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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3483-3488
IMMUNOBIOLOGY
Failure of gelsolin overexpression to regulate lymphocyte
apoptosis
S. Celeste Posey,
Maria Paola Martelli,
Toshifumi Azuma,
David J. Kwiatkowski, and
Barbara E. Bierer
National Heart, Lung, and Blood Institute, National Institutes of
Health, Bethesda, MD; Committee on Immunology, Division of Medical
Sciences, and the Departments of Medicine and Pediatrics, Harvard
Medical School, Boston, MA, Dana-Farber Cancer Institute, Boston, MA;
Genetics Laboratory, Hematology Division, Brigham and Women's
Hospital, Boston, MA.
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Abstract |
The actin regulatory protein gelsolin cleaves actin filaments in a
calcium- and polyphosphoinositide-dependent manner. Gelsolin has
recently been described as a novel substrate of the cysteinyl protease
caspase-3, an effector protease activated during apoptosis. Cleavage by
caspase-3 generates an amino-terminal fragment of gelsolin that can
sever actin filaments independently of calcium regulation. The
disruption of the actin cytoskeleton by cleaved gelsolin is
hypothesized to mediate many of the downstream morphological changes
associated with apoptosis. In contrast, overexpression of full-length
gelsolin has also been reported to inhibit apoptotic cell death
upstream of the activation of caspase-3, suggesting that gelsolin may
also act prior to commitment to cell death. The authors previously
observed that actin stabilization by the cell permeant agent
jasplakinolide enhanced cell death upon interleukin (IL)-2 or IL-3
withdrawal from growth-factor-dependent lymphocyte cell lines, and
hypothesized that actin polymerization could alter the activity of
gelsolin, thus enhancing apoptosis. Here the authors show that
constitutive overexpression of gelsolin did not, however, inhibit or
dramatically enhance apoptotic cell death upon growth-factor withdrawal, nor did it modify sensitivity to jasplakinolide. In contrast to previous reports, overexpression of gelsolin in Jurkat T
cells did not prevent or delay apoptosis induced by Fas ligation or
ceramide treatment. Overexpressed gelsolin protein was cleaved during
apoptosis, as seen previously in this and other cell types. In these
model systems, therefore, the level of gelsolin expression was not a
rate-limiting determinant in commitment to or time to the morphological
changes of apoptosis.
(Blood. 2000;95:3483-3488)
© 2000 by The American Society of Hematology.
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Introduction |
Apoptosis, a process of programmed cell death
characterized by defined biochemical and morphological changes, is
often divided into 2 phases, an initiation/commitment phase and a
downstream effector phase.1 A number of stimuli can trigger
different signaling pathways that culminate in cellular commitment to
apoptosis. The precise events that constitute irreversible commitment
are as yet unknown, but upon activation of a family of cysteinyl
proteases (caspases), commitment has occurred and cell death is
irreversible. In this regard, caspase-3 appears to play a critical role
as an executioner of the effector phase of apoptosis, since its
activation is often required for the downstream morphological changes
that define apoptosis, including membrane blebbing, DNA degradation, and nuclear condensation.1 The interrelationships between
commitment to apoptosis, activation of caspase-3, and the resulting
morphological changes are not fully understood.
One substrate of caspase-3 that has been recently identified is
gelsolin, an 80-kd actin-binding protein.2
Gelsolin consists of 6 repeating domains, with separate, distinct sites
for binding actin monomers and polymers.3 Through the
sequestration of actin monomers and the severing and capping of actin
filaments, gelsolin regulates the rates of both actin polymerization
and depolymerization. The activity of gelsolin itself is regulated by
calcium and polyphosphoinositide binding.4,5 Gelsolin contains a consensus caspase cleavage site (DQTD352G)
between the third and fourth repeating domains. Cleavage at this site
by caspase-3 generates an amino-terminal fragment that binds and severs
actin filaments independently of calcium regulation.2 Overexpression of this fragment generates many of the characteristic morphological changes of apoptosis, including DNA fragmentation, without activating the caspase cascade, and it has been suggested that
the cleavage of gelsolin is a critical element in the progression of
the "effector" phase of apoptosis.2 Interestingly, it
has also been reported that the overexpression of gelsolin prevented apoptosis induced by Fas ligation, by ceramide, and by dexamethasone in
Jurkat T cells by preventing the release of cytochrome c from the
mitochondrial membrane and the subsequent activation of
caspase-3.6,7 These results provide support for an
additional role for gelsolin in the upstream, commitment phase of
apoptosis or for the possibility that the interaction between caspase-3
and gelsolin is more complex than was initially described.
