The antitumor ether lipid ET-18-OCH₃ induces apoptosis through translocation and capping of Fas/CD95 into membrane rafts in human leukemic cells

The plasma membrane contains microdomains named membrane rafts, consisting of dynamic assemblies of cholesterol and sphingolipids.1-3 The presence of saturated hydrocarbon chains in sphingolipids allows for cholesterol to be tightly intercalated, leading to the presence of distinct liquid-ordered phases, membrane rafts, dispersed in the liquid-disordered matrix, and thereby more fluid, lipid bilayer.4 One key property of membrane rafts is that they can include or exclude proteins to varying degrees. Membrane rafts may serve as foci for recruitment and concentration of signaling molecules at the plasma membrane, and thus they have been implicated in signal transduction from cell surface receptors.3 

Antitumor ether phospholipids constitute a novel class of promising cancer chemotherapeutic drugs.5-7 The ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃; edelfosine) exerts a selective cytotoxic action against transformed cells,5-7 and has become the effective standard and prototype of the antitumor ether phospholipids. Encouraging clinical research on the use of ET-18-OCH₃ in purging leukemic bone marrow prior to autologous bone marrow transplantation has been reported.8,9ET-18-OCH₃ is a potent inducer of apoptosis in tumor cells, especially in leukemic cells, sparing normal cells.7,10,11Recent evidence has shown that ET-18-OCH₃–induced apoptosis is mediated by the intracellular activation of Fas/CD95 cell death receptor, independently of its ligand FasL, leading to clustering and subsequent capping of Fas.12 ET-18-OCH₃must be incorporated into the cell to exert its apoptotic action,7 and the ether lipid has been reported to be accumulated in the plasma membrane.13 Because ET-18-OCH₃ is a phospholipid and acts through plasma membrane–related processes, involving activation of cell surface Fas receptor, we investigated whether the Fas-mediated apoptotic effect of ET-18-OCH₃ on leukemic cells involved lipid rafts.

The human leukemic cell lines HL-60 (acute myeloid leukemia) and Jurkat (acute T-cell leukemia) were grown as described previously in RPMI-1640 culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS).7 Bone marrow aspirates, obtained from patients at the initial diagnosis and after signing informed consent, were kindly provided by the Hematology Department of the Rio Hortega Hospital (Valladolid, Spain). Mononuclear cells isolated by Ficoll-Hypaque density gradient centrifugation, consisting mostly of leukemia blasts (> 90%), were washed in phosphate-buffered saline (PBS), resuspended in cell culture medium, and used immediately for experimentation. ET-18-OCH₃ (INKEYSA, Barcelona, Spain) was prepared as described previously.7 To disrupt lipid rafts, cells (5 × 10⁵) were pretreated with methyl-β-cyclodextrin (MCD) (Sigma Chemical, St Louis, MO; 15 μg/mL) or filipin (Sigma; 1 μg/mL) for 1 hour at 37°C in serum-free medium before ether lipid addition.

Apoptosis was assessed by isolation of fragmented DNA as described previously.10,14 DNA was visualized after 1% agarose gel electrophoresis by ethidium bromide staining. For quantitative determination of apoptosis, cells (5 × 10⁵) were fixed overnight in 70% ethanol at 4°C. Cells were then incubated for 1 hour with 1 mg/mL RNase A and 20 μg/mL propidium iodide at room temperature, and analyzed with a Becton Dickinson (San Jose, CA) FACScan flow cytometer as described previously.11Apoptotic cells were calculated as the percentage of cells in the sub-G₁ region (hypodiploidy) in cell cycle analysis.

Untreated and ET-18-OCH₃–treated cells were settled onto slides coated with poly-l-lysine, fixed in 4% formaldehyde, stained with 8 μg/mL fluorescein isothiocyanate-labeled cholera toxin B subunit (FITC-CTx) (Sigma), and analyzed by confocal microscopy using a Zeiss LSM 310 laser scan confocal microscope (Oberkochen, Germany). For colocalization experiments FITC-CTx staining was performed as above, and then cells were incubated with 500 ng/mL antihuman Fas SM1/1 IgG_2a mouse monoclonal antibody (mAb) (Bender Medsystems, Vienna, Austria) for 1 hour at room temperature. Samples were further processed using CY3-conjugated antimouse antibody (Pharmacia, Uppsala, Sweden) (diluted 1:200 in PBS) for 1 hour at room temperature, and analyzed by confocal microscopy. Negative controls were prepared by either omitting the primary antibody or by using an irrelevant antibody, showing no fluorescence staining of the samples. Colocalization of CTx and Fas was analyzed by excitation of both fluorochromes in the same section.

