Inhibition of Glucose Metabolism Sensitizes Tumor Cells to Death Receptor-triggered Apoptosis through Enhancement of Death-inducing Signaling Complex Formation and Apical Procaspase-8 Processing*

Cristina Muñoz-PinedoDagger§, Carmen Ruiz-Ruiz, Carmen Ruiz de Almodóvar§, Carmen Palacios, and Abelardo López-Rivas

From the Instituto de Parasitología y Biomedicina, Consejo Superior de Investigaciones Cientificas, Calle Ventanilla 11, 18001 Granada, Spain

Received for publication, December 5, 2002, and in revised form, January 29, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tumors display a high rate of glucose uptake and glycolysis. We investigated how inhibition of glucose metabolism could affect death receptor-mediated apoptosis in human tumor cells of diverse origin. We show that both substitution of glucose for pyruvate and treatment with 2-deoxyglucose enhanced apoptosis induced by tumor necrosis factor (TNF)-alpha , CD95 agonistic antibody, and TNF-related apoptosis-inducing ligand (TRAIL). Inhibition of glucose metabolism enhanced killing of myeloid leukemia U937, cervical carcinoma HeLa, and breast carcinoma MCF-7 cells upon death receptor ligation. Caspase activation, mitochondrial depolarization, and cytochrome c release were increased under these conditions. Glucose deprivation-mediated sensitization to apoptosis was prevented in MCF-7 cells overexpressing BCL-2. Interestingly, the human B-lymphoblastoid cell line SKW6.4, a prototype for mitochondria-independent death receptor-induced apoptosis, was also sensitized to anti-CD95 and TRAIL-induced apoptosis under glucose-free conditions. Changes in c-FLIPL and cFLIPs levels were observed in some but not all the cell lines studied following glucose deprivation. Glucose deprivation enhanced death receptor-triggered formation of death-inducing signaling complex and early processing of procaspase-8. Altogether, these results suggest that the glycolytic pathway may be an important target for therapeutic intervention to sensitize tumor cells to selectively toxic soluble death ligands or death ligand-expressing cells of the immune system by facilitating the activation of initiator caspase-8.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Apoptotic cell death plays a fundamental role in normal development, tissue homeostasis, and pathological situations (1, 2). CD95 (Fas/Apo-1) receptor, a member of TNF1 receptor family (3, 4), is a potent activator of apoptosis upon interaction with its natural ligand CD95L, a type II integral membrane protein homologous to TNF (5). TRAIL, a recently identified member of the TNF family with homology to CD95L (6), induces apoptosis in different tumor cells upon binding to death-domain containing receptors, TRAIL-R1 and TRAIL-R2 (7-10). Although the expression of CD95L seems to be more restricted to lymphoid cells (5), TRAIL transcripts are detectable in many normal organs and tissues (6) suggesting that this ligand may be non-toxic to the majority of normal cells. Tumor cells frequently express significant levels of death receptors in their plasma membrane (11). However, sensitivity of tumor cells to death receptor-mediated apoptosis does not always correlate with death receptor expression (11, 12). In this respect, understanding the mechanisms that regulate tumor cell sensitivity to death ligand-induced apoptosis should be an important objective in the development of therapies to treat human malignancies.

Recent studies (13, 14) have revealed that apoptosis induced by various death receptor-independent treatments is blocked in ATP-depleted cells. Furthermore, although the ATP-dependent steps in apoptosis have not been completely elucidated, it has been suggested that the nuclear transport of pro-apoptotic factors could be one of the processes requiring an active energy metabolism (15). More controversial is the role of ATP in death receptor-mediated apoptosis as completely opposite results have been reported regarding the effect of ATP depletion on CD95-mediated apoptosis (14, 16, 17). Thus, some groups have reported (16, 17) that CD95-mediated apoptosis is prevented when cells are depleted of ATP. Under these conditions cell death caused by CD95 activation changes from apoptosis to necrosis (17). However, other data have indicated that although prevention of ATP generation completely inhibits caspase activation and apoptosis in response to chemotherapeutic drugs, ATP depletion does not affect CD95-induced apoptosis (14). Most of these studies were performed in glucose-free medium, in the presence of the mitochondrial inhibitor oligomycin, to prevent ATP production from both glycolysis and oxidative phosphorylation and thus achieve maximal depletion of cellular ATP (14, 16, 17). However, besides causing ATP depletion, inhibition of F0F1-ATPase by oligomycin may interfere in the apoptotic program by preventing cytosol acidification and cytochrome c release from mitochondria (18, 19). Furthermore, oligomycin can cause cell death by apoptosis or necrosis (14, 20).

An elevated number of tumor cells display a high rate of glycolysis under aerobic conditions, and some of them depend on the glycolytic flux to maintain the cellular levels of ATP and metabolism (21, 22). In vivo (23) as well as in vitro (23, 24) studies have revealed that sensitivity of tumor cells to TNF-alpha is increased under conditions of reduced glucose metabolism. These findings have led some investigators to propose the use of glucose anti-metabolites in combination with TNF-alpha as a possible anti-tumor treatment (24). However the mechanism underlying the facilitation of TNF-alpha action by 2-deoxy-D-glucose (2-DG) has not been characterized. More recently, it was reported that glucose deprivation enhances TRAIL-induced apoptosis by down-regulating the expression of cFLIP through the ceramide-AKT-FLIP pathway (25).

In this report, we have examined the effect of glucose deprivation on apoptosis induced upon activation of different death receptors of the TNF/nerve growth factor receptor family in human tumor cells whose ATP levels are largely dependent on the activity of the glycolytic pathway. The results obtained indicated that under conditions of glucose metabolism inhibition, death receptor-induced apoptosis was considerably enhanced in U937 myeloid leukemic, SKW6.4 B-lymphoblastoid, MCF-7 breast carcinoma and HeLa cervical carcinoma cells. Inhibition of glucose metabolism promoted the activation of both mitochondria-dependent and -independent pathways of death receptor-induced apoptosis. Finally, our data indicate that the sensitization mechanism probably involves the increased processing of apical procaspase-8 at the DISC upon death receptor activation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Antibodies-- RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Invitrogen. Etoposide, 4',6'-diamidino-2-phenylindole, RPMI 1640 glucose-free medium, 2-deoxy-D-glucose (2-DG), streptavidin-agarose beads, mouse anti-alpha -tubulin antibody, insulin, and LY294002 were obtained from Sigma. CH-11 monoclonal antibody (IgM) reacting with CD95 was from Upstate Biotechnology, Inc. (Lake Placid, NY). Human TRAIL and TNF-alpha were obtained from PeproTech (London, UK). Mouse anti-human caspase-8 monoclonal antibody was purchased from Cell Diagnostica (Münster, Germany). Rabbit anti-cleaved caspase-9 and anti-cleaved caspase-3 polyclonal antibodies were obtained from New England Biolabs (Beverly, MA). AKT and phospho-AKT (Ser-473) antibodies were from Cell Signaling (Beverly, MA). Anti-X chromosome-linked inhibitor of apoptosis protein and cellular inhibitor of apoptosis protein antibodies were donated by Dr. Douglas R. Green (La Jolla Institute for Allergy and Immunology, San Diego, CA). Anti-cFLIP antibody NF6 was provided by Dr. M. Peter (University of Chicago). Mouse monoclonal antibodies to BAX and cytochrome c were obtained from Pharmingen. Anti-AIF was a gift of Dr. Santos Susin (CNRS, Villejuif, France). Caspases inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (Z-VAD-FMK) was from Enzyme System Inc. (Dublin, CA). 3,3'-Dihexyloxacarbocyanine iodide(3) and dichlorodihydrofluorescein diacetate (H2DCFDA) were purchased from Molecular Probes (Eugene, OR). L-Buthionine-(SR)-sulfoximine was a generous gift from Dr. Isabel Fabregat, Universidad Complutense (Madrid, Spain).

