Fas Ligand-independent, FADD-mediated Activation of the Fas Death Pathway by Anticancer Drugs*
- From the INSERM U517, Pôle Biologie et Thérapie des Cancers (JE 515), Faculty of Medicine and Pharmacy, 7 boulevard Jeanne d’Arc, 21033 Dijon Cedex, France
Abstract
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 deathdomain) 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.
Footnotes
-
↵* This work was supported by grants from the Conseil Régional de Bourgogne and the Burgundy, Saône et Loire, Nièvre, and national committees of the Ligue Nationale Contre le Cancer and by Grant 9567 from the Association pour la Recherche contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵‡ Supported by a grant from the Ligue Nationale Contre le Cancer (Saône et Loire).
-
↵§ To whom correspondence should be addressed. Tel.: 33-3-80-39-32-84; Fax: 33-3-80-67-17-88; E-mail: mtboitre{at}u-bourgogne.fr.
- Abbreviations:
- TNF
-
tumor necrosis factor
- Fas-L
-
Fas ligand
- sFas-L
-
soluble Fas-L
- CDDP
-
cisplatin
- VB
-
vinblastine
- AU
-
arbitrary unit
- PBS
-
phosphate-buffered saline
- VP16
-
etoposide
-
- Received August 7, 1998.
- Revision received November 23, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
