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
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ABSTRACT |
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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)- 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- 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.
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- 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 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- 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.
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).
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-
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).
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).
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).
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-
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.
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- The protein synthesis inhibitor cycloheximide is a well known promoter
of death receptor-mediated apoptosis (32). It has been proposed that
cycloheximide facilitates TNF- 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.
, 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
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-
as a possible anti-tumor treatment (24).
However the mechanism underlying the facilitation of TNF-
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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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-
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).
m
(mitochondrial membrane potential) and ROS production were carried out
in a FACScan cytometer using the Cell Quest software.
-tubulin antibody.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[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- (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-
(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.
-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).
View larger version (21K):
[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.
View larger version (25K):
[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- (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.
View larger version (29K):
[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.
(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 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.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
-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.
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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.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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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.
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REFERENCES |
---|
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---|
1. |
Evan, G.,
and Littlewood, T.
(1998)
Science
281,
1317-1322 |
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 |
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 |
8. |
Pan, G.,
O'Rourke, K.,
Chinnaiyan, A. M.,
Gentz, R.,
Ebner, R.,
Ni, J.,
and Dixit, V. M.
(1997)
Science
276,
111-113 |
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 |
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 |
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 |
15. |
Yasuhara, N.,
Eguchi, Y.,
Tachibana, T.,
Imamoto, N.,
Yoneda, Y.,
and Tsujimoto, Y.
(1997)
Genes Cells
2,
55-64 |
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 |
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 |
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 |
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 |
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 |
32. |
Scaffidi, C.,
Schmitz, I.,
Zha, J.,
Korsmeyer, S. J.,
Krammer, P. H.,
and Peter, M. E.
(1999)
J. Biol. Chem.
274,
22532-22538 |
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 |
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 |
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 |
40. |
Fulda, S.,
Meyer, E.,
and Debatin, K. M.
(2000)
Cancer Res.
60,
3947-3956 |
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 |
42. |
Krueger, A.,
Schmitz, I.,
Baumann, S.,
Krammer, P.,
and Kirchhoff, S.
(2001)
J. Biol. Chem.
276,
20633-20640 |
43. |
Scaffidi, C.,
Schmitz, I.,
Krammer, P.,
and Peter, M.
(1999)
J. Biol. Chem.
274,
1541-1548 |
44. |
Vlahos, C.,
Matter, W.,
Hui, K.,
and Brown, R.
(1994)
J. Biol. Chem.
269,
5241-5248 |
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 |
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 |
47. |
Ruiz-Ruiz, C.,
Robledo, G.,
Font, J.,
Izquierdo, M.,
and Lopez-Rivas, A.
(1999)
J. Immunol.
163,
4737-4746 |
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 |
50. |
Linsinger, G.,
Wilhelm, S.,
Wagner, H.,
and Hacker, G.
(1999)
Mol. Cell. Biol.
19,
3299-3311 |
51. |
Shim, H.,
Chun, Y. S.,
Lewis, B. C.,
and Dang, C. V.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
1511-1516 |
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 |
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 |
57. |
Scaffidi, C.,
Volkland, J.,
Blomberg, I.,
Hoffmann, I.,
Krammer, P. H.,
and Peter, M. E.
(2000)
J. Immunol.
164,
1236-1242 |
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 |
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 |
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 |
67. |
Hueber, A. O.,
Bernard, A. M.,
Herincs, Z.,
Couzinet, A.,
and He, H. T.
(2002)
EMBO Rep.
3,
190-196 |
68. |
Vidalain, P. O.,
Azocar, O.,
Servet-Delprat, C.,
Rabourdin-Combe, C.,
Gerlier, D.,
and Manie, S.
(2000)
EMBO J.
19,
3304-3313 |
69. |
Ko, Y. G.,
Lee, J. S.,
Kang, Y. S.,
Ahn, J. H.,
and Seo, J. S.
(1999)
J. Immunol.
162,
7217-7223 |
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] |