Cell Lines and Cultures-- The human SKW6.4 B-lymphoblastoid cell line was provided by Dr. Katja Zimmermann (La Jolla Institute for Allergy and Immunology, San Diego, CA). The human breast tumor cell line MCF-7 was kindly donated by Dr. M. Ruiz de Almodovar (Department of Radiology, University of Granada). Human U937 myeloid leukemic, SKW6.4 B-lymphoblastoid, HeLa cervical carcinoma, and MCF-7 breast carcinoma cells were maintained in RPMI medium containing 10% fetal bovine serum, 1 mM glutamine, and gentamycin at 37 °C in a humidified 5% CO2, 95% air incubator. MCF-7 cells stably overexpressing human BCL-2 (MCF-7BCL-2) and mock-transfected cells (MCF-7neo) were generated as described (26).

Incubation under glucose-free conditions was performed by washing cells twice in glucose-free RPMI 1640 medium and incubating them in the same medium with 1 mM glutamine, 2 mM pyruvate, and 5% dialyzed FBS. Control cultures were incubated with 2 g/liter glucose instead of pyruvate. Treatment with 2-DG was performed in normal RPMI 1640 medium with 1 mM glutamine and 10% FBS.

Analysis of Apoptosis and Cell Viability-- Hypodiploid apoptotic cells were detected by flow cytometry according to published procedures (27). Basically, cells were washed with phosphate-buffered saline (PBS), fixed in cold 70% ethanol, and then stained with propidium iodide while treating with RNase. Quantitative analysis of sub-G1 cells was carried out in a FACScan cytometer using the Cell Quest software (BD Biosciences). Phosphatidylserine exposure on the surface of apoptotic cells was examined by flow cytometry after staining with annexin-V-FLUOS (Roche Molecular Biochemicals), following instructions provided by the manufacturer. Viable MCF-7 and HeLa cells were determined by the crystal violet method as described (26). Chromatin condensation was assessed after staining of cellular DNA with 4',6'-diamidino-2-phenylindole (alone or in combination with 1 µM propidium iodide) and viewing the cell preparations under a Zeiss Axiophot fluorescent microscope.

ATP Determination-- ATP was determined luminometrically as described (14).

Measurements of Mitochondrial Depolarization and Reactive Oxygen Species (ROS)-- For measurements of mitochondrial depolarization, cells were collected by centrifugation and resuspended in PBS with 40 nM 3,3'-dihexyloxacarbocyanine iodide(3) and 5 µM propidium iodide and incubated for 15 min at room temperature. Generation of ROS was quantified by adding H2DCFDA to the culture medium at a concentration of 5 µM, for the last 30 min of treatment. Similar results were obtained when staining with H2DCFDA was performed at room temperature in PBS. Quantitative analyses of Delta Psi m (mitochondrial membrane potential) and ROS production were carried out in a FACScan cytometer using the Cell Quest software.

Immunoblot Analysis of Proteins-- MCF-7 and HeLa cells were detached with RPMI/EDTA, washed with PBS, and collected by centrifugation. U937 and SKW6.4 cells were washed with PBS. Protein content was measured before lysing the cells in Laemmli sample buffer under reducing conditions. Cell lysates were sonicated, and proteins were resolved on SDS-polyacrylamide minigels and detected as described (26). For measurements of cytochrome c release from mitochondria and BAX redistribution, cells were lysed and cytosolic fractions were separated from mitochondria and nuclei as described previously (28). Equal protein loading in each lane was verified by incubating membranes with anti-alpha -tubulin antibody.

Isolation of the TRAIL DISC-- DISC precipitation was performed using biotin-tagged recombinant TRAIL (Bio-TRAIL) (29, 30), which was a gift from Nick Harper (MRC Toxicology Unit, University of Leicester, UK). U937 cells (75 × 106 cells/treatment) were washed twice in sterile PBS and incubated for 12 h in glucose-free RPMI 1640 medium with 1 mM glutamine, 2 mM pyruvate, and 5% dialyzed FBS. Control U937 cells were incubated in the same medium with 2 g/liter glucose instead of pyruvate. After this incubation, the same number of control and glucose-deprived cells were treated with Bio-TRAIL for the times indicated in the figure legends. DISC formation was then stopped, and unbound TRAIL was removed by washing the cells three times in ice-cold PBS. Cells were lysed in 3 ml of lysis buffer (30 mM Tris/HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, containing CompleteTM protease inhibitors (Roche Molecular Biochemicals)) for 30 min on ice followed by centrifugation at 15,000 × g for 10 min at 4 °C. To provide an unstimulated receptor control, Bio-TRAIL was added to lysates from untreated cells. The TRAIL DISC was then precipitated using 30 µl of streptavidin-agarose beads at 4 °C overnight. Precipitates were washed six times with lysis buffer, and receptor complexes were eluted with 30 µl of sample buffer. Western blotting was performed as described above.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glucose Depletion Enhances Apoptosis Induced by Death Receptor Activators but Not by Etoposide in U937 Myeloid Leukemic Cells-- To address the question of the effect of ATP depletion on death receptor-mediated apoptosis, we first tested whether glucose removal was sufficient to deplete intracellular ATP in U937 cells. Incubation of U937 cells in a glucose-free RPMI 1640 medium supplemented with pyruvate, glutamine, and 5% dialyzed FBS, as described under "Experimental Procedures," led to a marked drop in ATP levels (Fig. 1). The ATP decrease occurred more rapidly when cells were incubated under the same conditions with 1% dialyzed serum (Fig. 1a). Because viability of cultures upon glucose deprivation was better maintained in medium containing 5% dialyzed FBS, this serum concentration was used in all experiments shown in the present work. When U937 cells were incubated in the absence of glucose and pyruvate, cells died rapidly, probably due to the lack of a carbon source rather than lack of ATP (not shown). We next examined the effect of glucose deprivation on apoptosis induced either by the DNA-damaging drug etoposide or by activators of different death receptors. We observed that in cultures of U937 cells treated with the DNA-damaging drug etoposide under glucose-deprived conditions apoptosis was inhibited (Fig. 1b), in agreement with reported data (14). In contrast, when apoptosis was induced by ligation of CD95, TNFR, or TRAILR, we observed a marked increase in the percentage of apoptotic cells in glucose-free medium, as measured by sub-G1 DNA content (Fig. 1b). Inhibition of etoposide-induced apoptosis and enhancement of death receptor-induced apoptosis were also observed in cells incubated under glucose-free conditions with 1% FBS (not shown), in which the ATP content was very low (10% of normal level).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Inhibition of glucose metabolism markedly decreased ATP levels in human myeloid leukemic U937 cells and sensitized cells to death receptor-induced apoptosis. a, cells were washed twice in glucose-free medium and incubated for the indicated times in either glucose-containing or glucose-free, pyruvate-supplemented RPMI 1640 medium, with the stated concentrations of dialyzed FBS. ATP content was determined as indicated under "Experimental Procedures." Results are expressed as relative content of ATP as compared with cells incubated in glucose-containing medium. Error bars show S.D. of three experiments. b, U937 cells were incubated for 2 h in glucose-free or glucose-supplemented medium containing 5% FBS and treated for a further 14-h period in the same culture media with anti-CD95 (20 ng/ml), TNF-alpha (1 ng/ml), TRAIL (10 ng/ml), or etoposide (0.5 µg/ml). Percentage of total cell population with sub-G1 DNA content was determined. Error bars show S.D. of three experiments. c, U937 cells were incubated for 20 h in the presence or absence of 5 mM 2-DG and anti-CD95 (20 ng/ml), TNF-alpha (1 ng/ml), or TRAIL (10 ng/ml). Incubations were performed in the presence or absence of the caspases inhibitor Z-VAD-FMK (100 µM). Apoptosis was assessed by analysis of the percentage of total cells with hypodiploid DNA content. Error bars represent S.D. of three independent experiments.

