title: Survivin inhibition and DNA double-strand break repair: a molecular mechanism to overcome radioresistance in glioblastoma.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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, γ-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.
  
  
  RESULTS
  Following irradiation, we observed a nuclear accumulation and a direct interrelationship between Survivin, MDC1, γ-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 γ-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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Radiotherapy and Oncology, University of Frankfurt, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Reichert
authors.forename: Claus
authors.initials: C
authors.lastname: Rödel
authors.forename: Johanna
authors.initials: J
authors.lastname: Mirsch
authors.forename: Patrick N
authors.initials: PN
authors.lastname: Harter
authors.forename: Maja T
authors.initials: MT
authors.lastname: Tomicic
authors.forename: Michel
authors.initials: M
authors.lastname: Mittelbronn
authors.forename: Bernd
authors.initials: B
authors.lastname: Kaina
authors.forename: Franz
authors.initials: F
authors.lastname: Rödel
mesh_terms: D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects*; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D051747:DNA-Activated Protein Kinase; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000235:genetics* / Q000532:radiotherapy*; D006801:Humans; D047468:Immunoprecipitation; D051034:Inhibitor of Apoptosis Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011829:Radiation Dosage; D011836:Radiation Tolerance / Q000235:genetics*; D011839:Radiation, Ionizing; D000077022:Survivin; D014407:Tumor Cells, Cultured / Q000528:radiation effects
chemical_list: C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D034741:RNA, Small Interfering; D000077022:Survivin; D051747:DNA-Activated Protein Kinase; D020169:Caspases