REGULATION OF THE FAS RECEPTOR AND ITS LIGAND IN PEDIATRIC TUMORS

Fas是一种细胞表面受体，在被激活后诱导凋亡， 其自身配体或特异性单克隆抗体。 我们研究了Fas 神经母细胞瘤（NB）和外周原始神经外胚层中的mRNA水平 肿瘤（PNET）细胞系通过RT-PCR，并发现一致的高水平， PNET和NB中的低水平。 此外，抗Fas抗体诱导 PNET细胞凋亡，而NB细胞系中无凋亡。 然而，以前的研究 已经表明，各种细胞毒性和分化剂导致 NB细胞系的凋亡。 因此，我们假设， NB中的机器是完整的，并且NB细胞系未能经历 抗Fas诱导的细胞凋亡可能与Fas水平降低有关 受体，其导致与抗Fas抗体的相互作用不足。 先前的研究表明干扰素-γ增加Fas表达 在外周血单核细胞中，并增强细胞溶解活性 抗Fas抗体在结肠癌和乳腺癌细胞系中的表达。 我们 因此会尝试上调Fas受体， 干扰素-γ，并研究NB细胞对抗Fas的反应 抗体的 已知Fas受体与其配体的结合 导致胞浆内蛋白质聚集，如FADD 蛋白质，其结合Fas的胞质部分并抑制Fas的表达。 向下游分子传递信号。 我们将研究 FADD和Fas蛋白在对照未处理的NB细胞中，以及在 用干扰素-γ预处理并随后用干扰素-γ激发的细胞 抗Fas抗体诱导细胞凋亡的双重免疫荧光研究 标签。 FADD蛋白的加帽和与Fas的共定位 预期在用抗Fas抗体攻击的细胞中， 哪一个足够的正常序列的Fas蛋白可用于最佳的 约束力 在体内，凋亡是由Fas配体介导的， 通常在T淋巴细胞、巨噬细胞和一些其它细胞中表达。 理论上，细胞类型中Fas配体的上调可能会导致 这种细胞类型的凋亡与正反馈回路。 我们将 探讨Fas配体及其受体在NB中的可能诱导作用 细胞毒性剂如顺铂治疗的结果 和阿霉素。 我们还将调查 Fas受体-配体途径在细胞毒药物诱导细胞凋亡中的作用 使用抗Fas阻断抗体。

Fas is a cell surface receptor that induces apoptosis upon activation by its own ligand or a specific monoclonal antibody. We have studied Fas mRNA levels in neuroblastoma (NB) and peripheral primitive neuroectodermal tumor (PNET) cell lines by RT-PCR and found consistently high levels in PNET and low levels in NB. Furthermore, the anti-Fas antibody induced apoptosis in PNET but not in NB cell lines. However, previous studies have shown that a variety of cytotoxic and differentiating agents lead to apoptosis in NB cell lines. We therefore hypothesized that the apoptotic machinery in NB is intact and that the failure of NB cell lines to undergo anti-Fas induced apoptosis may be due to the low levels of the Fas receptor which lead to deficient interaction with the anti-Fas antibody. Previous studies have shown that interferon-gamma increases Fas expression in peripheral blood mononuclear cells and augments the cytolytic activity of the anti-Fas antibody in colon and breast carcinoma cell lines. We will therefore attempt to upregulate the Fas receptor with interferon-gamma and investigate the response of NB cells to the anti-Fas antibody. It is known that binding of the Fas receptor to its ligand results in clustering of intracytoplasmic proteins, such as the FADD protein, which bind to the cytosolic portion of Fas and transduce the signal to downstream molecules. We will investigate the localization of the FADD and the Fas proteins in control untreated NB cells, as well as in cells pre-treated with interferon-gamma and subsequently challenged with the apoptosis inducing anti-Fas antibody by double immunofluorescence labelling. Caping of the FADD protein and co-localization with the Fas protein is expected in cells challenged with the anti-Fas antibody in which sufficient Fas protein of normal sequence is available for optimal binding. In vivo, apoptosis is mediated by the Fas ligand, which is normally expressed in T lymphocytes, macrophages and a few other cells. Upregulation of the Fas ligand in a cell type could theoretically lead to apoptosis of this cell type with a positive feedback loop. We will investigate possible induction of the Fas ligand and its receptor in NB cells as the result of treatment with cytotoxic agents, such as cis-platin and adriamycin. We will also investigate the possible participation of the Fas receptor-ligand pathway in the cytotoxic drug-induced apoptosis using an anti-Fas blocking antibody.