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Originally published In Press as doi:10.1074/jbc.M511251200 on March 15, 2006

J. Biol. Chem., Vol. 281, Issue 21, 14833-14840, May 26, 2006
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Differential Targets and Subcellular Localization of Antitumor Alkyl-lysophospholipid in Leukemic Versus Solid Tumor Cells*

Teresa Nieto-Miguel{ddagger}1, Consuelo Gajate{ddagger}§2, and Faustino Mollinedo{ddagger}3

From the {ddagger}Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas-Universidad de Salamanca, Campus Miguel de Unamuno and the §Unidad de Investigación, Hospital Universitario de Salamanca, E-37007 Salamanca, Spain

Received for publication, October 17, 2005 , and in revised form, March 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Synthetic alkyl-lysophospholipids represent a family of promising anticancer drugs that induce apoptosis in a variety of tumor cells. Here we have found a differential subcellular distribution of the alkyl-lysophospholipid edelfosine in leukemic and solid tumor cells that leads to distinct anticancer responses. Edelfosine induced rapid apoptosis in human leukemic cells, including acute T-cell leukemia Jurkat and Peer cells, but promoted a late apoptotic response, preceded by G2/M arrest, in human solid tumor cells such as cervix epitheloid carcinoma HeLa cells and lung carcinoma A549 cells. c-Jun amino-terminal kinase (JNK) and caspase-3 were accordingly activated at earlier times in edelfosine-treated Jurkat cells as compared with drug-treated HeLa cells. Both leukemic and solid tumor cells took up this alkyl-lysophospholipid and expressed the two putative edelfosine targets, namely cell surface Fas death receptor (also known as APO-1 or CD95) and endoplasmic reticulum CTP: phosphocholine cytidylyltransferase. However, edelfosine was mainly located to plasma membrane lipid rafts in Jurkat and Peer leukemic cells and to endoplasmic reticulum in solid tumor HeLa and A549 cells. Edelfosine induced translocation of Fas, Fas-associated death domain-containing protein, and JNK into membrane rafts in Jurkat cells, but not in HeLa cells. In contrast, edelfosine inhibited phosphatidylcholine biosynthesis in both HeLa and A549 cells, but not in Jurkat or Peer leukemic cells, before the triggering of apoptosis. These data indicate that edelfosine targets two different subcellular structures in a cell type-dependent manner, namely cell surface lipid rafts in leukemic cells and endoplasmic reticulum in solid tumor cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Synthetic alkyl-lysophospholipids constitute a family of anticancer drugs, collectively named antitumor ether lipids, that unlike most conventional chemotherapeutic drugs do not target the DNA (1-4). The synthetic ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, edelfosine)4 has become the effective prototype of the synthetic alkyl-lysophospholipids and selectively induces apoptosis in both human tumor cell lines and primary tumor cell cultures from cancer patients (5). So far, two major targets have been proposed for the apoptotic action of edelfosine, namely the CTP:phosphocholine cytidylyltransferase (CCT) and the death receptor Fas (also known as APO-1 or CD95) (2, 4, 6, 7). Phosphatidylcholine (PtdCho) biosynthesis results predominantly via the Kennedy or CDP-choline pathway (8). Following its uptake into the cell, choline is phosphorylated by choline kinase, and then CCT catalyzes the transfer of choline from phosphocholine to cytidine 5'-diphosphate choline (CDP-choline), which is subsequently utilized by choline phosphotransferase along with diacylglycerol to form PtdCho (9). CCT has long been recognized as a key enzyme controlling the PtdCho biosynthetic pathway, acting as a rate-limiting and regulatory step for PtdCho biosynthesis (9), and has been shown to be necessary for cell survival in cultured mammalian cells (10, 11). This enzyme resides in the nucleus and cytoplasmic compartments and translocates to the endoplasmic reticulum when activated (12-14). Edelfosine has been reported to inhibit de novo PtdCho synthesis at the CCT step, leading to mitotic arrest and cell death in a number of cells, including HeLa cells (6, 15, 16). Increased expression of CCT, corresponding to CCT{alpha} isoform, in HeLa cells prevented edelfosine-induced apoptosis (6). In addition, the uptake and acylation of exogenously added lysophosphatidylcholine circumvented the requirement of CCT activity by providing an alternate route to PtdCho and prevented apoptosis induced by edelfosine (16, 17). On the other hand, we have recently shown that edelfosine induced a selective apoptotic response in leukemic cells through a novel mechanism of action involving the intracellular activation of the death receptor Fas and its recruitment together with downstream signaling molecules into lipid rafts, independently of its natural ligand FasL (7, 18-20). Cells lacking Fas were resistant to edelfosine-induced apoptosis, and the ectopic expression of Fas bestowed drug sensitivity (7, 18). Edelfosine has been recently shown to accumulate in lipid rafts (7, 16), and a fluorescent analog of the drug colocalized with Fas in aggregated plasma membrane raft microdomains in human leukemic cells (7). The antitumor effect of edelfosine in human leukemic cells has been shown to rely on its ability to recruit Fas death receptor into lipid rafts and to induce co-capping of Fas-containing raft membrane domains (7, 18, 19). Disruption of lipid rafts reduced the uptake of the alkyl-lysophospholipid and inhibited edelfosine-induced apoptosis (7, 16, 19). Here, we report that edelfosine shows two different subcellular localizations, namely in plasma membrane lipid rafts and endoplasmic reticulum, in a cell type-dependent manner, affecting two distinct targets and leading to apoptosis through different signaling mechanisms in human leukemic and solid tumor cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Cells were grown in RPMI 1640 (Jurkat, Peer, HL-60, and A549) or Dulbecco's modified Eagle's (HeLa) culture medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/ml of penicillin, and 24 µg/ml of gentamicin (7, 18).

