Actin Stabilization by Jasplakinolide Enhances Apoptosis Induced
by Cytokine Deprivation*
S. Celeste
Posey
§¶
and
Barbara E.
Bierer
¶**
From the
Department of Pediatric Oncology,
Dana-Farber Cancer Institute, the § Committee on Immunology,
Division of Medical Sciences, and the ** Department of Medicine, Harvard
Medical School, Boston, Massachussets 02115 and the ¶ NHLBI,
National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
Participation of the actin cytoskeleton in the
transduction of proliferative signals has been established through the
use of compounds that disrupt the cytoskeleton. To address the
possibility that actin also participates in the transduction of an
apoptotic signal, we have studied the response of the murine
interleukin 2 (IL-2)-dependent T cell line CTLL-20 to
treatment with the actin-binding compound jasplakinolide upon IL-2
deprivation. Like phalloidin, jasplakinolide stabilizes F-actin and
promotes actin polymerization. Treatment of CTLL-20 cells with
jasplakinolide, in the presence or absence of recombinant human IL-2,
altered actin morphology as assessed by confocal fluorescence
microscopy. Jasplakinolide was not toxic to CTLL-20 cells, nor was
apoptosis induced in the presence of exogenous recombinant human IL-2.
However, actin stabilization at the time of IL-2 deprivation enhanced
apoptosis by changing the time at which CTLL-20 cells committed to the
apoptotic pathway. This effect of jasplakinolide correlated with its
ability to stabilize polymerized actin, as treatment with a synthetic
analog of jasplakinolide with a greatly reduced ability to bind actin,
jasplakinolide B, did not enhance apoptosis. The enhancement occurred
upstream of the induction of caspase-3-like activity and could be
inhibited by the overexpression of the anti-apoptotic protein
Bcl-xL. These data suggest that the actin
cytoskeleton plays an active role in modulating lymphocyte apoptosis
induced by cytokine deprivation.
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INTRODUCTION |
The actin cytoskeleton appears to be intricately involved in
lymphocyte signal transduction (1, 2). It has long been recognized that
control of the actin cytoskeleton must be coordinated with control of
cell cycle events (3) and that actin-related events, such as adhesion,
receptor clustering, and receptor internalization, can affect mitogenic
signals (4-6). However, the role of the actin cytoskeleton in
transduction of intracellular signals leading to apoptosis in
lymphocytes remains poorly defined. Apoptosis is a regulated process by
which a cell undergoes a form of cell death characterized by cell
shrinkage, membrane blebbing, DNA cleavage, and nuclear condensation
(7, 8). Elucidation of apoptotic signal transduction pathways has
focused largely upon members of the Bcl family and upon the activation
of caspases; the mechanisms by which cellular damage results in the
changes in mitochondrial membrane potential thought to be required to initiate apoptosis are relatively unexplored.
There is compelling evidence to suggest that the disruption of
actin-based, integrin-mediated adhesion events is sufficient to trigger
apoptosis in endothelial and epithelial cell lines (reviewed in Ref.
9). However, in a model of lymphocyte apoptosis, suspension cells can
be used to segregate the role of the actin cytoskeleton in apoptotic
signal transduction from actin-dependent, integrin-mediated
survival signals. To study the possibility that the actin cytoskeleton
is involved in the transduction of an apoptotic signal triggered by
growth factor deprivation, we treated the nonadherent,
IL-21-dependent T
cell line CTLL-20 with the novel actin-binding cyclodepsipeptide jasplakinolide upon withdrawal from IL-2. Similar to phalloidin, jasplakinolide binds to and stabilizes actin microfilaments and can
promote actin polymerization in vitro (10). Jasplakinolide differs from phalloidin, however, in that it is permeant across cell
membranes and can therefore be easily used in vivo (10). Previous studies reported that jasplakinolide inhibited the growth of
prostate carcinoma cell lines in vitro (11), sensitized
Lewis lung carcinoma to radiation in vivo (12), and
prevented the self-renewal of acute myeloid leukemia cells (13). In
each of these reports, the effects of jasplakinolide were associated
with its ability to stabilize F-actin. None of these studies, however, addressed whether the inhibition of growth or sensitization was secondary to induction or enhancement of apoptosis.
We report that the addition of the actin-stabilizing compound
jasplakinolide to CTLL-20 cells enhanced apoptosis induced by IL-2
cytokine deprivation. Jasplakinolide was not toxic to cells, nor was
apoptosis induced in the presence of recombinant human IL-2 (rhIL-2).
The enhancement of apoptosis was time- and
concentration-dependent, occurred upstream of caspase
activation, and could be attenuated by the overexpression of the
anti-apoptotic protein Bcl-xL. Furthermore, actin
stabilization accelerated the time at which CTLL-20 cells committed to
the apoptotic program. Taken together, these data suggest that
modification of the actin cytoskeleton impacts upon signal transduction
leading to apoptosis.
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MATERIALS AND METHODS |
Reagents--
Jasplakinolide and its analog jasplakinolide B
were provided by the Drug Synthesis and Chemistry Branch, Developmental
Therapeutics Program, Division of Cancer Treatment, NCI, National
Institutes of Health (Bethesda, MD). Jasplakinolide and jasplakinolide
B were stored in Me2SO at
20 or
80 °C and diluted
into media immediately prior to use. rhIL-2 was kindly provided by
Hoffman-LaRoche (Nutley, NJ).
