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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 1891-1900
Protection From Apoptosis by Steel Factor But Not
Interleukin-3 Is Reversed Through Blockade of Calcium Influx
By
Jennifer L. Gommerman and
Stuart A. Berger
From the Wellesley Hospital Research Institute and Department of
Immunology, University of Toronto, Toronto, Ontario, Canada.
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ABSTRACT |
Steel factor (SLF), the ligand for the c-Kit receptor,
protects hemopoietic progenitors and mast cells from apoptosis. We show
here that protection of 32D-Kit cells or mast cells from apoptosis by
SLF is abrogated through concurrent inhibition of Ca2+
influx. In contrast, cell survival promoted by interleukin-3 is not
affected by Ca2+ influx blockers. In the presence of
blockers, increasing stimulation by SLF leads to greater levels of cell
death in the population, indicating that it is the combination of
activation by SLF with concurrent blockade of Ca2+ influx
that results in apoptosis. The p815 mastocytoma, which expresses a
mutated, constitutively active c-kit receptor, dies apoptotically in
the presence of Ca2+ influx blockers alone. Ionomycin
protects cells from SLF plus blocker-induced apoptosis, confirming
specificity for Ca2+ ion blockade in cell death
induction. Overexpression of bcl-2, which protects 32D-Kit cells from
factor withdrawal, does not protect cells from apoptosis by SLF plus
blocker. In contrast, caspase inhibitors YVAD-CHO, DEVD-FMK, and
Boc-Asp-FMK protect cells from SLF plus blocker-induced death. These
observations highlight the importance of SLF-stimulated
Ca2+ influx in the protection of cells from apoptosis and
demonstrate a new mechanism for inducing bcl-2 insensitive,
caspase-dependent apoptosis through the combination of SLF stimulation
with Ca2+ influx blockade.
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INTRODUCTION |
HEMATOPOIETIC CELLS are protected from
cell death by a variety of cytokines and growth factors. In the absence
of these protective signals, cells undergo a series of morphologic and
biochemical changes, including membrane blebbing, nuclear condensation,
cell shrinkage, and DNA fragmentation, culminating within 12 to 48 hours in apoptotic or programmed cell death.1
One documented mechanism for the protection of cells from apoptosis is
through the upregulation of bcl-2. The bcl-2 family of proteins are key
regulators of apototic death consisting of both anti-apoptotic and
pro-apoptotic members.2 Although the precise mechanism by
which bcl-2 family members influence apoptosis is unknown, recent
structural and biochemical evidence indicates that bcl-2 proteins
perform multiple functions that may influence cell death pathways.
These include physical interactions with numerous cytoplasmic proteins,
formation of ion channels, and regulation of the permeability
transition in mitochondria.3
For hematopoietic stem cells, myeloid progenitor cells, and mast cells,
two factors that protect cells from apoptosis are Steel factor (SLF),
the ligand for the c-Kit receptor tyrosine kinase, and interleukin-3
(IL-3).4-7 Upregulation of bcl-2 by IL-3 in myeloid cells
has been well documented.8-10 In contrast, SLF has only
been observed to upregulate bcl-2 in natural killer cells,11 indicating that the mechanism by which SLF
protects myeloid cells from apoptosis may differ from that of IL-3.
One biochemical change that has been associated with the induction of
apoptosis in a number of cell types is the deregulation of
intracellular Ca2+ concentrations.12,13
However, a general model explaining the role of Ca2+ in
apoptosis remains elusive. Excessive intracellular Ca2+
levels such as those induced by Ca2+ ionophore have been
shown to induce apoptosis in a number of experimental
systems.14,15 Apoptosis in splenocytes appears to involve a
Ca2+-dependent endonuclease,16 and
intracellular Ca2+ increases have been linked to apoptosis
of both activated T-cell hybridomas17 and immature
thymocytes.18 In contrast to these observations, some cells
seem to be protected from apoptosis by Ca2+ influx. For
instance, IL-3-dependent mast cells and cell lines are protected from
growth factor withdrawal-mediated apoptosis by addition of
Ca2+ ionophore,19 and programmed neuronal death
is also suppressed by increases in intracellular
Ca2+.20
SLF, unlike IL-3, stimulates the mobilization of Ca2+ from
internal stores followed by influx of Ca2+ from the
extracellular milieu.21 Given the importance of
intracellular Ca2+ in the apoptotic process, we have
investigated the effect of inhibitors of Ca2+ influx on
cell survival promoted by SLF and IL-3. We show here that blockade of
Ca2+ influx reverses the ability of SLF to protect cells
from apoptosis, but does not affect cell viability promoted by IL-3.
