From the Departments of Pathology and
¶ Otolaryngology, University of Pittsburgh School of Medicine and
University of Pittsburgh Cancer Institute,
Pittsburgh, Pennsylvania 15213
Received for publication, July 13, 2000, and in revised form, November 2, 2000
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ABSTRACT |
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Jurkat leukemic T cells are highly
sensitive to the extrinsic pathways of apoptosis induced via the death
receptor Fas or tumor necrosis factor-related apoptosis-inducing
ligand as well as to the intrinsic/mitochondrial pathways of
death induced by VP-16 or staurosporin. We report here that clonal
Jurkat cell lines selected for resistance to Fas-induced apoptosis were
cross-resistant to VP-16 or staurosporin. Each of the apoptotic
pathways was blocked at an apical phase, where common regulators of
apoptosis have not yet been defined. The Fas pathway was blocked at the
level of caspase-8, whereas the intrinsic pathway was blocked at the mitochondria. No processing or activity of caspases was detected in
resistant cells in response to either Fas-cross-linking or VP-16
treatment. Also, no apoptosis-associated alterations in the
mitochondrial inner membrane, outer membrane, or matrix were detected
in resistant Jurkat cells treated with VP-16. Thus, no changes in
permeability transition, loss in inner membrane cardiolipin, generation
of reactive oxygen species, or release of cytochrome c were
observed in resistant cells treated with VP-16. Further, unlike
purified mitochondria from wild type cells, those obtained from
resistant cells did not release cytochrome c or
apoptosis-inducing factor in response to recombinant Bax or truncated
Bid. These results identify a defect in mitochondria ability to release
intermembrane proteins in response to Bid or Bax as a mechanism of
resistance to chemotherapeuetic drugs. Further, the selection of
VP-16-resistant mitochondria via elimination of Fas-susceptible cells
may suggest the existence of a shared regulatory component between the
extrinsic and intrinsic pathways of apoptosis.
Susceptibility to apoptosis is an essential prerequisite for
successful treatment of tumor cells by cytotoxic T lymphocytes, natural
killer cells, radiation, or chemotherapy. However, resistance to
apoptosis has been established as one of the mechanisms responsible for
the failure of therapeutic approaches in many types of cancer, including hematopoietic malignancies. At least two pathways of caspase
activation have been delineated, including extrinsically and
intrinsically stimulated cascades (1-3). The extrinsic pathway involves apoptosis mediated by cell surface death receptors, such as
Fas, tumor necrosis factor receptor, or
TRAIL1 receptor. Fas
stimulation results in oligomerization of the receptors and recruitment
of the adapter protein Fas-associated death domain (FADD) and
caspase-8, forming a death-inducing signaling complex (DISC) (4).
Autoactivation of caspase-8 at the DISC is followed by activation of
effector caspases, including caspase-3, -6, and -7, which function as
downstream effectors of the cell death program. The intrinsic pathway
is mediated by diverse apoptotic stimuli, which converge at the
mitochondria. Release of cytochrome c from the mitochondria
to the cytoplasm initiates a caspase cascade. Cytosolic cytochrome
c binds to apoptosis protease-activating factor 1 (Apaf-1)
and procaspase-9, generating a DISC-like complex, "apoptosome" (5,
6). Within the apoptosome, caspase-9 is activated, leading to
processing of caspase-3. The two pathways of apoptosis, extrinsic/death
receptor and intrinsic/mitochondrial, converge on caspase-3 and
subsequently on other proteases and nucleases that drive the terminal
events of programmed cell death. The significance of Apaf-1, caspase-9,
and caspase-3 for the execution of apoptosis has been confirmed by
genetic studies in mice (7-9). Cells derived from Apaf-1, caspase-9,
and caspase-3 knockout mice demonstrated defects in response to a
variety of apoptotic stimuli. However, T lymphocytes from mice lacking
Apaf-1 or caspase-9 were susceptible to apoptosis signaled via Fas or
tumor necrosis factor receptors (10, 11). Also, cytochrome
c-deficient cells were susceptible to death
receptor-mediated apoptosis (12). These findings support the concept
that the two apoptotic pathways are discrete and function in parallel.
However, each of the pathways is amplified by activated components of
the "other" pathway. In cases of low initial caspase-8 activation,
the direct effect of caspase-8 on caspase-3 is amplified by a
mitochondrial loop (1). In this scenario, caspase-8 cleaves off an
N-terminal fragment of Bid, allowing the tBid to translocate to the
mitochondria and induce cytochrome c release (13-15). Also,
the intrinsic pathway is amplified by components of the death receptor
cascade, as cytostatic drugs have been reported to activate caspase-8
secondarily to mitochondrial damage (16, 17). Currently, cross-talk
between the extrinsic and intrinsic pathways has been observed mainly as an amplification loop at the level of execution, but not initiation, of each of the cascades. Also, tumor cell lines studied for resistance to apoptosis were resistant to either the death receptor or the mitochondrial pathway but not cross-resistant to both pathways (18).
Cross-resistance has been observed in cells overexpressing an
inhibitor, such as X-linked inhibitor of apoptosis (XIAP/hILP), which
targets downstream effector molecules, such as caspase-3, shared by
both pathways (19-22). Also, members of the Bcl-2 family, which play a
major role in control of the mitochondria-dependent apoptotic pathway (23), were reported to protect some cell lines and
tissues from Fas-induced apoptosis but not others (24, 25). However,
attenuation of Fas-mediated apoptosis by Bcl-2 might be mediated by
inhibition of the mitochondrial amplification loop (1).
In the current study, we demonstrate the selection of cells resistant
to a mitochondrial pathway (intrinsic) by elimination of Fas-sensitive
cells (extrinsic pathway). The selection of clonal cell lines
completely cross-resistant to inducers of the two apoptotic pathways
may suggest the existence of an upstream regulatory component shared by
the extrinsic and the intrinsic pathways.
