(Received for publication, December 7, 1995)
From the
COS cells are resistant to cell death induced either by
interleukin-1-converting enzyme (*ICE) and ICE homolog
(ICH-1
) overexpression or by serum deprivation. COS cells
deprived of serum undergo apoptosis after transfection with an ICE
expression construct, but not an ICH-1
construct.
ICE-mediated apoptosis of COS cells in serum-free medium is suppressed
by insulin-like growth factor (IGF)-1 and insulin. Viability of Rat-1
cell line (Rat-1/ICE) expressing low levels of ICE-LacZ fusion protein
is lower than those of cell lines expressing either both Bcl-2 and ICE
or mutant ICE
during serum deprivation.
Enzymatic activation and processing of ICE are observed in cells
induced to die by serum deprivation, which are suppressed by IGF-1.
IGF-1 or insulin suppresses ICE-mediated cell death without affecting
the expression levels of Bcl-2, Bcl-x, or Bax. Taken together, these
results indicate that ICE is activated by growth factor deprivation,
and IGF-1 is able to suppress ICE-mediated cell death through a
mechanism independent of the expression of Bcl-2, Bcl-x, or Bax.
Members of the mammalian ICE family are homologs of Caenorhabditis elegans programmed cell death gene product
Ced-3(1, 2, 3, 4, 5, 6, 7, 8) .
Overexpression of Ice or Ich-1 induces
apoptosis of cultured cells in cell type-specific
manner(2, 5) . ICE is a novel cysteine protease which
is inhibited by the product of crmA gene, a serpin encoded by
cowpox virus(9, 10, 11) . CrmA prevents or
delays apoptosis induced by various stimuli such as tumor necrosis
factor-
(TNF-
)(
)(7, 12) ,
anti-FAS antibody(7, 13, 14, 15) ,
and neurotrophic factor deprivation(16) . Moreover, peptide
inhibitors of the ICE protease family suppress death of motoneurons
during development and in culture(17) . Together, these
observations suggest that the ICE family plays an important role in
apoptosis.
Recent work has shown a correlation between the
regulation of Ice expression or ICE processing and apoptosis.
Increased Ice expression correlates with the onset of
apoptosis induced by the loss of extracellular matrix in epithelial
cells(18) . DNA damage-induced interferon regulatory factor
(IRF)-1 dependent T lymphocyte apoptosis is preceded by increase in Ice transcription, and it appears that impairment of Ice expression in IRF-1 (-/-) splenocytes may contribute
to the suppression of DNA damage-induced apoptosis(19) .
Furthermore, evidence of enzymatic activation of ICE has been observed
in cells induced to die by treatment of TNF- in the presence of
cyclohexamide(12) . HeLa cells treated with both TNF-
and
cycloheximide undergo apoptosis and secret mature interleukin-1
(IL-1
), which is mainly processed by ICE(12) . These
observations strongly suggest that ICE plays an important role in
controlling mammalian apoptosis, and furthermore, ICE may function at a
convergent step in apoptosis induced by various stimuli.
Recently, insulin-like growth factor (IGF-1) and its receptor have been implicated in playing an important role in tumorigenesis(20, 21, 22) . Inhibition of tumorigenesis was observed in nude mice injected with human FO-1 melanoma cells in which the number of IGF-1 receptors was markedly reduced by antisense. Some viral oncogenes may be capable of activating IGF-1 gene expression to promote transformation. Simian virus 40 large T (SV40 T) antigen activates the IGF-1 promoter and thereby increases the secretion of IGF-1(23) . This observation may explain the lack of transformation by SV40 T antigen in IGF-1 receptor-deficient mice(24) . Expression of c-Myb also increases the secretion of IGF-1(25) . In contrast, the Wilms' tumor gene, a tumor suppressor, has been reported to down-regulate the expression of the IGF-1 receptor and IGF-2(26) . These observations suggest that IGF-1 and IGF-1 receptors are important and may be essential components for cell transformation. Since increasing evidence suggests that suppression of apoptosis is a critical step in tumorigenesis, it is possible that IGF-1 could promote tumorigenesis by inhibiting apoptosis.
