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J. Biol. Chem., Vol. 277, Issue 1, 804-815, January 4, 2002
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,,,
,,,§¶¶
From the Division of Oncology Research, Mayo Clinic,
and § Department of Molecular Pharmacology, Mayo Graduate
School, Rochester, Minnesota 55905, the ¶ Institute of Cell and
Molecular Biology, University of Edinburgh, Edinburgh, EH9 3JR
Scotland, United Kingdom, the ** Division of Immunity and
Infection, Department of Rheumatology, Medical Research Council Centre
for Immune Regulation, University of Birmingham, Birmingham B15 2TT,
United Kingdom, and Elan Pharmaceuticals,
S. San Francisco, California 94080
Received for publication, February 12, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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MCF-7 human breast cancer cells are widely
utilized to study apoptotic processes. Recent studies demonstrated that
these cells lack procaspase-3. In the present study, caspase activation
and activity were examined in this cell line after treatment with the
microtubule poison paclitaxel. When cells were harvested 72 h
after the start of a 24-h treatment with 100 nM
paclitaxel, 37 ± 5% of the cells were nonadherent and displayed
apoptotic morphological changes. Although mitochondrial cytochrome
c release and caspase-9 cleavage were detectable by
immunoblotting, assays of cytosol and nuclei prepared from the
apoptotic cells failed to demonstrate the presence of activity that
cleaved the synthetic caspase substrates
LEHD-7-amino-4-trifluoromethylcoumarin (LEHD-AFC), DEVD-AFC, and
VEID-AFC. Likewise, the paclitaxel-treated MCF-7 cells failed to cleave
a variety of caspase substrates, including lamin A, Apoptosis is a morphologically and biochemically distinct form of
programmed cell death that occurs in many cell types after exposure to toxic stimuli (1, 2). Studies performed over the past 6 years have demonstrated that aspartate-directed cysteine proteases
called caspases play critical roles in the initiation and completion of
this process (3-8). In particular, caspase-mediated cleavages
destabilize structural components of the cytoskeleton, inactivate key
components of DNA repair pathways, and interrupt signal transduction
pathways involved in cell survival (8). At the same time, caspases
appear to activate a number of enzymes, including the caspase-activated
deoxyribonuclease (9), gelsolin (10, 11), and several kinases (8).
Collectively, these cleavages contribute to the stereotypic
morphological and biochemical changes (1) that constitute the process
of apoptosis.
Because of the importance of caspases in the apoptotic process, many
laboratories are currently performing enzymatic assays to assess the
activation of these proteases. In a typical assay, cells are lysed,
particulate matter is sedimented, and supernatants are incubated with
low molecular weight substrates that consist of a tetrapeptide or
pentapeptide coupled to a fluorogenic or chromogenic leaving group
(12-14). Previous results from this laboratory (13) and others (15),
for example, have used these types of assays to demonstrate that
multiple caspases with distinct substrate preferences are activated
during the process of apoptosis.
The MCF-7 human breast cancer line has been widely utilized to study
various components of the apoptotic machinery. Early studies
demonstrated that apoptotic DNA degradation in this cell line generates
high molecular weight DNA cleavage products but not internucleosomal
fragments (16). More recently, MCF-7 cells have been utilized to
investigate the cytotoxic effects initiated by ligation of various
death receptors (17-21), to examine the role of ceramide in
drug-induced apoptosis (22, 23), to study the actions of the
anticancer drug paclitaxel (24-26) and various hormones (24, 27-32),
and to evaluate the effect of cytoplasmic cytochrome c
injections on subsequent cell survival (33). Many of these studies were
performed under the assumption that MCF-7 cells contain a normal
complement of procaspases and scaffolding molecules required for their
activation. Additional studies, however, have revealed that MCF-7 cells
lack procaspase-3 polypeptide (33) as a consequence of a 47-bp deletion
within exon 3 of the procaspase-3 gene that alters the reading frame of
the message and results in an unstable truncated polypeptide (34).
Consistent with these observations, apoptotic MCF-7 cells resulting
from treatment with TNF- Previous studies from our laboratory have demonstrated that paclitaxel,
a chemotherapeutic agent that is widely used in the clinical treatment
of breast cancer (36, 37), induces apoptosis in MDA-MB-468 breast
cancer cells (38, 39). This process of paclitaxel-induced apoptosis was
accompanied by release of cytochrome c from mitochondria;
activation of caspases-3, -6 and -7; and cleavage of all caspase
substrates examined, including PARP, lamin A, lamin B1,
focal adhesion kinase, and topo I (39, 40). Previous studies (24, 40)
have demonstrated that MCF-7 cells also undergo paclitaxel-induced
apoptosis. Preliminary results, however, indicated that cleavage of
topo I, the single caspase substrate examined in our earlier study, was
undetectable in paclitaxel-treated MCF-7 cells (40).
In view of the widespread use of MCF-7 cells as a model system for the
study of apoptosis, we have reexamined the relationship between caspase
activation, substrate cleavage, and detection of caspase activity in
MCF-7 cells. Using a wide range of immunological probes, we demonstrate
in the present study that paclitaxel treatment in this cell line is
accompanied by release of cytochrome c to cytosol,
proteolytic cleavage of procaspases-9 and -7, and digestion of PARP and
ICAD, two polypeptides that can be cleaved by either caspase-3 or
caspase-7 (41, 42). In contrast, cleavage of additional polypeptides,
including topo I, the lamins, Materials--
Reagents were obtained from the following
suppliers: paclitaxel from Sigma; ECL and SuperSignalTM ULTRA enhanced
chemiluminescent reagents from Amersham Biosciences, Inc., and Pierce,
respectively; purified recombinant caspases-3 and -7 from Pharmingen
(San Diego, CA); DEVD-AFC from BioMol (Plymouth Meeting, PA); VEID-AFC,
LEHD-AFC, IETD-AFC, YVAD-AFC, and zVAD(OMe)-fmk from Enzyme Systems
Products (Dublin, CA); and zEK(bio)D-aomk (13) from the Peptide
Institute (Osaka, Japan).
Monoclonal antibodies to caspases-2, -3, and -7 as well as Cell Culture--
MDA-MB-468 cells (kindly provided by Dr. N. Davidson, Johns Hopkins Oncology Center, Baltimore, MD) were cultured
in improved minimal essential medium (Biofluids, Rockville, MD)
supplemented with 5% heat-inactivated fetal bovine serum, 100 units/ml
penicillin G, 100 µg/ml streptomycin, and 2 mM glutamine
in a humidified atmosphere containing 5% (v/v) CO2.
Untransfected MCF-7 cells (American Type Culture Collection, Manassas,
VA) were cultured in minimal essential medium containing Earle's
salts, 10% (v/v) fetal bovine serum, nonessential amino acids, 1 mM sodium pyruvate, and 10 µg/ml insulin according to the
supplier's instructions. MCF-7 cells were transfected with
procaspase-3 cDNA, the catalytically inactive C163S mutant, or
empty vector as described (46). After selection, cloned cell lines were
cultured in the same medium containing 750 µg/ml G418.
To assess the antiproliferative effects of paclitaxel on these cells,
aliquots containing 1200-1500 cells were plated in replicate 35-mm
plates and allowed to adhere for 14-16 h. Cells were then treated with
increasing concentrations of paclitaxel (added from a 1000-fold
concentrated stock in Me2SO) for 24 h, washed, and incubated in drug-free medium for 14 days to allow colony formation.
When log phase MCF-7 cells reached 40-60% confluence, the supernatant
was aspirated and replaced with the corresponding medium containing 100 nM paclitaxel, a concentration previously demonstrated to
induce apoptosis in breast cancer cell lines (38-40). Cells were
incubated for 24 h, washed, and incubated in drug-free medium for
48 h. At the completion of this incubation, nonadherent cells were
removed with the culture medium, and adherent cells were released by
trypsinization. Cells were sedimented at 200 × g for 10 min and processed for the various assays described below.