Notably, in the presence of Ca2+ influx blockers, higher
concentrations of SLF induce greater levels of cell death, indicating
that this form of apoptosis is dependent on cellular stimulation. We
also show that overexpression of bcl-2 does not protect cells from the
combination of SLF plus Ca2+ influx blockers, but caspase
inhibitors provide significant protection. Our results therefore show a
role for Ca2+ influx in SLF-mediated protection from cell
death and identify a new mechanism for inducing caspase-mediated
apoptosis through the combination of a growth signal with blockade of
Ca2+ influx.
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MATERIALS AND METHODS |
Cells.
32D-Kit cells (gift from Dr Mark Minden, Toronto, Ontario, Canada) are
an IL-3-dependent myelomonocytic cell line expressing c-kit.22 32D-Kit cells were grown in RPMI supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2% WEHI-3
supernatant, and 1 mg/mL G418 (GIBCO, Grand Island, NY).
Bone marrow-derived mast cells (BMMC) were generated as described
previously.23 They were cultured in OPTI-MEM (GIBCO)
supplemented with 10% heat-inactivated FBS and 2% WEHI-3 supernatant
as a source of IL-3. The P815 cell line is a murine
mastocytoma.24 P815 cells were grown in RPMI supplemented
with 10% heat-inactivated FBS. Bcl-2 gp+e retroviral producing NIH 3T3
packaging cells (gift from Dr Y. Ben-David, Toronto, Ontario, Canada)
contain an LXSN-based retroviral vector expressing genes for both
puromycin resistance and murine bcl-2. These were grown in Dulbecco's
modified Eagle's medium (DMEM) and supplemented with 10%
FBS and 2 µg/mL puromycin (Sigma, St Louis, MO).
32D-Kit-bcl-2 cells were generated by coculturing 32D-Kit cells with
packaging cells for 24 hours. Nonadherent 32D-Kit cells were then
removed, cultured for 48 hours, and then selected for bcl-2
overexpressing cells in the presence of 2 µg/mL puromycin. All cell
cultures also contained 55 µmol/L  -mercaptoethanol and antibiotics
(both Sigma).
Production of recombinant SLF.
Recombinant murine Steel Factor (SLF) was produced in soluble form in
Escherichia coli using the pFLAG.ATS, IPTG-inducible secretion
expression vector (InterScience, Markham, Ontario,
Canada). This vector includes an eight amino acid N-terminal FLAG
epitope. E coli containing the pFLAG.ATS.SLF plasmid
were grown to an OD600 of 0.4 to 0.5 in Luria Broth at
37°C and induced overnight with 33 µg/L
isopropyl--D-throgalactopyranoside (IPTG). A total of 1 mmol/L CaCl2 and 100 µmol/L phenylmethyl sulfonyl
fluoride (PMSF) were added to the bacterial supernatant and FLAG-SLF
was affinity-purified on a column of Anti-FLAG M1 mouse monoclonal
antibodies covalently attached to agarose gel. FLAG-SLF was eluted with
phosphate-buffered saline (PBS) + 2 mmol/L EDTA, collected,
concentrated, dialyzed against PBS, and checked for purity by silver
stain. Bioactivity of SLF was measured on both 32D-Kit cells and BMMC
by incubating 2.5 × 104 cells in 96-well plates with
varying concentrations of SLF for 36 hours, followed by a 6-hour pulse
with 3H-thymidine. Incorporated radioactivity was
determined by scintillation counting.
Other reagents.
Ca2+ channel blockers and ionomycin were obtained from
Sigma. YVAD-CHO ICE protease inhibitor peptide was obtained from
Amersham (Arlington Heights, IL). DEVD-FMK and Boc-Asp-FMK
were both obtained from Enzyme Systems (Dublin, CA).
Cell death assays.
BMMC or 32D-Kit cells (2.5 × 104) were placed in
96-well flat-bottom plates in a volume of 0.1 mL RPMI containing 0.5%
FBS. Cells were supplemented with either SLF or IL-3 plus
Ca2+ channel blocker. The proportion of dead cells was
determined after 18 or 24 hours in culture by counting cells that could
or could not exclude Trypan blue.