Because the ICE family is critical for apoptosis and the inhibition of apoptosis by growth factors, such as IGF-1, may be an important step in tumorigenesis, we investigated the potential interactions between growth factors and the ICE family. Here we show the regulation of ICE-mediated apoptosis by growth factors, which is different from the inability of ICH-1 to induce apoptosis of serum-deprived COS cells. ICE was activated in Rat-1 and COS-1 cells induced to die by serum deprivation, as evidenced by the processing of ICE protein. IGF-1 and insulin, but not EGF and FGF, suppressed ICE processing and ICE-mediated cell death.
To detect the expression of chimeric lacZ genes in transfected cells, cells were fixed with 1%
glutaraldehyde for 10 min, rinsed three times with phosphate-buffered
saline, and stained in X-gal buffer (0.5 mg/ml
5-bromo-4-chloro-3-indolyl -galactoside, 3 mM K
Fe(CN)
-
H
O, 3
mM K
Fe(CN)
-
H
O,
1 mM MgCl
, 10 mM KCl, 0.1% Triton X-100
in 0.1 M sodium phosphate buffer (pH 7.5)) at 37 °C for 3
h.
In the absence of growth factors, cells undergo apoptosis(5, 16) . To investigate the effects of individual growth factors on cell death, Rat-1 cells were induced to die by serum deprivation and the effect of growth factors on suppression of serum deprivation-induced cell death was examined (Fig. 1). 50% of Rat-1 cells underwent apoptosis after 2 days of incubation in serum-free defined medium (Fig. 1, A and B, Control). To dissect out the effects of individual growth factors on the suppression of apoptosis, growth factors were added individually to the serum-free defined medium. Addition of IGF-1 (100 ng/ml) or insulin (5 µg/ml) to the serum-free defined medium increased cell viability to 85 and 89%, respectively. While PDGF (10 ng/ml) showed weaker effects (70% viability), EGF (10 ng/ml) and bFGF (100 ng/ml) had no effects on cell survival. These results suggest that IGF-1 and insulin suppress Rat-1 cell death induced by serum deprivation.
Figure 1: Prevention of serum deprivation induced cell death by IGF-1 and insulin. A, Rat-1 cells were incubated in serum-free defined medium (Control) or defined medium supplemented with growth factors (either IGF-1: 100 ng/ml; insulin: 5 µg/ml; PDGF: 10 ng/ml; EGF: 10 ng/ml; or bFGF: 100 ng/ml) for 2 days and photographed using a light microscope. B, the viability of the same cells shown in A was determined by staining with 0.4% trypan blue. Each point indicates mean ± S.E. from at least three different experiments.
To examine whether ICE is activated when cells are
induced to die by serum deprivation and whether ICE-induced cell death
can be suppressed by growth factors, we established an cell culture
assay system. In contrast to Rat-1 cells, COS cells are resistant to
cell death induced by Ice and Ich-1 overexpression in the presence of 10% serum (5) and can
survive at least 5 days in the absence of serum. However, serum
deprivation after transfection with an Ice construct
(p
actM10Z) expressing murine ICE-LacZ fusion protein induces COS
cell death (Fig. 2A), indicating that serum in cell
culture medium suppresses ICE-induced cell death in COS cells. Serum
deprivation does not enhance the ability of ICH-1
to induce
COS cell death, showing different sensitivities of ICE and ICH-1
to the effects of growth factors (Fig. 2A). To
further characterize the effect of growth factors on the suppression of
ICE-induced COS cell death, COS cells were transfected with
p
actM10Z and maintained in serum-free defined medium supplemented
with growth factors for 2 days (Fig. 2B). Addition of
IGF-1 (100 ng/ml) or insulin (5 µg/ml), but not EGF (10 ng/ml), to
the defined medium suppressed ICE-induced COS cell death. PDGF (10
ng/ml) and FGF (100 ng/ml) showed weaker effects on ICE-induced cell
death. These results suggest that ICE is activated by serum
deprivation, and either IGF-1 or insulin is able to suppress
ICE-induced cell death.
