From the ¶ Instituto de Parasitología y Biomedicina,
Consejo Superior de Investigaciones Científicas, calle
Ventanilla 11, 18001 Granada, Spain and the Centro
Nacional de Sanidad Agropecuaria, carretera de Tapaste y Autopista
Nacional, San José de las Lajas, La Habana, Cuba
Received for publication, January 29, 2001
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ABSTRACT |
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The role of interferon (IFN)- Interferons (IFNs)1 are
a family of natural glycoproteins that share antiviral,
immunomodulatory, and anti-proliferative effects (1). In mice, a defect
in the transcriptional regulation of IFN-dependent genes
causes a marked increase in the number of myeloid cells and hematologic
alterations similar to chronic human myelogenous leukemia (2). In both
chronic and acute human myeloid leukemias, a decrease of IFN-modulated
transcriptional activity has been reported (3, 4). On the other hand,
the anti-tumor properties of IFNs against a variety of tumor cells such
as lymphomas, melanomas, and multiple myeloma has also been
demonstrated (5, 6). Furthermore, clinically and experimentally,
IFN- Apoptosis is an active form of cell death that plays a fundamental role
in normal development, tissue homeostasis, and pathological situations
(10-12). CD95 (Fas/Apo-1) receptor, a member of TNF/nerve growth
factor receptor family (13, 14), is a potent inducer of apoptosis in
the immune system upon interaction with its natural ligand CD95L, a
type II integral membrane protein homologous to TNF (15). TRAIL, a
recently identified member of the TNF family with homology to CD95L
(16), induces apoptosis (17) upon binding to death-domain containing
receptors, TRAIL-R1 and TRAIL-R2 (also known as DR4 and DR5,
respectively) (18-21). Although the expression of CD95L seems to be
more restricted to lymphoid cells (15), TRAIL transcripts are
detectable in many normal organs and tissues (16), suggesting that this
ligand may be non-toxic to normal cells.
Death receptors are expressed in many tumor cells that can therefore be
killed by the appropriate ligands (22). However, expression of death
receptors is not always sufficient to allow an apoptotic response since
there are examples of tumor cells, including myeloid leukemic cells,
that express significant levels of death receptors in the plasma
membrane but are resistant to death ligands (22, 23). Understanding the
mechanisms that sensitize tumor cells to death ligand-induced apoptosis
could therefore be an important objective in the development of
therapies to treat malignancies like human myelogenous leukemias. In
this respect, IFN- The above data prompted us to investigate the effects of IFN- Materials--
RPMI 1640 medium and fetal bovine serum
were obtained from Life Technologies, Inc. CH-11 monoclonal antibody
(mAb) reacting with CD95 was from Upstate Biotechnology Inc. (Lake
Placid, NY). Human IFN- Cell Culture--
The various human myeloid leukemic cell lines
used in this study were maintained in culture in RPMI 1640 medium
containing 10% fetal calf serum and 1 mM
L-glutamine, at 37 °C in a humidified 5%
CO2, 95% air incubator. Cell viability was determined by
the trypan blue dye exclusion method.
Determination of Apoptotic Cells--
Analysis by flow cytometry
of hypodiploid apoptotic cells was performed on a FACScan cytometer
using the Cell Quest software (Becton Dickinson, Mountain View, CA),
after extraction of the degraded DNA from apoptotic cells following a
recently described method (32).
Phosphatidylserine exposure on the surface of apoptotic cells was
detected by flow cytometry after staining with Annexin-V-FLUOS (Roche
Molecular Biochemicals).
Analysis of DNA cleavage into oligonucleosome-length fragments was
performed following a method described previously (33).
Cytochrome c Release from Mitochondria--
For measurements of
cytochrome c release from mitochondria, cells were lysed and
cytosolic fractions were separated from mitochondria as described (34).
Cytosolic proteins (40 µg of protein) were mixed with Laemmli buffer
and resolved on 12% SDS-polyacrylamide minigels. Cytochrome
c was determined by Western blot analysis as described below.
