Fas ligand-independent, FADD-mediated activation of the Fas death pathway by anticancer drugs
Fas ligand-independent, FADD-mediated activation of the Fas death pathway by anticancer drugs
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DOI：
10.1074/jbc.274.12.7987
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发表时间：
1999-03-19
影响因子：
4.8
通讯作者：
Dimanche-Boitrel, MT
中科院分区：
文献类型：
--
作者：
Micheau, O;Solary, E;Dimanche-Boitrel, MT
Fas受体(CD95，APO-1)是一种膜结合蛋白，它的三聚体通过细胞凋亡来触发细胞死亡。Fas受体激活的主要死亡途径涉及连接Fas受体和caspase级联的接头蛋白FADD(Fas相关死亡结构域)。已有研究表明，抗癌药物可增强肿瘤细胞表面Fas受体和Fas配体的表达。Fas配体-Fas受体相互作用对这些药物的细胞毒活性的贡献仍存在争议。在这里，我们表明，拮抗性抗Fas抗体ZB4和Fas-IgG分子都不能抑制药物诱导的三种不同细胞系的凋亡。在未经处理的U937人白血病细胞中发现了质膜上Fas配体的表达，但在未经处理的人结肠癌细胞株HT29和HCT116中仍未检测到Fas配体的表达，这种表达不会被各种细胞毒剂改变。激光共聚焦扫描显微镜观察到这些药物诱导了Fas受体的聚集，免疫共沉淀证实了FADD与FADD的相互作用，稳定表达的FADD使肿瘤细胞对药物诱导的细胞死亡和细胞毒性变得敏感，而通过瞬时转染反义构建物下调FADD的表达降低了肿瘤细胞对药物诱导的凋亡的敏感性，这些结果通过瞬时转染编码FADD显性阴性突变体或抑制FADD/caspase-8途径的MC159或E8病毒蛋白的构建体得到证实。这些结果表明，药物诱导的细胞死亡涉及Fas/FADD途径，其方式不依赖于Fas配体。
Trimerization of the Fas receptor (CD95, APO-1), a membrane bound protein, triggers cell death by apoptosis. The main death pathway activated by Fas receptor involves the adaptor protein FADD (for Fas-associated death domain) that connects Fas receptor to the caspase cascade. Anticancer drugs have been shown to enhance both Fas receptor and Fas ligand expression on tumor cells. The contribution of Fas ligand-Fas receptor interactions to the cytotoxic activity of these drugs remains controversial. Here, we show that neither the antagonistic anti-Fas antibody ZB4 nor the Fas-IgG molecule inhibit drug-induced apoptosis in three different cell lines. The expression of Fas ligand on the plasma membrane, which is identified in untreated U937 human leukemic cells but remains undetectable in untreated HT29 and HCT116 human colon cancer cell lines, is not modified by exposure to various cytotoxic agents. These drugs induce the clustering of Fas receptor, as observed by confocal laser scanning microscopy, and its interaction with FADD, as demonstrated by co-immunoprecipitation, Overexpression of FADD by stable transfection sensitizes tumor cells to drug-induced cell death and cytotoxicity, whereas down-regulation of FADD by transient transfection of an antisense construct decreases tumor cell sensitivity to drug-induced apoptosis, These results were confirmed by transient transfection of constructs encoding either a FADD dominant negative mutant or MC159 or E8 viral proteins that inhibit the FADD/caspase-8 pathway. These results suggest that drug-induced cell death involves the Fas/FADD pathway in a Fas ligand-independent fashion.