Figure 2:
Different sensitivities of ICE and ICH-1
to growth factors. A, COS-1 cells were transiently transfected
with either pactgal (lacZ alone), p
actM10Z (mIce-lacZ), or p
actH37Z (Ich-1
-lacZ) plasmids and incubated with
DMEM containing 10, 1, or 0% FCS. After 2 days, cells were incubated in
X-gal buffer as described under ``Experimental Procedures''
and examined for apoptosis based on the morphology of the blue cells. B, prevention of ICE-induced cell death by IGF-1 and insulin.
COS-1 cells are transiently transfected with p
actM10Z (mIce-lacZ) plasmid and maintained in serum-free medium
supplemented with growth factors for 2 days. Cells were fixed and
stained with X-gal buffer and examined for apoptosis. The
concentrations of growth factors are same as those in Fig. 1.
Data are from three independent experiments with mean ±
S.E.
To further characterize the effects of IGF-1
and insulin on ICE-induced cell death, Rat-1 cell lines expressing
either low levels of ICE-LacZ (Rat-1/ICE), both Bcl-2 and ICE-LacZ
(Rat-1/Bcl-2/ICE), or mutant ICE-LacZ (Rat-1/mICE),
in which the active site pentapeptide QACRG had been changed into
QACRS, were established. In the presence of 10% serum in the cell
culture medium, cells transfected with either p
actM10Z (ICE-lacZ) or pJ485 (mutant ICE
-lacZ) were selected with
G418 and individual clones were examined for the expression levels of
ICE-LacZ fusion proteins by Western blot analysis using
anti-
-galactosidase antibody (Fig. 3A). Expression
level of ICE-LacZ fusion protein in the Rat-1/ICE cells is at least
2-3-fold lower than mutant ICE
-LacZ fusion
protein of Rat-1/ICE
cells (Fig. 3A,
lane 2 and 3) in which mutant ICE
does not induce cell death and is almost similar to that of
ICE-LacZ protein in Rat-1/Bcl-2/ICE cells in which Bcl-2 inhibits cell
death inducing activity of ICE (Fig. 3A, lane 4).
Expression level of LacZ protein in Rat-1/ICE,
Rat-1/mICE
, and Rat-1/Bcl-2/ICE cells was also
examined by immunostaining using anti-
-galactosidase antibody and
is similar to that of Western blot analysis (data not shown).
Figure 3:
Western
blot analysis showing processing and activity of ICE-fusion protein in
dying Rat-1/ICE and COS cells. A, processing of ICE-LacZ
fusion protein in Rat-1/ICE cells. Cell lysates were prepared from live
cells attached to tissue culture plate (lanes 1-4) and
dead cells detached into cell culture medium (lane 5) as
described under ``Experimental Procedures'' and
electrophoresed on 8% SDS-polyacrylamide gel. Anti--galactosidase
antibody was used to detect the ICE-LacZ fusion protein. The molecular
sizes in kilodaltons are indicated at the left. The different
sizes of ICE-LacZ fusion proteins, p45-LacZ and p10-LacZ, in live and
in dead Rat-1/ICE cells, respectively, are indicated by the arrowheads to the right. Lane 1, Rat-1; lane 2, Rat-1/ICE; lane 3,
Rat-1/mICE
; lane 4, Rat-1/Bcl-2/ICE; lane 5, Rat-1/ICE (Dead). B, processing of
ICE in dying COS cells. COS cells are transiently transfected with
p
actM10T and incubated in serum-free defined medium without or
with IGF-1 (100 ng/ml) for 2 days. Live cells (Live) attached
to and dead cells (Dead) detached from the cell culture plate
were separately collected and analyzed on 12% SDS-polyacrylamide gel.
