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Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2342-2352
Nitric Oxide-Induced Apoptosis in Human Leukemic Lines Requires
Mitochondrial Lipid Degradation and Cytochrome C Release
By
Alexey Ushmorov,
Frank Ratter,
Volker Lehmann,
Wulf Dröge,
Volker Schirrmacher, and
Victor Umansky
From the Divisions of Immunochemistry and Cellular Immunology, Tumor
Immunology Program, German Cancer Research Center, Heidelberg, Germany.
 |
ABSTRACT |
We have previously shown that nitric oxide (NO) stimulates apoptosis
in different human neoplastic lymphoid cell lines through activation of
caspases not only via CD95/CD95L interaction, but also independently of
such death receptors. Here we investigated mitochondria-dependent
mechanisms of NO-induced apoptosis in Jurkat leukemic cells. NO donor
glycerol trinitrate (at the concentration, which induces apoptotic cell
death) caused (1) a significant decrease in the concentration of
cardiolipin, a major mitochondrial lipid; (2) a downregulation in
respiratory chain complex activities; (3) a release of the
mitochondrial protein cytochrome c into the cytosol; and (4) an
activation of caspase-9 and caspase-3. These changes were accompanied
by an increase in the number of cells with low mitochondrial
transmembrane potential and with a high level of reactive oxygen
species production. Higher resistance of the CD95-resistant Jurkat
subclone (APO-R) cells to NO-mediated apoptosis correlated with the
absence of cytochrome c release and with less alterations in
other mitochondrial parameters. An inhibitor of lipid peroxidation,
trolox, significantly suppressed NO-mediated apoptosis in APO-S Jurkat
cells, whereas bongkrekic acid (BA), which blocks mitochondrial
permeability transition, provided only a moderate antiapoptotic effect.
Transfection of Jurkat cells with bcl-2 led to a complete block of
apoptosis due to the prevention of changes in mitochondrial functions.
We suggest that the mitochondrial damage (in particular, cardiolipin
degradation and cytochrome c release) induced by NO in human
leukemia cells plays a crucial role in the subsequent activation of
caspase and apoptosis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE SMALL MOLECULE nitric oxide (NO) is
generated from a guanido nitrogen of L-arginine by at least three
distinct isoforms of NO synthase (NOS) encoded by three distinct
genes.1 A low level of NO synthesized by constitutive NOS
for short periods of time acts as a neurotransmitter and as a regulator
of blood pressure and platelet aggregation.2 In contrast, a
high level of NO, produced for long periods of time by inducible NOS
after lipopolysaccharide (LPS) and cytokine challenge, is
cytotoxic for pathogenes and tumor cells.3 It was shown
that NO-mediated cytotoxicity involved the inhibition of mitochondrial
respiration and DNA synthesis in tumor targets.4,5
Moreover, this cytotoxic effect was recently found to be associated
with apoptosis (programmed cell death) in normal6,7 and
tumor cells.8-11
Apoptosis results from the action of a genetically encoded suicide
program with characteristic biochemical and ultrastructural changes,
which can be induced by different stimuli such as tumor necrosis factor
(TNF), CD95 (FAS/APO-1) ligand, TNF-related apoptosis-inducing ligand
(TRAIL) (APO-2 ligand), shortage of growth factors,
oxygen, or certain metabolites.12 It is currently assumed
that the activation of a cascade of cytoplasmic cystein proteases
(caspases) is essential for apoptosis regardless of the initial death
signal.13 Recently, it has been found that alterations in
mitochondrial functions play a key role in the effector phase of
apoptosis induced by different agents.14-16 These
alterations include the disruption of mitochondrial transmembrane
potential (  m), the generation of reactive oxygen
species (ROS) and the opening of permeability transition (PT)
pores.14,17 In addition, the release of the 15-kD
mitochondrial protein cytochrome c, identified as apoptotic protease activating factor 2 (Apaf 2), induces the formation of the
complex between Apaf 1 and caspase-9 (Apaf 3). The latter becomes
activated under such conditions and in turn activates caspase-3, which
leads to DNA fragmentation and apoptosis.18-20 Cytochrome
c is normally present on the outer surface of the inner mitochondrial membrane and shuttles electrons between complexes III and
IV of the respiratory chain.18
Bcl-2 belongs to a growing family of proteins that can either block
(Bcl-2, Bcl-xL, etc) or promote (Bax, Bad, Bak, etc)
apoptosis. Bcl-2-related proteins are integrated in the outer
mitochondrial, outer nuclear, and endoplasmic reticular membranes with
the help of a carboxy-terminal membrane anchor.21,22 It was
demonstrated that Bcl-2 overexpression specifically prevents cells from
initiating apoptosis in response to a number of stimuli, including
NO,23-25 and that the antiapoptotic effect of Bcl-2
involved the normalization of mitochondrial functions. Thus, Bcl-2
blocked mitochondrial PT and prevented the release of caspase
activators (in particular, cytochrome c) from mitochondria and
the disruption of m induced by glucorticoids, DNA
damage, oxidants, or ceramide.21,23,26 Moreover, according
to recent data, high levels of Bcl-2 can delay cell death, even when
cytochrome c is already released into the cytosole.27
We have previously shown that NO triggers apoptosis in different human
neoplastic lymphoid cell lines and freshly isolated leukemic
lymphocytes through activation of caspases, including caspase-8, the most CD95 receptor-proximal
caspase.11 Furthermore, NO was able to induce activation of
caspase-8 not only via CD95/CD95L interaction, but also independently
of death receptors via direct caspase activation. Here we studied
mitochondria-dependent mechanisms of NO-mediated apoptosis in human
leukemia cells. Our results suggest that NO (at the concentration which
induces apoptotic death in Jurkat cells) causes catabolism of the major
mitochondrial phospholipid, cardiolipin, and the release of cytochrome
c into the cytosole, downregulates the activity of oxidative
phosphorylation complex I, III, and IV, induces significant changes in
mitochondrial transmembrane potential, and stimulates ROS production.
