|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4311-4320
Caspase Activation Is Required for Nitric Oxide-Mediated,
CD95(APO-1/Fas)-Dependent and Independent Apoptosis in Human
Neoplastic Lymphoid Cells
By
Katerina Chlichlia,
Marcus E. Peter,
Marian Rocha,
Carsten Scaffidi,
Mariana Bucur,
Peter H. Krammer,
Volker Schirrmacher, and
Victor Umansky
From the Division of Cellular Immunology and the Division of
Immunogenetics, Tumor Immunology Program, German Cancer Research
Center, Heidelberg, Germany.
 |
ABSTRACT |
Nitric oxide (NO), an important effector molecule
involved in immune regulation and host defense, was shown to induce
apoptosis in lymphoma cells. In the present report the NO donor
glycerol trinitrate was found to induce apoptosis in Jurkat cells that are sensitive to CD95-mediated kill. In contrast, a CD95-resistant Jurkat subclone showed substantial protection from apoptosis after exposure to NO. NO induced mRNA expression of CD95 (APO-1/Fas) and
TRAIL/APO-2 ligands. Moreover, NO triggered apoptosis in freshly isolated human leukemic lymphocytes which were also sensitive to
anti-CD95 treatment. The ability of NO to induce apoptosis was
completely blocked by a broad-spectrum ICE (interleukin-1 converting
enzyme)-protease/caspase inhibitor and correlated with FLICE/caspase-8 activation. This activation was abrogated in some neoplastic lymphoid cells but not in others by the inhibitor of protein
synthesis cycloheximide. Our results were confirmed using an in vitro
experimental model of coculture of human lymphoid target cells with
activated bovine endothelial cells generating NO as effectors.
Furthermore, the inhibition of endogenous NO production with the
inducible NO synthase inhibitor NG-monomethyl-L-arginine
caused a complete abrogation of the apoptotic effect. Our data provide
evidence that NO-induced apoptosis in human neoplastic lymphoid cells
strictly requires activation of caspases, in particular FLICE, the most
CD95 receptor-proximal caspase. Depending on the cell line tested this
activation required or was independent of the CD95 receptor/ligand
system.
| |
INTRODUCTION |
NITRIC OXIDE (NO) is an important
pleiotropic molecule involved in neurotransmission, regulation of
vascular homeostasis, and effector functions of macrophages and
endothelial cells.1,2 NO is derived from the oxidation of
L-arginine catalyzed by constitutive or inducible NO synthase (iNOS).
Large amounts of NO produced by iNOS in macrophages and endothelial
cells after challenge with lipopolysaccharide and cytokines, eg,
interferons, interleukin-1, and tumor necrosis factor- (TNF-),
were effective against various microbes and tumor cells.3,4
Cytokine-activated macrophages and vascular endothelial cells were
reported to kill tumor cells in vitro, and NO produced by these cells
was shown to play a major role in this antitumor
cytotoxicity.5-7 The regression of murine sarcoma liver
metastasis correlated with the expression of iNOS after treatment with
a macrophage activator.8 The cytotoxic effect of NO was
found to be associated with apoptosis (programmed cell death) in
normal9-12 and tumor cells.13-15 Furthermore,
the transfection of highly metastatic murine melanoma cells with iNOS caused partial apoptosis of these cells associated with suppression of
their tumorigenicity and metastatic potential.16
Collectively, these data show that NO production represents an
important host-defense mechanism against tumor cells.
Apoptosis is an active process of cellular self destruction with unique
morphologic and molecular characteristics. It can be induced by
external stimuli or by withdrawal of survival factors such as growth
factors or cytokines. Activation of a cascade of caspases (ICE-like
proteases) seems to be a common intracellular effector
pathway to which different apoptosis signals are
connected.17 Members of the TNF receptor superfamily of
cell-surface molecules play a critical role in apoptosis of lymphoid
cells.18 CD95L (APO-1L/FasL) and TNF- induce apoptosis
by binding to their respective death domain-containing receptors, CD95
and TNFR-1.19 Binding of CD95L or agonistic antibodies to
CD95 leads to a signal transduction cascade initiated by death
receptor-associated molecules such as FADD/MORT120-22 and
FADD-associated FLICE/MACH (caspase-8), a chimeric molecule containing
a death effector domain and a proteolytic ICE-like
domain.23,24 This leads to proteolytic
processing of caspases into active proteases. FLICE is the first in a
cascade of caspases activated by CD95.25
The CD95 system is critical for growth control of T cells. Elimination
of peripheral T cells involves the induction of a suicide or fratricide
mechanism triggered by CD95 ligand/receptor
interaction.26-28 Triggering of CD95 ligand/receptor
interaction in lymphoid and nonlymphoid cells may also be caused by
cellular stress-inducing agents, eg, by cytotoxic drugs.29
Another member of the TNF superfamily, named TRAIL/APO-2L, has recently
been isolated30-32 and, like CD95L, induces rapid apoptosis
in a variety of transformed cell lines.30-32 Human
receptors for TRAIL, named death receptor-4 and 5 (DR-4, DR-5), have
recently been identified.33-35
We have previously shown that activated bovine endothelial cells (BEC)
induced apoptosis in murine metastatic lymphoma cells. This effect was
mediated to a large extent by NO.14 Here we studied the
mechanism of NO-dependent apoptotic death using well-defined human T-
and B-cell lines as well as freshly isolated leukemic lymphocytes, and
investigated in particular the involvement of death receptors in this
process. Our results suggest that production of endogenous NO by
endothelial cells or exposure to exogenous NO triggers apoptosis in
human neoplastic lymphoid cells through activation of caspases,
including FLICE, the most CD95-upstream caspase. Interestingly, NO is
able to induce activation of FLICE not only via CD95/CD95L interaction
but also independently of death receptors via direct caspase
activation.
| |
MATERIALS AND METHODS |
Cell lines.