Analysis of Cell Cycle and Apoptosis by Flow Cytometry—Quantitation of apoptotic cells was determined by flow cytometry as the percentage of cells in the sub-G1 region (hypodiploidy) in cell cycle analysis as previously described (21). 5 x 105 cells were centrifuged and fixed overnight in 70% ethanol at 4 °C. Cells were washed three times with PBS and incubated for 1 h with 1 mg/ml of RNase A and 20 µg/ml of propidium iodide at room temperature. The proportion of cells in each phase of the cell cycle was then quantitated by flow cytometry in a Becton Dickinson FACSCalibur flow cytometer.

Fas Detection by Flow Cytometry—Fas cell surface expression was analyzed by immunofluorescence flow cytometry as previously described (7, 18) in a BD Biosciences FACSCalibur flow cytometer, using anti-Fas SM1/1 monoclonal antibody (Bender MedSystems, Vienna, Austria). Percent of Fas-positive cells was estimated using the P3X63 myeloma supernatant, kindly provided by F. Sánchez-Madrid (Hospital de La Princesa, Madrid, Spain), as a negative control.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)—Total RNA (5 µg) was primed with random hexamers and reversed transcribed with SuperScriptTM III reverse transcriptase (Invitrogen) in a 20-µl volume. The generated cDNA was amplified by using primers for human fas (forward 5'-ATAAGCCCTGTCCTCCAGGT-3' and reverse 5'-TGATGCCAATTACGAAGCAG-3'), cct{alpha} (forward 5'-ATCTCCACATCAGACATC-3' and reverse 5'-CACTCCTACTTCTTCTGATT-3'), and beta-actin (forward 5'-CTGTCTGGCGGCACCACCAT-3' and reverse 5'-GCAACTAAGTCATAGTCCGC-3'). A 25-µl PCR mixture contained 1 µl of the reverse transcription reaction, 10 pmol each primer, each deoxynucleotide triphosphate (0.2 mM), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 2.5 units of Taq DNA polymerase (Roche Applied Science). The PCR reaction profile was as follows: 95 °C for 30 s; 65 °C (fas and beta-actin) or 52°C (cct{alpha}) for 30 s; 72 °C for 90 s. After 30 cycles, the expected PCR products (670 bp for fas, 507 bp for cct{alpha}, and 254 bp for beta-actin) were size fractionated onto a 2% agarose gel and stained with ethidium bromide.

Western Blot—Cells (4-5 x 106) were lysed with 60 µl of 25 mM HEPES (pH 7.7), 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 20 mM beta-glycerophosphate, 0.1 mM sodium orthovanadate supplemented with protease inhibitors (1 mM phenylmethanesulfonyl fluoride, 20 µg/ml of aprotinin, 20 µg/ml of leupeptin). Proteins (35 µg) were run on SDS-polyacrylamide gels under reducing conditions, transferred to nitrocellulose filters, blocked with 5% (w/v) powder defatted milk in TBST (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20) for 90 min at room temperature, and incubated for 1 h at room temperature or overnight at 4 °C with C2.10 anti-116-kDa poly(ADP-ribose) polymerase monoclonal antibody (1:3000 dilution, BD Biosciences), CM1 anti-18-kDa active caspase-3 rabbit polyclonal antibody (1:5000 dilution, BD Biosciences), C-20 anti-48-kDa Fas rabbit polyclonal antibody (1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), SM1/23 anti-48-kDa Fas monoclonal antibody (1:500 dilution, Bender MedSystems), F-17 anti-42-kDa CCT{alpha} goat polyclonal antibody (1:500 dilution, Santa Cruz Biotechnology), or AC-15 anti-42-kDa beta-actin monoclonal antibody (1:5000 dilution, Sigma). Signals were developed using an enhanced chemiluminescence detection kit (Amersham Biosciences).

Edelfosine Uptake—Drug uptake was measured as previously described (5, 18) after incubating 106 cells/ml with 10 µM edelfosine + 0.05 µCi/ml of [3H]edelfosine for the indicated times and subsequent exhaustive washing with 2% bovine serum albumin-PBS. Edelfosine was from INKEYSA (Barcelona, Spain), and stock solutions were prepared as described previously (5). [3H]edelfosine (specific activity, 42 Ci/mmol) was synthesized by tritiation of the 9-octadecenyl derivative (Amersham Biosciences).

Solid Phase c-Jun Amino-terminal Kinase (JNK) Assay—A fusion protein between glutathione S-transferase and c-Jun (amino acids 1-223) was used as a substrate for JNK as described previously (21-23). The experimental conditions used have been shown to enable specific binding of JNK to the c-Jun amino-terminal domain (22). The phosphorylated proteins were resolved in SDS-10% polyacrylamide gels followed by autoradiography.

