Irradiation and various cytotoxic drugs enhance tyrosine phosphorylation and beta(1)-integrin clustering in human A549 lung cancer cells in a substratum-dependent manner in vitro.
OBJECTIVE
Interactions of cells with a substratum, especially extracellular matrix proteins, initiate clustering of integrin receptors in the cell membrane. This process represents the initial step for the activation of signaling pathways regulating survival, proliferation, differentiation, adhesion, and migration, and could, furthermore, be important for cellular resistance mediating mechanisms against radiation or cytotoxic drugs. The lack of data elucidating the impact of irradiation or cytotoxic drugs on this important phenomenon led to this study on human A549 lung cancer cells in vitro.


METHODS
The human lung carcinoma cell line A549 grown on polystyrene or fibronectin (FN) was irradiated with 0-8 Gy or treated with cisplatin (0.1-50 microM), paclitaxel (0.1-50 nM), or mitomycin (0.1-50 microM). Colony formation assays, immunofluorescence staining in combination with activation of integrin clustering using anti-beta(1)-integrin antibodies (K20), and Western blotting for tyrosine phosphorylation under treatment of cells with the IC(50) for irradiation (2 Gy; IC(50) = 2.2 Gy), cisplatin (2 microM), paclitaxel (5 nM), or mitomycin (7 microM) were performed.


RESULTS
Attachment of cells to FN resulted in a significantly reduced radio- and chemosensitivity compared to polystyrene. The clustering of beta(1)-integrins examined by immunofluorescence staining was only stimulated by irradiation, cisplatin, paclitaxel, or mitomycin in case of cell attachment to FN. By contrast, tyrosine phosphorylation, as one of the major events following beta(1)-integrin clustering, showed a 3.7-fold, FN-related enhancement, and treatment of cells with the IC(50) of radiation, cisplatin, paclitaxel, or mitomycin showed a substratum-dependent induction.


CONCLUSIONS
For the first time, a strong influence of irradiation and a variety of cytotoxic drugs on the clustering of beta(1)-integrins could be shown. This event is a prerequisite for tyrosine phosphorylation and, thus, the activation of cellular mechanisms regulating survival, proliferation, and adhesion. These data are not only important for the understanding of cellular resistance against cytotoxic agents but, furthermore, for tumor progression, anchorage-independent cell growth, and, possibly, the optimization of radiochemotherapeutic strategies.
Cordes
Nils
N
Institute of Radiobiology, German Armed Forces, Munich, Germany. nils_cordes@web.de
Beinke
Christina
C
Plasswilm
Ludwig
L
van Beuningen
Dirk
D
D000704:Analysis of Variance; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D015153:Blotting, Western; D002448:Cell Adhesion; D045744:Cell Line, Tumor / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival; D002945:Cisplatin / Q000494:pharmacology; D003114:Colony-Forming Units Assay; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D039641:Integrin beta Chains / Q000378:metabolism*; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000532:radiotherapy*; D018613:Microscopy, Confocal; D016685:Mitomycin / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D010766:Phosphorylation; D011137:Polystyrenes / Q000378:metabolism; D011829:Radiation Dosage; D011836:Radiation Tolerance; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*
D000903:Antibiotics, Antineoplastic; D000970:Antineoplastic Agents; D000972:Antineoplastic Agents, Phytogenic; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D039641:Integrin beta Chains; D011137:Polystyrenes; D011838:Radiation-Sensitizing Agents; D014443:Tyrosine; D016685:Mitomycin; D017239:Paclitaxel; D002945:Cisplatin