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Normal-Tissue Radioprotection by Overexpression of the Copper-Zinc and Manganese Superoxide Dismutase Genes

Radioprotektion des Normalgewebes durch Überexpression der Kupfer-Zink- und Mangan-Superoxiddismutase-Gene

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Background and Purpose:

Protection of normal tissue against radiation-induced damage may increase the therapeutic ratio of radiotherapy. A promising strategy for testing this approach is gene therapy-mediated overexpression of the copper-zinc (CuZnSOD) or manganese superoxide dismutase (MnSOD) using recombinant adeno-associated viral (rAAV2) vectors. The purpose of this study was to test the modulating effects of the SOD genes on human primary lung fibroblasts (HPLF) after irradiation.

Material and Methods:

HPLF were transduced with rAAV2 vectors containing cDNA for the CuZnSOD, MnSOD or a control gene. The cells were irradiated (1–6 Gy), and gene transfer efficiency, apoptosis, protein expression/activity, and radiosensitivity measured by the colony-forming assay determined.

Results:

After transduction, 90.0% ± 6.4% of the cells expressed the transgene. A significant fivefold overexpression of both SOD was confirmed by an SOD activity assay (control: 21.1 ± 12.6, CuZnSOD: 95.1 ± 17.1, MnSOD: 108.5 ± 36.0 U SOD/mg protein) and immunohistochemistry. CuZnSOD and MnSOD overexpression resulted in a significant radioprotection of HPLF compared to controls (surviving fraction [SF] ratio SOD/control > 1): CuZnSOD: 1.18-fold (95% confidence interval [CI]: 1.06–1.32; p = 0.005), MnSOD: 1.23-fold (95% CI: 1.07–1.43; p = 0.01).

Conclusion:

Overexpression of CuZnSOD and MnSOD in HPLF mediated an increase in clonogenic survival after irradiation compared to controls. In previous works, a lack of radioprotection in SOD-overexpressing tumor cells was observed. Therefore, the present results suggest that rAAV2 vectors are promising tools for the delivery of radioprotective genes in normal tissue.

Hintergrund und Ziel:

Der Ansatz, Normalgewebszellen gegen bestrahlungsinduzierte Schäden zu schützen, kann möglicherweise die therapeutische Breite strahlentherapeutischer Ansätze erhöhen. Ein potentieller Ansatz wäre die gentherapeutische Überexpression der Kupfer-Zink-(CuZnSOD) oder Mangan-Superoxiddismutase (MnSOD) mit rekombinanten Adeno-assoziierten viralen (rAAV2) Vektoren. Das Ziel dieser Studie war die Bestimmung der bestrahlungsmodulierenden Effekte der SOD-Gene auf humane primäre Lungenfibroblasten (HPLF).

Material und Methodik:

HPLF wurden mit rAAV2-Vektoren transduziert, die die cDNA für CuZnSOD, MnSOD oder ein Kontrollgen enthielten. Die Zellen wurden mit 1–6 Gy bestrahlt und deren Gentransfereffizienz, Apoptoserate, Proteinexpression/-aktivität und Strahlensensitivität mit einem Koloniebildungsassay bestimmt.

Ergebnisse:

Nach Transduktion exprimierten 90,0% ± 6,4% der Zellen das entsprechende Transgen. Für beide SOD konnte mittels Immunhistochemie sowie SOD-Aktivitätsassay eine signifikante Überexpression (fünffach; Kontrolle: 21,1 ± 12,6, CuZnSOD: 95,1 ± 17,1, MnSOD: 108,5 ± 36,0 U SOD/mg Gesamtprotein) gezeigt werden. Diese Überexpression führte zu einer signifikanten Radioprotektion der HPLF im Vergleich zu den Kontrollen (Überlebensfraktion-[SF-]Quotient SOD/Kontrolle > 1): CuZnSOD: 1,18-fach (95%-Konfidenzintervall [CI]: 1,06–1,32; p = 0,005), MnSOD: 1,23-fach (95%-CI: 1,07–1,43; p = 0,01).

Schlussfolgerung:

Im Vergleich zu den Kontrollen führte die Überexpression von CuZnSOD oder MnSOD in HPLF zu einem erhöhten klonogenen Überleben nach Bestrahlung. Da in vorherigen Arbeiten eine mangelnde Radioprotektion nach Überexpression der SOD-Gene in Tumorzellen beobachtet werden konnte, sind rAAV2-Vektoren erfolgversprechend für die Übertragung von radioprotektiven Genen in Normalgewebszellen.

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Authors and Affiliations

  1. Department of Radiation Oncology, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany

    Marlon R. Veldwijk, Carsten Herskind &  Frederik Wenz

  2. Pharmacology of Cancer Treatment (G402), German Cancer Research Center, Heidelberg, Germany

    Marlon R. Veldwijk, Leopold Sellner & W. Jens Zeller

  3. Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany

    Aleksandar Radujkovic

  4. Department of Experimental Surgery, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany

    Stephanie Laufs

  5. Molecular Oncology of Solid Tumors (G360), German Cancer Research Center, Heidelberg, Germany

    Stephanie Laufs

  6. Center for Tumor Diagnostic and Therapy, Paracelsus-Klinik, Osnabrück, Germany

    Stefan Fruehauf

  7. Department of Radiation Oncology, University Medical Center Mannheim, University of Heidelberg, 68135, Mannheim, Germany

    Frederik Wenz

Authors
  1. Marlon R. Veldwijk
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  2. Carsten Herskind
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  3. Leopold Sellner
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  4. Aleksandar Radujkovic
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  5. Stephanie Laufs
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  6. Stefan Fruehauf
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  7. W. Jens Zeller
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  8. Frederik Wenz
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Correspondence to Frederik Wenz.

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Veldwijk, M.R., Herskind, C., Sellner, L. et al. Normal-Tissue Radioprotection by Overexpression of the Copper-Zinc and Manganese Superoxide Dismutase Genes. Strahlenther Onkol 185, 517–523 (2009). https://doi.org/10.1007/s00066-009-1973-0

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  • DOI: https://doi.org/10.1007/s00066-009-1973-0

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