Regulation of the Fas Receptor and its Ligand in Pediatric Tumors

最明确的凋亡途径之一是由表面受体Fas介导的，当与Fas配体(FasL)连接时，Fas诱导形成死亡诱导信号复合体(DISC)。视盘由Fas受体、FADD和FLICE/caspase 8组成。caspase 8在视盘被激活，直接(I型细胞)或间接地通过从线粒体释放细胞色素c和激活Caspase9(II型细胞)来触发下游caspase的蛋白水解性激活。在II型细胞中，bcl2蛋白家族是重要的细胞凋亡抑制因子，抑制线粒体细胞色素c的释放。不同类型的肿瘤细胞都表达Fas和FasL，但并不是所有的肿瘤细胞都有Fas/FasL途径。由于细胞毒药物诱导细胞凋亡与其细胞内靶点无关，而Fas耐药肿瘤在体外对药物诱导的细胞凋亡表现出交叉耐药，我们假设Fas/FasL信号通路缺陷的肿瘤对治疗难以治疗，并表现出更具侵袭性的行为。我们研究了尤文斯肉瘤家族(ESF)和神经母细胞瘤(NB)肿瘤中Fas/FasL通路的信号成分，以了解导致其凋亡受损从而治疗失败的分子事件。我们发现许多ESF肿瘤在体外是Fas敏感的，而N-myc拷贝数高的NB细胞系是Fas耐药的。我们还证明，在NB细胞中，Fas信号通路需要线粒体(II型细胞)的参与，通过反义寡核苷酸下调bcl2，我们可以逆转它们对Fas介导的凋亡的抵抗。我们正在研究联合反义bcl2治疗和化疗对SCID小鼠NB模型的治疗效果。由于高N-myc拷贝数与体内侵袭性NB生物学有关，而侵袭性NB对现有的治疗方法无效，化疗药物和反义bcl2寡核苷酸联合治疗可能在体内被证明是有益的。我们的数据还表明，在体外具有Fas信号通路缺陷的NB细胞中，存在一种蛋白的表达，该蛋白最近被证明可以抑制Flice/caspase 8的激活，该蛋白被称为FLICE抑制蛋白(FLIP)。我们已经证明，翻转不仅存在于NB细胞系中，而且存在于侵袭性NB患者的肿瘤组织中。在Fas反应的ESFT细胞系中，我们发现我们不仅可以通过与Fas激活的抗体结合来诱导细胞凋亡，还可以通过诱导肿瘤细胞中Fas和FasL的表达增强来诱导细胞凋亡。这可以在翻译后用合成金属蛋白酶抑制剂(MMPI)处理后完成，MMPI可以抑制全长FasL分子的切割到其可溶形式，或者在转录上通过化疗药物上调Fas和FasL分子的合成来实现。我们通过FasL启动子激活实验证实了化疗药物对ESF肿瘤细胞中FasL的诱导。此外，我们还发现化疗药物可以通过激活核因子-kB转录因子来诱导FasL的表达。我们通过构建稳定的带有反义FasL序列的转基因细胞，阻断了FasL的合成，抑制了药物诱导的ESF肿瘤细胞的凋亡，进一步证实了FasL分子在化疗药物诱导ESF肿瘤细胞凋亡中的需求。这些发现支持化疗药物通过Fas/FasL途径诱导ESF肿瘤细胞凋亡。我们还发现，在体外，MMPI治疗与化疗药物具有协同作用，从而支持合成MMPI在治疗ESF肿瘤中的作用。-细胞凋亡，尤文肉瘤家族肿瘤(ESFT)，神经母细胞瘤，Fas/FasL，MMPIs，金属蛋白酶抑制剂，-人体组织，液体，细胞等。

One of the best defined apoptotic pathways is mediated by the surface receptor Fas, which upon ligation with the Fas ligand (FasL) induces the formation of a death-inducing signaling complex (DISC). The DISC consists of the Fas receptor, the adaptor molecule FADD and FLICE/caspase 8. Caspase 8 is activated at the DISC and triggers the proteolytic activation of downstream caspases, either directly (type I cells), or indirectly through release of cytochrome c from the mitochondria and activation of caspase 9 (type II cells). In type II cells, the bcl-2 family of proteins are important inhibitors of apoptosis inhibiting the release of cytochrome c from the mitochondria. Various tumor cell types express Fas and FasL, yet not all of them have a functional Fas/FasL pathway. Because cytotoxic drugs induce apoptosis, irrespective of their intracellular target, and Fas- resistant tumors show cross resistance to drug-induced apoptosis in vitro, we made the hypothesis that tumors with a defective Fas/FasL signaling pathway are refractory to treatment and exhibit a more aggressive behavior. We have investigated the signaling components of the Fas/FasL pathway in tumors of the Ewings sarcoma family (ESF) and in neuroblastoma (NB), in order to understand the molecular events that lead to their impaired apoptosis and hence treatment failures. This approach is expected to lead to the discovery of novel therapeutic agents in ESF tumors and NB.We have found that many ESF tumors are Fas- sensitive in vitro, whereas NB cell lines with high N-myc copy number are Fas-resistant. We also demonstrated that in NB cells, the Fas signaling pathway requires participation of mitochondria (type II cells) and by downregulating bcl-2 with antisense oligonucleotides we can reverse their resistance to Fas-mediated apoptosis. We are investigating the therapeutic efficacy of combined antisense bcl-2 treatment and chemotherapy in a NB model in SCID mice. Because high N- myc copy numbers have been associated with aggressive NB biology in vivo, and aggressive NB is refractory to existing therapeutic modalities, a combination treatment with chemotherapeutic agents and antisense bcl2 oligonucleotides may prove to be beneficial in vivo. Our data have also shown that NB cells with a defective Fas signaling pathway in vitro show expression of a protein that has been recently shown to inhibit the activation of FLICE/caspase 8 at the DISC and known as FLICE-inhibitory protein (FLIP). We have shown FLIP to be present not only in NB cell lines but also in tumor tissues from patients with aggressive NB. 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 inducing enhanced expression of Fas and FasL in tumor cells. This can be accomplished either post- translationally after treatment with synthetic metalloproteinase inhibitors (MMPIs) which inhibit the cleavage of the full length FasL molecule to its soluble form, or transcriptionally by upregulation of the synthesis of the the Fas and FasL molecules by chemotherapeutic agents. We confirmed the induction of FasL by chemotherapeutic agents in ESF tumor cells by FasL promoter activation experiments. Furthermore, we found that the chemotherapeutic agents can induce FasL expression through activation of the NF-kB transcription factor. We further confirmed the requirement of the FasL molecule for the induction of apoptosis by chemotherapeutic agents by generating stable transfectants with antisense FasL sequences which blocked FasL synthesis and inhibited the drug-induced apoptosis in ESF tumor cells. These findings support that chemotherapeutic agents use the Fas/FasL pathway to induce apoptosis in ESF tumors. We also showed that MMPI- treatment had a synergistic effect with chemotherapeutic agents in vitro, thus supporting a role for synthetic MMPIs in the treatment of ESF tumors. - apoptosis, Ewing's sarcoma family of tumors (ESFT), neuroblastoma, Fas/FasL, MMPIs, metalloproteinase inhibitors, - Human Tissues, Fluids, Cells, etc.