REGULATION OF THE FAS RECEPTOR AND ITS LIGAND IN PEDIATRIC TUMORS

“Fas/FasL凋亡途径在 肿瘤细胞生长和进展。我们研究了这条途径 神经母细胞瘤（NB）和尤文肉瘤家族肿瘤 （ESFT）为了了解导致 在这些肿瘤中受损的程序性细胞死亡（凋亡）。的 本项目的目标是确定治疗的原因 NB和ESFT的失败，并可能找到操纵 Fas/FasL系统用于治疗目的。我们的材料包括 建立的人肿瘤细胞系和石蜡包埋的肿瘤 组织切片。这些研究采用的方法包括： 体外细胞毒性测定，DNA片段化ELISA，TUNEL， 电子显微镜，免疫染色，蛋白质印迹，反向 北方转录聚合酶链反应（RT-PCR） 印迹和DNA测序。我们的数据显示，NB和 ESFT表达Fas和FasL。然而，表达和连接 Fas受体不足以驱动肿瘤细胞自杀， 因为并非所有的肿瘤细胞都具有功能性Fas信号传导， 通路具体来说，我们发现凋亡信号是 在ESFT中传播，但在NB细胞系中不传播，尽管最初的 Fas受体在后者中的参与。我们发现， 外源性神经酰胺类似物诱导bcl-2 NB中的下调、caspase 3激活和凋亡， 表明凋亡信号在线粒体被阻断， bcl-2可能在肿瘤中起重要抑制作用， 作用在Fas反应性ESFT细胞系中，我们发现我们可以 不仅通过与Fas活化抗体连接诱导细胞凋亡， 而且还通过上调Fas和FasL的表达， 在没有增加合成的情况下细胞表面。增加的表 Fas和FasL的表达在用 合成金属蛋白酶抑制剂（MMPIs），其抑制 将全长FasL分子切割成其可溶形式。在 除了诱导子宫内膜异位症外，MMPIs还使ESFT细胞对 化疗药物阿霉素。这些发现支持， MMPI可能在治疗Fas敏感性ESFT中发挥作用， 可能与传统的 化疗剂。"

"The Fas/FasL apoptotic pathway is important in tumor cell growth and progression. We have studied this pathway in neuroblastoma (NB) and the Ewing sarcoma family of tumors (ESFT) in order to understand the molecular events that lead to impaired programmed cell death (apoptosis) in these tumors. The objective of this project is to identify the reasons for treatment failure in NB and ESFT and possibly find ways to manipulate the Fas/FasL system for therapeutic purposes. Our material consists of established human tumor cell lines and paraffin-embedded tumor tissue sections. The methods employed for these studies consist of in vitro cytotoxic assays, DNA fragmentation ELISA, TUNEL, electron microscopy, immunostaining, Western blotting, reverse transcription polymerase chain reaction (RT-PCR), Northern blotting and DNA sequencing. Our data have shown that NB and ESFT express Fas and FasL. However, the expression and ligation of the Fas receptor is not sufficient to drive tumor cells to suicide, because not all tumor cells possess a functional Fas signaling pathway. Specifically, we found that the apoptotic signal is transmitted in ESFT, but not in NB cell lines, despite an initial engagement of the Fas receptor in the latter. Our finding that an exogenously administered synthetic ceramide analog induced bcl-2 downregulation, caspase 3 activation and apoptosis in NB, suggested that the apoptotic signal is blocked at the mitochondrial level in this tumor and that bcl-2 may play an important inhibitory role. In the Fas-responsive ESFT cell lines, we found that we can induce apoptosis not only by ligation with a Fas activating antibody, but also by upregulation of the expression of Fas and FasL on the cell surface in the absence of increased synthesis. Increased surface expression of Fas and FasL was accomplished after treatment with synthetic metalloproteinase inhibitors (MMPIs) which inhibit the cleavage of the full length FasL molecule to its soluble form. In addition to inducing apoptotis, MMPIs sensitized ESFT cells to the chemotherapeutic agent doxorubicin.These findings support that MMPIs may play a role in the treatment of Fas-sensitive ESFT, possibly in combination treatments with traditional chemotherapeutic agents."