Cell Lines and Cell Culture--
The IL-2-dependent
murine cell line CTLL-20 (14) was grown in RPMI 1640 medium (MediaTech,
Herndon, VA) supplemented with 10% heat-inactivated fetal calf serum
(Sigma), 10 mM HEPES (Sigma), 2 mM
L-glutamine (MediaTech), 100 units/ml penicillin and 100 µg/ml streptomycin (MediaTech), and 50 µM
2-mercaptoethanol (Sigma) (termed complete RPMI-10% (cRPMI-10%)) to
which had been added 1-2% IL-2-containing supernatant derived from
concanavalin A-stimulated rat splenocytes. Forty-eight hours prior to
an experiment, CTLL-20 cells were expanded in cRPMI-10% supplemented
with 100 units/ml rhIL-2. A CTLL-2 clone transfected with
pSFFVNeo-bcl-xL and consequently overexpressing
the Bcl-xL protein (Bcl-xL-CTLL-2) was kindly
provided by Dr. Craig Thompson (University of Chicago, Chicago, IL)
(15). Overexpression of the Bcl-xL protein was confirmed by
Western blot analysis (data not shown). Bcl-xL-CTLL-2 cells
were grown in cRPMI-10% supplemented with 100 units/ml rhIL-2. All
cell lines were free of mycoplasma as determined monthly by polymerase
chain reaction analysis (Mycoplasma PCR Primer Set, Stratagene, La
Jolla, CA).
For all apoptosis assays, CTLL-20 cells were washed three times in
cRPMI-10% and resuspended at a final density of 2 × 105 cells/ml in cRPMI-10% without rhIL-2 to which either
drug or vehicle had been added. Washed cells resuspended in cRPMI-10% with rhIL-2 (100 units/ml) served as controls. The final concentration of Me2SO in all treatment groups ranged from 0.02 to 0.1%,
as indicated.
Immunofluorescent Staining of Cytoskeleton--
Cells (1 × 106/sample) treated as indicated were fixed in 4%
paraformaldehyde and cytospun (Cytospin 3 cell preparation system, Shandon Scientific Ltd., Cheshire, United Kingdom) onto glass slides.
Cells were incubated with 50 µM NH4Cl for 5 min, permeabilized with a solution of 0.1% Triton X-100 in
phosphate-buffered saline for 2.5 min, and blocked with a 2% solution
of bovine serum albumin. Cells were then incubated with 30 µg/ml
anti-actin monoclonal antibody N350 (Amersham Pharmacia Biotech) for 30 to 45 min at room temperature. Cells were washed three times and
incubated with 15 µg/ml Texas Red-conjugated anti-mouse IgM antibody
(Jackson ImmunoResearch, West Grove, PA) for 30 min at room
temperature. All cells were stained with 1 µg/ml Hoechst 33258. Cytospun samples were mounted in a 0.1% (w/v) solution of
phenylenediamine in 90% glycerol. Slides were viewed by confocal
fluorescence microscopy (model TCS4D/DMIRBE, Leica Inc., Deerfield, IL)
equipped with argon and argon-krypton lasers for UV (351-364 nm) and
red (568 nm) excitation.
Hoechst Staining--
Cells were fixed and stained as described
(16). In brief, cells (2 × 105/sample) were fixed in
an excess volume of 3:1 (v/v) methanol/acetic acid, dried onto glass
slides, and stained with a 1 µg/ml Hoechst 33258 (Sigma) solution in
double distilled H2O blocked with nonfat dried milk. In one
set of experiments, a 20 or 50 µg/ml solution of propidium iodide
(Sigma) with 0.5 µg/ml RNase (Boehringer Mannheim) also blocked with
nonfat dried milk was used instead of Hoechst as a nuclear stain. The
percentage of cells undergoing apoptosis was determined by quantifying
the number of cells with and without condensed nuclei by fluorescence
microscopy (Zeiss Axioskop MC100; Carl Zeiss, Thornwood, NY, or Olympus
Bmax BX50, Olympus America, Melville, NY). Between 300 and 700 cells
were counted for each sample. The 95% confidence interval for each
sample within each experiment was determined according to the
statistical definition of the variance of a proportion in a binomial
experiment (17). The variance was computed as pq/n, where
p = the proportion apoptotic, q = 1
p, and n = the number of cells counted.
For the dose-response curve of jasplakinolide, the results of three
experiments were averaged and the data plotted as the mean ± S.D.
The IC50 was determined by logit analysis.
TUNEL Staining--
Terminal deoxynucleotide
transferase-mediated nick end labeling (TUNEL) was performed with the
Oncor (Gaithersburg, MD) ApopTag Plus kit according to the
manufacturer's instructions with modifications. Briefly, 2-4 × 106 cells/sample were fixed in 1% paraformaldehyde (in
phosphate-buffered saline) for 90 min. Cells were then permeabilized
with a buffer containing 0.1% (w/v) saponin and 1% FCS in
phosphate-buffered saline for 2 min. Following permeabilization, cells
were washed once with the ApopTag equilibration buffer, then incubated
with the TdT enzyme and digoxigenin-tagged dUTP at 37 °C for 30 min. Cells were washed once with the ApopTag stop/wash buffer and then incubated with the fluorescein isothiocyanate (FITC)-conjugated anti-digoxigenin antibody for 30 min. Cells were washed once with phosphate-buffered saline. Fluorescence of each sample was measured by
flow cytometry (FACScan; Becton Dickinson). The fluorescence of FITC
was measured at a wavelength of 535 nM and the data
analyzed using CellQuest. Data were gated based on forward and side
scatter to exclude cell debris; M1 regions of FITC-bright cells (and
therefore considered apoptotic) were selected for each histogram based
on the background levels of staining within each sample.
