Survivin inhibition and DNA double-strand break repair: a molecular mechanism to overcome radioresistance in glioblastoma.
Gliomas display prime examples of ionizing radiation (IR) resistant tumors. The IAP Survivin is reported to be critically involved in radiation resistance by anti-apoptotic and by caspase-independent mechanisms. The present study aimed to elucidate an interrelationship between Survivin's cellular localization and DNA damage repair in glioma cells.Cellular distribution and nuclear ... complex formation were assayed by immunoblotting, immunofluorescence staining and co-immunoprecipitation of Survivin bound proteins in LN229 glioblastoma cells. Apoptosis induction, survival and DNA repair following IR were assayed by means of caspase3/7 activity, clonogenic assay, ?-H2AX/53BP1 foci formation, single cell gel electrophoresis assay, and DNA-PKcs kinase assay in the presence of Survivin siRNA or over expression of Survivin-GFP.Following irradiation, we observed a nuclear accumulation and a direct interrelationship between Survivin, MDC1, ?-H2AX, 53BP1 and DNA-PKcs, which was confirmed by immunofluorescence co-localization. Survivin downregulation by siRNA resulted in an increased apoptotic fraction, decreased clonogenic survival and increased DNA-damage, as demonstrated by higher amount of DNA breaks and an increased amount of ?-H2AX/53BP1 foci post irradiation. Furthermore, we detected in Survivin-depleted LN229 cells a hampered S2056 (auto)phosphorylation and a significantly decreased DNA-PKcs kinase activity.Nuclear accumulation of Survivin and interaction with components of the DNA-double-strand break (DSB) repair machinery indicates Survivin to regulate DSB damage repair that leads to a significant improvement of survival of LN229 glioblastoma cells.
Mesh Terms:
Apoptosis, Caspases, Cell Survival, Comet Assay, DNA Breaks, Double-Stranded, DNA Damage, DNA Repair, DNA-Activated Protein Kinase, Gene Expression Regulation, Neoplastic, Glioblastoma, Humans, Immunoprecipitation, Inhibitor of Apoptosis Proteins, RNA, Small Interfering, Radiation Dosage, Radiation Tolerance, Radiation, Ionizing, Survivin, Tumor Cells, Cultured
Apoptosis, Caspases, Cell Survival, Comet Assay, DNA Breaks, Double-Stranded, DNA Damage, DNA Repair, DNA-Activated Protein Kinase, Gene Expression Regulation, Neoplastic, Glioblastoma, Humans, Immunoprecipitation, Inhibitor of Apoptosis Proteins, RNA, Small Interfering, Radiation Dosage, Radiation Tolerance, Radiation, Ionizing, Survivin, Tumor Cells, Cultured
Radiother Oncol
Date: Oct. 01, 2011
PubMed ID: 21852011
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