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Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 1891-1900
By
From the Wellesley Hospital Research Institute and Department of
Immunology, University of Toronto, Toronto, Ontario, Canada.
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.
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.
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 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- 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.
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
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.
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.
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.
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.
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.
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.
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.
Submitted August 4, 1997;
accepted October 30, 1997.
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
Introduction
Abstract
-mercaptoethanol and antibiotics
(both Sigma).
) 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.
) 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.
) 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.
(PLC-