Reagents--
Agonistic anti-Fas Ab (CH-11, IgM) was purchased
from Upstate Biotechnology Inc. (Lake Placid, NY). Staurosporin and
VP-16 were purchased from Sigma. Recombinant caspase-3 and caspase-8, anti-cytochrome c mAb, rabbit anti-caspase-3 Ab, and
anti-CD3 Preparation of GST-Bax and His-tagged tBid--
Mouse Bax-
Mouse tBid (amino acids 60-195) was cloned into a pET23dwHis vector
modified from the pET23d(+) vector (Novagen, WI), in fusion with the
6-histidine tag at its N terminus. Bacteria E. coli strain BL21(DE3) was transformed and cultured at 37 °C in Terrific Broth. The induction of expression was started at 0.8-1.0
A600 by the addition of 0.4 mM
isopropyl Cell Lines and Clones--
Jurkat T leukemic cell line was
obtained from the American Type Culture Collection (ATCC; Manassas,
VA). Jurkat cells were grown in RPMI 1640 medium containing 10% fetal
calf serum, 2 mM L-glutamine, and 100 units/ml
each penicillin and streptomycin (complete medium). The generation of
stable cell lines expressing epitope-tagged CrmA or Bcl-2 proteins has
been described previously (27-30). Transfected cell lines were
maintained in complete medium supplemented with 0.5 mg/ml G418 (Life Technologies).
To select for cells resistant to Fas-mediated apoptosis, wild-type
Jurkat T cell line was repeatedly exposed to the agonistic anti-Fas
mAb, CH-11 (200 ng/ml). Initial treatment induced apoptosis in 99% of
the cells. Surviving cells were expanded in culture, and following
several consecutive selections with anti-Fas Ab, the cultured cells
were further selected by flow cytometry cell sorting to include only
Fas-positive cells. Anti-Fas Ab was routinely added to the resistant
cell line approximately once a month. Clones of Fas-resistant Jurkat
cells were obtained by limiting dilution, and seven of the clonal cell
lines were subjected to analysis of mechanisms of resistance. Similar
features of resistance to apoptosis were found in all tested clones (as
described in this study).
Induction of Apoptosis--
Jurkat cells plated at 0.5-1 × 106 cells/ml in complete medium were treated with VP-16
(20-40 µM), agonistic anti-Fas Ab (200-500 ng/ml),
staurosporin (0.5-1 µM), or TRAIL (100 nM;
enhancer, 2 µg/ml) at 37 °C for varying lengths of time, as
indicated for each experiment. In several experiments, Jurkat cells
were treated with cycloheximide (10 µg/ml) for 4 h prior to the
addition of anti-Fas-Ab, CH-11.
Measurements of Apoptosis--
DNA fragmentation was assessed by
the JAM assay, in which loss of [3H]TdR-labeled DNA was
measured (31). DNA labeling of Jurkat target cells was performed by
incubation of the cells in the presence of 5 µCi/ml
[3H]TdR for 18-24 h at 37 °C. The cells were treated
with anti-Fas Ab, VP-16, or staurosporin for various lengths of time,
as indicated, and then harvested (Mach IIM, TOMTEC) 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
Cytofluorometric analyses of apoptosis were performed by costaining
with propidium iodide and fluorescein isothiocyanate-annexin V
conjugate (CLONTECH). Propidium iodide was used to
identify breaks in DNA as a feature of late apoptosis, and annexin V
was used to assess aberrant phosphatidylserine exposure (32). The staining was performed according to the manufacturer's directions.
Apoptosis-associated alterations in mitochondria were assessed by flow
cytometry analysis using special dyes designed to evaluate mitochondrial events. Disruption of the mitochondrial inner
transmembrane potential (
Generation of superoxide anions was measured by HE (Molecular Probes).
Cells were incubated at 37 °C for 15 min in the presence of HE (2 µM, fluorescence at 600 nm) followed by immediate
analysis of fluorochrome incorporation (34).
Decrease in mitochondrial mass was assessed by NAO (Molecular Probes),
reported to measure the inner mitochondrial cardiolipin content (35).
Cells were stained with 0.1 µM NAO in RPMI medium for 15 min at 37 °C. Cells were washed and immediately analyzed by flow cytometry.
Staining of cells with anti-APO2.7 served as an additional marker for
apoptotic mitochondria. This Ab was raised against apoptotic mitochondria and stains a p38 antigen exposed on apoptotic mitochondria (36).
Preparation of Cytosolic Extracts--
Cytosolic extracts were
prepared from clones of wild-type or resistant Jurkat cells, as
described previously (37). Briefly, cultured Jurkat cells were washed
twice with phosphate-buffered saline and then resuspended in ice-cold
buffer (20 mM HEPES, pH 7.0, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, 250 mM sucrose, and protease inhibitors). After incubation on
ice for 20 min, cells (2.5 × 106/0.5 ml) were
disrupted by Dounce homogenization (20 strokes). Nuclei were removed by
centrifugation at 650 × g for 10 min at 4 °C.
Cellular extracts were obtained as the supernatants resulting from
centrifugation at 14,000 × g at 4 °C for 30 min.
Caspase-3 activation was initiated by the addition of 10 µM horse heart cytochrome c and 1 mM dATP to 10 µl of cellular extract at 30 °C for
1 h. To enforce processing of caspase-8, recombinant caspase-3 was
added to the cell extracts. The extracts were boiled in sample buffer,
resolved by SDS-PAGE, and immunoblotted for processing of caspases.
Western Blot Analysis--
To generate whole cell extracts,
cells were lysed in 1% Nonidet P-40, 20 mM Tris-base, pH
7.4, 137 mM NaCl, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin. Proteins were resolved by SDS-PAGE and transferred to
polyvinylidene difluoride membranes, as described previously (28, 30).