Lipid Raft Isolation—Lipid rafts were isolated from 3-7 x 107 cells by nonionic detergent lysis and centrifugation on discontinuous sucrose gradients as described (19, 24). 1-ml fractions were collected from the top of the gradient, and 20 µl of each fraction were subjected to SDS-polyacrylamide gel electrophoresis, immunoblotting, and enhanced chemiluminescence detection. Location of GM1-containing lipid rafts was determined using cholera toxin (CTx) B subunit conjugated to horseradish peroxidase (Sigma) as described previously (24, 25). Proteins were identified using specific antibodies: anti-48-kDa Fas (C-20) and anti-46-kDa JNK1 (C-17) rabbit polyclonal antibodies (Santa Cruz Biotechnology), anti-29-kDa Fas-associated death domain-containing protein (FADD) (clone-1) monoclonal antibody (BD Biosciences), anti-120-kDa Fas death domain-associated protein (Daxx) rabbit polyclonal antibody (Alexis Corp., Lausen, Switzerland), and anti-22-kDa caveolin rabbit polyclonal antibody (BD Biosciences). Anti-JNK1 antibody reacted with both JNK1 and JNK3, leading to the two bands observed by Western blotting.

Cell Transfection and Fluorescence Microscopy—Cells were transfected with 3 µg of a plasmid encoding endoplasmic reticulum-targeted red fluorescence protein (erRFP) (26), kindly provided by F. X. Pimentel-Muiños (Centro de Investigación del Cáncer, Salamanca, Spain), using the jetPEITM reagent (Qbiogene), and then treated for 3 h with 20 µM edelfosine fluorescent analog PTE-edelfosine (all-[E]-1-O-[15'-phenylpentadeca-8',10',12',14'-tetraenyl]-2-O-methyl-rac-glycero-3-phosphocholine) (7), a kind gift from A. U. Acuña and F. Amat-Guerri (Consejo Superior de Investigaciones Científicas, Madrid, Spain). Colocalization of the distinct fluorochromes was analyzed using a fluorescence microscope (Axioplan 2; Carl Zeiss MicroImaging, Inc.) and a digital camera (ORCA-ER-1394; Hamamatsu). Lipid rafts were visualized using the raft marker fluorescein isothiocyanate-labeled cholera toxin (FITC-CTx) B subunit as described previously (7, 18).

PtdCho Biosynthesis—Cells (2-3 x 106) were washed with PBS and incubated with 1 µCi/ml of [methyl-14C]choline chloride (Amersham Biosciences) for 30 min in fetal calf serum-free culture medium. Labeled cells were washed three times with PBS and resuspended in the presence or absence of edelfosine in complete culture medium for the indicated times. After incubation, cells were washed with PBS and pelleted for extraction of lipids (27). Briefly, the pellet was extracted with a mixture of 0.125 ml of chloroform, 0.25 ml of methanol, and 0.1 ml of water. After 10 min at room temperature, 0.125 ml of chloroform and 0.125 ml of water were added. The tubes were shaken vigorously and then centrifuged to recover the lower organic and upper aqueous layers. Organic layers were dried under a stream of nitrogen, taken up in 20-40 µl of chloroform/methanol (2:1), applied to silica gel G-60 thin-layer chromatography plates (Merck), and developed with a solvent system of chloroform/methanol/acetic acid/water (60:30:8:5, v/v). Aqueous layers were evaporated at 40 °C under a stream of nitrogen, resuspended in 20-40 µl of ethanol/water (1:1, v/v), and spotted onto silica gel G-60 thin-layer chromatography plates that were developed in 95% ethanol/2% NH4OH (1:1, v/v) to separate water-soluble choline metabolites. Radioactive lipids were visualized using Fujifilm BAS-MS phosphorimaging and identified using internal controls. Appropriate authentic standards (100 µg) of PtdCho, choline, phosphocholine, and CDP-choline were also run in parallel, and their location was ascertained by iodine staining or Dragendorff reagent (Sigma). Spots corresponding to labeled PtdCho, choline, phosphocholine, and CDP-choline were scraped from the plates, and radioactivity was determined by liquid scintillation counting.


Figure 1
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  		FIGURE 1.
Effects of edelfosine on cell cycle distribution and activation of JNK and caspase-3 in leukemic and solid tumor cells. A, cells were treated with 10 µM edelfosine for the indicated times, and the proportion of cells in each phase of the cell cycle was quantitated by flow cytometry. Cells in the sub-G1 region represent apoptotic cells. Untreated control cells were run in parallel. Data are shown as means of four independent experiments ± S.E. B, cells were untreated or treated with 10 µM edelfosine for the indicated times and assayed for JNK activation as described under "Experimental Procedures." The position of phosphorylated glutathione S-transferase c-Jun (GST-c-Jun-(1-223)) is indicated. Experiments shown are representative of three performed. C, cells were untreated or treated with 10 µM edelfosine for the indicated times and analyzed by immunoblotting with anti-caspase-3 (SDS-12% polyacrylamide gels) and anti-poly(ADP-ribose) polymerase antibodies (SDS-8% polyacrylamide gels). The migration positions of the 18-kDa subunit of human active caspase-3 (p18 Caspase-3) as well as of full-length poly(ADP-ribose) polymerase (PARP) and its cleavage product p85 are indicated. Immunoblotting for beta-actin was used as an internal control for equal protein loading in each lane. Blots are representative of three experiments performed.