All of these biochemical alterations were blocked in bcl-2-transfected Jurkat T cells, which are completely resistant to the apoptotic effect
of NO.
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MATERIALS AND METHODS |
Cell lines.
Human leukemic T-cell lines (Jurkat APO-S and APO-R) were maintained in
5% CO2 at 37°C in RPMI 1640 medium (GIBCO-BRL,
Eggenstein, Germany) containing 5% fetal calf serum. CD95-resistant
(APO-R) Jurkat cells were derived from the parental CD95-sensitive
(APO-S) clone by long-term culture in the presence of a lethal dose of activating CD95 antibody.28 Northern and Western blot
analyses showed that APO-R Jurkat cells expressed CD95 mRNA, but failed to express CD95 protein. Both cell clones (APO-S and APO-R) were equally sensitive to CD95-independent apoptosis and expressed the same
cell surface markers.28 Jurkat cells transfected with empty
vector or containing bcl-2 were kindly provided by Dr M.E. Peter
(German Cancer Research Center, Heidelberg, Germany) and cultured as
described elsewhere.29 To evaluate apoptotic effects of NO,
all above-mentioned cells were treated with glycerol trinitrate (GTN;
Merck, Darmstadt, Germany), which is known to constitutively produce NO
in the incubation medium.
Antibodies and other reagents.
The monoclonal antibody (MoAb) against cytochrome c (clone
7H8.2C12) was purchased from PharMingen (Hamburg, Germany). The horseradish peroxidase-conjugated goat antimouse MoAbs were purchased from Dianova (Hamburg, Germany). The following reagents were from Molecular Probes, Inc (Eugene, OR):
10-N-nonyl-3,6-bis(dimethylamino)acridine (NAO),
3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)],
dihydroethidine (HE), and propidium iodide (PI). Bongkrekic acid (BA)
was kindly provided by Dr J.A. Duine (Delft University,
Delft, The Netherlands). Water-soluble analogue of vitamine E,
6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) and a
fluorogenic substrate for caspase 9 Ac-Leu-Glu-His-Asp-amido-trifluoromethyl-coumarin (LEHD-AFC) was from
Calbiochem (Bad Soden, Germany). A fluorogenic substrate for caspase 3 Asp-Glu-Val-Asp-aminomethyl-coumarin (DEVD-AMC) was purchased from
Bachem (Heidelberg, Germany). All other chemicals used were of
analytical grade and purchased from Sigma (Munich, Germany).
Apoptosis and cytotoxicity assay.
Apoptosis was assessed by determining DNA fragmentation (DNA-release
assay) after lysing the cells in a hypotonic solution (0.1% sodium
citrate, 0.1% Triton X-100) containing 50 µg/mL PI as
described30 and analyzed by flow cytometry using a FACScan analyzer with CELLQuest software (Becton Dickinson, Heidelberg, Germany). Cytotoxicity was evaluated after treatment of cells with 1 µg/mL PI for 5 minutes before fluorescence-activated cell sorting
(FACS) analysis. A total of 10,000 cells per sample was analyzed by
FACScan and CELLQuest software (Becton Dickinson).
Cytofluorometric analysis of mitochondrial transmembrane potential,
reactive oxygen species, and cardiolipin.
To measure m and ROS production, cells (7 × 105/mL) were incubated with DiOC6(3) (40 nmol/L
in phosphate-buffered saline [PBS]) and 5 µmol/L HE, respectively,
at 37°C for 30 minutes.17 For complete depletion of
m (positive control), a mitochondrial uncoupler
carbonyl cyanide m-chlorophenyl-hydrazone (CCCP, 50 µmol/L)
was used.17 The content of a main mitochondrial lipid cardiolipin was analyzed after incubation with specific dye NAO (100 nmol/L) for 30 minutes at 37°C.31 A total of 1 µg/mL
PI was added to the samples for 5 minutes before FACS analysis and measured at red fluorescence (FL3). Recordings were made only on PI
negative (viable) cells at green fluorescence (FL1) for DiOC6(3) and NAO and at red fluorescence (FL2) for HE.
Typically, 10,000 cells per sample were measured using FACScan and
CELLQuest software (Becton Dickinson).
Measurement of the oxidative phosphorylation (OXPHOS) activity.