Semisolid agar assays.
Cell death assays were plated as described above. After 18 hours of
incubation, cells were washed once in RPMI containing 10% FBS and
plated in 35-mm petri dishes along with 1 mg/mL G418, 25% WEHI-3
supernatant, and 0.3% agar (GIBCO). Colonies of 40 or more cells were
counted after 7 days at 37°C.
Analysis of DNA content.
Cells (1.25 × 106) were incubated for 18 or 24 hours
as described above. Cells were spun down and resuspended in
Vindelöv's reagent (3.4 mmol/L Tris [pH 8], 75 µmol/L
propidium iodide [from Sigma], 0.1% NP-40, 700 U/L RNAse [Sigma],
and 10 mmol/L NaCl). Cells were then analyzed by flow cytometry.
Western blotting.
For the Bcl-2 blot, 1 × 106 32D-Kit and 32D-Kit-bcl-2
cells were washed in PBS; resuspended in TBS lysis buffer containing 1% NP-40, 10% glycerol, protease inhibitors (500 µmol/L
sodium-orthovanadate, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 1 mmol/L PMSF [all Sigma]); and incubated at 4°C for 20 minutes.
Lysates were centrifuged at 12,000 rpm for 10 minutes, and the
supernatant was separated by 12% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose. The
blot was blocked with 5% skim milk powder and 0.1% TWEEN-20 in PBS,
probed with anti-bcl-2 antibodies (UBI, Lake Placid, NY) followed by a
horseradish peroxidase-labeled goat antimouse secondary antibody
(Jackson Immunoresearch, Mississauga, Ontario, Canada),
and developed with chemiluminescence reagents (Amersham). To confirm
equal protein loading, the blot was stripped by acid treatment and
reprobed with an antiactin monoclonal antibody (Sigma) followed by the same horseradish peroxidase-labeled goat antimouse secondary antibody (Jackson) and developed with chemiluminescence reagents.
For the PARP cleavage blot, 2 × 106 32D-Kit cells
were pretreated with combinations of 10 µmol/L econazole, SLF, IL-3,
and Boc-Asp-FMK. Cells were washed in PBS, resuspended in a hypotonic lysis buffer (20 mmol/L HEPES, 10 mmol/L KCl, 1 mmol/L EDTA, and 1 mmol/L dithiothreitol) with protease inhibitors aprotinin
and leupeptin (both at 1 µg/mL) and 1 mmol/L PMSF (all Sigma) and left at 4°C for 5 minutes before adding 0.1% NP40. Lysates were centrifuged at 6,000 rpm for 5 minutes at 4°C. The supernatant was
removed and centrifuged again at 10,000 rpm for 10 minutes at 4°C.
The nuclear pellets were resuspended in sample buffer containing 10%
-mercaptoethanol and 8 mol/L urea, boiled and sonicated for 10 seconds at a setting of 50%, resolved by 7.5% SDS-PAGE, and
transferred to nitrocellulose. The blot was probed and developed as
described above using a rabbit polyclonal anti-PARP antibody (UBI) that
was raised against a synthetic peptide corresponding to the region
C-terminal to the cleavage site of human PARP. The blot was then probed
with a goat antirabbit horseradish peroxidase-labeled secondary
antibody (Jackson) and then developed using chemiluminescence reagents.
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RESULTS |
Protection from apoptosis by SLF is reversed through blockade of
Ca2+ influx.
Ca2+ influx has been observed to both induce and protect
cells from apoptosis in different cell systems.14,15,19 To
investigate the role of Ca2+ influx in protection from
apoptosis, we determined the effect of Ca2+ influx blockers
on cell viability supported by SLF, a growth factor that mobilizes
Ca2+ or IL-3, a mitogenic cytokine that does not mobilize
Ca2+.21,25 For these experiments, we used
32D-Kit cells, an IL-3-dependent murine myelomonocytic cell line that
has been transfected with the c-kit receptor tyrosine kinase gene.
Expression of c-kit in these cells makes them mitogenically responsive
to SLF in vitro (Fig 1F) and renders them
tumorigenic in vivo.22 These cells die apoptotically within
12 to 24 hours upon removal of factor.10
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| Fig 1.