Human T- and B-lymphoid cell lines (Jurkat APO-S and APO-R, CEM, BJAB,
BL60, and K50) were maintained 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.36 Northern and Western blot analysis 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.36
BJAB B cells were stably transfected with a dominant negative FADD (FADD-DN) deletion mutant (kindly provided by Dr V.M. Dixit, Ann Arbor,
MI), which contains only the death domain.20 Western blot
analysis showed about 30-fold higher expression of the FADD mutant
protein compared with wild-type FADD in the BJAB FADD-DN transfectants
(data not shown). CEM is a T-cell leukemic cell line. The BL60 Burkitt
lymphoma cell line expresses low amounts of CD95 and fails to express
CD95L mRNA upon treatment with different apoptotic
stimuli.36 K50 is a stable transfectant of BL60 that expresses CD95 on the cell surface. Peripheral blood mononuclear cells
(PBMC) from a patient with T-cell acute lymphoblastic leukemia (T-ALL)
were freshly isolated using density gradient with Lymphoprep (Nycomed
Pharma, Oslo, Norway), and directly after isolation the cells were used
for further experiments. To evaluate apoptotic effects of NO, all
above-mentioned human lymphoid cells were treated with glycerol
trinitrate (GTN; Merck, Darmstadt, Germany), which is known to
constitutively produce NO in the incubation medium.
Bovine endothelial cells (BEC), kindly provided by B. Liliensiek and Dr
P. Nawroth (University of Heidelberg, Heidelberg, Germany), were
maintained in culture medium under standardized conditions.37 The activation of these cells to produce NO
and coculture conditions of BEC with lymphoid cells have been described elsewhere.14 Briefly, BEC were treated with human TNF-
(Knoll AG, Ludwigshafen, Germany) at a concentration of 1 nmol/L for 16 hours. Inhibitor of inducible NO synthase (iNOS)
NG-monomethyl-L-arginine (NMMA; Calbiochem, La Jolla, CA)
was added to the culture medium at a concentration of 0.5 mmol/L
simultaneously with TNF to block NO synthesis in stimulated BEC. An
effector/target ratio of 25:1 was used.
Antibodies and inhibitors.
Purified anti-CD95 (anti-APO-1) monoclonal antibody
(MoAb)38 was used for induction of apoptosis and for
cell-surface expression studies. F(ab )2 anti-CD95
blocking antibody was used as described elsewhere.27
zVAD-fmk (Bachem Biochemica, Heidelberg, Germany) was used for
inhibition of caspase activity at a final concentration of 20 µmol/L
in culture medium. Cycloheximide (CHX; Boehringer Mannheim, Mannheim,
Germany) was used for blocking of protein synthesis at concentrations
ranging from 0.1 to 1 µg/mL.
Apoptosis and cytotoxicity assays.
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 [BioRad, Munich,
Germany]) containing 50 µg/mL propidium iodide as
described39 and analyzed by flow cytometry using a FACScan
analyzer with CELLQuest software (Becton Dickinson, Heidelberg,
Germany). Cytotoxicity was evaluated after treatment of live cells with
1 µg/mL propidium iodide (Molecular Probes Inc, Eugene, OR) for 5 minutes before FACS analysis. Ten thousand cells per sample were
analyzed by flow cytometry (FACScan; Becton Dickinson) using CELLQuest
software.
Polymerase chain reaction (PCR) and Northern blot analysis.
Total RNA was isolated from APO-S Jurkat T cells at different time
points after addition of 0.2 mmol/L GTN using guanidium isothiocyanate.
Semi-quantitative PCR analysis of the CD95L expression was performed
using CD95L-specific primers and -actin primers as
described.36 The PCR products were electrophoresed through a 1.5% agarose gel, then blotted and hybridized with a
32P-labeled human CD95L fragment27 and as a
control with a -actin probe. The same PCR conditions were used to
amplify the TRAIL/APO-2 ligand using the following specific primers:
sense primer, CCC AAT GAC GAA GAG AGT ATG AA; and antisense primer, ACC
ATT TGT TTG TCG TTC TTT GTG together with the -actin specific
primers. The expected size of the amplified TRAIL-specific product was 422 bp. Northern blot analysis using CD95-specific fragments was performed as described elsewhere.40 Briefly, mRNA was
isolated from Jurkat cells at different time points after addition of
GTN according to Vennström and Bishop.41 mRNA (24 µg per lane) was electrophoresed through a 1% agarose-formaldehyde
gel and blotted onto a nylon membrane. Hybridization was performed with the same probes as described for PCR.
Densitometric quantification.
Levels of CD95L and TRAIL mRNA expression were quantified by
densitometry of autoradiograms using the Adobe Photoshop and the SCAN
Analysis programs for Macintosh (Munich, Germany).
Each relative expression value represents the ratio between the
densities of specific mRNA transcripts to -actin transcripts.
Western blotting.
Western blot detection of FLICE was performed as
described.42 Postnuclear supernatants equivalent to 1 × 106 cells were separated by 12% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. After electrophoresis all
samples were transferred to Hybond nitrocellulose membrane (Amersham
Buchler, Braunschweig, Germany), blocked with 2% bovine serum albumin
in phosphate-buffered saline (PBS)/Tween (PBS + 0.05% Tween-20) for at
least 1 hour, washed with PBS/Tween, and incubated with the MoAb C15
(directed against the p18 subunit of FLICE) in PBS/Tween for 16 hours
at 4°C. The blots were washed with PBS/Tween and incubated with
goat anti-mouse IgG2b (1:20,000) for 1 hour at room temperature. After washing with PBS/Tween the blots were developed with the
chemiluminescence method (ECL) following the manufacturer's protocol
(Amersham Buchler, Braunschweig, Germany).
| |
RESULTS |
NO induces higher apoptotic cell death in APO-S than in APO-R Jurkat
cells.
