Ionizing radiation induces up-regulation of functional beta1-integrin in human lung tumour cell lines in vitro.
OBJECTIVE
Cell-matrix interactions are in part mediated through the beta1-integrin pathway regulating cell survival, proliferation, adhesion and migration. This study was performed to elucidate alterations of expression of the beta1-integrin and its co-localized protein kinase, integrin-linked kinase (ILK), after exposure to ionizing radiation in two lung carcinoma cell lines in the presence or absence of different beta1-integrin-dependent matrix proteins.


METHODS
Exponentially growing A549 and SKMES1 cells grown on fibronectin, laminin, BSA or plastic were exposed to 2 Gy or 6 Gy. Besides colony formation assays (0.5-8 Gy) and immediate plating experiments, flow cytometry (for beta1-integrin) and immunoblotting (for beta1-integrin and ILK) were carried out to analyze the protein expression. The localization of both proteins plus filamentous (f-) actin was further examined by immunofluorescence staining and laser confocal scanning microscopy. Functionality of the beta1 receptor subunit after irradiation was investigated in adhesion assays.


RESULTS
A549 and SKMES1 cells grown on fibronectin or laminin demonstrated a significant increase in cell survival after irradiation compared to cells grown on BSA or plastic. Immediate plating of cells after irradiation on fibronectin did not show an improved survival. Flow cytometric and Western blot data showed a dose- and matrix-dependent induction of beta1-integrin and ILK expression after irradiation within 48 h. Adhesion to fibronectin or laminin compared to BSA or plastic was increased by 10-fold after irradiation, demonstrating these specific cell surface receptors to be functional. The staining of beta1-integrin and ILK in A549 cells confirmed the radiation-induced up-regulation of both proteins. Additionally, beta1-integrin and ILK co-localized with accumulated actin fibers at the cytoplasmic face of the cell membrane at confined areas.


CONCLUSIONS
Ionizing radiation strongly induced the expression of functional beta1-integrin and ILK in the two lung cancer cell lines, A549 and SKMES1, dependent on different matrices used. Additionally, the subcellular localization of both proteins was altered by irradiation, and the individual cellular radiosensitivity was reduced in the presence of an extracellular matrix. On the one hand, this may result in aggravated therapeutic approaches and on the other hand, cells could adhere more strongly in their environment by the increase in functional surface receptor density preventing metastasis. Concerning intravascular located tumour cells, beta1-integrin up-regulation might enable these cells to adhere to the endothelium, which represents a prerequisite for metastatic disease. Identification of such mechanisms will provide considerable insights into the understanding of tumorigenicity and metastatic phenotypes, possibly leading to new, optimized radiochemotherapeutic regimens.
Cordes
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N
Institute of Radiobiology, Federal Armed Forces Medical Academy, Neuherbergstrasse 11, 80937 Munich, Germany. cordes@firemail.de
Blaese
M A
MA
Meineke
V
V
Van Beuningen
D
D
D002448:Cell Adhesion / Q000528:radiation effects; D002470:Cell Survival / Q000528:radiation effects; D005109:Extracellular Matrix / Q000502:physiology; D006801:Humans; D019012:Integrin beta1 / Q000096:biosynthesis*; D008175:Lung Neoplasms / Q000503:physiopathology / Q000532:radiotherapy; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis*; D011836:Radiation Tolerance; D015398:Signal Transduction / Q000528:radiation effects; D013347:Subcellular Fractions / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay; D015854:Up-Regulation / Q000528:radiation effects; D000099200:Scaffold Protein ILK
D019012:Integrin beta1; D017346:Protein Serine-Threonine Kinases; D000099200:Scaffold Protein ILK