The activity of OXPHOS complexes was measured according to Hofhaus et
al32 with small modifications. Briefly, upon treatment with
GTN for 8 hours, 3 × 107 Jurkat cells were
permeabilized for 1 minute with 7.5 µg/mL digitonin in medium A (20 mmol/L HEPES, 250 mmol/L sucrose, 10 mmol/L MgCl2, pH 7.1),
washed twice in medium A and kept on ice. Oxygen consumption by
permeabilized cells resuspended in 0.75 mL of respiratory medium (medium A supplemented with 2 mmol/L adenosine diphosphate (ADP) and 2 mmol/L KH2PO4) was measured at 37°C using a
Clark-type oxygen electrode fitted to 2 mL water-jacketed closed
chamber. The activity of the OXPHOS complexes I-III was recorded after
incubation with 5 mmol/L glutamate and 5 mmol/L malate. To evaluate
complexes II-III activity, complex I was inhibited by 0.1 µmol/L
rotenone, and respiration was initiated by 5 mmol/L
glycerol-3-phosphate and 5 mmol/L succinate. After inhibition of
complex III with 100 nmol/L antimycin, 10 mmol/L ascorbate and 0.2 mmol/L N,N,N',N'-tetramethyl-p-phenylenediamine were added to drive
electrons directly to cytochrome c (complex IV). The activity
of complex IV was then blocked with 0.1 mmol/L potassium cyanide
(KCN) to evaluate cyanide resistant oxygen consumption. Data were expressed in fmol of O2 per minute per cell.
Determination of cytochrome c release.
The release of cytochrome c into the cytosol of Jurkat cells
treated with GTN for 8 hours was measured as described
elsewhere.33 Briefly, 6 × 107 cells were
washed in PBS and resuspended in 4 vol of ice-cold buffer containing 20 mmol/L HEPES, 250 mmol/L sucrose, 2 mmol/L EDTA, 20 µg/mL
phenylmethylsulfonyl fluoride, 2 µg/mL leupeptin, and 10 µg/mL
aprotinin, pH 7.1. Cells were disrupted on ice with 15 strokes of
Dounce homogenizer and centrifuged for 3 minutes at 3,000g to
remove nuclei and unbroken cells. The supernatants were then
centrifuged for 1 minute at 12,000g to isolate mitochondrial fraction. Resulting supernatants were centrifuged for 1 hour at 100,000g to sediment cell membranes. For Western blot detection of cytochrome c, the supernatants from the last centrifugation (fraction S100) and mitochondrial fractions were subjected to 12%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The proteins were then tranferred onto a polyvinylidene difluoride
membrane (Millipore, Bedford, MA) using a semidry blotting apparatus
(Bio-Rad, Munich, Germany). The membrane was blocked with a 5%
solution of nonfat dry milk in TBST buffer (25 mmol/L Tris-HCl, 137 mmol/L NaCl, 5 mmol/L KCl, 0.7 mmol/L CaCl2, 0.1 mmol/L
MgCl2, 0.05% (vol/vol) Tween 20, pH 7.4) and incubated overnight at 4°C with MoAb against cytochrome c. Blots were
extensively washed with TBST buffer and developed with goat antimouse
MoAb (dilution 1:2,500). After washing the membrane with TBST buffer, the immunoreactive bands were visualized by enhanced chemiluminescece (ECL) method following the manufacturer's protocol (Amersham, Braunscweig, Germany).
Caspase activity assay.
A total of 106 APO-S Jurkat cells (treated with GTN or
control) was washed with PBS and lysed in buffer A (100 mmol/L HEPES, 10% sucrose, 10 µmol/L leupeptin, 10 µmol/L aprotinin, 1 mmol/L EDTA, 5 mmol/L dithiothreitol, and 0.15%
3-cholamidopropyl-dimethylammonio-1-propansulfonate, pH 7.4). To
determine caspase-3 activity, 10 µL of cell lysates were incubated
with 20 µL (75 µmol/L, final concentration) of fluorogeneic peptide
DEVD-AMC (Bachem, Heidelberg, Germany) as described.34 The
product of reaction fluorescent AMC was measured after 1 hour of
incubation at excitation 355 nm, emission 485 nm using a plate reader
(Victor 1420, Wallac, Freiburg, Germany). For caspase-9, a fluorogenic
substrate LEHD-AFC (75 µmol/L, final concentration) was used and the
product of reaction AFC was measured with the same plate reader at
excitation 405 nm and emission 535 nm.35
| |
RESULTS |
NO inhibits the activity of OXPHOS complexes and induces cytochrome c
release.
We have previously shown that treatment of Jurkat cells with increasing
concentrations of the NO donor, GTN, caused a cytotoxic effect that was
due to apoptosis.11 The level of NO-induced apoptosis was
much higher in APO-S Jurkat cells that express CD95 on the cell surface
and are sensitive to CD95-mediated kill than in APO-R cells, which lack
CD95 expression and are resistant to anti-CD95 MoAb. To study
mitochondrial involvement, we measured the activity of complexes
of the mitochondrial electron transport chain (OXPHOS
complexes) and the presence or absence of the mitochondrial protein cytochrome c in the cytosol of both APO-S and APO-R
cells treated with NO for 8 hours. We showed previously that incubation with the NO donor, GTN, for 8 hours and its withdrawal for the next 16 hours was sufficient to achieve a maximal level of apoptosis 24 hours
after GTN treatment.11 Incubation with 0.2 mmol/L GTN for
24 hours caused 33% apoptosis in APO-S and 10% in APO-R cells. After
48 hours, the level of NO-induced apoptosis reached 52% in APO-S and
20% in APO-R cells.