To examine the potential role of the CD95/CD95L system in NO-dependent
cell death, two Jurkat clones with different sensitivity to CD95
agonistic antibodies were used as targets. The APO-S Jurkat cells
express CD95 on the cell surface and are very sensitive to
anti-CD95-induced kill, while the APO-R clone, lacking expression of
CD95 on the cell surface, is resistant to treatment with anti-CD95. After exposure to the NO donor glycerol trinitrate (GTN) for 24 hours
(at a concentration of 0.2 mmol/L), an increased percentage of
apoptotic cells (up to 35%) was observed in the APO-S Jurkat cells. In
contrast, the apoptotic fraction in the APO-R clone under the same
conditions was only 11% (Fig 1A). NO
induced apoptosis in a time-dependent manner. Treatment of APO-S cells
with GTN for 48 hours induced more than 50% apoptotic cells as
evidenced by DNA-release from the accumulation of subdiploid nuclei in
the treated cells (Fig 1A). The apoptotic effect of NO was elevated with increasing GTN concentrations up to 0.3 mmol/L (Fig 1B). A
cytotoxicity test performed in parallel using 0.2 mmol/L GTN showed the
same level of APO-S Jurkat cell death as the above DNA-release assay
(36% and 34% dead cells, respectively), indicating that the observed
NO-mediated cytotoxicity was caused by apoptosis. Concentrations of GTN
higher than 0.3 mmol/L led to a reduction in the percentage of
apoptotic cells (Fig 1B) and to an increase in the percentage of dead
cells as shown by cytotoxicity tests (data not shown).
Thus, APO-S Jurkat cells showed higher NO-mediated apoptosis than the
APO-R subclone. This difference was more pronounced at lower NO doses
(eg, 0.2 mmol/L, Fig 1B). The sensitivity to CD95-triggering correlated
with the sensitivity of cells to NO, and the loss of CD95 cell-surface
expression correlated with higher resistance to NO.
View larger version (13K):
[in this window]
[in a new window]
View larger version (15K):
[in this window]
[in a new window]
| Fig 1.
NO induces higher apoptotic cell death in APO-S than in
APO-R Jurkat cells. (A) Time-dependent apoptosis induced by NO donor GTN (0.2 mmol/L) in APO-S and APO-R Jurkat cells up to a time period of
48 hours. (B) Concentration-dependent apoptosis stimulated by GTN in
APO-S and APO-R Jurkat cells after 24 hours of incubation. Percentage
of apoptotic cells was measured by the DNA-release assay using
treatment with a hypotonic propidium iodide solution at 4°C
overnight in the dark followed by flow cytometric analysis. Experiments
were performed in triplicates. SD was less than 10%.
|
|
To test whether the presence of NO is needed for the whole period of
incubation to achieve a maximal apoptotic effect, APO-S Jurkat cells
were treated with 0.2 mmol/L GTN for 2, 4, 8, and 12 hours. Then the
cells were washed and fresh medium was added for the rest of the
incubation time until 24 hours. Incubation for at least 8 hours and
subsequent withdrawal of the NO donor for 16 hours resulted in the same
percentage of apoptotic cells as that observed in cells after 24 hours
of incubation in the presence of NO for the whole period (40% and 37%
apoptotic cells, respectively).
NO induces CD95L and TRAIL/APO-2L mRNA expression.
Because CD95L binding to the receptor triggers a whole cascade of
events leading to the characteristic features of apoptosis, we studied
the ability of NO to regulate CD95L mRNA expression. Semi-quantitative
PCR analysis of APO-S Jurkat cells showed NO-mediated upregulation of
CD95L mRNA. The highest increase of the ligand expression was reached
after 8 hours of incubation with GTN (Fig 2A). The NO-dependent induction of CD95L mRNA was transient and decreased upon prolonged incubation with the NO donor GTN (Fig 2A). The
PCR data were confirmed by Northern blot analysis using specific probes
for CD95L and -actin mRNA (data not shown). In contrast, NO was not
able to enhance expression of the CD95 receptor on the cell surface as
shown by FACS analysis using specific anti-CD95 antibodies (data not
shown).
View larger version (21K):
[in this window]
[in a new window]
View larger version (27K):
[in this window]
[in a new window]
| Fig 2.
Induction of CD95L (A) and TRAIL/APO-2L (B) mRNA
expression by GTN in APO-S Jurkat cells. Cells were treated for
different periods of time with 0.2 mmol/L GTN, and total RNA was
isolated. After semi-quantitative RT-PCR with specific primers for
CD95L or TRAIL/APO-2L, PCR products were run on a 1.5% agarose gel. APO-S cells stimulated with PMA (20 ng/mL; Sigma) and ionomycin (1 µmol/L; Calbiochem) for 6 hours were used as positive control. Both
pictures were scanned using the Adobe Photoshop and the Scan Analysis
programs. The ratio between the densities of the specific band to the
-actin band is shown in the histograms on the lower panel (indicated
in arbitrary units). (A) The amplified products for CD95L (447 bp) and
-actin (661 bp) are labeled. The gel was blotted and hybridized with
CD95L and -actin-specific probes. (B) The amplified products
specific for TRAIL (422 bp) and -actin (661 bp) are presented.
|
|
Furthermore, production of NO in culture medium could also stimulate
expression of another recently identified death cytokine, TRAIL/APO-2L.