We have recently demonstrated that pharmacological stabilization of the
actin cytoskeleton by the compound jasplakinolide enhanced apoptosis of
the interleukin (IL)-2-dependent T-cell line CTLL-20 induced by
cytokine deprivation.8 Incubation of the cells with
jasplakinolide after IL-2 deprivation decreased the time to commitment
to the apoptotic pathway. The enhancement of apoptosis occurred
upstream of the caspase cascade and could be inhibited by the
overexpression of the anti-apoptotic protein Bcl-xL.
CTLL-20 cells treated with jasplakinolide did not undergo apoptosis
when incubated in the presence of exogenous IL-2, arguing that this
compound was not toxic to the cells. These data suggested a specific
role for the actin cytoskeleton in the upstream "commitment" phase of apoptosis. On the basis of the reports of the involvement of
gelsolin in apoptosis, we hypothesized that jasplakinolide-mediated actin stabilization altered gelsolin activity and that the modulation of gelsolin in turn altered the apoptotic process. Here we tested this
hypothesis by overexpressing gelsolin in CTLL-20 cells, in the murine
IL-3 dependent pre-B cell line Ba/F3, and in the human T-cell line
Jurkat. As expected, gelsolin was cleaved during apoptosis; surprisingly, however, its overexpression did not inhibit apoptosis induced by cytokine deprivation, ceramide treatment, or Fas ligation. Furthermore, overexpression of gelsolin did not prevent the
jasplakinolide-mediated enhancement of apoptosis induced by cytokine deprivation.
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Materials and methods |
Reagents
The actin-binding compound jasplakinolide was provided by the Drug
Synthesis and Chemistry Branch, Developmental Therapeutics Program,
Division of Cancer Treatment, National Cancer Institute, National
Institutes of Health (Bethesda, MD). Jasplakinolide was stored in
Me2SO at  20°C or 80°C and diluted
into medium immediately prior to use. The human gelsolin expression
vector LKCG and the control vector LK444 have been previously
described.9 LKCG contains the complementary DNA for human
gelsolin driven by the  -actin promoter and contains a neomycin
resistance gene. The plasmids were linearized with ScaI (New England
Biolabs, Beverly, MA) and purified prior to transfection.
The murine antihuman gelsolin monoclonal antibody (mAb) GS-2C4 was
purchased from Sigma (St Louis, MO). C2-ceramide
(D-erythro-sphingosine, N-Acetyl-) was purchased from
Calbiochem (La Jolla, CA) and stored in Me2SO at
20°C. Antihuman Fas mAb 7C11 was kindly provided by Jerome
Ritz (Dana-Farber Cancer Institute, Boston, MA). Antihuman Fas mAb CH11
was purchased from Upstate Biotechnologies (Lake Placid, NY). Antiactin
mAb N350 was purchased from Amersham (Arlington Heights, IL).
Anti-caspase-3 mAb was purchased from Transduction Laboratories
(Lexington, KY).
Cell culture and transfection
The human leukemic T-cell line Jurkat was cultured in RPMI 1640 (MediaTech, Herndon, VA) supplemented with 10% heat-inactivated fetal
calf serum (Life Technologies, Gaithersburg, MD); 2 mmol/L L-glutamine,
10 mmol/L Hepes, 100 U/mL penicillin, and 100 µg/mL streptomycin
(MediaTech); and 50 µmol/L 2-mercaptoethanol (Sigma) (termed complete
media, or cRPMI-10%). The IL-2-dependent murine T-cell line CTLL-20
was cultured in cRPMI-10% supplemented with 3% IL-2-containing
supernatant derived from conconavalin A-stimulated rat splenocytes
(T-Stim) (Collaborative Biomedical Products, Bedford, MA). The
IL-3-dependent murine pre-B cell line Ba/F3 was cultured in cRPMI-10%
supplemented with 5% IL-3-containing Wehi culture supernatant. All
cells were free of mycoplasma as determined by routine
polymerase chain reaction analysis (Mycoplasma PCR Primers; Stratagene, La Jolla, CA).