 
Statistical Analyses—Unless otherwise indicated, the results given are the mean (± S.E.) of the number of experiments indicated. Statistical evaluation of the effect of edelfosine on PtdCho biosynthesis was performed by Student's t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Differences in the Induction of Apoptosis in Edelfosine-treated Leukemic and Solid Tumor Cells—Human T-cell acute lymphoblastic leukemia Jurkat and Peer cells (Fig. 1A), as well as human acute myeloid leukemia HL-60 cells (data not shown), underwent rapid apoptosis upon treatment with 10 µM edelfosine (>16% apoptosis following 6 h of drug treatment). However, cancer cell lines derived from human malignant solid tumors, such as cervix epitheloid carcinoma HeLa and lung carcinoma A549 cells, underwent first an arrest in G2/M before the onset of a late apoptotic response (Fig. 1A). This G2/M arrest preceding apoptosis was also evidenced by time lapse videomicroscopy in a variety of solid tumor cells.5 In addition, the ectopic expression of the antiapoptotic bcl-2 gene in HeLa cells prevented apoptosis but led to an arrest of cells in G2/M after treatment with edelfosine (data not shown). Edelfosine also triggered a persistent activation of JNK, a signaling pathway required for edelfosine-induced apoptosis (28, 29), before the onset of apoptosis in both leukemic Jurkat cells and solid tumor HeLa cells (Fig. 1B). Edelfosine-induced apoptosis is dependent on caspase-3 activation (30). We found activation of this caspase in edelfosine-treated cells, as assessed by cleavage of procaspase-3 into the p18 active form and cleavage of the typical caspase-3 substrate poly(ADP-ribose) polymerase, using a polyclonal anti-caspase-3 antibody that recognized the 18-kDa subunit of active caspase-3, but not the 32-kDa procaspase-3, and an anti-poly(ADP-ribose) polymerase monoclonal antibody that detected both the 116-kDa intact form and the 85-kDa cleaved form of poly-(ADP-ribose) polymerase (Fig. 1C). In keeping with the above timing differences in edelfosine-induced apoptosis between leukemic and solid tumor cells, we found an earlier JNK and caspase-3 activation in Jurkat cells as compared with HeLa cells (Fig. 1, B and C).


Figure 2
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  		FIGURE 2.
Expression of Fas and CCT and drug uptake in leukemic and solid tumor cells. Fas and CCT expression was assessed by Western blot (A) and RT-PCR (B). beta-Actin was used as a loading control. Experiments shown in panels A and B are representative of three performed. C, Fas cell surface expression was analyzed by immunofluorescence flow cytometry. Percent of antigen-positive cells was estimated using the P3X63 myeloma supernatant (X63) as a negative control. D, drug uptake was determined by incubating 106 cells with 10 nmol [3H]edelfosine for the indicated times as described under "Experimental Procedures." EDLF, edelfosine. Data in panels C and D are shown as means of three independent determinations ± S.E.

 
CCT and Fas Expression and Drug Uptake in Leukemic and Solid Tumor Cells—Because CCT, corresponding to CCT{alpha}, and Fas have been suggested as two putative edelfosine targets, we asked whether differences in expression of these two molecules could explain the different behavior of leukemic and solid tumor cells following edelfosine treatment. Western blot (Fig. 2A) and reverse transcription PCR (Fig. 2B) analyses demonstrated that Jurkat, Peer, HeLa, and A549 cells expressed both CCT and Fas at both mRNA and protein levels, even though CCT protein levels were somewhat more abundant in solid tumor cells and Fas protein in leukemic cells. Immunofluorescence flow cytometry of intact cells also indicated that Fas was present in the cell surface of both leukemic and solid tumor cells (Fig. 2C). In addition, both kinds of cell types incorporated edelfosine, although A549 and HeLa solid tumor cells took up higher amounts of the drug than Peer and Jurkat leukemic cells (Fig. 2D). Thus, the four tumor cell lines studied here fulfilled the two major requirements for edelfosine-induced apoptosis, namely ability to take up the drug and presence of the putative edelfosine targets Fas and CCT (2).

Edelfosine Is Not Differentially Metabolized in Tumor Cells—A hypothetical different rate of drug metabolism could account for the differences in the final apoptotic outcome between leukemic and solid tumor cells. However, edelfosine is metabolically a very stable compound because of the ether bonds present in its molecule structure (2, 4); accordingly, we found that the molecule remained largely intact (>98%) after 24 h of incubation of either Jurkat or HeLa cells with [3H]edelfosine as assessed by thin-layer-chromatography (data not shown). This lack of drug metabolism is in agreement with previous estimates showing that 98% of edelfosine remained unmodified in tumor cells in culture after 24 h of incubation (31). On these grounds, the lack of drug metabolism precludes the involvement of any putative distinct metabolic process for the drug in the differential action of edelfosine on both leukemic and solid tumor cells.