As shown in Fig 1A and B, the activity of
OXPHOS complexes in APO-S Jurkat cells treated for 8 hours with 0.2 mmol/L GTN was significantly reduced (down to 17% of the level in
untreated cells for complex III), whereas in APO-R cells, only a
comparatively small decrease in mitochondrial respiratory function was
seen. The substantial decrease in complex IV activity in APO-S cells (Fig 1B) was found to correlate with the detection of cytochrome c in the cytosol fraction (S100) after 4 and 8 hours of
incubation with GTN (Fig 1C) suggesting that the release of cytochrome
c is a possible reason for the suppression of complex IV
activity. In contrast, NO treatment did not induce any migration of
cytochrome c into the cytosol of APO-R cells (Fig 1C).
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| Fig 1.
Effect of NO on the activity of OXPHOS complexes (A and
B) and cytochrome c release (C) in APO-S and APO-R Jurkat
cells. After treatment with the NO donor GTN (0.2 mmol/L) for 8 hours,
3 × 107 cells were permeabilized with digitonin. Oxygen
consumption was measured at 37°C using a Clark type oxygen
electrode as described in Materials and Methods. To evaluate cytochrome
c release, 6 × 107 cells were homogenized on ice
and Western blot analysis with anticytochrome c MoAb was
performed. (A) Downregulation of OXPHOS complex activity under NO
treatment. O2 consumption is expressed as percentage of
untreated control. (B) NO-mediated inhibition of complex IV activity.
Data are expressed in fmol of consumed O2 per minute per
cell. Each bar represents the mean ± standard deviation (SD) of three
independent experiments. (C) Time-dependent release of cytochrome
c into the cytosol in APO-S, but not in APO-R cells. A
representative experiment of three is shown.
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NO induces caspase activation.
Because the release of cytochrome c can initiate an apoptotic
protease cascade,18-20 we tested NO-induced changes in the
activity of caspase-9 and caspase-3, which are the members of this
cascade that are the most proximal to cytochrome
c.19,20 It was found that NO treatment for 2 and 4 hours resulted in a very low level of activation of caspase-9
(Fig 2). However, after 8 hours, the activity of this enzyme was 4.7 times higher than in untreated cells
(P < .001). A similar level of caspase-9 activation was observed after 24 hours. Caspase-3 activity was also significantly upregulated only 8 hours after addition of GTN and remained activated at 24 hours (Fig 2).
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| Fig 2.
Activation of caspase-9 ( ) and caspase-3 ( ) by NO
in APO-S Jurkat cells. Cells were treated for different periods of time
with 0.2 mmol/L GTN. Cell lysates were then prepared and incubated with
fluorogeneic peptides: LEHD-AFC (for caspase-9) and DEVD-AMC (for
caspase-3). The products of reaction AFC or AMC were measured after 1 hour of incubation using a plate reader. Each point represents the mean ± SD of three independent experiments.
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Therefore, NO-induced caspase-9 and caspase-3 activation in APO-S
Jurkat cells, observed at 8 hours, follows detectable release of
cytochrome c into the cytosole.
NO stimulates an increase in the number of Jurkat cells with low
cardiolipin content, high ROS production, and low m.
Next, we measured the content of the main mitochondrial lipid,
cardiolipin (NAO staining), m (DiOC6(3)
staining) and ROS production (HE staining) in both APO-S and APO-R
cells treated with NO. All of these mitochondrial parameters were
tested in PI-negative live cells.
As shown in Fig 3A, the population of
untreated live (PI-negative) APO-S cells was homogeneous. After
incubation with the NO donor, GTN, live APO-S cells can be divided into
two subpopulations: one with low m
(DiOC6(3)low) and high ROS production
(HEhigh) and another with high m
(DiOC6(3)high) and low ROS production
(HElow) (12.3% and 82.3%, respectively at 24 hours).
After staining with NAO and HE, the live cells also formed two
subpopulations under NO treatment: NAOlow
HEhigh and NAOhigh HElow (14.2%
and 75.0%, respectively at 24 hours). These two subpopulations were
observed starting from 4 hours of GTN treatment. Interestingly, the
kinetics of NAOlow HEhigh and
DiOC6(3)low HEhigh cells were very
similar at the time points tested. This suggests that in APO-S cells,
NO stimulated the formation of a subpopulation with high ROS
production, as well as decreased cardiolipin content and
m. When APO-R cells were stained, we found that the
proportion of NAOlow HEhigh and
DiOC6(3)low HEhigh cells was much
lower than in APO-S cells (5.8% and 6.1%, respectively, at 24 hours;
Fig 3B). In contrast to APO-S cells, we observed in APO-R cells an
additional subpopulation with increased ROS production together with
high levels of cardiolipin content and m (7% to 9%
after 24 hours).
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| Fig 3.
NO-mediated changes in  m, ROS
production, and cardiolipin content in APO-S (A) and APO-R (B) Jurkat
cells. To measure m and ROS production, cells were
incubated with DiOC6(3) and HE, respectively, at 37°C
for 30 minutes. The content of the main mitochondrial lipid,
cardiolipin, was analyzed after incubation with NAO for 30 minutes at
37°C. Recordings were made only on PI negative (viable) cells at
green fluorescence (FL1) for DiOC6(3) and NAO and at red
fluorescence (FL2) for HE using FACScan and CELLQuest software. A
representative experiment of four is shown.