Whole Cell Lysates for Immunoblotting--
Sedimented cells were
washed once with ice-cold RPMI containing 10 mM HEPES (pH
7.4 at 4 °C); lysed by vigorous vortexing in 6 M
guanidine hydrochloride containing 250 mM Tris-HCl (pH 8.5 at 4 °C), 10 mM EDTA, 150 mM
Cell Fractionation, Caspase Assays, and Affinity
Labeling--
Subcellular fractions were prepared at 4 °C by a
minor modification of recently reported methods (39). In brief, cells
were homogenized in buffer A (25 mM HEPES (pH 7.5 at
4 °C), 5 mM MgCl2, 1 mM EGTA
supplemented immediately before use with 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, and 10 µg/ml
leupeptin) and then sequentially sedimented at 800 × g
for 15 min (nuclei plus cytoskeleton; see Ref. 48), 5000 × g for 15 min (mitochondria), and 280,000 × g for 60 min (lysosomes plus microsomes). Pellets obtained
at each step were resuspended in buffer A, sedimented again,
resuspended in buffer A, and stored in small aliquots at
Cleavage of DEVD-AFC, VEID-AFC, and LEHD-AFC was assayed as previously
described (13, 39). All reactions were run as end point assays using
100 µM substrate concentrations, 2-h reaction times at
37 °C, and 50-µg aliquots of protein from the indicated subcellular fractions. Standards containing 0-15 nmol of
7-amino-4-trifluoromethylcoumarin were utilized to determine the amount
of fluorochrome released. Control experiments indicated that the assays
were linear with respect to incubation time and enzyme content under
these conditions.
For affinity labeling of active caspases, aliquots containing 50 µg
of nuclear or cytosolic protein were incubated for 1 h at 20 °C
with 1 µM zEK(bio)D-aomk (13), diluted with 1/2 volume of 3× concentrated SDS sample buffer, heated to 95 °C for 3 min, and subjected to one-dimensional SDS-PAGE on 16% (w/v) acrylamide gels. Polypeptides were transferred to nitrocellulose, probed with
peroxidase-labeled streptavidin, and visualized using SuperSignalTM ULTRA chemiluminescent substrate. As a control, extracts prepared from
apoptotic chicken DU249 cells (E/X extracts; see Ref. 49) were
subjected to affinity labeling and applied to each blot.
Quantitation of Apoptotic Cells--
Adherent and nonadherent
cells were collected separately, sedimented at 200 × g
for 10 min, washed with ice-cold PBS, allowed to swell at 37 °C in
75 mM KCl as previously described (50), sedimented at
200 × g for 5 min, fixed in 3.7% formaldehyde in PBS
for 15 min at 21 °C, washed twice in PBS, stained with 1 µg/ml Hoechst 33258, and examined by fluorescence microscopy (51, 52). A
minimum of 500 cells/sample were scored for apoptotic changes
(chromatin condensation or nuclear fragmentation). Samples were
photographed using a Zeiss Axioplan microscope equipped with a N.A.
1.40 ×63 objective, a 365-nm excitation filter, and a 420-nm emission filter.
Localization of Caspase-3--
MDA-MB-468 cells and MCF-7/wt1
cells were treated with diluent or 100 nM paclitaxel,
washed, and incubated in drug-free medium for an additional 24-48 h.
Adherent and nonadherent cells were spun onto glass slides, air-dried,
fixed in 3% paraformaldehyde in PBS, permeabilized with TBST buffer
(150 mM NaCl, 50 mM Tris-HCl (pH 7.4 at
21 °C), 0.1% (w/v) Tween 20), and treated with 5.5% fetal bovine
serum in TBST to block nonspecific binding sites. Staining with
antiserum that recognizes a conformational epitope exposed only on
active caspase-3 followed by fluorescein-labeled anti-rabbit IgG was
performed according to the instructions of the supplier. After the last
wash, samples were stained with 1 µg/ml Hoechst 33258, mounted in
Vectashield (Vector Laboratories, Burlingame, CA) and examined using a
Zeiss LSM510 confocal microscope.
To perform transient transfections, MDA-MB-468 or parental MCF-7 cells
were transfected with 40 µg of plasmid encoding the C163S mutant of
procaspase-3 fused in frame with EGFP at its C terminus in the plasmid
pEGFP-N1 (CLONTECH) or the empty vector. Briefly,
log phase cells were trypsinized, washed twice in PBS, resuspended in
the buffer described by van den Hoff et al. (53), incubated
for 5 min with plasmid or empty vector, and permeabilized using a T840
square wave electroporator (BTX, San Diego, CA) delivering a 240-V
pulse for 10 ms. After return to tissue culture flasks for 24 h in
their respective media, cells were treated with diluent or 100 nM paclitaxel for 24 h followed by drug-free medium as described above. Adherent and nonadherent cells were then examined by
confocal microscopy.
Caspase-3 Deficiency in MCF-7 Cells but Not Other Breast Cancer
Cell Lines--
Previous studies have demonstrated that MCF-7 cells
lack caspase-3 (33, 34). To assess the possibility that down-regulation or deletion of caspase-3 or other components of the core apoptotic machinery might be a common feature in breast cancer cells, expression of Apaf-1 and procaspase-3, -6, -7, -8, and -9 was examined in 10 breast cancer cell lines (Fig. 1).
Results of this analysis confirmed that procaspase-3 was undetectable
in MCF-7 cells (top panel, lane
5). In contrast, procaspase-3 as well as Apaf-1 and procaspases-6, -7, -8, and -9 were detectable in all breast cancer cell
lines examined. Cytochrome c varied widely among the cell lines (bottom panel), possibly reflecting
differences in mitochondrial content, but was also universally
detectable.
Paclitaxel-induced Apoptosis in MCF-7 Cells--
In subsequent
experiments, the response of MCF-7 cells to paclitaxel was examined.
Initial experiments indicated that treatment of MCF-7 cells with
paclitaxel induced apoptosis in a concentration- and
time-dependent manner. In these experiments, cells were
incubated with increasing concentrations of paclitaxel for 24 h,
an exposure that mimics a widely utilized schedule for clinical
administration of this agent (36). Cells were then returned to
drug-free medium for 48 h and harvested. By 48 h after
removal of paclitaxel, 37 ± 5% of MCF-7 cells had detached from
their substratum. As indicated in Fig. 2,
A and B, paclitaxel also induced typical
apoptotic features, including chromatin condensation and nuclear
fragmentation, in a dose-dependent manner. As was the case
with MDA-MB-468 cells (39), paclitaxel-treated MCF-7 cells displaying
an apoptotic morphology were found exclusively in the detached cell
population. Treatment of MCF-7 cells with the broad spectrum caspase
inhibitor zVAD-fmk during the 48 h after the removal of paclitaxel
suppressed the appearance of apoptotic morphological features (Fig.
2C) as well as detachment of cells from the substratum (Fig.
2D), suggesting that caspases play a critical role in the
development of these paclitaxel-induced changes.
Failure to Detect Caspase Activity in MCF-7 Cytosol Despite Caspase
Activation--
In an attempt to determine which caspases might be
involved, cytosol and nuclei prepared from paclitaxel-treated MCF-7
cells were assayed for caspase activity using various fluorogenic
substrates. In these experiments, paclitaxel-treated MDA-MB-468 cells
served as a positive control. Activities that cleaved LEHD-AFC (a
preferred substrate of caspase-9) (8), DEVD-AFC (a well characterized substrate of caspases-3 and -7), and VEID-AFC (a preferred substrate of
caspase-6) were readily detected in cytosol and nuclei from nonadherent
(apoptotic) MDA-MB-468 cells (Fig. 3,
A-C). In contrast, activities that cleaved these substrates
did not detectably increase after paclitaxel treatment of MCF-7 cells.