Effect of SLF or IL-3 with Ca2+ influx
blockers on 32D-Kit cells. (A through D) 32D-Kit cells were incubated
with either SLF (500 ng/mL in all cases) (circles) or IL-3 (25% WEHI-3
conditioned medium in all cases) (squares) or both SLF and IL-3
(triangles) with varying amounts of econazole or ketotifen. In the case
of (A) and (C), the proportion of dead cells was determined after 18 hours in culture by counting cells that could or could not exclude
Trypan blue. For (B) and (D), colony formation was determined by
incubating cells in liquid culture with either SLF or IL-3 with
econazole or ketotifen followed by 7 days of incubation of cells in
semisolid medium. (E) 32D-Kit cells were incubated with varying amounts
of SLF. (Circles) Plus 7.5 µmol/L econazole. (Squares) No econazole.
(F) 32D-Kit cells were incubated with varying amounts of SLF for 36 hours followed by a 6-hour pulse with 3HdT thymidine,
harvesting, and counting. Error bars represent the standard error
determined from triplicate measurements.
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Although 32D-Kit cells are normally protected from apoptosis by SLF, we
observed, using Trypan blue exclusion analysis, that this protective
signal is converted to a death signal if the cells are co-incubated
with the Ca2+ influx blockers econazole or ketotifen (Fig
1A and C, circles). In contrast, death is not observed when 32D-Kit
cells are incubated with Ca2+ influx blocker plus the
mitogen IL-3 (Fig 1A and C, squares), and blockers do not accelerate
cell death due to withdrawal of protective factors (not shown).
Furthermore, simultaneous incubation of 32D-Kit cells with SLF,
Ca2+ influx blockers, and IL-3 still results in cell death
(Fig 1A, triangles), indicating that IL-3 is not protective for this
particular death signal. Induction of cell death by SLF plus
Ca2+ influx blocker increases with increasing
concentrations of SLF (Fig 1E). Therefore, taken together, the effect
of the Ca2+ influx blockers is not simply to counteract or
neutralize the protective effect of SLF. Rather, they combine with high
levels of SLF to induce cell death.
Failure to exclude Trypan blue is a useful but late cell death endpoint
that may not always correlate with other measures of cell viability. We
therefore also determined the effect of combining Ca2+
influx blockers with SLF or IL-3 on the clonogenic capacity of cells.
32D-Kit cells were exposed to blockers with either SLF or IL-3 for 18 hours. The cells were then collected and plated in semisolid agar in
the presence of IL-3, and colonies were counted 7 days later. As shown
in Fig 1B and D, overnight exposure to econazole or ketotifen in the
presence of IL-3 has little effect on the ability of cells to form
colonies. In contrast, exposure to SLF plus ketotifen or econazole
reduces clonogenicity by 10- or 1,000-fold, respectively. Therefore,
the specific combination of SLF with Ca2+ influx blockers
also results in clonogenic cell death.
Although 32D-Kit cells are factor-dependent, they grow continuously in
culture and are tumorigenic in vivo. We wished to determine if the
ability to induce cell death by SLF plus Ca2+ influx
blockers could also be observed in nontransformed cells. We therefore
determined the effect of combining Ca2+ influx blockers
with SLF or IL-3 on murine BMMC, which are also protected from
apoptosis and mitogenically stimulated by IL-3 and SLF. As shown in
Fig 2A, B, and C, exposure of BMMC to the combination of Ca2+ influx blockers ketotifen, econazole,
or Ni2+ with SLF but not IL-3 results in cell death. As
with 32D-Kit cells, greater stimulation by SLF leads to greater levels
of cell death in the population (Fig 2D). We therefore conclude that
cell death induced by the combination of SLF plus Ca2+
influx blockers is not limited to transformed cells such as 32D-Kit.
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| Fig 2.
Effect of SLF or IL-3 with Ca2+ influx
blockers on BMMC. (A through C) BMMC were incubated with either SLF
(500 ng/mL in all cases) (circles) or IL-3 (25% WEHI-3 conditioned
medium in all cases) (squares) or both SLF and IL-3 (triangles) with
varying amounts of econazole, ketotifen, or Ni2+. (D)
BMMC were incubated with IL-3 and varying amounts of SLF. (Circles)
Plus 2 mmol/L Ni2+. (Squares) No Ni2+. In
all cases, the proportion of dead cells was determined after 18 hours
in culture by counting cells that could or could not exclude Trypan
blue. Error bars represent the standard error determined from
triplicate measurements.
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Ca2+ influx blockers alone induce cell death in P815
mastocytoma cells.