Cell Extracts and Western Blot Analysis of Proteins--
Cells
were pelleted and lysed in Laemmli buffer. After sonication, proteins
were resolved on 7.5% SDS-polyacrylamide minigels for determination of
PARP cleavage or 12% SDS for analysis of other proteins, and
electrophoretically transferred onto Immobilon (Millipore). Membranes
were blocked with 5% milk powder in PBS plus 0.1% Tween 20 (PBS/Tween) for 1 h and washed with PBS/Tween. For protein
detection, immunoblots were probed with Reverse Transcriptase-Polymerase Chain Reaction
(RT-PCR)--
Total RNA was isolated from cells with Trizol reagent
(Life Technologies, Inc.) as recommended by the supplier. cDNAs
were synthesized from 2 µg of total RNA using a RT-PCR kit
(PerkinElmer Life Sciences) with the supplied oligo(dT) primer under
conditions described by the manufacturer. PCR reactions were performed
using the following primers: TRAIL-R1 sense,
5'-CTGAGCAACGCAGACTCGCTGTCCAC-3'; TRAIL-R1 antisense,
5'-TCCAAGGACACGGCAGAGCCTGTGCCAT-3'; TRAIL-R2 sense,
5'-GCCTCATGGACAATGAGATAAAGGTGGCT-3'; TRAIL-R2 antisense, 5'-CCAAATCTCAAAGTACGCACAAACGG-3'; BAK sense, 5'-CCTGTTTGAGAGTGGCATC-3'; BAK antisense, 5'-TCGTACCACAAACTGGCCCA-3'; IRF-1 sense,
5'-CTTAAGAACCAGGCAACCTCTGCCTTC-3'; IRF-1 antisense,
5'-GATATCTGGCAGGGAGTTCATG-3'; BCL-2 sense, 5'-AGATGTCCAGCCAGCTGC ACCTGAC-3'; BCL-2 antisense, 5'AGATAGGCACCAGGGTGAGCAAGCT-3'; IFN-
Sensitization by IFN-
It has been reported that IFN- Up-regulation of Caspase-8 and Down-regulation of BCL-2 in U937
Cells Treated with IFN-
To further characterize the cellular modulators of apoptosis that may
be responsible for the sensitizing action of IFN-
Interestingly, when we determined the expression of anti-apoptotic
BCL-2 protein, a marked decline in the cellular levels of this
anti-apoptotic protein was observed following IFN- Facilitation by IFN-
When analyzing BCL-2 protein levels in cells incubated in the presence
of IFN-
The above results indicated that, in U937 cells, IFN- In the present study, we have demonstrated that treatment of
different human myeloid leukemic cell lines with IFN- Caspase-8 is recruited in zymogen form to the DISC upon ligation of
CD95 at the cell surface, by either CD95L or agonistic CD95 antibodies
(66). This caspase also participates in the initial signaling complex
during TNF- In addition to the up-regulation of caspase-8, IFN- Besides its effect in the expression of BCL-2 in U937 cells, we have
also demonstrated here that IFN- Although the above observations open the possibility of using death
ligands as anti-leukemic agents in combination with IFN- as a sensitizing
agent in apoptosis induced by ligation of death receptors has been
evaluated in human myeloid leukemia cells. Incubation of U937 cells
with IFN-
sensitized these cells to apoptosis induced by tumor
necrosis factor-
, agonistic CD95 antibody, and tumor necrosis
factor-related apoptosis-inducing ligand. Other human myeloid leukemic
cells were also sensitized by IFN-
to death receptor-mediated
apoptosis. Treatment of U937 cells with IFN-
up-regulated the
expression of caspase-8 and potently synergized with death receptor
ligation in the processing of caspase-8 and BID cleavage.
Concomitantly, a marked down-regulation of BCL-2 protein was also
observed in cells incubated with IFN-
. Furthermore, the
caspase-dependent generation of a 23-kDa fragment of BCL-2
protein, the release of cytochrome c from mitochondria and
the activation of caspase-9 were also enhanced upon death receptor
ligation in IFN-
-treated cells. Ectopically expressed Bcl-2 protein
inhibited IFN-
-induced sensitization to apoptosis. In summary, these
results indicate that IFN-
sensitizes human myeloid leukemic cells
to a death receptor-induced, mitochondria-mediated pathway of apoptosis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
has been shown to enhance the anti-tumor effects of
anti-metabolite on cancer cells (5, 7). Positive anti-tumor effects
have also been obtained by immunotherapy with natural IFNs and
interleukins, particularly in combination strategies (8). In tumor cell
lines, IFN-
can induce or modulate cell death either as a single
agent or in combination with chemotherapeutic drugs (9).
and IFN-
can up-regulate the expression of a
number of apoptosis-related proteins in different types of cells (9, 24, 25). In certain cancer cells, including U937 myeloid leukemic cells, it has been reported that IFN-
induces sensitization to CD95-mediated apoptosis by up-regulating the expression of
ICE/caspase-1 (26, 27). However, more recent data have demonstrated
that caspase-1/ICE is not involved in the proteolytic cascade activated upon CD95 cross-linking at the cell surface by CD95L or CD95 antibody (28-30).