Edelfosine Translocates Fas and Apoptotic Molecules into Membrane Rafts in Leukemic Cells, but Not in Solid Tumor Cells—We have recently reported that edelfosine induces recruitment of Fas death receptor and additional downstream signaling molecules into lipid rafts in leukemic cells, this process being critical for the rapid induction of apoptosis (7). We then asked whether this process also took place in solid tumor cells. Lipid rafts were isolated based on their insolubility in Triton X-100 detergent and buoyant density on discontinuous sucrose density gradients as previously reported (19, 24). The distinct fractions from the gradient were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. The position of the membrane rafts in the sucrose gradient was determined using horseradish peroxidase-conjugated CTx B subunit that binds to ganglioside GM1 (32), mainly found in lipid rafts (33). GM1, used as a lipid raft marker, was enriched in the upper part of the sucrose gradient (Fig. 3, fractions 3-6) with a secondary localization at the bottom of the gradient, indicating a separation of lipid rafts (fractions 3-6) from the Triton X-100-soluble membranes. Using specific antibodies we found by Western blotting that Fas was translocated to the detergent-insoluble lipid rafts (fractions 4-6) together with other apoptotic signaling proteins, including FADD and JNK, following edelfosine treatment in Jurkat cells (Fig. 3A). However, Daxx, which has been reported to be involved in Fas-mediated apoptosis and JNK activation (34), was not recruited in membrane rafts (Fig. 3A). Interestingly, Fas, FADD, or JNK were not recruited into lipid rafts in edelfosine-treated HeLa cells following treatment with edelfosine for 14 or 24 h (Fig. 3B, and data not shown), indicating that the above recruitment of Fas and downstream signaling molecules into lipid rafts does not take place in HeLa cells. Caveolae represent a subset of lipid rafts that are enriched in caveolin and also function during signal transduction (35). Jurkat cells lack caveolin and caveolae (36, 37), whereas caveolin was detected in the detergent-insoluble fractions (Fig. 3B, fractions 3-6) in HeLa cells. This suggests that Jurkat and HeLa cells can differ in lipid raft composition. On the other hand, we have previously found that edelfosine promoted co-clustering of lipid rafts and Fas into "caps" on one or two poles of leukemic cells (7, 19). We corroborated these data in Jurkat and Peer cells, but we were unable to visualize these clusters in drug-treated HeLa cells (data not shown). Thus, these data suggest that edelfosine induces a redistribution of a subset of proteins in lipid rafts in Jurkat cells, but not in HeLa cells. In this regard, ongoing proteomic analysis indicates that a number of proteins are recruited into lipid rafts following edelfosine treatment of Jurkat cells, leading to an increased concentration of proteins in lipid rafts isolated from edelfosine-treated cells as compared with untreated cells (data not shown).


Figure 3
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  		FIGURE 3.
Differential recruitment of apoptotic signaling proteins into membrane rafts following edelfosine treatment in Jurkat versus HeLa cells. Jurkat (A) and HeLa (B) cells, untreated (Control) and treated with 10 µM edelfosine for 9 and 24 h respectively, were lysed in 1% Triton X-100 and fractionated by centrifugation on a discontinuous sucrose density gradient. An equal volume of each collected fraction was subjected to SDS-polyacrylamide gel electrophoresis before analysis of the indicated proteins by Western blotting using specific antibodies. Location of GM1-containing rafts was determined using CTx B subunit conjugated to horseradish peroxidase. Blots are representative of three separate experiments.

 
Differential Effect of Edelfosine on PtdCho Biosynthesis in Leukemic Versus Solid Tumor Cells—Because interruption of PtdCho biosynthesis at the CCT step by edelfosine has been reported as a major target in the cytotoxic action of the drug (6, 15), we analyzed the effect of edelfosine on PtdCho biosynthesis in the leukemic Jurkat and Peer cells as well as in the solid tumor HeLa and A549 cells by incubating cells with [14C]choline for 30 min followed by washing and incubation in the absence or presence of edelfosine for different times. This approach was used to eliminate any effects that edelfosine might have on the uptake of choline into the cells (38). We found that edelfosine inhibited [14C]choline incorporation into the PtdCho pool in both HeLa and A549 cells, but not in Jurkat and Peer cells, before the triggering of apoptosis (Fig. 4A). After 6 h of incubation, we observed a 53 and 44% decrease (p < 0.01) in the incorporation of radiolabeled choline into PtdCho in HeLa and A549 cells, respectively (Fig. 4A). The composition of the choline-derived metabolites in the water-soluble fraction in cells untreated and treated with edelfosine was analyzed (Fig. 4B). We found that the proportion of labeled CDP-choline was significantly reduced (p < 0.01) in edelfosine-treated HeLa cells compared with untreated control cells (Fig. 4B). This reduction in CDP-choline content in drug-treated HeLa cells was similar to the decrease (p < 0.01) in the PtdCho pool (Fig. 4B). Similar data were obtained with A549 cells (data not shown). This pattern of metabolic labeling is consistent with edelfosine inhibiting PtdCho synthesis at the CCT step, thus leading to a decrease in the CDP-choline and the ensuing PtdCho levels. CCT inhibition should lead to the accumulation of the choline and phosphocholine precursors. However, a slight increase in the choline levels was detected following edelfosine incubation, whereas the pool of phosphocholine was hardly enhanced (Fig. 4B). A similar pattern of choline metabolites, with a significant decrease of [14C]choline into CDP-choline and Ptd-Cho without a change in phosphocholine levels, has been also observed following edelfosine incubation with MCF-7 breast adenocarcinoma cells (39). Inhibition of CCT by alkyl-lysophospholipids in mammalian cells has been mainly detected by a reduction in the PtdCho and CDP-choline levels, whereas the pool of phosphocholine was either unaltered or slightly increased (39, 40). This could be in part due to the reported diverse effects of edelfosine on lipid metabolism and phospholipases C and D (39, 41, 42) that might affect labeled and unlabeled choline and phosphocholine pools.