Lipid rafts were isolated by using lysis conditions and centrifugation on discontinuous sucrose gradients as previously reported.15 In brief, cells (3.5 × 10⁷) were washed with ice-cold PBS and lysed for 30 minutes on ice in 1% Triton X-100 in TNEV buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM sodium orthovanadate) containing 1 mM phenylmethylsulfonyl fluoride. Cells were then homogenized with 10 strokes in a Potter-Elvehjem tissue grinder. Nuclei and cellular debris were pelleted by centrifugation at 1000 rpm for 8 minutes. Then, 1 mL cleared supernatant was mixed with 1 mL 85% sucrose in TNEV and transferred to the bottom of a Beckman 14 × 95-mm centrifuge tube. The diluted lysate was overlaid with 6 mL 35% sucrose in TNEV and finally 3.5 mL 5% sucrose in TNEV. The samples were centrifuged in an SW40 rotor at 38 000 rpm for 18 hours at 4°C in a Beckman Optima LE-80K ultracentrifuge (Beckman Instruments, Palo Alto, CA), and then 1-mL fractions were collected from the top of the gradient.

To determine the location of Fas and lipid rafts in the discontinuous sucrose gradient, 20 μL of the individual fractions was subjected to sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted as described previously.15,16After blocking for 1 hour at room temperature with 5% powdered defatted milk in TBST (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20), blots were incubated with anti-Fas rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:500 in TBST. Antibody reactivity was monitored with biotinylated anti–rabbit IgG, using an enhanced chemiluminescence detection (ECL) system (Amersham, Buckinghamshire, United Kingdom). The location of G_M1-containing lipid rafts was determined using CTx B subunit conjugated to horseradish peroxidase (Sigma) and an ECL system as described previously.15 

We tested whether ET-18-OCH₃ affects the distribution of membrane rafts using the raft marker FITC-CTx. CTx B subunit binds the oligosaccharide portion of ganglioside G_M1,17 which is thought to be mainly found in rafts.18 In unstimulated Jurkat cells, distribution of membrane rafts appeared homogeneous (Figure1A); however, after incubation of cells with ET-18-OCH₃ lipid rafts were redistributed in a time-dependent way to form dense patches (Figure 1A), indicating that membrane rafts had aggregated. Because we have recently reported that ET-18-OCH₃ induced clustering and capping of Fas in human leukemic Jurkat cells,12 we examined whether clustering of membrane rafts led to inclusion of Fas in the clustered rafts. As shown in Figure 1B, FITC-CTx staining colocalized with Fas in Jurkat cells treated with ET-18-OCH₃. Untreated Jurkat cells showed an extremely weak diffuse staining of Fas, whereas incubation of cells with ET-18-OCH₃ led to a dense patchy staining by confocal microscopy (Figure 1B), indicating clustering of Fas and corroborating our previous data.12 The Fas patches were colocalized with G_M1 glycosphingolipid-lipid rafts, indicating that Fas accumulated in membrane rafts in ET-18-OCH₃–treated Jurkat cells. This coclustering of membrane rafts and Fas occurred during the first 3 hours of ET-18-OCH₃ treatment, whereas ET-18-OCH₃–induced apoptosis of Jurkat cells was observed after 6 hours of incubation (2%, 3%, and 13% apoptotic cells after 3, 4, and 6 hours of ET-18-OCH₃ treatment, respectively). Thus, clustering of Fas-associated membrane rafts preceded triggering of apoptosis. Similar data were obtained with HL-60 cells (data not shown).

Because Fas labeling was negligible before treatment with ET-18-OCH₃ (Figure 1B), it was not possible to discern whether Fas was already located in lipid rafts in untreated cells or Fas translocated into the membrane rafts on cell treatment with the ether lipid. To work out this issue we isolated lipid rafts in both untreated and ET-18-OCH₃–treated Jurkat cells. Lipid rafts can be isolated based on their insolubility in Triton X-100 detergent and buoyant density on sucrose density gradients. Thus, Jurkat cells were lysed in 1% Triton X-100 lysis buffer and fractionated by discontinuous sucrose gradient centrifugation as previously described.15 The distinct fractions from the gradient were analyzed by SDS-PAGE and Western blotting. The position of the membrane rafts in the sucrose gradient was determined by the presence of the ganglioside G_M1, detected using the G_M1-specific ligand CTx B subunit (Figure2). G_M1 was enriched in the upper part of the sucrose gradient (fractions 4-6), with a secondary localization at the bottom of the gradient (fractions 10-12), indicating a separation of the lipid rafts (fractions 4-6) from the Triton X-100–soluble membranes (Figure 2). By Western blot analysis using a specific anti-Fas antibody, we found that Fas was located in the soluble fractions of the sucrose gradient (fractions 10-12) and not in the detergent-insoluble lipid raft region in untreated Jurkat cells, indicating that Fas is excluded from the lipid rafts in untreated Jurkat cells (Figure 2). However, after ET-18-OCH₃treatment, a significant portion of Fas was translocated into the lipid raft region (fractions 4-6) of the sucrose gradient (Figure 2). These data indicate that ET-18-OCH₃ treatment induces translocation of Fas into lipid rafts.