For transfection, 5 × 106 cells were resuspended in
500 µL RPMI plus 10% fetal calf serum and incubated in the
electroporation chamber for 15 minutes with 30 µg linearized plasmid
DNA at room temperature. Cells were electroporated at low resistance,
800 microfarads (µF), and 250 V (Cell-Porator) (Life Technologies) and resuspended in cRPMI-10%. After 48 hours, cells were plated at
1 × 104 cells/mL in 24-well plates in selection
medium consisting of cRPMI-10% plus 1 mg/mL G418 (Geneticin) (Life
Technologies) for Jurkat cells, cRPMI-10% plus 1 mg/mL G418 plus 5%
Wehi-derived supernatant for Ba/F3 cells, and cRPMI-10% plus 0.3 mg/mL
G418 plus 100 U/mL recombinant human IL-2 (kindly provided by
Hoffman-LaRoche, Nutley, NJ) for CTLL-20 cells. G418-resistant colonies
were screened for expression of gelsolin by Western blot, and stable
transfectants were maintained in selection medium.
Apoptosis assays
Washed cells were resuspended at a density of
2 × 105 cells/mL for the induction of apoptosis.
Apoptosis was induced by cytokine withdrawal, treatment with ceramide
(50 or 100 µmol/L), or treatment with anti-Fas mAb (100 ng/mL CH11 or
a 1:500 dilution of 7C11 ascites) for the indicated periods of time.
For cytokine withdrawal, stably transfected cytokine-dependent cell
lines CTLL-20 or Ba/F3 were washed 3 times in cRPMI-10% and then
resuspended in cytokine-free cRPMI-10%. For the induction of apoptosis
by ceramide treatment, cells were incubated in AIM-V serum free
lymphocyte medium (Life Technologies). AIM-V medium was supplemented
with 50 U/mL recombinant human IL-2 for experiments using
CTLL-20 cells to prevent cytokine-withdrawal-induced death.
Cell death was assayed by trypan blue exclusion, nuclear morphology, or
Annexin V immunofluorescent staining. For the assessment of nuclear
morphology, cells induced to undergo apoptosis were fixed in an excess
volume of 3:1 (vol/vol) methanol/acetic acid, dried onto glass slides,
and stained with 50 µg/mL propidium iodide (Sigma) blocked with dried
milk, and incubated with 0.5 µg/mL RNase (Boehringer Mannheim). The
percentage of cells undergoing apoptosis was determined by quantifying
the number of cells with and without condensed nuclei by fluorescence
microscopy (Olympus Bmax BX50) (Olympus America, Melville, NY). More
than 300 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. The variance was computed as pq/n,
where p = proportion apoptotic,
q = 1p, and n = the number of cells counted.
For Annexin-V immunofluorescence, cells were resuspended in 200 µL
binding buffer (ApoAlert AnnexinV-FITC kit) (ClonTech, Palo Alto, CA)
and then stained with 5 µL Annexin V conjugated to fluorescein
isothiocyanate (FITC) and 20 µL Via-Probe (7-amino-actinomycin D
[7-AAD]) (PharMingen, San Diego, CA) for a minimum of 15 minutes at
room temperature. Staining was assayed by flow cytometry (Epics XL,
Coulter, equipped with an argon laser emitting at 488 nm), measuring
Annexin V-FITC staining on the FL1 channel (emission wavelength 535 nm)
and 7-AAD fluorescence on the FL3 channel (emission wavelength 620 nm).