on
death receptor-induced apoptosis in the human U937 myeloid leukemic
cell line. We were particularly interested to ascertain whether IFN-
could enhance the sensitivity of these cells to TRAIL-induced
apoptosis, in view of the importance of TRAIL as a rather selective
anti-tumor protein (31). In this report we show that IFN-
sensitizes
U937 cells and other human myeloid leukemic cell lines to CD95-,
TNF-R-, and TRAIL-R-mediated apoptosis. Following treatment of U937
cells with IFN-
, we have observed a significant up-regulation of
caspase-8 and a marked down-regulation of BCL-2 protein. Furthermore,
we demonstrate that in U937 cells, IFN-
facilitates several
biochemical events involved in death receptor-induced
mitochondria-mediated apoptosis.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, human TNF-
and recombinant human TRAIL
were obtained from PreproTech EC Ltd (London, United Kingdom). Mouse
anti-BAX mAb, mouse anti-BAD mAb, and mouse anti-cytochrome
c mAb were obtained from PharMingen (San Diego, CA). Mouse
anti-BCL-2 mAb was from Dako (Glostrup, Denmark). Mouse anti-human
caspase-8 mAb and rabbit anti-caspase-9 polyclonal antibodies were
purchased from Cell Diagnostica (Münster, Germany) and StressGen
Biotechnologies Corp. (Victoria, Canada), respectively. Goat polyclonal
anti-caspase-3 antibody was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Rabbit polyclonal antiserum against PARP was
purchased from Roche Molecular Biochemicals (Mannheim, Germany). Mouse
anti-FADD mAb was from Transduction Laboratories (Lexington, KY).
Rabbit anti-BID polyclonal antibody was generously provided by Dr. X. Wang (Howard Hughes Medical Institute, Dallas, TX). Rabbit polyclonal antibodies to caspase-9 p37 fragment and caspase-3 p17 subunit were
obtained from New England Biolabs (Beverly, MA). Monoclonal antibody to
alpha-tubulin was purchased from Sigma (Poole, United Kingdom).
Benzyloxycarbonyl-Val-Ala-Asp- (OMe)fluoromethyl ketone (Z-VAD-fmk)
was from Enzyme System Inc. (Dublin, CA).
-tubulin mAb (1:40000), BAX
mAb (1 µg/ml), BAD mAb (1:500), cytochrome c mAb (0.5 µg/ml), polyclonal antiserum against PARP (1:2000), caspase-3
antibody (1:1000), caspase-8 mAb (1:200), caspase-9 antibody
(1:1000), caspase-9 (37-kDa fragment, 1:500), caspase-3 (17-kDa
subunit, 1:500), BID antibody (1:2000), or BCL-2 mAb (1:1000). After
washing, membranes were incubated with horseradish
peroxidase-conjugated anti-rabbit IgG (1:2000; Dako) or
horseradish peroxidase-conjugated anti-mouse Ig (1:2000, Dako).
Bound antibody was visualized by enhanced chemiluminescence (ECL,
Amersham Pharmacia Biotech), according to manufacturer's instructions.
-actin sense, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3'; and
-actin antisense, 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3', giving products of 506, 502, 266, 406, 367, and 661 base pairs, respectively. Expression of
-actin was used as a control of RNA integrity and equal gel loading.
Cycle conditions for all PCR reactions were 1 min at 95 °C, 1 min at
55 °C, and 1 min at 72 °C for 30 cycles. FLIP was analyzed by
RT-PCR as described recently (35).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Sensitizes U937 Human Myeloid Leukemic Cells to Death
Receptor-mediated Apoptosis--
Activation of death receptors in
tumor cells by appropriate ligands or agonistic antibodies results in
the death of target cells by apoptosis (22). However, some tumor
cells are not very sensitive to death receptor-mediated apoptosis
unless protein synthesis is inhibited (36). This is also the case for
the human promyelocytic cell line U937 (Fig.