In additional experiments, cleavage of YVAD-AFC (a preferred caspase-1
substrate), IETD-AFC (a preferred caspase-8 substrate), and zVAD-AFC (a
broad spectrum caspase substrate) was also undetectable in cytosol and
nuclei from paclitaxel-treated MCF-7 cells. Mixing experiments (Fig. 3D) failed to provide any evidence that cytosol from
apoptotic paclitaxel-treated MCF-7 cells contains a caspase inhibitor
or other substance that interfered with the tetrapeptide-based
fluorogenic assays. Instead, caspase activity was absent from the
cytosol. Consistent with this conclusion, affinity labeling with
zEK(bio)D-aomk, a reagent that reacts with a variety of active caspases
(13, 54), also failed to detect active caspase species in MCF-7 cytosol or nuclei (Fig. 3E). Collectively, these results raised
doubts about the conclusion that caspases participate in
paclitaxel-induced apoptosis in this model system.
In an attempt to resolve this apparent inconsistency, we examined
caspase activation and substrate cleavages in paclitaxel-treated MCF-7
cells. Previous studies (39, 55) demonstrated that paclitaxel treatment
results in mitochondrial release of cytochrome c followed by
activation of a caspase-9-initiated proteolytic cascade. Consistent with this model, cytochrome c was readily detected in
cytosol prepared from MCF-7 cells that had detached from their
substratum after paclitaxel treatment (Fig.
4A, upper
panel, lane 6). Although lower amounts
of cytochrome c were consistently detected in cytosol from
apoptotic MCF-7 cells compared with MDA-MB-468 cells (Fig. 4A, lanes 3 and 6), this
difference appeared to reflect the lower expression of cytochrome
c in MCF-7 cells (Fig. 1, bottom
panel). Moreover, release of cytochrome c in both
cell lines was accompanied by a decrease in procaspase-9 (Fig.
4C, upper panel, lanes
3 and 6) as well as the appearance of cleaved
caspase-9 species (Fig. 4D, upper
panel, lanes 3 and 6).
Likewise, the appearance of cytochrome c in the cytosol was
accompanied by cleavage of procaspase-7 (Fig. 4C,
third panel). Additional studies using antibodies
that recognize other procaspases (Fig. 4C) or cleaved
caspase species (Fig. 4D), however, revealed substantial
differences between the two cell lines. In particular, cleavage of
procaspase-6 was diminished in MCF-7 cells (Fig. 4C,
fourth panel). Moreover, cleavage of procaspase-8
(Figs. 4C, fifth panel) and
procaspase-10 (Fig. 4D, third panel)
to active caspase species (Fig. 4D, second and
third panels) was also markedly diminished. Thus,
only caspase-9 and possibly caspase-7 appeared to be activated in MCF-7
cells.
To provide further evidence for caspase activation in
paclitaxel-treated MCF-7 cells, we examined cleavage of a variety of polypeptide substrates. Paclitaxel treatment of MDA-MB-468 cells resulted in cleavage of PARP, ICAD, topo I, lamin B1,
lamins A and C,
To confirm that the altered pattern of substrate cleavages was due to
the absence of caspase-3, MCF-7 cell extracts were treated with
caspase-3 or caspase-7 (Fig. 5B). The addition of active caspase-3 to these lysates resulted in cleavage of PARP, topo I, lamin
B1, lamin A, Effect of Caspase-3 Transfection on Caspase-mediated Cleavages,
Caspase Activity, and Drug Sensitivity--
To determine whether the
lack of caspase-3 in MCF-7 cells contributed to the inability to detect
cleavage of fluorogenic substrates, MCF-7 cells were stably transfected
with wild type procaspase-3 or a catalytically inactive C163S mutant.
Indistinguishable results were obtained with two separate clones,
designated MCF-7/wt1 and MCF-7/wt2, each of which expressed wild type
procaspase-3 at levels comparable with those of MDA-MB-468 cells (Fig.
6A, inset).
Expression of procaspase-3 did not alter the sensitivity of MCF-7 cells
to paclitaxel as assessed using colony-forming assays (Fig.
6A) or morphological assessment of drug-induced apoptosis
(Fig. 6B). Moreover, expression of procaspase-3 did not
alter the release of cytochrome c to the cytosol (Fig.
6C, upper panel) or the amount of
cleaved caspase-9 detected in apoptotic MCF-7 cells (Fig.
6C, lower panel). After paclitaxel
treatment, procaspase-3 was quantitatively cleaved in procaspase-3
transfectants (Fig. 6D). Cleavage of procaspase-6, topo I,
lamin B1, lamin A, Sequestration of Cleaved Caspases after Activation in MCF-7
Cells--
In a final series of experiments, several approaches were
utilized to resolve the apparent discrepancy between caspase
activation, as assessed by substrate cleavages (Figs. 5A and
6D), and the lack of detectable caspase activity in cytosol
or nuclei of paclitaxel-treated MCF-7 cells (Figs. 3 and
6E). Whole cell lysates and cytosol were prepared from
caspase-3-transfected cells before and after paclitaxel treatment.
Immunoblot analysis revealed that procaspase-3 was readily detected in
cytosol of nonapoptotic cells (Fig. 7,
lanes 3, 4, 6,
7, 9, and 10). As expected,
procaspase-3 levels were diminished in the apoptotic cells (Fig. 7,
lanes 2, 5, 8, and 11). When the same blot were probed with a previously
characterized antiserum (43) raised against IETD, the peptide present
at the C terminus of the caspase-3 large subunit (8), species
corresponding to the 17- and 20-kDa forms of the caspase-3 large
subunit were readily detected in the whole cell lysates (Fig. 7,
lane 2, and data not shown). In contrast, cleaved
caspase-3 species were not detectable in cytosol from apoptotic MCF-7
transfectants (Fig. 7, lanes 5, 8, and
11). Similarly, a previously characterized antiserum that
recognizes the 35- and 19-kDa forms of active caspase-9 large subunit
(43, 57) detected cleaved caspase-9 in whole cell lysates (Fig. 7,
lane 2) but not in cytosol from transfected MCF-7
clones (Fig. 7A, lanes 5,
8, and 11).
To assess the fate of cleaved caspase species in these cells, MCF-7/wt1
cells were lysed under hypotonic conditions and subjected to
differential centrifugation (58) in order to isolate fractions enriched
in nuclei plus cytoskeleton (48), mitochondria, lysosomes plus
endoplasmic reticulum, and cytosol. MDA-MB-468 cells treated in a
similar fashion provided a basis for comparison. In nonapoptotic MDA-MB-468 cells, procaspases-3 and -9 were predominately found in the
cytosol (Fig. 8A,
lower panel, lanes 13 and
14). After induction of apoptosis with paclitaxel, cleaved
caspase species predominated in the nucleus plus cytoskeleton fraction
(Fig. 8A, lower panel, lane
6), although small amounts were also observed in cytosol and
the other cell fractions (Fig. 8A, lower
panel, lanes 9, 12, and
15). Consistent with these results, activity that cleaved
DEVD-AFC was detectable in fractions that contained cytosol, nuclei
plus cytoskeleton, or mitochondria from apoptotic MDA-MB-468 cells
(Fig. 8B). It is important to note, however, that the level
of activity did not precisely parallel the immunoreactive caspase-3
fragments (Fig. 8A), raising the possibility that part of
the cleaved caspase species present in fractions containing mitochondria and nuclei plus cytoskeleton might be inhibited.
Results obtained with MCF-7/wt1 cells were different in a number of
ways. First, more procaspase-3 and procaspase-9 were detected in
particulate fractions even before the induction of apoptosis (Fig.