Maximal level of TRAIL mRNA expression was also achieved after 8 hours
of incubation with GTN in APO-S Jurkat cells, but in contrast to CD95L
it remained stable up to 24 hours (Fig 2B).
Thus, we have shown that NO induces expression of mRNA for two death
cytokines, CD95L and TRAIL. The data suggest that CD95L and TRAIL
participate in the NO-dependent apoptotic cell death.
NO-induced apoptosis in freshly isolated human leukemic lymphocytes
requires caspase activity and involves the CD95 pathway.
NO was able to induce programmed cell death not only in lymphoid cell
lines but also in PBMC isolated from a patient with T-ALL
(Fig 3). Exposure of freshly isolated
leukemic cells to anti-CD95 MoAbs for 24 hours resulted in 79%
apoptotic cells, whereas treatment of these cells with 0.2 mmol/L and
0.3 mmol/L GTN induced 65% and 67% apoptotic cells, respectively
(spontaneous apoptosis was 38%). Pretreatment with the
F(ab)2 anti-CD95 blocking antibody (that
specifically blocks the interaction of CD95/CD95L) significantly
inhibited the observed NO-dependent apoptotic effect (Fig 3).
View larger version (17K):
[in this window]
[in a new window]
| Fig 3.
NO-induced apoptosis in freshly isolated human leukemic
cells requires caspase activity and involves the CD95 pathway. PBMC from a patient with T-ALL isolated by Lymphoprep separation were plated
at a density of 5 × 105 cells/mL and incubated for 24 hours with anti-CD95 (10 µg/mL), or broad-spectrum caspase-specific
inhibitor zVAD-fmk (zVAD; 20 µmol/L) or with
F(ab)2 anti-CD95 blocking antibody (1 µg/mL) and/or GTN at the concentrations indicated. Percentage of
apoptotic cells was measured by the DNA-release assay.
|
|
It is well documented that CD95- and TRAIL-signaling leads to
activation of caspases.43,44 Because the zVAD-fmk
broad-spectrum caspase inhibitor can completely block both signaling
pathways,43,44 we examined whether it could abrogate
NO-dependent apoptosis in human leukemic lymphocytes. It was shown that
this inhibitor completely blocked apoptosis triggered by NO as shown in
Fig 3. Thus, NO-mediated apoptosis in freshly isolated human leukemic
cells strictly required caspase activity and involved the CD95 system.
NO-dependent apoptosis is blocked by a broad-spectrum caspase
inhibitor and induces activation of FLICE.
We next examined whether the caspase inhibitor zVAD-fmk could abrogate
NO-induced cell death not only in freshly isolated human leukemic
lymphocytes, but also in different neoplastic lymphoid cell lines. As
shown in Fig 4, pretreatment with this
caspase inhibitor caused a complete abrogation of NO-mediated cell
death in these cells. Interestingly, zVAD-fmk inhibited also the low level of apoptosis induced by NO in APO-R Jurkat cells. In conclusion, we showed that NO-mediated apoptosis in human T- and B-cell lines strictly required caspase activity.
View larger version (22K):
[in this window]
[in a new window]
| Fig 4.
Blocking of NO-induced apoptosis in different neoplastic
lymphoid cells by the caspase inhibitor zVAD-fmk. Cells were plated at
a density of 3 × 105 cells/mL and incubated with 0.2 mmol/L GTN (APO-S and APO-R) or 0.25 mmol/L GTN (CEM, BJAB, BL60, and
K50) and/or with 20 µmol/L zVAD-fmk (zVAD). Percentage of
apoptotic cells was measured by the DNA-release assay. Data shown are
representative of three individual experiments. SD was less than 10%.
( ), Control; ( ), +GTN; ( ), +GTN +zVAD; (), +zVAD.
|
|
Because FLICE is the first in a cascade of caspases activated by
CD95,25 we tested whether NO would be able to induce FLICE cleavage into active subunits. It has been previously shown that FLICE
is activated upon triggering of CD95 leading to the release of the
active subunits p18 and p10 into the cytosol.25,42
Treatment of the APO-S cells with the NO-donor GTN for longer than 8 hours (up to 24 hours) resulted in accumulation of the active FLICE subunit p18 (Fig 5A). Moreover,
NO-dependent activation of FLICE could also be observed in the freshly
isolated leukemic cells after incubation with the NO donor GTN (data
not shown). Furthermore, increasing concentrations of the protein
synthesis inhibitor cycloheximide (CHX) could almost completely inhibit
the NO-induced activation of FLICE when APO-S cells were treated for 10 hours with GTN (Fig 5B). This correlated with a significant reduction
in the number of apoptotic cells (31.4% apoptotic cells in the
presence of GTN v 4.1% in the presence of GTN and 1.0 µg/mL
CHX for 24 hours). Therefore, protein synthesis is required for the
NO-dependent activation of FLICE during apoptosis in Jurkat cells.
View larger version (46K):
[in this window]
[in a new window]
| Fig 5.
NO-induced activation of FLICE in lymphoid cell lines.