Following probing with a specific primary Ab and horseradish
peroxidase-conjugated secondary Ab, the protein bands were detected by
enhanced chemiluminescence (Pierce).
Subcellular Fractionation--
After induction of apoptosis,
Jurkat cells were harvested in isotonic mitochondrial buffer (20 mM sucrose, 20 mM HEPES, 10 mM KCl,
1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 10 µg/ml
leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin) and Dounce-homogenized by 15-20 strokes (38). Samples were
transferred to Eppendorf centrifuge tubes and centrifuged at 650 × g for 5 min at 4 °C to eliminate nuclei and unbroken cells. The resulting supernatant was centrifuged at 10,000 × g for 30 min at 4 °C to obtain the heavy membrane pellet
(HM). The supernatant was further centrifuged at 100,000 × g for 1 h at 4 °C to yield the final soluble
cytosolic fraction, S-100. HM and S-100 subcellular fractions were
assessed for the presence of cytochrome c by Western blot analysis.
Mitochondria Purification--
Jurkat cells were suspended in
mitochondria buffer (MIB) composed of 0.3 M sucrose, 10 mM MOPS, 1 mM EDTA, and 4 mM
KH2PO4 and lysed by Dounce homogenization as
described previously (33). Briefly, nuclei and debris were removed by
10 min of centrifugation at 650 × g, and a pellet
containing mitochondria was obtained by two successive spins at
10,000 × g for 12 min. The washed mitochondria pellet
was resuspended in MIB and layered on a Percoll gradient consisting of
four layers of 10, 18, 30, and 70% Percoll in MIB. After
centrifugation for 35 min at 13,500 × g, the purified
mitochondria were collected at the 30/70 interface. Mitochondria were
diluted in MIB containing 1 mg/ml bovine serum albumin (at least a
10-fold dilution required to remove Percoll). The mitochondrial pellet was obtained by a 30-min spin at 20,000 × g and used
immediately in the cytochrome c release assay.
To assess insertion of Bax or tBid into the mitochondria membrane,
purified mitochondria were treated with 0.1 M
Na2CO3 (pH 11.5) for 20 min on ice to remove
alkali-sensitive proteins attached to the membrane (39).
Cytochrome c Release Assay--
Purified mitochondria (100 µg
of protein) were incubated with recombinant tBid or Bax at various
doses as indicated in 20 µl of MIB at 30 °C for 30 min.
Mitochondria were pelleted by centrifugation at 4000 × g for 5 min. The resulting supernatants or mitochondria were
mixed with lysis buffer and analyzed by SDS-PAGE and immunoblotting for
the presence of cytochrome c.
Cross-resistance to Fas- and VP-16-induced Apoptosis--
Selected
clonal T cell lines that survived in the presence of agonistic anti-Fas
Ab were also found to be completely resistant to apoptotic signals of
VP-16 known to initiate a mitochondrial cascade of apoptosis. The
degree of resistance to apoptosis induced through the extrinsic or the
intrinsic pathway was higher than that observed in Jurkat cells
engineered to overexpress either CrmA or Bcl-2. While
CrmA-overexpressing Jurkat cells were only partially resistant to
Fas-mediated apoptosis, complete resistance was observed in the
selected Jurkat cells (Fig.
1A). As expected, CrmA-Jurkat
cells were highly susceptible to VP-16-induced cell death, whereas the
Fas-resistant Jurkat cells were as resistant to VP-16 as Jurkat cells
overexpressing Bcl-2 (Fig. 1A). These results demonstrate
that the mechanism(s) responsible for resistance to apoptosis in the
selected Jurkat cells regulate(s) the two known pathways of apoptosis.
The cross-resistance to the two pathways of apoptosis was also
demonstrated by flow cytometry. As shown in Fig. 1B,
propidium iodide-positive and annexin-positive cells were detected in
Jurkat-sensitive cells treated either with anti-Fas Ab, staurosporin,
or VP-16 for 14 h. However, in resistant Jurkat cells treated
similarly with either Fas cross-linking or VP-16, no significant level
of apoptotic cells was observed. Few apoptotic cells were detected in
the resistant clones treated with staurosporin. All clones obtained by
limiting dilution of the parental resistant Jurkat cell line possessed
cross-resistance to both Fas- and VP-16-apoptotic signals.
Abrogation of Caspase Processing and Activation in Resistant Jurkat
Cells--
To determine the mechanism involved in resistance to both
Fas and VP-16, we tested the processing of endogenous caspases in sensitive and resistant clonal Jurkat cells in response to stimuli by
anti-Fas Ab, VP-16, or staurosporin. As shown in Fig.
2, cross-linking of Fas on sensitive
Jurkat cells induced processing of caspase-8, -3, -2, -7, and -9. No
such processing was observed in resistant Jurkat cells stimulated for
periods as long as 14, 24, or 48 h. Likewise, stimulation by VP-16
induced processing of these caspases in sensitive cells but not in the
resistant Jurkat cells treated for as long as 48 h. The observed
Fas-mediated activation of caspase-9 at 14 h poststimulation of
sensitive Jurkat cells results possibly from a mitochondrial
amplification loop mediated by caspase-8-cleaved Bid (13-15, 40).
Activation of caspase-8 in a late phase of the mitochondrial apoptotic
cascade, as seen for VP-16-treated sensitive Jurkat cells, has been
previously documented (17). These results demonstrate that caspases
known to be involved in either Fas-induced or VP-16-induced cascades
are not processed in resistant Jurkat cells. Caspase activation by
staurosporin was also significantly blocked in resistant cells as
compared with their sensitive counterpart. However,
staurosporin-induced processing of caspase-2 was detected in resistant
cells following 14 h of treatment. As shown in Fig. 1B
and corroborated by this analysis of caspase processing, apoptosis induced by staurosporin in resistant Jurkat cells was significantly, but not completely, blocked.