Inhibition of Glucose Metabolism Facilitates a Caspase-dependent Pathway of Death Receptor-induced Apoptosis in Different Human Tumor Cells-- It was previously reported that 2-DG, a non-metabolizable glucose analogue that inhibits glucose metabolism, enhanced TNF-alpha -induced apoptosis in U937 cells (24). We have confirmed these data and demonstrated that anti-CD95- and TRAIL-induced apoptosis was also clearly enhanced when U937 cells were incubated in the presence of 2-DG (Fig. 1c). At the concentration used (5 mM), 2-DG alone did not have a considerable effect on cell death. ATP loss upon 2-DG treatment occurred with similar kinetics to that observed in glucose removal experiments, although the decrease in ATP levels after 20 h of 2-DG treatment was 50% (not shown).

To establish further that the death process promoted by glucose metabolism inhibition was apoptosis, U937 cells were treated for 20 h with 2-DG in the presence of death receptor activators, and several features of apoptosis were examined. Results shown in Fig. 1c indicate that this death was dependent on caspase activation, as it was completely inhibited by the caspase inhibitor Z-VAD-FMK. Furthermore, results not shown indicated that there was an increase in the number of cells showing nuclear condensation and fragmentation in cultures incubated in the presence of both 2-DG and death receptor activator. In these experiments, 2-DG alone did not have a significant effect on cell death (not shown). Under these conditions, a low percentage of cells (<10% in all cases) was stained with propidium iodide, indicating loss of membrane integrity. However, these cells also displayed marked nuclear apoptotic changes suggesting that the observed changes in membrane permeability were probably due to secondary necrosis. Finally, we determined death receptor-induced translocation of phosphatidylserine to the external side of the plasma membrane when glucose uptake and utilization were inhibited by 2-DG. Results not shown indicated that annexin-V binding to cells upon death receptor triggering was also enhanced in the presence of 2-DG.

We next tested whether the regulation by glucose metabolism of death receptor-induced apoptosis was cell type-specific. Accordingly, we carried out some experiments on tumor cells of non-hematopoietic origin such as human breast tumor MCF-7 cells and human cervical carcinoma HeLa cells. MCF-7 cells are very resistant to CD95-mediated apoptosis mainly because they express very low levels of CD95 at the cell surface (26). Therefore, to analyze the role of glucose metabolism in death receptor-induced apoptosis in MCF-7 cells, we used TRAIL as an apoptosis trigger. Data shown in Fig. 2a demonstrate that both glucose withdrawal and 2-DG facilitated the activation by TRAIL of caspase-dependent loss of viability in cultures of breast tumor MCF-7 cells. Likewise, glucose removal from the medium strongly enhanced CD95-mediated cell death in cervical carcinoma HeLa cells (Fig. 2b).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2.   Inhibition of glucose metabolism facilitated a caspase-dependent death receptor-triggered apoptotic pathway in human breast tumor MCF-7 and cervical carcinoma HeLa cells. a, MCF-7 cells were incubated for 16 h either in glucose-free or glucose-containing medium or in the presence of 5 mM 2-DG, as described under "Experimental Procedures." Z-VAD-FMK (50 µM) and TRAIL (50 ng/ml) were added to some cultures. b, HeLa cells were incubated for 15 h in medium with or without glucose and various concentrations of CD95 antibody. Cell viability was estimated by the crystal violet method. Glucose removal caused a decrease in optical density that was due to inhibition of growth rather than cell death, as confirmed by microscopic observation (not shown). Error bars represent S.D. of three independent experiments.

Glucose Deprivation Facilitates Both Mitochondria-dependent and -independent Pathways of Death Receptor-mediated Apoptosis-- Death receptor-induced apoptosis can occur by at least two different pathways (31). One of them involving the mitochondria is facilitated when protein synthesis is inhibited and is inhibited in cells overexpressing anti-apoptotic BCL-2 family members (32). In this respect, death receptor-induced apoptosis in U937 cells is markedly enhanced in the presence of protein synthesis inhibitors (33) and inhibited in clones overexpressing BCL-2 (34). These results suggest that the mitochondrial pathway of apoptosis is required for death receptor-induced apoptosis in U937 cells. In order to understand the step in death receptor-triggered cell death that is modulated by glucose levels, we first examined whether or not inhibition of glucose metabolism may regulate the mitochondria-regulated apoptotic pathway. To this end we analyzed in U937 cells the depolarization of mitochondrial membrane and the release of cytochrome c into the cytosol, two mitochondrial parameters that have been largely implicated in death receptor-mediated apoptosis (35). Fig. 3a shows that although death receptor activation induced mitochondrial depolarization in U937 cells in the absence of 2-DG, this event was further stimulated upon ligation of death receptors in cells incubated in the presence of 2-DG. Likewise, release of cytochrome c into the cytosol of U937 cells was facilitated in the presence of glucose anti-metabolite (Fig. 3b).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3.   Glucose deprivation facilitates both mitochondria-dependent and -independent pathways of death receptor-mediated apoptosis. U937 cells were incubated for 20 h in the presence or absence of 5 mM 2-DG and anti-CD95 (20 ng/ml), TNF-alpha (1 ng/ml), or TRAIL (10 ng/ml). a, percentage of propidium iodide-negative population with low mitochondrial delta-psi is represented. b, cytochrome c release from mitochondria was assessed as described under "Experimental Procedures." n.t., not treated. c, mock-transfected (MCF-7neo) and BCL-2-overexpressing MCF-7 (MCF-7BCL-2) cells were incubated for 16 h in the absence or presence of glucose, as described under "Experimental Procedures," with or without TRAIL (50 ng/ml). Cell viability was estimated by the crystal violet method. Results show the average and range of 2 independent experiments. d, SKW6.4 cells were treated with the indicated doses of CD95 antibody or recombinant TRAIL, in glucose-free or glucose-containing medium, as described under "Experimental Procedures." Apoptosis was assessed by analysis of the percentage of total cells with sub-G1 DNA content. Results show the average and range of 2 independent experiments.

In breast tumor MCF-7 cells the mitochondrial pathway is also important in death receptor-triggered apoptosis because these cells are deficient in caspase-3 expression, which is required to activate the non-mitochondrial pathway after death receptor activation (31). Furthermore, in MCF-7 cells overexpressing BCL-2 or BCL-xL, both CD95 (26) and TRAILR-induced apoptosis (36) are considerably inhibited, indicating a role of mitochondria in death receptor-mediated apoptosis. In MCF-7BCL-2 cells, we examined the effect of glucose deprivation in TRAIL-induced apoptosis. As shown in Fig. 3c, TRAIL induced significant death when added to glucose-free mock-transfected cultures (MCF-7neo). Results presented in Fig. 3c demonstrate that overexpression of BCL-2 completely abrogated apoptosis induced by the death ligand in cells deprived of glucose. These results also suggest that in those cell lines in which mitochondria are involved in death receptor-induced apoptosis, glucose deprivation does not overcome BCL-2 protection.