Immunoprecipitations and immunoblotting
For immunoprecipitation, 2 × 107 cells were
lysed in 1 mL lysis buffer (1% Nonidet P-40 [Calbiochem], 150 mmol/L
NaCl, 25 mmol/L Tris pH 7.5, 1 mmol/L EDTA, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF)
(Boehringer Mannheim), and 1 mmol/L sodium orthovanadate). Postnuclear
lysates were rotated for 2 hours at 4°C with 2 µL anti-gelsolin
mAb and 20 µL Sepharose Protein G (Santa Cruz Biotechnology, Santa
Cruz, CA). Beads were washed 3 times with wash buffer (0.1% Nonidet
P-40, 150 mmol/L NaCl, 25 mmol/L Tris pH 7.5, 1 mmol/L EDTA, 1 mmol/L
sodium orthovanadate), and proteins were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Western blot assays were performed essentially as
described.10 In brief, whole-cell lysates were generated by
resuspending washed cells in SDS sample buffer and boiling for 10 minutes. Postnuclear lysates were generated by lysing washed cells in
lysis buffer (1% Nonidet P-40, 150 mmol/L NaCl, 25 mmol/L Hepes, 1 mmol/L EDTA pH 7.4, 1 mmol/L sodium orthovanadate, 10 µg/mL
aprotinin, 10 µg/mL leupeptin, 10 µg/mL soybean trypsin inhibitor),
incubating on ice for 20 minutes, and removing cell debris by
centrifugation at 15 300 rpm for 10 minutes. Supernatants were mixed
with 2 × SDS sample buffer and boiled for 10 minutes. Lysates
or immunoprecipitates were separated by SDS-PAGE (10% or 12%
acrylamide; Protogel) (National Diagnostics, Atlanta, GA). Proteins
were transferred to polyvinylidene membranes (Millipore, Bedford, MA),
which were blocked with a solution of 4% bovine serum albumin and then
incubated with the indicated antibody. Bound antibodies were detected
with the enhanced chemiluminescence system (Amersham Pharmacia Biotech)
according to the manufacturer's instructions.
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Results |
Overexpression of gelsolin in CTLL-20 or Ba/F3 cells did
not delay apoptosis in response to cytokine deprivation, nor did it
prevent the enhancement of apoptosis by jasplakinolide
To determine whether overexpression of gelsolin delayed or inhibited
apoptosis induced by cytokine deprivation, and to determine whether
overexpression of gelsolin prevented the enhancement of apoptosis
induced by actin polymerization, CTLL-20 cells were transfected with
the gelsolin expression or control vector by electroporation. Stably
transfected cell lines were selected by culture in G418 and screened by
Western blot analysis (Figure 1A). Endogenous gelsolin was
expressed at very low levels in CTLL-20 cells (data not shown).
Transfected cells were deprived of IL-2 and incubated for 15 hours in
the presence of either 100 nmol/L jasplakinolide or the
vehicle, 0.02% Me2SO. The percentage of apoptotic cells
was determined by nuclear morphology. Overexpression of human gelsolin
did not protect IL-2-deprived CTLL-20 cells from apoptosis, nor did it
prevent the enhancement of apoptosis observed when actin was stabilized
by the compound jasplakinolide (Figure 1B). Furthermore, the time of
commitment to cell death and the final percentage of cells that
underwent apoptosis were not changed by gelsolin overexpression (data
not shown).
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| Fig 1.
Effect of overexpression of gelsolin on timing of
apoptosis of growth factor-dependent cells induced by cytokine
deprivation.
Overexpression of gelsolin did not delay apoptosis of growth
factor-dependent cells induced by cytokine deprivation, or prevent its
enhancement by jasplakinolide, despite appropriate cleavage. (A) Whole
cell lysates (1.25 × 106 cell equivalents) of
transfected CTLL-20 cells were separated by SDS-PAGE and immunoblotted
for gelsolin and for actin. Serum was used as a positive control. (B)
Transfected CTLL-20 cells were incubated for 15 hours in the absence of
IL-2 and in the presence of 100 nmol/L jasplakinolide
(closed bars) or its vehicle, 0.02% Me2SO (open bars).
