the expression of indicated cell cycle proteins in nonirradiated (mock) or irradiated (6 Gy) A549 and CCD32 cells grown on polystyrene or FN are shown. Protein extracts of irradiated cells were isolated 2, 6, 12 and 24 h postradiation exposure. Detection of β-actin is shown to confirm equal loading of all lanes.
Figure 6
Figure 6
Cell cycle alterations by the PI3-K-specific inhibitor LY294002 (LY) were analysed in the presence or absence of FN using nonirradiated A549 and CCD32 controls in comparison with irradiated cells. Irradiation was delivered following an 18-h incubation with 50 μM of LY294002 in serum-free medium. Each data point represents the mean±s.d. of three independent experiments. LY294002 led to a strong decrease in G2 phase cells on polystyrene, which was partly prevented by FN. Radiation-dependent accumulation of cells in the G2 phase was completely prevented on polystyrene and markedly decreased on FN in comparison with irradiated cells not exposed to LY294002 as shown in Figure 4.
Figure 7
Figure 7
Cyclin D1 expression and pRb phosphorylation were investigated after a 12-h LY294002 (50 μM) incubation in A549 and CCD32 cells grown on polystyrene or FN in comparison with untreated controls (mock). Correlating the decreases of cyclin D1 expression and pRb phosphorylation with the DNA analysis shown in Figure 6 supported the important role of PI3-K-dependent growth factor pathways for the regulation of cell division and associated cell cycle proteins at FN presence and absence. Most interestingly, cells attached to FN did not demonstrate a downregulation of cyclin D1 expression or pRb phosphorylation, which indicates a strong influence of ECM on the cell cycle regulation via ILK-GSK-3β.
Figure 8
Figure 8
Schematic diagram of how integrin-linked kinase (ILK) and glycogen synthase kinase-3β (GSK-3β) might be involved in the acute radiation response with regard to cell survival, cell cycle progression and initiation of G2 phase arrest. Binding of cells to extracellular matrix (ECM) components via β1-integrins stimulates ILK and downstream targets protein kinase Bα/Akt (PKBα/Akt) (phosphorylation at amino-acid residues Ser473 and Thr308) and GSK-3β (phosphorylation at amino-acid residue Ser9). These events suppress apoptosis and promote survival by inhibiting Bad and caspase-9 and cell cycle transition by blocking proteolysis of cyclin D1. Facilitating growth factor binding to growth factor receptors (GFR) activates similar pathways downstream of the central regulator phosphatidylinositol-3 kinase (PI3-K). In bold letters, arrows and circles, we suggest and thereby support the hypothesis of direct ILK phosphorylation of GSK-3β when PI3-K is inhibited and cells are attached to ECM. Irradiation (IR) is able to activate this pathway, which then does not stimulate proliferation but rather blocks cells in the G2 phase possibly allowing damage repair.
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