(A) Time-dependent effect of NO in APO-S and BL60 cells. (B) Inhibition of FLICE activation upon treatment with protein synthesis inhibitor cycloheximide (CHX) in APO-S but not in BL60 cells. Cells were treated
with 0.2 mmol/L GTN for the time period indicated and/or with
CHX at different concentrations. Western blot analysis with anti-FLICE
MoAb was performed. The full-length FLICE and the p18 active cleavage
product are presented.
|
|
NO was also able to induce apoptosis and FLICE activation in BL60 and
K50 cells (Figs 4 and 5A). Both cells failed to express CD95L mRNA and
to stimulate expression of TRAIL/APO-2L mRNA upon treatment with GTN
(data not shown). Significant accumulation of the active FLICE subunit
p18 could be detected as early as 8 hours after exposure to GTN in both
cell lines (Fig 5A and data not shown). Interestingly, in contrast to
Jurkat cells, pretreatment of BL60 cells with CHX did not inhibit
NO-dependent activation of FLICE (Fig 5B). Our results provide evidence
that NO-mediated FLICE activation in Jurkat cells requires protein
synthesis consistent with upregulation of CD95L and TRAIL and the
involvement of the CD95 system. In contrast, the mechanism of
NO-mediated cell death in BL60 cells is independent of death ligand
production. Therefore, this apoptosis could not be blocked by CHX.
CD95-resistant lymphoid cells can be sensitive to NO-induced kill.
Because different human lymphoid cell lines differ in sensitivity
toward induction of apoptosis by CD95L and TRAIL,43,45 we
compared the sensitivity of different cells to anti-CD95 MoAb and to NO
treatment. Lymphoid cell lines which were resistant to CD95-induced
cell death (APO-R Jurkat cells and BJAB FADD-DN transfectants
expressing a dominant negative FADD mutant) were found to also be
resistant to NO-induced apoptosis, in contrast to the parental cells,
which were sensitive to both stimuli (Table 1). In different cell lines NO could induce apoptosis either via CD95
or it could be independent of death receptors. The CD95-dependent cell
lines APO-S Jurkat, CEM, T-ALL, and BJAB, which undergo cell death upon
treatment with anti-CD95, were also NO-sensitive. The CD95-independent
BL60 cell line, which is resistant to anti-CD95 treatment and which is
unable to produce CD95L, was nevertheless NO sensitive (Table 1).
View this table:
[in this window]
[in a new window]
|
Table 1.
Sensitivity of Different Human Lymphoid Cell Lines
Toward Apoptotic Cell Death Mediated by Anti-CD95 MoAb and NO
|
|
These data suggest that NO-mediated active cell death involves
CD95-dependent as well as independent apoptotic pathways.
NO produced by activated BEC induces apoptosis in CD95-sensitive B-
and T-cell lines.
Previously we have shown that TNF--activated BEC produced NO which
could induce apoptosis in murine lymphoma cells.14 This in
vitro experimental system is based on the direct contact of tumor
target cells with BEC effectors and thus resembles pathophysiological conditions in vivo. When we co-incubated APO-S or BJAB human lymphoid cells for 8 hours with activated BEC we detected significant apoptosis (Table 2). A specific inhibitor of iNOS,
NMMA, could decrease the apoptosis observed in malignant lymphoid cells
down to the background levels seen with nonactivated BEC. Therefore,
the observed apoptosis is completely caused by NO secretion by BEC and
not by the secretion of other cytotoxic molecules. It is also obvious from this experimental model that APO-R Jurkat cells were less sensitive to NO than the parental APO-S clone. Similarly, BJAB FADD-DN
cells showed protection from cell death initiated by NO, in contrast to
the parental nontransfected clone BJAB, which showed sensitivity to NO
(Table 2).
The apoptotic effect mediated by activated BEC in the CD95-sensitive
cells could be completely blocked by the caspase inhibitor zVAD-fmk
(Table 2). These data are consistent with the results presented in Fig
4 using exogenous NO. Treatment of the APO-S Jurkat T cells with the
protein synthesis inhibitor CHX or with the F(ab)2
anti-CD95 blocking antibody, which prevents binding of CD95L to the
receptor, could partially (approximately 40% in both cases) inhibit
NO-mediated active cell death (Table 2). The data indicate that protein
synthesis is required and that binding of CD95L to the receptor is, at
least in part, involved in the NO-induced apoptosis in these cells.
When BJAB cells were tested in the same way, GTN-induced apoptosis
could be blocked completely by zVAD-fmk or by CHX and only partially
(approximately 58%) by F(ab)2 anti-CD95.
Therefore, using a physiological test system in vitro blood vessel
endothelium interacting with lymphoid cells we could show that
endogenously produced NO induced apoptotic cell death in human
neoplastic lymphoid cells. This involved CD95/CD95L interaction and was
entirely dependent on caspase activation.
| |
DISCUSSION |
Apart from its many regulatory and effector functions NO, produced by
activated macrophages and endothelial cells, is an important cytotoxic
molecule responsible for lysis of tumor cells.6,7,46,47 Recently it was found that endogenously produced or exogenously added
NO could induce cytotoxicity through apoptosis in murine thymocytes,10 peritoneal macrophages,9
splenocytes,11 and tumor cells.13-15 However,
the underlying mechanism of the NO-induced apoptotic effect has not yet
been clearly elucidated.
In the present study we examined the involvement of the CD95/CD95L
system and signal transduction pathway in NO-induced apoptosis in human
neoplastic lymphoid cells. To address this question we compared
CD95-sensitive Jurkat cells and their CD95-resistant subclone,
characterized by lack of the CD95 receptor on the cell surface. We
found that APO-S cells were more susceptible to exogenous NO-induced
apoptosis than APO-R cells. A significant degree of cell death was
observed after exposure of APO-S cells to 0.3 mmol/L of the NO donor
GTN for 24 hours. Lower doses of GTN retarded induction of apoptosis.
Treatment of the cells for 8 hours with GTN and its subsequent
withdrawal for 16 hours was enough to achieve maximal apoptosis. It
thus seems that NO is required to initiate an apoptotic program and
then it is not needed thereafter.
