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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 2015-2023
IMMUNOBIOLOGY
Tumor-induced apoptosis of T lymphocytes: elucidation of
intracellular apoptotic events
Brian R. Gastman,
Daniel E. Johnson,
Theresa L. Whiteside, and
Hannah Rabinowich
From the Departments of Pathology, Pharmacology, Medicine, and
Otolaryngology, University of Pittsburgh School of Medicine and
University of Pittsburgh Cancer Institute, Pittsburgh, PA.
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Abstract |
Our recent studies suggest that human squamous cell carcinoma of the
head and neck (SCCHN) is capable of activating an intrinsic mechanism
of programmed-cell death in interacting lymphocytes in situ and in
vitro. The current study used Jurkat T-cell line as a model to
investigate intracellular apoptotic events in T cells interacting with
SCCHN. Apoptosis induced in T lymphocytes by tumor cells was in part
Fas-mediated, since it was partially, but significantly, inhibited in
the presence of anti-Fas ligand Ab or in Fas-resistant Jurkat cells.
The synthetic caspase inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) and
N-benzyloxycarbonyl-Asp-glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK), effectively blocked apoptosis of Jurkat cells co-incubated with SCCHN
cell lines, suggesting the involvement of caspases in tumor-induced apoptosis of lymphocytes. Overexpression of CrmA, an inhibitor of
caspase-1 and caspase-8, partially inhibited tumor-induced T-cell
death. Caspase-8 and caspase-3 were identified as effector molecules in
the execution of tumor-induced T-cell death, since the proform enzymes
were processed into active subunits during co-incubation of T cells
with tumor cells. Furthermore, co-incubation with tumor cells resulted
in cleavage of poly(ADP-ribose) polymerase (PARP), a common caspase-3
substrate, and in cleavage of TcR- chain, shown by us to be a T-cell
specific caspase-3 substrate. Overexpression of Bcl-2 did not provide
protection of T cells from SCCHN-induced DNA degradation. Instead, the
Bcl-2 protein was cleaved in the target T cells during their
co-incubation with tumor cells. These findings demonstrate that tumor
cells can trigger in T lymphocytes caspase-dependent apoptotic
cascades, which are not effectively protected by Bcl-2.
(Blood. 2000;95:2015-2023)
© 2000 by The American Society of Hematology.
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Introduction |
Mechanisms of tumor escape from immune destruction
include evasive strategies to avoid immune recognition,1 as
well as modulation and suppression of immune cell
functions.2-4 A previously unknown active mechanism of
tumor-induced immunosuppression has been suggested by the
identification of Fas ligand (FasL) expression on tumor cells.
Expression of FasL has recently been reported in solid tumors of
nonhematopoietic origin, including colon,1,2 hepatocellular
carcinoma,3 melanoma,4
astrocytoma,5,6 lung carcinoma,7 ovarian
carcinoma,8 esophageal carcinoma,9 glioblastoma,10 renal cell carcinoma,11 and
head and neck carcinoma.12 However, considerable
heterogeneity in surface expression of FasL was detected even within a
particular lineage of tumor.4,5,8,13,14 Moreover,
intracellular apoptotic events, which may help identify the apoptotic
pathway initiated, have not yet been elucidated in T cells interacting
with solid tumors.
Fas represents one member of a family of death receptors that might be
involved in death-signaling cascade induced in T cells by direct
engagement of death receptors on the lymphocytes with death ligands
expressed by the tumor cells. Activation-induced cell death (AICD)
represents a mechanism that involves engagement of death receptors on
lymphocytes by death ligands on the same lymphocyte (autocrine or
suicide) or on adjacent lymphocytes (paracrine or
fratricide).15,16 AICD is dependent on prior up-regulation in expression of Fas/FasL on T cells.15,17 Few reports have suggested that AICD may also be mediated by the up-regulation of the
death ligand, tumor necrosis factor-related apoptosis-inducing factor
(TRAIL), on activated lymphocytes.18,19 In addition to
AICD, such mechanisms of inducible sensitivity to Fas-mediated killing
have recently been suggested for stress-induced apoptosis in T cells
treated with DNA-damaging agents, including radiation or
chemotherapeutic agents.20,21 Death receptor-independent mechanisms of lymphocyte apoptosis may also be induced by inhibitory factors in the tumor microenvironment, such as TGF- or interleukin (IL)-10,22 alterations in redox status, or changes in
second messengers, such as ceramide or intracellular calcium. Since
various signaling cascades might be involved in tumor-induced apoptosis of lymphocytes, it is important to identify those common intracellular events that may serve as potential targets for therapeutic intervention.
Currently, it is not precisely clear what defines the point of no
return in the intracellular signaling cascade, the point at which the
cell is committed to apoptotic death. Blocking of early events in tumor-induced apoptosis of lymphocytes requires the
elucidation of the death pathways and effector molecules involved. Recent evidence suggests that distinct stimuli of apoptosis result in
activation of specific effector molecules. Thus, different sets of
caspases appear to participate in execution of apoptosis triggered by
death receptors, withdrawal of growth factors, or irradiation.23-25 Consistent with this notion, various
pathways of apoptosis are also differentially regulated by inhibitors
of apoptosis, including Bcl-2 family members cytokine
response-modifier gene A (CrmA), cellular FLICE-inhibitory protein or
inhibitor of apoptosis, which target different
caspases.26-28 The need to identify specific caspases that
might serve as potential targets for inhibition of apoptosis is further
indicated by studies performed in cells deficient in
caspase-3.29 Although caspase-3 has been shown to be one of
the major activated caspases present in apoptotic cells,30
its requirement in apoptosis is tissue-specific and can even be
stimulus-specific within the same cell type.29
The intracellular effector molecules involved in execution of
tumor-induced death of lymphocytes have not yet been elucidated. This
study is the first to identify caspases involved in tumor-induced death
of T cells and to assess the capability of inhibitors of apoptosis to
protect from tumor-induced apoptosis.
