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Nachweis einer strahleninduzierten Produktion von Tumor-Nekrose-Faktor alpha im Ewing-Sarkom RM 82 in vitro und in vivo

Ionizing radiation induces production of tumor necrosis factor α in the Ewing’s sarcoma cell line RM 82 in vitro and in vivo

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Zusammenfassung

Fragestellung

Die Expression von Zytokinen aus Tumor- und Normalgewebszellen spielt bei der Vermittlung der Strahlenwirkung auf Tumoren eine wichtige Rolle. Tumor-Nekrose-Faktor alpha (TNF α), ein pleiotropes, primär als Monokin beschriebenes Polypeptid, ist durch seine zytotoxische Wirkung auf Tumorzellen sowie durch die Induktion hämorrhagischer Nekrosen von Tumoren von besonderem Interesse. Die vorliegende Arbeit untersucht den Einfluß ionisierender Strahlen auf die tumorzelleigene Expression von TNF α an einer humanen Ewing-Sarkom-Zellinie in vitro und in vivo am Xenograft-Tumor der Nacktmaus.

Material und Methode

Der TNF-α-Protein-und-mRNA-Gehalt der Zellinie RM 82 wurde in vitro mittles “Enhanced Amplified Sensitivity Immunoassay” (EASIA) und semiquantitativer RT-PCR vor und nach Bestrahlung mit Einzeldosen zwischen 2 und 40 Gy zu unterschiedlichen Zeitpunkten (1 bis 72 Stunden nach Bestrahlung) bestimmt. Nach Etablierung der Zellinie auf der Nacktmaus wurde in vivo ebenfalls eine Zeit- und Dosiskorrelation zwischen Bestrahlung und TNF-α-mRNA-Produktion vorgenommen.

Ergebnisse

Die Zellinie RM 82 exprimiert in vitro einen TNF-α-Basisproteinspiegel von 20,1±4,3 pg/ml/106 Zellen. Nach Bestrahlung zeigt sich ein zeit- und dosisabhängiger Anstieg der TNF-α-Expression um das maximal 5,9 fache 24 Stunden nach Bestrahlung mit 20 Gy. Die TNF-α-Produktion auf mRNA-Ebene in vitro zeigt ihr Maximum nach bereits sechs bis zwölf Stunden. Die Eigenschaft der TNF-α-Expression von RM 82 blieb in vivo im Xenograft-Tumor erhalten. Die TNF-α-mRNA nimmt hier ebenfalls mit der Zeit und der Dosis zu: Die maximale Zunahme der Produktion liegt bei zwölf Stunden nach Bestrahlung mit 10 Gy.

Schlußfolgerungen

Die vorliegende Untersuchung zeigt in vitro eine zeit- und dosisabhängige Mehrproduktion von TNF α der Ewing-Sarkom-Linie RM 82 auf Protein- und mRNA-Ebene. Erstmals wird dieses Phänomen auch am In-vivo-System beschrieben. Damit steht ein Modell zur näheren Untersuchung der Bedeutung von TNF α als tumoreigenem “radiation response modifier” zur Verfügung.

Abstract

Aim

The expression of cytokines plays an important role in the transmission of the effects of ionizing radiation to tumor cells and normal tissue. Tumor necrosis factor alpha (TNF α), a pleiotropic monokine, is of special interest because of its cytotoxic effect on tumor cells and the induction of hemorrhagic necrosis in tumors. We examined the influence of ionizing radiation on TNF α production in a human Ewing’s sarcoma cell line in vitro and in vivo.

Methods

The protein and mRNA levels of the Ewing’s sarcoma cell line RM 82 were examined in vitro with „Enhanced Amplified Sensitivity Immunoassay” (EASIA) and semiquantitative RT-PCR before and after treatment with single doses of 2 to 40 Gy, 1 to 72 hours after irradiation. After successful transplantation to nude mice, the time and dose correlation of TNF α mRNA production was examined in vivo.

Results

In vitro, RM 82 had a basal protein level of TNF α of 20.1±4.3 pg/ml/106 cells. We observed a time- and dose-dependent increase of TNF α expression with a maximum of 125 pg/ml/106 (5.9fold) 24 hours after irradiation with 20 Gy. At the mRNA level, the maximal up-regulation occurred 6 to 12 hours after 10 Gy. In vivo, the xenograft tumor maintained the capacity of TNF α expression. Time-and dose-dependency in mRNA production showed a maximum increase 6 hours after treatment with 10 Gy.

Conclusions

The presented experiments show in vitro a dose- and time-dependent up-regulation of TNF α in the Ewing’s sarcoma cell line RM 82 on protein and mRNA level. For the first time this phenomenon was also observed in vivo in a human xenograft tumor. This tumor model could be used for further experiments to examine the role of TNF α as a biologic radiation response modifier in human tumors.

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Authors and Affiliations

  1. Klinik und Poliklinik für Strahlentherapie, Radioonkologie der Universität, Albert-Schweitzer-Straße 39, D-48129, Münster, Deutschland

    C. Rübe, C. Finke & N. Willich

  2. Zellbiologisches Labor der Klinik und Poliklinik für Orthopädie (Direktor: Prof. Dr. W. Winkelmann), Deutschland

    F. van Valen

  3. Gerhard-Domagk-Institut für Pathologie (Direktor: Prof. Dr. W. Böcker) der Westfälischen Wilhelms-Universität Münster, Deutschland

    K. L. Schäfer & B. Dockhorn-Dworniczak

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Rübe, C., Finke, C., van Valen, F. et al. Nachweis einer strahleninduzierten Produktion von Tumor-Nekrose-Faktor alpha im Ewing-Sarkom RM 82 in vitro und in vivo. Strahlenther. Onkol. 173, 407–414 (1997). https://doi.org/10.1007/BF03038316

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