Interestingly, NO-dependent apoptosis could be blocked in APO-S and
BJAB cells by the protein synthesis inhibitor CHX. This finding
suggests that protein synthesis is required (at least in part) and
correlates with the fact that NO has to be present in the medium for 8 hours to induce a maximal apoptotic effect.
These data are also in agreement with the observation that NO treatment
induces gene expression of CD95L and TRAIL/APO-2L and that the highest
increase in the mRNA expression of CD95L and TRAIL is reached after
treatment of the APO-S cells for 8 hours with the NO donor GTN.
Incubation of APO-S cells with GTN resulted in a strong increase in the
expression of CD95L and TRAIL mRNA. Although CD95L mRNA expression was
transient and reduced after prolonged (up to 24 hours) incubation of
the cells with NO, TRAIL expression was stable for the same time
period. Unlike CD95L, whose transcripts are predominantly restricted to
stimulated T cells and sites of immune privilege, TRAIL expression is
detected in many normal human tissues, suggesting that TRAIL should not be cytotoxic to most tissues in vivo.30-32 Jeremias et
al45 proposed that TRAIL-mediated apoptosis may play a role
in antileukemic growth control in APO-S Jurkat cells, although TRAIL
had only 25% of the activity of CD95L in inducing apoptosis. Such
TRAIL-mediated apoptosis could account to some extent for the
NO-induced apoptosis in lymphoid cells, and in particular in the
CD95-independent cell death observed in the APO-R Jurkat clone.
Because CD95 receptor expression remained unaltered after NO treatment,
it seems that NO triggers the death receptor systems by regulating the
expression of ligands involved in apoptosis like CD95L and
TRAIL/APO-2L. Signaling through CD95 involves the death
domain-associated molecule FADD20-22 and the upstream
caspase FLICE/MACH.23,24 Since NO failed to induce
significant apoptosis in the CD95-resistant BJAB cells expressing a
dominant negative FADD mutant and since the NO-induced apoptotic cell
death could be partially inhibited by a blocking antibody that
specifically inhibits the interaction of the CD95L with its receptor,
we conclude that CD95-mediated signaling can be involved in
NO-dependent death, at least to some extent. It is known that the CD95
system mediates activation-induced apoptotic cell death in T
cells26-28 as well as apoptosis induced by
drugs.29 It also plays a critical role in apoptosis induced
by viral proteins.40 Based on our observations, NO has to
be added to the list of inducers of apoptosis in which CD95 pathway is
involved.
Activation of the cascade of caspases seems to be a common
intracellular effector pathway to which different apoptosis signals are
connected.17 Caspases have also been implicated in
programmed cell death triggered by CD95L and
TRAIL/APO-2L.43,44 The tripeptide zVAD-fmk, a
broad-spectrum caspase inhibitor, is known to inhibit caspase
cleavage.48 We here showed complete inhibition of
NO-mediated apoptotic cell death by zVAD-fmk in different human
lymphoid cell lines and in freshly isolated leukemic cells. It has
recently been shown that proteolytic activity of caspases could be
stimulated also in HeLa cells and in macrophages upon treatment with NO
donors49 (Boggs S, personal communication, May
1997). Our data also provide evidence that NO induces
FLICE activation in human neoplastic lymphoid cells. FLICE is the most
upstream caspase in the CD95 signaling pathway and is activated upon
triggering of CD95 leading to the release of the active subunits p18
and p10 into the cytosol, where they can activate other
caspases.25 NO induced a clear accumulation of the p18
subunit of FLICE. Because the protein synthesis inhibitor CHX could
almost completely abrogate NO-mediated activation of FLICE and because
the kinetics of p18 accumulation correlated with the kinetics of CD95L
and TRAIL mRNA expression, we conclude that NO-mediated activation of
FLICE involves synthesis of CD95L and/or TRAIL in APO-S Jurkat
cells. It seems that NO leads to activation of caspases through
activation of CD95/CD95L and probably TRAIL death receptor systems.
Data on NO-induced apoptosis in different human malignant lymphoid cell
lines were also extended to PBMC from a patient with T-ALL. Freshly
isolated leukemic cells were sensitive both to anti-CD95 MoAb and to
GTN treatment. Addition of the F(ab)2 anti-CD95 blocking antibody decreased significantly the level of apoptosis triggered by NO. Moreover, NO donor GTN caused FLICE activation, and
caspase inhibitor zVAD-fmk was able to block NO-mediated apoptosis. This suggests that caspase activation is strictly required and CD95
pathway is also involved in the NO-dependent death of ex vivo isolated
human leukemic lymphocytes.
Interestingly, some lymphoid cells resistant to CD95-mediated apoptosis
(eg, BL60) were nevertheless sensitive to treatment with NO. Caspases
were shown to also mediate apoptosis in this case. It seems that NO can
use an alternative pathway of FLICE activation which does not require
CD95L or TRAIL/APO-2L protein synthesis because pretreatment of BL60
and K50 cells with CHX did not block the NO-induced FLICE activation.
Moreover, both cell lines failed to induce expression of CD95L and
TRAIL/APO-2L mRNA after GTN treatment. We suggest that this death
receptor-independent pathway of FLICE activation might involve
mitochondria that have recently been implicated in the apoptotic
process and in the activation of caspases.50 Significant
reduction of mitochondrial membrane potential through opening of
permeability transition pores, resulting in hyperproduction of reactive
oxygen species and caspase activation, have been postulated as a
crucial step in apoptosis.50,51
Mitochondrial alterations might be involved in
NO-mediated activation of caspases as it has been shown for the
cytotoxic drug betulinic acid, which may trigger CD95-independent
apoptosis via activation of caspases.52 To our knowledge NO
is the only apoptotic stimulus known thus far capable of inducing both
CD95-dependent and independent activation of ICE proteases.