8A, upper panel, lanes
4, 7, and 10). Second, the cleaved caspase species were detected exclusively in fractions containing nuclei plus cytoskeleton and mitochondria after treatment with paclitaxel (Fig. 8A, upper panel,
lanes 6 and 9). Thus, the failure to
detect appropriately cleaved caspase species in the cytosol of
MCF-7/wt1 cells (Fig. 7) does not reflect their destruction during the
cell fractionation procedure but rather their sequestration in
subcellular fractions that are sedimented during the preparation of
cytosol. On the other hand, activity capable of cleaving DEVD-AFC was
barely detectable (Fig. 8B) even in the MCF-7/wt1 fractions containing the largest amounts of cleaved caspase species (Fig. 8A). Identical results were obtained using LEHD-AFC and
VEID-AFC.2 Mixing experiments indicated that the
nucleus/cytoskeleton fraction from MCF-7/wt1 cells failed to inhibit
purified recombinant caspase-3 or DEVD-AFC cleavage activity in cytosol
from paclitaxel-treated MDA-MB-468 cells (data not shown; identical to
Fig. 3D), ruling out a diffusible inhibitor in this
fraction as well.
In further experiments, the localization of caspase-3 within MCF-7
cells was examined morphologically using two complementary approaches.
MDA-MB-468 cells again served as a basis for comparison. In the first
approach, cells were transiently transfected with cDNA encoding
procaspase-3 fused at its C terminus to EGFP. In nonapoptotic
MDA-MB-468 cells, this fusion protein was diffusely distributed
throughout the cytoplasm but largely excluded from nuclei (Fig.
9A). This pattern was distinct
from the distribution of EGFP alone, which was distributed throughout
the cells including the nuclei (data not shown). After paclitaxel
treatment, EGFP-labeled caspase-3 was still distributed diffusely in
apoptotic cells (Fig. 9B), although it was also concentrated
in punctate aggregates in some cells. The distribution of the
procaspase-3/EGFP fusion protein in MCF-7 cells paralleled that of
MDA-MB-468 cells prior to induction of apoptosis (Fig. 9D).
After paclitaxel treatment, however, the vast majority of the cleaved
caspase-3/EGFP was detected in large cytoplasmic aggregates with little
diffuse cytoplasmic labeling (Fig. 9E).
To evaluate the possibility that these observations resulted from the
use of transient transfection or the fusion construct, MDA-MB-468 cells
and MCF-7/wt1 cells were stained using an antiserum that recognizes
only cleaved caspase-3. Control experiments indicated that nonapoptotic
cells failed to stain with this serum.2 After paclitaxel
treatment, most apoptotic MDA-MB-468 cells displayed diffuse labeling
(short arrow, Fig. 9C). Although some punctate aggregates were observed (long arrow, Fig. 9C),
these were generally smaller than the aggregates observed after
procaspase-3/EGFP transfection (Fig. 9B). Apoptotic
MCF-7/wt1 cells, however, displayed striking punctate staining (Fig.
9F), consistent with the formation of cytoplasmic
aggregates. Additional fractionation experiments (Fig. 9G)
demonstrated that these cleaved caspase-3 species were resistant to
extraction with neutral detergent or 0.5 M NaCl but instead required In the present study, we have compared the apoptotic response
triggered by paclitaxel in MDA-MB-468, MCF-7, and caspase-3-transfected MCF-7 cells. Results of this analysis revealed that paclitaxel treatment of MCF-7 cells results in release of cytochrome c
to cytosol, proteolytic cleavage of procaspases-9 and -7, and digestion of some caspase substrates but not others. Surprisingly, activities that cleave widely utilized low molecular weight model substrates were
undetectable in cytosol and nuclei from these apoptotic cells. Transfection with wild type procaspase-3 partially restored the apoptotic digestion of caspase-3 substrates but did not restore detectable activities that cleave low molecular weight substrates. Instead, appropriately cleaved caspases-9 and -3 were sequestered in a
sedimentable fraction and were not active when assayed. These results
have important implications for current understanding of the fate of
activated caspases in apoptotic epithelial cells and for interpretation
of assays that are widely used to study caspase activation during apoptosis.
Previous studies from our laboratory (39) and others (55) have
suggested that paclitaxel induces apoptosis by activating the
cytochrome c/Apaf-1/caspase-9 pathway. In paclitaxel-treated MDA-MB-468 cells, however, we also observed proteolytic cleavage of
procaspase-8 and procaspase-10 (Fig. 4, C and D).
Some studies have raised the possibility that anticancer drugs might
induce apoptosis through a death receptor pathway (59, 60), whereas other studies have suggested that procaspase-8 might be activated downstream of caspase-3 in cytochrome c-initiated pathways
(61). The marked decrease in cleavage of procaspases-8 and -10 in MCF-7 cells strongly suggests that these cleavages occur downstream of
procaspase-3, providing additional support for the idea that paclitaxel-induced apoptosis primarily involves activation of the
cytochrome c/Apaf-1 pathway.
A variety of additional polypeptides are cleaved in paclitaxel-treated
MDA-MB-468 cells but not MCF-7 cells (Fig. 5A). These differences were observed despite cleavage (Fig. 4C) and
presumed activation (41) of caspase-7 in apoptotic MCF-7 cells.
Transfection of MCF-7 cells with procaspase-3 resulted in
apoptosis-associated cleavage of these caspase substrates (Fig.
6D). Even though caspases-3 and -7 have similar abilities to
cleave the tetrapeptide substrate DEVD-AFC (62, 63), these observations
suggest that the substrate specificities of these enzymes toward
macromolecular peptides are overlapping but nonidentical. Consistent
with this conclusion, recombinant caspase-7 was unable to cleave a
variety of caspase-3 substrates, including topo I, protein kinase C These observations differ from the conclusions of Jänicke
et al. (35), who reported that all caspase substrates except fodrin were cleaved normally in TNF- Tang and Kidd (65) have reported that additional apoptotic changes,
including detachment of cells from the substratum and fragmentation of
nuclei into apoptotic bodies, are also absent from staurosporine- or
TNF- Despite the presence of cleaved caspase-9 (Fig. 4D), as well
as cleavage of a number of presumed caspase substrates (Fig. 5A), we were unable to detect activities capable of cleaving
the tetrapeptide substrates DEVD-AFC, VEID-AFC, or LEHD-AFC in cytosol or nuclei from paclitaxel-treated MCF-7 cells (Fig. 3,
A-C). These results were in marked contrast to the
~50-fold increases in cleavage activities observed in the same
experiments using cell fractions from MDA-MB-468 cells. Although others
have reported detectable levels of DEVD-AFC cleavage activity after
treatment of MCF-7 cells with hydrogen peroxide (68), TNF- While the present studies were being prepared for publication, other
groups reported that DEVD-AFC cleavage activity was detectable in
cytosol prepared from procaspase-3-transfected MCF-7 cells after
treatment with cisplatin, doxorubicin, or etoposide (70-72). The
differences between these results and the present observations might
reflect different levels of expression of procaspase-3 or the use of
different apoptotic stimuli. Procaspase-3 levels in the recent
publications were not compared with those in similar cell lines. In
contrast, the transfectants utilized in the present study were shown to
express procaspase-3 at levels comparable with those of other breast
cancer lines (Fig. 6A, inset, and Fig. 1). In additional
experiments, we were unable to detect DEVD-AFC cleavage activity in
cytosol or nucleus plus cytoskeleton fractions from MCF-7/wt1 cells
after apoptosis was triggered with prolonged nocodazole or cisplatin
treatments.2 Similar results were obtained when the
procaspase-3-transfected cells of Faliero and Lazebnik (71) were
subjected to the same analysis.2 Collectively, these
observations suggest that the results shown in Figs. 6-8 are not
unique to paclitaxel-induced apoptosis or the transfected MCF-7
isolates we initially studied.