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Materials and methods |
Reagents
Agonistic anti-Fas antibody (Ab) (CH-11, immunoglobulin [Ig]M) was
purchased from Upstate Biotechnology (Lake Placid, NY); anti-Bcl-2
monoclonal antibody (mAb) (Bcl-2 100) from Santa Cruz Biotechnology
(Santa Cruz, CA); anti-caspase-3 mAb (CPP32, clone 19) from
Transduction Laboratories (Lexington, KY); and anti-caspase-8 mAb
(B9-2) from PharMingen (San Diego, CA). Anti-poly(adenosine diphosphate-ribose) polymerase (anti-PARP) mAb (C2.10) and
fluoromethyl ketone (FMK)-conjugated inhibitors of apoptosis,
including Z-VAD-FMK, Z-DEVD-FMK, and a control peptide
N-benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone (Z-FA-FMK), were
purchased from Enzyme Systems (Livermore, CA). Anti- mAb (6B10.2)
was purchased from Santa Cruz Biotechnology. Apoptosis terminal
deoxynucleotidyl transferase-mediated deoxyuridine triphosphate
nick-end labeling (TUNEL) kits were purchased from Boehringer-Mannheim (Indianapolis, IN); DiOC6 from Molecular Probes (Eugene, OR); and the etoposide VP-16 from Sigma (St Louis,
MO). Anti- 64 mAb (A9) was a generous
gift from Dr T. E. Carey (University of Michigan Cancer Center, Ann
Arbor). Goat anti-mouse Ig G (GAMIg)- conjugated magnetic beads were
purchased from PerSeptive Diagnostics (Cambridge, MA).
Cell lines
The squamous cell carcinoma of the head and neck (SCCHN) cell lines
PCI-13 and PCI-52 were established in our laboratory, as previously
described.31 All the cell lines used, including OSC-19,
SCC-68, and SCC-74, were routinely tested and confirmed to be negative
for the presence of mycoplasma (Gen Probe, San Diego, CA).
Fas-resistant Jurkat cells were obtained by multiple cycles of
treatment with agonistic anti-Fas Ab (CH-11, 200 ng/mL), followed by selection for Fas-positive cells by
fluorescence-activated cell sorter (FACS) sorting.
To generate stable cell lines expressing epitope-tagged proteins, the
CMV/Neo/CrmA-KT3 or CMV/Neo/Bcl-2-KT3, expression constructs prepared
as previously described,32 were introduced into Jurkat cells by electroporation (250V, 960 microfarads). As a control, the
CMV/Neo vector alone was electroporated into Jurkat cells. Following
electroporation, transfected cells were selected in media containing
0.5 mg/mL G418. After 2 weeks of selection, independent clones were
isolated by limiting dilution. Expression of the CrmA/KT3 or Bcl-2/KT3
proteins in independent clonal cell lines was confirmed by Western
blotting of whole cell lysates using anti-KT3 mAb.
Induction of apoptosis
To induce apoptosis or apoptosis-related changes in lymphocytes,
SCCHN cell lines were coincubated with Fas-sensitive or Fas-resistant Jurkat cells for 16 to 24 hours at a tumor-to-lymphocyte cell ratio
ranging from 20:1 to 80:1. To assess processing of intracellular proteins, including Bcl-2, caspase-3, and PARP, in the target cells,
Jurkat cells were negatively selected by removal of SCCHN cells by
epithelial-specific 64 mAb (A9) and
GAMIg-conjugated magnetic beads. Cocultures of SCCHN and Jurkat cells
were incubated with A9 mAb at 50 µg/107 cells/mL on ice
for 1 hour. The cells were washed 3 times in cold medium and subjected
to 2 cycles of incubation with and separation from GAMIg-coated
magnetic beads (30:1 beads-to-cell ratio). As assessed by flow
cytometry of cells stained with anti-64
integrin mAb, all tumor cells were removed by this procedure. Apoptotic
cells triggered by agonistic anti-Fas Ab (CH-11, 200 ng/mL) or VP-16
(20 µmol/L) served as controls.
Apoptosis detection assays
DNA fragmentation assays included the JAM assay, in
which loss of [3H]TdR-labeled DNA was measured, and the
terminal deoxynucleotidyl transferase-mediated
deoxyuridine-triphosphate (dUTP) nick-end labeling (TUNEL) assay, in
which the presence of breaks in DNA was assessed.8 DNA
labeling of Jurkat target cells for the JAM assay was performed by
incubation of the cells in the presence of 5 µCi/mL
[3H]TdR for 18 to 24 hours at 37°C. Tumor cells were
cocultured with [3H]TdR-labeled target cells for 16 hours
at 37°C at a tumor-to-lymphocyte cell ratio ranging from 20:1 to
80:1. At the end of the coincubation period, the cells were harvested
(Mach IIM, TOMTEC, Walloch, Gaithersburg, MD) onto glass fiber filters.