Our data show that stimulation of factor-dependent cells with SLF in
the presence of Ca2+ influx blockers leads to cell death.
There are many examples of cells that exhibit factor-independent growth
due to expression of receptor tyrosine kinases with activating
mutations. One example is the P815 mastocytoma,24 which
exhibits constitutive tyrosine phosphorylation of the c-Kit protein and
factor-independent growth.26 These properties have been
attributed to an activating point mutation in the c-kit cytoplasmic
domain.27 Our observations predict that these cells should
be susceptible to death induced by Ca2+ influx blockers in
the absence of SLF stimulus. As shown in
Fig 3, treatment of P815 cells with
Ca2+ channel blockers econazole, ketotifen, or
Ni2+ leads to cell death in the presence or absence of
added SLF. Thus, the constitutive signals in P815 cells are sufficient
to combine with Ca2+ channel blockers to induce cell death.
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| Fig 3.
Effect of SLF or IL-3 with Ca2+ influx
blockers on P815 mastocytoma cells. P815 cells were incubated with
either SLF (500 ng/mL;  ) or IL-3 (25% WEHI-3 conditioned medium;
 ) with Ca2+ channel blockers econazole, ketotifen, or
Ni2+. In all cases, the proportion of dead cells was
determined after 18 hours in culture by counting cells that could or
could not exclude Trypan blue. Error bars represent the standard error
determined from triplicate measurements.
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BMMC and 32D-Kit-induced death is apoptotic.
Visual inspection of 32D-Kit cells after treatment with SLF plus
Ca2+ influx blockers showed some morphologic
characteristics of apoptosis, including nuclear condensation and
membrane blebbing (Fig 4B). However, to
further substantiate that 32D-Kit and mast cells were undergoing
apoptosis, DNA content, which characteristically fragments and
decreases during apoptosis, was measured. As shown in
Fig 5A, C, and D and summarized in
Table 1, treatment of 32D-Kit cells with
SLF or IL-3 alone or IL-3 with econazole for 18 hours did not generate
a population of cells with subdiploid DNA content, whereas SLF plus
econazole generated a large population of cells with subdiploid DNA
(Fig 5B). 32D-Kit cells will undergo apoptosis when they are deprived
of growth factor. After 18 hours of growth factor withdrawal, 32D-Kit
cells exhibited a modest proportion of cells with subdiploid DNA
content. This population is substantially increased after 24 hours of
factor withdrawal (data not shown). Thus, the apoptotic process induced
by Ca2+ influx blocker plus SLF is more rapid than the
apoptosis observed from factor withdrawal.
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| Fig 4.
Morphology of 32D-Kit cells incubated with factor and
Ca2+ influx blockers. 32D-Kit cells were incubated for 18 hours with (A) SLF alone, (B) SLF + 8 µmol/L econazole, (C) IL-3
alone, or (D) IL-3 + 8 µmol/L econazole. (E) Cells were incubated
with no added factor.
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| Fig 5.
DNA content of 32D-Kit cells incubated with factor and
Ca2+ influx blockers. 32D-Kit cells were incubated for 18 hours with (A) SLF alone, (B) SLF + 8 µmol/L econazole, (C) IL-3
alone, or (D) IL-3 + 8 µmol/L econazole. (E) Cells were incubated
with no added factor. Cells were then resuspended in Vindelövs
solution containing propidium iodide and analyzed by flow
cytometry.
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Specificity for non-voltage-gated Ca2+ influx blockers.
Ca2+ influx after receptor activation is mediated by the
opening of store-operated Ca2+ channels
(SOC).28 It is likely that this channel is
the target for inhibition, because the efficacy with which the three
compounds, ketotifen, econazole, and Ni2+, induce cell
death in combination with SLF correlates with their ability to inhibit
the SOC.29 We have observed that the voltage-gated Ca2+ channel blockers verapamil and nifedipine are
ineffective in inducing cell death when combined with SLF (not shown).
This result suggests that the induction of cell death in combination
with SLF is specific for non-voltage-gated Ca2+ influx
blockers.
Ionomycin protects cells from SLF plus Ca2+ influx
blocker induced apoptosis.