Membrane rafts are enriched in cholesterol, and depletion of cellular cholesterol can disrupt rafts and destroy function.3 To explore the role of membrane rafts in ET-18-OCH₃–induced apoptosis, we disrupted membrane rafts by cell incubation with MCD or filipin in serum-free medium. MCD specifically removes cholesterol from the plasma membrane,19,20 and filipin is a polyene antibiotic that specifically complexes cholesterol and disrupts lipid raft function.21,22 HL-60 cells were used in these assays because they are prone to undergo ET-18-OCH₃–induced apoptosis and can be cultured for longer times than Jurkat cells in serum-free medium.10,14 We found that MCD or filipin inhibited ET-18-OCH₃–induced apoptosis in leukemic HL-60 cells (Figure 3A,B), indicating that ET-18-OCH₃–induced apoptosis required membrane raft integrity. Bone marrow cells derived from patients with acute myeloblastic leukemia or acute promyelocytic leukemia (M2 or M3, respectively, following the FAB classification) were positive for Fas (> 60% positive cells), and underwent apoptosis after ET-18-OCH₃ treatment (Figure 3C), corroborating our previous findings.7 In these M2 and M3 primary leukemic cells, Fas was clustered on ET-18-OCH₃ treatment in a similar way to that of Jurkat and HL-60 cells (data not shown), and ET-18-OCH₃–induced apoptosis of primary leukemic cells was also inhibited by MCD (Figure 3C). Furthermore, MCD-treated leukemic HL-60 cells did not show Fas clustering after ET-18-OCH₃treatment (Figure 3D).

The present findings indicate that translocation of Fas into membrane rafts and clustering of membrane raft–associated Fas trigger the apoptotic signaling cascade in leukemic cells treated with ET-18-OCH₃. The evidence for this conclusion is 3-fold: (1) Fas is translocated into membrane rafts following ET-18-OCH₃ treatment; (2) Fas is accumulated in aggregated rafts on ET-18-OCH₃ treatment; and (3) raft disruption inhibits both ET-18-OCH₃–induced apoptosis and Fas clustering. Fas has been shown to be required for ET-18-OCH₃–induced apoptosis because Fas⁺cells, but not Fas⁻ cells, are susceptible to undergoing ET-18-OCH₃–mediated apoptosis.12 Furthermore, Fas⁻ cells become sensitive to ET-18-OCH₃following Fas transfection.12 These data together with the results reported here indicate that clustering of membrane raft–associated Fas is required for antitumor ether lipid apoptosis. Clustered rafts are often bound to cytoskeleton.23 In this context, the association of Fas with ezrin24 and its capping during apoptosis have been reported recently.12,24The participation of membrane raft sphingolipid-cholesterol microdomains in ET-18-OCH₃–induced apoptosis can explain previous reports showing that addition of exogenous cholesterol can affect the cytotoxic action of the ether lipid.25 

The data reported herein showing that Fas is translocated into rafts on ET-18-OCH₃ treatment and that reorganization of membrane raft-associated Fas is required for ET-18-OCH₃–induced apoptosis indicate for the first time the association of Fas with membrane rafts and that antitumor ether lipids require membrane rafts for their proapoptotic activity. Thus, the present findings involve for the first time membrane rafts in cancer chemotherapy and Fas-mediated apoptosis. The generation of stabilized membrane lipid domains from a highly dispersed distribution may represent a general mode of regulating Fas activation. Membrane rafts could serve as platforms for coupling adaptor proteins required for Fas signaling.

We thank E. Del Canto-Jañez for excellent technical assistance and S. Callejo, E. Barbosa, and M. A. Ollacarizqueta for their help in confocal microscopy. We also thank Antonio Iglesias and Manuel Modolell for helpful discussions.