The results presented in Figs. 6-9 are consistent with the
observations of McFarlane et al. (73), who reported that
exogenous epitope-tagged caspases were bound to cleaved cytokeratins
after treatment of MCF-7 cells with high concentrations of
TNF- Further evaluation revealed that the cleavage of caspase-3 substrates
after paclitaxel treatment was less extensive in transfected MCF-7
cells than in MDA-MB-468 cells (cf. Fig. 5A and
6D) even though the transfected MCF-7 cells express as much
procaspase-3 as MDA-MB-468 cells (Fig. 6A,
inset). This disparity raises the possibility that caspase-3
might be inhibited or inactivated in situ before its
substrate cleavages are complete. Consistent with this possibility, we
have observed that DEVD-AFC cleaving activity is at the limit of
detection even when MCF-7/wt1 or MCF-7/wt2 fractions containing
properly cleaved caspases-3 and -9 are assayed (Fig. 8B,
nuclei plus cytoskeleton and mitochondria).2 It is possible
that the inability to detect caspase enzymatic activity when fractions
from parental MCF-7 cells are examined (Fig. 3) also reflects a process
that inhibits caspase activity. Consistent with this possibility, we
observed that the caspase inhibitor XIAP co-fractionated with cleaved
caspase species (Fig. 9G). An association between XIAP,
which can act as a ubiquitin ligase (74) for caspases, might also
explain the lower amounts of cleaved caspase-9 observed in MCF-7 cells
compared with MDA-MB-468 cells (Fig. 4D) even though similar
amounts of procaspase-9 have been cleaved in the two cell lines (Fig.
4C). The requirement for denaturing conditions to solubilize
XIAP (Fig. 9G) provides a potential explanation for the
inability to detect a soluble caspase inhibitor in MCF-7 cell fractions
(Fig. 3D and data not shown). On the other hand, this
requirement for denaturing conditions to solubilize XIAP and cleaved
caspase species (Fig. 9G)3 also precluded
coimmunoprecipitation experiments designed to determine whether XIAP
and cleaved caspases were physically associated. Thus, the conclusion
that caspases in MCF-7 cells are inhibited in addition to being
sequestered must be considered tentative. We also cannot at present
rule out the possibility that caspase-3 and -9 are bound to other
inhibitory polypeptides in the sedimentable fraction. In either case,
the fact that the inhibition of activity observed in these cellular
fractions is accompanied by diminished cleavage of caspase-3 substrates
in situ compared with other cell lines raises the
possibility that the inhibition of caspase activity observed in MCF-7
and MCF-7/wt1 cells might reflect a physiological process that serves
to limit caspase activity in certain cell types.
The observation that cleaved caspases can be sequestered and possibly
inhibited has two potentially important implications for methods that
are currently utilized to assess caspase activation. First, methods
that involve preparation of cytosol followed by enzymatic assay or
immunoblotting will potentially miss caspase activation. The MCF-7 line
represents the extreme of this phenomenon, but our results in
MDA-MB-468 cells indicate that a substantial portion of the cleaved
caspase species in this cell line are also found in the
nuclear/cytoskeleton fraction (Fig. 8A). Second, methods
that rely solely upon cleavage of tetrapeptide-based substrates also
have the potential to underestimate caspase activation. Both the
appearance of properly cleaved caspase species on immunoblots (Figs.
4D, 7, and 8A) and the cleavage of caspase
substrates in situ (Figs. 5A and 6D)
clearly indicate that caspases have been activated in MCF-7 cells, yet
assays based on tetrapeptide substrates detect little if any activity
(Figs. 3, A-C, 6E, and 8B). The ability to detect activity in the same fractions prepared from a
control cell line in the same experiments (Figs. 3, A-C,
6E, and 8B) argues that the difficulty in
detecting DEVD-AFC, VEID-AFC, and LEHD-AFC activity in MCF-7 low speed
pellets is not due to trivial technical difficulties. Instead, these
observations raise the possibility that inhibition of caspase activity
might occur in some subcellular fractions. This limitation must be kept
in mind when negative assays for caspase activation are encountered.
-catenin,
gelsolin, protein kinase C
, topoisomerase I, and procaspases-6, -8, and -10. Transfection of MCF-7 cells with wild type procaspase-3
partially restored cleavage of these polypeptides but did not result in
detectable activities that could cleave the synthetic caspase
substrates. Immunoblotting revealed that caspase-9, and -3, which were
proteolytically cleaved in paclitaxel-treated MCF-7/caspase-3 cells,
were sequestered in a salt-resistant sedimentable fraction rather than
released to the cytosol. Immunofluorescence indicated large cytoplasmic aggregates containing cleaved caspase-3 in these apoptotic cells. These
observations suggest that sequestration of caspases can occur in some
model systems, causing tetrapeptide-based activity assays to
underestimate the amount of caspase activation that has occurred
in situ.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
or staurosporine were reported to lack activity that cleaves DEVD-AFC,
a substrate of active caspase-3 (35). Despite this lack of caspase-3
protein and activity, cleavage of all caspase substrates except
-fodrin was initially reported to be normal in MCF-7 cells (35).
-catenin, gelsolin, protein kinase
C, and procaspases-6, -8, and -10, is undetectable after paclitaxel
treatment. Transfection of procaspase-3 into MCF-7 cells partially
restores the paclitaxel-induced cleavage of this second group of
polypeptides. Interestingly, however, cleavage of tetrapeptide
substrates such as DEVD-AFC, LEHD-AFC, and VEID-AFC remains extremely
low after paclitaxel treatment of procaspase-3-transfected MCF-7 cells.
Further studies using an antiserum that recognizes a neoepitope at the
carboxyl terminus of the caspase-3 large subunit (43) indicate that
procaspase-3 is cleaved appropriately in these cells but is not
released to the cytosol. Instead, it accumulates in a detergent- and
salt-resistant fraction that sediments at low speed. These observations
not only provide additional evidence that caspase-3 is required for the cleavage of a variety of nuclear and cytoplasmic substrates but also
indicate that sequestration of cleaved caspases can result in
underestimation of enzyme activity when caspase assays are performed
using widely accepted methods.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin
and gelsolin were purchased from Transduction Laboratories (Lexington,
KY). Monoclonal antibodies to cytochrome c and polyclonal serum against ICAD were obtained from Pharmingen. Polyclonal rabbit sera that recognize protein kinase C, procaspase-6, a neoepitope at
the amino terminus of the PARP 89-kDa fragment (44), glutathione S-transferase 
, and an active conformation of caspase-3
were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), Upstate Biotechnology, Inc. (Lake Placid, NY),
CLONTECH (Palo Alto, CA), Biotrin International
(Dublin, Ireland), and Cell Signaling Technology (Beverly, MA),
respectively. Monoclonal antibodies against PARP, topo I, and heat
shock protein 90 were kindly provided by Drs. G. Poirier (Laval
University School of Medicine, Ste-Foy, Quebec), Y-C. Cheng (Yale
University School of Medicine, New Haven, CT), and David Toft (Mayo
Clinic, Rochester, MN), respectively. Monoclonal antibodies that
recognize caspase-9 and Apaf-1 were a kind gift from Y. Lazebnik (Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY). Polyclonal sera that
recognize lamin A and lamin B1 were generated and
characterized as previously described (45). Rabbit antiserum that
recognizes caspase-10 was raised by injecting female New Zealand White
rabbits with the large subunit of recombinant human caspase-10, which
was expressed as a fusion protein with glutathione
S-transferase using the pGEX-2T plasmid (Amersham Pharmacia
Biotech), purified by affinity chromatography on glutathione-agarose,
released from the fusion protein with thrombin, subjected to SDS-PAGE,
and excised from the gel (43). Rabbit antisera that recognize the
neoepitopes generated upon activation of caspase-9 (PEPD), caspase-3
(IETD), and caspase-8 (VETD) were raised by synthesizing each peptide
coupled to the C terminus of cysteine and conjugating each pentapeptide
to keyhole limpet hemocyanin prior to injection. The caspase-9 and
caspase-3 neoepitope sera were previously characterized in detail (43). Characterization of the caspase-8 neoepitope serum is described in the
legend to Fig. 4B. Affinity-purified secondary antibodies coupled to peroxidase or fluorescein were from KPL (Gaithersburg, MD).