After incubation, cells were fixed and stained with propidium iodide
for quantification of apoptosis by nuclear morphology. Results are
representative of 2 independent experiments. (C, D) Whole cell lysates
(2 × 106 cell equivalents) of transfected Ba/F3
cells deprived of IL-3 for the indicated periods of time were separated
by SDS-PAGE and immunoblotted for human (C) or murine (D) gelsolin. The
time-dependent appearance of the 46 kd gelsolin cleavage
product is shown. In (D) there was a slight loss of the cell sample at
8 hours. Results are representative of 2 independent experiments. (E)
Parallel samples of transfected Ba/F3 cells deprived of IL-3 for the
indicated periods of time were fixed and stained with propidium iodide
for quantification of apoptosis by nuclear morphology. Closed circles
represent Ba/F3 cells that overexpress gelsolin, while open circles
represent the vector control. SDs were calculated as described in
"Materials and methods." The slight difference between the 2 cell
lines at 8 and 12 hours was not evident by trypan blue exclusion.
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Stable transfectants of the IL-3-dependent pre-B cell line Ba/F3 were
also generated (Figure 1C). Transfected Ba/F3 cells overexpressing
human gelsolin were deprived of IL-3 for the indicated period of time;
parallel samples were assayed for apoptosis and for cleavage of
gelsolin. Human gelsolin (Figure 1C) was cleaved at the same rate as
endogenous murine gelsolin (Figure 1D), confirming that human gelsolin
remained a substrate for murine caspases in a murine environment.
Consistent with the results observed in CTLL-20 cells, overexpression
of gelsolin did not inhibit nuclear condensation indicative of
apoptosis as measured by staining with propidium iodide (Figure 1E).
There was a modest time-dependent increase in apoptotic death in cells
overexpressing gelsolin following IL-3 deprivation; this increase may
reflect a modest acceleration in the effector phase of apoptosis,
consistent with an increase in levels of the amino-terminal fragment of
gelsolin following cleavage by caspases.2 However, this
difference was not reflected in cell death as measured by trypan blue
exclusion at these or later time points (data not shown). Thus, the
failure of gelsolin overexpression to inhibit apoptosis was not
specific to a cell line or to dependence on a particular cytokine.
Importantly, however, human gelsolin was able to interact with the
murine apoptotic machinery, as the exogenous human gelsolin was cleaved
at the same rate as the endogenous murine gelsolin (Figure 1C and 1D). In addition, the caspase machinery was not rate-limiting, as the endogenous gelsolin was cleaved at the same rate in Ba/F3 cell lines
that either did or did not overexpress gelsolin (Figure 1D).
Overexpression of gelsolin did not prevent apoptosis in response to
ceramide treatment or Fas ligation
While the role of gelsolin in apoptosis induced by cytokine
withdrawal has not been previously examined, its overexpression has
been reported to prevent ceramide-induced apoptosis in Jurkat T
cells.6 We therefore treated transfected CTLL-20 cells with vehicle (0.05% Me2SO) and 50 µmol/L or 100 µmol/L C2-ceramide while maintaining the cells in the
presence of 50 U/mL rhIL-2 to ensure growth-factor
sufficiency. The percentage of apoptotic cells was determined by
Annexin V-FITC staining (Figure 2), nuclear morphology, and/or trypan blue exclusion (data not shown).
Overexpression of gelsolin did not delay apoptosis in response to
ceramide treatment in CTLL-20 cells (Figure 2).
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| Fig 2.
Effect of overexpression of gelsolin on apoptosis of
CTLL-20 cells induced by C2-ceramide.
Overexpression of gelsolin did not prevent apoptosis of CTLL-20 cells
induced by C2-ceramide. Transfected CTLL-20 cells overexpressing
gelsolin (solid bars) or vector alone (hatched bars) were incubated for
12.5 hours in the presence of 50 U/mL recombinant human
IL-2 and in the presence of 100 µmol/L C2-ceramide
(closed or dark gray bars), 50 µmol/L C2-ceramide (gray
bars), or its vehicle, 0.05% Me2SO (open bars). The
percentage of apoptosis was determined by Annexin V-FITC staining as
described in "Materials and methods." Comparable results were
obtained by trypan blue exclusion (data not shown). These results are
representative of 3 independent experiments. Immunoblotting for
gelsolin (inset) demonstrated that the gelsolin CTLL-20 transfectants
were overexpressing gelsolin protein at the time of the experiment.