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APO-S Jurkat cells thus showed larger alterations in mitochondrial
functions than did APO-R cells. This correlated with higher sensitivity
of the APO-S cells to NO. There was a time-dependent increase in the
APO-S cell subpopulation with low cardiolipin content, low
m and high ROS production.
Jurkat cells transfected with bcl-2 are completely resistant to
apoptosis and show normal mitochondrial functions after NO treatment.
A growing number of studies suggested that overexpression of Bcl-2
could block apoptosis via mitochondrial effects.33,36,37 Therefore, we used bcl-2-transfected Jurkat cells
(J-Bcl-2)29 to investigate the effect of this protein on
NO-mediated apoptosis. Figure 4 shows that
Bcl-2 hyperexpression completely inhibited apoptosis in Jurkat cells on
treatment with GTN even when excess concentrations were used (up to 0.4 mmol/L). Control Jurkat cells stably transfected only with the neomycin
vector (J-Neo) showed a GTN concentration-dependent increase in the
percentage of apoptotic cells.
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| Fig 4.
Bcl-2 overexpression completely blocks NO-induced
apoptosis in Jurkat leukemic cells. Cells transfected with neomycin
vector only (J-Neo) or with bcl-2 expressing vector (J-Bcl-2) were
incubated with NO donor GTN at different concentrations for 24 hours.
Percentage of apoptotic cells was measured by the DNA-release assay
using treatment with a hypotonic PI solution at 4°C overnight in
the dark followed by flow cytometric analysis. Each bar represents the
mean ± SD of three independent experiments.
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Next, we tested mitochondrial functions in J-Bcl-2 cells subjected to
treatment with 0.3 mmol/L GTN. J-Neo cells (control) were found to be
NO susceptible and similar to APO-S cells with regard to suppression in
the activity of complexes I, II, and IV, as well as cytochrome
c release into the cytosol fraction (Fig 5A through C). In contrast, J-Bcl-2
cells were completely resistant to the above-mentioned mitochondrial
changes. Interestingly, in the latter cells, NO treatment even
stimulated to some extent the activity of mitochondrial respiratory
chain complexes (up to 145% of the level in untreated cells in the
case of complex I). Bcl-2 was also able to normalize the content of
cardiolipin, m and ROS production. As shown in
Fig 6A and B, a time-dependent upregulation
in the number of cells with low cardiolipin content, low
m, and high ROS production was found in J-Neo cells
(Fig 6A), but not in J-Bcl-2 cells (Fig 6B). Live (PI-negative) control J-Neo cells had 11.6% of cells with the phenotype NAOlow
HEhigh and DiOC6(3)low
HEhigh at 24 hours (Fig 6A), while less than 0.3% of
J-Bcl-2 cells had this phenotype (Fig 6B). Bcl-2 overexpressing cells
remained mostly homogenous after exposure to NO showing only a slight
increase in the HEhigh cell subpopulation without any
decrease in m and cardiolipin concentration
(Fig 6B).
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| Fig 5.
Effect of NO on the activity of OXPHOS complexes (A and
B) and cytochrome c release (C) in Jurkat cells transfected
only with neomycin (J-Neo) and in Bcl-2 overexpessing Jurkat cells
(J-Bcl-2). Evaluation of OXPHOS complex acitivity and cytochrome
c release was performed as described in the legend to Fig 1.
(A) Decrease of OXPHOS complex activity under NO treatment. Data were
expressed as a percentage to the untreated controls. (B) NO-mediated
suppression of complex IV activity. Data were expressed in fmol of
O2 per minute per cell. Each bar represents the mean ± SD
of three independent experiments. (C) Release of cytochrome c
into the cytosole in Neo, but not in Bcl-2 overexpressing cells. A
representative experiment of three is shown.
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| Fig 6.
NO-induced alterations in m, ROS
production, and cardiolipin content in Jurkat cells transfected only
with neomycin (J-Neo) (A) and in Bcl-2 overexpessing Jurkat cells
(J-Bcl-2) (B). To measure m, ROS production, and
cardiolipin content, cells were incubated with DiOC6(3),
HE, and NAO, respectively at 37°C for 30 minutes. Recordings were
made only on PI negative (viable) cells at green fluorescence (FL1) for
DiOC6(3) and NAO and at red fluorescence (FL2) for HE. A
representative experiment of four is shown.
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Thus, Bcl-2 overexpression prevents the damage of mitochondrial
functions induced by NO in human leukemia cells. This correlates with
the acquisition of complete resistance to NO-mediated apoptosis.
Trolox and BA partially inhibit NO-induced apoptosis in APO-S cells.
Although it seems established that the antiapoptotic effects of Bcl-2
are exerted at the mitochondrial level, the exact mechanism(s) of this
effect are still unclear. Bcl-2 can block lipid
peroxidation23,38 and the opening of mitochondrial PT
pores26 that occurs during apoptotic cell death induced by
different stimuli. To test the contribution of these Bcl-2-mediated
protective mechanisms under treatment with NO at apoptosis-inducing
concentrations, we used the lipid- and water-soluble vitamin E analogue
trolox, a potent inhibitor of lipid peroxidation,39 and BA,
which suppresses mitochondrial PT.40 We found that the
addition of trolox to the incubation medium substantially downregulated
the number of apoptotic APO-S cells induced by NO (from 34% to 9%
dead cells; P < .001; Fig 7).