The radioactivity of unfragmented DNA retained on the glass fiber
filters was measured by liquid scintillation counting. Specific DNA
fragmentation was calculated according to the following formula:
percentage of specific DNA fragmentation = 100 × (S-E)/S,
where S = retained DNA in the absence of effector cells
(spontaneous), and E = experimentally retained DNA in the presence of
tumor (effector) cells.
To identify fragmented DNA in Jurkat cells, a flow cytometry-based
TUNEL assay was performed (Boehringer-Mannheim).33 The cells (106/sample) were washed in phosphate-buffered saline
(PBS) and fixed in 2% paraformaldehyde for 30 minutes at room
temperature. After fixation, the cells were washed twice in PBS
containing .01% bovine serum albumin, and resuspended in TUNEL
reaction mixture containing fluorescein dUTP and terminal
deoxynucleotidyl transferase (TdT). Control cells were resuspended in
TUNEL reaction mixture containing fluorescein dUTP without TdT.
Fluorescein labels incorporated in DNA strand breaks were detected by
flow cytometry.
To identify fragmented DNA in CD3+ lymphocytes after
coincubation with tumor cells, the recovered cells were first stained with either phycoerythrin (PE)-labeled anti-CD3 mAb (Becton Dickinson, Bedford, MA) or PE-labeled isotype-matched control Ab, then fixed and
stained by TUNEL. Gated CD3+ cells were assessed for TUNEL staining.
Apoptosis-associated alterations in Jurkat cells were also evaluated by
staining with the potential-sensitive dye DiOC6, which is incorporated
into mitochondria.34 Loss in DiOC6 staining indicates
disruption of the mitochondrial inner transmembrane potential
associated with apoptosis.35 Cells were first stained for
DiOC6 (40 nmol/L, 15 minutes at 37°C), and then without fixation, stained by PE-anti-CD3 on ice. CD3+ cells were gated to
assess mitochondrial staining by DiOC6.
Intracellular caspase activity
Following apoptotic stimulation, Jurkat cells
(5 × 105) were resuspended in 50 µL of 10 µmol/L PhiPhiLux-G2D2
substrate solution (OncoImmunin, College Park, MD) in
RPMI-1640 supplemented with 10% FCS. After incubation
for 1 hour at 37°C avoiding direct light, the sample was diluted
with 0.5 mL of ice-cold flow cytometry dilution buffer (OncoImmunin).
Flow cytometric analysis was performed within 60 minutes of the end of
the incubation period.
Western blot analysis
Proteins were separated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis using 7.5% to 12% polyacrylamide gels and
transferred to polyvinylidene fluoride membranes, as previously described.36 Following probing with specific antibodies,
the protein bands were detected by enhanced chemiluminescence (Pierce, Rockford, IL).
Statistics
Statistical significance of results was determined by U
Mann-Whitney nonparametric test. Differences between
groups were considered significant at P < .05.
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Results |
Tumor-induced apoptosis in lymphocytes is partly Fas-mediated
To examine the ability of SCCHN cell lines to induce apoptosis in
lymphocytes, tumor cells were cocultured with lymphocytes at
tumor-to-lymphocyte ratios ranging from 20:1 to 80:1 and were examined
by various methods detecting the presence of apoptotic T lymphocytes. A
significant loss of DNA, as assessed by the JAM assay (Figure
1A), as well as fragmentation of DNA, as
evidenced by the TUNEL assay (Figure 1B), and altered polarization of
the mitochondria, as assessed by loss in DiOC6 staining (Figure 1C), were detected in the Jurkat target cells coincubated with various SCCHN
cells for 16 hours. To positively identify apoptotic T cells and
exclude apoptotic tumor cells, TUNEL and DiOC6 staining were each
performed in conjunction with CD3 staining. The presence of
TUNEL+ or DiOC6+ cells was determined in
CD3+ gated cell population (Figures 1B and 1C).
Tumor-induced apoptosis of Jurkat cells was dose-dependent (not shown)
and was first observed following 10 hours of coincubation, while death
induced by agonistic anti-Fas Ab (CH-11, 200 ng/mL) was detected as
early as 2 hours following the addition of the Ab.
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| Fig 1.
Tumor-induced apoptosis of Jurkat cells as assessed by
the JAM assay, flow cytometry TUNEL, and loss in DiOC6
staining of mitochondria.
(A) Apoptosis of Jurkat cells as assessed by the JAM assay. Various
SCCHN cell lines were coincubated with [3H]TdR-labeled
Jurkat cells at a 40:1 tumor-to-lymphocyte cell ratio for 16 hours.
Treatment with agonistic anti-Fas Ab (CH-11, 200 ng/mL) served as
positive control for apoptosis. Target cell death was determined by
measuring fragmentation of 3H-labeled target cell DNA. The
error bars represent the SEM of 8 replicates. (B) Apoptosis of Jurkat
cells as assessed by flow cytometry TUNEL. Fas-sensitive or
Fas-resistant Jurkat cells were treated with agonistic anti-Fas Ab
(CH-11, 200 ng/mL) or coincubated with PCI-13 (tumor-to-lymphocyte cell
ratio of 40:1) for 16 hours. The cells were then stained with
anti-CD3-PE, fixed, and stained for DNA breaks by TUNEL. TUNEL
staining was assessed by flow cytometry in CD3+-gated
cells. The percentage of TUNEL positive cells is indicated at the right
corners. (C) Apoptosis of Jurkat cells as assessed by loss in DiOC6
staining of mitochondria. Fas-sensitive Jurkat cells, incubated in
medium alone or with PCI-13 tumor cells (tumor-to-lymphocyte cell ratio
of 40:1) for 16 hours, were stained for DiOC6 (40 nmol/L, 15 minutes,
37°C) and then, without fixation, stained by PE-conjugated anti-CD3
Ab on ice. Results shown in A, B, and C were reproduced in at least 3 different experiments.