The human SKW6.4 B-lymphoblastoid cell line has been extensively used in studies of apoptosis activation by death receptors, particularly CD95. These cells do not require mitochondria-regulated events to undergo apoptosis upon death receptor activation (31). In order to get further insight into the mechanism of glucose depletion-promoted enhancement of death receptor-induced apoptosis, we examined the sensitivity of SKW6.4 cells to CD95 antibody and recombinant TRAIL. As shown in Fig. 3d, incubation of these cells in glucose-free medium increased their sensitivity to both CD95 antibody and TRAIL. Altogether, these results suggested that glucose deprivation must regulate a common step in the mitochondria-dependent and -independent pathways of apoptosis.

Enhancement of DISC Formation and Early Procaspase-8 Processing upon Death Receptor Activation under Conditions of Glucose Metabolism Inhibition-- Results not shown indicated that inhibition of glucose metabolism either by glucose deprivation or 2-DG treatment did not alter the levels of CD95 receptors in U937 cells, assessed by flow cytometry analysis. Therefore, we then tried to determine the step in the signaling pathway triggered upon death receptor activation that was enhanced under conditions of glucose deprivation. After culture in medium with or without glucose, U937 cells were further incubated in the same medium with CD95 antibody for various times. Following this treatment, caspase activation, cytochrome c release from mitochondria, and BAX redistribution from cytosol to a membranous compartment were examined. As shown in Fig. 4a, procaspase-8 cleavage was the first detectable event facilitated by glucose deprivation. In the presence of glucose, processing of procaspase-8 was first observed at 150 min after the addition of CD95 antibody. Interestingly, under glucose-free conditions procaspase-8 processing was clearly observed 45 min after CD95 activation. Regarding cytochrome c release from mitochondria, a small amount of cytosolic cytochrome c could be detected as early as 45 min after CD95 activation in the absence of glucose. At 90 min post-activation, cytochrome c was clearly observed in the cytosol of glucose-deprived U937 cells. At this time, other mitochondria-related events such as loss of BAX from the cytosol and caspase-9 processing were also stimulated upon CD95 activation in glucose-free medium, as was caspase-3 activation. In contrast, when cells were incubated in glucose-containing medium, cytochrome c release, BAX translocation, and processing of caspases were all barely detected at these times (Fig. 4a).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 4.   DISC formation and caspase-8 activation are the earliest detectable events in death receptor-induced apoptosis that are enhanced under glucose-free conditions. a, U937 cells were incubated for 6 h with or without glucose, as described under "Experimental Procedures." Cells were further incubated for the indicated times in the presence of CD95 antibody (500 ng/ml). Caspase activation was determined by Western blot as described. Cytosolic fractions were prepared for measurement of cytochrome c release from mitochondria and BAX redistribution as described under "Experimental Procedures." Total BAX levels in unfractionated extracts did not change under these conditions (not shown). b, U937 cells were incubated for 20 h in the presence or absence of 5 mM 2-DG and anti-CD95 (20 ng/ml), TNF (1 ng/ml), or TRAIL (10 ng/ml). At this time, caspase-8 processing was determined by Western blot. Arrows show the position of procaspase-8 (51-53 kDa) and the cleaved intermediate form of caspase-8 (41-43 kDa). n.t., not treated. c, MCF-7neo and MCF-7BCL-2 cells were incubated for 12 h in the presence or absence of glucose, as described under "Experimental Procedures." After this incubation, cells were treated for the indicated times with TRAIL (250 ng/ml), and procaspase-8 processing was examined by Western blot. Results show representative data from three independent experiments. d, U937 cells (75 × 106) incubated during 12 h with or without glucose were treated with Bio-TRAIL (250 ng/ml) for 15 and 30 min. Unstimulated receptor controls (u/s) represent the addition of Bio-TRAIL to an equivalent volume of lysate from unstimulated cells. TRAIL receptor complexes were precipitated with streptavidin-conjugated agarose beads and analyzed by Western blotting for the known TRAIL DISC components, FADD, and caspase-8. Lysates isolated from unstimulated control cells were included as a positive control for the expression of all these proteins in U937 cells. Data shown are representative of three independent experiments.

Processing of procaspase-8 to its 43-45-kDa intermediate fragments was also markedly stimulated in 2-DG-treated U937 cells upon ligation of CD95, TNFR, and TRAILR by their respective activators (Fig. 4b). In order to exclude the possibility of a mitochondria-operated feedback mechanism in glucose-regulated procaspase-8 processing, we determined TRAIL-induced procaspase-8 processing in MCF-7 cells overexpressing BCL-2. The lack of caspase 3 and the overexpression of BCL-2 make these cells rather refractory to mitochondria-mediated feedback stimulation of procaspase-8 processing (26, 37). To achieve maximal stimulation of procaspase-8 processing in these short term experiments, a higher dose of TRAIL (250 ng/ml) was used. In MCF-7BCL-2 cells activation of caspase-8 by TRAIL was still further enhanced when cells were previously deprived of glucose, similar to what could be observed in MCF-7 cells (Fig. 4c). Altogether, these results indicate apical procaspase-8 processing as the step regulated by glucose metabolism in death receptor-induced apoptosis.

To prove that activation of apical procaspase-8 processing is the step regulated by glucose deprivation in death receptor-induced apoptosis, we performed some experiments to examine TRAIL DISC formation in U937 cells that had been cultured for 12 h in medium containing either glucose or pyruvate. Results shown in Fig. 4d demonstrate that DISC formation was enhanced when cells were deprived of glucose prior to the addition of TRAIL. The amounts of both FADD and procaspase-8 bound to the TRAIL DISC were elevated in U937 cells that had been cultured under glucose-free conditions as compared with cells grown in the presence of glucose. Furthermore, activation of procaspase-8 processing at the DISC following TRAIL addition was observed only in glucose-deprived cells (Fig. 4d).