Analysis of Caspase-3-like Activation--
The activity of
caspase-3 in whole cell lysates (2 × 106
cells/sample) was determined using the CLONTECH
(Palo Alto, CA) ApoAlert CPP32 Colorimetric Assay kit according to the
manufacturer's instructions. Briefly, cells treated and incubated as
indicated were lysed in the kit lysis buffer and frozen at
80 °C
until use. Lysates were thawed on ice and centrifuged (14,000 rpm for
10 min at 4 °C) to remove debris. Supernatants were incubated with
the caspase-3 substrate (the peptide DEVD conjugated to the chromophore
p-nitroanilide) for 2 h at 37 °C. The absorbance of
each sample was measured at 405 nM using a 96 well
colorimetric plate reader (model 3550-UV; Bio-Rad).
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RESULTS |
Jasplakinolide Promoted Cell Shape Change and Redistribution of
Actin in CTLL-20 Cells--
It has previously been shown that
jasplakinolide enhances actin polymerization and alters the cellular
actin cytoskeleton (10, 11). To confirm that jasplakinolide altered the
actin cytoskeleton in CTLL-20 cells, cells were treated with
jasplakinolide (100 nM) or Me2SO (0.02%) for
8 h in the absence of IL-2. Cytospin preparations were stained by
indirect immunofluorescence using an anti-actin monoclonal antibody and
with Hoechst 33258 to assess nuclear morphology and analyzed by
confocal fluorescence microscopy. Jasplakinolide caused both cell shape
change and a redistribution of the actin cytoskeleton (Fig.
1). CTLL-20 cells were observed to be
typically smooth and round with an even peripheral distribution of
actin. Treatment with jasplakinolide induced protrusions of the cell
surface and resulted in a patchy appearance of cortical actin. In some
cells treated with jasplakinolide, cortical actin appeared only around
the nucleus (Fig. 1). The change in actin cytoskeletal distribution was
not a result of the apoptotic process, as it occurred prior to
commitment to apoptosis (see below), prior to the appearance of
condensed or apoptotic nuclei (Fig. 1), and in the presence of rhIL-2
when few, if any, cells were entering or undergoing apoptosis (data not
shown).

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Fig. 1.
Jasplakinolide dramatically alters the
morphology of the actin cytoskeleton in CTLL-20 cells. CTLL-20
cells were incubated with 100 nM jasplakinolide or 0.02%
Me2SO (DMSO) in the absence of rhIL-2 for
8 h. Actin morphology was visualized by staining with an
anti-actin monoclonal antibody and a Texas Red-conjugated
secondary anti-mouse-IgM antibody. Nuclei were stained with Hoechst
33258. Images are magnified × 1600. Photomicrographs shown are
representative of three independent experiments.
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Stabilization of the Actin Cytoskeleton Enhanced Apoptosis of
CTLL-20 Cells in Response to IL-2 Deprivation--
Previous reports
have suggested that the actin cytoskeleton may play a role in
transducing proliferative signals in lymphocytes (see, e.g.
Refs. 18-20). To determine whether actin might also be involved in
transducing an apoptotic signal, CTLL-20 cells were incubated in the
presence or absence of rhIL-2 with jasplakinolide or with vehicle,
Me2SO. Additionally, cells were also treated with
jasplakinolide B (Fig. 2A,
inset), an analog of jasplakinolide that has been reported to lack
actin-modulating activity (11); at a concentration of 1 µM, jasplakinolide B had no demonstrable effect on cell
shape or actin redistribution in CTLL-20 cells (data not shown). Cells
were harvested after 12 h of incubation with drug, and the
percentage of apoptotic cells was quantified by nuclear morphology
(Fig. 2A). The number of cells with condensed nuclei after
IL-2 deprivation (27%) increased 2-fold (56%) after the addition of
jasplakinolide at a concentration of 100 nM. At the
concentrations used, jasplakinolide was not toxic to cells, as the
percentages of apoptotic CTLL-20 cells incubated with rhIL-2 were
similar in the absence (1.1%) and presence (2.6%) of drug, a difference that was not statistically significant. Furthermore, the
effect of jasplakinolide on apoptosis was dependent upon its ability to
bind actin, as the non-actin binding analog jasplakinolide B did not
show a similar effect, even at a 10-fold higher concentration (Fig.
2A). In addition, 100 nM jasplakinolide also
enhanced apoptosis of the pre-B, IL-3-dependent Ba/F3 cell
line in response to IL-3 deprivation (data not shown), demonstrating
that the effect of drug on apoptosis was not specific to the CTLL-20
cell line nor to dependence on IL-2.

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Fig. 2.
Stabilization of actin by jasplakinolide
increased the number of CTLL-20 cells that have undergone
apoptosis. A, CTLL-20 cells were treated with 0.1%
Me2SO (DMSO), 100 nM
jasplakinolide, or 100 nM or 1 µM
jasplakinolide B (Jas B) upon withdrawal from IL-2
(solid columns). Cell populations similarly treated with
drug or vehicle were maintained in 100 units/ml rhIL-2 (open
columns). All cells were harvested after incubation for 12 h
and fixed. Percentage of apoptotic cells was determined by
Hoechst staining and counting condensed nuclei. Data shown
are percentage of apoptotic cells ± 2 S.D., calculated as
described under "Materials and Methods" and are representative of
two independent experiments. Inset, chemical structures of
jasplakinolide and its analog, jasplakinolide B, reproduced from Ref.