1). As shown in Fig. 1, U937 cells were
markedly sensitized to CD95-mediated death by co-treatment with the
protein synthesis inhibitor cycloheximide. In order to find a
physiologically relevant sensitizing agent, in this work we have
examined the ability of IFN-
to modulate the apoptotic response of
human U937 myeloid leukemic cells upon death receptor ligation in the
cell surface. IFN-
can induce an anti-proliferative response in a
variety of tumor cells (1). It could also activate an apoptotic program
or sensitize cells to apoptosis induced by other stimuli (27, 37, 38),
although the mechanism underlying the sensitization process remains
unclear. Results shown in Fig. 2 indicate
that pre-incubation of U937 cells with IFN-
(10 units/ml) for
24 h markedly sensitized these leukemic cells to apoptosis upon
death receptor activation. In these experiments we observed that
IFN-
facilitated the generation of hypodiploid apoptotic cells by a
subsequent treatment with TNF-
, CD95 agonistic antibody, or TRAIL
(Fig. 2a). This effect was paralleled by the externalization of phosphatidylserine in the plasma membrane of U937 cells (data not
shown). Other apoptotic features like activation of caspase-3 (Fig.
2b), DNA fragmentation in a ladder pattern (Fig.
2c), and PARP cleavage (Fig. 2d) were also
markedly enhanced by IFN-
. In the experiments involving CD95 IgM, an
irrelevant IgM antibody did not cooperate with IFN-
in the induction
of apoptosis (results not shown).
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Fig. 1.
Incubation of U937 cells with cycloheximide
sensitizes these leukemic cells to CD95-mediated death. U937 cells
were treated for 24 h with different doses of CD95 antibody CH-11
in the absence or presence of cycloheximide (0.25 µg/ml). After this
incubation, cell viability was assessed by the trypan blue dye
exclusion method. Data shown are representative of three separate
experiments.
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Fig. 2.
IFN- sensitizes U937
cells to death receptor-mediated apoptosis. Cells were
pre-incubated for 24 h with 10 units/ml IFN-
and treated for an
additional 24-h period with CD95 mAb (20 ng/ml), TNF-
(1 ng/ml), or
TRAIL (10 ng/ml). Apoptotic features were determined as described under
"Experimental Procedures." a, percentage of apoptotic
cells as determined by cytofluorimetric analysis of DNA content.
b, caspase-3 activation, measured by the generation of
17-kDa subunit. c, DNA fragmentation into
oligonucleosome-length fragments. d, proteolytic cleavage of
poly(ADP-ribose) polymerase. In a, error
bars represent S.D. from three independent experiments. In
b-d, the results illustrate a representative experiment
from at least three different experiments.
to death receptor-induced apoptosis was also
observed in two other human myeloid leukemic cell lines examined.
Results shown in Fig. 3 demonstrate that
incubation of either HL-60 (Fig. 3a) or THP-1 (Fig.
3b) cells with IFN-
sensitized them to a subsequent
treatment with TNF-
or CD95 antibody.
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Fig. 3.
Other myeloid leukemic cells are also
sensitized by IFN- to death receptor-induced
apoptosis. HL-60 (a) or THP-1 (b) cells were
incubated for 24 h with 10 units/ml IFN-
and treated for an
additional 36-h period with either CD95 mAb (20 ng/ml) or TNF-
(1 ng/ml). Apoptotic cells were determined by flow cytometry as described
under "Experimental Procedures." Percentage of hypodiploid
apoptotic cells is shown. The results illustrate a representative
experiment from at least three different experiments.
can elevate the expression of CD95 in
several acute myelogenous leukemic cell lines, including U937 cells
(39, 40) and that this effect could explain the enhanced sensitivity of
IFN-
-treated cells to apoptosis mediated by CD95 receptors. However,
U937 cells express a high number of CD95 molecules in their surface,
and in our experiments we have observed only a slight increase in CD95
expression in IFN-
-treated cells (data not shown). Furthermore, we
have determined by RT-PCR the expression of pro-apoptotic TRAIL
receptors (DR4 and DR5) in U937 cells treated with IFN-
(Fig.
4). As a control of IFN-
action, we
determined the expression of the transcription factor IRF-1, an
IFN-
-regulated gene (Fig. 4). Expression of TRAIL receptors was not
significantly elevated by IFN-
in U937 cells after 24 h (Fig.
4) or 48 h (data not shown). We also examined by RT-PCR the levels
of TRAIL decoy receptors DcR1 and DcR2 in cells incubated in the
presence of IFN-
for 24 h. Results not shown indicated that the
cellular levels of these anti-apoptotic receptors did not change upon
IFN-
treatment. Although we have not investigated the expression of
TNF-R in U937 cells, the above results suggested that the sensitization
to apoptosis observed in IFN-
-treated cells should be probably
related to changes in the intracellular levels of apoptosis regulators
rather than to an increase in the expression of death receptors.
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Fig. 4.