Role of cFLIP Levels and the PI3K/AKT Pathway in Glucose Deprivation-mediated Sensitization of Tumor Cells to Death Receptor-induced Apoptosis-- In order to examine the mechanism by which glucose deprivation enhances death receptor-induced apoptosis, we first determined whether this effect could be observed immediately after glucose withdrawal from the medium. For this purpose, we preincubated U937 cells in glucose-containing or glucose-free pyruvate-containing medium for different times, and we further treated them with a high dose of CD95 antibody. In Fig. 5a, we show that the minimum preincubation time in glucose-free medium required for the sensitizing effect was between 2 and 6 h, and cells were more clearly sensitized to anti-CD95-induced apoptosis at longer preincubation times. These results led us to investigate the possible changes in apoptosis-related biochemical parameters or proteins, in the cells depleted of glucose. Glucose metabolism is closely associated with the ability of cells to regulate their redox status as glutathione is reduced using NADPH+ that is formed through the pentose-phosphate pathway. On the other hand, in the absence of glucose, the use of glutamine as a carbon source could lead to the generation of ROS, which sensitizes several tumor cell lines to TNF-alpha (38, 39). In order to test if this was the case, we loaded U937 cells with the redox-sensitive probe H2DCFDA, and we determined the generation of ROS after 1, 3, 6, or 15 h of incubation by flow cytometry. We could not detect generation of ROS due to glucose deprivation at any of the time points studied (data not shown); instead, a more reducing state was always observed in the absence of glucose (Fig. 5b). Furthermore, treating the cells with the glutathione-depleting drug L-buthionine-(SR)-sulfoximine did not enhance cell death induced by anti-CD95 in glucose-containing medium (not shown), suggesting that depletion of glutathione is not the mechanism by which cells are sensitized to death receptor-triggered apoptosis in the absence of glucose. On the other hand, we could not detect any change in the protein levels of BCL-2, BAX, FADD, AIF, SMAC/DIABLO, X chromosome-linked inhibitor of apoptosis protein, and cellular inhibitor of apoptosis protein (not shown).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 5.   Role of cFLIP levels and PI3K/AKT pathway in glucose deprivation-mediated sensitization of tumor cells to death receptor-induced apoptosis. a, U937 cells preincubated in medium containing either glucose or pyruvate for the indicated times were further treated with anti-CD95 (500 ng/ml) for 150 min. Apoptosis was assessed by analysis of the percentage of total cells with sub-G1 DNA content. Error bars represent S.D. of 3 independent experiments. b, U937 cells were incubated for 6 h in medium with glucose or pyruvate and processed for determination of ROS as described under "Experimental Procedures." Results show a representative experiment from three independent determinations. c, U937 cells were washed and incubated in medium with or without glucose for 15 h, as described under "Experimental Procedures." After collecting the cells, cFLIPL and cFLIPs levels were determined by Western blotting as described under "Experimental Procedures." d, MCF-7 and U937 cells were preincubated with LY294002 for 1 h. After this incubation, TRAIL was added, and the cultures were further incubated for 15 h. Viable cells (MCF-7) were determined by the crystal violet method. In U937 cell cultures apoptosis was assessed by analysis of the percentage of total cells with hypodiploid DNA content. Results show the average and range of two independent experiments. Left panel also shows Western blot analysis of phospho-AKT and AKT levels in MCF-7 cells preincubated in the absence or presence of LY294002 (20 µM) for 1 h and treated with insulin (10 µg/ml) for 20 min to demonstrate the activity of the PI3K inhibitor. Right panel shows Western blot analysis of phospho-AKT and AKT levels in U937 cells treated with or without LY294002 (20 µM) for 16 h (lanes 2 and 3). Lane 1 is a control extract of MCF-7 cells stimulated with insulin for 20 min.

It has been reported that changes in sensitivity of cells to death ligands may be due to changes in cFLIP levels (25, 40). Because we have observed that sensitization following glucose deprivation occurred at an early stage in death receptor-induced apoptosis (Fig. 4), we analyzed cFLIPL and cFLIPs expression after glucose withdrawal in the cell lines used in our study. As a control for cells expressing both cFLIPL and cFLIPs, we included a Jurkat cell lysate (Fig. 5c). We observed that cFLIPL levels in U937, SKW6.4, and HeLa cells deprived of glucose declined after 6 h (not shown) and were clearly decreased by 15 h but did not change in MCF-7 cells (Fig. 5c). However, the role of cFLIPL in death receptor-mediated apoptosis remains controversial (41). In contrast to cFLIPL, cFLIPs levels could be critical in the regulation of procaspase-8 activation in the DISC (42), but its expression is more restricted to certain cell types (43). We analyzed cFLIPs levels following a 15-h deprivation of glucose in the various cell lines. As shown in Fig. 5c, cFLIPs was only detected in HeLa cells, and its expression was markedly reduced in glucose-deprived cells. Consequently, these results indicated that a relationship between a decrease in cFLIP levels upon glucose deprivation and sensitivity of the various cell types to death receptor-induced apoptosis could not be established as a general rule.

It has been reported recently (25) that in a human prostate adenocarcinoma cell line, glucose deprivation in combination with TRAIL caused sustained dephosphorylation and inactivation of AKT that subsequently down-regulated cFLIP expression in these cells. To examine whether inactivation of the PI3K/AKT pathway could be involved in the sensitization process triggered by glucose deprivation in our studies, we performed some experiments with LY294002, an inhibitor of the PI3K/AKT pathway (44). Results shown in Fig. 5d indicated that LY294002 at a concentration that inhibited insulin-induced phosphorylation of AKT at Ser-473 did not sensitize MCF-7 cells to TRAIL-induced apoptosis. Furthermore, in U937 cells grown in glucose-containing medium, we could not detect phosphorylated AKTSer-473, and LY294002 did not increase TRAIL-mediated apoptosis (Fig. 5d). In these cells, cFLIPL levels did not change after treatment with LY294002 for 16 h (data not shown). Accordingly, these results indicate that in these cells activity of the PI3K/AKT pathway does not play a prominent role in the regulation of sensitivity to death receptor-induced apoptosis. As a consequence of these findings, sensitization to death receptor-triggered apoptosis in MCF-7 and U937 cells deprived of glucose probably occurs through a mechanism not involving AKT inactivation.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of death receptors in cancer cells offers the possibility of killing these cells with the appropriate ligands, as novel anti-tumor agents (11). However, sensitivity of tumor cells to death ligands does not depend solely on receptor expression levels in the membrane of these cells as there are other anti-apoptotic mechanisms that can be operative (31, 45). In order to sensitize tumor cells to death receptor-mediated apoptosis, different studies have addressed the question of the mechanisms regulating the biochemical signaling elicited upon death receptor activation (24, 26, 40, 45-47). One important feature of many tumor cells is that they have a high rate of glycolysis, regardless of whether their oxygen supply is adequate or low (21, 22). This characteristic of tumor cells leads to a stringent dependence on extracellular glucose for maintaining intracellular ATP levels and may also be important in the acidification of the extracellular environment (48). Our results indicate that tumor cells of different types can be markedly sensitized to death receptor-mediated apoptosis either by glucose deprivation or by inhibition of glucose metabolism with 2-DG. Several conclusions can be extracted from our data. In the first place, intracellular ATP does not seem to be required for the induction of apoptosis upon ligation of either death receptor of the TNFR family investigated in our work. This is clearly indicated by the experiments carried out in U937 cells incubated in glucose-free medium and low serum. Although intracellular ATP dropped to 10% of normal levels under certain conditions, death receptor-induced apoptosis was still markedly potentiated. In this respect, recent studies (14, 16, 17) have produced controversial results regarding the role of ATP in CD95-mediated apoptosis. In these studies, depletion of cellular ATP was achieved in the presence of oligomycin to prevent mitochondrial generation of ATP. However, inhibition of F0F1-ATPase by oligomycin has been demonstrated to cause important alterations in the mitochondria-regulated pathway of apoptosis (18) and therefore must be avoided when analyzing the role of energy metabolism in apoptosis. On the other hand, although a role of ATP depletion has been claimed in the enhancement of CD95-mediated apoptosis of hepatocytes by fructose (49), recent results (50) obtained in Jurkat cells have suggested no implication of ATP loss in the increase of CD95-induced apoptosis by uncouplers of oxidative phosphorylation. Our data indicate that in the tumor cells studied in our report, similar enhancement of death receptor signaling of apoptosis was achieved under conditions producing very different effects on cellular ATP content. Furthermore, a 50% decrease in ATP levels, as obtained with 2-DG, still maximally sensitized the tumor cells to death receptor-activated apoptosis.