Western blot analysis was performed using anti-ICE antibody (left
panel) or anti-T7 tag antibody (right panel). p45-T7,
p30-T7, p20, and p10-T7 indicated by arrows are ICE detected
by anti-T7 tag and/or ICE antibodies. NS indicates nonspecific
protein recognized by anti-T7 tag antibody. C, suppression of
ICE activity by IGF-1. COS cells were co-transfected by p
actM10T
and pRc/CMV-hIL-1
(2:1 ratio) plasmids and maintained in the
defined medium without or with IGF-1 (100 ng/ml) for 2 days. The
hIL-1
was detected by Western blot analysis (13.5%
SDS-polyacrylamide gel) using anti hIL-1
antibody from the cell
lysate or from the defined medium which has been concentrated after
dialysis in distilled water.
Using
the cell lines described above, the effects of growth factors on
ICE-induced cell death were examined (Table 1). In the presence
of 10% serum, the viability of Rat-1 cells expressing low levels of ICE
was similar to that of control Rat-1 cells (96% versus 98%, Table 1). After serum deprivation, the viability of Rat-1/ICE
cells was 72%, which is lower than those of Rat-1 cells (93%),
Rat-1/Bcl-2/ICE cells (98%), and Rat-1/mICE cells (91%), showing that Rat-1 cells expressing ICE are more
sensitive to cell death induced by serum deprivation, as observed in
COS-1 cells transfected with p
actM10Z. This result suggests that
ICE activity is suppressed by serum and that ICE is activated upon
serum removal. The effects of individual growth factors on the
ICE-mediated cell death in the Rat-1/ICE cells were examined (Table 2). IGF-1 and insulin were added to the defined medium and
examined for the effects on the cell death. Quantitative analysis shows
that addition of increasing amounts of either IGF-1 or insulin to the
serum-free medium increased the viability of Rat-1/ICE cells from 72 to
94% at 0.8 nM IGF-1 and 91% at 0.34 µM insulin,
respectively. These results indicate that ICE-mediated cell death
triggered by serum deprivation is suppressed by IGF-1 or insulin,
consistent with the results observed in COS-1 cells (Fig. 2B).
To examine activation of ICE, Rat-1/ICE
cells were induced to undergo apoptosis by serum deprivation, stained
with X-gal buffer, and viability was determined based on morphology.
Rat-1 cells induced to die by Ice-lacZ expression are small
and round blue after X-gal staining(2) . Serum deprivation
induced the appearance of round blue cells in Rat-1/ICE culture (Fig. 4A, top), but not in Rat-1/mICE and Rat-1/Bcl-2/ICE culture which are resistant to serum
deprivation-induced cell death and show no color development after
X-gal staining (data not shown). Addition of IGF-1 to the defined
medium inhibited the appearance of round blue Rat-1/ICE cells (Fig. 4A, bottom). Rat-1/ICE cells in 10% serum are
also healthy and flat and do not show color development after X-gal
staining (data not shown). Western blot analysis (Fig. 3A,
lane 2 and 5) and immunostaining (data not shown) using
anti-
-galactosidase antibody showed that Rat-1/ICE cells contained
similar amounts of ICE-LacZ fusion protein in both the absence and
presence of IGF-1. These results show a correlation between appearance
of round blue cells and cell death in the Rat-1/ICE cells. In Rat-1/ICE
cells, the addition of increasing concentrations of IGF-1 or insulin to
the defined medium significantly decreased the numbers of round blue
cells by 20-fold (IGF-1: 100 ng/ml) and 13-fold (insulin: 5 µg/ml),
respectively (Fig. 4B).
Figure 4:
Activation and suppression of ICE.
Rat-1/ICE cells are maintained in the defined medium containing no
serum or the defined medium supplemented with either IGF-1 (100 ng/ml)
or insulin (5 µg/ml) for 1 day. After X-gal reaction, cells were
photographed in A, and the round blue cells in fixed area (90
mm) were counted (B). Data are from at least two
different experiments with mean ±
S.E.
ICE was isolated as an
heterodimeric enzyme composed of 10-kDa (p10) and 20-kDa (p20) subunits
derived from a 45-kDa precursor protein (p45) (10, 27) . To examine whether ICE is processed to be
activated by serum deprivation, Western blot analysis using
anti--galactosidase antibody was performed using cell lysates
prepared from cells undergoing apoptosis (Fig. 3A, lane
5). Most of the ICE-LacZ fusion protein detected in live Rat-1/ICE
cells (lane 2) is pro-ICE-LacZ (p45-LacZ), indicated by its
molecular mass (160 kDa). In contrast, after serum deprivation, a
different pattern of protein bands was detected (Fig. 3A,
lane 5) in which ICE is processed from p45-LacZ precursor protein
to 120 kDa, consistent with releasing of p10-LacZ from ICE-LacZ fusion
protein.