-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride; and prepared for electrophoresis as recently described in
detail (39). After protein was determined by the bicinchoninic acid
method (47), lyophilized samples were resuspended at a concentration of
5 mg of protein/ml in SDS sample buffer (4 M deionized
urea, 2% (w/v) SDS, 62.5 mM Tris-HCl (pH 6.8 at 21 °C), and 1 mM EDTA) and heated to 65 °C for 20 min. Aliquots
containing 50 µg of total cellular protein were subjected to SDS-PAGE
on gels with 5-15% (w/v) acrylamide gradients, transferred to
nitrocellulose or polyvinylidene difluoride, and probed with antibodies
using standard techniques (13).
70 °C in
buffer A containing 5 mM EDTA and 2 mM
dithiothreitol. In some experiments, spins at 800 and 280,000 × g were the only ones performed (39).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Examination of components of the core
apoptotic machinery in human breast cancer cell lines. Log phase
cultures of the indicated line were solubilized in SDS sample buffer.
Aliquots containing 50 µg of protein (quantitated by the nitric acid
method) (75) were subjected to SDS-polyacrylamide gel electrophoresis
followed by immunoblotting with reagents that recognize the indicated
polypeptides. Although deletion of components of the core death
machinery has been detected in other cell lines (76), MCF-7 cells are
the only breast cancer line observed to lack a procaspase.
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Fig. 2.
Characterization of paclitaxel-induced
apoptosis in MCF-7 cells. A, cells were treated with
the indicated concentration of paclitaxel for 24 h, washed,
incubated in drug-free medium for 48 h, fixed, stained with
Hoechst 33258, and examined by fluorescence microscopy to determine the
percentage of cells that were apoptotic. Insets, untreated
cells (upper inset) or MCF-7 cells that detached
during the 48 h after the removal of 100 nM paclitaxel
(lower inset) were stained with Hoechst 33258 and
examined by fluorescence microscopy. Arrowhead, example of
nucleus with condensed chromatin. Arrow, example of
fragmented nucleus. B, electron micrograph of nonadherent
MCF-7 cell harvested 72 h after the start of a 24-h treatment with
100 nM paclitaxel. Note the extensive vacuolization of
cytoplasm and fragmentation of the condensed nucleus. C and
D, effect of zVAD-fmk on development of apoptotic
morphological changes (C) and cell detachment
(D). MCF-7 cells were treated with 100 nM
paclitaxel (+) or diluent ( ) for 24 h, washed, and incubated for
48 h in fresh medium lacking () or containing (+) 100 µM zVAD-fmk. At the completion of the incubation, cells
were fixed, stained with Hoechst 33258, and scored as mitotic,
multinucleated, apoptotic, or normal interphase cells (C).
Only the first three categories of cells are shown in this graph.
Alternatively, nonadherent versus adherent cells were
counted (D). Error bars, mean ± S.D. of three separate experiments.
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Fig. 3.
Absence of detectable caspase activity or
affinity labeling in cytosol and nuclei of paclitaxel-treated MCF-7
cells. A-C, MCF-7 cells were treated with 100 nM paclitaxel (+) or diluent ( ) for 24 h, washed,
incubated in drug-free medium for 48 h, and harvested. The
280,000 × g supernatant (cytosol) and 800 × g pellet (nuclei plus cytoskeleton) from adherent
(A) and nonadherent (F) cells were assayed for
ability to cleave DEVD-AFC (panel A), VEID-AFC
(panel B), or LEHD-AFC (panel
C). Fractions from paclitaxel-treated MDA-MB-468 cells
served as a control. D, aliquots containing a constant
amount of cytosol from nonadherent paclitaxel-treated MDA-MB-468 cells
(30 µg of protein) were combined with aliquots of cytosol containing
0-60 µg of protein from nonadherent paclitaxel-treated MCF-7 cells.
After a 5-min preincubation, samples were incubated with DEVD-AFC for
2 h as described under "Experimental Procedures."
E, adherent (A) or nonadherent (F)
cells harvested 48 h after removal of diluent () or 100 nM paclitaxel (+) were fractionated as described for
panels A-C and subjected to affinity labeling
with zEK(bio)D-aomk. Two aliquots of DU249 apoptotic cell extracts
(EX) (49) were subjected to affinity labeling in parallel.
Because of the number of samples involved, nonadjacent lanes from two
simultaneously labeled blots (both containing labeled DU249 apoptotic
cell extracts) were juxtaposed to compose this panel.
Results are representative of two (E) or three
(A-D) separate experiments.
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Fig. 4.
Analysis of cytochrome c
release and caspase activation in paclitaxel-treated MCF-7
cells. A, after treatment of MCF-7 cells with
paclitaxel or diluent as described under "Experimental Procedures,"
aliquots containing 50 µg of cytosolic protein from adherent
(A) or nonadherent (F) cells were subjected to
SDS-PAGE followed by blotting with anti-cytochrome c
antibody. Paclitaxel-treated MDA-MB-468 cells served as a positive
control. To confirm equivalent loading of the samples, the blot was
reprobed with antiserum against GST . B, to characterize
antisera raised against the large subunit of caspase-10 and a
neoepitope at the C terminus of the caspase-8 large subunit (see
"Experimental Procedures"), aliquots of purified, recombinant
caspases-3, -7, -8, and -9 as well as the large subunit of caspase-10
were subjected to SDS-PAGE followed by transfer to nitrocellulose and
reaction with the indicated serum. C and D,
whole cell lysates were prepared from adherent (A) or
nonadherent (F) MCF-7 cells after treatment with paclitaxel
(+) or diluent (). After SDS-PAGE and transfer to nitrocellulose,
samples were probed with antisera that recognize the
indicated caspase species. Paclitaxel-treated MDA-MB-468 cells served
as a positive control. The two species of procaspase-8 and
procaspase-10 detected in these cells presumably reflect previously
reported splice variants (77-79). Arrows, cleaved caspase
species detected by the various sera. Note that activation of
procaspases-6, -8, and -10 is diminished in MCF-7 cells. Results are
representative of three separate experiments.
-catenin, gelsolin, and protein kinase C (Fig.
5A, lane 3). For many of these substrates, discrete fragments that
had the apparent molecular weights of previously reported caspase cleavage products (56) were detected (Fig. 5A,
arrows, and data not shown). A different pattern emerged in
paclitaxel-treated MCF-7 cells. PARP and ICAD were both cleaved after
paclitaxel treatment (Fig. 5A, lane
6). The resulting 89-kDa PARP fragment reacted with an
anti-neoepitope serum raised against the peptide GIDE (Fig.
5A, second panel), confirming that at
least one caspase capable of cleaving the sequence DEVD
GIDE (44) was
activated in paclitaxel-treated MCF-7 cells. In contrast, cleavage of
topo I, lamin B1, lamins A and C, -catenin, gelsolin,
and protein kinase C was undetectable in these cells.
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Fig. 5.