11 with permission. B, CTLL-20 cells were treated with 100 nM jasplakinolide or 0.02% Me2SO and either
withdrawn from (solid line) or maintained in (dotted
line) 100 units/ml rhIL-2 and incubated for 15 h. Cells were
then fixed in 1% paraformaldehyde for TUNEL staining. FL1-H indicates
the degree of FITC labeling of the cell populations. FITC-bright
populations were determined by comparison to the background levels of
staining within each sample. Data are representative of two independent
experiments.
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TUNEL staining and subsequent flow cytometry confirmed the results
obtained with Hoechst staining (Fig. 2B). The TUNEL assay labels the 3' hydroxyl ends of DNA remaining after cleavage by endonucleases (21); thus, cells that are stained FITC-bright are
considered apoptotic. In the experiment shown (Fig. 2B), the percentage of FITC-bright cells in the jasplakinolide-treated population (~70%) was greater than that in the
Me2SO-treated population (~40%) after 15 h of IL-2
deprivation. Again, treatment with jasplakinolide did not cause cell
death in the presence of rhIL-2, confirming that jasplakinolide is not
nonspecifically toxic to the cells. Both DNA content analysis by
propidium iodide staining followed by flow cytometry and DNA
fragmentation analysis by agarose gel electrophoresis confirmed that
jasplakinolide enhanced apoptosis upon IL-2 deprivation (data not
shown); polymerization of actin thus appeared to affect either the
apoptotic signal or the apoptotic process.
Stabilization of Actin Accelerated the Appearance of Apoptotic
CTLL-20 Cells after IL-2 Withdrawal--
Treatment with jasplakinolide
accelerated the appearance of apoptotic cells (Fig.
3A) after withdrawal of IL-2,
although the total proportion of cells (90-95%, Fig. 3A
and data not shown) that eventually completed apoptosis remained
unchanged. Condensed nuclear morphology indicative of apoptosis was not
observed in the Me2SO-treated cell population until 12 h after deprivation of IL-2, and most cells had undergone apoptosis by
24 h (Fig. 3A), consistent with time courses previously
reported (22, 23). Stabilization of actin accelerated the apoptotic
process: cells treated with jasplakinolide, unlike
Me2SO-treated cells, exhibited condensed nuclei at 8 h, and over 80% were dead by 18 h. The most dramatic difference
was observed approximately 12 h after withdrawal from IL-2, when
jasplakinolide treatment resulted in apoptosis of 50-60% of cells,
whereas only 10-30% of Me2SO-treated cells were apoptotic
(Fig. 3A and data not shown). CTLL-20 cells incubated with
jasplakinolide in the presence of rhIL-2 (100 units/ml) did not undergo
apoptosis during the time course of these experiments.

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Fig. 3.
Time course of apoptosis (A)
and dose-response (B) of CTLL-20 cells
treated with jasplakinolide upon IL-2 withdrawal. A,
CTLL-20 cells were treated with 100 nM jasplakinolide
(squares) or 0.02% Me2SO (circles)
and either deprived of IL-2 (closed symbols) or maintained
in 100 units/ml rhIL-2 (open symbols). Cells were harvested
at the indicated times, and the percentage of apoptotic cells was
determined by Hoechst staining and counting. Data are shown as
percentage of apoptotic cells ± 2 S.D. and are representative of
two independent experiments. B, CTLL-20 cells were incubated
for 12 h with the indicated concentrations of jasplakinolide
(closed squares) or 0.02% Me2SO
(DMSO) (closed circle) in the absence of IL-2
and then harvested for fixing and Hoechst staining. Data shown are from
three independent experiments (mean ± 2 S.D.).
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A dose-response curve of jasplakinolide demonstrated that
concentrations as low as 50 nM were sufficient to enhance
apoptosis, whereas concentrations of 25 nM and below had no
effect on the apoptotic process (Fig. 3B). The
IC50 for this enhancement was calculated as approximately
35 nM, a concentration similar to the reported
IC50 of 34 nM for inhibition of proliferation
of a prostate carcinoma cell line (11). At the concentrations used in
these experiments, the addition of Me2SO (0.02-0.1%) did
not increase the number of cells undergoing apoptosis after IL-2
deprivation compared with cells incubated in cRPMI-10% alone (data not shown).
Stabilization of Actin Altered the Time Course of Commitment to
Apoptosis--
To address whether actin stabilization altered the time
course of commitment to apoptosis, CTLL-20 cells treated with
jasplakinolide or Me2SO were deprived of IL-2 for varying
lengths of time, after which 100 units/ml rhIL-2 was added back to the
cultures. All samples were incubated for a total of 26 h and then
fixed for quantification of apoptotic cells (Fig.
4). All treatment groups could be rescued
by readdition of rhIL-2 within 8 h of deprivation, whereas neither
could be rescued 14.5 h after withdrawal. Notably, readdition of
rhIL-2 within 12 h of withdrawal was sufficient to rescue the
Me2SO-treated cells, but not those treated with jasplakinolide, suggesting that actin stabilization altered the time
course of commitment to apoptosis.

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Fig. 4.
Modulation of actin altered the time course
of commitment to apoptosis. CTLL-20 cells treated with 100 nM jasplakinolide (solid columns) or 0.02%
Me2SO (open columns) were deprived of IL-2 for
the indicated period of time. At the end of the allotted time of
deprivation, 100 units/ml rhIL-2 was added back to the cultures. All
samples were harvested 26 h after the beginning of the incubation
and fixed for staining with propidium iodide and quantification of
apoptosis by nuclear morphology. Data are shown as percentage of
apoptotic cells ± 2 S.D. and are representative of two
independent experiments.
