An essential role of integrin-linked kinase in the cellular radiosensitivity of normal fibroblasts during the process of cell adhesion and spreading

Abstract

Purpose: In addition to focal adhesion kinase (FAK), Paxillin and p130 Crk-associated substrate (p130Cas), integrin-linked kinase (ILK) mediates signals from β integrins for controlling, e.g., survival, adhesion and spreading. To evaluate the role of ILK in the cellular radiosensitivity at different stages of cell adhesion and spreading, ILK^{floxed/floxed (fl/fl)} and ILK^−/− mouse fibroblasts were examined.

Materials and methods: Cells were irradiated (0 – 4 Gy, X-rays) in suspension, after varying time periods on fibronectin (FN) or after 24 h on different matrix proteins. Irradiation was combined with phosphatidylinositol-3 kinase (PI3K) inhibition using Ly294002. Clonogenic radiation survival, cell adhesion, and kinetics of protein expression and phosphorylation during FN adhesion (ILK, v-akt murine thymoma viral oncogene homolog 1 (AKT), FAK, Paxillin, p130Cas) were examined.

Results: In suspension and during the first hour on FN, irradiated ILK^fl/fl cells survived significantly better than ILK^−/− cells in a PI3K- and serum-dependent manner. 24-h cell cultures on different matrix proteins showed no difference in radiosensitivity. During FN adhesion, which was slightly impaired in ILK^−/− cells, protein kinetics uncovered differences in AKT, FAK, Paxillin and p130Cas phosphorylation in the two cell lines. Phosphorylation of FAK, Paxillin and p130Cas was downregulated upon exposure to ionizing radiation in an ILK-independent manner.

Conclusions: These findings indicate a critical function of ILK in the cellular radiosensitivity during the early stages of adhesion to and spreading on FN. On the basis of the presented data, a precise correlation of adhesion-, serum- and PI3K-mediated changes in PI3K/AKT and FAK/Paxillin/p130Cas signaling cascades was not found. However, identifying the underlying mechanisms of adhesion- and spreading-related changes in the cellular radiosensitivity might be relevant for an optimization of radiotherapeutic strategies specifically targeting cells located at the invasive edge of a malignant tumor.

Keywords:

• Ionizing radiation
• ILK
• FAK
• Paxillin
• p130Cas