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Our results were in striking contrast to the results reported for
Jurkat cells.6 Therefore, by electroporation and selection in G418, stable transfectants of Jurkat T cells that overexpressed human gelsolin were generated. Expression of human gelsolin driven by
the heterologous promoter was higher than that of endogenous protein
(Figure 3A, 3B, 3C [insets]). As
expected, endogenous gelsolin co-immunoprecipitated with actin; the
amount of actin was dramatically increased in immunoprecipitates of
Jurkat cells transfected with and overexpressing gelsolin (Figure 3A).
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| Fig 3.
Effect of overexpression of gelsolin on apoptosis of
Jurkat T cells in response to ceramide treatment or to Fas ligation.
Overexpression of gelsolin did not prevent apoptosis of Jurkat T cells
in response to ceramide treatment or to Fas ligation, although gelsolin
was cleaved during apoptosis. (A) Both endogenous and overexpressed
gelsolin bound actin. Human gelsolin was immunoprecipitated from
lysates of transfected Jurkat cells (2 × 107
cells/sample). Precipitated proteins were separated by 10% SDS-PAGE on
a 10% gel, transferred to PVDF membrane, and immunoblotted for
gelsolin and actin as described. These results are representative of 2 independent experiments. (B) Transfected Jurkat T cells (solid bars
represent those that overexpress gelsolin, hatched bars represent the
vector control) were incubated with 50 µmol/L ceramide
(closed or gray bar) or its vehicle, 0.025% Me2SO (open
bars), for 12 hours. The percentage of apoptosis was determined by
trypan blue exclusion. Level of expression of gelsolin during this
experiment was determined by immunoblot (inset). (C) Transfected Jurkat
cells (solid bars represent those that overexpress gelsolin, hatched
bars represent the vector control) were incubated for 4 hours with
anti-Fas mAb (1:500 dilution of 7C11) (closed or gray bars) or with
medium alone (open bars), fixed, and stained with propidium iodide for
quantification of apoptosis by nuclear morphology. Immunoblot confirmed
the overexpression of gelsolin at the time of the experiment (inset).
Results are representative of 4 independent experiments.
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Transfected Jurkat cell lines were treated with 50 µmol/L C2-ceramide or vehicle control (0.025%
Me2SO) for 12 hours, and cell death was assayed by trypan
blue exclusion. Overexpression of gelsolin did not inhibit
ceramide-induced apoptosis in Jurkat T cells (Figure 3B). Transfected
Jurkat T-cell lines were treated with anti-Fas mAb and assayed for
apoptosis by nuclear morphology (Figure 3C), trypan blue exclusion, and
Annexin V-FITC staining (data not shown). Overexpression of gelsolin in
Jurkat T cells did not inhibit Fas-dependent apoptosis, as measured by
3 different methods, again suggesting that gelsolin is not
rate-limiting in the progression of apoptosis.
To confirm that the exogenous gelsolin was cleaved during
apoptosis, we conducted a time-course experiment in which transfected Jurkat T cells were incubated with anti-Fas mAb for the indicated amount of time and then assayed for both apoptosis and cleavage of
gelsolin. Exogenous gelsolin was cleaved during the apoptotic process
(Figure 4A), but the rate and extent of
cell death were not decreased by the overexpression of this caspase-3
substrate (Figure 4B). A slight increase in cell death of the Jurkat
T-cell line that overexpressed gelsolin was evident by
trypan blue exclusion, a finding that would be consistent with an
acceleration of the effector phase of apoptosis mediated by an increase
in the levels of the amino-terminal gelsolin fragment. However, this
increase was not measurable by Annexin-V FITC staining, and no
consistent, significant increase was seen in other experiments (Figure
3C and data not shown).
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| Fig 4.
Gelsolin and caspase-3 were cleaved during apoptosis
induced by Fas ligation.