Interestingly, incubation with trolox alone resulted in some increase
in apoptotic cell death (to 14%). BA could reduce the apoptotic
effects of GTN (from 34% to 25%; P < .05), although this
reduction was much lower than that observed with trolox. The fact that
the inhibitor of lipid peroxidation, trolox, can strongly downregulate
NO-induced apoptosis suggests an important role of lipid peroxidation
in this process in Jurkat cells. The protective effect of Bcl-2 may
involve the suppression of lipid degradation in NO-treated Jurkat
cells.
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| Fig 7.
Influence of trolox and BA on NO-mediated apoptosis in
APO-S Jurkat cells. Cells were incubated with NO donor GTN (0.2 mmol/L), trolox (5 mmol/L), and with BA (60 µmol/L) for 24 hours.
Percentage of apoptotic cells was measured by the DNA-release assay
using treatment with a hypotonic PI solution at 4°C overnight in
the dark followed by flow cytometric analysis. Each point represents
the mean ± SD of three independent experiments.
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We next investigated whether trolox and BA can affect the changes in
mitochondrial functions mediated by NO in Jurkat cells. The results of
these experiments, recorded for live (PI negative) APO-S cells, are
presented in Fig 8. After 24 hours of
incubation together with GTN, trolox was found to significantly reduce
the size of the subpopulation with low cardiolipin content
(NAOlow) as compared with the cell cultures treated with
GTN alone (from 30% to 10%; P < .01; Fig 8A). In contrast,
the number of cells with low m
(DiOC6(3)low) was only slightly decreased and
the proportion of HEhigh cells (high ROS production) at
this time point remained at the same level as in the cells incubated
with GTN alone. At an earlier time point (8 hours), trolox was able to
downregulate the quantity of NAOlow cells as compared with
the samples with GTN only (9% and 14%, respectively; P < .05), but it could induce almost no decrease in the proportion of
DiOC6(3)low and HEhigh cells (Fig
8A). When BA and GTN were added to APO-S cells for 24 hours, the number
of DiOC6(3)low cells was lower than in cell
cultures with GTN alone (22% and 28%, respectively; P < .05; Fig 8B). The proportion of NAOlow and
HEhigh cells remained unchanged under these culture
conditions.
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| Fig 8.
Effect of trolox (A) and BA (B) on m,
ROS production, cardiolipin content, and of trolox on the activity of
OXPHOS complex IV in NO-treated APO-S Jurkat cells. (A and B) Cells
were incubated for 8 and 24 hours with NO donor GTN (0.2 mmol/L) alone,
trolox (5 mmol/L) alone, BA (60 µmol/L) alone or with GTN together
with either trolox or BA. Flow cytometry analysis for
m [DiOC6(3)], ROS production (HE), and
for cardiolipin concentration (NAO) was performed as described in the
legend to Fig 2. ( ), DiOC6 (3)low; ( ),
HEhigh; (), NAOlow. (C) Cells were incubated
with NO donor GTN (0.2 mmol/L) alone, trolox (5 mmol/L) alone, and with
both agents for 8 hours. Activity of OXPHOS complex IV was measured as
described in the legend to Fig 1. Each bar represents the mean ± SD
of three independent experiments.
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As shown in Fig 8C, treatment of APO-S cells with the combination of
GTN and trolox for 8 hours resulted in the attenuation of the
GTN-mediated inhibitory effect on complex IV activity. Incubation with
trolox alone for the same period of time caused a decrease of complex
IV activity as compared with untreated control cells.
Thus, trolox, an inhibitor of lipid peroxidation, strongly suppressed
the NO-mediated apoptosis in APO-S Jurkat cells that correlated with
significant reduction of NO-induced changes in cardiolipin
concentration and OXPHOS complex IV activity. In contrast, treatment
with BA, an inhibitor of mitochondrial PT, caused only a moderate
suppression of NO-induced apoptosis and some decrease in the number of
APO-S cells with low m.