Selective cleavage of some substrates in
paclitaxel-treated MCF-7 cells and in vitro. A,
after MCF-7 cells were treated with 100 nM paclitaxel (+)
or diluent ( ) for 24 h, washed, and incubated for 48 h in
drug-free medium, whole cell lysates were prepared. Aliquots containing
50 µg of protein were subjected to SDS-PAGE, transferred to
nitrocellulose, and probed with reagents that recognize the indicated
polypeptides. Paclitaxel-treated MDA-MB-468 cells served as a positive
control for substrate cleavages. Arrows, cleavage products
that correspond in molecular weight to caspase-generated fragments
described in previous papers (56). The same samples shown in Fig. 4
were utilized for this experiment, which is representative of three
independent experiments. B, selective cleavage of some
caspase-3 substrates and not others by caspase-7. Cytosol (280,000 × g supernatant) and nuclei (800 × g
pellet) from MCF-7 cells were combined and treated with buffer,
purified recombinant caspase-3, or purified recombinant caspase-7.
After a 30-min incubation at 37 °C, samples were diluted with 3×
SDS sample buffer and subjected to SDS-PAGE. Blots were probed with
reagents that recognize the indicated polypeptides.
-catenin, gelsolin, protein kinase C, and procaspase-6 (Fig. 5B, lane 2, and
data not shown). In contrast, cleavage of PARP but not the other
substrates was observed when lysates were treated with caspase-7 (Fig.
5B, lane 3). Thus, the selective
cleavage of some caspase substrates but not others in paclitaxel-treated MCF-7 cells (Fig. 5A) appears to be
explained by the activation of caspase-7 but not caspase-3 in these
cells. Nonetheless, these results failed to explain the inability to detect active caspase species using activity assays and affinity labeling procedures (Fig. 3).
-catenin, gelsolin, and protein
kinase C was restored in transfectants containing wild type
procaspase-3 (Fig. 6D and data not shown), although the
cleavages were somewhat less complete than those observed in MDA-MB-468
cells (cf. Fig. 5A). Despite the cleavage of
these substrates, cytosol from transfected cells did not contain
detectable DEVD-AFC cleavage activity after paclitaxel treatment (Fig.
6E). Identical results were obtained when DEVD-AFC was
replaced with LEHD-AFC or
VEID-AFC.2
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Fig. 6.
Characterization of MCF-7 cells transfected
with wild type procaspase-3 (wt1, wt2) or the catalytically inactive
C163S mutant. A, sensitivity to paclitaxel as assessed
by colony-forming assays. The indicated cell lines were treated for
24 h with varying concentrations of paclitaxel, washed, and
incubated in drug-free medium for 14 days to allow colonies to form.
Note that wild type and transfected MCF-7 cells were equally sensitive
to the antiproliferative effects of paclitaxel. Inset,
comparison of procaspase-3 levels in two separate clones of MCF-7 cells
transfected with wild type procaspase-3 (wt1 and wt2) and a clone
transfected with the C163S mutant of procaspase-3. Untransfected
MDA-MB-468 cells are shown for comparison. Aliquots containing 50 µg
of total cellular protein from log phase cells were subjected to
SDS-PAGE followed by blotting with anti-procaspase-3. Nonadjacent lanes
from a single immunoblot were juxtaposed to produce this
panel. Blotting with anti-histone H1 (not shown) confirmed
equivalent loading of lanes. B, sensitivity as assessed by
induction of apoptosis. Cells were treated for 24 h with varying
concentrations of paclitaxel, washed, and incubated in drug-free medium
for 48 h. Adherent and nonadherent cells were separated, counted,
and stained with Hoechst 33258. Various cell lines are denoted by the
same symbols as in A. C, cytosol
(top panel) or whole cell lysates
(bottom panel) were prepared from nonadherent
cells harvested 72 h after the start of a 24-h paclitaxel
treatment. Aliquots containing 50 µg of protein were subjected to
SDS-PAGE followed by immunoblotting with reagents that recognize the
indicated polypeptides. D and E, analysis of
caspase substrate cleavage and cytosolic caspase activity in
caspase-3-transfected MCF-7 cells. The indicated cells were treated for
24 h with 100 nM paclitaxel (+) or diluent ( ),
washed, and incubated for 48 h in drug-free medium. Whole cell
lysates from adherent (A) or nonadherent (F)
MCF-7/wt1 cells were harvested and subjected to immunoblotting
(D). Similar blotting results were obtained with MCF-7/wt2
cells as well.2 Alternatively, cytosol was prepared and
assayed for ability to cleave DEVD-AFC (E). Similar results
were also obtained with VEID-AFC and LEHD-AFC.2 Results are
representative of three separate experiments.
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Fig. 7.
Failure to detect cleaved caspase species in
cytosol of caspase-3-transfected MCF-7 cells. The indicated clone
of MCF-7 cells was treated for 24 h with 100 nM
paclitaxel (+) or diluent ( ), washed, and incubated for 48 h in
drug-free medium. Aliquots containing 50 µg of total cellular
protein (lanes 1 and 2) or cytosolic
protein (228,000 × g supernatant; lanes
3-11) prepared from adherent (A) or nonadherent
(F) cells were subjected to SDS-PAGE followed by
immunoblotting with monoclonal antibodies that recognize procaspases-9
and -3 or previously characterized antisera that specifically recognize
cleaved forms of caspases-9 and -3 (43). An antiserum that
recognizes heat shock protein 90 (HSP90) was utilized to
confirm loading of cytosolic protein in lanes
3-11. Results are representative of three independent
experiments.
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Fig. 8.
Caspase localization and activity in
MCF-7/wt1 and MDA-MB-468 cells. A, distribution of
procaspases and cleaved caspases during cell fractionation. MCF-7/wt1
(top panel) and MDA-MB-468 cells
(bottom panel) were treated for 24 h with
100 nM paclitaxel (+) or diluent ( ), washed, and
incubated in drug-free medium until harvest. Adherent (A) or
nonadherent (F) cells were harvested for preparation of
whole cell lysates. Duplicate aliquots were lysed under hypotonic
conditions and fractionated by differential centrifugation as described
under "Experimental Procedures." Aliquots containing 50 µg of
protein were subjected to SDS-PAGE and probed with reagents that
recognize the indicated polypeptides. Note the different distributions
of procaspases and cleaved caspases in the two cell lines.
B, DEVD-AFC cleavage activity in selected subcellular
fractions. MDA-MB-468 cells (cross-hatched bars) and
MCF-7/wt1 cells (black bars) were treated for
24 h with 100 nM paclitaxel (+) or diluent (),
washed, and incubated in drug-free medium until harvest. Adherent
(A) or nonadherent (F) cells were harvested for
preparation of subcellular fractions by differential centrifugation.
Aliquots containing 50 µg of protein were assayed for the ability to
cleave DEVD-AFC as described under "Experimental Procedures."
Similar results were using with VEID-AFC and LEHD-AFC as
substrates.2 Results in each panel are
representative of at least three experiments.
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Fig. 9.
Active caspase-3 is localized in salt- and
detergent-resistant cytoplasmic aggregates in paclitaxel-treated MCF-7
cells. A, B, D, and E,
MDA-MB-468 cells (A and B) or MCF-7 cells
(D and E) were transiently transfected with C163S
procaspase-3 fused to EGFP. Localization was determined in cells
treated with diluent (A and D) or cells that
became nonadherent after paclitaxel treatment (B and
E). C and F, MDA-MB-468 cells and
MCF-7/wt1 cells that became apoptotic after paclitaxel treatment (see
"Experimental Procedures") were stained with a polyclonal antiserum
that recognizes only the active conformation of caspase-3. Nonapoptotic
cells failed to stain with this serum (not shown). Short
arrow in C, cell with diffuse staining. Long
arrow in C, cell with small punctate regions of
enhanced staining. G, MCF-7 cells were treated with 100 nM paclitaxel for 24 h followed by drug-free medium
for 48 h. The 12,000 × g pellet prepared from
nonadherent cells (lane 1) was treated for 30 min
at 4 °C with buffer A containing 1% (w/v) Nonidet P-40
(lanes 2 and 3), 0.5 M
NaCl and 25 µg/ml DNase I (lanes 4 and
5), or 4 M urea (lanes 6 and 7). Aliquots of 12,000 × g supernatants
(S) and pellets (P) resulting from these
extractions (50 µg of protein in each lane) were subjected to
SDS-PAGE followed by immunoblotting with reagents that detected the
indicated polypeptides.