(A) Transfected Jurkat cells were incubated for the indicated period of
time with 100 ng/mL CH11 anti-Fas mAb. Immunoblot analysis
of whole-cell lysates (1 × 106 cell equivalents)
demonstrated overexpression of gelsolin and its cleavage during
apoptosis. The gelsolin cleavage product is evident at approximately 46 kd. Results are representative of 4 independent
experiments. (B) Parallel samples of transfected Jurkat cells incubated
with 100 ng/mL CH11 anti-Fas mAb were assayed for the
percentage of cell deaths by trypan blue exclusion (closed circles,
cells overexpressing gelsolin, JK-Gsn-22; open circles, vector
controls, JK-Vec-33). The percentage of cell deaths of parallel samples
was confirmed by staining with Annexin V-FITC (solid bars, JK-Gsn-22;
hatched bars, JK-Vec-33). (C) Transfected Jurkat cells were treated
with anti-Fas mAb 100 ng/mL CH11 for the indicated periods
of time. Immunoblot analysis of postnuclear lysates
(1 × 106 cell equivalents) demonstrated cleavage of
caspase-3; one of the fragments of caspase-3 generated during apoptosis
is evident at approximately 17 kd. Results are
representative of 3 independent experiments.
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Overexpression of gelsolin had been reported to prevent the cleavage of
caspase-3 in Jurkat T cells during Fas-induced
apoptosis.6,7 In contrast to these results,6,7
no delay in caspase-3 cleavage was evident in the Jurkat T cells that
overexpressed gelsolin used here (Figure 4C). The cleavage product of
caspase-3 (p17) appeared 2 hours after Fas ligation in both gelsolin
and control Jurkat transfectants. Near-complete cleavage of caspase-3
was not apparent until 4 hours after Fas ligation (Figure 4C). Cleavage of both the endogenous and exogenous gelsolin occurred concurrently with caspase-3 cleavage, as did the appearance of dead cells assayed by
trypan blue exclusion (data not shown).
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Discussion |
We have shown that overexpression of gelsolin in 3 different
lymphocyte cell lines (CTLL-20, Ba/F3, and Jurkat) did not prevent or
delay cell death in response to cytokine deprivation, ceramide treatment, or Fas ligation. A slight increase in death in 2 experiments (Figures 1E and 4B) could reflect an acceleration of the apoptotic "effector" phase by the increased levels of the amino-terminal fragment of gelsolin, which would be suggested by the results of
Kothakota et al.2 However, as this increase was not seen in
all experiments or by all methods of assaying cell death, we are unable
to conclude that this is a consistent and biologically relevant result.
Our results differ from those of Ohtsu et al,6 who reported
that overexpression of gelsolin in Jurkat T cells delayed cell death in
response to treatment with ceramide, dexamethasone, and anti-Fas
antibody. Also in contrast to previous reports, we demonstrated that
overexpression of gelsolin did not affect the rate of activation of
caspase-3 in Jurkat T cells induced to undergo apoptosis by Fas
ligation.6,7 Furthermore, overexpression of gelsolin did
not modify the jasplakinolide-mediated enhancement of apoptosis of
CTLL-20 cells induced by cytokine deprivation, suggesting that the
enhancement of apoptosis by actin stabilization previously
described8 was not affected by an increase in the molar
content or actin-severing activity of gelsolin.
The observations of Ohtsu et al6 and Kamada et
al7 regarding the ability of gelsolin to inhibit
Fas-induced death may have differed from our observations because of
differences in the pathways of Fas signaling in the 2 different
populations of Jurkat cells under study. Scaffidi and
coworkers11 recently reported that the Fas apoptotic
pathway can proceed via 2 distinct pathways; in a given cell type, 1 of
these 2 pathways is predominant. Cells designated type I were shown to
cleave caspase-8 within 10 minutes of Fas ligation, while caspase-3
cleavage and subsequent activation were detected within 30 minutes. In
these cells, caspase-3 was activated independently of mitochondrial
permeability changes, and cell death was not inhibited by
overexpression of the anti-apoptotic proteins Bcl-2 or
Bcl-xL. In cells designated type II, however, caspase-8
cleavage was reported to appear only after 1 hour of anti-Fas
stimulation, and caspase-3 cleavage appeared weakly after 2 hours.