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To examine whether the Fas pathway was involved in the observed death
of Jurkat cells, anti-FasL neutralizing Ab was added to the coculture
of Jurkat and tumor cells (Figure 2A). In
the presence of FasL blocking Ab, apoptosis of Jurkat cells induced by
various SCCHN cells (results for PCI-13 cell line are presented) was
partially but significantly inhibited, suggesting that Fas signaling
was involved. To further investigate the involvement of the Fas pathway
in SCCHN-induced apoptosis of Jurkat cells, a Fas-resistant Jurkat cell
line was generated by selection of cells resistant to multiple cycles
of treatment with agonistic anti-Fas Ab followed by positive selection
of Fas-expressing cells using FACS cell sorting.8 When
[3H]TdR-labeled Fas-resistant cells were coincubated with
SCCHN cells, no DNA degradation was observed (Figure 2B).
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| Fig 2.
Involvement of Fas/FasL in tumor-induced apoptosis of T
cells.
(A) Inhibition of tumor-mediated apoptosis of Jurkat cells by anti-FasL
Ab. [3H]TdR-labeled Jurkat cells were treated for 2 hours
with anti-FasL Ab (4H9) or with a hamster IgG control at various
concentrations, as indicated. Target cell death was determined at 16 hours by measuring fragmentation of 3H-labeled DNA. The
error bars represent the SEM of 8 replicates. (B) Reduced sensitivity
of Fas-resistant Jurkat cells to PCI-13-mediated apoptosis.
Fas-resistant Jurkat cells were obtained as described in "Materials
and methods." [3H]TdR-labeled Fas-sensitive and
[3H]TdR-labeled Fas-resistant Jurkat cells were
coincubated with PCI-13 cells (tumor-to-Jurkat ratio, 80:1) or anti-Fas
(CH-11) Ab for 16 hours, and tested for apoptosis by JAM assay.
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Activation of caspases in T lymphocytes coincubated with tumor
cells
To investigate the role of caspases in tumor-induced apoptosis of T
cells, Jurkat target cells were pretreated with the irreversible peptide inhibitors Z-VAD-FMK or Z-DEVD-FMK.37-39 Whereas
Z-VAD-FMK is a pan-caspase inhibitor, Z-DEVD-FMK has an optimal
tetrapeptide recognition motif for caspase-2, caspase-3, and
caspase-7.40 In the presence of these inhibitors,
tumor-induced DNA degradation (Figure 3),
as well as trypan blue uptake in lymphocytes, was effectively
inhibited.
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| Fig 3.
Effects of peptide inhibitors of caspases on Fas- or
tumor-induced DNA degradation in Jurkat cells.
[3H]TdR-labeled Jurkat cells were pretreated with
Z-VAD-FMK or Z-DEVD-FMK at the indicated concentrations for 16 hours
and then coincubated with tumor at a 40:1 tumor-to-lymphocyte cell
ratio for an additional 16 hours. Jurkat cell apoptosis was determined
by measuring the loss of 3H-labeled DNA. The error bars
represent the SEM of 8 replicates. Similar results were obtained in the
absence of inhibitors or in the presence of the control inhibitor
Z-FA-FMK.
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To demonstrate activation of caspases in Jurkat cells coincubated with
tumor cells, the PhiPhiLux-G2D2 cell-permeable fluorescent substrate
for caspases was used. This substrate emits fluorescence when it is
cleaved within the sequence GDEVDGID, allowing the determination of
caspase-3-like activity by flow cytometry.41,42 As shown
in Figure 4, activity of caspases cleaving
at DEVD sequence was detected in Jurkat cells either treated by
agonistic anti-Fas Ab (38% positive cells) or cocultured with PCI-13
cells (31% positive cells).
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| Fig 4.
Flow cytometric analysis of caspase activity in tumor- or
Fas-stimulated T lymphocytes.
In (A) Jurkat cells were treated with anti-Fas Ab (CH-11, 200 ng/mL)
for 16 hours at 37°C. In (B) Jurkat cells were cocultured on PCI-13
monolayer (tumor-to-lymphocyte cell ratio of 40:1) for 16 hours. At the
end of the coincubation periods, the cells were treated with
anti-CD3-fluorescein isothiocyanate followed by incubation with 10 µmol/L PhiPhiLux-G2D2 for 1 hour. CD3-positive cells
were analyzed for caspase-3 cleavage activity of PhiPhiLux. Controls
indicate baseline fluorescence of substrate-loaded cells in the absence
of apoptotic stimulation. The percentage of positive cells for
PhiPhiLux fluorescence was as follows: 5% in control; 38% in Jurkat
treated with anti-Fas Ab; 31% in Jurkat coincubated with tumor
cells.