Expression of TRAIL receptors upon treatment
with IFN- . U937 cells were incubated for
24 h with 10 units/ml IFN-
. RT-PCR of TRAIL-R1, TRAIL-R2,
IRF-1, and
-actin were performed as described under "Experimental
Procedures."
--
Regulation of the expression and/or
activity of the death receptor-inducing signaling complex (DISC)
components could be a strategy used by tumor cells to escape from the
host immune system (41-43). Caspase-8, the most apical caspase
required in death receptor-mediated apoptosis (44), is also a cellular
target of oncogenic viruses, to protect transformed cells from death
receptor-induced apoptosis (45). The intracellular signaling mechanism
involved in the activation of apoptosis by death receptors comprises
different activities (46). The adapter protein FADD is responsible for coupling death receptors to the initiator caspase-8 (47). We have
examined the levels of both FADD and caspase-8 in U937 cells following
treatment with IFN-
. Results in Fig. 5
indicate that procaspase-8, but not FADD, was up-regulated in these
leukemic cells after 24 h of incubation in the presence of
IFN-
. This could be relevant in the mechanism of IFN-
-induced
sensitization of U937 cells to death receptor-mediated apoptosis as
overexpression of procaspase-8 by transfection has been reported to
facilitate apoptosis (48). On the contrary, the cellular levels of
procaspase-9, which plays an important role in the
mitochondria-mediated pathway of apoptosis, and procaspase-3,
responsible for many of the nuclear changes during apoptosis, did not
change in U937 cells treated with IFN-
for up to 48 h (Fig.
5).
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Fig. 5.
Effect of IFN- on
the expression of FADD and caspases. Cells were incubated for the
indicated times with 10 units/ml IFN-
. Expression levels of FADD and
caspase-8, -3, and -9 were determined by Western blot. Results are
representative of at least three independent experiments.
in U937 cells, we
analyzed the expression of the apoptosis inhibitor FLIP and several
pro-apoptotic members of the BCL-2 family. As shown in Fig.
6 (a and b),
IFN-
treatment did not modify the cellular levels of FLIP, BAK, BAX,
BAD, or BID, determined either by RT-PCR or Western blot analysis. In
these experiments, the cellular expression of IRF-1 mRNA was
up-regulated by IFN-
, serving as an internal control of IFN-
activity.
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Fig. 6.
Effect of IFN- on
the expression of FLIP and pro-apoptotic members of the BCL-2
family. a, levels of FLIP and BAK mRNA as detected
by RT-PCR, after treatment with 10 units/ml IFN-
. IRF-1 mRNA was
amplified as a control for IFN-
-mediated gene regulation.
b, protein levels of BAX, BAD, and BID, as detected by
Western blotting, after incubation of cells with 10 units/ml IFN-
for the indicated times. Results are representative of at least three
independent experiments.
treatment (Fig.
7). A decreased level of BCL-2 was
clearly observed after 24 h of IFN-
treatment and remained low
for at least 48 h. This effect was also observed at the mRNA
level (Fig. 7). Although we cannot exclude an IFN-
-induced decrease
of BCL-2 mRNA stability, these results may suggest a negative
regulation of BCL-2 gene transcription by IFN-
as described recently
(49). Reduction in the levels of BCL-2 could be an important event in
the regulation of cellular sensitivity to stress treatments, which
operate through a mitochondrial pathway of apoptosis (50). A decrease
in cellular BCL-2 protein levels could also sensitize type II cells to
CD95-mediated apoptosis (51). CD95 type II cells are also characterized
by a markedly increased sensitivity to death receptor-induced apoptosis upon inhibition of protein synthesis (52). Sensitivity of U937 cells to
death receptor-induced apoptosis is considerably enhanced by
cycloheximide treatment (Fig. 1) (53), which suggests that U937 cells
are probably type II cells (52).
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Fig. 7.
Down-regulation of BCL-2 following
IFN- treatment. U937 cells were incubated
for the indicated times with 10 units/ml IFN-
and the level of BCL-2
protein was determined by Western blot analysis (upper
panel). In the lower panel, cells were
treated for 24 h in absence or presence of IFN-
(10 units/ml)
and the expression of BCL-2 mRNA was determined by RT-PCR.
of a Death Receptor-mediated Mitochondrial
Pathway of Apoptosis in U937 Cells--
In order to ascertain the
steps in death receptor-mediated apoptosis that are affected by IFN-
treatment, we first examined the activation of caspase-8 by analyzing
the processing of this caspase into various specific proteolytic
fragments. As shown in Fig.