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The Effect of the Stabilization of Actin on Apoptosis Occurred
Upstream of Caspase Activation--
Because the stabilization of actin
by jasplakinolide altered the time of commitment to apoptosis, we
predicted that caspase activity would be detected at an earlier time
point in cells that had been treated with jasplakinolide. To test this
prediction, we investigated the time course of induction of
caspase-3-like activity. It has been previously demonstrated that
caspase-3 (CPP32), but not caspase-1 (ICE), is activated in CTLL-20
cells undergoing apoptosis after IL-2 deprivation (24). CTLL-20 cells
were incubated either with jasplakinolide (100 nM) or
Me2SO (0.02%) in the absence or presence (100 units/ml) of
rhIL-2 for 8 or 12 h (Fig. 5 and data not shown). Cells were lysed, and caspase-3-like activity was
assayed using as colorimetric substrate the peptide
DEVD-p-nitroanilide. Lysates of CTLL-20 cells deprived of
IL-2 for 21 h were used as positive controls for the maximal
A405 achievable within each determination of
caspase activity. Caspase-3-like activity was present in the
jasplakinolide-treated cells after 8 h of IL-2 deprivation,
although not in Me2SO-treated, IL-2 deprived cells (Fig.
5). After 12 h of IL-2 deprivation, both jasplakinolide and
Me2SO-treated cells contained the maximum caspase-3-like
activity detectable (data not shown). These data imply that the
stabilization of actin affected the apoptotic process upstream of the
caspase cascade, again suggesting that actin stabilization affected the transduction of an apoptotic signal and not just the downstream effector phase of the apoptotic process.

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Fig. 5.
Induction of caspase-3-like activity in cells
treated with jasplakinolide 8 h after IL-2 withdrawal.
Extracts of cells incubated with either 100 nM
jasplakinolide or 0.02% Me2SO (DMSO) in the
absence (solid columns) or presence (open
columns) of 100 units/ml of rhIL-2 for 8 h were incubated
with the caspase-3 substrate DEVD-p-nitroanilide. A cell
lysate from CTLL-20 cells incubated in the absence of IL-2 for 21 h (hatched column) was used as a positive control for
maximum caspase activity detectable with this assay. Data given are the
average of duplicate samples and are representative of two
experiments.
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The Overexpression of the Anti-apoptotic Protein Bcl-xL
Attenuated the Effect of Actin Stabilization on
Apoptosis--
Bcl-xL, a member of the Bcl-2 family, has
been shown to protect cells from apoptosis induced by cytokine
withdrawal (15). To test whether Bcl-xL inhibited the
enhancement of apoptosis by this actin-modulating agent, CTLL-2 cells
overexpressing Bcl-xL were treated with jasplakinolide (100 nM) or Me2SO (0.02%), deprived of IL-2 for the
indicated times, and fixed for Hoechst staining and quantification of
apoptosis (Fig. 6). Nontransfected
CTLL-20 cells treated with jasplakinolide and fixed 16 h after
IL-2 deprivation were included for comparison. Overexpression of
Bcl-xL delayed but did not prevent apoptosis of CTLL-2
cells, as previously observed (15). The stabilization of actin by
jasplakinolide increased only slightly the percentage of apoptotic
cells after 64 or 72 h of rhIL-2 deprivation, but the rate of
onset of apoptosis was unaffected. Thus, Bcl-xL
overexpression appeared to attenuate, if not ablate, the effects of
actin stabilization.

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Fig. 6.
Time course of Bcl-xL-CTLL-2
apoptosis in the presence of 100 nM jasplakinolide
(squares) or 0.02% Me2SO
(circles) when cells were withdrawn from IL-2
(closed symbols). The presence of rhIL-2 (100 units/ml; open symbols) prevented apoptosis. Data shown are percentage
of apoptotic cells determined by Hoechst counting ± 2 S.D. and
are representative of two independent experiments. Apoptosis of CTLL-20
cells treated with 100 nM jasplakinolide incubated in the
absence (solid column) or presence (open bar) of
100 units/ml rhIL-2 for 16 h is shown for comparison.
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DISCUSSION |
Jasplakinolide is a cyclodepsipeptide isolated from the marine
sponge, Jaspis johnstoni, originally found to be an
effective antifungal agent at micromolar concentrations (25) and later found to have antiproliferative activity in the nanomolar range in
acute myeloid leukemia and prostate carcinoma cells (11, 13).
Jasplakinolide has been shown to induce actin polymerization (10, 26,
27) and to bind F-actin competitively with phalloidin with a
dissociation constant (Kd) of approximately 15 nM (10); this agent has been used to investigate the
functions of actin microfilaments in a variety of cellular processes,
including ion transport, endocytosis, and adhesion (28-31). Here we
demonstrated that jasplakinolide effectively modified the actin
cytoskeleton in T lymphocytes (Fig. 1), a property that allowed us to
use this compound to investigate a potential role for actin in the
transduction of an apoptotic signal in these suspension cells.