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Tumor-mediated apoptosis in T cells overexpressing cytokine
response-modifier gene A
To further investigate the activation of caspases in apoptosis of
lymphocytes coincubated with tumor cells, Jurkat cells overexpressing the cowpox virus-encoded serpin, CrmA, were used as target
lymphocytes.32 CrmA is a potent inhibitor of caspase-1 and
caspase-8, and its inhibition of caspase-3 has been shown to be
indirect, resulting from inhibition of
caspase-8.38,40 Overexpression of CrmA in Jurkat cells resulted in partial inhibition of apoptosis induced by
either agonistic anti-Fas Ab, tumor cells, or VP-16, as assessed by the
JAM assay (Figure 5). While overexpression
of CrmA efficiently blocked apoptosis induced by OSC-68 cells, less
protection was observed in transfected Jurkat cells cocultured with
PCI-13 cells. The partial inhibition of PCI-13-induced apoptosis in
Jurkat cells overexpressing CrmA was confirmed by a TUNEL assay
performed in conjunction with CD3 staining (Figure
6). The ability of CrmA to inhibit
tumor-induced apoptosis of T cells suggests that caspase-8 is involved
in this apoptotic pathway. However, the observed partial inhibition by
CrmA suggests that, in addition to CrmA-sensitive caspases,
CrmA-insensitive caspases were also activated in Jurkat cells
following interaction with tumor cells.
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| Fig 5.
Effects of CrmA overexpression on the susceptibility of
Jurkat cells to tumor-mediated apoptosis.
CrmA or control Neo-Jurkat cells were labeled with
[3H]TdR and coincubated with tumor cells at a
tumor-to-lymphocyte cell ratio of 40:1. As controls, these cells were
treated with agonistic anti-Fas Ab (CH-11, 200 ng/mL) or VP-16 (20 µmol/L). Target cell death was determined at the
indicated time points by measuring loss in 3H-labeled DNA.
The error bars represent the SEM of 8 replicates.
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| Fig 6.
Tumor-induced apoptosis in Jurkat cells stably
transfected with CrmA, Bcl-2, or Neo vector alone.
Jurkat cells were coincubated with PCI-13 cells for 16 hours. The cells
were then stained with anti-CD3-PE, fixed, and stained for DNA breaks
by TUNEL. TUNEL staining was assessed by flow cytometry in
CD3+-gated cells. The percentage of TUNEL-positive cells is
indicated at the right corners.
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Activation of caspase-8 and caspase-3 in tumor-induced apoptosis of
T cells
Caspase-8 has been recognized as the most apical caspase activated
in the Fas or TRAIL pathways of cell death.43-45 As shown in Figure 7, the prodomain
of caspase-8 was processed in Fas-sensitive Jurkat cells, but not in
Fas-resistant Jurkat cells, that were treated with agonistic anti-Fas
Ab or coincubated with tumor cells. Owing to a fast turnover, active
subunits were not detected at 16 hours coincubation with tumor cells or
agonistic anti-Fas Ab.
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| Fig 7.
Tumor-induced activation of caspase-8.
Fas-sensitive or Fas-resistant Jurkat cells were treated with agonistic
anti-Fas Ab (CH-11, 200 ng/mL) or coincubated with tumor cells for 16 hours. At the end of the incubation period, Jurkat cells were
negatively selected by removal of tumor cells, lysed, and assessed by
Western blotting for loss in expression of caspase-8. Equal loading was
confirmed by equivalent detection of nonspecific bands on the same
membrane when stripped and probed for the presence of other caspases
(not shown).
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Caspase-3 has been shown to play a central executioner role in various
pathways of apoptosis. Processing of pro-caspase-3 into an active
enzyme results in cleavage of the pro-enzyme (p32) into p20, p17, and
p10 subunit proteins37,38; p17 is generated by further
cleavage of the p20 by catalytic activity, which is inhibited in the
presence of DEVD-FMK.46,47 The processing of caspase-3 in Jurkat cells triggered by agonistic anti-Fas Ab or
tumor cells was examined by Western blot analysis, using mAb, which
detects the presence of p32, p20, and p17, but not p10. Full processing
of the p32 pro-enzyme was observed in Neo-Jurkat cells treated with
agonistic anti-Fas Ab (Figure 8A). The
presence of CrmA inhibited the generation of active caspase-3 in
response to Fas crosslinking, while only partial inhibition was
observed in Fas-resistant Jurkat cells or in Jurkat cells
overexpressing Bcl-2. Interestingly, p17 active caspase-3 was generated
in the Neo-Jurkat cells, while only p20 was detected in either Bcl-2 or
Fas-resistant Jurkat cells. Coincubation of tumor cells with Fas-sensitive Jurkat cells, but not with Fas-resistant cells, resulted
in activation of caspase-3 (Figure 8B). The signal delivered by the
tumor cells was less potent than that of anti-Fas agonistic Ab, since
only partial processing of the p32 was observed as compared with Jurkat
cells treated with anti-Fas Ab. (Similar results were obtained in
untransfected or Neo-Jurkat cells.) However, both p20 and p17 were
generated in response to tumor-induced apoptosis (Figure 8B).
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| Fig 8.
Processing of caspase-3 in Jurkat cells treated with
agonistic anti-Fas Ab or coincubated with tumor cells.
In (A) Jurkat cells, including Fas-resistant (Fas-R), Neo control,
CrmA, and Bcl-2, were treated with CH-11 (200 ng/mL)
for 16 hours. At the end of the incubation period, the cells were lysed
and assessed by Western blotting for caspase-3 (anti-CPP32 mAb, 2.5 µg/mL). In (B) Fas-sensitive (Fas-S) and Fas-resistant Jurkat cells
were coincubated with tumor cell (20:1 tumor-to-Jurkat cell ratio) for
16 hours. At the end of the coincubation period, Jurkat lymphocytes
were negatively selected by removal of tumor cells, with the use of
epithelial-specific anti-64 mAb and
magnetic beads. Negatively selected Jurkat cells were lysed and
analyzed by Western blotting for processing of caspase-3.