To further assess activity of endogenous caspases, we compared
sensitive and resistant Jurkat cells for caspase activity in cleaving
known endogenous substrates. The substrates tested included the
following endogenous proteins: PARP, known to serve as substrate for
caspase-3 and -7 (41); XIAP, demonstrated recently by us (30) and
others (42) to be cleaved by caspase-3 and -7; TcR
Since caspase-8, the apical caspase of the Fas-apoptotic cascade, was
not processed in resistant Jurkat clones in response to Fas
cross-linking, we examined whether it would be processed by exogenously
added caspase-3. Caspase-8 has been shown to be cleaved by caspase-3
downstream of mitochondrial damage induced by
Caspase-3 has been identified as an executioner caspase in both the
Fas- and VP-16 apoptotic cascades. Therefore, we examined the
processing of endogenous caspase-3 in resistant Jurkat cells in the
presence of either exogenous recombinant caspase-8 or exogenous cytochrome c and dATP. As shown in Fig.
5, caspase-3 subunits were detected in
either sensitive or resistant Jurkat cells treated with recombinant
caspase-8 or cytochrome c and dATP to enforce processing of
prodomain caspase-3.
Fas-induced apoptosis in various cells is enhanced in the presence of
metabolic inhibitors such as cycloheximide (45). To investigate whether
an endogenous protein inhibitor was involved in the observed resistance
to the Fas-apoptotic cascade, wild-type and resistant Jurkat cells were
treated with cycloheximide prior to the addition of anti-Fas Ab. While
an increased percentage of apoptotic cells was detected in sensitive
Jurkat cells, this combined treatment did not induce apoptosis in
resistant Jurkat cells (data not shown). These observations were
further confirmed by assessment of caspase-8 processing. Combined
treatment of cycloheximide and anti-Fas Ab did not result in any
processing of caspase-8 in resistant cells, while it significantly
enhanced the processing seen in wild-type Jurkat cells (data not
shown). These findings suggest that the observed block in caspase-8
processing was not mediated by an endogenously metabolized inhibitor.
To further identify the site of blockage, we investigated whether
caspase-8 would be processed in response to signals via other death
receptors known to use caspase-8 activation as an apical event in their
cascade (46). To this end, sensitive and resistant Jurkat cells were
cross-linked by TRAIL (100 ng/ml; enhancer, 2 µg/ml) for 14 h at
37 °C. No processing of caspase-8 was detected in resistant cells,
whereas similar TRAIL cross-linking induced marked processing in
sensitive cells (data not shown). The arrest in caspase-8 activation in
both Fas and TRAIL receptor cascades, suggests that the block in the
apoptotic cascade in resistant cells is at the level of caspase-8 and
not at the receptor level. The block in caspase-8 activation was not
mediated by up-regulated expression of FLICE/caspase-8 Inhibitory
Protein (FLIP), since similar levels of FLIP-short or FLIP-long
proteins were detected in sensitive and resistant Jurkat cells (data
not shown). Also, similar levels of FADD expression were detected in
sensitive and resistant Jurkat cells (data not shown).
Lack of Apoptosis-associated Alterations in Mitochondria of
Resistant Jurkat Cells--
To determine the mechanism(s) of the
observed resistance of Jurkat cells to VP-16, we assessed
apoptosis-associated alterations within mitochondria using
fluorescent dyes, which specifically target mitochondrial components or
events. Sensitive and resistant Jurkat clones were treated for 8 h
with either anti-Fas Ab or VP-16 and assessed for incorporation of the
cationic lipophilic dye, CMXRos. Fas cross-linking in sensitive, but
not in resistant, Jurkat cells caused significant reduction in
incorporation of this dye (Fig.
6A). These results suggest
that alterations in permeability transition in response to VP-16 are
blocked in resistant Jurkat cells.
We also assessed mitochondrial changes by NAO, a mitochondria-specific
dye, which is independent of permeability transition. NAO is a probe
that interacts specifically with nonoxidized cardiolipin, a lipid that
is exclusively localized in the inner mitochondrial membrane (35). Loss
in cardiolipin, indicating loss in mitochondria matrix integrity, was
detected in sensitive cells but not in resistant cells treated with
either anti-Fas Ab or VP-16 (Fig. 6B). Also, no generation
of reactive oxygen species was detected in resistant Jurkat cells, as
assessed by flow cytometric analysis of HE staining (Fig.
6C).
It has been reported that the mAb APO2.7 detects an
apoptosis-associated increase in expression of a p38 mitochondrial
antigen (36). A population of cells expressing this antigen was
detected in sensitive, but not in resistant, Jurkat cells following Fas or VP-16 stimulation (Fig. 6D). These results suggest that
the block in the VP-16-induced cascade is at the level of the
mitochondria, since mitochondria-specific apoptotic events did not
occur in resistant Jurkat cells treated with VP-16.
Involvement of the mitochondria in apoptosis has been associated with
release of cytochrome c to the cytoplasm, where it initiates a caspase cascade (5, 6). Indeed, in wild-type Jurkat cells treated
with either anti-Fas Ab or VP-16, translocation of cytochrome c to the cytosol was observed (Fig.
7). However, no cytochrome c
release was detected in resistant Jurkat cells treated by each of these
stimuli. These observations suggest that in resistant Jurkat cells the
apoptotic mechanism responsible for cytochrome c release is
blocked.
