Involvement of raft aggregates enriched in Fas/CD95 death-inducing signaling complex in the antileukemic action of edelfosine in Jurkat cells.
BACKGROUND
Recent evidence suggests that co-clustering of Fas/CD95 death receptor and lipid rafts plays a major role in death receptor-mediated apoptosis.


RESULTS
By a combination of genetic, biochemical, and ultrastructural approaches, we provide here compelling evidence for the involvement of lipid raft aggregates containing recruited Fas/CD95 death receptor, Fas-associated death domain-containing protein (FADD), and procaspase-8 in the induction of apoptosis in human T-cell leukemia Jurkat cells by the antitumor drug edelfosine, the prototype compound of a promising family of synthetic antitumor lipids named as synthetic alkyl-lysophospholipid analogues. Co-immunoprecipitation assays revealed that edelfosine induced the generation of the so-called death-inducing signaling complex (DISC), made up of Fas/CD95, FADD, and procaspase-8, in lipid rafts. Electron microscopy analyses allowed to visualize the formation of raft clusters and their co-localization with DISC components Fas/CD95, FADD, and procaspase-8 following edelfosine treatment of Jurkat cells. Silencing of Fas/CD95 by RNA interference, transfection with a FADD dominant-negative mutant that blocks Fas/CD95 signaling, and specific inhibition of caspase-8 prevented the apoptotic response triggered by edelfosine, hence demonstrating the functional role of DISC in drug-induced apoptosis. By using radioactive labeled edelfosine and a fluorescent analogue, we found that edelfosine accumulated in lipid rafts, forming edelfosine-rich membrane raft clusters in Jurkat leukemic T-cells. Disruption of these membrane raft domains abrogated drug uptake and drug-induced DISC assembly and apoptosis. Thus, edelfosine uptake into lipid rafts was critical for the onset of both co-aggregation of DISC in membrane rafts and subsequent apoptotic cell death.


CONCLUSIONS
This work shows the involvement of DISC clusters in lipid raft aggregates as a supramolecular and physical entity responsible for the induction of apoptosis in leukemic cells by the antitumor drug edelfosine. Our data set a novel framework and paradigm in leukemia therapy, as well as in death receptor-mediated apoptosis.
Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas, Universidad de Salamanca, Salamanca, Spain. cgajate@usal.es
Consuelo
C
Gajate
Fernando
F
Gonzalez-Camacho
Faustino
F
Mollinedo
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D053181:Caspase 8 / Q000378:metabolism; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D019169:Jurkat Cells; D015458:Leukemia, T-Cell / Q000473:pathology*; D008563:Membrane Lipids / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010742:Phospholipid Ethers / Q000494:pharmacology*; D015398:Signal Transduction; D019014:fas Receptor / Q000378:metabolism*
D000970:Antineoplastic Agents; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D008563:Membrane Lipids; D010742:Phospholipid Ethers; D019014:fas Receptor; C026659:edelfosine; D053181:Caspase 8