Figure 4
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  		FIGURE 4.
Effect of edelfosine in PtdCho biosynthesis. [14C]Choline-labeled cells were grown in the absence (Cont) or presence of 10µM edelfosine (EDLF) for the indicated times, and then lipids were extracted and radioactivity associated with Ptd-Cho (A) or with the distinct choline metabolites (B) was determined. Data in panel B are percentages of 14C incorporation into the distinct choline metabolites with respect to untreated control cells (100%). Cho, choline; P-Cho, phosphocholine; CDP-Cho, CDP-choline. Data represent the means ± S.E. of three independent experiments. Asterisks indicate values that are significantly different from untreated control cells at p < 0.05 (*) and p < 0.01 (**) levels by Student's t-test.

 
Unlike solid tumor cells, we did not detect any significant decrease in either the CDP-choline or PtdCho pools in Jurkat and Peer cells following treatment with edelfosine (Fig. 4B, and data not shown). The levels of choline and phosphocholine pools were not modified in these leukemic cells upon drug incubation (Fig. 4B, and data not shown). These data indicate that PtdCho metabolism did not play a role in the induction of apoptosis in leukemic cells. Altogether, these results are consistent with the notion that edelfosine inhibits CCT, the key enzyme controlling the PtdCho biosynthetic pathway (15), in HeLa and A549 cells. However, this process does not play any role in edelfosine-induced apoptosis in Jurkat or Peer leukemic cells.

Subcellular Localization of Edelfosine in Leukemic and Solid Tumor Cells—The above data indicated that edelfosine affected two biological processes located in different subcellular structures in a cell type-dependent manner, namely reorganization of cell surface Fas and lipid rafts in leukemic cells and inhibition of endoplasmic reticulum-located PtdCho biosynthesis in solid tumor cells. Using the edelfosine fluorescent analog PTE-edelfosine that has been reported to preserve the apoptotic features of the parental edelfosine (7, 43), we were able to visualize the subcellular localization of the drug in both cell types. PTE-edelfosine behaved similarly to the parental edelfosine drug in its ability to promote apoptosis in the distinct cells studied. We have previously found that PTE-edelfosine at 20 µM induced an almost identical percentage of apoptosis as 10 µM edelfosine in Jurkat cells (7). We corroborated these data on all the cells assayed here. When Jurkat and Peer cells were incubated with 20 µM PTE-edelfosine for 9 h we found 31.6 and 30.1% apoptosis, respectively, similar to the apoptosis percentages rendered upon incubation with 10 µM edelfosine, namely 35.2 and 29.6% in Jurkat and Peer cells, respectively. In addition, Jurkat and Peer cells underwent 18 and 17.4% apoptosis, respectively, upon incubation with 10 µM PTE-edelfosine for 9 h. In HeLa cells, PTE-edelfosine elicited over 20 and 30% apoptosis when used at either 10 or 20 µM for 24 h, whereas 10 µM edelfosine induced 27.2% apoptosis under the same experimental conditions. A549 underwent 15% apoptosis upon incubation for 48 h with 20 µM PTE-edelfosine, a figure similar to that obtained with 10 µM parental edelfosine (Fig. 1A). In addition, incorporation of fluorescent PTE-edelfosine into the cancer cells was blocked by adding the parental drug edelfosine (data not shown). Thus, PTE-edelfosine behaves as a reliable fluorescent analog of edelfosine that allows us to visualize the subcellular location of the drug in situ. We found that edelfosine was mainly accumulated in the endoplasmic reticulum of HeLa and A549 cells (Fig. 5A), as assessed using a version of RFP targeted to endoplasmic reticulum (ER) lumen (erRFP), which completely colocalized with the ER marker calreticulin (26). We also found a predominant endoplasmic reticulum location for edelfosine in a variety of additional solid tumor cells, including glioblastoma cells (data not shown). However, only a minor portion of PTE-edelfosine colocalized with the endoplasmic reticulum in Jurkat cells, whereas most of the drug was concentrated in clusters at the plasma membrane of these cells (Fig. 5A). Likewise, PTE-edelfosine concentrated in clusters at the cell surface of Peer cells that were separated from the endoplasmic reticulum (Fig. 5A). Using the raft marker fluorescein isothiocyanate-CTx B subunit, we found that the drug co-clustered with lipid rafts in the plasma membrane of Jurkat and Peer cells (Fig. 5B). However, we were unable to detect these clusters in drug-treated HeLa cells. In addition, a combination of the fluorescence microscopic images regarding the location of PTE-edelfosine, lipid rafts, and endoplasmic reticulum in Peer cells highlighted a total colocalization between PTE-edelfosine and lipid rafts that was set apart from endoplasmic reticulum (Fig. 5C). Thus, we found that edelfosine localized in the same subcellular particulates as its two major drug targets, i.e. plasma membrane (Fas) and endoplasmic reticulum (activated CCT) in a cell type-specific way.