To further characterize the processing of ICE during cell
death, COS cells were transiently transfected with pactM10T (a p45
ICE expression plasmid containing T7 epitope at the carboxyl terminus
of ICE) and induced to die by growth factor deprivation. Floating
(dead) or adherent (live) cells from each cell culture plate were
collected and analyzed for the processing of ICE protein by Western
blot analysis using anti-ICE and anti-T7 tag antibodies (Fig. 3B). In the absence of IGF-1, ICE was processed
into p10, p20, and 30 kDa (p30) in the floating dying cells. In
contrast, ICE was not proteolytically processed in adherent live cells.
This result indicates that ICE is processed into its subunits in dying
cells and that the processing is suppressed by IGF-1.
One of the
enzymatic functions of ICE is to process pro-IL-1 (31 kDa) into
its bioactive mature IL-1
(17.5 kDa), a secreted form. To examine
enzymatic activation of ICE in growth factor-deprived cells, the
production of mature IL-1
was examined in COS cells (Fig. 3C). Because COS-1 cells do not produce IL-1
which are detectable by Western analysis, COS cells were transiently
co-transfected with p
actM10T and pRc/CMV-hIL-1
(a human
pro-IL-1
expression vector) and maintained in defined medium. In
the presence of IGF-1, pro-IL-1
(31 kDa) was detected in the cell
lysates and no mature IL-1
was detected. However, in the absence
of IGF-1, lower levels of pro-IL-1
were observed in cell lysates,
and mature IL-1
(17.5 kDa) was detected in the defined medium.
This result shows that IGF-1 suppresses the production of mature
IL-1
and suggests that IGF-1 affects the inhibition of ICE
enzymatic activity.
We have reported previously that expression of crmA in chicken dorsal root ganglion neurons suppresses cell death
induced by trophic factor removal, suggesting that ICE or ICE-like
proteases play critical roles in cell death induced by trophic factor
deprivation (16) . Here, we show that ICE is processed to be
activated during serum deprivation-induced cell death in
ICE-transfected immortalized Rat-1 fibroblasts and oncogene-transformed
COS-1 cells. ICE-transfected, but not ICH-1-transfected,
COS-1 cells can be induced to die by serum removal, thus, activity of
ICE might be more responsive to environmental clues than that of ICH-1.
We succeeded in establishing stable Rat-1 cell lines expressing a
mouse wild type Ice fused with an Escherichia coli lacZ gene. These cell lines, however, are unstable initially; the
expression of Ice is lost easily. After cloning, we obtained
several clones that appeared to stably express Ice. However,
we may have selected for cells which express high levels of ICE
inhibitor: since these cells do not turn blue after X-gal staining when
cultured in the presence of 10% serum, despite of the presence of
ICE-LacZ fusion protein detected by Western analysis and
immunocytochemistry. The dying Rat-1/ICE cells will turn blue when
cultured in the absence of serum under which condition significant
proportions of cell die. This is most likely due to the processing of
ICE: x-ray crystallographic analysis of the three-dimensional structure
of ICE has shown that ICE exists as a tetramer consisting of two p20
and two p10 subunits in a crystal lattice (28, 29) and
an evidence that the p10 and p20 subunits of ICE associate as oligomers
in transfected cells has been provided(30) . Since anti-ICE
antibodies currently available to us do not recognize the endogenous
activated p20 and p10 subunits of ICE, we followed the cleavage of an
ICE-LacZ fusion protein indirectly using an anti- galactosidase
antibody. After induction of apoptosis by serum deprivation, we
observed approximately 120-kDa protein, apparently p10-LacZ, on Western
analysis, consistent with the activation of ICE. Rat-1/ICE/Bcl-2 and
Rat-1/mutant ICE
cells, which do not die in the
absence of serum, do not turn blue after X-gal staining, suggesting
that induction of apoptosis, but not serum removal per se,
induces
-galactosidase activity. We hypothesize the reason that
Rat-1/ICE cells in the presence of 10% serum or IGF-1 do not turn blue
after X-gal staining is that ICE is in a stable complex either with
itself or with certain unknown inhibitors, which might prevent the
activity of
-galactosidase.