Analysis of Intermembrane Protein Release from Purified
Mitochondria--
It has recently been reported that the recombinant
proteins, Bax and tBid, induce cytochrome c release when
directly applied to purified mitochondria. To further characterize the
block in cytochrome c release in resistant cells treated
with VP-16, we investigated the response of purified mitochondria from
sensitive and resistant Jurkat cells to recombinant Bax (GST-Bax
To evaluate whether the resistance mechanism was specific for
cytochrome c or applied also to other intermembrane
proteins, we examined release of AIF from sensitive and resistant
mitochondria in response to recombinant tBid. As shown in Fig.
8C, AIF was released from sensitive, but not resistant,
mitochondria. These results suggest that the mitochondrial mechanism
responsible for release of intermembrane proteins is blocked in
resistant Jurkat cells.
To assess the ability of endogenous Bax or tBid to translocate to
the mitochondria, we examined the expression of these proteins within
the mitochondria of resistant cells. To this end, purified mitochondria
from sensitive and resistant Jurkat cells were treated with 0.1 M Na2CO3 (pH 11.5) for 20 min on
ice, to remove alkali-sensitive proteins that are attached but not
inserted into the mitochondrial outer membrane (39). The presence of
Bax within the alkali-resistant fraction of the treated mitochondria
was tested for by Western blot analysis. As shown in Fig.
9A, similar levels of
endogenous Bax were expressed in mitochondria of sensitive or resistant
Jurkat cells. The expression of mitochondrial VDAC and the absence of cytosolic In the present study, we describe the selection of Jurkat leukemic
T cells resistant to an intrinsic apoptotic pathway via elimination of
cells susceptible to the extrinsic Fas-mediated apoptotic cascade. The
block in the Fas cascade appears to be at the level of caspase-8, since
no processing or activation of caspase-8 was observed following
cross-linking of either Fas or TRAIL receptors, which were expressed at
a similar level on sensitive and resistant cells. However, caspase-8 in
resistant cells was processed in the presence of active recombinant
caspase-3, suggesting that it is potentially activable. Activation of
caspase-8 was not suppressed by up-regulation in expression of either
FLIP-long or FLIP-short or by a differential expression of FADD, since
similar levels of these proteins were detected in all sensitive and
resistant clones. The resistance to Fas-mediated apoptosis and the
block in caspase-8 activation was not affected by pretreatment of the cells with cycloheximide, a metabolism inhibitor that significantly increased susceptibility of sensitive Jurkat cells to Fas-mediated apoptosis. Although these findings suggest that caspase-8 inhibition is
not mediated by a metabolized protein inhibitor, the mechanism responsible for the block in caspase-8 activation in these resistant cells is not yet known. It is possible that additional regulatory components may be involved in the activation of caspase-8, which are
yet to be identified. Since caspase-8, the apical caspase in the death
receptor cascade, was not activated, it was expected that downstream
apoptotic events would not be executed. It was, however, surprising
that elimination of Fas-susceptible T cells by anti-Fas Ab would result
in selection of cells resistant to intrinsic apoptotic pathways.
The block in the intrinsic cascade was localized to the mitochondria,
since no apoptosis-associated alterations were detected in resistant
mitochondria treated with VP-16. We tested a broad range of
mitochondrial apoptotic events, including changes in the inner
membrane, the outer membrane, and the mitochondrial matrix. No changes
in inner membrane permeability transition, loss of mitochondrial inner
membrane cardiolipin, or reactive oxygen species generation were
detected in resistant cell mitochondria. Further, we identified a block
in the mechanism responsible for cytochrome c release in
response to an apoptotic signaling by VP-16 or in cell-free
mitochondria treated with recombinant Bax or tBid. The abrogation in
release of cytochrome c was not due to impaired
translocation of either Bax or tBid to the mitochondria. These
proapoptotic Bcl-2 family members were found inserted, rather than
attached, to the resistant mitochondria. The block in cytochrome c release applies also to another intermembrane protein,
AIF, reported to be released in response to apoptotic signaling (54, 55). Our results suggest that the block in resistant mitochondria to
specific death stimulation localizes to the mechanism responsible for
generating specific pores in the outer mitochondrial membrane, which
allow translocation of intermembrane proteins to the cytosol.
According to the currently accepted notion, which is based on genetic
studies in knockout mouse models (7-10, 12), the initiation phases of
the extrinsic and the intrinsic pathways of apoptosis are independent.
Cross-talk between the pathways would occur mainly as an amplification
loop once the cascade has been initiated. Fas-induced apoptosis is
mediated either by direct activation of downstream effector caspases or
via Bid-induced mitochondrial pathway (40). Thus, the observed
cross-resistance of Jurkat clones to Fas- and VP-16-mediated
apoptosis may be explained as two independent mechanisms involved
in blocking the initiation of two independent pathways. Since the
entire study was performed in clonal cell lines derived by limiting
dilution, each of the clones would encompass two independent mechanisms
of resistance. It is possible that in initial selection against cells
susceptible to Fas signaling, accumulation of type II cells dependent
on mitochondria for their Fas-mediated apoptosis took place (56),
whereas continuous selection resulted in cells deficient in
mitochondrial mechanism for releasing intermembrane proteins.
The identification of BAR, an apoptosis regulator, which can
mediate cross-talk between components of the "independent"
pathways, may suggest that in certain cells or apoptotic conditions,
common regulators of the two pathways may be functional. BAR was
identified by using a yeast-based screen for inhibitors of Bax-induced
cell death (57). The BAR protein contains a SAM domain, which is required for its interaction with Bcl-2 and Bcl-XL and for suppression of Bax-induced cell death in both mammalian cells and yeast. In addition, BAR contains a DED-like domain responsible for its
interaction with DED-containing procaspases and suppression of
Fas-induced apoptosis. Furthermore, BAR can bridge procaspase-8 and
Bcl-2 into a protein complex. Since BAR may serve as an inhibitor of Bax and at the same time bind to procaspase-8, we examined by reverse
transcription-polymerase chain reaction its level of expression in
sensitive and resistant Jurkat cells. We detected similar levels of BAR
mRNA in sensitive and resistant Jurkat cells (data not shown),
suggesting that BAR is not involved in the mechanism of resistance in
the Jurkat cells.