Other ion-blocking effects have been reported for both econazole and
ketotifen.29 To determine if specific blockade of
Ca2+ influx is critical for induction of cell death, we
examined the effect of the calcium ionophore ionomycin, on induction of
apoptosis. As shown in Fig 6, ionomycin
protects 32D-Kit cells from SLF plus Ca2+ influx
blocker-induced death in a concentration-dependent manner, with maximal
protection at 10 nmol/L (solid circles). Given the specificity of
ionomycin for Ca2+,30 these results indicate
that it is the specific blockade of Ca2+ influx that is
required for induction of apoptosis in combination with SLF.
Importantly, we also observed that higher concentrations of ionomycin,
which generate excessive levels of intracellular Ca2+,
resulted in the restoration of cell death. This shows that, in the
context of cell activation by SLF, both extremes of Ca2+
influx can induce cell death.
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| Fig 6.
Effect of ionomycin on SLF plus blocker-induced cell
death. 32D-Kit (,  ) or 32D-Kit-bcl-2 (,  ) cells were
incubated for 18 hours with 5 µmol/L econazole plus varying amounts
of ionomycin in the presence of either SLF (circles) or IL-3 (squares).
The proportion of dead cells was determined by Trypan Blue exclusion.
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Bcl-2 fails to protect 32D-Kit cells from SLF plus Ca2+
influx blocker-induced apoptosis.
The bcl-2 family of proteins have been strongly linked to protection of
cells from apoptosis induced by a wide variety of agents. Expression of
bcl-2 is correlated with proliferating cells31,32 and is
negatively regulated by the tumor suppressor p53,33 and overexpression of bcl-2 protects 32D cells from apoptotic death after
factor withdrawal.10 Given the importance of bcl-2 in regulating susceptibility to apoptosis, we were interested in the
effect that overexpression of this protein might have on the induction
of apoptosis by SLF plus Ca2+ influx blocker. We therefore
infected 32D-Kit cells with a retrovirus vector containing the bcl-2
gene,34 generated a bcl-2 overexpressing line
(Fig 7A), and tested the cells for
susceptibility to apoptosis. We confirmed that bcl-2 overexpression
protects 32D-Kit cells from apoptosis induced by factor withdrawal (Fig
7B). However, as also shown in Fig 7B, overexpression of bcl-2 in
32D-Kit cells fails to protect these cells from induction of apoptosis
by SLF plus econazole. These observations show that induction of cell death by SLF plus blocker occurs in a bcl-2-independent manner.
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| Fig 7.
Bcl-2 overexpression fails to protect 32D-Kit cells from
SLF plus Ca2+ influx blocker. (A) Western blot of cell
lysates from 32D-Kit (lane 1) or 32D-Kit-bcl-2 (lane 2) cells. Cell
lysates were separated by SDS-PAGE, transferred to nitrocellulose, and
probed with anti-bcl-2 antibodies (top panel). The blot was then
stripped and reprobed with an anti-actin monoclonal antibody to
demonstrate equal protein loading (bottom panel). (B) 32D-Kit () or
32D-Kit-bcl-2 ( ) cells were incubated for 18 hours with SLF or IL-3
plus 2.5, 5, or 10 µmol/L econazole, and the proportion of dead cells
was determined by Trypan Blue exclusion. In additional cultures, IL-3
alone or no factor was added and cell viability was similarly
determined.
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32D-Kit-bcl-2 cells were similarly protected from apoptosis by SLF plus
blocker with low levels of ionomycin (Fig 6); however, unlike the
32D-Kit cells, they did not demonstrate significant cell death at
higher levels of ionomycin. Taken together, these results therefore
confirm that bcl-2 can protect cells from both factor withdrawal and
high levels of intracellular Ca2+ but is unable to protect
cells from the combination of SLF plus Ca2+ influx
blockers.
Caspase inhibitors protect cells from Ca2+ influx blocker
plus SLF-induced death.
Members of the caspase family of intracellular proteases are common
effectors of apoptotic death induced by a wide variety of agents.
Current models of caspase involvement in apoptosis suggest that
pro-apoptotic stimuli activate a cascade of proteases, with ICE-like
caspases (caspase-1) acting upstream of CPP32-like caspases
(caspase-3).35,36 ICE-like caspases show cleavage specificity for substrates with aspartate in the P1 position and hydrophobic amino acids in the P4 position. These enzymes are preferentially inhibited by tetrapeptides such as the aldehyde YVAD-CHO.37 In contrast, CPP32-like caspases preferentially cleave substrates with acidic amino acids in the P4 position and are
inhibited by tetrapeptides such as DEVD-FMK and
Boc-Asp-FMK.38,39 To determine if caspases were involved in
the induction of apoptosis by SLF plus Ca2+ influx blocker,
these caspase inhibitors were tested for their protective ability. We
found that all three inhibitors protected 32D-Kit cells from apoptosis
induced by the topoisomerase inhibitor etoposide (not shown). We also
found that these inhibitors provided resistance to apoptosis induced by
SLF plus econazole (Fig 8), indicating that
apoptosis induced through the combination of SLF stimulation with a
Ca2+ influx blocker is likely mediated by a caspase cascade
involving both ICE-like and CPP32-like enzymes.
