The Journal of Experimental Medicine
Torrey Pines Biolabs
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Published 2 August 2004. doi:10.1084/jem.20040213
Rockefeller University Press, 0022-1007 $8.00
JEM, Volume 200, Number 3, 353-365
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Intracellular Triggering of Fas Aggregation and Recruitment of Apoptotic Molecules into Fas-enriched Rafts in Selective Tumor Cell Apoptosis

Consuelo Gajate1, Esther del Canto-Jañez1, A. Ulises Acuña2, Francisco Amat-Guerri3, Emilio Geijo4, Antonio M. Santos-Beneit1, Robert J. Veldman1, and Faustino Mollinedo1

1 Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (C.S.I.C.), Universidad de Salamanca, E-37007 Salamanca, Spain
2 Instituto de Química Física Rocasolano and 3 Instituto de Química Orgánica, C.S.I.C., E-28006 Madrid, Spain
4 Instituto de Neurociencias, C.S.I.C., Universidad Miguel Hernández, E-03550 Alicante, Spain

Address correspondence to Faustino Mollinedo, Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, CSIC, Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain. Phone: 34-923-294806; Fax: 34-923-294795; email: fmollin{at}usal.es

We have discovered a new and specific cell-killing mechanism mediated by the selective uptake of the antitumor drug 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, Edelfosine) into lipid rafts of tumor cells, followed by its coaggregation with Fas death receptor (also known as APO-1 or CD95) and recruitment of apoptotic molecules into Fas-enriched rafts. Drug sensitivity was dependent on drug uptake and Fas expression, regardless of the presence of other major death receptors, such as tumor necrosis factor (TNF) receptor 1 or TNF-related apoptosis-inducing ligand R2/DR5 in the target cell. Drug microinjection experiments in Fas-deficient and Fas-transfected cells unable to incorporate exogenous ET-18-OCH3 demonstrated that Fas was intracellularly activated. Partial deletion of the Fas intracellular domain prevented apoptosis. Unlike normal lymphocytes, leukemic T cells incorporated ET-18-OCH3 into rafts coaggregating with Fas and underwent apoptosis. Fas-associated death domain protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid were recruited into rafts, linking Fas and mitochondrial signaling routes. Clustering of rafts was necessary but not sufficient for ET-18-OCH3–mediated cell death, with Fas being required as the apoptosis trigger. ET-18-OCH3–mediated apoptosis did not require sphingomyelinase activation. Normal cells, including human and rat hepatocytes, did not incorporate ET-18-OCH3 and were spared. This mechanism represents the first selective activation of Fas in tumor cells. Our data set a framework for the development of more targeted therapies leading to intracellular Fas activation and recruitment of downstream signaling molecules into Fas-enriched rafts.

Key Words: CD95 • Fas signaling • death receptor • antitumor ether lipid • ET-18-OCH3

Abbreviations used in this paper: CTx, cholera toxin; DISC, death-inducing signaling complex; ET-18-OCH3, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; FADD, Fas-associated death domain protein; FasL, Fas ligand; JNK, c-Jun amino-terminal kinase; SMase, sphingomyelinase; TLC, thin layer chromatography; TRAIL, TNF-related apoptosis-inducing ligand; TUNEL, TdT-mediated dUTP nick-end labeling.


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