van Valen F, Kentrup-Lardong V, Truckenbrod B, Rübe C, Winkelmann W, Jürgens W W
Department of Orthopaedic Surgery, University of Münster, Germany.
J Cancer Res Clin Oncol. 1997;123(5):245-52. doi: 10.1007/BF01208634.
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.
本研究分析了来自尤因肉瘤（ES）/外周原始神经外胚层肿瘤（pPNET）的12种细胞系在暴露于γ射线和干扰素γ（IFNγ）之前和之后肿瘤坏死因子（TNF）α和可溶性TNF受体（sTNF-R）的产生情况。ES/pPNET细胞培养物的上清液在TNFα特异性扩增酶联免疫吸附测定（ELISA）、生物测定以及sTNF-Rp55和sTNF-Rp75 ELISA中进行检测。肿瘤细胞系释放少量的TNFα、大量的sTNF-Rp55（7/12细胞系）且不释放sTNF-Rp75。暴露于γ射线（5 Gy）要么诱导（3/12细胞系）要么上调（3/12细胞系）TNFα释放，而不改变sTNF-Rp55和sTNF-Rp75水平。用重组人IFNγ（rhIFNγ）预处理培养物显著增强了辐射反应性细胞系中TNFα的分泌，且对sTNF-Rp55和sTNF-Rp75水平无影响。rhIFNγ影响辐射反应的程度而非敏感性。分泌的TNFα具有生物活性，如对WEHI-164细胞的细胞毒性作用以及抗TNFα单克隆抗体对其活性的中和作用所示。赫比霉素A（一种酪氨酸特异性蛋白激酶抑制剂）而非钙泊三醇C（一种蛋白激酶C抑制剂）、H89（一种蛋白激酶A抑制剂）、AA-COCF3（一种磷脂酶A2特异性抑制剂）和MK-886（一种5-脂氧合酶特异性抑制剂）消除了γ射线刺激的TNFα释放。抗氧化剂N-乙酰半胱氨酸、去甲二氢愈创木酸和米帕林剂量依赖性地抑制γ射线介导的TNFα产生。总体而言，我们的研究结果表明，IFNγ预处理可增强ES/pPNET细胞在γ射线照射下生物活性TNFα的分泌，而不影响sTNF-R的释放。数据表明γ射线介导的导致TNFα产生的细胞内信号通路中需要蛋白酪氨酸激酶活性且活性氧起作用。