A Fas-interacting serine/threonine kinase capable of inducing
phosphorylation of FADD has recently been identified as one of the
homeodomain-interacting protein kinases (homeodomain-interacting protein kinase 3) involved in regulation of apoptosis via p53 (58, 59).
It is conceivable that such a common regulator of both apoptotic
pathways may be linked to the observed mechanism of cross-resistance
(58).
A common regulator of Fas- and
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain mAb (clone 610B10) were purchased from Pharmingen.
Anti-hILP/XIAP mAb (clone 28), anti-caspase-7 mAb (clone 51), and
anti-caspase-2 mAb (clone 47) were from Transduction Laboratories
(Lexington, KY); rabbit anti-caspase-8 Ab was from StressGen
Biotechnology (Victoria BC, Canada); caspase-8-specific mAb (clone 5F7)
was from Upstate Biotechnology, Inc.; anti-caspase-9 was from Oncogene (Cambridge, MA); anti-Bcl-2 mAb (clone 100), rabbit anti-Bcl-xL, and
goat anti-apoptosis-inducing factor (AIF) Ab were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA); anti-poly(ADP-ribose) polymerase
(PARP) mAb (clone C2-10) was from Enzyme Systems (Livermore, CA); and anti-voltage-dependent anion channel (VDAC) mAb
(clone 31HC) was from Calbiochem. Rabbit anti-Bid Ab was generated as described previously (26). Chloromethyl X-rosamine (CMXRos), nonyl
acridine orange (NAO), and hydroethidine (HE) were from Molecular
Probes, Inc. (Eugene, OR); phosphatidylethanolamine-conjugated anti-APO2.7, fluorescein isothiocyanate-annexin V and propidium iodide
were from CLONTECH (Palo Alto, CA); anti-
-actin
mAb (clone AC-15), horse heart cytochrome c, and dATP were
from Sigma; and the TRAIL kit was from Alexis (San Diego, CA).
TM
(amino acids 1-173) was cloned into pGEX-2T (Amersham Pharmacia
Biotech) in fusion with GST protein at its N terminus. Bacterial
Escherichia coli strain DH5
(Life Technologies, Inc.) was
transformed and cultured in LB medium. When
A600 reached 0.7-1.0, isopropyl
-D-thiogalactoside was added at 0.1 mM to induce the expression of fusion protein. Bacteria were harvested after
2-3 h of incubation at 37 °C and lysed by sonication in lysis
buffer (1% Triton X-100, 1 mM EDTA in PBS). After
centrifugation, the supernatant was incubated with preswollen
glutathione beads for 30 min at 4 °C. The beads were then
precipitated and washed by centrifugation. GST-BAX-
TM was eluted
with 5 mM glutathione in 50 mM Tris-HCl, pH
8.0.
-D-thiogalactoside at 37 °C for 2-3 h. The
bacterial pellets were resuspended and sonicated in the buffer
containing 5 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH 7.9. After centrifugation, the supernatants
were passed through a His-Bind nickel-agarose affinity chromatographic
column precharged with 50 mM NiSO4 (Novagen,
WI). The columns were washed with the washing buffer containing 60 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH 7.9. The proteins were eluted with the
elution buffer containing 400 mM imidazole, 500 mM NaCl, and 20 mM Tris-Cl, pH 7.9, and were
further purified using a Sephadex G-50 column balanced with
phosphate-buffered saline.
E)/S, where S
represents retained DNA in the absence of effector cells (spontaneous) and E represents experimentally retained DNA in the presence
of tumor (effector) cells.
m) was measured by the cationic
lipophilic fluorochrome CMXRos. Cells were incubated at 37 °C for 15 min in the presence of CMXRos (0.1 µM, fluorescence at
600 nm) followed by immediate analysis of fluorochrome incorporation
(33).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Resistance of clonal Jurkat cells to Fas and
VP-16 apoptotic signals. A, resistance of Jurkat cells
was assessed by the JAM assay as compared with Jurkat cells
overexpressing CrmA or Bcl-2. [3H]TdR-labeled clonal
Jurkat cell lines were treated with agonistic anti-Fas Ab (CH-11; 200 ng/ml) or VP-16 (20 µM) for 14 h. DNA degradation
was measured by loss of [3H]TdR-labeled DNA. The
error bars represent the S.E. of eight
replicates. B, resistance to Fas and VP-16 apoptotic signals
was assessed by flow cytometry. Clonal Jurkat cell lines were treated
with agonistic anti-Fas Ab or VP-16, as described above. The cells were
then stained with annexin V (2 µg/ml) and propidium iodide (5 µg/ml), and the presence of apoptotic cells was assessed by flow
cytometry.
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Fig. 2.
Abrogation of caspase processing in resistant
clonal Jurkat cells. Wild-type and resistant Jurkat cells were
treated with anti-Fas Ab (200 ng/ml), staurosporin (0.5 µM), or VP-16 (20 µM) for 14, 24, or
48 h. At the end of the treatment period, whole cell extracts were
separated by SDS-polyacrylamide gels, and resolved proteins were
transferred to a polyvinylidene difluoride membrane. The processing of
the indicated caspases was assessed by immunoblotting with specific Abs
as detailed under "Experimental Procedures." Equal amounts of
protein were loaded after quantification, and immunoblotting for
-actin served as an additional loading control. Each
panel represents the results of at least three
experiments.