Stabilization of actin microfilaments by jasplakinolide accelerated
commitment to apoptosis of CTLL-20 cells deprived of IL-2. Jasplakinolide did not induce apoptosis in the presence of IL-2 and was
thus not toxic to the cells. The enhancement of apoptosis was not
specific to CTLL-20 cells nor to IL-2 dependence, as treatment with
jasplakinolide also enhanced apoptosis of the pre-B,
IL-3-dependent cell line Ba/F3 upon growth factor
deprivation. Analysis of DNA content by flow cytometry, DNA
fragmentation, and TUNEL confirmed that the nuclear morphology observed
by fluorescence microscopy in jasplakinolide- and
Me2SO-treated, IL-2-deprived CTLL-20 cells was secondary to
the induction of apoptosis, not necrosis (Fig. 2B and data
not shown). The ability of jasplakinolide to enhance apoptosis
correlated with its ability to bind to and stabilize F-actin. In
addition, stabilization of actin appeared to sensitize the cells to
cytokine deprivation; treatment with jasplakinolide increased the
percentage of cells that underwent apoptosis at low concentrations of
IL-2 (data not shown). Finally, the stabilization of actin appeared to
affect the transduction of the apoptotic signal, as the enhancement
occurred upstream of commitment to apoptosis and induction of
caspase-3-like activity and could be attenuated by the overexpression
of the anti-apoptotic protein Bcl-xL.
The process of apoptosis has been divided into two phases, the
initiation/commitment phase and the downstream effector phase (32, 33).
Actin has long been postulated to play a role in the effector phase of
morphologic changes associated with apoptosis (34, 35). Recent reports
indicate that actin itself is cleaved during apoptosis (35-39);
cleavage of actin has been suggested to be both an effector of the
morphological changes associated with apoptosis as well as a mechanism
of DNase I activation (35, 36, 40, 41). Changes in the levels of total
cellular actin and of F-actin have also been associated with the
apoptotic process (38, 42). We offer evidence to suggest that actin
also plays a role in the initial phase of commitment to apoptosis,
independent of a requirement for adhesion.
The mechanism by which modulation of actin altered the apoptotic signal
and/or commitment is unclear. One possibility is that actin
stabilization by jasplakinolide has been demonstrated to affect ion
transport across the cell membrane in a variety of cell systems (28,
29). As apoptosis of CTLL-20 cells in response to IL-2 deprivation is
associated with intracellular acidification (43), it could be
postulated that an alteration in ion flux could alter the rate of cell
death. However, acidification has been shown not to be required for
progression to apoptosis (43), and treatment with calcium ionophore did
not prevent the effect of jasplakinolide (data not shown); we thus
consider this possibility unlikely.
An alternative explanation for the modification of the apoptotic
process by jasplakinolide stems from the observation that the effective
concentrations of jasplakinolide are far lower than the molar content
of actin in the cell and that the Kd of
jasplakinolide binding to F-actin (~15 nM) (see Ref. 10) is consistent with the IC50 of 34 nM for
inhibition of proliferation (11) and for the enhancement of apoptosis
(~35 nM) observed here. The discrepancy in effective drug
versus purported target concentrations would suggest either
that binding between jasplakinolide and F-actin is positively
cooperative (10) or that the effect of jasplakinolide results from drug
competition with an actin-binding protein present at much lower
concentrations in the cell. In this regard, gelsolin has also been
shown to displace phalloidin from actin filaments (44); whether
gelsolin and jasplakinolide compete for the same binding site(s) on
actin has not yet been investigated. Gelsolin is a well conserved
actin-regulatory protein that can bind actin monomers and sever actin
filaments (45), and gelsolin has recently been shown to be a substrate
of caspase-3 (46). In addition, overexpression of gelsolin has been
shown to inhibit apoptosis upstream of the caspase cascade (47). We are
currently investigating a role for gelsolin in this model system of apoptosis.
The actin cytoskeletal architecture, by virtue of its ability to
organize the cytoplasm by compartmentalizing and localizing proteins,
can act as a scaffolding element for signaling intermediates. The
enzymatic activities of a few actin-associated proteins, such as casein
kinase II (48) and GRK5 (49), have been reported to be modified by
binding directly to actin. Changes in actin polymerization that altered
cytoskeletal architecture could therefore affect either enzymatic
activity or substrate availability (see also Ref. 49). A number of
plausible candidate downstream effectors could thus be influenced by
stabilization of the actin cytoskeleton. The Rho family of GTPases
has been shown to be involved not only in the regulation of gene
transcription, cell cycle progression, and programmed cell death but
also in the regulation of actin rearrangement (50-52). In addition,
signaling intermediates including phosphatidylinositol 3-kinase
(phosphatidylinositol 3-kinase), protein kinase C-
, the
Wiskott-Aldrich syndrome protein, cofilin, and the proto-oncogenes VAV
and c-ABL have all been shown to influence cell survival, cell cycle
control, and/or the cell death program and have been directly or
indirectly implicated in the regulation of the actin cytoskeleton.
Pretreatment of CTLL-20 cells with wortmannin, an inhibitor of
phosphatidylinositol 3-kinase, did not affect apoptosis in response to
IL-2 withdrawal or the effect of jasplakinolide on this process.
Whether modification of actin by jasplakinolide impacts upon signaling
pathways other than that of phosphatidylinositol 3-kinase is the
subject of further investigation.
 |
ACKNOWLEDGEMENTS |
We thank Edward A. Sausville, Kimberly
L. K. Duncan, and Paul A. Janmey for invaluable discussions and
Hidde Ploegh and Abul K. Abbas for helpful advice. Our gratitude is
extended to Victor Ferrans and Zu-Xi Yu for assistance with confocal
microscopy. We also thank Robert Schlegel and David A. Thompson for
assistance with quantification of apoptosis by nuclear morphology.
 |
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.