8a, activation of either death
receptor clearly synergized with IFN-
in the stimulation of
procaspase-8 processing. By immunoblot analysis of caspase-8 in treated
cells, we detected both the 55- and 53-kDa inactive proforms
corresponding to caspase-8a and -8b as well as the 43- and 41-kDa
intermediate products corresponding to cleavage of both caspase-8a and
-8b between the large and small subunits. We also detected the presence
of the large 18-kDa subunit, which would lead upon combination with the
small 10-kDa subunit to the assembly of the active caspase (54).
Caspase-8 is the most upstream caspase required in death
receptor-mediated apoptosis (44), although it could also be activated
downstream of mitochondria through an amplification pathway regulated
by this organelle during apoptosis (52). One of the consequences of the
activation of caspase-8 at the DISC upon CD95 ligation is the cleavage
of BID, a BH3 domain-containing member of the BCL-2 family, to generate a 15-kDa fragment that translocates to mitochondria (55). Insertion of
cleaved BID into the mitochondrial membrane causes the release of
cytochrome c from mitochondria (55). We have examined the effect of IFN-
treatment on the cleavage of BID following death receptor activation. Results shown in Fig. 8b indicate that
IFN-
treatment did not affect the cellular level of BID, as observed previously (Fig. 6b), but it markedly facilitated the
cleavage of BID upon death receptor ligation, as determined by the
decrease in the level of intact BID in the cells. Results presented in Fig. 8 (a and b) also indicated that there was a
good correlation between the degree of caspase-8 activation and the
extent of intact BID protein loss, suggesting a possible cause-effect
relationship between both events.
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Fig. 8.
IFN- -mediated
sensitization of U937 cells to death receptor-induced caspase-8
activation, BID cleavage, and generation of 23-kDa BCL-2 fragment.
Cells were incubated with 10 units/ml IFN-
for 24 h and
subsequently treated for 24 h with CD95 mAb (20 ng/ml), TNF-
(1 ng/ml), or TRAIL (10 ng/ml). After this treatment, procaspase-8
processing was assayed by Western blot analysis (a). Bands
corresponding to intact proforms (55 and 53 kDa), cleaved intermediate
products (43 and 41 kDa), and large subunit (18 kDa) of caspase-8 are
marked with arrows. Results are representative of three
independent experiments. b, the cellular level of BID was
determined by Western blot following treatment with IFN-
and death
receptor activator.
-Tubulin expression was used as a control of
equal gel loading. c, following IFN-
treatment, Z-VAD-fmk
(100 µM) was added to some cultures 1 h before death
receptor activation. BCL-2 was detected by Western blotting.
Panels show two different exposures of the same immunoblot
membrane to demonstrate both the formation of 23-kDa fragment
(upper panel) and the down-regulation of BCL-2
(lower panel). The results shown are
representatives of four independent experiments.
and subsequently treated with TRAIL, TNF-
, or CD95
antibody, we observed the formation of a 23-kDa fragment of BCL-2
protein (Fig. 8c). In the presence of Z-VAD-fmk, an
inhibitor of multiple caspases, generation of the 23-kDa fragment of
BCL-2 was markedly abrogated (Fig. 8c). Processing by
caspases of the 26-kDa anti-apoptotic BCL-2 protein to produce a
pro-apoptotic 23-kDa fragment has been observed previously in different
cell types upon treatment with various apoptotic stimuli (56, 57). This
caspase-dependent cleavage of BCL-2 appears to promote
further caspase activation as part of a positive feedback loop for
executing cell death. This mechanism involves the localization of the
23-kDa fragment to mitochondria to induce cytochrome c
release from this organelle (57).
promoted the
generation of two different cytochrome c-releasing factors upon death receptor activation. To evaluate the possible involvement of
this mitochondria-derived factor in IFN-
-induced sensitization of
U937 cells to death receptor-mediated apoptosis, we determined the
release of cytochrome c into the cytosol of cells incubated with IFN-
and death receptor activators. In Fig.
9a, we show that cytosolic
cytochrome c was elevated following treatment of cells with
the various combinations of IFN-
and death receptor activators. Once
released from mitochondria, cytochrome c will bind to
Apaf-1, an event that triggers oligomerization of Apaf-1/cytochrome c in complexes that activate procaspase-9 (58). Fig.
9b illustrates the activation of procaspase-9 in cells
treated with IFN-
and subsequently incubated with death receptor
activators. Formation of 37-kDa fragment of caspase-9 was only observed
in those cultures subjected to combined treatments (Fig.