-chain, reported
by us to serve as substrate for caspase-3 and -7 (28); and the
anti-apoptosis proteins Bcl-2 and Bcl-xl, reported to convert to
proapoptosis regulators following cleavage by caspase-3 (43, 44). As
demonstrated in Fig. 3, no cleavage of
any of the substrates tested was detected in resistant Jurkat cells
treated with either anti-Fas Ab, VP-16, or staurosporin for 14 h.
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Fig. 3.
Abrogation of caspase cleaving activity of
endogenous substrates in resistant clonal Jurkat cells. Wild-type
and resistant Jurkat cells were treated with anti-Fas Ab (200 ng/ml),
staurosporin (0.5 µM), or VP-16 (20 µM) for
14 h. At the end of the treatment period, whole cell extracts were
separated by SDS-polyacrylamide gels, and resolved proteins were
transferred to a polyvinylidene difluoride membrane. The cleavage of
endogenous substrates was assessed by immunoblotting using specific
antibodies to PARP, XIAP, TcR -chain, Bcl-XL, or Bcl-2.
-radiation (17). As
shown in Fig. 4, processing of prodomain caspase-8 as well as the detection of the p20 subunit was similarly observed in extracts of either sensitive or resistant Jurkat cell lines
treated with recombinant caspase-3.
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Fig. 4.
Processing of endogenous caspase-8 in
extracts of resistant Jurkat cells by recombinant caspase-3.
Recombinant caspase-3 was added to extracts of sensitive or resistant
Jurkat cells at 0.3 or 1.5 µM for 30 min at 37 °C. The
proteins were resolved by SDS-PAGE and probed with anti-caspase-8 mAb
(5F7; UBI) (top). The membrane was stripped and reprobed
with polyclonal anti-caspase-8 (Ab3; StressGen), which detects the p20
subunit (bottom).
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Fig. 5.
Activation of endogenous caspase-3 in
extracts of resistant Jurkat cells by exogenous caspase-8 or cytochrome
c. A, wild-type and resistant Jurkat
cells were treated with agonistic anti-Fas Ab (200 ng/ml) for 12 h
and immunoblotted by anti-caspase-3 Ab. B, cytochrome
c (10 µg/ml) together with dATP (1 mM;
B) were added to cytosolic extracts of wild-type or
resistant Jurkat cells. C, recombinant caspase-8 was added
at the indicated doses to extracts of sensitive or resistant Jurkat
cells. The extracts (B and C) were incubated at
30 °C for 1 h and then assessed by immunoblot analysis for the
presence of the zymogen and active subunits of caspase-3.
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Fig. 6.
Lack of apoptosis-associated alterations in
mitochondria of resistant Jurkat cells treated with anti-Fas Ab or
VP-16. Wild-type and resistant Jurkat cells treated with anti-Fas
Ab (200 ng/ml) or VP-16 (20 µM) for 8 h were
assessed by flow cytometry for mitochondrial changes. Staining with
CMXRos (100 nM) served to assess changes in mitochondria
permeability transition (A); NAO staining (100 nM) served to assess loss in mitochondria cardiolipin
(B); HE staining (2 µM) served to assess the
presence of reactive oxygen species (C); and anti-APO.27 Ab
(2.5 µg/ml) served to detect a p38 antigen specific for apoptotic
mitochondria (D).
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Fig. 7.
Release of cytochrome c from
mitochondria to the cytoplasm in response to anti-Fas Ab, staurosporin,
or VP-16 is blocked in resistant Jurkat cells. Wild-type and
resistant Jurkat cells treated with anti-Fas Ab (200 ng/ml),
staurosporin (0.5 µM), or VP-16 (20 µM) for
8 h were assessed for redistribution of cytochrome c.
Following treatment, Jurkat cells were lysed and separated to an HM
fraction, which contains mitochondria, and a mitochondria-free S-100
fraction. The proteins were resolved by 15% SDS-PAGE and immunoblotted
by a cytochrome c-specific Ab. Equal protein loading was
ensured by protein quantification, and by reprobing the stripped
membranes with anti- -actin. Cytochrome c oxidase IV
served as a marker for mitochondrial fraction. The results shown are
representative of at least five independent experiments.
TM)
or tBid (His-tBid). These recombinant proteins have been previously demonstrated to efficiently induce cytochrome c release from
mitochondria (47-52). Purified mitochondria from sensitive or
resistant Jurkat cells were incubated for 30 min at 30 °C with
recombinant Bax or tBid. Following removal of mitochondria, the
resulting supernatant was tested by immunoblot analysis for the
presence of cytochrome c. As expected, cytochrome
c was released from mitochondria obtained from sensitive
Jurkat cells in response to either exogenous recombinant Bax or tBid
(Fig. 8, A and B).
No release of cytochrome c was detected in supernatants of
resistant mitochondria treated with these recombinant proteins. These
results suggest that the mitochondrial mechanism responsible for
release of cytochrome c in response to Bid, Bax, or cell
stimulation with VP-16 is impaired in mitochondria of resistant Jurkat
cells.
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Fig. 8.
Bid and Bax induce release of cytochrome
c from purified mitochondria of sensitive but not
resistant Jurkat cells. Purified mitochondria from wild-type or
resistant Jurkat cells were incubated with the indicated doses of
recombinant tBid (A and C) or recombinant Bax
(B) for 1 h at 30 °C. Mitochondria were then
pelleted by centrifugation, and the resulting supernatant
(Mit-Sup) was subjected to SDS-PAGE immunoblot analysis with
anti-cytochrome c Ab (A and B) and
anti-AIF Ab (C). Input of an equivalent amount of
mitochondria used for each treatment was verified by immunoblot
analysis of cytochrome c or AIF in control pellets of
wild-type and resistant Jurkat cells (Mit). The results
shown are representative of at least five independent
experiments.