Figure 5
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  		FIGURE 5.
Plasma membrane and endoplasmic reticulum localization of the fluorescent analog PTE-edelfosine in leukemic and solid tumor cells. A, cells were transfected with erRFP plasmid to visualize endoplasmic reticulum (ER) (red fluorescence) and then incubated with 20 µM PTE-edelfosine (blue fluorescence). Areas of colocalization between endoplasmic reticulum and PTE-edelfosine in the merge panels are pink. The corresponding differential interference contrast (DIC) microscopy images are also shown. B, Jurkat and Peer cells were incubated with 20 µM PTE-edelfosine (blue fluorescence, PTE-EDLF) for 3 h, and then its colocalization with membrane rafts was examined using fluorescein isothiocyanate-CTx B subunit (green fluorescence, Rafts) as previously described (7). C, Peer cells were transfected with erRFP plasmid to visualize endoplasmic reticulum (ER) (red fluorescence) and then incubated with 20 µM PTE-edelfosine (blue fluorescence) and fluorescein isothiocyanate-CTx B subunit (green fluorescence, Rafts). Areas of colocalization are shown in the merge panel. The corresponding DIC image is also shown. Images are representative of three independent experiments. Bar, 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have found that the antitumor alkyl-lysophospholipid edelfosine is differentially compartmentalized in leukemic and solid tumor cells, exerting different biological actions in both cell types that eventually lead to apoptosis. Thus, the mechanism of action of this antitumor alkyl-lysophospholipid is cell type-dependent. The data reported here lead us to propose a putative model in which the alkyllysophospholipid edelfosine is mainly localized at the cell surface lipid rafts in human leukemic cells and at the endoplasmic reticulum in human solid tumor cells, affecting processes taking place in both subcellular structures that eventually induce lipid raft- and endoplasmic reticulum-mediated cell death, respectively. Our previous (7, 19) and present data indicate that edelfosine induces a rather rapid and direct Fas-dependent apoptotic response in leukemic cells, whereas the induction of apoptosis in solid tumor cells is rather indirect and requires longer incubation times, being preceded by inhibition of PtdCho biosynthesis at the CCT step and a G2/M cell cycle arrest. This might explain the fact that leukemic cells are more sensitive to edelfosine than solid tumor cells. PtdCho is essential for maintaining membrane structure and for the survival of cultured cells because it is the major membrane phospholipid in animal cells and serves as a precursor for two other abundant membrane phospholipids, sphingomyelin (44) and phosphatidylethanolamine (45). In addition, cells must double their phospholipid mass to form daughter cells, and therefore PtdCho biosynthesis inhibition affects different phases of cell cycle (46). Thus, edelfosine-induced cell cycle arrest and the ensuing apoptosis in HeLa and A549 cells might be related to the inhibitory action of the drug on PtdCho biosynthesis. In this regard, inhibition of PtdCho synthesis at the CCT step by edelfosine has been considered a major physiological imbalance that accounts for the cytotoxic action of the drug (6). To be activated, CCT must translocate from an inactive nuclear reservoir to a functional site on the endoplasmic reticulum (13). Our findings show for the first time the main location of edelfosine in the endoplasmic reticulum in solid tumor cells, giving an explanation to previous reports that suggested a major role for the inhibition of PtdCho biosynthesis in the mechanism of action of this drug (6, 15). On the other hand, the endoplasmic reticulum regulates apoptosis both by sensitizing mitochondria to a variety of extrinsic and intrinsic death stimuli and by initiating cell death signals of its own (47). Endoplasmic reticulum stress has been shown to lead to an apoptosis signal-regulating kinase 1 (ASK1)-dependent JNK activation and cell death (48, 49), and this squares with the key role of JNK in edelfosine-induced apoptosis (28, 29) and the subcellular distribution reported here of the alkyl-lysophospholipid in the endoplasmic reticulum in solid tumor cells. Thus, our results suggest that the endoplasmic reticulum can be a major target for this drug in solid tumor cells that could lead to inhibition of PtdCho synthesis and to a putative endoplasmic reticulum stress-mediated cell death. However, in Jurkat and Peer leukemic cells, the drug was mostly located at the plasma membrane, colocalizing with membrane rafts, in agreement with previous studies (7, 16). Unlike HeLa and A549 solid tumor cells, T-lymphoid Jurkat and Peer leukemic cells did not show any inhibition in PtdCho biosynthesis following edelfosine treatment. In contrast, the alkyl-lysophospholipid promoted clustering of cell surface lipid rafts and recruitment of Fas and downstream signaling molecules, including FADD and JNK, in these membrane microdomains. We have recently shown that procaspase-8 and procaspase-10 were also translocated to Fas-enriched membrane rafts upon treatment of Jurkat leukemic cells with edelfosine (7). Thus, edelfosine induces the recruitment into lipid rafts of Fas, FADD, and procaspase-8, the three major components of the so-called death-inducing signaling complex involved in the triggering of an efficient apoptotic response (50). However, Daxx, which has been reported to participate in Fas-mediated apoptosis and JNK activation through a Fas-Daxx-JNK pathway (34), was not translocated to membrane rafts in edelfosine-treated Jurkat cells despite a rapid and persistent drug-induced JNK activation. Our results agree with reports showing that Fas-induced JNK activation is mediated by death-inducing signaling complex formation (51) or by extensive Fas receptor aggregation (52), but not by Daxx in T-lymphoid cells. The concentration of Fas and downstream signaling molecules in membrane rafts in edelfosine-treated Jurkat cells might lead to a potent and rapid apoptotic response, precluding any putative later event in the endoplasmic reticulum. In contrast, this translocation of Fas and downstream molecules into lipid rafts does not occur in HeLa cells; therefore, this allows later events to take place, including inhibition of PtdCho biosynthesis and cell cycle arrest that will lead ultimately to apoptosis. This suggests the existence of distinct lipid rafts in Jurkat and HeLa cells, differing in both composition and function. In this regard, Jurkat cells lack caveolin and plasma membrane domains with the characteristic features of caveolae, whereas HeLa cells express caveolin and have caveolae (36). Lipid rafts in cells appear to be heterogenous both in terms of their protein and their lipid content (53), and this diversity in the composition of lipid rafts might underlie their implication in diverse functions, including signal transduction, endocytosis, cholesterol trafficking, cell-cell communication, pathogen entry, etc. (35, 53, 54).