9b). These results suggested that IFN-
facilitated the
activation by death receptors of a mitochondria-operated pathway of
apoptosis in human myeloid leukemic U937 cells. It has been
demonstrated that Bcl-2/Bcl-XL can block cell death by
preventing the activation of the mitochondria-regulated pathway of
apoptosis (59-61). The importance of this pathway in IFN-
-induced
sensitization was further examined in U937 cells transfected with a
cDNA encoding anti-apoptotic Bcl-2. Results shown in Fig.
10 demonstrate that
U937Bcl-2 cells were markedly protected from apoptotic cell
death induced by the combination of IFN-
and the various death
receptor agonists.
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Fig. 9.
Death receptor-induced cytochrome
c release into the cytosol and caspase-9 processing
are enhanced by pre-incubation of U937 cells with
IFN- . Cells were pre-incubated with 10 units/ml IFN-
for 24 h and treated with CD95 mAb (20 ng/ml),
TNF-
(1 ng/ml), or TRAIL (10 ng/ml) for an additional 24-h period.
a, cytosolic fractions obtained as described under
"Experimental Procedures" were subjected to electrophoresis, and
cytochrome c was detected by Western blot analysis. The
mitochondrial Bcl-2 protein was undetectable in the cytosolic samples.
b, whole extracts of U937 cells, treated as described
previously, were analyzed for the formation of cleaved caspase-9
(37-kDa fragment) by Western blot.
-Tubulin levels were used as
control of equal gel loading. Results are representative of two
independent experiments.
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Fig. 10.
U937 cells ectopically expressing Bcl-2
protein are resistant to IFN- -induced
sensitization to death receptor-mediated apoptosis. Cells were
incubated for 24 h with 10 units/ml IFN-
and treated for
another 24-h period with either CD95 mAb (20 ng/ml), TNF-
(1 ng/ml),
or TRAIL (10 ng/ml). Hypodiploid apoptotic cells were determined as
described under "Experimental Procedures." Percentage of apoptotic
cells is shown. Results show a representative experiment from at least
three different experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
markedly enhances the sensitivity of these cells to death receptor-mediated apoptosis. In our report, we have also shown that IFN-
treatment up-regulates the expression of caspase-8 protein in U937 cells. Regulation by IFN-
of caspase expression and activation has been described in human erythroid progenitor cells as well as in colon and
breast tumor cells (9, 26, 62, 63). In myeloid leukemic cells, IFN-
caused an increased expression of caspase-1/ICE gene, following strong
induction of the IRF-1 gene (27), the product of which is a
transcriptional activator of the caspase-1 gene (64). However, recent
data have demonstrated that caspase-1/ICE is not involved in the
proteolytic cascade activated upon death receptor cross-linking at the
cell surface (30). Our results are the first demonstration of the
induction of caspase-8 protein in human myeloid leukemic cells upon
IFN-
treatment. In this report we have also shown that IRF-1 is
clearly induced by IFN-
in U937 cells. At present, we can only
speculate with the possibility of similarities between caspase-1 and -8 in terms of the mechanism regulating their expression by IFN-
(27,
65).
(44). Despite some recent controversy, several reports
have demonstrated the involvement of FADD and caspase-8 in
TRAIL-induced apoptosis of tumor cells (67-70). After recruitment to
the DISC, caspase-8 is autoprocessed to generate the active form that
can cleave other substrates, including executioner caspases. According
to the induced-proximity model for caspase-8 activation, a locally high
concentration of this caspase zymogen would promote its autoprocessing
and the release of the active caspase (71). It is therefore possible that the increased expression of caspase-8 found in IFN-
-treated U937 cells might facilitate formation of the DISC upon death receptor activation and subsequently activate an apoptotic program. This can be
especially important in CD95 type II cells, like the U937 cell line
(72), which show a reduced DISC formation upon CD95 engagement at the
cell surface (51). In these cells, mitochondria may function as
amplifiers of apoptosis activating caspase-9 and executioner caspases,
through the release of cytochrome c (52). This proposition
is in agreement with our results showing the cleavage of BID, an
increased release of cytochrome c from mitochondria, and the
activation of caspase-9 upon death receptor activation in
IFN-
-treated U937 cells. Regarding the role of IFN-
-induced caspase-8 elevation in the sensitization of U937 cells to TRAIL-induced apoptosis, a similar hypothesis could be proposed for TRAIL-R-mediated cell death (70). However, at present we can not exclude the possibility
of the processing of caspase-8 observed in our studies being the result
of both the formation of the DISC and its activation downstream of mitochondria.
