gms | German Medical Science

Objective: Ezrin (EZ) is a highly conserved actin-binding protein that crosslinks actin filaments to the plasma membrane via interaction with various surface receptors such as CD44 and EGFR. Through activation of downstream signal pathways EZ regulates cell scattering, survival and proliferation. EZ is overexpressed in some high-grade astrocytic tumors, thus being associated with malignancy and invasiveness of glial tumors. We intended to characterize the phenotype of EZ depleted glioma cells concerning proliferation, migration and clonogenic survival. In particular, we were interested in determining the correlation between ezrin expression levels and radiosensitivity.

Method: Retrovirus particle transduction of U343-MG and U373-MG cells was used to generate glioma cells with stable shRNA-mediated EZ-knockdown (KD). Following treatment with ionizing radiation EZ-KD and corresponding wild type (wt) cells were investigated with live cell or confocal laser scanning microscopy to analyze the intracellular EZ distribution. Additionally flow cytometry was applied to determine the proliferation index of cells using the live cell incorporation dye efluor670 and BrdU analysis.

Results: We observed a radiation dose dependent upregulation (wt cells) or downregulation (EZ-KD cells) of EZ protein levels in irradiated glioma cells after 6 days. Whereas in wt cells stable EZ upregulation was observed, EZ-KD cells restored their initial EZ level until 10 days post radiation if low radiation-doses were applied. The proliferation analysis revealed a putative EZ-dependent induction of a pool of slowly proliferating cells that were predominantly present in EZ-KD cells first observed at day 6 to 10 post radiation. In addition, immunoblot analysis of these cells showed constant Sox2 increase in EZ-KD cells.

Conclusions: Our data imply that irradiated EZ-KD cells are characterized by upregulation of the transcription factor Sox2, which in a small fraction of cells likely induce stem cell characteristics.