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Caspase substrates in T lymphocytes coincubated with tumor cells
PARP is a highly conserved nuclear enzyme, which is activated
following DNA damage and is implicated in DNA repair.48
Proteolytic cleavage of PARP has been shown to be linked to activation
of DEVD-specific caspases, including caspase-3 and
caspase-7.49 PARP cleavage was observed in transfected
Jurkat cells, including Neo, CrmA, and Bcl-2, following treatment with
agonistic anti-Fas Ab (Figure
9A). No PARP cleavage was
observed in Fas-resistant Jurkat cells, and the cleavage observed in
Jurkat cells overexpressing Bcl-2 or CrmA was significantly reduced as
compared with Neo control cells. Since no activation of caspase-3 was
observed in CrmA Jurkat cells, the PARP is probably cleaved by other
activated caspases (eg, caspase-7). The absence of PARP cleavage in the
Fas-resistant Jurkat cells suggests that the p20 caspase-3 detected in
Figure 8A is either not active or blocked. Similar to what occurs with Fas-ligation, coincubation with tumor cells induced PARP cleavage in
Fas-sensitive, but not in Fas-resistant, Jurkat cells (Figure 9B). These results suggest that PARP is a common substrate cleaved in
apoptotic lymphocytes induced by tumor cells or anti-Fas Ab. However,
the signal delivered by the tumor appears to be less potent than that
of anti-Fas Ab, as it induced only partial processing of PARP.
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| Fig 9.
Cleavage of PARP in apoptotic Jurkat cells.
In (A) Jurkat cells were treated with agonistic anti-Fas Ab; in (B)
Jurkat cells were coincubated with tumor cells. Experimental details
are similar to those described in Figure 8. C2-10 anti-PARP mAb was
used for blotting (Enzyme System, 1:7500).
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We have reported that reduced expression of TcR- chain in T cells
cocultured with tumor cells was related to T-cell
apoptosis.8 In that study, the loss in expression of
cytoplasmic TcR- chain, as assessed by flow cytometry, was prevented
in the presence of peptide inhibitors of caspases.8 We have
also identified the TcR- chain as a direct substrate for caspase-3
activity in a cell-free system, as well as in Fas-crosslinked Jurkat
cells.50 To further investigate the link between
caspase-mediated cleavage of -chain and the tumor-induced loss of
-chain expression, Jurkat cells coincubated with tumor cells for 24 hours were lysed and examined by Western blotting for the presence of
cleaved fragments. As shown in Figure 10,
a -chain fragment was detected in lysates of Fas-sensitive, but not
Fas-resistant, Jurkat cells coincubated with PCI-13 cells and probed
with the N-terminus-specific anti- Ab. Since the C-terminal
fragment of -chain is being cleaved off and the epitope recognition
site is presumably altered,50 a C-terminus-specific
anti- Ab would not detect the presence of a C-terminus deleted
product. Therefore, previous studies detected only a loss in expression
of full-length -chain.51,52 Using N-terminus-specific
anti- Ab, we were able to detect tumor-induced cleavage products of
-chain. These results demonstrate that -cleavage, shown
previously to be caspase-mediated,50 is initiated by
interaction with tumor cells.
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| Fig 10.
Tumor-induced -chain cleavage in Jurkat cells.
Fas-sensitive and Fas-resistant Jurkat cells were coincubated with
tumor cells for 16 hours at a tumor-to-lymphocyte cell ratio of 20:1.
Jurkat cells were then negatively selected by removal of tumor cells
with the use of epithelial-specific
anti-64 mAb and magnetic beads.
Negatively selected Jurkat cells were lysed and analyzed by Western
blotting for expression of -chain. N-terminus-specific anti- Ab
was used for probing.
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Tumor-mediated apoptosis in T cells overexpressing Bcl-2
The role of Bcl-2, an endogenous inhibitor of apoptosis, in
tumor-induced apoptosis of lymphocytes was assessed in Jurkat cells
stably transfected with a construct encoding epitope-tagged (KT3) Bcl-2
protein. Bcl-2 or Neo Jurkat cells were prelabeled with
[3H]TdR and coincubated with agonistic anti-Fas Ab (200 ng/mL, CH-11) tumor cells (40:1 tumor-to-lymphocyte cell ratio) or
VP-16 (20 µmol/L) for 8 to 24 hours. Apoptosis was
assessed by loss of labeled DNA as measured by the JAM assay (Figure
11). Although overexpression of Bcl-2
significantly inhibited DNA fragmentation induced in Jurkat cells by
VP-16, it did not inhibit Fas-mediated DNA fragmentation induced by
agonistic anti-Fas Ab or apoptosis induced by tumor cells. The failure
of overexpressed Bcl-2 to protect lymphocytes from tumor-induced DNA
fragmentation was also confirmed by a TUNEL assay performed in
conjunction with CD3 staining (Figure 6). In these experiments, a
similar proportion of apoptotic T cells was detected in Neo Jurkat
(50%) and in Bcl-2 Jurkat (46%) cocultured for 16 hours with PCI-13
tumor cells. These results indicate that the intracellular pathway of
death initiated in lymphocytes by interaction with tumor cells is not
effectively regulated by Bcl-2.
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| Fig 11.
Effects of overexpression of Bcl-2 in Jurkat cells on
their susceptibility to tumor-mediated DNA degradation.
