title: Regulation of the release of tumour necrosis factor (TNF)alpha and soluble TNF receptor by gamma irradiation and interferon gamma in Ewing's sarcoma/peripheral primitive neuroectodermal tumour cells.
abstract: This study analyses the production of tumour necrosis factor (TNF)alpha and soluble TNF receptor (sTNF-R) before and after exposure to gamma irradiation and interferon gamma (IFN gamma) in 12 cell lines derived from Ewing's sarcoma (ES)/peripheral primitive neuroectodermal tumours (pPNET). Supernatants from ES/pPNET cell cultures were tested in a TNF alpha-specific amplified enzyme-linked immunosorbent assay (ELISA), a bioassay, and sTNF-Rp55 and sTNF-Rp75 ELISA. The tumour cell lines released minimal amounts of TNF alpha, prominent amounts of sTNF-Rp55 (7/12 cell lines) and no sTNF-Rp75. Exposure to gamma irradiation (5 Gy) either induced (3/12) cell lines) or up-regulated (3/12 cell lines) TNF alpha release without changing sTNF-Rp55 and sTNF-Rp75 levels. Priming of cultures with recombinant human IFN gamma (rhIFN gamma) markedly enhanced TNF alpha secretion in the radiation-responsive cell lines and had no influence on sTNF-Rp55 and sTNF-Rp75 levels. rhIFN gamma affected the magnitude rather than the sensitivity of the radiation response. The TNF alpha secreted was bioactive, as shown by its cytotoxic effect of WEHI-164 cells, and neutralization of its activity by anti-TNF alpha monoclonal antibody. Herbimycin A (a tyrosine-specific protein kinase inhibitor) but not calphostin C (a protein kinase C inhibitor), H89 (a protein kinase A inhibitor), AA-COCF3 (a specific inhibitor of phospholipase A2) and MK-886 (a specific inhibitor of 5-lipoxygenase) abrogated gamma-irradiation-stimulated TNF alpha release. The antioxidants N-acetylcysteine, nordihydroguaiaretic acid and mepacrine dose-dependently inhibited gamma-irradiation-mediated TNF alpha production. Collectively our findings indicate that IFN gamma priming potentiates the secretion of bioactive TNF alpha by ES/pPNET cells in response to gamma irradiation without affecting sTNF-R release. The data suggest a requirement for protein tyrosine kinase activity and a role for reactive oxygen species in the gamma-irradiation-mediated intracellular signalling pathway leading to TNF alpha production.
authors.affiliation: Department of Orthopaedic Surgery, University of Münster, Germany.
authors.forename: F
authors.initials: F
authors.lastname: van Valen
authors.forename: V
authors.initials: V
authors.lastname: Kentrup-Lardong
authors.forename: B
authors.initials: B
authors.lastname: Truckenbrod
authors.forename: C
authors.initials: C
authors.lastname: Rübe
authors.forename: W
authors.initials: W
authors.lastname: Winkelmann
authors.forename: W W
authors.initials: WW
authors.lastname: Jürgens
mesh_terms: D000970:Antineoplastic Agents / Q000627:therapeutic use*; D000975:Antioxidants / Q000494:pharmacology; D001859:Bone Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000532:radiotherapy; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D007371:Interferon-gamma / Q000627:therapeutic use*; D017599:Neuroectodermal Tumors / Q000188:drug therapy / Q000378:metabolism* / Q000532:radiotherapy; D010524:Peripheral Nervous System Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000532:radiotherapy; D018714:Radiotherapy, Adjuvant / Q000379:methods; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D012512:Sarcoma, Ewing / Q000188:drug therapy / Q000378:metabolism* / Q000532:radiotherapy; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D000975:Antioxidants; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma