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Involvement of raft aggregates enriched in Fas/CD95 death-inducing signaling complex in the antileukemic action of edelfosine in Jurkat cells.

Gajate C, Gonzalez-Camacho F, Mollinedo F - PLoS ONE (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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

ABSTRACT

Background: Recent evidence suggests that co-clustering of Fas/CD95 death receptor and lipid rafts plays a major role in death receptor-mediated apoptosis.

Methodology/principal findings: 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/significance: 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.

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DISC formation in lipid rafts following Jurkat cell incubation with edelfosine.(A) Untreated control Jurkat cells (Control) and Jurkat cells treated with 10 µM edelfosine (EDLF) for 9 h were analyzed for lipid raft isolation on a discontinuous sucrose density gradient. Raft and non-raft fractions were analyzed by Western blotting for the indicated proteins using specific antibodies. The migration positions of the 55-kDa procaspase-8 as well as of the cleavage product p18 are denoted. Location of GM1-containing lipid rafts was determined using CTx B subunit conjugated to horseradish peroxidase. (B) Fas/CD95 was immunoprecipitated from the raft fraction of edelfosine-treated Jurkat cells. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted with Fas/CD95, FADD- and procaspase-8 specific antibodies, respectively. Raft fraction was also immunoprecipitated with P3X63 (X63) myeloma supernatant as a negative control. Experiments shown are representative of three performed.
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pone-0005044-g001: DISC formation in lipid rafts following Jurkat cell incubation with edelfosine.(A) Untreated control Jurkat cells (Control) and Jurkat cells treated with 10 µM edelfosine (EDLF) for 9 h were analyzed for lipid raft isolation on a discontinuous sucrose density gradient. Raft and non-raft fractions were analyzed by Western blotting for the indicated proteins using specific antibodies. The migration positions of the 55-kDa procaspase-8 as well as of the cleavage product p18 are denoted. Location of GM1-containing lipid rafts was determined using CTx B subunit conjugated to horseradish peroxidase. (B) Fas/CD95 was immunoprecipitated from the raft fraction of edelfosine-treated Jurkat cells. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted with Fas/CD95, FADD- and procaspase-8 specific antibodies, respectively. Raft fraction was also immunoprecipitated with P3X63 (X63) myeloma supernatant as a negative control. Experiments shown are representative of three performed.

Mentions: We have previously reported that edelfosine induced co-aggregates of rafts and Fas/CD95 in leukemia Jurkat T-cells and multiple myeloma cells [1], [4], [7]. The proapoptotic complex DISC was generated upon multiple myeloma cell treatment with edelfosine [7]. Here, we extend these results by finding that Fas/CD95 as well as downstream signaling molecules FADD and procaspase-8 were translocated into membrane rafts (Figure 1A), forming the proapoptotic complex DISC in lipid rafts (Figure 1B), upon edelfosine treatment of Jurkat leukemic T-cells. In contrast, co-immunoprecipitation assays conducted in untreated Jurkat cells rendered no DISC formation (data not shown). Lipid rafts were identified using cholera toxin (CTx) B subunit conjugated to horseradish peroxidase that binds to the oligosaccharide portion of ganglioside GM1 [22], [23], mainly found in lipid rafts [24].


Involvement of raft aggregates enriched in Fas/CD95 death-inducing signaling complex in the antileukemic action of edelfosine in Jurkat cells.

Gajate C, Gonzalez-Camacho F, Mollinedo F - PLoS ONE (2009)

DISC formation in lipid rafts following Jurkat cell incubation with edelfosine.(A) Untreated control Jurkat cells (Control) and Jurkat cells treated with 10 µM edelfosine (EDLF) for 9 h were analyzed for lipid raft isolation on a discontinuous sucrose density gradient. Raft and non-raft fractions were analyzed by Western blotting for the indicated proteins using specific antibodies. The migration positions of the 55-kDa procaspase-8 as well as of the cleavage product p18 are denoted. Location of GM1-containing lipid rafts was determined using CTx B subunit conjugated to horseradish peroxidase. (B) Fas/CD95 was immunoprecipitated from the raft fraction of edelfosine-treated Jurkat cells. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted with Fas/CD95, FADD- and procaspase-8 specific antibodies, respectively. Raft fraction was also immunoprecipitated with P3X63 (X63) myeloma supernatant as a negative control. Experiments shown are representative of three performed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2662410&req=5

pone-0005044-g001: DISC formation in lipid rafts following Jurkat cell incubation with edelfosine.(A) Untreated control Jurkat cells (Control) and Jurkat cells treated with 10 µM edelfosine (EDLF) for 9 h were analyzed for lipid raft isolation on a discontinuous sucrose density gradient. Raft and non-raft fractions were analyzed by Western blotting for the indicated proteins using specific antibodies. The migration positions of the 55-kDa procaspase-8 as well as of the cleavage product p18 are denoted. Location of GM1-containing lipid rafts was determined using CTx B subunit conjugated to horseradish peroxidase. (B) Fas/CD95 was immunoprecipitated from the raft fraction of edelfosine-treated Jurkat cells. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted with Fas/CD95, FADD- and procaspase-8 specific antibodies, respectively. Raft fraction was also immunoprecipitated with P3X63 (X63) myeloma supernatant as a negative control. Experiments shown are representative of three performed.
Mentions: We have previously reported that edelfosine induced co-aggregates of rafts and Fas/CD95 in leukemia Jurkat T-cells and multiple myeloma cells [1], [4], [7]. The proapoptotic complex DISC was generated upon multiple myeloma cell treatment with edelfosine [7]. Here, we extend these results by finding that Fas/CD95 as well as downstream signaling molecules FADD and procaspase-8 were translocated into membrane rafts (Figure 1A), forming the proapoptotic complex DISC in lipid rafts (Figure 1B), upon edelfosine treatment of Jurkat leukemic T-cells. In contrast, co-immunoprecipitation assays conducted in untreated Jurkat cells rendered no DISC formation (data not shown). Lipid rafts were identified using cholera toxin (CTx) B subunit conjugated to horseradish peroxidase that binds to the oligosaccharide portion of ganglioside GM1 [22], [23], mainly found in lipid rafts [24].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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

ABSTRACT

Background: Recent evidence suggests that co-clustering of Fas/CD95 death receptor and lipid rafts plays a major role in death receptor-mediated apoptosis.

Methodology/principal findings: 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/significance: 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.

Show MeSH
Related in: MedlinePlus