treatment caused
a marked decrease in the cellular levels of anti-apoptotic BCL-2
mRNA and protein in U937 cells. This could play a potentially important role in the sensitization process induced by IFN-
, as a
major mechanism for the anti-apoptotic action of BCL-2 is to prevent
the release of cytochrome c from mitochondria (59, 61). To
our knowledge, our results are the first indication of a
down-regulation of BCL-2 in human myeloid leukemic cells by IFN-
. In
agreement with our results, it has been recently demonstrated that
expression of Bcl-X(L), another member of the BCL-2 family of
anti-apoptotic proteins, is inhibited in U937 cells overexpressing the
interferon consensus sequence-binding protein (2). It has been reported
previously that IFN-
induced apoptosis in colon carcinoma cells,
which was correlated with the down-regulation of BCL-2 and the
up-regulation of BAX (73). On the other hand, the importance of BCL-2
in regulating IFN-
-induced apoptosis has been reported in HeLa cells
(74). In these studies, overexpression of BCL-2 blocked
interferon-induced double-stranded RNA-dependent protein
kinase-activated apoptosis.
promoted the
caspase-dependent cleavage of remaining BCL-2 protein to
generate a 23-kDa fragment, upon death receptor activation. A similar
proteolytic fragment of BCL-2 has been shown to be produced by
caspase-3 during staurosporine-induced apoptosis in breast tumor cells
(57). This caspase-generated BCL-2 fragment localizes to mitochondria
and promotes the release of cytochrome c (57), which
contributes to amplification of the caspase cascade (56). Therefore, in
human leukemic U937 cells, IFN-
not only diminished the levels of
anti-apoptotic mitochondrial membrane-operating BCL-2 protein but, by
promoting the generation of pro-apoptotic fragments of BID and BCL-2
upon death receptor activation, it could also favor
mitochondria-mediated apoptosis. This apoptotic pathway was prevented
in U937 cells that expressed ectopic Bcl-2, further supporting the role
of mitochondria in IFN-
-induced sensitization to death
receptor-mediated apoptosis.
, severe
toxicity has been observed in systemic anti-tumor treatments with
TNF-
or CD95 agonistic antibody. TNF-
causes a lethal
inflammatory response (75), and CD95 mAb produces lethal liver damage
(76). However, repeated systemic exposure of non-human primates to
elevated doses of TRAIL did not cause significant changes in clinical
parameters (31, 77), although it may affect normal human hepatocytes (78). Interestingly, in contrast to myeloid leukemic cells, treatment
of normal human monocytes with IFN-
or -
induces the down-regulation of pro-apoptotic TRAIL receptors, which renders these
cells resistant to TRAIL-mediated apoptosis (79). Altogether, these
data suggest that sensitizing regimes like IFN-
may be useful after
cautious studies, in therapeutic strategies with non-toxic death
receptor ligands or drugs for the treatment of certain human myeloid malignancies.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Faustino Mollinedo (Consejo Superior de Investigaciones Científicas, Salamanca, Spain) for THP-1 cells. U937 cells transfected with full-length human Bcl-2 (U937Bcl-2) were kindly donated by Dr. Carmen Garrido (Faculty of Medicine and Pharmacy, Dijon, France).
![]() |
FOOTNOTES |
---|
* This work was supported in part by Ministerio de Educación y Cultura Grants 1FD97-0514-C02-01 and SAF2000-0118-C03-01 and by Consejo Superior de Investigaciones Científicas/Ministerio de Ciencia, Technología y Medio Ambiente Grant 99CU0004 (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 Junta de Andalucia.
Both authors contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 34-958-80-51-88; Fax: 34-958-20-33-23; E-mail: alrivas@ipb.csic.es.
Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M100815200
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ABBREVIATIONS |
---|
The abbreviations used are:
IFN, interferon;
TNF, tumor necrosis factor;
TRAIL, tumor necrosis factor-related
apoptosis-inducing ligand;
ICE, interleukin-1--converting enzyme;
DISC, death-inducing signaling complex;
PARP, poly(ADP-ribose)
polymerase;
Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-(OMe)fluoromethyl ketone;
FLIP, FLICE-inhibitory protein;
IRF-1, interferon regulatory factor-1;
mAb, monoclonal antibody;
PCR, polymerase chain reaction;
RT, reverse
transcription;
PBS, phosphate-buffered saline;
FADD, fas-associated death domain.
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