REGULATION OF THE RELEASE OF TUMOR-NECROSIS-FACTOR (TNF)ALPHA AND SOLUBLE TNF RECEPTOR BY GAMMA-IRRADIATION AND INTERFERON-GAMMA IN EWINGS-SARCOMA PERIPHERAL PRIMITIVE NEUROECTODERMAL TUMOR-CELLS

This study analyses the production of tumour necrosis factor (TNF)alph a 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 tumour s (pPNET). Supernatants from ES/pPNET cell cultures were tested in a T NF alpha-specific amplified enzyme-linked immunosorbent assay (ELISA), a bioassay, and sTNF-R-p55 and sTNF-R-p75 ELISA. The tumour cell line s released minimal amounts of TNF alpha, prominent amounts of sTNF-R-p 55 (7/12 cell lines) and no sTNF-R-p75. Exposure to gamma irradiation (5 Gy) either induced (3/12) cell lines) or up-regulated (3/12 cell li nes) TNF alpha release without changing sTNF-R-p55 and sTNF-R-p75 leve ls. Priming of cultures with recombinant human IFN gamma (rhIFN gamma) markedly enhanced TNF alpha secretion in the radiation-responsive cel l lines and had no influence on sTNF-R-p55 and sTNF-R-p75 levels. rhIF N gamma affected the magnitude rather than the sensitivity of the radi ation 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-speci fic 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 A(2)) and MK-886 (a specific inhibitor of 5 -lipoxygenase) abrogated gamma-irradiation-stimulated TNF alpha releas e. The anti-oxidants N-acetylcysteine, nordihydroguaiaretic acid and m epacrine dose-dependently inhibited gamma-irradiation-mediated TNF alp ha production. Collectively our findings indicate that IFN gamma primi ng 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 in tracellular signalling pathway leading to TNF alpha production.