| |
DISCUSSION |
Recent studies performed in different cellular systems, including
cell-free extracts, clearly indicated a crucial role for mitochondrial
damage in many forms of apoptosis in mammalian cells. An early
disruption of m due to the formation of PT
pores14 and cytochrome c release from
mitochondria15,18 are considered the most important changes
in mitochondrial functions. Cytochrome c is normally located in
mitochondrial membranes and shuttles electrons between complexes III
and IV of the respiratory chain.18
It was already known that NO could inhibit mitochondrial respiration in
different types of cells.5,9,39 Moreover, NO has been
recognized to induce cell death via apoptosis,6-11 although the precise mechanism of this process is still obscure. We described previously that NO can induce apoptosis in different human neoplastic lymphoid cells through activation of caspases. This occurred not only
via CD95/CD95L interaction, but also independently of death receptors.11 In the present study, we addressed the
question whether alterations in mitochondrial functions might mediate
NO-induced caspase activation and subsequent apoptosis in human
malignant lymphoid cells, and if so, which type of alteration was
relevant. Using GTN (a donor of exogenous NO) at the concentrations
inducing apoptosis in CD95-sensitive (APO-S) Jurkat cells, we show for the first time, a release of cytochrome c from the mitochondria into the cytosol fraction as early as 4 hours after onset of the experiment. These data are in agreement with our earlier observation that incubation with GTN for at least 8 hours and its subsequent withdrawal for 16 hours was sufficient to achieve maximal apoptotis, ie, the latter became irreversible at this time point.11
It has been recently reported that released cytochrome c is
absolutely required (together with Apaf 1) for the activation of
caspase-9, which in turn cleaves and activates
caspase-3.18-20 Both caspases are considered the most
upstream members of the apoptotic protease cascade that is
triggered by cytochrome c.19,20 Here, we
compared the kinetics of both cytochrome c accumulation in the
cytosol and the activation of the above-mentioned caspases in
NO-treated APO-S Jurkat cells and found that the first process happened
earlier (at 4 hours) than the latter (at 8 hours). This suggests that
NO-mediated release of cytochrome c causes the activation of
the caspase cascade followed by apoptosis, as has already been shown
for CD95 and other apoptotic death signals.18,33
To elucidate the mechanism(s) of cytochrome c release upon NO
treatment of APO-S Jurkat cells, we studied the function of respiratory
chain complexes and the content of the major mitochondrial lipid,
cardiolipin, which plays a crucial role in cytochrome c attachment to the inner mitochondrial
membrane.21,41,42 In addition, we measured
m and ROS production. It was found that exposure to
NO for 8 hours significantly inhibited the activity of all complexes of
the mitochondrial electron transport chain. This correlated with
cytochrome c release and could be one of the reasons for the
observed suppression of complex IV activity (Fig 1B and C). Our data
are in agreement with other publications reporting on NO-mediated
inhibition of cytochrome c oxidase (complex IV) in different
cell types via binding to its heme moiety in a reversible
manner.43,44 This downregulation of complex IV activity
resulted in the upregulation of ROS synthesis45 followed by
the formation of the strong oxidant peroxynitrite anion
(ONOO-), which can induce irreversible inhibition of
complexes I and III, but not complex IV.46 The alternative
mechanism of a direct effect of NO on these complexes is also possible
in our system, as long-term exposure to NO in vitro has been recently
shown to block complex I activity in murine macrophages due to
S-nitrosylation of this enzyme.44
The inhibition of the activity of complexes I, II, and III (Fig 1A and
B) implies the cessation of ROS release from the electron transport
chain. However, we observed a steady increase of ROS production in
APO-S Jurkat T cells at 8 and 24 hours of NO treatment (Fig 2A). An
alternative source of ROS synthesis could be the peroxidation of
mitochondrial lipids (especially cardiolipin).47 Indeed,
Escames et al48 have recently reported on NO-mediated upregulation of this process in rat brain cells. Nevertheless, the role
of mitochondrial lipid degradation in the apoptotic effect of NO has
not yet been investigated. Here, we found a significant time-dependent
decrease in the content of cardiolipin, a phospholipid located in the
inner mitochondrial membrane (like cytochrome c). Cardiolipin
is necessary for complex IV activity49 and plays a crucial
role in the attachment of cytochrome c to the inner mitochondrial membrane.21,42 Thus, we suggest that the
observed degradation of cardiolipin upon NO treatment may be
responsible for cytochrome c release in APO-S Jurkat cells.
The critical role of lipid degradation in NO-mediated apoptosis in
APO-S Jurkat cells was confirmed by experiments with the vitamin E
analogue trolox. We found that this potent inhibitor of lipid
peroxidation39 significantly inhibited NO-induced apoptotic cell death and restored complex IV activity. A protective effect of
trolox on complex IV activity in NO-exposed rat astrocytes has also
been reported by Heales et al.50 This is probably due to
the prevention of cardiolipin oxidation.50 Indeed, trolox significantly reduced the number of NO-treated Jurkat cells with low
cardiolipin content without any effect on the quantity of cells with
low m and high ROS production, indicating that lipid peroxidation was required for this type of apoptosis.
Another possibility for mitochondrial damage during NO-mediated
apoptosis could be linked to the decrease of m due to
the opening of PT pores followed by the depolarization of the inner mitochondrial membrane and massive ROS production.51 This
mechanism of NO-induced apoptosis was shown in
thymocytes.52 We provide evidence for a time-dependent
increase in the number of live (PI-negative) APO-S Jurkat cells with
low m upon GTN treatment. To elucidate the role of PT
in the NO-induced apoptosis in our model, we used BA, which is known to
be a specific inhibitor of PT affecting the molecular conformation of
the adenine nucleotide translocator (a protein participating in the
formation of PT pores).17 BA was shown to suppress
dexamethasone-induced apoptosis in mouse thymocytes.17
However, when testing BA in combination with GTN, we observed only a
limited inhibitory effect on NO-induced apoptosis in APO-S Jurkat cells
(Fig 6). Under these conditions, only a moderate decrease in the number
of cells with low m was observed, thereby suggesting
that PT was not a major factor causing NO-induced reduction of
m and apoptosis in Jurkat cells.
