Fas ligand-independent, FADD-mediated activation of the Fas death pathway by anticancer drugs

Trimerization of the Fas receptor (CD95, APO-1), a membrane bound protein, triggers cell death by apoptosis. The main death pathway activated by Fas r eceptor involves the adaptor protein FADD (for Fas-associated death domain) that connects Fas receptor to the caspase cascade. Anticancer drugs have b een shown to enhance both Fas receptor and Fas ligand expression on tumor c ells. The contribution of Fas ligand-Fas receptor interactions to the cytot oxic activity of these drugs remains controversial. Here, we show that neit her the antagonistic anti-Fas antibody ZB4 nor the Fas-IgG molecule inhibit drug-induced apoptosis in three different cell lines. The expression of Fa s ligand on the plasma membrane, which is identified in untreated U937 huma n leukemic cells but remains undetectable in untreated HT29 and HCT116 huma n 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 demon strated by co-immunoprecipitation, Overexpression of FADD by stable transfe ction sensitizes tumor cells to drug-induced cell death and cytotoxicity, w hereas down-regulation of FADD by transient transfection of an antisense co nstruct decreases tumor cell sensitivity to drug-induced apoptosis, These r esults were confirmed by transient transfection of constructs encoding eith er a FADD dominant negative mutant or MC159 or E8 viral proteins that inhib it the FADD/caspase-8 pathway. These results suggest that drug-induced cell death involves the Fas/FADD pathway in a Fas ligand-independent fashion.