Our findings on apoptosis induced by NO donor GTN could be reproduced
using an in vitro experimental system mimicking a relevant in vivo
situation. Our model was based on coculture of lymphoid target cells
with TNF--activated blood vessel endothelial cells as effector
cells secreting NO. A similar system for murine and human lymphoma
cells has been established by us and other groups.6,7,14,15 A significant level of lymphoid cell apoptosis was observed which was
abrogated completely in the presence of the iNOS inhibitor NMMA. The
apoptotic fraction of the human lymphoid cell lines tested corresponded
with the cytotoxicity induced in K562 cells using similar coculture
conditions assessed by 51Cr release.15
Additionally, the caspase inhibitor zVAD-fmk was found to block the
observed programmed cell death completely. The protein synthesis
inhibitor CHX as well as a blocking antibody that inhibits binding of
CD95L to the receptor could partially abrogate this apoptotic effect.
Therefore, we conclude that apoptosis observed in our model system was
entirely caused by endogenously produced NO and that the activity of
caspases was strictly required. More studies will be necessary to
determine whether the mechanism of NO-mediated apoptosis is general or
restricted to certain cell types.
Taken together, our results show that the production of endogenous NO
by activated endothelial cells or exposure to exogenous NO exerts
strong apoptotic effects in human neoplastic lymphoid cells. The
observed cell death strictly requires caspase activity, in particular
FLICE, the most CD95 receptor-proximal caspase, and involves activation
of the CD95/CD95L system. Therefore, the cytotoxic effector molecule NO
triggers apoptosis using known signaling pathways and can thereby play
a critical role in host antitumor responses. Moreover, NO can induce
caspase activation and apoptosis using alternative pathways that are
independent of death receptor systems. This insight into the mechanism
of NO-mediated cell death may have clinical relevance because similar data were also obtained with freshly isolated human leukemic
lymphocytes. Thus, stimulation of the endogenous NO production could be
of importance for effective treatment of leukemia patients and NO donors might be promising new agents in therapeutic protocols.
| |
FOOTNOTES |
Submitted September 16, 1997;
accepted January 20, 1998.
Supported in part by the Deutsche Forschungsgemeinschaft (K.C. and
M.E.P.) and by Mildred Sheel Stiftung (V.U. and M.R.).
Address reprint requests to Victor Umansky, PhD, Division of Cellular
Immunology, Tumor Immunology Program, German Cancer Research Center,
D-69120 Heidelberg, Germany.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank B. Liliensiek and Dr P. Nawroth for providing the BEC, Dr V.M.
Dixit for providing the dominant negative FADD mutant, and Dr K. Khazaie for critical reading of the manuscript.
| |
REFERENCES |
1.
Lowenstein CJ,
Glatt CS,
Bredt DS,
Snyder SH:
Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme.
Proc Natl Acad Sci USA
89:6711,
1992[Abstract/Free Full Text]
2.
Nathan CF,
Xie Q-W:
Nitric oxide synthases: Rolles, tolls, and controls.
Cell
78:915,
1994[Medline]
[Order article via Infotrieve]
3.
Moncada S,
Palmer RJ,
Higgs EA:
Nitric oxide: Physiology, pathophysiology and pharmacology.
Pharmacol Rev
43:109,
1991[Medline]
[Order article via Infotrieve]
4.
Nathan CF,
Hibbs JB:
Role of nitric oxide synthesis in macrophage antimicrobial activity.
Curr Opin Immunol
3:65,
1991[Medline]
[Order article via Infotrieve]
5.
Stuehr DJ,
Nathan CF:
Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells.
J Exp Med
169:1543,
1989[Abstract/Free Full Text]
6.
Li L,
Nicolson GL,
Fidler IJ:
Direct in vitro lysis of metastatic tumor cells by cytokine-activated murine vascular endothelial cells.
Cancer Res
51:245,
1991[Abstract/Free Full Text]
7.
Li L,
Kilbourn RG,
Adams J,
Fidler IJ:
Role of nitric oxide in lysis of tumor cells by cytokine-activated endothelial cells.
Cancer Res
51:2531,
1991[Abstract/Free Full Text]
8.
Xie K,
Huang S,
Dong Z,
Gutman M,
Fidler J:
Direct correlation between expression of endogenous inducible nitric oxide synthase and regression of M5076 reticulum cell sarcoma hepatic metastases in mice treated with liposomes containing lipopeptide CGP 31362.
Cancer Res
55:3123,
1995[Abstract/Free Full Text]
9.
Albina JE,
Cui S,
Mateo RB,
Reichner JS:
Nitric oxide-mediated apoptosis in murine peritoneal macrophages.
J Immunol
150:5080,
1993[Abstract]
10.
Fehsel K,
Kr ncke K-D,
Meyer KL,
Huber H,
Wahn V,
Kolb-Bachofen V:
Nitric oxide induces apoptosis in mouse thymocytes.
J Immunol
155:2858,
1995[Abstract]
11.
Cui S,
Reichner JS,
Albina JE:
Nitric oxide (NO) induces apoptosis in ConA-stimulated splenocytes.
FASEB J
8:4470,
1994
12.
Stangel M,
Zettl UK,
Mix E,
Zielasek J,
Toyka KV,
Hartung HP,
Gold R:
H2O2 and nitric oxide-mediated oxidative stress induce apoptosis in rat skeletal muscle myoblasts.