4 M urea for efficient
solubilization.3 Although we
noted that the caspase inhibitor XIAP was also present in the 5000 × g pellet and displayed a similar extraction profile (Fig.
9G), the requirement for denaturing conditions to solubilize these polypeptides precluded immunoprecipitation studies to determine whether the two polypeptides were physically associated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
gelsolin, -catenin, and procaspase-6, when added to MCF-7 cell
lysates (Fig. 5B). These results indicate that cleavages of
certain substrates require active caspase-3.
- or staurosporine-treated MCF-7
cells. It is possible that these conflicting conclusions might reflect
differences in the apoptotic response of MCF-7 cells from different
sources (21, 64), the use of different stimuli, or the examination of
different substrates. The present observations, however, are in
agreement with the more limited results of Tang and Kidd (65) and
Kirsch et al. (26), who noted that cleavages of gelsolin and
Bcl-2 are diminished in apoptotic MCF-7 cells, and with the report of
Zheng et al. that lamin B1 is not cleaved during
CD95-mediated apoptosis in caspase-3
/ hepatocytes (66).
Our observations not only expand the list of
caspase-3-dependent substrates, but also confirm that
caspase-7 is unable to cleave these substrates in vitro.
-treated MCF-7 cells as a consequence of their caspase-3
deficiency (65). Interestingly, the absence of these features of the
cell death pathway appears to depend on the stimulus or the strain of
MCF-7 cells analyzed. In our hands, paclitaxel treatment results in
detachment of the parental MCF-7 cells from their substratum (Fig.
2D). The ability of zVAD-fmk to inhibit this process (Fig.
2D) suggests that detachment might depend on the activity of
another caspase in these caspase-3-deficient cells. Likewise,
paclitaxel-treated MCF-7 cells undergo nuclear fragmentation when they
become apoptotic (Fig. 2, A and B; Ref. 25)
despite the absence of caspase-3. These results are consistent with the
previous demonstration of nuclear fragmentation in other caspase-3-deficient cells (67).
(23, 27,
69), or agonistic anti-Fas antibodies (69), it is important to stress that these levels were extremely low in comparison with other lines
(69). Our subsequent experiments indicated that the paucity of DEVD-AFC
cleavage activity was not due to the presence of a soluble inhibitor or
interfering substance (Fig. 3D). In addition, cytosolic
DEVD-AFC cleavage activity was not restored by transfection of MCF-7
cells with procaspase-3 (Fig. 6E). Instead, the paucity of
cytosolic cleavage activity appeared to reflect, at least in part,
altered trafficking of cleaved caspase-3 and -9 species. Fractionation
studies demonstrated that these species were recovered in fractions
that sedimented at 
5000 × g rather than in cytosol (Figs. 7 and 8A). Two different morphological approaches
(Fig. 9, E and F) demonstrated that cleaved
caspase-3 species were present in cytoplasmic aggregates. This is in
contrast to the more diffuse localization of active caspase-3 seen in
MDA-MB-468 cells, particularly MDA-MB-468 cells expressing
physiological levels of procaspase-3 (Fig. 6A,
inset, and Fig. 9C). Collectively, these
experiments provide evidence for trafficking of the active caspase
species in MCF-7 cells to a subcellular compartment that resists
solubilization under the conditions usually employed to isolate
cytosol, a process we term "sequestration."
-related apoptosis-inducing ligand, an activator of the death
receptor pathway. The present results, however, extend the observations of McFarlane et al. in a number of ways. First, our
experiments have shown that trapping of cleaved caspases-3 and -9 in a
particulate fraction can also occur after activation of the
mitochondrial pathway. Second, we have shown that this trapping occurs
independent of the use of epitope tags to follow caspase species.
Third, we have examined the effect of this trapping on caspase activity in situ and in subcellular fractions prepared from apoptotic
MCF-7 cells. These experiments demonstrate that transfection of MCF-7 cells with procaspase-3 partially restores the cleavage of a number of
cellular polypeptides in situ (Fig. 6D) but does
not restore the ability of subcellular fractions to cleave tetrapeptide
substrates (Figs. 6E and 8B).
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. Guy Poirier, Y.-C. Cheng, David Toft, and Yuri Lazebnik for antibodies; Michael Heldebrant for assistance with confocal microscopy; the Electron Microscopy and Optical Morphology Shared Resources of the Mayo Clinic for assistance with Figs. 2B and 9, A-F; and Deb Strauss for secretarial assistance.
| |
FOOTNOTES |
|---|
* 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.This
work was supported in part by United States Public Health Service Grant R01 CA69008 (to S. H. K. and W. C. E.) and a grant from the Welcome Trust (to W. C. E.).
Present address: Division of Biological Sciences, University
of Wisconsin, Whitewater, WI 53190.
§§ Principal Fellow of the Welcome Trust.
¶¶ To whom correspondence should be addressed: Division of Oncology Research, 1301 Guggenheim, Mayo Clinic, 200 First St., S.W., Rochester, MN 55905. Tel.: 507-284-8950; Fax: 507-284-3906; E-mail: Kaufmann.Scott@Mayo.edu.
Published, JBC Papers in Press, October 24, 2001, DOI 10.1074/jbc.M108419200
2 T. J. Kottke and S. H. Kaufmann, unpublished results.
3 Additional experiments demonstrated that cleaved caspase-9 species exhibited the same behavior as cleaved caspase-3 species during the course of detergent, salt, and urea extraction (T. J. Kottke and S. H. Kaufmann, unpublished results).
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
TNF-, tumor necrosis factor-;
DEVD-AFC, aspartylglutamylvalinylaspartyl-7-amino-4-trifluoromethylcoumarin;
EGFP, enhanced green fluorescent protein;
LEHD-AFC, leucinylglutamylhistidylaspartyl-7-amino-4-trifluoromethylcoumarin;
YVAD-AFC, tyrosylvalinylalanylaspartyl-7-amino-4-trifluoromethylcoumarin;
PARP, poly(ADP-ribose) polymerase;
PBS, calcium- and magnesium-free
phosphate-buffered saline;
topo, topoisomerase;
VEID-AFC, valinylglutamylisoleucylaspartyl-7-amino-4-trifluoromethylcoumarin;
zEK(bio)D-aomk, N-(N
-benzyloxycarbonylglutamyl-N
-biotinyllysyl)
aspartic acid [(2,6-dimethylbenzoyl)oxy]methylketone;
zVAD(OMe)-fmk, N-(N-benzyloxycarbonylvalinylalanyl)
aspartic acid (O-methylester) fluoromethylketone;
ICAD, inhibitor of caspase-activated deoxyribonuclease.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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| 1. | Wyllie, A. H., Kerr, J. F. R., and Currie, A. R. (1980) Int. Rev. Cytol. 68, 251-306[Medline] [Order article via Infotrieve] |
| 2. | Arends, M. J., and Wyllie, A. H. (1991) Int. Rev. Exp. Pathol. 32, 223-254[Medline] [Order article via Infotrieve] |
| 3. | Cohen, G. M. (1997) Biochem. J. 326, 1-16 |
| 4. | Salvesen, G. S., and Dixit, V. M. (1997) Cell 91, 443-446[CrossRef][Medline] [Order article via Infotrieve] |
| 5. |
Cryns, V.,
and Yuan, J.
(1998)
Genes Dev.
12,
1551-1570 |