Supported by the National Institutes of Health, National
Institute of General Medical Sciences, National Research Service Award
T32 GM07753-19, by the National Institutes of Health, National Cancer
Institute, Ph.D. Program in Immunology Grant T32 CA09141, and by a
pre-Intramural Research Training Award fellowship sponsored by the
National Heart, Lung and Blood Institute.

To whom correspondence should be addressed: NHLBI, Bldg. 10, Rm. 5D49, 10 Center Dr., Bethesda, MD 20892. Tel.: 301-402-6786; Fax:
301-480-1792; E-mail: biererb{at}nih.gov.
The abbreviations used are:
IL, interleukin; rhIL-2, recombinant human IL-2; cRPMI-10%, complete RPMI-10%; TUNEL, terminal deoxynucleotide transferase-mediated nick end labeling; FITC, fluorescein isothiocyanate.
 |
REFERENCES |
-
Parsey, M. V.,
and Lewis, G. K.
(1993)
J. Immunol.
151,
1881-1893[Abstract/Free Full Text]
-
Rozdzial, M. M.,
Malissen, B.,
and Finkel, T. H.
(1995)
Immunity
3,
623-633[Medline]
[Order article via Infotrieve]
-
Vojtek, A.,
Haarer, B.,
Field, J.,
Gerst, J.,
Pollard, T. D.,
Brown, S.,
and Wigler, M.
(1991)
Cell
66,
497-505[Medline]
[Order article via Infotrieve]
-
Brock, M. A.,
and Chrest, F.
(1993)
J. Cell. Physiol.
157,
367-378[Medline]
[Order article via Infotrieve]
-
Caplan, S.,
Zeliger, S.,
Wang, L.,
and Baniyash, M.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
4768-4772[Abstract]
-
Valitutti, S.,
Dessing, M.,
Aktories, K.,
Gallati, H.,
and Lanzavecchia, A.
(1995)
J. Exp. Med.
181,
577-584[Abstract]
-
Wyllie, A. H.,
Kerr, J. F.,
and Currie, A. R.
(1980)
Int. Rev. Cytol.
68,
251-306[Medline]
[Order article via Infotrieve]
-
Kerr, J. F. R.,
Winterford, C. M.,
and Harmon, B. V.
(1994)
Cancer
73,
2013-2026[Medline]
[Order article via Infotrieve]
-
Frisch, S. M.,
and Ruoslahti, E.
(1997)
Curr. Opin. Cell Biol.
9,
701-706[CrossRef][Medline]
[Order article via Infotrieve]
-
Bubb, M. R.,
Senderowicz, A. M. J.,
Sausville, E. A.,
Duncan, K. L. K.,
and Korn, E. D.
(1994)
J. Biol. Chem.
269,
14869-14871[Abstract/Free Full Text]
-
Senderowicz, A. M. J.,
Kaur, G.,
Sainz, E.,
Laing, C.,
Inman, W. D.,
Rodriguez, J.,
Cres, P.,
Malspeis, L.,
Grever, M. R.,
Sausville, E. A.,
and Duncan, K. L. K.
(1995)
J. Natl. Cancer Inst.
87,
46-51[Abstract]
-
Takeuchi, H.,
Ara, G.,
Sausville, E. A.,
and Teicher, B.
(1998)
Cancer Chemother. Pharmacol.
42,
491-496[CrossRef][Medline]
[Order article via Infotrieve]
-
Fabian, I.,
Shur, I.,
Bleiberg, I.,
Rudi, A.,
Kashman, Y.,
and Lishner, M.
(1995)
Exp. Hematol.
23,
583-587[Medline]
[Order article via Infotrieve]
-
Gillis, S.,
and Smith, K. A.
(1977)
Nature
268,
154-156[Medline]
[Order article via Infotrieve]
-
Boise, L. H.,
McShan, C. L.,
and Thompson, C. B.
(1996)
J. Immunol. Methods
191,
143-148[CrossRef][Medline]
[Order article via Infotrieve]
-
Meikrantz, W.,
Gisselbrecht, S.,
Tam, S. W.,
and Schlegel, R.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
3754-3758[Abstract]
-
Walpole, R. E.,
and Myers, R. H.
(1989)
Probability and Statistics for Engineers and Scientists, Fourth Ed., pp. 257-261, MacMillan Publishing Co., New York
-
Phatak, P. D.,
and Packman, C. H.
(1994)
J. Cell. Physiol.
159,
365-370[Medline]
[Order article via Infotrieve]
-
Gomez, J.,
de Aragon, A. M.,
Bonay, P.,
Pitton, C.,
Garcia, A.,
Sliva, A.,
Fresno, M.,
Alvarez, F.,
and Rebollo, A.
(1995)
Eur. J. Immunol.
25,
2673-2678[Medline]
[Order article via Infotrieve]
-
Gomez, J.,
Garcia, A.,
Borlado, L. R.,
Bonay, P.,
Martinez-A, C.,
Silva, A.,
Fresno, M.,
Carrera, A. C.,
Eicher-Streiber, C.,
and Rebollo, A.
(1997)
J. Immunol.
158,
1516-1522[Abstract]
-
Allen, R. T.,
Hunter, W. J. I.,
and Agrawal, D. K.
(1997)
J. Pharmacol. Toxicol. Methods
37,
215-228[CrossRef][Medline]
[Order article via Infotrieve]
-
Duke, R. C.,
and Cohen, J. J.
(1986)
Lymphokine Res.
5,
289-299[Medline]
[Order article via Infotrieve]
-
Lu, Y.,
Tremblay, R.,
Jouishomme, H.,
Chakravarthy, B.,
and Durkin, J. P.
