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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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Related in: MedlinePlus

Edelfosine-induced apoptosis in Jurkat cells is mediated by FADD.Cells, stably transfected with control pcDNA3 empty vector (Vector) (A) or FADD-DN (B), were treated with 10 µM edelfosine for the indicated incubation times, and the proportion of cells in each phase of the cell cycle was quantified by flow cytometry. Cells in the sub-G1 region represent apoptotic cells. Untreated cells were run in parallel. Data are shown as means±SE of four independent experiments.
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pone-0005044-g004: Edelfosine-induced apoptosis in Jurkat cells is mediated by FADD.Cells, stably transfected with control pcDNA3 empty vector (Vector) (A) or FADD-DN (B), were treated with 10 µM edelfosine for the indicated incubation times, and the proportion of cells in each phase of the cell cycle was quantified by flow cytometry. Cells in the sub-G1 region represent apoptotic cells. Untreated cells were run in parallel. Data are shown as means±SE of four independent experiments.

Mentions: Using stable transfection in Jurkat cells with a dominant-negative form of the FADD adapter protein (FADD-DN), which lacks the death effector domain and prevents death receptor signaling [25], we found that blockade of Fas/CD95 downstream signaling abrogated edelfosine-induced apoptosis (about 80% inhibition after 48-h drug incubation) (Figure 4A and 4B). Conversely, transfection with a control pcDNA3 empty vector did not affect edelfosine-induced apoptosis (Figure 4A). Cells transfected with pcDNA3 control vector behaved similarly to intact nontransfected Jurkat cells regarding drug-induced apoptosis (data not shown).


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)

Edelfosine-induced apoptosis in Jurkat cells is mediated by FADD.Cells, stably transfected with control pcDNA3 empty vector (Vector) (A) or FADD-DN (B), were treated with 10 µM edelfosine for the indicated incubation times, and the proportion of cells in each phase of the cell cycle was quantified by flow cytometry. Cells in the sub-G1 region represent apoptotic cells. Untreated cells were run in parallel. Data are shown as means±SE of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005044-g004: Edelfosine-induced apoptosis in Jurkat cells is mediated by FADD.Cells, stably transfected with control pcDNA3 empty vector (Vector) (A) or FADD-DN (B), were treated with 10 µM edelfosine for the indicated incubation times, and the proportion of cells in each phase of the cell cycle was quantified by flow cytometry. Cells in the sub-G1 region represent apoptotic cells. Untreated cells were run in parallel. Data are shown as means±SE of four independent experiments.
Mentions: Using stable transfection in Jurkat cells with a dominant-negative form of the FADD adapter protein (FADD-DN), which lacks the death effector domain and prevents death receptor signaling [25], we found that blockade of Fas/CD95 downstream signaling abrogated edelfosine-induced apoptosis (about 80% inhibition after 48-h drug incubation) (Figure 4A and 4B). Conversely, transfection with a control pcDNA3 empty vector did not affect edelfosine-induced apoptosis (Figure 4A). Cells transfected with pcDNA3 control vector behaved similarly to intact nontransfected Jurkat cells regarding drug-induced apoptosis (data not shown).

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