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| Fig 8.
YVAD-CHO, DEVD-FMK, and Boc-Asp-FMK protect 32D-Kit cells
from SLF plus econazole. 32D-Kit cells were incubated with either SLF
() or SLF plus 10 µmol/L econazole (). The proportion of dead
cells was evaluated by Trypan Blue exclusion after 18 hours in
culture.
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Cleavage of PARP protein is observed with SLF treatment plus
Ca2+ influx blockade.
Poly(ADP-ribose) polymerase (PARP) is a nuclear repair enzyme that has
also been implicated in transcription enhancement during preinitiation
complex formation.40 A common endpoint in the caspase
cascade is the cleavage of this 116-kD nuclear protein into an
approximately 85-kD fragment.41-43 Because this cleavage event is likely mediated by the CPP32 subfamily of caspases and our
results indicate that CPP32-like caspases are involved in the induction
of apoptosis through the combination of SLF and Ca2+ influx
blockers, we evaluated the effect of this treatment on PARP. We first
confirmed that PARP cleavage is induced by exposure of 32D-Kit cells to
etoposide (not shown). As shown in Fig 9, an approximately 85-kD PARP cleavage product is observed in nuclear extracts when 32D-Kit cells are treated with SLF plus econazole but is
not detected in nuclear extracts from cells incubated with SLF alone,
IL-3 alone, or IL-3 plus econazole. Coincubation of cells with
Boc-Asp-FMK results in partial inhibition of PARP cleavage, in
agreement with our data showing partial protection from apoptosis by
this inhibitor. We therefore conclude that the combination of SLF plus
Ca2+ influx blocker initiates a cascade involving ICE-like
and CPP32-like caspases that ultimately results in cleavage of PARP.
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| Fig 9.
Western blot analysis of nuclear PARP. 32D-Kit cells were
incubated for 12 hours with either SLF (lane 1), IL-3 (lane 2), SLF
plus 10 µmol/L econazole (lane 3), IL-3 plus 10 µmol/L econazole (lane 4), or SLF plus 10 µmol/L econazole plus 100 µmol/L
Boc-Asp-FMK. Nuclear lysates from these cultures were separated by
7.5% SDS-PAGE, transferred to nitrocellulose, and probed with
anti-PARP antibodies.
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DISCUSSION |
An important property of many hematopoietic growth factors and
cytokines is the protection of cells from apoptosis. We have shown here
that blockade of Ca2+ influx reverses the ability of SLF
but not IL-3 to protect cells from apoptotic death, highlighting the
importance of Ca2+ influx in SLF-mediated cell survival.
However, our data indicate that the effect of Ca2+ influx
blockade extends beyond simple neutralization of the protective properties of Ca2+ influx. Because higher levels of SLF
stimulus lead to greater levels of cell death and induction of cell
death by SLF plus blockers occurs even in the presence of IL-3, this
suggests that activation by SLF is an essential component of this form
of apoptosis. Thus concurrent blockade of Ca2+ influx
effectively converts the SLF-mediated protective signal into a death
signal.
Several endpoints indicate that the cell death induced by the
combination of SLF and Ca2+ influx blockers is apoptotic.
These include specific morphologic characteristics such as membrane
blebbing and nuclear condensation, loss of membrane integrity as
indicated by failure to exclude Trypan Blue, DNA fragmentation,
protection by caspase inhibitors, and PARP cleavage. Although many of
these endpoints are also characteristic of apoptotic death caused by
factor withdrawal,5 an important difference is that,
whereas IL-3 or bcl-2 overexpression protects cells from factor
withdrawal, they do not protect these cells from apoptosis induced by
SLF plus Ca2+ influx blockade.