Activation of caspase-3 was dependent upon mitochondrial changes in
type II cells, and apoptosis was inhibited by Bcl-2 or
Bcl-xL. Caspase-8 overexpression converted a type II cell
line into a type I.11 Kamada and coworkers7
demonstrated that gelsolin overexpression inhibited cytochrome c
release upstream of caspase activation, suggesting that their cells
displayed a type II phenotype. Our Jurkat T cells also displayed type
II kinetics of caspase-3 activation (Figure 4C), as the cleavage
product of caspase-3 (p17) appeared after 2 hours of incubation with an
anti-Fas antibody, similar to those seen by Kamada et al.7
We do note that the kinetics of caspase-3 activation in their Jurkat T
cells were different from those seen here: in their vector-transfected cell lines, caspase-3 cleavage was apparent within 1 hour after Fas
ligation and appeared to be largely complete after 2 hours.7 The different rates of caspase-3 cleavage in these
2 populations of Jurkat T cells could be due to the higher
concentrations of anti-Fas mAb used by Kamada et al7 (1 µg/mL in Kamada et al; 0.1 µg/mL here),
or to clonal variation in the 2 Jurkat cell populations studied.
It is important to note that the demonstration of inhibition of
apoptotic death by a particular protein may depend upon the method used
to assay cell death. Following Fas ligation, caspase-3 is required for
blebbing, DNA degradation, and nuclear condensation but not for the
phosphatidylserine "flip" or for loss of membrane integrity as
measured by uptake of propidium iodide.12 Thus, an
inhibitor of caspase-3 activation, as gelsolin was proposed to
be,6 would appear to inhibit cell death if cell death were assayed by membrane blebbing, but not if death were assayed by Annexin
V staining. We used methods similar to those of Ohtsu et
al6 to detect apoptosis; both methods were sensitive to early morphologic changes. It remains possible that the discrepancy between previously published results and those presented here may be
due to different levels of (basal or transfected) expression of
gelsolin, or that the gelsolin overexpressed in Jurkat T cells by other
investigators was not cleaved during Fas-induced apoptosis. In their
system, it is possible that uncleaved, full-length gelsolin was able to
compete for substrates of the cleaved gelsolin that are required for
progression of apoptosis, thus acting as a "dominant interfering"
molecule. This competition could theoretically inhibit apoptosis. The
discrepancy could also be due to other, as yet undefined, differences
in apoptotic signaling following Fas ligation. That the regulation of
induction and progression of apoptosis is complex has been shown in
many systems, including Fas-induced death of Jurkat T
lymphocytes.11,13-15
Different roles for gelsolin have previously been attributed to
differences in the cellular environment and the apoptotic stimulus. Its
overexpression has been reported to prevent Fas- and ceramide-induced
death in Jurkat T cells,6 and its absence has been reported
to protect neutrophils from apoptosis induced by TNF .2
Additionally, the absence of gelsolin sensitized hippocampal neurons to
apoptosis induced by glutamate.16 We have confirmed that
gelsolin is cleaved during lymphocyte apoptosis, but we failed to show
that the quantitative increase in the generation of the active
amino-terminal fragment of gelsolin dramatically increased the rate of
the appearance of nuclear condensation or of cell death in lymphocytes
as might have been predicted from the results of Kothakota et
al.2 There did appear to be a variable, time-dependent
increase in cell death following cytokine deprivation (Figure 1E) and
after Fas ligation (Figure 4B), but this increase was not
consistent at all time points (Figure 1E, 4-hour time point) or in all
experiments (data not shown). In these lymphocyte cell lines,
therefore, gelsolin and the generation of the amino-terminal fragment
of gelsolin did not seem to be a significant or rate-limiting factor for apoptotic cell death.
| |
Footnotes |
Submitted September 22, 1999; accepted January 31, 2000.
S.C.P. was supported by a pre-Intramural Research Training Award from
the National Heart, Lung and Blood Institute of the National Institutes
of Health, Bethesda, MD. M.P.M. was supported by a fellowship from
Fondazione, "Istituto Pasteur Fondazione Cenci-Bolognetti,"
University of Rome "La Sapienza," Italy, and by a
supplemental fellowship from the National Institutes of Health, Bethesda, MD. D.J.K. was supported by grant HL54188 from the National Institutes of Health, Bethesda, MD.
Reprints: Barbara E. Bierer, National Heart, Lung and Blood
Institute, Bldg 10, Rm 5D49, 10 Center Dr, Bethesda, MD 20892; e-mail:
biererb{at}nih.gov.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction