TY - JOUR
T1 - Survivin inhibition and DNA double-strand break repair
T2 - A molecular mechanism to overcome radioresistance in glioblastoma
AU - Reichert, Sebastian
AU - Rödel, Claus
AU - Mirsch, Johanna
AU - Harter, Patrick N.
AU - Tomicic, Maja T.
AU - Mittelbronn, Michel
AU - Kaina, Bernd
AU - Rödel, Franz
N1 - Funding Information:
This work was supported by a grant of the German Research Foundation (DFG RO 3482/1-1) and the University of Frankfurt am Main within the Frankfurt Initiative for Neurooncological research (FIN). The authors gratefully acknowledge the excellent technical assistance of Mr. Julius Oppermann.
PY - 2011/10
Y1 - 2011/10
N2 - Background and purpose: 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. Material and 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. Conclusion: 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.
AB - Background and purpose: 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. Material and 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. Conclusion: 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.
KW - Apoptosis
KW - DNA-PKcs
KW - DNA-repair
KW - Glioblastoma
KW - Phospho-histone H2AX
KW - Survivin
UR - http://www.scopus.com/inward/record.url?scp=80655124597&partnerID=8YFLogxK
U2 - 10.1016/j.radonc.2011.06.037
DO - 10.1016/j.radonc.2011.06.037
M3 - Article
C2 - 21852011
AN - SCOPUS:80655124597
SN - 0167-8140
VL - 101
SP - 51
EP - 58
JO - Radiotherapy and Oncology
JF - Radiotherapy and Oncology
IS - 1
ER -