Other apoptotic signals have also been found to be bcl-2
insensitive.2 These signals include cell death induced by
tumor necrosis factor, Fas activation, activation-induced
cell death, and superantigen-mediated clonal deletion.44-48
It is therefore possible that disruption of calcium influx in the
context of activation may be an important component of such signals as
well. In support of this possibility, Kovacs and Tsokos49
showed that Fas activation inhibited anti-CD3-mediated Ca2+
influx in T cells without affecting Ca2+ release from
internal stores.
We found that ionomycin can protect cells from SLF plus blocker-induced
cell death, supporting our contention that specific blockade of
Ca2+ influx is required for apoptosis induction. Our
observation that higher levels of ionomycin restores cell death shows
that both extremes of Ca2+ influx can lead to apoptosis.
However, in both cases, cell activation is required, because only
minimal cell death is observed in the absence of SLF. As observed in
other cell systems,2,50 we found that IL-3 or bcl-2
overexpression protected cells from death induced by high
concentrations of ionomycin, highlighting the difference between
apoptosis caused by SLF plus Ca2+ influx blockade and
apoptosis caused by Ca2+ overload. In neurons, both low and
high levels of intracellular Ca2+ have been shown to lead
to apoptosis, whereas sustained levels of intracellular
Ca2+ generated through chronic depolarization lead to
protection from apoptotic death.51,52 These
observations are consistent with our data and may therefore indicate
that both high and low levels of intracellular Ca2+ can
affect the balance between survival and apoptosis in a variety of cell
types. One possible mediator of high Ca2+ death may be the
Ca2+-dependent phosphatase calcineurin, which has been
shown to potentiate apoptosis in T cells53 and B
cells.54
Our observation that caspase inhibitors protect cells from apoptosis
induced by SLF stimulation plus Ca2+ influx blockade,
coupled with the demonstration that PARP, a nuclear target of caspases
is cleaved by this treatment, indicates that this form of apoptosis is
effected through activation of a caspase cascade. However, the specific
molecular events responsible for activating this cascade remain to be
identified. SLF stimulation mobilizes Ca2+ through
activation of the enzyme phospholipase C-
(PLC-).55,56 It is therefore likely that PLC activation
is a minimal requirement. One prediction based on our observations is
that other signals that activate PLC should also be able to combine
with Ca2+ influx blockers to induce apoptosis. Our
preliminary observations indicate that this is indeed the case (J.L.G.
and S.A.B., unpublished data).
Ketotifen is a well-known anti-allergy and anti-asthmatic drug that
exhibits both histamine H1 receptor antagonism and inhibition of
Ca2+ influx.57,58 Given the importance of
Ca2+ influx for mast cell degranulation, inhibition of this
process is one accepted mechanism for its anti-allergic activity. Our observation that this compound also induces apoptosis when
coadministered with an activation signal such as SLF suggests that this
may be another mechanism by which ketotifen mediates its anti-allergic effects. Because increasing signal leads to increased apoptosis, this
form of cell death will be most effective when Ca2+ influx
blockers are combined with highly activated receptors. In support of
this possibility, we have observed that the p815 mastocytoma, which has
a constitutively activated c-Kit receptor,26,27 dies
apoptotically when exposed to Ca2+ influx blockers alone
(Fig 3).
Other investigators have proposed Ca2+ influx blockers as
antiproliferative agents.59-64 Our data suggest that these
compounds will be most effective when coupled with a signal such as SLF that mobilizes Ca2+. Therefore, the combination of
Ca2+ influx blockers with such activating signals may have
potent, but specific, antiproliferative or anti-inflammatory
properties.
| |
FOOTNOTES |
Submitted August 4, 1997;
accepted October 30, 1997.
Supported by grants from the National Cancer Institute of Canada and
the Leukemia Research Fund of Canada to S.A.B.
Address reprint requests to Stuart A. Berger, PhD, Wellesley Hospital
Research Institute, 160 Wellesley St E, Toronto, Ontario, Canada M4Y
1J3.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank M. Minden for the 32D-Kit cells, Y. Ben-David for the
bcl-2 retrovirus-producing cells, and R. Chow for technical assistance.
| |
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[Abstract]
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M. Haslauer, K. Baltensperger, and H. Porzig
Erythropoietin- and Stem Cell Factor-Induced DNA Synthesis in Normal Human Erythroid Progenitor Cells Requires Activation of Protein Kinase Calpha and Is Strongly Inhibited by Thrombin
Blood,
July 1, 1999;
94(1):
114 - 126.
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Abstract
Introduction
Abstract