Growth factor removal from COS
cells clearly induces the processing of exogenous ICE in dying cells.
Two subunits of p10 and p20 and an intermediate p30 are present in
dying cells, showing that ICE might be processed into an intermediate
p30 and, then, into p10 and p20. p30 is slightly more active than p45
in inducing cell death (2) and, thus, may be processed into p10
and p20 more easily than that of p45. In dying cells, the biological
consequence of ICE processing into its subunit p10 and p20 is the
activation of ICE. When COS cells were transfected with pactM10T
and pRc/CMV-hIL-1
expression vectors and induced to die by serum
removal, ICE was processed (Fig. 3B), and mature
IL-1
was produced and secreted into the defined medium (Fig. 3C). In addition, the presence of IGF-1 or
insulin suppressed both ICE-mediated cell death ( Fig. 2and Fig. 4) and the secretion of mature IL-1
in COS cells (Fig. 3C). These results suggest the correlation between
ICE-mediated apoptosis and pro-IL-1
processing and that IGF-1 may
inhibit ICE activation.
What would be the physiological relevance of
Rat-1/ICE cells? Tamura et al.(19) showed that Ice is induced in splenocytes following stimulation with
concanavalin A and that induction of Ice gene expression in
these mitogen-activated mature T lymphocytes enhances the
susceptibility to cell death induced by -radiation or other
DNA-damaging chemotherapeutic agents, such as adriamycin and etoposide.
In IRF-1-deficient mice, ICE is not induced after concanavalin A
treatment, and mitogen-activated mature T lymphocytes are much more
resistant to
-radiation and DNA-damaging agents, suggesting that
induction of ICE may be critically important for the sensitivity to
radiation-induced apoptosis in these cells. In addition, expression of
ICE correlated with the loss of extracellular matrix which regulates
apoptosis in mammary epithelial cells(18) . Rat-1/ICE cells may
mimic such conditions that ICE has been induced.
IGF-1 is a regulator of cell growth and metabolism(31) . What could be the downstream target molecules for the suppression activity of ICE-mediated cell death by IGF-1? IGF-1 and insulin preferentially activate cellular responses via IGF-1 and insulin receptors(31) , respectively, but appear to share a common downstream signal transduction pathway so far identified(32) . In addition, IGF-1 and insulin cross-react with each other receptors(31) . These observations suggest that the suppression of ICE-mediated cell death by IGF-1 and insulin is likely mediated by a common pathway. Even though the mechanism responsible for the suppression of ICE-mediated apoptosis by IGF-1 remains to be elucidated, several possibilities could be considered. The ICE-LacZ fusion protein is uncleaved in healthy Rat-1/ICE cells and, conversely, is cleaved in dying cells. This is consistent with the observation that ICE processing is required for its activity(27) . Thus, regulation of ICE cleavage appears to be an important step in induction of cell death. IGF-1 and insulin may function by enhancing the activity of survival factors which inhibit the activation of ICE. Bcl-2 family inhibits or delays the cell death induced by a variety of stimuli (33) and also suppresses ICE-induced cell death(5) . We observed that IGF-1 and insulin do not affect the expression levels of the Bcl-2 family members examined. The levels of Bcl-2(34) , Bcl-x(35) , and Bax (36) are similar in IGF-1 and insulin-treated and untreated cells on Western blot analysis (data not shown). Alternatively, inhibition of ICE activation by IGF-1 or insulin may be mediated through posttranslational modification of ICE and/or other cell survival effector molecules. Potential mechanism for suppression of ICE-mediated cell death by IGF-1 and insulin currently are under investigation.