Figure 1.
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.
Figure 2.
Ultrastructural localization of DISC embedded in lipid rafts during edelfosine treatment of leukemic cells.
(A–D) Electron microscopy images of Jurkat cells treated with 10 µM edelfosine (EDLF) for 9 h. Sections of edelfosine-treated cells were labeled with the raft marker GM1 using CTx B subunit (6-nm gold) alone (A), or in combination with anti-Fas/CD95 antibody (10-nm gold) (B). Drug-treated cells were also labeled with the raft marker GM1 using CTx B subunit (6-nm gold) (asterisk), anti-Fas/CD95 antibody (10-nm) (closed arrowhead), anti-FADD antibody (15-nm) (arrow) (C), and anti-procaspase-8 antibody (20-nm) (open arrowhead) (D). Lipid rafts are labeled on the external face of the membrane, whereas DISC components are located in the internal face of raft-enriched membrane domains. Bar, 400 nm.
Figure 3.
Edelfosine-induced apoptosis in Jurkat cells is mediated by Fas/CD95.
(A) Cells were transfected with Fas/CD95 shRNA (target sequence 1, Materials and Methods) (Fas shRNA) or neomycin-resistant scrambled sequence control vector (Vector), and the percentage of Fas/CD95 positive cells was assessed by flow cytometry using P3X63 (X63) myeloma supernatant as a negative control. (B) Jurkat cells transfected with neomycin-resistant scrambled sequence control vector (Vector) or Fas/CD95 shRNA (target sequence 1, Materials and Methods) (Fas shRNA) were treated with 10 µM edelfosine-induced apoptosis for the indicated incubation times and the proportion of cells in each phase of the cell cycle was quantitated by fluorescence flow cytometry. Cells in the sub-G1 region represent apoptotic cells. Untreated control cells were run in parallel. Data are shown as means±SE of four independent experiments.
Figure 4.
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.
Figure 5.
Edelfosine-induced apoptosis in Jurkat cells is mediated by caspase-8.
Cells were preincubated without or with 50 µM of z-IETD-fmk for 1 h, and then incubated in the absence or presence of 10 µM edelfosine (EDLF) for 24 h, and analyzed by flow cytometry to evaluate apoptosis. Untreated control cells were run in parallel. Data are shown as means±SE of three independent experiments.
Figure 6.
Involvement of DISC constituents in edelfosine-induced apoptosis as assessed by TUNEL assay.
(A) Jurkat cells transfected with neomycin-resistant scrambled sequence control vector (Vector) or Fas/CD95 shRNA (target sequence 1, Materials and Methods) (Fas shRNA) (upper panel), and with control pcDNA3 empty vector (Vector) or FADD-DN (middle panel), were treated with 10 µM edelfosine (EDLF)-induced apoptosis for 20 h, and analyzed by confocal microscopy for differential interference contrast (DIC), propidium iodide (PI) staining and TUNEL assay. Merging of PI and TUNEL panels (Merge) shows the apoptotic nuclei in yellow. Jurkat cells were also untreated (Control), treated with 10 µM edelfosine for 20 h (EDLF), or preincubated with 50 µM of z-IETD-fmk for 1 h followed by incubation in the presence of 10 µM edelfosine for 20 h (z-IETD-fmk+EDLF), and then analyzed by confocal microscopy for DIC, PI staining and TUNEL assay as above (lower panel). Data shown are representative of four independent experiments. Bar, 10 µm. (B–D) Histograms indicate the percentage of TUNEL-positive cells, as an estimate of cells undergoing apoptosis, following the experimental conditions shown in A (upper, middle and lower panels). For each experiment at least 120 cells were analyzed. Data are shown as means±SE of four independent experiments.
Figure 7.
Edelfosine accumulation in lipid rafts.
(A) Jurkat cells treated with 10 µM [3H]edelfosine for 9 h were lysed in 1% Triton and fractionated by centrifugation on a discontinuous sucrose density gradient. An equal volume of each collected fraction was subjected to SDS-PAGE and counted for radioactivity. The distribution patterns of GM1-containing rafts (upper panel) (fractions 3–5) and [3H]edelfosine (EDLF) (lower panel) over the gradient fractions are shown. Data are representative of three separate experiments. (B) Jurkat cells were incubated with 10 µM PTE-edelfosine (PTE-EDLF, blue fluorescence) for 9 h, and then its co-localization with membrane rafts was examined using fluorescein isothiocyanate-labeled CTx B subunit (green fluorescence for rafts). Areas of co-localization between membrane rafts and PTE-edelfosine in the merge panel are cyan. Data are representative of four independent experiments. Bar, 10 µm. (C) Higher magnifications of Jurkat cells treated as in B are shown. Data are representative of four independent experiments. Bar, 5 µm. (D, E) Jurkat cells were pretreated in the absence or presence of 2.5 mg/ml MCD for 30 min, and then drug uptake was determined after incubation with 10 µM [3H]edelfosine (EDLF) for 1 h (D), and apoptosis was analyzed by flow cytometry following incubation with 10 µM edelfosine for 24 h (E). Data are means±SE of three independent determinations.
Figure 8.
Schematic model for the involvement of DISC and lipid rafts in edelfosine-induced apoptosis in Jurkat cells.
This diagram portrays a currently plausible mechanism for the role of DISC recruitment in membrane rafts in drug-induced apoptosis based on the results reported in this work. Initially, Fas/CD95 death receptor is not located at the membrane raft regions of plasma membrane. Incubation of Jurkat cells with edelfosine (EDLF) leads to its accumulation in membrane rafts, inducing raft clustering and recruitment of Fas/CD95 into lipid rafts. This translocation and concentration of Fas/CD95 in rafts brings together FADD and procaspase-8, forming the DISC, through protein-protein homotypic interactions between their respective death domains (DD) and death effector domains (DED). Thus, lipid raft clusters act as scaffolds where DISC is concentrated, hence achieving caspase-8 activation and eventually apoptosis. These DISC-raft co-clusters would behave as a supramolecular and physical entity crucial for the death receptor-mediated regulation of apoptosis.