Although we cannot completely exclude a certain role of ATP loss in the increased sensitivity observed in our report, the above data suggest that changes in other metabolic parameters during glucose deprivation or inhibition of glucose metabolism could be more important to sensitize the tumor cells to death receptor-mediated apoptosis. In this respect, glucose deprivation has been demonstrated as a potent apoptosis inducer in several Myc-transformed cell types (51). This effect was mediated by induction of lactate dehydrogenase-A activity, whose gene is transcriptionally activated by c-Myc (52). It was suggested that constitutive generation of NAD+ and lactate by lactate dehydrogenase-A and the decrease in the regeneration of NADH by inhibition of glycolysis could alter the redox state in the cells and make them more susceptible to apoptosis (51). It is also interesting that c-Myc induces sensitization of cells to CD95-mediated apoptosis (53). It has also been described that in the absence of glucose there could be an increased production of reactive oxygen species arising from the metabolism of glutamine that sensitizes the cells to death receptor-mediated apoptosis, as reported in TNF-alpha -induced death of fibrosarcoma cells (39). However, our results indicate that neither the generation of peroxides nor the depletion of cellular glutathione is responsible for the sensitizing effect of glucose deprivation. Therefore, the connection between oncogenic transformation, glycolytic metabolism, and susceptibility to death receptor-mediated apoptosis remains to be elucidated. It is feasible that the increased expression of glycolytic enzymes that results from neoplastic transformation may be advantageous to tumor growth under certain conditions (52) but paradoxically increases the sensitivity of these cells to glucose deprivation (51) and death receptor-mediated apoptosis (present work).

The protein synthesis inhibitor cycloheximide is a well known promoter of death receptor-mediated apoptosis (32). It has been proposed that cycloheximide facilitates TNF-alpha -induced apoptosis by its action on glucose transport, although the mechanism underlying this effect was not revealed (54). Furthermore, activation of death receptor CD95 induced an early down-regulation of glucose transport in the cell, and this effect was suggested to be part of its mechanism of action (55). Therefore, in agreement with our results, it seems from the above data that a decrease in the glycolytic rate may favor the activation by death receptors of the apoptotic mechanism. In contrast, as shown by us and others (14), under conditions of reduced glycolysis genotoxic drug-induced apoptosis is inhibited.

Similar to cycloheximide-induced sensitization to CD95-mediated apoptosis (40), facilitation of death receptor-induced apoptosis by glucose metabolism inhibition seems to take place at an early stage in the signaling pathway (our results) (25). Thus, we detected an increased formation of the TRAIL DISC and processing of procaspase-8 as the earliest event analyzed in the sensitization process induced by glucose deprivation. In this respect, the mechanism of cycloheximide and glucose deprivation-induced sensitization to death receptor-mediated apoptosis has been reported recently (25, 40) to involve the down-regulation of the caspase-8 inhibitor cFLIP. Furthermore, the down-regulation of cFLIP in a human prostate adenocarcinoma cell line upon treatment with TRAIL in glucose-free medium was reported (25). Down-regulation of cFLIP levels occurred as a consequence of ceramide generation following glucose deprivation and treatment with TRAIL that led to inhibition of AKT (25). Although we have observed a decrease in cFLIPL levels in glucose-deprived cells in three of the cell lines analyzed in our work, the role of cFLIPL as a physiological caspase-8 inhibitor remains rather controversial (41). In contrast to cFLIPL, cFLIPS has been demonstrated to be a potent inhibitor of procaspase-8 activation in the DISC (42). However, our results and previous data (43) indicate that expression of cFLIPS is rather restricted to certain cell lines. We only could clearly observe cFLIPS expression in one (HeLa) of the four cell lines examined. Therefore, from our data we are not able to propose that down-regulation of cellular FLIP levels is a general mechanism for glucose deprivation-induced sensitization to death receptor-mediated apoptosis. It is possible that down-regulation of cFLIPs by glucose deprivation in certain cells (HeLa) could contribute to death receptor sensitization in these cells. In the other cell lines examined, the mechanism underlying the sensitization process may be different. In this respect, we studied the role of the PI3K/AKT pathway as a possible target for glucose deprivation-induced sensitization to apoptosis (25). However, our results indicate that at least in U937 and MCF-7 cells, prevention of AKT activation did not result in sensitization of these cells to TRAIL-induced apoptosis.

Both CD95 and FADD have been found to be phosphorylated (56), although phosphorylation, to date, has not been associated with modulation of death (57). A kinase involved in mediating glucose signaling in mammalian cells is protein kinase C (58). We have shown previously that activation of protein kinase C inhibits anti-CD95-induced apoptosis upstream of caspase-8 activation (47). On the other hand, the best characterized system of ATP sensing in mammals involves a kinase/phosphatase system, with a predominant role of the AMP-activated protein kinase, the mammalian analogue of the yeast kinase SNF-1 (59), that regulates the transcription of glucose-responsive genes in hepatocytes (60). One could speculate the possibility of a direct effect of these kinases on CD95 or FADD, although their role in glucose deprivation-mediated sensitization of death receptor-induced apoptosis needs further clarification. PED/PEA-15 is a protein whose overexpression and/or phosphorylation may inhibit DISC formation (61, 62). Interestingly enough, this protein is involved in the regulation of glucose transport (63). Whether the expression levels of this protein or the phosphorylation status may be affected by glucose is unknown at the moment. However, the data available suggest that this protein is a possible candidate to mediate glucose-regulated formation of DISC complex.

Membrane cholesterol biosynthesis is one of the metabolic pathways inhibited by glucose deprivation, through the phosphorylation of HMG-CoA reductase by AMP-activated protein kinase (64), and this phenomenon is associated with changes in membrane fluidity. Cholesterol has been implicated in membrane receptor signaling (for a review see Ref. 65). On the other hand, signaling from CD95 receptor (66, 67) and from CD40, another member of the TNFR family (68), has been shown to be initiated in membrane rafts, and TNF receptor 1 is found in caveolae-like domains (69). Consequently, a possible link between death receptor signaling and membrane composition cannot be excluded. Our results indicate a direct link between glucose metabolism of tumor cells of different origins and their sensitivity to death receptor-induced apoptosis, irrespective of the apoptotic pathway involved. Furthermore, because TRAIL has been proposed recently (70) as an anti-tumor agent and glucose anti-metabolites have been already assayed as part of combined anti-tumor therapies in clinical trials, the combination of both therapies could be a powerful way to selectively kill tumor cells. Finally, our data also suggest the necessity of a re-evaluation of previous results regarding the implication of ATP synthesis through glycolysis and/or respiration in the process of death receptor-induced apoptosis.

    ACKNOWLEDGEMENTS

We thank Dr. Isabel Fabregat, Dr. Marion MacFarlane, Dr. Douglas Green, and Nick Harper for invaluable advice and for the generous donation of reagents. We gratefully acknowledge Gema Robledo for excellent technical assistance.

    FOOTNOTES

* This work was supported in part by Grant SAF2000-0118-C03-01 from the Ministerio de Ciencia y Tecnología (to A. L.-R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a fellowship from the Ministerio de Educación y Cultura. Present address: La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121. E-mail: cmunoz@liai.org.

§ Recipient of Fellowship Exp. 00/9319 from the Ministerio de Educación y Cultura and Fondo de Investigación Sanitaria.