-actin in the alkali-treated mitochondria (Ins)
further suggest that the fractions tested represent pure mitochondria, and therefore, the translocation of endogenous Bax to the mitochondria is not impaired in resistant cells. These results also demonstrate that
in Jurkat cells, as shown previously for HeLa cells (39) and for the
hematopoietic cells FL5.12 (53), a basal level of Bax is present within
the mitochondria prior to induction of apoptosis. To assess the
presence of endogenous tBid in the mitochondria, extracts of sensitive
or resistant Jurkat cells were treated with recombinant caspase-8 (100 nM) for 1 h at 37 °C to generate endogenous tBid.
The extracts were then separated into cytosol (S-100) and mitochondria
(HM). As shown in Fig. 9B, endogenous tBid (15 kDa) generated by exogenous recombinant caspase-8 was detected in
mitochondrial fractions from both sensitive and resistant Jurkat cells.
The use of
-actin as a marker for cytosolic proteins and VDAC as a
marker for mitochondrial outer membrane proteins provides evidence for
the presence of endogenous tBid in the mitochondria. To further determine whether exogenous tBid added to purified mitochondria could
translocate into the mitochondria, purified mitochondria from sensitive
or resistant Jurkat cells were incubated with recombinant tBid for 30 min at 37 °C. The mitochondria were then treated with 0.1 M Na2CO3, and alkali-sensitive
(attached) or alkali-resistant (membrane-inserted) fractions were
assessed for the presence of tBid by Western blot analysis. As
demonstrated in Fig. 9C, tBid was found inserted into the
mitochondria membrane in both sensitive and resistant cells. In these
studies, cytochrome c oxidase IV, a mitochondrial inner
membrane enzyme, and VDAC, a mitochondrial outer membrane protein,
served as markers for the mitochondrial matrix as well as a gel loading
control. These results suggest that the block in cytochrome
c or AIF release in resistant mitochondria is not mediated
by impaired translocation of either Bax or tBid to the
mitochondria.
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Fig. 9.
Expression of Bax or tBid in mitochondria of
resistant Jurkat cells. A, purified mitochondria from
sensitive or resistant cells were treated with 0.1 M
Na2CO3 (pH 11.5) for 20 min on ice. The
alkali-resistant mitochondrial fraction (insert (Ins)) was
lysed, resolved by SDS-PAGE, and immunoblotted for the presence of Bax.
Results obtained with mitochondria purified in two different
experiments are shown. The blot membrane was stripped and reprobed with
anti-VDAC mAb, stripped again, and probed with anti- -actin. No
-actin was detected in purified mitochondria following alkali
treatment (not shown). B, extracts of sensitive or resistant
Jurkat cells were treated with recombinant caspase-8 (100 nM) for 1 h at 37 °C. The extracts were separated
into cytosol (S-100) and mitochondrial (HM) fractions as described
under "Experimental Procedures." These two cellular fractions were
resolved by SDS-PAGE and probed by immunoblotting for the presence of
tBid. The blot membrane was stripped and reprobed with anti-
-actin
mAb and subsequently with anti-VDAC mAb, which served as cellular
fractionation controls. C, purified mitochondria from
sensitive or resistant Jurkat cells were incubated with recombinant
His-tBid (100 nM) for 1 h at 30 °C. Treated and
untreated mitochondria were washed in MIB and further treated with 0.1 M Na2CO3 (pH 11.5) for 20 min on
ice. Alkali-sensitive (attached (Att)) and alkali-resistant
(insert (Ins)) fractions were lysed, resolved by SDS-PAGE,
and immunoblotted for the presence of tBid. The membrane was stripped
and reprobed with anti-cytochrome c oxidase IV Ab and
subsequently with anti-VDAC mAb as markers for mitochondrial matrix and
as loading controls.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-irradiation-induced apoptotic
cascade was also suggested in a previous study (60). Clones of Jurkat
cells were reported to have a deficiency in caspase-8 activation in
response to Fas cross-linking and in cytochrome c release
following irradiation. In the present study, we identified the
mitochondrial mechanism responsible for the release of intermembrane proteins as the target of the block observed in the intrinsic apoptotic
cascade. The biochemical nature of this block and the mechanism
responsible for the cross-resistance between the mitochondrial and the
death receptor cascades remain to be elucidated.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Daniel E. Johnson (University of Pittsburgh) for Jurkat cells transfected with CrmA or Bcl-2.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health (NIH) Grant RO1 CA 84134-01 (to H. R.), NCI, NIH Grant PO1DE 12321-01 (to H. R.), a grant from The Pittsburgh Foundation (to H. R.), American Cancer Society Grant RPG-98-288-01-CIM (to H. R.), Department of Defense Grant BC981056 (to H. R.), and a grant from the Pennsylvania Department of Health (to H. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors contributed equally to this work.
** To whom correspondence should be addressed: University of Pittsburgh Cancer Inst., W952 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15213. Tel.: 412-624-0289; Fax: 412-624-7737; E-mail: rabinow@pitt.edu.
Published, JBC Papers in Press, November 3, 2000, DOI 10.1074/jbc.M006222200
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ABBREVIATIONS |
---|
The abbreviations used are: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; AIF, apoptosis-inducing factor; Apaf-1, apoptosis protease-activating factor 1; BAR, bifunctional apoptosis regulator; CMXRos, chloromethyl X-rosamine; DISC, death-inducing signaling complex; FADD, Fas-associated death domain; FLIP, FLICE-inhibitory protein; HE, hydroethidine/hydroethidium; HM, heavy membrane; NAO, nonyl acridine orange; PARP, poly(ADP-ribose) polymerase; tBid, truncated Bid; VDAC, voltage-dependent anion channel(s); XIAP, X-linked inhibitor of apoptosis; Ab, antibody; mAb, monoclonal antibody; MIB, mitochondria buffer; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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