The current data would also be compatible with the notion that edelfosine is first accumulated in lipid rafts and then internalized into the endoplasmic reticulum, but the timing of these processes is largely dependent on the cell type. This view is consistent with recent evidence showing that HeLa cells incorporate edelfosine via raft- and dynamin-mediated endocytosis (55), whereas the drug remains in cell surface raft clusters in Jurkat cells (7) by the time apoptosis is induced. Thus, the concentration of edelfosine in membrane rafts in leukemic cells induces co-clustering of lipid rafts with Fas and downstream signaling molecules, leading to a rapid apoptotic response (7), and thereby any putative subsequent event at the endoplasmic reticulum level would be irrelevant in these cells. This indicates that edelfosine-induced apoptosis is mediated through the modification of the protein composition of lipid rafts in leukemic cells. In this regard, the induction of cell killing by edelfosine in the budding yeast Saccharomyces cerevisiae is mediated through the drug incorporation into lipid rafts and the subsequent selective modification of lipid raft composition, resulting in displacement of the essential plasma membrane protein Pma1p from lipid rafts (56).

The differential subcellular compartmentalization of edelfosine provides an explanation for the distinct responses induced by edelfosine in leukemic versus solid tumor cells and sets a new framework for the identification of novel sites of therapeutic intervention. The novel antitumor drug Aplidin is also incorporated into lipid rafts in leukemic cells, inducing recruitment of death receptors and downstream signaling molecules in lipid rafts and promoting a very rapid apoptotic response (57). These data suggest that lipid rafts are a major target in cancer therapy, particularly in leukemic cells. On the other hand, the fact that an antitumor drug can render different subcellular locations in a cell type-dependent manner leads to the notion that an antitumor drug is able to elicit distinct cell death-signaling processes that are specific for and triggered in each targeted subcellular structure, thus explaining, in part, the diverse actions of an antitumor drug in different cancer cells.


   FOOTNOTES
 
* This work was supported in part by grants from Fondo de Investigación Sanitaria and European Commission (FIS-FEDER 04/0843, 02/1199), Ministerio de Educación y Ciencia (SAF2005-04293), Fundación de Investigación Médica Mutua Madrileña (FMM), Fundación "la Caixa" (BM05-30-0), and Junta de Castilla y León (CSI04A05). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of a predoctoral fellowship from the Junta de Castilla y León. Back

2 Supported by the Ramón y Cajal Program from the Ministerio de Educación y Ciencia of Spain. Back

3 To whom correspondence should be addressed. Tel.: 34-923-294806; Fax: 34-923-294795; E-mail: fmollin{at}usal.es.

4 The abbreviations used are: edelfosine, ET-18-OCH3 or 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; CCT, CTP:phosphocholine cytidylyltransferase; CDP-choline, cytidine 5'-diphosphate choline; CTx, cholera toxin; FADD, Fas-associated death domain-containing protein; GM1, Galbeta1,3GalNAcbeta1,4(NeuAc{alpha}2,3)-Galbeta1,4Glc-ceramide; JNK,c-Junamino-terminalkinase;PBS,phosphate-bufferedsaline;PtdCho,phosphatidylcholine; PTE-edelfosine, all-[E]-1-O-[15'-phenylpentadeca-8',10',12',14'-tetraenyl]-2-O-methyl-rac-glycero-3-phosphocholine; Daxx, Fas death domain-associated protein. Back

5 F. Mollinedo, unpublished observations. Back



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 DISCUSSION
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