To whom correspondence should be addressed: Instituto de Parasitología y Biomedicina, Consejo Superior de Investigaciones Científicas, Calle Ventanilla 11, 18001 Granada, Spain. Tel.: 34-958-80-51-88, Fax: 34-958-20-39-11; E-mail: alrivas@ipb.csic.es.

Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M212392200

    ABBREVIATIONS

The abbreviations used are: TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TRAILR, tumor necrosis factor-related apoptosis-inducing ligand receptor; FLIP, FLICE-inhibitory protein; DISC, death-inducing signaling complex; 2-DG, 2-deoxy-D-glucose; ROS, reactive oxygen species; PI3K, phosphatidylinositol 3-kinase; FBS, fetal bovine serum; H2DCFDA, dichlorodihydrofluorescein diacetate; PBS, phosphate-buffered saline; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Evan, G., and Littlewood, T. (1998) Science 281, 1317-1322[Abstract/Free Full Text]
2. Thompson, C. B. (1995) Science 267, 1456-1462[Medline] [Order article via Infotrieve]
3. Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S. I., Sameshima, M., Hase, A., Seto, Y., and Nagata, S. (1991) Cell 66, 233-243[Medline] [Order article via Infotrieve]
4. Oehm, A., Behrmann, I., Falk, W., Pawlita, M., Maier, G., Klas, C., Li-Weber, M., Richards, S., Dhein, J., Trauth, B. C., Ponstingl, H., and Krammer, P. H. (1992) J. Biol. Chem. 267, 10709-10715[Abstract/Free Full Text]
5. Suda, T., Takahashi, T., Golstein, P., and Nagata, S. (1993) Cell 75, 1169-1178[Medline] [Order article via Infotrieve]
6. Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C. P., Nicholl, J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and Goodwin, R. G. (1995) Immunity 3, 673-682[Medline] [Order article via Infotrieve]
7. Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., and Ashkenazi, A. (1996) J. Biol. Chem. 271, 12687-12690[Abstract/Free Full Text]
8. Pan, G., O'Rourke, K., Chinnaiyan, A. M., Gentz, R., Ebner, R., Ni, J., and Dixit, V. M. (1997) Science 276, 111-113[Abstract/Free Full Text]
9. Sheridan, J. P., Marsters, S. A., Pitti, R. M., Gurney, A., Skubatch, M., Baldwin, D., Ramakrishnan, L., Gray, C. L., Baker, K., Wood, W. I., Goddard, A. D., Godowski, P., and Ashkenazi, A. (1997) Science 277, 818-821[Abstract/Free Full Text]
10. Walczak, H., Degli-Esposti, M. A., Johnson, R. S., Smolak, P. J., Waugh, J. Y., Boiani, N., Timour, M. S., Gerhart, M. J., Schooley, K. A., Smith, C. A., Goodwin, R. G., and Rauch, C. T. (1997) EMBO J. 16, 5386-5397[Abstract/Free Full Text]
11. Ashkenazi, A., and Dixit, V. M. (1999) Curr. Opin. Cell Biol. 11, 255-260[CrossRef][Medline] [Order article via Infotrieve]
12. Dirks, W., Schone, S., Uphoff, C., Quentmeier, H., Pradella, S., and Drexler, H. G. (1997) Br. J. Haematol. 96, 584-593[Medline] [Order article via Infotrieve]
13. Thakkar, N. S., and Potten, C. S. (1993) Cancer Res. 53, 2057-2060[Abstract]
14. Ferrari, D., Stepczynska, A., Los, M., Wesselborg, S., and Schulze-Osthoff, K. (1998) J. Exp. Med. 188, 979-984[Abstract/Free Full Text]
15. Yasuhara, N., Eguchi, Y., Tachibana, T., Imamoto, N., Yoneda, Y., and Tsujimoto, Y. (1997) Genes Cells 2, 55-64[Abstract/Free Full Text]
16. Eguchi, Y., Shimizu, S., and Tsujimoto, Y. (1997) Cancer Res. 57, 1835-1840[Abstract]
17. Leist, M., Single, B., Castoldi, A. F., Kuhnle, S., and Nicotera, P. (1997) J. Exp. Med. 185, 1481-1486[Abstract/Free Full Text]
18. Matsuyama, S., Xu, Q., Velours, J., and Reed, J. C. (1998) Mol. Cell 1, 327-336[Medline] [Order article via Infotrieve]
19. Matsuyama, S., Llopis, J., Deveraux, Q. L., Tsien, R. Y., and Reed, J. C. (2000) Nat. Cell Biol. 2, 318-325[CrossRef][Medline] [Order article via Infotrieve]
20. Wolvetang, E. J., Johnson, K. L., Krauer, K., Ralph, S. J., and Linnane, A. W. (1994) FEBS Lett. 339, 40-44[CrossRef][Medline] [Order article via Infotrieve]
21. Racker, E. (1972) Am. Sci. 60, 56-63[Medline] [Order article via Infotrieve]
22. Mathupala, S. P., Rempel, A., and Pedersen, P. L. (1997) J. Bioenerg. Biomembr. 29, 339-343[CrossRef][Medline] [Order article via Infotrieve]
23. Volland, S., Amtmann, E., and Sauer, G. (1992) Int. J. Cancer 52, 384-390[Medline] [Order article via Infotrieve]
24. Halicka, H. D., Ardelt, B., Li, X., Melamed, M. M., and Darzynkiewicz, Z. (1995) Cancer Res. 55, 444-449[Abstract]
25. Nam, S., Amoscato, A., and Lee, Y. (2002) Oncogene 21, 337-346[CrossRef][Medline] [Order article via Infotrieve]
26. Ruiz-Ruiz, C., Munoz-Pinedo, C., and Lopez-Rivas, A. (2000) Cancer Res. 60, 5673-5680[Abstract/Free Full Text]
27. Gong, J., Traganos, F., and Darzynkiewicz, Z. (1994) Anal. Biochem. 218, 314-319[CrossRef][Medline] [Order article via Infotrieve]
28. Chandra, J., Niemer, I., Gilbreath, J., Kliche, K. O., Andreeff, M., Freireich, E. J., Keating, M., and McConkey, D. J. (1998) Blood. 92, 4220-4229[Abstract/Free Full Text]
29. MacFarlane, M., Harper, N., Snowden, R., Dyer, M., Barnett, G., Pringle, J., and Cohen, G. (2002) Oncogene 21, 6809-6810[CrossRef][Medline] [Order article via Infotrieve]
30. Harper, N., Farrow, N., Kapstein, A., Cohen, G., and MacFarlane, M. (2001) J. Biol. Chem. 276, 34743-34752[Abstract/Free Full Text]
31. Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K. J., Debatin, K. M., Krammer, P. H., and Peter, M. E. (1998) EMBO J. 17, 1675-1687[Abstract/Free Full Text]
32. Scaffidi, C., Schmitz, I., Zha, J., Korsmeyer, S. J., Krammer, P. H., and Peter, M. E. (1999) J. Biol. Chem. 274, 22532-22538[Abstract/Free Full Text]
33. Noguchi, K., Naito, M., Kataoka, S., Yonehara, S., and Tsuruo, T. (1995) Cell Growth Differ. 6, 1271-1277[Abstract]