J Neuropathol Exp Neurol
55:36,
1996[Medline]
[Order article via Infotrieve]
13.
Xie K,
Huang S,
Dong Z,
Fidler J:
Cytokine-induced apoptosis in transformed murine fibroblasts involves synthesis of endogenous nitric oxide.
Int J Oncol
3:1043,
1993
14.
Umansky V,
Bucur M,
Schirrmacher V,
Rocha M:
Activated endothelial cells induce apoptosis in lymphoma cells: Role of nitric oxide.
Int J Oncol
10:465,
1997
15.
Geng Y-J,
Hellstrand K,
Wennmalm A,
Hansson GK:
Apoptotic death of human leukemic cells induced by vascular cells expressing nitric oxide synthase in response to  -interferon and tumor necrosis factor- .
Cancer Res
56:866,
1996[Abstract/Free Full Text]
16.
Xie K,
Huang S,
Dong Z,
Juang SH,
Gutman M,
Xie QW,
Nathan C,
Fidler IJ:
Transfection with the inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells.
J Exp Med
181:1333,
1995[Abstract/Free Full Text]
17.
Henkart PA:
ICE family proteases: Mediators of all apoptotic cell death?
Immunity
4:195,
1996[Medline]
[Order article via Infotrieve]
18.
Baker SJ,
Reddy EP:
Transducers of life and death: TNF receptor superfamily and associated proteins.
Oncogene
12:1,
1996[Medline]
[Order article via Infotrieve]
19.
Nagata S:
Apoptosis by death factor.
Cell
88:355,
1997[Medline]
[Order article via Infotrieve]
20.
Chinnaiyan AM,
Tepper CG,
Seldin MF,
O'Rourke K,
Kischkel FC,
Hellbardt S,
Krammer PH,
Peter ME,
Dixit VM:
FADD/MORT1 is a common mediator of CD95(Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis.
J Biol Chem
271:4961,
1996[Abstract/Free Full Text]
21.
Chinnaiyan AM,
O'Rourke K,
Tewari M,
Dixit VM:
FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis.
Cell
81:505,
1995[Medline]
[Order article via Infotrieve]
22.
Boldin MP,
Varfolomeev EE,
Pancer Z,
Mett IL,
Camonis JH,
Wallach D:
A novel protein that interacts with the death domain of Fas/APO-1 contains a sequence motif related to the death domain.
J Biol Chem
270:7795,
1995[Abstract/Free Full Text]
23.
Muzio M,
Chinnaiyan AM,
Kischkel FC,
O'Rourke K,
Shevchenko A,
Ni J,
Scaffidi C,
Bretz JD,
Zhang M,
Gentz R,
Mann M,
Krammer PH,
Peter ME,
Dixit VM:
FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex.
Cell
85:817,
1996[Medline]
[Order article via Infotrieve]
24.
Boldin MP,
Goncharov TM,
Goltsev YV,
Wallach D:
Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death.
Cell
85:803,
1996[Medline]
[Order article via Infotrieve]
25.
Medema JP,
Scaffidi C,
Kischkel FC,
Shevchenko A,
Mann M,
Krammer PH,
Peter ME:
FLICE is activated by association with the CD95 death-inducing signaling complex (DISC).
EMBO J
16:2794,
1997[Medline]
[Order article via Infotrieve]
26.
Brunner T,
Mogil RJ,
LaFace D,
Yoo NJ,
Mahboudi A,
Echeverri F,
Martin SJ,
Force WR,
Lynch DH,
Ware CF,
Green DR:
Cell autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas.
Nature
373:441,
1995[Medline]
[Order article via Infotrieve]
27.
Dhein J,
Walczak H,
B umler C,
Debatin K-M,
Krammer PH:
Autocrine T-cell suicide mediated by APO-1(Fas/CD95).
Nature
373:438,
1995[Medline]
[Order article via Infotrieve]
28.
Ju ST,
Panka DJ,
Cui H,
Ettinger R,
El-Khatib M,
Sherr DH,
Stanger BZ,
Marshak-Rothstein A:
Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation.
Nature
373:444,
1995[Medline]
[Order article via Infotrieve]
29.
Friesen C,
Herr I,
Krammer PH,
Debatin K-M:
Involvement of the CD95(APO-1/Fas) receptor/ligand system in drug-induced apoptosis in leukemia cells.
Nature Med
2:574,
1996[Medline]
[Order article via Infotrieve]
30.
Wiley SR,
Schooley K,
Smolak PJ,
Din WS,
Huang C-P,
Nicholl JK,
Sutherland GR,
Smith TD,
Rauch C,
Smith CA,
Goodwin RG:
Identification and characterization of a new member of the TNF family that induces apoptosis.
Immunity
3:673,
1995[Medline]
[Order article via Infotrieve]
31.
Pitti RM,
Marsters SA,
Ruppert S,
Donahue CJ,
Moore A,
Ashkenazi A:
Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family.
J Biol Chem
271:12687,
1996[Abstract/Free Full Text]
32.
Marsters SA,
Pitti RM,
Donahue CJ,
Ruppert S,
Bauer KD,
Ashkenazi A:
Activation of apoptosis by Apo-2 ligand is independent of FADD but blocked by CrmA.
Curr Biol
6:750,
1996[Medline]
[Order article via Infotrieve]
33.
Pan G,
O'Rourke K,
Chinnaiyan AM,
Gentz R,
Ebner R,
Ni J,
Dixit VM:
The receptor for the cytotoxic ligand TRAIL.
Science
276:111,
1997[Abstract/Free Full Text]
|
Abstract
Introduction
Abstract