Bcl-2 or control Neo-transfected Jurkat cells were labeled with
[3H]TdR and coincubated with tumor cells at a
tumor-to-lymphocyte cell ratio of 40:1. As controls, these cells were
treated with agonistic anti-Fas Ab (CH-11, 200 ng/mL) or VP-16 (20 µmol/L). Target cell apoptosis was determined at the
indicated time points by measuring loss in 3H-labeled DNA
in the JAM assay. The error bars represent the SEM of 8 replicates.
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Tumor-induced cleavage of Bcl-2 in T cells
To further investigate the failure of Bcl-2 to block Fas- or
tumor-mediated DNA degradation in lymphocytes, we performed Western blot analysis to assess possible changes in Bcl-2 expression following stimulation by either agonistic anti-Fas Ab or tumor cells. These studies revealed a cleaved fragment of Bcl-2 in Fas-sensitive, but not
in Fas-resistant Jurkat cells triggered by anti-Fas Ab (Figure
12A). Proteolytic cleavage of Bcl-2 has
recently been reported in apoptosis induced by alphaviruses,
Fas-ligation, or growth factors withdrawal.47,53 To test
whether Bcl-2 is also cleaved by apoptosis induced in Jurkat cells by
interaction with tumor cells, SCCHN PCI-13 cell line was cocultured
with Fas-sensitive Jurkat or with Fas-resistant Jurkat cells at a
tumor-to-lymphocyte cell ratio of 20:1 for 16 hours. At the end of the
incubation period, Jurkat target cells were negatively selected by
removal of tumor cells, lysed, and tested by Western blot analysis for expression of Bcl-2. Cleaved Bcl-2 was detected in Fas-sensitive Jurkat
cells coincubated with tumor cells, but not in Fas-resistant Jurkat
cells (Figure 12B). These results suggest that common effector molecules responsible for Bcl-2 cleavage were activated in T cells triggered by either tumor cells or anti-Fas agonistic Ab. Cleavage of
Bcl-2 may, in part, explain its ineffective protection of T cells from
apoptosis induced by tumor cells.
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| Fig 12.
Cleavage of Bcl-2 in Jurkat cells treated with agonistic
anti-Fas Ab or coincubated with tumor cells.
In (A), Fas-sensitive and Fas-resistant Jurkat cells were treated with
CH-11 (200 ng/mL) for 16 hours. At the end of the incubation period,
the cells were lysed and assessed by Western blotting for Bcl-2
expression (anti-Bcl-2 mAb 100, Santa Cruz). In (B), Fas-sensitive and
Fas-resistant Jurkat cells were coincubated with tumor cell (20:1
tumor-to-Jurkat cell ratio) for 16 hours. At the end of the
coincubation period, Jurkat lymphocytes were negatively selected by
removal of tumor cells, using epithelial-specific
anti-64 mAb and magnetic beads.
Negatively selected Jurkat cells were lysed and analyzed by Western
blotting for expression of Bcl-2. Better separation
between the Bcl-2 bands was observed in B than in A, owing to longer
electrophoresis.
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Discussion |
Our recent studies have suggested that coincubation of Jurkat T
cells or peripheral T lymphocytes of cancer patients with tumor cells
results in T-cell apoptosis.8,50 The biological relevance
of this finding became further apparent when similar observations were
made in vivo.12,54,55 An excessive death of lymphocytes at
the tumor site may represent a mechanism of tumor-induced
immunosuppression. Apoptosis of lymphocytes interacting with tumor
cells could result from a direct engagement of death receptors
expressed on lymphocytes with death ligands expressed on tumor cells.
Indeed, recent studies have suggested that apoptosis-inducing proteins,
such as FasL, are expressed on tumors and might be actively engaged in
modulation of the immune response in situ.1,3,4,8,56 However, additional mechanisms of induction of apoptosis may exist at
the tumor site, including up-regulation of death receptors and/or their
ligands on lymphocytes, leading to autocrine or paracrine stimulation
of cell death.13,14 The present study investigated the
intracellular effector molecules involved in the execution of the
apoptotic cascade induced in lymphocytes by tumor cells. Characterization of the molecular mechanisms involved in T-cell death
in the tumor microenvironment may have important implications for
therapeutic regimens, including vaccination strategies and adoptive
transfer of activated T cells into patients with cancer.
Evidence for the ability of human SCCHN cells to induce apoptosis in
interacting lymphocytes was obtained by various detection methods of
apoptosis, including degradation of DNA, as assessed by the JAM or
TUNEL assays; appearance of changes in mitochondria permeability
transition, as assessed by loss of DiOC6 staining; and enhanced
activity of caspase-3-like enzymes, as assessed by cleavage of a
cell-permeable fluorescent DEVD substrate. The Fas-FasL pathway appears
to be involved in T-cell apoptosis induced by SCCHN cells, since it was
partly inhibited by neutralizing anti-FasL Ab.
Ectopic expression of CrmA has been shown to inhibit apoptosis
triggered by ligation of death receptors, but not apoptosis triggered
by cytokine withdrawal or ionizing radiation.57,58 In the
present study, overexpression of CrmA provided partial protection to T
cells from tumor- or Fas-mediated apoptosis at an early period after
stimulation of apoptosis. Since caspase-8 is a major target of CrmA
inhibitory effect, the observed protection appears to be mediated by
direct inhibition of caspase-8, which actively participates in
execution of tumor-induced apoptosis of T cells. Overexpression of CrmA
also effectively inhibited the processing of pro-caspase-3 into active
subunits. Since CrmA is not an efficient inhibitor of caspase-3, the
inhibition of caspase-3 processing appears to represent an indirect
effect of CrmA-inhibited caspase-8.59,60 Although
overexpression of CrmA effectively inhibited the activation of
caspase-3, only partial inhibition of PARP cleavage was observed in
CrmA cells ligated by anti-Fas Ab. Since caspase-3 appears only as an
inactive proform in CrmA cells, the partial cleavage of PARP may be
executed by redundant caspases with cleavage site specificity similar
to that of caspase-3.