34. Monney, L., Otter, I., Olivier, R., Ravn, U., Mirzasaleh, H., Fellay, I., Poirier, G. G., and Borer, C. (1996) Biochem. Biophys. Res. Commun. 221, 340-345[CrossRef][Medline] [Order article via Infotrieve]
35. Brahma, C. A., Ian, T., Streets, K., Trautwein, C., Brenner, D. A., and Lemasters, J. J. (1998) Mol. Cell. Biol. 18, 6353-6364[Abstract/Free Full Text]
36. Srinivasula, S. M., Datta, P., Fan, X. J., Fernandes-Alnemri, T., Huang, Z., and Alnemri, E. S. (2000) J. Biol. Chem. 275, 36152-36157[Abstract/Free Full Text]
37. Slee, E. A., Keogh, S. A., and Martin, S. J. (2000) Cell Death Differ. 7, 556-565[CrossRef][Medline] [Order article via Infotrieve]
38. Obrador, E., Carretero, J., Pellicer, J. A., and Estrela, J. M. (2001) Curr. Pharm. Biotechnol. 2, 119-130[Medline] [Order article via Infotrieve]
39. Goossens, V., Grooten, J., and Fiers, W. (1996) J. Biol. Chem. 271, 192-196[Abstract/Free Full Text]
40. Fulda, S., Meyer, E., and Debatin, K. M. (2000) Cancer Res. 60, 3947-3956[Abstract/Free Full Text]
41. Chang, D., Xing, Z., Pan, Y., Algeciras-Schimnich, A., Barnhart, B., Yaish-Ohad, S., Peter, M., and Yang, X. (2002) EMBO J. 21, 3704-3714[Abstract/Free Full Text]
42. Krueger, A., Schmitz, I., Baumann, S., Krammer, P., and Kirchhoff, S. (2001) J. Biol. Chem. 276, 20633-20640[Abstract/Free Full Text]
43. Scaffidi, C., Schmitz, I., Krammer, P., and Peter, M. (1999) J. Biol. Chem. 274, 1541-1548[Abstract/Free Full Text]
44. Vlahos, C., Matter, W., Hui, K., and Brown, R. (1994) J. Biol. Chem. 269, 5241-5248[Abstract/Free Full Text]
45. Holmstrom, T. H., Schmitz, I., Soderstrom, T. S., Poukkula, M., Johnson, V. L., Chow, S. C., Krammer, P. H., and Eriksson, J. E. (2000) EMBO J. 19, 5418-5428[Abstract/Free Full Text]
46. Ossina, N. K., Cannas, A., Powers, V. C., Fitzpatrick, P. A., Knight, J. D., Gilbert, J. R., Shekhtman, E. M., Tomei, L. D., Umansky, S. R., and Kiefer, M. C. (1997) J. Biol. Chem. 272, 16351-16357[Abstract/Free Full Text]
47. Ruiz-Ruiz, C., Robledo, G., Font, J., Izquierdo, M., and Lopez-Rivas, A. (1999) J. Immunol. 163, 4737-4746[Abstract/Free Full Text]
48. Stubbs, M., McSheehy, P. M., Griffiths, J. R., and Bashford, C. L. (2000) Mol. Med. Today 6, 15-19[CrossRef][Medline] [Order article via Infotrieve]
49. Latta, M., Kunstle, G., Leist, M., and Wendel, A. (2000) J. Exp. Med. 191, 1975-1985[Abstract/Free Full Text]
50. Linsinger, G., Wilhelm, S., Wagner, H., and Hacker, G. (1999) Mol. Cell. Biol. 19, 3299-3311[Abstract/Free Full Text]
51. Shim, H., Chun, Y. S., Lewis, B. C., and Dang, C. V. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1511-1516[Abstract/Free Full Text]
52. Shim, H., Dolde, C., Lewis, B. C., Wu, C. S., Dang, G., Jungmann, R. A., Dalla-Favera, R., and Dang, C. V. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6658-6663[Abstract/Free Full Text]
53. Rohn, J. L., Hueber, A. O., McCarthy, N. J., Lyon, D., Navarro, P., Burgering, B. M., and Evan, G. I. (1998) Oncogene 17, 2811-2818[CrossRef][Medline] [Order article via Infotrieve]
54. Binder, C., Binder, L., Kroemker, M., Schulz, M., and Hiddemann, W. (1997) Exp. Cell Res. 236, 223-230[CrossRef][Medline] [Order article via Infotrieve]
55. Berridge, M. V., Tan, A. S., McCoy, K. D., Kansara, M., and Rudert, F. (1996) J. Immunol. 156, 4092-4099[Abstract]
56. Kennedy, N. J., and Budd, R. C. (1998) J. Immunol. 160, 4881-4888[Abstract/Free Full Text]
57. Scaffidi, C., Volkland, J., Blomberg, I., Hoffmann, I., Krammer, P. H., and Peter, M. E. (2000) J. Immunol. 164, 1236-1242[Abstract/Free Full Text]
58. Thams, P., Capito, K., and Hedeskov, C. J. (1988) Biochem. J. 253, 229-234[Medline] [Order article via Infotrieve]
59. Kemp, B. E., Mitchelhill, K. I., Stapleton, D., Michell, B. J., Chen, Z. P., and Witters, L. A. (1999) Trends Biochem. Sci. 24, 22-25[CrossRef][Medline] [Order article via Infotrieve]
60. da Silva Xavier, G., Leclerc, I., Salt, I. P., Doiron, B., Hardie, D. G., Kahn, A., and Rutter, G. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4023-4028[Abstract/Free Full Text]
61. Condorelli, G., Vigliotta, G., Cafieri, A., Trencia, A., Andalo, P., Oriente, F., Miele, C., Caruso, M., Formisano, P., and Beguinot, F. (1999) Oncogene 18, 4409-4415[CrossRef][Medline] [Order article via Infotrieve]
62. Xiao, C., Yang, B., Asadi, N., Beguinot, F., and Hao, C. (2002) J. Biol. Chem. 277, 25020-25025[Abstract/Free Full Text]
63. Condorelli, G., Vigliotta, G., Iavarone, C., Caruso, M., Tocchetti, C. G., Andreozzi, F., Cafieri, A., Tecce, M. F., Formisano, P., Beguinot, L., and Beguinot, F. (1998) EMBO J. 14, 3858-3866[CrossRef]
64. Clarke, P. R., and Hardie, D. G. (1990) EMBO J. 9, 2439-2446[Abstract]
65. Burger, K., Gimpl, G., and Fahrenholz, F. (2000) Cell. Mol. Life Sci. 57, 1577-1592[Medline] [Order article via Infotrieve]
66. Gajate, C., and Mollinedo, F. (2001) Blood 98, 3860-3863[Abstract/Free Full Text]
67. Hueber, A. O., Bernard, A. M., Herincs, Z., Couzinet, A., and He, H. T. (2002) EMBO Rep. 3, 190-196[Abstract/Free Full Text]
68. Vidalain, P. O., Azocar, O., Servet-Delprat, C., Rabourdin-Combe, C., Gerlier, D., and Manie, S. (2000) EMBO J. 19, 3304-3313[Abstract/Free Full Text]
69. Ko, Y. G., Lee, J. S., Kang, Y. S., Ahn, J. H., and Seo, J. S. (1999) J. Immunol. 162, 7217-7223[Abstract/Free Full Text]
70. Mohanti, B. K., Rath, G. K., Anantha, N., Kannan, V., Das, B. S., Chandramouli, B. A., Banerjee, A. K., Das, S., Jena, A., Ravichandran, R., Sahi, U. P., Kumar, R., Kapoor, N., Kalia, V. K., Dwarakanath, B. S., and Jain, V. (1996) Int. J. Radiat. Oncol. Biol. Phys. 35, 103-111[Medline] [Order article via Infotrieve]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.