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Suberoylanilide hydroxamic acid affects γH2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation

Histon-Deacetylasehemmer SAHA beeinflusst die γH2AX-Expression in Sarkom- und Normalgewebszelllinien nach Bestrahlung

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Abstract

Purpose

Osteosarcoma and atypical teratoid rhabdoid tumors are tumor entities with varying response to common standard therapy protocols. Histone acetylation affects chromatin structure and gene expression which are considered to influence radiation sensitivity. The aim of this study was to investigate the effect of the combination therapy with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and irradiation on atypical teratoid rhabdoid tumors and osteosarcoma compared to normal tissue cell lines.

Methods

Clonogenic assay was used to determine cell survival. DNA double-strand breaks (DSB) were examined by pulsed-field electrophoresis (PFGE) as well as by γH2AX immunostaining involving flow cytometry, fluorescence microscopy, and immunoblot analysis.

Results

SAHA lead to an increased radiosensitivity in tumor but not in normal tissue cell lines. γH2AX expression as an indicator for DSB was significantly increased when SAHA was applied 24 h before irradiation to the sarcoma cell cultures. In contrast, γH2AX expression in the normal tissue cell lines was significantly reduced when irradiation was combined with SAHA. Analysis of initial DNA fragmentation and fragment rejoining by PFGE, however, did not reveal differences in response to the SAHA pretreatment for either cell type.

Conclusion

SAHA increases radiosensitivity in tumor but not normal tissue cell lines. The increased H2AX phosphorylation status of the SAHA-treated tumor cells post irradiation likely reflects its delayed dephosphorylation within the DNA damage signal decay rather than chromatin acetylation-dependent differences in the overall efficacy of DSB induction and rejoining. The results support the hypothesis that combining SAHA with irradiation may provide a promising strategy in the treatment of solid tumors.

Zusammenfassung

Zielsetzung

Osteosarkome und atypische teratoide Rhabdoidtumore sind Tumorentitäten mit sehr variablem Ansprechen auf aktuelle Standardtherapien. Die Acetylierung von Histonen beeinflusst die Regulierung von Chromatinstruktur und Genexpression, beides Parameter, die eine wichtige Rolle bei der Strahlenempfindlichkeit von Zellen bzw. Geweben spielen. Die vorliegende Studie untersucht den Effekt des Histon-Deacetylasehemmers SAHA in Kombination mit einer Strahlentherapie auf die o.g. Tumorentitäten im Vergleich zu Normalgewebszelllinien (Osteoblasten, Fibroblasten) in vitro.

Methoden

Das zelluläre Überleben wurde im klonogenen Assay, DNA-Doppelstrangbrüche (DSB) mittels Durchflusszytometrie, Mikroskopie, Immunoblot und Pulsfeld-Gelelektrophorese (PFGE) untersucht.

Ergebnisse

SAHA führte zu einer signifikant erhöhten Strahlenempfindlichkeit in den Tumor-zelllinien, nicht aber in den Normalgewebszellen. Die γH2AX-Expression als Marker für DSB erhöhte sich signifikant, wenn die Tumorzellen 24 h vor der Bestrahlung SAHA ausgesetzt wurden. Im Gegensatz dazu kam es in den Normalgewebszellen zu einer signifikant niedrigeren γH2AX-Expression, wenn die Zellen nicht nur bestrahlt, sondern kombiniert mit SAHA behandelt wurden. Die Analyse der initialen DNA-Fragmentierung bzw. deren Reparatur mittels PFGE zeigte jedoch keine Unterschiede zwischen SAHA- und nicht-SAHA-behandelten Zellen, sowohl in den Normalgewebs- als auch in den Tumorzelllinien.

Schlussfolgerung

SAHA führt zu einer Radiosensitivierung von Tumor-, nicht aber von Normalgewebszellen. Der gesteigerte H2AX-Phosphorylierungsstatus nach Bestrahlung bei den SAHA-behandelten Tumorzellen scheint am ehesten durch eine verzögerte Dephosphorylierung nach DNA-Schädigung bedingt zu sein, weniger durch eine Chromatinacetylierungs-modifizierte DSB-Induktion oder Reparatur. Die Ergebnisse unterstützen die Hypothese, dass die Kombination von Bestrahlung mit dem Histon-Deacetylasehemmer SAHA eine neue, potenziell entwicklungsfähige Strategie in der Behandlung von soliden Tumoren darstellt.

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Acknowledgments

We would like to thank Sylvia Trinh, Ludmilla Frick, and Gabriele Becker for their excellent technical work. This work was supported by the Dietmar Hopp Stiftung, Germany.

Conflict of interest

The corresponding author states that there are no conflicts of interest.

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Authors and Affiliations

  1. Department of Pediatric Oncology, Hematology, Immunology, and Pulmology, University Children’s Hospital, Im Neuenheimer Feld 430, 69120, Heidelberg, Germany

    C. Blattmann M.D., S. Oertel, M. Thiemann, K.J. Weber, P. Schmezer, O. Zelezny, R. Lopez Perez, A.E. Kulozik, J. Debus & V. Ehemann

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  1. C. Blattmann M.D.
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  2. S. Oertel
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Correspondence to C. Blattmann M.D..

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Blattmann, C., Oertel, S., Thiemann, M. et al. Suberoylanilide hydroxamic acid affects γH2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation. Strahlenther Onkol 188, 168–176 (2012). https://doi.org/10.1007/s00066-011-0028-5

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  • DOI: https://doi.org/10.1007/s00066-011-0028-5

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