(1994)
J. Immunol.
153,
1495-1504[Abstract/Free Full Text]
-
Sarin, A.,
Wu, M.-L.,
and Henkart, P. A.
(1996)
J. Exp. Med.
184,
2445-2450[Abstract/Free Full Text]
-
Scott, V. R.,
Boehme, R.,
and Matthews, T. R.
(1988)
Antimicrob. Agents Chemother.
32,
1154-1157[Medline]
[Order article via Infotrieve]
-
Holzinger, A.,
and Meindl, U.
(1997)
Cell Motil. Cytoskeleton
38,
365-372[CrossRef][Medline]
[Order article via Infotrieve]
-
Lee, E.,
Shelden, E.,
and Knecht, D.
(1998)
Cell Motil. Cytoskeleton
39,
122-133[CrossRef][Medline]
[Order article via Infotrieve]
-
Furukawa, K.,
Smith-Swintosky, V. L.,
and Mattson, M. P.
(1995)
Exp. Neurol.
133,
153-163[CrossRef][Medline]
[Order article via Infotrieve]
-
Matthews, J. B.,
Smith, J. A.,
and Hrnjez, B. J.
(1997)
Am. J. Phys.
272,
C254-C262[Abstract/Free Full Text]
-
Shurety, W.,
Stewart, N. L.,
and Stow, J. L.
(1998)
Mol. Biol. Cell
9,
957-975[Abstract/Free Full Text]
-
Stewart, M. P.,
McDowall, A.,
and Hogg, N.
(1998)
J. Cell Biol.
140,
699-707[Abstract/Free Full Text]
-
Vaux, D. L.,
and Strasser, A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
2239-2244[Abstract/Free Full Text]
-
Cohen, G. M.
(1997)
Biochem. J.
326,
1-16[Medline]
[Order article via Infotrieve]
-
Brancolini, C.,
Benedetti, M.,
and Schneider, C.
(1995)
EMBO J.
14,
5179-5190[Abstract]
-
Kayalar, C.,
Ord, T.,
Testa, M. P.,
Zhong, L. T.,
and Bredesen, D. E.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
2234-2238[Abstract/Free Full Text]
-
Dipietrantonio, A.,
Hsieh, T.-c.,
and Wu, J. M.
(1996)
Biochem. Biophys. Res. Commun.
224,
837-842[CrossRef][Medline]
[Order article via Infotrieve]
-
Brown, S. B.,
Bailey, K.,
and Savill, J.
(1997)
Biochem. J.
323,
233-237[Medline]
[Order article via Infotrieve]
-
Guenal, I.,
Risler, Y.,
and Mignotte, B.
(1997)
J. Cell Sci.
110,
489-495[Abstract/Free Full Text]
-
Mashima, T.,
Naito, M.,
Noguchi, K.,
Miller, D. K.,
Nicholson, D. W.,
and Tsuruo, T.
(1997)
Oncogene
14,
1007-1012[CrossRef][Medline]
[Order article via Infotrieve]
-
Hall, A. K.
(1994)
Med. Hypotheses
43,
125-131[Medline]
[Order article via Infotrieve]
-
Chen, Z.,
Naito, M.,
Mashima, T.,
and Tsuruo, T.
(1996)
Cancer Res.
56,
5224-5229[Abstract]
-
Levee, M. G.
(1996)
Am. J. Phys.
271,
C1981-C1992[Abstract/Free Full Text]
-
Li, J.,
and Eastman, A.
(1995)
J. Biol. Chem.
270,
3203-3211[Abstract/Free Full Text]
-
Allen, P. G.,
and Janmey, P. A.
(1994)
J. Biol. Chem.
269,
32916-32923[Abstract/Free Full Text]
-
Hartwig, J. H.,
and Kwiatkowski, D. J.
(1991)
Curr. Opin. Cell Biol.
3,
87-97[Medline]
[Order article via Infotrieve]
-
Kothkakota, S.,
Azuma, T.,
Reinhard, C.,
Klippel, A.,
Tang, J.,
Chu, K.,
McGarry, T. J.,
Kirschner, M. W.,
Koths, K.,
Kwiatkowski, D. J.,
and Williams, L. T.
(1997)
Science
278,
294-298[Abstract/Free Full Text]
-
Ohtsu, M.,
Sakai, N.,
Fujita, H.,
Kashiwagi, M.,
Gasa, S.,
Shimizu, S.,
Eguchi, Y.,
Tsujimoto, Y.,
Sakiyama, Y.,
Kobayashi, K.,
and Kuzumaki, N.
(1997)
EMBO J.
16,
4650-4656[Abstract/Free Full Text]
-
Karino, A.,
Tanoue, S.,
Fukuda, M.,
Nakamura, T.,
and Ohtsuki, K.
(1996)
FEBS Lett.
398,
317-321[CrossRef][Medline]
[Order article via Infotrieve]
-
Freeman, J. L. R.,
De La Cruz, E. M.,
Pollard, T. D.,
Lefkowitz, R. J.,
and Pitcher, J. A.
(1998)
J. Biol. Chem.
273,
20653-20657[Abstract/Free Full Text]
-
Lores, P.,
Morin, L.,
Luna, R.,
and Gacon, G.
(1997)
Oncogene
15,
601-605[CrossRef][Medline]
[Order article via Infotrieve]
-
Hall, A.
(1998)
Science
279,
509-514[Abstract/Free Full Text]
-
Mackay, D. J. G.,
and Hall, A.
(1998)
J. Biol. Chem.
273,
20685-20688[Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.