In Jurkat cells coincubated with tumor cells, activation of caspase-3
differed from that in Jurkat cells treated with anti-Fas Ab. Whereas
Fas crosslinking by agonistic anti-Fas Ab resulted in processing of all
the detectable pro-caspase-3 (p32) into p17, only partial processing
of the pro-domain was observed in tumor-triggered T cells. Also, in
contrast to the generation of p17 alone in Fas-mediated apoptosis, both
p17 and p20 were detected in Jurkat cells coincubated with tumor cells.
Although the presence of caspase-3 active subunit, p20, was detected in
Fas-resistant Jurkat cells, its activity appears to be blocked, since
viability and growth of these cells were not affected by Fas
crosslinking. Furthermore, PARP or TcR- chain cleaving activity
was not detected in these cells.
We have recently reported that the -chain is a direct substrate for
caspase-3 in a cell-free system, as well as in cells triggered by
anti-Fas Ab.50 However, generation of -chain cleavage products in T cells interacting with tumor cells has not yet been reported. In this study, we obtained evidence demonstrating
that tumor-induced activation of intracellular caspases plays a role in -chain degradation. While in previous studies the use of
Cterminus-specific anti- Ab allowed only detection of loss in
full-length -chain, in the current study, the use of
N-terminus-specific anti- Ab revealed the presence of -chain
cleaved products in coculture of Jurkat and tumor cells. This
is the first direct evidence that tumor-induced alteration in
expression of -chain is mediated by protein degradation.
Bcl-2 has been shown to be a potent inhibitor of apoptosis induced by
various stimuli, including growth-factor deprivation, irradiation, and
cytotoxic drugs.61 Anti-apoptotic Bcl-2 family members bind
to mitochondria and inhibit cytochrome-c release.62,63 Recent studies confirmed the ability of Bcl-2 to also act downstream of
cytochrome-c release, binding to Apaf-1 or
caspase-9.26,64-66 However, Bcl-2 does not regulate
activity of caspases upstream of the mitochondria.67,68
Although Bcl-2 has been reported to protect hepatocytes, P815
mastocytoma, B-cell lymphoma, and certain clones of Jurkat cells from
Fas-mediated apoptosis, its ability to protect lymphocytes has been
controversial.69,70 In our studies, Jurkat cells
overexpressing Bcl-2 were not protected from tumor- or Fas-induced DNA
degradation. The cleavage of Bcl-2 may provide an explanation for its
ineffective protection. However, most of the intracellular Bcl-2
remained unprocessed, even at 24 hours after Fas crosslinking, when
90% of the cells were positive for apoptosis. Although the unprocessed
Bcl-2 did not protect Jurkat cells from ultimate DNA-degradation, it
was biologically active, as demonstrated by a partial processing of p32
caspase-3 or PARP in Bcl-2-Jurkat, as compared with full-processing in
Neo control-Jurkat. However, this inhibitory activity of Bcl-2 was not
sufficient to block the progress of the apoptotic cascade toward DNA
fragmentation. Recent studies suggest that Bcl-2 can interfere with
Fas-mediated apoptosis only in those cells where recruitment or
activation procaspase-8 is not efficient.68 This has been
suggested by a recently described mechanism of cross-talk between
caspase-8 and caspase-9 via mitochondria.71-73 This
cross-talk is mediated by BID, a proapoptotic member of the Bcl-2
family, which is cleaved by caspase-8, and its C-terminal fragment
translocates to the mitochondria and triggers cytochrome-c release.
Depletion of BID from cytosolic extracts disrupts the
ability of caspase-8 to trigger cytochrome-c release in
vitro.72 BID-induced cytochrome c/Apaf-1 pathway may serve
to amplify the caspase-8 cascade. Thus, it appears that anti-apoptotic
Bcl-2 family members can suppress Fas-induced apoptosis only in those
cells where the mitochondrial amplification loop is of significance.
The role of mitochondria in tumor-induced apoptosis of T lymphocytes is
currently under investigation.
In summary, tumor-induced apoptosis of lymphocytes may combine the
engagement of various apoptotic stimuli and pathways. Therefore, it is
important to identify those intracellular apoptotic events that might
be potential targets for therapeutic intervention in the
future. The current study is the first to identify
intracellular components involved in execution or inhibition of
apoptotic cascades induced in lymphocytes interacting with SCCHN cells.
| |
Footnotes |
Submitted May 6, 1999; accepted October 24, 1999.
Supported by grants from The Pittsburgh Foundation (H.R.); The Wendy
Will Case Cancer Fund, Inc (H.R.); American Cancer Association, Grant
RPG-98-288-01-CIM (H.R.); and The National Cancer Institute, Grant
PO1DE 12321-01 (T.L.W., H.R.).
Reprints: Hannah Rabinowich, University of Pittsburgh Cancer
Institute, W952 Biomedical Science Tower, 200 Lothrop Street,
Pittsburgh, PA 15213; e-mail: rabinow+{at}pitt.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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19 |
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Abstract
Introduction