We have earlier described a substantially lower NO-mediated apoptotic
cell death in the CD95-resistant Jurkat subclone (APO-R) as compared
with APO-S cells, which are sensitive to CD95-mediated kill.11 Interestingly, the basic level of mitochondrial
parameters studied here (eg, cardiolipin content, m,
ROS production, and the activity of OXPHOS complexes) was similar in
both cell lines. Nevertheless, NO failed to stimulate cytochrome
c release and decrease of cardiolipin concentration in APO-R
cells. In these cells, it induced only weak alterations in respiratory
complex activity, ROS production, and in m as
compared with the control values. This resistance correlated with a
substantial protection from apoptosis (Figs 1 and 2).
Bcl-2, which is expressed in the outer mitochondrial membrane, has been
shown to play a central role in the suppression of apoptosis in
different cell systems.21-25 In target cells, Bcl-2 prevent
not only the release of cytochrome c from
mitochondria21,33 by inhibition of lipid peroxidation (in
particular, cardiolipin),23,38 but also interfere with
cytochrome c already released into the cytosol.27
Other mechanisms of Bcl-2-related antiapoptotic effects at the
mitochondrial level are linked with (1) inhibition of PT and
stabilization of m17; (2) prevention of
caspase activation and cleavage of poly(ADP-ribose) polymerase
cleavage53; (3) regulation of proton flux36;
and (4) blocking of the proapoptotic effect of Bax
protein.54 Although an inverse correlation between Bcl-2
expression and sensitivity to NO-mediated apoptosis has recently been
shown in several cell lines,24,25 the mechanism of Bcl-2
protection from NO-mediated cell death is still elusive.
It has previously been reported that Jurkat cells express very low
levels of the Bcl-2 protein.29 In our experiments, we used
Jurkat cells transfected with bcl-2 and the same cell clone transfected
only with the corresponding neomycin vector (as a control). We found
that Bcl-2 overexpression resulted in a complete resistance to
apoptosis in GTN-treated cells at all concentrations tested (up to 0.5 mmol/L), whereas the control cells were sensitive to NO. Furthermore,
Bcl-2 was able to block the damage of mitochondrial functions observed
after exposure to GTN. It normalized the content of cardiolipin and ROS
production, m and OXPHOS complex activities, prevented cytochrome c release and lipid peroxidation in
mitochondria. From our data on the effect of BA, an inhibitor of
mitochondrial PT, and of trolox, an inhibitor of lipid peroxidation
(Fig 6), we suggest that Bcl-2-induced suppression of lipid
peroxidation plays a more important role in protection against
NO-mediated apoptosis than the inhibition of PT. However, the question
of which mechanism(s) are crucial for the suppression of NO-mediated cell death via Bcl-2 needs further investigation.
Taken together, our results show that NO-induced caspase activation and
subsequent apoptosis in human leukemia cells requires certain
alterations in mitochondrial functions, which include (1) a significant
decrease in the concentration of cardiolipin, a major mitochondrial
lipid; (2) a release of the mitochondrial protein cytochrome c
into the cytosol; and (3) a downregulation in respiratory chain complex
activities. Because NO-induced cell death was strongly suppressed by
trolox (an inhibitor of lipid peroxidation), we suggest that the
degradation of mitochondrial lipids can be an upstream step in this
process. We also found an increase in the number of cells with low
m and high ROS production, which may be considered as
secondary consequences of NO-mediated apoptosis. Bcl-2 overexpression
completely blocked the apoptotic effect of NO due to the prevention of
mitochondrial damage (including the normalization of cardiolipin
content and block of cytochrome c release). This new insight
into the mechanism of NO-induced cell death could be of importance for
effective treatment of leukemia patients by using NO donors or other
agents to induce apoptosis through damage of mitochondrial functions in
leukemic cells.
| |
ACKNOWLEDGMENT |
We thank Dr M.E. Peter for providing the Jurkat cells transfected with
empty vector or containing bcl-2, and Dr J.A. Duine for
providing BA.
| |
FOOTNOTES |
Submitted August 21, 1998; accepted November 19, 1998.
Supported in part by Grant No. 10-0980-Schi2, V.U. from the Dr. Mildred
Scheel Stiftung.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Victor Umansky, PhD, Division of Cellular
Immunology, Tumor Immunology Program, German Cancer Research Center,
Abteilung 710, lm Neuenheimer Feld 280, D-69120
Heidelberg, Germany; e-mail: V.Umansky{at}dkfz-heidelberg.de.
| |
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H.-R. Liu, L. Tao, E. Gao, B. L Lopez, T. A Christopher, R. N Willette, E. H Ohlstein, T.-L. Yue, and X.-L. Ma
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J. A. Bauer, B. H. Morrison, R. W. Grane, B. S. Jacobs, S. Dabney, A. M. Gamero, K. A. Carnevale, D. J. Smith, J. Drazba, B. Seetharam, et al.
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L. Piccotti, C. Marchetti, G. Migliorati, R. Roberti, and L. Corazzi
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P. Secchiero, A. Gonelli, C. Celeghini, P. Mirandola, L. Guidotti, G. Visani, S. Capitani, and G. Zauli
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C. C. Teixeira, K. Mansfield, C. Hertkorn, H. Ischiropoulos, and I. M. Shapiro
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TOP
ABSTRACT
INTRODUCTION